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• W4294549779 abstract "Normal myofibroblast differentiation is critical for proper skin wound healing. Neoexpression of α-smooth muscle actin (α-SMA), a marker for myofibroblast differentiation, is driven by transforming growth factor (TGF)-β receptor–mediated signaling. Hyaluronan and its three synthesizing enzymes, hyaluronan synthases (Has 1, 2, and 3), also participate in this process. Closure of skin wounds is significantly accelerated in Has1/3 double-knockout (Has1/3-null) mice. Herein, TGF-β activity and dermal collagen maturation were increased in Has1/3-null healing skin. Cultures of primary skin fibroblasts isolated from Has1/3-null mice had higher levels of TGF-β activity, α-SMA expression, and phosphorylation of p38 mitogen-activated protein kinase at Thr180/Tyr182, compared with wild-type fibroblasts. p38α mitogen-activated protein kinase was a necessary element in a noncanonical TGF-β receptor signaling pathway driving α-SMA expression in Has1/3-null fibroblasts. Myocardin-related transcription factor (MRTF), a cofactor that binds to the transcription factor serum response factor (SRF), was also critical. Nuclear localization of MRTF was increased, and MRTF binding to SRF was enhanced in Has1/3-null fibroblasts. Inhibition of MRTF or SRF expression by RNA interference suppresses α-SMA expression at baseline and diminished its overexpression in Has1/3-null fibroblasts. Interestingly, total matrix metalloproteinase activity was increased in healing skin and fibroblasts from Has1/3-null mice, possibly explaining the increased TGF-β activation. Normal myofibroblast differentiation is critical for proper skin wound healing. Neoexpression of α-smooth muscle actin (α-SMA), a marker for myofibroblast differentiation, is driven by transforming growth factor (TGF)-β receptor–mediated signaling. Hyaluronan and its three synthesizing enzymes, hyaluronan synthases (Has 1, 2, and 3), also participate in this process. Closure of skin wounds is significantly accelerated in Has1/3 double-knockout (Has1/3-null) mice. Herein, TGF-β activity and dermal collagen maturation were increased in Has1/3-null healing skin. Cultures of primary skin fibroblasts isolated from Has1/3-null mice had higher levels of TGF-β activity, α-SMA expression, and phosphorylation of p38 mitogen-activated protein kinase at Thr180/Tyr182, compared with wild-type fibroblasts. p38α mitogen-activated protein kinase was a necessary element in a noncanonical TGF-β receptor signaling pathway driving α-SMA expression in Has1/3-null fibroblasts. Myocardin-related transcription factor (MRTF), a cofactor that binds to the transcription factor serum response factor (SRF), was also critical. Nuclear localization of MRTF was increased, and MRTF binding to SRF was enhanced in Has1/3-null fibroblasts. Inhibition of MRTF or SRF expression by RNA interference suppresses α-SMA expression at baseline and diminished its overexpression in Has1/3-null fibroblasts. Interestingly, total matrix metalloproteinase activity was increased in healing skin and fibroblasts from Has1/3-null mice, possibly explaining the increased TGF-β activation. Skin wound healing is a complex event that involves various types of cells working collaboratively at different stages of the process. Disruption of the skin initiates hemostasis, in which platelets aggregate within the wound site to stop the bleeding, followed by inflammation, in which leukocytes, monocytes, and macrophages migrate to the wound site to destroy invading pathogens and produce a variety of inflammatory and profibrotic cytokines.1Singer A.J. Clark R.A. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4607) Google Scholar In response to these stimuli, epidermal keratinocytes migrate from the wound edges toward the center to help seal the open defect. Fibroblasts are also mobilized, first migrating to the wound edges and then into the provisional matrix.1Singer A.J. Clark R.A. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4607) Google Scholar,2Rodrigues M. Kosaric N. Bonham C.A. Gurtner G.C. Wound healing: a cellular perspective.Physiol Rev. 2019; 99: 665-706Crossref PubMed Scopus (620) Google Scholar A subset of fibroblasts transforms into myofibroblasts in later stages of proliferation. Myofibroblast differentiation is critical for functional fibroblast maturation, allowing the cells to produce key components of granulation tissues, such as collagen and fibronectin.2Rodrigues M. Kosaric N. Bonham C.A. Gurtner G.C. Wound healing: a cellular perspective.Physiol Rev. 2019; 99: 665-706Crossref PubMed Scopus (620) Google Scholar Nascent expression of α-smooth muscle actin (α-SMA) is a well-established hallmark for myofibroblast differentiation.3Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases.J Pathol. 2003; 200: 500-503Crossref PubMed Scopus (1234) Google Scholar Incorporation of α-SMA into the actin cytoskeleton allows myofibroblasts to form stress fibers that connect with the extracellular matrix via focal adhesions, and to undergo contraction, which is important during wound closure, scar formation, and tissue remodeling.3Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases.J Pathol. 2003; 200: 500-503Crossref PubMed Scopus (1234) Google Scholar Transforming growth factor (TGF)-β receptor signaling is the most potent pathway in regulating α-SMA gene expression and myofibroblast differentiation.4Akhurst R.J. Hata A. Targeting the TGFbeta signalling pathway in disease.Nat Rev Drug Discov. 2012; 11: 790-811Crossref PubMed Scopus (1031) Google Scholar TGF-β1 is the predominant isoform among the three TGF-β ligands (TGF-β1, TGF-β2, and TGF-β3) that bind to the TGF-β receptor II. On ligand binding, the TGF-β receptor II homodimer forms a heterotetrameric complex with the TGF-β receptor I homodimer, resulting in the phosphorylation of the GS domain of TGF-β receptor I and the activation of its kinase activity, which, in turn, phosphorylates receptor-regulated Smads, including Smad2 and Smad3. Phosphorylated Smad2 or Smad3 dissociates from TGF-β receptor I and forms a complex with Smad4, which translocates to the nucleus and activates transcription of target genes.4Akhurst R.J. Hata A. Targeting the TGFbeta signalling pathway in disease.Nat Rev Drug Discov. 2012; 11: 790-811Crossref PubMed Scopus (1031) Google Scholar In addition to this well-recognized Smad-dependent canonical pathway, the activation of the TGF-β receptor may also activate several other Smad-independent signaling pathways, such as mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), p38 MAPK, phosphatidylinositol 3-kinase/AKT, and RhoA.5Kim S.I. Choi M.E. TGF-beta-activated kinase-1: new insights into the mechanism of TGF-beta signaling and kidney disease.Kidney Res Clin Pract. 2012; 31: 94-105Crossref PubMed Scopus (49) Google Scholar Among all these so-called noncanonical pathways, p38 MAPK has been shown to have a crucial role in regulating tissue fibrosis in various organs, including kidney,6Stambe C. Nikolic-Paterson D.J. Hill P.A. Dowling J. Atkins R.C. p38 Mitogen-activated protein kinase activation and cell localization in human glomerulonephritis: correlation with renal injury.J Am Soc Nephrol. 2004; 15: 326-336Crossref PubMed Scopus (80) Google Scholar eye,7Meyer-ter-Vehn T. Han H. Grehn F. Schlunck G. Extracellular matrix elasticity modulates TGF-beta-induced p38 activation and myofibroblast transdifferentiation in human tenon fibroblasts.Invest Ophthalmol Vis Sci. 2011; 52: 9149-9155Crossref PubMed Scopus (28) Google Scholar and cardiac muscle.8Molkentin J.D. Bugg D. Ghearing N. Dorn L.E. Kim P. Sargent M.A. Gunaje J. Otsu K. Davis J. Fibroblast-specific genetic manipulation of p38 mitogen-activated protein kinase in vivo reveals its central regulatory role in fibrosis.Circulation. 2017; 136: 549-561Crossref PubMed Scopus (164) Google Scholar One mechanism by which p38 MAPK regulates fibroblast activation and fibrotic processes is via phosphorylation of transcription factors, such as serum response factor (SRF) and myocardin-related transcription factor A (MRTF-A), and subsequent modulation of their functions.9Ronkina N. Lafera J. Kotlyarov A. Gaestel M. Stress-dependent phosphorylation of myocardin-related transcription factor A (MRTF-A) by the p38(MAPK)/MK2 axis.Sci Rep. 2016; 6: 31219Crossref PubMed Scopus (12) Google Scholar,10Martin-Garrido A. Brown D.I. Lyle A.N. Dikalova A. Seidel-Rogol B. Lassegue B. San Martin A. Griendling K.K. NADPH oxidase 4 mediates TGF-beta-induced smooth muscle alpha-actin via p38MAPK and serum response factor.Free Radic Biol Med. 2011; 50: 354-362Crossref PubMed Scopus (77) Google Scholar SRF is a mammalian transcription factor that binds to a consensus sequence CArG box [CC(A/T)6GG], also known as serum response element, that is found in smooth muscle cell–specific contractile genes.11Strauch A.R. Hariharan S. Dynamic interplay of smooth muscle alpha-actin gene-regulatory proteins reflects the biological complexity of myofibroblast differentiation.Biology (Basel). 