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Effects of Progranulin on Blastocyst Hatching and Subsequent Adhesion and Outgrowth in the Mouse
Abstract
Using cDNA microarray methodology, we have shown previously that transcripts of progranulin gene (Grn, also known as acrogranin), a recently identified autocrine growth factor, were upregulated in mouse blastocysts adhered to the filter membrane in an in vitro-culture system. In the present study, we investigated the expression and effects of progranulin on blastocyst hatching, adhesion, and embryo outgrowth during the peri-implantation period in the mouse. During this period, substantial amounts of Grn mRNA were present in both inner cell mass (ICM) and trophectoderm. Progranulin was localized exclusively to the surface of the trophectoderm in early and pre- and postadhesion blastocysts as well as in trophoblast cells and ICM of outgrowth embryos, being secreted as a single, 88-kDa form into the surrounding medium. NIH3T3 cells that had been transfected with a progranulin expression construct secreted the 88-kDa form of the protein, from which a 68-kDa form could be generated by deglycosylation. In vitro treatment of blastocysts with recombinant progranulin promoted blastocyst hatching, adhesion, and outgrowth, whereas rabbit anti-mouse progranulin immunoglobulin G reduced the incidence of blastocyst hatching, adhesion, and outgrowth. Studies of bromodeoxyuridine incorporation and immunodissection of the ICM revealed that progranulin was effective on the trophectoderm but not on the ICM. These results indicate that progranulin is an important factor for the processes of blastocyst hatching, adhesion, and outgrowth, and they suggest that the effects of progranulin on blastocyst adhesion and outgrowth may have been triggered by the previous action of progranulin to induce hatching of the blastocysts.
... Furthermore, the expression of PGRN has been reported in preimplantation embryos with the highest levels in the blastocyst stage (117). In addition to blastocyst outgrowth, PGRN can promote the adhesion of blastocyst (118). The maternal and embryonic expression of PGRN suggests its role in the implantation and placentation process. ...
... It has also been documented that the expression of PGRN is increased during human embryo implantation, and dysregulated PGRN expression is associated with pregnancy-related diseases such as abnormal placental angiogenesis (122). Qin et al. (118) have indicated that PGRN is secreted from the mammalian blastocysts into the surrounding medium and it is involved in the preparation of the blastocyst for hatching and implantation. In this regard, they reported that adding exogenous PGRN to the culture medium could considerably increase blastocyst hatching, an essential process for implantation (118). ...
... Qin et al. (118) have indicated that PGRN is secreted from the mammalian blastocysts into the surrounding medium and it is involved in the preparation of the blastocyst for hatching and implantation. In this regard, they reported that adding exogenous PGRN to the culture medium could considerably increase blastocyst hatching, an essential process for implantation (118). Overall, according to the results of studies, PGRN might be an effective factor for embryo implantation, however, to clarify the exact role of this adipokine in implantation further studies are required. ...
Embryo implantation is a complex process in which multiple molecules acting together under strict regulation. Studies showed the production of various adipokines and their receptors in the embryo and uterus, where they can influence the maternal-fetal transmission of metabolites and embryo implantation. Therefore, these cytokines have opened a novel area of study in the field of embryo-maternal cross-talk during early pregnancy. In this respect, the involvement of adipokines has been widely reported in the regulation of both physiological and pathological aspects of the implantation process. However, the information about the role of some recently identified adipokines is limited. This review aims to highlight the role of various adipokines in embryo-maternal interactions, endometrial receptivity, and embryo implantation, as well as the underlying molecular mechanisms.
... Inhibition of progranulin inhibits the onset of blastocoel formation in mice, whereas stimulation enhances the differentiation and proliferation of murine trophoblast cells [5]. It further stimulates blastocyst hatching and excites trophoblast adhesion and outgrowth in vitro [8]. At later stages of murine placental development (days 7.5-8.5, ...
... As observed by Qin et al. in mice, we detected the glycosylated as well as the deglycosylated forms of progranulin in Western blot analysis. Qin et al. found in the outgrowing part of murine blastocysts only the glycosylated form of progranulin and assumed an association between the grade of glycosylation and the proliferative as well as invasive capacity of the cells [8]. ...
... The concentrations we measured were in the range of normal plasma levels in healthy adults [18,19]. Despite the data for a functional relevance regarding murine blastocyst hatching and adhesion as well as outgrowth and proliferation of trophectoderm cells in vitro [8], we did not have any data for the function of progranulin in later stages of placentation. In our study, the proliferation of BeWo cells was significantly stimulated by progranulin at physiologic concentrations. ...
Expression of the glycoprotein progranulin has been recently identified in rodent trophoblast cells during early embryonic development. The aim of our study was to describe the expression pattern of progranulin in human placental tissue specimens by immunostaining. We further analyzed the influence of progranulin on invasion and migration of isolated first trimester villous trophoblast cells. The effect of progranulin on cell proliferation was investigated using the human choriocarcinoma derived cell lines BeWo and Jeg-3. Cells were tested with recombinant human progranulin at various concentrations (0.1, 0.2 and 1.0 µg/ml). The strongest expression of progranulin was observed in the villous trophoblast cells, particularly in the syncytiotrophoblast. The intensity of staining in these cells was higher in the first trimester than in the third trimester. In contrast, the staining of the extravillous trophoblast cells and of the villous and decidual stroma was only weak. Using an ELISA technique, we also detected progranulin in amniotic fluid of the early second trimester. Isolated human first trimester trophoblast cells also expressed and secreted progranulin. Progranulin significantly stimulated the cell proliferation of BeWo cells, but it did not influence the amount of trophoblast cell migration and invasion in vitro. Furthermore, it did not promote the cell proliferation of Jeg-3 cells. Our results suggested that progranulin, although it is mainly synthesized and secreted by villous trophoblast cells, may not primarily act on the villous trophoblast cells in a paracrine or autocrine manner. The observed effect of progranulin on cell proliferation in BeWo cells may indicate a growth stimulating effect also on the small part of proliferating extravillous trophoblast cells during placental development.
... Recent work suggests that progranulin, a secreted glycoprotein and a growth factor, is involved in mouse trophoblast proliferation (Diaz-Cueto et al. 2000, Qin et al. 2005), placental development, and endometrial decidualization (Daniel et al. 2003). Progranulin, also known as granulin–epithelin precursor (Zanocco-Marani et al. 1999), proepithelin (Plowman et al. 1992), PC cellderived growth factor (Zhou et al. 1993), or acrogranin (Baba et al. 1993), is a 66–88 kDa protein (Zhou et al. 1993) comprising 7.5 granulin/epithelin consensus sequences, each of these consisting of a conserved 12-cysteine motif (Bhandari et al. 1992). ...
... Moreover, mutations in the progranulin gene were found to cause neurodegenerative frontotemporal dementia, suggesting roles for progranulin in the maintenance of healthy neurons (Baker et al. 2006, Cruts et al. 2006). Progranulin transcripts have been detected in rodent oocytes, spermatocytes, and spermatogonia (Daniel et al. 2000, Diaz-Cueto et al. 2000, Suzuki & Nishiahara 2002) and have been found in the embryo throughout pre-implantation development (Diaz-Cueto et al. 2000, Qin et al. 2005). After fertilization, the levels of progranulin mRNA slowly decline until the four-cell stage embryo in the mouse and rise again in the eightcell stage embryo (Diaz-Cueto et al. 2000). ...
... After fertilization, the levels of progranulin mRNA slowly decline until the four-cell stage embryo in the mouse and rise again in the eightcell stage embryo (Diaz-Cueto et al. 2000). Progranulin can promote mouse blastocyst formation by enhancing trophoblast proliferation, blastocyst cavitation, and expansion (Diaz-Cueto et al. 2000, Qin et al. 2005). Progranulin also stimulates hatching, adhesion, and outgrowth of the mouse blastocyst in vitro (Qin et al. 2005). ...
Embryo implantation in the mink is preceded by a variable but obligate period of delay in development. Under the influence of progesterone and unknown luteal factors, the mink embryo implants 11-13 days following its exit from diapause. Recent work suggests that progranulin, a growth factor and secreted glycoprotein, is involved in trophoblast proliferation, placental development and endometrial differentiation in the mouse. Using the mink model of delayed implantation and endotheliochorial placentation, we examined the spatiotemporal distribution of progranulin in trophoblast and endometrium during pre- and early post-implantation gestation in vivo. A partial sequence of the mink progranulin gene was cloned and sequenced. Comparative sequence analysis revealed that exons 1 and 2 of mink progranulin share 86.6, 82.4, and 94.9% of nucleic acid sequence identity with the human, mouse, and dog sequences respectively, and indicated that the invariable residues of the cysteine-rich motifs of progranulin are well conserved in the mink sequence. By in situ hybridization, we show that mink progranulin transcript is present in the cytotrophoblast and in epithelial and stromal endometrial cells at the site of implantation and during early placental formation. Immunohistochemistry revealed the progranulin protein to be strongly expressed in endometrial luminal and glandular epithelium around the time of implantation. In the incipient labyrinth, progranulin expression is localized to cytotrophoblasts and fetal capillaries, as well as to the hypertrophied maternal endothelial cells. This study demonstrates that high levels of progranulin expression correspond to active cell proliferation, remodeling, and angiogenesis occurring during the establishment of the placenta in the mink.
... GRN is a pleiotropic glycoprotein and growth factor with proliferative, invasive, and anti-inflammatory properties [17]. Despite the fact that a couple of studies have attributed GRN a role within blastocyst development and implantation of mice and mink [12,13,31], very little is known of its function in human endometrium. In our study, GRN mRNA expression is considerably upregulated in women with EM compared with that in healthy controls, which may contribute to its role in inflammation and cell proliferation process. ...
... Moreover, our findings of GRN protein expression show a significant continuous reduction in expression level the higher the degree of the disease. While Qin et al. demonstrated that the addition of recombinant GRN in blastocyst culture media promoted blastocyst hatching, adhesion, and outgrowth, rabbit anti-mouse GRN IgG reduced that effect [31]. The reduction of GRN in the endometrium of women with EM may have a negative effect on blastocyst development, potentially contributing to a reduction of implantation rates in affected women, as seen in women with EM according to the severity of the disease [37]. ...
Purpose
Endometriosis (EM) is a common gynecological disease affecting 10–15% of women of reproductive age. However, molecular mechanisms and pathogenesis are still not completely understood. Furthermore, due to the absence of a reliable clinical biomarker, the only viable method for the often-delayed definitive diagnosis is laparoscopic surgery. Our objective was to analyze molecular differences of selected endometrial proteins and genes of women suffering from different stages of EM compared with healthy women to evaluate potential clinical biomarkers.
Methods
We analyzed eutopic endometrial tissue samples from women undergoing a laparoscopic surgery (n = 58). mRNA gene expression of progranulin (GRN), neurogenic locus notch homolog protein (NOTCH3), fibronectin (FN1), and PTEN-induced kinase 1 (PINK1) was analyzed using qRT-PCR. Protein expression was determined using ELISA and immunohistochemistry.
Results
Significant differences in gene expression between the different stages of the disease were noted for GRN, NOTCH3, FN1, and PINK1 (p < 0.05). The endometrium of women with minimal EM (ASRM I) showed the highest mRNA expression. Protein levels of GRN and FN1 on the other hand were significantly decreased in the endometrium of women with EM compared with those of healthy controls. Furthermore, for GRN and FN1, we could detect a correlation of protein expression with the severity of the disease.
Conclusion
Our findings suggest a potential use of GRN and FN1 as clinical biomarkers to detect endometriosis. In addition, GRN, NOTCH3, FN1, and PINK1 could potentially be useful to differentiate between the underlying stages of the disease. However, a validation with a larger study population is needed.
... PGRN can stimulate the cell proliferation of BeWo cells, indicating a growth-stimulating effect during the development of the placenta (19). Furthermore, PGRN is a key factor in blastocyst hatching, adhesion and outgrowth (20). The process of mouse blastocyst formation is delayed when PGRN expression is inhibited (21). ...
... This fluctuation may be associated with placental secretion (27). However, although PGRN has been linked to blastocyst formation in reproductive development and PE pathology in several studies (17,(20)(21)(22)(23), its specific role in the development of placental blood vessels, particularly in the placental labyrinth, remains to be elucidated. ...
Progranulin (PGRN) is a secreted growth factor involved in pleiotropic functions, particularly angiogenesis. A distinctly different placental expression of PGRN has been reported between normal pregnancies and pregnancies with complications, such as pre‑eclampsia or fetal growth restriction. However, the role of PGRN in placental vascular development remains to be elucidated. In the present study, PGRN‑knockout mice (PGRN‑/‑) were used to investigate the role of PGRN in the development of placental blood vessels and placental formation. Placental weights and pup body weights were significantly lower in the PGRN‑/‑ mice compared with the wild‑type mice. Reduced labyrinthine layer areas and aberrant vascularization were also observed via hematoxylin and eosin staining of PGRN‑/‑ mice at embryonic day 14.5 (E14.5) and E17.5. In addition, the morphological data obtained via immunohistochemistry, immunofluorescence staining and western blotting demonstrated decreased expression levels of the blood vessel markers α‑smooth muscle actin and CD31 in PGRN‑/‑ placentas. Furthermore, vasodilator endothelial nitric oxide synthase was reduced in the PGRN‑/‑ placenta. These results indicated that PGRN serves an essential role in the normal angiogenesis of the placental labyrinth in mice.
... For this, the positivity sites of PCNA were compared to those of PGRN and HNF4α. PGRN has already been described as present in developing organs (LI et al., 1996;), including the liver (HE;), and as a growth factor and inductor of cell proliferation in the process of incubation, adhesion and growth of blastocysts (DANIEL et al., 2003; QIN et al., 2005). Thus, PGRN could also be acting in proliferation, and not only in differentiation of the developing liver. ...
... The results demonstrated that there is a peak of PCNA positivity at E12.5, and that positivity is lower at the other periods. Since the embryo is in development, with high cell proliferation, and PGRN has already been described as precursor of cell proliferation (LI et al., 1996; DANIEL et al., 2003; QIN et al., 2005), a peak of positivity was expected for PCNA simultaneously with the peak for HNF4α and PGRN, thus showing that PGRN has a function in cell proliferation during hepatogenesis, and this did not happen. Thus, it seems that first there is a peak of cell proliferation at E12.5, which can be induced by HNF4α, which is highly positive from E12.5 to E14.5. ...
