Polycystin-2 and Phosphodiesterase 4C Are Components of a Ciliary A-Kinase Anchoring Protein Complex That Is Disrupted in Cystic Kidney Diseases

Polycystin-2 and phosphodiesterase 4C are components of a ciliary A-kinase anchoring protein complex that is disrupted in cystic kidney diseases

Yun-Hee Choia,1, Akira Suzukia,1, Sachin Hajarnisa, Zhendong Maa, Hannah C. Chapinb, Michael J. Caplanb, Marco Pontoglioc, Stefan Somlod, and Peter Igarashia,e,2

Departments of aInternal Medicine and ePediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390; Departments of bCellular and Molecular Physiology and dInternal Medicine and Genetics, Yale University School of Medicine, New Haven, CT 06520; and cInstitut National de la Santé et de la Recherche Médicale U567, Centre National de la Recherche Scientiﬁque Unité Mixte de Recherche 8104, Université Paris-Descartes, Institut Cochin, 75014 Paris, France

Edited* by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved May 23, 2011 (received for review October 28, 2010) Polycystic kidney disease (PKD) is a genetic disorder that is primary cilia have an abnormality in planar cell polarity that may characterized by cyst formation in kidney tubules. PKD arises from initiate cyst formation (7). Primary cilia have been shown to abnormalities of the primary cilium, a sensory organelle located on regulate several intracellular signaling pathways that control the cell surface. Here, we show that the primary cilium of renal planar cell polarity, including Wnt/β-catenin signaling (8, 9); epithelial cells contains a protein complex comprising adenylyl however, the mechanism by which the loss of renal cilia produces cyclase 5/6 (AC5/6), A-kinase anchoring protein 150 (AKAP150), kidney cysts remains poorly understood. and protein kinase A. Loss of primary cilia caused by deletion of The intracellular second messenger cAMP has been implicated Kif3a results in activation of AC5 and increased cAMP levels. Polycystin-2 in the growth and expansion of kidney cysts (10). Renal cAMP (PC2), a ciliary calcium channel that is mutated in human PKD, concentrations are elevated in animal models of PKD (8). interacts with AC5/6 through its C terminus. Deletion of PC2 Treatment of embryonic kidney explants from Pkd1 mutant mice increases cAMP levels, which can be corrected by reexpression of with 8-Br-cAMP results in tubular dilation (11). Moreover, cAMP wild-type PC2 but not by a mutant lacking calcium channel activity. increases the proliferation of ADPKD cyst epithelial cells by ac- Phosphodiesterase 4C (PDE4C), which catabolizes cAMP, is also tivating the B-Raf/MEK/ERK pathway (12). This effect appears located in renal primary cilia and interacts with the AKAP150 com- to be Ca2+ dependent because treatment with Ca2+ ionophores plex. Expression of PDE4C is regulated by the transcription factor inhibits the mitogenic response to cAMP, whereas Ca2+ channel hepatocyte nuclear factor-1β (HNF-1β), mutations of which pro- blockers promote proliferation (10). Subcellular compartmen- duce kidney cysts. PDE4C is down-regulated and cAMP levels are talization of cAMP signaling is mediated by A-kinase anchoring increased in HNF-1β mutant kidney cells and mice. Collectively, proteins (AKAP), which tether adenylyl cyclases (AC) that syn- these ﬁndings identify PC2 and PDE4C as unique components of thesize cAMP with downstream effectors such as protein kinase

