A novel E box/AT-rich element is required for muscle-specific expression of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene (original) (raw)
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Journal of Biological Chemistry, 1996
The rabbit cardiac/slow twitch muscle sarcoplasmic reticulum Ca 2؉ -ATPase (SERCA2) gene encodes a Ca 2؉ transport pump whose expression is regulated during skeletal and cardiac muscle development and in response to various pathophysiological and hormonal states. Employing transient transfection analyses in Sol8 muscle cells, we have identified two positive regulatory regions, one distal (؊1810 base pair (bp) to ؊1110 bp) and one proximal (؊284 bp to ؊72 bp), within the SERCA2 promoter. The proximal promoter region from ؊284 bp to ؊80 bp was shown to confer muscle-specific expression to a heterologous promoter in Sol8 cells. This region is highly GC-rich containing the consensus sequence for four Sp1 elements (GGGCGG) and three Sp1like elements (GGGAGG). DNase I footprint analysis with Sol8 nuclear extracts and purified Sp1 protein showed the protection of the seven Sp1 binding sites. In addition, site-directed mutagenesis of the Sp1 consensus sites demonstrated that Sp1 sites are essential for the muscle-specific expression of the SERCA2 promoter. Furthermore, we demonstrate that cotransfection of an Sp1 expression vector together with SERCA2-CAT constructs can up-regulate SERCA2 promoter activity. These results imply that the Sp1 transcription factor plays an important role in the transcriptional regulation of SERCA2 within muscle cells.
Journal of Biological Chemistry, 1996
The rabbit cardiac/slow twitch muscle sarcoplasmic reticulum Ca 2؉-ATPase (SERCA2) gene encodes a Ca 2؉ transport pump whose expression is regulated during skeletal and cardiac muscle development and in response to various pathophysiological and hormonal states. Employing transient transfection analyses in Sol8 muscle cells, we have identified two positive regulatory regions, one distal (؊1810 base pair (bp) to ؊1110 bp) and one proximal (؊284 bp to ؊72 bp), within the SERCA2 promoter. The proximal promoter region from ؊284 bp to ؊80 bp was shown to confer muscle-specific expression to a heterologous promoter in Sol8 cells. This region is highly GC-rich containing the consensus sequence for four Sp1 elements (GGGCGG) and three Sp1like elements (GGGAGG). DNase I footprint analysis with Sol8 nuclear extracts and purified Sp1 protein showed the protection of the seven Sp1 binding sites. In addition, site-directed mutagenesis of the Sp1 consensus sites demonstrated that Sp1 sites are essential for the muscle-specific expression of the SERCA2 promoter. Furthermore, we demonstrate that cotransfection of an Sp1 expression vector together with SERCA2-CAT constructs can up-regulate SERCA2 promoter activity. These results imply that the Sp1 transcription factor plays an important role in the transcriptional regulation of SERCA2 within muscle cells.
Proceedings of the National Academy of Sciences, 2004
Transient elevations of cytosolic Ca 2؉ are a common mechanism of cellular signaling. In striated muscle, the sarco(endo)plasmic reticulum Ca 2؉ ATPase (SERCA) plays an important role in terminating Ca 2؉ transients by returning cytosolic Ca 2؉ to intracellular stores. Stored Ca 2؉ can then be released again for subsequent signaling. We down-regulated SERCA2 gene expression in cultured cardiac myocytes by means of endogenous transcription of small interfering RNA encoded by an exogenous cDNA template. The cDNA template was delivered by adenovirus vector. Reduction of SERCA expression in all myocytes in culture was documented by immunochemistry, real-time RT-PCR, and determination of ATP-dependent Ca 2؉ transport. The reduction of SERCA2 expression was associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na ؉ ͞Ca 2؉ exchanger, indicating that intracellular store deficiency was compensated for by Ca 2؉ fluxes through the plasma membrane. In fact, SERCA silencing was followed by increased transcription of Na ؉ ͞ Ca 2؉ exchanger, TRPC4, TRPC5, and related transcriptional factors, such as stimulating protein 1, myocyte enhancer factor 2, and nuclear factor of activated cells 4, through activation of calcineurin. This finding demonstrates that the observed compensation occurs through transcriptional crosstalk and the remodeling of Ca 2؉ signaling pathways. The wide significance of this regulatory mechanism is related to its general involvement in Ca 2؉ signaling dynamics and in cardiac development and hypertrophy.