Transplants are the only effective therapy for the treatment of advanced liver diseases such as cirrhosis. Given the limited number of organ donors, regenerative medicine has sought for sources of cells and tissues for replacement therapy. Embryonic stem cells are a promising source of material for transplantation because of their exclusive property of being expanded indefinitely in culture, thus, they are a source of replacement tissue. Moreover, they are capable of differentiating into practically all cell types, and may be utilized in replacement therapy in various diseases. The liver bud has bipotent stem cells that have not yet differentiated into hepatocytes or biliary duct cells; however, they have great potential of proliferation and differentiation. Thus, the challenge is to identify methods that promote their differentiation in specific and functional strains. This study aimed to evaluate the role of the progranulin growth factor PGRN during the liver development of rats F344, since this growth factor could be utilized in protocols of differentiation of stem cells of the liver bud in functional hepatocytes. The results showed that PGRN is present during different periods of hepatogenesis in F344 rats, and that this growth factor should be involved in the process of differentiation of hepatoblasts into hepatocytes after activation by HNF4α, however, PGRN seems not to exert a cellular proliferation function during the hepatogenesis. Thus, PGRN can be used in future protocols of liver cell differentiation directed toward cellular therapy in Regenerative Medicine.
... For this, the positivity sites of PCNA were compared to those of PGRN and HNF4α. PGRN has already been described as present in developing organs (LI et al., 1996;), including the liver (HE;), and as a growth factor and inductor of cell proliferation in the process of incubation, adhesion and growth of blastocysts (DANIEL et al., 2003; QIN et al., 2005). Thus, PGRN could also be acting in proliferation, and not only in differentiation of the developing liver. ...
... The results demonstrated that there is a peak of PCNA positivity at E12.5, and that positivity is lower at the other periods. Since the embryo is in development, with high cell proliferation, and PGRN has already been described as precursor of cell proliferation (LI et al., 1996; DANIEL et al., 2003; QIN et al., 2005), a peak of positivity was expected for PCNA simultaneously with the peak for HNF4α and PGRN, thus showing that PGRN has a function in cell proliferation during hepatogenesis, and this did not happen. Thus, it seems that first there is a peak of cell proliferation at E12.5, which can be induced by HNF4α, which is highly positive from E12.5 to E14.5. ...
Os transplantes sao a unica terapia eficaz para o tratamento de doencas hepaticas avancadas, como a cirrose. Dado o numero limitado de doadores de orgaos, a medicina regenerativa tem procurado fontes de celulas para a terapia de substituicao. As celulas embrionarias sao uma fonte promissora de material para o transplante devido a sua propriedade exclusiva de ser expandida indefinidamente em cultura, assim, elas sao uma fonte de tecido de substituicao. Alem disso, sao capazes de se diferenciar em praticamente todos os tipos celulares, e podem ser utilizadas na terapia de substituicao em varias doencas. O broto hepatico tem celulas-tronco (CT) bipotenciais que ainda nao se diferenciam em hepatocitos ou celulas do ducto biliar, contudo, elas tem um grande potencial de proliferacao e de diferenciacao. Desse modo, o desafio e identificar metodos que promovam sua diferenciacao em linhagens especificas e funcionais. Este estudo teve como objetivo avaliar o papel do fator de crescimento progranulina (PGRN) durante o desenvolvimento hepatico em ratos F344, uma vez que a PGRN poderia ser utilizada em protocolos de diferenciacao de CT do broto hepatico em hepatocitos funcionais. Os resultados mostraram que PGRN esta presente durante diferentes periodos da hepatogenese em ratos F344, e que a mesma deve estar envolvida no processo de diferenciacao de hepatoblastos em hepatocitos apos ativacao por HNF4α, no entanto, a PGRN parece nao desempenhar uma funcao de proliferacao celular durante a hepatogenese. Assim, a PGRN pode ser usada em futuros protocolos de diferenciacao de celulas hepaticas voltadas para a terapia celular na medicina regenerativa.
... Acrogranin activates both the PI3K and ERK pathway, and promotes the expression of cyclin D1 and cyclin B ). In addition, acrogranin has been involved in cell adhesion; antibodies against acrogranin block blastocyst adhesion whereas addition of purified acrogranin increases it (Qin et al. 2005). It has been reported that acrogranin as a glycoprotein of 67 kDa is expressed in guinea pig spermatozoa, solely located in the acrosome region (Anakwe & Gerton 1990). ...
... Given the sperm-egg interaction takes place at the level of the equatorial segment of spermatozoa (Flesch & Gadella 2000), our findings that ADAM15 and acrogranin physically interact in a protein complex in guinea pig spermatozoa (Fig. 5) and that the sperm-egg interaction is interfered with an antibody against acrogranin (Fig. 7), or with a peptide against the small putative regions of desintegrin domain of ADAM15 (Pasten-Hidalgo et al. 2008), support the idea that the ADAM15/acrogranin complex might be important as a mediator of gamete interaction during fertilization. Moreover, our results suggest that ADAM15-acrogranin complex in spermatozoa acrogranin might be an accessory protein of ADAM15 that participates in cell adhesion, this is supported by the fact that, besides spermatozoa, the ADAM15/Acrogranin complex is also present in two breast cancer cell lines (Fig. 9), and by the fact that blastocyst adhesion is blocked by antibodies against acrogranin whereas addition of purified acrogranin increases their adhesion (Qin et al. 2005). Considering that acrogranin lacks a transmembrane region, we suggest that one of the functions of ADAM15 in the complex is to serve as an anchor for acrogranin on the sperm surface. ...
Mammalian fertilization is completed by direct interaction between sperm and egg. This process is primarily mediated by both adhesion and membrane-fusion proteins found on the gamete surface. ADAM1, 2, and 3 are members of the ADAMs protein family, and have been involved in sperm-egg binding. In this study, we demonstrate the proteolytic processing of ADAM15 during epididymal maturation of guinea pig spermatozoa to produce a mature form a size of 45 kDa. We find that the size of the mature ADAM15, 45 kDa, in cauda epididymal spermatozoa indicates that the pro-domain and metalloprotease domain are absent. In addition, using indirect immunofluorescence, ADAM15 was found throughout the acrosome, at the equatorial region and along the flagellum of guinea pig spermatozoa. After acrosome reaction, ADAM15 is lost from the acrosomal region and retained in the equatorial region and flagellum. In this study, we also report the first evidence of a complex between ADAM15 and acrogranin. By immunoprecipitation, we detected a protein band of 65 kDa which co-immunoprecipated together ADAM15. Analysis of the N-terminal sequence of this 65 kDa protein has revealed its identity as acrogranin. In addition, using cell-surface labeling, ADAM15 was found to be present on the cell surface. Assays of heterologous fertilization showed that the antibody against acrogranin inhibited the sperm-egg adhesion. Interestingly, ADAM15 and acrogranin were also found associated in two breast cancer cell lines. In conclusion, our results demonstrated that ADAM15 and acrogranin are present on and associated with the surface of guinea pig spermatozoa; besides both proteins may play a role during sperm-egg binding.
... To deglycosylate GRN (Qin, Diaz-Cueto et al. 2005 ...
... The former species predominated in cell lysates, whereas the latter was the major variant secreted into conditioned media (Figure 2.1a). When digested with PNGase F (an enzyme capable of deglycosylating the Nlinked glycans of GRN, (Zhou, Gao et al. 1993;Qin, Diaz-Cueto et al. 2005)), immunoreactivity in both cell lystates and conditioned media shifted toward the molecular mass predicted for non-glycosylated mouse GRN (~65 kDa; Figure 2.1a). ...
... PGRN, which occurs in the trophoblasts of the preimplantation embryo (Diaz-Cueto et al. 2000;Qin et al. 2005), stimulates blastocyst formation and implantation (Diaz-Cueto et al. 2000;Qin et al. 2005) as well as the subsequent formation of the placenta (Desmarais et al. 2008;Stubert et al. 2011). It might be expected therefore that the development in grn −/− mice would be severely disrupted. ...
... PGRN, which occurs in the trophoblasts of the preimplantation embryo (Diaz-Cueto et al. 2000;Qin et al. 2005), stimulates blastocyst formation and implantation (Diaz-Cueto et al. 2000;Qin et al. 2005) as well as the subsequent formation of the placenta (Desmarais et al. 2008;Stubert et al. 2011). It might be expected therefore that the development in grn −/− mice would be severely disrupted. ...
Mutation of human GRN, the gene encoding the secreted glycoprotein progranulin, results in a form of frontotemporal lobar degeneration that is characterized by the presence of ubiquitinated inclusions containing phosphorylated and cleaved fragments of the transactivation response element DNA-binding protein-43. This has stimulated interest in understanding the role of progranulin in the central nervous system, and in particular, how this relates to neurodegeneration. Progranulin has many roles outside the brain, including regulation of cellular proliferation, survival, and migration, in cancer, including cancers of the brain, in wound repair, and inflammation. It often acts through the extracellular signal-regulated kinase and phopshatidylinositol-3-kinases pathways. The neurobiology of progranulin has followed a similar pattern with proposed roles for progranulin (PGRN) in the central nervous system as a neuroprotective agent and in neuroinflammation. Here we review the structure, biology, and mechanism of progranulin action. By understanding PGRN in a wider context, we may be better able to delineate its roles in the normal brain and in neurodegenerative disease.
... Gene expression analysis by qRT-PCR revealed that knockdown of maternal Cdx2 led to a 77.5% reduction in the transcript levels of the TE-associated gene Eomes in 8-cell stage embryos, whereas a reduction in the Fgfr2 transcript and an increase in the transcripts of the pluripotency gene Oct4 (Pou5f1-Mouse Genome Informatics) became significant only after blastulation ( Fig. 2A,B; Fig. 4; see Fig. S3A in the supplementary material). Notably, Hand1, which is expressed in the TE and regulates trophoblast cell differentiation into trophoblast giant cells ( Cross et al., 1995), and Grn, which is an important factor for blastocyst hatching, adhesion and outgrowth (Qin et al., 2005), were overexpressed in Cdx2deficient blastocysts (Fig. 4). ...
... To determine why the normally shaped Cdx2-deficient blastocysts failed to hatch from the zonae pellucidae, we performed gene expression studies with qRT-PCR or immunocytochemistry on E4.0 Cdx2-deficient blastocysts. We found that Grn (an autocrine growth factor) and ISP1 (a tryptase; Prss28-Mouse Genome Informatics), which have been reported to play crucial roles in the hatching process (O' Sullivan et al., 2001;Qin et al., 2005), were not reduced in Cdx2-deficient blastocysts ( Fig. 4; see Fig. S3B in the supplementary material). However, further examination revealed the cause of this hatching failure. ...
The separation of the first two lineages - trophectoderm (TE) and inner cell mass (ICM) - is a crucial event in the development of the early embryo. The ICM, which constitutes the pluripotent founder cell population, develops into the embryo proper, whereas the TE, which comprises the surrounding outer layer, supports the development of the ICM before and after implantation. Cdx2, the first transcription factor expressed specifically in the developing TE, is crucial for the differentiation of cells into the TE, as lack of zygotic Cdx2 expression leads to a failure of embryos to hatch and implant into the uterus. However, speculation exists as to whether maternal Cdx2 is required for initiation of TE lineage separation. Here, we show that effective elimination of both maternal and zygotic Cdx2 transcripts by an RNA interference approach resulted in failure of embryo hatching and implantation, but the developing blastocysts exhibited normal gross morphology, indicating that TE differentiation had been initiated. Expression of keratin 8, a marker for differentiated TE, further confirmed the identity of the TE lineage in Cdx2-deficient embryos. However, these embryos exhibited low mitochondrial activity and abnormal ultrastructure, indicating that Cdx2 plays a key role in the regulation of TE function. Furthermore, we found that embryonic compaction does not act as a 'switch' regulator to turn on Cdx2 expression. Our results clearly demonstrate that neither maternal nor zygotic Cdx2 transcripts direct the initiation of ICM/TE lineage separation.
... To deglycosylate GRN [46], spinal cord homogenates as well as lysates and conditioned media from HEK293 cells transfected with recombinant mouse GRN were dissolved in 0.5% SDS, heat denatured (100°C for 10 min), then digested with Peptide N-glycosidase F (New England Biolabs Inc., Ipswich, MA) at 37°C for 60 min in 50 mM sodium phosphate buffer (pH 7.5) containing 1% NP-40 nonionic detergent. To proteolytically digest recombinant GRN to granulin peptides, conditioned medium was incubated with human neutrophil elastase (Sigma-Aldrich) in 0.1 M Tris-HCl buffer, pH 8.3, 0.96 M NaCl at 37°C [29,62]. ...
... The former species predominated in cell lysates, whereas the latter was the major variant secreted into conditioned media (Fig 1a). When digested with Peptide N-glycosidase F (an enzyme capable of deglycosylating the N-linked glycans of GRN, [46,61]), immunoreactivity in both cell lystates and conditioned media shifted toward the molecular mass predicted for non-glycosylated mouse GRN (~65 kDa; Fig. 1a). These data suggested that the ~78 kDa and ~88 kDa species corresponded to immature and mature N-glycosylated GRN variants, respectively [51]. ...
Progranulin (proepithelin) is a pleiotropic growth-factor associated with inflammation and wound repair in peripheral tissues. It also has been implicated in the response to acute traumatic brain injury as well as to chronic neurodegenerative diseases. To determine whether changes in progranulin expression also accompany acute spinal cord injury, C57BL/6 mice were subjected to mid-thoracic (T9 level) contusion spinal cord injury and analyzed by immunohistochemical and biochemical methods. Whereas spinal cord sections prepared from non-injured laminectomy control animals contained low basal levels of progranulin immunoreactivity in gray matter, sections from injured animals contained intense immunoreactivity throughout the injury epicenter that peaked 7-14 days post injury. Progranulin immunoreactivity colocalized with myeloid cell markers CD11b and CD68, indicating that expression increased primarily in activated microglia and macrophages. Immunoblot analysis confirmed that progranulin protein levels rose after injury. On the basis of quantitative polymerase chain reaction analysis, increased protein levels resulted from a tenfold rise in progranulin transcripts. These data demonstrate that progranulin is dramatically induced in myeloid cells after experimental spinal cord injury and is positioned appropriately both spatially and temporally to influence recovery after injury.
... For blastocyst adhesion and outgrowth assays, single two-cell-stage embryos were placed in 5-l drops of KSOM medium (Chemicon, Temecula, CA) in the presence of 3% fetal bovine serum and covered with mineral oil as described (15). The embryos were then transferred to freshly prepared media every 24 h. ...
... When trophoblast cells had grown outward from the adhered blastocysts and the primary giant trophoblast cells became visible, these embryos were designated as outgrowth blastocysts. The proportions of blastocysts undergoing adhesion and outgrowth were estimated at 24 and 48 h of BDNF treatment, respectively (15,16). The proportions of hatched blastocysts showing adhesion or outgrowth were used to estimate the implantation capacity of the blastocysts in vitro. ...
