Wnt Signaling in Polycystic Kidney Disease

Thomas Benzing,* Matias Simons,† and Gerd Walz* *Renal Division, University Hospital Freiburg, Freiburg, Germany; and †Department of Molecular Cell and Developmental Biology, Mount Sinai School of Medicine, New York, New York

Wnt signaling cascades activate morphogenetic programs that range from cell migration and proliferation to cell fate determination and stem cell renewal. These pathways enable cells to translate environmental cues into the complex cellular programs that are needed to organize tissues and build organs. Wnt signaling is essential for renal development; however, the specific molecular underpinnings involved are poorly understood. Recent research has revealed an unexpected intersection between Wnt signaling and polycystic kidney disease. Some polycystic kidney disease proteins, such as Inversin and Bardet-Biedl syndrome family members, were found to use components of the Wnt signaling cascade to orient cells along a secondary polarity axis within the plane of the epithelium. These spatial cues may be needed to position nascent tubules with a defined geometry. J Am Soc Nephrol 18: 1389–1398, 2007. doi: 10.1681/ASN.2006121355

nts comprise a large family of secreted glycopro- olism and endocytosis. The extracellular domain of LRP5 and teins (reviewed in Cadigan and Liu [1]) that coor- LRP6 consists mainly of four YWTD ␤-propeller domains, each W dinate the development of conjoining cells and flanked by EGF-like repeats (reviewed in He et al. [5]). Mice that thereby control the patterning of organs in vertebrates. Wnts lack both LRP5 and LRP6 receptors fail to form a primitive may play a regenerative role in adult cells, because Wnts seem streak and its derivatives, a phenotype similar to that of Wnt3- to control stem cell fate in several tissues (2,3). Misregulated deficient animals (6). The cytoplasmic domain of either LRP5 or Wnts signaling contributes to human disease such as limb LRP6 targeted to the plasma membrane constitutively activates malformation, bone abnormalities, and cancer (reviewed in ␤-catenin–dependent Wnt signaling, confirming a role of Logan and Nusse [4]). Numerous genetic and biochemical stud- LRP5/6 in Wnt signaling (7–10). Because the association be- ies have delineated the multiple pathways by which Wnt mol- tween Fz and LRP stimulates ␤-catenin signaling (11,12), Wnt ecules and their downstream effectors exert these diverse func- molecules may co-recruit Fz and LRP to initiate ␤-catenin– tions. This review provides an overview of the key molecules dependent signaling. The association of Fz and LRP results in that are involved in Wnt signaling, describes the role of Wnt dual phosphorylation of LRP6 by glycogen synthase kinase-3␤ signaling in renal development, and discusses how defects in and casein kinase 1 (CK1) (13) and the subsequent recruitment Wnt signaling may underlie polycystic kidney disease (PKD). of Axin and Dsh to the intracellular domains of LRP6 and Fz, respectively. Both LRP–Axin interaction and Dsh phosphoryla- ␤-Catenin–Dependent (canonical) Wnt tion stabilize cytosolic ␤-catenin, which translocates to the nu- Signaling cleus and complexes with transcription factors of the LEF/TCF Canonical Wnt signaling requires Frizzled (Fz) and LDL- family to activate gene expression. related protein (LRP) co-receptors (Figure 1). Direct binding of Wnt to a cysteine-rich domain has been shown for several Fz ␤ receptors, including Drosophila Fz1/Fz2 and mouse Fz8 (re- -Catenin–Independent (Noncanonical) Wnt viewed in Cadigan and Liu [1]). Fz induces phosphorylation of Signaling ␤ Dishevelled (Dsh), which inhibits the APC–Axin–glycogen The noncanonical Wnt signaling pathway is -catenin inde- synthase kinase 3 complex from phosphorylating ␤-catenin, pendent but shares several components (e.g., Fz, Dsh) with the allowing it to escape proteasomal degradation. LRPs are evo- canonical pathway. The noncanonical pathway has been di- lutionarily conserved single-pass transmembrane proteins that vided into several distinct branches, including the planar cell mediate various biologic functions, such as cholesterol metab- polarity (PCP) and the Wnt/calcium pathway (reviewed in Veeman et al. [14]). This review focuses on the PCP pathway that has been best studied in flies but is increasingly recognized

