p38 MAPK mediates acid-induced transcription of PEPCK in LLC-PK₁-FBPase⁺cells

Abstract

LLC-PK(1)-FBPase(+) cells are a gluconeogenic and pH-responsive renal proximal tubule-like cell line. On incubation with acidic medium (pH 6.9), LLC-PK(1)-FBPase(+) cells exhibit an increased rate of ammonia production as well as increases in glutaminase and phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels and enzyme activities. The increase in PEPCK mRNA is due to an enhanced rate of transcription that is initiated in response to intracellular acidosis. The involvement of known MAPK activities (ERK1/2, SAPK/JNK, p38) in the associated signal transduction pathway was examined by determining the effects of specific MAPK activators and inhibitors on basal and acid-induced PEPCK mRNA levels. Transfer of LLC-PK(1)-FBPase(+) cultures to acidic medium resulted in specific phosphorylation, and thus activation, of p38 and of activating transcription factor-2 (ATF-2), respectively. Anisomycin (AI), a strong p38 activator, increased PEPCK mRNA to levels comparable to those observed with acid stimulation. AI also induced a time-dependent phosphorylation of p38 and ATF-2. SB-203580, a specific p38 inhibitor, blocked both acid- and AI-induced PEPCK mRNA levels. Western blot analyses revealed that the SB-203580-sensitive p38alpha isoform is strongly expressed. The octanucleotide sequence of the cAMP-response element-1 site of the PEPCK promotor is a perfect match to the consensus element for binding ATF-2. The specificity of ATF-2 binding was proven by ELISA. We conclude that the SB-203580-sensitive p38alpha-ATF-2 signaling pathway is a likely mediator of the pH-responsive induction of PEPCK mRNA levels in renal LLC-PK(1)-FBPase(+) cells.

CCAAT/Enhancer-binding Protein α Mediates Induction of Hepatic Phosphoenolpyruvate Carboxykinase by p38 Mitogen-activated Protein Kinase

“…Our study shows that both p38 and JNK are activated in the livers of db/db diabetic mice. Studies have indicated that p38 activation enhances expression of PEPCK in both hepatocytes and kidney cells (21,22). Knocking down endogenous p38 in the liver reduced PEPCK mRNA level and hepatic glucose production in mice (23).…”

“…Activation of p38 has been observed in several tissues of diabetic animals and human subjects (16 -20). Early studies reported that p38 stimulates activating transcription factor 2 (ATF2), which increases PEPCK promoter activation in liver and kidney cells (21,22). While we were preparing this manuscript, Cao et al (23) reported that p38 mediates cAMPstimulated PEPCK and G6Pase gene expression and gluconeogenesis.…”

Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media

“…For Western blotting, cells were harvested in freshly prepared ice-cold RIPA-buffer (Feifel et al, 2002). the lysate was centrifuged at 10,000 g and 4°C for 5 min.…”

“…eRK1 and eRK2 are members of the MAPK family, which belong to a specific transcriptional activation cascade involved in many cell regulating processes. eRK1/2 are activated by specific phosphorylation that initiates the transcription of genes leading to cell growth and proliferation (Feifel et al, 2002;Schramek, 2002).…”

Oxidative Stress Induces Phosphoenolpyruvate Carboxykinase Expression in H4IIE Cells

“…In kidney cells, activation of p38 MAP kinase by culture in acidic media induces PEPCK mRNA expression. 19) Like the kidney, the liver is a major gluconeogenic organ, but it is unclear whether oxidative stress has a role independent of its actions on insulin signaling in the increased expression of the hepatic PEPCK gene.…”