an AKAP complex in primary cilia and reveal a common mechanism A (PKA), phosphodiesterases (PDE), and exchange factors di- MEDICAL SCIENCES for dysregulation of cAMP signaling in cystic kidney diseases aris- rectly activated by cAMP (Epac) (13). Receptor-mediated ago- ing from different gene mutations. nists of adenylyl cyclase or nonselective phosphodiesterase inhibitors increase cAMP levels in cyst epithelial cells and stim- cyclic AMP | PKD2 | vHNF1 | TCF2 | intraflagellar transport ulate fluid secretion and proliferation (14, 15). Conversely, drugs that inhibit cAMP synthesis reduce cyst formation in animal olycystic kidney disease (PKD) is the most common genetic models and are currently being evaluated in clinical trials of hu- Pcause of kidney failure in humans (1). PKD is characterized man ADPKD (16). However, the mechanism that is responsible by kidney enlargement and progressive loss of renal function due for the elevation of cAMP levels in PKD is not known. to the accumulation of numerous fluid-filled cysts in the renal parenchyma. The cysts arise from renal tubules as a consequence Results of disturbances in cell proliferation, apoptosis, differentiation, Loss of Primary Cilia Activates cAMP Signaling. To investigate the fluid secretion, and planar cell polarity (2). The autosomal role of the primary cilium in the regulation of cAMP signaling, dominant form of PKD (ADPKD) is caused by mutations of we generated renal epithelial cell lines lacking primary cilia. Kif3aF/– fl PKD1 or PKD2, which encode the membrane proteins polycystin-1 mice carrying one null allele and one oxed allele of Kif3a (PC1) and polycystin-2 (PC2), respectively (3). PC1 and PC2 are the ciliogenic gene were crossed with mice expressing localized in the primary cilium, a whip-like, sensory organelle temperature-sensitive mutant SV40 large T antigen, and condi- that projects from the surface of most cells (1, 2). In the kidney, tionally immortalized renal epithelial cell lines were established. Kif3a Kif3aF/– primary cilia are located on the apical surface of renal tubular To delete , cells were infected with a retrovirus Kif3a−/− epithelial cells and project into the tubule lumen. Renal cilia are encoding self-excising Cre recombinase. The resulting immotile but bend in response to fluid flow and may have a mechanosensory function (4). In addition to PKD, several other human genetic disorders, collectively called the ciliopathies, are Author contributions: Y.-H.C., A.S., Z.M., and P.I. designed research; Y.-H.C., A.S., S.H., Z.M., H.C.C., and M.J.C. performed research; M.P. and S.S. contributed new reagents/ caused by mutations in proteins that are localized in the primary analytic tools; Y.-H.C., S.H., Z.M., and P.I. analyzed data; and Y.-H.C., A.S., S.H., and P.I. cilium and/or basal body (5). wrote the paper. The synthesis and maintenance of primary cilia requires The authors declare no conflict of interest. fl intra agellar transport, in which multiprotein complexes are *This Direct Submission article had a prearranged editor. transported along the ciliary axoneme by kinesin-II and dynein 1Y.-H.C. and A.S. contributed equally to this work. fi motor proteins. We have previously shown that kidney-speci c 2To whom correspondence should be addressed. E-mail: peter.igarashi@utsouthwestern. inactivation of the KIF3A subunit of kinesin-II results in the loss edu. of renal cilia and produces kidney cysts in mice (6). Analysis of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. precystic tubules in Kif3a mutant mice revealed that cells lacking 1073/pnas.1016214108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1016214108 PNAS | June 28, 2011 | vol. 108 | no. 26 | 10679–10684 Downloaded by guest on September 28, 2021 cells lacked KIF3A protein and primary cilia, whereas cilia were phosphorylated PKA substrates (RRXXS/T) revealed increased – present on the parental Kif3aF/ cells (Fig. 1A and Fig. S1 A–C). staining in the nuclei of cyst epithelial cells compared with the – − − Compared with Kif3aF/ cells, Kif3a / cells contained higher levels predominantly cytoplasmic staining in wild-type renal tubules of cAMP under basal conditions and following treatment with (Fig. 1F). As a positive control, staining was also increased in − − – forskolin, a diterpene that maximally activates adenylyl cyclases cultured Kif3a / cells compared with Kif3aF/ cells (Fig. S1D). (Fig. 1B). The increase in cAMP levels was sufficient to activate Similarly, staining with an antibody against phospho-CREB, a cAMP-dependent signaling, as indicated by cAMP response ele- specific PKA substrate, showed increased nuclear staining in − − ment binding protein (CREB) reporter assays (Fig. 1C). Kif3a / kidney cysts (Fig. 1G). Costaining with fluorescent lectins cells also exhibited enhanced phosphorylation of the PKA sub- showed that cAMP signaling was activated in cysts derived from strate Kemptide (Fig. 1 D and E). both proximal tubules and collecting ducts (Fig. 1 F and G). In addition to primary cilia, KIF3A is also located in the cytoplasm where it may have other functions (Fig. S1C). To verify AC5/6 and AKAP150 Are Localized in Renal Primary Cilia. cAMP is that the effects on cAMP were due to the loss of primary cilia, synthesized by adenylyl cyclase, which comprises a family of nine – ciliated Kif3aF/ cells were deciliated with dibucaine (17). Treat- membrane-associated isoenzymes with distinct tissue distribu- ment with dibucaine resulted in the removal of cilia from ∼95% tions and subcellular localizations. Adenylyl cyclase 6 (AC6) has of cells (Fig. S1 E and F) and increased cAMP levels (Fig. S1G). previously been localized in primary cilia in renal tubular cells – As a negative control, dibucaine had no effect on the already and cholangiocytes (19, 20). In Kif3aF/ renal epithelial cells, − − elevated cAMP levels in nonciliated Kif3a / cells. Because the adenylyl cyclases 5 and 6 (AC5/6) colocalized with acetylated − − formation of primary cilia depends on cell confluence (18), we tubulin, a marker of the primary cilium (Fig. 2A). In Kif3a / also compared cAMP levels in confluent and nonconfluent cells. cells, cilia were absent, and AC5/6 remained associated with the cAMP levels were higher in nonconfluent (nonciliated) mIMCD3 plasma membrane. Because the antibody to AC5 also recognizes cells compared with confluent (ciliated) cells (Fig. S1H). Treat- AC6, we confirmed the localization of AC5 using epitope-tagged ment with dibucaine increased cAMP levels in confluent cells to proteins. Consistent with the endogenous staining results, Flag- approximately the same level as nonconfluent cells. Although tagged AC5 and AC6 were localized in the plasma membrane these treatments may have additional effects, the most parsimo- and primary cilia of transfected renal epithelial cells (Fig. 2B). nious explanation for these findings is that the loss of primary cilia To determine whether AC5 mediates the increase in cAMP stimulates cAMP-dependent signaling. levels in Kif3a mutant cells, we treated the cells with NKY80, a To determine whether the loss of cilia activates cAMP sig- selective AC5 inhibitor. Treatment with NKY80 reduced the − − naling in vivo, we analyzed cystic kidneys from kidney-specific magnitude of the increase in CREB reporter activity in Kif3a / Kif3a mutant mice (6). Staining with an antibody that recognizes cells. In contrast, the elevated CREB reporter activity was not

