Regulation of Cytosolic NADP+-Dependent Isocitrate Dehydrogenase Expression

Abstract

The long-range goal of this project is to investigate the mechanisms involved in the regulation of the cytosolic NADP+- dependent isocitrate dehydrogenase (IDH1) expression. This dissertation focuses on the central hypothesis that the expression of IDH1 is modulated by regulators of mammary epithelial differentiation and metabolic effectors in bovine mammary epithelium. To test this hypothesis, we examined the mRNA expression of IDH1 in late pregnancy and at various stages of lactation in bovine mammary tissue and demonstrated that IDH1 mRNA levels increased by 2.3 fold after parturition compared to late pregnancy and remained constant thereafter. Then, we investigated the effects of extracellular matrix and lactogenic hormones on the expression of IDH1 in cultured BME-UV bovine mammary epithelial cells. We found that the expression of IDH1 mRNA increased in parallel with beta-casein expression during cell differentiation induced by extracellular matrix. Fetal calf serum and insulin repressed, whereas prolactin stimulated the expression of IDH1 mRNA in a dose-dependent fashion. The inhibitory effects of insulin on IDH1 mRNA levels were antagonized by cotreatment with prolactin. In contrast, treatment with prolactin in the presence of extracellular matrix further increased IDH1 mRNA and protein accumulation. Prolactin-induced IDH1 expression was inhibited by the mitogen-activated protein kinase (MAPK) pathway inhibitors PD98059 and U0126, and Janus tyrosine kinase 2 (JAK2), suggesting that both MAPK and JAK2 contribute to regulation of IDH1 expression by prolactin. Moreover, we demonstrated that the levels of IDH1 transcripts were reduced when BME-UV cells were treated with alpha-ketoglutarate and palmitic acid. Finally, we report that the trans-10, cis-12 CLA, but not cis-9, trans-11 CLA isomer, repress prolactin-induced IDH1 mRNA as well as protein accumulation. Taken together, these data suggest that the expression of IDH1 is modulated by regulators of mammary epithelial differentiation and metabolic effectors including lactogenic hormones, extracellular matrix and nutrients.