The G60S Cx43 mutant enhances keratinocyte proliferation and differentiation Jared M. Churko 1 , John J. Kelly 1 , Andrew MacDonald 2 , Jack Lee 2 , Jacinda Sampson 3 , Donglin Bai 2 and Dale W. Laird 1,2 1 Department of Anatomy and Cell Biology, University of Western Ontario, London, ON, Canada; 2 Department of Physiology and Pharmacology, University of Western Ontario, London, ON, Canada; 3 Department of Neurology, University of Utah School of Medicine, Salt Lake City, UT, USA Correspondence: Dale W. Laird, Department of Anatomy and Cell Biology, University of Western Ontario, London, ON, Canada N6A-5C1, Tel.: 001-519-661-2111 ext.86827, Fax: 001-519-850-2562, e-mail: Dale.laird@schulich.uwo.ca Abstract: Transient knock-down of the gap junction protein Cx43 by antisense and siRNA, or gap junction block with mimetic peptides, have been shown to enhance epidermal wound healing. However, patients with oculodentodigital dysplasia (ODDD) express mutant Cx43 that leads to a chronic reduction in gap junctional intercellular communication. To determine whether mutant Cx43 in keratinocytes would impact upon the wound healing process, we localized Cx43 in human and mouse skin tissue expressing mutant Cx43 and assessed the ability of primary keratinocytes derived from a mouse model of ODDD to proliferate, migrate and differentiate. In the epidermis from an ODDD patient and in the epidermis of mice expressing the G60S mutant or in keratinocytes obtained from mutant mice, Cx43 was frequently found within intracellular compartments and rarely localized to punctate sites of cell–cell apposition. Primary keratinocytes derived from G60S mutant mice proliferated faster but migrated similarly to keratinocytes derived from wild-type control mice. Keratinocytes derived from mutant mice expressed abundant Cx43 and higher levels of involucrin and loricrin under low calcium conditions. However, after calcium-induced differentiation, similar levels of Cx43, involucrin and loricrin were observed. Thus, we conclude that during wound healing, mutant Cx43 may enhance keratinocyte proliferation and promote early differentiation of keratinocytes. Key words: Cx43 – G60S – keratinocytes – oculodentodigital dysplasia – proliferation Accepted for publication 8 May 2012 Introduction A gap junction channel is composed of two inter-docked hemi- channels with each hemichannel containing six oligomerized connexin (Cx) subunits. Gap junctions regulate the direct intercel- lular passage of ions and small metabolites from cell to cell, and various Cx43 mutants have been shown to disrupt cell to cell communication (1, 2). Mutant Cx43 expression has been specifi- cally linked to the disease called oculodentodigital dysplasia (ODDD) (3) and patients with ODDD commonly develop fusion of the digits and malformations of the bones, eyes and teeth (4). To investigate how mutant Cx43 leads to this disease, we have used the ODDD mouse model expressing a G60S mutant Cx43 to study how mutant Cx43 impacts the development and function of the eyes (5), teeth (6) and heart (7). However, mutant Cx43 may also disrupt skin homoeostasis and regeneration as Cx43 has pre- viously been shown to be highly expressed during epidermal development and in the steady-state adult epidermis (8). During epidermal wound healing, Cx43 is dynamically regu- lated. Twenty-four hours after injury, Cx43 expression is reduced at the wounded edge (9–11), and the S368 phosphorylated species of Cx43 is specifically expressed in the stratum basale (12). Two to three days after wounding, Cx43 is reported to be absent at the wound border but up-regulated in the nascent proliferating kerati- nocyte zone (9, 11, 13). At 6 and 7 days after wounding, Cx43 expression is observed in the reformed epidermis (9, 11). Because the expression of Cx43 dynamically changes during cutaneous wound healing, Cx43 expression is hypothesized to modulate the ability of keratinocytes to repair the damaged epithelium. To test this hypothesis, siRNAs and mimetic peptides against Cx43 have been used to transiently modulate wound healing. When Cx43 lev- els were inhibited by an antisense oligodeoxynucleotide against Cx43 (14) or when blocking the interaction between gap junction channels using a gap junction mimetic peptide, Gap27 (15), an increase in keratinocyte proliferation was observed. In addition, rodent wounds treated with antisense Cx43 exhibited reduced inflammation and enhanced fibroblast migration, all of which have been postulated to accelerate wound healing (14, 16). While these studies have shown that transient decreases in Cx43 expression and function can accelerate wound healing, chronic reductions in Cx43 gap junction function had not been studied until recently. Using the G60S ODDD mouse model, we showed that in contrast to transient Cx43 knock-down, full exci- sion wounds performed on the backs of G60S mice healed slower than wild-type (WT) control littermate mice (17). In addition, fibroblasts derived from the G60S mice and from patients with ODDD demonstrated defects in fibroblast proliferation, migration and differentiation (17). However, mouse wounds heal primarily through contraction, and this delay in wound healing may not translate to a beneficial advantage in human wound healing. In addition, keratinocytes also play a significant role in wound heal- ing and how mutant Cx43 impacts upon Cx43 localization within human ODDD epidermis as well as the ability of kerati- nocytes to proliferate, migrate and differentiate has not been investigated to date. Materials and methods Cell lines and mice All animal experiments were approved by the Animal Use Subcommittee of the University Council on Animal Care at the 612 ª 2012 John Wiley & Sons A/S Experimental Dermatology, 2012, 21, 612–618 DOI: 10.1111/j.1600-0625.2012.01532.x www.blackwellpublishing.com/EXD Original Article