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Pdgfrα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts

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Pdgfrα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Sriram, Sriniwas, Jennifer A. Tran, Xiaoqing Guo, Audrey E. K. Hutcheon, Hetian Lei, Andrius Kazlauskas, and James D. Zieske. 2017. “PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts.” Investigative Ophthalmology & Visual Science 58 (2): 1179-1186. doi:10.1167/ iovs.16-20016. http://dx.doi.org/10.1167/iovs.16-20016. Published Version doi:10.1167/iovs.16-20016 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:32072016 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Cornea PDGFRa Is a Key Regulator of T1 and T3’s Differential Effect on SMA Expression in Human Corneal Fibroblasts Sriniwas Sriram,1 Jennifer A. Tran,1 Xiaoqing Guo,1 Audrey E. K. Hutcheon,1 Hetian Lei,1 Andrius Kazlauskas,2 and James D. Zieske1 1The Schepens Eye Research Institute/Massachusetts Eye and Ear and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States 2F. Hoffmann-La Roche AG, Basel, Switzerland Correspondence: Sriniwas Sriram, PURPOSE. The goal of this study was to examine the mechanism behind the unique differential Schepens Eye Research Institute, 20 action of transforming growth factor b3 (TGF-b3) and TGF-b1 on SMA expression. It was our Staniford Street, Boston, MA 02114, hypothesis that platelet-derived growth factor receptor a (PDGFRa) played a key role in USA; determining TGF-b3’s response to wounding. [email protected]. METHODS. A stable cell line, human corneal fibroblast (HCF)-P, was created from HCFs by Submitted: May 27, 2016 Accepted: January 12, 2017 knocking down PDGFRa expression using a lentivirus-delivered shRNA sequence. A three- dimensional (3D) in vitro model was constructed by culturing HCF or HCF-P on poly-transwell Citation: Sriram S, Tran JA, Guo X, et membranes for 4 weeks in the presence and absence of 0.1 ng/mL TGF-b1or-b3. At the end al. PDGFRa is a key regulator of T1 of 4 weeks, the constructs were processed for immunofluorescence and reverse and T3’s differential effect on SMA expression in human corneal fibro- transcription–quantitative polymerase chain reaction (RT-qPCR). In addition, HCF and HCF- blasts. Invest Ophthalmol Vis Sci. P cell migration was evaluated. 2017;58:1179–1186. DOI:10.1167/ RESULTS. In HCF, TGF-b3 treatment resulted in significantly lower a-smooth muscle actin iovs.16-20016 (SMA) mRNA expression and immunolocalization when compared to TGF-b1, while in HCF- P, both TGF-b1and-b3 treatment increased the SMA mRNA expression and immunolocal- ization compared to both the untreated HCF-P control and TGF-b3-treated HCF. Human corneal fibroblast-P also had a lower migration rate and construct thickness when compared to HCF. CONCLUSIONS. These results show that TGF-b3 decreases SMA in HCF, while remarkably increasing SMA in HCF-P, thus indicating that the presence or absence of PDGFRa elicits contrasting responses to the same TGF-b3 treatment. Understanding the role of PDGFRa in TGF-b3’s ability to stimulate SMA may potentially help in understanding the differential functions of TGF-b1 and TGF-b3 in corneal wound healing. Keywords: TGFb3, corneal scarring, PDGF, 3D cell culture fter a corneal injury, the cornea may scar (fibrosis) and effect in skin, lung, and kidney,5–7 and in some cases, even A become opaque, thus impairing vision and even leading to stimulates scar-free healing.8,9 blindness. The process of corneal wound healing is complex Like the TGFbs, there is also an extensive literature set and involves a variety of cytokines, growth factors, and associating platelet-derived growth factors (PDGFs) with proteases, which interact to regulate key phases of corneal fibrosis in different scarring models.10 The intrinsic affinity of healing. One such growth factor, transforming growth factor b each PDGF isoform for the two PDGFR subunits determines the (TGFb), has emerged as a key component of the corneal type of receptor dimer that is assembled. Platelet-derived wound-healing response.1 Transforming growth factor b has growth factor B is the universal ligand, whereas PDGFAB and been found to regulate the transformation of quiescent PDGFC assemble and activate only PDGFRa homodimers and keratocytes into activated fibroblasts, upregulate the secretion PDGFRab heterodimers. Platelet-derived growth factor D of extracellular matrix (ECM) materials, and enhance the activates PDGFRb homodimers, while PDGFA only activates formation of stress fibers.2 Furthermore, TGFb directly stimu- PDGFRa homodimers.11 