Na(+)/Ca(2+) exchanger isoforms in nervous tissue and isoform-specific regulation by PKA
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Na+/Ca2+ exchanger isoforms in nervous tissue and isoform-specific regulation by PKAAbstract
In many tissues including nervous tissue, cytoplasmic Ca2+ concentration is regulated by the plasma membrane Na+/Ca{2+ exchanger. I have measured Na+/Ca2+ exchanger activity in brain-derived primary astrocyte cultures by using Na+-dependent45Ca2+ influx. Multiple Na+/Ca2+ exchanger genes have been identified in the rat brain tissue. I used RT-PCR cloning and RNase protection to show that NCX1 mRNA was the main transcript expressed in primary astrocyte and neuron cultures compared to NCX2. The NCX1 gene has previously been demonstrated to undergo alternative splicing of the primary transcript in the carboxyl end of the intracellular loop of the Na+/Ca2+ exchanger protein to produce multiple tissue-specific isoforms. This alternative splicing is generated by six exons present in the gene (A, B, C, D, E and F), among which exons A and B are mutually exclusive in their expression and the other four are "cassette" type exons. Using RT-PCR cloning, I demonstrated the presence of four NCX1 isoforms (BDEF, BDF, BDE and BD) in the cultured astrocytes and two NCX1 isoforms (ADF and AD) in the cultured neurons. To quantitate the relative mRNA amounts of these NCX1 isoforms, I developed a new method, quantitative end-labeled RT-PCR (QERT-PCR). Using this method, I have demonstrated that three exon B-containing isoforms (BDEF, BDF and BD) are predominant in the primary astrocyte culture and each represents about 23% of the total QERT-PCR product. Interestingly, QERT-PCR analysis of the C6 astrocytoma cell line has shown a similar pattern for the predominant isoforms as in the primary astrocyte culture. In the primary neuron culture, QERT-PCR results demonstrate that two exon A-containing isoforms (ADF and AD) are the predominant representing 57% and 37% of the NCX1 transcripts, respectively. While alternative splicing of NCX1 would predict that up to 32 different Na+/Ca2+ exchanger mRNAs may be produced by various combination of exons A to F, by using QERT-PCR, I have demonstrated there are a total of only 16 isoforms including the 3 predominant ones in the primary astrocyte culture. Each of these isoforms contain either exon A or B and all have exon D in the sequence. To determine the functional significance underlying the different isoforms in these cells, a representative astrocytic isoform (BD) and neuronal isoform (AD) have been subcloned into the full-length NCX1 cDNA. When RNA from these constructs were expressed in Xenopus oocytes, both isoforms showed the Na+/Ca2+ exchanger activity. Importantly, the activity of the AD isoform increased by 31-54% after activating the protein kinase A (PKA) pathway whereas the activity of the BD isoform did not change. Function of chimeric constructs containing parts of exon A and B sequences have been examined, demonstrating that the carboxyl terminal 20 amino acids of exon A were important to enable PKA to increase the Na+/Ca2+ exchanger activity. Additionally, experiments with exon D deletion construct showed that this exon was also necessary for the PKA-dependent modulation of the Na+/Ca2+ exchanger activity.Description
University of Maryland, Baltimore. Microbiology and Immunology. Ph.D. 1998Keyword
Biology, Molecularisoform-specific regulation
PKA
Cyclic AMP-Dependent Protein Kinases
Nerve Tissue
Sodium-Calcium Exchanger--genetics
Identifier to cite or link to this item
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Pro-inflammatory Cytokine IFNγ and Microbiome-Derived Metabolites Dictate Epigenetic Switch between FOXP3 Isoforms in Celiac DiseaseSerena, Gloria; Fasano, Alessio; Shea-Donohue, Terez (2017)Celiac disease (CD) is an autoimmune enteropathy triggered by gluten and characterized by a strong Th1/Th17 immune response in the small intestine. Treg cells are CD4+ CD25++ cells that regulate the immune response and maintain immune homeostasis. Given its role in controlling the differentiation and function of Treg cells, FOXP3 has been considered their master transcription factor. Two main isoforms of FOXP3 have been described. Conversely to its counterpart FOXP3 full length (FL), the alternatively spliced isoform FOXP3 ∆2 cannot properly down-regulate Th17 driven immune response. Since the active state of CD has been associated with impairments in Treg cell function, we aimed at determining whether imbalances between the FOXP3 isoforms may be associated with the development of the disease. Intestinal biopsies from patients with active CD showed increased expression of the less functional FOXP3 ∆2 isoform over FL, while both isoforms were similarly expressed in non-celiac control subjects (HC). Conversely to what we saw in the intestine, peripheral blood mononuclear cells (PBMC) from HC subjects did not show the same balance between the two isoforms. We therefore hypothesized that the intestinal microenvironment may play a role in modulating the alternative splicing of FOXP3 isoforms. The pro-inflammatory intestinal microenvironment of active patients have been reported to be enriched in butyrate producing bacteria, while high concentrations of lactate have been shown to characterize the preclinical stage of the disease. We show that the combination of IFNγ (the main pro-inflammatory cytokine produced in the intestine of active CD patients) and butyrate triggers the balance between FOXP3 isoforms in HC subjects, while the same does not occur in CD patients. Furthermore we report that lactate significantly increases both isoforms in CD patients when compared to control subjects. Collectively these findings highlight the importance of the ratio between FOXP3 isoforms in CD and, for the first time, mechanistically associate the alternative splicing process with microbial derived metabolites.
