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• W2051208987 abstract "Hyaluronan (HA) promotes transforming growth factor (TGF)-β1-driven myofibroblast phenotype. However, HA can also have disease-limiting activity. Bone morphogenetic protein-7 (BMP7) is an antifibrotic cytokine that antagonizes TGF-β1, and isolated studies have demonstrated that HA can both mediate and modulate BMP7 responses. In this study, we investigated whether BMP7 can modulate HA in a manner that leads to prevention/reversal of TGF-β1-driven myofibroblast differentiation in human lung fibroblasts. Results demonstrated that BMP7 prevented and reversed TGF-β1-driven myofibroblast differentiation through a novel mechanism. BMP7 promoted the dissolution and internalization of cell-surface HA into cytoplasmic endosomes. Endosomal HA co-localized with the HA-degrading enzymes, hyaluronidase-1 and hyaluronidase-2 (Hyal2). Moreover, BMP7 showed differential regulation of CD44 standard and variant isoform expression, when compared with TGF-β1. In particular, BMP7 increased membrane expression of CD44v7/8. Inhibiting CD44v7/8 as well as blocking Hyal2 and the Na+/H+ exchanger-1 at the cell-surface prevented BMP7-driven HA internalization and BMP7-mediated prevention/reversal of myofibroblast phenotype. In summary, a novel mechanism of TGF-β1 antagonism by BMP7 is shown and identifies alteration in HA as critical in mediating BMP7 responses. In addition, we identify Hyal2 and CD44v7/8 as new potential targets for manipulation in prevention and reversal of fibrotic pathology.Background: Bone morphogenetic protein-7 (BMP7) can prevent/reverse fibrosis, but mechanisms are unclear.Results: BMP7 prevents/reverses myofibroblast formation through cell-surface hyaluronan internalization into catalytic endosomes. Hyaluronidase-2 and variant CD44v7/8 mediate this process.Conclusion: Alterations in hyaluronan are critical in prevention/reversal of myofibroblast differentiation.Significance: A novel mechanism of BMP7 action is described and identifies CD44v7/8 and hyaluronidase-2 as potential targets in preventing fibrotic pathology. Hyaluronan (HA) promotes transforming growth factor (TGF)-β1-driven myofibroblast phenotype. However, HA can also have disease-limiting activity. Bone morphogenetic protein-7 (BMP7) is an antifibrotic cytokine that antagonizes TGF-β1, and isolated studies have demonstrated that HA can both mediate and modulate BMP7 responses. In this study, we investigated whether BMP7 can modulate HA in a manner that leads to prevention/reversal of TGF-β1-driven myofibroblast differentiation in human lung fibroblasts. Results demonstrated that BMP7 prevented and reversed TGF-β1-driven myofibroblast differentiation through a novel mechanism. BMP7 promoted the dissolution and internalization of cell-surface HA into cytoplasmic endosomes. Endosomal HA co-localized with the HA-degrading enzymes, hyaluronidase-1 and hyaluronidase-2 (Hyal2). Moreover, BMP7 showed differential regulation of CD44 standard and variant isoform expression, when compared with TGF-β1. In particular, BMP7 increased membrane expression of CD44v7/8. Inhibiting CD44v7/8 as well as blocking Hyal2 and the Na+/H+ exchanger-1 at the cell-surface prevented BMP7-driven HA internalization and BMP7-mediated prevention/reversal of myofibroblast phenotype. In summary, a novel mechanism of TGF-β1 antagonism by BMP7 is shown and identifies alteration in HA as critical in mediating BMP7 responses. In addition, we identify Hyal2 and CD44v7/8 as new potential targets for manipulation in prevention and reversal of fibrotic pathology. Background: Bone morphogenetic protein-7 (BMP7) can prevent/reverse fibrosis, but mechanisms are unclear. Results: BMP7 prevents/reverses myofibroblast formation