Differential expression of chaperonin containing T-complex polypeptide (CCT) subunits during fetal and adult skin wound healing

Abstract

Integumentary wound healing in early fetal life is regenerative and proceeds without scar formation. Expressomic analysis of this phenomenon by differential display has previously determined that the eta subunit of the cytosolic chaperonin containing T-complex polypeptide (CCT) is downregulated in the healing fetal wound milieu. We now report that no other CCT subunit shares this distinct pattern of gene regulation as determined by limiting dilution reverse transcriptase polymerase chain reaction (RT-PCR); all seven of the remaining CCT subunits demonstrate no change in messenger RNA (mRNA) expression in healing fetal wounds compared to unwounded control tissue. The alpha subunit, however, did evidence reduced message levels in healing adult wound tissue. We herein report on the cloning and sequence of the complementary DNA (cDNA) for rabbit CCT-alpha and confirm its wound specific decrease in adult tissues through quantitative real-time RT-PCR assay. We also confirm that quantitative evaluation of CCT-alpha and CCT-zeta mRNA expression shows no change in healing fetal wounds.

Chaperonin containing T-complex polypeptide (CCT) subunit expression in oral mucosal wounds and fibroblasts

“…1). These results are somewhat at variance with our observations from adult cutaneous wounds at a similar time point; in skin wounds, CCT-alpha is reduced, while CCT-beta remains invariant (Satish et al 2008).…”

“…We noted that no other chaperonin subunits share this specific pattern of downregulation at the tissue level (Satish et al 2008), and have further observed that fibroblasts from fetal skin tissues express substantially less CCT-eta mRNA than do fibroblasts from adult skin (Satish et al 2010a, b). These observations led us to investigate the pattern of CCT subunit expression in mucosal tissues and fibroblasts to see if comparable patterns would emerge.…”

“…Primer sets to each of these four chaperonin subunits were designed within the coding regions and used in initial RT-PCR assays on 10 ng of adult mucosal control total RNA to verify that each of the respective CCT subunits were detectable in adult tissue and resulted in only a single amplicon of the expected molecular weight. Primers and probes for rabbit CCT-alpha, -beta, -eta, -zeta, and GAPDH have been previously reported (Kathju et al 2006;Satish et al 2008;Satish et al 2010a, b). All Taqman primers/probes were designed using Primer Express software (Applied Biosystems, Foster City, CA).…”

“…All primers and fluorocoupled Taqman probes were purchased from Integrated DNA Technologies (Coralville, IA) wounded mucosal tissues (versus unwounded control), and also examine the level of CCT message expression in fibroblasts derived from adult mucosal tissues versus adult skin. We have previously described the probes and primers used to assay for CCT subunits -alpha, -beta, -eta, and -zeta in semi-quantitative limiting dilution RT-PCR as well as quantitative real-time RT-PCR (Darden et al 2000;Satish et al 2008;Satish et al 2010a, b).…”

“…Studies from our laboratory suggest that the chief cellular cytosolic chaperone, the chaperonin containing Tcomplex polypeptide (CCT), plays a significant role in differentiating scarless fetal healing from the scirrhous healing of adult skin wounds (Darden et al 2000;Satish et al 2008;Satish et al 2010a, b). The CCT protein is a large (900 kD) barrel-shaped hexadecameric protein complex that has the ability to bind and engulf unfolded/ misfolded proteins (Hartl and Hayer-Hartl 2002;Fenton and Horwich 2003), assisting them to achieve proper conformation.…”