Chapter
Introduction
1
General introduction
Autosomal Dominant Polycystic Kidney Disease, ADPKD, is a common inherited disorder that aff ects the kidneys. Progressive development of renal cysts ultimately results in chronic renal failure. To develop therapies for ADPKD patients, further insight into the mechanism of cyst formation is required. We set out to investigate the molecular and cellular processes that are disrupted in ADPKD. In the next section, normal function of the kidney and renal epithelium is discussed. This is followed by an overview of the literature available on the structural and functional properties of polycystin-1 and polycystin-2, the proteins responsible for ADPKD.
Finally, our data on polycystin-1 and polycystin-2 function and ADPKD cyst formation is outlined and discussed in the remainder of the thesis.
The kidneys
The kidneys are highly specialized organs that primarily function to Þ lter the blood to maintain its proper composition and osmolarity (Figure 1). Our total blood volume is Þ ltered through the kidneys every 45 minutes (1). The kidneys are therefore very metabolically active and consume approximately 20-25% of all oxygen used by the body at rest. The smallest functional unit of the kidney is the nephron (Figure 1, right panel). The nephron is composed of diff erent segments each with their own speciÞ c function. The glomerulus Þ lters the primary urine from the blood. This primary urine is then processed via reabsorption and
secretion during its transport through the proximal tubule, Henle’s loop, the distal tubule, and the collecting duct. Virtually all organic nutrients, such as glucose and amino acids, are reabsorbed. Reabsorption of water and ions is regulated according to requirement. Thanks to this reabsorption process a relatively small amount of urine is ultimately produced and transported to the bladder where it can leave the body.
Structure and function of the renal epithelium
The diff erent segments in the nephron, the proximal and distal tubules, and collecting ducts, are lined by specialized cells that can be discriminated by their morphology and physiology, optimized for their function (Figure 2; 1). Proximal tubule epithelial cells are cubic with numerous microvilli at the apical membrane.
This so-called brush border dramatically increases the surface of the proximal tubule cells thereby facilitating the reabsorption and secretion process. The majority of all the water, nutrients, and ions, is reabsorbed by the proximal tubule epithelial cells. Since this is an active process, proximal tubule cells are also the most metabolically active cells, containing enlarged and increased amounts of mitochondria to generate the necessary energy. Numerous vesicles are present in the cytoplasm giving the cells a “foamy” appearance. During the passage through Henle’s loop, water and solutes are exchanged via an osmotic gradient. The
squamous epithelial cells of the thin segments of Henle’s loop are freely permeable to water. These cells are replaced by cuboidal cells with almost no microvilli where Henle’s loop interchanges into the distal tubule. Distal tubule cells secrete solutes rather than reabsorb them. Therefore, distal tubule and also collecting ducts are relatively impermeable to water. Hormones regulate reaborption in these nephron segments. Collecting duct cells become more permeable to water due
Figure 2. Schematic representation of the diff erent segments of the nephron (left panel). Immunohistochemical images of the glomerulus, the proximal tubules, Henle’s loop, distal tubules, and collecting ducts are depicted of a normal hu- man kidney. Cell nuclei are stained with hematoxylin (blue) and the extra-cellular matrix is visualized using Sirius Red (pink/red). The right panel shows immunohistochemical images of the proximal and distal tubules, and collecting ducts, with their average tubule diameter and a schematic representation of the specialized tubule lining epithelial cells in each segment. Average tubule diameter data from ref. 1. Nephron image adapted from www.nephrohus.org.
Figure 1. Schematic image of the kidney anatomy in the human body (left panel) and cross-section of a normal kidney (middle panel) with the smallest functional unit of the kidney, the nephron, depicted in the right panel. The diff erent segments of the nephron are indicated. Kidney anatomy from www.encyclopedia.farlex.com and from www.nucleusinc.
com. Nephron image (right panel) adapted from www.nephrohus.org.
to translocation of aquaporin channels to the apical membrane under control of Anti-Diuretic Hormone (ADH) or Arginine Vasopressin (AVP). Reabsorption of Na+ (and accompanying water and K+ secretion) is regulated by aldosterone that is released from the adrenal cortex via the Renin-Angiotensin System (RAS). ATP- dependent active transport of Ca2+ is mediated by Para-Thyroid Hormone (PTH).
The cuboidal to columnal cells of the collecting duct can be divided into two types, the principal cells and the intercalated cells. Principal cells, most abundant and devoid of microvilli, serve to maintain water and Na+ balance. Intercalated cells do show some microvilli and maintain acid-base balance.
