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SDF-1 Increases Recruitment of Osteoclast Precursors by Upregulation of Matrix Metalloproteinase-9 Activity
Abstract
Although chemokines play essential roles in the trafficking and homing of many circulating hematopoietic cell types, their potential influences on osteoclast (OC) recruitment or bone remodeling are not well known. Therefore, chemokine receptor expression was analyzed by RNase protection assay during OC formation induced by RANKL in a murine mononuclear cell line (RAW 264.7). Relatively high CXCR4 expression was detected in RAW cells (pre-OCs), whereas CXCR4 levels were downregulated during RAW-OC development. SDF-1, the unique ligand for CXCR4, stimulated RAW cell production of matrix metalloproteinase (MMP)-9 activity, a matrix-degrading enzyme essential for pre-OC migration into the developing bone marrow cavity. Induced MMP-9 activity in RAW cells was associated with their increased MMP-dependent transmigration through a collagen gel in response to SDF-1. We conclude that SDF-1 stimulation of MMP-9 activity in pre-OCs may be a key aspect of their recruitment to bone and migration within the marrow to sites for OC differentiation and bone resorption.
... One gene that was very highly upregulated was the chemokine CCL9 (also called MIP-1␥, CCF18, Scya9, or MRP-2). 12 Recent cell culture studies by several groups [13][14][15][16][17] suggest that chemokines and their receptors could play roles in extravasation, migration, and/or survival of osteoclastic cells in vivo. In addition, CCL3 (MIP-1␣) has been implicated in local osteolysis due to myeloma 18 and prosthesis wear particles. ...
... CXCL12 (SDF-1) has been shown to exert significant chemotactic activity on RAW cells, which express high levels of its putative receptor, CXCR4. 15 Further work in RAW cells showed an induction of the tissue remodeling enzyme MMP9 by CXCL12, 16 suggesting a role for CXCL12 in promoting the extravasation of osteoclast precursors and guiding their migration to appropriate bone resorption sites. Our findings are not inconsistent with those results, in that the near-constant levels of CXCL12 and CXCR4 in our time-course may be sufficient for these activities without necessitating additional transcription. ...
Osteoclasts differentiate from hematopoietic precursors under systemic and local controls. Chemokines and receptors direct leukocyte traffic throughout the body and may help regulate site-specific bone resorption. We investigated bone gene expression in vivo during rapid osteoclast differentiation induced by colony-stimulating factor 1 (CSF-1) in Csf1-null toothless (tl/tl) rats. Long-bone RNA from CSF-1-treated tl/tl rats was analyzed by high-density microarray over a time course. TRAP (tartrate-resistant acid phosphatase)-positive osteoclasts appeared on day 2, peaked on day 4, and decreased slightly on day 6, as marrow space was expanding. TRAP and cathepsin K mRNA paralleled the cell counts. We examined all chemokine and receptor mRNAs on the arrays. CCL9 was strongly induced and peaked on day 2, as did its receptor, CCR1, and regulatory receptors c-Fms (CSF-1 receptor) and RANK (receptor activator of nuclear factor kappaB). Other chemokines and receptors showed little or no significant changes. In situ hybridization and immunohistochemistry revealed CCL9 in small, immature osteoclasts on day 2 and in mature cells at later times. Anti-CCL9 antibody inhibited osteoclast differentiation in culture and significantly suppressed the osteoclast response in CSF-1-treated tl/tl rats. While various chemokines have been implicated in osteoclastogenesis in vitro, this first systematic analysis of chemokines and receptors during osteoclast differentiation in vivo highlights the key role of CCL9 in this process.
... Even though very little is known about how this recruitement occurs, several studies have described the capacity of mononucleated osteoclasts precursors (pre-OCs) to transmigrate through endothelial cells (transendothelial migration). (8,9) On the other hand, MMOCs are supposed to form directly on the bone surface by the fusion of mononucleated progenitors and are considered as highly migratory cells when found on the bone surface or through collagen. (1,(10)(11)(12)(13)(14)(15) Together, these data describe MMOCs as highly motile cells but never as transmigrated cells, even though they did exhibit several features usually involved in cell invasion that can be implicated in their recruitment to the bone surface. ...
... MMPs are involved in cell invasion and are known to be important for cell migration through extracellular matrices as well as for monocyte transmigration. (8,30,33) A MMP inhibitor caused a dose-dependent decrease in MMOC transmigration ( Fig. 3E) but had no effect on MMOC adhesion or podosome belt formation (Fig. 3F), suggesting that MMPs are important actors in MMOC transmigration, independent of the actin cytoskeleton organization. ...
Even though it is assumed that multinucleated osteoclasts are migrating cells on the bone surface to be resorbed, we show that they can also selectively transmigrate through layers of cells usually found in the bone microenvironment. This activity is associated with c-src and MMPs and can be stimulated by bone metastatic breast cancer cells, a process blocked by bisphosphonate treatment.
Osteoclasts have an hematopoietic origin and are bone-resorbing cells. Monocytic precursors migrate to the bone surface where they fuse to form multinucleated osteoclasts able to migrate over the bone surface. We studied whether multinucleated osteoclasts were also able to transmigrate through tissues.
Murine spleen-derived and green fluorescent protein (GFP)-Raw derived osteoclasts were seeded on osteoblasts and several other cell types. The cells were fixed for 20 minutes, 4 or 12 h after osteoclast seeding, and stained with phalloidin to visualize actin using confocal microscopy. Drugs such as PP2 and GM6001, inhibitors of c-src and matrix metalloproteinases (MMPs), respectively, and risedronate were used to determine osteoclast transmigration regulating factors.
We observed by confocal microscopy that multinucleated osteoclasts specifically transmigrate through confluent layers of various cell types present in the bone microenvironment in vitro. This is an efficient process associated with c-src and MMPs but is independent of podosomes. Moreover, conditioned medium from bone metastatic breast cancer cells stimulates osteoclast transmigration in vitro, a process inhibited by bisphosphonate treatment.
Our data describe a new property of mature multinucleated osteoclasts to transmigrate through various cell types. The ability to control this highly regulated osteoclast transmigration process may offer new therapeutic strategies for bone diseases associated with an imbalance in bone remodeling caused by excessive osteoclast resorption.
... An efficient uptake of NPs is essential for the intracellular delivery of cargo [184]. RAW 264.7 cells are reported to express CXCR4 receptor [185]. Hence, the first goal was to identify if the uptake we observed was because of the ability to target CXCR4 receptor. ...
... (44) Besides its function in hematopoiesis, CXCL12 is known to be a potent chemoattractant for pre-osteoclasts to bone and supports their migration to BM sites for osteoclast differentiation. (45)(46)(47) In several skeletal diseases where a bias toward postmenopausal women is found, eg, rheumatoid arthritis or osteoarthritis, CXCL12 levels are elevated locally in the joints as well as in peripheral blood. (48,49) Also, elevated plasma levels of CXCL12 are a marker for disease severity in women with postmenopausal osteoporosis. ...
Estrogen has pronounced effects on the immune system, which also influences bone homeostasis. In recent years, stromal cells in lymphoid organs have gained increasing attention as they not only support the regulation of immune responses, but also affect bone remodeling. A conditional knockout mouse model where estrogen receptor alpha (ERα) is deleted in Ccl19‐expressing stromal cells (Ccl19‐Cre ERαfl/fl mice) was generated and bone densitometry was performed to analyze the importance of stromal cell specific ERα signaling on the skeleton. Results showed that female Ccl19‐Cre ERαfl/fl mice display reduced total bone mineral density and detailed x‐ray analyses revealed that ERα expression in Ccl19 expressing stromal cells is important for trabecular but not cortical bone homeostasis. Further analysis showed that the trabecular bone loss is caused by increased osteoclastogenesis. Additionally, the bone formation rate was reduced, however the expression of osteoprogenitor genes was not altered. Analysis of the bone marrow stromal cell compartment revealed a deletion of ERα in a subgroup of CXCL12‐abundant reticular (CAR) cells resulting in increased secretion of the pro‐osteoclastogenic chemokine CXCL12. In conclusion, this study reveals the importance of ERα signaling in CAR cells for bone health. This article is protected by copyright. All rights reserved.
... MMPs are involved in bone resorption by removing organic bone matrix and generating collagen fragments that could activate osteoclasts [55]. Additionally, MMPs' activity is thought to act over preosteoclast recruitment to the bone tissue and migration within the marrow to sites for osteoclast differentiation and bone resorption [56,57]. Collagenase-mediated tissue destruction has been suggested to have a role in foreign-body host response [54] and in active osteoclastic activity in peri-implant lesions [27,58]. ...
Objectives:
The aim of this retrospective study was to analyze peri-implant marginal bone loss levels/rates and peri-implant sulcular fluid levels/rates of metalloproteinase-8 in three timeframes (6 months post-surgery-restoration delivery (T0)-and 6 (T6) and 24 (T24)-months post-loading) and to evaluate if there is a correlation between peri-implant sulcular fluid levels of metalloproteinase-8 and peri-implant marginal bone loss progression.
Materials and methods:
Two cohorts of patients undergoing implant surgery between January 2017 and January 2019 were selected in this retrospective study. A total of 39 patients received 39 implants with a laser-microtextured collar surface, and 41 subjects received 41 implants with a machined/smooth surface. For each patient, periapical radiographs and a software package were used to measure marginal bone loss rates. Implant fluid samples were analyzed by an enzyme-linked immunosorbent assay (ELISA) test. The modified plaque index, probing depth, and bleeding on probing were also recorded.
Results:
High marginal bone rates at T24 were strongly associated with elevated rates between T0 and T6. The levels of metalloproteinase-8 were significantly more elevated around implants with marginal bone loss, in relation to implants without marginal bone loss. Marginal bone loss (MBL) rates at 24 months were associated with initial bone loss rates and initial levels of metalloproteinase-8.
Conclusions:
Peri-implant marginal bone loss progression is statistically correlated to peri-implant sulcular fluid levels of metalloproteinase-8. Moreover, the initial high levels of marginal bone loss and metalloproteinase-8 can be considered as indicators of the subsequent progression of peri-implant MBL: implants with increased marginal bone loss rates and metalloproteinase-8 levels at 6 months after loading are likely to achieve additional marginal bone loss values.
... In support of this observation numerous studies have documented that the chemokine CXCL12 induced MMP synthesis in different cell types and facilitated tumor cell adhesion and colonization [181][182][183][184][185]. ...
Influence of the chemokine CXCL12 on the
progression and the signalling in colorectal
cancer
... CXCL12/CXCR4 protein expression was dose-dependently activated in both cell lines with all metal exposures (202). Upregulation of CXCL12 in preosteoclasts has been associated with increased osteoclastic activity in several bone diseases, which may participate in eventual periprosthetic osteolysis (203)(204)(205). When AMD3100 was administered to block the interaction of CXCL12 with CXCR4, a partial reduction in TNF-α expression was observed. ...
Chemokines play an important role in normal bone physiology and the pathophysiology of many bone diseases. The recent increased focus on the individual roles of this class of proteins in the context of bone has shown that members of the two major chemokine subfamilies—CC and CXC—support or promote the formation of new bone and the remodeling of existing bone in response to a myriad of stimuli. These chemotactic molecules are crucial in orchestrating appropriate cellular homing, osteoblastogenesis, and osteoclastogenesis during normal bone repair. Bone healing is a complex cascade of carefully regulated processes, including inflammation, progenitor cell recruitment, differentiation, and remodeling. The extensive role of chemokines in these processes and the known links between environmental contaminants and chemokine expression/activity leaves ample opportunity for disruption of bone healing by environmental factors. However, despite increased clinical awareness, the potential impact of many of these environmental factors on bone-related chemokines is still ill defined. A great deal of focus has been placed on environmental exposure to various endocrine disruptors (bisphenol A, phthalate esters, etc.), volatile organic compounds, dioxins, and heavy metals, though mainly in other tissues. Awareness of the impact of other less well-studied bone toxicants, such as fluoride, mold and fungal toxins, asbestos, and chlorine, is also reviewed. In many cases, the literature on these toxins in osteogenic models is lacking. However, research focused on their effects in other tissues and cell lines provides clues for where future resources could be best utilized. This review aims to serve as a current and exhaustive resource detailing the known links between several classes of high-interest environmental pollutants and their interaction with the chemokines relevant to bone healing.
... MMP-2 and -9 degrade collagen during periodontal homeostasis and in pathologic conditions (29). Furthermore, MMP-9 may act over preosteoclast recruitment and migration (30) and also removes the collagenous layer from the bone surface before the demineralization process, initiating osteoclast resorption (31). Some studies reported increased levels of MMP2 and/ or MMP9 mRNA in periapical inflammatory lesions (7,28); in addition, others found increased protein expression or gelatinolytic activity (26,32). ...
