Abstract
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 (α-SMA) expression, a gene product well known to play a critical role in adult wound healing. Fetal fibroblasts were found to constitutively express less α-SMA than adult cells. Reduction of CCT-eta with siRNA had minimal effect on cellular beta-actin but markedly decreased α-SMA; in contrast, reduction of CCT-beta had minimal effect on either actin isoform. Direct inhibition of α-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 α-SMA expression.
Highlights
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]
Immunoblotting demonstrated that Containing T-Complex Polypeptide Subunit Eta (CCT-eta) protein expression was substantially elevated in adult fibroblasts compared to fetal fibroblasts, whereas CCT-beta protein expression showed no difference between the two cell types (Figures 1c and 1d)
Since one of the main phenotypic attributes of fibroblastic cells relevant to their role in wound healing and scar formation is their ability to migrate into a wound bed, we investigated the migration profiles of fetal and adult fibroblasts in a wellestablished in vitro assay [35], both at baseline and in response to several growth factors known to be important in the adult wound healing process (EGF and platelet derived growth factor (PDGF))
Summary
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]. 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. 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]. The recapitulation of a fetal pattern of wound healing in adult tissues would be of enormous clinical significance, as it would allow for mitigation of scar formation and diminish the attendant morbidity
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