A B C P=0.0003 3.5 P=0.05 P=0.04 18 45 P=0.003 700 a b 16 40 600 3 35 14 500 2.5 30 12 25 400 2 10 20 300 1.5 8 15 6 200 1 Relative CREB cAMP (pmol/mg) 4 10 reporter activity 0.5 5 100 2 c 0 0 0 0 d F/- -/- F/- -/- F/- -/-F/- -/- 0.6 P=0.001 D E 0.5 -/- F/- -/- F/--/- F/- -/-F/- NC 0.4 Nonphosphorylated 0.3 Phosphorylated 0.2 PKA activity 0.1 0 F G F/- -/- a b c a b c

d e f d e f C C C C C C

Fig. 1. Activation of cAMP signaling in Kif3a mutant cells and kidneys. (A) Immunostaining of KIF3A (red) and acetylated tubulin (green) shows that KIF3A – − − and primary cilia (arrows) are present in Kif3aF/ cells (a and b) but absent in confluent Kif3a / cells (c and d). Nuclei are counterstained with DAPI (blue). − − (Scale bars: 20 μm.) (B) Basal (open bars, Left axis) and forskolin-stimulated (closed bars, Right axis) cAMP levels are 11-fold and 1.5-fold higher in Kif3a / cells – − − than in Kif3aF/ cells. (C) Basal (open bars, Left axis) and forskolin-stimulated (closed bars, Right axis) CREB reporter activity is twofold higher in Kif3a / cells than in Kif3aF/– cells. (D) Agarose gel electrophoresis of phosphorylated and nonphosphorylated Kemptide shows that PKA activity is higher in Kif3a−/− cells than in Kif3aF/– cells. Each lane represents a different cell clone. NC, negative control. (E) Quantification of phosphorylated Kemptide shows that basal − − PKA activity is increased 1.7-fold in Kif3a / cells. (F) Immunostaining of phosphorylated PKA substrates (red) shows increased nuclear staining (arrows) in kidney cysts from kidney-specific Kif3a knockout mice (d) compared with wild-type kidneys (a) at age P19. Collecting ducts are stained with dolichos biflorus agglutinin (DBA) (green, b and e). C, cysts. (Scale bars: 10 μm.) (G) Immunostaining of phospho-CREB (red) shows increased nuclear staining (arrows) in kidney cysts from kidney-specific Kif3a knockout mice (d) compared with wild-type kidneys (a). Proximal tubules are stained with lotus tetragonolobus agglutinin (LTA) (green, b and e). (Scale bars: 10 μm.)