Introduction:
Matrix metalloproteinases (MMPs) are the major class of enzymes responsible for degradation of extracellular matrix components and participate in the pathogenesis of periapical inflammatory lesions. MMP expression may be regulated by DNA methylation. The purpose of the present investigation was to analyze the expression of MMP2 and MMP9 in periapical granulomas and radicular cysts and to test the hypothesis that, in these lesions, their transcription may be modulated by DNA methylation.
Methods:
Methylation-specific polymerase chain reaction was used to evaluate the DNA methylation pattern of the MMP2 gene in 13 fresh periapical granuloma samples and 10 fresh radicular cyst samples. Restriction enzyme digestion was used to assess methylation of the MMP9 gene in 12 fresh periapical granuloma samples and 10 fresh radicular cyst samples. MMP2 and MMP9 messenger RNA transcript levels were measured by quantitative real-time polymerase chain reaction.
Results:
All periapical lesions and healthy mucosa samples showed partial methylation of the MMP2 gene; however, periapical granulomas showed higher MMP2 mRNA expression levels than healthy mucosa (P = .014). A higher unmethylated profile of the MMP9 gene was found in periapical granulomas and radicular cysts compared with healthy mucosa. In addition, higher MMP9 mRNA expression was observed in the periapical lesions compared with healthy tissues.
Conclusions:
The present study suggests that the unmethylated status of the MMP9 gene in periapical lesions may explain the observed up-regulation of messenger RNA transcription in these lesions.
... Metalloproteiases (MMPs) (Yu et al., 2003) are able to, first, transmigrate through such cells layers by forming large actin rich protrusions and, second, to spread over matrix and underneath the invaded cells ( Figure 13) . The actin rich protrusion observed in transmigrating OCs reaches a diameter of at least 5µm ( Figure 13) and therefore seems bigger than podosome-like structures observed in 3D-migrating macrophages (Van Goethem et al., 2010) and invadopodia observed in metastatic cells (Linder, 2007). ...
Bone remodeling is a physiological process by which old bone is replaced by new bone. Osteoclasts are multinucleated giant cells of the monocytic lineage. Their function is bone resorption, the first step of bone remodeling. The work of this thesis is in continuity with a theme long developed in our laboratory, that of the actin cytoskeleton organization in bone-resorbing osteoclasts. Our first study investigated the role of the podosome organization in osteoclast spreading, adhesion and migration. Our results showed that podosome patterning into rings exerted outward tension upon the substrate and thereby triggered cell migration. Through cycles of assembly, growth and alternating disassembly, rings promote a saltatory mode of migration universal to all osteoclasts.The main objective of this thesis, however, was dedicated to finding new genes that govern podosome patterning in resorption-related processes such as osteoclast migration and sealing zone formation. To find such new genes, we employed a differential transcriptomic analysis of osteoclasts and osteoclast-like cells that exhibit podosomes but are unable to resorb bone. Among a list of six genes highly and exclusively expressed in osteoclasts, we chose to investigate RhoE, a constitutively active GTP-binding protein known for its regulation of actin structures. We provided evidence, using primary RhoE-deficient osteoclasts, that RhoE activity is essential to bone resorption. We unveiled a new role for RhoE in the control of actin turnover in podosomes through a Rock-antagonistic function. Finally, we demonstrated that the role of RhoE in osteoclasts is essential to their migration and sealing zone formation.
... Cathepsin-K and MMP-9 are the two main proteases found in the cytoplasmic region of osteoclasts (Delaisse et al. 2000;Yu et al. 2003). They play important roles in osteoclast-mediated bone resorption. ...
The purpose of this study was to investigate the effect of zoledronate (ZA) on osteoclast functions and viability in the tibiae of 8-week-old male mice. After weekly intravenous administration of ZA (125 μg/kg body weight) for 8 weeks, the mice were fixed by transcardial perfusion of 4 % paraformaldehyde under anesthesia, and their tibiae were extracted for histochemical analysis. Compared with the control group, many tartrate-resistant acidic phosphatase-positive osteoclasts were found on the surface of the trabecular bone, but cartilage cores were obviously increased in the metaphysis of the ZA group. Osteoclasts of both groups showed similar expression of cathepsin K and matrix metalloproteinase-9. However, hardly any expression of c-src, a gene necessary for ruffled border formation and bone resorption, was found in osteoclasts of the ZA group. Moreover, no expression of CD44 or osteopontin (OPN) was observed in osteoclasts of the ZA group. Taken together, our findings suggest that ZA administration decreases the bone resorption ability of osteoclasts by inhibiting c-src expression and suppressing osteoclast adhesion by interfering with CD44/OPN binding.
... MMPs are involved in bone resorption which includes the initiation of bone resorption 9) and the migration of pre-OCs 2,6) and osteoclasts 10) . Pre-OCs expressed MMP-9 and its expression was increased by SDF-1 2,6) . This suggests that SDF-1 increases recruitment of pre-OCs by up-regulation of MMP-9. ...
... Matrix metalloproteinases (MMPs) are a group of endopeptidases that regulate osteoclast migration and bone resorption123 . Proteinases mobilize bone matrix proteins and determine where and when bone resorption should be initiated. ...
CD44, MT1-MMP, and MMP9 are implicated in the migration of osteoclast and bone resorption. This study was designed to determine the functional relationship between CD44 and MT1-MMP in the activation of pro-MMP9. We used osteoclasts isolated from wild-type and CD44-null mice. Results showed that MT1-MMP is present in multiple forms with a molecular mass ~63, 55, and 45 kDa in the membrane of wild-type osteoclasts. CD44-null osteoclasts demonstrated a 55 kDa active MT1-MMP form in the membrane and conditioned medium. It failed to activate pro-MMP9 because TIMP2 binds and inhibits this MT1-MMP (~55 kDa) in CD44-null osteoclasts. The role of MT1-MMP in the activation of pro-MMP9, CD44 expression, and migration was confirmed by knockdown of MT1-MMP in wild-type osteoclasts. Although knockdown of MMP9 suppressed osteoclast migration, it had no effects on MT1-MMP activity or CD44 expression. These results suggest that CD44 and MT1-MMP are directly or indirectly involved in the regulation of pro-MMP9 activation. Surface expression of CD44, membrane localization of MT1-MMP, and activation of pro-MMP9 are the necessary sequence of events in osteoclast migration.
... One gene that was very highly upregulated was the chemokine CCL-9 (also called MIP-1γ, CCF18, Scya9, or MRP-2) 12 . Recent cell culture studies by several groups [13][14][15][16][17] suggest that chemokines and their receptors could play roles in extravasation, migration, and/or survival of osteoclastic cells in vivo. In addition, CCL3 (MIP-1α) has been implicated in local osteolysis due to myeloma 18 and prosthesis wear particles 19 . ...
... 20 SDF-1 stimulation of MMP-9 has an important role in osteoclast differentiation/bone resorption activity. 21 Macrophage inflammatory protein (MIP)-1a and b produced by myeloma cells induce RANKL expression in stromal cells. 22 Recently, we reported that CXCL13 stimulates RANKL expression in bone marrow stromal/preosteoblast cells. ...
Paget's disease of bone (PDB) is a chronic focal skeletal disorder that affects 2-3% of the population over 55 years of age. PDB is marked by highly localized areas of bone turnover with increased osteoclast activity. Evidence suggests a functional role for measles virus nucleocapsid protein (MVNP) in the pathogenesis of PDB. In the present study, we identified elevated levels (∼180-fold) of CXCL5 mRNA expression in bone marrow cells from patients with PDB compared with that in normal subjects. In addition, CXCL5 levels are increased (five-fold) in serum samples from patients with PDB. Furthermore, MVNP transduction in human bone marrow monocytes significantly increased CXCL5 mRNA expression. Real-time PCR analysis showed that CXCL5 stimulation increased (6.8-fold) RANKL mRNA expression in normal human bone marrow-derived stromal (SAKA-T) cells. Moreover, CXCL5 increased (5.2-fold) CXCR1 receptor expression in these cells. We further showed that CXCL5 treatment elevated the expression levels of phospho-ERK1/2 and phospho-p38. CXCL5 also significantly increased phosphorylation of CREB (cAMP response element-binding) in bone marrow stromal/preosteoblast cells. Chromatin immuneprecipitation (ChIP) assay confirmed phospho-CREB binding to RANKL gene promoter region. Further, the suppression of p-CREB expression by the inhibitors of ERK1/2, p38 and PKA significantly decreased CXCL5 stimulation of hRANKL gene promoter activity. Thus, our results suggest that CREB is a downstream effector of CXCL5 signaling and that increased levels of CXCL5 contribute to enhanced levels of RANKL expression in PDB.Laboratory Investigation advance online publication, 25 February 2013; doi:10.1038/labinvest.2013.5.
... Under inflammatory conditions, bone resorptive mediators, like interleukin-1 and prostaglandin E2, induce a marked expression of RANKL and MMPs, such as MMP-13, -3 and -2 by osteoblasts and MMP-9 by activated osteoclasts and leukocytes [1,28]. Additionally, MMP-9 activity is thought to act over preosteoclast recruitment and migration [29]. High levels of oxidants in tissues perturb the normal redox balance and shift cells into a state of oxidative stress303132. ...
Oxidative stress and matrix metalloproteinases -9 and -2 are involved in periodontal breakdown, whereas gingival crevicular fluid has been reported to reflect apical status. The aim of this study was to characterize oxidant balance and activity levels of MMP -2 and -9 in apical lesions and healthy periodontal ligament; and second, to determine whether potential changes in oxidant balance were reflected in gingival crevicular fluid from asymptomatic apical periodontitis (AAP)-affected teeth at baseline and after endodontic treatment.
Patients with clinical diagnosis of AAP and healthy volunteers having indication of tooth extraction were recruited. Apical lesions and healthy periodontal ligaments, respectively, were homogenized or processed to obtain histological tissue sections. Matrix metalloproteinase -9 and -2 levels and/or activity were analyzed by Immunowestern blot, zymography and consecutive densitometric analysis, and their tissue localization was confirmed by immunohistochemistry. A second group of patients with AAP and indication of endodontic treatment was recruited. Gingival crevicular fluid was extracted from AAP-affected teeth at baseline, after endodontic treatment and healthy contralateral teeth. Total oxidant and antioxidant status were determined in homogenized tissue and GCF samples. Statistical analysis was performed using STATA v10 software with unpaired t test, Mann-Whitney test and Spearman's correlation.
Activity of MMP-2 and MMP-9 along with oxidant status were higher in apical lesions (p < 0.05). Total oxidant status correlated positively with matrix metalloproteinase-2 and lesion size (p < 0.05). Gingival crevicular fluid showed significantly lower levels of total antioxidant status in diseased teeth at baseline compared to controls and endodontically-treated groups.
Apical lesions display an oxidant imbalance along with increased activity of matrix metalloproteinase-2 and -9 and might contribute to AAP progression. Oxidant imbalance can also be reflected in GCF from AAP-affected teeth and was restored to normal levels after conservative endodontic treatment. These mediators might be useful as potential biomarkers for chair-side complementary diagnostic of apical status in GCF.
... Previous studies based on induced experimental periodontitis in mice have demonstrated elevated levels of MMP-2, -9, -1 and RANKL that correlated with the expression of IL-1b, TNF-a and IF-g during alveolar bone loss (Garlet et al. 2006). Additionally, MMP-9 activity is thought to act over preosteoclast recruitment to the bone tissue and migration within the marrow to sites for osteoclast differentiation and bone resorption (Yu et al. 2003 Fig. 3. Pro-matrix metalloproteinases (MMP)-9 activation in MMP-13-treated gingival tissue explants and controls from chronic periodontitis patients. Explants from diseased gingiva were cultured for 24 h and MMP-13 was added to the media for 0.5, 2, 14 and 24 h. ...
Matrix metalloproteinases (MMP)-13 can initiate bone resorption and activate proMMP-9 in vitro, and both these MMPs have been widely implicated in tissue destruction associated with chronic periodontitis. We studied whether MMP-13 activity and TIMP-1 levels in gingival crevicular fluid (GCF) associated with progression of chronic periodontitis assessed clinically and by measuring carboxy-terminal telopeptide of collagen I (ICTP) levels. We additionally addressed whether MMP-13 could potentiate gelatinase activation in diseased gingival tissue.
In this prospective study, GCF samples from subjects undergoing clinical progression of chronic periodontitis and healthy controls were screened for ICTP levels, MMP-13 activity and TIMP-1. Diseased gingival explants were cultured, treated or not with MMP-13 with or without adding CL-82198, a synthetic MMP-13 selective inhibitor, and assayed by gelatin zymography and densitometric analysis.
