Gabriela Rodríguez

Transcriptional Analysis of the Human Cardiac Calsequestrin Gene in Cardiac and Skeletal Myocytes

Calsequestrin is the main calcium-binding protein inside the sarcoplasmic reticulum of striated muscle. In mammals, the cardiac calsequestrin gene (casq2) mainly expresses in cardiac muscle and to a minor extent in slow-twitch skeletal muscle and it is not expressed in non-muscle tissues. This work is the first study on the transcriptional regulation of the casq2 gene in cardiac and skeletal muscle cells. The sequence of the casq2 genes proximal promoter (180 bp) of mammals and avians is highly conserved and contains one TATA box, one CArG box, one E-box, and one myocyte enhancer factor 2 (MEF-2) site. We cloned the human casq2 gene 5′-regulatory region into a luciferase reporter expression vector. By functional assays we showed that a construct containing the first 288 bp of promoter was up-regulated during myogenic differentiation of Sol8 cells and had higher transcriptional activity compared with longer constructs. In neonatal rat cardiac myocytes, the larger construct containing 3.2 kb showed the highest transcriptional activity, demonstrating that the first 288 bp are sufficient to confer muscle specificity, whereas distal sequences may act as a cardiac-specific enhancer. Electrophoretic mobility shift assay studies demonstrated that the proximal MEF-2 and CArG box sequences were capable of binding MEF-2 and serum response factor, respectively, whereas the E-box did not show binding properties. Functional studies demonstrated that site-directed mutagenesis of the proximal MEF-2 and CArG box sites significantly decreased the transcription of the gene in cardiac and skeletal muscle cells, indicating that they are important to drive cardiac and skeletal muscle-specific transcription of the casq2 gene.

Induction of cell differentiation activates transcription of the Sarco/Endoplasmic Reticulum calcium-ATPase 3 gene (ATP2A3) in gastric and colon cancer cells.

The Sarco/Endoplasmic Reticulum Ca2+‐ATPases (SERCAs), pump Ca2+ into the endoplasmic reticulum lumen modulating cytosolic Ca2+ concentrations to regulate various cellular processes including cell growth. Previous studies have reported a downregulation of SERCA3 protein expression in gastric and colon cancer cell lines and showed that in vitro cell differentiation increases its expression. However, little is known about the transcriptional mechanisms and transcription factors that regulate SERCA3 expression in epithelial cancer cells. In this work, we demonstrate that SERCA3 mRNA is upregulated up to 45‐fold in two epithelial cancer cell lines, KATO‐III and Caco‐2, induced to differentiate with histone deacetylase inhibitors (HDACi) and by cell confluence, respectively. To evaluate the transcriptional elements responding to the differentiation stimuli, we cloned the human ATP2A3 promoter, generated deletion constructs and transfected them into KATO‐III cells. Basal and differentiation responsive DNA elements were located by functional analysis within the first −135 bp of the promoter region. Using site‐directed mutagenesis and DNA‐protein binding assays we found that Sp1, Sp3, and Klf‐4 transcription factors bind to ATP2A3 proximal promoter elements and regulate basal gene expression. We showed that these factors participated in the increase of ATP2A3 expression during cancer cell differentiation. This study provides evidence for the first time that Sp1, Sp3, and Klf‐4 transcriptionally modulate the expression of SERCA3 during induction of epithelial cancer cell differentiation.

ATP2A3 gene as an important player for resveratrol anticancer activity in breast cancer cells

The Ca2+‐ATPases from the Sarco/endoplasmic reticulum (SERCA) are fundamental for maintaining intracellular [Ca2+] homeostasis by pumping Ca2+ into the endoplasmic reticulum (ER) of eukaryotic cells. SERCA enzymes are encoded by three different genes (ATP2A1‐3), whose expression occurs in a tissue and development stage‐specific manner. It has been reported alterations in the expression of SERCA2 and SERCA3 pumps in different types of cancer: oral, lung, colon, stomach, central nervous system, thyroid, breast, and prostate. Resveratrol (RSV), a phytoalexin produced by a wide variety of plants in response to stress situations can modulate cellular processes involved in all stages of carcinogenesis. In this work, we used breast cancer cell lines (MCF‐7 and MDA‐MB‐231) to evaluate mRNA levels of ATP2A2 and ATP2A3 genes in response to RSV treatment. Our results demonstrate that RSV treatment induced the expression of ATP2A3 gene in both cell lines in a time and concentration‐dependent manner, while the expression of ATP2A2 gene remained unaffected. The RSV‐induced expression of SERCA3 in these breast cancer cell lines produced decreased cell viability, triggered apoptosis and changes in cytosolic Ca2+ levels, as well as changes in the capacity for Ca2+ release by the ER. These data suggest an important participation of SERCA3 genes in RSV‐mediated anti‐tumor effect in breast cancer cell lines. Nevertheless, further research is needed to elucidate the molecular mechanisms underlying this effect.

