Regulation of alternative splicing of protein kinase C beta by insulin.

Insulin regulates a diverse array of cellular signaling processes involved in the control of growth, differentiation, and cellular metabolism. Insulin increases glucose transport via a protein kinase C (PKC)-dependent pathway in BC3H-1 myocytes, but the function of specific PKC isozymes in insulin action has not been elucidated. Two isoforms of PKC beta result via alternative splicing of precursor mRNA. As now shown, both isoforms are present in BC3H-1 myocytes, and insulin induces alternative splicing of the PKC beta mRNA thereby switching expression from PKC beta I to PKC beta II mRNA. This effect occurs rapidly (15 min after insulin treatment) and is dose-dependent. The switch in mRNA is reflected by increases in the protein levels of PKC beta II. High levels of 12-0-tetradecanoylphorbol-13-acetate, which are commonly used to deplete or down-regulate PKC in cells, also induce the switch to PKC beta II mRNA following overnight treatment, and protein levels of PKC beta II reflected mRNA increases. To investigate the functional importance of the shift in PKC beta isoform expression, stable transfectants of NIH-3T3 fibroblasts overexpressing PKC beta I and PKC beta II were established. The overexpression of PKC beta II but not PKC beta I in NIH-3T3 cells significantly enhanced insulin effects on glucose transport. This suggests that PKC beta II may be more selective than PKC beta I for enhancing the glucose transport effects of insulin in at least certain cells and, furthermore, that insulin can regulate the expression of PKC beta II by alternative mRNA splicing.

Tumor necrosis factor-alpha induces the expression of carbonic anhydrase II in pancreatic adenocarcinoma cells.

TNF is a 17kD cytokine classically known for its cytotoxic effects on malignant cells. More recent cell culture studies demonstrated TNF induced cytostasis associated with the expression of a terminally differentiated phenotype. This was best characterized in malignant hematopoietic models, although a similar action on cells derived from solid tumors is now increasingly recognized. In the present study, six day exposure to TNF (40 ng/ml) stimulated morphologic changes in a human pancreatic adenocarcinoma cell line (HPAC), including increased cellular homogeneity, decreased nuclear to cytoplasmic ratio and detachment from the cell monolayer. Proliferation and DNA synthesis were reversibly inhibited while cellular viability was maintained. Parallel to the changes in morphology and growth was the delayed appearance of carbonic anhydrase II (CA II, E.C. 4.2.1.1), an accepted marker for pancreatic cells of ductal origin. A concomitant increase in the steady-state level of CA II mRNA was also observed over the time-course of TNF exposure. These results suggest a novel role for TNF in the induction of a more terminally differentiated ductal cell phenotype in a human pancreatic carcinoma model.