Published online ahead of print. Publication date available at www.jasn.org. to play a critical role in vertebrate organogenesis (reviewed in references 15–17). There are three classes of PCP signaling Address correspondence to: Dr. Gerd Walz, Renal Division, University Hospital Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany. Phone: ϩ49-761-270- molecules: The upstream Fat/Dachsous (Ds) PCP proteins, the 3250; Fax: ϩ49-761-270-3245; E-mail: [email protected] PCP core proteins, and the downstream PCP effectors (Figure

Figure 1. Canonical Wnt signaling. Canonical Wnt signaling induces the stabilization of ␤-catenin. In the absence of Wnt signaling, ␤-catenin forms a complex with Axin and APC, is phosphorylated by glycogen synthase kinase 3␤ (GSK-3␤) and casein kinase 1 (CK1), and is targeted for proteasomal degradation by the ubiquitin-ligase transductin repeat-containing protein (␤-TrCP). Wnt molecules seem to trigger the association of Frizzled (Fz), a seven-membrane–spanning receptor with LDL-related protein 5 (LRP5) or LRP6. This complex formation results in recruitment and phosphorylation of Dishevelled (Dsh), which physically interacts with the C-terminal domain of Fz. Wnt-induced phosphorylation of the C-terminal domain of LRP receptors by GSK-3␤ and CK1 recruits Axin to the plasma membrane, preventing the degradation of ␤-catenin; the microtubule actin cross-linking factor 1 (MACF1) is required for translocation of Axin to LRP5/6 (75). Together with transcription factors of the LEF/TCF family, ␤-catenin activates target genes of the canonical Wnt signaling pathway. Catenins, which can also be released from cadherin-based cell adhesions, seem to integrate extracellular cues to control morphogenesis and tissue homeostasis (76,77).

2). Ds is expressed in a gradient in the Drosophila eye and wing, example, mechanosensory cells display a tissue polarization whereas Fat shows a graded activity and is expressed uni- from the inner to the outer edge. In this manner, Vangl2 (the formly in most tissues. These two proteins have therefore been mouse Stbm ortholog) localizes to the inner cell cortex of sen- suggested to provide a long-range “upstream” signal for the sory cells, and Dsh localizes to the stereocilia-forming outer cell core PCP proteins (18). By an unknown mechanism, the signal cortex (19,20). This scenario resembles the localization patterns triggers the asymmetric distribution of PCP core proteins that in Drosophila cells. In contrast, mFz3 and mFz6 co-localize with include Fz, Dsh, Flamingo/Starry Night (Fmi, Stan), Strabis- Vangl2, which differs from the findings in Drosophila. In addi- mus/Van Gogh (Stbm/Vang), Prickle (Pk), and Diego (Dgo). tion, there is no evidence that PCP signaling requires Wnt in At least in the Drosophila wing, this means that Fz, Dsh, and Drosophila, whereas several reports demonstrate that Wnt mol- Dgo move to one side of the cell (distal), whereas Pk and Stbm ecules initiate PCP signaling in C. elegans, Xenopus, and ze- accumulate at the proximal plasma membrane. Fmi is found on brafish. Vertebrates are endowed with numerous Fz and Wnt both the anterior and the posterior plasma membrane, engag- isoforms (at least 10 Fz and 19 Wnt) suggesting redundancy ing in homophilic interactions. PCP effectors such as Inturned and, perhaps, more flexibility. In fact, recent findings suggest (In), Fuzzy (Fy), and RhoA then reorganize the cytoskeleton to that the receptor context can determine the Wnt signaling cas- ensure proper cell morphogenesis. In some instances (e.g.,in cade. For example, Wnt5a can inhibit ␤-catenin–dependent the Drosophila eye), the morphogenetic processes also involve PCP-dependent gene expression mediated by the c-Jun N-ter- signaling through activation of the orphan tyrosine kinase Ror2 minal kinase (JNK) family of transcription factors. The resulting but activates canonical Wnt signaling in the presence of Fz4 effects on tissue architecture can vary, ranging from orientation (21). The localization of Fz to different membrane domains of wing hairs to ommatidial rotation to regulation of mitotic represents another level of regulation of Wnt pathway speci- spindle orientation in the fly. In vertebrates, analogous path- ficity. In the Drosophila wing, Fz is primarily localized to the ways regulate many aspects of development, including conver- apical domain, where it activates the PCP pathway. Fz2, in gent extension and neural tube closure, inner ear development, turn, is localized basolaterally and signals only in the canonical hair orientation in mammals, and the formation of cilia (15). It pathway. It is interesting that overexpression of Fz leads to has to be pointed out, however, that there are differences be- sequestering of Dsh to the apical domain, inhibiting the canon- tween vertebrate and invertebrate PCP. In the inner ear, for ical pathway (22). This example nicely illustrates the intercon- J Am Soc Nephrol 18: 1389–1398, 2007 Wnt Signaling in PKD 1391

