Published online before print May 1, 2003, doi: 10.1161/01.RES.0000074881.56564.46
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© 2003 American Heart Association, Inc.
Cellular Biology |
Receptor Tyrosine Kinase Axl Modulates the Osteogenic Differentiation of Pericytes
From the Wellcome Trust Centre for Cell-Matrix Research (G.C., A.W., R.P.B.-H., A.E.C.), School of Biological Sciences, and the Cardiovascular Research Group (G.C., M.Y.A., V.O., J.O., A.E.C.), School of Medicine, University of Manchester, Manchester, UK; the Department of Pharmacology (B.C.V.), Amgen Inc, Thousand Oaks, Calif; and the Department of Genetics and Developmental Biology (Y.-W.F.), University of Connecticut Health Center, Farmington, Conn.
Correspondence to Dr Ann E Canfield, University of Manchester, Wellcome Trust Centre for Cell-Matrix Research, 2.205, Stopford Building, Oxford Road, Manchester M13 9PT, UK. E-mail ann.canfield{at}man.ac.uk
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Abstract |
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Vascular pericytes undergo osteogenic differentiation in vivo and in vitro and may, therefore, be involved in diseases involving ectopic calcification and osteogenesis. The purpose of this study was to identify factors that inhibit the entry of pericytes into this differentiation pathway. RNA was prepared from pericytes at confluence and after their osteogenic differentiation (mineralized nodules). Subtractive hybridization was conducted on polyA PCR-amplified RNA to isolate genes expressed by confluent pericytes that were downregulated in the mineralized nodules. The subtraction product was used to screen a pericyte cDNA library and one of the positive genes identified was Axl, the receptor tyrosine kinase. Northern and Western blotting confirmed that Axl was expressed by confluent cells and was downregulated in mineralized nodules. Western blot analysis demonstrated that confluent pericytes also secrete the Axl ligand, Gas6. Immunoprecipitation of confluent cell lysates with an anti-phosphotyrosine antibody followed by Western blotting using an anti-Axl antibody, demonstrated that Axl was active in confluent pericytes and that its activity could not be further enhanced by incubating the cells with recombinant Gas6. The addition of recombinant Axl-extracellular domain (ECD) to pericyte cultures inhibited the phosphorylation of Axl by endogenous Gas6 and enhanced the rate of nodule mineralization. These effects were inhibited by coincubation of pericytes with Axl-ECD and recombinant Gas6. Together these results demonstrate that activation of Axl inhibits the osteogenic differentiation of vascular pericytes.
Key Words: pericytes • calcification • osteogenesis • Axl receptor tyrosine kinase • Gas6
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Introduction |
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Pericytes are an integral part of the microvasculature, participating in local and systemic homeostasis.1–3 Cells expressing the pericyte marker (3G5) have also been identified in arteries and arterioles.4 There is now compelling evidence that pericytes can differentiate into other cell types such as chondrocytes and osteoblasts, thereby providing a source of undifferentiated mesenchymal cells in situations of growth and repair, and in diseases involving ectopic calcification and osteogenesis.5–7 Indeed, recent studies have highlighted the potential role of pericytes and pericyte-like cells in the calcification of arteries, arterioles, and capillaries.6,8–10
Morphological studies have demonstrated that pericytes provide a source of osteoblasts and chondrocytes in vivo.5 Furthermore, when loaded in diffusion chambers and implanted into athymic mice, pericytes form a well-organized matrix of bone and cartilage.11 The temporal and spatial pattern of bone formation by pericytes in diffusion chambers is reminiscent of that observed after the implantation of freshly isolated marrow, cultured marrow cells, or bone-derived cells. Interestingly, Bianco and his colleagues12 recently highlighted the similarities between bone marrow stromal cells and pericytes and suggested that these cells may even be the same entity. However, the molecular cues that trigger the osteogenic differentiation of pericytes are still not fully understood.
