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

Sriram, SriniwasTran, Jennifer A.Guo, XiaoqingHutcheon, AudreyLei, HetianKazlauskas, AndriusZieske, James2017-04-062017Sriram, 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.http://nrs.harvard.edu/urn-3:HUL.InstRepos:32072016Purpose The goal of this study was to examine the mechanism behind the unique differential action of transforming growth factor β3 (TGF-β3) and TGF-β1 on SMA expression. It was our hypothesis that platelet-derived growth factor receptor α (PDGFRα) played a key role in determining TGF-β3's response to wounding. Methods: A stable cell line, human corneal fibroblast (HCF)-P, was created from HCFs by knocking down PDGFRα 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 membranes for 4 weeks in the presence and absence of 0.1 ng/mL TGF-β1 or -β3. At the end of 4 weeks, the constructs were processed for immunofluorescence and reverse transcription–quantitative polymerase chain reaction (RT-qPCR). In addition, HCF and HCF-P cell migration was evaluated. Results: In HCF, TGF-β3 treatment resulted in significantly lower α-smooth muscle actin (SMA) mRNA expression and immunolocalization when compared to TGF-β1, while in HCF-P, both TGF-β1 and -β3 treatment increased the SMA mRNA expression and immunolocalization compared to both the untreated HCF-P control and TGF-β3-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-β3 decreases SMA in HCF, while remarkably increasing SMA in HCF-P, thus indicating that the presence or absence of PDGFRα elicits contrasting responses to the same TGF-β3 treatment. Understanding the role of PDGFRα in TGF-β3's ability to stimulate SMA may potentially help in understanding the differential functions of TGF-β1 and TGF-β3 in corneal wound healing.en-USTGFβ3corneal scarringPDGF3D cell culturePDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal FibroblastsJournal Article2017-04-0610.1167/iovs.16-20016