Produção Nacional
Revisado por pares
NF-κB and GATA-Binding Factor 6 Repress Transcription of Caveolins in Bladder Smooth Muscle Hypertrophy
2019; Elsevier BV; Volume: 189; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2018.12.013
ISSN1525-2191
AutoresChellappagounder Thangavel, Cristiano Mendes Gomes, Stephen A. Zderic, Elham Javed, Sankar Addya, Jagmohan Singh, Sreya Das, Ruth Birbe, Robert B. Den, Satish Rattan, Deepak A. Deshpande, Raymond B. Penn, Samuel Chacko, Ettickan Boopathi,
Tópico(s)Caveolin-1 and cellular processes
ResumoCaveolins (CAVs) are structural proteins of caveolae that function as signaling platforms to regulate smooth muscle contraction. Loss of CAV protein expression is associated with impaired contraction in obstruction-induced bladder smooth muscle (BSM) hypertrophy. In this study, microarray analysis of bladder RNA revealed down-regulation of CAV1, CAV2, and CAV3 gene transcription in BSM from models of obstructive bladder disease in mice and humans. We identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in mice with obstruction-induced BSM hypertrophy, and in men with benign prostatic hyperplasia. DNA affinity chromatography and chromatin immunoprecipitation assays revealed a greater increase in binding of GATA-binding factor 6 (GATA-6) and NF-κB to their cognate binding motifs on CAV1, CAV2, and CAV3 promoters in obstructed BSM relative to that observed in control BSM. Knockout of NF-κB subunits, shRNA-mediated knockdown of GATA-6, or pharmacologic inhibition of GATA-6 and NF-κB in BSM increased CAV1, CAV2, and CAV3 transcription and promoter activity. Conversely, overexpression of GATA-6 decreased CAV2 and CAV3 transcription and promoter activity. Collectively, these data provide new insight into the mechanisms by which CAV gene expression is repressed in hypertrophied BSM in obstructive bladder disease. Caveolins (CAVs) are structural proteins of caveolae that function as signaling platforms to regulate smooth muscle contraction. Loss of CAV protein expression is associated with impaired contraction in obstruction-induced bladder smooth muscle (BSM) hypertrophy. In this study, microarray analysis of bladder RNA revealed down-regulation of CAV1, CAV2, and CAV3 gene transcription in BSM from models of obstructive bladder disease in mice and humans. We identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in mice with obstruction-induced BSM hypertrophy, and in men with benign prostatic hyperplasia. DNA affinity chromatography and chromatin immunoprecipitation assays revealed a greater increase in binding of GATA-binding factor 6 (GATA-6) and NF-κB to their cognate binding motifs on CAV1, CAV2, and CAV3 promoters in obstructed BSM relative to that observed in control BSM. Knockout of NF-κB subunits, shRNA-mediated knockdown of GATA-6, or pharmacologic inhibition of GATA-6 and NF-κB in BSM increased CAV1, CAV2, and CAV3 transcription and promoter activity. Conversely, overexpression of GATA-6 decreased CAV2 and CAV3 transcription and promoter activity. Collectively, these data provide new insight into the mechanisms by which CAV gene expression is repressed in hypertrophied BSM in obstructive bladder disease. Caveolae are 50- to 100-nm (diameter) flask-shaped invaginations of the cell plasma membrane; they contain high levels of sphingolipids and cholesterols, and are present in muscle, endothelia, and adipocytes.1Gabella G. Quantitative morphological study of smooth muscle cells of the guinea-pig taenia coli.Cell Tissue Res. 1976; 170: 161-186Crossref PubMed Scopus (94) Google Scholar, 2Razani B. Woodman S.E. Lisanti M.P. Caveolae: from cell biology to animal physiology.Pharmacol Rev. 2002; 54: 431-467Crossref PubMed Scopus (844) Google Scholar, 3Galbiati F. Razani B. Lisanti M.P. Emerging themes in lipid rafts and caveolae.Cell. 2001; 106: 403-411Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar Caveolae have been shown to be necessary for multiple cellular processes, which include transcytosis, lipid metabolism, and receptor trafficking.3Galbiati F. Razani B. Lisanti M.P. Emerging themes in lipid rafts and caveolae.Cell. 2001; 106: 403-411Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar, 4Fielding C.J. Fielding P.E. Relationship between cholesterol trafficking