Expression of platelet-derived growth factor (PDGF)-A, PDGF-B and the PDGF-alpha receptor, but not the PDGF-beta receptor, in human malignant melanoma in vivo

There has been considerable interest in the potential role of growth factors in the initiation and development of cutaneous malignant melanoma (CMM). Platelet-derived growth factor (PDGF) has been shown to be secreted by melanoma cell lines and by metastatic melanoma in vivo. PDGF also has been reported to stimulate the development of tumour stroma and new blood vessels. We studied the expression of PDGF and its receptors by both immunohistochemistry (IHC) and in situ hybridization (ISH) in primary and metastatic melanoma and in normal skin specimens. Cryostat sections were incubated with 35S-labelled riboprobes and antibodies for PDGF-AA, PDGF-alpha receptor, PDGF-BB and PDGF-beta receptor. Both primary and metastatic melanoma exhibited significant expression of PDGF-AA, PDGF-BB and PDGF-alpha receptor by both IHC and ISH, compared with only background expression in normal skin. We did not observe expression of PDGF-beta receptor in melanoma. Our results suggest that PDGF may function as an autocrine growth factor, as well as an angiogenesis factor, in CMM tumour development. This expression of the PDGF-alpha receptor rather than the beta receptor may be unique among solid tumours.     

Lens-specific expression of PDGF-A alters lens growth and development

The vertebrate lens provides an in vivo model to study the molecular mechanisms by which growth factors influence development decisions. In this study, we have investigated the expression patterns of platelet-derived growth factor (PDGF) and PDGF receptors during murine eye development by in situ hybridization. Postnatally, PDGF-A is highly expressed in the iris and ciliary body, the ocular tissues closest to the germinative zone of the lens, a region where most proliferation of lens epithelial cells occurs. PDGF-A is also present in the corneal endothelium anterior to the lens epithelium in embryonic and early postnatal eyes. PDGF-B is expressed in the iris and ciliary body as well as in the vascular cells which surround the lens during early eye development. In the lens, expression of PDGF-alpha receptor (PDGF-alphaR), a receptor that can bind both PDGF-A and PDGF-B, is restricted to the lens epithelium throughout life. The expression of PDGF-alphaR in the lens epithelial cells and PDGF (A- and B-chains) in the ocular tissues adjacent to the lens suggests that PDGF signaling may play a key role in regulating lens development. To further examine how PDGF affects lens development in vivo, we generated transgenic mice that express human PDGF-A in the lens under the control of the alphaA-crystallin promoter. The transgenic mice exhibit lenticular defects that result in cataracts. The percentage of surface epithelial cells in S-phase is increased in transgenic lenses compared to their nontransgenic littermates. Higher than normal levels of cyclin A and cyclin D2 expression were also detected in transgenic lens epithelium. These results together suggest that PDGF-A can induce a proliferative response in lens epithelial cells. The lens epithelial cells in the transgenic mice also exhibit characteristics of differentiating fiber cells. For example, the transgenic lens epithelial cells are slightly elongated, contain larger and less condensed nuclei, and express fiber-cell-specific beta-crystallins. Our results suggest that PDGF-A normally acts as a proliferative factor for the lens epithelial cells in vivo. Elevated levels of PDGF-A enhance proliferation, but also appear to induce some aspects of the fiber cell differentiation pathway.     

Expression of platelet-derived growth factor transcripts in medulloblastomas and ependymomas

We used Northern blot analysis to measure the expression of mRNA for platelet-derived growth factor subunit A (PDGF-A), PDGF-B and the PDGF-alpha receptor (PDGFR-alpha) and PDGF-beta receptor (PDGFR-beta) in ependymomas and medulloblastomas. We analyzed tissue from 5 patients for each tumor type, looking specifically for components of an autocrine or paracrine system in these tumors. PDGF-A was expressed in all tumors, PDGFR-alpha, which binds all 3 PDGF isoforms, was only found in ependymomas. Thus only ependymomas appeared to have a potential for using PDGFR-alpha autocrine loops. PDGF-B was expressed only in ependymomas, although the PDGFR-beta was expressed in both medulloblastomas and ependymomas. Again, therefore, only ependymomas appear to have a potential autocrine loop with PDGFR-beta. These data suggest that ependymomas have the biochemical prerequisites for autocrine and/or paracrine loops using PDGFR-alpha or PDGFR-beta systems. In this they resemble other glial tumors such as anaplastic astrocytomas and glioblastomas. Medulloblastomas do not appear to have the ligand and/or receptor for either the PDGFR-alpha or PDGFR-beta autocrine loop.     

