PDGF C Is A Selective α Platelet-Derived Growth Factor Receptor Agonist That Is Highly Expressed in Platelet α Granules and Vascular Smooth Muscle
Objective— The platelet-derived growth factor (PDGF) family consists of four members, PDGF A, PDGF B, and 2 new members, PDGF C and PDGF D, which signal through the α and β PDGF receptor (PDGFR) tyrosine kinases. This study was performed to determine the receptor specificity and cellular expression profile of PDGF C.
Methods and Results— PDGF C growth factor domain (GFD) was shown to preferentially bind and activate α PDGFR and activate β PDGFR when it is co-expressed with α PDGFR through heterodimer formation. An investigation of PDGF C mRNA and protein expression revealed that during mouse fetal development, PDGF C was expressed in the mesonephric mesenchyme, prefusion skeletal muscle, cardiac myoblasts, and in visceral and vascular smooth muscle, whereas in adult human tissues expression was largely restricted to smooth muscle. Microarray analysis of various cell types showed PDGF C expression in vascular smooth muscle cells, renal mesangial cells, and platelets. PDGF C mRNA expression in platelets was confirmed by real-time polymerase chain reaction, and PDGF C protein was localized in α granules by immuno-gold electron microscopy. Western blot analysis of platelets identified 55-kDa and 80-kDa PDGF C isoforms that were secreted on platelet activation.
Conclusions— Taken together, our results demonstrated for the first time to our knowledge that like PDGF A and B, PDGF C is likely to play a role in platelet biology.
- platelet-derived growth factor
- platelet-derived growth factor receptor
- smooth muscle
- platelet α granule
Platelet-derived growth factor (PDGF) originally identified in platelets and in serum as a mitogen for fibroblasts, smooth muscle cells, and glia cells has been linked to the cause of a number of human diseases, including atherosclerosis, tissue fibrosis, and cancer.1 The PDGF family consists of 4 members, the well-characterized PDGF A and PDGF B, and 2 new members, PDGF C and PDGF D, that signal through the α and β PDGF receptor (PDGFR) tyrosine kinases.1–5 Biosynthesis and processing of the PDGFs result in the formation of full-length disulfide-linked homodimers PDGF AA, BB, CC, and DD and the heterodimer PDGF AB. Although PDGF AA, BB, and AB undergo additional processing, it is not required for their biological activity.1 In contrast, PDGF CC and DD require proteolytic cleavage to remove their N-terminal CUB domain that generates the biologically active growth factor domain (GFD).2–4 The PDGF A and C chains selectively bind α PDGFR, whereas PDGF D preferentially binds β PDGFR and PDGF B displays similar affinity for both receptors.1–5 PDGFR activation requires PDGF-induced receptor dimerization, leading to transphosphorylation on tyrosine. PDGF AA induces only α/α receptor dimers, PDGF AB induces α/α and α/β receptor dimers, and PDGF BB induces all 3 receptor dimer combinations.1 A unique property of PDGF C and PDGF D is that their binding specificity is restricted to one PDGFR subtype, but they can induce transphosphorylation of both receptors when they are co-expressed in the same cell.3–5 We have previously demonstrated that this PDGFR transactivation induced by PDGF DD is accompanied by α/β heterodimer formation. Here, we show PDGF CC has the same ability to induce PDGFR heterodimerization.3
A number of studies by us and others have investigated the expression of PDGF C in normal and diseased tissues and cell lines.6–11 Here we show that during embryonic development, PDGF C is widely expressed in mesenchymal precursors and myoblasts of smooth and skeletal muscle. We found that in adult human tissues, PDGF C mRNA and protein expression was largely restricted to visceral and vascular smooth muscle. Interestingly, among the cell types evaluated, a high level of PDGF C expression was observed in platelets, where it was stored in α granules and released on platelet activation. We believe that this identification of PDGF C in platelets will be critical in understanding the biology of this new member of the PDGF family.
