Inflammation, caveolae and CD38-mediated calcium regulation in human airway smooth muscle

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Biochimica et Biophysica Acta xxx (2013) xxx–xxx

BBAMCR-17122; No. of pages: 6; 4C: 4

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bbamcr

Inflammation, caveolae and CD38-mediated calcium regulation inhuman airway smooth muscle☆

OFVenkatachalem Sathish a,b, Michael A. Thompson a, Sutapa Sinha a, Gary C. Sieck a,b,

Y.S. Prakash a,b, Christina M. Pabelick a,b,⁎a Departments of Anesthesiology, Mayo Clinic, Rochester, MN 55905, USAb Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA

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☆ Funding support: Supported by NIH R01 grants HL09HL56470 (Prakash), and HL074309 (Sieck).⁎ Corresponding author at: Department of Anesthesio

Clinic College of Medicine, Rochester, MN 55905, USA. T507 255 7300.

E-mail address: [email protected] (C.M. Pa

0167-4889/$ – see front matter © 2013 Published by Elsehttp://dx.doi.org/10.1016/j.bbamcr.2013.11.011

Please cite this article as: V. Sathish, et al., IBiochim. Biophys. Acta (2013), http://dx.doi

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Article history:Received 2 August 2013Received in revised form 11 November 2013Accepted 14 November 2013Available online xxxx

Keywords:Plasma membraneADP ribosyl cyclaseLungAsthmaCavin

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RThe pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) increases expression of CD38 (amembrane-associated bifunctional enzyme regulating cyclic ADP ribose), and enhances agonist-induced intracellular Ca2+

([Ca2+]i) responses in human airway smooth muscle (ASM). We previously demonstrated that caveolae andtheir constituent protein caveolin-1 are important for ASM [Ca2+]i regulation, which is further enhanced byTNFα. Whether caveolae and CD38 are functionally linked in mediating TNFα effects is unknown. In this regard,whether the related cavin proteins (cavin-1 and -3) thatmaintain structure and function of caveolae play a role isalso not known. In the present study, we hypothesized that TNFα effects on CD38 expression and function inhuman ASM involve caveolae. Caveolar fractions from isolated human ASM cells expressed CD38 and its expres-sion was upregulated by exposure to 20 ng/ml TNFα (48 h). ASM cells expressed cavin-1 and cavin-3, whichwere also upregulated by TNFα. Knockdown of caveolin-1, cavin-1 or cavin-3 (using siRNA) all significantlyreduced CD38 expression and ADP-ribosyl cyclase activity in the presence or absence of TNFα. Furthermore,caveolin-1, cavin-1 and cavin-3 siRNAs reduced [Ca2+]i responses to histamine under control conditions, andblunted the enhanced [Ca2+]i responses in TNFα-exposed cells. These data demonstrate that CD38 is expressedwithin caveolae and its function is linked to the caveolar regulatory proteins caveolin-1, cavin-1 and -3. The linkbetween caveolae and CD38 is further enhanced during airway inflammation demonstrating the important roleof caveolae in regulation of [Ca2+]i and contractility in the airway.

© 2013 Published by Elsevier B.V.

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1. Introduction

Intracellular Ca2+ concentration ([Ca2+]i) plays an important role inairway smooth muscle (ASM) contraction and relaxation. Agonist-induced [Ca2+]i responses involve Ca2+ influx and sarcoplasmic reticu-lum (SR) Ca2+ release [1–6].

Inflammatory cytokines, such as tumor necrosis factor-alpha (TNFα)and interleukins such as IL-13 have been implicated as mediators in thepathophysiology of reactive airway diseases such as asthma [7–9] andchronic obstructive pulmonary disease (COPD) [10,11]. For example,we and others have shown that TNFα enhances ASM contractility byincreasing agonist-induced [Ca2+]i responses [12–18]. We and othershave shown that in ASM, SR Ca2+ release involves both inositoltrisphosphate receptor [1–3] and ryanodine receptor channels [4,19].Several studies, including our own, have shown that the second

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nflammation, caveolae and C.org/10.1016/j.bbamcr.2013.1

messenger cyclic-ADP-ribose (cADPR) is involved in Ca2+ release viaryanodine receptor channels [6,20]. cADPR is synthesized and degradedby the bifunctional ectoenzyme CD38 via ADP-ribosyl cyclase andcADPR hydrolase activities, respectively [21]. Indeed, the CD38/cADPRpathway has been implicated in [Ca2+]i regulation in several smoothmuscle types including ASM [6,22–28]. Other studies suggest that theCD38/cADPR signaling pathway contributes to TNFα-induced augmen-tation of [Ca2+]i responses in ASM [29].

