DETECTION OF MAP KINASE PHOSPHATASE-3 AND ITS ALTERNATIVE SPLICE VARIANT IN ADRENOCORTICAL H295R CELL LINE: REGULATION BY ANGIOTENSIN II

M M. MORI SEQUEIROS, JM COHEN SABBAN; P MELE; S NUDLER; F CORNEJO MACIEL; P MALOBERTI; C PAZ

Munich

Conferencia; Adrenal Cortex Conference; 2018

Felix Beuschlein, Ludwig-Maximilians-University of Munich and University Hospital of Zürich Martin Fassnacht, University Hospital Würzburg Martin Reincke, Ludwig-Maximilians-University of Munich

MAP kinase phosphatases (MKP) recognize MAP kinase (MAPK) family members and promote their dephosphorylation and inactivation. Thus, MKP regulate MAPK-dependent processes such as proliferation, differentiation and apoptosis. MKP-3 is induced by different proliferative stimuli and specifically dephosphorylates ERK1/2, although it has also been reported to dephosphorylate non-MAPK substrate transcription factor P-foxo-1. Here we analyze the expression and regulation by angiotensin II (AII) of MKP-3 in adrenocortical cell line H295R. We also study the effect of AII on P-foxo-1 dephosphorylation and the induction of target p21. MKP-3S, a shorter variant lacking exon 2 and resulting from alternative splicing of the gene, has been described in some human tissues and cellular lines, mainly of tumoral origin. Using a set of primers for the simultaneous detection of both transcripts by RT-PCR, two signals were detected by electrophoretic analyses of RT-PCR products, corresponding to DNA fragments with the expected size for MKP-3L and S. Cells were serum-starved for 24 h and stimulated with AII (10-8 M) for different times, and total RNA was processed for the evaluation of each transcript by real time PCR using two sets of specific primers. MKP-3L and S mRNA levels were both up-regulated by AII. MKP-3L messenger levels reached a significant ~1.5-fold increase after 30 min stimulation, the earliest time point analyzed, and slightly declined over the next 4 h. MKP-3S mRNA levels were also significantly increased by ~1.8-fold after 30 min AII stimulation, with a temporal profile similar to that of MKP-3L. P-ERK1/2 levels detected after AII stimulation were in accordance with MKP-3 action in the late ERK-dephosphorylation phase. P-foxo-1 levels increased rapidly after stimulation and decreased concomitantly with MKP-3 protein induction. Moreover, p21 was also induced by AII, which is in agreement with the dephosphorylation and activation of P-foxo-1. In summary, our data suggest that AII regulates the levels of P-ERK, MKP-3 and MKP-3-linked events. This regulation of MKP-3 involves mRNA increase in two MKP-3 transcripts. Since MKP-3S protein lacks important regulatory sites, it is expected to display different properties and function from MKP-3L. Thus, L and S variants of MKP-3 proteins could differentially regulate ERK-dependent events in adrenocortical cells upon AII stimulation.