Active sites demonstrated increased ICTP levels and MMP-13 activity (p<0.05) in progression subjects. The MMP-9 activation rate was elevated in MMP-13-treated explants (p<0.05) and MMP-13 inhibitor prevented MMP-9 activation.
MMP-13 could be implicated in the degradation of soft and hard supporting tissues and proMMP-9 activation during progression of chronic periodontitis. MMP-13 and -9 can potentially form an activation cascade overcoming the protective TIMP-1 shield, which may become useful for diagnostic aims and a target for drug development.
... (2) Murine and human monocytes have been shown to chemotax toward a variety of bone matrix peptides. (3,4) Osteoclasts also exhibit migratory ability toward M-CSF and transforming growth factor GTPases act as molecular switches to control actin-dependent structures and processes in OCG and in mature osteoclast function, including chemotaxis, actin filament elongation, actin ring and sealing zone formation, and cell polarity. (7)(8)(9)(10) Filamins are actin-binding proteins that cross-link F-actin into high-angle orthogonal networks, and their homodimeric structure and multiple springlike hinge regions impart flexibility and elasticity to the cross-linked actin cytoskeleton. ...
Osteoclastogenesis (OCG) results from the fusion of monocytes after stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). Migration of monocytes into close proximity precedes critical fusion events that are required for osteoclast formation. Cellular migration requires leading-edge actin cytoskeleton assembly that drives cellular locomotion. Filamin A (FLNa) cross-links F-actin filaments in the leading edge of migrating cells and also has been shown to regulate signal transduction during cell migration. However, little is known about the possible role of FLNa in osteoclastogenesis. Our objective in this study was to investigate the role of FLNa in osteoclastogenesis. Bone marrow monocytes isolated from the tibiae and femora of wild type (WT) and Flna-null mice were cultured for 6 days with M-CSF and RANKL, and osteoclasts were identified by tartrate-resistant acid phosphatase (TRACP) staining. The Flna-null mouse skeletal phenotype was characterized using dual-energy X-ray absorptiometry (DXA) to analyze the skeleton, as well as tests on blood chemistry. Osteoclast levels in vivo were quantified by counting of TRACP-stained histologic sections of distal femora. To elucidate the mechanisms by which Flna regulates osteoclastogenesis, migration, actin polymerization, and activation of Rho GTPases, Rac1, Cdc42, and RhoA were assessed in monocytes during in vitro OCG. Deficiencies in migration were rescued using constitutively active Rac1 and Cdc42 TAT fusion proteins. The RANKL signaling pathway was evaluated for activation by monitoring nuclear translocation of NF kappaB and c-jun and expression of key osteoclast genes using quantitative real-time polymerase chain reaction (qRT-PCR). Our results show that Flna-null monocytes formed fewer osteoclasts in vitro, and those that were formed were smaller with fewer nuclei. Decreased OCG was reflected in vivo in TRACP-stained histologic bone sections. Flna-null monocytes experienced impaired migratory ability. When OCG was performed at increasing starting cellular plating densities in order to decrease intercellular distances, there was progressive rescue of Flna-null osteoclast formation comparable with WT levels, confirming that Flna regulates monocyte migration prefusion. Activation of the actin cytoskeleton regulators Rac1, Cdc42, and RhoA and actin free-barbed end generation were partially or completely abrogated in Flna-null monocytes; however, monocyte migration was restored on rescuing with constitutively active Rac1 and Cdc42 TAT fusion proteins. We conclude that filamin A is required for osteoclastogenesis by regulating actin dynamics via Rho GTPases that control monocyte migration.
... Third, MMP-9 cleaves SDF-1 at a Ser 4 –Lys 5 peptide bond that inactivates the protein[37]. SDF-1 itself enhances the expression of MMP-9 by BMDSC, thus promoting its own degradation[38]. The HSC mobilizing agent, granulocyte-colony stimulating factor (G-CSF), promotes HSC migration from the osteoblastic niche toward the vascular niche in a similar way by inducing neutrophil elastase degradation of SDF-1[39]. ...
The bone marrow constitutes an unique microenvironment for cancer cells in three specific aspects. First, the bone marrow actively recruits circulating tumor cells where they find a sanctuary rich in growth factors and cytokines that promote their proliferation and survival. When in the bone marrow, tumor cells profoundly affect the homeostasis of the bone and the balance between osteogenesis and osteolysis. As a consequence, growth and survival factors normally sequestered into the bone matrix are released, further fueling cancer progression. Second, tumor cells actively recruit bone marrow-derived precursor cells into their own microenvironment. When in the tumors, these bone marrow-derived cells contribute to an inflammatory reaction and to the formation of the tumor vasculature. Third, bone marrow-derived cells can home in distant organs, where they form niches that attract circulating tumor cells. Our understanding of the contribution of the bone marrow microenvironment to cancer progression has therefore dramatically improved over the last few years. The importance of this new knowledge cannot be underestimated considering that the vast majority of cancer treatments such as cytotoxic and myeloablative chemotherapy, bone marrow transplantation and radiation therapy inflict a trauma to the bone marrow microenvironment. How such trauma affects the influence that the bone marrow microenvironment exerts on cancer is still poorly understood. In this article, the reciprocal relationship between the bone marrow microenvironment and tumor cells is reviewed, and its potential impact on cancer therapy is discussed.
... 36 CXCL12-stimulated bone marrow and peripheral blood CD34 þ cells secrete MMP-2 and MMP-9. 37 Similarly, this chemokine induces the expression MMP-9 by monocytes 38,39 and purified mature polyploid human megakaryocytes in a phosphatidyl-inositol 3-kinase-dependent fashion. 40,41 The CXCL12-CXCR4 axis also induces rhabdomyosarcoma cells to produce MMP-2. ...
The mechanisms responsible for prostate cancer metastasis are incompletely understood at both the cellular and molecular levels. In this regard, chemokines are a family of small, cytokine-like proteins that induce motility of neoplastic cells, leukocytes and cancer cells. The current study evaluates the molecular mechanisms of CXCL12 and CXCR4 in prostate cancer cell migration and invasion. We report that functional CXCR4 is significantly expressed by prostate cancer cell lines, LNCaP and PC3, when compared with normal prostatic epithelial cells (PrEC). As measured using motility and invasion chamber assays, prostate cancer cells migrated and invaded through extracellular matrix components in response to CXCL12, at rates that corresponded to CXCR4 expression. Anti-CXCR4 antibodies (Abs) significantly impaired the migration and invasive potential of PC3 and LNCaP cells. CXCL12 induction also enhanced collagenase-1 (metalloproteinase-1 (MMP-1)) expression by LNCaP and PC3 cells. Collagenase-3 (MMP-13) was expressed by prostate cancer cells, but it was not expressed by PrEC cells or modulated by CXCL12. CXCL12 increased MMP-2 expression by LNCaP and PC3; however, MMP-9 expression was elevated only in PC3 cells after CXCL12-CXCR4 ligation. PC3 cells also expressed high levels of stromelysin-1 (MMP-3) after CXCL12 stimulation. CXCL12 also significantly increased stromelysin-2 (MMP-10) expression by LNCaP cells. Stromelysin-3 (MMP-11) was expressed by LNCaP cells, but not by PC3 or PrEC cells and CXCL12 induced PC3 MMP-11 expression. Membrane type-1 MMP (MMP-14) was not expressed by PrEC or LNCaP cells, but CXCL12 significantly enhanced MMP-14 expression by PC3 cells. These studies reveal important cellular and molecular mechanisms of CXCR4/CXCL12-mediated prostate cancer cell migration and invasion.
... In the bone marrow SDF-1 is constitutively produced by osteoblasts, fibroblasts and endothelial cells. Osteoblast-produced SDF-1 may be a mechanism for the selective attraction of circulating osteoclast precursors into bone and their migration within marrow to appropriate perivascular stromal sites for RANKL differentiation into restorative osteoclasts (38,39). Since the formation of the marrow required the coordinated action of osteoblasts and osteoclastic precursors, the absence of osteoblasts producing SDF-1 or hematopoietic cells expressing CXCR4 could limit osteoclastic and subsequent HSC recruitment into the marrow cavity. ...
Bone marrow stromal cells are critical regulators of hematopoiesis. Osteoblasts are part of the stromal cell support system in bone marrow and may be derived from a common precursor. Several studies suggested that osteoblasts regulate hematopoiesis, yet the entire mechanism is not understood. It is clear, however, that both hematopoietic precursors and osteoblasts interact for the production of osteoclasts and the activation of resorption. We observed that hematopoietic stem cells (HSCs) regulate osteoblastic secretion of various growth factors, and that osteoblasts express some soluble factors exclusively in the presence of HSCs. Osteoblasts and hematopoietic cells are closely associated with each other in the bone marrow, suggesting a reciprocal relationship between them to develop the HSC niche. One critical component regulating the niche is stromal-derived factor-1 (SDF-1) and its receptor CXCR4 which regulates stem cell homing and, as we have recently demonstrated, plays a crucial role in facilitating those tumors which metastasize to bone. Osteoblasts produce abundant amounts of SDF-1 and therefore osteoblasts play an important role in metastasis. These findings are discussed in the context of the role of osteoblasts in marrow function in health and disease.
... 42 SDF1α has also been described to induce MMP9 secretion in other tumor models. 43,44 For the definitive test of whether SDF1α is really involved in the process of homing of MM cells in vivo, we performed an immediate homing analysis. 4F-benzoylTN14003 blocked the immediate homing of 5T2MM cells by 40% and that of 5T33MM cells by 25%, confirming the important role of SDF1α in the homing of MM cells to the bone marrow. ...
Multiple myeloma (MM) is a lethal plasma cell cancer characterized by the monoclonal growth of cells in the bone marrow. To reach the bone marrow, MM cells need to be attracted by chemokines. Recently, it has been shown that chemokines can also be involved in the growth of several cancer types. Stromal cell derived factor 1a (SDF1alpha) or CXCL12 is known to play an important role as a chemokine for hematopoietic progenitor cells and human MM cells. We studied the effects of SDF1a in the 5TMM murine model.
The in vitro effects of SDF1alpha were analyzed by gelatin zymography, adhesion, migration, proliferation, and chemoinvasion assays and by blockade with the CXCR4 inhibitor, 4F-benzoyl-TN14003. In vivo, diseased mice were treated with either vehicle or 4F-benzoyl-TN14003.
In vitro SDF1alpha was capable of attracting both 5T2MM and 5T33MM cells and inducing a 1.6-fold increase in MMP9 production by the 5TMM cells, which was correlated with an increased invasive capacity. In addition, SDF1alpha induced a 20% increase in DNA synthesis in the 5TMM cells. All these effects could be blocked by the CXCR4 inhibitor, 4Fbenzoyl- TN14003. An in vivo study in the 5T33MM model showed that blocking CXCR4 led to a 20% reduction in bone marrow tumor load.
These data demonstrate that SDF1alpha/CXCR4 is involved in the homing and the expansion of MM cells. Blocking CXCR4 could be useful in synergy with other anti-neoplastic treatments targeting the bone marrow microenvironment.
... Activation of MMPs break down the physical barriers of metastasis, thus promoting invasion by cancer cells [49]. Several studies have documented that CXCL12 induces MMP synthesis in different cell types [62,75,81,127,148], and facilitates tumor cell adhesion and colonization. ...
The chemokine stroma-derived factor (SDF-1/CXCL12) plays multiple roles in tumor pathogenesis. It has been demonstrated that CXCL12 promotes tumor growth and malignancy, enhances tumor angiogenesis, participates in tumor metastasis, and contributes to immunosuppressive networks within the tumor microenvironment. Therefore, it stands to reason that the CXCL12/CXCR4 pathway is an important target for the development of novel anti-cancer therapies. In this review, we consider the pathological nature and characteristics of the CXCL12/CXCR4 pathway in the tumor microenvironment. Strategies for therapeutically targeting the CXCL12/CXCR4 axis also are discussed.
... Colocalization of actin/MMP-9 in podosomes (Fig. 4A) but not in the actin ring (Fig. 5A) may provide an insight into the role of MMP-9 in specific functions of osteoclast. MMP-9 and cathepsin-K are the main proteases involved in osteoclast migration and bone resorption (Blavier and Delaisse, 1995a; Delaisse et al., 2000; Yu et al., 2003a). Cysteine proteinase cathepsin K solubilizes the mineralized matrix during bone resorption by the polarized resorbing osteoclasts. ...
