Molecular characterization of pannexins in the lens.

PURPOSE: Cell communication in the lens is critical for the life-long homeostasis of this tissue. Abundant gap junctions and cell-cell fusions are reported to be indispensable to the metabolic requirements and optical properties of the highly interconnected syncytial lens tissue. The expression of the recently characterized Panx1 and Panx2 gap junction proteins in the lens is, therefore, rather intriguing. Co-expression of pannexins and abundant connexins in the lens suggests that the two gap junction protein families have distinct roles in cell communication. METHODS: Panx1 and Panx2 expression was studied by in situ hybridization and quantitative RT-PCR. We examined properties and tissue distribution of Panx1 isoforms by Western blot analysis. Immunohistochemistry was used to visualize lens regions that accumulate Panx1 to study intercellular localization and spatial relationship with lens connexin gap junctions. RESULTS: Panx1 and Panx2 expression peaked in lens epithelial cells prior to differentiation. We detected one ubiquitously expressed Panx1 isoform and two additional isoforms that were only detected in the lens and the retina. Our results indicated that the ubiquitous 58 kDa and the oligomeric 120 kDa isoforms were plasma membrane-bound, resistant to Triton X-100 treatment, and was likely associated with cholesterol-enriched membrane microdomains. Immunohistochemistry revealed Panx1-specific punctuate labeling in the plasma membrane, and intensive labeling of the organelles in the epithelial and immature fiber cells. In addition, we detected Panx1 immunoreactivity in blood endothelial cells of the tunica vasculosa lentis capillaries and in blood erythrocytes. CONCLUSIONS: Despite similarity in detergent solubility of pannexins and connexins, the lack of spatial co-localization in the lens membranes suggested a distinct, non-redundant to connexin function for these proteins in the membrane.

Microarray analysis of fiber cell maturation in the lens.

The mammalian lens consists of an aged core of quiescent cells enveloped by layers of mature fully elongated cells and younger, continuously elongating transcriptionally active cells. The fiber cell maturation is initiated when fiber cells cease to elongate. The process of maturation represents a radical switch from active elongation to a life-long quiescence and has not been studied previously. It may also include critical stages of preparation for the organelle removal and denucleation. In the present study, we used laser capture microdisection (LCM) microdissection and RNA amplification to compare global gene expression profiles of young elongating and mature, non-elongating fiber cells. Analysis of microarray data from three independent dye-swap experiments identified 65 differentially expressed genes (FDR<0.1) with greater than 2-fold change in expression levels. Microarray array results for a group of randomly selected genes were confirmed by quantitative RT-PCR. These microarray results provide clues to understanding the molecular pathways underlying lens development. The identified changes in the profile of gene expression reflected a shift in cell physiology characterizing the lens fiber maturation.

Distinct organization of the candidate tumor suppressor gene RFP2 in human and mouse: multiple mRNA isoforms in both species- and human-specific antisense transcript RFP2OS.

In the present study, we describe the human and mouse RFP2 gene structure, multiple RFP2 mRNA isoforms in the two species that have different 5' UTRs and a human-specific antisense transcript RFP2OS. Since the human RFP2 5' UTR is not conserved in mouse, these findings might indicate a different regulation of RFP2 in the two species. The predicted human and mouse RFP2 proteins are shown to contain a tripartite RING finger-B-box-coiled-coil domain (RBCC), also known as a TRIM domain, and therefore belong to a subgroup of RING finger proteins that are often involved in developmental and tumorigenic processes. Because homozygous deletions of chromosomal region 13q14.3 are found in a number of malignancies, including chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), we suggest that RFP2 might be involved in tumor development. This study provides necessary information for evaluation of the role of RFP2 in malignant transformation and other biological processes.