Cell adhesion components in renal epithelium
To facilitate the reabsorption and secretion process, it is essential that renal epithelial cells are able to maintain a gradient between the apical (tubule lumen) and basal membrane. This makes selective water and ion transport possible across the epithelial cell. The gradient is achieved by the formation of cell-cell adhesion complexes (Figure 3). Tight junctions form a barrier between the apical and basal side of the epithelium (2). Tight junctions are composed of speciÞ c transmembrane proteins, including claudin and occludin, and interact with the actin cytoskeleton via intermediates such as ZO-1. Through these intermediates interaction with intra-cellular signaling pathways is also facilitated.
Adherens junctions mediate cell-cell adhesion and are composed of cadherins, of which E-cadherin is the most well known. E-cadherin is a transmembrane glycoprotein that forms stable homophilic interactions thereby establishing cell- cell adhesion. The complex is tightly linked to the actin cytoskeletion via α- and β-catenin. Since β-catenin is also a crucial component of the Wnt/TCF signaling pathway, adhesion and signaling are coupled through β-catenin.
Desmosomes mediate cell-cell adhesion via desmosomal cadherins, such as desmocollin and desmoglein. Desmosomes are connected to the keratin Þ laments of the cystoskeleton via plakoglobin and desmoplakin.
Integrins are cell surface receptors that can be regulated by their extra-cellular ligands. As heterodimers, they mediate cell-matrix interactions at focal contacts.
These focal contacts are linked to the actin cytoskeleton. Integrins connecting to the intermediate Þ laments of the cytoskeleton are called hemi-desmosomes.
Components of cell adhesion complexes can regulate signal transduction pathways and thereby establish a link between the (extra-)cellular signal and the intra- cellular transduction pathways that are subsequently activated to generate a cellular response.
ADPKD cyst formation
Proper function of the renal epithelium is thus essential for renal function. Patients suff ering from renal failure, require renal transplantation or renal dialysis, when donor kidneys are not available. One of the major causes of renal failure is the formation of ß uid-Þ lled cysts due to ADPKD. In ADPKD, cysts are formed by
defective functioning renal epithelial cells (Figure 4). Normal renal epithelial cells are tightly regulated to ensure proper function of the kidney. Due to an as yet unknown mechanism, a subset of epithelial cells undergoes a signiÞ cant change that enables them to escape the regulatory mechanisms. These cells expand and grow out to form an isolated cyst. Cyst enlargement then occurs via ß uid accumulation in the cyst lumen due to mislocalization of ion channels.
Progressive development of cysts and accompanying Þ brosis of the surrounding
Figure 3. Schematic representation of the major cell adhesion complexes in renal epithelial cells: the tight junction, the adherens junctions, desmosomes, focal adhesion complexes, and hemi-desmosomes. Image based on ref. 2.
Figure 4. Schematic representation of cyst formation in ADPKD. Since ADPKD is an autosomal inherited disorder, all cells lining the tubule have one mutated PKD1 or PKD2 allele and one unaff ected allele. During life, an additional trig- ger, whether this is somatic inactivation of the second allele or another mechanism, occurs in a small subset of cells (left panel). These cells then no longer fall under the strict regulation which normally control renal tubule epithelial cells and start to grow out, ultimately forming an isolated cyst (middle panels). Cyst enlargement then occurs via ß uid-accumula- tion in the cyst lumen due to mislocalization of ß uid transporter channels (right panel).
tissue disrupts renal function and ultimately results in chronic renal failure in 50% of patients by the age of 50-60 years (Figure 5A). Although cyst formation in ADPKD is considered the major threat, the disease manifests itself with other clinical manifestations as well, such as liver cysts, pancreatic cysts, cardiovascular abnormalities, and cerebral aneurysms.
In addition to the autosomal dominant inherited disorder, there is also an autosomal recessive variant called Autosomal Recessive Polycystic Kidney Disease, ARPKD (Figure 5B). ARPKD is much less frequent than ADPKD and is characterized by severe and early onset polycystic kidney disease. This thesis focussed on the frequently occurring variant, ADPKD. In the next section, genetic inheritance of ADPKD is discussed, followed by an overview on the structural and functional properties of the gene products responsible for ADPKD.