2013; 2: 555-586PubMed Google Scholar Studies showed that p38 MAPK mediates TGF-β–induced SRF expression.12Turner N.A. Blythe N.M. Cardiac fibroblast p38 MAPK: a critical regulator of myocardial remodeling.J Cardiovasc Dev Dis. 2019; 6: 27Crossref PubMed Scopus (44) Google Scholar The full transcriptional activity of SRF requires its binding with various cofactors, including MRTF-A.13Miano J.M. Long X. Fujiwara K. Serum response factor: master regulator of the actin cytoskeleton and contractile apparatus.Am J Physiol Cell Physiol. 2007; 292: C70-C81Crossref PubMed Scopus (365) Google Scholar At baseline, MRTF-A is sequestered in the cytoplasm as a result of its binding to monomeric globular actin (G-actin). In response to activation of Rho/Rho-associated protein kinase (ROCK) signaling or growth factor stimulation, G-actin polymerizes into filamentous actin and decreases in amount, thereby releasing MRTF from cytosolic sequestration. MRTF then relocates to the nucleus, where it binds to SRF, which triggers serum response element–dependent transcription of the α-SMA gene.14Olson E.N. Nordheim A. Linking actin dynamics and gene transcription to drive cellular motile functions.Nat Rev Mol Cell Biol. 2010; 11: 353-365Crossref PubMed Scopus (688) Google Scholar,15Miralles F. Posern G. Zaromytidou A.I. Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL.Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar In addition, MRTF can be phosphorylated at certain residues by kinases, such as ERK and p38 MAPK, which affects the subcellular localization of MRTF.16Panayiotou R. Miralles F. Pawlowski R. Diring J. Flynn H.R. Skehel M. Treisman R. Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity.Elife. 2016; 5: e15460Crossref PubMed Scopus (59) Google Scholar Hyaluronan (HA) is a linear glycosaminoglycan composed of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine.17Fraser J.R. Laurent T.C. Laurent U.B. Hyaluronan: its nature, distribution, functions and turnover.J Intern Med. 1997; 242: 27-33Crossref PubMed Scopus (1456) Google Scholar HA is the only glycosaminoglycan that is not sulfated, not linked to a core protein, and not synthesized by the Golgi pathway. Instead, HA is normally synthesized on the cytoplasmic surface of the plasma membrane by three HA synthases (Has1, Has2, and Has3), and extruded into the extracellular space.18Itano N. Kimata K. Mammalian hyaluronan synthases.IUBMB Life. 2002; 54: 195-199Crossref PubMed Scopus (300) Google Scholar Has2 is the predominant HAS in most tissues, including the skin, and the only one for which genetic depletion results in embryonic lethality because of defects in cardiac development.19Camenisch T.D. Spicer A.P. Brehm-Gibson T. Biesterfeldt J. Augustine M.L. Calabro Jr., A. Kubalak S. Klewer S.E. McDonald J.A. Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme.J Clin Invest. 2000; 106: 349-360Crossref PubMed Scopus (705) Google Scholar Has1 and Has3 synthesize HA with an estimated molecular mass of 2 × 105 to ∼2 × 106 Da. On the other hand, large HA molecules (>2 × 106 Da) are synthesized by Has2.20Itano N. Sawai T. Yoshida M. Lenas P. Yamada Y. Imagawa M. Shinomura T. Hamaguchi M. Yoshida Y. Ohnuki Y. Miyauchi S. Spicer A.P. McDonald J.A. Kimata K. Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.J Biol Chem. 1999; 274: 25085-25092Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar HA is the most abundant glycosaminoglycan in the skin,17Fraser J.R. Laurent T.C. Laurent U.B. Hyaluronan: its nature, distribution, functions and turnover.J Intern Med. 1997; 242: 27-33Crossref PubMed Scopus (1456) Google Scholar and the roles of HA and hyaluronan synthases in regulating tissue injury and repair have been extensively studied. A previous study from our laboratory showed that wound closure was accelerated in Has1 and Has3 double-knockout (Has1/3-null) mice.21Mack J.A. Feldman R.J. Itano N. Kimata K. Lauer M. Hascall V.C. Maytin E.V. Enhanced inflammation and accelerated wound closure following tetraphorbol ester application or full-thickness wounding in mice lacking hyaluronan synthases Has1 and Has3.J Invest Dermatol. 