In a recent study [25], we demonstrated that the extent of store-operated Ca2+ entry (SOCE) in human ASM cells is modulated by thelevel of CD38 expression, which suggest that CD38 effects do not neces-sarily involve cADPR. Accordingly, themechanisms that regulate plasmamembrane CD38 levels become important. In this regard, we previouslyreported [30] the presence of caveolae and their constituent proteinscaveolin-1 and -2 in human ASM as well as the role of these plasmamembranemechanisms in [Ca2+]i regulation. The importance of caveo-lae lies in the facts that they express a number of [Ca2+]i and otherregulatory proteins, and that ASM caveolae and caveolin-1 expressionare increased following TNFα exposure [14]. Based on these observa-tions, we hypothesized that CD38 is expressed within caveolae, andmodulated by caveolar proteins, providing an avenue for TNFα toupregulate CD38 in airway inflammation.

D38-mediated calcium regulation in human airway smooth muscle,1.011

http://dx.doi.org/10.1016/j.bbamcr.2013.11.011

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2. Materials and methods

2.1. Isolation of human ASM cells

The techniques for isolation of humanASMcells have been previous-ly described [30,31]. Briefly, in accordance with previously approvedprocedures and considered exempt from Human Subjects classificationby theMayo Clinic Institutional Review Board, human 3rd to 6th gener-ation bronchi were obtained from discarded surgical specimens andASM cells enzymatically dissociated as described previously [30,31].Cells were seeded into culture flasks, maintained under normal condi-tions (21% O2, 5% CO2; 37 °C) and used for experiments under serumstarved conditions prior to passage 3 of subculture. Validation of ASMcell phenotype was performed by western analysis and RT-PCR forsmooth muscle markers [30,31].

2.2. CD38 overexpression

As recently described [25], full length CD38was generated using RT-PCR and primers designed from published sequence (Accession #D84276) that include the endogenous translation start and stop codons(Forward primer sequence: 5′-ACCCCGCCTGGAGCCCTATG-3′ Reverseprimer sequence: 5′-GCTAAAACAACCACAGCGACTGG-3′). A green fluo-rescent protein (GFP)-tagged CD38 DNA construct was constructedusing standard procedures, and ASM cells were transfected with GFP-CD38 or Lipofectamine™ 2000 (Invitrogen) alone according to manu-facturer's protocol. Cells were incubated with the transfection mix inDMEM/F12 media without serum for 24 h and analyzed for CD38expression and function as previously described [25]. For [Ca2+]i stud-ies, DMEM/F12 containing 10% FBS was added 4 h post-transfectionand maintained for 24 h. This medium was replaced with serum freeDMEM/F12 media for an additional 48 h to drive cells to quiescenceprior to [Ca2+]i measurements.

2.3. Preparation of caveolar membranes

We have previously described techniques for preparation ofcaveolin-rich membranes from human ASM cells [30]. Briefly, ASMcells were homogenized in cold buffer A (0.25 M sucrose, 1 mM EDTA,and 20 mM Tricine, pH 7.8), layered onto a 30% Percoll gradient, andcentrifuged at 84,000 ×g for 30 min. The crude membrane fractionwas then sonicated, resuspended and normalized to equal proteinconcentration and a linear 20% to 10% OptiPrep gradient was layeredon top for re-centrifugation. The upper membrane layer (containingcaveolae) was collected for further experimentation. Equal volumes ofcaveolae enriched fraction were loaded for western blot analysis toaccount for comparable measurements of caveolar proteins withincaveolae.

2.4. Caveolin-1, cavin-1, and cavin-3 knockdown

The technique for knocking down of caveolar proteins in humanASM using siRNA has also previously been described [30,32]. siRNAduplex oligonucleotides were purchased from Dharmacon (Lafayette,CO). ASM cells at 60% confluence were transfected using 50 nM siRNAand Lipofectamine in DMEM F/12 without FBS. Fresh growth mediumwas added 6 h following transfection and cells processed after 48 h.Efficacy of siRNA knockdown was verified by western analysis.