through cell-surface hyaluronan internalization into catalytic endosomes. Hyaluronidase-2 and variant CD44v7/8 mediate this process. Conclusion: Alterations in hyaluronan are critical in prevention/reversal of myofibroblast differentiation. Significance: A novel mechanism of BMP7 action is described and identifies CD44v7/8 and hyaluronidase-2 as potential targets in preventing fibrotic pathology. Bone morphogenetic protein-7 (BMP7) is a member of the transforming growth factor-β (TGF-β) superfamily that demonstrates reduced expression in progressive renal diseases. Studies in animal models of renal disease, including ischemic reperfusion injury, unilateral urethral obstruction, nephrotoxin-induced glomerulonephritis, and streptozotocin-induced diabetic nephropathy, have shown that BMP7 can both prevent and reverse renal fibrosis (1.Hruska K.A. Guo G. Wozniak M. Martin D. Miller S. Liapis H. Loveday K. Klahr S. Sampath T.K. Morrissey J. Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction.Am. J. Physiol. Renal. Physiol. 2000; 279: F130-F143Crossref PubMed Google Scholar2.Zeisberg M. Hanai J. Sugimoto H. Mammoto T. Charytan D. Strutz F. Kalluri R. BMP-7 counteracts TGF-β1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury.Nat. Med. 2003; 9: 964-968Crossref PubMed Scopus (1180) Google Scholar, 3.Vukicevic S. Basic V. Rogic D. Basic N. Shih M.S. Shepard A. Jin D. Dattatreyamurty B. Jones W. Dorai H. Ryan S. Griffiths D. Maliakal J. Jelic M. Pastorcic M. Stavljenic A. Sampath T.K. Osteogenic protein-1 (bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat.J. Clin. Invest. 1998; 102: 202-214Crossref PubMed Scopus (280) Google Scholar4.Wang S. Chen Q. Simon T.C. Strebeck F. Chaudhary L. Morrissey J. Liapis H. Klahr S. Hruska K.A. Bone morphogenic protein-7 (BMP-7), a novel therapy for diabetic nephropathy.Kidney Int. 2003; 63: 2037-2049Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Studies in animal models of pulmonary and liver fibrosis have demonstrated similar benefits (5.Kinoshita K. Iimuro Y. Otogawa K. Saika S. Inagaki Y. Nakajima Y. Kawada N. Fujimoto J. Friedman S.L. Ikeda K. Adenovirus-mediated expression of BMP-7 suppresses the development of liver fibrosis in rats.Gut. 2007; 56: 706-714Crossref PubMed Scopus (120) Google Scholar, 6.Myllärniemi M. Lindholm P. Ryynänen M.J. Kliment C.R. Salmenkivi K. Keski-Oja J. Kinnula V.L. Oury T.D. Koli K. Gremlin-mediated decrease in bone morphogenetic protein signaling promotes pulmonary fibrosis.Am. J. Respir. Crit. Care Med. 2008; 177: 321-329Crossref PubMed Scopus (135) Google Scholar7.Yang G. Zhu Z. Wang Y. Gao A. Niu P. Tian L. Bone morphogenetic protein-7 inhibits silica-induced pulmonary fibrosis in rats.Toxicol. Lett. 2013; 220: 103-108Crossref PubMed Scopus (49) Google Scholar). However, BMP7 has also been implicated in progression of bone metastasis in osteotropic cancers and in promoting ectopic bone formation, and thus it is not currently viable as a clinical therapy (8.Buijs J.T. Petersen M. van der Horst G. van der Pluijm G. Bone morphogenetic proteins and its receptors; therapeutic targets in cancer progression and bone metastasis?.Curr. Pharm. Des. 2010; 16: 1291-1300Crossref PubMed Scopus (30) Google Scholar). Dissecting the mechanisms by which BMP7 prevents and reverses fibrosis may provide more suitable therapeutic targets. There are data indicating that the actions of BMP7 are, in part, related to antagonism of the biological effects of TGF-β1, a pro-fibrotic mediator (9.Rees J.R. Onwuegbusi B.A. Save V.E. Alderson D. Fitzgerald R.C. In vivo and in vitro evidence for transforming growth factor-β1-mediated epithelial to mesenchymal transition in esophageal adenocarcinoma.Cancer Res. 2006; 66: 9583-9590Crossref PubMed Scopus (109) Google Scholar, 10.Luo D.D. Phillips A. Fraser D. Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.Am. J. Pathol. 2010; 176: 1139-1147Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar11.Motazed R. Colville-Nash P. Kwan J.T. Dockrell M.E. BMP-7 and proximal tubule epithelial cells: activation of multiple signaling pathways reveals a novel anti-fibrotic mechanism.Pharm. Res. 2008; 25: 2440-2446Crossref PubMed Scopus (35) Google Scholar). BMP7 has been shown to attenuate TGF-β1-dependent Smad3 signaling (10.Luo D.D. Phillips A. Fraser D. Bone morphogenetic protein-7 inhibits proximal tubular epithelial cell Smad3 signaling via increased SnoN expression.Am. J. Pathol. 2010; 176: 1139-1147Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). However, other studies have shown that Smad-independent, MAPK-signaling events are central to TGF-β1-driven renal fibrosis (11.Motazed R. Colville-Nash P. Kwan J.T. Dockrell M.E. BMP-7 and proximal tubule epithelial cells: activation of multiple signaling pathways reveals a novel anti-fibrotic mechanism.Pharm. Res. 2008; 25: 2440-2446Crossref PubMed Scopus (35) Google Scholar, 12.Simpson R.M. Wells A. Thomas D. Stephens P. Steadman R. Phillips A. Aging fibroblasts resist phenotypic maturation because of impaired hyaluronan-dependent CD44/epidermal growth factor receptor signaling.Am. J. Pathol. 2010; 176: 1215-1228Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Hence, the anti-fibrotic mechanisms of BMP7 are not yet fully elucidated. Hyaluronan (HA) 2The abbreviations used are: HAhyaluronanQPCRquantitative PCRbHAPBbiotinylated HA-binding proteinEIPA5-(N-ethyl-N-isopropyl) amilorideEGFREGF receptorHyal1hyaluronidase-1Hyal2hyaluronidase-2NHE1Na/H exchangerαSMAα-smooth muscle actin. is a connective tissue glycosaminoglycan, which in vivo is present as a high molecular mass component of extracellular matrices. It is synthesized by three mammalian HA synthase isoenzymes (HAS1, HAS2, and HAS3), and its breakdown is mediated by the hyaluronidase (Hyal) group of enzymes. HA regulates cellular processes through interaction with cell-surface receptors (principally CD44) and has been implicated in numerous biological processes, including embryonic development, wound healing, chronic inflammation, and tumor progression (13.DeGrendele H.C. Estess P. Picker L.J. Siegelman M.H. CD44 and its ligand hyaluronate mediate rolling under physiologic flow: a novel lymphocyte-endothelial cell primary adhesion pathway.J. Exp. Med. 1996; 183: 1119-1130Crossref PubMed Scopus (364) Google Scholar14.DeGrendele H.C. Estess P. Siegelman M.H. Requirement for CD44 in activated T cell extravasation into an inflammatory site.Science. 1997; 278: 672-675Crossref PubMed Scopus (478) Google Scholar, 15.Cuff C.A. Kothapalli D. Azonobi I. Chun S. Zhang Y. Belkin R. Yeh C. Secreto A. Assoian R.K. Rader D.J. Puré E. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation.J. Clin. Invest. 2001; 108: 1031-1040Crossref PubMed Scopus (260) Google Scholar16.Clark R.A. Alon R. Springer T.A. CD44 and hyaluronan-dependent rolling interactions of lymphocytes on tonsillar stroma.J. Cell Biol. 1996; 134: 1075-1087Crossref PubMed Scopus (126) Google Scholar). Therefore, HA has an important role in maintaining tissue homeostasis. hyaluronan quantitative PCR biotinylated HA-binding protein 5-(N-ethyl-N-isopropyl) amiloride EGF receptor hyaluronidase-1 hyaluronidase-2 Na/H exchanger α-smooth muscle actin. Alterations in HA generation and turnover have also been associated with promotion of disease states. Increased expression of HA has been demonstrated in numerous fibrotic conditions associated with organ dysfunction, including IgA nephropathy, diabetic nephropathy, pulmonary fibrosis, and cirrhotic liver disease (17.Nishikawa K. Andres G. Bhan A.K. McCluskey R.T. Collins A.B. Stow J.L. Stamenkovic I. Hyaluronate is a component of crescents in rat autoimmune glomerulonephritis.Lab. Invest. 