Norman D. Rosenblum

GLI3-dependent transcriptional repression of Gli1, Gli2 and kidney patterning genes disrupts renal morphogenesis

Truncating mutations in Gli3, an intracellular effector in the SHH-SMO-GLI signaling pathway, cause renal aplasia/dysplasia in humans and mice. Yet, the pathogenic mechanisms are undefined. Here, we report the effect of decreased SHH-SMO signaling on renal morphogenesis, the expression of SHH target genes and GLI binding to Shh target genes. Shh deficiency or cyclopamine-mediated SMO inhibition disrupted renal organogenesis, decreased expression of GLI1 and GLI2 proteins, but increased expression of GLI3 repressor relative to GLI3 activator. Shh deficiency decreased expression of kidney patterning genes (Pax2 and Sall1) and cell cycle regulators (cyclin D1 and MYCN). Elimination of Gli3 in Shh–/– mice rescued kidney malformation and restored expression of Pax2, Sall1, cyclin D1, MYCN, Gli1 and Gli2. To define mechanisms by which SHH-SMO signaling controls gene expression, we determined the binding of GLI proteins to 5′ flanking regions containing GLI consensus binding sequences in Shh target genes using chromatin immunoprecipitation. In normal embryonic kidney tissue, GLI1 and/or GLI2 were bound to each target gene. By contrast, treatment of embryonic kidney explants with cyclopamine decreased GLI1 and/or GLI2 binding, and induced binding of GLI3. However, cyclopamine failed to decrease Gli1 and Gli2 expression and branching morphogenesis in Gli3-deficient embryonic kidney tissue. Together, these results demonstrate that SHH-SMO signaling controls renal morphogenesis via transcriptional control of Gli, renal patterning and cell cycle regulator genes in a manner that is opposed by GLI3.

Polycystin-1 C-terminal tail associates with β-catenin and inhibits canonical Wnt signaling

Polycystin-1 (PC1), the product of the PKD1 gene mutated in the majority of autosomal dominant polycystic kidney disease (ADPKD) cases, undergoes a cleavage resulting in the intracellular release of its C-terminal tail (CTT). Here, we demonstrate that the PC1 CTT co-localizes with and binds to beta-catenin in the nucleus. This interaction requires a nuclear localization motif present in the PC1 CTT as well as the N-terminal portion of beta-catenin. The PC1 CTT inhibits the ability of both beta-catenin and Wnt ligands to activate T-cell factor (TCF)-dependent gene transcription, a major effector of the canonical Wnt signaling pathway. The PC1 CTT may produce this effect by reducing the apparent affinity of the interaction between beta-catenin and the TCF protein. DNA microarray analysis reveals that the canonical Wnt signaling pathway is activated in ADPKD patient cysts. Our results suggest a novel mechanism through which PC1 cleavage may impact upon Wnt-dependent signaling and thereby modulate both developmental processes and cystogenesis.

Systems biology of autosomal dominant polycystic kidney disease (ADPKD): computational identification of gene expression pathways and integrated regulatory networks

To elucidate the molecular pathways that modulate renal cyst growth in ADPKD, we performed global gene profiling on cysts of different size (<1 ml, n = 5; 10-20 ml, n = 5; >50 ml, n = 3) and minimally cystic tissue (MCT, n = 5) from five PKD1 human polycystic kidneys using Affymetrix HG-U133 Plus 2.0 arrays. We used gene set enrichment analysis to identify overrepresented signaling pathways and key transcription factors (TFs) between cysts and MCT. We found down-regulation of kidney epithelial restricted genes (e.g. nephron segment-specific markers and cilia-associated cystic genes such as HNF1B, PKHD1, IFT88 and CYS1) in the renal cysts. On the other hand, PKD1 cysts displayed a rich profile of gene sets associated with renal development, mitogen-mediated proliferation, cell cycle progression, epithelial-mesenchymal transition, hypoxia, aging and immune/inflammatory responses. Notably, our data suggest that up-regulation of Wnt/beta-catenin, pleiotropic growth factor/receptor tyrosine kinase (e.g. IGF/IGF1R, FGF/FGFR, EGF/EGFR, VEGF/VEGFR), G-protein-coupled receptor (e.g. PTGER2) signaling was associated with renal cystic growth. By integrating these pathways with a number of dysregulated networks of TFs (e.g. SRF, MYC, E2F1, CREB1, LEF1, TCF7, HNF1B/ HNF1A and HNF4A), our data suggest that epithelial dedifferentiation accompanied by aberrant activation and cross-talk of specific signaling pathways may be required for PKD1 cyst growth and disease progression. Pharmacological modulation of some of these signaling pathways may provide a potential therapeutic strategy for ADPKD.