Expression and Purification of Human PDGF C
The full-length PDGF C cDNA was polymerase chain reaction (PCR) amplified from human heart total RNA (Ambion) and used to construct mammalian expression vectors. PDGF C full length (FL) (codons 23 to 345) and PDGF C-GFD (codons 230 to 345) were subcloned into pSecTag2A (Invitrogen) that provided a signal sequence at the N-terminus as well as Myc and His tags at the C-terminus. These vectors were then transfected into 293T cells in the presence of 10% fetal bovine serum (FBS) using standard procedures. After 24 hours, the cells were washed and secreted proteins were collected into serum free medium over 48 hours. Both PDGF C-FL and PDGF C-GFD were purified from conditioned medium using Ni-NTA-agarose according to the manufacturers’ instructions (Qiagen).
Anti-PDGF C monoclonal antibodies were generated using standard techniques.12 BALB/c mice were immunized with 10 μg of recombinant purified PDGF C-FL or PDGF C-GFD protein, and additional boosts were administered every 2 weeks until a high serum titer was achieved. Spleens of mice were isolated, and their splenocytes were fused with P3X cells. Positive hybridomas were identified and cloned. Ascites fluids were prepared, and purified IgG was obtained using protein G chromatography. In the present study, monoclonal antibodies 51C352–7 and 53CBD402–2 were raised against PDGF C-FL and PDGF C-GFD, respectively. Anti-PDGF C polyclonal antibodies were prepared by immunizing New Zealand White rabbits with an N-terminal peptide corresponding to residues 29 to 48 (N-pepC) or an internal peptide corresponding to residues 230 to 250 (C-pepC), as previously described.2 N-pepC and C-pepC total IgGs were obtained by standard protein A purification followed by affinity chromatography using peptide-conjugated Sephadex columns.
Primary human umbilical vein endothelial cells, aortic smooth muscle cells, skin fibroblasts, lung fibroblasts, and renal mesangial cells were purchased from Cambrex. The hepatocellular carcinoma cell line HepG2 and osteosarcoma cell line MG63 were purchased from ATCC. The 32DαR and CHO-βR cell lines have been described previously.13–15
PDGFR Phosphorylation Assays
Cells were grown to confluence, starved, and then stimulated with 200 ng/mL PDGF AA, BB (R&D systems), PDGF CC-FL, and PDGF CC-GFD as described.3,15 Whole-cell lysates were prepared, incubated with PDGFR antibodies (3979 for αPDGFR and 1C7D5 for βPDGFR),15 and precipitated with protein A/G agarose. For Western blot analysis, sample buffer (reducing or non-reducing) was added, and proteins were separated using 4% to 20% SDS-PAGE and detected with the indicated antibodies as previously described.3,15
PDGFR Heterodimerization Assay
The PDGFR heterodimerization assay was performed using MG63 cells as previously described.3
PDGF C In Situ Hybridization
Tissue samples were fixed with 4% formaldehyde and embedded in paraffin wax. Digoxigenin-labeled antisense and sense riboprobes were synthesized from the DNA templates (codon 156 to 320) using reagents supplied by Roche Molecular Biochemicals. The in situ hybridization experiments were performed as previously described.16 The signal was amplified using TSA-Plus kit (Perkin-Elmer Life Sciences) and detected with BCIP/NBT substrate (Vector Laboratories).
PDGF C Immunohistochemistry Analysis
Frozen sections were incubated with a 1:25 dilution of anti-PDGF C antibody C-pepC and detected by anti-rabbit EnVision-PO kit (Dako). Peroxidase activity was detected by DAB substrate followed by hematoxylin counterstain.
Slides were observed with an Olympus BX50 microscope (Olympus US) using DIC illumination. The microscope was equipped with a NIKON DXM1200 digital camera (Technical Instruments). Digitized images (1280×1024 pixel resolution) were acquired using ACT-1 software (Nikon USA).