1993; 68: 146-153PubMed Google Scholar18.Johnsson C. Tufveson G. Wahlberg J. Hällgren R. Experimentally-induced warm renal ischemia induces cortical accumulation of hyaluronan in the kidney.Kidney Int. 1996; 50: 1224-1229Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 19.Lewis A. Steadman R. Manley P. Craig K. de la Motte C. Hascall V. Phillips A.O. Diabetic nephropathy, inflammation, hyaluronan and interstitial fibrosis.Histol. Histopathol. 2008; 23: 731-739PubMed Google Scholar, 20.Sano N. Kitazawa K. Sugisaki T. Localization and roles of CD44, hyaluronic acid and osteopontin in IgA nephropathy.Nephron. 2001; 89: 416-421Crossref PubMed Scopus (38) Google Scholar, 21.Zaman A. Cui Z. Foley J.P. Zhao H. Grimm P.C. Delisser H.M. Savani R.C. Expression and role of the hyaluronan receptor RHAMM in inflammation after bleomycin injury.Am. J. Respir. Cell Mol. Biol. 2005; 33: 447-454Crossref PubMed Scopus (87) Google Scholar, 22.Bjermer L. Lundgren R. Hällgren R. Hyaluronan and type III procollagen peptide concentrations in bronchoalveolar lavage fluid in idiopathic pulmonary fibrosis.Thorax. 1989; 44: 126-131Crossref PubMed Scopus (109) Google Scholar, 23.Stickel F. Poeschl G. Schuppan D. Conradt C. Strenge-Hesse A. Fuchs F.S. Hofmann W.J. Seitz H.K. Serum hyaluronate correlates with histological progression in alcoholic liver disease.Eur. J. Gastroenterol. Hepatol. 2003; 15: 945-950Crossref PubMed Scopus (47) Google Scholar24.Nyberg A. Engström-Laurent A. Lööf L. Serum hyaluronate in primary biliary cirrhosis–a biochemical marker for progressive liver damage.Hepatology. 1988; 8: 142-146Crossref PubMed Scopus (138) Google Scholar). We have previously shown that changes in HA synthesis and macromolecular organization are key in directing TGF-β1-driven differentiation of fibroblasts to myofibroblasts, the principal effector cells in fibrosis (12.Simpson R.M. Wells A. Thomas D. Stephens P. Steadman R. Phillips A. Aging fibroblasts resist phenotypic maturation because of impaired hyaluronan-dependent CD44/epidermal growth factor receptor signaling.Am. J. Pathol. 2010; 176: 1215-1228Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 25.Meran S. Luo D.D. Simpson R. Martin J. Wells A. Steadman R. Phillips A.O. Hyaluronan facilitates transforming growth factor-β1-dependent proliferation via CD44 and epidermal growth factor receptor interaction.J. Biol. Chem. 2011; 286: 17618-17630Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 26.Webber J. Meran S. Steadman R. Phillips A. Hyaluronan orchestrates transforming growth factor-β1-dependent maintenance of myofibroblast phenotype.J. Biol. Chem. 2009; 284: 9083-9092Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar27.Midgley A.C. Rogers M. Hallett M.B. Clayton A. Bowen T. Phillips A.O. Steadman R. Transforming growth factor-β1 (TGF-β1)-stimulated fibroblast to myofibroblast differentiation is mediated by hyaluronan (HA)-facilitated epidermal growth factor receptor (EGFR) and CD44 co-localization in lipid rafts.J. Biol. Chem. 2013; 288: 14824-14838Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). TGF-β1 stimulation directs HAS2-driven assembly of HA into pericellular coats, which promotes membrane redistribution of CD44. This results in its physical co-localization with the epidermal growth factor receptor (EGFR) and subsequent selective activation of the MAPK/ERK signaling pathway necessary for myofibroblast formation (12.Simpson R.M. Wells A. Thomas D. Stephens P. Steadman R. Phillips A. Aging fibroblasts resist phenotypic maturation because of impaired hyaluronan-dependent CD44/epidermal growth factor receptor signaling.Am. J. Pathol. 