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Platelet-derived growth factor induces cellular growth in cultured chick ventricular myocytes
1Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Insights
Platelet-derived growth factor (PDGF) directly stimulates embryonic cardiac myocyte growth. The mitogen-activated protein kinase (MAPK) pathway is crucial for this PDGF-induced myocardial growth.
Area of Science:
- Cardiovascular Biology
- Cellular Signaling
- Developmental Biology
Background:
- Platelet-derived growth factor (PDGF) is known to stimulate cell growth.
- Its specific effects on cardiac myocytes and the underlying signaling pathways are not well understood.
Purpose of the Study:
- To investigate the direct effect of PDGF on embryonic cardiac myocytes.
- To elucidate the intracellular signaling pathways involved in PDGF-mediated cardiac myocyte growth.
Main Methods:
- Primary culture of chick embryonic ventricular myocytes.
- Assays for cellular growth, DNA synthesis, and PDGF binding.
- Analysis of c-fos mRNA induction, AP-1 binding activity, and MAPK/STAT activation.
- Measurement of intracellular calcium and L-type calcium channel current.
Main Results:
- PDGF-AB and PDGF-BB significantly increased cardiac myocyte number and DNA synthesis.
- PDGF-BB induced c-fos mRNA, AP-1 binding, and MAPK activation.
- MAPK inhibition attenuated PDGF-induced growth and c-fos induction.
- PDGF did not affect STAT phosphorylation, intracellular calcium, or calcium currents.
Conclusions:
- PDGF directly promotes the growth of embryonic cardiac myocytes.
- The MAPK signaling cascade plays a key role in PDGF-induced embryonic myocardial growth.
Objectives:
Platelet-derived growth factor (PDGF) stimulates growth in various types of cells, but little is known about its effect on cardiac myocytes. Therefore, we examined whether PDGF had a direct effect on cardiac myocytes and investigated their intracellular signaling pathways.
Methods:
A primary culture of chick embryonic (Hamburger and Hamilton stage 36) ventricular myocytes was prepared. Cellular growth was estimated by 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromo-2'-deoxyuridine incorporation assay. The number of PDGF binding sites was measured by binding assay. Induction of c-fos mRNA was analyzed by Northern blot analysis. The binding activity of activator protein (AP)-1 was examined by electrophoretic mobility shift assay. The activation of mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription (STATs) was analyzed by Western blot analysis, immunoprecipitation, and immunocytochemistry. Furthermore, intracellular Ca2+ concentration ([Ca2+]i) was measured with indo-1 and L-type Ca(2+)- channel current (ICa) was recorded with the patch clamp technique.
Results:
PDGF-AB and -BB, but not PDGF-AA, increased viable cell number (5 ng/ml of PDGF-AA, -AB, -BB: 101 +/- 4%, 115* +/- 4%, 122* +/- 4%, respectively, n = 4, *P < 0.05) and DNA synthesis (104 +/- 11%, 202* +/- 18%, 295* +/- 25%, respectively, n = 4, *P < 0.05). Scatchard analysis demonstrated that the maximal number of PDGF-AA, -AB, -BB binding sites was 5 +/- 1, 63 +/- 12, 126 +/- 24 fmol/10(6) cells, respectively. PDGF-BB provoked induction of c-fos mRNA and increases in binding activity to the AP-1 site. PDGF-BB also induced tyrosine phosphorylation and nuclear translocation of MAPK. The c-fos induction, the increased AP-1 binding activity and the acceleration of DNA synthesis were all attenuated by genistein (100 microM) or MAPK kinase inhibitor (10 or 50 microM PD98059). Interestingly, protein kinase C inhibitor (250 nM calphostin C) attenuated the increases of AP-1 binding activity to some extent, but did not inhibit the c-fos induction at all. The phosphorylation states of STATs were not significantly affected by PDGF-BB. PDGF-BB did not alter [Ca2+]i or ICa.
Conclusions:
We conclude that PDGF can exert direct effects on embryonic cardiac myocytes and induce their growth. MAPK cascade may play an important role in the PDGF-induced embryonic myocardial growth.