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PBX1 enhanced HF-iPSC generation and activated the NANOG promoter. a, b Quantification of the number of alkaline phosphatasepositive colonies 32 days after lentivirus vector-mediated transduction with SOMKP and SOMK vectors into HF-MSCs. c Expression of endogenous pluripotency genes in HF-iPSCs SOMK and HF-iPSCs SOMKP was assessed by qPCR. The value in iPSCs SOMK as the control group was set as 1.0. d, e Quantification of the number of alkaline phosphatase-positive colonies 32 days after lentivirus vector-mediated transduction by SOMK with scrambled shRNA and SOMK with shRNA against PBX1. f Transduced HF-MSCs were harvested on days 7, 14, 21, and 28 to assess the expression of endogenous NANOG by qPCR. Data are shown as fold induction compared with the SOMK control on day 7. g Dual-luciferase reporter gene assays were used to assess the activation of the NANOG promoter in transduced HF-MSCs on days 7, 14, 21, and 28. The value in SOMKtransduced HF-MSCs as the control group was set as 1.0. *P < 0.05; **P < 0.01; ***P < 0.001
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Background:
PBX homeobox 1 (PBX1) is involved in the maintenance of the pluripotency of human embryonic and hematopoietic stem cells; however, the effects of PBX1 in the self-renewal and reprogramming of hair follicle mesenchymal stem cells (HF-MSCs) are unclear. The AKT/glycogen synthase kinase (GSK) 3β pathway regulates cell metabolism, prolifer...
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... into NOD-SCID mice (Fig. 4f). Moreover, HF-iPSCs SOMKP exhibited a normal male chromosome type (46XY), similar to HF-MSCs, and no chromosomal aberrations were found (Fig. 4g). Interestingly, compared with SOMK transduction, SOMKP transduction significantly increased both HF-iPSC colony formations, from 50.67 ± 3.84 to 79 ± 8.02 (P < 0.05; Fig. 5a, b), and increased the expression levels of the endogenous OCT4, LIN28, SOX2, and NANOG genes (P < 0.05, P < 0.01; Fig. 5e). In contrast, knockdown of PBX1 with PBX1 shRNA significantly decreased SOMKinduced HF-iPSC colony formation, from 52 ± 5.5 to 28 ± 4.5 (P < 0.05; Fig. 5d, e). NANOG is a core TF involved in the maintenance of the ...
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... and no chromosomal aberrations were found (Fig. 4g). Interestingly, compared with SOMK transduction, SOMKP transduction significantly increased both HF-iPSC colony formations, from 50.67 ± 3.84 to 79 ± 8.02 (P < 0.05; Fig. 5a, b), and increased the expression levels of the endogenous OCT4, LIN28, SOX2, and NANOG genes (P < 0.05, P < 0.01; Fig. 5e). In contrast, knockdown of PBX1 with PBX1 shRNA significantly decreased SOMKinduced HF-iPSC colony formation, from 52 ± 5.5 to 28 ± 4.5 (P < 0.05; Fig. 5d, e). NANOG is a core TF involved in the maintenance of the pluripotent state in hESCs and reprogramming of somatic cells into iPSCs. Thus, we next evaluated the expression of the ...
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... HF-iPSC colony formations, from 50.67 ± 3.84 to 79 ± 8.02 (P < 0.05; Fig. 5a, b), and increased the expression levels of the endogenous OCT4, LIN28, SOX2, and NANOG genes (P < 0.05, P < 0.01; Fig. 5e). In contrast, knockdown of PBX1 with PBX1 shRNA significantly decreased SOMKinduced HF-iPSC colony formation, from 52 ± 5.5 to 28 ± 4.5 (P < 0.05; Fig. 5d, e). NANOG is a core TF involved in the maintenance of the pluripotent state in hESCs and reprogramming of somatic cells into iPSCs. Thus, we next evaluated the expression of the NANOG by qPCR. The results showed that NANOG expression in HF-iPSCs induced by either SOMK or SOMKP transduction increased over time from day 14 to day 28. SOMKP ...