Control of sarcoplasmic/endoplasmic-reticulum Ca2+ pump expression in cardiac and smooth muscle
Biochemical Journal, 1999
Cardiac muscle expresses sarcoplasmic\endoplasmic-reticulum Ca# + pump isoform SERCA2a ; stomach smooth muscle expresses SERCA2b. In 2-day-old rabbits, cardiac muscle contained levels of SERCA2 protein that were 100-200-fold those in the stomach smooth muscle. In nuclear run-on assays, the rate of SERCA2 gene transcription in heart nuclei was not significantly higher than in the stomach smooth-muscle nuclei. However, the SERCA2 mRNA levels (meanpS.E.M.) were (29p4)-fold higher in the heart. In both tissues the SERCA2 mRNA was associated with polyribosomes. In a sucrose-density-gradient sedimentation Abbreviations used : DTT, dithiothreitol ; PNS, postnuclear supernatant ; RT-PCR, reverse-transcriptase-mediated PCR ; SERCA, sarcoplasmic/ endoplasmic-reticulum Ca 2 + ATPase ; UTR, untranslated region. 1 To whom correspondence should be addressed (e-mail groverak!fhs.csu.mcmaster.ca). velocity experiment on polyribosomes, there was no difference in the sedimentation pattern of SERCA2 mRNA between the two tissues, suggesting that the translation efficiency of SERCA2 RNA in the two tissues is quite similar. Thus the main difference in the control of SERCA2 expression in the two tissues is posttranscriptional and pretranslational.
Cell Calcium, 2003
Adult SERCA2 b/b mice expressing the non-muscle Ca 2+ transport ATPase isoform SERCA2b in the heart instead of the normally predominant sarcomeric SERCA2a isoform, develop mild concentric ventricular hypertrophy with impaired cardiac contractility and relaxation [Circ. Res. 89 (2001) 838]. Results from a separate study on transgenic mice overexpressing SERCA2b in the normal SERCA2a context were interpreted to show that SERCA2b and SERCA2a are differentially targeted within the cardiac sarcoplasmic reticulum (SR) [J. Biol. Chem. 275 ]. Since a different subcellular distribution of SERCA2b could underlie alterations in Ca 2+ handling observed in SERCA2 b/b , we wanted to compare SERCA2b distribution in SERCA2 b/b with that of SERCA2a in wild-type (WT). Using confocal microscopy on immunostained fixed myocytes and BODIPY-thapsigargin-stained living cells, we found that in SERCA2 b/b mice SERCA2b is correctly targeted to cardiac SR and is present in the same SR regions as SERCA2a and SERCA2b in WT. We conclude that there is no differential targeting of SERCA2a and SERCA2b since both are found in the longitudinal SR and in the SR proximal to the Z-bands. Therefore, alterations in Ca 2+ handling and the development of hypertrophy in adult SERCA2 b/b mice do not result from different SERCA2b targeting.
Molecular and Cellular Biochemistry
In a previous study we described the inhibitory action of a cytosolic protein fraction from heart muscle on ATP-dependent Ca2+ uptake by sarcoplasmic reticulum (SR); further, this inhibition was shown to be blocked by an inhibitor antagonist, also derived from the cytosol (Narayanan et al. Biochim Biophys Acta 735: 53-66, 1983). The present study investigated the ontogenetic expression of the activities of Ca2+ transport inhibitor and inhibitor antagonist in heart cytosol during fetal and postnatal development of the rat. The SR Ca2+ transport inhibitor activity was undetectable in the cytosol of fetal (15- or 20-days gestation) rat heart but was manifested in the cytosol as early as one day after birth and increased progressively thereafter to reach almost adult levels within the first two weeks of postnatal development. The activity of the SR Ca2+ transport inhibitor antagonist was barely detectable in the near-term (20 days gestation) fetus but increased substantially during earl...
