The roles of caveolin-1 and MAP kinase pathways in cellular transformation and differentiation
Engelman, Jeffrey Adam
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Caveolae are small (∼50--100nm) invaginations of the plasma membrane. Signal transduction molecules are highly enriched in these domains. Caveolae are most highly expressed in terminally-differentiated cell types such as adipocytes, endothelial cells, muscle cells, and epithelial cells. Caveolins are a family of proteins that constitute the principal components of caveolae. Recent experiments have demonstrated that caveohn-1 functionally interacts with many signaling molecules.;Caveolae are abundant in NIH 3T3 fibroblasts, but are absent in fibroblasts transformed by a wide variety of activated oncogenes such as H-Ras (G12V) and v-Abl. In these cells, caveolin-1 mRNA and protein expression are lost or reduced. However, it was unknown whether the down-regulation of caveolin-1 protein and caveolar structures contributes to their transformed phenotype.;The data in this thesis support the hypothesis that caveolin-1 protein is downregulated in response to oncogenic cellular transformation and that this downregulation contributes to the transformed phenotype. Caveolin-1 was expressed in H-Ras (G12V) and v-Abl transformed cells under the control of an inducible expression system. Expression of caveolin-1 abrogated the ability of these cells to grow in soft agar. Furthermore, induction of caveolin-1 led to the appearance of caveolae as determined by electron microscopy. We have examined the expression of caveolin-1 in a multitude of excised tumors from a variety of mouse models of mammary carcinoma. In all cancer types examined, caveohn-1 protein was absent.;We discovered that p42/44 MAP kinase activation inhibits caveolin-1 expression. In contrast, caveolin-1 is dramatically upregulated during the course of differentiation of 3T3-L1 fibroblasts to adipocytes. Treatment of 3T3-L1 fibroblasts with inhibitors of the p42/44 MAP Kinase and the p38 MAP Kinase in fibroblasts did not affect caveolin-1 expression. However, we discovered that inhibitors of p38, but not p42/44, MAP kinase completely block 3T3-L1 adipocyte differentiation. Furthermore, we constructed a 3T3-L1 cell line harboring an inducible dominant-negative p38. Expression of the dominant-negative p38 inhibited adipogenesis. We show that adipogenic transcription factors, such as C/EBPbeta, are direct p38 targets and thereby provide at least one mechanism by which p38 promotes adipogenesis.;In addition, we constructed 3T3-L1 cell lines harboring an inducible, constitutively active MKK6 (a specific activator of p38). Activation of p38 was sufficient to trigger 3T3-L1 differentiation in the absence of normally required hormonal stimulation. However, activation of p38 in the adipocytes led to apoptosis. (Abstract shortened by UMI.).