EGF receptor signaling is involved in expression of osmoprotective TonEBP target gene aldose reductase under hypertonic conditions.
Renal medullary cells adapt to their hyperosmotic environment by enhanced expression of various osmoprotective genes. Although it is clearly established that TonEBP contributes to the expression of these genes, neither the precise signaling mechanism by which hypertonicity activates TonEBP is completely understood, nor is it known whether a membrane-bound osmosenser, corresponding to yeast and bacteria, is present in mammalian cells. We found evidence that metalloproteinase (MMP)-dependent activation of the epidermal growth factor receptor (EGFR) signals to TonEBP and stimulates the expression of the TonEBP target gene aldose reductase (AR) under hypertonic conditions. Phosphorylation of EGFR and the downstream MAP kinases ERK1/2 and p38 was significantly enhanced by high NaCl in Madin-Darby canine kidney (MDCK) cells. Conversely, the broad-spectrum MMP inhibitor GM6001 or the EGFR inhibitor AG1478 diminished phosphorylation of EGFR, p38, and ERK1/2, the induction of AR mRNA and protein, and AR promoter reporter activity in response to hypertonicity. Accordingly, neutralizing antibodies against the putative EGFR ligand transforming growth factor-alpha (TGF-alpha) abolished AR induction during osmotic stress. Furthermore, tonicity-induced phosphorylation of p38 and ERK1/2 and expression of AR were reduced significantly in MDCK cells transfected with a dominant-negative Ras construct. These effects were not caused by reduced nuclear abundance of TonEBP during osmotic stress; however, inhibition of EGFR or p38 diminished TonEBP transactivation activity under hypertonic conditions. The contribution of MMP/EGFR signaling in vivo was confirmed in C57BL/6 mice, in which treatment with GM6001 was associated with reduced AR induction following dehydration. Taken together, these results indicate that osmotic stress induces MMP-dependent activation of EGFR, likely via shedding of TGF-alpha, and downstream activation of Ras and the MAP kinases p38 and ERK1/2, which stimulate TonEBP transactivation activity. This EGFR-Ras-MAPK pathway contributes to TonEBP transcriptional activation and targets gene expression during osmotic stress, thus establishing a membrane-associated signal input that contributes to the regulation of TonEBP activity.
Küper
Christoph
C
Department of Physiology, University of Munich, Munich, Germany.
Steinert
Daniela
D
Fraek
Maria-Luisa
ML
Beck
Franz-X
FX
Neuhofer
Wolfgang
W
D000449:Aldehyde Reductase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D004285:Dogs; D066246:ErbB Receptors / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006982:Hypertonic Solutions / Q000494:pharmacology*; D007668:Kidney / Q000166:cytology*; D007679:Kidney Medulla / Q000187:drug effects / Q000201:enzymology; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008297:Male; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009997:Osmotic Pressure / Q000502:physiology; D011480:Protease Inhibitors / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014882:Water-Electrolyte Balance / Q000502:physiology*
D004151:Dipeptides; D006982:Hypertonic Solutions; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; C492200:Nfat5 protein, mouse; D011480:Protease Inhibitors; D014157:Transcription Factors; D000449:Aldehyde Reductase; D066246:ErbB Receptors; D045726:Metalloproteases