Growth and cell density-dependent expression of stathmin in C2 myoblasts in culture

Stathmin is a 19-kDa, ubiquitous cytoplasmic phosphoprotein whose expression is strongly regulated during tissue development and maturation and which was proposed as a general relay integrating diverse intracellular signaling pathways. Since myoblasts tend to align and differentiate in vitro toward myotubes above a certain density in culture, we examined the expression of stathmin as a function of cell density in the C2 myogenic cell line. Whereas stathmin was hardly detectable in low-to medium-density cultures corresponding to less than 1 microgram soluble protein/cm2, it became expressed to a stable level above this threshold of cell density. This cell density effect on stathmin expression was not mediated by a diffusible factor, since myogenic C2 or fibroblastic 3T3 cells grown at low and high density within the same culture flask displayed the same pattern of density-dependent stathmin expression, significant stathmin levels being observed only in the dense moiety of the flask. Interestingly, culture conditions which indirectly perturb cell-cell contacts, such as low Ca2+ or incubation with cytoskeleton disrupting agents such as nocodazole or cytochalasin D, prevented the expression of stathmin in C2 cells even at high density. More directly, anti-E-cadherin immunoglobulins, interfering with direct cell-cell contacts of the E-cadherin expressing S180 sarcoma-derived 2B2 cells, also prevented the expression of stathmin in these cells even at high density. Altogether, our results indicate that cell-cell contacts, probably mediated by adhesion molecules such as cadherins, are responsible for the high-density-induced expression of stathmin, which might then participate, in particular in the case of myogenic cells, in the control of the proliferation of cells and of their entry into the differentiation process.