Molecular cloning of gene sequences regulated by platelet-derived growth factor

We have screened a cDNA library for gene sequences that are regulated by platelet-derived growth factor (PDGF) in BALB/c-3T3 cells. Of 8000 clones screened, less than 14 independent PDGF-inducible sequences were found. Two of these (KC and JE) were studied in detail. By hybrid-selection and translation the KC and JE mRNAs encode 10,000 and 19,000 dalton polypeptides, respectively. In the absence of PDGF, the JE and KC sequences correspond to low abundance mRNAs. One hour after addition of PDGF their abundance level can be increased 10- to 20-fold. Within 4 hr, a 60-fold induction of JE can be attained. Nanogram per ml quantities of pure PDGF regulate these sequences whereas μg/ml quantities of chemically unrelated mitogens (EGF, insulin, or platelet-poor plasma) have either a weak or an undetectable effect. Inhibitors of protein synthesis block the progression of quiescent 3T3 cells through G1 into S phase; however these drugs do not block the induction of KC and JE by PDGF. This result indicates that these sequences correspond to “early genes” which are not induced as a consequence of cell growth, but rather are directly regulated by PDGF.     

BALB/c-3T3 fibroblasts resistant to growth inhibition by beta interferon exhibit aberrant platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor signal transduction.

Several lines of evidence now exist to suggest an interaction between the platelet-derived growth factor (PDGF) growth-stimulatory signal transduction pathway and the beta interferon (IFN-beta) growth-inhibitory signal transduction pathway. The most direct examples are inhibition of PDGF-mediated gene induction and mitogenesis by IFN-beta and the effects of activators and inhibitors of the IFN-inducible double-stranded RNA-dependent eIF2 kinase on expression of PDGF-inducible genes. To further investigate the nature of this PDGF/IFN-beta interaction, we selected BALB/c-3T3 cells for resistance to growth inhibition by IFN-beta and analyzed the phenotypes of resulting clonal lines (called IRB cells) with respect to PDGF signal transduction. Although selected only for IFN resistance, the IRB cells were found to be defective for induction of growth-related genes c-fos, c-myc and JE in response to PDGF. This block to signal transduction was not due to loss or inactivation of PDGF receptors, as immunoprecipitation of PDGF receptors with antiphosphotyrosine antibodies showed them to be present at equal levels in the BALB/c-3T3 and IRB cells and to be autophosphorylated normally in response to PDGF. Furthermore, treatment with other peptide growth factors (PDGF-AA, fibroblast growth factor, and epidermal growth factor) also failed to induce c-fos, c-myc, or JE expression in IRB cells. All of these growth factors, however, were able to induce another early growth-related gene, Egr-1. The block to signaling was not due to a defect in inositol phosphate metabolism, as PDGF treatment induced normal calcium mobilization and phosphotidylinositol-3-kinase activation in these cells. Activation of protein kinase C by phorbol esters did induce c-fos, c-myc, and JE in IRB cells, indicating that signalling pathways distal to this enzyme remained intact. We have previously shown that IFN-inducible enzyme activities, including double-stranded RNA-dependent eIF2 kinase and 2',5'-oligoadenylate synthetase, are normal in IRB cells. The finding that the induction of multiple growth-related genes by several independent growth factors is inhibited in these IFN-resistant cells suggests that there is a second messenger common to both growth factor and IFN signaling pathways and that this messenger is defective in these cells.     

Transforming growth factor-beta-induced gene expression of monocyte chemoattractant JE in mouse osteoblastic cells, MC3T3-E1

A recent study demonstrated that PDGF-inducible JE is an inflammatory cytokine that directs chemotactic activity of monocytes. Accumulation of monocyte/macrophage lineage cells at site of bone tissue sites is very important for formation of multinucleate osteoclasts, which mediate bone resorption. Since transforming growth factor-beta (TGF-beta) is a potent regulator in bone remodeling, we examined whether TGF-beta induced JE gene expression in mouse osteoblastic cells, MC3T3-E1. TGF-beta induced a maximum JE mRNA expression at 3 hr after initiation of the cytokine treatment. This maximal expression was observed in when TGF-beta was used at a concentration of 1 ng/ml. The chemotactic activity for human monocytes was detected in conditioned medium of TGF-beta-treated cells, and the chemotactic activity was neutralized by anti-JE serum treatment.     

