Altered Trafficking and Epithelial Cell Polarity in Disease

374 Review TRENDS in Cell Biology Vol.12 No.8 August 2002 Altered trafficking and epithelial cell polarity in disease

Mary-Pat Stein, Angela Wandinger-Ness and Tamara Roitbak

Establishment and maintenance of a polarized epithelium relies on the non-polarized expression of normally polarized integration of signaling cascades, acquisition of specialized trafficking circuits molecules. Thus, identifying the signals and and establishment of a unique cytoarchitecture. Defects in any of these molecular machinery required to appropriately processes can adversely affect cell polarity and cause defects in specific organs maintain the polarized expression of newly and systemic disease. Mutations that disrupt the proper transport of individual synthesized, endocytosed or transcytosed proteins, plasma membrane proteins, or inactivate components of the epithelial-specific is essential to expose the potential underlying trafficking machinery, have severe functional consequences. Links between causes of human diseases. renal diseases and defects in trafficking, differentiation or signaling, highlight Specific cytoplasmic signal sequences or the delicate balance between these parameters which, when altered, conformational determinants mediate the transport precipitates a loss of renal function. of newly synthesized molecules from the trans-Golgi to either the basolateral or apical membrane domains The barrier and transport functions provided by (Fig. 1). Basolateral delivery is specified by either epithelia depend on their highly polarized phenotype. tyrosine-based sorting signals (e.g. YXXφ, where φ Polarization of epithelial cells is a complex process denotes a bulky hydrophobic amino acid, or NPXY) directed by external cues such as cell–cell and or dileucine motifs [2]. These sorting signals cell–extracellular matrix (ECM) interactions [1]. promote clustering of the molecules and their These interactions initiate the formation of association with vesicle-associated adaptor proteins specialized junctions that demarcate apical and [3], leading to the inclusion of proteins exiting the basolateral plasma membrane domains. Once Golgi in discrete, basolaterally targeted vesicles. polarity is established, a diverse array of cellular Similarly, apical sorting and retention signals are machinery is used to ensure maintenance of epithelial encoded in protein transmembrane domains, polarity, with specialized intracellular trafficking attached lipid moieties, carbohydrate domains pathways directing the domain-specific targeting of or C-terminal PDZ domains [4,5]. Sorting of newly synthesized, endocytosed and transcytosed apically targeted proteins into detergent-insoluble molecules. The accurate delivery of molecules to their glycosylphosphatidylinositol–cholesterol (DIG) appropriate membrane domains depends on the membrane rafts, mediated either by interaction of differentiation state of the cell. Consequently, defects their glycosylphosphatidylinositol or glycan moieties in trafficking pathways that are used to maintain with DIG components, or by glycan interaction with epithelial polarity or alterations in epithelial putative carbohydrate-specific receptors in the differentiation can cause disease in organs in which trans-Golgi network (TGN) [6–11], facilitates apical epithelial cell polarity is crucial; for example, the targeting. In kidney and intestinal epithelia, both liver, kidney and intestines. DIG-dependent and DIG-independent routes of Here, we focus on diseases resulting from the transport have been documented [11]. Hepatocytes mistargeting of proteins or the loss of polarity, either can use a third route for apical transport, in which of which can lead to the inappropriate expression or many (but perhaps not all) apical plasma membrane loss of molecules from specific cell-surface domains proteins are first delivered to the basolateral with pathological consequences (Table 1). Although membrane and subsequently reach the apical many significant diseases result from point membrane by transcytosis [12,13]. mutations that lead to protein misfolding and Maintenance of epithelial cell polarity not only retention in the endoplasmic reticulum (ER), requires regulated insertion of membrane proteins discussion of diseases that affect de novo biosynthesis into the apical and basolateral domains, but also is beyond the scope of this review. tight control of endocytic recycling and transcytosis Mary-Pat Stein (Fig. 1). Similar to de novo basolateral sorting, Angela Wandinger-Ness* Molecular sorting endocytosis and transcytosis of cell-surface molecules Tamara Roitbak Molecular Trafficking Defects in the ability to sort or transport molecules to depends largely on cytoplasmic sorting cassettes. Laboratory, Dept of their appropriate cellular destinations can render Pathological conditions can arise as a result of the Pathology, University of epithelial cells non-functional with respect to barrier inappropriate removal or insertion of itinerant New Mexico School of and transport functions, thereby causing disease. proteins, stressing the importance of proper endocytic Medicine, Albuquerque, NM 87131, USA. Genetic mutations affecting either sorting signals or and transcytotic transport in the maintenance of *e-mail: [email protected] epithelial trafficking machinery can result in the polarized protein expression.