CD44 and MMP-9 are implicated in cell migration. In the current study, we tested the hypothesis that actin polymerization is critical for CD44 surface expression and MMP-9 activity on the cell surface. To understand the underlying molecular mechanisms involved in CD44 surface expression and MMP-9 activity on the cell surface, osteoclasts were treated with bisphosphonate (BP) alendronate, cytochalasin D (Cyt D), and a broad-spectrum MMP inhibitor (GM6001). BP has been reported to block the mevalonate pathway, thereby preventing prenylation of small GTPase signaling required for actin cytoskeleton modulation. We show in this study that osteoclasts secrete CD44 and MMP-9 into the resorption bay during migration and bone resorption. Results indicate that actin polymerization is critical for CD44 surface expression and osteoclast function. In particular, the surface expression of CD44 and the membrane activity of MMP-9 are reduced in osteoclasts treated with alendronate and Cyt D despite the membrane levels of MMP-9 being unaffected. Although GM6001 blocked MMP-9 activity, osteoclast migration, and bone resorption, the surface levels of CD44 were unaffected. We suggest that the surface expression of CD44 requires actin polymerization. Disruption of podosome and actin ring structures by Cyt D and alendronate not only resulted in reduced localization of MMP-9 in these structures but also in osteoclast migration and bone resorption. These results suggest that inhibition of actin polymerization by alendronate and Cyt D is effective in blocking CD44/MMP-9 complex formation on the cell surface, secretion of active form of MMP-9, and osteoclast migration. CD44/MMP-9 complex formation may signify a unique motility-enhancing signal in osteoclast function.
... They originate from hematopoietic precursors of the monocyte-macrophage lineage that emigrate from the peripheral circulation to the bone remodeling site. These osteoclast precursors express chemokine receptors, such as CCR2 and CCR5, and therefore, chemotactic signals provided by chemokines such as MCP-1/CCL2, MIP-1a/CCL3, SDF-1/CXCL12 are essential to guide their migration to bone tissues89101112 . In addition, MCP-1, MIP- 1a and SDF-1 are also able to induce their differentiation into osteoclasts, activate their bone resorption activity, and promote osteoclast survival [12,15161718 . ...
Orthodontic tooth movement is achieved by the remodeling of alveolar bone in response to mechanical loading, and is supposed to be mediated by several host mediators, such as chemokines. In this study we investigated the pattern of mRNAs expression encoding for osteoblast and osteoclast related chemokines, and further correlated them with the profile of bone remodeling markers in palatal and buccal sides of tooth under orthodontic force, where tensile (T) and compressive (C) forces, respectively, predominate. Real-time PCR was performed with periodontal ligament mRNA from samples of T and C sides of human teeth submitted to rapid maxillary expansion, while periodontal ligament of normal teeth were used as controls. Results showed that both T and C sides exhibited significant higher expression of all targets when compared to controls. Comparing C and T sides, C side exhibited higher expression of MCP-1/CCL2, MIP-1alpha/CCL3 and RANKL, while T side presented higher expression of OCN. The expression of RANTES/CCL5 and SDF-1/CXCL12 was similar in C and T sides. Our data demonstrate a differential expression of chemokines in compressed and stretched PDL during orthodontic tooth movement, suggesting that chemokines pattern may contribute to the differential bone remodeling in response to orthodontic force through the establishment of distinct microenvironments in compression and tension sides.
During an inflammatory response, a large number of distinct mediators appears into the affected tissues or into the blood circulation. These include acute phase proteins such as serum amyloid A (SAA), cytokines and chemokines and proteolytic enzymes. Although these molecules are generated within a cascade sequence in specific body compartments allowing for independent action, their co-appearance in space and time during acute or chronic inflammation points toward important mutual interactions. Pathogen-associated molecular patterns lead to fast induction of the pro-inflammatory endogenous pyrogens, which are evoking the acute phase response. Interleukin-1, tumor necrosis factor-α and interferons simultaneously trigger different cell types, including leukocytes, endothelial cells and fibroblasts for tissue-specific or systemic production of chemokines and matrix metalloproteinases (MMPs). In addition, SAA induces chemokines and both stimulate secretion of MMPs from multiple cell types. As a consequence, these mediators may cooperate to enhance the inflammatory response. Indeed, SAA synergizes with chemokines to increase chemoattraction of monocytes and granulocytes. On the other hand, MMPs post-translationally modify chemokines and SAA to reduce their activity. Indeed, MMPs internally cleave SAA with loss of its cytokine-inducing and direct chemotactic potential whilst retaining its capacity to synergize with chemokines in leukocyte migration. Finally, MMPs truncate chemokines at their NH2- or COOH-terminal end, resulting in reduced or enhanced chemotactic activity. Therefore, the complex interactions between chemokines, SAA and MMPs either maintain or dampen the inflammatory response.
Objective:
P. gingivalis is a gram-negative anaerobic bacterium and a major periodontal pathogen. LPS produced by P. gingivalis promotes osteoclast formation. TECK is a CC chemokine whose expression is increased in gingival epithelial cells exposed to P. gingivalis LPS. In this study, we investigated the effect of TECK in osteoclastogenesis induced by P. gingivalis LPS.
Designs:
Real time reverse transcriptase polymerase chain reaction (RTPCR) analysis and western blotting were performed to confirm TECK in MG63, human osteoblast cell line and primary murine osteoblasts and CCR9 in RAW 264.7 cells and murine bone marrow macrophages (BMMs) as osteoclast precursors. P. gingivalis LPS-treated BMMs and Raw 264.7 cells were cultured with or without TECK or TECK antibody to examine the effect of TECK on osteoclast formation. Cocultures with murine osteoblasts and bone marrow cells were also treated with or without TECK or TECK antibody. Luciferase assay and western blotting were used to determine whether TECK-CCR9 induced osteoclastogenesis was mediated through NFATc1 or NF-kB signaling.
Results:
TECK was shown to be expressed by osteoblasts, and its receptor, CCR9, by osteoclast precursors. TECK increased P. gingivalis LPS-induced osteoclast numbers in an in vitro osteoclast formation assay using osteoclast precursors. The enhanced osteoclast formation by TECK was mediated by NFATc1, but not by NF-kB signaling.
Conclusion:
TECK may be a novel regulator of osteoclast formation induced by P. gingivalis LPS in periodontitis.
MM cells produce factors which stimulate bone resorption and suppress new bone formation. In addition, MMBD has a major role in pathogenesis of MM. OCL can both stimulate MM cell growth and tumor-associated angiogenesis which both enhance growth of tumor cells and chemoresistance. Further, OCL release growth factors from the bone marrow microenvironment, which amplify the growth effects of other growth factors produced by cells in the marrow microenvironment. OCL can also produce growth factors that directly stimulate MM cell growth. The recent results showing that mature OB can suppress MM cell growth suggests that suppression of OB differentiation in MM plays a key role in tumor progression, and if it can be reversed, and OB differentiation induced in patients, this would further suppress tumor growth and improve both bone quality and the quality of life of the patients. The availability of new anabolic agents that are safe for use on MM patients and stimulate new bone formation while not stimulating MM cell growth may reverse the devastating effects of MMBD. Thus, identification of agents that target both MM cells and the microenvironment should provide new therapeutic modalities that suppress or eradicate the tumor and restore bone health in patients with MM. © Springer Science+Business Media New York 2013. All rights are reserved.
Chemokines comprise several subfamilies of small proteins with conserved cysteine residues and common structural features. Chemokines interact with signaling receptors to elicit effects on cell migration, proliferation, and survival. Both CXC and CC subfamily chemokines promote bone formation developmentally and in response to hormonal and mechanical stimuli. Effects on homing of progenitor cells may be involved in effects on osteoblastogenesis. CXC and CC chemokines are also implicated in processes leading to osteoclastogenesis and bone resorption, with promotion of the migration of mononuclear osteoclast precursor cells being an important action. Chemokines contribute to the bone loss resulting from arthritis, both through promoting inflammation and osteoclastogenesis and attenuating cartilage repair. Both the positive effects of chemokines on bone formation and the bone loss resulting from inflammatory reactions to wear particles are factors in the success or failure of implants. In primary tumors of bone as well as cancers metastasizing to bone, chemokines facilitate interactions between tumor cells and the bone microenvironment through effects on angiogenesis, tumor growth, and invasion. Development of therapeutic agents that could target deleterious effects of chemokines, including effects on bone, has been slow, although a number of compounds for various disease indications are currently being evaluated.
Chronic apical periodortitis is a prevalent disease among Chilean population and a frequent cause of tooth loss. It represents a manifestation of ocalized tissue injury with well-defhed signs of chronic inflammation, destruction of apical periodontium, bone nesorption and formation of an apical granuloma or cyst.
CXCR4, the chemokine receptor for CXCL12, also known as SDF-1 (stromal cell derived factor-1), has been shown to play a pivotal role in bone metastasis, inflammatory and autoimmune conditions but has not been investigated in periprosthetic osteolysis. We co-cultured osteoblast-like cells with increasing concentrations of metallic (Co-35Ni-20Cr-10Mo and Co-28Cr-6Mo) and Co-ions simulating wear debris. Real-time Polymerase Chain Reaction (RT-PCR) and Western Blotting were used to quantify gene and protein expression of CXCR4. The expression of TNF-α and the effects of AMD3100 (bicyclam) on both CXCR4 and TNF-α expression among these cells was investigated. RT-PCR showed an increase in CXCR4 mRNA (7.5 fold for MG63 and 4.0 fold for SaOs-2 cells) among cells co-cultured with metal alloy particles. Western blotting showed a time-dependent increase in protein expression of CXCR4. The attempted blockade of CXCR4 by its known competitive receptor agonist AMD3100 led to a significant inhibition TNF-α mRNA expression. Immunohistochemistry showed CXCR4 positivity among patients with failed metal-on-metal (M-o-M) hip replacements and radiographic evidence of osteolysis. Our data collectively suggest that the CXCR4 chemokine is upregulated in a dose- and time-dependent manner in the presence of metallic wear debris.
Bone metastases development requires multistep and multicellular machinery consisting not only of processes shared with any types of metastases (formation of a pre-metastatic niche, chemotaxis of tumor cells into the host tissue, tumor cells escape from the microvasculature), but also biological interactions that are strictly related to bone microenvironment (bone marrow colonization by cancer cells, osteomimicry, deregulation of bone homeostasis). The aim of this review is to depict the complex sequence of events from early cancer cells dissemination to the onset of clinically detectable bone lesions with particular attention to those molecular targets that have been investigated in order to find out new therapeutical approaches helpful in reducing clinical sequelae of skeletal metastases.
Received: 29/Jun/2008 Chemokines are a small group of related chemoattractant peptides that play an essential role in the development and homeostatic maintenance of the immune system. They control the recruitment of cells needed for the induction and activation of innate and adaptive immune re- sponses. Stromal cell-derived factor 1(SDF-1) and its receptor (CXCR4) have role in regula- tion of trafficking of normal hematopoietic stem cells (HSCs) in their homing/ retention in bone marrow, also they control lymphocyte trafficking, angiogenesis, cell adherent or migration. In addition, chemokines and their receptors involved in several auto- immune diseases such as inflammation, HIV. In fact, chemokines are involved directly or indirectly in almost every as- pect of tumorigenesis. They mediate survival and metastatic spread of tumors, promote new blood vessel formation (neovascularization). SDF- 1/ CXCR4 axis for migration, enhanced re- sistance to apoptosis and an increased capacity for drug resistance. .A number of therapeutic strategies have been proposed to target almost every step of the chemokine or chemokine receptor involvement in tumors. Yet, despite occasional success stories, most of them appear to be ineffective or impractical,. The strategy would only be effective if it also promoted anti- tumor activity and more study is needed to clear the tumor relapse mechanism..
The function of the bone marrow is to coordinate hematopoiesis, i.e., the continuous production of mature blood cells throughout
life. Hematopoiesis often is envisioned as a pyramidal/hierarchical process with the least differentiated cells, i.e., those
of greatest maturation potential at the top, hierarchically speaking, and cells that have undergone terminal differentiation
at the bottom. The two major lineages of terminally differentiated blood cells are those derived from the myeloid and lymphoid
progenitors. Myeloid cells include red blood cells, platelets, and cells that provide cellular immunity, e.g., macrophages
and granulocytes. Lymphoid cells comprise T, B, and natural killer cells, and play a major role in coordinating humoral immunity.
Introduction:
Bone marrow-derived cells (BMDC) localize in premetastatic niche through chemokines and integrins signals and establish clusters that precede the arrival of even single metastatic tumor cell at distant site. CSCs demonstrate an increased metastatic propensity and would seem likely candidates for the acquisition of migratory capabilities and propagation of heterogeneous tumor cell populations to different target organs. Sonic Hedgehog (SHH), FOXM1 and Notch pathways and signaling molecules such as integrin and chemokine could dictate their fate.
