Histone Deacetylase 2 Is Upregulated in Normal and Keloid Scars

glyceraldehyde-3-phosphate dehydrogenase histone acetyltransferase histone deacetylase immunohistochemistry transforming growth factor-β TO THE EDITOR Repair following cutaneous injury is essential to return function, form, and integrity to the tissue. Scar formation is the inevitable and currently unpreventable consequence of tissue damage, and misregulation can lead to the development of pathological scarring as in hypertrophic and keloid scars. In developed countries alone, approximately 100 million people each year will be left with a scar following surgery; moreover, there are thought to be 11 million people with keloid scars (Sund, 2000Sund B. New Developments in Wound Care. PJB Publications, London2000Google Scholar). This represents a significant burden to both health-care providers and individuals. Currently, the pathogenesis of keloid scars is not fully understood, but there are a number of clues as to the etiology. There is an obvious genetic component, and they have also been linked to hormonal influences, wound infection, and skin tension (Wolfram et al., 2009Wolfram D. Tzankov A. Pulzl P. et al.Hypertrophic scars and keloids—a review of their pathophysiology, risk factors, and therapeutic management.Dermatol Surg. 2009; 35: 171-181Crossref PubMed Scopus (466) Google Scholar). Given the burden presented and our current poor ability to reduce or prevent normal and pathological scarring, clearly an improved understanding of the molecular mechanisms underlying scar formation is essential, and research into novel treatment strategies is justified. Histone deacetylases (HDACs) and counteracting histone acetyltransferases (HATs) are epigenetic modifying enzymes that are accepted to, respectively, remove and add acetyl groups to histones, and in this manner influence gene expression (acetylation is generally an activating, transcription-promoting modification; Kuo and Allis, 1998Kuo M.H. Allis C.D. Roles of histone acetyltransferases and deacetylases in gene regulation.Bioessays. 1998; 20: 615-626Crossref PubMed Scopus (1067) Google Scholar). The objective of this work was to characterize the expression profiles of specific HDACs in normal and keloid scars; this has not been previously investigated, and there is a lack of consensus about the role of histone (de)acetylation in various fibrotic conditions. For example, HDAC inhibitors used in vitro suppressed myofibroblast differentiation (Glenisson et al., 2007Glenisson W. Castronovo V. Waltregny D. Histone deacetylase 4 is required for TGFbeta1-induced myofibroblastic differentiation.Biochim Biophys Acta. 2007; 1773: 1572-1582Crossref PubMed Scopus (134) Google Scholar; Mannaerts et al., 2010Mannaerts I. Nuytten N.R. Rogiers V. et al.Chronic administration of valproic acid inhibits activation of mouse hepatic stellate cells in vitro and in vivo.Hepatology. 2010; 51: 603-614Crossref PubMed Scopus (98) Google Scholar) and decreased collagen production in keloid fibroblasts (Diao et al., 2011Diao J.S. Xia W.S. Yi C.G. et al.Trichostatin A inhibits collagen synthesis and induces apoptosis in keloid fibroblasts.Arch Dermatol Res. 2011; 303: 573-580Crossref PubMed Scopus (52) Google Scholar); in animal models, HDAC inhibition decreased heart, kidney, and liver fibrosis (Pang et al., 2009Pang M. Kothapally J. Mao H. et al.Inhibition of HDAC activity attenuates renal fibroblast activation and interstitial fibrosis in obstructive nephropathy.Am J Physiol Renal Physiol. 2009; 297: F996Crossref PubMed Scopus (181) Google Scholar; Iyer et al., 2010Iyer A. Fenning A. Lim J. et al.Antifibrotic activity of an inhibitor of histone deacetylases in DOCA-salt hypertensive rats.Br J Pharmacol. 2010; 159: 1408-1417Crossref PubMed Scopus (119) Google Scholar; Marumo et al., 2010Marumo T. Hishikawa K. Yoshikawa M. et al.Histone deacetylase modulates the proinflammatory and -fibrotic changes in tubulointerstitial injury.Am J Physiol Renal Physiol. 2010; 298: F133-F141Crossref PubMed Scopus (114) Google Scholar). Conversely, HATs were overexpressed in fibrotic lesions of scleroderma patients (Bhattacharyya et al., 2005Bhattacharyya S. Ghosh A.K. Pannu J. et al.Fibroblast expression of the coactivator p300 governs the intensity of profibrotic response to TGFbeta.Arthritis Rheum. 2005; 52: 1248-1258Crossref PubMed Scopus (73) Google Scholar), and HAT inhibition was found to be anti-fibrotic (Li et al., 2008Li H.L. Liu C. de Couto G. et al.Curcumin prevents and reverses murine cardiac hypertrophy.J Clin Invest. 2008; 118: 879-893Crossref PubMed Scopus (8) Google Scholar). To investigate HDAC expression in human skin scars, three types of skin tissue were assessed by immunohistochemistry (IHC): normal human skin, normal scar tissue from patients undergoing melanoma re-excision approximately 2–3 weeks after the original wound, and keloid scar tissue from revision procedures (age of scar >6 months). Tissue samples were batch analyzed for HDAC1, HDAC2, HDAC4, and HDAC7. Scar-associated fibroblasts in both normal and keloid scars showed a significant and striking upregulation of HDAC2, but not HDAC1, 4, or 7 (Figure 1). Within the epidermis, HDAC expression was unchanged in scar versus non-scar regions (data not shown). Our observation that HDAC2 was upregulated in scar tissue was substantiated using a mouse model of wound repair. Specifically, 4-mm excisional wounds were made to the shaved dorsal skin of anesthetized adult male mice (CD-1; 6–8 weeks; protocol approved by an