Successful implantation of the blastocyst and subsequent placental development is essential for reproduction. Expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5, together with their receptor, tyrosine kinase B (TrkB), in trophectoderm cells of blastocyst suggests their potential roles in implantation and placental development. Here we demonstrated that treatment with BDNF promoted blastocyst outgrowth, but not adhesion, in vitro and increased levels of the cell invasion marker matrix metalloproteinase-9 in cultured blastocysts through the phosphatidylinositol 3-kinase pathway. After implantation, BDNF and neurotrophin-4/5 proteins as well as TrkB were expressed in trophoblast cells and placentas during different stages of pregnancy. Both TrkB and its ligands were also expressed in decidual cells. Treatment of cultured trophoblast cells with the TrkB ectodomain, or a Trk receptor inhibitor K252a, suppressed cell growth as reflected by decreased proliferation and increased apoptosis, whereas an inactive plasma membrane nonpermeable K252b was ineffective. Studies using the specific inhibitors also indicated the importance of the phosphatidylinositol 3-kinase/Akt pathway in mediating the action of TrkB ligands. In vivo studies in pregnant mice further demonstrated that treatment with K252a, but not K252b, suppressed placental development accompanied by increases in trophoblast cell apoptosis and decreases in placental labyrinth zone at midgestation. In vivo K252a treatment also decreased fetal weight at late gestational stages. Our findings suggested important autocrine/paracrine roles of the BDNF/TrkB signaling system during implantation, subsequent placental development, and fetal growth by increasing trophoblast cell growth and survival.
... Progranulin is a pluripotent growth factor with important roles in several physiological processes. Progranulin is expressed in both the embryo and placenta, where it modulates embryo growth [1] and implantation [2], as well as placenta formation [3]. In adult tissues, progranulin regulates tissue regeneration [4,5], promotes angiogenesis [6], modulates the immune response [7,8] and is implicated in host defense against bacterial infections [8,9]. ...
Progranulin is a pleiotropic growth factor with important physiological roles in embryogenesis and maintenance of adult tissue homeostasis. While-progranulin deficiency is associated with a broad range of pathological conditions affecting the brain, such as frontotemporal dementia and neuronal ceroid lipofuscinosis, progranulin upregulation characterizes many tumors, including brain tumors, multiple myeloma, leiomyosarcoma, mesothelioma and epithelial cancers such as ovarian, liver, breast, bladder, adrenal, prostate and kidney carcinomas. The increase of progranulin levels in tumors might have diagnostic and prognostic significance. In cancer, progranulin has a pro-tumorigenic role by promoting cancer cell proliferation, migration, invasiveness, anchorage-independent growth and resistance to chemotherapy. In addition, progranulin regulates the tumor microenvironment, affects the function of cancer-associated fibroblasts, and modulates tumor immune surveillance. However, the molecular mechanisms of progranulin oncogenic function are not fully elucidated. In bladder cancer, progranulin action relies on the activation of its functional signaling receptor EphA2. Notably, more recent data suggest that progranulin can also modulate a functional crosstalk between multiple receptor-tyrosine kinases, demonstrating a more complex and context-dependent role of progranulin in cancer. Here, we will review what is currently known about the function of progranulin in tumors, with a focus on its molecular mechanisms of action and regulation.
... PGRN is known as protein with the molecular mass of about 68.5 <kDa [16] which contains seven and one-half copies of granulin repeats [17]. As a multifunctional growth factor [18], PGRN is proved to be associated with tumorigenesis [19], neurodegeneration [20], wound healing [21] and early embryogenesis [22]. In regard of inflammation modulation, PGRN has been shown to promote proliferation of Treg cells and IL-10 secretion, and inhibit neutrophil degranulation, at least partly, through directly binding to TNF receptors (TNFRs) and antagonizing TNF-mediated pro-inflammatory signaling pathway [23,24]. ...
Background:
Macrophage M1 polarization plays a pivotal role in inflammatory diseases. Progranulin (PGRN) has potential anti-inflammation action, however, the effect of PGRN on macrophage M1 polarization has been poorly studied. Our study aimed to investigate the effect of PGRN on lipopolysaccharide (LPS)-induced macrophage M1 polarization and clarify the underlying mechanisms.
Methods:
RAW264.7 cells were polarized to M1 macrophage by LPS with or without recombinant PGRN (rPGRN) and tumor necrosis factor alpha antibody (anti-TNF-α). A cell counting kit-8 assay (CCK-8), flow cytometry, Quantitative Real-Time PCR assay (q-PCR), Western blot assay and enzyme-linked immunosorbent assay (ELISA) were used to determine the effect of different treatments on cell proliferation, expression of surface phenotype marker and expressions and secretion of inflammatory cytokines. The activation of NF-κB/mitogen-activated protein kinase (MAPK) pathways and the nuclear translocation of NF-κB p65 were detected by Western blot and immunofluorescence respectively. THP-1 and primary bone marrow-derived monocytes (BMDMs) were also used to demonstrate effect of PGRN on expressions and secretion of inflammatory cytokines induced by LPS.
Results:
In RAW264.7 cells, rPGRN at concentrations below 80 ng/ml significantly promoted cell proliferation in dose dependent fashion. rPGRN significantly inhibited LPS-induced change of phenotype (CD86/CD206 ratio) and function (tumor necrosis factor (TNF-α) and inducible nitric oxide synthase (iNOS) expressions). LPS-stimulated secretion of TNF-α and activated phosphorylation of IKKα/β, IкBα, p65, JNK and p38 and the nucleus translocation of NF-кB p65 were also significantly downregulated by rPGRN. In addition, recombinant TNF-α (rTNF-α) significantly boosted TNF-α and iNOS expression vs the control group. Moreover, anti-TNF-α significantly inhibited LPS-induced TNF-α and iNOS expression. In THP-1 and BMDM cells, reversing effect of rPGRN on LPS-enhanced expressions of TNF-α and iNOS and secretion of TNF-α was further demonstrated.
Conclusions:
PGRN down-regulates LPS-induced macrophage M1 polarization in phenotype and function via NF-κB/MAPK signaling pathways.
... It stimulates growth factor-related signaling pathways such as the phosphorylation of shc, p44/42 mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase B/AKT and the p70S6 kinase [27,28], and promotes the development and progression of many tumors. PGRN is also involved in many physiological processes such as wound repair [5], embryo development [6], blastocyst hatching [29] and male-specific differentiation of the neonatal hypothalamus [30,31]. Due to the important roles of PGRN in so many physiological and pathological processes, a sensitive, reliable method is needed for exploring the transcriptional regulation of PGRN expression. ...
Progranulin has multiple functions in several physiological and pathological processes, including embryonic development, wound repair, tumorigenesis, inflammation and neurodegeneration. To investigate the transcriptional regulation of the PGRN gene, a luciferase knock-in reporter system was established in HEK293 cells by integrating luciferase gene in the genome controlled by the endogenous PGRN promoter using CRISPR/Cas9. PCR results demonstrated the site-specific integration of the exogenous luciferase gene into the genome. To validate the novel luciferase knock-in system, a CRISPR/Cas9 transcription activation/repression system for the PGRN gene was constructed and applied to the knock-in system. In addition, phorbol ester (phorbol 12-myristate, 13-acetate), previously reported as activating the expression of PGRN, was applied to the system. The results indicated that luciferase activity was directly correlated with the activity of the PGRN endogenous promoter. This novel system will be a useful tool for investigating the transcriptional regulation of PGRN, and it has great potential in screening the drugs targeting PGRN.
... Progranulin has been implicated in physiological processes throughout life. It is involved in the growth of preimplantation embryos [28], in embryo implantation [29], and in the formation of the placenta [30]. In adulthood progranulin has growth factorlike roles in wound healing [27] and angiogenesis [27,31]. ...
The purpose of this brief overview of the progranulin protein is to provide a sense of the range and extent of the roles of progranulin in normal physiology and pathology. Progranulin has received attention due to its role in neurodegeneration, where mutation of a single copy of GRN, the gene encoding progranulin, results in frontotemporal dementia, whereas viral delivery of progranulin to the brains of mice exhibiting Parkinson's or Alzheimer's disease phenotypes inhibits the progression of the neurodegenerative phenotypes. Of equal importance, progranulin protects tissues against the harmful effects of poorly controlled inflammation and promotes tissue regeneration after injury at a multitude of sites throughout the body. Progranulin is overexpressed by many types of cancer and contributes to their progression. Given suitable analytical methods and model systems, progranulin offers a wealth of research possibilities.
... Many factors have been proved to function in embryo implantation in the past few decades, including embryonic cell number, trypsin-like proteinases, progranulin, prostanoid pathways, uterine function and the leukaemia inhibitory factor, zygote genomic expression and epigenetic changes, such as DNA methylation and histone deacetylation [6][7][8][9][10][11][12][13][14]. However, none of them has been identified as the determinant factor for embryo hatching. ...
Background:
Hatching is crucial for mammalian embryo implantation, since difficulties during this process can lead to implantation failure, ectopic pregnancy and consequent infertility. Despite years of intensive researches, how internal and external factors affecting embryo hatch are still largely unclear.
Methods:
The effects of parental genetic material and oxygen concentration on hatch process were examined. Fertilized and parthenogenetic mouse preimplantation embryos were cultured in vitro under 5 and 20% oxygen for 120 h. Zona pellucida drilling by Peizo micromanipulation were performed to resemble the breach by sperm penetration.
Results:
Firstly, parthenogenetic embryos had similarly high blastocyst developmental efficiency as fertilized embryos, but significantly higher hatch ratio than fertilized embryos in both O2 concentrations. 5% O2 reduced the hatch rate of fertilized embryos from 58.2 to 23.8%, but increased that of parthenogenetic embryos from 81.2 to 90.8% significantly. Analogously, 5% O2 decreased the ratio of Oct4-positive cells in fertilized blastocysts, whereas increased that in parthenogenetic blastocysts. Additionally, 5% O2 increased the total embryonic cell number in both fertilized and parthegenetic embryos, when compared to 20% O2, and the total cell number of fertilized embryos was also higher than that of parthegenetic embryos, despite O2 concentration. Real-time PCR revealed that the expression of key genes involving in MAPK pathway and superoxide dismutase family might contribute to preimplantation development and consequent blastocyst hatch in vitro. Finally, we showed that fertilized and parthenogenetic embryos have diverse hatch dynamics in vitro, although the zona pellucida integrity is not the main reason for their mechanistic differences.
Conclusion:
Both parental genetic material and O2 concentration, as the representative of intrinsic and extrinsic factors respectively, have significant impacts on mouse preimplantation development and subsequent hatch dynamics, probably by regulating the gene expression involving in MAPK pathway and superoxide dismutase family to control embryonic cell proliferation and allocation of ICM cells.
... Cellular proliferation was first demonstrated by the PGRN derivative epithelin 1 (granulin A) on BALB/MK mouse epidermal keratinocytes [3]. Later, a study in mouse embryos revealed that exogenous PGRN added to cultures increases the number of trophectoderm cells compared to the controls, besides exhibiting other important effects on embryos [24,38]. Moreover, to emphasize how important this protein is as a growth factor, PGRN purified from the culture medium of the PC cell line (highly tumorigenic cells) promotes proliferation of 3T3 mouse embryo fibroblast cells [7]. ...
Cancer cells have defects in regulatory mechanisms that usually control cell proliferation and homeostasis. Different cancer cells share crucial alterations in cell physiology, which lead to malignant growth. Tumorigenesis or tumor growth requires a series of events that include constant cell proliferation, promotion of metastasis and invasion, stimulation of angiogenesis, evasion of tumor suppressor factors, and avoidance of cell death pathways. All these events in tumor progression may be regulated by growth factors produced by normal or malignant cells. The growth factor progranulin has significant biological effects in different types of cancer. This protein is a regulator of tumorigenesis because it stimulates cell proliferation, migration, invasion, angiogenesis, malignant transformation, resistance to anticancer drugs, and immune evasion. This review focuses on the biological effects of progranulin in several cancer models and provides evidence that this growth factor should be considered as a potential biomarker and target in cancer treatment.
... Concurrently, as ICM cells proliferate, they pile up on one another creating a rudimentary ICM "stalk" like colony that is obvious by light microscopy. During the third 24 hours of OG culture, the ICM colony is taller and more obvious, surrounded by a monolayer of trophoblast that proliferates and expands in area 23,24,38,39 . Establishment of the ICM colony is essential for embryonic stem cell (ESC) derivation. ...
With readily available transcriptome-wide data, understanding the role of each expressed gene is an essential next step. Although RNAi technologies allow for genome-wide screens in cell culture, these approaches cannot replace strategies for discovery in the embryo. Here we present, for the first time, a knockdown screen in mouse preimplantation embryos. Early mammalian development encompasses dynamic cellular, molecular and epigenetic events that are largely conserved from mouse to man. We assayed 712 genes for requirements during preimplantation. We identified 59 genes required for successful development or outgrowth and implantation. We have characterized each phenotype and revealed cellular, molecular, and lineage specific defects following knockdown of transcript. Induced network analyses demonstrate this as a valid approach to identify networks of genes that play important roles during preimplantation. Our approach provides a robust and efficient strategy towards identification of novel phenotypes during mouse preimplantation and facilitates functional annotation of the mammalian transcriptome.
... PGRN has been shown to be involved in gameto-and embryogenesis as well as neonatal development in rodents [17][18][19]. Moreover, PGRN enhances trophoblast proliferation, blastocyst cavitation, and expansion in mouse models [18,20]. After the adhesion of the blastocyst, PGRN amount increases compared to the blastocysts before the adhesion [21]. ...
Aims:
To investigate the possible pathophysiological associations between progranulin (PGRN) and preeclampsia (PE), early-onset PE (EOPE) and late-onset PE (LOPE).
Study design:
A cross-sectional study was designed to include consecutive patients with uncomplicated pregnancy (n = 28), EOPE (n = 30) and LOPE (n = 22). Maternal levels of serum PGRN were measured with the use of an enzyme-linked immunosorbent assay kit.
Results:
The mean serum PGRN level was significantly higher in women with PE compared to the control group (54.17 ± 4.20 pg/ml versus 42.37 ± 5.64 pg/ml, p < 0.001), in the LOPE group compared to the control group (51.63 ± 4.61 pg/ml versus 42.37 ± 5.64 pg/ml, p < 0.001) and also in women with EOPE compared to women with LOPE (56.03 ± 2.68 pg/ml versus 51.63 ± 4.61 pg/ml, p < 0.001). Serum PGRN was negatively correlated with gestational age at birth (r = -0.669, p = 0.001) and birth weight (r = -0.653, p = 0.001); and positively correlated with systolic (r = 0.653, p = 0.001) and diastolic blood pressure (r = 0.601, p = 0.001), C-reactive protein (r = 0.519, p = 0.001), uterine artery pulsatility (r = 0.441, p = 0.001) and resistance indices (r = 0.441, p = 0.001).
Conclusions:
Serum PGRN levels increase significantly in women with PE as an indirect sign of placental dysfunction. This increase is even more prominent in women with EOPE. The serum PGRN in the third trimester is positively correlated with gestational age at birth and birth weight.
