The differential effects of 12-O-tetradecanoylphorbol-13-acetate on the gap junctions and connexins of the developing mammalian lens.
The differential effects of 12-O-tetradecanoylphorbol-13-acetate on the gap junctions and connexins of the developing mammalian lens.
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DOI：
10.1006/dbio.1997.8703
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发表时间：
1997-11
影响因子：
2.7
通讯作者：
E. Tenbroek;C. Louis;Ross G. Johnson
中科院分区：
文献类型：
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作者：
E. Tenbroek;C. Louis;Ross G. Johnson
原代羊晶状体上皮细胞表达缝隙连接蛋白connexin43 （Cx43）和connexin49 (Cx49，又称MP70)，这是小鼠connexin50的同源物。相比之下，分化的纤维样细胞（称为透镜状细胞）的晶状体培养表达Cx49和connexin46 (Cx46)，但不表达Cx43。为了研究蛋白激酶C （PKC）对晶状体细胞间隙连接的调节作用，我们用PKC激活剂12- o - tetradecanoylphorol -13-acetate （β - tpa）处理分化晶状体培养物。在10分钟内，β - tpa显著抑制了路西法黄染料在上皮细胞而非透镜状细胞之间的转移。这种抑制与Cx43的磷酸化相关，随后Cx43从细胞界面逐渐消失。蛋白激酶抑制剂staurosporine可阻止Cx43磷酸化和细胞间连接中Cx43的丢失。在用β - tpa处理培养物2-6小时后，Cx49从上皮细胞界面上消失，并且在β - tpa处理24小时后，纯化的上皮膜组分免疫印迹上检测到的Cx49水平也显著降低。β - tpa诱导的上皮细胞接触区和分离膜上Cx49的丢失与Cx49 mRNA的消失有关。与上皮连接蛋白相反，透镜状连接蛋白Cx49和Cx46甚至不受延长β - tpa处理的影响。尽管透镜状染料转移对β - tpa不敏感，但在透镜状细胞中检测到显著水平的pkc - α（一种β - tpa敏感的异构体）。晶状体间隙连接对β - tpa的反应取决于分化阶段和表达的连接蛋白的补体。β - tpa对晶状体上皮细胞Cx43和Cx49的不同作用表明，在哺乳动物晶状体发育过程中，这些连接蛋白的调控存在根本差异。
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.