Phosphorylation of cAMP responsive element binding protein after treatment of mesangial cells with high glucose plus TGFβ or PMA

Jeffrey I. Kreisberg, Robert A. Radnik, Suzanne H. Kreisberg

Research output: Contribution to journalArticlepeer-review

39 Scopus citations

Abstract

We recently showed that mesangial cells treated with high glucose plus TGFβ or PMA demonstrated activation of a cAMP-response element (CRE) located in the 5' flanking region of the fibronectin gene. Gel shift mobility assays with a CRE oligonucleotide revealed multiple complexes that did not change in mobility or abundance under conditions of high glucose plus TGFβ or PMA. Here we show that treatment with cycloheximide to inhibit protein synthesis also did not change the DNA/protein complexes. These observations led us to conclude that post-translational modification of transcription factors may be responsible for the activation of the fibronectin gene observed under our experimental conditions. We identified the proteins complexed to CRE as CRE binding protein (CREB) and activating factor 1 (ATF1). This was accomplished by supershift assays and immunoblots. Two hours of high glucose plus TGFβ or 30 minutes of PMA caused a twofold elevation in phosphorylated CREB. Neither high glucose nor TGFβ alone caused phosphorylation of CREB. ATF-1 was not phosphorylated. We also show that high glucose plus TGFβ and PMA activated protein kinase Cα; however, none of the agents tested stimulated intracellular cAMP levels, indicating that phosphorylation of CREB was independent of protein kinase A activation. These results demonstrate cross-talk between the protein kinase C and protein kinase A pathways in that agents which activate the protein kinase C pathway can stimulate phosphorylation of proteins that commonly serve as substrates for protein kinase A.

Original languageEnglish (US)
Pages (from-to)805-810
Number of pages6
JournalKidney international
Volume50
Issue number3
DOIs
StatePublished - 1996
Externally publishedYes

ASJC Scopus subject areas

  • Nephrology

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