TGF- as differentiating factor for cultured smooth muscle cells

¹ Dept of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

^* To whom correspondence should be addressed. E-mail: nstephe{at}ms.umanitoba.ca.

	Abstract

In cultured smooth muscle cells starved by removal of 10% FBS for 7 days, growth arrest was seen; 30% became elongated and demonstrated super contractility. Study of conditioned medium suggested the differentiating factor was TGF.

SDS-PAGE was carried out on conditioned medium from the arrested cells. Two protein bands were identified as MMP-2 and TGF-1. To determine second messenger signaling by SMAD2, Western blotting and confocal microscopy were employed.

Conditioned medium from arrested cultures showed presence of MMP-2 and TGF-1 as revealed by SDS-PAGE, 68kDa and 25kDa bands were seen. Differentiation was confirmed by up-regulation of marker proteins, smooth muscle type myosin heavy chain and myosin light chain kinase. Confirmation was obtained by down-regulating these proteins with decorin treatment which reduces levels of active TGF, and an adenoviral dominant-negative vector coding for a mutated type II TGF-receptor. Activation of second messenger signaling was demonstrated by presence of phosphorylated SMAD2 and SMAD4 immunocytochemically.

TGF- is the likely differentiating factor responsible for the development of these super-contractile smooth muscle cells.

Such cells developing in vivo after cessation of an asthmatic attack, could contribute to the non-specific hyperreactivity of airways seen in patients.

Keywords:  Decorin, double mass spectrometry, matrix metalloproteinase-2, SMAD-2, TGF-1, TGF-RII

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