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... SOMK or SOMKP transduction increased over time from day 14 to day 28. SOMKP transduction significantly increased NANOG expression on days 14 (P < 0.05), 21 (P < 0.001), and 28 (P < 0.05) compared with SOMK transduction. However, knockdown of PBX1 with Pbx1 shRNA significantly decreased NANOG expression during reprogramming (P < 0.01, P < 0.001; Fig. 5f). Dual-luciferase assays showed that compared with SOMK, SOMKP transduction significantly increased NANOG promoter activities by 1.74-, 1.46-, and 1.25-fold during reprogramming of HF-MSCs into HF-iPSCs on days 7 (P < 0.01), 14 (P < 0.01), and 21 (P < 0.05), whereas knockdown of PBX1 significantly decreased NANOG promoter activities on ...
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... transduction significantly increased NANOG promoter activities by 1.74-, 1.46-, and 1.25-fold during reprogramming of HF-MSCs into HF-iPSCs on days 7 (P < 0.01), 14 (P < 0.01), and 21 (P < 0.05), whereas knockdown of PBX1 significantly decreased NANOG promoter activities on days 7 (P < 0.001), 14 (P < 0.001), 21 (P < 0.01), and 28 (P < 0.05; Fig. 5g). These findings suggested a role for PBX1 in the activation of the pluripotency-related gene NANOG during iPSC reprogramming. f, g Western blot analysis the levels of phospho-AKT, phospho-GSK3β, cyclin D1, p16, p21, and β-catenin proteins in HF-MSCs EGFP and HF-MSCs PBX1 . GAPDH and HISTONE were used as endogenous controls for equal ...
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Background
Current understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem...
Citations
... From extensive previous studies, we already know that PBX1 acts in proliferation in stem cells and during organogenesis [54][55][56][57]. This is a good starting point to make us question whether PBX1 is involved in cancer cell proliferation as well. ...
... PBX1 was already associated with cell survival and cell death in non-tumoral circumstances. In hair follicle mesenchymal stem cells, PBX1 expression reduces mitochondrialmediated apoptosis through AKT/GSK3β pathway activation [55]. However, Pbx1 seems to mediate apoptosis in mice's midface morphogenesis [73]. ...
... However, PBX1 alone is not sufficient to induce p21 expression since HOXA1 was the only participant in the trimeric complex that could bind directly to the p21 promoter and could induce p21 expression by itself [96]. Moreover, PBX1 expression decreased p21 protein levels in hair follicle mesenchymal stem cells [55], supporting its role as a regulator of p21 that might act both activating or repressing its expression. Regarding the interaction between PBX1 and p27, to date, no other study that would be able to corroborate or contradict the results found by Li et al. could be found [72]. ...
Pre-B-cell leukemia homeobox transcription factor 1 (PBX1) was first identified as part of a fusion protein resulting from the chromosomal translocation t(1;19) in pre-B cell acute lymphoblastic leukemias. Since then, PBX1 has been associated with important developmental programs, and its expression dysregulation has been related to multifactorial disorders, including cancer. As PBX1 overexpression in many cancers is correlated to poor prognosis, we sought to understand how this transcription factor contributes to carcinogenesis, and to organize PBX1’s roles in the hallmarks of cancer. There is enough evidence to associate PBX1 with at least five hallmarks: sustaining proliferative signaling, activating invasion and metastasis, inducing angiogenesis, resisting cell death, and deregulating cellular energetics. The lack of studies investigating a possible role for PBX1 on the remaining hallmarks made it impossible to defend or refute its contribution on them. However, the functions of some of the PBX1’s transcription targets indicate a potential engagement of PBX1 in the avoidance of immune destruction and in the tumor-promoting inflammation hallmarks. Interestingly, PBX1 might be a player in tumor suppression by activating the transcription of some DNA damage response genes. This is the first review organizing PBX1 roles into the hallmarks of cancer. Thus, we encourage future studies to uncover the PBX1’s underlying mechanisms to promote carcinogenesis, for it is a promising diagnostic and prognostic biomarker, as well as a potential target in cancer treatment.
