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Differential regulation of cell functions by CSD peptide subdomains Reese, Charles; Dyer, Shanice; Perry, Beth; Bonner, Michael; Oates, James; Hofbauer, Ann; Sessa, William; Bernatchez, Pascal; Visconti, Richard P; Zhang, Jing; Hatfield, Corey M; Silver, Richard M; Hoffman, Stanley; Tourkina, Elena Sep 8, 2013

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RESEARCH Open AccessDifferential regulation of cell functions by CSDpeptide subdomainsCharles Reese1, Shanice Dyer1, Beth Perry1, Michael Bonner1, James Oates1, Ann Hofbauer1, William Sessa3,Pascal Bernatchez4, Richard P Visconti2, Jing Zhang2, Corey M Hatfield1, Richard M Silver1, Stanley Hoffman1,2and Elena Tourkina1,5*AbstractBackground: In fibrotic lung diseases, expression of caveolin-1 is decreased in fibroblasts and monocytes. Theeffects of this deficiency are reversed by treating cells or animals with the caveolin-1 scaffolding domain peptide(CSD, amino acids 82–101 of caveolin-1) which compensates for the lack of caveolin-1. Here we compare thefunction of CSD subdomains (Cav-A, Cav-B, Cav-C, Cav-AB, and Cav-BC) and mutated versions of CSD (F92A andT90A/T91A/F92A).Methods: Migration toward the chemokine CXCL12 and the associated expression of F-actin, CXCR4, and pSmad2/3 were studied in monocytes from healthy donors and SSc patients. Fibrocyte differentiation was studied usingPBMC from healthy donors and SSc patients. Collagen I secretion and signaling were studied in fibroblasts derivedfrom the lung tissue of healthy subjects and SSc patients.Results: Cav-BC and CSD at concentrations as low as 0.01 μM inhibited the hypermigration of SSc monocytes andTGFβ-activated Normal monocytes and the differentiation into fibrocytes of SSc and Normal monocytes. While CSDalso inhibited the migration of poorly migrating Normal monocytes, Cav-A (and other subdomains to a lesserextent) promoted the migration of Normal monocytes while inhibiting the hypermigration of TGFβ-activatedNormal monocytes. The effects of versions of CSD on migration may be mediated in part via their effects onCXCR4, F-actin, and pSmad 2/3 expression. Cav-BC was as effective as CSD in inhibiting fibroblast collagen I andASMA expression and MEK/ERK signaling. Cav-C and Cav-AB also inhibited collagen I expression, but in many casesdid not affect ASMA or MEK/ERK. Cav-A increased collagen I expression in scleroderma lung fibroblasts. Full effectson fibroblasts of versions of CSD required 5 μM peptide.Conclusions: Cav-BC retains most of the anti-fibrotic functions of CSD; Cav-A exhibits certain pro-fibrotic functions.Results obtained with subdomains and mutated versions of CSD further suggest that the critical functional residuesin CSD depend on the cell type and readout being studied. Monocytes may be more sensitive to versions of CSDthan fibroblasts and endothelial cells because the baseline level of caveolin-1 in monocytes is much lower than inthese other cell types.Keywords: Caveolin-1, Monocytes, Fibrocytes, Fibroblasts, Scleroderma (SSc), Migration, TGFβ* Correspondence: tourkine@musc.edu1Department of Medicine/Division of Rheumatology and Immunology,Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC29425, USA5Division of Rheumatology and Immunology, Department of Medicine,Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 912MSC 637, Charleston, SC 29425, USAFull list of author information is available at the end of the article© 2013 Reese et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Reese et al. Respiratory Research 2013, 14:90http://respiratory-research.com/content/14/1/90BackgroundCaveolin-1, a protein associated with plasma membraneinvaginations known as caveolae and with other cellularmembranes, is a promising therapeutic target in ILDs.We and others have shown that caveolin-1 is deficient inthe lung tissue of SSc and IPF patients and in cells iso-lated from the lung tissue and blood of these patients in-cluding fibroblasts, monocytes, and neutrophils [1-3].Similarly, caveolin-1 is also deficient in mice in whichILD has been induced with bleomycin or irradiation[2,3].Caveolin-1 binds to and thereby inhibits the functionof kinases in several major families including PKC,MAPK, Src, and G protein [4-7] and regulates signalingand cell functions induced by the major pro-fibroticcytokine, TGFβ [1,8,9]. The effects of caveolin-1 de-ficiency in cells and in animals can be reversed eitherby using adenovirus encoding full-length caveolin-1 orusing the caveolin-1 scaffolding domain peptide (CSD;amino acids 82–101 of caveolin-1) [1,2]. When CSD issynthesized in fusion with the Antennapedia Internaliza-tion Sequence, it can enter cells and inhibit kinases justlike full-length caveolin-1 [10,11]. CSD was reported [4]to bind to target kinases through consensus sequences(ΦXΦXXXXΦ and ΦXXXXΦXXΦ) where Φ stands forany of the aromatic amino acids (F, W, or Y) and Xstands for any amino acid. Later studies suggested thatthe initial definition of the consensus sequences wasoverly stringent and that the consensus sequences forcaveolin binding domains (CBDs) are ΦXZXXXXΦand ZXXXXΦXXZ where Z stands for F, W, Y, I, V,or L [12].Given the large number of signaling molecules thatcontain CBDs and the heterogeneity of the primarysequences of these CBDs, it is extremely likely thatsubdomains of CSD will differ from each other and fromCSD in their ability to regulate the activity of these ki-nases and therefore will have distinctive effects on cellbehavior. Indeed, previous studies on CSD subdomainshave given distinct results depending on the peptide be-ing studied. For example, in experiments using endothe-lial cells, amino acids 89–95, 82–95, and 89–101 allinhibited eNOS production and it was therefore con-cluded that 89–95 was the key sequence involved in thisprocess [11,13]. In contrast, 86–101, but not 88–101,inhibited the activity of PKC isoforms purified fromtransfected H5 insect cells [5]. Similarly, CSD, but not84–92 or 93–101, inhibited the activity of MEK andERK purified from bacterial extracts [14].In order to identify CSD subdomains that may bemore useful than full-length CSD in treating human dis-eases, here we have compared the ability of CSD andseveral subdomains and mutated versions (each attachedto the Antennapedia Internalization Sequence) to reverseeffects associated with low caveolin-1 on the behavior ofmonocytes (migration toward CXCL12; expression ofCXCR4 and F-actin and Smad 2/3 activation; differenti-ation into fibrocytes) and fibroblasts (collagen I andASMA expression, MEK/ERK activation). For these exper-iments, cells were isolated from both normal subjects andscleroderma patients. Overall, the Cav-BC peptide (aminoacids 89–101) was as effective as, and sometimes moreeffective than, full-length CSD. Interestingly, the Cav-Apeptide (amino acids 82–88) in some cases exacerbatedeffects associated with low caveolin-1. While not surpris-ing, it is noteworthy that the patterns of relative activitythat we observed with CSD subdomains and with mutatedversions of CSD differed from those obtained in a study ofCSD regulation of eNOS-mediated NO release in endo-thelial cells [11].MethodsSubjects for monocyte studiesUnder a protocol approved by the Institutional ReviewBoard for Human Research for a Rheumatology Re-search Repository, patients with SSc-ILD were recruitedfrom the Scleroderma Clinic at the Medical Universityof South Carolina (MUSC). All patients fulfilled the Ameri-can College of Rheumatology (formerly the AmericanRheumatism Association, ARA) criteria for SSc [15] andhad evidence of SSc-ILD as previously defined [16]. Demo-graphic data for SSc patients and normal healthy donorsare summarized in Table 1.Monocyte isolationMonocytes were isolated by standard methods [16,17].Following centrifugation on density 1.083 Histopaquecushions, monocytes were enriched by immunodep-letion using a Dynal Monocyte Negative Isolation Kit(Invitrogen, Carlsbad, CA) resulting in a cell popula-tion about 95% Mac-1+ monocytes.Monocyte migrationAssays were performed as previously described [17].Briefly, CXCL12 (100 ng/ml in RPMI 1640 with 1% BSA)was placed into the lower wells of Neuro Probe MultiwellChemotaxis Chambers (Neuro Probe, Gaithersburg, MD)fitted with 5-μm pore size polycarbonate filters. 