Table 1. Diseases resulting from altered trafficking or cell polaritya

Classification Disease Defect Refs Sorting signals Sucrase–isomaltase deficiency type IV N- and O-linked glycans [9,10,19–22] Cystic fibrosis CFTR PDZ-binding domains [23–25] Wilson disease (copper toxicity) ATP7B [26,27] Familial hypercholesterolemia LDLR [14–18] Liddle’s syndrome ENaC [30–32] Nephrogenic diabetes insipidus AQP2 [28,29] Trafficking machinery Lowe syndrome Phosphatidylinositol 5-phosphatase [38–41] Tuberous sclerosis Tuberin (Rab5 GAP) and hamartin [46–49] Differentiation ADPKD, ARPKD Dedifferentiation [73–77,84–87]

aAbbreviations: ADPKD, autosomal dominant polycystic kidney disease; AQP2, aquaporin-2; ARPKD, autosomal recessive polycystic kidney disease; ATP7B, a P-type ATPase; CFTR, cystic fibrosis transmembrane conductance regulator; ENaC, amiloride-sensitive epithelial Na+ channel; GAP, GTPase-activating protein; LDLR, low-density lipoprotein receptor.

Sorting signal defects IV results in mispolarization of SI from its normal Familial hypercholesterolemia (FH) is an autosomal intestinal brush-border membrane to the basolateral dominant disorder resulting from the inability of membrane in intestinal epithelial cells [19]. patients to remove low-density lipoprotein (LDL) Recognition of glycosylation and incorporation into from their plasma, leading to elevated levels of serum DIGs enables efficient apical transport of SI and cholesterol bound to LDL, premature atherosclerosis dipeptidyl peptidase IV (DPPIV), another apically and coronary heart disease. Defects in LDL uptake targeted intestinal enzyme. Sorting of SI is dependent are attributed to genetic variants of the LDL receptor on a heavily O-glycosylated stalk region of the protein (LDLR) that affect receptor internalization, and on the transmembrane domain [10]. Inhibition of LDL binding and appropriate targeting of LDLR in O- or N-linked glycosylation results in random hepatocytes [14–16]. The inappropriately targeted distribution of SI and DPPIV, respectively, on both mutant is of interest for this review as it results in the apical and basolateral membranes [9,10]. In addition, loss of LDLR from the basolateral (sinusoidal) surface a P-domain or trefoil motif [20] in SI interacts with and expression on the apical membrane. the O-glycosylated stalk region and provides a spatial LDLR is normally sorted and transported to the determinant for SI sorting and apical targeting [21]. basolateral surface of hepatocytes. Two tyrosine-based A point mutation in the P-domain is also linked to sorting motifs facilitate the basolateral-membrane non-polarized membrane targeting [22]. The targeting of LDLR: a membrane proximal motif importance of post-translational modifications and containing a single tyrosine residue that is also conformational determinants in the apical sorting of crucial for the endocytosis of LDLR; and a more distal SI is clearly apparent and illustrates how disease C-terminal motif containing a crucial G–Y amino acid might result from defects in protein modification. pair [16,17]. A naturally occurring point mutation Proper apical trafficking of the cystic fibrosis identified in the FH–Turku LDLR allele leads to transmembrane conductance regulator (CFTR), substitution of the glycine residue in this motif with a cAMP-activated chloride channel, is dependent on aspartic acid (i.e. D–Y) [18]. This mutation results in two C-terminal domains [5] and a C-terminal the predominant expression of LDLR on the apical PDZ-binding motif [23]. Interactions between the bile canalicular surface, with only low levels of LDLR PDZ domains of CFTR and the Golgi-associated being expressed on the basolateral membrane. The CAL protein (CFTR-associated ligand) promote Golgi importance of proper basolateral targeting of LDLR retention, whereas interactions between CFTR and in hepatic cells is demonstrated by the severe NHE-RF (Na+/H+ exchange regulatory factor) [24] consequences resulting from its apical expression in promote apical plasma membrane trafficking. patients with FH, namely predisposition to heart Although the majority of CFTR mutations result in failure and early death. ER misfolding and premature degradation, Defects in cytoplasmic apical sorting signals are approximately 10% of all CFTR mutations are also linked to human disease. Congenital C-terminal mutants or deletions that result in loss sucrase–isomaltase (SI) deficiency (CSID) is an of apical sorting determinants and mispolarization of autosomal recessive intestinal disorder that afflicts CFTR. Loss of polarized CFTR results in cystic affected individuals with osmotic-fermentive diarrhea, fibrosis, a lethal autosomal recessive disease with cramps and abdominal pain, upon ingestion of di- impaired chloride secretion and significant and oligo-saccharides. Five different CSID phenotypes pathological consequences for lung epithelia resulting from genetic defects in SI have been function [25]. described: types I and II result in misfolding and Wilson disease, an autosomal recessive disease retention of SI in the ER, and the SI in types III and V resulting from defective localization of a P-type is enzymatically inactive or degraded. Only phenotype ATPase (ATP7B) in renal epithelia, is another human http://tcb.trends.com 376 Review TRENDS in Cell Biology Vol.12 No.8 August 2002