BMP-2 and OP-1 exert direct and opposite effects on renal branching morphogenesis

The bone morphogenetic proteins, BMP-2 and OP-1, are candidates for growth factors that control renal branching morphogenesis. We examined their effects in embryonic kidney explants and in the mIMCD-3 cell model of collecting duct morphogenesis (mIMCD-3 cells are derived from the terminal inner medullary collecting duct of the SV40 mouse). Osteogenic protein-1 (OP-1), at a dose of 0.25 nM, increased explant growth by 30% (P = 0.001). In contrast, 100-fold greater concentrations of OP-1 (28 nM) decreased explant growth by 10% (P < 0.001). BMP-2 was entirely inhibitory (maximum inhibition of 7% at 5 nM, P < 0.0004). In an in vitro model for branching morphogenesis utilizing the kidney epithelial cell line, mIMCD-3, low doses of OP-1 (< 0.5 nM) increased the number of tubular structures formed by 28 +/- 5% (P = 0.01), whereas concentrations > 0.5 nM decreased that number by 22 +/- 8% (P = 0.02). All concentrations of BMP-2 (0.05-10 nM) were inhibitory (maximum inhibition at 10 nM of 88 +/- 3%, P < 0.0001). Stimulatory doses of OP-1 increased tubular length (P = 0.003) and the number of branch points/structure (3.2-fold increase, P = 0.0005) compared with BMP-2. To determine the molecular basis for these effects, we demonstrated that BMP-2 is bound to mIMCD-3 cells by the type I serine/threonine kinase receptor, ALK-3, and that OP-1 bound to an approximately 80-kDa protein using ligand-receptor affinity assays. To demonstrate that OP-1 can exert both stimulatory and inhibitory effects within a developing kidney, embryonic explants were treated with agarose beads saturated with 2 microM OP-1. OP-1 decreased the number of ureteric bud/collecting duct branches adjacent to the beads by 58 +/- 1% (P < 0.0001). In contrast, the number of branches in tissue distal to the OP-1 beads was enhanced, suggesting a stimulatory effect at lower doses of OP-1. We conclude that OP-1 and BMP-2 directly control branching morphogenesis and that the effects of OP-1 are dependent on its local concentration within developing kidney tissue.

Canonical WNT/β-catenin signaling is required for ureteric branching

WNT/beta-catenin signaling has an established role in nephron formation during kidney development. Yet, the role of beta-catenin during ureteric morphogenesis in vivo is undefined. We generated a murine genetic model of beta-catenin deficiency targeted to the ureteric bud cell lineage. Newborn mutant mice demonstrated bilateral renal aplasia or renal dysplasia. Analysis of the embryologic events leading to this phenotype revealed that abnormal ureteric branching at E12.5 precedes histologic abnormalities at E13.5. Microarray analysis of E12.5 kidney tissue identified decreased Emx2 and Lim1 expression among a small subset of renal patterning genes disrupted at the stage of abnormal branching. These alterations are followed by decreased expression of genes downstream of Emx2, including Lim1, Pax2, and the ureteric tip markers, c-ret and Wnt 11. Together, these data demonstrate that beta-catenin performs essential functions during renal branching morphogenesis via control of a hierarchy of genes that control ureteric branching.

