HNF-1beta regulates transcription of the PKD modifier gene Kif12.

Hepatocyte nuclear factor-1beta (HNF-1beta) is a transcription factor that regulates gene expression in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1beta lead to a syndrome of inherited renal cysts and diabetes and are also a common cause of sporadic renal dysplasia. The full complement of target genes responsible for the functions of HNF-1beta, however, is incompletely defined. Using a functional genomics approach involving chromatin immunoprecipitation and promoter arrays, combined with gene expression profiling, we found that an HNF-1beta target gene in the kidney is kinesin family member 12 (Kif12), a gene previously identified as a candidate modifier gene in the cpk mouse model of polycystic kidney disease. Mutations of HNF-1beta inhibited Kif12 transcription in both cultured cells and knockout mice by altering co-factor recruitment and histone modification. Because kinesin-12 family members participate in orienting cell division, downregulation of Kif12 may underlie the abnormal planar cell polarity observed in cystic kidney diseases.

Mutations of HNF-1beta inhibit epithelial morphogenesis through dysregulation of SOCS-3.

Hepatocyte nuclear factor-1beta (HNF-1beta) is a Pit-1, Oct-1/2, Unc-86 (POU) homeodomain-containing transcription factor expressed in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1beta cause maturity-onset diabetes of the young, type 5 (MODY5), which is characterized by early-onset diabetes mellitus and congenital malformations of the kidney, pancreas, and genital tract. Knockout of HNF-1beta in the mouse kidney results in cyst formation. However, the signaling pathways and transcriptional programs controlled by HNF-1beta are poorly understood. Using genome-wide chromatin immunoprecipitation and DNA microarray (ChIP-chip) and microarray analysis of mRNA expression, we identified SOCS3 (suppressor of cytokine signaling-3) as a previously unrecognized target gene of HNF-1beta in the kidney. HNF-1beta binds to the SOCS3 promoter and represses SOCS3 transcription. The expression of SOCS3 is increased in HNF-1beta knockout mice and in renal epithelial cells expressing dominant-negative mutant HNF-1beta. Increased levels of SOCS-3 inhibit HGF-induced tubulogenesis by decreasing phosphorylation of Erk and STAT-3. Conversely, knockdown of SOCS-3 in renal epithelial cells expressing dominant-negative mutant HNF-1beta rescues the defect in HGF-induced tubulogenesis by restoring phosphorylation of Erk and STAT-3. Thus, HNF-1beta regulates tubulogenesis by controlling the levels of SOCS-3 expression. Manipulating the levels of SOCS-3 may be a useful therapeutic approach for human diseases induced by HNF-1beta mutations.

The role for HNF-1beta-targeted collectrin in maintenance of primary cilia and cell polarity in collecting duct cells.

Collectrin, a homologue of angiotensin converting enzyme 2 (ACE2), is a type I transmembrane protein, and we originally reported its localization to the cytoplasm and apical membrane of collecting duct cells. Recently, two independent studies of targeted disruption of collectrin in mice resulted in severe and general defects in renal amino acid uptake. Collectrin has been reported to be under the transcriptional regulation by HNF-1alpha, which is exclusively expressed in proximal tubules and localized at the luminal side of brush border membranes. The deficiency of collectrin was associated with reduction of multiple amino acid transporters on luminal membranes. In the current study, we describe that collectrin is a target of HNF-1beta and heavily expressed in the primary cilium of renal collecting duct cells. Collectrin is also localized in the vesicles near the peri-basal body region and binds to gamma-actin-myosin II-A, SNARE, and polycystin-2-polaris complexes, and all of these are involved in intracellular and ciliary movement of vesicles and membrane proteins. Treatment of mIMCD3 cells with collectrin siRNA resulted in defective cilium formation, increased cell proliferation and apoptosis, and disappearance of polycystin-2 in the primary cilium. Suppression of collectrin mRNA in metanephric culture resulted in the formation of multiple longitudinal cysts in ureteric bud branches. Taken together, the cystic change and formation of defective cilium with the interference in the collectrin functions would suggest that it is necessary for recycling of the primary cilia-specific membrane proteins, the maintenance of the primary cilia and cell polarity of collecting duct cells. The transcriptional hierarchy between HNF-1beta and PKD (polycystic kidney disease) genes expressed in the primary cilia of collecting duct cells has been suggested, and collectrin is one of such HNF-1beta regulated genes.

Proteolytic cleavage and nuclear translocation of fibrocystin is regulated by intracellular Ca2+ and activation of protein kinase C.

