Thrombin stimulates c-sis gene expression in microvascular endothelial cells

We have determined whether expression of the c-sis gene product, platelet-derived growth factor (PDGF), is regulated in cultured renal microvascular endothelial cells by factors to which vascular endothelial cells may be exposed at sites of perivascular cellular proliferation. Thrombin exposure increased endothelial cell levels of c-sis message by 3-5-fold over a time course that peaked at 4 h after exposure. Similarly, thrombin-exposed microvascular endothelial cells released increased amounts of PDGF activity into their media. The thrombin effect was not mediated through the proteolytic activity of thrombin, as proteolytically inactive thrombin stimulated the c-sis expression as well as native thrombin. This stimulation was mimicked by exposure of cells to biologically active phorbol esters, suggesting that thrombin action may be mediated through activation of kinase C (Ca2+/phospholipid-dependent enzyme). Thus, thrombin regulates the expression and release of PDGF activity from endothelial cells in culture and may act in vivo to stimulate mitogen release from endothelial cells, thereby inducing proliferation of perivascular cells.     

Regulation of platelet-derived growth factor A and B chain gene expression in bone marrow stromal cells

MBA-2, bone marrow-derived endothelial stromal cells, express platelet-derived growth factor (PDGF) A and B chain mRNAs and secrete PDGF activity that is induced by TGF-beta. Either chain of the PDGF molecule could modulate hematopoiesis and stromal cell growth. Intracellular pathways that regulate PDGF expression in the marrow microenvironment are unknown. In the present study, we examined the mechanisms that mediate PDGF A and B chain mRNA induction by TGF-beta and the role of protein kinase C (PKC) and cyclic AMP in PDGF regulation. TGF-beta was tested in parallel with PMA, an activator of phorbol ester-dependent PKC isoforms. Both PMA (10^?7M) and TGF-beta (2.5 ng/ml) increased PDGF A and B chain mRNA levels. The serine/threonine protein kinase inhibitor, H7, blocked PDGF A and B chain mRNA induction in response to TGF-beta. However, down-regulation of PKC by prolonged incubation with PMA failed to abolish TGF-beta induction of PDGF A and B chain mRNAs. These findings indicate that induction of PDGF A and B chain mRNAs can be mediated via phorbol ester-dependent PKC pathway. In contrast, H7-sensitive protein kinase(s) other than phorbol ester-sensitive protein kinase C mediate the effect of TGF-beta. Agents that increase cAMP were also tested for their effect on PDGF gene expression. TGF-beta-mediated induction of PDGF A and B chain mRNAs was markedly inhibited by cAMP. cAMP also blocked stimulation of PDGF A chain mRNA by PMA. The positive and negative signaling mechanisms involved in modulating PDGF in the microenvironment may be important for determining hematopoietic and stromal cell responses in vivo. © 1995 Wiley-Liss, Inc.     

Cell-specific cyclic AMP-mediated induction of the PDGF receptor.

Cyclic AMP (cAMP) cooperates with a wide variety of polypeptide growth factors to synergistically stimulate the proliferation of many vertebrate cell types. However, the cellular mechanisms underlying these cooperative interactions are for the most part unknown. We have identified one such mechanism by observing that (i) cultured rat Schwann cells proliferate in response to platelet-derived growth factor (PDGF) only if simultaneously cultured in the presence of agents that elevate intracellular cAMP and (ii) this unmasked PDGF response is accounted for by a dramatic cAMP-mediated induction of PDGF receptor mRNA and protein. cAMP-mediated induction of the PDGF receptor results in enhanced, ligand dependent receptor autophosphorylation, and in enhanced PDGF activation of c-fos gene expression. In addition, this induction is unique to those cells, such as Schwann cells, for which cAMP is itself mitogenic. These results indicate that the synergistic proliferative effect obtained from the combination of cAMP and polypeptide growth factors may in large result from the cAMP-mediated induction of growth factor receptors.     

