We previously reported that an altered growth response to HC and resistance of keloid fibroblasts to HC downregulation of collagen and elastin are observed only in fibroblasts from the keloid nodule, findings that support the hypothesis that keloid fibroblasts are an epigenetically distinct subpopulation (Russell et al., 1978
; Russell et al., 1995
; Russell et al., 1989
). Here we provide further evidence for that hypothesis. The differential expression of several fibrosis-associated genes, including the Wnt inhibitor SFRP1, MMP3, DPT, JAG1, CTGF, and IGFBP5 is confined to fibroblasts cultured from the keloid nodule. Immunohistochemical measurements confirmed that decreased levels of SFRP1 and SFRP2 and increased levels of IGFBP5 are confined to active keloid tissue.
The hypothesis that differences between normal and keloid fibroblasts in culture are due to differences in vivo
aging is not supported by our studies. While the number of in vitro
population doublings may not accurately reflect the number of divisions undergone in vivo
(Cristofalo et al., 2004
; Maier and Westendorp, 2009
), detailed analysis of the replicative lifespan of two keloid and two normal strains aged in the presence or absence of HC revealed no consistent differences to support the hypothesis that fibroblasts cultured from keloids underwent more population doublings than fibroblasts from normal dermis or scar in the formation of the tumor. HC has been reported to extend (Cristofalo and Rosner, 1979
) or have no effect (Didinsky and Rheinwald, 1981
) on the replicative lifespan of normal fibroblasts. We observed little effect of HC on the lifespan of either normal or keloid fibroblasts.
It is routine to compare patterns of gene expression in cultured cells at low passage number to minimize loss of an in vivo
phenotype (Feghali and Wright, 1999
; Smith et al., 2008
; Tuan et al., 2008
). It has been reported that simply culturing cells results in loss of a difference in α1β1 integrin collagen receptor expression between fibroblasts from keloids and normal skin (Szulgit et al., 2002
). However, some characteristics of an altered program are retained for many generations in culture. An example is the persistent downregulation of Fli1, a suppressor of collagen transcription, in scleroderma fibroblasts in vivo
and in vitro
(Asano et al., 2007
; Wang et al., 2006
). We have found that the altered pattern of gene expression in keloid fibroblasts, including failure of HC to downregulate collagen, elastin, and CTGF, decreased expression of SFRP1 and MMP3, and increased expression of IGFBP5 and JAG1 in standard culture medium is not abolished for at least 80% of the replicative lifespan. While not identifying a mechanism, these findings support the hypothesis of an epigenetically regulated program of fibrosis. Furthermore, persistence of the stimulatory effect of HC on growth of normal fibroblasts throughout the culture lifetime contradicts the notion that as normal fibroblasts age they act like keloid fibroblasts.
Recent inhibitor studies have provided evidence that epigenetic alterations occur during activation of wound healing and fibrosis. TSA blocks transforming growth factor β-mediated myofibroblastic differentiation (Glenisson et al., 2007
) and induction of collagen gene expression (Ghosh et al., 2007
; Rombouts et al., 2002
) in human skin fibroblasts. TSA also prevents accumulation of extracellular matrix in a mouse model of bleomycin-induced skin fibrosis (Huber et al., 2007
). TSA and 5-aza-dC have been reported to reverse epigenetic repression of the Fli1 gene and to decrease collagen expression in scleroderma fibroblasts (Wang et al., 2006
). Gene profiling studies have revealed no differences in expression of Fli1 in keloid fibroblasts; thus Fli1 does not appear to play a role in the keloid program of fibrosis.
Our findings support an altered program of DNA methylation and histone acetylation that could account for the stable pattern of differential gene expression in keloid fibroblasts in culture. These epigenetically distinct fibroblasts may have been produced or selected in the wound-healing environment of genetically predisposed individuals. While not irreversible, patterns of DNA methylation and histone modifications can be replicated over many cell generations in vivo
and in vitro
by complex albeit incompletely understood mechanisms involving chromatin architecture, long-range gene interactions and a complex network of trans-acting proteins and noncoding RNAs (Margueron and Reinberg, 2010
). The observation that TSA-induced reversal of SFRP1 gene silencing is associated with decreased expression of profibrotic IGFBP5 and JAG1 supports a role for differential histone acetylation of the SFRP1 gene or of a gene(s) that regulates SFRP1 expression in keloids. SFRP1 is best known as an inhibitor of Wnt signaling and increased Wnt signaling has been reported to play a role in the pathogenesis of keloids (Sato, 2006
) and several other fibrotic disorders including pulmonary and renal fibrosis (He et al., 2009
; Morrisey, 2003
). SFRP1 and SFRP2 have recently been reported to inhibit bone morphogenetic protein signaling (Misra and Matise, 2010
). Increased BMP signaling has been implicated in fibrotic disorders such as fibrodysplasia ossificans progressiva (Kaplan et al., 2009
). Therefore, silencing of SFRP1 may be important in the fibrosis signature displayed by keloid fibroblasts. While these inhibitor studies do not identify causal relationships between the expression of different genes, they provide additional evidence for an epigenetically altered program in keloid fibroblasts. Further elucidation of this program may be achieved by determining individual gene and genome-wide patterns of DNA methylation and histone modification. Manipulation of expression of specific epigenetically modified genes may identify causal relationships. Characterization of an epigenetically altered program in cultured fibroblasts may reveal mechanisms leading to keloid formation and suggest strategies to treat or prevent keloids and possibly other fibrotic disorders that disproportionately affect individuals of African ancestry.