HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-4585fjev1 20/7/1000    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kang, B.-N. Articles by Kannan, M. S. Search for Related Content PubMed PubMed Citation Articles by Kang, B.-N. Articles by Kannan, M. S.

Transcriptional regulation of CD38 expression by tumor necrosis factor- in human airway smooth muscle cells: role of NF-B and sensitivity to glucocorticoids

Bit-Na Kang^*, K. G. Tirumurugaan^*, Deepak A. Deshpande^*, Yassine Amrani, Reynold A. Panettieri, Timothy F. Walseth and Mathur S. Kannan^*,,1

^* Department of Veterinary and Biomedical Sciences,

Pharmacology and

Pediatrics, University of Minnesota, St. Paul, Minnesota, USA; and

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

¹Correspondence: Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Ave., St. Paul, MN 55108, USA. E-mail: kanna001{at}umn.edu

SPECIFIC AIMS

The enzyme ADP-ribosyl cyclase, which converts ßbeta;-NAD to the calcium-mobilizing second messenger molecule cyclic ADP-ribose, is integral to CD38, a type II transmembrane glycoprotein. In human airway smooth muscle (HASM) cells, CD38 expression and ADP-ribosyl cyclase activity are increased by tumor necrosis factor-alpha (TNF-), an inflammatory cytokine, resulting in augmented calcium responses to agonists, i.e., hyperresponsiveness. Glucocorticoids are used in the management of airway hyperresponsiveness, and the presence of the putative glucocorticoid response elements in the CD38 gene provides for transcriptional regulation of its expression. The transcription factor NF-B has a key role in the actions of TNF- and glucocorticoids. The aim of the study was to determine the role of NF-B in the regulation of CD38 expression and its function in HASM cells.

PRINCIPAL FINDINGS

1. NF-B has a central role in the regulation of CD38 expression
NF-B mediates the effects of TNF- on ASM and other cell types. However, the role of NF-B in mediating TNF--induced CD38 expression in ASM is unclear. TNF--induced NF-B activation was inhibited by SN-50, an inhibitor of NF-B activation (Fig. 1 ). TNF--induced CD38 expression was significantly attenuated in the presence of SN-50 or after transient transfection with a deletion mutant of inhibitor B (IB)- encoding amino acids 37–317 (IBN), or a dominant negative IB kinase (IKK)-ßbeta; with Ser-177 and Ser-181 replaced by alanine (IKK-AA) (Fig. 1) . Transient transfection with the C-terminal deletion mutant encoding amino acids 1–242 (IBC), on the other hand, did not inhibit TNF--induced CD38 expression. These findings support the hypothesis that NF-B mediates the effects of TNF- on CD38 expression.

View larger version (27K): [in this window] [in a new window]   Figure 1. A) Representative Western blot image showing the presence of p50 subunit of NF-B in the nuclear extracts prepared from HASM cells. C: control cells; TNF-: cells treated with 50 ng/ml TNF- for 22 h; SN50: cells pretreated with 18 µM SN50 followed by 50 ng/ml TNF- for 22 h; and SN50M: cells pretreated with 18 µM SN50M followed by 50 ng/ml TNF- for 22 h. Note decreased amount of p50 in nuclear extracts prepared from SN50-treated cells indicating decreased NF-B activation. Nuclear actin is shown as an internal control; n = 3. B) Results of quantitative real-time PCR shown as fold change in CD38 mRNA expression in HASM cells. Note significant (P<0.05) attenuation of CD38 expression in cells pretreated with SN50; n = 3. C) Results of transient transfection using the various IB mutants on TNF--induced CD38 expression in HASM cells. Lanes 1 and 2: untreated and TNF--treated cells after electroporation; lanes 3 and 4: cells transfected with the vector alone; lanes 5 and 6: cells transfected with the N-terminal deletion mutant; lanes 7 and 8: cells transfected with the C-terminal deletion mutant; and lanes 9 and 10: cells transfected with the IKK dominant negative mutant. Note attenuation of TNF--induced CD38 expression in the presence of N-terminal deletion and IKK dominant negative mutants and increased expression in the presence of the C-terminal deletion mutant. Representative of 3 experiments.

2. Dexamethasone attenuates TNF--induced CD38 expression and ADP-ribosyl cyclase activity
The inflammatory cytokines TNF-, IFN-, and interleukin (IL)-1ßbeta; and the Th-2 cytokine IL-13 increase the expression of CD38 in HASM cells. This results in increased ADP-ribosyl cyclase activity, production of cyclic ADP-ribose (cADPR), and agonist-induced intracellular calcium responses. The augmented intracellular calcium responses are attenuated by an antagonist of cADPR, indicating that CD38/cADPR signaling contributes to cytokine-induced changes in calcium homeostasis in HASM cells. Cloning and nucleotide sequence analysis from our laboratory has revealed one putative NF-B consensus binding site and four glucocorticoid response elements within the 5' untranslated region of the CD38 gene (Acc. No. DQ091293), providing evidence for transcriptional regulation of its expression. We investigated the effects of dexamethasone, a glucocorticoid, on CD38 expression and ADP-ribosyl cyclase activity in HASM cells stimulated with TNF-. Exposure of HASM cells to TNF- resulted in a significant increase in CD38 expression and ADP-ribosyl cyclase activity. TNF--induced CD38 expression was significantly attenuated by pretreatment of HASM cells with dexamethasone over a concentration range of 10 nM to 1 µM (Fig. 2 ). However, a significant decrease in ADP-ribosyl cyclase activity was seen only at 1 µM of dexamethasone. Dexamethasone did not inhibit basal expression of CD38 in HASM cells.

View larger version (18K): [in this window] [in a new window]   Figure 2. A) Dexamethasone inhibits TNF--induced CD38 mRNA expression in HASM cells. Results of quantitative real-time PCR shown as fold change in CD38 mRNA expression in control cells and in cells treated with 50 ng/ml TNF- for 22 h alone, or TNF- in the presence of 0.01, 0.1, and 1 µM dexamethasone. C + D refers to cells exposed to 1 µM dexamethasone. Different letters denote significant (P<0.05) differences between treatments in this and other figures. Note significant inhibition of TNF--induced CD38 expression in the presence of 0.1 and 1 µM dexamethasone; n = 5. Values are mean ± SE in this and other figures. B) Nuclear extracts were prepared from HASM cells and assayed for NF-B binding to 32P end-labeled NF-B oligonucleotide probe by EMSA. Effect of different concentrations of dexamethasone pretreatment (1 h) on TNF- (10 ng/ml)-induced NF-B activation was also measured. Specificity of NF-B binding was determined using 100-fold excess of unlabeled probe as well as a nonspecific competitor (activating protein-2). Specificity of NF-B complex is demonstrated by gel supershift using p65 or the p50 antibody. Note TNF--induced NF-B activation is decreased by 1 µM dexamethasone as well as by the NF-B inhibitory peptides (SN-50 and SN-50M). Representative of 3 assays.

Glucocorticoids are often used to reverse airway hyperresponsiveness resulting from exposure to inflammatory mediators. To simulate this, we investigated the effect of dexamethasone after the induced expression of CD38 by TNF-. The magnitude of CD38 expression in cells exposed to TNF- for 48–72 h was fivefold higher than in cells exposed for 22 h. On withdrawal of TNF-, there was a rapid decline in CD38 expression possibly reflecting termination of continued transcription and mRNA stability, and in the presence of dexamethasone, there was a greater decline in expression (data not shown). The ADP-ribosyl cyclase activity, i.e., CD38 protein, also declined on withdrawal of TNF-, with no further decline in the presence of dexamethasone (data not shown).

The mechanisms of action of glucocorticoids are complex and involve transcription factors including NF-B. The activation of NF-B results from the phosphorylation and ubiquitylation of the inhibitory IB subunit and the nuclear translocation of NF-B. NF-B regulates transcription of IB by binding to its promoter elements. The nuclear export signal within the IB facilitates the expulsion of the NF-B-IB complex, thereby terminating NF-B binding to DNA elements. Exposure to TNF- resulted in nuclear translocation of NF-B, as shown by electrophoretic mobility shift assays (Fig. 2B ). The specificity of the NF-B was confirmed by gel supershift using specific antibodies against the p65 and p50 subunits of NF-B. Dexamethasone attenuated TNF--induced NF-B activation only at the highest concentration used (1 µM). In cells treated with TNF-, there was a significant elevation of IB expression, which increased further in the presence of dexamethasone. These findings suggest that the inhibition of CD38 expression by glucocorticoids is due to decreased NF-B expression and its activation, the latter due to increased IB expression. Glucocorticoid effects through mechanisms independent of NF-B activation also appear to contribute to TNF--induced CD38 expression.