mistrafficking of AQP2 from the plasma membrane to Polarized lysosomes have recently been identified [29]. Mutants Exocytosis Endocytosis of AQP2 (E258K, 721delG, 763–772del, 812–818del and 727∆G) heteroligomerize with wild-type AQP2 and redirect wild-type AQP2 to lysosomes. Additional analyses to determine how these mutations trigger altered trafficking to lysosomes could reveal new sorting signals for lysosomal trafficking or might identify novel cellular mechanisms utilized for protein quality control. Basolaterally As documented in this section, defects in either targeted ] vesicles N apical or basolateral sorting signals can have Apically Golgi dramatic functional consequences on polarized ] targeted vesicles sorting in epithelia, resulting in either target N organ-specific or systemic disease.

Defects in endocytosis or retention signals Some sorting signals specify internalization or Apical recycling Apical Tight junction Hemi- Microtubules endosome trafficking retention of molecules at the cell surface. Genetic desmosomes Actin variation in these signals can also result in Polycystin1/2 Rab GTPase Basolateral Basolateral endosome trafficking pathological conditions owing to prolonged or E-cadherin Motor Cytokeratins protein shortened cell-surface expression of receptors. For Extracellular Endocytic Transcytosis Desmosomes matrix vesicle example, Liddle’s syndrome is a hereditary form of Transcytotic vesicle arterial hypertension resulting from inappropriate stabilization of the amiloride-sensitive epithelial TRENDS in Cell Biology Na+ channel (ENaC) on apical membranes. Liddle’s Fig. 1. A unique cytoarchitecture and specialized membrane-trafficking pathways are key syndrome patients demonstrate, among other determinants in epithelial cell polarity. Proper sorting of proteins to unique apical and basolateral symptoms, severe (salt-sensitive) hypertension owing plasma membrane domains is essential to the polarized absorptive and secretory functions of fully to constitutive activation of apical ENaCs and differentiated epithelial cells. The fidelity of polarized transport is ensured by sorting signals that are + decoded by epithelial-specific trafficking machinery in both the exocytic and endocytic pathways. excessive Na reabsorption by kidney epithelia [30]. A unique polarized epithelial cytoarchitecture is established through cell–cell and cell–ECM adhesion ENaCs are composed of three protein subunits and through formation of specialized adhesion complexes, including tight junctions, adherens (α, β and γ), which form a channel that is crucial for junctions, desmosomes and hemidesmosomes. These specialized junctions orchestrate cytoskeletal + organization and constitute a scaffold for polarized transport and integrated signaling that are crucial Na and fluid reabsorption in the lung, colon and for the maintenance of epithelial polarity. Abbreviation: N, nucleus. kidney. ENaCs normally turnover rapidly in Madin–Darby canine kidney (MDCK) epithelial cells disease associated with mislocalization or (1–2 h [31]), suggesting that tight control of ENaC mistargeting of an apically expressed cell-surface activity is required for normal kidney function. A receptor. ATP7B mediates hepatic copper clearance to conserved PY motif in the C-termini of the β and the bile canaliculus. Immunofluorescence studies of γ subunits dramatically affects turnover of ENaCs [31] ATP7B suggest that when copper levels are high, and facilitates the interaction of ENaC with Nedd4, a ATP7B redistributes from the Golgi to intracellular ubiquitin protein ligase. Point or missense mutations vesicles [26] and the plasma membrane [27]. resulting in loss of the PY motif by truncation of the Mislocalization mutants of ATP7B have been C-termini of β- and γ-ENaC subunits have been identified [26], and mutations appear to inhibit either described in cohorts of Liddle’s patients [30,32]. Loss the sorting of ATP7B into apically targeted vesicles or of the PY motif might inhibit ENaC ubiquitination the transport of ATP7B–copper complexes to the bile and endocytosis, thereby resulting in continuous canalicular surface for excretion. A more complete ENaC activation. Liddle’s syndrome illustrates how understanding of the regulated sorting and transport defects in the regulation of temporal expression of of ATP7B will be required to fully appreciate how cell-surface receptors can cause human disease. genetic mutations affect trafficking of ATP7B in Wilson disease patients. Molecular trafficking machinery and polarity Another human disease resulting from improper It is relatively clear how organ-specific disease can trafficking is autosomal dominant nephrogenic arise as a consequence of alterations in the sorting diabetes insipidus (NDI). NDI is characterized by signals of individual plasma membrane proteins. polyuria and polydipsia which, when left unchecked, However, it is also important to consider defects in the cause stunted growth, mental retardation and, trafficking machinery as possible underlying causes ultimately, renal insufficiency [28]. Most NDI cases of altered epithelial cell polarity and disease. Studies result from either X-linked defects in the vasopressin in non-polarized and specialized cell types and in receptor or autosomal recessive defects in aquaporin-2 genetic model systems have already pinpointed (AQP2). However, mutations that result in several human diseases that are caused by