... PBX1 can activate NANOG transcription by binding to the NANOG promoter alone or in combination with OCT4 and KLF4, enhancing the self-renewal capability of human embryonic stem cells (Chan et al. 2009). Our research group found that PBX1 can increase hHF-MSCs proliferation and selfrenewal, potentially by activated PI3K/AKT and increasing phosphorylation of GSK3b (Jiang et al. 2019). The typical growth signaling PI3K/AKT pathway regulates various cellular functions, including cell proliferation, survival, migration, and metabolism (Han et al. 2013). ...
... Western blot analysis of signaling pathway-related proteins P5 hHF-MSCs were seeded in a 10-cm culture dish, and 10 lg/mL TAT-PBX1 or isopyknic PBS was added to DMEM/F12 medium. The cells were incubated from P5 to P10, and then a western blot was performed as described previously (Jiang et al. 2019). The primary antibodies used were AKT (pan) (C67E7) rabbit mAb (CST USA, 1:1000), phospho-AKT (Ser473) rabbit mAb (CST USA, 1:1000), cyclin D1(92G2) rabbit mAb (CST USA, 1:1000), p16 rabbit mAb (ProteinTech Group Inc, USA, 1:1000), p21 Waf1/ Cip1 (12D1) rabbit mAb (CST, 1:1000), and GAPDH mouse mAb (ProteinTech Group Inc, USA, 1:10,000). ...
... Internalization of TAT-PBX1 promotes hHF-MSC proliferation and delays senescence by activating AKT pathway signaling Previous study showed that ectopic expression of PBX1 using retroviral vectors promoted cell proliferation through activation of the AKT signaling pathway in hHF-MSCs (Jiang et al. 2019). In this study, hHF-MSCs treated with the TAT-PBX1 fusion protein showed higher expression p-AKT compared with that in the PBS-treated control group, which indicates that TAT-PBX1 activated AKT after cellular uptake (Fig. 3a, b). ...
Objectives
To express a TAT-PBX1 fusion protein using a prokaryotic expression system and to explore potential effects of TAT-PBX1 in the proliferation and senescence of human hair follicle-derived mesenchymal stem cells.ResultsThe TAT-PBX1 fusion was produced in inclusion bodies and heterogenously expressed in Rosetta (DE3) cells. Immunofluorescence staining showed that TAT-PBX1 fusion proteins were internalized by human hair follicle-derived mesenchymal stem cells. The growth rate of cells was increased after treatment with more than 5.0 μg/mL of TAT-PBX1. The rate of senescence-associated β-galactosidase positive cells was reduced in the 10.0 μg/mL TAT-PBX1 group (28%) than the 0 μg/mL control group (60%). Cells treated with the TAT-PBX1 fusion protein showed higher expression of p-AKT (1.22-fold that of the control), which indicates that TAT-PBX1 activated AKT pathway after cellular uptake.Conclusions
The TAT-PBX1 fusion protein increased the proliferation of hair follicle mesenchymal stem cells and delayed their senescence by activating the AKT pathway following internalization by cells.
... A 1500-bp DNA fragment containing cyclin D1 promoter region was synthesized and cloned into pGL3-Basic plasmid by ZooNBio Biotechnology, and then 3 plasmids, including control plasmid pRL-TK, recombinant plasmid pGL3-CD117p or pGL3-Basic, and MSCV-puro-ELK1 or MSCV-puro, were transfected into HEK293T cells, and cells were cultured for 48 hours. Luciferase reporter assay was performed as described previously [18]. The activity of firefly luciferase and Renilla luciferase was assayed using the Trans Detect Double-Luciferase Reporter Assay Kit (TransGen Biotech) in a multifunctional chemiluminometer. ...