Genetics of ADPKD
To date, two genes have been identiÞ ed which cause ADPKD: the Polycystic Kidney Disease 1 (PKD1) and the Polycystic Kidney Disease 2 (PKD2) gene (3-6;
Figure 5B). Mutation analysis shows that the majority of all ADPKD cases results from a mutation in the PKD1 gene (~85%) with the minority caused by a mutation in the PKD2 gene (~15%; 7). The PKD1 gene is located on chromosome 16 and contains 46 exons spanning 52 kb of genomic DNA that is transcribed into a 14.1 kb messenger RNA transcript. A large part of the PKD1 gene is reiterated on the same chromosome. These homologous DNA fragments encode for several transcripts that are speculated to be pseudo-genes. However, litt le data have been obtained about their precise function. These repeated regions are not conserved among lower mammals, like mice.
The PKD2 gene localizes to chromosome 4 and contains 15 exons spanning 68 kb of genomic DNA (5;6;8). The gene encodes a 5.4 kb mRNA transcript that is translated into polycystin-2.
Structural properties of polycystin-1
As stated previously, the PKD1 gene encodes for polycystin-1. Sequential analysis of polycystin-1 using UniProt (Universal Protein Resource; www.uniprot.org) reveals it is a large transmembrane protein with an extra-cellular N-terminus and intra- cellular C-terminus that contains several domains involved in cell-cell and cell- matrix interactions (Figure 6; 4). The leucin-rich repeats (LRR) ß anked by cystein- rich repeats are implicated in protein-protein interactions, whereas the S. cerevisiae cell-wall integrity and stress-response component 1 homology domain, WSC, is involved in carbohydrate binding. The C-type lectin and Low Density Lipoprotein receptor class A (LDL) domains are involved in Ca2+, sugar and lipid binding.
Along the N-terminal region various PKD domains characteristic for the polycystin protein family can be identiÞ ed. The PKD/REJ domain is homologous to sea urchin Receptor for egg jelly (suREJ), a sperm glycoprotein that is involved in fusion of
Figure 5. A) Normal (left ) and polycystic kidneys (middle and right panel). The polycystic kidneys have been dissected due to end-stage renal failure. Numerous ß uid-Þ lled cysts are visible. The right panel shows a cross-section of a polycystic kidney. Extensive Þ brosis can be seen as yellowish tissue surrounding the cysts. Normal and polycystic kidney image from www.pkdcure.org. B) Schematic representation of the subdivision between autosomal dominant and autosomal re- cessive polycystic kidney disease (ADPKD and ARPKD) with the genes mutated and the encoded proteins depicted.
Figure 6. Predicted protein structure of polycystin-1 and polycystin-2.
Figure 7. Schematic representation of canocial Wnt/β-catenin signaling. In the absence of Wnt, β-catenin is only found in E-cadherin based adhesion junctions (left panel). Cytoplasmic free β-catenin is phosphorylated by a complex containing GSK3β, APC, and axin. Phosphorylated β-catenin is then rapidly degraded via the ubiquitin pathway. Upon stimula- tion by Wnt, the GSK3β/APC/axin complex is inhibited and cytoplasmic free β-catenin is stabilized and subsequently translocated to the nucleus (right panel). Binding of β-catenin to the TCF/LEF (T-cell Factor/Lymphoid Enhancing Factor) family of transcription factors then results in transactivation of speciÞ c target genes.
Figure 8. Schematic representation of the Activator Protein-1 (AP-1) transcription factor activation. AP-1 transcription factors are composed of multiple homo- or heterodimers. The heterogeneity of AP-1 is thought to provide a mechanism to regulate a broad spectrum of cellular responses, since AP-1 can be activated by a variety of stimuli, such as growth factors and genotoxic stress. These stimuli aff ect intracellular signaling pathways resulting in activation of one or more AP-1 components including Jun, ATF, and Fos family members.
the male sperm to the egg during fertilization. The G-protein coupled receptor proteolytic site (GPS) encodes a putative site for proteolytic cleavage of polycystin- 1. The Polycystin/Lipoxygenase/α-Toxin (PLAT) or Lipoxygenase homology (LH2) domain is typical for membrane or lipid associated proteins and encodes a β-sandwich 3D-structure. The predicted intra-cellular C-terminus of polycystin-1 contains a coiled-coil domain that is implicated in protein-protein interactions. The polycystin interaction domain reported by Qian et al. (9) and by Tsiokas et al. (10) is also depicted in Figure 6. Based on the protein structure, it has been postulated that polycystin-1 may function as a receptor that translates extracellular signals into an intracellular response. However, a potential ligand has yet to be identiÞ ed.
