and maturation of actin, a protein essential to both motility and contractility. However, in vitro biochemical studies of the interaction between a-SMA and CCT have suggested that the beta subunit is more directly essential to this dynamic pairing than eta [51]. Per this previous evidence, then, we might expect to see a greater effect on a-SMA with CCT-beta siRNA than with CCT- eta; instead the opposite obtains. In fact, we observe effective inhibition of a-SMA using CCT-eta siRNA only, whereas cells treated with CCT-beta siRNA do not show a significant difference in a-SMA levels. This remarkable observation highlights the specific importance of the eta subunit to a-SMA expression (and the cellular characteristics which derive from it). In fact, both cellular motility and contractility have previously been well connected to actin expression, and there is also evidence for a particular role for the a-SMA isoform. Tsai et al. [52] found that ultrasonic treatment of tendon-derived fibroblasts resulted in increased a-SMA and a corresponding increase in cell motility. Conversely, treatment of tendon fibroblasts with glucocorticoids diminished a-SMA expression with a correlated decrease in cell motility [53]. Our own findings are in accordance with these results, with an experimentally-induced specific reduction of a-SMA resulting in decreased fibroblast motility. Further, adult fibroblasts, with higher levels of both CCT-eta and a-SMA, were clearly better able to migrate in response to growth factor stimulation than the relatively eta- and a-SMA-deficient fetal fibroblasts. There is a strong case as well for the importance of a-SMA to fibroblast contractility. Chen et al. [54] examined a-SMA expression and cellular traction force (CTF) in fibroblasts stimulated with TGF-b. They found that both a-SMA levels and CTF increased in a dose-dependent manner. The TGF-b-induced increase in CTF was reversed by an siRNA specific to a-SMA. Other agents that inhibited TGF-b-induced a-SMA expression (eg. basic fibroblast growth factor and a TGF-b type I receptor inhibitor) also resulted in reduced CTF. These observations were found to be unrelated to non-muscle myosin II and beta-actin expression, and clearly implicate a-SMA as an important mediator of fibroblast/myofibroblast contractility as well as motility. In this context, and coupled with the previously noted finding that a-SMA expression correlates with contracting, scar-forming wounds but is absent from scarlessly healing wounds, our results build upon a growing body of evidence that fibroblast motility and contractility are important determinative features of wound contraction and scar formation, and that a critical element in this process is a-SMA. Since CCT-eta is shown herein to be essential for a-SMA protein accumulation, we posit that reduction of CCT-eta regulates fibroblast motility and contractility through its downstream reduction of a-SMA. The reduction of CCT-eta seen in fetal wound tissues, reflected also in the relative paucity of CCT-eta in fetal fibroblasts, may therefore be a crucial component of the scarless nature of fetal wound repair. Although our findings demonstrate the specific importance of the CCT-eta subunit to fibroblast biology and define its effect on a-SMA, the question of whether this effect is through perturbation of CCT holoenzyme function or is a CCT-independent phenom- References 1. Clark RAF (1996) Wound Repair: overview and general considerations. In: Clark RAF, ed. The molecular and cellular biology of wound repair. 2nd ed. New York: Plenum Press. pp 3–50. 2. Hantash BM, Zhao L, Knowles JA, Lorenz PH (2008) Adult and fetal wound healing. Front Biosci 13: 51–61. 3. Colwell AS, Longaker MT, Lorenz PH (2005) Mammalian fetal organ regeneration. Adv Biochem Eng Biotechnol 93: 83–100. 4. Wilgus TA (2007) Regenerative healing in fetal skin: A review of the literature. Ostomy Wound Manage 53: 16–31. CCT-eta & Fibroblast Phenotype enon remains unsettled. Since reduction of CCT-beta had no effect on fibroblast motility and contractility (but would be expected to disturb CCT holoenzyme function in a manner similar to CCT-eta depletion), it remains possible and perhaps even likely that our observations derive from a CCT-eta monomer-specific function. CCT-eta has been shown to have a specific biologic activity separate from its role in the greater CCT complex: CCT-eta was recently found to be a co-factor for the soluble guanylyl cyclase (sGC), the chief intracellular secondary mediator of nitric oxide (NO) signaling [26]. Eta acts as an inhibitor of sGC; the net result of a downregulation of eta, therefore, would be a relative increase in sGC activity, resulting in increased cGMP and amounting to an increased functional NO signal. NO has previously been found to inhibit the PDGF-induced migration of fibroblasts and pericytes [55] and to inhibit the trans-endothelial migration of T cells [56], but likely acts in cell specific fashion through a variety of downstream molecular mediators. Although NO has been well studied in adult wound healing and is thought to favorably affect wound healing in general [57,58,59], its role in fibroblast physiology and especially in fetal wound repair is still relatively unexplored. The interaction of CCT-eta with sGC may represent one molecular mechanism which can (under certain circumstanc- es) recapitulate an NO-dependent physiology. Surprisingly, our results show that downregulating CCT-eta actually decreased cGMP levels in adult fibroblasts (data not shown). Since steady state cGMP levels are obviously the product of countervailing synthetic and degradative processes, it remains possible that decreasing CCT-eta is allowing for actual increased cGMP production but that this is overwhelmed by other downstream factors. On balance, however, with reduced cGMP levels it seems unlikely that the effects we observe by inhibiting CCT-eta on fibroblast contractility and motility are operating through a process that recapitulates nitric oxide signaling. Of course, both contractility and motility are extremely complex properties that result from the summation of numerous factors within cells; more experimentation will be required to further elucidate which other specific interactions may contribute particularly to fibroblast behavior and to the different properties of fetal versus adult cells. Acknowledgments We thank Jianxin Chen, Torin Yeager and Nicholas Kucher of Dr. Wang’s laboratory for their technical support in completing the traction force experiments. We also extend our thanks to Ms. Mary O’Toole of the Center for Genomic Sciences for her assistance in preparing this manuscript. Author Contributions Conceived and designed the experiments: LS SK. Performed the experiments: LS SJ. Analyzed the data: LS JHCW SK. Contributed reagents/materials/analysis tools: LS JHCW SK. Wrote the paper: LS SK. Critically reviewed paper: JCP GDE. 5. Gabbiani G (2003) The myofibroblast in wound healing and fibrocontractive diseases. J Pathol 200: 500–503. 6. Desmouliere A, Chaponnier C, Gabbiani G (2005) Tissue repair, contraction, and the myofibroblast. Wound Repair Regen 13: 7–12. 7. Burton K, Park JH, Taylor DL (1999) Keratocytes generate traction forces in two phases. Mol Biol Cell 10: 3745–3769. 8. DePalma RL, Krummel TM, Durham LA, 3rd, Michna BA, Thomas BL, et al. (1989) Characterization and quantitation of wound matrix in the fetal rabbit. Matrix 9: 224–231. PLoS ONE | www.plosone.org 13 April 2010 | Volume 5 | Issue 4 | e10063