Real-Time Quantitative PCR Expression Analysis
RNA from human platelets was prepared as previously described.17 Probes and primers for each of the PDGF and PDGFR family members and the control human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used have been described.6 Real-time quantitative PCR was performed using an ABI Prism 7700 sequence-detection system (PE Applied Biosystems) using Taqman reagents (PE Applied Biosystems). Equal quantities of RNA were used as template in real-time reactions with gene-specific reagents to obtain threshold cycle (Ct) values with a total of 40 cycles.
Total RNAs were extracted from human primary cells and cell lines, and cRNA was synthesized and labeled with using a low RNA input fluorescent linear amplification kit (Agilent Technologies). A total of 2 sets of experiments were performed. In one set, individually transcribed RNA labeled with Cy5 was competitively hybridized to a human oligonucleotide microarray (Agilent Technologies) against a reference sample (consisting of a pool of all the RNAs labeled with Cy3). The second set was repeated under the same hybridization conditions with reversed fluorescence labeling. The combined data were analyzed using strictly defined statistical methods and the RESOLVER package of software tools (Rosetta Biosoftware).
Preparation of Washed Platelet Lysates
Human blood was drawn from healthy donors, and resting platelets were isolated as previously described.18 The platelet pellets were dissolved in HEPES-Tyrode buffer, lysed with 2× triton buffer (2% Triton X-100, 100 mmol/L Tris-HCl, pH 8.0, 5 mmol/L EDTA, 250 mmol/L NaCl, 20% glycerol, 8 mmol/L Na3VO4, and a cocktail of protease inhibitors; Roche), and analyzed using standard immunoprecipitation and Western blot techniques as described.
Immuno-Gold Staining of PDGF C in Platelets
Platelets were obtained and fixed in 1.25% glutaraldehyde (Fluka AG) diluted in 0.1 mol/L phosphate buffer (pH7.2) for 30 minutes at 25°C. Samples were washed and resuspended in phosphate buffer. Immuno-gold labeling was performed as described previously using C-pepC antibody.19 Observation was made with a JEOL JEM-1010 transmission electron microscope at 80 KV (JEOL).19
Characterization of Human PDGF C
Recombinant PDGF C-FL and PDGF C-GFD that lacks the N-terminal CUB domain were expressed in 293T cells as secreted proteins and purified from conditioned media. The subunit structure and purity of each of the recombinant proteins was assessed by SDS-PAGE followed by Coomassie blue staining. Under non-reducing conditions, PDGF C-FL and PDGF C-GFD migrated at 90 kDa and 32 kDa, respectively. SDS-PAGE analysis of the reduced proteins confirmed they were disulfide-linked dimers whose monomeric forms migrated at 55 kDa for PDGF C-FL and 23 kDa for PDGF C-GFD (Figure IA, available online at http://atvb.ahajournals.org)
PDGFR Activation by PDGF CC
To test the activity of recombinant PDGF CC, we analyzed the induction of PDGFR phosphorylation in cells expressing either one or both receptor isoforms. Cells were stimulated with 200 ng/mL of PDGF, PDGFR was immunoprecipitated from cell lysates, and phosphorylation was analyzed by Western blotting with anti-phosphotyrosine antibodies. In 32D-αR cells that express only αPDGFR, PDGF CC-GFD induced receptor phosphorylation to a level comparable to that seen with PDGF AA and PDGF BB, whereas PDGF CC-FL was found to be inactive (Figure IB, available online at http://atvb.ahajournals.org). In CHO-βR cells expressing only the βPDGFR isoform, both PDGF CC-GDF and FL were inactive as compared with the PDGF BB control (Figure IC, available online at http://atvb.ahajournals.org). We next tested PDGF-induced PDGFR phosphorylation in MG63 cells expressing both receptor isoforms. As expected, αPDGFR phosphorylation was induced by PDGF AA, BB, and CC-GFD. Interestingly, PDGF CC-GFD, like PDGF BB, was able to activate βPDGFR in these cells (Figure ID and IE, available online at http://atvb.ahajournals.org). These results demonstrate that PDGF CC-GDF was able to directly activate αPDGFR, whereas βPDGFR activation only occurred when both receptor subtypes were co-expressed, which is consistent with previous reports.4,20
PDGFR Heterodimer Formation Induced by PDGF CC
The fact that PDGF CC induces βPDGFR phosphorylation only in cells that co-express αPDGFR implicates that a α/β receptor heterodimerization occurs, but a direct association between these receptor subtypes has not been demonstrated. Therefore, we used a 2-site enzyme-linked immunosorbent assay to measure α/β PDGFR heterodimerization using specific antibodies by first capturing βPDGFR from cell lysates on plastic, followed by detection of αPDGFR in the heterodimer complex. As shown in Figure 1, neither PDGF AA nor PDGF CC-FL induced receptor heterodimer formation at concentration as high as 900 ng/mL. In contrast, PDGF CC-GFD, like PDGF BB, was able to induce α/β receptor heterodimer formation. These results indicated that PDGF CC-GFD transactivates βPDGFR through complex formation with αPDGFR.
