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

Tirumurugaan KG, Kang BN, Panettieri RA, Foster DN, Walseth TF, Kannan MS - Respir. Res. (2008)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St, Paul, MN, USA. drthiru20@yahoo.com

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-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.

Conclusion: 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.

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Related in: MedlinePlus

The cloned 3 kb cd38 promoter showing the location of the putative binding sites for NF-κB, AP-1 and GR (labeled NF-κB, AP-1 and GRE). Location of the putative binding sites for NF-κB, AP-1, and GRE on the cd38 promoter, the 3 kb (Full) promoter, the truncated 1.8 kb promoter (Truncated), and promoter constructs with mutations in the binding sites for NF-κB or AP1–4 or both binding sites (NF-AP-mut, NF-mut and AP-mut). The promoter was cloned in front of a luciferase reporter gene in the pGL3 plasmid and was used to transfect HASM cells.
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Figure 7: The cloned 3 kb cd38 promoter showing the location of the putative binding sites for NF-κB, AP-1 and GR (labeled NF-κB, AP-1 and GRE). Location of the putative binding sites for NF-κB, AP-1, and GRE on the cd38 promoter, the 3 kb (Full) promoter, the truncated 1.8 kb promoter (Truncated), and promoter constructs with mutations in the binding sites for NF-κB or AP1–4 or both binding sites (NF-AP-mut, NF-mut and AP-mut). The promoter was cloned in front of a luciferase reporter gene in the pGL3 plasmid and was used to transfect HASM cells.

Mentions: The EMSA studies revealed that TNF-α increased the binding of nuclear proteins to the putative NF-κB site, and to some of the putative AP-1 sites in the cd38 promoter. Likewise, dexamethasone increased the binding of nuclear proteins selectively to some of the putative cd38 GREs. To investigate whether TNF-α modulates cd38 promoter activity directly, HASM cells were transiently transfected with a cd38 promoter-driven luciferase reporter construct. In the initial studies, we used the 3 kb promoter (Figure 7) and a truncated 1.8 kb promoter that lacks the NF-κB site and the AP1–4 site that exhibited very strong binding following TNF-α treatment. HASM cells were transiently transfected with the promoter constructs and luciferase activity was determined following exposure to TNF-α. TNF-α caused an increase in luciferase activity of the 3 kb promoter, but not the truncated 1.8 kb promoter, and dexamethasone decreased the TNF-α-induced activation of the 3 kb promoter (Figure 8)). To determine the transcription factor binding sites within the 3 kb promoter that are involved in the regulation of CD38 expression, HASM cells were transfected with site directed mutated constructs. For these studies, cd38 promoter luciferase constructs mutated at the NF-κB site or the AP1–4 site, or at both of these sites were used. Following exposure to TNF-α, luciferase activity was abolished in the promoter constructs with mutations of either the NF-κB or the AP1–4 sites, or mutation in both the sites (Figure 8). The EMSA results and the decreased activation of the promoter with mutations (that lack the NF-κB and the dominant AP1–4 binding sites) confirm a functional role for NF-κB and AP1–4 in the transcriptional regulation of CD38. Glucocorticoid regulation also involves binding to cd38 GREs and inhibition of NF-κB- and AP-1-dependent transcription.


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

Tirumurugaan KG, Kang BN, Panettieri RA, Foster DN, Walseth TF, Kannan MS - Respir. Res. (2008)

The cloned 3 kb cd38 promoter showing the location of the putative binding sites for NF-κB, AP-1 and GR (labeled NF-κB, AP-1 and GRE). Location of the putative binding sites for NF-κB, AP-1, and GRE on the cd38 promoter, the 3 kb (Full) promoter, the truncated 1.8 kb promoter (Truncated), and promoter constructs with mutations in the binding sites for NF-κB or AP1–4 or both binding sites (NF-AP-mut, NF-mut and AP-mut). The promoter was cloned in front of a luciferase reporter gene in the pGL3 plasmid and was used to transfect HASM cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2278140&req=5

Figure 7: The cloned 3 kb cd38 promoter showing the location of the putative binding sites for NF-κB, AP-1 and GR (labeled NF-κB, AP-1 and GRE). Location of the putative binding sites for NF-κB, AP-1, and GRE on the cd38 promoter, the 3 kb (Full) promoter, the truncated 1.8 kb promoter (Truncated), and promoter constructs with mutations in the binding sites for NF-κB or AP1–4 or both binding sites (NF-AP-mut, NF-mut and AP-mut). The promoter was cloned in front of a luciferase reporter gene in the pGL3 plasmid and was used to transfect HASM cells.
Mentions: The EMSA studies revealed that TNF-α increased the binding of nuclear proteins to the putative NF-κB site, and to some of the putative AP-1 sites in the cd38 promoter. Likewise, dexamethasone increased the binding of nuclear proteins selectively to some of the putative cd38 GREs. To investigate whether TNF-α modulates cd38 promoter activity directly, HASM cells were transiently transfected with a cd38 promoter-driven luciferase reporter construct. In the initial studies, we used the 3 kb promoter (Figure 7) and a truncated 1.8 kb promoter that lacks the NF-κB site and the AP1–4 site that exhibited very strong binding following TNF-α treatment. HASM cells were transiently transfected with the promoter constructs and luciferase activity was determined following exposure to TNF-α. TNF-α caused an increase in luciferase activity of the 3 kb promoter, but not the truncated 1.8 kb promoter, and dexamethasone decreased the TNF-α-induced activation of the 3 kb promoter (Figure 8)). To determine the transcription factor binding sites within the 3 kb promoter that are involved in the regulation of CD38 expression, HASM cells were transfected with site directed mutated constructs. For these studies, cd38 promoter luciferase constructs mutated at the NF-κB site or the AP1–4 site, or at both of these sites were used. Following exposure to TNF-α, luciferase activity was abolished in the promoter constructs with mutations of either the NF-κB or the AP1–4 sites, or mutation in both the sites (Figure 8). The EMSA results and the decreased activation of the promoter with mutations (that lack the NF-κB and the dominant AP1–4 binding sites) confirm a functional role for NF-κB and AP1–4 in the transcriptional regulation of CD38. Glucocorticoid regulation also involves binding to cd38 GREs and inhibition of NF-κB- and AP-1-dependent transcription.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St, Paul, MN, USA. drthiru20@yahoo.com

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-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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus