Glucocorticoid regulation of CD38 expression in human airway smooth muscle cells: role of dual specificity phosphatase 1

Abstract

The enzymatic activity of CD38, ADP-ribosyl cyclase, synthesizes the calcium mobilizing molecule cyclic ADP-ribose from beta-NAD. In human airway smooth muscle (HASM) cells, CD38 expression is augmented by the inflammatory cytokine, TNF-alpha, causing increased intracellular calcium response to agonists. The transcriptional and posttranscriptional regulation of CD38 expression involves signaling through MAPKs and requires activation of NF-kappaB and activator protein-1 (AP-1). The cytokine-augmented CD38 expression is decreased by anti-inflammatory glucocorticoids due to inhibition of NF-kappaB activation and other mechanisms. In this study, we investigated glucocorticoid regulation of CD38 expression in HASM cells through the MKP-1. In HASM cells, dexamethasone and TNF-alpha induced MKP-1 expression (both mRNA and protein) rapidly. Dexamethasone decreased TNF-alpha-induced phosphorylation of the major MAPKs, i.e., ERK, p38, and JNK, and decreased the activation of NF-kappaB and AP-1. Dexamethasone also decreased CD38 expression induced by TNF-alpha, and part of this effect was attributable to decreased transcript stability. In cells transfected with MKP-1-specific small interfering RNAs (siRNAs), there was significant attenuation of MKP-1 expression and partial, but nonsignificant, reversal of dexamethasone inhibition of CD38 expression. These results indicate that regulation of CD38 expression in HASM cells by glucocorticoids involves decreased signaling through MAPKs and activation of transcription factors. The glucocorticoid effects on decreased CD38 expression and function result from regulation through transcription and transcript stability.

Fig. 1.

Dexamethasone (Dex) increases MKP-1 expression. Human airway smooth muscle (HASM) cells were treated with 1–1,000 nM dexamethasone for 1 h, and MKP-1 mRNA and protein levels were measured by RT-PCR and Western blot analysis, respectively. A: note that dexamethasone increases the expression of MKP-1 mRNA over the entire concentration range used in the study. B: a representative Western blot showing MKP-1 protein expression in the presence of different concentrations of dexamethasone and in response to TNF-α with or without 100 nM dexamethasone. The band intensities (density units) of the control and dexamethasone-treated samples were normalized to actin expression. Density units represent expression in the treated samples relative to expression in the control sample. Representative of 3 separate experiments.

Fig. 2.

Inhibition of TNF-α-induced activation of MAPKs by dexamethasone in HASM cells. HASM cells were treated with TNF-α in the presence or absence of dexamethasone (1–1,000 nM). Representative Western blots showing expressions of phosphorylated (p-) and total MAPKs [p-p44/42 (p-ERK) and p44/42 (ERK), p-p38 and p38, and p-JNK and JNK]. A: cells were pretreated with 100 nM dexamethasone and collected at 15, 30, and 60 min after TNF-α treatment. Note rapid activation of the MAPKs and a decrease in their activation in the presence of dexamethasone (highlighted by square boxes). B: cells were pretreated with 1–1,000 nM dexamethasone and collected 15 min after TNF-α treatment. The band intensities of the phosphorylated MAPKs were normalized to the total MAPKs and shown as density units. Note dexamethasone concentration-dependent decrease in the activation of MAPKs. Representative of 3 separate experiments.

Fig. 3.

Dexamethasone decreases NF-κB and activator protein-1 (AP-1) activation following exposure to TNF-α. EMSAs demonstrating DNA binding of NF-κB to consensus oligonucleotide probe and AP-1 to cd38 putative oligonucleotide (AP-1–4) probe. Nuclear proteins were extracted from HASM cells treated with TNF-α in the presence of 1–1,000 nM dexamethasone. The specific complexes formed can be competed by 100-fold molar excess of unlabeled specific oligonucleotide probes [NF-κB consensus (NF-κB con) or AP-1–4]. A nonspecific oligonucleotide probe (SP1) was used as an internal control. A: a representative EMSA for NF-κB binding to consensus oligonucleotide sequences. Note increased NF-κB activation by TNF-α, which is decreased in the presence of dexamethasone (at ≥100 nM) (vertical arrows). Note gel supershift in the presence of anti-p65 and -p50 antibodies (labeled p65 and p50), demonstrating specificity of the assay (horizontal arrows). B: a representative EMSA for AP-1 binding to cd38 putative oligonucleotide sequences. Note increased AP-1 activation by TNF-α, which is decreased by dexamethasone (at ≥10 nM) (vertical arrows). Note gel supershift in the presence of anti-c-Jun antibody (labeled Jun) (horizontal arrow). These results are representative of 4 experiments. T, TNF-α. FP, Free Probe.

Fig. 4.

Effect of dexamethasone on TNF-α-induced CD38 expression in growth-arrested HASM cells. A: a representative gel image showing CD38 expression induced by TNF-α with or without dexamethasone (D) (1–1,000 nM). B: ADP-ribosyl cyclase activity shown as pmol·mg⁻¹·h⁻¹ in lysates of HASM cells treated with TNF-α (T) and dexamethasone. Note significant decreases in ADP-ribosyl cyclase activity in cells exposed to dexamethasone in a concentration-dependent manner. Results are expressed as means ± SE of 4–6 different experiments. C, control. * Significant decrease in ADP-ribosyl cyclase activity.

Fig. 5.

Effect of MKP-1 downregulation in HASM cells by transient transfection with SMARTpool small interfering RNA (siRNA) on CD38 expression. A, top trace: representative gel image showing MKP-1 expression in HASM cells following transient transfection with nontargeting siRNA or MKP-1 siRNA. The band intensities of MKP-1 are normalized to GAPDH and shown as density units. A, bottom trace: MKP-1 mRNA expression normalized to GAPDH expression in HASM cells. C, control (unstimulated cells); T, cells exposed to 40 ng/ml TNF-α for 24 h; D, cells exposed to 100 nM dexamethasone for 1 h; D+T, cells exposed to dexamethasone for 1 h followed by TNF-α for 24 h. Note significant decrease in MKP-1 expression in cells transfected with MKP-1 siRNA. Results are expressed as means ± SE of 3 different experiments. B, top trace: representative gel image of CD38 expression in HASM cells following transient transfection with nontargeting siRNA or MKP-1 siRNA. The band intensities of CD38 are normalized to GAPDH and shown as density units. B, bottom trace: CD38 expression in HASM cells following transfection with nontargeting siRNA or MKP-1 siRNA. Results are expressed as means ± SE of 3 different experiments.

Fig. 6.

Effect of dexamethasone on CD38 transcript stability. HASM cells were treated with TNF-α in the presence of 100 nM dexamethasone for 12 h. Cells were washed, replaced with fresh media containing actinomycin D to inhibit further transcription, and collected at 0 min, 15 min, 30 min, 1 h, 2 h, and 6 h for the determination of CD38 mRNA expression. Gel images show CD38 mRNA expression. GAPDH expression is used as an internal control. Note a rapid decline in the mRNA levels in the presence of dexamethasone (indicated by arrows). Representative of 3 experiments.