10680 | www.pnas.org/cgi/doi/10.1073/pnas.1016214108 Choi et al. Downloaded by guest on September 28, 2021 A a b c B a b

d e f c d

Fig. 2. Localization of AC5/6 and AKAP150 in renal primary cilia. (A) Kif3aF/− cells were stained with antibodies that recognize both AC5 and AC6 (red, b) or AC6 only (red, e) and costained with antiacetylated tubulin antibody P=0.017 C (green, a and d). The merged image shows colocalization in E 0.14 P=0.008 μ a b c 0.12 primary cilia (c and f). (Scale bars: 5 m.) (B) IMCD3 cells 0.1 were stably transfected with Flag-tagged AC5 or AC6 and 0.08 stained with anti-Flag antibody (green). Flag-tagged AC5 (a 0.06 and b) and AC6 (c and d) are localized in plasma membrane 0.04 (a and c) and primary cilia (arrows in b and d). (Scale bars: Relative CREB reporter activity 0.02 a and c,10μm; b and d,20μm.) (C) Treatment with NKY80, de f 0 a speciﬁc inhibitor of AC5, produces a dose-dependent − − NKY80 (mM) 0 0.1 0.2 0.4 0 0.1 0.2 0.4 decrease in CREB reporter activity in Kif3a / cells but has F/- -/- F/− 0.45 no effect in Kif3a cells. Error bars indicate SD (n = 3). (D) D P=0.005 − − 0.4 Transfection of Kif3a / cells with siRNA to AC5 reduces 0.35 CREB reporter activity, whereas siRNA to AC6 or scrambled 0.3 0.25 siRNA (control) have no effect. Expression of siRNAs does F/– g h i 0.2 not affect CREB reporter activity in Kif3a cells (n = 3). (E) 0.15 mIMCD3 cells were costained with anti-AKAP150 (red in

Relative CREB reporter activity 0.1 0.05 b, e,andh) and antibodies (green) against acetylated tu- 0 bulin (a), γ-tubulin (d), or α-tubulin (g). Merged images siRNA Control AC5 AC6 Control AC5 AC6 show localization of AKAP150 in primary cilia (c), cen- F/- -/- trosomes (f), and mitotic spindles (i). (Scale bars: 5 μm.)

– affected by NKY80 in Kif3aF/ cells (Fig. 2C). Similarly, knock- N-terminal PC2 deletion mutant did not localize to the cilium down of AC5 with siRNA normalized CREB reporter activity in and did not affect CREB activity. − − Kif3a / cells, whereas knockdown of AC6 had no effect (Fig. 2D). To verify that PC2 regulates cAMP signaling in vivo, cAMP The A-kinase anchoring protein AKAP150 (also called AKAP79) levels were measured in the kidneys of mice following kidney-

has previously been localized in cholangiocyte primary cilia and specific deletion of Pkd2 (23). Kidneys from 14- and 21-d-old MEDICAL SCIENCES has been shown to interact with AC5/6 (21). We found that mice were cystic and contained elevated levels of cAMP com- AKAP150 was also localized in primary cilia in renal epithelial pared with wild-type littermates (Fig. 3D). The cysts arose from cells (Fig. 2E). In addition, AKAP150 was found in centrosomes, collecting ducts, and the cells lining the cysts showed increased − − mitotic spindles, and the plasma membrane (Fig. 2E). In Kif3a / nuclear expression of phosphorylated PKA substrates, including B C cells that lack primary cilia, expression of AKAP150 was main- phospho-CREB (Fig. S2 and ). cAMP levels were also ele- Pkd2−/− tained in other cellular compartments. Taken together, these vated in cultured renal epithelial cells that were pro- Kif3a−/− E findings suggest that an AKAP150 complex containing AC5/6 is duced using a similar approach as cells (Fig. 3 ). Pkd2+/− Pkd2−/− located in renal primary cilia. Antibody staining showed that and cells contained primary cilia (Fig. 3F, a and d). AC5/6 was present in +/− Polycystin-2 Interacts with AC5/6 and Regulates cAMP Levels. primary cilia in Pkd2 cells but was not detected in the cilia of −/− Mutations of PKD2, which encodes the protein PC2, are found Pkd2 cells (Fig. 3F, b and e). Quantitative real-time RT-PCR in 15–20% of patients with ADPKD (3). PC2 is a Ca2+-permeable showed that the expression of AC5 mRNA transcripts was down- regulated by 60% and AC6 mRNA was up-regulated by 40% in channel that is located in the primary cilia of renal epithelial cells −/− 2+ fl fl Pkd2 cells (Fig. S2D). However, Western blot analysis showed where it is required for Ca entry induced by uid ow (22). −/− 2+ that the total amount of AC5/6 protein was unchanged in Pkd2 Because AC5 and AC6 are Ca -sensitive enzymes, we tested E the hypothesis that PC2 regulates cAMP through interactions cells (Fig. S2 ). These results suggest that PC2 may be required with AC5/6. Immunoprecipitation of endogenous AC5/6 in for the ciliary localization of AC5/6. Next, we examined whether the Ca2+ channel activity of PC2 mIMCD3 renal epithelial cells resulted in coprecipitation of − − plays a role in regulating cAMP signaling. Pkd2 / cells were native PC2 (Fig. 3A). To verify these interactions and identify the stably transfected with wild-type PC2 or a PC2 mutant, D511V, interacting region of PC2, we performed immunoprecipitation which lacks Ca2+ channel activity (24). Expression of wild-type − − using tagged proteins. Expression of GFP fusion proteins con- PC2 in Pkd2 / cells reduced forskolin-stimulated cAMP levels, taining the C-terminal and N-terminal domains of PC2, and whereas cAMP levels remained elevated following expression of immunoprecipitation of epitope-tagged AC5/6, revealed that the the PC2-D511V mutant, despite similar levels of protein expres- C terminus of PC2 was required for the interaction with the sion (Fig. 3G). Antibody staining confirmed that the PC2-D511V B AKAP150 complex (Fig. 3 ). Expression of a PC2 mutant mutant, similar to wild-type PC2, is localized in the primary cilium lacking the C-terminal interaction domain stimulated CREB (Fig. S2F). These results indicate that the Ca2+ channel activity of reporter activity, which suggests that the interaction may be PC2 is important for regulating cAMP signaling. important for regulating cAMP signaling (Fig. 3C). Immuno- fluorescence confocal microscopy verified that the mutant pro- HNF-1β–Regulated PDE4C Interacts with the AKAP150 Complex in tein localized to the primary cilium (Fig. S2A). In contrast, an Primary Cilia. In addition to stimulation of cAMP synthesis, the