25 μl ofcell suspension (1 × 106 cells/ml) with or without TGFβpretreatment (45 min, 10 ng/ml in RPMI 1640 with 1%BSA) was placed in the upper wells. Peptides were addedto cells before they were placed in the upper chamber.After incubation for 2.5 h at 37°C in a 5% CO2 incubator,filters were removed, fixed, and stained with DAPI(Invitrogen, Carlsbad, CA). Cells on the underside of themembrane were photographed and counted in six highpower fields per filter.Reese et al. Respiratory Research 2013, 14:90 Page 2 of 18http://respiratory-research.com/content/14/1/90F-actin, CXCR4, and pSmad 2/3 levelsWere evaluated by immunocytochemistry in monocytesthat were isolated as described above, cultured overnightin 6-well plates (1 × 105 cells per well) on coverslips inRPMI 1640/ 20% FCS, and sequentially treated withRPMI 1640/ 1% BSA with or without 10 ng/ml TGFβ,then with the same medium containing 5 μM of the in-dicated peptides as previously described [16]. Cells werethen fixed, labeled with FITC phalloidin (Sigma-Aldrich),rabbit anti-CXCR4 (Santa Cruz Biotechnology sc-9046),rabbit anti pSmad 2/3 (Cell Signaling 3102) Alexa Fluor®555-conjugated secondary antibodies (Invitrogen), andcounterstained with the nuclear stain DAPI. Stainingwas quantified in arbitrary units by image analysis of tencells in each category in terms of the average fluo-rescence intensity ± s.e.m.Smad western blotsMonocytes (2 × 106 cells per well) incubated as describedabove for pSmad 2/3 immunocytochemistry were nextwashed twice with PBS, then extracted with SDS-PAGEsample buffer. Smad 2/3 and pSmad 2/3 levels were de-termined by Western blotting using rabbit anti-Smad2/3 (Cell Signaling 3102), rabbit anti-pSmad 2/3 (CellSignaling 3101), and mouse monoclonal anti-GAPDH(EMD Millipore MAB374, clone 6C5) as a loadingcontrol.Monocyte to fibrocyte differentiationTotal PBMC from 40 ml of peripheral blood were platedin eight wells of fibronectin-coated six-well plates inDMEM/ 20% FCS with supplements for 12 days [18].Medium was changed on day 5. Peptides were added onday 2 and again on day 5 after the medium was changed.Specific peptides used are described in the FigureLegends. Images were acquired on day 12 and fibrocyteswere quantified in terms of elongated cells per 10 × field,all of which were collagen I+. For immunocytochemistry,the same methods were used except that the wellscontained coverslips.Subjects for fibroblast studiesFibroblasts were derived from lung tissue obtained atautopsy from SSc patients (SLF) and from normal sub-jects (NLF) and cultured as previously described [7].Cells were used in passages 2–4. SSc lung tissue wasobtained from the Division of Pathology and LaboratoryMedicine at the Medical University of South Carolina(MUSC). These SSc patients fulfilled the criteria ofAmerican College of Rheumatology for the diagnosis ofSSc with lung involvement. Normal human lung tissuewas obtained from the Brain and Tissue Bank for De-velopmental Disorders (Baltimore, MD) or from theNational Disease Research Interchange (Philadelphia,Table 1 Clinical features of SSc patients involved inmonocyte experimentsRace/Smoking Gender Patients ControlsCaucasian M 2 9Caucasian F 7 12African-American M 3 2African-American F 3 9Asian F 0 1Smoker 0 2Former Smoker 2 3Age: Mean ± SD (range) Patients 52.3 ± 12.8 (27–79)Controls 43.0 ± 11.9 (18–64)Disease Limited Cutaneous 3Diffuse Cutaneous 9Overlap 3Disease duration: Mean ± SD (range), yr 4.3 ± 2.8 (1-10)Pulmonary Involvement (ILD) 15/15 (100%)Pulmonary HTN 3/15 (20%)GI Involvement 15/15 (100%)Cardiac Involvement 2/15 (13.3%)Renal Involvement 1/15 (6.7%)Autoantibodies: ANA+ 15/15 (100%)Scl-70+ 6/15 (40%)Anti-centromere 1/15 (6.7%)Table 2 Clinical features of autopsy samples used infibroblast experimentsRace/Smoking Gender Patients ControlsCaucasian M 0 1Caucasian F 4 2African-American M 0 0African-American F 1 0Smoker 1 0Former Smoker 1 1Age: Mean ± SD (range) Patients 46.6 ± 17.4 (21-62)Controls 56.6 ± 8.5 (46-65)Disease: Limited Cutaneous 4Diffuse Cutaneous 1Overlap 0Pulmonary Involvement (ILD) 5/5 (100%)GI Involvement 4/5 (80%)Cardiac Involvement 4/5 (80%)Renal Involvement 1/5 (20%)Reese et al. Respiratory Research 2013, 14:90 Page 3 of 18http://respiratory-research.com/content/14/1/90Figure 1 (See legend on next page.)Reese et al. Respiratory Research 2013, 14:90 Page 4 of 18http://respiratory-research.com/content/14/1/90PA). The study was approved by the Institutional ReviewBoard for Human Subject Research at the MUSC asnon-human research. The demographics of these sub-jects are provided in Table 2.Fibroblast collagen I secretion and signalingAs previously described [8], aliquots of culture mediumor of cell layer, representing material derived from thesame number of cells, was probed on Western blotsusing the following primary antibodies and appropriatesecondary antibodies: rabbit antibodies against ERK 1/2(9102), pERK 1/2 (9106), MEK 1/2 (9122), and pMEK1/2 (9121) from Cell Signaling (Beverly, MA); mousemonoclonal anti-ASMA (A2547, clone 1A4) from Sigma(Saint Louis, MO), and mouse monoclonal anti-actin(MAB1501) from Millipore (Temecula, CA) and goatanti-collagen I (AB758) from Millipore.Fibroblast immunocytochemistryNLF and SLF were cultured on coverslips and stained aspreviously described [2,7] using the ASMA and pERKantibodies indicated above and appropriate secondaryantibodies tagged with Alexa Fluor® 488. Nuclei werestained with DAPI. Images were acquired using a Zeiss510SML Laser Confocal Microscope (excitation S490/20,emission D528/38) fitted with an oil-immersion ob-jective (40 × /1.4).PeptidesTo compare the activity of CSD (amino acids 82–101 ofcaveolin-1) to its subdomains, CSD and five subdomainsnamed by Bernatchez et al. [11] [ Cav-A (aa 82–88),Cav-B (aa 89–95), Cav-C (aa 96–101), Cav-AB (aa 82–95)and Cav-BC (aa 89–101)] were synthesized in fusionwith the Antennapedia Internalization Sequence. Weroutinely refer to these fusions simply as CSD, Cav-A,Cav-B, Cav-C, Cav-AB, and Cav-BC. The AntennapediaInternalization Sequence alone was routinely used asControl peptide, when tested scrambled CSD gavesimilar results to the Antennapedia Internalization Se-quence alone. In addition, mutated CSD peptides inwhich F92 was converted to A and in which T90, T91,and F92 were all converted to A (referred to respectivelyas 92A and 90-92A) were synthesized. CSD, Cav-A, Cav-B,Cav-C, Cav-AB, Cav-BC, 92A, 90-92A, and control pep-tides were dissolved at 10 mM in 100% DMSO, diluted10-fold with water as previously described, and furtherdiluted as appropriate for each experiment [2,11,16,17].Statistical analysesImmunoreactive bands were quantified by densitometryusing ImageJ 1.32 NIH software. Raw densitometric datawere processed and analyzed using Prism 3.0 (GraphPadSoftware Inc.). Student’s t-test was used to evaluate data.In all Figures, *** indicates p < 0.001, ** indicates p < 0.01,and * indicates p < 0.05.ResultsCSD and its subdomains differ in their ability to inhibitthe migration of normal and SSc monocytesWe previously demonstrated that SSc monocytes andTGFβ-treated normal monocytes are hypermigratory to-ward the CXCR4 ligand CXCL12 and that this migrationis inhibited by CSD [17]. To evaluate the possible role ofregulation