Dataset Information

Inflammation, caveolae and CD38-mediated calcium regulation in human airway smooth muscle.

ABSTRACT: The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF?) increases expression of CD38 (a membrane-associated bifunctional enzyme regulating cyclic ADP ribose), and enhances agonist-induced intracellular Ca(2+) ([Ca(2+)]i) responses in human airway smooth muscle (ASM). We previously demonstrated that caveolae and their constituent protein caveolin-1 are important for ASM [Ca(2+)]i regulation, which is further enhanced by TNF?. Whether caveolae and CD38 are functionally linked in mediating TNF? effects is unknown. In this regard, whether the related cavin proteins (cavin-1 and -3) that maintain structure and function of caveolae play a role is also not known. In the present study, we hypothesized that TNF? effects on CD38 expression and function in human ASM involve caveolae. Caveolar fractions from isolated human ASM cells expressed CD38 and its expression was upregulated by exposure to 20ng/ml TNF? (48h). ASM cells expressed cavin-1 and cavin-3, which were also upregulated by TNF?. Knockdown of caveolin-1, cavin-1 or cavin-3 (using siRNA) all significantly reduced CD38 expression and ADP-ribosyl cyclase activity in the presence or absence of TNF?. Furthermore, caveolin-1, cavin-1 and cavin-3 siRNAs reduced [Ca(2+)]i responses to histamine under control conditions, and blunted the enhanced [Ca(2+)]i responses in TNF?-exposed cells. These data demonstrate that CD38 is expressed within caveolae and its function is linked to the caveolar regulatory proteins caveolin-1, cavin-1 and -3. The link between caveolae and CD38 is further enhanced during airway inflammation demonstrating the important role of caveolae in regulation of [Ca(2+)]i and contractility in the airway.

SUBMITTER: Sathish V

PROVIDER: S-EPMC3926667 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Inflammation, caveolae and CD38-mediated calcium regulation in human airway smooth muscle.

Sathish Venkatachalem V Thompson Michael A MA Sinha Sutapa S Sieck Gary C GC Prakash Y S YS Pabelick Christina M CM

Biochimica et biophysica acta 20131122 2

The pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) increases expression of CD38 (a membrane-associated bifunctional enzyme regulating cyclic ADP ribose), and enhances agonist-induced intracellular Ca(2+) ([Ca(2+)]i) responses in human airway smooth muscle (ASM). We previously demonstrated that caveolae and their constituent protein caveolin-1 are important for ASM [Ca(2+)]i regulation, which is further enhanced by TNFα. Whether caveolae and CD38 are functionally linked in mediating ...[more]

PMID: 24275509

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Project description:CD38 is expressed in human airway smooth muscle (HASM) cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-alpha). CD38 has a role in airway hyperresponsiveness, a hallmark of asthma, since deficient mice develop attenuated airway hyperresponsiveness compared to wild-type mice following intranasal challenges with cytokines such as IL-13 and TNF-alpha. Regulation of CD38 expression in HASM cells involves the transcription factor NF-kappaB, and glucocorticoids inhibit this expression through NF-kappaB-dependent and -independent mechanisms. In this study, we determined whether the transcriptional regulation of CD38 expression in HASM cells involves response elements within the promoter region of this gene.We cloned a putative 3 kb promoter fragment of the human cd38 gene into pGL3 basic vector in front of a luciferase reporter gene. Sequence analysis of the putative cd38 promoter region revealed one NF-kappaB and several AP-1 and glucocorticoid response element (GRE) motifs. HASM cells were transfected with the 3 kb promoter, a 1.8 kb truncated promoter that lacks the NF-kappaB and some of the AP-1 sites, or the promoter with mutations of the NF-kappaB and/or AP-1 sites. Using the electrophoretic mobility shift assays, we determined the binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-kappaB, AP-1, and GRE sites, and the specificity of this binding was confirmed by gel supershift analysis with appropriate antibodies.TNF-alpha induced a two-fold activation of the 3 kb promoter following its transfection into HASM cells. In cells transfected with the 1.8 kb promoter or promoter constructs lacking NF-kappaB and/or AP-1 sites or in the presence of dexamethasone, there was no induction in the presence of TNF-alpha. The binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-kappaB site and some of the six AP-1 sites was increased by TNF-alpha, and to some of the putative cd38 GREs by dexamethasone.The EMSA results and the cd38 promoter-reporter assays confirm the functional role of NF-kappaB, AP-1 and GREs in the cd38 promoter in the transcriptional regulation of CD38.

Dataset Information

Inflammation, caveolae and CD38-mediated calcium regulation in human airway smooth muscle.

Publications

Inflammation, caveolae and CD38-mediated calcium regulation in human airway smooth muscle.

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