2.5. Measurement of ADP-ribosyl cyclase activity

The fluorescence-based assay of the conversion of the NAD analog,NGD, to the non-hydrolyzable fluorescent product cGDPR as an indexof ADP-ribosyl cyclase activity has been previously described [33]. Brief-ly, normal medium was replaced with HBSS containing 400 μM NGDwith or without TNFα. HBSS with NGD in cells without cells served as

Please cite this article as: V. Sathish, et al., Inflammation, caveolae and CBiochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbamcr.2013.1

background. After 24 h (during which cells were maintained understandard culture conditions), an aliquot of medium was removedand fluorescence measured (excitation 305 nm, emission 410 nm;Amersham spectrofluorometer). Changes in fluorescence above back-ground were considered as valid conversions from NGD to cGDPR.

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2.6. Western blot analysis

Standard techniques were used to separate proteins of interest bySDS-PAGE, transfer to PVDFmembranes, blockingwith 5%milk, incuba-tion overnight with 1 μg/ml primary antibodies and detection of bandswith either horseradish peroxidase-conjugated secondary antibodies orchemiluminescence substrate (Supersignal West Pico, Pierce ChemicalCo., Rockford, IL). Signal was developed using Supersignal West PicoChemiluminescent substrate or far-red fluorescent dye-conjugatedantibodies. Blots were imaged on a Kodak Image Station 4000MM(Carestream Health, New Haven, CT) or a LiCor OdysseyXL system,and quantified using densitometry.

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RO2.7. Fluorescence [Ca2+]i imaging

These techniques have been extensively described [25,34,35]. Cellswere incubated for 45 min in 5 μM fura-2/AM. ASM cells were initiallyperfused with 2.5 mM Ca2+ HBSS and baseline fluorescence levelswere established. The [Ca2+]i responses of 15–30 cells per chamberwere achieved using software-defined regions. The ratiometric mea-surements of 510 nm emissions following 340/380 nm excitationswere recorded every 750 ms using a videofluorescence imaging system(Molecular Devices, Sunnyvale, CA). In regard to the histamine-induced[Ca2+]i responses, the amplitudes of [Ca2+]i responses were calculatedby subtracting the baseline [Ca2+]i level from the peak [Ca2+]iresponses.[22,25].

2.8. Materials

Antibodies to caveolin-1 (Abcam, #ab18199), CD38 (Epitomics,#2935-1), cavin-1 (Novus Biologicals, #NB100-60635), cavin-3 (Abcam,#ab76676), andGAPDH (Cell Signaling, #2118)were purchased as listed.DMEM, antibiotic/antimycotic mixture as well as Fura-2 were obtainedfrom Invitrogen, Grand Island, NY. Unless mentioned otherwise allchemicals were purchased from Sigma-Aldrich, St. Louis, MO.

2.9. Statistical analysis

Experimentswere performedusing ASMcells derived from at least 5different patients, although not all protocols were performed in eachsample. Each protocol was repeated at least 5 times. Statistical analysiswas performed using unpaired Student's t test or 2-way ANOVA withrepeated measures when necessary (Bonferroni correction for repeatedmeasures). Data are shown asmean ± SEM. Statistical significance wasset at p b 0.05.

3. Results

3.1. Caveolar protein expression and upregulation with TNFα

We previously demonstrated caveolin-1 expression and its upregu-lation within caveolar fractions following TNFα exposure [14]. In thepresent study, we found that CD38 as well as the caveolar structuralregulatory proteins cavin-1 and cavin-3 are expressed within caveolarfractions of human ASM cells (Fig. 1). All these proteins are significantlyupregulated in the presence of TNFα (Fig. 1; p b 0.05).



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Fig. 1. Caveolar expression of CD38. In caveolar fractions of humanASM cells, considerableCD38 expression was observed at baseline. Caveolar fractions expressed the constitutiveprotein caveolin-1 as well as the structural regulatory proteins cavin-1 and cavin-3 thatdetermine caveolin-1 insertion into plasma membrane (and thus invagination) and cave-olin internalization, respectively. Overnight exposure to TNFα significantly increasedexpression of all these proteins (CD38, caveolin-1, cavin-1, and cavin-3). * in bar graphindicates significant TNFα effect (p b 0.05).

Fig. 2. Effect of caveolar protein knockdown on CD38 expression in human ASM cells.siRNA knockdown of caveolin-1, cavin-1, or cavin-3 all significantly reduced caveolarCD38 expression in human ASM at baseline. TNFα-induced increase in CD38 expressionwas substantially blunted by siRNA against caveolin-1, cavin-1, and particularly cavin-3(A). The siRNA efficacy of all three caveolar proteins was verified using western blot anal-ysis (B). Values aremean ± SEM. * indicates significant siRNA effect, # indicates significanteffect of TNFα (p b 0.05).