The processes involved in pericyte differentiation can be investigated using an in vitro model system in which cultured pericytes undergo a well-defined pattern of growth and differentiation, resulting in the formation of large multicellular nodules and deposition of a mineralized matrix.11,13 The osteogenic differentiation of pericytes in vitro occurs in standard growth medium and can be enhanced by culturing the cells in the presence of ß-glycerophosphate13 or by isolating the cells in the presence of dexamethasone (A.E. Canfield, unpublished data, 2002). Using this model system, it has been shown that pericyte differentiation is accompanied by the stage-specific expression of a panel of proteins known to be involved in skeletogenesis and the ectopic calcification of blood vessels (ie, alkaline phosphatase, bone sialoprotein, Cbfa-1, STRO-1, osteocalcin, osteonectin, osteopontin, type I collagen, thrombospondin-1, and matrix Gla protein).5,6,8–10,11,13–15
Little is known about the factors that inhibit the entry of pericytes into this differentiation pathway. Therefore, we have used subtractive hybridization to identify genes that are switched off as pericytes differentiate into bone-forming cells and deposit a mineralized matrix, as these genes may be involved in regulating osteogenesis and the calcification of blood vessels in vivo.16 We now demonstrate that one of these genes encodes Axl (also known as UFO or Ark), the prototype of a distinctive family of receptor tyrosine kinases that is involved in regulating cell survival, adhesion, differentiation, migration, and proliferation in a cell- and tissue-specific manner.17–19 We confirm that this receptor is specifically expressed by confluent pericytes and that its expression is downregulated in postconfluent cultures containing mineralized nodules. We also demonstrate that pericytes secrete the Axl ligand, Gas6, and we show that inhibiting the activity of endogenous Axl in pericytes enhances their differentiation along the osteogenic pathway. These results are discussed in the context of the possible role of Axl-Gas6 interactions in regulating osteogenesis and diseases involving ectopic calcification.
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Materials and Methods |
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Cell Culture
Pericytes were isolated from adult bovine retinal microvessels and cultured in Eagle’s Minimum Essential Medium with 20% fetal calf serum, 50 µg/mL ascorbic acid-2-phosphate, 1 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, nonessential amino acids, and penicillin and streptomycin (standard growth medium). Cells were characterized as pericytes as described.11
Preparation of RNA
RNA was isolated from confluent pericytes, postconfluent cultures containing mineralized nodules and individually picked mineralized nodules using RNAzol.11
Subtractive Hybridization
Polyadenylated (PolyA) complementary DNA (cDNA) with an average length of 400 bases was generated from RNA prepared from confluent pericytes and from individually picked mineralized nodules obtained after 6 to 10 weeks.11 Subtractive hybridization was performed to isolated cDNAs specifically expressed by confluent cells and that were downregulated in the mineralized nodules.15,16 Twenty-five to 50 ng of 32P labeled PolyA cDNAs, obtained from the final round of subtraction (S5), was used to screen a custom-made Lambda ZAP-II pericyte cDNA library (Stratagene Ltd, UK). Positive phagemids were isolated from Lambda ZAP-II vector using ExAssist/SOLR system, and plasmids of interest were purified and sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit and an ABI 377 automated sequencer (Applied Biosystems Inc, UK).
Southern Blotting
Amplified cDNAs (5 to 10 µL) were electrophoresed in 1% agarose gels in Tris-Acetate buffer, denatured in 0.5 mol/L sodium hydroxide/1.5 mol/L sodium chloride, neutralized in 0.5 mol/L Tris-HCl pH 8.0/1.5 mol/L sodium chloride, and transferred to Hybond N membranes (Amersham Pharmacia, UK). Hybridization probes were prepared by random labeling 50 ng of S5 cDNA with [
-32P]-dCTP. Filters were hybridized with 32P-labeled probes at 65°C overnight, washed at high stringency (0.2xSSC/0.1% SDS at 65°C), and exposed to X-ray film at -80°C.
