BRADYKININ STIMULATES THE ERK-]ELK-1-]FOS AP-1 PATHWAY IN MESANGIAL CELLS/

Among its diverse biological actions, the vasoactive peptide bradykini n (BK) induces the transcription factor AP-1 and proliferation of mesa ngial cells (S. S. El-Dahr, S. Dipp, I. V. Yosipiv, and W. H. Baricos. Kidney Int. 50: 1850-1855, 1996). In the present study, we examined t he role of protein tyrosine phosphorylation and the mitogen-activated protein kinases, ERK1/2, in mediating BK-induced AP-1 and DNA replicat ion in cultured rat mesangial cells. BK (10(-9) to 10(-7) M) stimulate d a rapid increase in tyrosine phosphorylation of multiple proteins wi th an estimated molecular mass of 120-130, 90-95, and 44-42 kDa. Immun oblots using antibodies specific for ERK or tyrosine-phosphorylated ER K revealed a shifting of p42 ERK2 to a higher molecular weight that co rrelated temporally with an increase in tyrosine-phosphorylated ERK2. Genistein, a specific tyrosine kinase inhibitor, prevented the phospho rylation of ERK2 by BK. In-gel kinase assays indicated that BK-induced tyrosine phosphorylation of ERK2 is accompanied by fourfold activatio n of its phosphotransferase activity toward the substrate PHAS-I (P < 0.05). Furthermore, BK stimulated a 2.5-fold increase (P < 0.05) in ph osphorylation of Elk-1, a transcription factor required for growth fac tor-induced c-fos transcription. In accord with the stimulation of Elk -1 phosphorylation, BK induced c-fos gene expression and the productio n of Fos/AP-1 complexes. In addition, thymidine incorporation into DNA increased twofold (P < 0.05) following BK stimulation. Each of these effects was blocked by tyrosine kinase inhibition with genistein or he rbimycin A. Similarly, antisense oligodeoxynucleotide targeting of ERK 1/2 mRNA inhibited BK-stimulated DNA synthesis. In contrast, protein k inase C inhibition or depletion had no effect on BK-induced c-fos mRNA , AP-1-DNA binding activity, or DNA synthesis. Collectively, these dat a demonstrate that BK activates the ERK-->Elk-1-->AP-1 pathway and tha t BK mitogenic signaling is critically dependent on protein tyrosine p hosphorylation.