of cell death and/or apoptosis by TGFβ orCSD in their effects on migration, we studied cell death(measured by propidium iodide staining) and apoptosis(measured in terms of cell surface annexin V labeling).We found that neither TGFβ nor CSD affected eithercell death or apoptosis even though in a positive controlexperiment validating the method, H2O2 (a known in-ducer of cell death and apoptosis) did have the expectedeffects (Figure 1A,C). To further validate these observa-tions, apoptosis was also evaluated by TUNEL labeling.Again, essentially no apoptosis was observed in controlcells or in cells treated with CSD, TGFβ, or both re-agents even though in a positive control TUNEL label-ing was observed (Figure 1B,D). These observations(See figure on previous page.)Figure 1 Effects of CSD and TGFβ on monocyte migration do not involve cell death/apoptosis. (A) Cell death/apoptosis was evaluated byflow cytometry using Molecular Probes kit V13241. Briefly, cells were treated with CSD, TGFβ, or both reagents (see Methods). Cells were thenincubated with propidium iodide (PI) and fluorescent annexin V and analyzed by flow cytometry. Gates were set using unstained cells. A typicalexperiment is shown in which live cells (PI-negative) were gated from dead cells (PI-positive) indicated as Live/Dead. Live cells were further gatedinto apoptotic cells (annexin V-positive) and non-apoptotic cells (annexin V-negative) indicated as Annexin V: Minus/Plus. To validate the assay,the indicated cells were treated with 20 mM H2O2 (a known inducer of cell death/apoptosis). (B) Apoptosis was evaluated by TUNEL labelingusing the In Situ Cell Death Detection Kit, Fluorescein (Product No. 11684795910, Roche, Indianapolis, IN). Briefly, cells on coverslips were treatedwith CSD, TGFβ, or both reagents (see Methods); fixed; permeabilized; DNA strand breaks fluorescently labeled using TUNEL reagent; and imagedby fluorescent microscopy. A typical experiment is shown in which essentially no cells were labeled except when permeabilized cells weretreated with DNase prior to TUNEL labeling. Nuclei were counter-stained using DAPI. (C) Flow cytometry quantification. Average values ± s.e.m.are presented summarizing the results of four independent experiments performed using cells from different subjects. PI + indicates thepercentage of dead PI-positive cells; Annexin V + indicates the percentage of the PI-negative cell population that is annexin V-positive (i.e. apoptotic).(D) TUNEL quantification. Three independent experiments were performed using cells from different subjects. The percentage of fluorescent,TUNEL-positive cells was always < 2% except when DNA strand breaks were generated using DNase.Reese et al. Respiratory Research 2013, 14:90 Page 5 of 18http://respiratory-research.com/content/14/1/90strongly suggest that the effects of CSD and TGFβ onmigration are not mediated via effects on cell death orapoptosis.When we examined the effect of CSD subdomains onthe migration of normal monocytes, we found that whileall peptides used at the standard concentration of 5 μM[2,10,11,16,17] inhibited TGFβ-induced hypermigration,only CSD inhibited the low level of migration observedin the absence of TGFβ (Figure 2). Indeed, otherpeptides, especially Cav-A, appeared to increase thisbackground migration (Figure 2, Table 3). While thehypermigration of SSc monocytes was similar to theTable 3 Quantification and statistical significance of monocyte migration and pSmad 2/3 staining dataMonocytes Peptide Migration Statistical significance pSmad 2/3 Statistical significanceNormal Control 20.1 ± 2.5 26.3 ± 1.1Normal CSD 12.6 ± 2.1 p < 0.05 vs Normal/Control 16.4 ± 0.5 p < 0.01 vs Normal/ControlNormal Cav-A 48.4 ± 7.8 p < 0.01 vs Normal/Control 31.0 ± 1.1 p < 0.05 vs Normal/ControlNormal Cav-B 35.4 ± 4.5 p < 0.01 vs Normal/Control 16.6 ± 1.6 p < 0.01 vs Normal/ControlNormal Cav-C 27.1 ± 2.1 19.1 ± 1.5 p < 0.01 vs Normal/ControlNormal Cav-AB 37.8 ± 8.7 p < 0.05 vs Normal/Control 26.8 ± 1.4Normal Cav-BC 32.6 ± 5.1 p < 0.05 vs Normal/Control 18.4 ± 1.2 p < 0.01 vs Normal/ControlNormal + TGFβ Control 99.9 ± 11.7 p < 0.001 vs Normal/Control 43.1 ± 0.9 p < 0.01 vs Normal/ControlNormal + TGFβ CSD 18.7 ± 4.5 p < 0.01 vs Normal + TGFβ/Control 21.2 ± 1.2 p < 0.01 vs Normal + TGFβ/ControlNormal + TGFβ Cav-A 47.4 ± 5.5 p < 0.01 vs Normal + TGFβ/Control 37.7 ± 1.2Normal + TGFβ Cav-B 47.6 ± 4.4 p < 0.01 vs Normal + TGFβ/Control 40.7 ± 1.2Normal + TGFβ Cav-C 36.2 ± 3.0 p < 0.01 vs Normal + TGFβ/Control 43.0 ± 1.1Normal + TGFβ Cav-AB 37.7 ± 3.5 p < 0.01 vs Normal + TGFβ/Control 39.0 ± 1.5Normal + TGFβ Cav-BC 25.9 ± 5.7 p < 0.01 vs Normal + TGFβ/Control 18.7 ± 1.4 p < 0.01 vs Normal + TGFβ/ControlSSc Control 303 ± 32 p < 0.001 vs Normal/Control 54.1 ± 1.0 p < 0.01 vs Normal/ControlSSc CSD 77 ± 10 p < 0.01 vs SSc/Control 37.6 ± 0.7 p < 0.01 vs SSc/ControlSSc Cav-A 334 ± 44 49.1 ± 3.1SSc Cav-B Not Done 48.4 ± 1.5SSc Cav-C Not Done 49.3 ± 1.2SSc Cav-AB Not Done 46.1 ± 3.0SSc Cav-BC 138 ± 24 p < 0.02 vs SSc/Control 36.6 ± 1.4 p < 0.01 vs SSc/ControlData from Figure 2 (migration) and Figure 3 (image analyses of pSmad 2/3 staining) are presented in terms of average value ± s.e.m. The statistical significance ofthe indicated comparisons were determined using Students’ t test.Figure 2 Effects of CSD and its subdomains on Normal and SSc monocyte migration in vitro. Normal monocytes (A) were isolated fromhealthy donors, SSc monocytes (C) were isolated from SSc patients. The migration of these cells and Normal monocytes pretreated with TGFβ(B) toward CXCL12 was quantified in the presence of 5 μM of CSD or the indicated subdomains of CSD as described in the Methods. TheAntennapedia Internalization Sequence alone was routinely used as the Control peptide; when tested scrambled CSD attached to theAntennapedia Internalization Sequence gave similar results. Each symbol represents the results obtained with cells from an individual donor.Reese et al. Respiratory Research 2013, 14:90 Page 6 of 18http://respiratory-research.com/content/14/1/90hypermigration of TGFβ-treated monocytes in that bothwere inhibited by CSD and Cav-BC, they differed dra-matically in their response to Cav-A which inhibitedTGFβ-induced monocyte hypermigration but slightly in-creased SSc monocyte hypermigration (Figure 2, Table 3).Cav-B, Cav-C, and Cav-AB also inhibited TGFβ-inducedmonocyte hypermigration, although not as effectively asCSD and Cav-BC (Figure 2, Table 3).Because TGFβ enhanced monocyte migration and thiseffect was inhibited by CSD and each subdomain, we ex-amined the effect of CSD and its subdomains on canon-ical TGFβ signaling via the activation of Smad 2/3.Figure 3 Effects of CSD and its subdomains on pSmad 2/3 expression in normal, TGFβ-treated, and SSc monocytes. As described in theMethods, monocytes were isolated, plated on coverslips, treated with the indicated reagents (TGFβ, CSD, or its subdomains), fixed, and stained forpSmad 2/3 and with the nuclear stain DAPI. Representative images are shown selected from 20 to 60 cells observed from four donors in eachcategory. (A) Normal monocytes treated with TGFβ and CSD; (B) SSc monocytes treated with CSD and its subdomains; (C) Normal monocytestreated with TGFβ and CSD subdomains. The Antennapedia Internalization Sequence alone was routinely used as the Control peptide; whentested scrambled CSD attached to the Antennapedia Internalization Sequence gave similar results.Reese et al. Respiratory Research 2013, 14:90 Page 7 of 18http://respiratory-research.com/content/14/1/90Immunocytochemical analyses of activated Smad 2/3(i.e. pSmad 2/3) levels in monocytes showed a strongcorrelation between ability to migrate and pSmad 2/3levels under several conditions (Figure 3, quantified inTable 3). For example, the