... Blastocyst attachment and outgrowth assays were performed as described previously (Qin et al., 2005). In brief, blastocysts were obtained by flushing the uterine horns with Ham's F-12 medium on day 4 morning of pregnancy and transferred in 96-well plates precoated with fibronectin (10 mg/ml; Sigma), containing Ham's F-12 (supplemented with 0.4% BSA) plus 0, 1, 10, 50, 100, 200, 1,000 ng/ml of recombinant mouse CXCL14 (rmCXCL14) protein (R&D Systems). ...
CXCL14, a member of chemokine family, was previously known to participate in many pathophysiological events, such as leukocytes recruitment and tumor suppression. However, it remained largely unknown whether CXCL14 is a physiological player during early pregnancy. In this regard, our recent global gene microarray analysis has observed an implantation-specific expression profile of CXCL14 mRNA during early pregnancy in mice, showing its higher levels at implantation sites compared to inter-implantation sites, implicating a potential role of CXCL14 in the periimplantation events. In the present investigation, using Northern blot, in situ hybridization and immunostaining, we further demonstrated that uterine CXCL14 expression was specifically induced at embryo implantation site and expanded with subsequent decidualization process in a spatiotemporal manner. The implanting embryo also showed a highlighted expression of CXCL14 in the blastocyst trophectoderm and its derived ectoplacental cones (EPCs) during postimplantation development. In vitro functional study revealed that CXCL14 could significantly inhibit both primary and secondary trophoblast attachment and outgrowth, correlated with a stage-dependant downregulation of MMP-2 and/or MMP-9 activity. Moreover, it was found that biotinylated CXCL14 could specifically bind to trophoblast cells in vitro and in vivo, suggesting trophoblast cell, perhaps expressing the unidentified CXCL14 receptor, is a bioactive target of CXCL14. Collectively, our findings provide evidences supporting the contention that CXCL14 is an important paracrine/autocrine modulator regulating trophoblast outgrowth at the maternal–fetal interface during the process of pregnancy establishment. This study is clinically related since CXCL14 is also highly expressed in human receptive endometrium and trophoblasts.
... PGRN is a multifunctional protein that has been implicated in early embryonic development [14,15], bone development [16], inflammation [17][18][19] and wound repair [20]. Mutational loss of a single copy of the human GRN gene results in a form of early onset dementia, called frontotemporal lobar dementia that is characterized by neuronal atrophy in the frontal and temporal cerebral lobes [21,22]. ...
Citation: Zhang Y, Bateman A (2011) The Glycoprotein Growth Factor Progranulin Promotes Carcinogenesis and has Potential Value in Anti-cancer Therapy. J Carcinogene Mutagene S2:001. Abstract Progranulin (PGRN) is a secreted glycoprotein growth factor with tumorigenic roles in a variety of tumors includ-ing, among others, breast, ovarian, prostate, bladder, and liver cancer. In some patients, for example with breast, ovarian or liver cancers, high PGRN expression in tumors correlated with a worse outcome. Studies using cell lines and animal models provide evidence that PGRN promotes tumor cell proliferation, migration and survival, and in-duces drug resistance. Increasing or decreasing PGRN production enhances or inhibits respectively the growth of PGRN-sensitive tumors in vivo. PGRN activity is associated with p44/42 mitogen-activated protein kinase as well as phosphatidylinositol 3-kinases signaling pathways. In addition, PGRN may stimulate the formation of the tumor stroma. As an extracellular regulator of tumorgenesis, PGRN is a potential therapeutic target and biomarker of prog-nosis in the treatment of various cancers.
... In humans, it plays an important role in childhood growth and stimulates anabolic processes also in adults. Both the insulin and IGF-1 receptors (IGF-1R) are expressed in the trophectoderm cells and trophoblast tissue [26]. IGF plays a critical role in promotion of survival and proliferation in a diversity of cell types, including both embryonic and adult stem cells. ...
Regenerative medicine is a novel clinical branch aiming at the cure of diseases by replacement of damaged tissues. The crucial use of stem cells makes this area rich of challenges, given the poorly understood mechanisms of differentiation. One highly needed and yet unavailable technology should allow us to monitor the exact (metabolic) state of stem cells differentiation to maximize the effectiveness of their implant in vivo. This is challenged by the fact that not all relevant metabolites in stem cells differentiation are known and not all metabolites can currently be continuously monitored. To bring advancements in this direction, we propose the enhancement and integration of two available technologies into a general pipeline. Namely, high-throughput biochip for gene expression screening to pre-select the variables that are most likely to be relevant in the identification of the stem cells’ state and low-throughput biochip for continuous monitoring of cell metabolism with highly sensitive carbon nanotubes-based sensors. Intriguingly, additionally to the involvement of multidisciplinary expertise (medicine, molecular biology, computer science, engineering, and physics), this whole query heavily relies on biochips: it starts in fact from the use of high-throughput ones, which output, in turn, becomes the base for the design of low-throughput, highly sensitive biochips. Future research is warranted in this direction to develop and validated the proposed device.
... PGRN can promote anchorage-independent growth (He et al., 2003;Jones et al., 2003) and proliferation of uterine smooth cells and mouse blastocysts (Qin et al., 2005;Matsumura et al., 2006). We examined whether this is also the case in gastric epithelial cells and the role of PGRN in the proliferation gastric cancer cells. ...
Helicobacter pylori is a major human pathogen associated with gastric diseases such as chronic active gastritis, peptic ulcer, and gastric carcinoma. The growth factor progranulin (PGRN) is a secreted glycoprotein that functions as an important regulator of cell growth, migration, and transformation. We aimed to determine the molecular mechanisms by which H. pylori upregulates the expression of PGRN and the relationship between H. pylori infection and production of PGRN in controlling cell proliferation and migration. Levels of PGRN were examined in gastric tissues from patients and in vitro in gastric epithelial cells. Cell proliferation was measured by colony formation assay. Cell migration was monitored by wound healing migration assay. PGRN protein levels were increased in patients with gastritis and gastric cancer tissue. Infection of gastric epithelial cells with H. pylori significantly increased PGRN expression in a time-dependent manner. Blockade of the p38 and MEK1/2 pathway by inhibitor inhibited H. pylori-mediated PGRN upregulation. Activation of p38 and MEK1/2 pathway by H. pylori was also identified. Knockdown of PGRN attenuated the H. pylori-induced proliferative activity and migration of cancer cells. These findings suggest that the upregulation of PGRN in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.
... The signalling pathways associated with PGRN in neurons are unknown, but in non-neuronal cell types it activates growth factor-related signal transduction pathways including the phosphorylation of shc, p44/42 mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase B/AKT, and the p70S6 kinase [26-28], and, by so doing, contributes to carcinogenesis in numerous tumour types [29-39]. PGRN is involved in wound repair and inflammation [40-43], and plays an important role in early embryonic development [44-46]. The ability of PGRN to regulate critical proliferative, survival and motility signals in a diverse range of non-neuronal cell types suggests that it may support similar functions in nerve cells. ...
Progranulin is a secreted high molecular weight growth factor bearing seven and one half copies of the cysteine-rich granulin-epithelin motif. While inappropriate over-expression of the progranulin gene has been associated with many cancers, haploinsufficiency leads to atrophy of the frontotemporal lobes and development of a form of dementia (frontotemporal lobar degeneration with ubiquitin positive inclusions, FTLD-U) associated with the formation of ubiquitinated inclusions. Recent reports indicate that progranulin has neurotrophic effects, which, if confirmed would make progranulin the only neuroprotective growth factor that has been associated genetically with a neurological disease in humans. Preliminary studies indicated high progranulin gene expression in spinal cord motor neurons. However, it is uncertain what the role of Progranulin is in normal or diseased motor neuron function. We have investigated progranulin gene expression and subcellular localization in cultured mouse embryonic motor neurons and examined the effect of progranulin over-expression and knockdown in the NSC-34 immortalized motor neuron cell line upon proliferation and survival.
In situ hybridisation and immunohistochemical techniques revealed that the progranulin gene is highly expressed by motor neurons within the mouse spinal cord and in primary cultures of dissociated mouse embryonic spinal cord-dorsal root ganglia. Confocal microscopy coupled to immunocytochemistry together with the use of a progranulin-green fluorescent protein fusion construct revealed progranulin to be located within compartments of the secretory pathway including the Golgi apparatus. Stable transfection of the human progranulin gene into the NSC-34 motor neuron cell line stimulates the appearance of dendritic structures and provides sufficient trophic stimulus to survive serum deprivation for long periods (up to two months). This is mediated at least in part through an anti-apoptotic mechanism. Control cells, while expressing basal levels of progranulin do not survive in serum free conditions. Knockdown of progranulin expression using shRNA technology further reduced cell survival.
Neurons are among the most long-lived cells in the body and are subject to low levels of toxic challenges throughout life. We have demonstrated that progranulin is abundantly expressed in motor neurons and is cytoprotective over prolonged periods when over-expressed in a neuronal cell line. This work highlights the importance of progranulin as neuroprotective growth factor and may represent a therapeutic target for neurodegenerative diseases including motor neuron disease.
... It has been suggested that PGRN is a steroid-regulated growth factor and mediates the mitogenic effect of estrogens in breast cancer tumorgenesis [13;14]. PGRN is expressed in the acrosome of the sperm [15] and oocytes [16] and modulates the development of early embryos in vitro [17]. PRGN has been also implicated in wound healing and inflammation181920. ...
Progranulin (PGRN) is a growth modulating factor released by a variety of cells. This molecule has gained the attention of the neuroscience community with recent discoveries of multifunctional roles of PGRN in normal brain and neurodegenerative disorders. We focus on novel roles of PGRN as a sex steroid-responsible gene in the developing and adult rodent brain. While the developing brain is feminine by default, hormone exposure, including androgen and estrogen, induces masculinization during the critical period. We have shown that PGRN is a sex steroid-responsible gene that may be involved in masculinization of the perinatal rat brain. We also found that in adult rats PGRN gene expression was up-regulated by estrogen in the hippocampus, suggesting that PGRN may mediate the mitogenic effects of estrogen in the active area of neurogenesis. Since it has been recently reported that mutations in PGRN gene are responsible for a type of frontotemporal lobar degeneration in humans, PGRN appears to be also involved in modulating neurodegeneration. Together, PGRN gene expression is induced by estrogen in both developing and adult brains, and it may play multifunctional roles in the organization of functional masculinization in the developing brain and the maintenance of adult brain function.
... Blastocyst attachment and outgrowth assays were performed as described previously (Qin et al., 2005). In brief, blastocysts were obtained by flushing the uterine horns with Ham's F-12 medium on day 4 morning of pregnancy and transferred in 96-well plates precoated with fibronectin (10 mg/ml; Sigma), containing Ham's F-12 (supplemented with 0.4% BSA) plus 0, 1, 10, 50, 100, 200, 1,000 ng/ml of recombinant mouse CXCL14 (rmCXCL14) protein (R&D Systems). ...
CXCL14, a member of chemokine family, was previously known to participate in many pathophysiological events, such as leukocytes recruitment and tumor suppression. However, it remained largely unknown whether CXCL14 is a physiological player during early pregnancy. In this regard, our recent global gene microarray analysis has observed an implantation-specific expression profile of CXCL14 mRNA during early pregnancy in mice, showing its higher levels at implantation sites compared to inter-implantation sites, implicating a potential role of CXCL14 in the periimplantation events. In the present investigation, using Northern blot, in situ hybridization and immunostaining, we further demonstrated that uterine CXCL14 expression was specifically induced at embryo implantation site and expanded with subsequent decidualization process in a spatiotemporal manner. The implanting embryo also showed a highlighted expression of CXCL14 in the blastocyst trophectoderm and its derived ectoplacental cones (EPCs) during postimplantation development. In vitro functional study revealed that CXCL14 could significantly inhibit both primary and secondary trophoblast attachment and outgrowth, correlated with a stage-dependant downregulation of MMP-2 and/or MMP-9 activity. Moreover, it was found that biotinylated CXCL14 could specifically bind to trophoblast cells in vitro and in vivo, suggesting trophoblast cell, perhaps expressing the unidentified CXCL14 receptor, is a bioactive target of CXCL14. Collectively, our findings provide evidences supporting the contention that CXCL14 is an important paracrine/autocrine modulator regulating trophoblast outgrowth at the maternal-fetal interface during the process of pregnancy establishment. This study is clinically related since CXCL14 is also highly expressed in human receptive endometrium and trophoblasts.
... The exogenous addition of recombinant progranulin to eight-cell stage mouse embryos grown in culture ex vivo accelerates the onset of cavitation, stimulates the rate of blastocoel expansion, and leads to an increase in the number of trophectoderm cells compared to controls [13]. Conversely, the use of a progranulin function-blocking antibody arrests growth and prohibits embryo implantation [13,14]. These results are consistent with the growth-promoting activity of progranulin upon epithelial cells in vitro. ...
Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor) that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian progranulin may be represented by two co-orthologues in zebrafish.
The cDNAs encoding two zebrafish granulin precursors, progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of progranulin. Chromosomal localization of the four progranulin (grn) genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial compartments of various organs.
In support of the duplication-degeneration-complementation model of duplicate gene retention, partitioning of expression between grna and grnb was observed in the intermediate cell mass and yolk syncytial layer, respectively. Taken together these expression patterns suggest that the function of an ancestral grn gene has been devolved upon four paralogues in zebrafish.
... It stimulates growth factor-related signaling pathways such as the phosphorylation of shc, p44/42 mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase B/AKT, and the p70 S6 kinase (24,35,65) and contributes to carcinogenesis in breast (51), ovarian (16,26), renal (18), hepatocellular (10), and prostate (44) cancers, gliomas (33), and multiple myelomas (63). Physiologically, pgrn is involved in wound repair (25,67) and is expressed during development (8) where it regulates cavitation in preimplantation embryos (17), blastocyst hatching (48), and malespecific differentiation of the neonatal hypothalamus (56,58). ...
