Saxon M L, Zhao X, Black J D
Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263.
J Cell Biol. 1994 Aug;126(3):747-63. doi: 10.1083/jcb.126.3.747.
The mechanisms underlying control of cell growth and differentiation in epithelial tissues are poorly understood. Protein kinase C (PKC) isozymes, members of a large family of serine/threonine kinases of fundamental importance in signal transduction, have been increasingly implicated in the regulation of cell growth, differentiation, and function. Using the rat intestinal epithelium as a model system, we have examined PKC-specific activity as well as individual PKC isozyme expression and distribution (i.e., activation status) in epithelial cells in situ. Increased PKC activity was detected in differentiating and functional cells relative to immature proliferating crypt cells. Immunofluorescence and Western blot analysis using a panel of isozyme-specific antibodies revealed that PKC alpha, beta II, delta, epsilon, and zeta are expressed in rat intestinal epithelial cells and exhibit distinct subcellular distribution patterns along the crypt-villus unit. The combined morphological and biochemical approach used permitted analysis of the activation status of specific PKC isozymes at the individual cell level. These studies showed that marked changes in membrane association and level of expression for PKC alpha, beta II, delta, and zeta occur as cells cease division in the mid-crypt region and begin differentiation. Additional changes in PKC activation status are observed with acquisition of mature function on the villus. These studies clearly demonstrate naturally occurring alterations in PKC isozyme activation status at the individual cell level within the context of a developing tissue. Direct activation of PKC in an immature intestinal crypt cell line was shown to result in growth inhibition and coincident translocation of PKC alpha from the cytosolic to the particulate subcellular fraction, paralleling observations made in situ and providing further support for a role of intestinal PKC isozymes in post-mitotic events. PKC isozymes were also found to be tightly associated with cytoskeletal elements, suggesting participation in control of the structural organization of the enterocyte. Taken together, the results presented strongly suggest an involvement of PKC isoforms in cellular processes related to growth cessation, differentiation, and function of intestinal epithelial cells in situ.
上皮组织中细胞生长和分化的控制机制目前仍知之甚少。蛋白激酶C（PKC）同工酶是丝氨酸/苏氨酸激酶大家族的成员，在信号转导中至关重要，越来越多地参与细胞生长、分化和功能的调节。我们以大鼠肠上皮为模型系统，检测了原位上皮细胞中PKC的特异性活性以及各个PKC同工酶的表达和分布（即激活状态）。相对于未成熟的增殖隐窝细胞，在分化细胞和功能细胞中检测到PKC活性增加。使用一组同工酶特异性抗体进行的免疫荧光和蛋白质印迹分析表明，PKCα、βII、δ、ε和ζ在大鼠肠上皮细胞中表达，并沿隐窝-绒毛单元呈现出不同的亚细胞分布模式。所采用的形态学和生化方法相结合，使得能够在单个细胞水平上分析特定PKC同工酶的激活状态。这些研究表明，随着细胞在隐窝中部区域停止分裂并开始分化，PKCα、βII、δ和ζ的膜结合和表达水平发生显著变化。随着绒毛上成熟功能的获得，还观察到PKC激活状态的其他变化。这些研究清楚地证明了在发育中的组织背景下，单个细胞水平上PKC同工酶激活状态的自然发生改变。在未成熟的肠隐窝细胞系中直接激活PKC被证明会导致生长抑制，同时PKCα从胞质转移到颗粒状亚细胞组分，这与原位观察结果相似，并为肠PKC同工酶在有丝分裂后事件中的作用提供了进一步支持。还发现PKC同工酶与细胞骨架成分紧密相关，表明其参与肠上皮细胞结构组织的控制。综上所述，所呈现的结果强烈表明PKC同工型参与了与原位肠上皮细胞生长停止、分化和功能相关的细胞过程。