http://tcb.trends.com Review TRENDS in Cell Biology Vol.12 No.8 August 2002 377 alterations in molecular trafficking machinery. For mutations in adaptors with more specialized example, defects in cell-type-specific rab GTPases functions might cause cell-type-specific diseases [42]. (such as Rab27a) and their interacting partners For example, AP-3 adaptor complex defects interfere (myosin V) can influence vesicle trafficking, leading to with specialized lysosome formation and result in secretion defects that affect melanosome, platelet, human bleeding and pigmentation disorders [37,42]. immune cell and neuronal function [33–35]. Similarly, In epithelia, two adaptor proteins have been observed defects in adaptor complexes that are required for to promote basolateral-membrane targeting: µ1B and vesicle coat formation have been associated with AP-4 [3,43]. Absence or depletion of µ1B or AP-4 pigmentation, bleeding and immune disorders results in apical missorting of select basolateral [36,37]. In the following section, we consider how membrane proteins in animal kidney epithelia defects in epithelial-specific components that are [3,43,44], making it of interest to determine whether known to be important in microdomain-mediated human diseases might also be precipitated by µ1B or sorting, vesicle budding, transport and fusion might AP-4 adaptor protein defects. cause disease. Combined evidence from model system Rab GTPases and their interacting partners are studies and disease mechanism analyses indicates important for constitutive vesicle docking and fusion that when mutant, these factors cause defects in in all cells [45]. Defects in these factors are known to polarized transport or recycling. Therefore, defects cause human disease in non-polarized cells [33], and in components of the epithelial-specific transport emerging evidence suggests that their aberrant machinery warrant further consideration as causes function in polarized cells might also cause disease. of human disease. Such deficits could result in In brain and epithelial tissues, the TSC1 and embryonic lethality owing to failed organ development, TSC2 genes encode hamartin, a protein of unknown or cause disease as a result of aberrant trafficking of function, and tuberin, a putative GTPase-activating key cellular proteins. protein (GAP) for Rab5, respectively [46]. Mutations in either gene result in tuberous sclerosis, an Trafficking machinery required for membrane polarity autosomal dominant disorder exhibiting a broad Membrane microdomains spectrum of symptoms, including renal cysts and Transport of newly synthesized proteins from the tumors [47,48]. The cystic phenotype might, in part, TGN to the plasma membrane is initiated by the be explained by the Golgi retention of polycystin-1 incorporation of cargo into membrane microdomains (a protein important for signaling and cell adhesion) within the TGN. The generation of membrane in the absence of proper tuberin function [49]. Defects microdomains and their