Northern Blotting
RNA (15 µg) prepared from at least two different populations of pericytes was electrophoresed in formaldehyde-containing 1.0% agarose gels and transferred to Hybond N. The filters were prehybridized for 1 hour, and hybridized for 16 hours in hybridization solution containing 5x SSPE (0.9 mol/L NaCl, 0.04 mol/L Sodium Phosphate, 0.005 mol/L EDTA); 5x Denhardt’s solution (0.1% (wt/vol) bovine serum albumin, 0.1% (wt/vol) Ficoll, 0.1% (wt/vol) polyvinylpyrrolidone); 0.5% (wt/vol) SDS and 20 µg/mL denatured salmon sperm DNA at 65°C. The bovine cDNA probe for Axl was isolated from the pericyte cDNA library as part of this study. For 18S ribosomal RNA detection, a 44-mer oligonucleotide sequence was used (5'-cgtggtcaccatggtaggcacggcgactaccatcgaaagttgat-3'). 32P-labeling of cDNA probes was achieved by random priming (Ready-To-Go labeling system, Amersham Pharmacia). After hybridization, filters were washed and exposed to X-Ray films.
Immunoblotting
Conditioned medium (18 mL) was collected from cells (6x25 cm2 flasks) cultured in serum-free medium for 48 hours, dialyzed overnight at 4°C in 50 mmol/L Ammonium chloride, freeze-dried, and resuspended in 600 µL of 3x Laemmelli sample buffer (LSB). A sample of control, serum-free, unconditioned medium was prepared for analysis in the same way. Cell layer/matrix extracts were prepared from confluent pericytes, pooled mineralized nodules, confluent pericytes incubated in serum-free medium for 48 hours, and confluent pericytes incubated in serum-free medium for 48 hours followed by the addition of recombinant human Gas6 (rhGas6, 100 ng/mL) for 15 minutes.14 Samples were applied to SDS-polyacrylamide gels under reducing conditions, and transferred to nitrocellulose (Biorad, UK) or Hybond-P membranes (Amersham Pharmacia). Immunoblot analysis was performed using specific primary antibodies: goat anti-Axl polyclonal (Santa Cruz, USA), rabbit anti-rhGas6 polyclonal, and rabbit anti-phosphotyrosine monoclonal (PY20; Transduction Labs, USA). Secondary antibodies were conjugated to horseradish peroxidase (Dako, Denmark). Results were visualized by enhanced chemiluminescence and autoradiography (ECL, ECL Plus, Amersham Pharmacia). Membranes were re-probed after submersion in stripping buffer (100 mmol/L 2-ßmercaptoethanol, 2% (wt/vol) SDS, 62.5 mmol/L Tris-HCl pH 6.7) for 30 minutes at 50°C and washing several times in 1x phosphate buffered saline/0.1% Tween 20 (PBST). Finally, blots were stained with India ink to confirm equal loading. Experiments were performed using samples prepared from at least 3 different populations of cells.
Immunoprecipitation
Pericytes (4x25 cm2 tissue culture flasks) at 80% confluence were gently rinsed 3 times with PBS containing 1 mmol/L sodium orthovanadate. Cells were extracted with 2 mL of lysis buffer (0.15 mmol/L sodium chloride; 20 mmol/L Tris pH 7.6; 0.5% (wt/vol) sodium deoxycholate; 1% (v/v) Ipegal; 0.1% (wt/vol) sodium dodecyl sulfate; 50 mmol/L sodium fluoride; 1 mmol/L EDTA) containing 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethlysulfonylflouride (PMSF), 1 mmol/L sodium pyrophosphate, and 1x solution of complete mini protease inhibitor cocktail (Roche, UK) for 20 minutes at 4°C. Clarified lysates were precleared with 40 µL of protein G-agarose (Perbio Science Ltd, UK) and 0.25 µg of mouse IgG per ml lysate for 1 hour at 4°C. After a brief centrifugation, 1 mL lysate was incubated for 1 hour at 4°C with 1.4 µg/mL of PY99 anti-phosphotyrosine monoclonal (Santa Cruz, USA) or mouse IgG. Precipitation was performed at 4°C for 1 hour using 40 µL of protein G-agarose per sample. The beads were gently washed twice with 1 mL lysis buffer and resuspended in 80 µL 3X LSB. Triplicate experiments were conducted using different populations of pericytes.
