Caveolin-1 regulation of store-operated Ca(2+) influx in human airway smooth muscle

Abstract

Caveolae, plasma membrane invaginations with constitutive caveolin proteins, harbour proteins involved in intracellular calcium ([Ca(2+)](i)) regulation. In human airway smooth muscle (ASM), store-operated Ca(2+) entry (SOCE) is a key component of [Ca(2+)](i) regulation, and contributes to increased [Ca(2+)](i) in inflammation. SOCE involves proteins Orai1 and stromal interaction molecule (STIM)1. We investigated the link between caveolae, SOCE and inflammation in ASM. [Ca(2+)](i) was measured in human ASM cells using fura-2. Small interference RNA (siRNA) or overexpression vectors were used to alter expression of caveolin-1 (Cav-1), Orai1 or STIM1. Tumour necrosis factor (TNF)-α was used as a representative pro-inflammatory cytokine. TNF-α increased SOCE following sarcoplasmic reticulum Ca(2+) depletion, and increased whole-cell and caveolar Orai1 (but only intracellular STIM1). Cav-1 siRNA decreased caveolar and whole-cell Orai1 (but not STIM1) expression, and blunted SOCE, even in the presence of TNF-α. STIM1 overexpression substantially enhanced SOCE: an effect only partially reversed by Cav-1 siRNA. In contrast, Orai1 siRNA substantially blunted SOCE even in the presence of TNF-α. Cav-1 overexpression significantly increased Orai1 expression and SOCE, especially in the presence of TNF-α. These results demonstrate that caveolar expression and regulation of proteins such as Orai1 are important for [Ca(2+)](i) regulation in human ASM cells and its modulation during inflammation.

FIGURE 1

Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within the plasma membrane (top row; inset in composite panel magnified 10×). STIM1 was primarily expressed in the intracellular compartments (bottom row; inset in composite panel magnified 10×). Cav-1 secondary stain was Alexa488 (green), while STIM1 or Orai1 were visualised using Alexa568 (red). Composite represents overlap of green and red images from same cell. b) Cav-1, STIM1 and Orai1 expression were also determined in isolated plasma membrane (PM) and microsomal (MS) fractions from ASM cells. Cellular fractions confirmed that Cav-1 and Orai1 were highly expressed in PM fractions, while STIM1 expression was limited to the heavy MS fraction. Scale bar: 10 µm.

FIGURE 2

Effect of caveolin (Cav)-1 overexpression and tumour necrosis factor (TNF)-α on human airway smooth muscle (ASM) cells. a) Confocal fluorescence imaging demonstrated that overexpression of Cav-1 using Cav-1-mRed significantly increased Cav-1 expression compared with vehicle (control-mRed transfection). b) Western blots analysis showed that exposure to TNF-α increased Cav-1 (as shown before). This effect was further enhanced in the presence of Cav-1-mRed. Data are presented as mean±se. ^*: significant Cav-1-mRed effect; ^#: significant TNF-α effect (all p<0.05).

FIGURE 3

Effect of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α, caveolin (Cav)-1 small interference RNA (siRNA) and Cav-1 overexpression (Cav-1-mRed) on store-operated Ca²⁺ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Representative intracellular Ca²⁺ ([Ca²⁺]_i) tracings demonstrating SOCE in the different experimental groups. After removal of extracellular Ca²⁺, sarcoplasmic reticulum Ca²⁺ stores were depleted with cyclopiazonic acid (CPA). Subsequent rapid introduction of extracellular Ca²⁺ resulted in activation of SOCE (in the continued presence of CPA). b) Overnight exposure to TNF-α significantly increased SOCE in comparison with vehicle control. In cells transfected with Cav-1 siRNA, SOCE was significantly decreased compared with vehicle control and TNF-α. Overexpression of Cav-1 using Cav-1-mRed significantly increased SOCE compared with vehicle control. Exposure to TNF-α further enhanced SOCE in the presence of Cav-1-mRed. Data are presented as mean±se. ^*: significant TNF-α effect; ^#: significant siRNA effect; ^¶: significant Cav-1-mRed effect (all p<0.05).

FIGURE 4

Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM cell lysates. In Cav-1 small interference RNA (siRNA)-transfected cells, Orai1 expression significantly decreased in both in cell lysate and caveolar fractions (a and b). In Cav-1-mRed-transfected cells, Orai1 expression significantly increased in both in cell lysate and caveolar fractions in the presence of TNF-α. Interestingly, STIM1 expression was very low within caveolar membrane fractions in comparison to Orai1 and did not show significant changes (b). Data are presented as mean±se. ^*: significant TNF-α effect; ^#: significant Cav-1 siRNA effect; ^¶: significant Cav-1-mRed effect (all p<0.05).

FIGURE 5

Effect of Orai1 small interference RNA (siRNA) on intracellular Ca²⁺ ([Ca²⁺]_i) and store-operated Ca²⁺ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth muscle α-actin served as loading control. b) Orai1 siRNA significantly reduced histamine-induced [Ca²⁺]_i responses compared with vehicle and negative control. c) Orai1 siRNA significantly reduced SOCE compared with vehicle control. Exposure to 5 µM Cav-1-specific caveolin scaffolding domain peptide (CSD; 6 h) significantly reduced SOCE (confirming the Cav-1 siRNA effect on SOCE in figure 3). Combination of Orai1 siRNA and CSD also significantly reduced SOCE compared with vehicle control but not in comparison with Orai1 siRNA or CSD alone. Cav-1-mRed significantly increased SOCE, which was decreased by Orai1 siRNA. Orai1 siRNA significantly reduced SOCE in the presence and absence of tumour necrosis factor (TNF)-α, confirming the importance of Orai1 during airway inflammation. Data are presented as mean±se. ^*: significant Orai1 siRNA effect; ^#: significant CSD effect; ^¶: significant Cav-1-mRed effect; ^§: significant TNF-α effect (all p<0.05).

FIGURE 6

Effect of stromal interaction molecule (STIM)1 overexpression and caveolin (Cav)-1 small interference RNA (siRNA) on store-operated Ca²⁺ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Overexpression of STIM1 using green fluorescent protein (GFP)-STIM1 significantly increased SOCE when compared with vehicle control (transfection control). STIM1 siRNA significantly reduced SOCE when compared with vehicle control. Cav-1 siRNA transfection partially reduced the GFP-STIM1-mediated increase in SOCE. b) GFP-STIM1 transfection significantly increased STIM1 expression confirmed by Western blot analysis. Data are presented as mean±se. ^*: significant GFP-STIM1 effect; ^#: significant Cav-1 siRNA effect; ^¶: significant STIM1 siRNA effect (all p<0.05).

FIGURE 7

Schematic of tumour necrosis factor (TNF)-α effects on caveolae and store-operated Ca²⁺ entry (SOCE) components (stromal interaction molecule (STIM)1 and Orai1). Caveolae in airway smooth muscle (ASM) cell membranes express caveolin (Cav)-1 as well as major SOCE player Orai1. Sarcoplasmic reticulum (SR) contains STIM1 and other Ca²⁺ regulatory proteins. Exposure to TNF-α increases the expression of Cav-1 as well as Orai1, with more caveolae being formed or with more regulatory proteins within caveolae. These changes contribute to increased [Ca²⁺]_i in ASM.