Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-alpha 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-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.


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-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.


RESULTS
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.


CONCLUSIONS
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.
Tirumurugaan
Krishnaswamy G
KG
Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St, Paul, MN, USA. drthiru20@yahoo.com
Kang
Bit Na
BN
Panettieri
Reynold A
RA
Foster
Douglas N
DN
Walseth
Timothy F
TF
Kannan
Mathur S
MS
D051997:ADP-ribosyl Cyclase 1 / Q000235:genetics*; D016535:Bronchial Hyperreactivity / Q000503:physiopathology; D002478:Cells, Cultured; D003907:Dexamethasone / Q000378:metabolism / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D005938:Glucocorticoids / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D014132:Trachea / Q000166:cytology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology*
D005938:Glucocorticoids; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011965:Receptors, Glucocorticoid; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D003907:Dexamethasone; D008156:Luciferases; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1