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Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone

BACKGROUND: 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 has a role in airway hyperresponsiveness, a hallmark of asthma, since deficient mice develop attenuated airway hyper...

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Autores principales: Tirumurugaan, Krishnaswamy G, Kang, Bit Na, Panettieri, Reynold A, Foster, Douglas N, Walseth, Timothy F, Kannan, Mathur S
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2278140/
https://www.ncbi.nlm.nih.gov/pubmed/18341691
http://dx.doi.org/10.1186/1465-9921-9-26
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author Tirumurugaan, Krishnaswamy G
Kang, Bit Na
Panettieri, Reynold A
Foster, Douglas N
Walseth, Timothy F
Kannan, Mathur S
author_facet Tirumurugaan, Krishnaswamy G
Kang, Bit Na
Panettieri, Reynold A
Foster, Douglas N
Walseth, Timothy F
Kannan, Mathur S
author_sort Tirumurugaan, Krishnaswamy G
collection PubMed
description BACKGROUND: 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 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. METHODS: 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-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. RESULTS: TNF-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. CONCLUSION: The 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.
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spelling pubmed-22781402008-04-02 Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone Tirumurugaan, Krishnaswamy G Kang, Bit Na Panettieri, Reynold A Foster, Douglas N Walseth, Timothy F Kannan, Mathur S Respir Res Research BACKGROUND: 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 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. METHODS: 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-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. RESULTS: TNF-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. CONCLUSION: The 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. BioMed Central 2008 2008-03-14 /pmc/articles/PMC2278140/ /pubmed/18341691 http://dx.doi.org/10.1186/1465-9921-9-26 Text en Copyright © 2008 Tirumurugaan et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Tirumurugaan, Krishnaswamy G
Kang, Bit Na
Panettieri, Reynold A
Foster, Douglas N
Walseth, Timothy F
Kannan, Mathur S
Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone
title Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone
title_full Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone
title_fullStr Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone
title_full_unstemmed Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone
title_short Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone
title_sort regulation of the cd38 promoter in human airway smooth muscle cells by tnf-α and dexamethasone
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2278140/
https://www.ncbi.nlm.nih.gov/pubmed/18341691
http://dx.doi.org/10.1186/1465-9921-9-26
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