Patients with chemotherapy or radiation therapy often generate anemia and low immunity due to the therapy-induced bone marrow (BM) suppression. To enhance hematopoietic regeneration during the disease treatment urgently need to be solved. Fibroblast growth factors (FGFs) play important regulatory roles in hematopoietic stem and progenitor cell (HSPC) expansion in vitro and in vivo by FGF receptor (FGFR1-4)-mediated signaling pathway. FGFR3 is an important member of FGFR family and its regulatory function in hematopoiesis is largely unknown. Using knock out (KO) mice of FGFR3, we found that loss of FGFR3 does not affect HSPC functions, linage differentiation during steady-state hematopoiesis, but FGFR3 deletion accelerates HSPC expansion and hematopoiesis recovery via a cell-autonomous manner under 5-FU-induced BM suppression. Our results showed that FGFR3 inactivation accelerates BM suppression-induced HSPC expansion by upregulating FGFR1 and its downstream transcriptional factor, ELK, which regulates the expression of Cyclin D1 gene at the level of transcription. Further studies confirmed that loss of FGFR3 in hematopoietic cells inhibits in vivo leukemogenesis under BM suppression. Our data firstly found a novel hematopoietic regulatory mechanism by which FGFR3 deletion promotes HSPC expansion under BM suppression, and also provided a promising approach to enhance anti-leukemia and hematopoietic regeneration by inhibiting FGFR3 functions in HSPCs combined with leukemic chemotherapy.
... The reason behind this phenomenon is that PBX1 knockdown reduced p-AKT expression, which leads to a similar consequence as AKT knockdown. The result is consistent with our group's research which showed that PBX1 enhances HF-MSCs proliferation by activating AKT and downregulation of p16 and p21 [49]. These results also suggested that there was a feedback loop between PBX1 and AKT signaling. ...
Stem cells derived from elderly donors or harvested by repeated subculture exhibit a marked decrease in proliferative capacity and multipotency, which not only compromises their therapeutic potential but also raises safety concerns for regenerative medicine. NANOG—a well-known core transcription factor—plays an important role in maintaining the self-renewal and pluripotency of stem cells. Unfortunately, the mechanism that NANOG delays mesenchymal stem cell (MSC) senescence is not well-known until now. In our study, we showed that both ectopic NANOG expression and PBX1 overexpression (i) significantly upregulated phosphorylated AKT (p-AKT) and PARP1; (ii) promoted cell proliferation, cell cycle progression, and osteogenesis; (iii) reduced the number of senescence-associated- β -galactosidase- (SA- β -gal-) positive cells; and (iv) downregulated the expression of p16, p53, and p21. Western blotting and dual-luciferase activity assays showed that ectopic NANOG expression significantly upregulated PBX1 expression and increased PBX1 promoter activity. In contrast, PBX1 knockdown by RNA interference in hair follicle- (HF-) derived MSCs that were ectopically expressing NANOG resulted in the significant downregulation of p-AKT and the upregulation of p16 and p21. Moreover, blocking AKT with the PI3K/AKT inhibitor LY294002 or knocking down AKT via RNA interference significantly decreased PBX1 expression, while increasing p16 and p21 expression and the number of SA- β -gal-positive cells. In conclusion, our findings show that NANOG delays HF-MSC senescence by upregulating PBX1 and activating AKT signaling and that a feedback loop likely exists between PBX1 and AKT signaling.