Expression of PDGF and their receptors in human retinal pigment epithelial cells and fibroblasts: regulation by TGF-beta

Platelet derived growth factors (PDGF) are known to be associated with vitreoretinal disorders such as proliferative vitreoretinopathy (PVR). We have studied the expression of PDGF and their receptors in human retinal pigment epithelial cells (HRPE) and choroid fibroblasts (HCHF), and the regulation of PDGF and its receptors by various cytokines and growth factors. RT-PCR analyses showed enhanced expression of PDGF-A and PDGF-B mRNA in HRPE treated with TGF-beta, but not with other cytokines. A minimal increase was observed in PDGF-A mRNA in TGF-beta treated HCHF cells. PDGF-R alpha mRNA, which was expressed prominently in HCHF and at very low levels in HRPE, was not affected by any of the agents. PDGF-R beta was not detectable in either HRPE or HCHF. HRPE secreted PDGF-AA and AB constitutively, and this secretion was significantly enhanced by TGF-beta. In contrast, HCHF cultures did not secrete detectable levels of any of the three isoforms of PDGF (AA, AB, BB). All three human recombinant PDGF isoforms enhanced HCHF cell proliferation significantly, while only a minimal increase was observed in HRPE. PDGF isoforms also induced HCHF cell elongation and promoted migration of HCHF in an in vitro wound assay. The results presented in this study demonstrate that TGF-beta activated RPE cells produce PDGF that may act on fibroblasts and other mesenchyme derived cells which express PDGF receptors. These studies indicate that the promotion of the proliferation and migration of mesenchymal cells by RPE cell derived PDGF may facilitate the formation of fibrovascular tissues associated with PVR.     

Type beta transforming growth factor in human platelets: release during platelet degranulation and action on vascular smooth muscle cells

A specific radioimmunoassay for type beta transforming growth factor (TGF-beta) was developed and used to show that human platelets treated with thrombin release TGF-beta as a consequence of degranulation. The thrombin concentrations required to induce release of TGF-beta parallel those concentrations that release the alpha-granule marker, beta-thromboglobulin. Related studies showed that TGF-beta acts on early passage, explant cultures of bovine aortic smooth muscle cells by inhibiting the effect of mitogens on proliferation of subconfluent cell monolayers yet synergizing with mitogens to stimulate growth of the same cells when cultured in soft agar. The results show that primary cultures of bovine aortic smooth muscle cells and established normal rat kidney cells behave similarly with regard to TGF-beta action. Moreover, the data suggest that platelet-mediated proliferation of aortic smooth muscle cells in vivo may not result solely from the stimulatory effect of platelet-derived growth factor (PDGF), but rather from an interaction of platelet factors which has the intrinsic ability to limit as well as stimulate mitosis.     

Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.     

The cell cycle dependence of c-sis gene expression: artifactual conclusions in cells prepared by chemical but not physical techniques

The c-sis oncogene encoding the B-chain of platelet-derived growth factor (PDGF) may be involved in an autocrine growth stimulation of tumours expressing the PDGF receptor, such as glioblastomas and sarcomas. To investigate whether expression of c-sis RNA is regulated in a cell cycle dependent manner, human A172 glioblastoma cells were synchronized by either centrifugal elutriation or chemical blockage with the DNA synthesis inhibitors hydroxyurea or aphidicolin. In non-perturbed elutriated cells, c-sis RNA levels were lower in the S phase of the cell cycle than in the G1 phase. In contrast, the chemically synchronized cells revealed a transient rise in c-sis RNA shortly after drug release, in early S phase. The RNA changes occurring after release from drug inhibition represent cell recovery from drug induced metabolic disturbances rather than true cell cycle dependent effects.     

Transforming growth factor-beta induces expression of vascular endothelial growth factor in human retinal pigment epithelial cells: involvement of mitogen-activated protein kinases

Vascular endothelial growth factor (VEGF) is a major agent in choroidal and retinal neovascularization, events associated with age-related macular degeneration (AMD) and diabetic retinopathy. Retinal pigment epithelium (RPE), strategically located between retina and choroid, plays a critical role in retinal disorders. We have examined the effects of various growth factors on the expression and secretion of VEGF by human retinal pigment epithelial cell cultures (HRPE). RT-PCR analyses revealed the presence of three isoforms of mRNA corresponding to VEGF 121, 165, and 189 that were up regulated by TGF-beta1. TGF-beta1, beta2, and beta3 were the potent inducers of VEGF secretion by HRPE cells whereas bFGF, PDGF, TGF-alpha, and GM-CSF had no effects. TGF-beta receptor type II antibody significantly reversed induction of VEGF secretion by TGF-beta. In contrast activin, inhibin and BMP, members of TGF-beta super family, had no effects on VEGF expression in HRPE. VEGF mRNA levels and protein secretion induced by TGF-beta were significantly inhibited by SB203580 and U0126, inhibitors of MAP kinases, but not by staurosporine and PDTC, protein kinase C and NF-kappaB pathway inhibitors, respectively. TGF-beta also induced VEGF expression by fibroblasts derived from human choroid of eye. TGF-beta induction of VEGF secretion by RPE and choroid cells may play a significant role in choroidal neovascularization (CNV) in AMD. Since the secretion of VEGF by HRPE is regulated by MAP kinase pathways, MAP kinase inhibitors may have potential use as therapeutic agents for CNV in AMD.     