2012; 132: 198-207Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar A further in vitro study found that Has2 was overexpressed in Has1/3-null fibroblasts (possibly as a compensatory response for loss of the other two Has enzymes), which resulted in an increase in HA synthesis and size of the pericellular HA coat.22Wang Y. Lauer M.E. Anand S. Mack J.A. Maytin E.V. Hyaluronan synthase 2 protects skin fibroblasts against apoptosis induced by environmental stress.J Biol Chem. 2014; 289: 32253-32265Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar In addition, Has1/3-null fibroblasts were more resistant to apoptosis induced by environmental stress, such as serum starvation or UVB irradiation. The enhanced apoptosis resistance appeared to be Has2 dependent because it could be significantly abrogated by inhibition of Has2 expression.22Wang Y. Lauer M.E. Anand S. Mack J.A. Maytin E.V. Hyaluronan synthase 2 protects skin fibroblasts against apoptosis induced by environmental stress.J Biol Chem. 2014; 289: 32253-32265Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar These findings suggest that myofibroblasts may have a slower turnover rate in Has1/3-null mice, leading to their accumulation and acceleration of wound closure in Has1/3-null skin. An unanswered question is whether fibroblast-to-myofibroblast conversion might be dysregulated in Has1/3-null wounds (perhaps affecting the production of extracellular matrix) and what role HA and Has2 may have in this dysregulation. Studies in other tissues, such as the lung, showed that HA and Has2 have an important regulatory effect on pulmonary fibroblast differentiation and function.23Li Y. Jiang D. Liang J. Meltzer E.B. Gray A. Miura R. Wogensen L. Yamaguchi Y. Noble P.W. Severe lung fibrosis requires an invasive fibroblast phenotype regulated by hyaluronan and CD44.J Exp Med. 2011; 208: 1459-1471Crossref PubMed Scopus (271) Google Scholar This was the motivation behind seeking a better mechanistic understanding about what happens to myofibroblast differentiation in the healing skin wounds of Has1/3-null mice. The present study shows an enhanced myofibroblast differentiation in Has1/3-null fibroblasts (as monitored by α-SMA expression). This enhancement occurred via a noncanonical TGF-β receptor signaling pathway (that extends from the cell membrane to the nucleus) mediated by p38 MAPK and resulting in enhanced function of MRTF/SRF. Interestingly, the enhanced myofibroblast differentiation observed in Has1/3-null mice appeared to be independent of HA or Has2. The findings from this study provide insight into an interesting phenotype in which constitutive up-regulation of a p38 MAPK-mediated, noncanonical TGF-β receptor signaling has a critical role in myofibroblast differentiation and in Has1/3-null skin wounds. C57BL/6J mice were obtained from JAX Laboratories (Bar Harbor, ME). Has1 and Has3 double-knockout mice (Has1/3 null) were generated by intercrossing Has1−/− and Has3−/− mice to generate mice nullizygous for both alleles, as previously described.21Mack J.A. Feldman R.J. Itano N. Kimata K. Lauer M. Hascall V.C. Maytin E.V. Enhanced inflammation and accelerated wound closure following tetraphorbol ester application or full-thickness wounding in mice lacking hyaluronan synthases Has1 and Has3.J Invest Dermatol. 