2010; 176: 1215-1228Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 27.Midgley A.C. Rogers M. Hallett M.B. Clayton A. Bowen T. Phillips A.O. Steadman R. Transforming growth factor-β1 (TGF-β1)-stimulated fibroblast to myofibroblast differentiation is mediated by hyaluronan (HA)-facilitated epidermal growth factor receptor (EGFR) and CD44 co-localization in lipid rafts.J. Biol. Chem. 2013; 288: 14824-14838Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Numerous reports have indicated that HA assembly and its conformation within the pericellular matrix are crucial in determining its subsequent biological actions. For example, interleukin-1β (IL-1β)-mediated formation of HA pericellular protrusions promotes fibroblast immune activation through fibroblast-monocyte interactions, whereas pericellular HA cable-like structures are shown to be anti-inflammatory and prevent monocyte-dependent inflammatory cytokine production (28.Meran S. Martin J. Luo D.D. Steadman R. Phillips A. Interleukin-1β induces hyaluronan and CD44-dependent cell protrusions that facilitate fibroblast-monocyte binding.Am. J. Pathol. 2013; 182: 2223-2240Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 29.Selbi W. de la Motte C.A. Hascall V.C. Day A.J. Bowen T. Phillips A.O. Characterization of hyaluronan cable structure and function in renal proximal tubular epithelial cells.Kidney Int. 2006; 70: 1287-1295Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Thus, it is clear that under specific biological contexts HA may also have the ability to function in the prevention of disease. There is some evidence that HA can both mediate and modulate BMP7 responses. In chondrocytes, BMP7-dependent Smad1 signaling was mediated through CD44-Smad1 interactions (30.Andhare R.A. Takahashi N. Knudson W. Knudson C.B. Hyaluronan promotes the chondrocyte response to BMP-7.Osteoarthritis Cartilage. 2009; 17: 906-916Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Other studies demonstrated that pericellular HA augments BMP7 Smad signaling (31.Peterson R.S. Andhare R.A. Rousche K.T. Knudson W. Wang W. Grossfield J.B. Thomas R.O. Hollingsworth R.E. Knudson C.B. CD44 modulates Smad1 activation in the BMP-7 signaling pathway.J. Cell Biol. 2004; 166: 1081-1091Crossref PubMed Scopus (65) Google Scholar). In renal epithelial cells, BMP7 has also been shown to induce the formation of HA cables (32.Selbi W. de la Motte C. Hascall V. Phillips A. BMP-7 modulates hyaluronan-mediated proximal tubular cell-monocyte interaction.J. Am. Soc. Nephrol. 2004; 15: 1199-1211Crossref PubMed Scopus (52) Google Scholar). However, whether HA plays a specific role in BMP7-mediated antagonism of TGF-β1 actions is not known. In this work, we demonstrate a novel mechanism in which BMP7 reverses TGF-β1 induction of myofibroblast differentiation in human lung fibroblast cultures by internalizing pericellular HA into catalytic endosomes within the cytoplasm and preventing TGF-β1-driven pericellular HA accumulation. The CD44 variant isoform, CD44v7/8, and membrane-bound Hyal2 are both critical for this process of HA internalization and thus potentially present new targets for study and intervention in fibrosis. All reagents were purchased from Sigma (Poole, UK) or Life Technologies and Invitrogen (Paisley, UK) unless otherwise stated. Reverse transcription reagents, siRNA transfection reagents, and quantitative PCR (QPCR) primers and reagents were purchased from Invitrogen and Applied Biosystems (Cheshire, UK). Other reagents used were recombinant human BMP7 (Merck Millipore, Darmstadt, Germany), recombinant human TGF-β1 (R&D Systems, Abingdon, UK), and the selective NHE1 inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA; Sigma). Human lung fibroblasts (AG02262) were purchased from Coriell Cell Repositories (Coriell Institute for Medical Research, Camden, NJ). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and F12 containing 5 mm glucose, 2 mmol/liter l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and supplemented with 10% fetal bovine serum (Biological Industries Ltd., Cumbernauld, UK). The cells were maintained at 37 °C in a humidified incubator in an atmosphere of 5% CO2, and fresh growth medium was added to the cells every 3 to 4 days until the cells were ready for experimentation. The cells were incubated in serum-free medium for 48 h before use in all experiments, and all experiments were performed under serum-free conditions unless otherwise stated. All experiments were undertaken using cells at passage 6–10. Phenotypic differentiation of fibroblasts was induced by stimulation with 10 ng/ml TGF-β1 for 72 h according to time course and dose-response experiments and according to our previous protocols (33.Meran S. Thomas D. Stephens P. Martin J. Bowen T. Phillips A. Steadman R. Involvement of hyaluronan in regulation of fibroblast phenotype.J. Biol. Chem. 2007; 282: 25687-25697Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Time course and dose-response experiments were also initially done to determine the optimum concentration and incubation times with BMP7. A dose of 400 ng/ml BMP7 was subsequently used in all experiments. Two experimental cell systems were utilized to determine effects of BMP7 and related alterations in HA on TGF-β1 responsiveness, a prevention model and a reversal model of fibrosis. For the prevention model, cells were either treated with TGF-β1 and BMP7 at the same time (prevention I) or cells were pretreated with BMP7 prior to stimulation with TGF-β1 (prevention II). For the reversal model, cells were first stimulated with TGF-β1 to induce differentiation and then subsequently treated with BMP7. These treatment conditions and incubation times are more clearly demonstrated in Table 1.TABLE 1Experimental cell treatment systemsTreatment conditions0–72 h72–144 hUntreated controlTGF-β110 ng/ml TGF-β110 ng/ml TGF-β1BMP7400 ng/ml BMP7400 ng/ml BMP7Prevention I10 ng/ml TGF-β1 and 400 ng/ml BMP710 ng/ml TGF-β1 and 400 ng/ml BMP7Prevention II400 ng/ml BMP710 ng/ml TGF-β1Reversal10 ng/ml TGF-β1400 ng/ml BMP7 Open table in a new tab An ELISA-like assay (HA ELISA) was commercially purchased (Corgenix, Broomfield, CO) and used to assess the concentrations of intracellular HA, HA in conditioned cell culture medium, and on the cell surface. Briefly, conditioned cell culture medium was removed and transferred into Eppendorf microcentrifuge tubes (soluble HA) and kept on ice. Cells were then incubated with trypsin/EDTA for 5 min at room temperature, transferred into microcentrifuge tubes, and centrifuged at 4000 × g for 5 min at 4 °C. The supernatant was transferred to Eppendorf microcentrifuge tubes, and trypsin was deactivated by heating to 90 °C for 5 min (cell-surface HA) and then kept on ice. The cell pellet was resuspended in dilute (10% v/v) RIPA buffer and kept on ice for 10 min. The solution was centrifuged again, and the supernatant transferred to fresh microcentrifuge tubes (intracellular HA). Samples were then analyzed by Corgenix HA ELISA kits (Corgenix), according to the manufacturer's protocol. RT-QPCR was used to assess α-SMA (ACTA2), CD44, Hyal1, Hyal2, HAS1, HAS2, and HAS3 mRNA expressions. Primers and probes for these genes were commercially designed and purchased from Applied Biosystems. The cells were grown in 35-mm dishes and washed with PBS before lysis with TRI Reagent and RNA purification according to the manufacturer's protocol. Reverse transcription was done using the high capacity cDNA reverse transcription