Platelet-derived growth factor induces interleukin-1 receptor gene expression in Balb/c 3T3 fibroblasts

Our previous studies have demonstrated that platelet-derived growth factor (PDGF) modulated cellular responses to interleukin-1 (IL-1). In this communication, we show that PDGF regulates expression of IL-1 receptor (IL-1 R) gene. Treatment of quiescent cultures of Balb/c 3T3 fibroblasts with PDGF produced 20-30-fold stimulation of IL-1 R mRNA with a concomitant increase in cell surface 125I-binding. IL-1 R mRNA accumulation occurred after an initial lag period and with a time course preceding the increase in 125I-IL-1 binding to cells. Induction of IL-1 R mRNA was blocked by inhibitors of protein synthesis, suggesting that a product of a gene expressed immediately after PDGF addition is required for IL-1 R gene expression. These latter data provide evidence for an ordered sequence of expression of PDGF-inducible "immediate early" gene(s) and IL-1 R gene. These results suggest that in connective tissues, PDGF may be an important determinant in initiating and maintaining cellular responses to IL-1. Such responses may have important consequences in the actions of IL-1 under normal and pathological conditions such as arthritis and atherosclerosis.     

The stimulatory effect of PDGF on vascular smooth muscle cell migration is mediated by the induction of endogenous basic FGF

The migration of arterial smooth muscle cells from the media to the intima is a crucial event for the development of the atherosclerotic lesion, and platelet derived growth factor (PDGF) is thought to play an important role in this process. Here we report that the spontaneous migration of bovine smooth muscle (BSM) cells is dependent on endogenously produced basic fibroblast growth factor (bFGF). PDGF stimulates the migration of BSM cells and its effect is abolished by affinity purified anti-bFGF antibody. PDGF induces bFGF mRNA in BSM cells. These results indicate that the effect of PDGF on the migration of BSM cells may be mediated by the induction of endogenous bFGF.     

Differential regulation of interleukin-6, macrophage inflammatory protein-1, and JE/MCP-1 cytokine expression in macrophage cell lines.

Activated macrophages produce a number of proinflammatory cytokines including IL-6, JE, MIP-1 alpha and MIP-1 beta. The induction requirements for production of either IL-6 or the MIP-1 related inflammatory proteins (MIP-1 alpha, MIP-1 beta, and JE) have been analyzed independently using fibroblasts, monocytes, or endothelial cells. However, little is known about the regulation of these cytokines in macrophages. Since activated macrophages produce prostaglandins (PGE2) which may participate in the autoregulation of cytokine production by stimulation of adenylate cyclase and the induction of cAMP-dependent signal pathways, we determined the effects of PGE on the production of IL-6 and MIP-1-related proteins. Murine macrophage cell lines were incubated with PGE1, PGE2, cholera toxin, or dibutyryl cAMP in the presence of absence suboptimal doses of LPS. Pharmacologic agents alone did not induce IL-6 production but incubation of macrophages with combinations of adenylate cyclase stimulators and LPS or dcAMP and LPS led to the dose-dependent enhancement of IL-6 secretion and mRNA expression. In contrast, PGE1 inhibits LPS-induced JE, MIP-1 alpha, and MIP-1 beta mRNA expression and this inhibition is partially dependent on a cAMP-mediated pathway of signal transduction. In previous work we demonstrated that IFN-gamma and PMA do not stimulate the production of IL-6 by macrophages. Here we show that incubation of macrophages with either IFN-gamma or PMA induces the expression of JE, MIP-1 alpha and MIP-1 beta mRNA expression. JE mRNA expression is much more responsive to the stimulatory effects of IFN-gamma than are the MIP-1 genes. Finally, PGE inhibits PMA and IFN-gamma-induced JE and MIP-1-related mRNA expression.     

Erythromycin and clarithromycin modulation of growth factor-induced expression of heparanase mRNA on human lung cancer cells in vitro.

