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Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle
L A Pukac1, M E Ottlinger, M J Karnovsky
1Harvard Medical School, Department of Pathology, Boston, Massachusetts 02115.
Abstract:
We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.
Insights
Heparin inhibits vascular smooth muscle cell growth by selectively targeting protein kinase C-dependent gene expression pathways early in the cell cycle.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Heparin is known to inhibit vascular smooth muscle cell (VSMC) growth.
- Previous studies indicated heparin inhibits c-fos and c-myc protooncogene mRNA induction by phorbol 12-myristate 13-acetate (PMA) but not epidermal growth factor (EGF).
- This suggests different signaling pathways are involved in mitogen-stimulated gene expression.
Purpose of the Study:
- To investigate the molecular mechanisms of heparin's inhibition of VSMC growth.
- To determine if heparin selectively inhibits protein kinase C (PKC)-dependent signaling pathways.
- To elucidate the role of cell cycle phase in heparin's inhibitory effects.
Main Methods:
- Down-regulation of protein kinase C (PKC) in VSMC.
- Stimulation of VSMC with PMA, EGF, dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187.
- Measurement of c-fos and c-myc mRNA levels.
- Assessment of DNA synthesis and cell proliferation.
Main Results:
- PMA-induced c-fos and c-myc mRNA were suppressed in PKC down-regulated VSMC, while EGF induction was not, indicating distinct pathways.
- Heparin inhibited serum-stimulated c-fos mRNA in control VSMC but not in PKC down-regulated VSMC.
- Heparin did not inhibit c-fos mRNA stimulation by dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, or A23187.
- Heparin inhibited DNA synthesis and proliferation when added during the G0/G1 phase of the cell cycle.
Conclusions:
- Heparin selectively inhibits PKC-dependent, but not PKC-independent, signaling pathways for gene expression.
- Heparin acts early in the cell cycle (G0/G1 phase) to inhibit VSMC proliferation.
- These findings provide insight into the molecular mechanisms of heparin's anti-proliferative effects on VSMC.