turnover must be tightly in other epithelial-specific rab GTPases – for example regulated to ensure proper trafficking of proteins. Rab17 (which plays a role in kidney development [50]) Lowe syndrome is an X-linked disorder that results and Rab18 – have been linked to mouse mutations, from mutations in OCRL1 (oculocerebrorenal including leaden, twirler and ataxia [51]. In addition, syndrome of Lowe), a gene encoding an inositol mutations in Rab23 cause an embryonic lethal ‘open polyphosphate 5-phosphatase [38]. Lack of Ocrl1 in brain’ phenotype [52]. Further clarification of the Lowe syndrome patients causes progressive kidney functions of epithelial-specific rab proteins and their failure, blindness and mental retardation. The interacting partners will be crucial for identifying Ocrl1 phosphatase removes the 5′-phosphate group additional causes of kidney disease as well as of other from phosphatidylinositol 4,5-bisphosphate polarized cell disorders.

[PtdIns(4,5)P2]. PtdIns(4,5)P2 5-phosphatases are Targeted delivery of Golgi-derived transport important regulators of vesicular trafficking [39]. vesicles to the basolateral membrane of mammalian Recent work localizing Ocrl1 to the TGN [40], and the cells is mediated by a protein complex termed observation of extracellular release of numerous the ‘exocyst’, first identified in yeast [53]. The lysosomal enzymes by the kidney epithelia of Lowe mammalian exocyst, constituted by Sec6 and Sec8 syndrome patients, suggest that Ocrl1 plays an (commonly denoted Sec6/8), localizes near tight important role in protein trafficking [41]. It will be of junctions where it interfaces with GTPases such as significant interest to establish how PtdIns(4,5)P2 RalA and Cdc42 to coordinate polarized trafficking and other phosphoinositides mediate the sorting and [53–55]. Studies in model systems demonstrate that targeting of proteins from the TGN to the cell surface. mutations in RalA and Cdc42 disrupt repletion of the readily releasable synaptic vesicle pool and epithelial Budding, transport and docking of vesicles basolateral trafficking, respectively [56–58]. Primary Vesicle budding selects proteins for incorporation into autosomal dominant polycystic kidney disease distinct transport vesicles that target proteins to the (ADPKD) is associated with defective basolateral apical or basolateral domains [11]. Protein coats targeting that is apparently caused by depletion of called adaptor complexes (APs) associate with Sec6/8 [59]. Based on model system studies, forming transport vesicles by binding to cytoplasmic components of the mammalian exocyst might also domains on cargo molecules. Animal model studies control the cystogenic and tubulogenic potential of suggest that mutations in the ubiquitous AP-1 and kidney epithelia [53,60]. Epithelial cells transiently AP-2 adaptors are embryonic lethal, whereas lose their polarity during tubulogenesis; components http://tcb.trends.com 378 Review TRENDS in Cell Biology Vol.12 No.8 August 2002