Structural properties of polycystin-2
Sequential analysis of polycystin-2 using UniProt reveals it is a transmembrane protein with homology to polycystin-1. The numerous PKD domains scatt ered along the protein are characteristic for both proteins (PKD domains in polycystin-2 not depicted in Figure 6). Unlike polycystin-1, polycystin-2 also has high homology to the family of Transient Recepter Potential ion channels (TRP; Figure 3; 8). Both N- and C-termini of polycystin-2 are predicted to localize intra-cellularly. The C- terminal region contains an EF hand domain that is implicated in Ca2+ binding and a coiled-coil domain thought to be involved in protein-protein interactions. The polycystin interaction domain reported by Qian et al. (9) and by Tsiokas et al. (10), via which polycystin-2 can form homodimers or heterodimers with polycystin-1, is also depicted.
Sub-cellular localization of polycystin-1
Polycystin-1 is ubiqitously expressed with highest expression during development.
In adult kidney, polycystin-1 is expressed in distal tubules and collecting duct (11-14). Sub-cellular localization studies have shown that polycystin-1 localizes in the plasma membrane of renal epithelial cells at sites of cell adhesion complexes such as adhesion junctions and desmosomes (15-19). Furthermore, in primary cells from ADPKD patients polycystin-1 and the integral adhesion junction component, E-cadherin, were depleted from the plasma membrane as a result of the increased phosphorylation of polycystin-1 (18). Loss of E-cadherin from these adhesion junctions was compensated by upregulation of N-cadherin. Disruption of these adhesion complexes did not have marked eff ects on β-catenin association.
Silberberg et al. recently reported that desmosomes were mislocalized in ADPKD cells and tissue (19). These data indicate that polycystin-1 plays a major role in cell adhesion, since it is essential for proper composition and localization of adhesion junctions and desmosomes.
Regulation of polycystin-1 plasma membrane localization and stability
Plasma membrane localization of polycystin-1 has been reported to depend on tuberin, the protein defect in tuberous sclerosis2, TSC2, an inherited disorder that also manifests with polycystic kidney disease (20). Interestingly, polycystin-1 stability can be regulated via interaction with the human homologue of Drosophila Seven in Absentia, Siah-1 (21). Siah-1 contains a RING domain and promotes degradation of polycystin-1 via the ubiquitin-dependent proteasome pathway.
Stability of polycystin-1 has also been reported to depend on polycystin-2 and vice versa (10).
The role of polycystin-1 in cellular signaling
Besides the role of polycystin-1 in cell adhesion, several reports have been published on polycystin-1 function in cellular signalling pathways. Mostly,
signaling pathways modulated by polycystin-1 have been identiÞ ed using reporter constructs. In this way, the Wnt/β-catenin signaling pathway was initially identiÞ ed as a potential target of polycystin-1 (22). In addition, polycystin-1 has also been shown to activate several transcription factors, including Activator Protein-1 (AP- 1), calcineurin/nuclear factor of activated T-cells (NFAT), and Janus kinase-signal transducers and activators of transcription (JAK-STAT; 23-27). These data are outlined in the following sections.
Polycystin-1 and Wnt/β-catenin signaling
Overexpression of the C-terminal region of polycystin-1 in human embryonic kidney 293T, HEK293T, cells has been shown to activate a reporter construct containing the promoter region of the Siamois gene, a known target gene of Wnt/β-catenin signaling (22). The Wnt/β-catenin signaling pathway is involved in cell proliferation, diff erentiation, polarity, migration, and survival (reviewed in 28). β-catenin is a crucial component. Upon stimulation by Wnt, cytoplasmic free β-catenin is stabilized and subsequently translocated to the nucleus (Figure 7). Binding of β-catenin to the TCF/LEF (T-cell Factor/Lymphoid Enhancing Factor) family of transcription factors then results in transactivation of speciÞ c target genes. Wnt/β-catenin signaling is an intriguing pathway for ADPKD, since polycystin-1 has been reported to be located at adherens junctions and may therefore modulate β-catenin activity similar to the way E-cadherin can modulate Wnt/β-catenin signaling: β-catenin can be released from E-cadherin based adhesion junctions and thus contribute to the cytoplasmic free pool for Wnt/β-catenin signaling. However, no cytoplasmic accumulation or nuclear translocation of β- catenin, a major hallmark of Wnt activation, was detected upon expression of the C-terminal polycystin-1 construct (26; this thesis). Moreover, β-catenin was not up- regulated or detected in the nucleus, in renal cystic tissue from ADPKD patients, indicating that Wnt/β-catenin signaling is not relevant for ADPKD.