Fibrocystin, a type I membrane protein of unknown function, is the protein affected in the autosomal recessive form of polycystic kidney disease. Here we show that fibrocystin undergoes regulated proteolysis. Several proteolytic cleavages occur within the predicted ectodomain, whereas at least one cleavage occurs within the cytoplasmic portion. The latter generates a C-terminal intracellular fragment that harbors the nuclear localization signal KRKVSRLAVTGERTATPAPKIPRIT and translocates to the nucleus. Proteolytic cleavage of fibrocystin occurs constitutively in long term cultures of polarized inner medullary collecting duct cells (mIMCD-3). Activation of protein kinase C and release of intracellular Ca2+ are required for proteolysis under these conditions. In short term cultures of human embryonic kidney 293 cells (HEK-293), proteolytic cleavage of fibrocystin can be elicited by stimulation of intracellular Ca2+ release or activation of protein kinase C. These results identify a novel Ca2+-dependent pathway that signals from fibrocystin located in the cell membrane to the nucleus.

Essential roles for the FE65 amyloid precursor protein-interacting proteins in brain development.

Targeted deletion of two members of the FE65 family of adaptor proteins, FE65 and FE65L1, results in cortical dysplasia. Heterotopias resembling those found in cobblestone lissencephalies in which neuroepithelial cells migrate into superficial layers of the developing cortex, aberrant cortical projections and loss of infrapyramidal mossy fibers arise in FE65/FE65L1 compound null animals, but not in single gene knockouts. The disruption of pial basal membranes underlying the heterotopias and poor organization of fibrillar laminin by isolated meningeal fibroblasts from double knockouts suggests that FE65 proteins are involved in basement membrane assembly. A similar phenotype is observed in triple mutant mice lacking the APP family members APP, APLP1 and APLP2, all of which interact with FE65 proteins, suggesting that this phenotype may be caused by decreased transmission of an APP-dependent signal through the FE65 proteins. The defects observed in the double knockout may also involve the family of Ena/Vasp proteins, which participate in actin cytoskeleton remodeling and interact with the WW domains of FE65 proteins.

Roles of HNF-1beta in kidney development and congenital cystic diseases.

Hepatocyte nuclear factor-1beta (HNF-1beta) is a Pit-1/Oct-1/Unc-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1beta produce maturity-onset diabetes of the young type 5 (MODY5) and are associated with congenital cystic abnormalities of the kidney. Transgenic mice expressing mutant HNF-1beta under the control of a kidney-specific promoter develop kidney cysts and renal failure, which is similar to the phenotype of humans with MODY5. Similarly, kidney-specific deletion of HNF-1beta using Cre/loxP recombination results in renal cyst formation. HNF-1beta directly regulates the Pkhd1 promoter. HNF-1beta mutant mice show decreased expression of Pkhd1, the gene that is mutated in humans with autosomal-recessive polycystic kidney disease (ARPKD). These studies demonstrate that HNF-1beta is required for the development of the mammalian kidney. They establish a previously unrecognized link between two renal cystic diseases, MODY5 and ARPKD, and suggest that the mechanism of cyst formation in humans with mutations of HNF-1beta involves down-regulation of PKHD1 gene transcription.

Elucidating the function of primary cilia by conditional gene inactivation.

PURPOSE OF REVIEW: This review discusses recent experimental approaches to determine the function of primary cilia by conditional inactivation of genes crucial for cilia formation. RECENT FINDINGS: A functional role in the sensing of fluid flow was recently assigned to the primary cilia. This discovery shed light onto how cells sense dynamic fluid movements. Conditional inactivation of primary cilia formation in later ontogenic stages demonstrated the crucial role renal primary cilia play in the control of cell proliferation. SUMMARY: Primary cilia can act as flow sensors, transmitting signals by means of calcium influx into the cells. Structures based on primary cilia are also crucial for the function of photoreceptor cells and it can be expected that additional functions of these organelles will be determined in the future. An important experimental approach to elucidate the involvement of primary cilia in other physiological processes is to specifically inactivate genes crucial for formation of primary cilia. Morphological and physiological changes induced by the loss of primary cilia will help determine additional roles primary cilia play in physiology and organ development.

Role of the hepatocyte nuclear factor-1beta (HNF-1beta) C-terminal domain in Pkhd1 (ARPKD) gene transcription and renal cystogenesis.

