Identification of Weight-bearing-responsive Elements in the Skeletal Muscle Sarco(endo)plasmic Reticulum Ca2+ ATPase (SERCA1) Gene

Abstract

The skeletal muscle sarco(endo)plasmic reticulum calcium ATPase (SERCA1) gene is transactivated as early as 2 days after the removal of weight-bearing (Peters, D. G., Mitchell-Felton, H., and Kandarian, S. C. (1999) Am. J. Physiol. 276, C1218-C1225), but the transcriptional mechanisms are elusive. Here, the rat SERCA1 5 flank and promoter region (؊3636 to ؉172 base pairs) was comprehensively examined using in vivo somatic gene transfer into rat soleus muscles (n ‫؍‬ 804) to identify region(s) that are both necessary and sufficient for sensitivity to weight-bearing. In all, 40 different SERCA1 reporter plasmids were constructed and tested. Several different regions of the SERCA1 5 flank were sufficient to confer a transcriptional response to 7 days of muscle unloading when placed upstream of a heterologous promoter. Two of these regions were analyzed further because they were necessary for the unloading response of ؊3636 to ؉172, as demonstrated using internal deletion constructs. Deletion analysis of these regions (؊1373 to ؊1158 and ؊330 to ؉172) suggested that unloading responsiveness corresponded to CACC sites and E-boxes. Mutagenesis of cis-elements in the first region showed that a specific CACC box (؊1262) was involved in SERCA1 transactivation and a nearby E-box (؊1248) was also implicated. Constructs containing trimerized CACC sites and E-boxes showed that the presence of both elements is required to activate transcription. This is the first identification of specific ciselements required for the regulation of a Ca 2؉ handling gene by changes in muscle loading condition.The sarco(endo)plasmic reticulum is the major organelle that regulates the Ca 2ϩ signaling associated with a multitude of cellular processes (1, 2). The sarco(endo)plasmic reticulum Ca 2ϩ ATPase (SERCA) 1 proteins translocate Ca 2ϩ from the cytosol to the sarcoplasmic reticulum or endoplasmic reticulum lumen, thereby reducing intracellular Ca 2 and refilling the sarcoplasmic/endoplasmic reticulum Ca 2ϩ stores. The cloning, expression, and functional characterization of three SERCA genes and their splicing variants have been described (3-12).At least one of the SERCA gene products is expressed in every mammalian tissue studied, emphasizing the fundamental role of this protein in cellular function. Products of the SERCA1 gene are the predominant isoforms expressed in skeletal muscle (SERCA1a adult, SERCA1b neonatal) (4, 9), and one product of the SERCA2 gene is the predominant isoform expressed in cardiac myocytes (SERCA2a (3)). The alternative splicing product of SERCA2 (SERCA2b (3)) and the products of the SERCA3 gene (3, 11) are ubiquitously expressed in muscle and non-muscle tissue but in much lower levels. SERCA expression is much higher in striated muscle than in smooth muscle or non-muscle to accommodate the large calcium fluxes associated with excitation-contraction coupling.Contractile activity has a marked effect on SERCA expression in striated muscle. Increased contractile activity leads to decreases in SERCA1 and SERCA2a express...

Large-scale mRNA analysis of female skeletal muscles during 60 days of bed rest with and without exercise or dietary protein supplementation as countermeasures

“…Multiple cellular processes are associated with Ca 2ϩ signaling regulated by the sarcoplasmic reticulum (SR) (14). SERCA1, protein and mRNA, have previously been shown to be upregulated following short-term unloading in soleus (55,60), but little is known about the transcriptional regulation of this gene. The upregulation of other calcium handling mRNAs, such as the ryanodine receptor (RYR1), involved in calcium release, and calsequestrin (CS), involved in calcium storage, has been previously reported following unloading (10,29).…”

In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage

“…Among non-viral techniques, intramuscular injection of naked plasmid DNA is a safe and simple approach for gene delivery. Since the pioneering work of Wolff et al [1], this technique has been widely used and has revealed important information about transcriptional mechanisms regulating gene expression in skeletal muscle cells [2][3][4]. Recently, gene transfer by electroporation, equally termed gene electrotransfer, has been demonstrated to greatly improve in vivo naked plasmid DNA transfer into skeletal muscle [5][6][7][8][9].…”

Sp3 Proteins Negatively Regulate β Myosin Heavy Chain Gene Expression during Skeletal Muscle Inactivity

“…A recent study has implicated multiple CACC elements in regulation of the sarco(endo)plasmic reticulum Ca 2ϩ ATPase (SERCA1) gene under NWB conditions (29). However the proteins which bound to the CACC elements were not identified.…”