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. We have previously investigated gene expression in healing fetal wounds in vivo with multiple expressomic techniques, including differential display [11], PCR suppression subtraction hybridization [12] and fetal wound specific microarrays (Kathju et al., manuscript in preparation). These investigations have allowed us to compare the fetal (and adult) wound expressomes without any preconcep- tions as to which gene products may be important. Multiple candidate genes have been found to be differentially and specifically regulated in healing fetal wounds; this report focuses on the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta), which was noted to be specifically underexpressed in fetal wounds by differential display RT-PCR [11]. The CCT molecule is the major cytosolic chaperonin in eukaryotes and has been estimated to interact with up to 15% of all cellular proteins. The structure of the CCT holoenzyme is unique among chaperonins, consisting of a pair of identical rings; each ring is composed of eight different 60-kDa subunits: alpha, beta, gamma, delta, epsilon, eta, theta and zeta [16] which are coded by eight different genes [17,18,19]. CCT’s role as a chaperonin has been best elucidated in its interactions with the cytoskeletal proteins actin and tubulin [20], but it has also been found to assist in the folding of multiple other proteins, including cyclin E, myosin, transducin and Von Hippel Lindau tumor suppressor among others. Alteration of CCT expression thus has the potential to impose pleiotropic secondary effects on cells. The majority of studies on CCT function have thus far focused on the holoenzyme, however, an increasing body of evidence suggests that the individual subunits of CCT have an independent function and significance. Roobol and Carden have found that not all CCT subunits co-localize identically within cells, suggesting individual subunit functionalization [21]. Genetic studies have identified several discrete CCT subunit genes as the loci for mutations resulting in neurological defects: a 143 bp deletion in CCT-gamma causes the no tectal neuron phenotype in zebrafish [22]; a missense mutation in CCT-delta causes the mutilated foot phenotype in rats [23]; a missense mutation in CCT-epsilon is responsible for mutilating sensory neuropathy with spastic paraplegia in humans [24]. Individual subunits have also been identified as the biological partners of various proteins; yeast two- hybrid analysis has identified the EBNA-3 nuclear protein from Epstein-Barr virus as binding to CCT-epsilon [25], and signifi- cantly the CCT-eta subunit has been found to serve as a co-factor for the soluble guanylyl cyclase [26]. CCT is ubiquitously expressed in all eukaryotic tissues thus far examined, and Kubota et al. describe that in mouse tissues all CCT subunits appear to be coordinately regulated [27]. Cyrne et al. [28] note that the CCT-eta and -gamma mRNAs are co- regulated during ciliary biogenesis and sexual reproduction in Tetrahymena. However, in other systems no such co-regulation is apparent. Himmelspach et al. [29] working in a plant system, find a light dependent reduction of CCT-epsilon but not CCT-alpha. Few reports examine all eight major CCT subunits. The only examination of CCT function in wound healing thus far has centered on CCT-eta. Previous studies from our laboratory have shown the specific reduction of CCT-eta mRNA in the healing fetal wound mileu [11] and subsequently we have also shown that no other CCT subunit shares this distinct pattern of gene regulation [14]. In contrast, Koulikovska et al. [30] have shown that increased expression of CCT-eta correlated with the increased expression of alpha smooth muscle actin (a-SMA) in an adult rabbit model of corneal wounding. The substantial decrease CCT-eta & Fibroblast Phenotype of CCT-eta in a fetal wound healing mileu (in contrast to adult wounds) lead us to hypothesize that CCT-eta might be a critical determinant of the distinct behavior of these two phenotypes and that the regulation of CCT-eta expression might modulate the healing response of adult wounds. We have focused our efforts on what effects specific modulation of CCT-eta levels might have on fibroblast physiology. Since fibroblasts are the ultimate effectors of scar deposition and contraction, and since wound healing (in adults) requires that they migrate into a wound bed and contract the wound substance, we have directed our studies to the examination of fibroblast motility and contractility and the role of CCT subunits therein. We first demonstrate that fetal fibroblasts express substantially less CCT-eta subunit compared to adult fibroblasts, and that they have inherently distinct characteristics of cellular locomotion and traction. Most particularly, we employ siRNAs directed against two discrete subunits (CCT-eta and CCT-beta) to demonstrate that only downregulation of the former has marked effects on the motility and contractility of adult fibroblasts, in each case shifting the adult fibroblast profile towards a more fetal-like state. We next examined expression of cellular actin, long understood as the major cytoskeletal element in cellular locomotion and traction, and known to be a major substrate of the CCT holoenzyme. Fibroblasts are known to express two actin isoforms (namely b- and c- actin) which are similarly expressed in all eukaryotic cell types. However, under certain conditions fibro- blasts may also express the alpha-smooth muscle isoform of actin (a-SMA), eg. when stimulated by serum in tissue culture or when stimulated during adult wound healing in vivo to function as ‘‘myofibroblasts,’’ the derivative cell type most closely associated with wound contraction and scar formation. The presence of a- SMA has also been found to closely correlate with the appearance of scar formation even in fetal tissues that have already transitioned to the adult scar-forming phenotype in late gestation, whereas a-SMA is largely absent from earlier scarlessly healing fetal wounds [31,32]. Thus, a-SMA expression appears to be an important differentiating feature between scarring and scarless wounds, and offers a potential mechanistic connection between CCT function, fibroblast physiology, and scar contracture. We now report that fetal fibroblasts express less constitutive a- SMA than adult cells, and that reduction of CCT-eta markedly diminishes a-SMA protein levels, whereas reduction of CCT-beta has no such effect. Direct reduction of a-SMA leads to a similar decrease in both basal and growth-factor induced motility as seen with CCT-eta depletion, again causing adult fibroblasts to mimic a more fetal pattern of behavior. Materials and Methods Materials Human epidermal growth factor (EGF) was obtained from Collaborative Biomedical Products (Bedford, MA). Human platelet derived growth factor (PDGF-BB) was purchased from R&D Systems (Minneapolis, MN). Antibodies against CCT-eta (cat # MCA2179) and CCT-beta (cat # MCA2275) were purchased from Serotec Inc. (Raleigh, NC). Antibody against a- SMA was purchased from Sigma Chemical Corp. (St Louis, MO). Antibody against GAPDH (cat # ab8245) was purchased from Abcam, Inc. (Cambridge, MA). RPMI-1640 medium, Trypsin- EDTA, OptiMEM and Lipofectamine 2000 were obtained from Invitrogen Corp. (Carlsbad, California). Antibiotic and antimy- cotic solution was purchased from Sigma Chemical Corp. Fetal bovine serum was purchased from Gemini Bio-Products (West Sacramento, CA). PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e10063