2012; 132: 198-207Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar All mice were maintained in accordance with guidelines of the American Association for the Accreditation of Laboratory Animal Care. All wounding and tissue harvesting procedures were preapproved by our Institutional Animal Care and Use Committee. C57BL/6J wild-type (WT) mice or Has1/3-null mice (aged 8 to 10 weeks) were anesthetized with ketamine/xylazine, and the fur was shaved from the upper back. Then, 5-mm full-thickness excisional wounds (two wounds per mouse) were generated down to the fascia using sterile biopsy punches. At designated time points after wounding (day 0, day 1, day 3, day 5, day 7, and day 10), mice were anesthetized, and wounds were harvested along with unwounded skin for further preservation, processing, and analysis. Primary mouse dermal fibroblasts were isolated from the skin of 2- to 3-day–old pups from WT C57BL/6J mice, per our established protocol.22Wang Y. Lauer M.E. Anand S. Mack J.A. Maytin E.V. Hyaluronan synthase 2 protects skin fibroblasts against apoptosis induced by environmental stress.J Biol Chem. 2014; 289: 32253-32265Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Briefly, the entire trunk skin was removed and incubated overnight with 0.25% trypsin without EDTA, followed by mechanical separation of epidermis from dermis. To isolate fibroblasts, the dermis was finely diced and incubated with 400 U/mL of collagenase type I (Worthington Biochemical, Lakewood, NJ) for 30 minutes and with DNAase-I (100 U/mL) for 10 minutes at 37°C. The digested tissue suspension was passed through a 100-μm cell strainer to remove undigested tissue pieces. Hair follicles were removed by centrifugation at 9 × g for 3 minutes. The fibroblasts were collected and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA), 1% penicillin/streptomycin, and 1.0 g/L of glucose. The cells were maintained at 37°C in a humidified incubator with 5% CO2, and fresh growth medium was added every other day. For experiments, cells from passage 1 or 2 were used at subconfluent density. Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN). TGF-β receptor inhibitor SB431542 (catalog number 1614), p38 MAPK inhibitor SB202190 (catalog number 1264), ERK1/2 inhibitor U0126 (catalog number 1144), and the broad-spectrum matrix metalloproteinase (MMP) inhibitor marimastat (catalog number 2631) were purchased from TOCRIS Bioscience (Bristol, UK). Epidermal growth factor receptor (EGFR) inhibitor AG1478 (catalog number 658548) was purchased Calbiochem/Millipore-Sigma (San Diego, CA). Mink lung epithelial cells stably expressing a plasminogen activator inhibitor-1 promoter-luciferase construct [a kind gift from Daniel Rifkin (Department of Cell Biology, NYU Grossman School of Medicine, New York, NY)] were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 200 μg/mL geneticin (G418; Life Technologies), and passaged every 2 to 3 days. ON-TARGETplus SMARTpool siRNAs and nontargeted scrambled siRNA were purchased from Dharmacon/Horizon Discovery (Cambridge, UK): p38-αMAPK (Mapk14; catalog number L-040125-00-0005), p38-βMAPK (Mapk11; catalog number L-050928-00-0005), MRTF (catalog number L-054350-00-0005), SRF (catalog number L-009800-00-0005), Has2 (catalog number L-042589-01-0005), and nontargeted scrambled siRNA (catalog number D-001810-01-05). Reverse transfection was used to transfect siRNA into the primary mouse skin fibroblasts, as published previously.22Wang Y. Lauer M.E. Anand S. Mack J.A. Maytin E.V. Hyaluronan synthase 2 protects skin fibroblasts against apoptosis induced by environmental stress.J Biol Chem. 2014; 289: 32253-32265Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Briefly, for a 6-well tissue culture plate format, 30 pmol of siRNA was mixed with 8 μL of Lipofectamine RNAiMAX (Life Technologies) in 500 mL of Opti-MEM. The final duplex concentration was 100 nmol/L. The siRNA/transfection reagent mixture was added to the plates first, followed by fibroblasts seeded at a density of 2.2 × 105 per well. The cells were refed with fresh antibiotic-free medium at 24 hours after transfection. The knockdown efficacy of targeted gene expression by RNA interference (RNAi) was confirmed by either quantitative real-time PCR analysis of mRNA levels or Western blot analysis of protein abundances. For collagen imaging, Histochoice-fixed paraffin sections (5 μm thick) were stained using a Masson trichrome staining kit (Thermo Fisher Scientific, Pittsburgh, PA), according to the manufacturer's protocol. For visualization of α-SMA, sections were incubated overnight at 4°C with a rabbit anti-mouse α-SMA antibody (1:200; Abcam, Cambridge, MA) and processed using the rabbit ABC staining system (Santa Cruz Biotechnology, Dallas, TX) following the manufacturer's instructions. All treated sections