that are crucial for vesicle fusion, such as SNARE and These results suggest that IFs are not only important exocyst components, are removed from the cell for epithelial architecture but might also play a role in membrane and stored in intracellular vacuoles [61]. apical membrane trafficking through interactions No known defects in epithelial SNARE proteins have with components of the membrane trafficking been identified and a Sec8 knockout mouse was machinery such as syntaxin 3. embryonic lethal, suggesting that these trafficking components are essential for establishment and Differentiation and polarity: the polycystic kidney maintenance of epithelial polarity. Nevertheless, as disease paradigm intimated by the results in ADPKD cells, loss of Epithelial cells establish polarity early in proper localization of the Sec6/8 complex or SNARE organogenesis in response to cell–cell and cell–ECM proteins, through altered signaling or second-site cues. Ligation of adhesion receptors, including mutations, should be considered as possible causes homotypic binding of E-cadherin molecules and of human disease. integrin binding to ECM, signals cells to establish The recent completion of the human genome specialized intercellular junctions, alter their gene sequence and the identification of numerous new expression programs, rearrange their cytoarchitecture trafficking components present new opportunities to and establish specialized membrane-transport analyze the correlation of these components with circuits leading to and from newly formed apical and genetic loci implicated in human diseases [42,62]. basolateral plasma membrane domains [1]. Once Such analyses have the potential to unveil the polarity is achieved, maintenance continues to underlying causes of as-yet-uncharacterized depend on the interplay between signals regulating diseases affecting epithelial target organs; the epithelial cell differentiation, the ability of cells to Online Mendelian Inheritance in Man website maintain their unique cytoarchitecture, and the is a powerful resource in this regard (http://www.ncbi. efficiency of sorting and transport of proteins and nlm.nih.gov:80/entrez/query.fcgi?db=OMIM). lipids to the cell membrane (Fig. 2). Changes in any one of these features might shift epithelial polarity Cytoskeleton components required for trafficking and and differentiation (along a continuum) between a polarity fully differentiated epithelial phenotype and a Actin non-polarized fibroblast-like phenotype, and can The actin cytoskeleton plays an important role in the result in human disease. Consideration of the establishment and maintenance of epithelial cell mechanisms underlying loss of polarity in cystic polarity [1,63,64]. Intestinal microvilli rely heavily on kidney diseases, which encompass changes in actin for proper membrane organization. Enterocytes signaling, differentiation or membrane trafficking, from patients with microvilli inclusion disease (MVI) is instructive in illustrating this point (Fig. 2). display decreased levels of actin, myosin and vinculin, ADPKD is a prevalent genetic disorder affecting and loss of microvilli [65,66]. The loss of apical 1 in 800 individuals. It is characterized by the formation microvilli is paralleled by an increase in subapical of large, fluid-filled cysts in the kidney and, in some microvilli inclusions that contain numerous apically cases, in the liver. The continuous accumulation of targeted proteins [67]. Similarities between the cysts over time leads to loss of renal function, kidney subapical microvilli inclusions and the vacuolar failure and death. Aberrant cell-surface polarity of apical compartment (VAC) induced in subconfluent or select membrane proteins including epidermal calcium-starved MDCK cells [61,68] suggest that growth factor receptor [72], Na+–K+ ATPase [73], α β defects in apical trafficking result in vesicular integrin receptor 5 1 and desmosomal proteins intermediates halted before apical membrane (A. Charron and A. Wandinger-Ness, unpublished), as exocytosis. MVI could, therefore, represent a disease well as aberrant secretion of lysosomal enzymes and associated with loss of epithelial cell polarity that is growth factors, are hallmarks of ADPKD cells [73]. caused by alterations in cytoskeletal architecture, These observations led to early speculation that and that consequently affects polarized trafficking. ADPKD might be precipitated by a defect in polarized sorting [73]. Identification of the gene mutated in the Intermediate filaments (IFs) majority (85%) of ADPKD cases has focused much It was previously assumed that IFs, formed from attention on its protein product: polycystin-1, a type I and type II keratin, were only involved in 462-kDa membrane-spanning protein suggested to mechanical functions required for cell integrity. function in signaling and adhesion and, in concert However, defects in the cytokeratin CK8 have been with polycystin-2, to effect calcium transport [74]. associated with a predisposition to liver cirrhosis and There is now a growing consensus that altered cryptogenic liver disease [69], highlighting the polycystin-1 signaling and/or complex formation additional importance of IFs for apical membrane precipitates return to a more immature, integrity [70]. Interestingly, villus enterocytes and dedifferentiated phenotype, possibly through hepatocytes from CK8 knockout mice lacked IFs and activation of fetal gene transcription [75–77]. displayed a loss of syntaxin 3, alkaline phosphatase, The localization of polycystin-1 to adherens SI and CFTR from the apical domain of these cells [71]. junctions and focal adhesions in normal kidney,