Figure 2. The planar cell polarity (PCP) signaling pathway. In Drosophila, the “upstream” PCP proteins Fat (Ft) and Dachsous (Ds), two proto-cadherins, engage in heterophilic interactions and initiate the asymmetric subcellular distribution of the PCP core proteins Fz, Dsh, Strabismus/Van Gogh (Stbm), and Prickle (Pk). The atypical, seven-pass transmembrane cadherin Flamingo/ Starry Night (Fmi) forms homomers and can be found at the anterior and posterior plasma membrane. In contrast, the Fz/Dsh accumulates at the posterior plasma membrane, where it interacts with the ankyrin-repeat protein Diego (Dgo). The four-pass transmembrane protein Stbm interacts with Pk and is found in the anterior plasma membrane. Inhibition of the Fz/Dsh complex by Stbm/Pk may reinforce the asymmetric subcellular distribution of the Fz/Dsh/Dgo complex, which can trigger the effector cascades Daam1/Rho/ROK to initiate cytoskeletal changes or transcriptional changes via the Cdc42/Rac/c-Jun N-terminal kinase pathway. Asymmetric localization of Fz may also be maintained by polarized transport of Fz along microtubules that have their plus ends preferentially at the posterior cell cortex (57). The type 2 transmembrane Golgi protein Four-Jointed (Fj) has been proposed to regulate Ds (see references [14,15] for further details). Most functional and genetic interactions, established in the Drosophila wing or eye, are likely modified in mammalian cells and may vary between tissues. nection of both Wnt pathways and shows that activity of one triggers the outgrowth of the UB. GDNF activates at least three pathway might suppress the activity of the other. signaling pathways that are essential for UB branching: The Ras/extracellular signal–regulated kinase, phosphatidylinosi- Renal Development tol 3-kinase/AKT, and the PLC-␥ pathway. The subsequent Canonical Wnt signaling induces transcriptional changes that branching of the UB is contingent on several transcription direct proliferation, stem cell renewal, and establishment of factors (Hox11, WT1, Emx-2, and Sall1) that act at least in part apical-basolateral cell polarity. Noncanonical Wnt signaling through regulation of GDNF expression. A comprehensive re- regulates a broad array of morphogenetic cell and tissue func- view by Costantini (25) summarized the current knowledge of tions, including convergent extension and directed cell divi- the cellular mechanisms that drive branching morphogenesis of sion. Although it remains unclear how Wnt-directed signaling the UB. During mouse embryogenesis, the UB rapidly branches programs specify cell lineage, patterning, and growth of tissue in the MM during embryonic day 11.5 (E11.5) to 15.5, followed that compose kidney embryogenesis, genetic evidence clearly by a slower phase of branching shortly before birth. From E15.5 establishes a role for Wnt molecules in renal development. The until birth, the UB extensively elongates, giving rise to the long mammalian kidney originates from mesodermal tissue that lies unbranched collecting ducts of the outer medulla (26). A similar ventral to the paraxial mesoderm and dorsal to intermediate elongation process flanked by (rapid) UB branching has been mesoderm (reviewed by Perantoni and by Dressler [23,24]). The reported for the development of the human kidney. Branching intermediate mesoderm forms the pronephric duct, an epithe- UB cells extend the lumen of the UB while maintaining their lial tube that extends caudally until it reaches the cloaca. polarity (27). Localized cell proliferation seems to transform the Shortly before it reaches the cloaca, the pronephric duct (pri- rounded UB ampulla to a branched structure, perhaps sup- mary nephric duct) develops an outgrowth, the ureteric bud