Increased expression of platelet-derived growth factor beta receptor gene in hypoxic rat lungs

The changes of platelet-derived growth factor (PDGF) beta receptor gene expression in hypoxic rats lungs was examined. Northern blots analysis revealed that normal lungs expression PDGF-beta receptor mRNA, with the longer of hypoxia the level of the mRNA increased rapidly. It reached a maximum at day 4, and was 1.34 fold as compared with the control (P < 0.05). Immunohistochemistry investigation showed that PDGF-beta receptor mainly distributed on smooth muscle cells and endothelial cells of middle and small arterial trees in rat lung. With hypoxia, the distribution of PDGF-beta receptors did not change, but it was more intense and reached a maximum at day 7, and was 2.40 fold as compared with the control (P < 0.05). The results suggested that increased expression of PDGF receptor gene may play a role in hypoxic pulmonary vascular remodeling.     

Cloning and expression of Xenopus HGF-like protein (HLP) and Ron/HLP receptor implicate their involvement in early neural development

Endoglin was first identified on a cell line derived from pre-B acute lymphoblastic leukemia. This 180-kDa homodimeric glycoprotein was then shown to be primarily expressed on endothelial cells and to bind the beta 1 and beta 3 isoforms of TGF-beta with high affinity. We now demonstrate that pre-B leukemic cells express a functional TGF-beta 1 receptor complex. The levels of mRNA for these receptors and for TGF-beta 1 were quantitated by PCR. HOON, G2, and NALM-6 cell lines express similar levels of mRNA for TGF-beta 1 and for TGF-beta receptor I (R-I) and receptor II (R-II). HOON cells express ten times more endoglin than G2 and NALM-6 cells, whereas all three cell lines have low levels of betaglycan relative to other cell types. The receptors were identified by affinity labeling with 125I-labeled TGF-beta 1, chemical cross-linking, and specific immunoprecipitation. Endoglin, R-II, and R-I were co-precipitated by Abs to either endoglin or R-II, indicating that these proteins are forming a receptor complex on leukemic cells; no betaglycan could be immunoprecipitated. The receptor complex is functional, as demonstrated by inhibition of proliferation of HOON cells (80%) and NALM-6 cells (60%) with 25 pM TGF-beta 1. Furthermore, the motility of HOON and NALM-6 cells on immobilized fibronectin, which appears to be alpha 4 beta 1-integrin mediated, was stimulated two- to threefold by TGF-beta 1. These results suggest that active TGF-beta 1 produced in the bone marrow microenvironment might stimulate the motility of normal pre-B cells and the peripheral dissemination of leukemic pre-B cells.     

PDGF-alpha receptor subunit expression down-regulated by IL-1beta in human periodontal ligament cells

The responses of cells to the distinct PDGF isoforms have been correlated directly to the relative numbers of specific PDGF receptor subunits on the cell surface. The modulation of PDGF-alpha receptor subunits, the major subunit expressed in human periodontal ligament (PDL) cells, by cytokines present in the periodontal wound site, such as interleukin-1 (IL-1), may be an important factor influencing regenerative outcomes. The purpose of the present study was to examine the effects of IL-1 beta on PDGF-alpha receptor subunit expression in human PDL cells. Primary cultures of human PDL cells were treated with IL-1 beta over a range of concentrations. We assessed PDGF-alpha receptor subunits by examining the mitogenic responses of cells to PDGF-AA, specific binding of 125I-labeled PDGF-AA, immunofluorescent analysis of PDGF-alpha receptor subunits, and PDGF-alpha receptor subunit mRNA levels using Northern blot analysis. The results demonstrate a significant concentration-dependent decrease in 3H-thymidine incorporation in response to PDGF-AA following IL-1 beta treatment (p < 0.001). This decreased response correlated directly with IL-1-induced decreases in 125I-labeled PDGF-AA binding (p < 0.01), the numbers of immunolabeled PDGF-alpha receptor subunits, and in PDGF-alpha receptor subunit mRNA levels. However, when combined with TGF-beta, IL-1 beta did not show additional down-regulation in proliferative response to PDGF-AA or PDGF-alpha receptor subunits beyond that achieved with these factors individually. These experiments identify IL-1 beta, along with TGF-beta, as significant inhibitors of PDGF stimulation in human PDL cells, acting through the down-regulation of PDGF-alpha receptor subunit expression.     

