Attendants Medical Research Institute (FAMRI; CMP, VS). Acknowledgments. Figure 1. Caveolae facilitate plasma membrane-sarcoplasmic reticulum.
Caveolin-1, Cavins and CD38-Mediated Ca2+ Regulation in Airway inflammation Venkatachalem Sathish, PhD,1,2 Michael A Thompson, BS,1 Gary C. Sieck, PhD,2,1 Y S Prakash, MD, PhD,1,2 Christina M Pabelick, MD,1,2 Departments of Anesthesiology1 and Physiology & Biomedical Engineering2 Mayo Clinic, Rochester, MN 55905
Introduction
Inflammatory cytokines, such as tumor necrosis factoralpha (TNFα) are mediators in the pathophysiology of reactive airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). TNFα has been shown to enhance airway smooth muscle (ASM) contractility, and augment agonist-induced [Ca2+]i responses. The mechanisms underlying TNFα-induced enhancement of [Ca2+]i are being actively investigated. We have previously shown in ASM, that SR Ca2+ release involves both IP3R and RyR channels. Furthermore, the CD38/cADPR signaling pathway important for SR Ca2+ release through RyR channels may contribute to TNFαinduced augmentation of [Ca2+]i responses in ASM (1). Our previous studies demonstrated that caveolae and their constituent proteins are present in human ASM. In addition, caveolae express proteins involved in [Ca2+]i regulation. Cavin proteins thought to help with maintenance of the shape of caveolae and function as a link between plasma membrane and SR. There is currently no information on caveolar expression of CD38 and its role in [Ca2+]i regulation.
Hypothesis
Role of Caveolae in ASM
Cavins, TNFα and [Ca2+]i Response in ASM
TNFα, Caveolar Proteins and ADPR Cyclase Activity
Figure 7. Incubation with the caveolin-1 specific inhibitor peptide (CSD, 6h, 5µM ) reduced peak [Ca2+]i responses to 10 µM histamine. This effect was still present in cells overexpressing CD38, cells exposed to TNFα or the combination of both demonstrating the important role of caveolin-1 in [Ca2+]i regulation. #significant CSD effect, *significant TNFα effect, % significant CD38 overexpression effect.
Figure 5: Compared to vehicle control (ASM cells exposed to Figure 3. We have previously shown that caveolin-1 is involved in Figure 1. Caveolae facilitate plasma membrane-sarcoplasmic reticulum interactions vis-à-vis [Ca2+]i regulation. (SR: Sarcoplasmic reticulum; IP3R: IP3 receptor channel; RyR: Ryanodine receptor channel; SERCA: SR Ca2+ ATPase; SOCE: Store-operated Ca2+ entry; Cav-1: Caveolin-1). Exposure to pro-inflammatory cytokines such as TNFα results in altered caveolin expression (and perhaps increased caveolae), contributing to enhanced [Ca2+]i responses (2,3,4).
Caveolae and CD38 play an important role in TNFαinduced airway inflammation
Caveolar Expression of CD38
[Ca2+]
I
regulation in human ASM cells (3). As with caveolin-1, siRNA transfection targeting cavin-1 and cavin-3 significantly reduced peak [Ca2+]i responses to 10 µM histamine compared to control. Overnight exposure to TNFα increased peak [Ca2+]i responses to 10 µM histamine. This TNFα effect were largely abrogated in siRNA treated cells. *significant siRNA effect, #significant TNFα effect
TNFα, Caveolar Proteins and CD38 Expression
Lipofectamine only), transfection with caveolin-1 siRNA, cavin-1 siRNA and cavin-3 siRNA decreased the activity of ADP ribosyl cyclase (enzyme involved in cADPR formation). TNFα significantly increased ADP-ribosyl cyclase activity compared to non-exposed controls. Inhibition of the caveolar protein expression shown above using siRNAs significantly attenuated TNFα-induced increase in ADP-ribosyl cyclase activity. *significant from vehicle, #significant from TNFα
CD38 overexpression, TNFα and ADPR Cyclase Activity
Methods Isolation of Human ASM Cells: Human bronchial smooth muscle cells were enzymatically dissociated from human lung tissue incidental to lung surgery (approved by Mayo IRB). Cells were plated in sterile culture flasks and grown in a 95% air/ 5% CO2 humidified incubator using DMEM F/12. Preparation of Caveolar Membranes: ASM cells were homogenized in sucrose buffer, layered onto a 30% Percoll gradient, and centrifuged. The PM fraction was sonicated, resuspended in a 23% solution of OptiPrep. A linear 20% to 10% OptiPrep gradient was layered on top and centrifuged. The upper membrane layer containing caveolae used. siRNA Knockdown/Overexpression: ASM cells at 60% confluence were transfected using 50nM siRNA/ GFP-tagged DNA construct and Lipofectamine in DMEM F/12. [Ca2+]i imaging: Cells were loaded with 5 µM fura 2-AM (60 min, room temperature) and imaged using real-time fluorescence microscopy (1). ADP Ribosyl Cyclase Activity: The fluorescence-based assay converting the NAD analog NGD to the non-hydrolyzable fluorescent product cGDPR as an index of ADP-ribosyl cyclase activity was used.
Caveolin-1 Modulates CD38/TNFα-Mediated [Ca2+]i
Summary & Conclusions • CD38 is expressed within caveolae of human airway smooth muscle, and exposure to TNFα increases CD38 and caveolar protein expression (caveolin-1, cavin-1 and cavin-3) • Like caveolin-1, cavins (cavin-1 and cavin-3) are involved in [Ca2+]i regulation of human ASM cells under control conditions and during TNFα exposure • Inhibition of caveolin-1, cavin-1 and cavin-3 expression reduces CD38 expression as well as its function • CD38 overexpression increases its activity which is further augmented in the presence of TNFα • Suppression of caveolin-1 activity (using CSD) significantly reduces [Ca2+]i responses to agonists even in cells overexpressing CD38. • Overall, these data demonstrate the important role of caveolae in the regulation of CD38 and [Ca2+]i regulation in airway smooth muscle
References 1) Sieck GC, et al. AJP 294: 378-385, 2008; 2) Prakash YS, et al. AJP 293: 1118-1126, 2007; 3) Sathish V, et al. AJP 301:607-614, 2011; 4) Sathish V, et al Eur Res J, 2012 (In Press)
Figure 2. Caveolar membrane fractions express CD38 and the constitutive proteins caveolin-1, cavin-1 (PTRF) and -3 (SBRC) (we previously found that human ASM expresses only caveolin-1 and -2, not 3). Overnight exposure to TNFα (20ng/ml) substantially increased expression of all these proteins.
Figure 4: Role of caveolar proteins in CD38 expression. Human ASM cells transfected with caveolin-1 siRNA, cavin-1 siRNA and cavin-3 siRNA decreased the expression of CD38. TNFα exposure significantly increased CD38 expression in human ASM cells. This TNFα-mediated effect was reduced by siRNAs targeting caveolar proteins. *significant from vehicle. #significant from TNFα
Figure 6: In human ASM cells, TNFα significantly increased ADP-ribosyl cyclase activity compared to non-exposed controls. Transfection of human ASM cells with GFP-CD38 significantly increased ADP-ribosyl cyclase activity compared to control cells. Exposure to TNFα further enhanced cyclase activity. * indicates significant effect of TNFα and #significant effect of GFP-CD38
Acknowledgments This study was supported by NIH grants HL090595 (Pabelick), HL074309 (GCS) and HL088029 (Prakash), and the Flight Attendants Medical Research Institute (FAMRI; CMP, VS)