ported by migration of epithelial cells toward the forming (UB), which invades the surrounding metanephric mesen- branch poles. Several growth factors are known to regulate chyme (MM). The basic principle of renal development is the growth and branching. FGF-stimulated cell migration was re- reciprocal interaction between two tissues, the UB and the MM. cently described for the branching morphogenesis of the Dro- The transcription factors Pax-2, Pax-8, and Lim-1 control ex- sophila air sac (28). GDNF stimulates chemotaxis of cultured pression of the GDNF receptors c-ret/Gfr␣1, allowing the pro- tubular epithelial cells in vitro; however, redirecting GDNF nephric duct to respond to mesenchymal GDNF. GDNF, regu- expression from the mesenchyme to the UB is sufficient to lated by a complex network of positive and negative stimuli promote nearly normal kidney development, arguing against a (e.g., fibroblast growth factor-7 [FGF-7], FGF-10, retinoid acid), significant role of GDNF in chemoattraction and UB guidance 1392 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1389–1398, 2007

(29). After the MM condenses over the tip of the UB, a small epithelial nephrons (37). Because Wnts can activate JNK in number of mesenchymal cells convert to epithelial cells to form renal tissues (38), PCP-dependent JNK activation may be nec- a vesicle adjacent to the UB, the precursor of the proximal essary to maintain the integrity of the developing nephron. nephron segment. The conversion of the mesenchymal cells involves the formation of adherens and tight junctions. A crucial event is the recruitment of the Par6–Par3–atypical protein Wnt Signaling in PKD kinase C (aPKC; PAR) complex to sites of cell–cell contacts and Efforts to uncover the pathogenesis of PKD have provided the formation of tight junctions, which establishes the basolat- novel insight into the cellular mechanisms that are required for eral and apical domain of a cell (reviewed in Etienne-Mannev- normal renal embryogenesis. Ectopic activation of the Wnt ille and Hall [30]). The genetic programs that drive these events signaling cascade is associated with PKD. For example, target- are poorly understood, but it is clear that signaling cascades ing of constitutively active ␤-catenin to the kidney of transgenic such as the Wnt pathway provide both permissive and instruc- mice results in severe polycystic disease that affects all seg- tive information. ments of the nephron (39). A similar phenotype ensues when the degradation of ␤-catenin is prevented by deletion of APC Wnt Signaling in Renal Development (40). Abnormal ␤-catenin levels have also been reported in The first demonstration that Wnt are involved in mesenchy- KIF3A-deficient tubular epithelial cells, suggesting that mislo- mal-to-epithelial transition came from Wnt4-deficient mice. calization of the polycystin complex and/or the absence of cilia These mice experience renal agenesis because they cannot un- results in dysregulation of ␤-catenin–dependent signaling (41). dergo mesenchymal-to-epithelial transition (31). Later it was Whereas canonical Wnt signaling seems mandatory for early shown that another Wnt, Wnt9b, functions even earlier than renal development, persistent ␤-catenin signaling seems to trig- Wnt4. Wnt9b is expressed in the UB; it is essential for the early ger cyst formation at later developmental stages. Inversin, a inductive response of the MM, and causes condensation of protein that is mutated in nephronophthisis type II (reviewed mesenchymal cells (32). Wnt4 is not required for the conden- by Hildebrandt and Otto [42]), seems to facilitate a switch from sation of mesenchymal cells, but is essential for their subse- canonical to noncanonical Wnt signaling (43,44). Inversin tar- quent conversion to a polarized epithelium. It is thought that gets cytoplasmic Dsh for ubiquitin-dependent degradation and these Wnt act via the canonical Wnt pathways. This is sup- blocks canonical Wnt signaling. However, membrane-associ- ported by the fact that lithium, which inhibits glycogen syn- ated Dsh is spared, and both Inversin and Dsh co-localize to the thase kinase-3␤ and activates canonical Wnt signals, promotes plasma membrane of polarized tubular epithelial cells. Inversin condensation of the MM (33). In addition, Wnt1, a Wnt mole- is required for C/E movements during Xenopus