CONCLUSIONS AND SIGNIFICANCE

In HASM, NF-B mediates TNF--induced CD38 expression by direct transactivation and/or secondarily through NF-B-dependent genes. Glucocorticoids inhibit CD38 expression either through NF-B (decreased expression of NF-B and increased expression of IB) and/or directly by binding to response elements within cd38 to cause inhibition of transcription. These results demonstrate the pivotal role for NF-B in the regulation of CD38 expression in human airway smooth muscle cells and a model depicting the key findings are incorporated in Fig. 3 .

View larger version (30K): [in this window] [in a new window]   Figure 3. A model showing the regulation of CD38 expression by the inflammatory cytokine TNF- and glucocorticoids in human airway smooth muscle cells. Activation of TNF receptor 1 (TNFR1) by TNF causes receptor trimerization, recruitment of an adaptor protein complex and a downstream signaling cascade, resulting in IB phosphorylation, dissociation of the p50/p65 subunits of NF-B and their nuclear translocation to activate transcription of NF-B-dependent genes. Two possible modes of transcriptional regulation of cd38 are shown. A) Binding of the p50/p65 subunits of NF-B to response elements activates transcription of potential NF-B-dependent genes, which secondarily activate cd38 expression; B) Direct binding to response elements resulting in transactivation of cd38. Glucocorticoid (GC) binding to its receptor (GR) causes dissociation of the GR/heat shock protein complex, nuclear translocation of the GR homodimer and binding to glucocorticoid response element (GRE). Down-regulation of cd38 expression results from: transrepression of NF-B and potential NF-B-dependent genes; activation of IB transcription; direct binding to GRE motif within the cd38 to cause inhibition of transcription. Model also shows direct protein-protein interaction involving the GCR (GC bound to its receptor)/NF-B, which may result in mutual repression.

The CD38/cyclic ADP-ribose signaling contributes to normal airway function and to cytokine-mediated airway smooth muscle hyperresponsiveness. Therefore, elucidation of how this signaling pathway is regulated in inflammatory airway diseases such as asthma is important. Future investigations must address whether regulation of CD38 expression in human airway smooth muscle cells by NF-B results from binding to motifs within the CD38 gene (as shown in Fig. 3B ) or secondarily through NF-B-dependent genes (as shown in Fig. 3A ). Also, transrepression of cd38 through the glucocorticoid response element needs to be determined.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4585fje

This article has been cited by other articles:

				B. N. Kang, J. A. Jude, R. A. Panettieri Jr., T. F. Walseth, and M. S. Kannan Glucocorticoid regulation of CD38 expression in human airway smooth muscle cells: role of dual specificity phosphatase 1 Am J Physiol Lung Cell Mol Physiol, July 1, 2008; 295(1): L186 - L193. [Abstract] [Full Text] [PDF]

				O. Tliba, G. Damera, A. Banerjee, S. Gu, H. Baidouri, S. Keslacy, and Y. Amrani Cytokines Induce an Early Steroid Resistance in Airway Smooth Muscle Cells: Novel Role of Interferon Regulatory Factor-1 Am. J. Respir. Cell Mol. Biol., April 1, 2008; 38(4): 463 - 472. [Abstract] [Full Text] [PDF]

				A. G. P. Guedes, J. A. Jude, J. Paulin, H. Kita, F. E. Lund, and M. S. Kannan Role of CD38 in TNF-{alpha}-induced airway hyperresponsiveness Am J Physiol Lung Cell Mol Physiol, February 1, 2008; 294(2): L290 - L299. [Abstract] [Full Text] [PDF]

				J. A. Jude, M. E. Wylam, T. F. Walseth, and M. S. Kannan Calcium Signaling in Airway Smooth Muscle Proceedings of the ATS, January 1, 2008; 5(1): 15 - 22. [Abstract] [Full Text] [PDF]

				K. G. Tirumurugaan, J. A. Jude, B. N. Kang, R. A. Panettieri, T. F. Walseth, and M. S. Kannan TNF-{alpha} induced CD38 expression in human airway smooth muscle cells: role of MAP kinases and transcription factors NF-{kappa}B and AP-1 Am J Physiol Lung Cell Mol Physiol, June 1, 2007; 292(6): L1385 - L1395. [Abstract] [Full Text] [PDF]

				A. G. P. Guedes, J. Paulin, L. Rivero-Nava, H. Kita, F. E. Lund, and M. S. Kannan CD38-deficient mice have reduced airway hyperresponsiveness following IL-13 challenge Am J Physiol Lung Cell Mol Physiol, December 1, 2006; 291(6): L1286 - L1293. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-4585fjev1 20/7/1000    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kang, B.-N. Articles by Kannan, M. S. Search for Related Content PubMed PubMed Citation Articles by Kang, B.-N. Articles by Kannan, M. S.