Rac1/osmosensing scaffold for MEKK3 contributes via phospholipase C-γ1 to activation of the osmoprotective transcription factor NFAT5

Abstract

Separate reports that hypertonicity activates p38 via a Rac1-OSM-MEKK3-MKK3-p38 pathway and that p38α contributes to activation of TonEBP/OREBP led us to the hypothesis that Rac1 might activate TonEBP/OREBP via p38. The present studies examine that possibility. High NaCl is hypertonic. We find that siRNA knockdown of Rac1 reduces high NaCl-induced increase of TonEBP/OREBP transcriptional activity (by reducing its transactivating activity but not its nuclear localization). Similarly, siRNA knockdown of osmosensing scaffold for MEKK3 (OSM) also reduces high NaCl-dependent TonEBP/OREBP transcriptional and transactivating activities. Simultaneous siRNA knockdown of Rac1 and OSM is not additive in reduction of TonEBP/OREBP transcriptional activity, indicating a common pathway. However, siRNA knockdown of MKK3 does not reduce TonEBP/OREBP transcriptional activity, although siRNA knockdown of MKK6 does. Nevertheless, the effect of Rac1 on TonEBP/OREBP is also independent of MKK6 because it occurs in MKK6-null cells. Furthermore, we find that siRNA knockdown of Rac1 or OSM actually increases activity (phosphorylation) of p38, rather than decreasing it, as previously reported. Thus, the effect of Rac1 on TonEBP/OREBP is independent of p38. We find instead that phospholipase C-γ1 (PLC-γ1) is involved. When transfected into PLC-γ1-null mouse embryonic fibroblast cells, catalytically active Rac1 does not increase TonEBP/OREBP transcriptional activity unless PLC-γ1 is reconstituted. Similarly, dominant-negative Rac1 also does not inhibit TonEBP/OREBP in PLC-γ1-null cells unless PLC-γ1 is reconstituted. We conclude that Rac1/OSM supports TonEBP/ OREBP activity and that this activity is mediated via PLC-γ1, not p38. osmotic stress | MAPK H ypertonicity (e.g., high NaCl) activates the transcription factor TonEBP/OREBP (also known as NFAT5), resulting in increased expression of osmoprotective genes (1). Hypertonicity increases TonEBP/OREBP activity by increasing its abundance (1), transactivating activity (1), nuclear localization (1), and phosphorylation (2, 3). The regulatory increase of phosphorylation depends on both increased kinase activity and reduced phosphatase activity (4, 5). The stress-activated MAP kinase p38 has been extensively studied in this regard, but understanding its role has been complicated because hypertonicity activates two different p38s, namely p38α (MAPK14) and p38δ (MAPK13), which have opposite effects on TonEBP/OREBP activity: p38α increases TonEBP/OREBP activity and p38δ decreases it (6). Furthermore, the phosphospecific antibodies commonly used to measure p38 activity do not directly distinguish between p38α activity and p38δ activity, nor do some forms of inhibition that have been used (6). In this article, we will continue to refer to "p38" unless p38α and p38δ are distinguished. Hypertonicity activates p38α via the upstream kinase MKK3 (MAP2K3) or MKK6 (MAP2K6) (7), and p38α and MEKK3 (MAP3K3) contribute to hypertonicity-induced activation of TonEBP/OREBP (6, 8, 9). Thus, p38α contributes to To...

Inhibitory phosphorylation of GSK-3β by AKT, PKA, and PI3K contributes to high NaCl-induced activation of the transcription factor NFAT5 (TonEBP/OREBP)

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