and signaling in rafts and caveolae.Biochim Biophys Acta. 2003; 1610: 219-228Crossref PubMed Scopus (112) Google Scholar Caveolins (CAVs) are a family of 21- to 24-kDa integral membrane proteins involved in the formation of caveolae-rich membrane domains5Cohen A.W. Hnasko R. Schubert W. Lisanti M.P. Role of caveolae and caveolins in health and disease.Physiol Rev. 2004; 84: 1341-1379Crossref PubMed Scopus (736) Google Scholar with three mammalian isoforms identified as CAV1, CAV2, and CAV3.5Cohen A.W. Hnasko R. Schubert W. Lisanti M.P. Role of caveolae and caveolins in health and disease.Physiol Rev. 2004; 84: 1341-1379Crossref PubMed Scopus (736) Google Scholar CAV1 and CAV2 are widely expressed, whereas CAV3 is found predominantly in skeletal, cardiac, and smooth muscle cells (SMCs).5Cohen A.W. Hnasko R. Schubert W. Lisanti M.P. Role of caveolae and caveolins in health and disease.Physiol Rev. 2004; 84: 1341-1379Crossref PubMed Scopus (736) Google Scholar All three isoforms of CAV proteins are detected in bladder smooth muscle (BSM).6Sullivan M.P. Cristofaro V. Radisavljevic Z.M. Yalla S.V. Regional distribution and molecular interaction of caveolins in bladder smooth muscle.BJU Int. 2012; 110: E1163-E1172Crossref PubMed Scopus (6) Google Scholar Caveolae are also characterized by the presence of recently described cavin family members; interaction of CAV1 and cavin-1 at the plasma membrane is required for caveolae formation.7Bastiani M. Liu L. Hill M.M. Jedrychowski M.P. Nixon S.J. Lo H.P. Abankwa D. Luetterforst R. Fernandez-Rojo M. Breen M.R. Gygi S.P. Vinten J. Walser P.J. North K.N. Hancock J.F. Pilch P.F. Parton R.G. MURC/cavin-4 and cavin family members form tissue-specific caveolar complexes.J Cell Biol. 2009; 185: 1259-1273Crossref PubMed Scopus (207) Google Scholar, 8Hill M.M. Bastiani M. Luetterforst R. Kirkham M. Kirkham A. Nixon S.J. Walser P. Abankwa D. Oorschot V.M. Martin S. Hancock J.F. Parton R.G. PTRF-cavin, a conserved cytoplasmic protein required for caveola formation and function.Cell. 2008; 132: 113-124Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar Previous studies have established that these specialized lipid microdomains are involved in the compartmentalization of several signaling elements, such as growth factors, G-protein–coupled receptors, Src family protein kinases, and nitric oxide synthases, all of which can interact with CAV proteins and with caveolae.9Liu P. Ying Y. Ko Y.G. Anderson R.G. Localization of platelet-derived growth factor-stimulated phosphorylation cascade to caveolae.J Biol Chem. 1996; 271: 10299-10303Crossref PubMed Scopus (337) Google Scholar, 10Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. Identification of peptide and protein ligands for the caveolin-scaffolding domain: implications for the interaction of caveolin with caveolae-associated proteins.J Biol Chem. 1997; 272: 6525-6533Crossref PubMed Scopus (805) Google Scholar Previous studies have suggested a role for caveolae in smooth muscle signaling, and loss of CAV1 in these cells leads to failure of caveolae formation.11Patel H.H. Zhang S. Murray F. Suda R.Y. Head B.P. Yokoyama U. Swaney J.S. Niesman I.R. Schermuly R.T. Pullamsetti S.S. Thistlethwaite P.A. Miyanohara A. Farquhar M.G. Yuan J.X. Insel P.A. Increased smooth muscle cell expression of caveolin-1 and caveolae contribute to the pathophysiology of idiopathic pulmonary arterial hypertension.FASEB J. 2007; 21: 2970-2979Crossref PubMed Scopus (104) Google Scholar Both CAV1 and CAV3 can induce caveolae formation, whereas CAV2 requires CAV1 to reach the plasma membrane.2Razani B. Woodman S.E. Lisanti M.P. Caveolae: from cell biology to animal physiology.Pharmacol Rev. 2002; 54: 431-467Crossref PubMed Scopus (844) Google Scholar, 12Stehr M. Adam R.M. Khoury J. Zhuang L. Solomon K.R. Peters C.A. Freeman M.R. Platelet derived growth factor-BB is a potent mitogen for rat ureteral and human bladder smooth muscle cells: dependence on lipid rafts for cell signaling.J Urol. 2003; 169: 1165-1170Crossref PubMed Scopus (50) Google Scholar CAV3 is required for the assembly of membrane caveolae.13Campostrini G. Bonzanni M. Lissoni A. Bazzini C. Milanesi R. Vezzoli E. Francolini M. Baruscotti M. Bucchi A. Rivolta I. Fantini M. Severi S. Cappato