Changes in three types of ubiquitin mRNA and ubiquitin-protein conjugate levels during lens development.

Ubiquitin is a small, highly conserved protein that covalently attaches to other proteins to form a unique branched protein structure. The best characterized function of this post-translational modification is to mark the modified protein for degradation by the proteasome. To investigate whether ubiquitin genes are regulated in lens development, the authors analyzed the levels of three ubiquitin mRNAs (UbA(52), UbB and UbC) in freshly dissected fiber and epithelial cells, and in epithelial explants induced to differentiate ex vivo. Explants, comprising the capsule and adherent epithelial cells, were dissected from lenses of 3 day old Sprague Dawley rats and cultured +/-bFGF to induce differentiation. Quantitative competitive RT-PCR was used to determine the mRNA levels in fresh and cultured cells. UbA(52), UbB and UbC mRNAs were 3.2 (P < 0.0001), 5.0 (P < 0.0001) and 6.8 (P < 0.0001) fold higher, respectively, in freshly dissected epithelial cells than in differentiated fiber cells. Immunological spot assays showed that ubiquitin protein is over two fold as high in rat pup lens epithelial cells as in fiber cells. The ubiquitin protein in fiber cells of adult rat is lower than that in adult epithelium and in pup fiber cells, indicating that ubiquitin content further decreased during lens fiber maturation. Western blots showed a greater amount of protein-conjugated ubiquitin (MW > 81 kD) in epithelial cells than in fiber cells, demonstrating a parallel pattern between the expression of ubiquitin mRNA, the level of ubiquitin protein and the level of conjugates in the cells. Epithelial cell explant cultures permit study of cells initiating differentiation. In contrast to fully differentiated fiber cells, explant cultures induced to initiate differentiation underwent differential up-regulation of ubiquitin gene expression. UbA(52) and UbB mRNA levels in +bFGF (differentiating) explant cultures were 2.6 (P < 0.001) and 1.4 (P < 0.001) fold higher, respectively, than those of -bFGF cultures. UbC mRNA content was similar in explants cultured with or without bFGF. Dissection of the isolated epithelial cells into regions representing distinct populations gave results consistent with this observation of the explant results. UbA(52), UbB and UbC mRNAs are 2.0, 2.2 and 1.76 fold higher, respectively, in the peripheral (initiating differentiation) than in the central (undifferentiated) region of epithelial cells. These results together indicate that UbA(52) and UbB mRNAs are transiently increased during the initiation and early stages of differentiation. However, UbC mRNA appears to be relatively unaffected at the earliest stage in this differentiation model and may have a different distribution than UbA(52) and UbB in the anterior lens cells. These data are consistent with an important role for ubiquitin during the early stages of lens differentiation. The selective expression indicates that the three genes have specific differentiation related functions.

Yang S ，Wang-Su ST ，Cai H ，Wagner BJ ... -  《EXPERIMENTAL EYE RESEARCH》

Expression and regulation of alpha-, beta-, and gamma-crystallins in mammalian lens epithelial cells.

Wang X ，Garcia CM ，Shui YB ，Beebe DC ... -  《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》

Identification of a novel gene product preferentially expressed in rat lens epithelial cells.

Cai H ，Howells RD ，Wagner BJ 《-》

Ubiquitin-proteasome pathway function is required for lens cell proliferation and differentiation.

Guo W ，Shang F ，Liu Q ，Urim L ，Zhang M ，Taylor A ... -  《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》

alpha6 Integrin is regulated with lens cell differentiation by linkage to the cytoskeleton and isoform switching.

The developing chicken embryo lens provides a unique model for examining the relationship between alpha6 integrin expression and cell differentiation, since multiple stages of differentiation are expressed concurrently at one stage of development. We demonstrate that alpha6 integrin is likely to mediate the inductive effects of laminin on lens differentiation as well as to function in a matrix-independent manner along the cell-cell interfaces of the differentiating cortical lens fiber cells. Both alpha6 isoform expression and its linkage to the cytoskeleton were regulated in a differentiation-specific manner. The association of alpha6 integrin with the Triton-insoluble cytoskeleton increased as the lens cells differentiated, reaching its highest levels in the cortical fiber region where the lens fiber cells are formed. In this region of the lens alpha6 integrin was uniquely localized along the cell-cell borders of the differentiating fiber cells, similar to beta1. alpha6beta4, the primary transmembrane protein of hemidesmosomes, is also expressed in the lens, but in the absence of hemidesmosomes. Differential expression of alpha6A and alpha6B isoforms with lens cell differentiation was seen at both the mRNA and the protein levels. RT-PCR studies demonstrated that alpha6B was the predominant isoform expressed both early in development, embryonic day 4, and in the epithelial regions of the day 10 embryonic lens. Isoform switching, with alpha6A now the predominant isoform, occurred in the fiber cell zones. Immunoprecipitation studies showed that alpha6B, which is characteristic of undifferentiated cells, was expressed by the lens epithelial cells but was dramatically reduced in the lens fiber zones. Expression of alpha6B began to drop as the cells initiated their differentiation and then dropped precipitously in the cortical fiber zone. In contrast, expression of the alpha6A isoform remained high until the cells became terminally differentiated. alpha6A was the predominant isoform expressed in the cortical fiber region. The down-regulation of alpha6B relative to alpha6A provides a developmental switch in the process of lens fiber cell differentiation.

Walker JL ，Menko AS 《DEVELOPMENTAL BIOLOGY》