Addition of Axl-Extracellular Domain to Pericyte Cultures
Pericytes were seeded in 24-well plates (3 to 5x104 cells per well) and cultured in standard growth medium. When cells were (1) confluent (days 5 to 7; 2 experiments) or (2) displayed signs of early nodule formation (
day 14; 4 experiments), 2 to 4 µg/mL of Axl-extracellular domain (Axl-ECD) or 4 µg/mL bovine serum albumin (BSA) were added and the incubations continued for 7 or 14 days. Fresh Axl-ECD or BSA was added every time the medium was changed (ie, twice a week). In some experiments, cells were incubated in the presence of both Axl-ECD and rhGas6 (200 ng/mL). Cultures were then fixed and stained with Von Kossa’s reagent11 or extracted in lysis buffer and analyzed by SDS/PAGE and immunoblotting. The total number of nodules and the number of nodules that were mineralized (as evidenced by positive staining with von Kossa’s reagent) were counted.14 Experiments were conducted in triplicate using different preparations of cells.
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Results |
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Axl Gene Expression Is Downregulated During Pericyte Differentiation
RNA was prepared from confluent pericytes (Figure 1A) and from pericytes present in mineralized nodules (Figure 1B). PolyA PCR was performed to produce cDNA representative of the entire polyadenylated mRNA present in the two samples. Subtractive hybridization was then conducted to isolate cDNAs specifically expressed by confluent pericytes and downregulated in mineralized nodules. Amplified cDNAs from confluent pericytes (Figure 1C, track 1), pericytes in mineralized nodules (track 2), and the final subtraction product (S5) (track 3) were electrophoresed on an agarose gel and subjected to Southern hybridization using 32P-labeled cDNA probes. Figure 1C, bottom, confirms that the samples were equally loaded onto the gel. Figure 1C, top, demonstrates that 32P-labeled S5 cDNA hybridized with cDNA prepared from confluent cells and with the final subtraction product but not with cDNA from cells in mineralized nodules. Hybridization of the same filter with cDNA derived from pooled mineralized nodules demonstrated a strong interaction with samples of cDNA also derived from mineralized nodules (not shown). When probed with 32P-labeled cDNA from confluent pericytes, cDNAs present in both confluent and nodular samples bound the probes, but only weak hybridization was observed with subtracted products (not shown). The above results confirm that the final subtraction product, S5, contains a panel of cDNAs specifically expressed by confluent pericytes that are downregulated as cells deposit a mineralized matrix. This product was used to screen a custom-made pericyte cDNA library. Positive plaques were identified, collected, purified, and sequenced. BLAST search comparisons with sequences from the National Center for Biotechnology Information revealed that the sequence of one of these clones was the bovine orthologue of Axl, a receptor tyrosine kinase (RTK).17,18
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Differential Expression of Axl During the Osteogenic Differentiation of Pericytes
To examine the pattern of expression of Axl during pericyte differentiation, RNA was prepared from confluent pericytes and postconfluent cultures containing mineralized nodules and examined initially by Northern blotting. Figure 2A, top, confirms that Axl mRNA is expressed by confluent pericytes and is reduced after confluence. Hybridization of the same filter with 18S ribosomal cDNA confirms that equal amounts of RNA were loaded on the gel (Figure 2A, bottom).