Histone deacetylase inhibitors induce the expression of tumor suppressor genes Per1 and Per2 in human gastric cancer cells

Period circadian regulator (Per)1 and Per2 genes are involved in the molecular mechanism of the circadian clock, and exhibit tumor suppressor properties. Several studies have reported a decreased expression of Per1, Per2 and Per3 genes in different types of cancer and cancer cell lines. Promoter methylation downregulates Per1, Per2 or Per3 expression in myeloid leukemia, breast, lung, and other cancer cells; whereas histone deacetylase inhibitors (HDACi) upregulate Per1 or Per3 expression in certain cancer cell lines. However, the transcriptional regulation of Per1 and Per2 in cancer cells by chromatin modifications is not fully understood. The present study aimed to determine whether HDACi regulate Per1 and Per2 expression in gastric cancer cell lines, and to investigate changes in chromatin modifications in response to HDACi. Treatment of KATO III and NCI-N87 human gastric cancer cells with sodium butyrate (NaB) or Trichostatin A (TSA) induced Per1 and Per2 mRNA expression in a dose-dependent manner. Chromatin immunoprecipitaion assays revealed that NaB and TSA decreased lysine 9 trimethylation on histone H3 (H3K9me3) at the Per1 promoter. TSA, but not NaB increased H3K9 acetylation at the Per2 promoter. It was also observed that binding of Sp1 and Sp3 to the Per1 promoter decreased following NaB treatment, whereas Sp1 binding increased at the Per2 promoter of NaB- and TSA-treated cells. In addition, Per1 promoter is not methylated in KATO III cells, while Per2 promoter was methylated, although NaB, TSA, and 5-Azacytidine do not change the methylated CpGs analyzed. In conclusion, HDACi induce Per1 and Per2 expression, in part, through mechanisms involving chromatin remodeling at the proximal promoter of these genes; however, other indirect mechanisms triggered by these HDACi cannot be ruled out. These findings reveal a previously unappreciated regulatory pathway between silencing of Per1 gene by H3K9me3 and upregulation of Per2 by HDACi in cancer cells.

The cardiac calsequestrin gene transcription is modulated at the promoter by NFAT and MEF-2 transcription factors

Calsequestrin-2 (CASQ2) is the main Ca2+-binding protein inside the sarcoplasmic reticulum of cardiomyocytes. Previously, we demonstrated that MEF-2 and SRF binding sites within the human CASQ2 gene (hCASQ2) promoter region are functional in neonatal cardiomyocytes. In this work, we investigated if the calcineurin/NFAT pathway regulates hCASQ2 expression in neonatal cardiomyocytes. The inhibition of NFAT dephosphorylation with CsA or INCA-6, reduced both the luciferase activity of hCASQ2 promoter constructs (-3102/+176 bp and -288/+176 bp) and the CASQ2 mRNA levels in neonatal rat cardiomyocytes. Additionally, NFATc1 and NFATc3 over-expressing neonatal cardiomyocytes showed a 2-3-fold increase in luciferase activity of both hCASQ2 promoter constructs, which was prevented by CsA treatment. Site-directed mutagenesis of the -133 bp MEF-2 binding site prevented trans-activation of hCASQ2 promoter constructs induced by NFAT overexpression. Chromatin Immunoprecipitation (ChIP) assays revealed NFAT and MEF-2 enrichment within the -288 bp to +76 bp of the hCASQ2 gene promoter. Besides, a direct interaction between NFAT and MEF-2 proteins was demonstrated by protein co-immunoprecipitation experiments. Taken together, these data demonstrate that NFAT interacts with MEF-2 bound to the -133 bp binding site at the hCASQ2 gene promoter. In conclusion, in this work, we demonstrate that the Ca2+-calcineurin/NFAT pathway modulates the transcription of the hCASQ2 gene in neonatal cardiomyocytes.

Calcium-independent release of [3H]spermine from chick retina

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [3H]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [3H]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca2+-independent manner. Inhibition of Na+/K+-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na+ electrochemical gradients, since nigericin and veratrine did not induce release in Na+ containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.