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cell-adhesion molecule E-cadherin in primary (a) Differentiation Signaling ADPKD cells, and loss of stable protein complex (CellÐcell and (Polycystins, cAMP, cell-ECM signals) Wnt, catenins) formation [77,80]. The consequent downregulation of E-cadherin is accompanied by an increase in the normally mesenchymal N-cadherin, in both primary ADPKD cells and cysts in situ. These data, among others, imply a close cooperation between polycystin-1 and Wnt signaling cascades Cellular Trafficking architecture (Rab GTPases, [81,82]. Because polycystin-1, together with (Actin, microtubule, motor proteins, polycystin-2, is implicated in the control of intermediate filament, SNARES, Sec6/8) + junction complexes) Ca2 currents and numerous other intracellular signaling cascades, it is probable that further (b) ARPKD fibrocystin study will reveal the hierarchy and contributions Differentiation Cilium of integrative signaling to the establishment and Polaris Cytoarchitecture maintenance of renal epithelial polarity [83,84]. Cystin Polycystin L Tuberous sclerosis, another autosomal dominant Ca2+ flux disease resulting in cysts and tumors of the kidney, ADPKD can be caused by defects in the GAP for Rab5– tuberin. Signaling T.J. (-) 2+ rab13 Tuberin Ca flux Interestingly, the lack of tuberin results in a (Ð) AE Hamartin Polycystin1/2 E-cadherin defect in the basolateral membrane expression of Sec6/8 Tuberous sclerosis polycystin-1, and its intracellular retention in Trafficking (+) Microtubules the Golgi complex [49]. Therefore, it appears that BE Desmosomes either altered signaling or localization of polycystin-1, as a consequence of altered trafficking, can N Golgi precipitate loss of kidney epithelial polarity and (+) (+) function (Fig. 2b). N Autosomal recessive polycystic kidney disease (+) ECM (+) (ARPKD) afflicts 1 in 6000 individuals with massive renal cystic disease during embryogenesis, and is Differentiation state accompanied by some of the same defects in cell ARPKDTSC ADPKD polarity that are associated with ADPKD [85]. The Undifferentiated Fully differentiated (fibroblast-like) (epithelial) fetal manifestation of ARPKD suggests a defect in