Fig. 3. Effect of caveolar protein knockdown on ADP-ribosyl cyclase activity. siRNA knock-down of caveolin-1, cavin-1 or cavin-3 significantly reduced ADP-ribosyl cyclase activity.TNFα enhancement of cyclase activity was reduced to an even greater extent by siRNAsagainst all three proteins. Values are mean ± SEM. * indicates significant siRNA effect,# indicates significant effect of TNFα (p b 0.05).

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siRNA suppression of caveolin-1, cavin-1, or cavin-3 expression allsignificantly reduced CD38 expression in human ASM (Fig. 2A,p b 0.05). Surprisingly, cavin-3 siRNA was most effective in reducingCD38 expression. The significant increase in CD38 expression followingTNFα exposurewas still blunted by knockdown of caveolin-1, cavin-1 orcavin-3 (Fig. 2A, p b 0.05), and to a greater extent compared to non-TNFα exposed cells. Here too, cavin-3 siRNA was most effective. Trans-fection with small interference RNA (siRNA) specific for caveolin-1,cavin-1 or cavin-3 resulted in significant reduction in specific proteinexpression. Vehicle (Lipofectamine) or negative siRNA had no signifi-cant effect on protein expression (Fig. 2B, p b 0.05).

3.3. Effect of caveolar protein knockdown on ADP-ribosyl cyclase activity

Compared to baseline, exposure to TNFα significantly enhancedADP-ribosyl cyclase activity (Fig. 3, p b 0.05) consistent with increasedCD38 expression. siRNA-induced suppression of caveolin-1, cavin-1,and cavin-3 all significantly reduced ADP-ribosyl cyclase activity inASM cells with or without TNFα exposure (Fig. 3, p b 0.05), withproportionately greater effects in TNFα-exposed cells.

3.4. Effect of caveolin-1, cavin-1 and cavin-3 siRNAs on [Ca2+]i response toagonist

In fura-2 loaded control (i.e. non-transfected, non-TNFα exposed,Lipofectamine only) ASM cells, baseline [Ca2+]i was ~100–180 nM(mean 154 ± 14 nM). Exposure to 10 μM histamine produced charac-teristic biphasic [Ca2+]i responses, which we have also reported previ-ously [14]. Consistent with previous results [14,15], exposure to TNFα

Please cite this article as: V. Sathish, et al., Inflammation, caveolae and CD38-mediated calcium regulation in human airway smooth muscle,Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbamcr.2013.11.011


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significantly increased [Ca2+]i responses to histamine compared tocontrol (Fig. 4C,D; p b 0.05). In cells exposed to caveolin-1, cavin-1 orcavin-3 siRNA, [Ca2+]i responses to histamine were significantlyreduced in comparison to vehicle (Fig. 4 A,B, p b 0.05). In cells exposedto TNFα, caveolin-1, cavin-1, and cavin-3 siRNAs blunted the enhance-ment of [Ca2+]i responses due to this cytokine (Fig. 4C,D; p b 0.05).

3.5. CD38 overexpression and [Ca2+]i responses

Overexpression of CD38 increased [Ca2+]i responses to histaminecompared to vehicle (Lipofectamine only; Fig. 5, p b 0.05). This enhanc-ing effectwas as pronounced aswith exposure to TNFα. The combinationof CD38 overexpression and TNFα exposure even further enhanced[Ca2+]i responses (Fig. 5, p b 0.05). To investigatewhether the increased[Ca2+]i responses in the presence of CD38 overexpression and/or TNFαare linked to changes in caveolar proteins, we used caveolin-1, cavin-1or cavin-3 siRNA under these experimental conditions. Downregulationof caveolin-1, cavin-1 or cavin-3 using siRNA significantly reduced[Ca2+]i responses under all these experimental conditions (control,TNFα, CD38 overexpression, and CD38 overexpression plus TNFα;Fig. 5, p b 0.05).