R. Crotti L. J Schwartz P. DiFrancesco D. Barbuti A. The expression of the rare caveolin-3 variant T78M alters cardiac ion channels function and membrane excitability.Cardiovasc Res. 2017; 113: 1256-1265Crossref PubMed Scopus (14) Google Scholar CAV2 interacts with CAV1 and forms hetero-oligomeric complexes within caveolae. All three isoforms of CAV genes are expressed in bladder and vascular SMCs. CAV1, CAV2, and CAV3 proteins interact with each other to form an oligomeric complex that lines the membranes and provides structural stability to the inverted ω-shaped membrane invaginations.14Woodman S.E. Cheung M.W. Tarr M. North A.C. Schubert W. Lagaud G. Marks C.B. Russell R.G. Hassan G.S. Factor S.M. Christ G.J. Lisanti M.P. Urogenital alterations in aged male caveolin-1 knockout mice.J Urol. 2004; 171: 950-957Crossref PubMed Scopus (69) Google Scholar Loss of CAV1 gene expression has been shown to impair smooth muscle contraction in bladder and airways.14Woodman S.E. Cheung M.W. Tarr M. North A.C. Schubert W. Lagaud G. Marks C.B. Russell R.G. Hassan G.S. Factor S.M. Christ G.J. Lisanti M.P. Urogenital alterations in aged male caveolin-1 knockout mice.J Urol. 2004; 171: 950-957Crossref PubMed Scopus (69) Google Scholar, 15Cao G. Yang G. Timme T.L. Saika T. Truong L.D. Satoh T. Goltsov A. Park S.H. Men T. Kusaka N. Tian W. Ren C. Wang H. Kadmon D. Cai W.W. Chinault A.C. Boone T.B. Bradley A. Thompson T.C. Disruption of the caveolin-1 gene impairs renal calcium reabsorption and leads to hypercalciuria and urolithiasis.Am J Pathol. 2003; 162: 1241-1248Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 16Keshavarz M. Schwarz H. Hartmann P. Wiegand S. Skill M. Althaus M. Kummer W. Krasteva-Christ G. Caveolin-1: functional insights into its role in muscarine- and serotonin-induced smooth muscle constriction in murine airways.Front Physiol. 2017; 8: 295Crossref PubMed Scopus (6) Google Scholar Loss of CAV1 is associated with disruption of M3 muscarinic acetylcholine receptor activity in bladder and serotonergic and cholinergic activity in airways.16Keshavarz M. Schwarz H. Hartmann P. Wiegand S. Skill M. Althaus M. Kummer W. Krasteva-Christ G. Caveolin-1: functional insights into its role in muscarine- and serotonin-induced smooth muscle constriction in murine airways.Front Physiol. 2017; 8: 295Crossref PubMed Scopus (6) Google Scholar, 17Lai H.H. Boone T.B. Yang G. Smith C.P. Kiss S. Thompson T.C. Somogyi G.T. Loss of caveolin-1 expression is associated with disruption of muscarinic cholinergic activities in the urinary bladder.Neurochem Int. 2004; 45: 1185-1193Crossref PubMed Scopus (46) Google Scholar CAV3 expression is associated with contractile protein expression in rat aortic SMCs, and loss of CAV3 is associated with down-regulation of myocardin, which has been shown to be important for the smooth muscle contractile phenotype.18Ackers-Johnson M. Talasila A. Sage A.P. Long X. Bot I. Morrell N.W. Bennett M.R. Miano J.M. Sinha S. Myocardin regulates vascular smooth muscle cell inflammatory activation and disease.Arterioscler Thromb Vasc Biol. 2015; 35: 817-828Crossref PubMed Scopus (80) Google Scholar, 19Gutierrez-Pajares J.L. Iturrieta J. Dulam V. Wang Y. Pavlides S. Malacari G. Lisanti M.P. Frank P.G. Caveolin-3 promotes a vascular smooth muscle contractile phenotype.Front Cardiovasc Med. 2015; 2: 27Crossref PubMed Scopus (10) Google Scholar Caveolae modulate receptor-mediated contractile responses in the bladder, and the expression of all three CAV proteins in BSM supports a central role for caveolae in the regulation of selective G-protein–coupled receptor signaling.14Woodman S.E. Cheung M.W. Tarr M. North A.C. Schubert W. Lagaud G. Marks C.B. Russell R.G. Hassan G.S. Factor S.M. Christ G.J. Lisanti M.P. Urogenital alterations in aged male caveolin-1 knockout mice.J Urol. 2004; 171: 950-957Crossref PubMed Scopus (69) Google Scholar, 17Lai H.H. Boone T.B. Yang G. Smith C.P. Kiss S. Thompson T.C. Somogyi G.T. Loss of caveolin-1 expression is associated with disruption of muscarinic cholinergic activities in the urinary bladder.Neurochem Int. 2004; 45: 1185-1193Crossref PubMed Scopus (46) Google Scholar, 20Cristofaro V. Peters C.A. Yalla S.V. Sullivan M.P. Smooth muscle caveolae differentially regulate specific agonist induced bladder contractions.Neurourol Urodyn. 