Hepatocyte nuclear factor-1beta (HNF-1beta) is a homeodomain-containing transcription factor that regulates tissue-specific gene expression in the kidney and other epithelial organs. Mutations of HNF-1beta produce congenital cystic abnormalities of the kidney, and previous studies showed that HNF-1beta regulates the expression of the autosomal recessive polycystic kidney disease (ARPKD) gene, Pkhd1. Here we show that the C-terminal region of HNF-1beta contains an activation domain that is functional when fused to a heterologous DNA-binding domain. An HNF-1beta deletion mutant lacking the C-terminal domain interacts with wild-type HNF-1beta, binds DNA, and functions as a dominant-negative inhibitor of a chromosomally integrated Pkhd1 promoter. The activation of the Pkhd1 promoter by wild-type HNF-1beta is stimulated by sodium butyrate or coactivators CREB (cAMP-response element)-binding protein (CBP) and P/CAF. The interaction with CBP and P/CAF requires the C-terminal domain. Expression of an HNF-1beta C-terminal deletion mutant in transgenic mice produces renal cysts, increased cell proliferation, and dilatation of the ureter similar to mice with kidney-specific inactivation of HNF-1beta. Pkhd1 expression is inhibited in cystic collecting ducts but not in non-cystic proximal tubules, despite transgene expression in this nephron segment. We conclude that the C-terminal domain of HNF-1beta is required for the activation of the Pkhd1 promoter. Deletion mutants lacking the C-terminal domain function as dominant-negative mutants, possibly by preventing the recruitment of histone acetylases to the promoter. Cyst formation correlates with inhibition of Pkhd1 expression, which argues that mutations of HNF-1beta produce kidney cysts by down-regulating the ARPKD gene, Pkhd1. Expression of HNF-1alpha in proximal tubules may protect against cystogenesis.

Mechanical stimuli induce cleavage and nuclear translocation of the polycystin-1 C terminus.

Polycystin-1, which is encoded by a gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), is involved in cell-matrix interactions as well as in ciliary signaling. The precise mechanisms by which it functions, however, remain unclear. Here we find that polycystin-1 undergoes a proteolytic cleavage that releases its C-terminal tail (CTT), which enters the nucleus and initiates signaling processes. The cleavage occurs in vivo in association with alterations in mechanical stimuli. Polycystin-2, the product of the second gene mutated in ADPKD, modulates the signaling properties of the polycystin-1 CTT. These data reveal a novel pathway by which polycystin-1 transmits messages directly to the nucleus.

Mutation of hepatocyte nuclear factor-1beta inhibits Pkhd1 gene expression and produces renal cysts in mice.

Hepatocyte nuclear factor-1beta (HNF-1beta) is a Pit-1, Oct-1/2, UNC-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the liver, kidney, and other organs. Humans with autosomal dominant mutations of HNF-1beta develop maturity-onset diabetes of the young type 5 (MODY5) and congenital cystic abnormalities of the kidney. Autosomal recessive polycystic kidney disease (ARPKD) is an inherited cystic disorder that produces renal failure in infants and children and is caused by mutations of PKHD1. The proximal promoter of the mouse Pkhd1 gene contains an evolutionarily conserved HNF-1-binding site that is located near a region of deoxyribonuclease hypersensitivity. HNF-1beta and the structurally related HNF-1alpha bind specifically to the Pkhd1 promoter and stimulate gene transcription. Mutations of the HNF-1 site or expression of a dominant-negative HNF-1beta mutant inhibit Pkhd1 promoter activity in transfected cells. Transgenic mice expressing a dominant-negative HNF-1beta mutant under the control of a kidney-specific promoter develop renal cysts, similarly to humans with MODY5. Pkhd1 transcripts are absent in the cells lining the cysts but are present in morphologically normal surrounding tubules. These studies identify a link between two cystic disease genes, HNF1beta (MODY5) and PKHD1 (ARPKD). HNF-1beta directly regulates the transcription of Pkhd1, and inhibition of PKHD1 gene expression may contribute to the formation of renal cysts in humans with MODY5.

A transcriptional network in polycystic kidney disease.

Mutations in cystic kidney disease genes represent a major genetic cause of end-stage renal disease. However, the molecular cascades controlling the expression of these genes are still poorly understood. Hepatocyte Nuclear Factor 1beta (HNF1beta) is a homeoprotein predominantly expressed in renal, pancreatic and hepatic epithelia. We report here that mice with renal-specific inactivation of HNF1beta develop polycystic kidney disease. We show that renal cyst formation is accompanied by a drastic defect in the transcriptional activation of Umod, Pkhd1 and Pkd2 genes, whose mutations are responsible for distinct cystic kidney syndromes. In vivo chromatin immunoprecipitation experiments demonstrated that HNF1beta binds to several DNA elements in murine Umod, Pkhd1, Pkd2 and Tg737/Polaris genomic sequences. Our results uncover a direct transcriptional hierarchy between HNF1beta and cystic disease genes. Interestingly, most of the identified HNF1beta target gene products colocalize to the primary cilium, a crucial organelle that plays an important role in controlling the proliferation of tubular cells. This may explain the increased proliferation of cystic cells in MODY5 patients carrying autosomal dominant mutations in HNF1beta.