gastrulation, cule primarily implicated in the canonical Wnt pathway, res- suggesting that Inversin facilitates noncanonical Wnt signaling cues Wnt9b deficiency, supporting the role of canonical Wnt by interacting with membrane-associated Dsh. Containing N- signaling during early MM induction. With the use of a re- terminal ankyrin repeats, Inversin shares its domain architec- porter mouse that drives ␤-catenin/TCF-dependent ␤-gal ex- ture with Diversin, the mammalian homologue of Dgo. Diver- pression (34), extensive ␤-catenin–dependent signaling is also sin blocks canonical but facilitates noncanonical Wnt signaling detectable in the branching UB tips but declines toward the (45,46), and both Diversin and Dgo interact with Pk and Stbm, more distal nephron segments. Wnt11 is expressed at the UB providing evidence for a PCP multiprotein complex (15,43,46). tips and helps to maintain GDNF levels and normal branching Knockdown of Inversin in zebrafish causes pronephric cysts. morphogenesis but has no effect on the induction of the MM. This phenotype is rescued by Diversin (43), further supporting Although other Wnt (Wnt7b and Wnt6) are expressed in the a role for noncanonical PCP signaling during normal renal UB, none extends into the ureteric tips. Wnt11 (silberblick) development. Additional evidence for this hypothesis comes triggers convergence/extension movements during zebrafish from the genetic interaction between Bardet-Biedl syndrome gastrulation (35) and is required for PCP signaling during (BBS) proteins and the PCP core protein Vangl2. BBS is a rare Xenopus cardiogenesis (36). These findings suggest that Wnt11 pleiotropic disorder that is characterized by multiple symp- initiates noncanonical Wnt signaling during UB branching, per- toms, including obesity, mental retardation, retinal degenera- haps involving conversion/extension (C/E) movements to tion, and cystic kidneys (reviewed in Blacque and Leroux [47]). elongate the collecting ducts during mid-embryogenesis. C/E BBS1, BBS4, and BBS6 (Ϫ/Ϫ) mice display abnormalities that movements that extend the body axis during vertebrate gastru- are consistent with abnormal PCP signaling, including incom- lation involve oriented cell division, cell migration, changes of plete neural tube closure and disrupted cochlear sterociliary cell shape, and intercalation of cells (17). The spindle alignment bundles; these phenotypic changes are enhanced by Vangl2 is randomized in Wnt11-mutant zebrafish, supporting a role of mutations (48). Although BBS members belong to a diverse Wnt-mediated PCP signaling in oriented cell division. It is group of proteins, they localize to the cilium/basal body com- interesting that mice with a hypomorphic Wnt9b allele develop plex (CBC) and seem to participate in microtubule-dependent cysts (16), suggesting that Wnt9b either plays a dual role in intracellular trafficking. BBS4 interacts with p150glued, a sub- canonical and noncanonical Wnt signaling or provides permis- unit of dynactin that links dynactin with the dynein motor sive signals to allow normal PCP signaling. Combined deletion protein, and mutations of all BBS proteins seem to affect the of JNK isotypes decreases the expression of Pax2, Shh, and dynein-driven, microtubular transport (49). Although it has bone morphogenic protein-4 and results in deformed renal been hypothesized that BBS proteins target PCP proteins to the J Am Soc Nephrol 18: 1389–1398, 2007 Wnt Signaling in PKD 1393

CBC, it remains unknown why this subcellular localization of Communication of Extracellular Cues to the PCP proteins is needed to execute PCP programs (47,48). Centrosome The cilium originates from one of the two centrioles that form the centrosome and microtubule-organizing center Oriented Cell Division during Renal (MTOC) in most cells. In tubular epithelial cells, the mother Development centriole moves to the apical membrane of polarized cells, During proliferation of tubular epithelial cells and elongation transforms into the basal body, and nucleates the microtu- of the tubular segments, the dividing cells precisely align along bular skeleton of the cilium. How cilia are inserted into a the anterior-posterior axis of the growing nephron (50). This predefined region of the apical membrane is not well under- exact alignment is partially lost in animal models of PKD, and stood. Because