Changes in Three Types of Ubiquitin mRNA and Ubiquitin-protein Conjugate Levels During Lens Development

Abstract

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 (UbA52, 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.UbA52, 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>81kD) 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. UbA52 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. UbA52, 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 UbA52 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 UbA52 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.