Choi et al. PNAS | June 28, 2011 | vol. 108 | no. 26 | 10681 Downloaded by guest on September 28, 2021 Fig. 3. Polycystin-2 interacts with AC5/ A C D 250 6 and regulates cAMP levels. (A) Immu- 1 202 658 968 Full length PC2 Myc N TM C 200 noprecipitation of mIMCD3 cell lysates AC 5/6 PC2 Kif3a rabbit IgG

with anti-AC5/6, anti-PC2, or anti-Kif3a PC2 dC-PC2 Myc N TM 150 followed by immunoblot analysis with Kif3a Myc TM C anti-PC2 and anti-Kif3a shows coimmu- dN-PC2 100 noprecipitation of endogenous PC2 and cAMP (pmol/mg) 50 + + Flag-AC6 AC5/6. (B) HEK293 cells were cotrans- B P=0.011 + + Flag-AC5 0 fected with plasmids encoding GFP fu- 0.7 P8 P14 P21 sion proteins containing the C-terminal GFP-PC2-C 0.6 IP : 3.5 P=0.009 180 or N-terminal domains of PC2, Flag- 0.5 α-Flag E tagged AC5 or AC6, and His-tagged 3 160 His-AKAP79 0.4 140 AKAP79/150. Cell lysates were immuno- 2.5 0.3 120 precipitated with anti-Flag antibody Flag 0.2 2 100 and subjected to immunoblot analysis. Total lysate 0.1 1.5 80 (Upper two panels) AC5 and AC6 in- 60

GFP-PC2-C CREB reporterRelative activity 0 1 teract with AKAP79/150 and the C ter- GFP-PC2-N WT-PC2 dC-PC2 dN-PC2 cAMP (pmol/mg) 40 minus of PC2, but not the N terminus. 0.5 20 (Lower two panels) Total amount of a 0 0 F b c -/- Flag-tagged and GFP-tagged proteins. +/- +/- -/- (C) Schematic diagram of PC2 mutants lacking the C terminus (dC-PC2) or the G N terminus (dN-PC2) (Upper). Expression 450 of dC-PC2 in mIMCD3 cells increases 400 350 CREB reporter activity threefold, whereas 300 expression of wild-type PC2 or dN-PC2 d e f 250 has no effect (Lower). Error bars indicate 200