the orientation of the mitotic spindle requires cell divisions are randomly oriented. Oriented cell division is cues from cadherin- and integrin-based signaling, it is tempt- one of the outputs of PCP signaling and mandatory to organize ing to speculate that similar cues position the basal body. tissue in the plane of an epithelial cell layer (51,52). Random- Recent findings demonstrate how focal adhesion proteins ized cell divisions in PKD suggest that proteins that are mu- may communicate with the centrosome (58). HEF1, a mul- Cas tated in PKD contribute to the orientation of the dividing axis. tidomain scaffold protein that shares with p130 an amino- Because most PKD proteins localize to the CBC, it seems obvi- terminal SH3 domain, localizes to integrin-based focal adhe- ous to assume that the CBC plays a crucial role in oriented cell sions, where it interacts with focal adhesion kinase (Figure division. However, most Drosophila cells lack cilia, and deletion 3). During the G2/M phase of the cell cycle, however, HEF1 of the centrioles does not impede development of the fruit fly. translocates to the centrosome, where it activates Aurora A, Centriole-deficient flies reach maturity and succumb to defects a protein kinase that promotes activation of cyclinB/Cdk1, in mechanosensation as a result of a lack of cilia on sensory and transitions through G2. Another protein that shuttles neurons (53,54), suggesting that centrioles, at least in the fly, between focal adhesions and the centrosome is the GRK- orient the microtubular cytoskeleton that is required for cilio- interactor 1–associated kinase p21-activated kinase, which genesis but are not essential to deliver other microtubule-de- binds and phosphorylates Aurora A. It is interesting that pendent cargo molecules to their subcellular destinations. nephrocystin, a protein that is mutated in nephronophthisis These findings also emphasize the importance of positional type I, shares with HEF1 an amino-terminal SH3 domain and cues from cell–cell and cell–matrix contacts such as the adher- interacts with several focal adhesion components, including p130Cas, tensin, and Pyk2 (59). Nephrocystin contains two ens junction and focal adhesions. In fact, recent data suggest phosphorylatable acidic clusters that flank the SH3 domain. that the orientation of cell division in mammalian cells is dom- Interaction of the amino-terminal acidic cluster with the inated by cell adhesion and traction forces that are applied connecter protein phosphaturin acidic cluster sorting protein during interphase (reviewed in Thery and Bornens [55]). In 1 seems to recruit nephrocystin to the CBC (60), thereby cultured cells, adhesive properties during interphase seem to providing another link between focal adhesions and the encode the information that controls the orientation of subse- centrosome. It is interesting that nephrocystin also interacts quent cell divisions. Similarly, the site of neurite formation is with Inversin (61). Although, it has not been tested whether determined by the orientation of the spindle poles and there- nephrocystin affects the function of Inversin in PCP signal- fore is specified before the mitosis of the mother neuron. Be- ing, it is tempting to speculate that the Inversin/nephrocys- cause inhibition of actin polymerization inhibits neurite out- tin complex could link PCP signaling to focal adhesions and growth, reorganization of the actin cytoskeleton seems to organization of the actin cytoskeleton. precede the organization of the microtubular cytoskeleton. The sequential specification of the actin cytoskeleton followed by the orientation of microtubules is supported by the function of Intersection between Apico-Basal and Planar the PCP effectors In and Fy. Deletion of either In or Fy causes Cell Polarity defects in ciliogenesis in Xenopus embryos in combination with At this point, there is no unifying theme in ciliogenesis. PCP and Hedgehog signaling abnormalities (56), linking both One problem but perhaps also an advantage is that cilia are pathways to ciliogenesis. On the subcellular level, the defect studied in many tissues and species; the underlying mecha- seems to be a disordered apical cytoskeleton that fails to align nisms of cilia formation likely vary significantly between the ciliary microtubules. Although the CBC complex may pro- various organisms. Most of what is known about ciliogenesis vide spatial cues to control and/or stabilize the localization of stems from studies in Chlamydomonas, a unicellular organism PCP proteins, signaling upstream of the core PCP proteins (62). However, assembly and maintenance of cilia in multi- involving the actin cytoskeleton seems to control ciliogenesis. cellular organisms may require additional cues. For example, Because the polarized transport of Fz requires microtubular kidney epithelial cells do not form cilia until they are em- arrays along the apical plane of Drosophila wing epithelium bedded in a confluent monolayer and fully polarized (Figure (57), it will be interesting to see how the “upstream” PCP 4). The evolutionary conserved PAR complex, converts ini- proteins instruct In and Fy to control microtubule-dependent tial polarity cues into the establishment of apical and baso- ciliogenesis without affecting the microtubular transport of lateral membrane domain (reviewed in Suzuki and Ohno core PCP proteins. [63]). The PAR complex consists of three essential compo- 1394 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1389–1398, 2007

Figure 3. Signaling from the cell matrix. In interphase, the nephronophthisis proteins nephrocystin (NPHP1) and Inversin (nephrocystin-2) can be localized at the plasma membrane (43,78,79). Inversin localizes to the spindle poles during ana-/ metaphase (80) that coincides with translocation of HEF1 to the centrosome (81). Whether NPHP1 displays similar dynamics is unknown. It is interesting that polycystin-2, an ion channel of the transient receptor potential (TRP) family that is mutated in patients with autosomal dominant polycystic kidney disease, interacts with mammalian Diaphanous-related formin 1 protein (mDia1), which mediates association of polycystin-2 with the mitotic spindle during cell division (82). Although neither mechanisms nor functional consequences have been elucidated, it is tempting to speculate that the shuttling of proteins from the cell cortex to the centrosome communicates the information needed to position dividing cells correctly.

nents: Par6, one of three Ras-related Rho-GTPases that con- fore, it has been postulated that the PCP signaling cassette trol microtubule–cortex interactions and coordinate the can provide such a fine tuning of the ciliary complex (56). For growth of cortical microtubules (64,65); Par3, a PDZ domain– example, Inversin mutations do not disrupt ciliogenesis but containing scaffold protein that interacts with the antero- rather affect the normal tilting of the cilia on the ventral grade intraflagellar transport motor protein kinesin-2, (66); node, a structure that establishes normal left–right asymme- and aPKC. Because the future basal body has to travel to the try during mouse development (68). Recently, a molecular apical plasma membrane (Figure 4), it is perhaps not sur- link between the PAR complex and PCP signaling was es- prising that the PAR polarity complex plays a crucial role in tablished by demonstrating that apical Fz can be phosphor- the ciliogenesis of MDCK cells (66). Recent data demonstrate ylated and regulated by aPKC (69). Fz, in turn, triggers that the tumor suppressor gene product pVHL binds Par6 phosphorylation of duboraya, a mediator of noncanonical and controls the direction of microtubule formation (67). Wnt signaling that regulates ciliogenesis and left–right pat- Because VHL mutations lead to defective ciliogenesis and terning in zebrafish (70). However, PCP components also premalignant kidney cysts, it is conceivable that pVHL me- affect the PAR complex and apico-basolateral polarity. For diates the PAR-dependent contributions to ciliogenesis. The example, Dsh together with the tumor suppressor product of signal for cilia formation after polarization of epithelial cell lethal giant larvae, a negative regulator of the PAR complex, layers remains unclear. Conceptually, the PAR complex are required for normal apical-basal polarity in the Xenopus needs to establish an apical membrane before the mother ectoderm (71). It is intriguing that overexpression of Par3/ centriole can find its correct location to form the basal body Bazooka, a protein that is essential for the apico-basolateral and nucleate the cilium. Hence, any interference with the polarity of the Drosophila embryo, causes PCP defects of the specification of the apical membrane should result in defec- Drosophila wing without affecting the apico-basolateral po- tive ciliogenesis. However, the precise positioning of the larity (72). These findings suggest that the PAR proteins basal body, for example, and the angle of the cilium relative display tissue- and development-specific functions. In cili- to the apical membrane may require additional cues. There- ated epithelia, the PAR proteins might therefore interact J Am Soc Nephrol 18: 1389–1398, 2007 Wnt Signaling in PKD 1395