enhanced ability of TGFβ-treated normal monocytes and SSc monocytes to mi-grate is accompanied by an increase in pSmad 2/3 levels.The abilities of CSD and Cav-BC to strongly inhibit themigration of TGFβ-treated normal monocytes and SScmonocytes are accompanied by a major decrease inpSmad 2/3 levels. The activation of Normal monocytemigration by Cav-A is accompanied by an increase inpSmad 2/3 level. On the other hand, various CSDsubdomains enhance the migration of Normal mono-cytes or inhibit the migration of TGFβ-treated normalmonocytes while having no effect or an opposite effecton pSmad 2/3 level. In summary, these studies demon-strate that while CSD and its subdomains can affectTGFβ signaling through pSmad 2/3, it is likely that boththeir effects on TGFβ signaling and their well-knowneffects on other signaling cascades together regulate mo-nocyte migration.To further validate these results, select experimentswere repeated and analyzed by Western blot (Figure 4).In accord with the immunocytochemical data, the pSmad2/3 level was much higher in SSc monocytes than in Nor-mal monocytes. Interestingly, the Smad 2/3 level was in-creased to an even greater extent than the pSmad 2/3level in SSc monocytes (p < 0.001), suggesting that the in-crease in pSmad 2/3 is driven by the increase in Smad 2/3.Also in accord with the immunocytochemical data, thepSmad 2/3 level in Normal monocytes was increased byTGFβ treatment and this increase was substantiallyblocked by CSD. In this case, the increase in pSmad 2/3level occurred in the absence of any change in Smad 2/3level.CSD and its subdomains differ in their effects on CXCR4expression and F-actin staining in normal and SScmonocytesTo evaluate the mechanisms underlying the differentialeffects of CSD and its subdomains on migration, wecompared the effects of these peptides on the expressionof CXCR4 and F-actin. Treatment of monocytes fromhealthy donors with TGFβ significantly increased CXCR4expression and this increase was completely inhibited byCSD (Figure 5A, Table 3). In contrast, TGFβ only slightlyincreased F-actin staining (Figure 5A) and this increasewas not fully reversed by CSD. When other peptides wereexamined (Figure 5B, Table 3), the results were more com-plicated because, in many cases, the peptide affectedCXCR4 and F-actin expression in the absence of TGFβ.Cav-A, Cav-B, Cav-C, and to a lesser extent Cav-AB pro-moted F-actin staining and this effect was decreased byTGFβ. Cav-BC, like CSD, had little effect on F-actin stai-ning. Cav-A, and to a lesser extent Cav-B and Cav-C, in-creased CXCR4 expression in normal cells while eachpeptide inhibited CXCR4 expression similarly in TGFβ-treated cells (Figure 5B, Table 3).Experiments using SSc monocytes revealed a muchhigher level of CXCR4 expression [16] and F-actin stain-ing than in normal monocytes and a somewhat differentpattern of sensitivity to caveolin-1 peptides (Figure 6,Table 3). As previously shown, CSD inhibited CXCR4expression. Cav-BC also inhibited CXCR4 expression asdid Cav-B, Cav-C, and Cav-AB to a lesser extent. In con-trast, as in healthy monocytes, Cav-A increased CXCR4expression (Figure 6). CSD treatment also inhibited F-actin staining. In addition, the effect of CSD on the cyto-skeleton results in smaller cells. Cav-B and Cav-AB andto a lesser extent Cav-C and Cav-BC somewhat inhibitedF-actin staining.In summary, for each type of monocyte (Normal,Normal + TGFβ, SSc) the expression of CXCR4 andF-actin, particularly CXCR4, are predictive of the level ofFigure 4 Smad 2/3 expression and activation in monocytes. Theindicated monocytes were isolated and treated with CSD (or controlpeptide) as described in the Methods (Smad Western blots). TGFβindicates TGFβ-treated Normal monocytes. Smad 2/3 expressionand activation (i.e. pSmad 2/3 levels) were evaluated by Westernblotting (50 μg total protein per lane). The data shown are theaverage ± s.e.m. in arbitrary units of the densitometric quantificationof three independent experiments with cells from different subjects.The levels of Smad 2/3 and pSmad 2/3 in Normal monocytestreated with control peptide (normalized against the GAPDH loadingcontrol) were set to 100 arbitrary units. Indications of statisticalsignificance for TGFβ-treated Normal monocytes and SSc monocytesare versus Normal monocytes. The indication of statisticalsignificance for TGFβ-treated Normal monocytes treated with CSD isversus TGFβ-treated Normal monocytes treated withcontrol peptide.Reese et al. Respiratory Research 2013, 14:90 Page 8 of 18http://respiratory-research.com/content/14/1/90migration. Nevertheless, CXCR4 and F-actin levels arenot sufficient to explain the different ability of each typeof monocyte to migrate strongly suggesting that CXCR4and F-actin levels are not the only differences betweenNormal, Normal + TGFβ, and SSc monocytes.CSD and its subdomains differ in their ability to inhibitmonocyte to fibrocyte differentiation in vitroMonocytes and monocyte-derived fibrocytes are believedto participate in lung fibrosis both as sources of cytokinesand as the precursors of myofibroblasts [19-26]. Given thatcaveolin-1 levels regulate monocyte functions, we exam-ined the effect of CSD and it subdomains on monocyte tofibrocyte differentiation in 12-day cultures (Figure 7). BothCSD and Cav-BC significantly inhibited differentiation atthe routine 5 μM and even at the lowest concentrationtested (0.01 μM). In contrast, control Antennapedia pep-tide and Cav-A had no effect at 5 μM.When 12-day cultures were stained for collagen I(Figure 8), we observed a similar level of staining innormal and SSc fibrocytes which was not decreased byCSD or Cav-BC treatment (even though these treat-ments did decrease the number of fibrocytes present).However, when the cultures were stained for ASMA,we observed a high level of staining in SSc fibrocytes,but not in normal fibrocytes, and this accumulation ofASMA in SSc fibrocytes was almost completely blockedby CSD or Cav-BC. During a 21-day culture, as wasshown previously [24,27], ASMA could be detected innormal fibrocyte cultures (data not shown), indicating thatby the criterion of ASMA expression, SSc monocytes dif-ferentiate into fibrocytes more rapidly than do normalmonocytes. The combined results highlight Cav-BC as anexcellent candidate to be a subdomain of CSD active inthe amelioration of lung fibrosis in vivo because it inhibitsprofibrotic features of monocytes as well as their migra-tion and differentiation into fibrocytes.Dose-dependent effects of csd and subdomains onmonocyte migrationGiven the Results highlighting the functional importanceof Cav-A and Cav-BC, we examined the dose-dependenceFigure 5 Effects of TGFβ, CSD, and its subdomains on F-actinand CXCR4 expression in normal monocytes. As described in theMethods, normal monocytes were isolated, plated on coverslips,treated with the indicated reagents (TGFβ, CSD, or its subdomains),fixed, and stained for CXCR4 and F-actin and with the nuclear stainDAPI. Representative images are shown selected from 20 to 60 cellsobserved from four donors in each category. (A) Normal monocytestreated with TGFβ and CSD; (B) Normal monocytes treated withTGFβ and CSD subdomains. The Antennapedia InternalizationSequence alone was routinely used as the Control peptide; whentested scrambled CSD attached to the Antennapedia InternalizationSequence gave similar results.Reese et al. Respiratory Research 2013, 14:90 Page 9 of 18http://respiratory-research.com/content/14/1/90of CSD, Cav-A, and Cav-BC on the migration of nor-mal monocytes with and without TGFβ treatment(Figure 9). Even when diluted an additional 500-fold(to 0.01 μM), CSD, Cav-A, and Cav-BC strongly inhibitedthe migration of TGFβ-activated normal monocytes. Forunactivated normal monocytes, throughout