Progranulin (pgrn; granulin-epithelin precursor, PC-cell-derived growth factor, or acrogranin) is a multifunctional secreted glycoprotein implicated in tumorigenesis, development, inflammation, and repair. It is highly expressed in macrophage and monocyte-derived dendritic cells. Here we investigate its regulation in myeloid cells. All-trans retinoic acid (ATRA) increased pgrn mRNA levels in myelomonocytic cells (CD34(+) progenitors; monoblastic U-937; monocytic THP-1; progranulocytic HL-60; macrophage RAW 264.7) but not in nonmyeloid cells tested. Interleukin-4 impaired basal expression of pgrn in U-937. Differentiation agents DMSO, and, in U-937 only, phorbol ester [phorbol 12-myristate,13-acetate (PMA)] elevated pgrn mRNA expression late in differentiation, suggestive of roles for pgrn in more mature terminally differentiated granulocyte/monocytes rather than during growth or differentiation. The response of pgrn mRNA to ATRA differs in U-937 and HL-60 lineages. In U-937, ATRA and chemical differentiation agents greatly increased pgrn mRNA stability, whereas, in HL-60, ATRA accelerated pgrn mRNA turnover. The initial upregulation of pgrn mRNA after stimulation with ATRA was independent of de novo protein synthesis in U-937 but not HL-60. Chemical blockade of nuclear factor-kappaB (NF-kappaB) activation impaired ATRA-stimulated pgrn expression in HL-60 but not U-937, whereas in U-937 it blocked PMA-induced pgrn mRNA expression, suggestive of cell-specific roles for NF-kappaB in determining pgrn mRNA levels. We propose that: 1) ATRA regulates pgrn mRNA levels in myelomonocytic cells; 2) ATRA acts in a cell-specific manner involving the differential control of mRNA stability and differential requirement for NF-kappaB signaling; and 3) elevated pgrn mRNA expression is characteristic of more mature cells and does not stimulate differentiation.
... Mutations in the progranulin gene cause neurodegenerative frontotemporal dementia, suggesting roles for progranulin in the maintenance of healthy neurons ( Baker et al. 2006;Cruts et al. 2006). Progranulin is involved in wound repair and inflammation ( Zhu et al. 2002;He et al. 2003;Ong et al. 2006) and plays an important role in embryonic development ( Cadieux et al. 2005) where it regulates cavitation in preimplantation embryos ( Diaz-Cueto et al. 2000), blastocyst hatching ( Qin et al. 2005) and male-specific differentiation of the neonatal hypothalamus ( Suzuki et al. 1998Suzuki et al. , 2000). Progranulin is a mitogen for fibroblasts (Zanocco-Marani et al. 1999;He et al. 2003). ...
The growth factor progranulin (granulin-epithelin precursor, PC-derived growth factor or acrogranin) regulates proliferation and migration and is implicated in cancer, development, wound repair and neurodegenerative diseases. Under most conditions fibroblasts do not express progranulin in vivo, however its expression is activated following wounding. We hypothesised that progranulin is part of a fibroblast stress response. Fibroblasts in culture were exposed to two physiologically and clinically relevant microenvironmental stresses; hypoxia (1% oxygen) and acidosis, both of which increase progranulin expression. The greatest increases occurred when hypoxia and acidosis were combined. Increased progranulin expression is not a direct response to apoptosis since it occurred under conditions of pH and hypoxia under which cell viability remained high. Low concentrations of progranulin (2 nM) protected fibroblasts from apoptosis induced by extreme acidosis (pH 5.0 and 4.0). We propose that progranulin is part of a fibroblast stress response and is cytoprotective to acidotic stress.
Critical reprogramming factors resided predominantly in the oocyte or male pronucleus can enhance the efficiency or the quality of induced pluripotent stem cells (iPSCs) induction. However, few reprogramming factors exist in the male pronucleus had been verified. Here, we demonstrated that granulin ( Grn ), a factor enriched specifically in male pronucleus, can significantly improve the generation of iPSCs from mouse fibroblasts. Grn is highly expressed on Day 1, Day 3, Day 14 of reprogramming induced by four Yamanaka factors and functions at the initial stage of reprogramming. Transcriptome analysis indicates that Grn can promote the expression of lysosome‐related genes, while inhibit the expression of genes involved in DNA replication and cell cycle at the early reprogramming stage. Further verification determined that Grn suppressed cell proliferation due to the arrest of cell cycle at G2/M phase. Moreover, ectopic Grn can enhance the lysosomes abundance and rescue the efficiency reduction of reprogramming resulted from lysosomal protease inhibition. Taken together, we conclude that Grn serves as an activator for somatic cell reprogramming through mitigating cell hyperproliferation and promoting the function of lysosomes.
Conserved in female reproduction across all mammalian species is the estrous cycle and its regulation by the hypothalamic-pituitary-gonadal/HPG axis, a collective of intersected hormonal events that are crucial for ensuring uterine fertility. Nonetheless, knowledge of direct mediators that synchronously shape the uterine microenvironment for successive yet distinct events such as transit of sperm and support for progressive stages of preimplantation embryo development, remain principally deficient. Towards understanding the timed endometrial outputs that permit luminal events as directed by the estrous cycle, we used cattle as a model system to uniquely surface-sample and study temporal shifts to in vivo endometrial transcripts that encode for proteins destined to be secreted. The results revealed the full quantitative profile of endometrial components that shape the uterine luminal microenvironment at distinct phases of the estrous cycle (estrus, metestrus, diestrus and proestrus). In interpreting this comprehensive log of stage-specific endometrial secretions, we define the 'uterine secretory cycle' and extract predictive understanding of recurring physiological actions regulated within the uterine lumen in anticipation of sperm and preimplantation embryonic stages. This repetitive microenvironmental preparedness to sequentially provide operative support was a stable intrinsic framework, with only limited responses to sperm or embryos if encountered in the lumen within the cycling time period. In uncovering the secretory cycle and unraveling realistic biological processes, we present novel foundational knowledge of terminal effectors controlled by the HPG axis to direct a recurring sequence of vital functions within the uterine lumen.
Progranulin is a growth factor with pro-tumorigenic activity. We recently demonstrated that in mesothelioma progranulin regulates cell migration, invasion, adhesion, and in vivo tumor formation by modulating a complex signaling network involving multiple receptor tyrosine kinase (RTK)s. Progranulin biological activity relies on EGFR and RYK, a co-receptor of the Wnt signaling pathway, which are both required for progranulin-induced downstream signaling. However, the molecular mechanism regulating the functional interaction among progranulin, EGFR and RYK are not known. In this study we demonstrated that progranulin directly interacted with RYK by specific ELISA (KD=0.67). Using immunofluorescence and proximity ligation assay we further discovered that progranulin and RYK colocalized in mesothelioma cells in distinct vesicular compartments. Notably, progranulin-dependent downstream signaling was sensitive to endocytosis inhibitors, suggesting that it could depend on RYK or EGFR internalization. We discovered that progranulin promoted RYK ubiquitination and endocytosis preferentially through caveolin-1-enriched pathways, and modulated RYK stability. Interestingly, we also showed that in mesothelioma cells RYK complexes with the EGFR, contributing to the regulation of RYK stability. Collectively, our results suggest a complex regulation of RYK trafficking/activity in mesothelioma cells, a process that is concurrently regulated by exogenous soluble progranulin and EGFR.
Introduction:
Functional disorders of the villous trophoblast may result in preeclampsia through the release of endothelial activating substances. Progranulin is an anti-inflammatory, pro-angiogenic cytokine with TNF-α antagonizing activity. The trophoblastic expression of progranulin is increased during preeclampsia. The aim of the study was to investigate the impact of placental progranulin synthesis on endothelial cell activation.
Methods:
Placental progranulin expression was modified by transduction of an adenoviral vector. Primary isolated human umbilical venous endothelial cells (HUVECs) were incubated with conditioned medium of first trimester placental explants. Functional studies on HUVECs included assays for proliferation, viability, cytotoxicity and analyzes of Intercellular adhesion molecule-1 (ICAM-1) and E-selectin expression.
Results:
Placental progranulin expression was more than 10-fold higher by using an adenoviral-mediated overexpression system (Ad.PGRN) compared to control vector (Ad.CTRL) and untreated controls. Incubation of HUVECs with conditioned placental medium revealed a dose-dependent increase of cytotoxicity, reduced cell proliferation and viability and resulted in an increase of ICAM-1 and E-selectin expression. Overexpression of progranulin (Ad.PGRN) antagonized the ICAM-1 expression induced by conditioned medium. However progranulin did not influence the effects on cell proliferation, viability, cytotoxicity and E-selectin expression in HUVECs.
Discussion:
Regulation of gene expression in human placental explants is possible by usage of an adenoviral vector system. The increase of endothelial ICAM-1 expression following the incubation with placental conditioned medium was partly reversed by overexpression of placental progranulin. It is suggested that up-regulation of the placental progranulin expression is an endogenous anti-inflammatory mechanism that partially antagonizes the endothelial cell activation during preeclampsia.
Progranulin is a 67-88 kDa glycoprotein, also known as acrogranin, PC-cell-derived growth factor, granulin-epithelin precursor, and proepithelin. This protein is present in a variety of mouse, rat, and human tissues. Progranulin, which is a growth factor, mediates cell cycle progression and cell migration in normal and pathological conditions. In several types of cancers, progranulin expression is upregulated, whereas function-interfering mutations in the granulin gene in humans have been linked to a subset of heritable cases of frontotemporal lobar degeneration. Also, progranulin has important effects on mouse preimplantation embryo development in vitro, including regulation of the appearance of the epithelium in the developing mouse blastocyst and growth of trophectoderm. Furthermore, progranulin promotes mouse blastocyst hatching, adhesion, and outgrowth in vitro. In this chapter, we describe some of the techniques that may be useful in the study of progranulin in embryo development.
Objectives:
Progranulin (PGRN) promotes cell growth and cell cycle progression in several cell types and contributes to tumorigenesis in diverse cancers. We have recently reported PGRN expression in islets and tumors developed in an MEN1 transgenic mouse. Here we sought to investigate PGRN expression and regulation after exposure to hypoxia as well as its effects on pancreatic islet cells and neuroendocrine tumors (NETs) in MEN1 mice.
Methods:
Gene and protein expression were analyzed by quantitative polymerase chain reaction, immunohistochemistry, and Western blot. We also investigated PGRN expression in samples from patients carrying pancreatic NETs associated or not with the multiple endocrine neoplasia 1 syndrome, using enzyme-linked immunosorbent assay and immunohistochemistry analysis.
Results:
Progranulin is upregulated in tumors and islets of the MEN1 mouse as well as in the serum of patients with pancreatic NETs associated with glucagonoma syndrome. In normal mice islets and pancreatic tumors, PGRN expression was strongly potentiated by hypoxia. Progranulin promotes cell proliferation in islet cells and βTC-6 cells, a process paralleled by activation of the mitogen-activated protein kinase signaling cascade.
Conclusions:
Our findings identify PGRN as an effective inducer of pancreatic islet cell proliferation and a possible important factor for pancreatic endocrine tumor development.
The placenta is a transient organ consisting of cell types that are unique to eutherian mammals. It is fetal in origin and shares just half of the genome with that of the maternal uterus. Although existing for only 9 months, there are constant morphological and biological fluctuations within the placenta proper and at the interface between the placenta and the maternal endomyometrium. It is at the latter interface that unique fetomaternal tissue remodeling, hormonal regulation, and immunological interactions are regulated in such a delicate balance, so that appropriate maternal support can be delivered to the embryo and the mother does not illicit an immunologic rejection response to the growing gestational structures. Proliferative disorders including tumors arising from the placenta have distinct genetic, biological, and immunological properties that are drastically different from those of the maternal neoplasms. Recent findings of genomic imprinting including imprinted X chromosome inactivation in the placenta and its implication in the pathogenesis of gestational trophoblastic diseases raised some fundamental questions in mammalian biology and oncology.
Stress is normal during early embryogenesis and transient, elevated stress is commonplace. Stress in the milieu of the peri-implantation embryo is a summation of maternal hormones, and other elements of the maternal milieu, that signal preparedness for development and implantation. Examples discussed here are leptin, adrenaline, cortisol, and progesterone. These hormones signal maternal nutritional status and provide energy, but also signal stress that diverts maternal and embryonic energy from an optimal embryonic developmental trajectory. These hormones communicate endocrine maternal effects and local embryonic effects although signaling mechanisms are not well understood. Other in vivo stresses affect the embryo such as local infection and inflammation, hypoxia, environmental toxins such as benzopyrene, dioxin, or metals, heat shock, and hyperosmotic stress due to dehydration or diabetes. In vitro, stresses include shear during handling, improper culture media and oxygen levels, cryopreservation, and manipulations of the embryo to introduce sperm or mitochondria. We define stress as any stimulus that slows stem cell accumulation or diminishes the ability of cells to produce normal and sufficient parenchymal products upon differentiation. Thus stress deflects downwards the normal trajectories of development, growth and differentiation. Typically stress is inversely proportional to embryonic developmental and proliferative rates, but can be proportional to induction of differentiation of stem cells in the peri-implantation embryo. When modeling stress it is most interesting to produce a ‘runting model’ where stress exposures slow accumulation but do not create excessive apoptosis or morbidity. Windows of stress sensitivity may occur when major new embryonic developmental programs require large amounts of energy and are exacerbated if nutritional flow decreases and removes energy from the normal developmental programs and stress responses. These windows correspond to zygotic genome activation, the large mRNA program initiated at compaction, ion pumping required for cavitation, the differentiation of the first lineages, integration with the uterine environment at implantation, rapid proliferation of stem cells, and production of certain lineages which require the highest energy and are most sensitive to mitochondrial inhibition. Stress response mechanisms insure that stem cells for the early embryo and placenta survive at lower stress exposures, and that the organism survives through compensatory and prioritized stem cell differentiation, at higher stress exposures. These servomechanisms include a small set of stress enzymes from the 500 protein kinases in the kinome; the part of the genome coding for protein kinases that hierarchically regulate the activity of other proteins and enzymes. Important protein kinases that mediate the stress response of embryos and their stem cells are SAPK, p38MAPK, AMPK, PI3K, Akt, MEK1/2, MEKK4, PKA, IRE1 and PERK. These stress enzymes have cytosolic function in cell survival at low stress exposures and nuclear function in modifying transcription factor activity at higher stress exposures. Some of the transcription factors (TFs) that are most important in the stress response are JunC, JunB, MAPKAPs, ATF4, XBP1, Oct1, Oct4, HIFs, Nrf2/KEAP, NFKB, MT1, Nfat5, HSF1/2 and potency-maintaining factors Id2, Cdx2, Eomes, Sox2, Nanog, Rex1, and Oct4. Clearly the stress enzymes have a large number of cytosolic and nuclear substrates and the TFs regulate large numbers of genes. The interaction of stress enzymes and TFs in the early embryo and its stem cells are a continuing central focus of research. In vitro regulation of TFs by stress enzymes leads to reprogramming of the stem cell when stress diminishes stem cell accumulation. Since more differentiated product is produced by fewer cells, the process compensates for fewer cells. Coupled with stress-induced compensatory differentiation of stem cells is a tendency to prioritize differentiation by increasing the first essential lineage and decreasing later lineages. These mechanisms include stress enzymes that regulate TFs and provide stress-specific, shared homeostatic cellular and organismal responses of prioritized differentiation.
Aims:
The glycoprotein progranulin directly binds to TNF-receptors and thereby can antagonize the inflammatory effects of TNF-α. Here we analyzed the impact of both cytokines on cytotoxicity and viability of trophoblast cells.