cAMP (pmol/mg) 150 -PKD2 pcDNA SD (n = 3). (D) cAMP levels in kidneys WT D511V-PKD2 fi 100 from kidney-speci c Pkd2 knockout 50 mice (■) are increased eightfold at 0 Forskolin (min) 0 30 180 postnatal day (P) 14 and P21 compared (100μM) with kidneys from wild-type mice (▲). − − − Error bars indicate SD (n = 3). (E) Forskolin-stimulated cAMP levels (closed bars, Right axis) are threefold higher in Pkd2+/ renal epithelial cells than in Pkd2 / − − − cells. Basal cAMP levels (open bars, Left axis) are only slightly increased (n = 3). (F) Pkd2+/ cells (a–c) and Pkd2 / cells (d–f) are costained with antiacetylated − − tubulin (green) and anti-AC5/6 (red). Higher-magnification images (Insets) show that the ciliary localization of AC5/6 is decreased in Pkd2 / cells (e vs. b). (Scale − − bars: 5 μm.) (G) cAMP levels were measured in Pkd2 / cells that stably express wild-type PC2 (●), D511V mutant PC2 (▲), or a control plasmid (■). Expression of wild-type PC2 reduces cAMP levels, whereas cAMP remains elevated in cells expressing the PC2-D511V mutant (Left). Immunoblot analysis confirms that wild- type PC2 and the PC2-D511V mutant are expressed at similar levels (Right).

– increased steady-state levels of cAMP in Kif3aF/ cells could also because no binding was detected on the promoter of another arise from decreased cAMP degradation. Hydrolysis of cyclic PDE4 family member, PDE4A. We identified two consensus nucleotides is mediated by PDEs, a multigene family of enzymes HNF-1β binding sites in the PDE4C promoter located 582 and with distinct subcellular localizations and substrate specificities. 860 bp upstream from the translation start site (Fig. S4 A and B). Antibody staining revealed that a cAMP-specific member of the The sequence located 582 bp upstream was evolutionarily con- PDE family, PDE4C, was localized in the primary cilia of renal served between mouse and human. Luciferase reporter assays epithelial cells (Fig. 4A). To determine whether PDE4C interacts showed that HNF-1β activated the PDE4C promoter (Fig. S4C), with other components of the ciliary AKAP150 complex, coim- and mutations of either binding site or expression of dominant- munoprecipitation was performed. Expression of either Flag- negative mutant HNF-1β inhibited promoter activity (Fig. S4 D tagged PDE4C or Flag-tagged AKAP150 followed by immuno- and E). These findings demonstrate that HNF-1β directly regu- precipitation with anti-Flag antibody resulted in coprecipitation lates PDE4C gene transcription. of endogenous PDE4C, AKAP150, a regulatory subunit of protein kinase A (PKA-RIIα), and AC5/6, which indicated that cAMP Levels Are Elevated in HNF-1β Mutant Cells and Kidneys. Next, PDE4C was a component of the AKAP150 complex (Fig. 4B). we examined whether the HNF-1β–dependent transcription of Because cilia constitute only a very minor portion of the cell, the PDE4C plays a role in the regulation of cAMP signaling. Ex- strength of the coimmunoprecipitation signal suggested that the pression of dominant-negative mutant HNF-1β in renal epithe- protein interactions may also occur outside the primary cilium. lial cells decreased the levels of PDE4C mRNA transcripts and To examine this possibility, we performed coimmunoprecipita- inhibited the expression of PDE4C protein in the primary cilia tion on nonconfluent mIMCD3 cells that lack primary cilia. The (Fig. 4 A and D). Consistent with down-regulation of PDE4C, PC2-AC5/6-AKAP150-PDE4C complex was detected in both cAMP levels and CREB reporter activity were increased in cells confluent mIMCD3 cells and nonconfluent cells. However, the expressing dominant-negative mutant HNF-1β (Fig. 4 E and F). abundance of the complex was higher in confluent cells that The increase in cAMP signaling was not due to changes in the formed primary cilia (Fig. S1I). expression or ciliary localization of AC5/6 and AKAP150 (Fig. We previously identified PDE4C in a genome-wide screen for S4 F–H). To confirm these findings in vivo, cAMP levels were genes that were regulated by the transcription factor HNF-1β in measured in mice following kidney-specific deletion of HNF-1β the kidney (25). This result was of interest because mutations (27). Deletion of HNF-1β produced cystic kidneys that contained of HNF-1β produce kidney cysts in humans and mice (26, 27). higher levels of cAMP compared with wild-type kidneys (Fig. Chromatin immunoprecipitation and DNA microarray analysis 4G). To verify that down-regulation of PDE4C was sufficient to (ChIP-on-chip) identified PDE4C as a potential HNF-1β target increase cAMP levels, we reduced its expression in wild-type gene (Fig. S3). ChIP assays showed that HNF-1β binds to the renal epithelial cells using siRNAs. Knockdown of PDE4C with PDE4C promoter in chromatin from mIMCD3 cells and mouse four different siRNAs increased CREB reporter activity, and the kidney (Fig. 4C). Binding to the PDE4C promoter was specific, magnitude of the increase correlated with the degree of PDE4C