the dose curveCSD inhibited migration while Cav-A promoted migrationand Cav-BC slightly promoted migration.CSD and its subdomains differ in their ability to inhibitcollagen I and ASMA expression and MEK/ERK signalingin NLF and SLFAs in our previous studies, CSD inhibited collagen Iexpression in both NLF and SLF, but inhibited ASMAexpression only in SLF. Cav-C, Cav-AB, and Cav-BCinhibited collagen I expression in both cell types(Figure 10AB) with Cav-BC being most effective, buthad different effects on ASMA expression. In particular,Cav-C was similar to CSD in inhibiting ASMA expres-sion in only SLF, Cav-BC inhibited ASMA expression inboth cell types, while Cav-AB did not affect either celltype (Figure 10AB). Cav-A and Cav-B did not inhibitcollagen I or ASMA expression. In fact, Cav-A clearlyincreased collagen I expression in SLF and slightly in-creased collagen I expression in NLF. As we previouslydemonstrated that MEK/ERK signaling regulates colla-gen I and ASMA expression [2,7], we determined the ef-fect of CSD subdomains on the activation of thesekinases. Cav-BC (like CSD) inhibited MEK and ERK ac-tivation in both cell types (Figure 10AB). Cav-C wassomewhat less effective but still inhibited MEK and ERKin both cell types. Cav-AB slightly inhibited MEK inNLF and ERK in both NLF and SLF, while Cav-A andCav-B were inactive.To validate these observations and to learn moreabout the distribution of ASMA and activated ERK, weexamined their expression and distribution by fluores-cent microscopy (Figure 10CD). For ASMA (Figure 10C),as in Figure 10AB, CSD inhibited its expression only inSLF while Cav-BC inhibited its expression in both celltypes. For activated ERK (Figure 10D), as in Figure 10AB,inhibition of expression by CSD, Cav-BC, and Cav-C wasFigure 6 Effects of CSD and its subdomains on F-actin andCXCR4 expression in SSc monocytes. As described in theMethods, SSc monocytes were isolated, plated on coverslips, treatedwith the indicated reagents (CSD or its subdomains), fixed, andstained for CXCR4 and F-actin and with the nuclear stain DAPI. TheAntennapedia Internalization Sequence alone was routinely used asthe Control peptide; when tested scrambled CSD attached to theAntennapedia Internalization Sequence gave similar results.Representative images are shown selected from 20 to 60 cellsobserved from four donors in each category.Figure 7 Different effects of CSD and its subdomains onfibrocyte differentiation. PBMC were incubated for fibrocytedifferentiation in vitro as described in the Methods in the presenceof the indicated peptides at the indicated concentrations. Thenumber of cells per field identified as fibrocytes by their spindle-shaped morphology was quantified. The AntennapediaInternalization Sequence alone was routinely used as the Controlpeptide; when tested scrambled CSD attached to the AntennapediaInternalization Sequence gave similar results. These data representthe average ± s.e.m. of six independent fields for each conditionfrom five normal and five SSc donors. For comparisons of CSD andCav-BC with Control peptide ** indicates p < 0.01 and * indicatesp < 0.05. For Normal cells/Control peptide vs SSc cells/Controlpeptide p < 0.01.Reese et al. Respiratory Research 2013, 14:90 Page 10 of 18http://respiratory-research.com/content/14/1/90apparent. The slight inhibition observed in Figure 10ABfor Cav-AB could not be detected by fluorescent micros-copy. In addition, fluorescent microscopy revealed achange in subcellular distribution induced by Cav-A and,to a lesser extent, Cav-AB. Cav-A and Cav-AB caused ac-tivated ERK to translocate to the nucleus in NLF, but notin SLF.To further study the effects of CSD, Cav-BC, andCav-C, we determined the dose-dependence of their ef-fects on the expression of collagen I, ASMA, activatedERK, and activated MEK in NLF and SLF (Figure 11). Ingeneral, the effects that we observed using 5 μM peptide(Figure 10) were almost absent at 1 μM. These experi-ments validated the observation that CSD and Cav-Cblock ASMA expression in SLF but not in NLF becausethis effect was observed in cells treated with both 3 and5 μM peptide (Figure 11). In contrast, Cav-BC inhibitedASMA expression similarly in both cell types at all con-centrations tested.Given that CSD and related peptides must be used at3 μM to affect fibroblast function but strongly affectmonocyte function at 0.01 μM, we hypothesized that thiseffect results from a much lower caveolin-1 concentra-tion in monocytes than in fibroblasts. Thus proportion-ally lower concentrations of peptide would be needed toreverse the effects of low caveolin-1 in SSc monocytescompared to SSc fibroblasts. To test this hypothesis, wecompared the levels of caveolin-1 in normal monocytes,fibroblasts, and endothelial cells. Endothelial cells wereincluded because the literature [12] shows that, like fibro-blasts, micromolar CSD is required to affect the functionof these cells. As predicted, the level of caveolin-1 is farless in monocytes than in either fibroblasts or endothelialcells (Figure 12), strongly supporting the idea that the ex-treme sensitivity of monocytes to CSD is due to the lowbaseline concentration of caveolin-1 in these cells.Specific amino acids involved in the function of CSDAlanine screening revealed that amino acids 90 to 92 ofcaveolin-1 (particularly 92) are critical amino acids inCSD in the regulation of eNOS-dependent NO releasefrom endothelial cells (Bernatchez, 2005). To determinewhether these same amino acids are critical in the abilityof CSD to regulate monocyte migration and differen-tiation and to regulate collagen I, pERK, pMEK, andFigure 8 ASMA staining is observed in SSc fibrocytes, but notnormal fibrocytes, and is blocked by CSD and Cav-BC. PBMCwere incubated for fibrocyte differentiation in vitro on coverslips asdescribed in the Methods in the presence of the indicated peptidesat 0.1 μM, then stained for collagen I and ASMA and counterstainedwith the nuclear stain DAPI. The Antennapedia InternalizationSequence alone was routinely used as the Control peptide; whentested scrambled CSD attached to the Antennapedia InternalizationSequence gave similar results. Note the increased number offibrocytes in SSc cultures, the inhibition of fibrocyte differentiationby CSD and Cav-BC in both SSc and normal cultures, the expressionof ASMA in SSc fibrocytes but not in normal fibrocytes, theinhibition of ASMA expression by CSD and Cav-BC, and the similarlevels of collagen I expression in fibrocytes in all cases. Similar resultswere obtained in four independent experiments.Figure 9 Dose dependence of effects of CSD and its subdomainson the migration in vitro of Normal monocytes with and withoutTGFβ activation. The migration toward CXCL12 of Normal monocyteswith and without TGFβ activation was quantified as described in theMethods in the presence of the indicated concentration of CSD and itssubdomains. The Antennapedia Internalization Sequence alone wasroutinely used as the Control peptide; when tested scrambled CSDattached to the Antennapedia Internalization Sequence gave similarresults. The results represent the average ± s.e.m of four independentexperiments. For comparisons of CSD, Cav-A, and Cav-BC with Controlpeptide ** indicates p < 0.01 and * indicates p < 0.05. For Normal cells/Control peptide vs TGFβ cells/Control peptide p < 0.001.Reese et al. Respiratory Research 2013, 14:90 Page 11 of 18http://respiratory-research.com/content/14/1/90Figure 10 Inhibition of collagen I and ASMA expression and MEK/ERK activation in fibroblasts by CSD and its subdomains. Serum-starved NLF and SLF were incubated for an additional 6 h in fresh serum-free medium containing 5 μM of either CSD, Control peptide, or theindicated subdomain of CSD. The Antennapedia Internalization Sequence alone was routinely used as the Control peptide; when testedscrambled CSD attached to the Antennapedia Internalization Sequence gave similar results. The levels of collagen I in the culture medium and ofpMEK, MEK, pERK, ERK, ASMA, and actin (loading control) in the cell layer were determined by Western blotting as described in the Methods (A).