Expression of PDGF C mRNA and Protein in Embryonic and Adult Tissues
To localize PDGF C expression, we next performed in situ hybridization experiments on embryonic and adult tissues. On mouse E14 and E18 embryos, PDGF C mRNA was detected in vascular smooth muscle cells of arteries and veins and in visceral smooth muscle in the small intestine (Figure 2A, 2B, and 2D). Moreover, PDGF C was expressed in prefusion myoblasts of the developing skeletal muscles at E14, but it was not detectable in fully formed skeletal muscle fibers at a later stage of development on E18 (Figure 2A and 2B). PDGF C was also expressed in the mesonephric mesenchyme in the developing kidney (Figure 2C). In adult human tissues, PDGF C mRNA was detected in vascular smooth muscle cells of arteries and veins in every organ and tissue studied, including kidney, breast, and colon (Figure 3A through 3C). Moreover, PDGF C was found to be expressed in visceral smooth muscle in the colon (Figure 3C). These observations were confirmed by IHC analysis of the same tissues using C-pepC polyclonal antibody. As shown in Figure 3D through 3F, PDGF C protein was detected in vascular smooth muscle cells in human kidney, breast, and colon and in visceral smooth muscle of the gastrointestinal tract. The signal was specific because the staining was abolished by preincubation of C-pepC with the peptide against which it was raised (data not shown).
PDGF C mRNA and Protein Is Highly Expressed in Platelets
PDGF C expression in normal adult tissues was found to be quite highly restricted to visceral and vascular smooth muscle (Figure 3). Similarly, PDGF A and/or B are expressed in fibroblasts and vascular smooth muscle cells, especially after tissue injury.1 However, a fundamental source of PDGF A and B released at the site of tissue damage is the platelet where they were originally discovered.1 Based on these observations, we chose to look at the expression of PDGF C in the relevant cell types, including platelets. To determine the relative level of PDGF C mRNA expression, microarray experiments were performed and the PDGF C hybridization signal observed in individual cell types was compared with a reference standard that was generated by pooling RNA from each of the cell sources. Consistent with the expression pattern observed in tissues, PDGF C was highly expressed in primary human aortic smooth muscle cells, lung fibroblasts, and renal mesangial cells, whereas no expression was detected in human umbilical vein endothelial cells, white blood cells, or liver cells (Figure IIA, available online at http://atvb.ahajournals.org). Interesting, a high level of PDGF C mRNA was also detected in platelets.
To confirm platelet expression of PDGF C, Taqman assays were performed using platelet RNA isolated from normal healthy donors. As expected, platelets expressed PDGF A and B with an average Ct value of 23.7 and 28.1, respectively, as compared with a Ct of >40 for the minus RT controls (data not shown). Consistent with our microarray data, PDGF C was readily detected (Ct=27.9), whereas PDGF D mRNA was undetectable in platelets (Figure IIB, available online at http://atvb.ahajournals.org). The lack of detection of α PDGFR and PDGF D mRNA was not caused by the design of the probe or primers, because we have used them in other studies.6 GAPDH was used as a positive control.