Methods:
Isolated villous first trimester human trophoblast cells and the human choriocarcinoma cell line BeWo were treated with recombinant human progranulin and TNF-α. Analyses were performed by LDH- and MTT-assay and measurement of caspase-8-activity.
Results:
Progranulin treatment showed some cytoprotective effects on isolated trophoblast cells. However, TNF-α-induced apoptosis was not antagonized by addition of progranulin. Effects were similar, but more pronounced in BeWo cells.
Conclusion:
The cytoprotective activity of progranulin on trophoblast cells in vitro was only weak and of doubtful biologic relevance. It was not able to antagonize TNF-α. Future studies should focus on possible paracrine activities of progranulin.
Neuromuscular disorders are a broad group of inherited conditions affecting the structure and function of the motor system with polymorphic clinical presentation and disease severity. Although individually rare, collectively neuromuscular diseases have an incidence of 1 in 3,000 and represent a significant cause of disability of the motor system. The past decade has witnessed the identification of a large number of human genes causing muscular disorders, yet the underlying pathogenetic mechanisms remain largely unclear, limiting the developing of targeted therapeutic strategies. To overcome this barrier, model systems that replicate the different steps of human disorders are increasingly being developed. Among these, the zebrafish (Danio rerio) has emerged as an excellent organism for studying genetic disorders of the central and peripheral motor systems. In this review, we will encounter most of the available zebrafish models for childhood neuromuscular disorders, providing a brief overview of results and the techniques, mainly transgenesis and chemical biology, used for genetic manipulation. The amount of data collected in the past few years will lead zebrafish to became a common functional tool for assessing rapidly drug efficacy and off-target effects in neuromuscular diseases and, furthermore, to shed light on new etiologies emerging from large-scale massive sequencing studies.
Aims:
The expression of the anti-inflammatory glycoprotein progranulin and the hypoxia-induced transcription factor 1α (HIF-1α) in the villous trophoblast was compared between placentae from patients with preeclampsia (PE), fetal growth restriction (FGR), and normal controls.
Study design:
Matched pairs analysis of third trimester placentae specimens (mean gestational age 36+2) was performed by semiquantitative measurements of the immunohistochemical staining intensities for progranulin and HIF-1α expression (PE n=13, FGR n=9 and controls n=11). Further, placental progranulin mRNA expression was analyzed by qRT-PCR on term placentae (n=3 for each group).
Results:
Compared to controls, villous trophoblast revealed a significantly higher expression of progranulin in cases of PE (P<0.05) and FGR (P<0.01). Similar results were shown for HIF-1α expression (P<0.01 for PE and <0.05 for FGR). The increase of the progranulin protein was not accompanied by an increase of the progranulin mRNA in term placentae.
Conclusions:
Increased expression of progranulin protein in villous trophoblast cells in cases of PE and FGR may result from disturbed placental development and, therefore, may be of pathogenetic importance. The increase was correlated to HIF-1α expression. Further evaluation of this potential mechanism of regulation is required.
Progranulin is a fascinating multifunctional protein, which has been implicated in cell growth, wound repair, tumorigenesis, inflammation, neurodevelopment, and more recently in neurodegeneration. The mechanism of action of this protein is still largely unknown, but the knowledge about the cellular effects on various cell types is expanding. In the current review, we will summarize what is known about the cell biology of progranulin. A better understanding of the biology of progranulin will impact diverse areas of research.
Citation Lemons AR, Naz RK. Contraceptive vaccines targeting factors involved in establishment of pregnancy. Am J Reprod Immunol 2011; 66: 13–25
Current methods of contraception lack specificity and are accompanied with serious side effects. A more specific method of contraception is needed. Contraceptive vaccines can provide most, if not all, the desired characteristics of an ideal contraceptive. This article reviews several factors involved in the establishment of pregnancy, focusing on those that are essential for successful implantation. Factors that are both essential and pregnancy-specific can provide potential targets for contraception. Using database search, 76 factors (cytokines/chemokines/growth factors/others) were identified that are involved in various steps of the establishment of pregnancy. Among these factors, three, namely chorionic gonadotropin (CG), leukemia inhibitory factor (LIF), and pre-implantation factor (PIF), are found to be unique and exciting molecules. Human CG is a well-known pregnancy-specific protein that has undergone phase I and phase II clinical trials, in women, as a contraceptive vaccine with encouraging results. LIF and PIF are pregnancy-specific and essential for successful implantation. These molecules are intriguing and may provide viable targets for immunocontraception. A multiepitope vaccine combining factors/antigens involved in various steps of the fertilization cascade and pregnancy establishment may provide a highly immunogenic and efficacious modality for contraception in humans.
Progranulin (PGRN) encoded by the GRN gene, is a secreted glycoprotein growth factor that has been implicated in many physiological and pathophysiological processes. PGRN haploinsufficiency caused by autosomal dominant mutations within the GRN gene leads to progressive neuronal atrophy in the form of frontotemporal lobar degeneration (FTLD). This form of the disease is associated with neuronal inclusions that bear the ubiquitinated TAR DNA Binding Protein-43 (TDP-43) molecular signature (FTLD-U). The neurotrophic properties of PGRN in vitro have recently been reported but the role of PGRN in neurons is not well understood. Here we document the neuronal expression and functions of PGRN in spinal cord motoneuron (MN) maturation and branching in vivo using zebrafish, a well established model of vertebrate embryonic development.
Whole-mount in situ hybridization and immunohistochemical analyses of zebrafish embryos revealed that zfPGRN-A is expressed within the peripheral and central nervous systems including the caudal primary (CaP) MNs within the spinal cord. Knockdown of zfPGRN-A mRNA translation mediated by antisense morpholino oligonucleotides disrupted normal CaP MN development resulting in both truncated MNs and inappropriate early branching. Ectopic over-expression of zfPGRN-A mRNA resulted in increased MN branching and rescued the truncation defects brought about by knockdown of zfPGRN-A expression. The ability of PGRN to interact with established MN developmental pathways was tested. PGRN over-expression was found to reverse the truncation defect resulting from knockdown of Survival of motor neuron 1 (smn1). This is involved in small ribonucleoprotein biogenesis RNA processing, mutations of which cause Spinal Muscular Atrophy (SMA) in humans. It did not reverse the MN defects caused by interfering with the neuronal guidance pathway by knockdown of expression of NRP-1, a semaphorin co-receptor.
Expression of PGRN within MNs and the observed phenotypes resulting from mRNA knockdown and over-expression are consistent with a role in the regulation of spinal cord MN development and branching. This study presents the first in vivo demonstration of the neurotrophic properties of PGRN and suggests possible future therapeutic applications in the treatment of neurodegenerative diseases.
The growth factor progranulin (PGRN) regulates cell division, survival, and migration. PGRN is an extracellular glycoprotein bearing multiple copies of the cysteine-rich granulin motif. With PGRN family members in plants and slime mold, it represents one of the most ancient of the extracellular regulatory proteins still extant in modern animals. PRGN has multiple biological roles. It contributes to the regulation of early embryogenesis, to adult tissue repair and inflammation. Elevated PGRN levels often occur in cancers, and PGRN immunotherapy inhibits the growth of hepatic cancer xenografts in mice. Recent studies have demonstrated roles for PGRN in neurobiology. An autosomal dominant mutation in GRN, the gene for PGRN, leads to neuronal atrophy in the frontal and temporal lobes, resulting in the disease frontotemporal lobar dementia. In this review we will discuss current knowledge of the multifaceted biology of PGRN.
Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. In this study, goldfish granulin was one of the most commonly identified transcripts in the differential cross-screening of macrophage cDNA libraries and was preferentially expressed in proliferating macrophages. Unlike mammalian granulins, which possess 7.5 repeats of a characteristic signature of 12 cysteine residues, the goldfish granulin encoded a putative peptide possessing only 1.5 cysteine repeats. Northern blot and real-time PCR analyses indicated that goldfish granulin was expressed only in the hematopoietic tissues of the goldfish, specifically the kidney and spleen, and in activated peripheral blood mononuclear cells. We expressed granulin using a prokaryotic expression system and produced an affinity-purified rabbit anti-goldfish granulin IgG. Recombinant goldfish granulin induced a dose-dependent proliferative response of goldfish macrophages that was inversely related to the myeloid differentiation stage of the cells studied. The highest proliferative response was observed in macrophage progenitor cells and monocytes. This proliferative response of macrophages was abrogated by the addition of anti-granulin IgG. These results indicate that goldfish granulin is a growth factor that positively modulates cell proliferation at distinct junctures of macrophage differentiation.
Epithelin 1 and 2 were originally purified from rat kidneys based on their ability to inhibit the growth of A-431 human epidermoid carcinoma cells (Shoyab, M., McDonald, V.L., Byles, C., Todaro, G.J., and Plowman, G.D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7912-7916). This study presents the complete amino acid sequence of these two growth factors and the cloning of their cDNA from rat, mouse, and human sources. Epithelins 1 and 2 are 56- and 57-amino acid polypeptides, respectively, and share 47% amino acid sequence identity with the conserved spacing of 12 cysteine residues. Molecular cloning revealed that both proteins are encoded by a single precursor that contains 7 1/2 copies of this novel 12-cysteine motif, 2 of which represent the known active molecules. Recombinant expression in COS cells demonstrated that the epithelin 1 protein was mitogenic on rodent keratinocytes and fibroblasts. In contrast, epithelin 2 had no activity on these cells, but at high concentrations was capable of antagonizing the growth proliferative activities of epithelin 1. Northern analysis shows the epithelin mRNA to be expressed in many types of epithelial cells. The broad expression profile of epithelin transcripts, along with the opposing activities of the two mature protein products, implicates these factors as natural mediators of epithelial homeostasis.
The biogenesis of the sperm-specific organelle, the acrosome, was investigated using an acrosomal glycoprotein as a marker of development. This component, which we have named acrogranin, was purified from an acid extract of guinea pig testes by standard chromatographic procedures. The molecular weight of reduced acrogranin was determined to be 67,000 by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunization of female rabbits with purified acrogranin produced an antiserum that recognized a single protein with Mr = 67,000 in an acid extract of guinea pig testes. By indirect immunofluorescence, acrogranin was found only in the acrosome of mature sperm. In haploid spermatids, acrogranin was localized in the developing acrosome and, weakly, in the cytoplasm. Acrogranin was also detected in the cytoplasm and juxtanuclear region in putative proacrosomal granules of meiotic cells (pachytene spermatocytes). Detergent extracts from different purified germ cell populations contained only the Mr = 67,000 form of acrogranin, but sperm extracts had four lower Mr immunoreactive forms not present in the testicular extracts. By two-dimensional gel electrophoresis, acrogranin was found to be an acidic glycoprotein. Analysis of glycosylated and trifluoromethanesulfonic acid-deglycosylated acrogranin indicated that the antibody recognized polypeptide determinants. After highly enriched germ cell populations were labeled overnight with [35S]methionine and extracted with detergent, anti-acrogranin immunoprecipitated a single protein of Mr = 67,000. The synthesis of acrogranin by pachytene spermatocytes and round spermatids was similar, but the synthesis of the glycoprotein by condensing spermatids was markedly reduced. These studies demonstrate that acrosome biogenesis, as determined by the synthesis of a specific acrosomal component, begins during meiosis and continues through the early stages of spermiogenesis.
The mammalian embryo cannot develop without the placenta. Its specialized cells (trophoblast, endoderm, and extraembryonic
mesoderm) form early in development. They attach the embryo to the uterus (implantation) and form vascular connections necessary
for nutrient transport. In addition, the placenta redirects maternal endocrine, immune, and metabolic functions to the embryo's
advantage. These complex activities are sensitive to disruption, as shown by the high incidence of early embryonic mortality
and pregnancy diseases in humans, as well as the numerous peri-implantation lethal mutations in mice. Integration of molecular
and developmental approaches has recently produced insights into the molecules that control these processes.
Temporal and cell-type specific distribution of transforming growth factor alpha (TGF alpha) precursor (proTGF alpha) was examined in the mouse uterus during the periimplantation period, and after steroid hormone treatments of ovariectomized adult mice by immunohistochemistry using antibodies that recognize the precursor forms of the growth factor. These studies were complemented by immunoblot analysis of proTGF alpha in separated uterine cell-type preparations. The specificity of the antibodies used in these studies was confirmed by use of pancreas or lactating mammary glands from transgenic mice in which mutated proTGF alpha, lacking recognition sites for proteolytic cleavages, was targeted for expression under a tissue-specific enhancer/promoter. Analysis of histochemical studies revealed accumulation of immunoreactive proTGF alpha primarily in luminal and glandular epithelial cells on Day 1 of pregnancy or pseudopregnancy followed by little or no accumulation on Days 2 and 3. However, immunoreactive proTGF alpha started to reappear in the luminal epithelium on the morning of Day 4 and became more prominent in the afternoon. In pregnant mice, immunostaining persisted in these cells at the implantation sites during the time of attachment reaction (2130 h on Day 4), but disappeared by morning of Day 5. Immunostaining appeared to be situated at the apical border of the luminal epithelium. No positive immunostaining could be detected in the nonreceptive uterus on Day 5 or 6 of pseudopregnancy. Consistent with the immunohistochemistry results, Western blot analysis detected two species of precursor proteins (14.5 and 17 kDa) in isolated luminal epithelial cell-enriched preparations on Day 4, but not on Day 5, of pseudopregnancy. The results suggest that proTGF alpha accumulates in the luminal epithelium of the receptive uterus prior to implantation. The effects of ovarian steroids on uterine accumulation of proTGF alpha were examined in ovariectomized adult mice by immunohistochemistry and immunoblotting. Whereas an injection of estradiol-17 beta (E2) or progesterone (P4) had little or a modest effect on epithelial accumulation of proTGF alpha, P4 priming for several days resulted in distinct accumulation of proTGF alpha in epithelial cells. The superimposition of an E2 treatment on P4 priming showed a biphasic response, with an initial gradual loss of immunostaining through 12 h followed by a return by 24 h of E2 treatment. The combined hormone treatment schedule employed here is similar to the situation of inducing implantation with E2 in P4-primed delayed implanting mice. The results suggest a paracrine/"juxtacrine" role for this growth factor in implantation.
