PPARgamma-independent mechanisms of Src kinase activation and EGFR transactivation in response to thiazolidinediones

The thiazolidinediones (TZDs), a drug family used in the improvement of type II diabetes, are synthetic ligands for the peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear hormone receptor that mediates the expression of genes important in glucose homeostasis, energy metabolism, and cell proliferation and differentiation. TZDs also mediate, independently of PPARgamma, activation of the mitogen-activated protein kinases (MAPKs) and the signal transduction pathways contributing to their activation have only recently begun to be explored. Interestingly, TZDs mediate the activation of different MAPK subfamilies; however, the mechanisms responsible for this differential activation are not fully understood. The goal of this work was examine mechanisms regulating signal transduction pathways contributing to the activation of MAPKs in response to TZDs.;Here we demonstrate that two TZDs, ciglitazone and troglitazone, activated the non-receptor tyrosine kinase Src by affecting known regulatory tyrosine phosphorylation in the protein. Activation of Src involved protein tyrosine phosphatase (PTPase)-mediated dephosphorylation of a specific tyrosine residue known to negatively regulate Src's kinase activity. Disruption of lipid rafts, distinct plasma membrane structures that function to localize signaling proteins, prevented dephosphorylation of this site; however, no significant amount of Src or PTPase activity was observed in lipid rafts before and after TZD treatment. A PTPase, putatively identified as phosphatidylinositol polyphosphate-5 phosphatase, was shown to be constitutively associated with Src protein. In contrast to the activation of Src by both TZDs, epidermal growth factor receptor (EGFR) transactivation and extracellular signal-regulated kinase (Erk) activation were only observed with ciglitazone. Both TZDs mediated a transient increase in cytosolic calcium concentrations, suggestive of endoplasmic reticulum (ER) store release; however, only ciglitazone induced a secondary calcium influx, indicative of capacitative calcium entry (CCE). Ciglitazone-induced EGFR transactivation and Erk activation were prevented by either removal of extracellular calcium or by 2-aminoethyl diphenyl-borinate (2-APB), a known CCE inhibitor. Collectively, these studies provide additional information of the PPARgamma-independent signaling pathways affected by TZDs contributing to the activation MAPKs. Ultimately, this knowledge may help us better understand both the pharmacological actions and potential toxic side effects associated with these compounds.