Kathju1* Abstract Chaperonin Containing T-Complex Polypeptide Subunit Eta (CCT-eta) Is a Specific Regulator of Fibroblast Motility and Contractility Latha Satish1, Sandra Johnson1, James H-C. Wang2, J. Christopher Post1, Garth D. Ehrlich1, Sandeep 1 Center for Genomic Sciences, Allegheny-Singer Research Institute, Allegheny General Hospital, Pittsburgh, Pennsylvania, United States of America, 2 MechanoBiology Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America Integumentary wounds in mammalian fetuses heal without scar; this scarless wound healing is intrinsic to fetal tissues and is notable for absence of the contraction seen in postnatal (adult) wounds. The precise molecular signals determining the scarless phenotype remain unclear. We have previously reported that the eta subunit of the chaperonin containing T- complex polypeptide (CCT-eta) is specifically reduced in healing fetal wounds in a rabbit model. In this study, we examine the role of CCT-eta in fibroblast motility and contractility, properties essential to wound healing and scar formation. We demonstrate that CCT-eta (but not CCT-beta) is underexpressed in fetal fibroblasts compared to adult fibroblasts. An in vitro wound healing assay demonstrated that adult fibroblasts showed increased cell migration in response to epidermal growth factor (EGF) and platelet derived growth factor (PDGF) stimulation, whereas fetal fibroblasts were unresponsive. Downregulation of CCT-eta in adult fibroblasts with short inhibitory RNA (siRNA) reduced cellular motility, both basal and growth factor-induced; in contrast, siRNA against CCT-beta had no such effect. Adult fibroblasts were more inherently contractile than fetal fibroblasts by cellular traction force microscopy; this contractility was increased by treatment with EGF and PDGF. CCT-eta siRNA inhibited the PDGF-induction of adult fibroblast contractility, whereas CCT-beta siRNA had no such effect. In each of these instances, the effect of downregulating CCT-eta was to modulate the behavior of adult fibroblasts so as to more closely approximate the characteristics of fetal fibroblasts. We next examined the effect of CCT-eta modulation on alpha-smooth muscle actin (a-SMA) expression, a gene product well known to play a critical role in adult wound healing. Fetal fibroblasts were found to constitutively express less a-SMA than adult cells. Reduction of CCT-eta with siRNA had minimal effect on cellular beta-actin but markedly decreased a-SMA; in contrast, reduction of CCT-beta had minimal effect on either actin isoform. Direct inhibition of a-SMA with siRNA reduced both basal and growth factor-induced fibroblast motility. These results indicate that CCT-eta is a specific regulator of fibroblast motility and contractility and may be a key determinant of the scarless wound healing phenotype by means of its specific regulation of a-SMA expression. Citation: Satish L, Johnson S, Wang JH-C, Post JC, Ehrlich GD, et al. (2010) Chaperonin Containing T-Complex Polypeptide Subunit Eta (CCT-eta) Is a Specific Regulator of Fibroblast Motility and Contractility. PLoS ONE 5(4): e10063. doi:10.1371/journal.pone.0010063 Editor: Sudha Agarwal, Ohio State University, United States of America Received January 19, 2010; Accepted March 16, 2010; Published April 30, 2010 Copyright: 2010 Satish et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by Allegheny Singer Research Institute, Allegheny General Hospital and Pittsburgh Tissue Engineering Institute (PTEI). This work was funded from the grants awarded to S.K. (K08 DE014780; United States Department of Defense, W81XWH-08-2-0032), J.C.P. (DC 05659), G.D.E. (DC04173) and to L.S. (3M Fellowship). Funding support for cellular traction force microscopy work was provided from the grant awarded to J.H-C.W. (NIHR01 AR049921). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: skathju@wpahs.org Introduction Adult mammalian tissues respond to injury by healing with scar formation [1,2]; in contrast, mammalian fetuses demonstrate an ability to heal without scar, a process that has been likened to regeneration [3,4]. Although scar formation allows for the rapid sealing of an injured area, it can frequently prove the source of persistent pathology in the organism, eg. restricting movement, narrowing viscera etc. At the phenotypic level adult and fetal wound healing differ in multiple important respects: adult wound healing is marked by a prominent initial acute inflammatory response, which is absent in fetal wound healing, and fetal wound healing displays no accumulation of intermediary granulation tissue as found in healing adult wounds [4]. Most importantly for this study, healing adult wounds are characterized by a marked contraction of the wound substance, thought to be mediated by tissue fibroblasts (and their cellular derivatives, myofibroblasts), whereas in fetal wounds no such contraction occurs. It has been suggested that fibroblasts/ myofibroblasts effect wound contrac- tion either by acting together as a contractile unit, or more likely by acting individually to apply traction to a wound in the process of cell locomotion [5,6,7]. Multiple studies have sought to identify the key molecular agents responsible for scarless wound healing, and have examined the roles of various growth factors, cytokines, extracellular matrix (ECM) proteins [8], chaperonins and homeobox genes among others [2,8,9,10,11,12,13,14,15]. Despite observed differences in expression, however, the critical molecular mechanisms by which scarless fetal wound healing distinguishes itself from scirrhous wound healing remain unclear. The recapitulation of a fetal PLoS ONE | www.plosone.org 1 April 2010 | Volume 5 | Issue 4 | e10063