Polycystin-1 activates Activator Protein-1
The C-terminal region of polycystin-1 has also been reported to activate the Activator Protein-1 (AP-1) transcription factors (23,24,26; this thesis). AP-1 transcription factors are composed of multiple homo- or heterodimers that regulate key cellular processes such as cell proliferation, diff erentiation, and survival (29-31). The heterogeneity of AP-1 is thought to provide a mechanism to regulate a broad spectrum of cellular responses, since AP-1 can be activated by a variety of stimuli, such as growth factors and genotoxic stress (Figure 8). These stimuli aff ect intracellular signaling pathways resulting in activation of one or more AP-1 components including Jun, ATF, and Fos family members. Both the activity and expression levels of these transcription factors can be regulated by Mitogen Activated Protein Kinases (MAPKs), including Extra-cellular signal- Regulated Kinase (ERK), p38, and c-Jun N-terminal kinase (JNK; Figure 9; 32).
Phosphorylation of c-Jun at Ser73 results in increased transactivation potential and stability. Transactivation capacity of ATF2 is regulated via phosphorylation at Thr71 and Thr69 (33). Interestingly, active phsophorylated ATF2 has been shown to bind to the promoter region of both PKD1 and PKD2 using chromatin immuno-precipation (34). Therefore, PKD1 and PKD2 are also target genes of AP-1, suggesting the presence of a feedback loop.
Figure 9. Schematic representation of mitogen-activated protein kinase, MAPK, signaling, one of the major regulators of AP-1 activity. MAPKs can be regulated by a variety of stimuli, such as growth factors and and gentoxic stress, resulting in activation of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs, including Extra-cellular signal-Regulated Kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Depending on the MAPKs that are activated, diff erent transcription factors are modulated and thus diff erent cellular responses achieved. Image adapted from www.
cellsignal.com.
Polycystin-1 activates Calcineurin/NFAT
Calcineurin is a ubiquitous Ser/Thr-phosphatase that is activated upon sustained increases in intracellular Ca2+. Polycystin-1 can activate calcineurin dependent dephosphorylation and nuclear translocation of the transcription factor NFAT (nuclear factor of activated T-cells) through Gα(q) -mediated activation of phospholipase C (PLC; 27). Calcineurin/NFAT transactivation regulates diff erentiation and apoptosis in various cell types (35). Interestingly, NFAT can regulate transcription by interaction with AP-1 transcription factors. Moreover, calcineurin/NFAT has been reported to crosstalk with Wnt/Ca2+ signalling during embryonic development (36). Therefore, NFAT is at the intersection of several signaling pathways, further linking polycystin-1 function to signaling.
Polycystin-1 activates JAK-STAT and is involved in cell cycle regulation
Polycystin-1 can not only modulate AP-1 and NFAT transactivation, but overexpression of a full-length polycystin-1 construct has also been reported to transactivate Janus kinase-signal transducers and activators of transcription (JAK- STAT) with concomitt ant upregulation of p21(waf1) and cell cycle arrest in G0/G1 (25). Activation of JAK-STAT requires both polycystin-1 and polycystin-2. Mouse embryos lacking Pkd1 have defective STAT1 phosphorylation and p21(waf1) induction. These data, together with data from Bolett a et al. (37) demonstrating that polycystin-1 overexpression inhibits cell growth, indicate that polycystin- 1 functions in both signaling and cell cycle regulation. Indeed, depletion of
polycystin-1 has been reported to increase cell proliferation. Kim et al. (38) showed this was caused by premature G1/S-phase transition, whereas Nishio et al. (39) reported that the increased cell proliferation correlated with JNK activation.
Polycystin-1 is proteolytically cleaved
Qian et al. demonstrated that polycystin-1 undergoes cleavage at the G-protein coupled receptor proteolytic site (GPS; 40). Proteolytic cleavage of polycystin-1 requires the REJ domain. PKD1-associated mutations in the REJ domain disrupt cleavage and eliminate the ability of polycystin-1 to induce AP-1 activity. In addition, Chauvet et al. reported that polycystin-1 undergoes proteolytic cleavage releasing its C-terminal tail (41). The C-terminal tail then translocates to the nucleus and modulates expression of target genes. Cleavage of the C-terminal tail occurs in vivo under regulation of mechanical stimuli. Furthermore, signaling activity of the C-terminal tail is modulated by polycystin-2. Recently, it has been reported that the C-terminal tail of polycystin-1 interacts with the Na+,K+-ATPase α-subunit, thereby modulating Na+,K+-ATPase mediated renal tubular ß uid and electrolyte transport (42).