TRENDS in Cell Biology epithelial differentiation and establishment of polarity. Mutant fibrocystin, a large membrane Fig. 2. Pleiotropic causes of altered cell polarity. (a) Defects in polarity can arise by point mutations protein of unknown function [86], causes severe that disrupt sorting signals in individual plasma membrane proteins or by functional inactivation of human ARPKD, resulting in up to 30% mortality select transport machinery components (e.g. coat proteins, rab GTPases, SNAREs, sec6/8), which in infants [87]. Two mouse models of ARPKD, alter the sorting of whole classes of proteins. More global changes in cellular differentiation, signaling or cytoarchitecture can also result in deficits in membrane protein polarity, with CPK and Tg737 (orpk) mice, both have defects dramatic consequences for target organ function. This diagram shows the interplay between the in proteins that are required for cilia formation mechanisms required for epithelial polarity. (b) A series of unlinked diseases, all of which result in [88–90]. Cilia play important roles in sensory cystic kidney and, to some extent, liver disease, illustrate the importance of integrated signaling, differentiation and trafficking in the control of polarity. In animal models, ARPKD results from perception, fluid movement and embryonic mutations affecting proteins required for cilia formation (e.g. cystin, polaris), the absence of patterning. Disruption of polaris (encoded by which is thought to cause defects in epithelial differentiation and severe fetal cystic disease. TG737), a protein associated with cystic kidney ADPKD results from mutations affecting basolaterally localized polycystin-1 and polycystin-2. disease, and cystin (encoded by CPK), a hydrophilic Polycystin-1 is speculated to integrate numerous intracellular signaling cascades, possibly by controlling calcium flux in conjunction with polycystin-2. The cohort of ‘mispolarized’ proteins protein localized with polaris in renal epithelia, in ADPKD parallels those proteins normally observed to exhibit an altered polarity during might abrogate cilia development [88,90,91]. development or reestablishment of polarity, implying that altered polycystin-1 signaling causes Although it remains to be determined whether ADPKD cells to assume a dedifferentiated phenotype with altered polarity. Tuberous sclerosis is caused by mutations in two interacting proteins, one of which is a Rab5 GAP (tuberin) that is required and how cilia influence the functional differentiation for the transport of polycystin-1 to the plasma membrane. In the emerging picture, epithelial of polarized epithelia, the phenotype of Tg737 differentiation and polarity are dictated by a delicate balance between cytoarchitecture, signaling and and CPK mice suggest that epithelial cilia trafficking. Abbreviations: AE, apical endosome; BE, basolateral endosome; ADPKD, autosomal dysfunction has dramatic pathological consequences dominant polycystic kidney disease; ARPKD, autosomal recessive polycystic kidney disease; ECM, extracellular matrix; GAP, GTPase-activating protein; N, nucleus; T.J., tight junction; that lead to polycystic kidney disease. Similar to TSC, tuberous sclerosis. differentiation signals generated by ligation of E-cadherin and integrin receptors, cilia might and its inclusion in protein complexes constituting signal epithelial differentiation by sensing the these cell–cell and cell–matrix adhesions, suggest a apical environment. Loss of these signals, because of central role for polycystin-1 in orchestrating renal defects in cilia formation, might hinder complete epithelial polarity [78,79] (Fig. 2b). Mutant differentiation of epithelia, resulting in ARPKD. polycystin-1 expression is associated with Thus, as depicted in Fig. 2b, loss of renal epithelial decreased cell-surface localization of the epithelial cell polarity can be precipitated by genetic defects

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that impinge on the fidelity of polarized trafficking, Concluding remarks proper signaling or expression of a fully differentiated The generation and maintenance of epithelial polarity Acknowledgements phenotype. Some of these changes are attributed to are complex processes that involve cell signaling, Studies on ADPKD, matrix the altered localization or function of polycystin-1. changes in cell architecture and a diversity of microfibrils and endocytic Multiple polycystins, such as polycystin L, which intracellular trafficking pathways. Here, we have membrane traffic are supported by NIDDK R01 have an apical distribution and cation channel summarized current knowledge about human diseases 50141, PKD Foundation activity but, as yet, no disease etiology, might also be that result from genetic defects in the signals and 12A2R, NSF MCB9982161, involved [92]. It is anticipated that further work on molecular machinery necessary for epithelial polarity and AHA 0040211N to A.W.N. T.R. is supported the mechanistic origins of these diseases, as well as on and differentiation. In addition, we have described how by an NKF fellowship the less well-characterized medullary cystic kidney the balance between cell signaling, differentiation and F758. We thank Robert [93] and polycystic liver [94] diseases, will offer trafficking must be maintained for proper epithelial Bacallao, Keith Mostov pivotal insights into the hierarchical cascades cell function. Future challenges entail identifying how and Pedro Salas for critical reading of the governing the establishment and maintenance of a individual pathways are hierarchically integrated and manuscript. fully polarized epithelium. held in balance in a fully polarized epithelium.

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