2007; 26: 71-80Crossref PubMed Scopus (37) Google Scholar Earlier studies have described loss of CAV1, CAV2, and CAV3 expression in BSM from mice, rabbit, and human with partial bladder outlet obstruction (PBOO) and in aged rat bladders.21Polyak E. Boopathi E. Mohanan S. Deng M. Zderic S.A. Wein A.J. Chacko S. Alterations in caveolin expression and ultrastructure after bladder smooth muscle hypertrophy.J Urol. 2009; 182: 2497-2503Crossref PubMed Scopus (32) Google Scholar, 22Boopathi E. Gomes C.M. Goldfarb R. John M. Srinivasan V.G. Alanzi J. Malkowicz S.B. Kathuria H. Zderic S.A. Wein A.J. Chacko S. Transcriptional repression of caveolin-1 (CAV1) gene expression by GATA-6 in bladder smooth muscle hypertrophy in mice and human beings.Am J Pathol. 2011; 178: 2236-2251Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 23Lowalekar S.K. Cristofaro V. Radisavljevic Z.M. Yalla S.V. Sullivan M.P. Loss of bladder smooth muscle caveolae in the aging bladder.Neurourol Urodyn. 2012; 31: 586-592Crossref PubMed Scopus (37) Google Scholar Thus, it is possible that the decreased BSM contraction observed in obstructed bladder could be due to loss of CAV protein expression in these tissues. Given the clear effect of reduced CAV protein expression in regulating BSM contraction, loss of CAV gene expression is anticipated to have significant impact in the pathophysiology of the BSM. Although several studies have described changes in CAV protein expression under various pathologic conditions,21Polyak E. Boopathi E. Mohanan S. Deng M. Zderic S.A. Wein A.J. Chacko S. Alterations in caveolin expression and ultrastructure after bladder smooth muscle hypertrophy.J Urol. 2009; 182: 2497-2503Crossref PubMed Scopus (32) Google Scholar, 22Boopathi E. Gomes C.M. Goldfarb R. John M. Srinivasan V.G. Alanzi J. Malkowicz S.B. Kathuria H. Zderic S.A. Wein A.J. Chacko S. Transcriptional repression of caveolin-1 (CAV1) gene expression by GATA-6 in bladder smooth muscle hypertrophy in mice and human beings.Am J Pathol. 2011; 178: 2236-2251Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 23Lowalekar S.K. Cristofaro V. Radisavljevic Z.M. Yalla S.V. Sullivan M.P. Loss of bladder smooth muscle caveolae in the aging bladder.Neurourol Urodyn. 2012; 31: 586-592Crossref PubMed Scopus (37) Google Scholar the mechanisms responsible for mediating these changes in smooth muscle have not been elucidated. Therefore, it is important to understand the transcriptional regulation of CAV genes and elucidate the mechanism of CAV gene repression during BSM remodeling in PBOO either induced surgically in mice or caused by benign prostatic hyperplasia (BPH) in men. Identifying the transcriptional machinery critical for CAV gene transcription may also be useful in regenerative medicine for tissue engineering using stem cells. Herein, we identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in BSM from models of obstructive bladder disease in mice and humans using a combination of protein purification by DNA affinity chromatography, mutational analysis, chromatin immunoprecipitation (ChIP), and promoter reporter assay. Herein, we demonstrate, for the first time, that GATA and κB motifs are required for CAV promoter repression, and GATA-binding factor 6 (GATA-6) and NF-κB repress CAV1, CAV2, and CAV3 gene expression through their cognate binding sites on these promoters. Moreover, both in a mouse model of PBOO as well as in men with BPH-induced PBOO, enhanced binding of GATA-6 and NF-κB to GATA and κB motifs on CAV promoters in hypertrophic BSM was evident and shown to be associated with the loss of CAV gene expression. Furthermore, both in vitro cell culture and in vivo mouse models were used to examine the effects of small-molecule inhibitors of NF-κB and GATA-6 on CAV gene expression. We demonstrate, for the first time, that GATA-6 inhibitor, K-7174, and NF-κB inhibitor, BAY 11-7082, up-regulate CAV1, CAV2, and CAV3 mRNA and protein expression in human BSM cells. Most important, i.p. administration of small-molecule inhibitors of NF-κB and GATA-6 increase CAV1, CAV2, and CAV3 mRNA and protein expression in murine BSM tissue. Being transcriptional repressors of CAV1, CAV2, and CAV3 gene transcription, GATA-6 