institutional ethics committee and the UK Home Office). Wound tissue was harvested after 3, 7, or 14 days and analyzed for HDAC2 by IHC. On Day 3, HDAC2-positive cells were found at the wound margins, whereas on Days 7 and 14 highly expressing cells were abundant in the wound bed (Figure 2a). This approach demonstrated that HDAC2 is upregulated in normal scar tissue in the mouse as it is in human. To begin to understand how HDAC2 expression may be regulated in this setting, primary cultures of normal human dermal fibroblasts (nHDFs, < passage 15) were either (1) treated with transforming growth factor (TGF)β1 (0, 0.1, 0.5, 1ngml-1) or (2) cultured at varying densities, and the effects on HDAC2 expression were observed by western blot analysis. TGFβ1 was able to trigger a concentration-dependent upregulation of HDAC2 (Figure 2b). Intriguingly, this effect was not observed in all patients: only two of the four primary cell populations tested responded in this way. Future work will investigate the determinants of this variable response and its influence on the efficacy of different targeted therapeutics. The same two nHDF isolations showed that HDAC2 expression correlates positively with cell density (Figure 2b), which would be high in a developing scar in vivo. Experiments using murine Swiss 3T3 fibroblasts (< passage 12) confirmed that TGFβ1 can increase HDAC2 expression, and, interestingly, also revealed that HDAC1 and HDAC7 were TGFβ1-responsive in these cells (Figure 2c). This seemingly contradictory finding that only HDAC2 was upregulated in in vivo scars, whereas TGFβ1 stimulation of cultured fibroblasts increased HDAC1 and 7, as well as 2, may indicate that the timing and duration of TGFβ1 exposure are important factors in the regulation of specific family members; alternatively, there may be confounding negative influences on the expression of specific HDACs in the heterogeneous in vivo setting. Our in vivo studies on mouse and human skin wounds revealed that HDAC2 is significantly overexpressed in both normal and keloid scar tissue. The failure of HDAC2 to distinguish between normal and keloid scars leads us to speculate that, in keloids, expression may fail to return to normal levels at the completion of the healing process, and thus may contribute to persistent growth and/or alternative differentiation of these cells. Our ongoing hypothesis is that pharmacological inhibition of HDACs will decrease skin fibrosis. Early, but promising, findings in various animal models of fibrosis certainly indicate that this may be the case in other organ systems (Pang et al., 2009Pang M. Kothapally J. Mao H. et al.Inhibition of HDAC activity attenuates renal fibroblast activation and interstitial fibrosis in obstructive nephropathy.Am J Physiol Renal Physiol. 2009; 297: F996Crossref PubMed Scopus (181) Google Scholar; Iyer et al., 2010Iyer A. Fenning A. Lim J. et al.Antifibrotic activity of an inhibitor of histone deacetylases in DOCA-salt hypertensive rats.Br J Pharmacol. 2010; 159: 1408-1417Crossref PubMed Scopus (119) Google Scholar; Marumo et al., 2010Marumo T. Hishikawa K. Yoshikawa M. et al.Histone deacetylase modulates the proinflammatory and -fibrotic changes in tubulointerstitial injury.Am J Physiol Renal Physiol. 2010; 298: F133-F141Crossref PubMed Scopus (114) Google Scholar). As inflammation tends to exacerbate scarring, it will be interesting to determine whether these results are owing to HDAC inhibitors acting directly on the fibroblasts, or acting indirectly, as anti-inflammatories (Han and Lee, 2009Han S.B. Lee J.K. Anti-inflammatory effect of Trichostatin-A on murine bone marrow-derived macrophages.Arch Pharm Res. 2009; 32: 613-624Crossref PubMed Scopus (59) Google Scholar). Regardless of the mode of action, if correct, the pathway from bench to bedside for HDAC inhibitors in the treatment of skin scars is anticipated to be relatively smooth, as there are 13 HDAC inhibitors already in use clinically (Paris et al., 2008Paris M. Porcelloni M. Binaschi M. et al.Histone deacetylase inhibitors: from bench to clinic.J Med Chem. 2008; 51: 1505-1529Crossref PubMed Scopus (352) Google Scholar). Currently, there are no successful treatments that prevent or eliminate scar tissue; however, counteracting TGFβ1, for example, using recombinant TGFβ3 (Avotermin, Renovo, UK), has shown potential success as an anti-scarring treatment (So et al., 2011So K. McGrouther D.A. Bush J.A. et al.Avotermin for scar improvement following scar revision surgery: a randomized, double-blind, within-patient, placebo-controlled, phase II clinical trial.Plast Reconstr Surg. 2011; 128: 163-172Crossref PubMed Scopus (73) Google Scholar). Our finding that TGFβ1 treatment increased HDAC expression suggests that HDAC inhibitors equally have the potential to be anti-fibrotic, depending on the extent to which TGFβ1 relies on HDAC2 to exert its effects. People with scars face many physical, psychological, esthetic, and social consequences that may be associated with substantial emotional and financial cost (Brown et al., 2008Brown B.C. McKenna S.P. Siddhi K. et al.The hidden cost of skin scars: quality of life after skin scarring.J Plast Reconstr Aesthet Surg. 2008; 61: 1049-1058Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). This research implicates HDACs in skin scarring, and suggests they may be novel therapeutic targets for the prevention of normal and pathological scarring. This work was supported by the University of London Central Research Fund and St George's, University of London.