Hair loss, or alopecia, is a prevalent condition in modern society that imposes substantial mental and psychological burden on individuals. The types of hair loss, include androgenetic alopecia, alopecia areata, and telogen effluvium; of them, androgenetic alopecia is the most common condition. Traditional treatment modalities mainly involve medical options, such as minoxidil, finasteride and surgical interventions, such as hair transplantation. However, these treatments still have many limitations. Therefore, exploring the pathogenesis of hair loss, specifically focusing on the development and regeneration of hair follicles (HFs), and developing new strategies for promoting hair regrowth are essential. Some emerging therapies for hair loss have gained prominence; these therapies include low-level laser therapy, micro needling, fractional radio frequency, platelet-rich plasma, and stem cell therapy. The aforementioned therapeutic strategies appear promising for hair loss management. In this review, we investigated the mechanisms underlying HF development and regeneration. For this, we studied the structure, development, cycle, and cellular function of HFs. In addition, we analyzed the symptoms, types, and causes of hair loss as well as its current conventional treatments. Our study provides an overview of the most effective regenerative medicine-based therapies for hair loss.
Stemness pertains to the intrinsic ability of mesenchymal stem cells (MSCs) to undergo self-renewal and differentiate into multiple lineages, while simultaneously impeding their differentiation and preserving crucial differentiating genes in a state of quiescence and equilibrium. Owing to their favorable attributes, including uncomplicated isolation protocols, ethical compliance, and ease of procurement, MSCs have become a focal point of inquiry in the domains of regenerative medicine and tissue engineering. As age increases or ex vivo cultivation is prolonged, the functionality of MSCs decreases and their stemness gradually diminishes, thereby limiting their potential therapeutic applications. Despite the existence of several uncertainties surrounding the comprehension of MSC stemness, considerable advancements have been achieved in the clarification of the potential mechanisms that lead to stemness loss, as well as the associated strategies for stemness maintenance. This comprehensive review provides a systematic overview of the factors influencing the preservation of MSC stemness, the molecular mechanisms governing it, the strategies for its maintenance, and the therapeutic potential associated with stemness. Finally, we underscore the obstacles and prospective avenues in present investigations, providing innovative perspectives and opportunities for the preservation and therapeutic utilization of MSC stemness.
Graphical Abstract
Hair follicle (HF) homeostasis is regulated by various signaling pathways. Disruption of such homeostasis leads to HF disorders, such as alopecia, pigment loss, and hair aging, which is causing severe health problems and aesthetic concerns. Among these disorders, hair aging is characterized by hair graying, hair loss, hair follicle miniaturization (HFM), and structural changes to the hair shaft. Hair aging occurs under physiological conditions, while premature hair aging is often associated with certain pathological conditions. Numerous investigations have been made to determine the mechanisms and explore treatments to prevent hair aging. The most well-known hypotheses about hair aging include oxidative stress, hormonal disorders, inflammation, as well as DNA damage and repair defects. Ultimately, these factors pose threats to HF cells, especially stem cells such as hair follicle stem cells, melanocyte stem cells, and mesenchymal stem cells, which hamper hair regeneration and pigmentation. Here, we summarize previous studies investigating the above mechanisms and the existing therapeutic methods for hair aging. We also provide insights into hair aging research and discuss the limitations and outlook.
Prenatal hypoxia (PH) is one of the most common complications of obstetrics and is closely associated with many neurological disorders such as depression, anxiety, and cognitive impairment. Our previous study found that Zfp462 heterozygous (Het) mice exhibit significant anxiety-like behavior. Interestingly, offspring mice with PH also have anxiety-like behaviors in adulthood, accompanied by reduced expression of Zfp462 and increased expression of miR-377-3p; however, the exact regulatory mechanisms remain unclear. In this study, western blotting, gene knockdown, immunofluorescence, dual-luciferase reporter assay, immunoprecipitation, cell transfection with miR-377-3p mimics or inhibitors, quantitative real-time PCR, and rescue assay were used to detect changes in the miR-377-3p-Zfp462-Pbx1 (pre-B-cell leukemia homeobox1) pathway in the brains of prenatal hypoxic offspring to explain the pathogenesis of anxiety-like behaviors. We found that Zfp462 deficiency promoted Pbx1 protein degradation through ubiquitination and that Zfp462 Het mice showed downregulation of the protein kinase B (PKB, also called Akt)-glycogen synthase kinase-3β (GSK3β)-cAMP response element-binding protein (CREB) pathway and hippocampal neurogenesis with anxiety-like behavior. In addition, PH mice exhibited upregulation of miR-377-3p, downregulation of Zfp462/Pbx1-Akt-GSK3β-CREB pathway activity, reduced hippocampal neurogenesis, and an anxiety-like phenotype. Intriguingly, miR-377-3p directly targets the 3′UTR of Zfp462 mRNA to regulate Zfp462 expression. Importantly, microinjection of miR-377-3p antagomir into the hippocampal dentate gyrus of PH mice upregulated Zfp462/Pbx1-Akt-GSK3β-CREB pathway activity, increased hippocampal neurogenesis, and improved anxiety-like behaviors. Collectively, our findings demonstrated a crucial role for miR-377-3p in the regulation of hippocampal neurogenesis and anxiety-like behaviors via the Zfp462/Pbx1-Akt-GSK3β-CREB pathway. Therefore, miR-377-3p could be a potential therapeutic target for anxiety-like behavior in prenatal hypoxic offspring.