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The expression of Axl by cultured pericytes was further investigated by Western blotting. Protein samples (20 µg) extracted from confluent pericytes and from purified mineralized nodules were separated by SDS/PAGE and examined by immunoblotting using specific antibodies (Figure 2B). Axl was identified as a band migrating with an apparent molecular weight of 140 kDa in the confluent pericyte extract (Figure 2B, lane 1). In contrast, little or no Axl was detected in extracts of pooled mineralized nodules (Figure 2B, lane 2).
Pericytes Express and Secrete Gas6
Gas6 is the predominant ligand for Axl and other RTKs of the Axl family. Gas6 activates Axl through binding interactions at the cell surface, which are then transduced across the plasma membrane resulting in Axl autophosphorylation.19–21 The expression and secretion of Gas6 by pericytes in vitro was examined by immunoblotting. Conditioned medium was collected from confluent pericytes cultured for 48 hours under serum-free conditions. After concentrating the samples by dialysis and freeze-drying, they were separated by SDS/PAGE and analyzed by immunoblotting. Figure 2C demonstrates the presence of Gas6 in the pericyte-conditioned medium (lane 1). Gas6 was not detected in serum-free, unconditioned medium (lane 2).
Demonstration That Axl Is Active in Confluent Pericytes
To determine whether endogenous Axl is autophosphorylated in confluent pericytes, protein extracts from confluent cells and postconfluent cultures were immunoblotted sequentially using an anti-phosphotyrosine antibody, PY20 (Figure 3A) and an anti-Axl antibody (Figure 3B). Several tyrosine-phosphorylated proteins were detected in these extracts (Figure 3A). Interestingly, there was a marked reduction in the level of all phosphorylated proteins present in the postconfluent samples compared with confluent samples (Figure 3A, compare lanes 1 and 2), although approximately equal amounts of protein were loaded (Figure 3C). Of particular interest was a band migrating with an apparent molecular weight of 140 kDa in confluent extracts (Figure 3A, lane 1). The intensity of this band was also markedly reduced after confluence (lane 2). That this band was Axl was confirmed by stripping and re-probing the membrane with an anti-Axl antibody (compare Figure 3A, lanes 1 and 2 with Figure 3B, lanes 1 and 2). Densitometric analysis of these blots revealed that, after correcting for loading, total Axl remained unchanged in these samples, whereas phosphorylated Axl was 7-fold greater in the confluent samples compared with the postconfluent samples.
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Extracts were also immunoprecipitated with the anti-phosphotyrosine antibody, PY99, and then immunoblotted for Axl. Figure 3D confirms that endogenous Axl is autophosphorylated, as the anti-Axl antibody reacted with a tyrosine-phosphorylated protein of Mr 140 to 150 kDa (Figure 3D, lane 1). No bands were observed when the same sample was immunoprecipitated with mouse IgG and immunoblotted for Axl (Figure 3D, lane 2).
We next asked whether phosphorylation of Axl could be further induced by incubation of the cells with rhGas6. Confluent pericytes cultured in serum-free medium were incubated with rhGas6 (100 ng/mL) and Axl phosphorylation was determined by immunoblotting. The results in Figures 3A and 3B demonstrate that control cells and cells incubated with rhGas6 had very similar levels of phosphorylated Axl (compare lanes 3 and 4), suggesting that Axl activity was already at (or near) its maximal level in these cells.
