Research

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

Krishnaswamy G Tirumurugaan^1* , Bit Na Kang^1* , Reynold A Panettieri² , Douglas N Foster³ , Timothy F Walseth⁴ and Mathur S Kannan^1,5

¹  Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA

²  Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA

³  Department of Animal Science, University of Minnesota, St. Paul, MN, USA

⁴  Department of Pharmacology, College of Medicine, University of Minnesota, Minneapolis, MN, USA

⁵  Department of Pediatrics, College of Medicine, University of Minnesota, Minneapolis, MN, USA

author email corresponding author email* Contributed equally

Respiratory Research 2008, 9:26doi:10.1186/1465-9921-9-26

Published:	14 March 2008

Abstract

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.