Areas covered:
In this review, the molecular mechanisms of premetastatic niche onset are summarized.
Expert opinion:
Premetastatic niche is defined as a fertile microenvironment that forms in metastatic target organ and facilitates the invasion, survival and/or proliferation of metastatic tumor cells, providing a novel mechanism for the promotion of metastasis. Drugs targeting premetastatic niche could represent a new promising therapeutic approach in the treatment of bone metastases.
Starting first from Paget's "seed and soil" to the latest hypothesis about metastatic process involving the concept of a premetastatic niche, a large amount of data suggested the idea that metastatization is a multistep coordinated process with a high degree of efficiency. A specific subpopulation of cells with tumor-initiating and migratory capacity can selectively migrate toward sites that are able to promote survival, and/or proliferation of metastatic tumor cells through a microenvironment modification. Bone plays a pivotal role in this process, acting not only as a preferential site for cancer cells' homing and proliferation, due to a complex interplay between different cellular phenotypes such as osteoblasts and osteoclasts, but also as a source of bone marrow precursors that are able to facilitate the metastatic process in extra-skeletal disease. Moreover, bone microenvironment has the unique capacity to retain cancer stem cells in a quiescent status, acting as a reservoir that is able to cause a metastatic spread also many years after the resection of the primary tumor. To add a further level of complexity, these mechanisms are strictly regulated through the signalling through several soluble factors including PTH, vitamin D or calcium concentration. Understanding this complexity represents a major challenge in anti-cancer research and a mandatory step towards the development of new drugs potentially able not only to reduce the consequences of bone lesions but also to target the metastatization process from the "bone pre-neoplastic niche" to "visceral pre-neoplastic niches".
The stem cell microenvironment (in vivo known as niche) is a specific space in the bone marrow (BM), which nurses hematopoietic stem cells and regulates their self-renewal and differentiation using extrinsic cues, such as secreted factors. The niche plays a major role in regulating the number of blood cells and also protects stem cells against excessive proliferation. Till date, several possible secreted regulators of HSC function have been reported. Many of these were originally isolated from stromal cells and the cell lines isolated from hematopoietic tissues. These secreted factors act in concert and not only regulate HSC, but also the niche cells. It has also become clear that deregulation of the niche function is a potential cooperating factor during the development of hematological malignancies. An understanding of how the niche participates in HSC maintenance and repair through soluble factors can offer new opportunities for the development of novel therapeutic tools against hematological malignancies.
Matrix metalloproteinase-8 (MMP-8) is a central mediator in chronic periodontitis. Recently developed MMP-8-deficient mice show an impaired polymorphonuclear neutrophil response and more severe alveolar bone loss in Porphyromonas gingivalis-induced experimental periodontitis. The main mediators involved in neutrophil and monocyte/macrophage recruitment and in bone loss include lipopolysaccharide-induced CXC chemokine (LIX/CXCL5), stromal-derived factor-1/CXC chemokine ligand 12 (SDF1/CXCL12) and RANKL. Therefore, the aim of this study was to characterize the expression of LIX/CXCL5, SDF1/CXCL12 and RANKL in Porphyromonas gingivalis-induced experimental periodontitis in MMP-8⁻/⁻ (knockout) and wild-type mice.
MMP-8 null and WT P. gingivalis-infected and uninfected mice were included. Histopathological changes were assessed and LIX/CXCL5, SDF1/CXCL12 and RANKL were immunodetected and quantified.
Typical histopathological features of chronic periodontitis were seen in P. gingivalis-infected groups. LIX/CXCL5 expression was restricted to the gingival papilla in all four groups. Significantly lower expression of LIX/CXCL5 was seen in the knockout group compared with the wild-type infected group (p < 0.05). SDF1/CXCL12 and RANKL expression was mainly localized to the alveolar crest, including inflammatory leukocytes, vascular endothelium, osteoblasts and osteoclasts. Significant increases of SDF1/CXCL12 and RANKL were seen in both knockout and wild-type P. gingivalis-infected groups compared with uninfected groups (p < 0.05).
RANKL and SDF1/CXCL12 are up-regulated in P. gingivalis-induced periodontitis and they appear to be associated with the pathogenesis of the disease. MMP-8 is associated with a reduced expression of LIX/CXCL5 in the P. gingivalis-induced experimental periodontitis model.
Osteoclasts are derived from macrophage-lineage precursors. ED1 is an antibody that can recognize this lineage of cells. Matrix metalloproteinase 9 (MMP9) is essential for the migration of osteoclasts and their precursors during osteoclastogenesis. The aim of this research was to investigate differentiation and recruitment of osteoclasts during the early phase of experimental tooth movement in rats. The upper three molars of Wistar rats at one side were moved mesially, using Ni-Ti coil springs of 10 cN, for 6, 12, 24, 36, 48, 72, 96, and 120 h. The contralateral sides served as controls. Immunohistochemical staining using ED1 and MMP9 antibodies was performed. ED1(+) and MMP9(+) mononuclear and multinuclear cells were counted and statistically analysed. After force application, the number of ED1(+)/MMP9(+) multinuclear cells first increased in the bone marrow. At compressed areas, the number of ED1(+) mononuclear cells decreased; this was followed by an increase in the number of ED1(+/)MMP9(+) mononuclear and multinuclear cells. At tension areas, the number of ED1(+)/MMP9(+) multinuclear cells decreased while the number of ED1(+) mononuclear cells remained stable. It was concluded that force application induces osteoclast differentiation within the bone marrow. These osteoclasts probably migrate subsequently into the compressed PDL. Pre-existing osteoclasts disappear at the tension areas while the number of mononuclear macrophage-lineage cells remains stable.
Osteoclasts (OCs) are the cells uniquely responsible for dissolving both the organic and inorganic components of bone during bone development and remodeling throughout life. These cells originate from hematopoietic precursors of the monocyte/macrophage lineage that are present both in the bone marrow and peripheral circulation, and their numbers and/or activity are frequently increased in a wide array of clinical disorders that are associated with excessive bone loss and affect millions of people (1). For many years, investigations into how OCs develop and function have been hampered by the considerable difficulties associated with isolating and culturing these normally rare cells. Whereas cell lines have frequently provided an invaluable research tool and are widely used to decipher mechanisms involved in osteoblast (OB) differentiation and bone formation, no immortalized cell lines for mature OCs exist and the few pre-OC cell lines that have been reported either do not undergo full OC differentiation (2,3) or involve coculture systems and cells that may not be readily available to all researchers (4–6). To compound the problem further, it has proven difficult or impossible until recently to generate reliably bone-resorptive OCs expressing mature OC characteristics from primary bone marrow or circulating precursor cells in vitro. However, recent breakthroughs have now made the latter possible owing to the identification of the key signal, receptor activator of nuclear factor κB ligand (RANKL), that is responsible for the full development and activation of OCs both in vitro and in vivo (7–9).
Stromal derived factor-1 (SDF-1 or CXCL12) controls many aspects of stem cell function including trafficking and proliferation. Previously, it was demonstrated that DNA-damaging agents such as irradiation, cyclophosphamide or 5-fluorouracil increase the expression of SDF-1 by osteoblasts in murine marrow. Here, the production of SDF-1 by osteoblasts in vitro in response to cytokines known to be particularly important in bone physiology was examined using primary human osteoblasts (HOBs), mixed marrow stromal cells (BMSCs), and by, mouse, rat and human osteoblast-like cell lines. From these studies, it was determined that the expression of SDF-1 is an early feature of osteoblastic induction that may be modulated by IL-1beta, PDGF-BB, VEGF, TNF-alpha and PTH. Each of these factors increased SDF-1 synthesis, while TGF-beta1 decreased SDF-1 secretion. Of note, the biodistribution of SDF-1 in culture was equally distributed between the medium and detergent-soluble and -insoluble fractions of the cultures. Immunohistochemistry of developing bones demonstrated that SDF-1 was also a feature of early bone development first beginning in the perichondrium and moving into the marrow cavity of the developing bone analogue. As SDF-1 expression increases in response to PTH in vitro, animals were treated with an anabolic regime of PTH for 21 days. Under these conditions, significant increases in SDF-1 mRNA expression were observed near the growth plate and epiphysis regions of the long bones. Yet, in serum, immunodetectable SDF-1 levels were significantly reduced (24%) in the PTH-treated animals (Vehicle: 408 +/- 25 vs. PTH 308 +/- 20 SDF-1 pg/ml). Together, these data suggest a possible mechanism for localizing stem cells into a developing marrow where increased expression of SDF-1 in the local marrow environment along with decreased SDF-1 in the serum may create a homing gradient.
Osteoclasts are multinucleated cells that derive from hematopoietic progenitors in the bone marrow which also give rise to monocytes in peripheral blood, and to the various types of tissue macrophages. Osteoclasts are formed by the fusion of precursor cells. They function in bone resorption and are therefore critical for normal skeletal development (growth and modeling), for the maintenance of its integrity throughout life, and for calcium metabolism (remodeling). To resorb bone, the osteoclasts attach to the bone matrix, their cytoskeleton reorganizes, and they assume polarized morphology and form ruffled borders to secrete acid and collagenolytic enzymes and a sealing zone to isolate the resorption site. Identification of the osteoclastogenesis inducer, the receptor activator of nuclear factor-kappaB ligand (RANKL), its cognate receptor RANK, and its decoy receptor osteoprotegerin (OPG), has contributed enormously to the dramatic advance in our understanding of the molecular mechanisms involved in osteoclast differentiation and activity. This explosion in osteoclast biology is reflected by the large number of reviews which appeared during the last decade. Here I will summarize the "classical" issues (origin, differentiation, and activity) in a general manner, and will discuss an untouched issue (multinucleation) and a relatively novel aspect of osteoclast biology (osteoimmunology).
The specialized interaction between embryonic and maternal tissues is unique to mammalian development. This interaction begins with invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. The transient tumor-like behavior of cytotrophoblasts, which peaks early in pregnancy, is developmentally regulated. Likewise, in culture only early-gestation human cytotrophoblasts invade a basement membrane-like substrate. These invasive cells synthesize both metalloproteinases and urokinase-type plasminogen activator. Metalloproteinase inhibitors and a function-perturbing antibody specific for the 92-kD type IV collagen-degrading metalloproteinase completely inhibited cytotrophoblast invasion, whereas inhibitors of the plasminogen activator system had only a partial (20-40%) inhibitory effect. We conclude that the 92-kD type IV collagenase is critical for cytotrophoblast invasion.
Several chemotactic agonists including interleukin-8 (IL-8) and related cytokines have been shown to activate and attract leukocytes via seven-transmembrane domain, GTP-binding protein-coupled receptors. A cDNA clone, LESTR, encoding a protein of 352 amino acids, corresponding to a novel receptor of this type, was isolated from a human blood monocyte cDNA library. The sequence of the deduced protein, LESTR (leukocyte-derived seven-transmembrane domain receptor), has 92.6% identity with that of a recently reported bovine neuropeptide Y (NPY) receptor, boLCR1 (Rimland, J., Xin, W., Sweetnam, P., Saijoh, K., Nestler, E. J., and Duman, R. S. (1991) Mol. Pharmacol. 40, 869-875). LESTR, however, is more similar (> 34%) to the IL-8 receptors, IL-8R1 and IL-8R2, than to several NPY receptors of different origin (< 20%). In the monocyte library, LESTR cDNA fragments were about 20 times as frequent as cDNA coding for IL-8R1 and IL-8R2, and much higher levels of LESTR- than IL-8R-specific mRNA were found in human blood neutrophils and lymphocytes. LESTR transcripts, by contrast, were low or undetectable in several neuroblastoma cell lines that are widely used to study NPY functions. Transfected cells expressing high levels of LESTR mRNA did not bind radiolabeled NPY, IL-8, NAP-2, GRO alpha, PF4, IP10, MCP-1, MCP-3, MIP-1 alpha, HC14, I309, RANTES, C3a, or LTB4. NPY also failed to bind to neutrophils, monocytes, and lymphocytes, to elicit responses in vitro such as Ca2+ changes, shape change, chemotaxis, enzyme release, and the respiratory burst, and to induce leukocyte accumulation upon injection in rats and rabbits. Although the ligand for LESTR could not be identified among a large number of chemotactic cytokines, the high expression in white blood cells and the marked sequence relation to IL-8R1 and IL-8R2 suggest that LESTR may function in the activation of inflammatory cells.