4. Discussion

In this study, we present novel data demonstrating that caveolarCD38 expression is linked to TNFα-induced augmentation of [Ca2+]iresponses to agonist stimulation in ASM. In this regard, we found thatCD38 expression within caveolae is regulated by the cavin proteinsthat happen to be important for caveolar structure (cavin-1) and traf-ficking (cavin-3). Exposure to TNFα upregulates such mechanisms,resulting in enhanced caveolar CD38 which in turn leads to increasedADP ribosyl cyclase activity and [Ca2+]i. However, such enhancementof CD38 is not unidirectional in the sense that caveolae in turn canregulate CD38 function as well. This is demonstrated by the findingsthat inhibition of caveolin-1, cavin-1 and cavin-3 expression leads toblunting of CD38 effects on [Ca2+]i responses in the presence or absenceof cytokine. The relevance of these findings lies in the recognition thatboth CD38 [6,22–28] and caveolae [14,32,36] are important regula-tors of ASM [Ca2+]i and contractility, and increased expression of

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Fig. 4. Effect of caveolin-1, cavin-1 and cavin-3 siRNAs on intracellular Ca2+ ([Ca2+]i) responseLipofectaminevehicle only), caveolin-1, cavin-1 and cavin-3 siRNA treated cells showed significa[Ca2+]i responses to histamine. In the presence of TNFα, all caveolin-1 siRNA, cavin-1 siRNA anmine (C and D). Values are mean ± SEM. * indicates significant siRNA effect, # indicates signifi


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either mechanism contributes to increased airway contractility[14,25,29,32,36]. Accordingly, structural and functional linkage be-tween the two highlights a potentially synergistic pathway by whichcytokines or other inflammatory factors can enhance airway contractil-ity in the setting of airway diseases.

4.1. CD38 in ASM

We and others have demonstrated that CD38 exists in ASM, and isinvolved in regulation of [Ca2+]i and contractility [25,29,37–39].Furthermore, the link between CD38 and cADPR, synthesized by ADP-ribosyl cyclase and degraded by cADPR hydrolase, has also been well-established in vitro [37,40,41] as well as in vivo using CD38 knockoutmice [26,42,43]. Intracellular cADPR levels can be modulated via CD38expression and function, for example by neurotransmitters and cyto-kines (IL-1, TNFα) [29,44,45]. In human ASM cells, we previouslyreported that TNFα increases CD38 expression as well as activity asdetermined using an NGD assay: effects inhibited by CD38 siRNA [25].Furthermore, we have demonstrated that the enzymatic machineryfor cADPR metabolism exists in ASM [46], and is important for [Ca2+]iregulation by showing that exogenous cADPR modulates SR Ca2+

release via ryanodine channels [6]. In human ASM, cADPR produc-tion occurs with several bronchoconstricting agents (acetylcholine,endothelin-1 and histamine) [20,22]. And finally, beyond cADPR,CD38 also has the potential to modulate [Ca2+]i via effects on Ca2+

influx mechanisms such as TRPC3 [25]. Overall, these data link CD38and [Ca2+]i regulation in ASM. Furthermore, they demonstrate thatthese relationships can play a role in cytokine enhancement of [Ca2+]iin ASM. The current study now demonstrates that such links involvecaveolar CD38 with an additional role for caveolin-1 in regulatingCD38 effects.

4.2. TNFα, CD38 and ASM

The important role of TNFα in airway inflammation and hyperreac-tivity has been well-recognized [8,47,48]. Here, several targets andmechanisms for TNFα effects, including [Ca2+]i regulatory pathways,have been identified in ASM [13,49]. Relevant to this study, we andothers [14,50] have shown that TNFα enhances caveolin-1 expression

s to agonist in human ASM cells. Compared to control cells (non-transfected, treated withntly reduced [Ca2+]i responses to 10 μMhistamine (Aand B). TNFα significantly enhancedd cavin-3 siRNA treated cells still showed significantly smaller [Ca2+]i responses to hista-cant effect of TNFα (p b 0.05).



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Fig. 5. Role of caveolin-1, cavin-1 and cavin-3 in the regulation of CD38 effects. Overex-pression of CD38 using a GFP-tagged construct resulted in a significant increase in[Ca2+]i responses to 10 μM histamine. This effect is as pronounced as the TNFα-inducedincrease in [Ca2+]i responses. Combination of TNFα and GFP-CD38 even further enhancedthe increase in [Ca2+]i responses. Under these different conditions, caveolin-1, cavin-1 orcavin-3 siRNA treated cells showed significantly reduced [Ca2+]i responses under allconditions (vehicle; CD38 overexpression, TNFα, CD38 overexpression plus TNFα)suggesting that caveolar proteins modulate CD38 effects. Values are mean ± SEM. # indi-cates significant siRNA effect, * indicates significant effect of TNFα (compared tocontrol), % indicates significant CD38 effect (p b 0.05).