Mesenchymal stem cells (MSCs) have favourable outcomes in the treatment of kidney diseases. Pre-B-cell leukaemia transcription factor 1 (PBX1) has been reported to be a regulator of self-renewal of stem cells. Whether PBX1 is beneficial to MSCs in the treatment of haemorrhagic shock (HS)-induced kidney damage is unknown. We overexpressed PBX1 in rat bone marrow-derived mesenchymal stem cells (rBMSCs) and human bone marrow-derived mesenchymal stem cells (hBMSCs) to treat rats with HS and hypoxia-treated human proximal tubule epithelial cells (HK-2), respectively. The results indicated that PBX1 enhanced the homing capacity of rBMSCs to kidney tissues and that treatment with rBMSCs overexpressing PBX1 improved the indicators of kidney function, alleviated structural damage to kidney tissues. Furthermore, administration with rBMSCs overexpressing PBX1 inhibited HS-induced NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and the release of proinflammatory cytokines, and further attenuated apoptosis. We then determined whether NF-κB, an important factor in NLRP3 activation and the regulation of inflammation, participates in HS-induced kidney damage, and we found that rBMSCs overexpressing PBX1 inhibited NF-κB activation by decreasing the p-IκBα/IκBα and p-p65/p65 ratios and inhibiting the nuclear translocation and decreasing the DNA-binding capacity of NF-κB. hBMSCs overexpressing PBX1 also exhibited protective effects on HK-2 cells exposed to hypoxia, as shown by the increase in cell viability, the mitigation of apoptosis, the decrease in inflammation, and the inhibition of NF-κB and NLRP3 inflammasome activation. Our study demonstrates that MSCs overexpressing PBX1 ameliorates HS-induced kidney damage by inhibiting NF-κB pathway-mediated NLRP3 inflammasome activation and the inflammatory response.
Pre-B-cell leukemia homeobox transcription factor 1 (PBX1) is a member of the TALE (three-amino acid loop extension) family and functions as a homeodomain transcription factor (TF). When dimerized with other TALE proteins, it can act as a pioneer factor and provide regulatory sequences via interaction with partners. In vertebrates, PBX1 is expressed during the blastula stage, and its germline variations in humans are interrelated with syndromic anomalies of the kidney, which plays an important role in hematopoiesis and immunity among vertebrates. Herein, we summarize the existing data on PBX1 functions and the impact of PBX1 on renal tumors, PBX1-deficient animal models, and blood vessels in mammalian kidneys. The data indicated that the interaction of PBX1 with different partners such as the HOX genes is responsible for abnormal proliferation and variation of the embryonic mesenchyme, while truncating variants were shown to cause milder phenotypes (mostly cryptorchidism and deafness). Although such interactions have been identified to be the cause of many defects in mammals, some phenotypic variations are yet to be understood. Thus, further research on the TALE family is required.