Heparanase activity is correlated with the metastatic potential of several cancer cells and is a key enzyme in the breakdown of tissue barriers. It is also involved in the regulation of growth factor and cytokine activity. However, little is known about the factors that induce heparanase in cancer cells. We investigated the effect of three growth factors, platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF), on heparanase mRNA induction in lung cancer cells in vitro. In addition, we examined the effect of erythromycin (EM) and clarithromycin (CAM), which are 14-membered ring macrolide antibiotics that act as biological response modifiers, on the expression of heparanase mRNA induced by growth factors. PDGF, HGF and bFGF stimulated cell migration activity and enhanced the expression of heparanase mRNA in the human lung adenocarcinoma cell line A549. Via different mechanisms, EM and CAM modulate the induction by these factors of heparanase mRNA expression on A549 cells. EM also significantly suppressed A549 cell migration induced by PDGF and HGF, and CAM significantly suppressed A549cell migration induced by bFGF. The results suggest that the growth factors PDGF, HGF and bFGF are important inducers of heparanase in potentially invasive and metastatic cancer cells. The suppressive effect of heparanase mRNA expression by EM and CAM may have interestingtherapeutic applications in the prevention of metastasis.     

The effect of LPS on expression of the early "competence" genes JE and KC in murine peritoneal macrophages

The expression of early "competence" genes has been examined in murine peritoneal macrophages treated with bacterial lipopolysaccharide (LPS). This set of genes (e.g., c-myc, c-fos, r-fos, JE, and KC) were first described in BALB/c 3T3 cells treated with platelet-derived growth factor. We have previously reported that LPS induces the rapid and transient expression of both c-myc and c-fos in macrophages. In the present report, we present evidence demonstrating that the mRNA for JE and KC are also induced in macrophages after treatment of LPS. The r-fos gene was not detectably induced by LPS under the experimental conditions used in this study. The induction of JE and KC were dependent upon the dose of LPS and exhibited different time courses. mRNA for both KC and JE was induced within 30 min from the initiation of treatment. Although mRNA for JE continued to accumulate for up to 24 hr, mRNA for KC was optimally seen after 60 min and had disappeared by 4 hr. c-fos, JE, and KC mRNA were all inducible by a variety of structurally diverse but functionally similar agents (e.g., heat killed Listeria monocytogenes, maleyl-bovine serum albumin, and fucoidan). Interferon-gamma, a potent but functionally distinct stimulus of macrophage activation, did not effect the expression of JE or KC mRNA. The expression of mRNA for c-fos could be readily induced by treatment of macrophages with phorbol myristate acetate (PMA) alone and that for JE by PMA plus the inophore A23187; mRNA for KC was largely unaffected by these agents. These results suggest that expression of the c-fos and JE genes are regulated by products of polyphosphoinositide hydrolysis. The difference between c-fos or JE and KC raises the possibility that LPS may stimulate at least two independent routes of early gene expression. LPS does not promote macrophage proliferative activity alone, and in fact inhibits the proliferative response to the macrophage growth factor colony-stimulating factor 1. Taken together these findings suggest that the products of these genes may function in the acquisition of competence for highly differentiated functions in addition to that for cell division.     

Adherence-dependent increase in human monocyte PDGF(B) mRNA is associated with increases in c-fos, c-jun, and EGR2 mRNA

Adherence is an important initial step in the transition of a circulating monocyte to a tissue macrophage. This differentiation is accompanied by an augmented capacity to generate growth factors. We hypothesized that adherence itself might be an important trigger for a sequence of gene activation culminating in cells with increased mRNA encoding profibrotic growth factors such as platelet-derived growth factor B subunit (PDGF[B]) and transforming growth factor-beta (TGF- beta). After in vitro adherence, human monocytes had a biphasic increase in PDGF(B) mRNA with peaks at 6 h and 13 d. No increase in TGF- beta mRNA was observed. The 6-h increase in PDGF(B) mRNA was adherence dependent, and in addition, was abrogated when the cytoskeletal integrity was compromised by cytochalasin D. The 6-h increase in PDGF(B) mRNA was unaltered by adherence in the presence of the monocyte stimulus lipopolysaccharide. Adherence to either fibronectin or collagen-coated plastic had little consistent effect on PDGF(B) mRNA accumulation. The increased PDGF(B) mRNA observed in adherent monocytes was accompanied by increases in mRNAs of the early growth response genes c-fos (maximal at 20 min), c-jun, and EGR2 (maximal at 6-24 h). The increase in c-jun and EGR2, but not c-fos, mRNA was also abrogated by cytochalasin D. These observations suggest that adherence results in increases of c-fos, c-jun, EGR2, and PDGF(B) mRNA. In addition, the increases in c-jun, EGR2, and PDGF(B) may depend on cytoskeletal rearrangement. Modulation of these events at the time of adherence offers a mechanism by which differential priming of the cells may be accomplished.