Heparin suppresses the induction of c-fos and c-myc mRNA in murine fibroblasts by selective inhibition of a protein kinase C-dependent pathway.

Abstract

Heparin is a complex glycosaminoglycan that inhibits the proliferation of several cell types in culture and in vivo. To begin to define the mechanism(s) by which heparin exerts its antiproliferative effects, we asked whether heparin interferes with the expression of the growth factor-inducible protooncogenes c-fos and c-myc. We show that heparin suppressed the induction of c-os and c-myc mRNA by serum in murine (BALB/c) 3T3 fibroblasts. Using purified mitogens, we further show that suppression was most marked when protooncogene expression was induced by phorbol 12-myristate 13-acetate, an activator of protein kinase C. By contrast, there was little or no suppression when the cells were stimulated by epidermal growth factor, which, in these cells, utilizes a protein kinase C-independent pathway for the induction of gene expression. Heparin also inhibited the change in cell morphology induced by the phorbol ester but had no effect on the morphological change induced by epidermal growth factor and agents that raise intracellular cAMP. Heparin did not inhibit intracellular protein kinase C activity, phorbol ester-induced down-regulation of protein kinase C, or phosphorylation of the 80-kDa intracellular protein kinase C substrate. These results suggest that heparin inhibits a protein kinase C-dependent pathway for cell proliferation and suppresses the induction of c-fos and c-myc mRNA at a site distal to activation of the kinase.The proliferation of higher eukaryotic cells is regulated primarily by extracellular factors that provide growthstimulatory and growth-inhibitory signals (1). Over the last decade, a number of growth-promoting factors have been described. Considerable progress has been made in understanding the signal transduction mechanisms utilized by various growth factors and in identifying nuclear events that are regulated by growth factors. Among the nuclear events is

Cellular Effects of Heparin on the Production and Release of Tissue Factor Pathway Inhibitor in Human Endothelial Cells in Culture

“…It is known, for example, that heparin inhibits the PKC-␣-dependent pathway of mitogenesis in smooth muscle cells and fibroblasts, but because it does not directly affect PKC activity or the phosphorylation step, it is believed that the heparin block is distal to the activation of PKC. 56 On the other hand, there are other cellular responses elicited by PMA, such as stimulation of phospholipase D, which can occur independently of PKC. 57 Therefore, further work is needed to elucidate the nature of the mechanism(s) involved in the release and redistribution of TFPI in ECs in culture.…”

Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription.

“…al., 1989 ;Reilly et al, 1989;Pukac et al, 1990) . The antiproliferative response was not associated with a decrease in the level of inducible protein kinase C (PKC) activity (Wright et al, 1989;Pukac et al, 1990) suggesting that the intracellular GAG blocked events distal to PKC. Wright et al (1989) and Pukac et al (1990) found that the effects of heparin on cell proliferation, induced by TPA or serum, correlates with attenuated levels of cfos and c-myc mRNA.…”

“…The antiproliferative response was not associated with a decrease in the level of inducible protein kinase C (PKC) activity (Wright et al, 1989;Pukac et al, 1990) suggesting that the intracellular GAG blocked events distal to PKC. Wright et al (1989) and Pukac et al (1990) found that the effects of heparin on cell proliferation, induced by TPA or serum, correlates with attenuated levels of cfos and c-myc mRNA. The expression of these immediate early, or competence class genes, including cjun, is known to be stimulated by various mitogens and growth factors Kelly et al, 1983 ;Greenberg and Ziff, 1984;Kruijer et al ., 1984;Mdller et al, 1984;Greenberg et al, 1985 ;Verma, 1986;Gilman et al ., 1986;Almendral et al, 1988;Chiu et al, 1988;Lamph et al, 1988;Curran and Franza, 1988;Verma and Sassone-Corsi, 1989;Vogt and Bos, 1989), and to precede the onset of protein synthesis required for entry into S phase or the development ofa more differentiated cellular phenotype .…”

“…EVERAL laboratories have recently demonstrated that cell matrix glycosaminoglycans (GAGs)' and heparin potently suppress entry of cells into S-phase in response to mitogens by arresting them early in the cell cycle (Reilly et al, 1986;Benitz et al ., 1986;Wright et al ., 1989;Reilly et al, 1989 ;Jackson et al, 1991) . Inhibition of cell growth was not due to the sequestration of serum nutrients, mitogens, and growth factors (Reilly et al, 1986;Benitz et al, 1986) but rather involved the high affinity binding of heparin to specific cell surface receptors (Castellot et al ., 1985;Resink et al ., 1989) and internalization of the GAGs characterized by a ti/2 of 15-20 min in primary cell cultures .…”

Regulation of Vascular Smooth Muscle Proliferation by Heparin

“…The early G 1 block observed in this work may result from effects of heparin on cell cycle regulatory molecules other than p27 kip1 . For example, the induction of c-Fos and c-Myc in murine fibroblasts (76) and c-Fos induction in mesangial cells (77,78) is blocked by heparin. In addition, heparin has been shown to inhibit cell cycle by inhibition of multiple pathways such as mitogen activated protein kinase (MAPK), calmodulin-dependent protein kinase II, and protein kinase C (PKC).…”