The primary cilium
Polycystin-1 may not only transfer extra-cellular signals into the cells via cell adhesion based complexes and proteolytic cleavage, but also via an organelle called the primary cilium. The primary cilium is an appendage protruding from the plasma membrane and can be found in a wide variety of cell types including renal epithelial cells (Figure 10). The cilium is formed at the plasma membrane where the centrosome is localized when the cell is not dividing. The centrosome acts as a docking site, the so-called basal body, where ciliary proteins are
transported into the ciliary body, called the axoneme. The structural integrity of the ciliary axoneme is maintained by several cytoskeletal proteins, including specially modiÞ ed, acetylated α-tubulin. The microtubule Þ laments form a characteristic 9+0 composition (Figure 10, right panel). Along this cytoskeletal axis specialized proteins, including intra-ß agellar transport (IFT), kinesins, and dyneins, facilitate transportation of ciliary proteins from the basal body into the axoneme. The precise function of the primary cilium has not yet been identiÞ ed, but data suggest that the cilium acts as a mechano-sensor that responds to ß uid ß ow passing through the renal tubule lumen.
Figure 10. Schematic representation of the primary cilium. The structural integrity of the ciliary axoneme is maintained by several cytoskeletal proteins, including specially modiÞ ed acetylated α-tubulin. These microtubule Þ laments form a characteristic 9+0 composition (right panel; image taken from www.133.100.213.46/photo/T-Cilium). Along this cytoskeletal axis specialized proteins, including intra-ß agellar transport (IFT), kinesins, and dyneins, facilitate transport of ciliary proteins from the basal body into the axoneme.
Polycystin-1 function in the primary cilium
IdentiÞ cation of polycystin-1 as a structural component of the primary cilium has had considerable implication for the functional properties assigned to polycystin-1.
In vitro experiments showed that mechanical stimulation of the primary cilium in renal epithelial cells resulted in a Ca2+ inß ux (43). This Ca2+ inß ux was hypothesized to be caused by polycystin-1 in its function as a Ca2+ permeable ion channel or by polycystin-1 mediated activation of polycystin-2 channel function (44,45). Thus, mechano-stimulation of the primary cilium gives rise to a Ca2+ increase in the cell.
The mechano-sensory function of the primary cilium may be crucial for adequate function of the tubule and possibly regulate tubule lumen size in vivo. This is an intriguing hypothesis since patients with a mutation in polycystin-1 show aberrant renal tubule lumen size resulting in cyst formation.
Sub-cellular localization of polycystin-2
Polycystin-2, like polycystin-1, is ubiqitously expressed. Since patients with mutations in either polycystin-1 or polycystin-2 show clinically indistinguishable phenotypes, it has been hypothesized that polycystin-1 and polycystin-2 have overlapping localizations and functions. Indeed, polycystin-1 and polycystin-2 are coordinately expressed in the kidney (13). Newby et al. in fact immuno-precipitated a polycystin-1-polycystin-2 heterodimer complex in renal epithelial cells (46).
Moreover, polycystin-2 has also been identiÞ ed as a structural component of the primary cilium. It is likely that part of the overlapping functions of polycystin-1 and polycystin-2 lie in their presence in the primary cilium.
However, polycystin-2 shows a broader sub-cellular localization patt ern than polycystin-1. In the cytoplasm, polycystin-2 has been localized to endoplasmic reticulum (ER) and Golgi compartments (47). Similar to polycystin-1, polycystin- 2 was detected at the plasma membrane in E-cadherin based adherens junctions, and also in desmosomes (15-18; 48). Traffi cking of polycystin-2 between ER, Golgi and plasma membrane compartments has been shown to be mediated by interaction with adaptor proteins that recognize an acidic cluster in the carboxy- terminal domain of polycystin-2, phosphofurin acidic cluster sorting protein (PACS)-1 and PACS-2 (49). Binding to PACS-1 and PACS-2 is regulated by casein kinase II mediated phosphorylation of polycystin-2. Hidaka et al. identiÞ ed another protein involved in traffi cking of polycystin-2, polycystin-2 interactor, Golgi- and endoplasmic reticulum-associated protein with a molecular mass of 14 kDa (PIGEA-14; 50). Intracellular traffi cking of polycystin-2 has been shown to depend also on an N-terminal glycogen synthase kinase 3 phosphorylation site (51). Recently, Geng et al. reported that polycystin-2 traffi cs to the primary cilium independent of polycystin-1 via an N-terminal RVxP motif (52).
