endothelin-1 and manumycin

We have shown previously that chronic exposure to endothelin-1 (ET-1) may stimulate GLUT1-mediated glucose transport in 3T3-L1 adipocytes via both protein kinase C (PKC)- and mitogen-activated protein kinase (p42/p44 MAPK)-dependent pathways. In the present study, by using a luciferase reporter driven by Glut1 promoter and enhancers (pLuc-GT1/E1/E2) and various constitutively active and dominant negative mutants of PKC isoforms, we identified PKCepsilon as the PKC isoform involved. In addition, we provide evidence that there is no direct interaction between ET-1 activated PKCepsilon and MAPK, at least at the kinase activity level. Furthermore, investigations employing deletion mutants of pLuc-GT1/E1/E2 to locate the putative ET-1 responsive sites and inhibitory agents to suppress the activities of putative transcription factors suggested that transcription factors CREB, Sp1 and NF-kappaB were involved. In summary, the results of this study indicate that ET-1 induction of Glut1 transcription involves distinct PKCepsilon- and MAPK-dependent pathways, as well as downstream transcription factors CREB, Sp1 and NF-kappaB.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Cyclic AMP Response Element-Binding Protein; Endothelin-1; Gene Expression Regulation; Glucose Transporter Type 1; Indoles; Maleimides; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Polyenes; Polyunsaturated Alkamides; Protein Isoforms; Protein Kinase C-epsilon; Protein Kinase Inhibitors; Sp1 Transcription Factor; Transcription, Genetic

Angiotensin II (AngII) induces cardiac hypertrophy through activating a variety of protein kinases. In this study, to understand how cardiac hypertrophy develops, we examined AngII-evoked signal transduction pathways leading to the activation of extracellular signal-regulated protein kinases (ERKs), which are reportedly critical for the development of cardiac hypertrophy, in cultured cardiac myocytes isolated from neonatal rats. Inhibition of protein kinase C (PKC) with calphostin C or down-regulation of PKC by pretreatment with a phorbol ester for 24 h abolished AngII-induced activation of Raf-1 and ERKs, and addition of a phorbol ester conversely induced a marked increase in the activities of Raf-1 and ERKs. Pretreatment with two chemically and mechanistically dissimilar tyrosine kinase inhibitors, genistein and tyrphostin, did not attenuate AngII-induced activation of ERKs. In contrast, genistein strongly blocked insulin-induced ERK activation in cardiac myocytes. Although pretreatment with manumycin, a Ras farnesyltransferase inhibitor, or overexpression of a dominant-negative mutant of Ras inhibited insulin-induced ERK activation, neither affected AngII-induced activation of ERKs. Overexpression of a dominant-negative mutant of Raf-1 completely suppressed ERK2 activation by AngII, endothelin-1, and insulin. These results suggest that PKC and Raf-1, but not tyrosine kinases or Ras, are critical for AngII-induced activation of ERKs in cardiac myocytes.

Topics: Angiotensin II; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Endothelin-1; Enzyme Activation; Genistein; Insulin; Isoflavones; Mitogen-Activated Protein Kinase 1; Myocardium; Polyenes; Polyunsaturated Alkamides; Protein Kinase C; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; ras Proteins; Rats; Rats, Wistar; Signal Transduction; Tetradecanoylphorbol Acetate