kit according to the manufacturer's protocol (Applied Biosystems). This kit uses the random primer method for initiating cDNA synthesis. As a negative control, reverse transcription was done with sterile H2O replacing the RNA sample. QPCR was done using the ViiA7 real time QPCR system from Applied Biosystems in a final volume of 20 μl per sample as follows: 1 μl of reverse transcription product, 1 μl of target gene primers and probe, 10 μl of TaqMan Fast Universal PCR MasterMix, and 8 μl of sterile RNase-free water. Amplification was done using a cycle of 95 °C for 1 s and 60 °C for 20 s for 40 cycles. QPCR was simultaneously performed for ribosomal RNA (primers and probe commercially designed and purchased from Applied Biosystems) as a standard reference gene. For assessment of CD44 variant isoform expression, custom primer pairs were designed and purchased from Invitrogen. Primer pairs used are shown in Table 2.TABLE 2Forward and reverse primer pairs for CD44 standard and variant isoformsCD44sForward 5′-GCTACCAGAGACCAAGACACA-3′Reverse 5′-GCTCCACCTTCTTGACTCC-3′CD44v7Forward 5′-GAATCCCTGCTACCACAGCCTC-3′Reverse 5′-TCTCCCATCCTTCTTCCTGCTT-3′CD44v8Forward 5′-ATGTGTCTTGGTCTCGCGTT-3′Reverse 5′TCCCTGCTACCAATATGGACTC-3′CD44v3/7Forward 5′-TTATCTCCAGCACCACAGCCTC-3′Reverse 5′-TCTTGGTCTCCCATCCTTCTTC-3′CD44v7/8Forward 5′AGGAAGAAGGATGGATATGGACT-3′Reverse 5′-GTCTTGGTCTCGCGTTGTCA-3′ Open table in a new tab QPCR for custom primer pairs was done with the ViiA7 real time QPCR system from Applied Biosystems in a final volume of 20 μl per sample as follows: 1 μl of reverse transcription product; 0.6 μl of 10 μm target gene forward primer and 0.6 μl of 10 μm target gene reverse primer; 10 μl of Power SYBR Green Mastermix; and 7.8 μl of sterile RNase-free water. Amplification used a cycle of 95 °C for 15 s and 60 °C for 1 min for 40 cycles, followed by a melt-curve stage at 95 °C for 15 s, 60 °C for 1 min, and a final step of 95 °C for 15 s. QPCR was simultaneously performed for GAPDH mRNA expression (custom primers designed and purchased from Applied Biosystems) as a standard reference gene. As a negative control, PCR was performed with sterile H2O replacing the cDNA sample. The comparative CT method was used for relative quantification of gene expression. The CT (threshold cycle where amplification is in the linear range of the amplification curve) for the standard reference gene (rRNA/GAPDH) was subtracted from the target gene CT to obtain the ΔCT. The mean ΔCT values for replicate samples were then calculated. The expression of the target gene in experimental samples relative to expression in control samples was then calculated using the following equation: 2−(ΔCT(1) − ΔCT(2)), where ΔCT(1) is the mean ΔCT calculated for the experimental samples, and ΔCT(2) is the mean ΔCT calculated for the control samples. Transient transfection of fibroblasts was done with specific siRNA nucleotides (Applied Biosystems) targeting Hyal1, Hyal2, or CD44v7/8. Transfection was done using Lipofectamine 2000 transfection reagent (Invitrogen) in accordance with the manufacturer's protocol. Briefly, cells were grown to 70% confluence in antibiotic-free medium in either 35-mm dishes or 8-well Permanox chamber slides. Transfection reagent (2% v/v) was diluted in Opti-MEM reduced growth medium (GIBCO) and left to incubate for 5 min at room temperature. The specific siRNA oligonucleotides were diluted in Opti-MEM reduced growth medium to achieve a final concentration of 30 nm. The transfection agent and siRNA mixtures were then combined and incubated at room temperature for an additional 20 min. The newly formed transfection complexes were subsequently added to the cells and incubated at 37 °C with 5% CO2 for 24 h in serum-free medium before experimentation. As