Epithelial cells in primary ovine lens cultures express the gap junction proteins connexin43 (Cx43) and connexin49 (Cx49; a.k.a. MP70), a homologue of mouse connexin50. In contrast, lens cultures of differentiated, fiber-like cells (termed lentoid cells) express Cx49 and connexin46 (Cx46), but not Cx43. To investigate the regulation of lens cell gap junctions by protein kinase C (PKC), differentiating lens cultures were treated with the PKC activator 12-O-tetradecanoylphorbol-13-acetate (β-TPA). Within 10 min, β- TPA significantly inhibited the transfer of Lucifer Yellow dye between epithelial, but not lentoid, cells. This inhibition was correlated with the phosphorylation of Cx43 and was followed by the gradual disappearance of Cx43 from cell interfaces. The protein kinase inhibitor staurosporine prevented Cx43 phosphorylation and the loss of Cx43 from intercellular junctions. Following treatment of cultures with β-TPA for 2-6 hr, Cx49 disappeared from epithelial cell interfaces, and by 24 hr of β-TPA treatment, levels of Cx49 detected on immunoblots of purified epithelial membrane fractions had also diminished significantly. The β-TPA-induced loss of Cx49 both from regions of epithelial cell contact and from isolated membranes was correlated with the disappearance of Cx49 mRNA. In contrast to the epithelial connexins, the lentoid connexins Cx49 and Cx46 were unaffected by even extended β-TFA treatment. In spite of lentoid dye transfer being refractory to β-TPA, significant levels of PKC-α (a β-TPA-sensitive isoform) were detected in the lentoid cell. The response of lens gap junctions to β-TFA depends upon the stage of differentiation and the complement of connexins expressed. The contrasting effects of β-TPA on Cx43 and Cx49 in lens epithelial cells indicate a fundamental difference in the regulation of these connexin proteins in the developing mammalian lens.
Bibliographical noteFunding Information:
The authors thank Dr. Todd Starich for his helpful advice and both Ding I. Yang and Grant Churchill for the many hours they spent on the road to obtain the fresh lenses used in these studies. This research was supported by National Institutes of Health Grants GM-46277 and EY-05684, by National Eye Institute Training Grant EY-07133-01, and by National Cancer Institute Training Grant CA-09607.
- Gap junctions
- β- TPA