CCT-eta & Fibroblast Phenotype Figure 11. siRNA against a-SMA inhibits both basal and EGF-induced cell migration in adult fibroblasts. Cells were incubated in the presence or absence of EGF(1 nM) +/2 siRNA against a-SMA in an in vitro wound healing assay. In all experiments a non-specific control siRNA was used as a control. Cell motility is displayed as a relative percentage of baseline motility in the absence of EGF or siRNA exposure (100%). Active siRNA versus a-SMA reduced both basal and EGF-induced motility; a non-specific control siRNA had no such effect. Values are means 6 SEM of eight independent studies, each performed in duplicate. Statistical analyses were performed with Student’s t test. doi:10.1371/journal.pone.0010063.g011 effects seen, but the results were essentially identical to those with single constructs alone. The reduction of CCT-eta with siRNA significantly reduced the basal and growth factor-induced migration of adult fibroblastic cells. This observation is in itself striking: although many hundreds of proteins are certainly involved in the complicated process of cellular locomotion, reduction of this one protein appears to have a significant biological effect. No such effect was seen when the CCT-beta subunit was decreased, suggesting that this may be a property specific to the eta subunit and distinct from its role as a component of the CCT holoenzyme. By reducing the adult fibroblasts’ ability to respond to growth factor induction, it may be considered that selective downregulation of CCT-eta shifts the cells’ functional phenotype toward a condition approximating that of fetal fibroblasts. The results presented here are for EGF and PDGF, but a similar pattern obtains also for transforming growth factor-beta, a highly pro-fibrotic agent which can be similarly counteracted in its effects on fibroblast locomotion by our siRNA constructs (Satish et al., manuscript in preparation). Thus CCT- eta may prove an attractive target through which the biological end effects of multiple scar-promoting growth factors and cytokines may be blocked concomitantly. Another cellular characteristic highly pertinent to wound healing and scar formation is contractility, which we have assayed through measurement of applied cellular traction force (CTF); this assay has been found to correlate well with other established methods such as contraction of fibroblast populated collagen lattices [49]. We note that fetal fibroblasts exert less traction than adult, consistent with the disparate characteristics of healing fetal versus adult wounds. We note also that adult fibroblasts become even more contractile upon treatment with the growth factors EGF and PDGF, even as they similarly become relatively hypermotile. Although downregulation of CCT-eta does not appear to have any effect on cellular basal traction, it does appear to eliminate the ability of the cells to respond to a growth factor stimulus. The end result, as with cellular migration, is to shift the fibroblasts’ behavioral profile in a direction more resembling that of fetal cells. Once again, CCT-beta has no such effect, highlighting again the distinctive importance of the eta subunit to this characteristic. Although the above described observations demonstrate a clear role for CCT-eta in fibroblast physiology, and offer a rationale for why it is differentially regulated in fetal wounds and cells versus adult, they do not detail the exact molecular mechanism by which it acts. This question is a complicated one, since it is difficult to discriminate between the eta subunit’s known role in the CCT holoenzyme and its potential functions independent of said holoenzyme. The CCT chaperonin complex itself stands at the nexus of a large network as it has been found to interact with a wide range of proteins implicated in such disparate cellular processes as endocytosis, mitochondrial function, and even chromatin remodeling, among others [50]; disruption of the CCT complex (such as might be accomplished by depletion of any given subunit) may therefore have pleiotropic effects. Of immediate apparent relevance is the role of CCT in the synthesis PLoS ONE | www.plosone.org 12 April 2010 | Volume 5 | Issue 4 | e10063