The effects of sodium arsenite on focal adhesion formation and dynamics in H9C2 cells.

Focal adhesions (FAs) attach the extracellular matrix to the actin cytoskeleton in cells. FAs are functionally dynamic, regulate many cellular processes and are composed of scaffold proteins, numerous protein kinases, phosphatases and their respective substrates. The environmental toxicant arsenic causes multiple effects in cells, suggesting that it exerts its effects by altering signal transduction pathways. We hypothesized that sodium arsenite exposure disrupts signaling pathways involved in FA regulation, causing a disturbance in FA organization and dynamics, protein tyrosine phosphorylation and the cellular processes in which these events are involved. H9C2 cells were exposed to sublethal doses of sodium arsenite for up to 48 hours. In response to sodium arsenite, FAs localized only to the cell periphery and tyrosine phosphorylation of paxillin and FAK decreased. Cell migration, adhesion and spreading were also inhibited in a dose-dependent manner. Treatment with pervanadate, a tyrosine phosphatase inhibitor, blocked the effects of sodium arsenite on FAK and paxillin tyrosine phosphorylation, FA distribution, and cell migration and adhesion rates. Sodium arsenite caused an increase in cell tyrosine phosphatase activity, increased expression of the tyrosine phosphatase SHP-2, and increased interaction between SHP-2 and the Rho family guanine nucleotide exchange factor, Vav. Vav's phosphorylation was reduced and this was correlated with an inactivation of the RhoA-Rho kinase pathway. The inactivation of this pathway caused the amount of RhoA-GTP to be reduced and the Rho kinase substrate MYPT1 was not activated. FA dynamics were analyzed in live cells transfected with a GFP-paxillin fusion protein. Sodium arsenite reduced motility of and distance traveled by FAs. The recruitment of FA proteins to magnetic beads coated with RGD peptide was delayed and the contraction of collagen gel lattices was reduced in sodium arsenite-treated cells. Infection of H9C2 cells with a constitutively active RhoA adenovirus expression vector prevented the sodium arsenite-dependent reduction in collagen gel lattice contraction and stimulated the recruitment of FA proteins to RGD-coated magnetic beads. These results indicate that sodium arsenite affects FA proteins' tyrosine phosphorylation, organization and dynamics. These affects may be caused by sodium arsenite's ability to increase SHP-2 expression resulting in inactivation of the RhoA-Rho kinase pathway.PhDHealth and Environmental SciencesToxicologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125260/2/3186795.pd