Multiple Sp1 Binding Sites in the Cardiac/Slow Twitch Muscle Sarcoplamsic Reticulum Ca[IMAGE]-ATPase Gene Promoter Are Required for Expression in Sol8 Muscle Cells (original) (raw)
Related papers
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.
1998
The cardiac/slow twitch sarcoplasmic reticulum (SR) Ca 2+-ATPase gene (SERCA2) encodes a calcium transport pump whose expression is regulated in a tissue-and development-specific manner. Previously we have identified two distinct positive regulatory regions (bp-284 to-72 and-1815 to-1105) as important for SERCA2 promoter activity. Here we demonstrate that the SERCA2 distal promoter region functions like an enhancer by activating a heterologous promoter (TK) in a muscle cell-specific manner. Through deletion analysis a core enhancer region was delimited to the-1467 to-1105 bp fragment. We identified the E box/AT-rich element located at-1115 bp as critical for maximal enhancer activity. Gel mobility shift studies revealed that this E box/AT-rich element specifically binds a protein which is induced during Sol8 myogenesis. This region includes two other cis-acting elements, CArG and MCAT, which also bind specific nuclear protein complexes from Sol8 myotubes. Mutagenesis of each of these sites resulted in decreased SERCA/TK-CAT promoter activity. Based on these data, we propose that the E box/AT-rich element may contribute along with CArG and MCAT elements to the overall activation and regulation of the SERCA2 gene promoter.
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.
PLOS ONE, 2015
The sarcoplasmic/endoplasmic reticulum Ca 2+ ATPases (SERCAs) are the main Ca 2+ pumps which decrease the intracellular Ca 2+ level by reaccumulating Ca 2+ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca 2+ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca 2+ -transients were evoked by applications of 120 mM KCl. The significantly higher [Ca 2+ ]i measured at the 20 th and 40 th seconds after the beginning of KCl application (112±3 and 110±3 nM vs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca 2+ -uptake capability which was quantified by extracting the maximal pump rate (454±41 μM/s vs. 144±24 μM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 μM/s) and the amount of calcium released (843±75 μM vs. 576±80 μM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca 2+ ]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.
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.
Muscle-specific transcriptional regulation of the cardiac/slow-twitch SERCA2 gene
1 The abbreviations used are: bp, base pair(s); tss, transcription start site; DLM, dorsal longitudinal muscle; DVM, dorso-ventral muscle; dcm, direct control muscles, specifically the basalare and pterale I and II; TT, tergotrochanter muscle; PCR, polymerase chain reaction; X-gal, 5-bromo-4-chloro-3-indolyl -D-galactopyranoside.
The Biochemical journal, 1996
Expression of the muscle-specific 2a isoform of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2) requires activation of an otherwise inefficient splice process at the 3'-end of the primary gene transcript. We provide evidence that SERCA2 splicing is a specifically regulated process, rather than the result of an increase in general splice efficiency or a decrease in polyadenylation efficiency at the 5'-most polyadenylation site. This is indicated by the fact that changes in general splice and polyadenylation efficiency, as observed during B-cell maturation, did not affect SERCA2 splicing. Furthermore, expression and overexpression studies did not support the hypothesis that changes in the level of the alternative splice factor ASF/SF2 or other arginine and serine rich proteins are sufficient to obtain the regulation of muscle- and neuronal-specific splicing.
AJP: Cell Physiology, 2002
Stomach smooth muscle (SSM) and left ventricular muscle (LVM) express the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump gene SERCA2. Alternative splicing yields two major isoforms, SERCA2a in LVM and slow twitch muscle and SERCA2b in SSM and most other tissues. The splices have different 3′-untranslated regions (UTR) and also encode proteins that differ slightly in their COOH-terminal domains. SERCA2 transcription rates are similar in the two tissues, yet LVM has a much higher level of SERCA2 mRNA than SSM. To understand the control of SERCA2 RNA expression, we inhibited transcription and showed that the half-life of SERCA2 mRNA is significantly longer ( P < 0.05) in primary cultures of LVM cells than in SSM cells. Nuclear SERCA2 mRNA levels were also higher in LVM than in SSM. In vitro decay assays using synthetic RNA corresponding to the 3′-UTR of SERCA2a and -2b showed that nuclear extracts produced a faster decay of SERCA2 RNA than cytoplasmic extracts and that nuclea...
The Journal of physiology, 2011
Sarcoplasmic reticulum Ca2+ ATPases (SERCAs) play a major role in muscle contractility by pumping Ca2+ from the cytosol into the sarcoplasmic reticulum(SR) Ca2+ store, allowing muscle relaxation and refilling of the SRwith releasableCa2+.Decreased SERCAfunction has been shown to result in impaired muscle function and disease in human and animal models. In this study,we present a newmouse modelwith targeted disruption of the Serca2 gene in skeletal muscle (skKO) to investigate the functional consequences of reduced SERCA2 expression in skeletal muscle. SkKO mice were viable and basic muscle structure was intact. SERCA2 abundance was reduced inmultiplemuscles, and by asmuch as 95% in soleusmuscle, having the highest content of slow-twitch fibres (40%). The Ca2+ uptake rate was significantly reduced in SR vesicles in total homogenates. We did not find any compensatory increase in SERCA1 or SERCA3 abundance, or altered expression of several other Ca2+-handling proteins. Ultrastructural analysis revealed generally well-preserved muscle morphology, but a reduced volume of the longitudinal SR. In contracting soleus muscle in vitro preparations, skKO muscles were able to fully relax, but with a significantly slowed relaxation time compared to controls. Surprisingly, the maximal force and contraction rate were preserved, suggesting that skKO slow-twitch fibres may be able to contribute to the total muscle force despite loss of SERCA2 protein. Thus it is possible that SERCA-independent mechanisms can contribute to muscle contractile function.