The central fragment of Reelin, generated by proteolytic processing in vivo, is critical to its function during cortical plate development.

Reelin is a large extracellular protein that controls cortical development. It binds to lipoprotein receptors very-low-density lipoprotein receptor and apolipoprotein-E receptor type 2, thereby inducing phosphorylation of the adapter Dab1. In vivo, Reelin is cleaved into three fragments, but their respective function is unknown. Here we show the following: (1) the central fragment is necessary and sufficient for receptor binding in vitro and for Dab1 phosphorylation in neuronal cultures; (2) Reelin does not bind the protocadherin cadherin-related neuronal receptor (CNR1) as reported previously; (3) Reelin and its central fragment are equally able to rescue the reeler phenotype in a slice culture assay; and (4) anti-receptor antibodies can induce Dab1 phosphorylation but do not correct the reeler phenotype in slices. These observations show that the function of Reelin is critically dependent on the central fragment generated by processing but primarily independent of interactions with CNR1 and on the N-terminal region. They also indicate that events acting in parallel to Dab1 phosphorylation might be required for full activity.

Receptor clustering is involved in Reelin signaling.

The Reelin signaling cascade plays a crucial role in the correct positioning of neurons during embryonic brain development. Reelin binding to apolipoprotein E receptor 2 (ApoER2) and very-low-density-lipoprotein receptor (VLDLR) leads to phosphorylation of disabled 1 (Dab1), an adaptor protein which associates with the intracellular domains of both receptors. Coreceptors for Reelin have been postulated to be necessary for Dab1 phosphorylation. We show that bivalent agents specifically binding to ApoER2 or VLDLR are sufficient to mimic the Reelin signal. These agents induce Dab1 phosphorylation, activate members of the Src family of nonreceptor tyrosine kinases, modulate protein kinase B/Akt phosphorylation, and increase long-term potentiation in hippocampal slices. Induced dimerization of Dab1 in HEK293 cells leads to its phosphorylation even in the absence of Reelin receptors. The mechanism for and the sites of these phosphorylations are identical to those effected by Reelin in primary neurons. These results suggest that binding of Reelin, which exists as a homodimer in vivo, to ApoER2 and VLDLR induces clustering of ApoER2 and VLDLR. As a consequence, Dab1 becomes dimerized or oligomerized on the cytosolic side of the plasma membrane, constituting the active substrate for the kinase; this process seems to be sufficient to transmit the signal and does not appear to require any coreceptor.

Kidney-specific inactivation of the KIF3A subunit of kinesin-II inhibits renal ciliogenesis and produces polycystic kidney disease.

Polycystic kidney disease (PKD) is the most common genetic cause of renal failure in humans. Several proteins that are encoded by genes associated with PKD have recently been identified in primary cilia in renal tubular epithelia. These findings have suggested that abnormalities in cilia formation and function may play a role in the pathogenesis of PKD. To directly determine whether cilia are essential to maintain tubular integrity, we conditionally inactivated KIF3A, a subunit of kinesin-II that is essential for cilia formation, in renal epithelia. Constitutive inactivation of KIF3A produces abnormalities of left-right axis determination and embryonic lethality. Here we show that tissue-specific inactivation of KIF3A in renal tubular epithelial cells results in viable offspring with normal-appearing kidneys at birth. Cysts begin to develop in the kidney at postnatal day 5 and cause renal failure by postnatal day 21. The cyst epithelial cells lack primary cilia and exhibit increased proliferation and apoptosis, apical mislocalization of the epidermal growth factor receptor, increased expression of beta-catenin and c-Myc, and inhibition of p21(CIP1). These results demonstrate that the absence of renal cilia produces both the clinical and cell biological findings associated with PKD. Most generally, the phenotype of Kif3a mutant mice suggests a role for primary cilia in the maintenance of lumen-forming epithelial differentiation.

Regulation of kidney-specific Ksp-cadherin gene promoter by hepatocyte nuclear factor-1beta.

