Abstract:
Ochratoxin A is recognized as a strong nephrotoxin accumulating and initiating cellular damage in kidney proximal tubule cells. OTA exerts its effects both on cell sur vival and cell death via the deregulation of mitogen activated protein kinase (MAPK) and phosphotidylinositide 3-kinase/Akt (PI3K/Akt) pathways in a time- and dose dependent manner. Previous research showed that OTA activates PI3K/Akt in part by c-MET in HK-2 cells, however, little is known about the upstream regulator of MAPK/ERK pathway. Even though MAPKs are regulated through various upstream regulators, our recent findings indicates that an unknown GPCR is the most potent candidate. In this study, we portray the outcomes of OTA induced GPCR activation. Using EPAC biosensor and FRET technology, we demonstrate the increase in cellular cAMP levels as an early response to OTA exposure. However, it become clear that cAMP switches to a decreasing trend with Gαi activation in later time points with the help of cAMP modulators. ERK1/2 phosphorylation levels decrease as cAMP levels increase in early time points whereas ERK1/2 phosphorylation increase as cAMP lev els decrease in later time points of OTA exposure. Moreover, we also show receptor desensitization process upon OTA exposure with the help of β-arrestin localization on endosomes possessing Rab9 and Rab11. β-arrestin mediated ERK1/2 activation as a response to OTA also takes place on endosomes. We demonstrated that ERK1/2 acti vation is upregulated by β-arrestin 2 and dowregulated by β-arrestin 1 in a reciprocal manner. All together these results are the first to demonstrate OTA-mediated GPCR signaling activation.
