C-Peptide Increases Na,K-ATPase Expression via PKC- and MAP Kinase-Dependent Activation of Transcription Factor ZEB in Human Renal Tubular Cells

Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in primary human renal tubular cells (HRTC) in control and hyperglycemic conditions.HRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium ((86)Rb(+)) uptake and Na,K-ATPase activity were determined. Abundance of Na,K-ATPase was determined by Western blotting in intact cells or isolated basolateral membranes (BLM). DNA binding activity was determined by electrical mobility shift assay (EMSA). Culturing of HRTCs for 5 days with 1 nM, but not 10 nM of human C-peptide leads to increase in Na,K-ATPase α(1)-subunit protein expression, accompanied with increase in (86)Rb(+) uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α(1)-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to1 nM, but not 10 nM of C-peptide increased PKCε phosphorylation as well as phosphorylation and abundance of nuclear ERK1/2 regardless of glucose concentration. Exposure to 1 nM of C-peptide increased DNA binding activity of transcription factor ZEB (AREB6), concomitant with Na,K-ATPase α(1)-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α(1)-subunit expression and/or ZEB DNA binding activity in HRTC were abolished by incubation with PKC or MEK1/2 inhibitors and ZEB siRNA silencing.Despite activation of ERK1/2 and PKC by hyperglycemia, a distinct pool of PKCs and ERK1/2 is involved in regulation of Na,K-ATPase expression and activity by C-peptide. Most likely C-peptide stimulates sodium pump expression via activation of ZEB, a transcription factor that has not been previously implicated in C-peptide-mediated signaling. Importantly, only physiological concentrations of C-peptide elicit this effect

Prostaglandin D(2), its metabolite 15-d-PGJ(2), and peroxisome proliferator activated receptor-γ agonists induce apoptosis in transformed, but not normal, human T lineage cells

Prostaglandin D(2) (PGD(2)) is abundantly produced by mast cells, platelets, and alveolar macrophages and has been proposed as a key immunoregulatory lipid mediator. 15-Deoxy-Δ12,14-PGJ(2) (15-d-PGJ(2)), a key PGD(2) metabolite, is under intense study as a potential anti-inflammatory mediator. Little is known about PGD(2) or the role of 15-d-PGJ(2), if any, in regulating the activities of human T lineage cells. In this report we demonstrate that both PGD(2) and 15-d-PGJ(2) have potent antiproliferative effects, and in fact kill human T lymphocyte lines derived from malignant cells by an apoptotic mechanism. Interestingly, normal human T cells were not similarly affected. Although the T lymphocyte lines express mRNA for the PGD(2) receptor (DP-R), a potent DP receptor agonist, BW245C, did not inhibit the proliferation or viability of the cells, suggesting an alternative mechanism of action. PGD(2) and 15-d-PGJ(2) can bind to the peroxisome proliferator activated receptor-γ (PPAR-γ) which is implicated in lipid metabolism and apoptosis. Exposure to synthetic PPAR-γ ligands (e.g. ciglitazone, troglitazone) mimicked the inhibitory responses of PGD(2) and 15-d-PGJ(2), and induced apoptosis in the transformed T cells consistent with a PPAR-γ-dependent mechanism. These observations suggest that PPAR-γ ligands (which may include PGD(2)) provide strong apoptotic signals to transformed, but not normal T lymphocytes. Thus, the efficacy of utilizing PPAR-γ and its ligands as therapeutics for human T cell cancers needs to be further evaluated