Cell and Molecular Biology of Kidney Development

Abnormalities of kidney and urinary tract development are the most common cause of end-stage kidney failure in childhood in the United States. Over the past 20 years, the advent of mutant and transgenic mice and the manipulation of gene expression in other animal models has resulted in major advances in identification of the cellular and molecular mechanisms that direct kidney morphogenesis, providing insights into the pathophysiology of renal and urologic anomalies. This review focuses on the molecular mechanisms that define kidney progenitor cell populations, induce nephron formation within the metanephric mesenchyme, initiate and organize ureteric bud branching, and participate in terminal differentiation of the nephron. Highlighted are common signaling pathways that function at multiple stages during kidney development, including signaling via Wnts, bone morphogenic proteins, fibroblast growth factor, sonic hedgehog, RET/glial cell-derived neurotrophic factor, and notch pathways. Also emphasized are the roles of transcription factors Odd1, Eya1, Pax2, Lim1, and WT-1 in directing renal development. Areas requiring future investigation include the factors that modulate signaling pathways to provide temporal and site-specific effects. The evolution of our understanding of the cellular and molecular mechanisms of kidney development may provide methods for improved diagnosis of renal anomalies and, hopefully, targets for intervention for this common cause of childhood end-stage kidney disease.

Smad1, β-catenin and Tcf4 associate in a molecular complex with the Myc promoter in dysplastic renal tissue and cooperate to control Myc transcription

Renal dysplasia, the major cause of childhood renal failure in humans, arises from perturbed renal morphogenesis and molecular signaling during embryogenesis. Recently, we discovered induction of molecular crosstalk between Smad1 and β-catenin in the TgAlk3QD mouse model of renal medullary cystic dysplasia. Our finding that Myc, a Smad andβ -catenin transcriptional target and effector of renal epithelial dedifferentiation, is misexpressed in dedifferentiated epithelial tubules provided a basis for investigating coordinate transcriptional control by Smad1 and β-catenin in disease. Here, we report enhanced interactions between a molecular complex consisting of Smad1, β-catenin and Tcf4 and adjacent Tcf- and Smad-binding regions located within the Myc promoter in TgAlk3QD dysplastic renal tissue, and Bmp-dependent cooperative control of Myc transcription by Smad1, β-catenin and Tcf4. Analysis of nuclear extracts derived from TgAlk3QD and wild-type renal tissue revealed increased levels of Smad1/β-catenin molecular complexes, and de novo formation of chromatin-associated Tcf4/Smad1 molecular complexes in TgAlk3QD tissues. Analysis of a 476 nucleotide segment of the 1490 nucleotide Myc genomic region upstream of the transcription start site demonstrated interactions between Tcf4 and the Smad consensus binding region and associations of Smad1, β-catenin and Tcf4 with oligo-duplexes that encode the adjacent Tcf- and Smad-binding elements only in TgAlk3QD tissues. In collecting duct cells that express luciferase under the control of the 1490 nucleotide Myc genomic region, Bmp2-dependent stimulation of Myc transcription was dependent on contributions by each of Tcf4, β-catenin and Smad1. These results provide novel insights into mechanisms by which interacting signaling pathways control transcription during the genesis of renal dysplasia.

Endostatin regulates branching morphogenesis of renal epithelial cells and ureteric bud

Endostatin (ES) inhibits endothelial cell migration and has been found to bind to glypicans (Gpcs) on both endothelial cells and renal epithelial cells. We examined the possibility that ES might regulate epithelial cell morphogenesis. The addition of ES to cultured epithelial cells causes an inhibition of both hepatocyte growth factor- and epidermal growth factor-dependent process formation and migration. In contrast, ES does not inhibit epidermal growth factor-dependent morphogenesis in renal epithelial cells derived from Gpc-3 −/mice, whereas expression of Gpc-1 in these cells reconstitutes ES responsiveness. Gpc-3 −/mice have been shown to display enhanced ureteric bud (UB) branching early in development, and cultured UB cells release ES into the media, suggesting that ES binding to Gpcs may regulate UB branching. The addition of ES inhibits branching of the explanted UB, whereas a neutralizing Ab to ES enhances UB outgrowth and branching. Thus, local expression of ES at the tips of the UB may play a role in the regulation of UB arborization.