2000
The cardiac sarco(endo)plasmic reticulum Ca 2ϩ -ATPase gene (ATP2A2) encodes the following two different protein isoforms: SERCA2a (muscle-specific) and SERCA2b (ubiquitous). We have investigated whether this isoform specificity is required for normal cardiac function. Gene targeting in mice successfully disrupted the splicing mechanism responsible for generating the SERCA2a isoform. Homozygous SERCA2a Ϫ/Ϫ mice displayed a complete loss of SERCA2a mRNA and protein resulting in a switch to the SERCA2b isoform. The expression of SERCA2b mRNA and protein in hearts of SERCA2a Ϫ/Ϫ mice corresponded to only 50% of wild-type SERCA2 levels. Cardiac phospholamban mRNA levels were unaltered in SERCA2a Ϫ/Ϫ mice, but total phospholamban protein levels increased 2-fold. The transgenic phenotype was characterized by a Ϸ20% increase in embryonic and neonatal mortality (early phenotype), with histopathologic evidence of major cardiac malformations. Adult SERCA2a Ϫ/Ϫ animals (adult phenotype) showed a reduced spontaneous nocturnal activity and developed a mild compensatory concentric cardiac hypertrophy with impaired cardiac contractility and relaxation, but preserved -adrenergic response. Ca 2ϩ uptake levels in SERCA2a Ϫ/Ϫ cardiac homogenates were reduced by Ϸ50%. In isolated cells, relaxation and Ca 2ϩ removal by the SR were significantly reduced. Comparison of our data with those obtained in mice expressing similar cardiac levels of SERCA2a instead of SERCA2b indicate the importance of the muscle-specific SERCA2a isoform for normal cardiac development and for the cardiac contraction-relaxation cycle. (Circ Res. 2001;89:838-846.)
Regulation and rate limiting mechanisms of Ca2+ ATPase (SERCA2) expression in cardiac myocytes
Molecular and Cellular Biochemistry, 2012
Involvement of the calcineurin/NFAT pathway in transcription of cardiac sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) was demonstrated (Prasad and Inesi, Am J Physiol Heart Circ Physiol 300(1):H173-H180, 2011) by upregulation of SERCA2 following calcineurin (CN) activation by cytosolic Ca(2+), and downregulation of SERCA2 following CN inhibition with cyclosporine (CsA) or CN subunits gene silencing. We show here that in cultured cardiac myocytes, competitive engagement of the CN/NFAT pathway is accompanied by downregulation of SERCA2 and Ca(2+) signaling alterations. In fact, SERCA2 downregulation occurs following infection of myocytes with adenovirus vectors carrying luciferase or SERCA1 cDNA under control of NFAT-dependent promoters, but not under control of CMV promoters that do not depend on NFAT. SERCA2 downregulation is demonstrated by comparison with endogenous transcription and protein expression standards such as GAPDH and actin, indicating prominent SERCA2 involvement by the CN/NFAT pathway. Transcription of genes involved in hypertrophy, triggered by adrenergic agonist or by direct protein kinase C (PKC) activation with phorbol 12-myristate 13-acetate (PMA), is also prominently dependent on CN/NFAT. This is demonstrated by CN inhibition with CsA, CN subunits gene silencing with siRNA, displacement of NFAT from CN with 9,10-Dihydro-9,10[1',2']-benzenoanthracene-1,4-dione (INCA-6), and myocyte infection with vectors carrying luciferase cDNA under control of NFAT-dependent promoter. We show here that competitive engagement of the CN/NFAT pathway by endogenous genes involved in hypertrophy produces downregulation of SERCA2, reduction of Ca(2+) transport and inadequate Ca(2+) signaling. It is most interesting that, in the presence of adrenergic agonist, specific protein kinase C (PKC) inhibition with 3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (Gö 6983) prevents development of hypertrophy and maintains adequate SERCA2 levels and Ca(2+) signaling.
Journal of Cardiovascular Diseases & Diagnosis, 2013
Deregulated or enhanced calcium ion (Ca 2+) influx across an unstable sarcolemma has been proposed to directly affect cardiac hypertrophic remodelling, vascular proliferative diseases and degenerative muscle disorders. Aberrant intracellular handling is partly due to a defect in Sarcoplasmic Reticulum (SR) function. Decreased Ca 2+ uptake in cardiac, vascular and skeletal myocytes is associated with a decrease in the expression and activity of the fast sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA2a or SERCA1a isoforms). SERCA2a gene transfer was successfully used in heart failure; this approach holds further therapeutic promises in vascular proliferative diseases and dystrophin-deficient muscular diseases. The growing family of human SERCA isoforms comprises at least 14 mRNA and proteins with different functional characteristics and cell-specific expression. This review focuses on the biological role and therapeutic potential of different isoforms of SERCA in the physiology and pathology of cardiac, vascular and skeletal muscle cells.