Tenbroek E M, Louis C F, Johnson R
Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108, USA.
Dev Biol. 1997 Nov 1;191(1):88-102. doi: 10.1006/dbio.1997.8703.
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 (beta-TPA). Within 10 min, beta-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 beta-TPA for 2-6 hr, Cx49 disappeared from epithelial cell interfaces, and by 24 hr of beta-TPA treatment, levels of Cx49 detected on immunoblots of purified epithelial membrane fractions had also diminished significantly. The beta-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 beta-TPA treatment. In spite of lentoid dye transfer being refractory to beta-TPA, significant levels of PKC-alpha (a beta-TPA-sensitive isoform) were detected in the lentoid cell. The response of lens gap junctions to beta-TPA depends upon the stage of differentiation and the complement of connexins expressed. The contrasting effects of beta-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.
原代绵羊晶状体培养物中的上皮细胞表达缝隙连接蛋白连接蛋白43（Cx43）和连接蛋白49（Cx49，又称MP70，是小鼠连接蛋白50的同源物）。相反，分化的纤维样细胞（称为类晶状体细胞）的晶状体培养物表达Cx49和连接蛋白46（Cx46），但不表达Cx43。为了研究蛋白激酶C（PKC）对晶状体细胞缝隙连接的调节作用，用PKC激活剂12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（β - TPA）处理正在分化的晶状体培养物。在10分钟内，β - TPA显著抑制了荧光素黄染料在上皮细胞之间的转移，但对类晶状体细胞没有影响。这种抑制作用与Cx43的磷酸化相关，随后Cx43逐渐从细胞界面消失。蛋白激酶抑制剂星形孢菌素可阻止Cx43的磷酸化以及Cx43从细胞间连接的丢失。用β - TPA处理培养物2 - 6小时后，Cx49从上皮细胞界面消失，到β - TPA处理24小时时，纯化的上皮细胞膜组分免疫印迹检测到的Cx49水平也显著降低。β - TPA诱导的Cx49从上皮细胞接触区域和分离膜中的丢失与Cx49 mRNA的消失相关。与上皮连接蛋白不同，即使延长β - TPA处理时间，类晶状体连接蛋白Cx49和Cx46也不受影响。尽管类晶状体染料转移对β - TPA不敏感，但在类晶状体细胞中检测到了显著水平的PKC - α（一种对β - TPA敏感的同工型）。晶状体缝隙连接对β - TPA的反应取决于分化阶段和所表达的连接蛋白的组成。β - TPA对晶状体上皮细胞中Cx43和Cx49的不同作用表明，在发育中的哺乳动物晶状体中，这些连接蛋白的调节存在根本差异。