Expression of platelet-derived growth factor ligands and receptors by rat aortic endothelium in vivo

In situ hybridization and immunostaining were used to study the time course of expression for platelet-derived growth factor (PDGF) ligands and receptors in endothelium of the rat aorta after injury. The PDGF-A and -B chains were expressed in endothelial cells at the wound edge within 4 h after injury, but no expression was detectable in uninjured endothelium. PDGF alpha-receptor was expressed in a pattern similar to the PDGF-A chain, while expression of PDGF beta-receptor was not detected at any time. Expression of the PDGF-B chain remained elevated in endothelial cells at the leading edge even at later measurements when these cells had stopped replicating. Smooth muscle cells (SMCs), which are absent from the intima of the normal aorta and are known to express PDGF beta-receptors, were predominantly found to migrate into the intima near the endothelial leading edge where PDGF-B was expressed. These data suggest a paracrine role for endothelial PDGF in SMC migration.     

Platelet-derived growth factor (PDGF)-signaling mediates radiation-induced apoptosis in human prostate cancer cells with loss of p53 function

Platelet-derived growth factor (PDGF) signals a diversity of cellular responses in vitro, including cell proliferation, survival, transformation, and chemotaxis. PDGF functions as a "competence factor" to induce a set of early response genes expressed in G1 including p21WAF1/CIP1, a functional mediator of the tumor suppressor gene p53 in G1/S checkpoint. For PDGF-stimulated cells to progress beyond G1 and transit the cell cycle completely, progression factors in serum such as insulin and IGF-1 are required. We have recently shown a novel role of PDGF in inducing apoptosis in growth-arrested murine fibroblasts. The PDGF-induced apoptosis is rescued by insulin, suggesting that G1/S checkpoint is a critical determinant for PDGF-induced apoptosis. Because recent studies suggest that radiation-induced signal transduction pathways interact with growth factor-mediated signaling pathways, we have investigated whether activation of the PDGF-signaling facilitates the radiation-induced apoptosis in the absence of functional p53. For this study we have used the 125-IL cell line, a mutant p53-containing, highly metastatic, and hormone-unresponsive human prostate carcinoma cell line. PDGF signaling is constitutively activated by transfection with a p28v-sis expression vector, which was previously shown to activate PDGF alpha- and beta- receptors. Although the basal level of p21WAF1/CIP1 expression and radiation-induced apoptosis were not detectable in control 125-IL cells as would be predicted in mutant p53-containing cells, activation of PDGF-signaling induced expression of p21WAF1/CIP1 and radiation-induced apoptosis. Our study suggests that the level of "competence" growth factors including PDGF may be one of the critical determinants for radiation-induced apoptosis, especially in cells with loss of p53 function at the site of radiotherapy in vivo.     

Dopamine D1/D2 antagonist combinations as antagonists of the discriminative stimulus effects of cocaine

Platelet-derived growth factor (PDGF) exists as a dimer composed of two homologous but distinct peptides termed PDGF-A and -B chains, and may exist as AA, AB, and BB isoforms. The PDGF-B chain has been implicated as a mediator of renal vascular rejection by virtue of up-regulated expression of its receptor, PDGF beta-receptor, in affected arteries. A role for PDGF-A chain in mediating intimal proliferation has been suggested in human atherosclerosis (Rekhter MD, Gordon D: Does platelet-derived growth factor-A chain stimulate proliferation of arterial mesenchymal cells in human atherosclerotic plaques? Circ Res 1994, 75:410), but no studies of this molecule in human renal allograft injury have been reported to date. We used two polyclonal antisera to detect expression of PDGF-A chain and one monoclonal antibody to detect PDGF-B chain by immunohistochemistry in fixed, paraffin-embedded tissue from 1) normal adult kidneys, 2) a series of renal transplant biopsies chosen to emphasize features of vascular rejection, and 3) allograft nephrectomies. Immunohistochemistry was correlated with in situ hybridization on adjacent, formalin fixed tissue sections from nephrectomies utilizing riboprobes made from PDGF-A and -B chain cDNA. PDGF-A chain is widely expressed by medial smooth muscle cells of normal and rejecting renal arterial vessels of all sizes by immunohistochemistry and in situ hybridization. PDGF-A chain is also expressed by a population of smooth muscle cells (shown by double immunolabeling with an antibody to alpha-smooth muscle actin) comprising the intima in chronic vascular rejection. In arteries demonstrating acute rejection, up-regulated expression of PDGF-A chain by endothelial cells was detected by both immunohistochemistry and in situ hybridization. In contrast, PDGF-B chain was identified principally in infiltrating monocytes within the rejecting arteries, similar to its localization in infiltrating monocytes in human atherosclerosis. Although less prominent than the case for PDGF-A chain, PDGF-B chain also was present in medial and intimal smooth muscle cells in both rejecting and nonrejecting renal arteries. PDGF-A and -B chains have now been localized at both the mRNA and protein levels to the intimal proliferative lesions of vascular rejection. These peptides, which are known stimuli for smooth muscle cell migration and proliferation in experimental vascular injury, may have similar stimulatory effects on smooth muscle cells in an autocrine and/or paracrine manner to promote further intimal expansion and lesion progression in this form of human vasculopathy.     

