title: Rac1/osmosensing scaffold for MEKK3 contributes via phospholipase C-gamma1 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.
authors.affiliation: Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA. xiazhou@usuhs.mil
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Zhou
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Izumi
authors.forename: Maurice B
authors.initials: MB
authors.lastname: Burg
authors.forename: Joan D
authors.initials: JD
authors.lastname: Ferraris
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D005786:Gene Expression Regulation / Q000235:genetics*; D055785:Gene Knockdown Techniques; D006801:Humans; D008156:Luciferases; D048371:MAP Kinase Kinase 3 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D048308:Mitogen-Activated Protein Kinase 14 / Q000378:metabolism; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D051966:Phospholipase C gamma / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein
chemical_list: D008840:Microfilament Proteins; D009479:Neuropeptides; C492200:Nfat5 protein, mouse; D034741:RNA, Small Interfering; C484602:Rac1 protein, mouse; D014157:Transcription Factors; C480498:osmosensing scaffold for MEKK3 protein, mouse; D008156:Luciferases; D048308:Mitogen-Activated Protein Kinase 14; D048371:MAP Kinase Kinase 3; C482531:Map2k3 protein, mouse; D051966:Phospholipase C gamma; C117399:Plcg1 protein, mouse; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein