Luigi Gnudi

Multiple Signal Transduction Pathways Link Na+/K+-ATPase to Growth-related Genes in Cardiac Myocytes THE ROLES OF Ras AND MITOGEN-ACTIVATED PROTEIN KINASES

We showed before that in neonatal rat cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophic growth and transcriptional regulations of genes that are markers of cardiac hypertrophy. In view of the suggested roles of Ras and p42/44 mitogen-activated protein kinases (MAPKs) as key mediators of cardiac hypertrophy, the aim of this work was to explore their roles in ouabain-initiated signal pathways regulating four growth-related genes of these myocytes,i.e. those for c-Fos, skeletal α-actin, atrial natriuretic factor, and the α3-subunit of Na+/K+-ATPase. Ouabain caused rapid activations of Ras and p42/44 MAPKs; the latter was sustained longer than 90 min. Using high efficiency adenoviral-mediated expression of a dominant-negative Ras mutant, and a specific inhibitor of MAPK kinase (MEK), activation of Ras-Raf-MEK-p42/44 MAPK cascade by ouabain was shown. The effects of the mutant Ras, an inhibitor of Ras farnesylation, and the MEK inhibitor on ouabain-induced changes in mRNAs of the four genes indicated that (a) skeletal α-actin induction was dependent on Ras but not on p42/44 MAPKs, (b) α3 repression was dependent on the Ras-p42/44 MAPK cascade, and (c) induction of c-fos or atrial natriuretic factor gene occurred partly through the Ras-p42/44 MAPK cascade, and partly through pathways independent of Ras and p42/44 MAPKs. All ouabain effects required extracellular Ca2+, and were attenuated by a Ca2+/calmodulin antagonist or a protein kinase C inhibitor. The findings show that (a) signal pathways linked to sarcolemmal Na+/K+-ATPase share early segments involving Ca2+ and protein kinase C, but diverge into multiple branches only some of which involve Ras, or p42/44 MAPKs, or both; and (b) there are significant differences between this network and the related gene regulatory pathways activated by other hypertrophic stimuli, including those whose responses involve increases in intracellular free Ca2+ through different mechanisms.

Inhibition of DNA Synthesis by a Farnesyltransferase Inhibitor Involves Inhibition of the p70s6k Pathway

Previously, the protein farnesyltransferase inhibitor (FTI), L-744,832, has been shown to inhibit the proliferation of a number of tumor cell lines in vitro in a manner that correlated with the inhibition of the mitogen-activated protein kinase cascade. Here we show that FTI inhibits p70s6kphosphorylation in mammary tumors in vivo in transgenic mice. Furthermore, in a mouse keratinocyte cell line, FTI inhibits p70s6k phosphorylation and activity and inhibits PHAS-1 phosphorylation in vitro in both rapidly growing cells and in growth factor-stimulated quiescent cells. Dominant-negative Ras expression inhibits p70s6k stimulation by epidermal growth factor, demonstrating a requirement for Ras activity during p70s6k activation. FTI does not inhibit protein kinase B phosphorylation on Ser473, indicating that FTI does not act by inhibiting phosphatidylinositol 3-kinase. FTI also inhibits DNA synthesis in keratinocytes, and inhibition of DNA synthesis correlates closely with p70s6k inhibition. Rapamycin, an inhibitor of p70s6k and PHAS-1 phosphorylation, causes a 30–45% reduction in DNA synthesis in keratinocytes, while FTI induces an 80–90% reduction in DNA synthesis. These observations suggest that alteration of p70s6k and PHAS-1 function by FTI are responsible for a substantial portion of the growth-inhibitory properties of FTI. Together, these data demonstrate that p70s6k and PHAS-1 are novel downstream targets of FTI and suggest that the anti-tumor properties of FTI are probably due to the inhibition of multiple mitogenic pathways.

Over-expression of GLUT4 selectively in adipose tissue in transgenic mice: Implications for nutrient partitioning

In summary, over-expression of GLUT4 selectively in fat causes increased flux of glucose into adipocytes and leads to increases in either the replication of immature pre-adipocytes or their differentiation into mature adipocytes resulting in an increase in fat cell number. This is the first model in which obesity is accounted for entirely by adipocyte hyperplasia and, therefore, is useful for studying the mechanisms involved in controlling fat cell number in vivo. GLUT4 over-expression in adipocytes of transgenic animals also increased whole- body insulin sensitivity. However, GLUT4 over-expression exclusively in adipocytes did not protect them from insulin resistance in vivo induced by high-fat feeding, in spite of the fact that insulin resistance was prevented at the level of the adipocyte. Interestingly, GLUT4 over-expression in fat protected the animals from developing further obesity when fed on a high-fat diet. It is possible that this failure to increase adiposity further is due to enhanced partitioning of glucose into fat, which may result in decreased glucose supply to muscle. This in turn may cause diversion of lipid to muscle to be oxidized as fatty acid. This diversion of lipid could result in protection against increased fat deposition in adipocytes. Further studies will be required in order to understand the molecular mechanisms by which GLUT4 over-expression in adipose tissues affects nutrient partitioning between muscle and adipose tissue and what the consequences of this are for whole-body fuel metabolism.