Browsing by Subject "Kidney Diseases, Cystic"

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    Role of Primary Cilium-Mediated Signaling in Development, Disease and Cancer
    (2018-07-27) Somatilaka, Bandarigoda N.; Amatruda, James F.; Mukhopadhyay, Saikat; Kim, James; Wu, Jiang I.
    The primary cilium serves as an antenna in most vertebrate cells. Dysfunction of the primary cilium is associated with a variety of diseases, called "ciliopathies". Mechanisms underlying ciliogenesis have been well studied. However, deciphering the role of cilia in cellular signaling pathways is critical in understanding the basis behind the varied ciliopathic phenotypes, and remains a frontier mostly unexplored. Primary cilia are required for vertebrate Shh signaling. The final output of the Shh pathway is the formation of Gli transcriptional activators or repressors, both of which occur in a cilia-dependent manner. However, the role of cilia-generated signaling during development, particularly that of the poorly understood negative regulation and basal suppression of sonic hedgehog (Shh) signaling and its relationship to hyperproliferative conditions such as cystic kidney disease and carcinogenesis is not well understood. Here, I studied the role of primary cilium-generated signaling in the context of development and disease. Particularly, I focused on the role of poorly understood negative regulation of Shh signaling and ciliary trafficking in the context of cellular signaling pathways. My studies provide key insights into the role of: (a) novel mechanisms for trafficking adenylyl cyclases to cilia in Shh signaling during neural tube development; (b) primary cilium-generated signaling in cystic kidney disease; (c) and role of basal suppression of Shh pathway and cilia in Shh-subtype medulloblastoma initiation, progression and prognosis. In addition, I contributed to other projects on the role of negative regulation of Shh signaling in limb and skeletal development, and regulation of ciliary trafficking of membrane proteins. Overall, my projects highlight the importance of cilium-generated signaling, particularly that of negative regulation of Shh signaling in embryonic development, cystic kidney disease, and Shh-subtype medulloblastoma.
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    Sumo and Ubiquitin Ligases Regulate Hepatocyte Nuclear Factor - 1 Beta Transcriptional Activity
    (2007-05-27) McNally, Brian Timothy; Igarashi, Peter
    Hepatocyte nuclear factor-1beta (HNF-1β ) is a homeodomain-containing transcription factor that regulates tissue-specific gene expression in the kidney, liver, and other organs. Humans with heterozygous mutations of HNF-1β develop maturity-onset diabetes of the young type 5 (MODY5) associated with cystic abnormalities of the kidney. A yeast two-hybrid screen was performed to identify proteins that interact with HNF-1β The SUMO E2 ligase, Ubc9, SUMO E3 ligases, PIAS1 and PIASgamma , and the ubiquitin E3 ligase, Arkadia, were isolated as putative interacting proteins. SUMOylation of transcription factors affects their subcellular localization, intracellular transport, stability, and transcriptional activity. Ubiquitination of transcription factors has been shown to alter their stability and transcriptional activity. Immunostaining of the adult mouse kidney showed that HNF-1β co-localized with Ubc9, PIAS1, and PIASgamma in the nuclei of renal tubular epithelial cells. Immunostaining also showed that Arkadia localized to the nuclei of renal tubules. Overexpression of Ubc9, PIAS1, or PIASgamma inhibited the activity of the Pkhd1 promoter, a known HNF-1β target. Expression of catalytically inactive Ubc9 or PIASgamma mutants also inhibited Pkhd1 promoter activity, suggesting that repression of HNF-1β transcriptional activity is SUMOylation-independent. In contrast to the repressive effect of SUMO ligases, overexpression of Arkadia stimulated Pkhd1 promoter activity in an HNF-1β;-dependent fashion. Immunoprecipitation of epitope-tagged SUMO or ubiquitin resulted in co-precipitation of HNF-1β , indicating that HNF-1β is both SUMOylated and ubiquitinated in vivo. In summary, the E2 and E3 SUMO ligases Ubc9, PIAS1, and PIASgamma repressed HNF-1β transcriptional activity via SUMOylation-dependent and -independent mechanisms. The E3 ubiquitin ligase Arkadia stimulated HNF-1β transcriptional activity.Together, these results demonstrated that HNF-1β is covalently modified by SUMO and ubiquitin, and these modifications had opposing effects on HNF-1β transcriptional activity. In addition, insights into the regulation of HNF-1β and ubsequent effects on target gene expression may lead to a greater understanding of the mechanisms that lead to renal cystogenesis.