Figure 4. Relationship between centrosome and cellular polarity of tubular epithelial cells. (A) The centrosome, consisting of the mother and daughter centrioles, migrates to the apical membrane (83). (B) Cadherin-mediated cell–cell contacts reorganize the actin cytoskeleton. Although the centrosome (microtubule-organizing center [MTOC]) organizes the microtubular cytoskeleton in migrating cells (e.g., fibroblasts), the centrosome organizes few microtubules of tubular epithelial cells during interphase. Most microtubules are ordered in either longitudinal arrays along the apical-basal axis of the cell with their plus ends toward the apical membrane or in a network parallel to the apical membrane (84). (C) The cadherin-based adherens junctions (AJ) facilitate recruitment of the Par6–Par3–atypical protein kinase C (PAR) complex. The PAR complex initiates apical-basolateral polarization and recruits the CRB complex (Drosophila: Crumbs) to the apical membrane (tight junction [TJ]). Pals1 (Drosophila: Discs Lost [Dlt]), a membrane-associated guanylate kinase (MAGUK)/PDZ domain–containing scaffold protein, binds to the carboxy-terminal CRB and to the PDZ domain of Par6. The basolateral membrane is controlled by a complex that consists of Scribble, a leucine-rich PDZ protein, the MAGUK protein mDlg (Drosophila: Discs Large [Dlg]), and the WD40-repeat protein mLgl (Drosophila: Lethal Giant Larvae [Lgl]). The CRB/PAR complex excludes the Lgl/Dgl/Scribble complex from the apical membrane, whereas the Lgl/Dgl/ Scribble complex prevents the accumulation of the PAR complex at the basolateral membrane (85,86). Dsh is required for Lgl-mediated specification of the basolateral membrane and establishment of apical-basolateral polarity in Xenopus (71). Whereas an actomyosin-based cortical process is necessary to localize the PAR complex to tight junctions, Par3 can also interact with KIF3A. This interaction couples the PAR complex to kinesin II–powered, microtubular transport processes that are required for ciliogenesis (66) as well as for axon specification in developing hippocampal neurons (87,88). 1396 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1389–1398, 2007 with components of the PCP pathway to regulate coopera- proteins 5 and 6 in Wnt/beta-catenin signaling: Arrows tively cell polarity, ciliogenesis, and ciliary function. point the way. Development 131: 1663–1677, 2004 6. Kelly OG, Pinson KI, Skarnes WC: The Wnt co-receptors Conclusions Lrp5 and Lrp6 are essential for gastrulation in mice. Devel- opment 131: 2803–2815, 2004 Although the cilium seems to serve as platform for several 7. Tamai K, Zeng X, Liu C, Zhang X, Harada Y, Chang Z, He signaling cascades such as the Hedgehog pathway (reviewed X: A mechanism for Wnt coreceptor activation. Mol Cell 13: in Zariwala et al. and Badano et al. [73,74]), it is unclear which 149–156, 2004 components of the Wnt signaling cascades require an intact 8. Mao B, Wu W, Davidson G, Marhold J, Li M, Mechler BM, cilium. Clearly, in Drosophila, most cells lack a cilium; there- Delius H, Hoppe D, Stannek P, Walter C, Glinka A, Niehrs fore, canonical and noncanonical Wnt signaling during Dro- C: Kremen proteins are Dickkopf receptors that regulate sophila development have to function independent of this Wnt/beta-catenin signalling. Nature 417: 664–667, 2002 organelle. Ciliary bending can augment Inversin expression, 9. Mao B, Wu W, Li Y, Hoppe D, Stannek P, Glinka A, Niehrs suggesting that flow-induced signaling modulates Wnt sig- C: LDL-receptor-related protein 6 is a receptor for Dick- naling. It is interesting that the synchronized elongation of kopf proteins. Nature 411: 321–325, 2001 collecting ducts at approximately E15.5 coincides with the 10. 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