Phenotypic analysis of retinal leukocyte infiltration during combined cyclosporin A and nasal antigen administration of retinal antigens: delay and inhibition of macrophage and granulocyte infiltration

The development of interstitial pulmonary fibrosis is associated with a variety of inflammatory mediators, including peptide growth factors and cytokines. In the work presented here, we have asked whether or not platelet-derived growth factor (PDGF)-A and -B genes and proteins are expressed in anatomic and temporal patterns consistent with this factor playing a role in the disease process. Using an established rat model of asbestos-induced fibroproliferative lung disease, we demonstrate elevated levels of PDGF-A and -B mRNAs in total lung RNA immediately after a single 5-h exposure to approximately 1,000 fibers/ml of chrysotile asbestos. In situ hybridization revealed the PDGF-A and -B in RNAs primarily in macrophages and bronchiolar-alveolar epithelial cells at sites of initial fiber deposition and lung injury. There was clear evidence of PDGF-A and -B mRNAs in interstitial cells as well. The pattern of in situ hybridization was entirely consistent with the appearance (established by immunohistochemistry) of PDGF-A and -B proteins by 24 h post-exposure in the same cell types. Both mRNAs and proteins remained detectable at the fiber deposition sites for almost 2 wk post-exposures. These findings are consistent with our previous studies showing increased mesenchymal cell proliferation and fibroproliferative lesions that progress at the sites where PDGF-A and -B are expressed. Although it is clear that multiple growth factors are produced simultaneously at sites of initial injury, we suggest that the PDGF isoforms could be playing a central role in the disease process based upon their potent mitogenic effects upon mesenchymal cells.     

Stimulated activation of platelet-derived growth factor receptor in vivo in balloon-injured arteries: a link between angiotensin II and intimal thickening

BACKGROUND: Growth factors such as platelet-derived growth factor (PDGF) have been postulated to be important mediators of neointimal formation in balloon-injured artery. Binding of growth factors to their receptors activates intrinsic receptor tyrosine kinase, resulting in tyrosine phosphorylation of receptors themselves and cellular substrate proteins. We investigated in vivo activities of growth factors by determining the extent of tyrosine phosphorylation of growth factor receptors and substrate proteins in injured artery. METHODS AND RESULTS: Rat balloon-injured carotid artery was analyzed for phosphotyrosine content of PDGF alpha- and beta-receptors, epidermal growth factor (EGF) receptors, and insulin receptor substrate-1 (IRS-1) by immunoprecipitation and anti-phosphotyrosine Western blot. The development of intimal thickening after deendothelializing balloon catheterization of rat carotid artery was accompanied by transient twofold to threefold increases in the extent of tyrosyl phosphorylation of PDGF alpha- and beta-receptors but not EGF receptor or IRS-1. The AT1 angiotensin II (Ang II) receptor antagonist TCV-116 markedly inhibited both tyrosyl phosphorylation of PDGF alpha- and beta-receptors and intimal thickening. The AT1 antagonist reduced mRNA levels of both PDGF-A and -B chains in injured arteries. CONCLUSIONS: The present study provides direct evidence for increased PDGF activities in injured artery in situ and the involvement of Ang II in stimulated activation of PDGF receptors. These results are consistent with the pathogenetic role for PDGF in intimal thickening.     

