Abstract

BackgroundCD38 is expressed in human airway smooth muscle (HASM) cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-α). 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-α. Regulation of CD38 expression in HASM cells involves the transcription factor NF-κB, and glucocorticoids inhibit this expression through NF-κB-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.MethodsWe 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-κB 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-κB and some of the AP-1 sites, or the promoter with mutations of the NF-κB 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-κB, AP-1, and GRE sites, and the specificity of this binding was confirmed by gel supershift analysis with appropriate antibodies.ResultsTNF-α 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-κB and/or AP-1 sites or in the presence of dexamethasone, there was no induction in the presence of TNF-α. The binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB site and some of the six AP-1 sites was increased by TNF-α, and to some of the putative cd38 GREs by dexamethasone.ConclusionThe EMSA results and the cd38 promoter-reporter assays confirm the functional role of NF-κB, AP-1 and GREs in the cd38 promoter in the transcriptional regulation of CD38.

Highlights

  • CD38 is expressed in human airway smooth muscle (HASM) cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-α)

  • NF-κB, AP-1 and Glucocorticoid Receptor binding to the cd38 promoter To investigate the transcriptional regulation of CD38 expression in HASM cells, we cloned a putative 3 kb promoter fragment

  • Using the electrophoretic mobility shift assay (EMSA), we examined whether transcription factors from HASM nuclear extracts or recombinant human glucocorticoid receptor (GR) proteins can bind to these putative binding sites following exposure of cells to TNF-α and dexamethasone

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Summary

Introduction

CD38 is expressed in human airway smooth muscle (HASM) cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-α). CD38 is a pleiotropic protein that has enzymatic and receptor functions [1,2,3] It is a ~45-kDa glycosylated transmembrane protein, with an extracellular domain that has an enzyme activity which generates cyclic ADPribose (cADPR) and ADPR from nicotinamide adenine dinucleotide (NAD) [1]. Previous studies from our laboratory showed that CD38 expression and its enzymatic activities are augmented by TNF-α and IL-13, cytokines that are implicated in the pathogenesis of inflammatory airway diseases such as asthma [5,8]. The regulation of CD38 expression by TNF-α requires NF-κB activation and involves MAPK signaling in ASM cells [9,10]

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