(B) Densitometric quantification combining data from three experiments similar to A performed with three independent pairs of NLF and SLF.For comparisons of CSD and related peptides with Control peptide*** indicates p < 0.001, ** indicates p < 0.01, and * indicates p < 0.05. For NLF/Control peptide vs SLF/Control peptide for each chart in (B) p < 0.01. Immunofluorescent detection of effects of CSD and its subdomains on pERK(C) and ASMA (D) expression in NLF and SLF. Cells were cultured on coverslips under the conditions described above, then fixed and stained todetect the indicated proteins. Nuclei were counterstained with DAPI.Figure 11 Dose-dependence of inhibition of collagen I and ASMA expression and MEK/ERK activation in fibroblasts by CSD and itssubdomains. NLF and SLF were cultured as described in Figure 8 with 0, 1, 3, or 5 μM of the indicated peptides. Collagen I in the medium andpERK, ph-MEK, ASMA and actin (loading control) in the cell layer were detected by Western blotting. Blots were quantified densitometrically. Thelevels of collagen I, pERK, pMEK, and ASMA in cells treated with no peptide (divided by the level of actin) were set to 100 arbitrary units. The datapresented are the average of the densitometric quantification of three independent experiments.Reese et al. Respiratory Research 2013, 14:90 Page 12 of 18http://respiratory-research.com/content/14/1/90ASMA expression in fibroblasts, we compared the activ-ities of CSD and the mutated versions of CSD describedas 92A and 90-92A.Compared to CSD, both 92A and 90-92A were par-tially active in their ability to inhibit the migration ofTGFβ-treated Normal monocytes and in their ability toinhibit the differentiation of Normal monocytes intofibrocytes (Figure 13AB). Although CSD inhibits the mi-gration of Normal monocytes that had not been treatedwith TGFβ, 92A and 90-92A had no effect on thesepoorly migrating cells.A range of outcomes were observed with fibroblastsdepending on the target protein and whether NLF orSLF were being studied (Figure 13C-G). 92A and 90-92A were fully as active as CSD in inhibiting collagen Iexpression in both NLF and SLF and in inhibiting pMEKand pERK in NLF. In contrast, 92A and 90-92A were in-active compared to CSD in inhibiting pMEK, pERK, andASMA expression in SLF. None of CSD, 92A, or 90-92Ainhibited ASMA expression in NLF (i.e. cells whosebaseline expression of ASMA is low). Thus, just as therelative activities of Cav-A, Cav-B, Cav-C, Cav-AB, andCav-BC are context dependent, the importance of aminoacids 90 to 92 in the regulation of cell behavior is alsocontext dependent in that it depends on the cell typeand readout being studied.DiscussionIn this study, we have examined cellular mechanisms in-volved in lung fibrosis that are affected when the defi-ciency of caveolin-1 in fibrotic lung tissue is reversedusing specific subdomains of the CSD peptide and mu-tated versions of the CSD peptide. These critical cellularmechanisms are the migration of monocytes into dam-aged tissue and their differentiation into fibrocytes andthe expression of collagen I by fibroblasts. Studies ondownstream molecular mechanisms through which theseversions of CSD mediate their effects are also presen-ted. More detailed studies on molecular mechanismare underway.We previously showed that monocytes and fibroblastsfrom SSc patients are deficient in caveolin-1, and thattreatment of these cells with the CSD peptide compen-sates for this deficiency thereby reversing a number ofpathological features of these cells. Our goal in thisstudy was to determine whether different subdomains ofCSD have different abilities to regulate parameters asso-ciated with fibrosis in monocytes and fibroblasts withthe idea that a particular subdomain might be a more ef-fective treatment for SSc than full-length CSD. Our re-sults are summarized in Table 4.Migration toward CXCL12 is enhanced several-fold inTGFβ-treated normal monocytes and even more in SScmonocytes [17]. While CSD inhibits migration in allthree cell populations and at very low doses, thesubdomains have different effects (Table 4). Cav-BC alsostrongly inhibits at very low doses the migration of cellsthat migrate well (TGFβ-treated and SSc monocytes).Lesser inhibition of migration in TGFβ-treated monocyteswas obtained with the other subdomains while Cav-Aslightly enhanced SSc monocyte migration. Cav-A, andother subdomains to a lesser extent, also enhanced themigration of normal monocytes (i.e. not TGFβ-treated).Because TGFβ treatment strongly enhanced monocytemigration, we also examined canonical TGFβ signalingvia Smad 2/3. In many cases, the effects of CSD and itssubdomains on monocyte migration and Smad 2/3 acti-vation were similar. For example, CSD and Cav-BCstrongly inhibit the migration of monocytes that migratewell (i.e. TGFβ-treated Normal monocytes and SScmonocytes) and also strongly inhibit Smad 2/3 activationin these cells. Likewise, the enhancement of migrationthat occurs in Normal monocytes treated with Cav-Awas accompanied by an enhancement of Smad 2/3 acti-vation in these cells. In other cases, the effects ofsubdomains on migration and Smad 2/3 activation werenot similar. For example, several subdomains inhibitedmigration in TGFβ-treated Normal monocytes withoutaffecting Smad 2/3 activation; several subdomains en-hanced migration in Normal monocytes while inhibitingSmad 2/3 activation. An additional striking observationFigure 12 Monocytes contain little caveolin-1 compared tofibroblasts and endothelial cells. Human umbilical veinendothelial cells (Endo), NLF (Fib), and Normal monocytes (Mono)were extracted using SDS-PAGE sample buffer. (A) 10 μg each Endoand Fib extract and 30 μg Mono extract were Western blotted forcaveolin-1 and actin (loading control). Similar results were obtainedwhen GAPDH was used as the loading control (not shown).(B) Three times as much of each sample was loaded to allow thedetection of caveolin-1 in Mono.Reese et al. Respiratory Research 2013, 14:90 Page 13 of 18http://respiratory-research.com/content/14/1/90revealed by these studies is that while the levels ofactivated Smad 2/3 are very high in SSc monocytes com-pared to Normal monocytes, the enhancement in thelevel of total Smad 2/3 is even more pronounced. Insummary, we find that while CSD and its subdomainscan affect TGFβ signaling via pSmad 2/3, it is likely thattheir effects on TGFβ signaling combine with their well-known effects on other signaling cascades to regulatemonocyte migration.We report here the novel observation that the differ-entiation of SSc monocytes into spindle-shaped, ASMA-positive, collagen I-positive fibrocytes is enhancedcompared to normal monocytes (Figure 9). In both celltypes CSD and Cav-BC inhibit monocyte differentiation atFigure 13 Effects of mutated CSD on monocyte and fibroblast functions. The activity of CSD and mutated forms (92A and 90-92A) werecompared. (A,B) Monocytes. (C-F) Fibroblasts. (A) Monocyte migration experiments were performed with Normal monocytes and TGFβ-treatedNormal monocytes and the indicated peptides (0.1 μM) as described in the Methods. Indication of statistical significance for TGFβ-treated Normalmonocytes with Control peptide is versus Normal monocytes with Control peptide. Indications of statistical significance for TGFβ-treated Normalmonocytes treated with CSD, 92A, or 90-92A is versus TGFβ-treated Normal monocytes treated with Control peptide. (B) Fibrocyte differentiationexperiments were performed with Normal monocytes and the indicated peptides (0.1 μM) as described in the Methods. (C) The levels ofcollagen I in the culture medium and of pMEK, pERK, ASMA, and actin (loading control) in the cell layer were determined by Western blotting asdescribed in the Methods using NLF and SLF treated with the indicated peptides as described in the legend to Figure 10. (D-G) Densitometricquantification combining data from three experiments similar to (C) performed with three independent pairs of NLF and SLF. (D) Collagen I.