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Muscle-specific and fiber type-specific regulation of the gene encoding the human slow-twitch skeletal muscle-specific isoform of troponin ICorin, Shari Jill; Wade, Robert P. (1995)Muscle-specific contractile proteins are encoded by multigene families, most of whose members are differentially expressed in fast versus slow twitch myofibers. This fiber type-specific gene regulation occurs by unknown mechanisms, and is not observed within cultured myocytes. Hence, the only means by which to study fiber type-specific gene regulation has been by generating numerous lines of transgenic mice, which is expensive and laborious. The goal of these studies was to develop an improved system by which the molecular mechanisms of fiber type-specific gene regulation could be dissected. The gene encoding the human slow twitch skeletal muscle-specific isoform of troponin I (TnI{dollar}\sb{lcub}\rm s{rcub}{dollar}) was chosen as a model gene, because expression of TnI{dollar}\sb{lcub}\rm s{rcub}{dollar} is largely restricted to slow twitch myofibers in adult mammals. Structural analysis showed that the TnI{dollar}\sb{lcub}\rm s{rcub}{dollar} gene contains nine exons spanning 12.5 kilobases. Transcriptional analysis revealed two transcription initiation sites. A muscle-specific promoter and a muscle-specific enhancer were identified 5' to the TnI{dollar}\sb{lcub}\rm s{rcub}{dollar} transcription initiation region. Next, an assay by which to identify DNA elements involved in fiber type-specific gene regulation was developed: this assay employs gene transfer into the muscles of live rats. A plasmid-borne luciferase reporter gene fused to various muscle-specific contractile gene promoters was differentially expressed when injected into slow versus fast twitch rat muscle: the luciferase gene was preferentially expressed in slow muscle when fused to a TnI{dollar}\sb{lcub}\rm s{rcub}{dollar} promoter, and conversely, was preferentially expressed in fast muscle when fused to a fast troponin C promoter. In contrast, the luciferase gene was equally well-expressed by both muscle types when fused to a non-fiber type-specific skeletal actin promoter. Deletion analysis of the TnI{dollar}\sb{lcub}\rm s{rcub}{dollar} promoter region revealed that the 157 base pair muscle-specific enhancer conferred slow muscle-preferential activity upon a minimal thymidine kinase promoter. Transgenic analysis confirmed the role of this enhancer in restricting gene expression to slow twitch myofibers. This delineation of a fiber type-specific control element represents a significant advance toward understanding fiber type-specific gene regulation at the molecular level. Hence, somatic gene transfer may be used to rapidly define elements that direct myofiber type-specific gene expression, prior to the generation of transgenic mice.
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Developmental regulation of the human slow troponin I isoform gene in skeletal and cardiac musclesZhu, Lei; Wade, Robert P. (1996)The differentiation and maturation of striated muscle involves numerous alterations in the pattern of contractile protein isoform gene expression which ultimately lead to the mature phenotype of distinct muscles. The molecular mechanisms controlling these processes remain poorly understood. Troponin I (TnI) is one of the contractile proteins that is subjected to this complex regulation. Three TnI isoform genes are differentially expressed in slow and fast skeletal myofibers and adult cardiac muscle. We first studied the expression profile of the three TnI isoform genes during murine development. Detailed analysis showed that the slow isoform of TnI (TnI{dollar}\rm \sb{lcub}S{rcub}){dollar} is the predominant isoform found in early development of both skeletal and cardiac muscles. The differential expression pattern of TnI isoform genes occurs at late fetal and postnatal stages. During this period, TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} becomes restricted to slow twitch skeletal myofibers and the conductive tissues of the heart. Next, we focused on the regulation of the human TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} gene, in an effort to identify cis-regulatory elements which govern its skeletal muscle fiber type-specific gene expression as well as those regulating TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} gene expression in the developing heart. Transgenic mice harboring 4,200 bp of the 5{dollar}\sp\prime{dollar} flanking sequence of the human TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} gene exhibited proper transgene expression in adult slow twitch myofibers and in the developing heart with the notable exception of aberrantly low level of expression in fetal ventricles. In vitro transfection assays had identified two enhancer elements, an upstream USE and an intron 1 INE, and sequence within the first 95 bp of upstream from the transcription start site which play important roles in the cell type-specific expression. Additional lines of transgenic mice driven by various combinations of the cis-regulatory elements from the TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} gene were generated to further define their in vivo functions. Our results suggested that separate regulatory elements regulate the human TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} gene in skeletal versus developing cardiac muscles. The USE or INE is able to activate the {dollar}-{dollar}95 promoter in the developing skeletal muscle. In addition, activity of the INE is down-regulated during postnatal development and it appears not to be involved in slow skeletal muscle fiber type-specificity. The differences between the activities of these two enhancer elements support our hypothesis that the INE may be required for proper developmental regulation, and the USE is the primary determinant for slow fiber restricted gene expression. However, in contrast to in vitro expression data, even the combination of the two enhancers is not sufficient to activate the {dollar}-{dollar}95 promoter in cardiac muscle. Our studies show that aspects of the spatial and temporal expression pattern of a tissue-specific gene during development can be conferred by separate and distinct regulatory elements. Moreover, expression of the TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} gene in the embryonic and fetal heart may be governed by a complex mechanisms some of which are not readily amenable to in vitro study.