The multiple localization patt erns of polycystin-2 compared to polycystin-1, suggest that polycystin-2 function only partially overlaps with polycystin-1 and that polycystin-2 may have functions independent of polycystin-1 as well.
Polycystin-2 functions as an ion channel
In conjunction with the structural predictions, polycystin-2 has been reported to function as a Ca2+-permeable nonselective cation channel (48,53). Activation of polycystin-2 results in increases in intracellular Ca2+ via release from extra-cellular or intra-cellular stores, depending on the plasma membrane or cytoplasmic localization respectively.
Polycystin-2 can form homo- or heterodimers with polycystin-1 and with transient receptor potential channel 1, TRPC1 (9,10,46,54). Polycystin-2 channel activity was regulated by polycystin-1 (55,56). Channel activity could also be modulated by phosphorylation at Ser812 (57). This amino acid position is a potential
phosphorylation site for casein kinase II (CKII). Furthermore, channel activity was also modulated by interaction of polycystin-2 with α-actinin, an actin-binding protein that is essential for cytoskeletal organization, cell adhesion, proliferation, and migration (58). In addition, Montalbett i et al. demonstrated that polycystin- 2 channel activity could be modulated by hydrostatic and osmotic pressure and was dependent on interaction with the actin cytoskeleton (59). Recently, EGF was reported to induce polycystin-2 activity (60). This synergistic eff ect was achieved by physical interaction of polycystin-2 with the EGF receptor (EGFR). Furthermore, Ma et al. proposed that polycystin-2 is normally under negative regulation of phosphatidylinositol-4,5-bisphosphate (PIP(2)).
The two-hit model for cyst formation
Several theories have been postulated to describe the mechanism of cyst formation, including the two-hit model and the haplo-insuffi ciency model (Figure 11). An alternative working hypothesis for these two models, the stop-signal hypothesis, does not focus on the primary defect but rather on the cellular consequence of the defect. In the following sections, these three models are outlined.
The two-hit model predicts that both alleles of PKD1 or PKD2 need to be aff ected to result in ADPKD. Since all cells have inherited one aff ected allele (germ-line mutation), a somatic mutation in the other normal allele is required for cyst formation. Somatic mutations in kidney epithelial cells are frequent and increase exponentially in life (61). The two-hit model predicts that cysts will develop only from cells that have acquired a somatic mutation in the second allele. This hypothesis adequately explains the phenotypic variation observed within families, since individuals can acquire diff erent second hit mutations during life. It also Þ ts well in line with the observation that cysts originate from less than 1% of total nephrons. Furthermore, Qian et al. (62) have demonstrated that cyst-lining cells derived from a single cyst are of clonal origin. However, where the two-hit model predicts that cysts are devoid of any functional polycystin-1 or polycystin- 2, expression of both polycystin-1 and polycystin-2 has been reported in cysts.
Whether these proteins are functional or abnormally expressed mutant products remains to be determined.
The haplo-insuffi ciency model for cyst formation
The haplo-insuffi ciency model postulates that levels of polycystin-1 and polycystin-2 need to be tightly regulated for proper function. Cook et al. have demonstrated that inactivation of one allele due to an inherited mutation results in increased stochastic variations in expression levels (63). These variations in expression level may ultimately lead to late-onset disease manifestations. This may also be the case in ADPKD. The haplo-insuffi ciency model is in line with the observation that polycystin-1 and polycystin-2 can be detected in cysts. Moreover, mouse models have shown that both too low (knock-out) and too high expression (transgenic) of polycystin-1 results in ADPKD (64-72).
The stop-signal model for cyst formation
Another approach to describe cyst formation is the stop-signal hypothesis
(reviewed in 73). This model describes cysts formation as a defect in normal tubule formation. Kidney structures develop from metanephrogenic mesenchyme. The mesenchyme diff erentiates into highly specialized epithelial cells and is organized in such a way that the cells form a tubular structure surrounding a lumen. This transformation process is tightly regulated and in this respect, cyst formation can be seen as a defect in tubule formation and maintenance. Due to the lack of a stop-signal that tells cells when the tubule architecture and lumen are of adequate size, a cyst can arise. The stop-signal model Þ ts well in line with the fact that polycystin-1 and polycystin-2 both localize in the primary cilium, an organelle that is thought to act as a mechano-sensor, translating signals from the tubule lumen into the epithelial cell. The fact that less than 1% of all nephrons will give rise to a cyst implies that additional genetic or environmental factors are required to trigger cyst formation in aff ected cells. To date, these factor(s) have not yet been identiÞ ed.