and NF-κB are likely involved in the suppression of all three CAV genes in PBOO-induced BSM hypertrophy in men and in mice. Identification of GATA-6 and NF-κB as key regulatory molecules involved in CAV gene repression in obstructive disease implicates these proteins as potential targets for therapeutic intervention. All animal studies were performed with approval from the Institutional Animal Care and Use Committees at the Children's Hospital of Philadelphia (Philadelphia, PA) and the University of Pennsylvania (Philadelphia, PA). All animal experiments were performed in compliance with the standards for care and use of animals, as affirmed in the Guide for the Care and Use of Laboratory Animals,24Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council: Guide for the Care and Use of Laboratory Animals: Eighth Edition. Washington, DC, National Academies Press, 2011Google Scholar published by the NIH (Bethesda, MD). Partial surgical ligation of the urethra was performed on adult male mice, as described previously.25Austin J.C. Chacko S.K. DiSanto M. Canning D.A. Zderic S.A. A male murine model of partial bladder outlet obstruction reveals changes in detrusor morphology, contractility and myosin isoform expression.J Urol. 2004; 172: 1524-1528Crossref PubMed Scopus (48) Google Scholar The identical procedure was followed for sham-operated animals until the suture was tied down, and then the suture was removed and the abdomen was closed. Mice were euthanized at 2 weeks of PBOO, the bladders were harvested, the mucosa and serosa were removed from the muscle layer, and the muscle tissue was snap frozen in liquid nitrogen for biochemical and molecular biological studies. RNA was isolated from sham and PBOO murine BSM using a TRIzol Plus RNA Purification System (Life Technologies Corp., Carlsbad, CA), as per the manufacturer's direction. An RNeasy minikit (Qiagen, Germantown, MD) was used to purify the RNA. An Agilent bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA) with an RNA 6000 nanochip was used to determine the quality of RNA. Reverse transcription of total RNA to cDNA was performed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA), as per the manufacturer's instruction. Microarray experiments of bladder tissue RNA from sham-operated control and PBOO mice were performed using Illumina Genome-Wide Expression Bead Chips (MouseWG-6 version 2.0 Expression Bead Chip; Illumina, San Diego, CA). The chips were scanned after completion of hybridization by the Illumina bead array scanner. Illumina microarray data were analyzed by Genome Studio 3.0 and GeneSpring 14.4 software (Agilent Technologies, Inc.). Differentially expressed genes (DEGs) between sham and PBOO mice were identified using GeneSpring GX 14.4 on the basis of a P value (P ≤ 0.05). The microarray data from the intensity data (IDAT) file were exported to Microsoft Excel (Microsoft, Redmond, WA), and the data were analyzed at the Cancer Genomics Centre of Thomas Jefferson University (Philadelphia, PA). The fold ratio between PBOO and sham-operated control was generated using the average of normalized gene expression values of two biological replicates of each group. Differentially expressed genes were chosen as candidates for further studies based on the statistical significance of P ≤ 0.05 using the t-test (unpaired) and subsequently limited by an absolute fold change of ≥1.4. A volcano plot was constructed to look at fold change, and statistical significance simultaneously. The probe sets were grouped based on their expression pattern using hierarchical clustering algorithm and heat maps were generated from a differentially expressed gene list. The microarray data were deposited to the National Center for Biotechnology Information's Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo; accession number GSE107427). Canonical pathways in microarray data analysis were predicted using Ingenuity Pathway Analysis software version 8.0 (IPA; Qiagen; https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis, last accessed April 29, 2018). Expression data sets containing gene identifiers (Entrez Gene ID) and their corresponding expression values as fold changes in the mouse bladder from sham and PBOO groups were uploaded into the IPA. Differentially