Axl-ECD Promotes Mineralization of Pericyte Nodules
The results presented demonstrate that Axl and its ligand Gas6 are expressed by confluent pericytes and that the expression of Axl and its phosphorylation are markedly downregulated as pericytes form mineralized nodules. To investigate further the role of Axl in matrix mineralization, pericytes were cultured in standard growth medium until the early stages of nodule formation (day 14). At this time, duplicate cultures were incubated in the presence of 2 or 4 µg/mL of Axl-ECD for up to 14 days. Control cultures were incubated in the presence of 4 µg/mL of BSA for the same time period. Cells were then fixed and stained with Von Kossa’s reagent (Figure 4). At this time point, mineralization was barely detected in control cultures (Figure 4A). In contrast, Axl-ECD was found to enhance the size, maturity, and mineralization of pericyte nodules compared with control cultures (compare Figures 4A and 4B). Thus, 12% to 25% nodules were mineralized in the control cultures, compared with 75% to 90% in the cultures incubated with Axl-ECD. However, the total number of nodules was reduced in the presence of Axl-ECD (13 to 21 per field in controls compared with 5 to 10 per field with Axl-ECD). These results were dose-dependent: less nodules were mineralized in the presence of 2 µg/mL of Axl-ECD compared with 4 µg/mL of Axl-ECD (results not shown). Furthermore, the effect of Axl-ECD on mineralization was inhibited by coincubation with rhGas6. It is noteworthy that the level of mineralization observed when 14-day cultures were incubated with Axl-ECD (4 µg/mL) for a further 14 days is comparable to that observed when pericytes are incubated in standard growth medium for 8 to 10 weeks (see Doherty et al11). Similar results were obtained when confluent cultures were incubated with Axl-ECD for 14 days (results not shown), supporting the suggestion that it is the latter stages of pericyte differentiation that are modulated by Axl-ECD and not early nodule formation.
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Modulation of Pericyte Differentiation by Axl-ECD Is Associated With Decreased Phosphorylation of Endogenous Axl
We hypothesized that Axl-ECD was exerting its effect by inhibiting the activation of Axl by endogenous Gas6. To test this hypothesis, confluent cultures were incubated for a further 7 days in the presence of either Axl-ECD (lane 1) or Axl-ECD+rhGas6 (lane 2) and the effect of these reagents on Axl autophosphorylation determined (Figure 5). Accordingly, a marked decrease in phosphorylation of a 140-kDa protein corresponding to full-length Axl was observed in cultures incubated with Axl-ECD compared with cultures incubated with both Axl-ECD and rhGas6 (Figure 5A, compare lane 1 and 2). However, the level of Axl in both samples was similar (Figure 5B). Equal loading was confirmed by staining the gels with India ink (Figure 5C).
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Discussion |
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This study demonstrates that modulation of Axl signaling is a key event in pericyte differentiation and matrix mineralization. First, we have shown that Axl and its ligand Gas6 are expressed by bovine retinal pericytes. Second, levels of Axl mRNA and protein, and its phosphorylation status, are modulated during the osteogenic differentiation of pericytes with high expression detected at confluence when the receptor is phosphorylated. Finally, we demonstrate that the addition of recombinant Axl-ECD to the culture medium reduces Axl phosphorylation and this decrease is accompanied by a concomitant increase in the mineralization of pericyte nodules. These effects were inhibited by the addition of recombinant Gas6. Together, these studies suggest that the ability to modulate the Axl signaling pathway in vivo may lead to a novel therapeutic approach to halt or prevent ectopic calcification.
Axl is the prototype of a distinctive family of tyrosine kinase receptors (RTKs).17,18 The extracellular domain of Axl contains two immunoglobulin-like repeats and two fibronectin type III repeats.17 Intrinsic kinase activity, associated with RTKs, is localized in the cytoplasmic portion of Axl.17 Autophosphorylation occurs through high-affinity interactions with Gas6, a secreted Gla-containing protein that shares significant amino acid homology with Protein S, a negative regulator of the coagulation cascade.20,21 Axl has previously been detected in vitro in several cell types, including bone marrow stromal cells,22 chondrocytes,23 immature mast cells,24 endothelial cells,21,25,26 and vascular smooth muscle cells (VSMCs).27 In vivo, Axl and Gas6 have been localized to articular cartilage,23 rheumatoid arthritic synovium and synovial fluid,28 and in the neointima of arteries after balloon injury.29,30 In these locations, it has been suggested that Axl-Gas6 interactions may modulate cell survival, differentiation, adhesion, migration, and/or proliferation in a cell- and tissue-specific manner.19
We are the first to show that Axl is expressed at high levels in confluent pericytes, and that its expression is modulated during differentiation. We also demonstrate that at confluence, endogenous Axl is phosphorylated independent of treatment of pericytes with exogenous rhGas6. Furthermore, as reported for several other cell types,20,27 the addition of Axl-ECD to cultured pericytes downregulates Axl phosphorylation. Together, these results suggest that endogenous Axl activity and phosphorylation are optimal in confluent pericytes and are promoted by interactions with Gas6, which is secreted by pericytes into the medium.
