Like Cx43, in both rat tail and mouse back epidermis Cx26 and Cx30 were down-regulated at the wound edge within 6 hours of wounding. However, they both became highly up-regulated directly behind the wound edge through several epidermal layers and for up to 500 µm from the wound (figure 1.5.B-C). Following wound closure expression levels remained high in the hyperproliferative epidermis during differentiation, but returned to normal following completion of full barrier restoration (Coutinho et al. 2003; Goliger & Paul 1995).
Both Cx26 and Cx30 have been implicated in the pathology of chronic wounds, which are characterised by hyperproliferative epidermis peripheral to the wound and by a bulb of non-migratory keratinocytes at the wound edge (Herrick et al. 1992). Brander et al reported that Cx26 and Cx30 were both expressed at the wound edge of a mixed selection of chronic wounds, and for some distance peripherally, whereas in healing wounds both were down-regulated at the wound edge (Brandner et al. 2004). Additionally Cx26 and Cx30 were found to be significantly up-regulated at the wound edge and up to 4 mm peripherally in both diabetic foot ulcers and venous leg ulcers, and to a lesser extent in pressure ulcers (Sutcliffe et al, manuscript submitted). These finding suggest that high expression at the wound edge may be detrimental to healing. This is
supported by experiments where Cx26 was overexpressed ectopically in the epidermis of transgenic mice and impaired healing (Djalilian et al. 2006). Homozygous mutations were lethal due to impaired skin barrier function, but a heterozygous mutation resulted in a hyper-thickened epidermis, though differentiation of the epidermis was normal. However, these mice exhibited very poor wound healing compared to wild type littermates, and interestingly this was alongside an increased inflammatory response. Cx26 expression also remained elevated at the wound edge in the transgenic mice following injury, and maintained keratinocytes in a hyperproliferative state even after re- epithelialisation was complete. The re-epithelialising epidermis also displayed hyperkeratosis, parakeratosis, hypogranulosis and acanthosis, and the authors proposed that Cx26 down-regulation is necessary for the return of full skin barrier function (Djalilian et al. 2006). Alternatively, Cx30 deletion in mice was not observed to cause any abnormalities in skin, and wounding experiments conducted on the tail revealed no improvement or delay in healing compared to wild type litter mates (Teubner et al. 2003; Kretz et al. 2003). The effect of overexpressing wild type Cx30 on skin formation and wound healing remains to be determined.
Cx26 and Cx30 are normally expressed at very low levels, but are highly expressed in regions of hyperproliferative epidermis during healing in rodents (Goliger & Paul 1995; Coutinho et al. 2003). Interestingly, high levels of Cx26 and Cx30 are also found in hyperproliferative epidermis such as viral warts, psoriasis and porokeratosis (Hivnor et al. 2004; Lemaître et al. 2006; Lucke et al. 1999; Labarthe et al. 1998). Thus, both Cx26 and Cx30 have been suggested to be markers of hyper-thickened epidermis. Furthermore, many of the hyperproliferative skin diseases where Cx26 and Cx30 expression are increased are also associated with abnormalities in differentiation, prompting authors to suggest they may be involved in abnormal differentiation, or at least reliable markers (Labarthe et al. 1998; Lucke et al. 1999). Like other differentiation markers connexins are expressed in specific layers of epidermis, and induction of differentiation of keratinocytes in vitro with calcium resulted in changes in the connexins expressed (Brissette et al. 1994). Additionally, treatment of human epidermis with retinoic acid, known to significantly modify keratinocyte differentiation, also modified connexin expression, particularly
increasing expression of Cx26 (Masgrau-Peya et al. 1997). Cx26 and Cx30 mutations have also been linked to hyperproliferative and poorly differentiated epidermis through connexin related diseases, where patients frequently present with hyperkeratosis and parakeratosis (Richard, White, et al. 1998; Lamartine et al. 2000; Kelsell et al. 2000). Expression of one Cx26 human mutation (R143W) that appears to prevents Cx26 trafficking, in keratinocytes in vitro resulted in hyper-thickened organotypic cultures in comparison to wild type Cx26. Surprisingly though, this was not in conjunction with abnormal differentiation (Man et al. 2007). Expression of another, encoding a stop mutation (35delG) that effectively knocks out Cx26, instead reduced the thickness of keratinocyte organotypic cultures. However, a Cx30 mutation that causes hidrotic ectodermal dysplasia, G11R, caused no apparent differences (Wiszniewski et al. 2001). Alternatively, expression of a different hidrotic ectodermal dysplasia Cx30 mutation (A88V) driven by the endogenous promoter caused mild hyperkeratosis in transgenic mice (Bosen et al. 2014), though its effect on differentiation and wound healing remains to be tested. A further loss of function Cx26 dominant mutation was expressed in a rat epidermal keratinocyte line, alongside overexpression of wild type Cx26 (Thomas et al. 2007). Surprisingly, neither caused a difference in epidermal thickness or differentiation of the keratinocytes. Moreover, neither altered proliferation or migration of the cells. Oppositely, over-expression of wild type Cx26 in a human keratinocyte cell line (nTERT) resulted in significantly increased proliferation in organotypic cultures (Man et al. 2007). It also increased migration rates compared to vector only controls. Similarly, overexpression of Cx30 in nTERT keratinocytes caused a significant increase in proliferation and migration. Enhanced cell proliferation was also observed in head and neck squamous cancer cells where full length and partial length Cx30 was over-expressed (Ozawa et al. 2009). Nevertheless,
in vivo observations do not support a direct association between proliferation
and either connexin. While Goliger et al observed Cx26 in hyperproliferative epidermis during healing, they noted that it was present predominantly in the differentiated layers rather than in the proliferative layers (Goliger & Paul 1995). Double labelling for Cx26 and Ki67, a marker for proliferation, in human hyperproliferative psoriatic lesional epidermis similarly showed most Cx26 expressing cells did not express Ki67 (Lucke et al. 1999). Additionally a negative correlation between the connexins Cx26 and Cx30, and Ki67 was
reported during wound healing in the mouse epidermis (Coutinho et al. 2003). These conflicting results means that while it is clear Cx26 and Cx30 are involved in the wound healing process, the roles they play in proliferation, differentiation and migration are elusive, and their ultimate importance is still highly contentious.