Because PDGF A and B are localized to the α granules in platelets, we hypothesized that PDGF C protein may also be stored in the α granules and released on platelet activation. To investigate this hypothesis, we generated polyclonal antibodies N-pepC and C-pepC directed against PDGF C peptides and the monoclonal antibody 51C352–7 raised against recombinant PDGF C-FL, as described in the Methods. The specificity of these antibodies was first tested by Western blot analysis using recombinant PDGF C-FL and PDGF-GFD expressed in 293T cells. As a control, PDGF D-FL expressed under similar conditions was included in the analysis. The recombinant PDGF C and D proteins used in these experiments each contain a myc tag at their carboxyl terminus. As a control for the amount of protein loaded, Western blot analysis with myc antibody revealed a similar level of detection of each PDGF protein (Figure III, available online at http://atvb.ahajournals.org). As expected, N-pepC detected only PDGF C-FL protein, whereas C-pepC and 51C352–7 detected both PDGF C-FL and PDGF C-GFD. The PDGF C antibodies did not cross-react with PDGF D.
To directly assess the cellular localization of PDGF C in platelets, we used C-pepC and gold-labeled secondary antibody to immunostain resting platelets, followed by electron microscopy analysis. As shown in Figure 4, gold particles were observed in the α granules of platelets, implicating this organelle as a storage site for PDGF C. We also observed gold particles in the surface-connected canalicular system, suggesting some of PDGF C might be bound to or localized close to the membrane.
To further characterize PDGF C protein in human platelets, cell lysates and supernatants collected before and after activation with 2.5 μmol/L TRAP were subjected to Western blot analysis with PDGF C antibodies. As expected for a α granule protein, PDGF C was detected at a much higher level in the supernatant after platelet activation (Figure 5A). Polypeptides of 55-kDa (p55) and 80-kDa (p80) in size, released by platelets, were detected by N-pepC, C-pepC, and 51C352–7, establishing their identity as PDGF C (Figure 5A).
Serum Cleavage of Platelet PDGF C
It has been reported that recombinant PDGF C-FL can be cleaved by FBS to release the biologically active PDGF C-GFD.4 To further characterize PDGF C in platelets, secreted proteins were immunoprecipitated using C-pepC before incubation with 1%, 5%, and 10% FBS at room temperature overnight. The mixture was separated by SDS-PAGE followed by Western blotting with antibody 53CBD402–2 generated against purified recombinant PDGF C-GFD. As shown in Figure 5C, incubation with 5% and 10% FBS resulted in the generation of PDGF C-GFD that was detected by 53CBD402–2 as a 23-kDa band. As a control, FBS treatment of recombinant PDGF C-FL was shown to generate PDGF-GFD that was the same size as that detected from platelets (Figure 5B and 5C). These results demonstrate that p55 contains a proteolytic cleavage site similar to that in recombinant PDGF C-FL, consistent with the 2 proteins being highly related.
PDGF C is a new member of the PDGF family that has been shown to be unique in its ability to bind and/or activate α and β PDGFR, but there is little known about its role in normal physiological or pathophysiological processes. Consistent with previous reports,2,4 we demonstrated that PDGF C-FL is biologically inactive, whereas PDGF C-GFD preferentially activates α PDGFR. However, if β PDGFR is co-expressed with α PDGFR, then PDGF-GFD will induce heterodimerization and phosphorylation of both PDGFR isoforms. In an attempt to gain further insight into the in vivo role of PDGF C, we investigated its expression pattern in embryonic and adult tissues using ISH and IHC. Previous studies have demonstrated that PDGF C expression is widespread during early stages of mouse embryogenesis.11,21 We found in the later stages of development at E18, the most prominent expression of PDGF C mRNA was in vascular smooth muscle of arteries and veins, in visceral smooth muscle in the gut, and in the mesonephric mesenchyme of the developing kidney. We also found PDGF C expression in the prefusion myoblasts of developing skeletal muscle at E14, but not in fully formed skeletal muscle at E18. In all adult tissues examined, including kidney, breast, and colon, staining was largely restricted to vascular smooth muscle of arteries and veins and visceral smooth muscle in the colon. Immunohistochemistry analysis of the same tissues revealed that the pattern of PDGF C protein expression was indistinguishable from that of mRNA.