were mounted in Vectamount Permanent Mounting Medium (Vector Laboratories, Inc., Burlingame, CA). To quantify the expression of newly synthesized collagen or α-SMA, histologic skin sections (stained with either Masson trichrome or anti–α-SMA antibody) were scanned at ×20 or ×10 on a Leica (Wetzlar, Germany) slide scanner. The digitized images were analyzed using IPLab Spectrum software version 3.1.2 (Vienna, VA). Specifically, a software drawing tool was used to paint over all positive areas of interest (blue stain for new collagen, or brown areas for α-SMA immunostain) within the dermis. These painted areas were then summed and expressed as a percentage of total dermal area. The primary antibodies for Western blot analysis purchased from Cell Signaling Technology (Danvers, MA) were as follows: rabbit anti–phosphorylated p38 MAPK specific for epitopes Thr180/Tyr182 (catalog number 4511), total p38 MAPK (catalog number 9212), rabbit anti–megakaryoblastic leukemia 1 (anti-MKL1)/MRTF-A (catalog number 14760), rabbit anti–poly (ADP-ribose) polymerase (catalog number 9542), anti–glyceraldehyde-3-phosphate dehydrogenase (catalog number 2118), and rabbit anti-SRF (catalog number 5147). Rabbit anti–α-smooth muscle actin antibody was purchased from Abcam (catalog number 5694). Secondary antibodies, including goat anti-rabbit and goat anti-mouse IgG conjugated with horseradish peroxidase, were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Following treatment, cells were scraped off the culture plates and lysed in radioimmunoprecipitation assay buffer supplemented with protease inhibitor cocktails (Millipore, Burlington, MA). They were diluted in 4X NuPAGE LDS sample buffer (Life Technologies) and boiled at 70°C for 10 minutes. Equal amounts of proteins were separated by electrophoresis on a 4% to 12% gradient polyacrylamide gel (Life Technologies), followed by transfer to polyvinylidene difluoride membranes (Immobilon-P; Millipore). They were then blocked in 5% nonfat milk in Tris-buffered saline with 0.05% Tween-20 for 1 hour at room temperature, and incubated with primary antibodies at 4°C overnight, followed by incubation with the appropriate secondary antibody for 1 hour at room temperature. Signals were developed using an enhanced chemiluminescence lighting Western blotting detection reagent kit (GE Health Care, Piscataway, NJ). Protein bands of interest were imaged and digitally quantified using one-dimensional analysis software (Gel Logic 5.0; Carestream Health, Inc., Rochester, NY). The membranes were stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase as loading controls. RNA was prepared from treated fibroblasts using TRIzol reagent (Life Technologies), per the manufacturer's instruction. The extracted RNA was then reverse transcribed into cDNA using a random primer and Superscript III Reverse Transcriptase (Life Technologies). For real-time PCR, assay reagents and the following TaqMan gene expression probes were purchased from Applied Biosystems/Life Technologies (Foster City, CA): acta2 (reference number Mm01546133_m1), SRF (reference number Mm00491032_m1), Mkl1 (MRTF-A; reference number Mm00461840_m1), and eukaryotic 18S ribosomal RNA endogenous control (FAM/MGB probe; catalog number 4332641). The mRNA expression levels were measured in triplicate and calculated using the 2–ΔΔCT method, and levels were presented as a fold difference relative to the untreated normal control. Mink lung epithelial cells, permanently transfected with a truncated plasminogen activator inhibitor-1 promoter fused to a firefly luciferase reporter construct, were employed for this assay.24Jurukovski V. Dabovic B. Todorovic V. Chen Y. Rifkin D.B. Methods for measuring TGF-b using antibodies, cells, and mice.Methods Mol Med. 2005; 117: 161-175PubMed Google Scholar Mink lung epithelial cells were plated at a density of 4.0 × 105 cells/mL in a 96-well plate; 4 hours later, the media were removed and replaced with conditioned media from primary murine dermal fibroblasts, or with protein lysates from skin tissues. After a 16-hour incubation, samples/standards were removed and the mink lung epithelial cells were washed 2× with phosphate-buffered saline. Then, 25 μL of 1× lysis buffer (reference number E397A; Promega, Madison, WI) was