Master of Science / Department of Biochemistry / Dolores J. Takemoto / The vertebrate lens relies on gap junction-mediated intercellular communication to maintain cellular homeostasis and lenticular transparency. Differentiation of cuboidal lens epithelial cells into cortical fiber cells involves the degradation of endogenous gap junction protein, connexin 43 (Cx43) and the up-regulation of connexin 46 (Cx46). Cx46 may also be involved in the hypoxia response in other tissues; a function that can possibly be attributed to unique phosphorylation sites at the cytoplasmic C-terminus. In this study, we have developed a mammalian (rabbit) lens epithelial cell (RLEC) culture model that overexpresses Cx46 to ascertain the role of Cx46 in differentiation and oxidative stress response. The cell line N/N1003A was stably transfected with a GFPCx46 plasmid construct, and analyzed for differentiation markers including endogenous gap junction protein isoforms (Cx43 and Cx50). Western blot analysis and visual observation determined that the stable overexpression of Cx46 (sCx46OE) induced the degradation of Cx43 and elicited morphological changes indicative of fiber cell elongation. Total RNA from RLEC culture was isolated and analyzed for mRNA levels using RT-PCR. Comparable levels of Cx43 transcript were present in wild type, transient Cx46OE (tCx46OE), and sCx46OE which suggests a post-transcriptional regulation of Cx43 degradation. Treatment of sCx46OE with proteasome inhibitors restored Cx43 protein levels, and scanning confocal microscopy supported our hypothesis that Cx43 is degraded in differentiating lens cells by way of a ubiquitinmediated proteasomal pathway. It is our conclusion that Cx46 has application in hypoxic conditioning and differentiation in addition to its conventional role as a gap junction protein.

Doctor of Philosophy / Graduate Biochemistry Group / Dolores J. Takemoto / Eukaryotic cells possess a unique way to communicate with each other by passing metabolites and small molecules through protein pores that connect adjacent cells. Although there are many types and families of protein pores, connexins comprise a unique family. Six connexin monomers assemble into a hemichannel, which is transported to the cell membrane. An opposing cell membrane containing compatible connexin hemichannels is located and connected, forming an intercellular dodecameric protein complex. This results in a protein channel that connects two separate cytoplasmic compartments to each other. This type of channel is known as a gap junction. Connexin expression and function is commonly tissue specific. Of the 21 known human connexins, less than half are currently well characterized. Three connexins are expressed in the lens, connexin 43 (Cx43), 46 (Cx46), and 50 (Cx50). Of these three, Cx46 and Cx50 both have major functions in the mature lens. Cx46 functions as a major gap junction channel, which maintains mature lens homeostasis, while Cx50 possesses growth control properties in the lens. Cx46 expression is modulated in breast and bone tumors, and during ischemia. It is hypothesized that Cx46 provides resistance to hypoxia mediated cell death by prolonging survival. In this study, Cx46 expression was detected in human Y79 retinoblastoma cells. Decreasing the expression of Cx46 in nude mice carrying Y79 xenografts slowed early stage tumor growth. Y79 cells in culture survive for over 72 hours in 1% oxygen in vitro. C46 was upregulated in cultured lens cells when grown under hypoxia. Human lens epithelial cells, rabbit N/N1003A lens cells, and Y79 cells proliferated in 1% oxygen until Cx46 expression was depleted by use of siRNA. Protection from hypoxia-induced cell death was provided by transfection with the C-terminus of Cx46. We further determined that the promoter activity of Cx46 was increased in 1% oxygen. These results indicate that Cx46 would increase in response to hypoxia and suggest a role for Cx46 in protection from hypoxia. The studies demonstrate a novel function for Cx46 in cell survival during hypoxia.