expressed genes are mapped to genetic networks available in the Ingenuity database in IPA and were then ranked by score. IPA identifies biological networks, functional pathways, and global functions of a particular data set based on the known interaction/relations among target genes/proteins. The relationships between the molecules are represented as a graph, which is also referred to as a network. The nodes in the network represent molecules or genes, and the edge (line) represents the relationship between the molecules or genes. All edges are supported by at least one reference from the literature, from a textbook, or from canonical information stored in the Ingenuity Pathway Knowledge Base. The intensity of the node color indicates the degree of up-regulation (red) or down-regulation (green). Nodes are shown using various shapes, and these shapes represent the functional class of the gene product. Experiments using human bladder tissues were approved by the University of Pennsylvania Institutional Review Board (approval number 803645). Frozen human bladder tissue samples (collected through University of São Paulo School of Medicine institutional review board protocol number 811/04) were obtained from the University of São Paulo (São Paulo, Brazil) by C.M.G. Bladder tissue biopsy specimens were collected from patients, aged 62 to 78 years, who underwent suprapubic prostatectomy to treat BPH. All patients had severe lower urinary tract symptoms and were preoperatively characterized as having overt bladder outlet obstruction. Bladder outlet obstruction was assessed via multichannel urodynamics using the bladder outlet obstruction index. A detailed description of BSM obtained from control and BPH patients has been published previously.22Boopathi E. Gomes C.M. Goldfarb R. John M. Srinivasan V.G. Alanzi J. Malkowicz S.B. Kathuria H. Zderic S.A. Wein A.J. Chacko S. Transcriptional repression of caveolin-1 (CAV1) gene expression by GATA-6 in bladder smooth muscle hypertrophy in mice and human beings.Am J Pathol. 2011; 178: 2236-2251Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 26Chang S. Gomes C.M. Hypolite J.A. Marx J. Alanzi J. Zderic S.A. Malkowicz B. Wein A.J. Chacko S. Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein.Am J Physiol Renal Physiol. 2010; 298: F1416-F1423Crossref PubMed Scopus (48) Google Scholar Control samples were acquired from patients undergoing ureteral reimplantation and from nondiseased bladder tissue from patients with bladder cancer undergoing cystectomy. The reasons for ureteral reimplantation in the control group were distal ureteral stenosis and vesicoureteral reflux in one patient each. No patients in the control group exhibited lower urinary tract symptoms. They all had an American Urological Association symptom score of <8 and had no clinical symptoms of BPH. Age-matched control individuals were used for comparison with the BPH group. Serosa and mucosa were removed from murine bladder before the tissue was frozen in liquid nitrogen. Total protein was extracted from the frozen bladder tissue and subjected to Western blot analysis, as previously described.22Boopathi E. Gomes C.M. Goldfarb R. John M. Srinivasan V.G. Alanzi J. Malkowicz S.B. Kathuria H. Zderic S.A. Wein A.J. Chacko S. Transcriptional repression of caveolin-1 (CAV1) gene expression by GATA-6 in bladder smooth muscle hypertrophy in mice and human beings.Am J Pathol. 2011; 178: 2236-2251Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar Briefly, protein samples were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were probed with the following primary antibodies: anti-rabbit CAV1, CAV2, and CAV3, NF-κB c-Rel, and GATA-6 (Abcam, Cambridge, MA), and NF-κB p50 (Santa Cruz Biotechnology, Dallas, TX). Membranes were washed with phosphate-buffered saline/Tween before incubation with species-specific secondary antibodies. Target proteins were visualized using enhanced chemiluminescence, as described previously.22Boopathi E. Gomes C.M. Goldfarb R. John M. Srinivasan V.G. Alanzi J. Malkowicz S.B. Kathuria H. Zderic S.A. Wein A.J. Chacko S. Transcriptional repression of caveolin-1 (CAV1) gene expression by GATA-6 in bladder smooth muscle hypertrophy in mice and human beings.Am J Pathol. 