CCT-eta & Fibroblast Phenotype Figure 10. siRNA against a-SMA specifically decreases both both basal and EGF- induced mRNA and protein levels of a-SMA in adult fibroblasts. (a) qRT-PCR analysis of a-SMA mRNA levels showed effective inhibition of both basal expression and EGF-induction in siRNA- transfected adult fibroblasts. Results are expressed as relative quotient (RQ) of measured a-SMA mRNA and were calculated as a percentage of baseline control levels (100%). Values are means 6 SEM of six independent studies, each performed in duplicate. Statistical analyses were performed with Student’s t test. Ntx- no transfection; EGF-EGF treatment (1 nM); siRNA-treatment with a-SMA siRNA; Ctr- treatment with a non-specific control siRNA. (b) Western blot results using a-SMA antibody (1:500) showed effective reduction of a-SMA protein levels when siRNA was administered but no decrease when non-specific control siRNA was employed. GAPDH was used as a loading control. A representative immunoblot of up to four similar such blots is shown for each analysis. doi:10.1371/journal.pone.0010063.g010 confirming previous analyses that these subunits (and indeed, all CCT subunits) likely derive from a common ancestor gene (Satish et al., submitted). Of the eight subunits that comprise the CCT holoenzyme, however, only CCT-eta was found to be reduced in healing fetal wounds, suggesting that it might play a unique role in the physiology pertinent thereto. Since CCT-beta is the subunit most closely evolutionarily related to –eta, and since –beta displayed no evident change in expression in healing fetal (or adult) wound tissues, we have investigated CCT-beta as the most appropriate ‘‘control’’ subunit for –eta and its potentially unique importance to fibroblast behavior. The reduction of CCT-eta in a healing fetal wound is apparent as early as 12 hours post-wounding. A wound milieu has multiple cell types within it, and it is as yet uncertain which manifest the decrease most, and whether a part of the decrease might result from migration of cells into or out of the wounded area. In fetal wounds, this question is less complicated, as there is no significant infiltration of inflammatory cells, and fibroblasts and epithelial cells/keratinocytes continue to comprise the main cellular populations. Our particular interest is in determining the mechanisms behind scarlessness versus scarring, and the ultimate end effector cells in this process must be fibroblastic [38], although of course subject to influence from other cell types. Accordingly, we based the current study on fibroblasts derived from rabbit fetal and adult skin and focused on the role of CCT-eta in fibroblast migration and cellular traction force, two key determinants of the fibrotic phase of dermal repair. Our own observations with immunohistochemical analysis of healing fetal and adult wound tissues indicate that fetal fibroblasts express little CCT-eta in vivo, whereas adult wound fibroblasts appear to stain more heavily, consistent with our in vitro results (Satish et al., manuscript in preparation). Cell migration plays a vital role in wound healing, tissue morphogenesis, angiogenesis, and metastasis [39] and has been previously studied in vitro using a variety of methods, eg., on various mechanical substrates, 3-D vs. planar migration etc. Using a widely accepted planar migration assay we first explored whether the migratory capabilities of fetal and adult fibroblasts differed either at baseline or in response to growth factors implicated in wound healing. This question has yielded conflicting answers in previous studies: some suggest that fetal fibroblasts display enhanced migratory activity compared to the adult cells [40,41,42] whereas others do not [43]. It has been reported that serum can both stimulate [44] and inhibit [45] fetal and adult fibroblast motility; others find fibroblast migration is entirely independent of serum factors [39]. Complicating the picture, many investigators use fetal fibroblast cell lines rather than primary cell cultures [46,47,48]. Our results with low passage primary cultures of fetal and adult fibroblasts showed no difference in the basal motility of these cells, but demonstrated a markedly different response to the fibrogenic growth factors PDGF and EGF. Adult fibroblasts dramatically increased their migration in response to these agents but fetal fibroblasts remained insensitive to this stimulus. This distinct physiology may prove relevant to the differing wound conditions these cells might be expected to encounter: adult cells would presumably find it advantageous to migrate rapidly to seal off an exposed wound site, whereas fetal cells, programmed for function in the protected uterine environment, may not biologically recognize any such imperative. To selectively manipulate CCT subunit levels in our cells of interest we used siRNAs, which proved effective in reducing the targeted mRNAs and proteins. The results presented here derive from the use of single siRNA constructs; we did undertake similar experiments with multiple siRNAs to attempt to maximize the PLoS ONE | www.plosone.org 11 April 2010 | Volume 5 | Issue 4 | e10063