added to each well for 10 minutes, followed by 100 μL luciferase assay substrate (reference number E151A; Promega) in luciferase assay buffer (reference number E152A; Promega) for 5 minutes at room temperature with rotation. Luciferase activity was read using a spectrophotometer and quantified by luminescence (relative light units). Total MMP activities of skin fibroblasts or healing skin tissues from either WT or Has1/3-null mice were assayed using an MMP activity kit (catalog number ab112147; Abcam), according to the manufacturer's instruction. Briefly, equal amounts of protein lysate from cells or skin were added to 96-well plates (50 μL final volume; 25 μL assayed in duplicate) followed by 4-amino phenyl mercuric acetate. The sample/4-amino phenyl mercuric acetate mixtures were incubated at 37°C for up to 2 hours and transferred to a new 96-well plate, and MMP red substrate working solution was added in a 1:1 ratio. The mixture was incubated in the dark for 30 minutes, and the fluorescent intensity was read at excitation/emission wavelengths of 540/590 nm. Fibroblasts were scraped and lysed in radioimmunoprecipitation assay buffer25Wang Y. Mack J.A. Maytin E.V. CD44 inhibits alpha-SMA gene expression via a novel G-actin/MRTF-mediated pathway that intersects with TGFbetaR/p38MAPK signaling in murine skin fibroblasts.J Biol Chem. 2019; 294: 12779-12794Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar supplemented with a protease inhibitor cocktail (EMD/Millipore, Gibbstown, NJ). A 10 μL aliquot of anti-SRF antibody (catalog number 5147; Cell Signaling Technology), or of IgG control (catalog number 2729; Cell Signaling Technology), was added to 500 μg/500 μL of each lysate and incubated at 4°C overnight. After incubation, 20 μL of pre-equilibrated protein A/G agarose beads (number SC2003; Santa Cruz Biotechnology) was added to each sample and incubated with rotation at 4°C for 2 hours, followed by washing three times with radioimmunoprecipitation assay buffer. Proteins were eluted by heating the beads in 45 μL of 5× Laemmli sample buffer at 95°C for 5 minutes, followed by centrifugation at 20,000 × g for 15 seconds. The supernatant containing eluted proteins was transferred to a new tube, and eluted proteins were probed for SRF and MRTF using Western blot analysis (see above). The results were expressed as means ± SD or SEM. Data were analyzed using GraphPad Prism 9 software (GraphPad Software, San Diego, CA). Statistical comparisons between two groups were done by two-tailed unpaired t-test, with a significance level of P < 0.05. The Has1/3-null mice appear to be grossly normal, with good fertility and normal life spans. Skin wounds of Has1/3-null mice close faster than in WT mice, and the early inflammatory phase (neutrophil/macrophage infiltration) was accelerated.21Mack J.A. Feldman R.J. Itano N. Kimata K. Lauer M. Hascall V.C. Maytin E.V. Enhanced inflammation and accelerated wound closure following tetraphorbol ester application or full-thickness wounding in mice lacking hyaluronan synthases Has1 and Has3.J Invest Dermatol. 2012; 132: 198-207Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar To characterize changes that occur later in the process (tissue fibrosis and remodeling), excisional wounds were generated in WT or Has1/3-null mice using 5-mm skin biopsy punches, and the healing skin was harvested 10 days after wounding. Collagen deposition, a marker for dermal repair and maturation, was visualized by Masson trichrome staining and analyzed semiquantitatively. As shown in Figure 1A, new (blue-stained) collagen was significantly more abundant in 10-day he" @default.
• W4294549779 created "2022-09-04" @default.
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• W4294549779 date "2022-12-01" @default.
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• W4294549779 title "Transforming Growth Factor-β Receptor–Mediated, p38 Mitogen-Activated Protein Kinase–Dependent Signaling Drives Enhanced Myofibroblast Differentiation during Skin Wound Healing in Mice Lacking Hyaluronan Synthases 1 and 3" @default.
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• W4294549779 doi "https://doi.org/10.1016/j.ajpath.2022.08.003" @default.
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