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Originally published as MBC in Press, 10.1091/mbc.E06-03-0243 on September 6, 2006

Vol. 17, Issue 11, 4896-4910, November 2006

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Sphingosine 1-Phosphate Induces Myoblast Differentiation through Cx43 Protein Expression: A Role for a Gap Junction-dependent and -independent FunctionFormula

R. Squecco*, C. Sassoli{dagger}, F. Nuti{ddagger}, M. Martinesi{ddagger}, F. Chellini{dagger}, D. Nosi{dagger}, S. Zecchi-Orlandini{dagger}, F. Francini*, L. Formigli{dagger}, and E. Meacci{ddagger}

Departments of {ddagger}Biochemical Sciences, {dagger}Anatomy, Histology, and Forensic Medicine, and *Physiological Sciences, University of Florence, Interuniversity Institute of Myology, Florence I-50134, Italy

Submitted March 28, 2006; Accepted August 28, 2006
Monitoring Editor: Asma Nusrat

Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43{Delta}130–136 reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P.

Formula The online version of this contains supplemental material at MBC Online (http://www.molbiolcell.org).

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-03-0243) on September 6, 2006.

Address correspondence to: Elisabetta Meacci (elisabetta.meacci{at}unifi.it)

Abbreviations used: S1P, sphingosine 1-phosphate; wtCx43, wild-type connexin 43; DNCx43, dominant negative connexin 43; PBS, phosphate buffer solution; FCS, fetal calf serum; DM, differentiation medium; HS, horse serum; MAPK, mitogen-activated protein kinase; ERK1/2, p44/42 MAPK; siRNA, short interfering RNA; GFP, green fluorescent protein; MHC, myosin heavy chain.




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