Fig. 4.

PDE4C is localized in primary cilia and is regulated by the transcription factor HNF-1β. (A) Immunostaining of acetylated tubulin (green) and PDE4C (red) shows that PDE4C is localized in primary cilia in uninduced 53A cells (a–c) but is not detectable in cilia following treatment with mifepristone to induce expression of dominant-negative mutant HNF-1β (d–f). (B) mIMCD3 cells were transfected with either Flag-tagged PDE4C (Left) or Flag-tagged AKAP150 (Right), and cell lysates were immunoprecipitated with anti-Flag antibody. Immunoblot analysis shows coimmunoprecipitation of PDE4C, AKAP150, PKA regulatory subunit, and AC5/6. (C) ChIP of the promoter region of PDE4C using an antibody to HNF-1β (Right lane) and primers P1 and P2 (Upper and Middle panels). No product is seen using control IgG (Center lane) or primers that amplify an irrelevant region of the promoter (Lower). (D) PDE4C mRNA transcripts are reduced by 80% in 53A cells that are treated with mifepristone (Mif) to induce expression of dominant-negative mutant HNF-1β. Error bars indicate SD (n = 3). (E) cAMP levels are two- to 4.2-fold higher in mifepristone-treated 53A cells (▲) compared with uninduced cells (■) (n = 3). (F) Basal (open bars, Left axis) and forskolin-stimulated (closed bars, Right axis) CREB reporter activity is twofold and threefold higher in mifepristone-treated 53A cells (n = 3). (G) cAMP levels are 5.6-fold higher in kidneys from kidney-specific HNF-1β knockout mice compared with wild-type littermates (age P7, n = 2). (H) mIMCD3 cells were transfected with four different PDE4C siRNAs or a scrambled siRNA (control), and CREB reporter activity was measured. siRNAs 1, 2, and 4 reduced PDE4C mRNA levels (Right) and increased CREB reporter activity 1.3- to 5.4-fold (Left). (I) Immunostaining of acetylated tubulin (green) and PDE4C (red) in Pkd2^+/− cells (a–c) and Pkd2^−/− cells (d–f) shows that the ciliary localization of PDE4C is decreased in Pkd2^−/− cells (e and f). (Scale bars: 30 μm.)