The signals that lead to the downregulation of Axl expression after confluence remain to be defined. It has been documented that other RTKs undergo regulated proteolysis releasing a soluble extracellular domain, thus modulating receptor function and activity. In vitro experiments demonstrate that Axl is also actively cleaved at the cell surface, in a process that is independent of protein synthesis and cell trafficking pathways.31,32 In addition, soluble Axl-ECD has been detected in the media of tumor cell lines, but it is not known whether regulated Axl proteolysis occurs in vivo.32 The expression of Axl is also modulated by several different growth factors and cytokines, including angiotensin II, thrombin, and basic fibroblast growth factor.30 It is not clear whether growth factors also modulate Axl expression by pericytes.
Axl-Gas6 interactions may modulate pericyte differentiation in a number of ways. First, Axl-Axl and Axl-Gas6 interactions on adjacent cells can mediate cell aggregation and cell adhesion.25,33,34 As the osteogenic differentiation of pericytes is associated with changes in adhesive interactions between cells and the extracellular matrix and between neighboring cells, the involvement of Axl and Gas6 with such changes should be examined further.
Second, Axl-Gas6 interactions are known to protect a range of cell types from apoptotic death.23,26,28,35–39 Apoptosis is associated with the transition from cartilage to bone during endochondral ossification40,41 and in the formation of calcified atherosclerotic plaques in the vasculature.42–44 Interestingly, in the in vitro VSMC model of matrix calcification used by Proudfoot et al,44 these workers demonstrated that cell death occurred before the onset of calcification and that the degree of mineralization was dependent on the level of VSMC apoptosis. Together, these data suggest that the downregulation of Axl-Gas6 interactions in postconfluent pericytes results in the removal of a survival signal in these cells, thereby promoting matrix mineralization. Moreover, treatment with Axl-ECD could accelerate mineralization by depleting the media of endogenous Gas6 and removing the inherent survival signal.
Finally, there is good evidence to suggest that Axl-Gas6 interactions play a role in cell migration associated with normal tissue homeostasis, wound healing events, and disease. Thus, Axl-mediated chemotaxis in response to Gas6 gradients has been described in vitro for VSMCs27,38 and migratory GnRH neurons.39,45 In vivo, concomitant upregulation of both Axl and Gas6 in balloon-injured arteries occurs when VSMCs migrate from the media to the intima.30 Therefore, it is possible that Axl elicits similar responses to Gas6 in pericyte cultures, thereby stimulating the migration of these cells during the early stages of growth and differentiation in vitro.
In conclusion, our results support the suggestion that Axl signaling can impact cell behavior, thereby regulating cell differentiation in both normal and diseased states. We have discussed the possibility that downregulation of Axl-Gas6 interactions during pericyte differentiation could induce pericyte apoptosis, thereby promoting matrix mineralization in vitro. It is also possible that Axl-mediated cell-cell interactions may provide an adhesive function in the absence of Axl kinase activity. Modification of intact Axl or expression of alternative Gas6 splice variants may also act to modify the activity and function of Axl. Further studies are, therefore, required to fully define the mechanism by which Axl-Gas6 interactions modulate the osteogenic differentiation of pericytes.
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Acknowledgments |
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The financial support of the Medical Research Council, UK, and the British Heart Foundation, UK, is gratefully acknowledged.
Received November 7, 2002; revision received April 16, 2003; accepted April 17, 2003.
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References |
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