PC cell line is a highly tumorigenic insulin-independent variant from the teratoma-derived adipogenic cell line 1246. Culture medium of PC cells contains a growth promoting activity for 3T3 cells and producer cells. PC cell-derived growth factor (PCDGF) was purified to homogeneity from PC cell-conditioned medium as an apparent 88-kDa protein by chromatography on heparin-Sepharose, Sephacryl S-200, and phenyl-Sepharose. Digestion with peptide-N-glycosidase F yielded an apparent 68-kDa protein component indicating that PCDGF is a glycoprotein containing about 20 kDa of carbohydrate. Partial sequence from Edman degradation of peptide fragments obtained by digestion of PCDGF with cyanogen bromide and trypsin demonstrates that PCDGF contains regions of sequence identity to that deduced from the granulin or epithelin precursor cDNAs. Granulins are small polypeptides purified from granulocyte extracts with no apparent biological functions. Epithelins are cell growth modulators purified as small molecular mass 6-kDa polypeptides from kidney extracts. The existence of a large molecular mass precursor for granulin or epithelin has been predicted based upon recently cloned cDNAs encoding these biomolecules within a 63.5-kDa protein with putative glycosylation sites. No biological activity has previously been attributed to the precursor. The present results indicate that PCDGF is a potential precursor for epithelin and/or granulin, that this 88-kDa protein is secreted and glycosylated, and that it can function as a mitogen for 3T3 cells as well as an autocrine growth factor for PC cells.
Myeloid leukaemia inhibitory factor (LIF) is expressed at highest concentrations in the maternal endometrial glands at about the stage of blastocyst implantation. LIF is also expressed by the extraembryonic membranes of the early mouse embryo. Embryos of different ages were cultured with, or without, LIF, and embryo growth in vivo and in vitro was examined to determine whether LIF is important for embryo development. Supplementing embryo culture media with 1000 U recombinant human LIF ml-1 increased the number of eight-cell mouse embryos developing beyond the hatched blastocyst stage in vitro from 62.1% to 85.1% (P < 0.05). LIF significantly increased the number of embryos hatching (33.8% versus 7.65% for controls 96 h after hCG injection, P < 0.001), completely hatching (85.1% versus 62.1%, P < 0.05), and exhibiting trophoblast outgrowth (13.5% versus 0% 120 h after hCG treatment, 85.1% versus 47.0% 144 h after hCG treatment, P < 0.001) in vitro. LIF-treated embryos also displayed a significantly greater area of trophoblast outgrowth than did controls as early as day 5 in culture (P < 0.005). These data show that LIF enhances mouse eight-cell embryo development in vitro, as seen by the accelerated rate of embryo hatching and trophoblast outgrowth. In addition, enhanced embryo survival in vivo is shown, following the transfer of LIF-treated embryos into a pseudopregnant recipient female. Expression of mRNA encoding LIF was detected in endometrial cells cultured in monolayer from uteri of day 3 pregnant females, explaining the known embryotrophic effects of endometrial coculture. This expression was not enhanced significantly by treatment with oestradiol (3.7 x 10(-5) mol l-1) or progesterone (3.2 x 10(-6) mol l-1) or both hormones. These results indicate that LIF could have a dual action in early embryogenesis as an embryotrophin and as a factor required for embryo implantation. Multiple roles for LIF are consistent with the expression of this factor at embryonic, extraembryonic and maternal sites during early embryogenesis.
Transcription of endogenous genes in preimplantation 1- and 2-cell mouse embryos was determined by monitoring the incorporation of BrUTP by plasma membrane-permeabilized embryos. Incorporation is observed starting by mid-S phase in the 1-cell embryo and increases progressively; the amount of incorporation by the 1-cell embryo in G2 is about 20% that of the 2-cell embryo in G2. Incorporation by the male pronucleus is always about four to five times greater than that of the female pronucleus. Nevertheless, the amount of incorporation by the female pronucleus present in parthogenetically activated eggs is similar to the total amount of incorporation in inseminated eggs, i.e., the transcriptional capacity of the female pronucleus is not inherently less than that of the male pronucleus. Inhibiting the first round of DNA replication does not prevent the initiation of transcription in the 1-cell embryo, but does inhibit the extent of BrUTP incorporation by 35%. The transcriptional machinery of the 1-cell embryo appears to be rate-limiting, since the total amount of BrUTP incorporation by parthenogenetically activated and dispermic eggs is similar to that in monospermic eggs; trispermic eggs incorporate BrUTP to only about 60% the level of monospermic eggs. A transcriptionally repressive state may start to develop in the 2-cell embryo, since inhibiting the second round of DNA replication results in an 50% increase in BrUTP incorporation. Trapoxin treatment, which induces histone hyperacetylation, enhances incorporation by 2-cell embryos 1.8-fold and suggests that histone hyperacetylation can relieve this repression.
In the preimplantation mouse embryo, activation of the embryonic genome is accompanied by a transient enrichment of histone H4 acetylated at lysines 5, 8, and 12 at the nuclear periphery (Worrad et al., 1995: Development 121:2949-2959). In the present report, we use laser-scanning confocal microscopy and a new panel of antibodies to define the distribution of specific acetylated isoforms of the other three core histones in mouse embryos at the 1- to 4-cell stage. We find that histone H3 acetylated at lysine 9 and/or 18 (H3.Ac9/18) and the single acetylated form of H2A (H2A.Ac5) become transiently enriched at the nuclear periphery in the 2-cell embryo. In contrast, H3.Ac14, H3.Ac23, and acetylated H2B, like H4.Ac16, remain distributed throughout the nucleoplasm. The staining intensity with antisera to H3.Ac9/18, even at the periphery was weak compared to that obtained with antisera to acetylated H4. A brief period of culture, however, in the presence of the inhibitor of histone deacetylases trichostatin A (TSA) or trapoxin increased labeling. Thus, the steady-state level of H3.Ac9/18 at the nuclear periphery and H3.Ac14 and H3.Ac23 in the nuclear interior is relatively low, but turnover remains high. The localization of selected acetylated isoforms of H3 and H2A at the nuclear periphery was independent of ongoing transcription or of cytokinesis, but did require DNA replication. We propose a model in which the selective, replication-dependent acetylation and deacetylation of zygotic chromatin at the nuclear periphery mediates the programming of zygotic transcription.
Annually, 1.25 million individuals suffer burns in the United States and 6.5 million experience chronic skin ulcers, often from diabetes, pressure or venous stasis. Growth factors are essential mediators of wound repair, but their success as therapeutics in wound treatment has, so far, been limited. Therefore, there is a need to identify new wound-response regulatory factors, but few have appeared in recent years. Progranulin (also called granulin or epithelin precursor, acrogranin or PC-derived growth factor) is a growth factor involved in tumorigenesis and development. Peptides derived from progranulin have been isolated from inflammatory cells, which led to suggestions that progranulin gene products are involved in the wound response, but this remains undemonstrated. We report that in murine transcutaneous puncture wounds, progranulin mRNA is expressed in the inflammatory infiltrate and is highly induced in dermal fibroblasts and endothelia following injury. When applied to a cutaneous wound, progranulin increased the accumulation of neutrophils, macrophages, blood vessels and fibroblasts in the wound. It acts directly on isolated dermal fibroblasts and endothelial cells to promote division, migration and the formation of capillary-like tubule structures. Progranulin is, therefore, a probable wound-related growth factor.
The growth factor progranulin (acrogranin/PC-derived growth factor/granulin-epithelin precursor) promotes onset of blastocyst cavitation and is required for neonatal hypothalamic sexual differentiation. Little is known, however, of the range of developmental processes in which it is involved. We used in situ hybridization to investigate progranulin expression in murine embryos. Progranulin mRNA is expressed in maternal and embryonic components during early establishment of pregnancy. Abundant expression is observed in the early decidualizing uterine stroma and glands. In the embryo, the trophoblast giant cells at the interface of placental exchange sites (both choriovitelline and chorioallantoic placenta) show strong expression. The gastrulating epiblast and mesenchyme (intraembryonic and extraembryonic mesenchyme) all revealed activity. The allantois and yolk sac mesenchyme (site of early hemopoiesis) were positive, as were later phases of active vessel formation (pia mater of brain, epicardium of the heart). In the urogenital system, it was expressed in Sertoli cells and in kidney tubules. It was highly expressed in proliferating epidermal cells. During epidermal appendage formation, the early epithelial bud was positive, but the forming duct and differentiating adjacent mesenchyme was negative. It is widely distributed during central nervous system development and the peripheral nervous system (dorsal root ganglia and sympathetic ganglia). Based on the pattern of progranulin gene expression, we propose proliferative and developmental roles for progranulin in establishing pregnancy, during gastrulation, and during embryonic development of the epidermis, nervous system, blood vessel, formation, and spermatogenesis.
Conceptus implantation to the mother's uterus is a complex series of events involving coordinated expression of numerous genes at both the embryonic and the uterine sides. Since there are no suitable in vivo or in vitro experimental models, sequential changes occurring during the peri-implantation periods have not been well characterized. Using GeneChip technology and a recently introduced murine in vitro model of implantation, the expression of embryonic genes was examined before and after attachment to the uterine stromal cells. Instead of RNA or mRNA, amplified cRNA was subjected to the GeneChip analysis because amounts of mRNA in each blastocyst were minimal. Among 6,500 gene transcripts examined, changes in mRNA levels for 802 genes were identified. Of these detections, transcripts previously unsuspected were changes in a group of tumor suppressor and stress-induced genes, whose transcripts increased as embryos attached to the membrane. Validity of the data was evaluated using reverse transcription-polymerase chain reaction and in situ hybridization analyses, both of which confirmed developmental changes in selected gene expressions during pre- and post-attachment periods. The present data suggest that GeneChip technology would be very useful for finding genes previously unsuspected, and this method should be used as an initial step, particularly as a screening tool, toward the dissection of complex mechanisms such as the processes of implantation.
Epithelin 1 and 2 were originally purified from rat kidneys based on their ability to inhibit the growth of A-431 human epidermoid carcinoma cells (Shoyab, M., McDonald, V. L., Byles, C., Todaro, G. J., and Plowman, G. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7912-7916). This study presents the complete amino acid sequence of these two growth factors and the cloning of their cDNAs from rat, mouse, and human sources. Epithelins 1 and 2 are 56- and 57-amino acid polypeptides, respectively, and share 47% amino acid sequence identity with the conserved spacing of 12 cysteine residues. Molecular cloning revealed that both proteins are encoded by a single precursor that contains 7 1/2 copies of this novel 12-cysteine motif, 2 of which represent the known active molecules. Recombinant expression in COS cells demonstrated that the epithelin 1 protein was mitogenic on rodent keratinocytes and fibroblasts. In contrast, epithelin 2 had no activity on these cells, but at high concentrations was capable of antagonizing the growth proliferative activities of epithelin 1. Northern analysis shows the epithelin mRNA to be expressed in many types of epithelial cells. The broad expression profile of epithelin transcripts, along with the opposing activities of the two mature protein products, implicates these factors as natural mediators of epithelial homeostasis.
Temporal and cell-type specific distribution of transforming growth factor alpha (TGF alpha) precursor (proTGF alpha) was examined in the mouse uterus during the periimplantation period, and after steroid hormone treatments of ovariectomized adult mice by immunohistochemistry using antibodies that recognize the precursor forms of the growth factor. These studies were complemented by immunoblot analysis of proTGF alpha in separated uterine cell-type preparations. The specificity of the antibodies used in these studies was confirmed by use of pancreas or lactating mammary glands from transgenic mice in which mutated proTGF alpha, lacking recognition sites for proteolytic cleavages, was targeted for expression under a tissue-specific enhancer/promoter. Analysis of histochemical studies revealed accumulation of immunoreactive proTGF alpha primarily in luminal and glandular epithelial cells on Day 1 of pregnancy or pseudopregnancy followed by little or no accumulation on Days 2 and 3. However, immunoreactive proTGF alpha started to reappear in the luminal epithelium on the morning of Day 4 and became more prominent in the afternoon. In pregnant mice, immunostaining persisted in these cells at the implantation sites during the time of attachment reaction (2130 h on Day 4), but disappeared by morning of Day 5. Immunostaining appeared to be situated at the apical border of the luminal epithelium. No positive immunostaining could be detected in the nonreceptive uterus on Day 5 or 6 of pseudopregnancy. Consistent with the immunohistochemistry results, Western blot analysis detected two species of precursor proteins (14.5 and 17 kDa) in isolated luminal epithelial cell-enriched preparations on Day 4, but not on Day 5, of pseudopregnancy. The results suggest that proTGF alpha accumulates in the luminal epithelium of the receptive uterus prior to implantation. The effects of ovarian steroids on uterine accumulation of proTGF alpha were examined in ovariectomized adult mice by immunohistochemistry and immunoblotting. Whereas an injection of estradiol-17 beta (E2) or progesterone (P4) had little or a modest effect on epithelial accumulation of proTGF alpha, P4 priming for several days resulted in distinct accumulation of proTGF alpha in epithelial cells. The superimposition of an E2 treatment on P4 priming showed a biphasic response, with an initial gradual loss of immunostaining through 12 h followed by a return by 24 h of E2 treatment. The combined hormone treatment schedule employed here is similar to the situation of inducing implantation with E2 in P4-primed delayed implanting mice. The results suggest a paracrine/"juxtacrine" role for this growth factor in implantation.
Uterine endometrium is the anatomic prerequisite for the continuation of the species. During the reproductive years, its main purpose is to communicate with, receive, nourish, and protect the implanting blastocyst. However, roles for it prepubertally and in the peri- and postmenopause periods remain undefined. In ovulatory cycles, in the absence of implantation, a series of events is initiated in endometrium to promote its uniform and efficient shedding and regeneration during the subsequent cycle. Disorders of endometrial cyclic development can result in infertility, repetitive miscarriage, poor placentation, and dysfunctional uterine bleeding. Mechanisms underlying normal endometrial development are beginning to be elucidated, as is the ''cross-talk'' between the implanting blastocyst and the maternal decidua. This article reviews endometrial growth factors and proteins that participate in endometrial growth, development, shedding, and tissue remodeling, as well as blastocyst-decidua interactions. Several growth factors and cytokines are discussed in detail in later articles but are mentioned here for completeness.
Preimplantation mammalian embryos in culture secrete autocrine growth factors into the surrounding medium that, in turn, stimulate the development of the embryos. The full complement of these factors is unknown. Since one hallmark of embryo development is the formation of an epithelium, the trophectoderm, we tested the hypothesis that one such embryo-derived growth factor is acrogranin (epithelin/granulin precursor), a factor that possesses growth-regulatory activities principally toward epithelial cells. We found that acrogranin mRNA was expressed in preimplantation mouse embryos with the transcript levels rising to their highest point in blastocysts, coincident with the appearance of the trophectoderm. Indirect immunofluorescence confocal microscopy of preimplantation mouse embryos at different developmental stages revealed that acrogranin immunostaining was most concentrated in the trophectoderm of blastocysts. Immunoblotting and immunoprecipitation experiments demonstrated that the embryos secreted acrogranin into the surrounding medium. To determine how altering the levels of acrogranin in the culture medium surrounding the embryos might affect embryonic growth and development, acrogranin protein levels in the culture medium were decreased with a function-blocking antibody or increased by adding the purified acrogranin to the medium. In both a concentration-dependent and a reversible manner, affinity-purified anti-acrogranin antibody significantly inhibited the development of eight-cell embryos to the blastocyst stage compared to controls (no added immunoglobulin or nonspecific IgG). Furthermore, embryo cell numbers were significantly decreased in the presence of the highest concentrations of acrogranin antibody compared to control embryos. Exogenous acrogranin added to cultures of eight-cell embryos accelerated the time for the onset of cavitation, as well as stimulating the rate of blastocoel expansion and increasing the number of trophectoderm cells compared to controls. These results indicate that acrogranin can regulate the appearance of the epithelium in the developing mouse blastocyst, the growth of the trophectoderm, and/or the function of the embryonic epithelium.