(E) pMEK. (F) pERK. (G) ASMA. Indications of statistical significance for SLF plus Control peptide are versus NLF plus Control peptide. Indications ofstatistical significance for NLF plus 92A, 90-92A, or CSD are versus NLF plus Control peptide. Indications of statistical significance for SLF plus 92A,90-92A, or CSD are versus SLF plus Control peptide.Reese et al. Respiratory Research 2013, 14:90 Page 14 of 18http://respiratory-research.com/content/14/1/90very low doses, while Cav-A has no effect even at a highdose (Figure 9, Table 4). In summary, Cav-BC and Cav-Aare of particular interest. Cav-BC, like CSD, inhibits thepathological hypermigration of TGFβ-treated and SScmonocytes and the differentiation into fibrocytes of bothNormal and SSc monocytes. Unlike CSD, Cav-BC doesnot inhibit the migration of Normal monocytes. Cav-Aenhances the migration of both Normal and SSc mono-cytes although it partially inhibits the migration of TGFβ-treated monocytes and has no effect on differentiation.We previously reported that collagen I expression andMEK/ERK signaling in fibroblasts (both NLF and SLF) isinhibited by CSD but that ASMA expression is onlyinhibited by CSD in cells expressing it at high levels (i.e.SLF) [2]. Here we report that Cav-BC, like CSD, inhibitscollagen I expression and MEK/ERK signaling in bothNLF and SLF. However, unlike CSD, Cav-BC also in-hibits ASMA expression in both cell types. The functionof Cav-A in fibroblasts is also noteworthy, causing an in-crease in collagen I expression in SLF, a slight increasein collagen I in NLF, and the nuclear translocation ofERK in NLF. Thus, in both monocytes and fibroblastsCav-BC is the subdomain most similar in function toCSD while Cav-A has a variety of distinct, potentiallypro-fibrotic, functions. In addition to the differencesbetween the effect of each peptide on the expression ofcollagen I and ASMA and the differences between NLFand SLF in their response to a given peptide, our resultswere also very different than those of Bernatchez et al.[11] who examined the sensitivity of eNOS activity inendothelial cells to these same peptides. As shown inTable 4, eNOS activity was most strongly inhibited byintact CSD and Cav-B and was also inhibited by Cav-ABand Cav-BC. Therefore, Table 4 demonstrates that theability of each peptide to regulate the expression of aparticular target protein depends on the target proteinand on the cell type being studied.To further explore differences between particular celltypes and particular target proteins in terms of how theyare affected by different versions of CSD, we studied twomutated versions of CSD (92A and 90-92A) that werepreviously shown to be totally ineffective in inhibitingeNOS-mediated generation of NO in endothelial cells[11]. In contrast, 92A and 90-92A were partially activein inhibiting monocyte migration or differentiation intofibrocytes. Moreover, 92A and 90-92A were essentiallyas effective as CSD in inhibiting Collagen I expression inNLF and SLF and in inhibiting MEK and ERK activationin NLF. In contrast, they were almost inactive in inhibitingMEK and ERK activation and ASMA expression in SLF.Table 4 Differential effects of CSD subdomains on monocyte CXCR4 and F-actin expressionCells Peptide CXCR4 Statistical significance F-actin Statistical significanceNormal Control 23.2 ± 0.8 32.1 ± 2.6Normal CSD 20.6 ± 1.8 26.8 ± 1.7Normal Cav-A 40.6 ± 1.9 p < 0.01 vs Normal/Control 85.8 ± 3.6 p < 0.01 vs Normal/ControlNormal Cav-B 34.6 ± 1.2 p < 0.01 vs Normal/Control 93.9 ± 2.2 p < 0.01 vs Normal/ControlNormal Cav-C 28.7 ± 0.8 p < 0.01 vs Normal/Control 73.7 ± 5.0 p < 0.01 vs Normal/ControlNormal Cav-AB 23.9 ± 0.8 42.0 ± 2.1 p < 0.05 vs Normal/ControlNormal Cav-BC 20.9 ± 0.7 34.5 ± 1.4Normal + TGFβ Control 50.1 ± 2.6 p < 0.01 vs Normal/Control 40.7 ± 1.4 p < 0.05 vs Normal/ControlNormal + TGFβ CSD 23.7 ± 0.8 p < 0.01 vs Normal + TGFβ/Control 35.4 ± 2.0 p < 0.05 vs Normal + TGFβ/ControlNormal + TGFβ Cav-A 21.9 ± 0.6 p < 0.01 vs Normal + TGFβ/Control 49.0 ± 3.6Normal + TGFβ Cav-B 32.4 ± 1.1 p < 0.01 vs Normal + TGFβ/Control 75.2 ± 3.7 p < 0.01 vs Normal + TGFβ/ControlNormal + TGFβ Cav-C 27.6 ± 0.8 p < 0.01 vs Normal + TGFβ/Control 54.7 ± 4.5 p < 0.01 vs Normal + TGFβ/ControlNormal + TGFβ Cav-AB 22.3 ± 0.6 p < 0.01 vs Normal + TGFβ/Control 47.5 ± 4.7Normal + TGFβ Cav-BC 20.4 ± 0.8 p < 0.01 vs Normal + TGFβ/Control 38.7 ± 1.6SSc Control 43.8 ± 2.6 p < 0.01 vs Normal/Control 129.2 ± 11.9 p < 0.01 vs Normal/ControlSSc CSD 27.6 ± 1.4 p < 0.01 vs SSc/Control 58.3 ± 2.8 p < 0.01 vs SSc/ControlSSc Cav-A 51.4 ± 2.6 138.1 ± 7.3SSc Cav-B 32.6 ± 1.6 p < 0.01 vs SSc/Control 60.9 ± 5.7 p < 0.01 vs SSc/ControlSSc Cav-C 31.5 ± 1.5 p < 0.01 vs SSc/Control 100.0 ± 8.6SSc Cav-AB 30.9 ± 2.3 p < 0.01 vs SSc/Control 79.5 ± 5.1 p < 0.01 vs SSc/ControlSSc Cav-BC 27.7 ± 0.8 p < 0.01 vs SSc/Control 92.9 ± 4.5 p < 0.05 vs SSc/ControlImage analyses of Figures 5 and 6 are quantified in terms of average fluorescence intensity ± s.e.m. and the statistical significance of the indicated comparisonsdetermined using Students’ t test.Reese et al. Respiratory Research 2013, 14:90 Page 15 of 18http://respiratory-research.com/content/14/1/90These observations further support the conclusion thatthe ability of versions of CSD to regulate the expression ofa particular target protein depends on the target proteinand on the cell type being studied.Table 3 allows us to speculate on whether CSD and itssubdomains regulate monocyte migration through theireffects on CXCR4 or F-actin expression. Our observa-tions can be summarized as: 1) The enhanced migrationof TGFβ-treated normal monocytes is correlated primar-ily with increased CXCR4 expression while the enhancedmigration of SSc monocytes is correlated both with en-hanced CXCR4 and F-actin expression; 2) For each typeof monocyte (Normal, Normal + TGFβ, SSc), CSD and itsubdomains had essentially parallel effects on migrationand CXCR4 expression; 3) In cells that migrate well(Normal + TGFβ and SSc monocytes), Cav-BC andCSD are the most effective inhibitors of migration dueto their inhibition of CXCR4 expression. In contrast,TGFβ-treated and SSc monocytes differ greatly in thatCav-A inhibits CXCR4 expression and migration inTGFβ-treated monocytes while having no effect on theseparameters in SSc monocytes; 4) In cells that do notmigrate well (Normal monocytes), Cav-A promotesmigration due to its positive effects on both CXCR4 andF-actin levels. 