Cyclosporin A-Dependent Downregulation of the Na+/Ca2+ Exchanger Expression
ABSTRACT Cyclosporin A (CsA) is an immunosuppressive drug commonly given to transplant patients. Its application is accompanied by severe side effects related to calcium, among them hypertension and nephrotoxicity. The Na+/Ca2+ exchanger (NCX) is a major calcium regulator expressed in the surface membrane of all excitable and many nonexcitable tissues. Three genes, NCX1, NCX2, and NCX3 code for Na+/Ca2+ exchange activity. NCX1 gene products are the most abundant. We have shown previously that exposure of NCX1-transfected HEK 293 cells to CsA, leads to concentration-dependent reduction of Na+/Ca2+ exchange activity and surface expression, without a reduction in total cell-expressed NCX1 protein. We show now that the effect of CsA on NCX1 protein expression is not restricted to transfected cells overexpressing the NCX1 protein but exhibited also in cells expressing endogenously the NCX1 protein (L6, H9c2, and primary smooth muscle cells). Exposure of NCX2- and NCX3-transfected cells to CsA results also in reduction of Na+/Ca2+ exchange activity and surface expression, though the sensitivity to the drug was lower than in NCX1-transfected cells. Studying the molecular mechanism of CsA-NCX interaction suggests that cyclophilin (Cyp) is involved in NCX1 protein expression and its modulation by CsA. Deletion of 426 amino acids from the large cytoplasmic loop of the protein retains the CsA-dependent downregulation of the truncated NCX1 suggesting that CsA-Cyp-NCX interaction involves the remaining protein domains.
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ABSTRACT: The Na(+)-Ca(2+) exchanger (NCX) is a major Ca(2+)-regulating protein encoded by three genes: NCX1, NCX2, and NCX3. They share a sequence homology of approximately 65%. NCX1 protein is expressed ubiquitously, and NCX2 and NCX3 are expressed almost exclusively in the brain. We have shown previously (Kimchi-Sarfaty et al., 2002) that treatment of NCX1-transfected human embryonic kidney (HEK) 293 cells with the immunosuppressive cyclosporin A (CsA) and its nonimmunosuppressive analog PSC833 (valspodar) results in down-regulation of surface expression and transport activity of the protein without a decrease in expression of cell NCX1 protein. In this study, we show that cyclosporin A and PSC833 treatment of NCX2- and NCX3-transfected HEK 293 cells also resulted in dose-dependent down-regulation of surface expression and transport activity of the two brain NCX proteins; however, whereas CsA had no effect on total cell NCX protein expression, PSC833 reduced mRNA and cell protein expression of NCX2 and NCX3. Moreover, tacrolimus (FK506), which had no effect on NCX1 protein expression, down-regulated NCX2 and NCX3 surface expression and transport activity without any significant effect on cell protein expression. Sirolimus (rapamycin) had no effect on NCX2 and NCX3 protein expression, yet it reduced NCX2 and NCX3 transport activity. Because all of the experimental conditions in our studies were identical, presumably the different drug response is related to structural differences between NCX isoforms. Clinical studies suggested that immunosuppressive regimes of patients who have received transplants resulted in complications related to Ca(2+). Expression of NCX genes is tissue-specific. Hence, our results can potentially provide a tool for choosing the immunosuppressive protocol to be used.Molecular pharmacology 05/2008; 73(4):1254-63. DOI:10.1124/mol.107.041582 · 4.13 Impact Factor
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ABSTRACT: The calcineurin inhibitors-cyclosporine and tacrolimus-are the mainstay of immunosuppressive therapy in solid organ transplantation. These drugs produce severe adverse drug effects (ADEs) such as nephrotoxicity, posttransplantation diabetes mellitus, and hypertension. Accumulated evidence suggests that the development of type 2 diabetes, hypertension, and renal failure may be associated with specific DNA genotypes. In this review, the genes involved with the development of these disease processes are compared with those implicated in calcineurin inhibitor-induced ADEs. The renin-angiotensin system genes, cytokine-encoding genes, and plasminogen activator inhibitor type 1 genes have been implicated in calcineurin inhibitor-induced nephrotoxicity, as well as in development of renal failure. A number of genes are implicated in contributing to diabetes, and these include the vitamin D receptor gene, VDR; hepatocyte nuclear factor genes, HNF; transcription factor 7-like 2 gene, TCF7L2; angiotensin-converting enzyme gene, ACE; cytokines; peroxisome proliferator-activated receptor gamma gene, PPARG; and others. Studies have suggested that the VDR, PPARG, HNF1A, and adenosine 5'-triphosphate-binding cassette ABCC8 (which encodes the sulfonylurea receptor) genes are associated with calcineurin inhibitor-induced diabetes. The genes encoding for the angiotensin-converting enzyme, endothelial constitutive nitric oxide synthase, and cytochrome P450 3A isoenzyme have been involved in the development of hypertension and in calcineurin inhibitor-induced hypertension. The genetic study of disease states can be the stepping stones for thoroughly understanding the genetic basis of ADEs. Gene polymorphisms are implicated in the development of diseases and corresponding disease-like ADEs. The disease-associated genes provide candidate genes for exploring ADEs and may provide genomic biomarkers for assessing the risk for developing severe calcineurin inhibitor-related ADEs as well as for developing preventive strategies.Pharmacotherapy 02/2010; 30(2):195-209. DOI:10.1592/phco.30.2.195 · 2.66 Impact Factor
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ABSTRACT: The Na(+)-Ca(2+) exchanger (NCX) is a major Ca(2+) regulating protein. It is almost ubiquitously expressed. Cyclophilins (Cyps) make up a class of proteins that are involved in protein folding via their peptidyl prolyl cis-trans isomerase (PPIase) and chaperone domains. They are also the cellular receptors of cyclosporin A (CsA). Binding of CsA to cyclophilins inhibits both PPIase and chaperone activities. We have shown that treatment of transfected HEK 293 cells expressing the Na(+)-Ca(2+) exchanger NCX1 with CsA results in downregulation of surface expression and transport activity, without any reduction in the total level of cell NCX1 protein [Kimchi-Sarfaty, C., et al. (2002) J. Biol. Chem. 277 (4), 2505-2510]. In this work, we show that knockdown of cell CypA using targeting siRNA (without any CsA treatment) results in a reduction in the level of NCX1 surface expression, a decrease in the level of Na(+)-dependent Ca(2+) uptake, and no change in the total amount of cell NCX1 protein in NCX1.5-transfected HEK 293 cells and nontransfected H9c2 cells that express NCX1.1 naturally. It also reduced Na(+)-dependent Ca(2+) fluxes measured by changes in Fluo-4 AM fluorescence in single NCX1.5-transfected HEK 293 and single H9c2 cells. Knockdown of CypB had no significant effect on either transport activity, surface expression, NCX1 cell protein expression, or Ca(2+) fluxes. Overexpression of CypA or its R55A mutant, which exhibits a substantially reduced PPIase activity, alleviated the reduction of NCX1 surface expression caused by CsA treatment, suggesting that the PPIase domain was probably not mandatory for NCX1 functional expression. We suggest that CypA plays a role in the functional expression of NCX1 protein.Biochemistry 09/2010; 49(35):7634-42. DOI:10.1021/bi1008722 · 3.02 Impact Factor