The stop-signal model presents a simple and att ractive working model describing the defects that lead to cyst formation without tackling the precise predisposing molecular (genetic) mechanism. Moreover, both the two-hit model and the haplo- insuffi ciency model that focus more on the molecular defect in cyst formation, are
Figure 11 . Schematic diagram of the two-hit, the haplo-insuffi ciency, and the stop-signal models of cyst formation.
compatible with the stop-signal hypothesis that describes the cellular consequence of the molecular defect.
Cilia and polycystic kidneys: senses and polarity
Although the precise function of the primary cilium and the role of polycystins in this compartment has not yet been fully uncovered, it is clear that there is a link between ciliary proteins and polycystic kidney disease. Primary cilia have been proposed to act as mechanosensors, responding to ß uid ß ow inside the tubule lumen. Besides polycystin-1 and polycystin-2, Þ brocystin, responsible for autosomal recessive polycystic kidney disease (ARPKD), nephrocystin-1, KIF3a, and IFT88, all localize in the primary cilium and have been linked to polycystic kidney disease (73,74). Interestingly, the cilium is not only present in renal epithelial cells, but can be found in an extremely wide variety of cell types (www.
members.global2000.net/bowser/cilialist). The presence and function of cilia in diff erent cell types, may also account for the extra-renal manifestations of ADPKD, including liver and pancreatic cysts, and cardiovascular abnormalities. During embryonic development, nodal cilia are crucial for left -right axis determination.
Pennekamp et al. have shown that polycystin-2 localizes to these nodal cilia and is an important factor determining left -right axis formation (72). Taken together, these data suggest that polycystins play an important role in determining cell polarity in nodal cilia during development and in primary cilia in diff erentiated cells. Cell polarity is required for proper function of the renal cell epithelium, the cells that are defective in polycystic kidney disease. As outlined previously, it is essential that renal epithelial cells are able to maintain a gradient between the apical (tubule lumen) and basal membrane. This apical-to-basal polarity enables selective water and ion transport across the epithelial cell. Therefore, the connection between cilia and polycystic kidney disease may lie in cell polarity as much as in sensing ß uid ß ow.
All roads lead to polycystic kidneys?
Although there seems to be an undeniable link between cilia and polycystic kidney disease, it is also clear that numerous routes can lead to polycystic kidney disease.
For instance, transgenic overexpression of TGFα, Erb-B2, an Epidermal Growth Factor Receptor (EGFR) related receptor tyrosine kinase, and T24ras, a component of EGFR signaling, all result in polycystic kidney disease (76-78). Transgenic mice expressing c-myc and prothymosin A (ProT), a target gene of c-myc, also develop polycystic kidney disease (79,80). In addition, mouse models for the transcription factor AP-2B (81), bcl-2 (82), Makorin-1 (83), β-catenin (84), Apc (85), Rho GDP dissociation inhibitor, Rho GDIα (86) all have been reported to develop polycystic kidney disease. Whether these factors are also involved in ADPKD and therefore can be linked to polycystin-1 and polycystin-2 functions, remains to be determined.
Polycystic kidney disease may develop as a secondary eff ect in some of these cases.
Outline of the thesis
We set out to investigate the functions of polycystin-1 and polycystin-2 in order to gain further insight into the molecular and cellular processes that are disrupted in ADPKD cyst formation. Potential signaling pathways modulated by polycystin-1 were identiÞ ed using a widely implemented approach based on reporter constructs. A C-terminal polycystin-1 construct was co-expressed in cells with luciferase reporter constructs to detect Wnt/β-catenin signaling or AP-1 activation (Chapter 2). Results were conÞ rmed using immunoß uorescence microscopy and western blot analysis to detect activated signaling components in normal and cystic cells and tissue. Using this approach we excluded Wnt/
β-catenin signaling and identiÞ ed AP-1 activation as a potential player in ADPKD cyst formation. The role of AP-1 in cyst formation was then further analyzed in ADPKD cystic tissue using immunohistochemical and western blot analysis (Chapter 3). Since AP-1 is regulated by MAPK’s, we explored the potential role of MAPK signaling by analyzing cystic epithelial cells derived from human ADPKD patients and from mouse renal cysts (Chapter 4). In Chapter 5, we report down-regulation of PKD1 and PKD2 aft er cellular stress as a potential factor in cyst formation. Chapter 6 gives an overview of the data so far on EGF/EGFR and AVP/cAMP signalling as potential targets for therapeutic intervention in ADPKD.
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