Reactive oxygen species mediates the apoptosis induced by transforming growth factor beta(2) in human lens epithelial cells

Transforming growth factor beta(2) (TGF-beta(2)), a growth regulator of human lens epithelial cells (HLECs), also regulates the death of these cells. Dose-response analysis showed that the TGF-beta(2) concentration needed to induce HLECs death (100 pg/ml) was 10 times that needed to inhibit growth in these cells (10 pg/ml). TGF-beta(2)-induced apoptosis in HLECs was preceded by an induction of reactive oxygen species (ROS) and a decrease in glutathione in the intracellular content, indicating that this factor induces oxidative stress in HLECs. Studies performed to analyze the levels of c-fos mRNA, a gene whose expression is modulated by the redox state, demonstrated that only high, apoptotic concentrations of TGF-beta(2) (100 pg/ml) produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of hydrogen peroxide. Finally, the cell death induced by TGF-beta(2) in HLECs was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells, whereas these agents did not modify the growth-inhibitory effect elicited by TGF-beta(2) in these cells. The results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-beta(2) in HLECs.     

The cell cycle dependence of c-sis gene expression: artifactual conclusions in cells prepared by chemical but not physical techniques

Abstract. The c-sis oncogene encoding the B-chain of platelet-derived growth factor (PDGF) may be involved in an autocrine growth stimulation of tumours expressing the PDGF receptor, such as glioblastomas and sarcomas. To investigate whether expression of c-sis RNA is regulated in a cell cycle dependent manner, human A172 glioblastoma cells were synchronized by either centrifugal elutriation or chemical blockage with the DNA synthesis inhibitors hydroxyurea or aphidicolin. In non-perturbed elutriated cells, c-sis RNA levels were lower in the S phase of the cell cycle than in the G₁ phase. In contrast, the chemically synchronized cells revealed a transient rise in c-sis RNA shortly after drug release, in early S phase. The RNA changes occurring after release from drug inhibition represent cell recovery from drug induced metabolic disturbances rather than true cell cycle dependent effects.     

Heparin and dibutyryl cAMP modulate gene expression in stimulated human saphenous vein smooth muscle cells

Summary Increased expression of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) A chain, and tissue plasminogen activator (tPA) by smooth muscle cells (SMC) has been postulated to mediate the progression of intimal hyperplasia. We tested whether heparin would suppress the expression of these genes in stimulated human saphenous vein SMC. Quiescent cultured human saphenous vein SMC were stimulated for 4 h with heat-inactivated fetal bovine serum (10% by vol) in the presence or absence of heparin (1 to 250μg/ml). Heparin (50μg/ml) attenuated the induction by serum of bFGF mRNA, tPA mRNA, and tPA secretion. Nonanticoagulant heparin also attenuated serum induction of bFGF and tPA mRNA levels. To further study the role of second messenger signaling, a more specific mode of SMC stimulation was used with thrombin (3 U/ml) in the presence or absence of dibutyryl cyclic AMP (Bu₂-cAMP; 0.5 mM). In contrast to heparin, which had no effect on PDGF expression, Bu₂-cAMP decreased the induction by thrombin of PDGF-A chain mRNA levels. In thrombin-stimulated SMC, Bu₂-cAMP significantly decreased secretion of PDGF-AA protein. Thrombin, however, caused an increase in bFGF mRNA levels which was potentiated by Bu₂-cAMP with associated potentiation by Bu₂-cAMP of intracellular bFGF protein levels. The induction of tPA mRNA and tPA secretion by thrombin was sharply blocked by Bu₂-cAMP. These results suggest that heparin reduces intimal hyperplasia at least partly via partial inhibition of SMC gene expression.