Chaperonin containing T-complex polypeptide subunit eta (CCT-eta) is a specific regulator of fibroblast motility and contractility

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 (alpha-SMA) expression, a gene product well known to play a critical role in adult wound healing. Fetal fibroblasts were found to constitutively express less alpha-SMA than adult cells. Reduction of CCT-eta with siRNA had minimal effect on cellular beta-actin but markedly decreased alpha-SMA; in contrast, reduction of CCT-beta had minimal effect on either actin isoform. Direct inhibition of alpha-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 alpha-SMA expression.

Figure 1. CCT-eta but not CCT-beta protein and mRNA are differentially expressed in fetal versus adult fibroblasts.

RNA and protein extracted from fetal and adult fibroblasts were subjected to qRT PCR (a & b) and Western blot (c & d) analyses respectively. CCT-eta mRNA was significantly more abundant in adult fibroblasts when compared to fetal fibroblasts (a); there was no significant difference in CCT-beta message levels between fetal and adult fibroblasts (b). Values are means ± SEM of three independent studies performed in duplicate. Statistical analyses were performed using Student's t test. NS = non-significant. Equal amounts of protein loaded from fetal and adult fibroblasts showed that adult fibroblasts express significantly greater CCT-eta protein(c). In contrast CCT-beta protein levels were not different between fetal and adult fibroblasts (d). Blots shown here are representative of at least three different experiments.

Figure 2. Cell migration of adult but not fetal fibroblasts is responsive to EGF and PDGF induction.

Primary cultures of fibroblasts obtained from fetal and adult rabbit skin were tested for motility in an in vitro wound healing assay. Cells were treated with increasing concentrations of EGF (1 nM and 10 nM) and PDGF (40 nM, 100 nM, 200 nM). The values are normalized to baseline motility and shown as EGF- and PDGF-induced cell motility at each concentration. Fetal and adult fibroblasts had essentially identical baseline motility, but only adult cells responded to growth factor stimulation. The values are mean ± SEM of six independent studies each performed in triplicate. Statistical analyses were performed by Student's t-test.

Figure 3. siRNAs against CCT-eta and CCT-beta decrease both basal and EGF- induced mRNA and protein levels of their targets in fibroblasts.

(a & b) qRT-PCR analysis of CCT-eta and CCT-beta 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 CCT-eta or CCT-beta mRNA and were calculated as a percentage of baseline control levels (100%). Values are means ± 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 CCT-eta/CCT-beta siRNA; Scr- treatment with scrambled control siRNA. (c & d) Western blot results using CCT-eta and CCT-beta antibody (1∶500) showed effective reduction of CCT-eta and CCT-beta protein levels when siRNA was administered but no decrease when scrambled 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.

Figure 4. siRNA against CCT-eta decreases EGF - induced fibroblast migration, whereas siRNA against CCT-beta does not.

Cells were incubated in the presence or absence of EGF(1 nM) +/− siRNA against CCT-eta (a) or CCT-beta (b) in an in vitro wound healing assay. In all experiments a subunit -specific scrambled siRNA sequence was used as control. Cell motility is displayed as a relative percentage of baseline motility in the absence of EGF or siRNA exposure (100%). Active siRNA versus CCT-eta reduced both basal and EGF-induced motility; siRNA versus CCT-beta and scrambled controls had no effect. Values are means ± SEM of six independent studies, each performed in triplicate. Statistical analyses were performed with Student's t test.

Figure 5. siRNA against CCT-eta decreases PDGF-induced fibroblast migration, whereas siRNA against CCT-beta does not.

Cells were incubated in the presence or absence of PDGF (200 nM) +/− siRNA against CCT-eta (a) or CCT-beta (b) in an in vitro wound healing assay. In all experiments a subunit -specific scrambled siRNA sequence was used as control. Cell motility is shown as a percentage of baseline migration in the absence of PDGF or siRNA exposure. As with EGF, active siRNA targeting CCT-eta inhibited basal and PDGF-induced motility, whereas CCT-beta siRNA and scrambled controls did not. Values are means ± SEM of six independent studies, each performed in triplicate. Statistical analyses were performed with Student's t test.

Figure 6. Adult fibroblasts are more contractile than fetal fibroblasts.

(a) Fetal fibroblasts are less contractile than adult fibroblasts as determined by traction force microscopy. Each bar represents mean ± SEM of more than 20 cells from two independent experiments. Statistical analyses were performed using Student's t test. (b) PDGF treatment of adult fibroblasts results in an increase in the observed cumulative traction force; EGF treatment results in a similar although smaller increase. Each bar represents mean ± SEM of more than 25 cells from two different experiments. Statistical analyses were performed using Student's t test.

Figure 7. siRNA against CCT-eta but not CCT-beta reduces PDGF-induced cellular traction force in adult fibroblasts.

Adult fibroblasts transfected with CCT-eta (a) or CCT-beta siRNA (b) along with pDSRed2-C1 were quantified for microdisplacement fields of red fluorescent cells on the green fluorescent substrate. Each assay was repeated twice with more than 30 cells quantified in each experiment. CCT-eta siRNA abolished the increased cellular traction force seen with PDGF treatment (200 nM), whereas CCT-beta siRNA and scrambled controls did not. Values are means ± SEM of two independent experiments with statistical analyses performed using Student's t test.

Figure 8. mRNA and protein levels show that α-SMA level is significantly increased in adult fibroblasts in comparison to fetal fibroblasts.

RNA and protein extracted from fetal and adult fibroblasts were subjected to qRT PCR (a) and Western blot (b) analyses respectively. The α-SMA mRNA levels were significantly more abundant in adult fibroblasts when compared to fetal fibroblasts (a). Values are means ± SEM of three independent studies performed in duplicate. Statistical analyses were performed using Student's t test. NS = non-significant. Equal amounts of protein loaded from fetal and adult fibroblasts showed that adult fibroblasts express significantly greater α-SMA protein (b). GAPDH was used as loading control.

Figure 9. siRNA downregulation of CCT-eta but not CCT-beta reduces α-SMA protein levels in adult fibroblasts.

(a & b)Western blot showed effective reduction of both CCT-eta and α-SMA protein levels when CCT-eta siRNA was administered, leaving beta-actin largely unaffected. In contrast, downregulating CCT-beta did not lead to significant reduction in either α-SMA or beta-actin levels. GAPDH was used as loading internal control. A representative immunoblot of up to three similar such blots are shown for each analysis. Ntx- no transfection; EGF-EGF treatment (1 nM); siRNA-treatment with CCT-eta/CCT-beta siRNA; Scr- treatment with scrambled control siRNA.

Figure 10. siRNA against α-SMA specifically decreases both both basal and EGF- induced mRNA and protein levels of α-SMA in adult fibroblasts.

(a) qRT-PCR analysis of α-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 α-SMA mRNA and were calculated as a percentage of baseline control levels (100%). Values are means ± 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 α-SMA siRNA; Ctr- treatment with a non-specific control siRNA. (b) Western blot results using α-SMA antibody (1∶500) showed effective reduction of α-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.

Figure 11. siRNA against α-SMA inhibits both basal and EGF-induced cell migration in adult fibroblasts.

Cells were incubated in the presence or absence of EGF(1 nM) +/− siRNA against α-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 α-SMA reduced both basal and EGF-induced motility; a non-specific control siRNA had no such effect. Values are means ± SEM of eight independent studies, each performed in duplicate. Statistical analyses were performed with Student's t test.