By the cDNA subtraction between cDNA preparations from androgenized and intact neonate rats hypothalami, granulin/ epithelin (grn) gene was identified as a gene enriched by androgenization. Strong grn mRNA signals were detected by in situ hybridization in the ventromedial nucleus (VMH) and the arcuate nucleus (ARC) of the hypothalamus of a 5-day-old male. The grn gene expression level in the hypothalamus was similar between males and females at birth. At 10 days of age, this level was maintained in males, but decreased to 1/4 in females. Thus, grn can be the gene of which expression in the VMH and ARC is sustained by endogenous androgen in male neonates.
Mouse pre-implantation development appears to be under the control of paracrine and autocrine growth factors. The epithelium of the oviduct and the uterus together, with the population of macrophages and lymphocytes present in the reproductive tract from the onset of pregnancy, are thought to be the major sources of paracrine growth factors targeted to the developing embryos. Some of the growth factors are synthesized by both uterine epithelial cells and activated lympho-hematopoietic cells, suggesting a partial overlap of the regulatory signals used by the reproductive and lympho-hematopoietic systems. Such growth factors may be the long sought-after mediators of the synchrony between the pre-implantation embryo and the sex-steroid hormone-induced changes in the uterus.
Hatching, attachment and outgrowth of mouse blastocysts in vitro are dependent upon the presence of specific free amino acids in the medium. When grown in Eagle's Basal Medium (BME) containing 1% dialyzed fetal calf serum, 67% of blastocysts hatch. Omission of histidine, methionine, threonine, tryptophan, tyrosine, or valine from BME significantly reduces the incidence of hatching. For attachment to occur, cystine and lysine are also required, and for trophoblastic outgrowth, every essential amino acid except isoleucine is required. Omission of glutamine reduces the extent of outgrowth. Addition of higher (10X–20X BME) concentrations of cystine, histidine, or lysine increases the extent of outgrowth, but addition of 10−5–10−2M concentrations of non-essential amino acids does not stimulate either hatching, attachment, or outgrowth.
When all essential amino acids are at optimal concentrations, nearly 100% hatching occurs in a chemically defined medium in the absence of a macromolecular fixed nitrogen source. Extensive trophoblastic outgrowth and inner cell mass growth occur at optimal amino acid concentrations, but only in the presence of serum. Under these conditions, a collagen substrate is not necessary for growth and differentiation of the inner cell mass into endoderm and ectoderm in vitro. These requirements indicate that during post-blastocyst development in vitro the mouse embryo gradually becomes dependent upon specific exogenous fixed nitrogen sources, including essential amino acids and a non-dialyzable component from serum.
A rapid procedure has been devised to count the numbers of outer trophectoderm (TE) and inner cell mass (ICM) cells of mouse blastocysts by differentially labelling their nuclei in situ with polynucleotide-specific fluorochromes. The TE nuclei were labelled with propidium iodide (PI) by permeabilising the cells using selective antibody-mediated complement lysis (Solter and Knowles, '75). The blastocysts were then fixed in ethanol and the ICM nuclei labelled with bisbenzimide. These two fluorochromes have widely different fluorescent spectra. Thus, by using fluorescence microscopy with appropriate filter combinations, the PI-labelled TE nuclei appeared pink or red; the bisbenzimide-labelled ICM nuclei, blue or unlabelled.
The total numbers of blastocyst nuclei and the numbers of ICM nuclei counted by differential labelling were similar to the numbers detected after spreading the nuclei of intact blastocysts or immunosurgically isolated ICMs by air-drying (Tarkowski '66).
Differential labelling of TE and ICM nuclei in situ has two important advantages—that the numbers of both these cell types can be determined for individual blastocysts and that spatial relationships are partially preserved so that regional interactions can be studied.
Spermatogenesis is a unique system of differentiation involving cellular remodeling and the biogenesis of sperm-specific organelles. To study the biogenesis of one such organelle, the acrosome, we have been examining the gene expression, biosynthesis, and targeting of specific acrosomal proteins during mammalian spermatogenesis. An acrosomal marker that we recently purified and began characterizing is acrogranin, a 67,000-molecular-weight glycoprotein originally isolated from guinea pig testes. This glycoprotein is detected in pachytene spermatocytes and is found later in the acrosomes of developing spermatids and sperm. Immunoblotting of several tissues and immunofluorescent localization in frozen sections of guinea pig testes suggested that acrogranin was a germ cell-specific glycoprotein that was expressed meiotically and post-meiotically. However, Northern blot analysis demonstrated that the mRNA for acrogranin was ubiquitously expressed in all guinea pig and mouse tissues examined. Furthermore, the primary structures of guinea pig and mouse acrogranins, deduced from the cDNA sequences, reveal that this glycoprotein is a cysteine-rich molecule with a motif that is tandemly repeated seven times, very similar to that of the human epithelin/granulin precursor. We conclude that guinea pig and mouse acrogranins are homologues of the precursor of the human and rat epithelin/granulin peptides previously demonstrated to have growth-modulating properties.
Embryo implantation is a complex developmental process requiring precise coordination between mother and offspring to ensure success. Implantation failure is clinically relevant to in vitro fertilization programs and to an understanding of diseases of pregnancy like preeclampsia. Basic and clinical research have identified a number of proteins involved in peri-implantation development, but an understanding of the implantation process and its cellular and molecular components is just beginning. This review will focus on the implantation and development of the murine embryo and placenta. The significance of ectopic expression and targeted mutagenesis models to these processes will be discussed.
During early development, a subset of fetal (placental) cytotrophoblasts exhibits tumor-like behavior and invades the uterus. To access a supply of maternal blood, they invade arterioles and form heterotypic interactions with, and replace, resident maternal endothelium, creating a hybrid uterine vasculature. Recently, it has become clear that invading cytotrophoblasts transform their adhesion receptor phenotype to resemble the endothelial cells they replace. Furthermore, they express vasculogenic factors and receptors. Is this a form of vasculogenesis?
Temporal and spatial distribution of the sites of DNA replication were examined in 1-cell mouse embryos. Embryos were labelled with bromodeoxyuridine (BrdU) at hourly intervals after fertilisation, and the incorporation of BrdU was examined by laser-scanning confocal microscopy following immunostaining with an anti-BrdU antibody. DNA replication first started uniformly in both the male and female pronuclei in the intranuclear region and then was observed in the peripheral regions of nucleus and nucleolus. These changes, however, occurred asynchronously in that the female pronucleus required a longer time to complete replication in the intranuclear region but not in the peripheral regions. Inhibiting transcription with alpha-amanitin had no effect on the temporal and spatial patterns of DNA replication. Treatment of the embryos with trapoxin, a specific inhibitor of histone deacetylase, accelerated the completion of replication in the peripheral regions but not in the intranuclear region. These results suggest that DNA replication is temporally and spatially regulated in the 1-cell embryos and that acetylation of histones, but not transcription, is involved in the regulation of DNA replication.
During human pregnancy specialized placental cells of fetal origin, termed cytotrophoblasts, invade the uterus and its blood vessels. This tumor-like process anchors the conceptus to the mother and diverts the flow of uterine blood to the placenta. Previously, we showed that the expression of molecules with important functional roles, including a number of extracellular matrix integrin receptors, is precisely modulated during cytotrophoblast invasion in situ. Here we exploited this observation to study the role of the focal adhesion kinase (FAK), which transduces signals from the extracellular matrix and recruits additional signaling proteins to focal adhesions. Immunolocalization studies on tissue sections showed that FAK is expressed by cytotrophoblasts in all stages of differentiation. Because extracellular matrix-induced integrin clustering results in FAK (auto)phosphorylation on tyrosine 397 (Y397FAK), we also localized this form of the molecule. Immunolocalization experiments detected Y397FAK in a subset of cytotrophoblasts near the surface of the uterine wall. To assess the functional relevance of this observation, we used an adenovirus strategy to inhibit cytotrophoblast expression of FAK as the cells differentiated along the invasive pathway in vitro. Compared to control cells transduced with a wild-type virus, cytotrophoblasts that expressed antisense FAK exhibited a striking reduction in their ability to invade an extracellular matrix substrate. When cytotrophoblast differentiation was compromised (hypoxia in vitro, preeclampsia in vivo), Y397FAK levels associated with the plasma membrane were strikingly lower, although total FAK levels did not change. Together our results suggest that (auto)phosphorylation of Y397 on FAK is a critical component of the signaling pathway that mediates cytotrophoblast migration/invasion.
The mechanisms of sexual differentiation of the brain by sex steroids seem to be conserved throughout the mammalian species, although there may be some species differences. In rats, sex-dependent differentiation of the brain occurs in a sex steroid-dependent manner during the perinatal period known as the critical period. Androgen exposure during the perinatal period results in the development of structural and functional sexually dimorphic characteristics in the brain; the absence of testicular androgen leads the central nervous system to develop passively in a primarily female fashion, while the presence of androgen induces the masculinization of the brain. We attempted to characterize sex steroid-inducible genes that are involved in the sexually dimorphic function of the brain. Following the cDNA subtraction between hypothalami of 5-day-old intact and neonatally androgenized female rats, a granulin (grn) precursor gene was identified. The grn gene encodes a 6-kDa polypeptide known as a growth modulating factor of epithelial cells in vitro. Exogenous estrogen, as well as androgen, induced grn gene expression in the neonatal hypothalamus. In the brain of a 5-day-old male rat, grn mRNA was expressed in the ventromedial hypothalamic nucleus and the arcuate nucleus of the hypothalamus. Throughout the critical period for sexual differentiation of the brain, grn gene expression remained high in males, while in females it gradually decreased. Antisense oligodeoxynucleotide (ODN) complementary to grn mRNA was synthesized and infused into the third ventricle of male rats at 2 days of age. Two different control treatments were used; the first consisted of a control sequence ODN that had virtually no homology to known mRNAs, and the second consisted of vehicle alone. After maturation, the subject animals that were treated with antisense ODN of grn displayed significantly lower scores than the control males in various parameters assessing sexual behavior, i.e., mount, intromission, and ejaculation. The present results suggest that the grn gene, the expression of which is induced by sex steroids in the neonatal hypothalamus, plays a crucial role in the functional masculinization of the rat brain.
Current traditional pathological parameters, including staging and grading, are not sufficient in predicting outcome in patients with renal cell carcinoma (RCC). Acrogranin is an epithelial growth factor and has been demonstrated to play a role in teratocarcinogenesis and tumorigenesis. The aim of this study was to examine levels of acrogranin in renal cancer.
Western blot analysis was performed on renal tissue protein lysates. In addition, immunohistochemical (IHC) analysis of acrogranin expression was conducted on tissue sections of various histological types and grades of RCC.
Western analysis showed that acrogranin levels were low in benign renal tissue and increased in malignant renal tissue. In addition, IHC revealed that high-grade RCC exhibited higher levels of expression than low-grade RCC and normal tissue.
These data suggest that acrogranin may be a functional important growth factor in RCC and may be a potential molecular marker for high-grade RCC.
PC-cell-derived growth factor (PCDGF, progranulin) is a novel autocrine growth factor that is overexpressed in human breast cancer cell lines. We have examined immunohistochemical PCDGF expression in 206 paraffin-embedded human breast lesions and investigated its association with clinicopathological variables. PCDGF staining was observed in breast carcinoma, whereas it was almost always negative in benign breast epithelium. PCDGF expression was more common in invasive ductal carcinoma (80% cases positive) than in invasive lobular carcinoma (53% positive). PCDGF staining was almost never observed in lobular carcinoma in situ. Ductal carcinoma in situ expressed PCDGF in 66% of the cases, and this expression correlated strongly with nuclear grade. Similar correlation was observed between PCDGF expression and histologic grade of invasive ductal carcinoma. Average Ki-67 index of PCDGF-negative/weakly positive invasive carcinomas (30.3) was significantly lower than that of strongly PCDGF-positive tumors (48.8, P=0.01). A larger percentage of tumors that expressed PCDGF with a staining intensity of 2+ or 3+ were p53 positive (44%) than were PCDGF-negative tumors (25%), P=0.02. PCDGF expression was independent of c-erbB-2 overexpression and of ER and PR status. Our study provides the first evidence of high incidence of PCDGF expression in human breast cancer in which it correlates with clinicopathological variables such as tumor grade, proliferation index, and p53 expression. These characteristics, as well as the virtual absence of expression in benign breast tissue, suggest an important role of PCDGF in breast cancer pathogenesis and make it a potential novel target for the treatment of breast cancer.
Conceptus implantation to the uterine endometrium represents a complex series of events, including synchronized development of conceptus and uterus through up- and/or down-regulation of numerous gene products. In a previous study using the DNA microarray technique, we had discovered evidence that increase in a transcript for mesenchymal morphogen, epimorphin, was noted as the conceptus attached to the matrix in vitro (Qin et al., 2003). In the present study, the expression and potential function of epimorphin in developing conceptuses was investigated through the use of reverse transcription-polymerase chain reaction (RT-PCR), whole mount in situ hybridization/immunohistochemistry, and in vitro blastocyst culture. RT-PCR and in situ hybridization analysis revealed that epimorphin mRNA was expressed weakly in murine conceptuses during early developmental stages (1 cell to post-adhesion blastocyst stages) and higher levels of epimorphin transcripts were observed in both inner cell mass (ICM) and trophectoderm of outgrowing blastocysts. Immunohistochemical analysis confirmed that epimorphin was localized in outgrowing trophoblast cells and ICM. Treating blastocysts in culture with a 115 kDa form of recombinant epimorphin promoted trophoblast outgrowth (P < 0.05), but a 34 kDa form of recombinant epimorphin had no effect. Treatment with a function inhibitor, rat anti-mouse epimorphin IgM, reduced the number of embryos progressing to blastocyst outgrowth to the levels similar to those observed with plain culture medium. Reverse transcription-polymerase chain reaction (RT-PCR) analysis also revealed that epimorphin increased the expression of a trophoblast cell differentiation marker, placental lactogen-1 (PL-1), mRNA (P < 0.01). These results suggest that epimorphin is involved in trophoblast outgrowth, a process required for conceptus implantation into the endometrium.
Regulation of embryo outgrowth by a morphogenic factor, epimorphin, in the mouse
- JW Qin
- Y Takahashi
- K Isuzugawa
- M Imai
- S Yamamoto
- Y Hrai
- K Imakawa