10682 | www.pnas.org/cgi/doi/10.1073/pnas.1016214108 Choi et al. Downloaded by guest on September 28, 2021 A B C PDE4C CDS abc P1 P2

Chr 8 pCMV-Flag PDE4C-Flag pCMV-FlagAKAP150-Flag PDE4C Input IgG HNF1β

d ef AKAP150 mIMCD3 cells IP: α-Flag Mouse Kidney PKA-RIIα mIMCD3 cells AC5/6

P<0.005 0.2 P<0.005 P<0.005 2 80 D 1.4 E 800 F G 0.18 1.8 70 1.2 700 0.16 1.6 60 1 600 0.14 1.4 50 500 0.12 1.2 0.8 0.1 1 40 400 0.6 0.08 0.8 30 cAMP (pmol/ml) cAMP 300 (pmol/ml) cAMP 0.4 0.06 0.6 20 200 0.04 0.4 0.2 10 100 0.02 0.2 Relative CREB reporter activity 0 0 0 0 0 Control Mutant

Normalized fold expression of mRNA Mif (-) Mif (+) 0 30 180 (min) EtOH Mif EtOH Mif forskolin (100 μM)

a c H 0.35 I b 0.3

0.25 PDE4C siRNA 0.2 control 1 2 34 4 0.15 PDE4C d e f 0.1 Actin 0.05

Relative CREB reporter activity 0 Control 1 2 3 4 PDE4C siRNA

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 MEDICAL SCIENCES 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.)

knockdown (Fig. 4H). Next, we examined PDE4C expression These findings support the concept that the primary cilium is − − in Pkd2 / mutant kidney cells. Similar to previous findings in a subcellular cAMP signaling compartment. – − − − Pkd2WS25/ mutant mice (28), we found that Pkd2 / and Pkd2+/ Previous studies have shown that primary cilia on cholangio- renal epithelial cells had comparable levels of PDE4C mRNA cytes contain components of the cAMP signaling machinery, in- transcripts (Fig. S2D). However, ciliary staining of PDE4C was cluding AKAP150; AC4, AC6, and AC8; protein kinase A; and − − not detected in Pkd2 / cells (Fig. 4I), indicating that the ciliary Epac2 (21). Here, we show that a protein complex comprising localization of the AKAP150 complex containing PDE4C is de- AKAP150, AC5/6, and PKA exists in primary cilia in renal epi- pendent on PC2. thelial cells (Fig. S5). Moreover, we identify PC2 and PDE4C as unique components of the ciliary AKAP complex. Under normal Discussion conditions, the ciliary AKAP complex constrains cAMP signaling: fl PDE4C promotes the hydrolysis of cAMP, and PC2, functioning cAMP promotes cyst expansion by stimulating uid secretion 2+ 2+ and increasing cell proliferation (10). Drugs that reduce cAMP as a Ca entry channel, may mediate local accumulation of Ca that inhibits the activity of Ca2+-sensitive AC5 and AC6. PC2 also levels inhibit cyst growth in experimental animals and retard appears to be required for the ciliary localization of the AKAP150 kidney enlargement in humans with ADPKD (2, 8, 29). How- complex. Consistent with the latter finding, the N terminus of PC2 ever, the molecular mechanism that is responsible for the accu- contains a 15-amino acid sequence that mediates trafficking to the mulation of cAMP in PKD is not known. Here, we show that cilium (30). In addition to the primary cilium, the AKAP150 Kif3a deletion of the ciliogenic gene results in the loss of the complex is also located in the cytoplasm where it is also likely to primary cilium and produces increased cAMP levels. Because be important for proper regulation of cAMP signaling. KIF3A may have functions outside the primary cilium, we used Primary cilia have been shown to contain G protein-coupled two additional approaches, pharmacological deciliation and receptors (GPCR) that signal through cAMP, including receptors growth under nonconfluent conditions, to confirm that primary for somatostatin and odorants (31). Recently, the type 2 vaso- cilia are required for the proper regulation of cAMP signaling. pressin receptor (V2R) has been identified in primary cilia on

