Effects of constitutively active and dominant negative MAPK kinase (MKK) 3 and MKK6 on the pH-responsive increase in phosphoenolpyruvate carboxykinase mRNA

Metabolic acidosis is partially compensated by a pronounced increase in renal catabolism of glutamine. This adaptive response is sustained, in part, through increased expression of phosphoenolpyruvate carboxykinase ( PEPCK). Previous inhibitor studies suggested that the pH-responsive increase in PEPCK mRNA in LLCPK1-FBPase(+) cells is mediated by a p38 mitogen-activated protein kinase ( MAPK). These cells express high levels of the upstream kinase MAPK kinase (MKK) 3 but relatively low levels of the alternative upstream kinase MKK6. To firmly establish the role of the p38 MAPK signaling pathway, clonal lines of LLC-PK1-FBPase(+) cells that express constitutively active (ca) and dominant negative (dn) forms of MKK3 and MKK6 from a tetracycline-responsive promoter were developed. Western blot analyses confirmed that 0.5 mu g/ml doxycycline was sufficient to block transcription and that removal of doxycycline led to pronounced and sustained expression of the caMKKs and dnMKKs. Expression of caMKK6 ( but not caMKK3) caused an increase in phosphorylation of p38 MAPK and an increase in the level of PEPCK mRNA that closely mimicked the effect of treatment with acidic medium (pH 6.9, 10 mM HCO3-). Only caMKK6 activated transcription of a PEPCK-luciferase reporter construct. Co-expression of both dnMKKs blocked the increases in phosphorylation of p38 MAPK and PEPCK mRNA. The latter effect closely mimicked that of the p38 MAPK inhibitor SB203580. The expression of either dnMKK3 or dnMKK6 was less effective than co-expression of both dnMKKs. Thus, the pH-responsive increase in PEPCK mRNA in the kidney is mediated by the p38 MAPK signaling pathway and involves activation of MKK3 and/or MKK6.