A key event in bone resorption is the recruitment of osteoclasts to future resorption sites. We follow here the migration of preosteoclasts from the periosteum to the developing marrow cavity of fetal mouse metatarsals in culture, and investigate the role of proteinases and demineralization in this migration. Our approach consisted in testing inhibitors of proteinases and demineralization on the migration kinetics. Migration was monitored by histomorphometry and the (pre)osteoclasts were identified by their tartrate resistant acid phosphatase (TRAP) activity. At the time of explantation, TRAP+ cells (all mononucleated) are detected only in the periosteum, and the core of the diaphysis (future marrow cavity) consist of calcified cartilage. Upon culture, TRAP+ cells (differentiating progressively into multinucleated osteoclasts) migrate through a seam of osteoid and a very thin and discontinuous layer of mineral, invade the calcified cartilage and transform it into a "marrow' cavity; despite the passage of maturing osteoclasts, the osteoid develops into a bone collar. The migration of TRAP+ cells is completely prevented by matrix metalloproteinase (MMP) inhibitors, but not by a cysteine proteinase inhibitor, an inhibitor of carbonic anhydrase, or a bisphosphonate. The latter three drugs inhibit, however, the resorptive activity of mature osteoclasts at least as efficiently as do the MMP inhibitors, as assessed in cultures of calvariae and radii. Furthermore, in situ hybridizations reveal the expression of 2 MMPs, gelatinase B (MMP-9 or 92 kDa type IV collagenase) in (pre)osteoclasts, and interstitial collagenase (MMP-13) in hypertrophic chondrocytes. It is concluded that only MMPs appear obligatory for the migration of (pre)osteoclasts, and that this role is distinct from the one MMPs may play in the subosteoclastic resorption compartment. We propose that this new role of MMPs is a major component of the mechanism that determines where and when the osteoclasts will attack the bone.
Hematopoietic progenitor cells migrate in vitro and in vivo towards a gradient of the chemotactic factor stromal cell-derived factor-1 (SDF-1) produced by stromal cells. This is the first chemoattractant reported for human CD34+ progenitor cells. Concentrations of SDF-1 that elicit chemotaxis also induce a transient elevation of cytoplasmic calcium in CD34+ cells. SDF-1-induced chemotaxis is inhibited by pertussis toxin, suggesting that its signaling in CD34+ cells is mediated by seven transmembrane receptors coupled to Gi proteins. CD34+ cells migrating to SDF-1 include cells with a more primitive (CD34+/CD38- or CD34+/DR-) phenotype as well as CD34+ cells phenotypically committed to the erythroid, lymphoid and myeloid lineages, including functional BFU-E, CFU-GM, and CFU-MIX progenitors. Chemotaxis of CD34+ cells in response to SDF-1 is increased by IL-3 in vitro and is lower in CD34+ progenitors from peripheral blood than in CD34+ progenitors from bone marrow, suggesting that an altered response to SDF-1 may be associated with CD34 progenitor mobilization.
Interleukin-1 (IL-1) greatly induces osteoclast formation and stimulates bone resorption of mouse calvaria in culture. In the presence of soluble IL-6 receptor (sIL-6R), IL-6 similarly induces osteoclast formation, but the potency of IL-6 in inducing bone resorption in organ culture is weaker than that of IL-1. To study the differences in bone-resorbing activity between IL-1 and IL-6, we examined the effects of the two cytokines on the induction of matrix metalloproteinases (MMPs). In mouse calvarial cultures, IL-1 markedly enhanced the messenger RNA (mRNA) expression of MMP-13 (collagenase 3), MMP-2 (gelatinase A), MMP-9 (gelatinase B), and MMP-3 (stromelysin 1), which associated with increases in bone matrix degradation. A hydroxamate inhibitor of MMPs significantly suppressed bone-resorbing activity induced by IL-1. Gelatin zymography showed that both pro- and active-forms of MMP-2 and MMP-9 were detected in the conditioned medium collected from calvarial cultures, and IL-1 markedly stimulated both pro- and active-forms of the two gelatinases. IL-6 with sIL-6R also stimulated mRNA expression and biological activities of these MMPs, but the potency was much weaker than that of IL-1. Conditioned medium collected from IL-1-treated calvariae degraded native type I collagen, but 3/4- and 1/4-length collagen fragments were not detected, suggesting that both collagenases and gelatinases synergistically degraded type I collagen into smaller fragments. In mouse osteoblastic cells, the expression ofMMP-2, MMP-3, and MMP-13 mRNAs could be detected, and they were markedly enhanced by IL-1alpha on days 2 and 5. IL-6 with sIL-6R also induced expression of MMP-13 and MMP-2 mRNAs on day 2, but the expression was rather transient. These results demonstrate that the potency of induction of MMPs by IL-1 and IL-6 is closely linked to the respective bone-resorbing activity, suggesting that MMP-dependent degradation of bone matrix plays a key role in bone resorption induced by these cytokines.
and acts as a decoy receptor in the RANK-RANKL signaling system (Fig. 8). In conclusion, osteoblasts/stromal cells are involved in all of the processes of osteoclast development, such as differentiation, survival, fusion, and activation of osteoclasts (Fig. 8). Osteoblasts/stromal cells can now be replaced with RANKL and M-CSF in dealing with the whole life of osteoclasts. RANKL, RANK, and OPG are three key molecules that regulate osteoclast recruitment and function. Further studies on these key molecules will elucidate the molecular mechanism of the regulation of osteoclastic bone resorption. This line of studies will establish new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment and functions such as osteopetrosis, osteoporosis, metastatic bone disease, Paget's disease, rheumatoid arthritis, and periodontal bone disease.
Hematopoietic stem cell homing and engraftment require several adhesion interactions, which are not fully understood. Engraftment of nonobese/severe combined immunodeficiency (NOD/SCID) mice by human stem cells is dependent on the major integrins very late activation antigen-4 (VLA-4); VLA-5; and to a lesser degree, lymphocyte function associated antigen-1 (LFA-1). Treatment of human CD34(+) cells with antibodies to either VLA-4 or VLA-5 prevented engraftment, and treatment with anti-LFA-1 antibodies significantly reduced the levels of engraftment. Activation of CD34(+) cells, which bear the chemokine receptor CXCR4, with stromal derived factor 1 (SDF-1) led to firm adhesion and transendothelial migration, which was dependent on LFA-1/ICAM-1 (intracellular adhesion molecule-1) and VLA-4/VCAM-1 (vascular adhesion molecule-1). Furthermore, SDF-1-induced polarization and extravasation of CD34(+)/CXCR4(+) cells through the extracellular matrix underlining the endothelium was dependent on both VLA-4 and VLA-5. Our results demonstrate that repopulating human stem cells functionally express LFA-1, VLA-4, and VLA-5. Furthermore, this study implies a novel approach to further advance clinical transplantation.
Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737-744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.
Despite the discovery of thrombopoietin (TPO) and its contribution to megakaryocytopoiesis, the exact mechanisms and sites of platelet production are unknown. It has been shown that mature megakaryocytes (MKs) functionally express the stromal-derived factor 1 (SDF-1) receptor, CXCR4. SDF-1-induced migration of mature MKs through endothelial cell layers results in increased platelet production. Because the migration of polyploid MKs from the bone marrow microenvironment requires remodeling of the perivascular extracellular matrix, it was hypothesized that mature polyploid MKs may express matrix metalloproteinases (MMPs), facilitating their exit into the bone marrow extravascular space. In this report, it is demonstrated that SDF-1 induces the expression and release of gelatinase B (MMP-9) by purified mature polyploid human MKs and an adeno-CXCR4-infected megakaryocytic cell line. Neutralizing antibody to MMP-9, but not MMP-2, blocked SDF-1-induced migration of MKs through reconstituted basement membrane, suggesting that expression of MMP-9 is critical for MK migration. Incubation of mature MKs with a synthetic MMP inhibitor, 5-phenyl-1,10-phenanthrolene, resulted in the inhibition of platelet formation, suggesting that the expression of MMPs is not only critical for megakaryocyte migration but also for subsequent platelet release. Confirming these results, adeno-SDF-1 injection into normal mice resulted in increased platelet counts, a process that could be blocked by a synthetic MMP inhibitor. These results suggest mobilization of MKs involves sequential expression and activation of chemokine receptors such as CXCR4, MMP-9, followed by transendothelial migration. MMP inhibitors may have potential use in the treatment of thrombotic and myeloproliferative disorders. (Blood. 2000;96:4152-4159)
Hematopoietic stem cell homing and engraftment require several adhesion interactions, which are not fully understood. Engraftment of nonobese/severe combined immunodeficiency (NOD/SCID) mice by human stem cells is dependent on the major integrins very late activation antigen–4 (VLA-4); VLA-5; and to a lesser degree, lymphocyte function associated antigen–1 (LFA-1). Treatment of human CD34+cells with antibodies to either VLA-4 or VLA-5 prevented engraftment, and treatment with anti–LFA-1 antibodies significantly reduced the levels of engraftment. Activation of CD34+ cells, which bear the chemokine receptor CXCR4, with stromal derived factor 1 (SDF-1) led to firm adhesion and transendothelial migration, which was dependent on LFA-1/ICAM-1 (intracellular adhesion molecule–1) and VLA-4/VCAM-1 (vascular adhesion molecule–1). Furthermore, SDF-1–induced polarization and extravasation of CD34+/CXCR4+ cells through the extracellular matrix underlining the endothelium was dependent on both VLA-4 and VLA-5. Our results demonstrate that repopulating human stem cells functionally express LFA-1, VLA-4, and VLA-5. Furthermore, this study implies a novel approach to further advance clinical transplantation.
Despite the discovery of thrombopoietin (TPO) and its contribution to megakaryocytopoiesis, the exact mechanisms and sites of platelet production are unknown. It has been shown that mature megakaryocytes (MKs) functionally express the stromal-derived factor 1 (SDF-1) receptor, CXCR4. SDF-1–induced migration of mature MKs through endothelial cell layers results in increased platelet production. Because the migration of polyploid MKs from the bone marrow microenvironment requires remodeling of the perivascular extracellular matrix, it was hypothesized that mature polyploid MKs may express matrix metalloproteinases (MMPs), facilitating their exit into the bone marrow extravascular space. In this report, it is demonstrated that SDF-1 induces the expression and release of gelatinase B (MMP-9) by purified mature polyploid human MKs and an adeno-CXCR4–infected megakaryocytic cell line. Neutralizing antibody to MMP-9, but not MMP-2, blocked SDF-1–induced migration of MKs through reconstituted basement membrane, suggesting that expression of MMP-9 is critical for MK migration. Incubation of mature MKs with a synthetic MMP inhibitor, 5-phenyl-1,10-phenanthrolene, resulted in the inhibition of platelet formation, suggesting that the expression of MMPs is not only critical for megakaryocyte migration but also for subsequent platelet release. Confirming these results, adeno-SDF-1 injection into normal mice resulted in increased platelet counts, a process that could be blocked by a synthetic MMP inhibitor. These results suggest mobilization of MKs involves sequential expression and activation of chemokine receptors such as CXCR4, MMP-9, followed by transendothelial migration. MMP inhibitors may have potential use in the treatment of thrombotic and myeloproliferative disorders.
The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 (fusin, LESTR) are likely to be involved in the trafficking of hematopoietic progenitor and stem cells, as suggested by the reduced bone marrow hematopoiesis in SDF-1–deficient mice and the chemotactic effect of SDF-1 on CD34+ progenitor cells. Migration of leukemic cells might also depend on the expression of chemokine receptors. Therefore, we analyzed expression of CXCR-4 on mobilized normal CD34+ progenitors and leukemic cells. In addition, SDF-1–induced transendothelial migration across a bone marrow endothelial cell layer was assessed in vitro. By flow cytometry, CXCR-4 was found to be expressed in significant amounts on circulating CD34+ hematopoietic progenitor cells, including more primitive subsets (CD34+/CD38− and CD34+/Thy-1+ cells). In accordance with the immunofluorescence data, CD34+ progenitors efficiently migrated across endothelium in response to SDF-1 containing conditioned medium from the stromal cell line MS-5. Leukemic blasts (mostly CD34+) from patients with acute myeloblastic leukemia (AML) expressed variable amounts of CXCR-4, which was functionally active, as demonstrated by a positive correlation between the SDF-1–induced transendothelial migration and the cell surface density of CXCR-4 (r = 0.97). Also recombinant SDF-1β induced migration of CXCR-4–positive leukemic blasts. The effect of both conditioned medium and recombinant SDF-1 was inhibited by a CXCR-4 blocking antibody. In contrast, CD34+ leukemic cell lines (KG1, KG1a, Kasumi-1, MOLM-1) expressed low levels or were negative for CXCR-4, and did not migrate. By reverse transcriptase-polymerase chain reaction (RT-PCR), however, basal levels of CXCR-4 mRNA were also detected in all leukemic cell lines. We conclude that CXCR-4 is expressed on CD34+cells including more primitive, pluripotent progenitors, and may therefore play a role in the homing of hematopoietic stem cells. CXCR-4 expressed in variable amounts on primary AML leukemic cells is functionally active and may be involved in the trafficking of malignant hematopoietic cells.