5) A comparison of the data obtained withvarious combinations of cell type and peptide makes itclear that other factors besides CXCR4 and F-actinexpression control migration. For example, levels ofCXCR4 and F-actin in SSc monocytes in the presence ofCav-BC are similar to their levels in Normal monocytesin the presence of Cav-B yet the SSc monocytes exhibitfour-fold higher migration than the Normal monocytes.Thus, the enhanced migration of SSc monocytes com-pared to Normal monocytes must involve more differ-ences between these cell types than simply their CXCR4and F-actin levels.These studies have revealed that monocytes are muchmore sensitive to CSD and its subdomains than arefibroblasts. In studies using fibroblasts [2] and endothe-lial cells [11], 5 to 10 μM CSD was used to compensatefor a loss of caveolin-1. The current studies demonstratethat for fibroblasts this level of CSD and subdomains isrequired, because little or no effect is observed at 1 μM.In contrast, in experiments using monocytes, we reportthat CSD and Cav-BC are as active at 0.01 μM as theyare at 5 μM. These observations raise the possibility thatmonocytes are more sensitive to CSD and its subdo-mains than are fibroblasts and endothelial cells becausethe baseline level of caveolin-1 in monocytes is muchlower than in these other cell types. Indeed, we havedemonstrated in Figure 12 that this is the case. Thisfurther suggests that while the use of CSD or itssubdomains may have a therapeutic effect in human pa-tients by reversing the profibrotic and proinflammatoryeffects of low caveolin-1 in monocytes and fibrocytes,CSD and subdomains may not have side effects in fibro-blasts and endothelial cells because the small increase incaveolin-1 function in these cells that already containcaveolin-1 at high levels is not likely to have an appre-ciable effect on their function.Although many authors have proposed that TGFβ isthe major cytokine responsible for the pathology of SScand have used TGFβ-treated cells as a model for SSc,the current results demonstrate both differences andsimilarities between SSc and Normal + TGFβ monocytes(Tables 3, 4, and 5). These observations suggest thatpathways other than TGFβ are involved in the path-ology of SSc and that the sensitivity of these pathways toCSD and to its subdomains differ. Therefore, it is notsurprising, for example, that Cav-A inhibits the migra-tion of Normal + TGFβ monocytes, but slightly enhancesthe migration of Normal and SSc monocytes.ConclusionsThese studies have revealed Cav-BC to be an anti-inflammatory, anti-fibrotic subdomain of CSD that mayTable 5 Differential effects of CSD subdomains on monocyte migration, fibrocyte differentiation, and fibroblastcollagen and ASMA expressionPeptide Monoycte migration Fibrocyte differentiation Protein expression in fibroblasts and endothelial cellsNormal TGFβ SSc Normal SSc Collagen NLF Collagen SLF ASMA NLF ASMA SLF eNOS EndControl 20.1 99.9 303 30 54 100 200 100 260CSD ↓ ↓↓ ↓↓ ↓ ↓ ↓↓ ↓↓ No Effect ↓↓ ↓Cav-A ↑↑ ↓ No Effect No Effect No Effect No Effect ↑ No Effect No Effect No EffectCav-B ↑ ↓ Not Done Not Done Not Done No Effect No Effect No Effect No Effect ↓Cav-C ↑ ↓ Not Done Not Done Not Done ↓ ↓↓ No Effect ↓ No EffectCav-AB ↑ ↓ Not Done Not Done Not Done ↓↓ ↓↓ No Effect No Effect ↓Cav-BC ↑ ↓↓ ↓ ↓ ↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓The Control Peptide row shows the baseline values for each cell type for Monocyte Migration (in Cells/hpf from Figure 2 and Table 3), for Fibrocyte Differentiation(in Elongated cells/field from Figure 7), and for Protein Expression in Fibroblasts (in Arbitrary Units from Figure 10). eNOS expression in Endothelial Cell data arefrom ref. [11]. The other rows show increases and decreases from these baseline levels caused by CSD and related peptides (↑↑ = > 100% increase, ↑ = 35 to 100%increase, No Effect = 35% decrease to 35% increase, ↓ = 35 to 65% decrease, ↓↓ = 65 to 100% decrease.Reese et al. Respiratory Research 2013, 14:90 Page 16 of 18http://respiratory-research.com/content/14/1/90be useful in treating fibrotic lung diseases in human pa-tients. In contrast, Cav-A has certain pro-inflammatory,pro-fibrotic functions which may make it a useful treat-ment for other diseases such as wound healing. Thesestudies have also revealed major differences betweenNormal monocytes activated with TGFβ and SSc mono-cytes that suggest that the pathology of this disease is morecomplex than simply hyperactivated TGFβ signaling. Fu-ture studies will expand upon the peptide-specific and celltype-specific differences in signal transduction alreadyobserved that must underlie these complex observa-tions. Finally, we observed that monocytes are much moresensitive to CSD and its subdomains than are fibroblasts,suggesting that in vivo monocytes and fibrocytes will beselectively affected by CSD treatment without the treat-ment having significant side effects on other cell types.AbbreviationsASMA: α-smooth muscle actin; CXCL12: C-X-C chemokine ligand 12;CXCR4: C-X-C Chemokine receptor type 4; eNOS: Endothelial Nitric OxideSynthase; ERK: Extracellular signal-regulated kinases; ILD: Interstitial lungdisease; IPF: Idiopathic pulmonary fibrosis; MEK: MAPK/ERK kinase; NO: Nitricoxide; SSc: Systemic sclerosis, scleroderma; TGFβ: Transforming growth factorβ; PI: Propidium iodide.Competing interestsWhile none of the authors have received any financial benefit from thiswork, Drs. Hoffman and Tourkina are the Inventors on a use patent(# 8,058,227) issued to the Medical University of South Carolina on thecaveolin-1 scaffolding domain peptide as a treatment for fibrotic diseases.Drs. Hoffman and Tourkina are also the founders of a company,FibroTherapeutics, Inc., which has an option to license this patent fromMUSC for the purpose of developing a drug based on the caveolin-1scaffolding domain peptide.Authors’ contributionsCR participated in study design, monocyte isolation and all studies on thesecells, data interpretation, and manuscript preparation. SD and BP performedmonocyte isolation and studies. MB participated in monocyte to fibrocytedifferentiation studies and manuscript preparation. JO participated indata interpretation and manuscript preparation. AH performedimmunocytochemical analyses on human cells. RPV participated in studydesign, data interpretation and manuscript preparation. JZ participated inimmunocytochemical studies on monocytes. CMH analyzed patientdemographics. RMS participated in data interpretation and manuscriptpreparation. SH participated in study design, data interpretation, manuscriptpreparation and performed statistical analyses. ET participated in studydesign, human lung fibroblasts studies, data interpretation, and manuscriptpreparation. All authors have read and approved the final manuscript.AcknowledgmentsThis work was supported by grants: NIH NIAMS R01 AR062078, R03AR056767, and K01 AR054143 (to ET); USARMY/USAMRAA W81XWH-11-1-0508, NIH NHLBI R01 HL73718, NIH NCCAM R21 AT004450, and an SCTR PilotProject (to SH), NIH NIAMS P60 AR049459 (Multidisciplinary Clinical ResearchCenter, to RMS), and an NIH NCRR Construction Grant C06 RR015455. ET alsoreceived a grant and the Marta Max Award from the SclerodermaFoundation. SH also received support as a Co-Investigator on grants NIHNCRR P20 RR016434, NIH NCRR P20 RR016434-09S2, NIH NCRR P20RR021949, and a grant from the Leducq Foundation.The authors thank Drs. Robert Strieter and Marie Burdick for advice andassistance on monocyte to fibrocyte differentiation and Kelley Gibson andDana Rosson for recruiting donors and collecting blood samples.Author details1Department of Medicine/Division of Rheumatology and Immunology,Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC29425, USA. 2Department of Regenerative Medicine and Cell Biology, MedicalUniversity of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA.3Department of Pharmacology and Vascular Biology and TherapeuticsProgram, Yale University School of Medicine, New Haven, CT 06520, USA.4Department of Anesthesiology, The James Hogg Research Centre, Heart andLung Institute at St. Paul’s Hospital, Pharmacology and Therapeutics,University of British Columbia, St. Paul’s Hospital, Vancouver, British Columbia,Canada. 5Division of Rheumatology and Immunology, Department ofMedicine, Medical University of South Carolina, 96 Jonathan Lucas Street,Suite 912 MSC 637, Charleston, SC 29425, USA.Received: 5 March 2013 Accepted: 2 September 2013Published: 8 September 2013References1. 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