To understand the role of
connexin43 (
Cx43) in epidermal differentiation, we reduced
Cx43 levels by
RNA-mediated interference knockdown and impaired its functional status by overexpressing loss-of-function
Cx43 mutants associated with the human disease
oculodentodigital dysplasia (
ODDD) in rat epidermal keratinocytes. When
Cx43 expression was knocked down by 50-75%, there was a coordinate 55-65% reduction in Cx26 level, gap junction-based
dye coupling was reduced by 60%, and transepithelial resistance decreased. Importantly, the overall growth and differentiation of
Cx43 knockdown organotypic epidermis was severely impaired as revealed by alterations in the levels of the
differentiation markers loricrin and
involucrin and by reductions in vital and cornified layer thicknesses. Conversely, although the expression of
Cx43 mutants reduced the coupling status of rat epidermal keratinocytes by approximately 80% without altering the levels of endogenous
Cx43 or Cx26, their ability to differentiate was not altered. In addition, we used a mouse model of
ODDD and found that newborn mice harboring the loss-of-function
Cx43(G60S) mutant had slightly reduced
Cx43 levels, whereas Cx26 levels, epidermis differentiation, and barrier function remained unaltered. This properly differentiated epidermis was maintained even when
Cx43 and Cx26 levels decreased by more than 70% in 3-week-old mutant mice. Our studies indicate that
Cx43 and Cx26 collectively co-regulate epidermal differentiation from basal keratinocytes but play a more minimal role in the maintenance of established epidermis. Altogether, these studies provide an explanation as to why the vast majority of
ODDD patients, where
Cx43 function is highly compromised, do not suffer from
skin disease.