PDGF A and B were originally identified in platelets and were later shown to be more widely expressed in other cell types, including vascular smooth muscle and endothelial cells.1 Therefore, we chose to investigate the expression of PDGF C in platelets as compared with other cell types. As expected from the expression pattern observed in tissue sections, PDGF C expression was found in vascular smooth muscle and renal mesangial and fibroblast cell lines but not in endothelial cells, white blood cells, or hepatocytes consistent with previous reports.4,10 Interestingly, the level of PDGF C mRNA detected in platelets was equivalent or higher than that observed in any of the other cell types. To confirm and extend these observations, real-time PCR performed on platelet RNA revealed a high level of expression for PDGF C, PDGF A, and PDGF B, but no expression was observed for PDGF D. Like PDGF A and B, PDGF C protein was shown by immuno-gold electron microscopy to be stored in platelet α granules. Western blot analysis demonstrated that 2 PDGF C isoforms, p55 and p80, were stored in platelets and released on activation by the thrombin receptor agonist TRAP. Both isoforms were detected by antibodies directed against epitopes present at the amino terminus or in the GFD, consistent with their being noncleaved full-length PDFG C containing both CUB and GFD domains. Whereas p55 is the same size as recombinant PDGF C-FL, p80 is significantly larger, indicating either that there are additional posttranslational modifications of p55 or that alternative exon usage results in the synthesis of a novel isoform. Both possibilities are supported by the fact that PDGF C-FL has 3 putative N-linked glycosylation sites at positions 25, 55, and 254 and that mRNAs of 2.9 and 3.8 kb have been observed in many different tissues.2,4,10
Unlike PDGF A and B, PDGF C is synthesized and released from cells in an inactive form that requires proteolytic processing to remove the N-terminus to generate the biologically active PDGF C-GFD.2,4 Because of the size of platelet PDGF C and the presence of an N-terminal epitope in common with PDGF C-FL, it is also likely to require proteolytic processing for activation. The physiologically relevant protease responsible for PDGF C activation has not been identified, but it could be present in platelets because proteases in serum are capable of performing this function in vitro.2,4 However, we found this to be unlikely because extended incubation of activated platelets did not result in further processing of p55 or p80 (data not shown). However, incubating partially purified p55 or recombinant PDGF C-FL with serum resulted in the generation of the p23 PDGF C-GDF (Figure 5C). This observation is in agreement with data reported by Li et al,2 in which a fibrinolytic protease such as plasmin, which is present in serum but not in plasma, is capable of cleaving PDGF C. Thus, the latent form of PDGF C could be activated by locally released fibrinolytic and coagulation proteases at the site of ongoing thrombosis.
The identification of PDGF C as a platelet α granule secretory protein implies that it has overlapping functions with the other platelet PDGF isoforms, especially PDGF AB, which has the same PDGFR specificity. It is important to note that PDGF C is likely to be inactive on release from platelets; therefore, its action may be delayed or more distal to that of the other isoforms. It is thought that platelet PDGF A and B participate in the wound healing process after injury by facilitating tissue remodeling and promoting angiogenesis through the recruiting pericytes.1 In this regard, PDGF CC is a potent mitogen for smooth muscle cells, pericytes, and fibroblasts.2,4 More importantly, topical application of PDGF CC potently induces angiogenesis in the chick chorioallantoic membrane and mouse cornea, and it enhances diabetic wound healing in a mouse model.4,20 This study demonstrates that PDGF C is likely to play a role in platelet biology, but a greater understanding of the biological role of PDGF C relative to the other PDGF family members will require further studies, including a PDGF C gene knockout.
We thank Dr Pamela Conley and Diana Vincent for providing platelet RNA and Dr Uma Sinha for discussions.
- Received October 30, 2003.
- Accepted January 26, 2004.
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