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and signaling in humanASM. Studies in humanASM, including our own,have already shown that TNFα increases CD38 expression [25,29,35].Furthermore, the CD38/cADPR signaling pathway is involved in TNFα-induced increase in airway responsiveness [29]. For example, adenovi-ral mediated anti-sense CD38 transfection leads to blunted [Ca2+]iresponses to agonist [51], while CD38 overexpression leads to enhanced[Ca2+]i responses as well as store-operated Ca2+ influx [25]. Thepresent study shows that TNFα induced alterations in CD38 are linkedto changes in caveolar proteins which in turn can regulate CD38function.

4.3. Caveolae in ASM

Caveolae are flask-shaped invaginations of the plasma membranethat are enriched in cholesterol, sphingolipids, and integral membraneproteins called caveolins. Caveolin-enriched domains harbor a rangeof proteins and facilitate binding of intracellular as well as extracellularmoieties, and therefore can have profound effects on signaling betweenplasma membrane regulatory components and intracellular structures.There is currently no information on caveolar localization of CD38 inany tissue. In this regard, the results of the current study are novel. Byco-expressing CD38 alongwith agonist receptors andmultiple regulato-ry proteins such as agonist receptors and influx channels, caveolae mayfacilitate interactions between these proteins and thus enhance [Ca2+]iand other signaling. Accordingly, mechanisms that regulate caveolarstructure and the protein composition of caveolae become importantin the context of regulatory proteins such as CD38.

The results of the present study show that in human ASM, caveolarCD38 expression is regulated by caveolin-1 as well as two cavins thatthemselves modulate caveolar structure and function. ASM from differ-ent species has now been shown to express caveolin-1 (and to a lesserextent caveolin-2) [30,52–54]. We and others have also shown thatcaveolin-1 expression and its effects on [Ca2+]i per se are enhanced inthe presence of cytokines such as TNFα. Such effects are mediated, atleast in part, by caveolin-1 itself, as well as signaling intermediates suchas MAPK and NFκB. Thus caveolin-1 is an integral aspect of caveolar ef-fects in ASM. What is novel in this study is the demonstration thatcaveolin-1 regulates the expression and functionof an important enzymesuch as CD38 that is not simply due to altered caveolar levels per se.

In addition to caveolin-1, two relatively novel regulatory proteins,cavin-1 (or PTRF) and cavin-3 (or SRBC) also appear to be importantfor CD38 expression and its activity. There is currently very little


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information on cavin expression patterns in ASM, or their role in ASMcaveolar expression/function or other pathways. Here, the results ofthe present study showing robust cavin-1 and cavin-3 expression inhuman ASM are also novel, as are the data that TNFα substantiallyincreases expression of both proteins. A primary role of these proteinsis control of caveolin-1 levels within caveolae (by enhancing plasmamembrane insertion vs. internalization). Accordingly, the overall levelof caveolin-1 (a relatively stable protein when in the plasma mem-brane) is likely driven by a balance between these regulators. Here,the increase in cavin-3 with TNFα is surprising since that should leadto reduced plasma membrane caveolin-1, and not an increase as hasbeen previously reported [55]. While the focus of the present studywas not on the role of cavins in caveolin control per se, it is possible tohypothesize that the relative contribution of cavin-1 (insertion) vs.cavin-3 (internalization) is different. However, what is more interestingis that cavin-3 in particular appears to regulate caveolar CD38 levels.Given cavin-3's role, this cannot be a result of increased caveolin-1 (orcaveolae) butmust reflect an entirely differentmechanism that remainsto be explored. Nonetheless, the effects of cavins on CD38 expression(and consequent function) demonstrate the importance of caveolarproteins in regulation of CD38, particularly in the presence of cytokines.

In conclusion, the present study demonstrates that TNFα inducedaugmentation of [Ca2+]i responses to agonist in human ASM is mediat-ed via changes in caveolar CD38, driven by effects of caveolin-1 as wellas the regulatory proteins cavin-1 and cavin-3. Such changes mayfurther lead to increased cADPR levels, and thus increased [Ca2+]iresponses due to release via ryanodine channels, which can contributeto the enhanced airway contractility that occurs with inflammation.

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Inflammation, caveolae and CD38-mediated calcium regulation in human airway smooth muscle