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Gbeta gamma mediate differentiation of vascular smooth muscle cells
H P Reusch1, M Schaefer, C Plum
1Institute of Clinical Pharmacology and Toxicology and the Institute of Pharmacology, Benjamin Franklin Medical Center, Freie Universität Berlin, Berlin 14195, Germany.
Abstract:
Proliferation and subsequent dedifferentiation of vascular smooth muscle (VSM) cells contribute to the pathogenesis of atherosclerosis and postangioplastic restenosis. The dedifferentiation of VSM cells in vivo or in cell culture is characterized by a loss of contractile proteins such as smooth muscle-specific alpha-actin and myosin heavy chain (SM-MHC). Serum increased the expression of contractile proteins in neonatal rat VSM cells, indicating a redifferentiation process. RNase protection assays defined thrombin as a serum component that increases the abundance of SM-MHC transcripts. Additionally, serum and thrombin transiently elevated cytosolic Ca(2+) concentrations, led to a biphasic extracellular signal-regulated kinase (ERK) phosphorylation, up-regulated a transfected SM-MHC promoter construct, and induced expression of the contractile proteins SM-MHC and alpha-actin. Pertussis toxin, N17-Ras/Raf, and PD98059 prevented both the serum- and thrombin-induced second phase ERK phosphorylation and SM-MHC promoter activation. Constitutively active Galpha(q), Galpha(i), Galpha(12), and Galpha(13) failed to up-regulate SM-MHC transcription, whereas Gbetagamma concentration-dependently increased the SM-MHC promoter activity. Furthermore, the Gbetagamma scavenger beta-adrenergic receptor kinase 1 C-terminal peptide abolished the serum-mediated differentiation. We conclude that receptor-mediated differentiation of VSM cells requires Gbetagamma and an intact Ras/Raf/MEK/ERK signaling.
Insights
Vascular smooth muscle cell differentiation, crucial for cardiovascular health, is promoted by thrombin via Gbetagamma and Ras/Raf/MEK/ERK signaling pathways. This process involves increased expression of key contractile proteins like SM-MHC.
Area of Science:
- Cardiovascular Biology
- Cell Signaling
- Molecular Medicine
Background:
- Vascular smooth muscle cell (VSM) dedifferentiation contributes to atherosclerosis and restenosis.
- Dedifferentiated VSM cells lose contractile proteins, including smooth muscle-specific alpha-actin and myosin heavy chain (SM-MHC).
Purpose of the Study:
- To investigate the molecular mechanisms regulating VSM cell redifferentiation.
- To identify serum components and signaling pathways involved in VSM cell differentiation.
Main Methods:
- RNase protection assays to quantify SM-MHC transcripts.
- Measurement of cytosolic Ca(2+) concentrations and ERK phosphorylation.
- Analysis of SM-MHC promoter construct activity.
- Inhibition studies using pertussis toxin, dominant-negative Ras/Raf, PD98059, and Gbetagamma scavengers.
Main Results:
- Serum and thrombin induce redifferentiation of neonatal rat VSM cells, increasing contractile protein expression.
- Thrombin increases SM-MHC transcripts; both serum and thrombin activate ERK signaling and up-regulate SM-MHC promoter activity.
- Ras/Raf/MEK/ERK pathway and Gbetagamma signaling are essential for serum- and thrombin-induced VSM cell differentiation.
Conclusions:
- Receptor-mediated differentiation of VSM cells is dependent on Gbetagamma subunits and the Ras/Raf/MEK/ERK signaling cascade.
- Understanding these pathways offers potential therapeutic targets for cardiovascular diseases involving VSM cell dysfunction.