Requirement for phosphatidylinositol 3'-kinase activity in platelet-derived growth factor-stimulated tyrosine phosphorylation of p125 focal adhesion kinase and paxillin

The role of phosphatidylinositol 3'-kinase (PI 3'-kinase) activity in platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin has been examined. The tyrosine phosphorylation of p125FAK and paxillin in response to PDGF was markedly inhibited by wortmannin in a dose-dependent manner. PDGF-stimulated PI 3'-kinase activity, membrane ruffle formation, and tyrosine phosphorylation of p125FAK and paxillin were all inhibited by the same low concentrations of wortmannin (>90% inhibition at 40nM). In contrast, tyrosine phosphorylation of p125FAK and paxillin in response to bombesin, endothelin, and phorbol 12,13-dibutyrate was not inhibited by wortmannin in these cells. Furthermore, LY294002, an inhibitor of PI 3'-kinase structurally unrelated to wortmannin, also inhibited PDGF-stimulated p125FAK tyrosine phosphorylation. PDGF was shown to stimulate the tyrosine phosphorylation of p125FAK in porcine aortic endothelial (PAE) cells transfected with the wild type PDGF-beta receptors, but not in PAE cells transfected with PDGF-beta receptors in which the PI 3'-kinase binding sites (Tyr-740/751) were replaced by phenylalanine. PDGF-stimulated, PI 3'-kinase-dependent tyrosine phosphorylation of p125FAK was not inhibited by rapamycin, and thus it was dissociated from the activation of p70 S6 kinase, previously identified as a molecular downstream target of PI 3'-kinase. Thus, we have identified a PI 3'-kinase-dependent signal transduction pathway in the action of PDGF, which leads to the phosphorylation of p125FAK and paxillin.     

Excessive activation of tyrosine kinases leads to inhibition of proliferation in a thyroid carcinoma cell line

Autocrine stimulation of growth is a hallmark of many tumor cell lines. In this work we investigated the synthesis and secretion of growth factors and the expression of their corresponding receptors in HTC-TSHr thyroid carcinoma cells. These cells synthesize epidermal growth factor (EGF) receptors and platelet-derived growth factor beta (PDGF beta) receptors and in addition transforming growth factor alpha (TGF alpha), PDGF-A and PDGF-B chains, respectively. Addition of EGF or PDGF-BB to the culture medium resulted in growth inhibition of HTC-TSHr cells. In contrast, treatment of the cells with low concentrations of neutralizing anti-TGF alpha antibodies or tyrosine kinase inhibitors led to stimulation of cell proliferation. Low concentrations of neutralizing anti-PDGF-B antibodies did not affect growth of the cells. As expected, cell proliferation was inhibited when high concentrations of either neutralizing anti-TGF alpha antibodies or anti-PDGF-B antibodies were applied. PDGF-AA did not influence growth of HTC-TSHr cells. We conclude that growth of HTC-TSHr thyroid carcinoma cells is influenced by two autocrine loops between TGF alpha and EGF receptors and between PDGF-B and PDGF beta receptors. However, our data suggest that excessive activation of tyrosine kinase receptors in these cells results in a relative inhibition rather than stimulation of growth.     

The in vitro effect of platelet-derived growth factor isoforms on the proliferation of bovine corneal stromal fibroblasts depends on cell density

PURPOSE: Recently, it has been shown that corneal stromal fibroblasts express the mRNA for PDGF-beta-type receptors, while corneal epithelial cells express the mRNA for the PDGF B-chain, suggesting a role of PDGF isoforms in the regulation of corneal homeostasis and wound healing via an unidirectional epithelial to stromal paracrine interaction. The purpose of this study was to characterize the proliferative response of cultured bovine corneal stromal fibroblasts to PDGF isoforms. METHODS: Bovine corneal stromal fibroblasts were seeded at a cell density of 60 cells/mm2 (low density) and 120 cells/mm2 (high density) and were cultured under serum-free conditions. Except for corresponding controls, PDGF AA, BB and AB (obtained by separate expression of cloned genes in E. coli) were added in concentrations ranging from 3.125 to 100 ng/ml. Cell numbers were determined after an incubation period of 6 days using a cell counter. RESULTS: Stromal fibroblasts, when cultured at a high density, revealed constant cell numbers during the whole incubation period. Under these culture conditions, stimulation with PDGF AA, BB and AB led to a significant dose-dependent increase in cell proliferation. When cultured at a low cell density, stromal fibroblasts revealed a significant reduction of cell numbers after 6 days of incubation. This reduction was prevented by PDGF AA and AB isoforms in a dose-dependent manner. In contrast, PDGF BB was not effective. CONCLUSION: The results of the "high-density" assays suggest that PDGF isoforms act as mitogens for stromal fibroblasts during wound healing, when density of fibroblasts is high. The results of the "low-density" assays support the idea that PDGF AA and AB can prevent cell loss during corneal homeostasis when density of keratocytes is low.