Merosin (laminin-2/4)-driven survival signaling: complex modulations of Bcl-2 homologs.

We have shown previously that the promotion of myofiber survival by the basement membrane component merosin (laminin-2 [alpha2beta1gamma1]/laminin-4 [alpha2beta2gamma1]) is dependent on the activity of the tyrosine kinase Fyn, whereas myofiber anoikis induced by merosin deficiency is dependent on the stress-activated protein kinase p38alpha. To further understand such merosin-driven survival signaling, we analyzed the expression of five Bcl-2 homologs (Bcl-2, Bcl-X(L), Bax, Bak, Bad) and one non-homologous associated molecule (Bag-1) in normal and merosin-deficient myotubes, with or without pharmacological inhibitors for Fyn and p38. Herein, we report that (1) merosin deficiency induces anoikis and causes decreased Bcl-2, Bcl-X(L), and Bag-1 levels, increased Bax and Bak levels, and decreased Bad phosphorylation; (2) Bcl-2, Bcl-X(L), Bag-1, and Bad phosphorylation are also decreased in anoikis-dying, Fyn-inhibited myotubes; (3) the inhibition of p38alpha in Fyn-inhibited and/or merosin-deficient myotubes protects against anoikis and increases Bcl-2 levels above normal, in addition to restoring Bad phosphorylation and Bag-1 levels to normal; (4) the overexpression of merosin in deficient myotubes also rescues from anoikis and increases Bcl-2 levels and Bad phosphorylation above normal, in addition to restoring Bcl-X(L), Bag-1, Bax, and Bak levels to normal; and (5) Bcl-2 overexpression is sufficient to rescue merosin-deficient myotubes from anoikis, even though the expression/phosphorylation levels of the other homologs analyzed are not restored to normal. These results indicate that merosin-driven myofiber survival signaling affects complex, differential modulations of individual Bcl-2 homologs. These further suggest that Bcl-2 can play a major role in suppressing myofiber anoikis.

Merosin-integrin promotion of skeletal myofiber cell survival: Differentiation state-distinct involvement of p60Fyn tyrosine kinase and p38alpha stress-activated MAP kinase.

Myofiber survival and suppression of anoikis depend in large part on the merosin (laminin-2/-4)-integrin alpha7beta1D cell adhesion system; however, the question remains as to the nature of the signaling molecules/pathways involved. In the present study, we investigated this question using the C2C12 cell model of myogenic differentiation and its merosin- and laminin-deficient derivatives. Herein, we report that: 1) of four members of the Src family of tyrosine kinases studied (p60Src, p53/56Lyn, p59Yes, or p60Fyn), the expression and activity of p60Fyn are found in myotubes exclusively; 2) a severe decrease of p60Fyn activity correlates with myotube apoptosis/anoikis induced by pharmocological compounds (herbimycin A or PP2) which inhibit tyrosine kinases of the Src family, by merosin deficiency and by beta1 integrin inhibition; 3) myoblast survival depends on Fak and the MEK/Erk pathway, in contrast to myotubes; 4) the PI3-K pathway is not involved in either myoblast or myotube survival; and 5) p38alpha SAPK stimulation and activity (but not that of p38beta) are required in the progression of myotube apoptosis/anoikis induced by p60Fyn inhibition, merosin deficiency or beta1 integrin-inhibition; however, p38 is not involved in myoblast apoptosis. Taken together, these results suggest that the promotion of myotube survival by the merosin-alpha7beta1D adhesion system involves p60Fyn, and that disruptions in this cell adhesion system induce myotube apoptosis/anoikis through a p38alpha SAPK-dependent pathway.