Interleukin-1 (IL-1) greatly induces osteoclast formation and stimulates bone resorption of mouse calvaria in culture. In the presence of soluble IL-6 receptor (sIL-6R), IL-6 similarly induces osteoclast formation, but the potency of IL-6 in inducing bone resorption in organ culture is weaker than that of IL-1. To study the differences in bone-resorbing activity between IL-1 and IL-6, we examined the effects of the two cytokines on the induction of matrix metalloproteinases (MMPs). In mouse calvarial cultures, IL-1 markedly enhanced the messenger RNA (mRNA) expression of MMP-13 (collagenase 3), MMP-2 (gelatinase A), MMP-9 (gelatinase B), and MMP-3 (stromelysin 1), which associated with increases in bone matrix degradation. A hydroxamate inhibitor of MMPs significantly suppressed bone-resorbing activity induced by IL-1. Gelatin zymography showed that both pro- and active-forms of MMP-2 and MMP-9 were detected in the conditioned medium collected from calvarial cultures, and IL-1 markedly stimulated both pro- and active-forms of the two gelatinases. IL-6 with sIL-6R also stimulated mRNA expression and biological activities of these MMPs, but the potency was much weaker than that of IL-1. Conditioned medium collected from IL-1-treated calvariae degraded native type I collagen, but 3/4- and 1/4-length collagen fragments were not detected, suggesting that both collagenases and gelatinases synergistically degraded type I collagen into smaller fragments. In mouse osteoblastic cells, the expression of MMP-2, MMP-3, and MMP-13 mRNAs could be detected, and they were markedly enhanced by IL-1α on days 2 and 5. IL-6 with sIL-6R also induced expression of MMP-13 and MMP-2 mRNAs on day 2, but the expression was rather transient. These results demonstrate that the potency of induction of MMPs by IL-1 and IL-6 is closely linked to the respective bone-resorbing activity, suggesting that MMP-dependent degradation of bone matrix plays a key role in bone resorption induced by these cytokines.
To elucidate the mechanisms by which haemopoietic progenitor cells lodge in the bone marrow, we examined the secretion of chemoattractants for haemopoietic progenitor cells by bone marrow and lung endothelial cells. The bone marrow endothelial cells, but not lung endothelial cells, secreted chemoattractants for the haemopoietic progenitor cell line, FDCP-2, and normal haemopoietic progenitor cells. Checkerboard analysis demonstrated that the conditioned medium of the bone marrow endothelial cells had chemotactic activity and random motility-stimulating activity. The bone marrow endothelial cells expressed stromal-cell-derived factor-1 (SDF-1) mRNA and produced SDF-1 protein, whereas the lung endothelial cells did not. Adhesion of FDCP-2 cells to the bone marrow endothelial cells was partially inhibited by anti-SDF-1 antibody. These findings suggest that the chemoattractants for haemopoietic progenitor cells including SDF-1 and random motility-stimulating factor(s) selectively secreted by the bone marrow endothelial cells may contribute to the homing of haemopoietic progenitor cells to bone marrow.
The gelatin-degrading matrix metalloproteinase (MMP) activities and their inhibitors produced by rabbit articular chondrocytes have been characterized by gel substrate analysis ('zymography') after electrophoresis on non-reducing sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Differentiated chondrocytes in confluent primary culture produced constitutively only one gelatinase which presented the main characteristics of proMMP-2 ('72 kDa type IV procollagenase'). It had an apparent Mr of 66,000 (unreduced), which was partially or totally converted to 61,000 by, respectively, trypsin or APMA treatment; exogenous TIMP (tissue inhibitor or metalloproteinases) inhibited the conversion triggered by APMA but not that induced by trypsin. This proMMP-2 was also the predominant gelatinase found, together with its 61 kDa activation product, in extracts of articular cartilage. Differentiated chondrocytes simultaneously produced MMP inhibitors which on reverse zymograms were distributed over two bands with Mr of 27,500 and 20,400, resistant to both pH 2 and 100 degrees C, corresponding, respectively, presumably, to TIMP and TIMP-2. Interleukin-1 (IL1) and tumor necrosis factor alpha (TNF alpha) did not affect the production of the proMMP-2 nor of the two species of TIMP. However, IL1 induced the coordinated production of 91 and 55 kDa gelatinases. The 91 kDa activity is likely to correspond to proMMP-9. It could be converted to a 81 kDa gelatinase by trypsin or APMA treatment, in a process that was inhibited in both cases by exogenous TIMP. The 55 kDa gelatinolytic activity most probably represents the sum of the activities of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). It was sequentially converted to lower size forms (49 to 35 kDa) by either trypsin or APMA; that conversion was inhibited by TIMP, with the exception, however, of the first steps (from 55 to 49, then to 42 kDa) induced by trypsin. The 55 kDa and its conversion forms were all active on both gelatin and casein. TNF alpha did also stimulate the production of proMMP-9, although less efficiently than IL1, but it did not induce, or very poorly, that of the 55 kDa proMMP-1/proMMP-3 activity. Low levels of proMMP-9 and of its 81 kDa product of activation were also found in extracts of cartilage. With increasing passage number and cell dedifferentiation, confluent chondrocytes produced increasing amounts of proMMP-2 and of the two species of TIMP. A spontaneous low production of proMMP-9 and proMMP-1/proMMP-3 was only occasionally observed in cultures of dedifferentiated chondrocytes, accompanying a spontaneous low production of procollagenase.(ABSTRACT TRUNCATED AT 400 WORDS)
The identification of the plasminogen activator (PA) types present in bone and the regulation of their activity by parathyroid hormone (PTH) were investigated in cultures of fetal mouse calvariae with the use of either a chromogenic substrate or a zymographic assay. PA was detected essentially in the tissue extracts of the explanted bones, with only 1-2% of the total activity released in the surrounding culture media. From their electrophoretic behavior compared to PAs of other mouse tissues and from their response to a specific antibody raised against the tissue type PA (tPA), two major molecular species, of 70 and 48 kD were identified as tPA and urokinase (uPA), respectively, a third minor species of 105 kD being likely to correspond to complexes between tPA and an inhibitor; the culture fluids, moreover, contained enzymatically active degradation products of uPA of 42 and 29 kD. The PA activity of the bone extracts was only minimally affected by the addition of fibrinogen fragments to the chromogenic assays. PTH induced bone resorption and stimulated in parallel the accumulation of PA in the tissue; other bone-resorbing agents, 1,25-dihydroxyvitamin D3 and prostaglandin E2, had similar effects. Densitometric scanning of the zymograms of the bone extracts indicated that PTH stimulated only the production of tPA and had no effect on that of uPA. However, PTH also enhanced the release of uPA (both the 48 kD and the 29 kD forms) from the bones into the media. Although inhibiting bone resorption, calcitonin had no effect on the PTH-induced accumulation of PA in bone or on the release of tPA, but it prevented the PTH-induced accumulation of 29 kD uPA in the culture fluids. Thus these studies support the view that tPA and possibly also uPA may have a role in the physiology of bone; the nature of this role remains to be elucidated, however.
The osteoclast is the specialized multinucleated cell primarily responsible for the degradation of the organic and inorganic components of bone matrix. The functional and developmental relationship between osteoclasts and foreign body giant cells is unclear. The osteoclast plasma membrane ruffled border juxtaposed to the bone surface is a unique morphologic characteristic of active osteoclasts. In the studies reported here giant cell formation was induced in response to a variety of materials implanted onto the richly vascularized chick chorioallantoic membrane. Light and electron microscopic techniques were used to examine the morphologic characteristics of the giant cells. In addition, immunohistochemical methods were used to demonstrate the appearance of a 150 kD cell surface antigen on chicken osteoclasts recognized by monoclonal antibody 121F. Giant cells that formed in response to mineralized bone particles exhibited ruffled borders and stained positively with the 121F antibody. Many giant cells that formed in response to hydroxyapatite possessed ruffled borders similar to but not as extensive as those observed on giant cells formed on bone. Immunohistochemical localization of the 121F antigen on these cells suggested that the antigen was present, but staining intensity was reduced compared to that of bone-associated giant cells. The formation of mineral matrix complexes by the adsorption to hydroxyapatite of bone extract or osteocalcin enhanced ruffled borders and the presence of the 121F antigen on elicited giant cells. In contrast, giant cells that formed on non-resorbable materials, such as Sepharose beads, mica, and methacrylate, lacked ruffled borders and were negative for the 121F antigen. It appears that expression of the 121F osteoclast antigen correlates with the appearance and extent of ruffled membranes on giant cells. Furthermore, it appears that giant cell ruffled membrane development and the presence of the 121F osteoclast antigen are related to giant cell formation in response to resorbable materials that are subject to extracellular dissolution. Expression of this antigen may be indicative of the developmental and/or functional state of giant cells (osteoclasts) that form on resorbable substrates. In addition, components of the bone matrix, including osteocalcin, in association with bone mineral, lead to elevated levels of this osteoclast antigen.
Osteoblasts have been shown to modulate osteoclast activity, but the reverse process has not been investigated. In the current study conditioned medium (CM) was collected from osteoclasts and osteoclast-like cells and its effects on osteoblast alkaline phosphatase (ALPase) activity and collagen synthesis ([3H]proline hydroxylation) were determined. In primary chick osteoblasts, cultured chick embryo frontal bones, and UMR-106-01 cells, collagen synthesis and ALPase activity, but not [3H]thymidine incorporation, were inhibited by CM from chick marrow-derived giant cells, which possess some of the phenotypic characteristics of osteoclasts. However, collagen synthesis in chick embryo fibroblasts was not affected by giant cell CM. CM collected from cultures of chicken osteoclasts and human osteoclastoma cells and marrow-derived giant cells inhibited collagen synthesis in UMR-106-01 cells, but the effects of ALPase activity varied with the cell type. In contrast, mononuclear cell and fibroblast CM did not alter collagen synthesis. Initial characterization studies demonstrate that the inhibitor is a heat-labile factor with a molecular weight greater than 3500. In summary, authentic osteoclasts, tumor osteoclast-like cells, and chicken and human multinucleated giant cells produce a soluble factor that alters osteoblast collagen synthesis, suggesting that osteoclasts play a role in the modulation of osteoblast activity.
Much has been learned about the cell biology and molecular biology of the osteoclast in the last 5 yr. The osteoclast appears to be derived from CFU-GM, the committed monocyte-granulocyte precursor cell. This cell then differentiates into more committed precursors for the osteoclast. The role of the marrow microenvironment appears to be critical for murine osteoclast formation, although in human systems it appears to be nonessential but acts to enhance osteoclast formation and resorption. The osteoclast has been shown to be a secretory cell capable of producing both stimulators and inhibitors of osteoclast formation and resorption. The identification of the role of protooncogenes, such as c-fos and pp60c-src, in osteoclast differentiation and bone resorption has provided important insights into the regulation of normal osteoclast activity. Studies such as these should help us to dissect the pathophysiology of abnormal osteoclastic activity, such as seen in hypercalcemia of malignancy, osteopetrosis, and Paget's disease of bone. Future research is needed to further delineate the signaling pathways involved in osteoclastic bone resorption in response to cytokines and hormones, as well as to identify the molecular events required for commitment of multipotent precursors to the osteoclast lineage. Development of osteoclast cell lines may be possible and would greatly enhance our understanding of the biology of the osteoclast. Utilization of current model systems to examine the effects of cytokines and hormones on osteoclast precursors in vitro and in vivo and the ability to obtain large numbers of highly purified osteoclasts for production of osteoclast cDNA libraries should lead to important new discoveries in osteoclast biology.
Circulating CD34+ cells were isolated from leukapheresis products collected from patients with ovarian cancer. CD34− contaminating cells, identified immediately after immunoselection, ranged from 5% to 25% in five different experiments and were predominantly CD3+ T-lymphocytes (range 2–12%), CD3−/CD16+/CD56+ natural killer cells (range 2–11%) and rare mature CD15+/CD11b+ granulocytes (range 1–2%). CD34+ cells were cultured in liquid medium in the presence of interleukin-3, granulocyte-macrophage colony stimulating factor, stem cell factor, granulocyte colony stimulating factor and a powerful proliferation with prevalent differentiation along the granulocytic/monocytic lineage was obtained. After 10 d of culture a small but consistent number of early multinucleated osteoclasts were identified with a frequency of one cell per 700 granulocytic/monocytic cells, as revealed by cytologic examination. This observation was confirmed by staining for tartrate-resistant acid phosphatase activity which revealed red multinucleated elements with a frequency comparable to that reported above. Conversely, no osteoclasts were observed in those cultures in which macrophage overgrowth was obtained by culturing CD34+ cells until day 35.