a control, cells were transfected with negative control siRNA (a scrambled sequence that bears no homology to the human genome) (Applied Biosystems). Cells were grown to 70% confluence in 8-well Permanox chamber slides. The culture medium was removed, and the cells washed with sterile PBS before fixation in 4% paraformaldehyde for 10 min at room temperature. After fixation, cells were permeabilized with 0.1% (v/v) Triton X-100 in PBS for 10 min at room temperature. Slides were blocked with 1% bovine serum albumin (BSA) for 1 h before a further washing step with 0.1% (w/v) BSA in PBS. Subsequently, the slides were incubated with the primary antibody diluted in 0.1% BSA and PBS for 2 h at room temperature. When visualizing HA, biotinylated HA-binding protein (bHABP) was used in place of primary antibody. After a further washing step, slides were incubated with AlexaFluor 488-conjugated and/or AlexaFluor 555-conjugated secondary antibodies for 1 h at room temperature. Avidin-FITC was used in place of a secondary antibody when visualizing HA. Cell nuclei were stained with Hoechst solution. Cells were then mounted and analyzed by confocal and fluorescent microscopy. A co-localization scatter plot was used to quantify the level of co-localization present between distinct antibody stains in selected experiments. The following primary antibodies were used: mouse anti-human α-SMA (Sigma); rat anti-human CD44 antibody (Merck Millipore; A020); mouse anti-human EGFR antibody (Merck Millipore); biotinylated bHABP (Seikagaku Corp., Tokyo, Japan); mouse anti-human EEA1 antibody (Santa Cruz Biotechnology Inc., Heidelberg, Germany); mouse anti-human Cav1 antibody (Sigma); rabbit anti-human Hyal1 antibody and rabbit anti-human Hyal2 antibody (gift from Dr. Robert Stern, Department of Pathology, University of California, San Francisco); mouse anti-human CD44v7/8 antibody (Thermo Fisher Scientific, Leicestershire, UK); and cholera toxin subunit B Alexa Fluor 594 conjugate (Molecular Probes, Invitrogen). The following secondary antibodies were used: rabbit anti-rat AlexaFluor 555, goat anti-mouse AlexaFluor 488, goat anti-rabbit AlexaFluor 488, goat anti-rabbit AlexaFluor 555 (Merck Millipore); and avidin-FITC (Vector Laboratories, Burlingame, CA). Specific primer pairs were designed and purchased (Invitrogen) to target CD44v7 and CD44v8. Two primer sets were used for CD44v7 analysis as follows: set one forward 5′-TCAATGCTTCAGCTCCACCT-3′ and reverse 5′-TCTCCCATCCTTCTTCCTGCTT-3′; set two forward 5′-GAATCCCTGCTACCACAGCCTC-3′ and reverse 5′-CAAAGCCAAGGCCAAGAGGGATGC-3′. The following two primer sets were used for CD44v8 analysis as follows: set one forward 5′-TCAATGCTTCAGCTCCACCT-3′ and reverse 5′-TCCCTGCTACCATATGGACTC-3′; set two forward 5′-ATGTGTCTTGGTCTCGCGTT-3′ and reverse 5′-CAAAGCCAAGGCCAAGAGGGATGC-3′. Fragments were amplified using the Phusion High Fidelity DNA polymerase system (New England Biolabs, Inc.). Briefly, 4 μl of 5× High Fidelity buffer, 0.4 μl of dN" @default.
• W2051208987 created "2016-06-24" @default.
• W2051208987 creator A5011921917 @default.
• W2051208987 creator A5016299316 @default.
• W2051208987 creator A5029931631 @default.
• W2051208987 creator A5032471667 @default.
• W2051208987 creator A5042687613 @default.
• W2051208987 creator A5042764711 @default.
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• W2051208987 date "2015-05-01" @default.
• W2051208987 modified "2023-10-18" @default.
• W2051208987 title "Hyaluronan Regulates Bone Morphogenetic Protein-7-dependent Prevention and Reversal of Myofibroblast Phenotype" @default.
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• W2051208987 doi "https://doi.org/10.1074/jbc.m114.625939" @default.
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