Recently, it has been suggested that lates the transformation of activated fibroblasts into myofibro- these PDGF receptors were activated through an autocrine blasts, which are filled with a smooth muscle actin (SMA) that loop leading to keratocyte proliferation and myofibroblast forms microfilaments and are a major source of light scattering differentiation.12 In addition, PDGFRa co-localizes with SMA, in corneal scars.3,4 and PDGF-blocking antibodies inhibit the TGFb-induced Transforming growth factor b isoforms, in particular, TGF-b1 differentiation of keratocytes to myofibroblasts.12 and -b3 (T1 and T3, respectively), have been shown to play These studies led to the logical supposition that PDGFs different roles that are key in determining the wound-healing contribute to SMA expression by differentially activating the outcome. For instance, T1 has been found to be a regulator of PDGFRs. Therefore, in this study, we utilized our three- fibrosis and scarring, while T3 does not stimulate this scarring dimensional (3D) cell-culture model13 to study the relationship Copyright 2017 The Authors iovs.arvojournals.org j ISSN: 1552-5783 1179 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Absence of PDGFRa Makes TGFB3 Stimulate SMA IOVS j February 2017 j Vol. 58 j No. 2 j 1180 TABLE. List of SYBR Green Primer Sequences Used for RT-qPCR Growth Factor Forward Sequences Reverse Sequences SMA GGTGACGAAGCACAGAGCAA CAGTTGGTGATGATGCCATGTT PDGFA GGTGGTCACAGGTGCTTTTT AAACCACTTAAGGCTCTCAGGA PDGFB TGAGAAAGATCGAGATTGTGCG GGGCTTCGGGTCACAGG PDGFC GAATGTCAATGTGTCCCAAGCA TGCTCCCTCTGCACACACAG PDGFD ATTTGGCAGGCCATCATAGA CCACAATTGACAACTGGCTTC GAPDH GAAGGTGAAGGTCGGAGTCA GACAAGCTTCCCGTTCTCAG between T1’s and T3’s interaction with PDGFR and determined processed for reverse transcription–quantitative polymerase if this interaction is responsible for their differential stimulation chain reaction (RT-qPCR) and immunofluorescence. of SMA expression. Reverse Transcription–Quantitative Polymerase Chain Reaction MATERIALS AND METHODS Total RNA was extracted (Trizol; Thermo Fisher Scientific) Generation of Stable PDGFRa Knockdown Cell from the constructs, and cDNA was synthesized (High Capacity Line (HCF-P) cDNA Reverse Transcription Kit; Applied Biosystems, Carlsbad, Human corneal fibroblasts (HCFs) were isolated as previously CA, USA) according to manufacturer’s protocols. The cDNA described14 from human corneas obtained from the National was combined with qPCR master mix (KAPA SYBR Fast qPCR Disease Research Interchange (NDRI; Philadelphia, PA, USA). master mix; KAPA Biosystems, Wilmington, MA, USA) and primers (Table: SMA,19 PDGFA, PDGFB, PDGFC, PDGFD, or All research adhered to the tenets of the Declaration of 20 Helsinki. Once isolated, HCFs were plated on six-well plates glyceraldehyde 3-phosphate dehydrogenase [GAPDH] ), and and grown to 75% confluency in media (Eagle’s minimum then amplified (Mastercycler ep realplex real-time PCR system; essential media [EMEM]; American Type Culture Collection Eppendorf, Hauppauge, NY, USA) with the following thermal [ATCC], Manassas, VA, USA) with 10% serum (fetal bovine cycling conditions: 2 minutes at 508C, 10 minutes at 958C, 40 serum [FBS]; Atlanta Biologicals, Flowery Branch, GA, USA) cycles of 15 seconds at 958C, and 1 minute at 608C. GAPDH, an and 1% Antibiotic-Antimycotic (ABAM; Thermo Fisher Scientif- endogenous control, was used to normalize target genes. The ic, Waltham, MA, USA). primers for each gene were prepared by the CCIB DNA Core To generate the HCF-P cell line, HCFs were infected with a Facility at Massachusetts General Hospital (Cambridge, MA, PDGFRa shRNA lentivirus, as previously described.15 In brief, USA), and the sequences are given in the Table below. The HCFs were grown to ~70% confluence in six-well cell culture relative gene expression of the growth factors was calculated 21 plates. Media with lentivirus particles (pCMV-dR8.91, VSV-G/ by using the comparative Ct method. pMD2.G, hairpin-pLKO.1 vector containing the PDGFRa shRNA oligo [Dana Farber Cancer Institute/Harvard Medical Migration Assay School, Boston, MA, USA]), and Polybrene (4 lg/mL; Sigma- Aldrich Corp., St. Louis, MO, USA), which was used to improve Human corneal fibroblasts or HCF-Ps were cultured in six-well the transduction efficiency, was prepared. This media was cell culture plates. After growing to confluence, a scratch was made in the middle of the well using the end of a 200-lL applied to HCF, incubated overnight at 378C with 5% CO2, and then the cells were cultured with puromycin (2 lg/mL;
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