Kidney-specific cadherin (Ksp-cadherin) is a tissue-specific member of the cadherin family that is expressed exclusively in the kidney and developing genitourinary tract. Recent studies have shown that the proximal 250 bp of the Ksp-cadherin gene promoter are sufficient to direct tissue-specific gene expression in vivo and in vitro. The proximal 120 bp of the promoter are evolutionarily conserved between mouse and human and contain a DNase I hypersensitive site that is kidney cell specific. At position -55, the promoter contains a consensus recognition site for hepatocyte nuclear factor-1 (HNF-1). Mutations of the consensus HNF-1 site and downstream GC-boxes inhibit promoter activity in transfected cells. HNF-1alpha and HNF-1beta bind specifically to the -55 site, and both proteins transactivate the promoter directly. Expression of Ksp-cadherin is not altered in the kidneys of HNF-1alpha-deficient mice. However, expression of a gain-of-function HNF-1beta mutant stimulates Ksp-cadherin promoter activity in transfected cells, whereas expression of a dominant-negative mutant inhibits activity. These studies identify Ksp-cadherin as the first kidney-specific promoter that has been shown to be regulated by HNF-1beta. Mutations of HNF-1beta, as occur in humans with inherited renal cysts and diabetes, may cause dysregulated Ksp-cadherin promoter activity.

A minimal Ksp-cadherin promoter linked to a green fluorescent protein reporter gene exhibits tissue-specific expression in the developing kidney and genitourinary tract.

Ksp-cadherin is a unique, tissue-specific member of the cadherin family of cell adhesion molecules that is expressed exclusively in tubular epithelial cells in the kidney and developing genitourinary (GU) tract. Transgenic mice carrying 3425 bp of the Ksp-cadherin 5' flanking region linked to a lacZ reporter gene express beta-galactosidase exclusively in the kidney, although the expression pattern is incomplete (Am J Physiol 277: F599-F610, 1999). To further define the region that mediates tissue-specific expression, transgenic mice carrying 1341 bp or 324 bp of the 5' flanking region linked to a green fluorescent protein (GFP) reporter gene were produced. Transgenic mice carrying 1341 bp of the 5' flanking region expressed GFP in all embryonic tissues that endogenously express Ksp-cadherin, including the ureteric bud, Wolffian duct, Müllerian duct, and developing tubules in the mesonephros and metanephros. In the adult kidney, GFP was highly expressed in thick ascending limbs of Henle's loops and collecting ducts and weakly expressed in proximal tubules and Bowman's capsules. Transgenic mice carrying 324 bp of the 5' flanking region exhibited expression exclusively in tubular epithelial cells in the developing kidney and GU tract. Immunoblot analysis showed that the expression of GFP was restricted to the kidney in adult mice. Taken together, these results demonstrate that 324 bp of the Ksp-cadherin 5' flanking region is sufficient to direct epithelial-specific expression in the developing kidney and GU tract. Transgenic mice that express GFP in the mesonephros, metanephros, ureteric bud, and sex ducts may be useful for cell lineage studies.

Platelet-derived growth factor mediates tyrosine phosphorylation of the cytoplasmic domain of the low Density lipoprotein receptor-related protein in caveolae.

The low density lipoprotein (LDL) receptor gene family represents a class of multifunctional, endocytic cell surface receptors. Recently, roles in cellular signaling have also emerged. For instance, the very low density lipoprotein receptor (VLDLR) and the apolipoprotein receptor-2 (apoER2) function in a developmental signaling pathway that regulates the lamination of cortical layers in the brain and involves the activation of tyrosine kinases. Furthermore, the cytoplasmic domain of the LDL receptor-related protein (LRP) was found to be a substrate for the non-receptor tyrosine kinase Src, but the physiological significance of this phosphorylation event remained unknown. Here we show that tyrosine phosphorylation of LRP occurs in caveolae and involves the platelet-derived growth factor (PDGF) receptor beta and phosphoinositide 3-kinase. Receptor-associated protein, an antagonist of ligand binding to LRP, and apoE-enriched beta-VLDL, a ligand for LRP, reduce PDGF-induced tyrosine phosphorylation of the LRP cytoplasmic domain. In the accompanying paper (Loukinova, E., Ranganathan, S., Kuznetsov, S., Gorlatova, N., Migliorini, M., Ulery, P. G., Mikhailenko, I., Lawrence, D. L., and Strickland, D. K. (2002) J. Biol. Chem. 277, 15499-15506) Loukinova et al. further demonstrate that one form of PDGF, PDGF-BB, binds specifically to LRP and that phosphorylation of LRP requires the activation of Src family kinases. Taken together, these findings provide a biochemical basis for a cellular signaling pathway that involves apoE and LRP.