Caveolin-1 in cytokine-induced enhancement of intracellular Ca(2+) in human airway smooth muscle

Abstract

Diseases such as asthma are characterized by airway hyperresponsiveness. Enhanced airway smooth muscle (ASM) intracellular Ca(2+) ([Ca(2+)](i)) response to agonist stimulation leading to increased airway constriction has been suggested to contribute to airway hyperresponsiveness. Caveolae are flask-shaped plasma membrane invaginations that express the scaffolding protein caveolin and contain multiple proteins important in [Ca(2+)](i) signaling (e.g., agonist receptors, ion channels). We recently demonstrated that caveolae and caveolin-1 are important in [Ca(2+)](i) regulation in human ASM. Proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-13 modulate [Ca(2+)](i) in ASM. We hypothesized that cytokine upregulation of caveolar signaling in ASM contributes to enhanced agonist-induced [Ca(2+)](i) in inflammation. Enzymatically dissociated human ASM cells were exposed to medium (control), 20 ng/ml TNF-α, or 50 ng/ml IL-13 for 24 h. Caveolae-enriched membrane fractions displayed substantial increase in caveolin-1 and -2 expressions by TNF-α and IL-13. Transfection with caveolin-1-mRed DNA substantially accelerated and increased plasma membrane caveolin-1 expression by TNF-α and to a lesser extent by IL-13. Caveolin-1 enhancement was inhibited by nuclear factor-κB and mitogen-activated protein kinase inhibitors. In fura 2-loaded ASM cells, [Ca(2+)](i) responses to 1 μM ACh, 10 μM histamine, or 10 nM bradykinin were all exaggerated by TNF-α as well as IL-13 exposure. However, disruption of caveolae using caveolin-1 suppression via small-interfering RNA resulted in significant blunting of agonist-induced [Ca(2+)](i) responses of vehicle and TNF-α-exposed cells. These functional data were correlated to the presence of TNFR(1) receptor (but not the IL-4/IL-13 receptor) within caveolae. Overall, these results indicate that caveolin-1 plays an important role in airway inflammation by modulating the effect of specific cytokines on [Ca(2+)](i).

Fig. 1.

Effect of proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin-13 (IL-13) on caveolin-1, -2, and -3 expression in caveolae-enriched fractions of human airway smooth muscle (ASM) cells. ASM cell plasma membrane fractions enriched in caveolae were obtained as described previously (33). Exposure for 24 h to 20 ng/ml TNF-α significantly increased caveolin-1, and to a lesser extent caveolin-2, expression. Exposure to IL-13 also increased caveolin-1 and -2 expression compared with control, but to a lesser extent compared with TNF-α. Caveolin-3 was not expressed within caveolar membrane fractions of human ASM, even with cytokine exposure (positive controls such as rat heart or human diaphragm containing caveolin-3 not shown). AU, arbitrary units. Values are means ± SE. *Significant TNF-α effect compared with vehicle control. #Significant IL-13 effect compared with vehicle control. @Significant difference between TNF-α and IL-13 (P < 0.05).

Fig. 2.

Effect of TNF-α on caveolin-1 expression and intracellular Ca²⁺ ([Ca²⁺]_i) responses. A: Western analysis demonstrated increased caveolin-1 with TNF-α exposure, an effect that was significantly reduced in the presence of either 2 μM PD-98059 [mitogen-activated protein (MAP) kinase inhibitor] or 20 μM SN-50 or 1 μM CAY-10512 [both nuclear factor-κB (NF-κB) inhibitors], respectively. B: [Ca²⁺]_i responses under the same experimental conditions as shown above. While TNF-α significantly increased [Ca²⁺]_i responses, additional exposure to PD-98059, SN-50, or CAY-10512 significantly inhibited [Ca²⁺]_i responses, suggesting a functional role for MAP kinases and NF-κB as underlying mechanisms. Values are means ± SE. *Significant difference from control. #Significant inhibitor effect vs. TNF-α treatment (P < 0.05).

Fig. 3.

Effect of caveolin-1 small-interfering RNA (siRNA) on TNF-α enhancement of [Ca²⁺]_i responses of ASM cells to agonist stimulation. Exposure for 24 h to TNF-αsignificantly increased [Ca²⁺]_i responses to ACh (1 μM), histamine (10 μM), and bradykinin (10 nM) compared with buffer (vehicle) control (A). In cells transfected with caveolin-1 siRNA, [Ca²⁺]_i responses to all agonists were significantly decreased compared with transfection vehicle (Lipofectamine) controls (B). Caveolin-1 siRNA also blunted [Ca²⁺]_i responses to agonist in the presence of TNF-α; however, in the case of histamine and bradykinin (but not ACh), there remained a residual enhancing effect of TNF-α on [Ca²⁺]_i. Values are means ± SE. *Significant TNF-α effect. #Significant cav-1 siRNA effect compared with Lipofectamine control. @Significant cav-1 siRNA effect in the presence of TNF-α (compared to TNF-α in Lipofectamine alone) (P < 0.05).

Fig. 4.

Effect of caveolin-1 siRNA on IL-13 enhancement of [Ca²⁺]_i responses to agonist stimulation. Exposure to IL-13 significantly increased [Ca²⁺]_i responses to ACh, histamine, and bradykinin compared with control (with a greater effect on responses to ACh) (A). In cells transfected with caveolin-1 siRNA, [Ca²⁺]_i responses to all agonists were decreased significantly compared with control (B). However, even with caveolin-1 siRNA, exposure to IL-13 increased the [Ca²⁺]_i responses to all agonists. The effect of caveolin-1 siRNA on IL-13 enhancement of [Ca²⁺]_i was not as pronounced as with TNF-α. Values are means ± SE. *Significant IL-13 effect. #Significant cav-1 siRNA effect compared with Lipofectamine control. @Significant cav-1 siRNA effect in the presence of IL-13 (compared with IL-13 in Lipofectamine alone) (P < 0.05).

Fig. 5.

Effect of caveolin-1 overexpression on cytokine effects on [Ca²⁺]_i responses. Overexpression of caveolin-1 using cav-1-mRed significantly increased [Ca²⁺]_i responses to ACh, histamine, and bradykinin compared with nontransfected controls (A). Exposure to TNF-α increased [Ca²⁺]_i responses to all agonists (B). The presence of cav-1-mRed further enhanced the effect of TNF-α on [Ca²⁺]_i. The effect of cav-1-mRed on IL-13-induced increase in [Ca²⁺]_i responses was minimal (C). Values are means ± SE. *Significant mRed cav-1 effect. #Significant cytokine effect compared with Lipofectamine control (P < 0.05).

Fig. 6.

Caveolar expression of TNF receptor 1 (TNFR₁) and IL-4α receptor (IL-4Rα). Both TNFR₁ and IL4Rα were expressed in whole ASM cell lysates and were significantly increased when cells were exposed to TNF-α or IL-13, respectively (A). However, only TNFR₁ was expressed within caveolar membrane fractions, with increased expression following TNF-α exposure (B). Such differential expression may underlie the different effects of altered caveolin-1 expression on TNF-α vs. IL-13 enhancement of [Ca²⁺]_i. Values are means ± SE. *Significant cytokine effect vs. control (P < 0.05).