Cx26 regulates proliferation of repairing basal airway epithelial cells.

The recovery of an intact epithelium following injury is critical for restoration of lung homeostasis, a process that may be altered in cystic fibrosis (CF). In response to injury, progenitor cells in the undamaged areas migrate, proliferate and re-differentiate to regenerate an intact airway epithelium. The mechanisms regulating this regenerative response are, however, not well understood. In a model of circular wound injury of well-differentiated human airway epithelial cell (HAEC) cultures, we identified the gap junction protein Cx26 as an important regulator of cell proliferation. We report that induction of Cx26 in repairing HAECs is associated with cell proliferation. We also show that Cx26 is expressed in a population of CK14-positive basal-like cells. Cx26 silencing in immortalized cell lines using siRNA and in primary HAECs using lentiviral-transduced shRNA enhanced Ki67-labeling index and Ki67 mRNA, indicating that Cx26 acts a negative regulator of HAEC proliferation. Cx26 silencing also markedly decreased the transcription of KLF4 in immortalized HAECs. We further show that CF HAECs exhibited deregulated expression of KLF4, Ki67 and Cx26 as well enhanced rate of wound closure in the early response to injury. These results point to an altered repair process of CF HAECs characterized by rapid but desynchronized initiation of HAEC activation and proliferation.

Cx26 affects the in vitro reconstruction of human epidermis.

To study the function of connexins in human keratinocytes, we have used a three-dimensional culture system, in which a tissue is reconstructed using cells from the outer root sheet of hair follicles. This tissue reproduces in vitro the histological organisation of human epidermis in situ and the normal distribution of several keratinocyte markers. Furthermore, it shows characteristics of a differentiating epidermis, including the expression of connexin26. Connexin26 protein expression is increased under physiological and pathological conditions resulting in increased keratinocyte turnover. Loss of this protein in keratinocytes, obtained from patients carrying a stop mutation, resulted in a reduced stratification of the in vitro reconstructed tissue, probably due to a lower proliferation and migration capacity of the keratinocytes, although dye coupling and persistence of other gap junctions is maintained. No changes were seen in tissues reconstructed with keratinocytes from patients carrying a non stop mutation of connexin30. The data indicate that, at least in vitro, connexin26 affects the function of human keratinocytes, independently of obvious changes in coupling.

Differential expression of connexins during stratification of human keratinocytes.

To assess whether gap junctions and connexins change during keratinocyte differentiation, we have studied epidermal equivalents obtained in organotypic cultures of keratinocytes from the outer root sheath of human hair follicles. These reconstituted tissues exhibit a number of differentiation and proliferation markers of human epidermis, including gap junctions, connexins, and K6 and Ki67 proteins. Immunostaining and northern blots showed that gap junctions of the epidermal equivalents were made of Cx26 and Cx43. Cx26 was expressed in all keratinocyte layers, throughout the development of the epidermal equivalents. In contrast, Cx43 was initially observed only in the basal layer of keratinocytes and became detectable in the stratum spinosum and granulosum only after the epidermal equivalents had thickened. The levels of Cx26 and its transcript markedly increased as a function of stratification of the epidermal equivalents, whereas those of Cx43 remained almost constant. Microinjection of Lucifer Yellow into individual keratinocytes showed that gap junctions were similarly permeable at all stages of development of the epidermal equivalents. The data show that epidermal equivalents (i) feature a pattern of connexins typical of an actively renewing human interfollicular epidermis, and (ii) provide a model that reproduces the tridimensional organization of intact epidermis and that is amenable for experimentally testing the function of junctional communication between human keratinocytes.