Choi et al. PNAS | June 28, 2011 | vol. 108 | no. 26 | 10683 Downloaded by guest on September 28, 2021 renal epithelial cells where it colocalizes with AC5/6 (31). Treat- 23, 27). H-2Kb-tsA58 mice expressing temperature-sensitive mutant SV40 ment of isolated cilia with vasopressin in the presence of the large T antigen were obtained from Charles River. All experiments involving phosphodiesterase inhibitor IBMX results in increased cAMP animals were performed under the auspices of the University of Texas production, which highlights the potential importance of ciliary Southwestern Institutional Animal Care and Use Committee. phosphodiesterases. The AKAP150 complex identified in the present study contains PDE4C, which may function to negatively Antibody Staining. Cells were grown on coverslips and fixed with 4% paraformaldehyde in PBS or ice-cold methanol, then permeabilized in PBS con- regulate cAMP signaling that is coupled to GPCRs such as V2R. The identification of an AKAP complex containing PC2 and taining 0.2% Triton X-100. Kidneys were fixed with 4% paraformaldehyde PDE4C in the primary cilium reveals a common mechanism for and then cryosectioned. Antibody staining and immunofluorescence mi- dysregulation of cAMP signaling in cystic kidney diseases arising croscopy were performed as described previously (6). from different gene mutations. Mutations of Kif3a lead to loss of cilia, disinhibition of AC5, and increased cAMP levels, indicating Reporter Gene Assays. Cells were transfected with pCRE-Luc or PDE4C pro- that the ciliary localization of the complex is necessary for the moter-reporter plasmids using Effectene (Qiagen), Lipofectamine 2000 ’ proper regulation of cAMP signaling. Missense mutations of (Invitrogen), or FuGene (Roche) according to the manufacturer s directions. Pkd2 that inhibit the channel activity of PC2 (e.g., D511V) may Cotransfection with pRL-TK (Promega) was used to control for transfection fi μ reduce the local Ca2+ within the cilium and activate Ca2+- ef ciency. After 48 h, the cells were lysed in 500 L of passive lysis buffer μ inhibitable adenylyl cyclases, such as AC5/6. Previous observa- (Promega), freeze-thawed once, and centrifuged. Supernatants (20 L) were − tions that cAMP concentrations are elevated in Pkd2+/ vascular added to 96-well plates, and Photinus and Renilla luciferase activities were 2+ measured using the Dual-Luciferase Reporter Assay System (Promega) smooth muscle cells and in wild-type cells in which [Ca ]i is ’ lowered with verapamil or BAPTA-AM, further support the according to the manufacturer s directions. tight link between Ca2+ and cAMP signaling (32). cAMP Assays. cAMP levels were measured using an enzyme immunoassay Our studies also reveal a unique role of the transcription factor (Assay Designs) according to the manufacturer’s directions. Protein concen- HNF-1β in the regulation of cAMP signaling. HNF-1β plays a tration was determined with the Coomassie Plus Bradford Assay (Pierce). central role in the transcriptional regulation of cystic disease Pkd2 Pkhd1 genes such as and (27, 33). Here, we show that wild- Statistical Analysis. Statistical analysis was performed using two-tailed un- type HNF-1β activates transcription of PDE4C, whereas muta- ’ β PDE4C paired Student t test. For multiple comparisons, ANOVA and Dunnett s post tions of HNF-1 inhibit the expression of and increase hoc test of significance were performed using GraphPad Prism software. cAMP levels. Because HNF-1β also regulates the transcription of fi fi < Pkd2 fi Statistical signi cance was de ned as P 0.05. Additional methods can be (27), down-regulation of PC2 and impaired ciliary traf ck- found in SI Methods. ing of the AKAP complex may also contribute to the elevation in cAMP. Drugs that reduce cAMP levels are currently in clinical ACKNOWLEDGMENTS. We thank Patricia Cobo-Stark, Rajiv Parmar, and trials and may be effective in inhibiting cyst growth in humans Yimei Gong for expert technical assistance and Fangming Lin for assistance with mutations of HNF-1β. with the generation of cell lines. We thank Leo Tsiokas, John Scott, Daniel Silver, and Ron Taussig for providing reagents. We acknowledge support from Methods National Institutes of Health Grants R01DK042921 (to P.I.), R01DK067565 (to P.I.), University of Texas Southwestern O’Brien Kidney Research Core Center Animals. Mice with kidney-specific inactivation of Kif3a, Pkd2, and HNF-1β Grant P30DK079328, the PKD Foundation (A.S. and Z.M.), and the National (Tcf2) were generated by Cre/loxP recombination as described previously (6, Kidney Foundation (A.S.).

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