These observations suggest that circulating progenitors have a multilineage potential in vitro and contribute to the clarification of osteoclast development in humans; additionally, they provide the basis for the future development of optimized osteoclast culture techniques in liquid medium and the basic culture system, to test the distinct activity of 1,25(OH)2D3, parathyroid hormone, interleukin-11 and of other cytokines on osteoclast development in humans.
The osteoclasts are the cells responsible for bone resorption. Matrix metalloproteinases (MMPs) appear crucial for this process. To identify possible MMP expression in osteoclasts, we amplified osteoclast cDNA fragments having homology with MMP genes, and used them as a probe to screen a rabbit osteoclast cDNA library. We obtained a cDNA of 1,972 bp encoding a polypeptide of 582 amino acids that showed more than 92% identity to human, mouse, and rat membrane-type 1 MMP (MT1-MMP), a cell surface proteinase believed to trigger cancer cell invasion. By northern blotting, MT1-MMP was found to be highly expressed in purified osteoclasts when compared with alveolar macrophages and bone stromal cells, as well as with various tissues. In situ hybridization on bone sections showed that MT1-MMP is expressed also in osteoclasts in vivo. Antibodies recognizing MT1-MMP reacted with specific plasma membrane areas corresponding to lamellipodia and podosomes involved, respectively, in migratory and attachment activities of the osteoclasts. These observations highlight how cells might bring MT1-MMP into contact with focal points of the extracellular matrix, and are compatible with a role of MT1-MMP in migratory and attachment activities of the osteoclast.
Homozygous mice with a null mutation in the MMP-9/gelatinase B gene exhibit an abnormal pattern of skeletal growth plate vascularization and ossification. Although hypertrophic chondrocytes develop normally, apoptosis, vascularization, and ossification are delayed, resulting in progressive lengthening of the growth plate to about eight times normal. After 3 weeks postnatal, aberrant apoptosis, vascularization, and ossification compensate to remodel the enlarged growth plate and ultimately produce an axial skeleton of normal appearance. Transplantation of wild-type bone marrow cells rescues vascularization and ossification in gelatinase B-null growth plates, indicating that these processes are mediated by gelatinase B-expressing cells of bone marrow origin, designated chondroclasts. Growth plates from gelatinase B-null mice in culture show a delayed release of an angiogenic activator, establishing a role for this proteinase in controlling angiogenesis.
The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 (fusin, LESTR) are likely to be involved in the trafficking of hematopoietic progenitor and stem cells, as suggested by the reduced bone marrow hematopoiesis in SDF-1-deficient mice and the chemotactic effect of SDF-1 on CD34+ progenitor cells. Migration of leukemic cells might also depend on the expression of chemokine receptors. Therefore, we analyzed expression of CXCR-4 on mobilized normal CD34+ progenitors and leukemic cells. In addition, SDF-1-induced transendothelial migration across a bone marrow endothelial cell layer was assessed in vitro. By flow cytometry, CXCR-4 was found to be expressed in significant amounts on circulating CD34+ hematopoietic progenitor cells, including more primitive subsets (CD34+/CD38- and CD34+/Thy-1+ cells). In accordance with the immunofluorescence data, CD34+ progenitors efficiently migrated across endothelium in response to SDF-1 containing conditioned medium from the stromal cell line MS-5. Leukemic blasts (mostly CD34+) from patients with acute myeloblastic leukemia (AML) expressed variable amounts of CXCR-4, which was functionally active, as demonstrated by a positive correlation between the SDF-1-induced transendothelial migration and the cell surface density of CXCR-4 (r = 0.97). Also recombinant SDF-1beta induced migration of CXCR-4-positive leukemic blasts. The effect of both conditioned medium and recombinant SDF-1 was inhibited by a CXCR-4 blocking antibody. In contrast, CD34+ leukemic cell lines (KG1, KG1a, Kasumi-1, MOLM-1) expressed low levels or were negative for CXCR-4, and did not migrate. By reverse transcriptase-polymerase chain reaction (RT-PCR), however, basal levels of CXCR-4 mRNA were also detected in all leukemic cell lines. We conclude that CXCR-4 is expressed on CD34+ cells including more primitive, pluripotent progenitors, and may therefore play a role in the homing of hematopoietic stem cells. CXCR-4 expressed in variable amounts on primary AML leukemic cells is functionally active and may be involved in the trafficking of malignant hematopoietic cells.
Monocytes and macrophages are capable of degrading both the mineral and organic components of bone and are known to secrete local factors which stimulate host osteoclastic bone resorption. Recent studies have shown that monocytes and macrophages, including those isolated from neoplastic and inflammatory lesions, can also be induced to differentiate into cells that show all the cytochemical and functional characteristics of mature osteoclasts, including lacunar bone resorption. Monocyte/macrophage-osteoclast differentiation occurs in the presence of osteoblasts/bone stromal cells (which express osteoclast differentiation factor) and macrophage-colony stimulating factor and is inhibited by osteoprotegerin. Various systemic hormones and local factors (e.g. cytokines, growth factors, prostaglandins) modulate osteoclast formation by controlling these cellular and humoral elements. Various pathological lesions of bone and joint (e.g. carcinomatous metastases, arthritis, aseptic loosening) are associated with osteolysis. These lesions generally contain a chronic inflammatory infiltrate in which macrophages form a significant fraction. One cellular mechanism whereby pathological bone resorption may be effected is through generation of increased numbers of bone-resorbing osteoclasts from macrophages. Production of humoral factors which stimulate mononuclear phagocyte-osteoclast differentiation and osteoclast activity is also likely to influence the extent of pathological bone resorption.
The mechanism(s) underlying the release of stem/progenitor cells from bone marrow into the circulation is poorly understood. We hypothesized that matrix metalloproteinases (MMPs), especially gelatinases, which are believed to participate in the proteolysis of basement membranes and in the migration of leukocytes, may facilitate this process. First, we investigated whether CD34(+) stem/progenitor cells express gelatinases A (MMP-2) and/or B (MMP-9) and whether growth factors and cytokines (granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], stem cell factor [SCF], macrophage colony-stimulating factor [M-CSF], interleukin-3 [IL-3], IL-6, IL-8, and tumor necrosis factor-alpha [TNF-alpha]) are able to modulate their expression. Next, we examined the transmigration of these stem/progenitor cells through reconstituted basement membrane (Matrigel) and its modulation by growth factors and cytokines. CD34(+) cells were obtained from steady-state bone marrow and peripheral blood (from leukapheresis products collected either in steady-state hematopoiesis or after mobilization with G-CSF plus chemotherapy or G-CSF alone). We found that peripheral blood CD34(+) cells, regardless of whether they were mobilized or not, strongly expressed both gelatinases (MMP-2 and MMP-9) in contrast to steady-state bone marrow CD34(+) cells, which did not. However, all the growth factors and cytokines tested could induce MMP-2 and MMP-9 secretion by the latter cells. Moreover, the stimulatory effects of G-CSF and SCF on both MMP-2 and MMP-9 secretion were found to be significantly higher in CD34(+) cells isolated from bone marrow than in those from peripheral blood. In addition TNF-alpha, GM-CSF, and IL-6 increased the secretion of a partially active form of MMP-2. Basal transmigration of bone marrow CD34(+) cells through Matrigel was lower than that of peripheral blood CD34(+) cells (P <.0001), but growth factors and cytokines increased it by 50% to 150%. Positive correlations were established between expression of gelatinases and CD34(+) cell migration (r >.9). The stimulatory effect of G-CSF was significantly greater on the migration of CD34(+) cells from bone marrow than on those from peripheral blood (P =.004). Moreover, CD34(+) cell migration was reduced to approximately 50% by antibodies to MMP-2 and MMP-9, tissue inhibitors of metalloproteinases (rhTIMP-1 and -2), and o-phenanthroline. TNF-alpha-induced gelatinase secretion and migration of CD34(+) cells and of clonogenic progenitors (colony-forming unit-granulocyte-macrophage [CFU-GM], burst-forming unit-erythroid [BFU-E], colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM], and colony-forming unit-megakaryocyte [CFU-MK]) were dose-dependent. Therefore, this study demonstrated that CD34(+) cells that are circulating in peripheral blood express both MMP-2 and MMP-9 and transmigrate through Matrigel. In contrast, CD34(+) cells from steady-state bone marrow acquire similar properties after exposure to growth factors and cytokines, which upregulate expression of gelatinases and transmigration of these cells when they enter the bloodstream. Hence, we suggest that growth factors and cytokines induce release of stem/progenitor cells from bone marrow into peripheral blood during mobilization, as well as during steady-state hematopoiesis, by signaling through gelatinase pathways.
Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.
Interactions between osteoclast progenitors and stromal cells derived from mesenchymal stem cells (MSCs) within the bone marrow are important for osteoclast differentiation. In vitro models of osteoclastogenesis are well established in animal species; however, such assays do not necessarily reflect human osteoclastogenesis. We sought to establish a reproducible coculture model of human osteoclastogenesis using highly purified human marrow-derived MSCs (hMSCs) and CD34+ hematopoietic stem cells (HSCs). After 3 weeks, coculture of hMSCs and HSCs resulted in an increase in hematopoietic cell number with formation of multinucleated osteoclast-like cells (Ocls). Coculture of hMSCs with HSCs, transduced with a retroviral vector that expresses enhanced green fluorescent protein, produced enhanced green fluorescent protein+ Ocls, further demonstrating that Ocls arise from HSCs. These Ocls express calcitonin and vitronectin receptors and tartrate-resistant acid phosphatase and possess the ability to resorb bone. Ocl formation in this assay is cell contact dependent and is independent of added exogenous factors. Conditioned medium from the coculture contained high levels of interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), and macrophage-colony stimulating factor. IL-6 and LIF were present at low levels in cultures of hMSCs but undetectable in cultures of HSCs alone. These data suggest that coculture with HSCs induce hMSCs to secrete cytokines involved in Ocl formation. Addition of neutralizing anti-IL-6, IL-11, LIF, or macrophage-colony stimulating factor antibodies to the coculture inhibited Ocl formation. hMSCs seem to support Ocl formation as undifferentiated progenitor cells, because treatment of hMSCs with dexamethasone, ascorbic acid, and beta-glycerophosphate (to induce osteogenic differentiation) actually inhibited osteoclastogenesis in this coculture model. In conclusion, we have developed a simple and reproducible assay using culture-expanded hMSCs and purified HSCs with which to study the mechanisms of human osteoclastogenesis.
To elucidate the mechanisms by which haemopoietic progenitor cells lodge in the bone marrow, we examined the secretion of chemoattractants for haemopoietic progenitor cells by bone marrow and lung endothelial cells. The bone marrow endothelial cells, but not lung endothelial cells, secreted chemoattractants for the haemopoietic progenitor cell line, FDCP-2, and normal haemopoietic progenitor cells. Checkerboard analysis demonstrated that the conditioned medium of the bone marrow endothelial cells had chemotactic activity and random motility-stimulating activity. The bone marrow endothelial cells expressed stromal-cell-derived factor-1 (SDF-1) mRNA and produced SDF-1 protein, whereas the lung endothelial cells did not. Adhesion of FDCP-2 cells to the bone marrow endothelial cells was partially inhibited by anti-SDF-1 antibody. These findings suggest that the chemoattractants for haemopoietic progenitor cells including SDF-1 and random motility-stimulating factor(s) selectively secreted by the bone marrow endothelial cells may contribute to the homing of haemopoietic progenitor cells to bone marrow.
Although chemokines were originally defined as host defense proteins it is now clear that their repertoire of functions extend well beyond this role. For example chemokines such as MGSA have growth regulatory properties while members of the CXC chemokine family can be mediators or inhibitors of angiogenesis and may be important targets for oncology. Recent work shows that the chemokine receptor CXCR4 and its cognate ligand SDF play important roles in the development of the immune, circulatory and central nervous systems. In addition, chemokine receptors play an important role in the pathogenesis of the AIDS virus, HIV-1. Taken together these findings expand the biological importance of chemokines from that of simple immune modulators to a much broader biological role than was at first appreciated and these and other properties of the chemokine receptor family are discussed in detail in this review.
- Mbalaviele G.
Advances in bone biology: The osteoclast
- G D Roodman
- Sato T.