Protein kinase A is a negative regulator of renal branching morphogenesis and modulates inhibitory and stimulatory bone morphogenetic proteins.

Protein kinase A (PKA) regulates morphogenetic responses to bone morphogenetic proteins (BMPs) during embryogenesis. However, the mechanisms by which PKA regulates BMP function are unknown. During kidney development, BMP-2 and high doses of BMP-7 inhibit branching morphogenesis, whereas low doses of BMP-7 are stimulatory (Piscione, T. D., Yager, T. D., Gupta, I. R., Grinfeld, B., Pei, Y., Attisano, L., Wrana, J. L., and Rosenblum, N. D. (1997) Am. J. Physiol. 273, F961–F975). We examined the interactions between PKA and these BMPs in embryonic kidney explants and in the mouse inner medullary collecting duct-3 model of collecting duct morphogenesis. H-89, an inhibitor of PKA, stimulated branching morphogenesis and enhanced the stimulatory effect of low doses of BMP-7 on tubule formation. Furthermore, H-89 rescued the inhibition of tubulogenesis by BMP-2 (or high doses of BMP-7) by attenuating BMP-2-induced collecting duct apoptosis. In contrast, 8-bromo-cAMP, an activator of PKA, inhibited tubule formation and attenuated the stimulatory effects of low doses of BMP-7. To determine mechanisms underlying the interdependence of BMP signaling and PKA activity, we examined the effect of PKA on the known signaling events in the BMP-2-dependent Smad1 signaling pathway and the effect of BMP-2 on PKA activity. PKA did not induce endogenous Smad1 phosphorylation, Smad1-Smad4 complex formation, or Smad1 nuclear translocation. In contrast, BMP-2 increased endogenous PKA activity and induced phosphorylation of the PKA effector, cAMP-response element-binding protein, in a PKA-dependent manner. We conclude that BMP-2 induces activation of PKA and that PKA regulates the effects of BMPs on collecting duct morphogenesis without activating the known signaling events in the BMP-2-dependent Smad1 signaling pathway.

Elevated SMAD1/β-catenin molecular complexes and renal medullary cystic dysplasia in ALK3 transgenic mice

Renal dysplasia, the most frequent cause of childhood renal failure in humans, arises from perturbations in a complex series of morphogenetic events during embryonic renal development. The molecular pathogenesis of renal dysplasia is largely undefined. While investigating the role of a BMP-dependent pathway that inhibits branching morphogenesis in vitro, we generated a novel model of renal dysplasia in a transgenic (Tg) model of ALK3receptor signaling. We report the renal phenotype, and our discovery of molecular interactions between effectors in the BMP and WNT signaling pathways in dysplastic kidney tissue. Expression of the constitutively active ALK3 receptor ALK3QD, in two independent transgenic lines caused renal aplasia/severe dysgenesis in 1.5% and 8.4% of hemizygous and homozygous Tg mice, respectively, and renal medullary cystic dysplasia in 49% and 74% of hemizygous and homozygous Tg mice, respectively. The dysplastic phenotype, which included a decreased number of medullary collecting ducts, increased medullary mesenchyme, collecting duct cysts and decreased cortical thickness, was apparent by E18.5. We investigated the pathogenesis of dysplasia in these mice, and demonstrated a 30% decrease in branching morphogenesis at E13.5 before the appearance of histopathogical features of dysplasia, and the formation of β-catenin/SMAD1/SMAD4 molecular complexes in dysplastic renal tissue. Increased transcriptional activity of a β-catenin reporter gene in ALK3QD;Tcf-gal mice demonstrated functional cooperativity between the ALK3 and β-catenin-dependent signaling pathways in kidney tissue. Together with our results in the dysplastic mouse kidney, our findings that phospho-SMAD1 and β-catenin are overexpressed in human fetal dysplastic renal tissue suggest that dysregulation of these signaling effectors is pathogenic in human renal dysplasia. Our work provides novel insights into the role that crucial developmental signaling pathways may play during the genesis of malformed renal tissue elements.