2011; 178: 2236-2251Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar Equal loading of the protein was confirmed by probing the membranes with anti–glyceraldehyde-3-phosphate dehydrogenase antibody (Abcam). Bands on the immunoblot were quantified by densitometry using an Alpha Innotech FluroChem 8800 Image system (ProteinSimple, San Jose, CA). The 5′ upstream promoter regions of CAV1 (1.3 kb), CAV2 (1.0 kb), and CAV3 (1.0 kb) were isolated from human genomic DNA. The PCR product amplified from human genomic DNA using the gene-specific primers was cloned into pGL-4 basic luciferase reporter vector (pGL-4.1; Promega, Madison, WI). Specific mutations were introduced within the CAV promoters using the Quick-change site-directed mutagenesis kit (Stratagene, La Jolla, CA). Mutations were generated at the κB and GATA binding site in the human CAV1, CAV2, and CAV3 promoters, yielding the mutant constructs, as per the manufacturer's direction (Stratagene). Adenovirus expressing GATA-6 and NF-κB protein was obtained from Vector Biolabs (Malvern, PA). Primary BSM cells were prepared from mouse bladders, as described previously.22Boopathi E. Gomes C.M. Goldfarb R. John M. Srinivasan V.G. Alanzi J. Malkowicz S.B. Kathuria H. Zderic S.A. Wein A.J. Chacko S. Transcriptional repression of caveolin-1 (CAV1) gene expression by GATA-6 in bladder smooth muscle hypertrophy in mice and human beings.Am J Pathol. 2011; 178: 2236-2251Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar Briefly, the BSM was dissected into small pieces after removing the urothelial and serosal layers from 8-week–old mice. Collagenase (Sigma-Aldrich Co, St. Louis, MO), was used to dissociate the cells from the muscle tissue, and the isolated cells were cultured in smooth muscle growth medium-2 (Lonza Walkersville, Inc., Walkersville, MD). Isolated cells were identified as SMCs by the presence of smooth muscle myosin heavy chain and smooth muscle 22 (SM22) using immunofluorescence and immunoblot analyses. Human primary BSM cells were obtained from Lonza Walkersville, Inc. Transfection of murine and human BSM cells was performed by electroporation using Amaxa Nucleofector II (Lonza Walkersville, Inc.), as per the manufacturer's instructions. Renilla luciferase reporter plasmid (pRL-SV40) was cotransfected with firefly luciferase promoter reporter construct in all transfections of murine and human BSM cells as an internal control for assessing variations in transfection efficiency. pGL-4 basic vector without the promoter insert was used as a negative control for firefly luciferase promoter reporter (pGL-4 promoter reporter constructs). At the end of the experiment, cell lysates were prepared, and both firefly and Renilla luciferase activities were measured with the Dual-Luciferase Reporter Assay system (Promega). The TATA box in the human CAV3 promoter was predicted using the Matrix Family Library version 8.4 of Genomatix MatInspector software version 8.0 (Intrexon Bioinformatics Germany GmbH, Munich, Germany; http://www.genomatix.de/online_help/help_matinspector/matinspector_help.html). Bioinformatics program AliBaba2 version 2 (GeneXplain GmbH, Wolfenbüttel, Germany; http://gene-regulation.com/pub/programs/alibaba2/index.html), which uses the Transcription Factor Database (http://gene-regulation.com/pub/programs.html), was used to identify GATA-6 and NF-κB transcription factor binding sites on the CAV2 and CAV3 promoters. A promoter pull-down approach was used to identify NF-κB and GATA-6 transcription factor binding sites on CAV2 and CAV3 promoters. The DNA affinity column was prepared, as described previously.27Masternak K. Muhlethaler-Mottet A. Villard J. Zufferey M. Steimle V. Reith W. CIITA is a transcriptional coactivator that is recruited to MHC class II promoters by multiple synergistic interactions with an enhanceosome complex.Genes Dev. 2000; 14: 1156-1166Crossref PubMed Google Scholar To maximize the DNA binding to the affinity column, the human CAV2 and CAV3 promoters were subdivided into three smaller regions (1 to −350, −350 to −700, and −700 to −1000 bp). PCR-amplified promoter regions were end labeled with T4 polynucleotide kinase and were coupled to cyanogen bromide–activated Sepharose 4B (Sigma-Aldrich) individually, as described previously.
Referência(s)