Ligand-Activated Ahr Signaling Leads to Disruption of Nephrogenesis and Altered Wilms' Tumor Suppressor Mrna Splicing
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Oncogene (2003) 22, 2160–2171 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Ligand-activated Ahr signaling leads to disruption of nephrogenesis and altered Wilms’ tumor suppressor mRNA splicing M Hadi Falahatpisheh and Kenneth S Ramos* Center for Environmental and Rural Health, Texas A & M University, College Station, TX, USA The aryl hydrocarbon receptor (Ahr), a member of the nuclear-binding protein activity has been implicated in large basic helix–loop–helix (bHLH) and PAS homology anomalies of kidney development (Avner, 1993). Wilms’ domain superfamily, is a highly conserved transcriptional tumor suppressor gene (wt1) has been characterized as a regulator involved in mammalian development. In the key regulatory gene in mesenchymo-epithelial transition present study, a murine metanephros organ culture system and differentiation during nephrogenesis. wt1 encodes a was employed to evaluate the role of the Ahr signaling in Cis2-His2 zinc finger DNA-binding protein that med- nephrogenesis in vitro. Ahr and Wilms’ tumor suppressor iates transcriptional repression/activation of target (wt1) mRNAs were detected by in situ hybridization and genes by binding DNA sequences containing the 50- 0 RT–PCR during the course of renal development. GCGGGGGCG-3 as well as (TCC)n repeats. Both Treatment with 3 lm BaP, a hydrocarbon ligand of Ahr, transcriptional repression and activation depend on the inhibited glomerulogenesis and branching morphogenesis architecture of the promoter, and involve distinct of metanephric kidneys. Deficits in the epithelialization of domains of the WT1 protein (Drumond et al., 1992; mesenchymal cells were evidenced by inhibition of the Wang et al., 1992). formation of podocyte foot processes and glomerular The wt1 gene contains 10 exons spanning approxi- basement membranes. Hydrocarbon treatment markedly mately 50 kb (Gessler et al., 1992). As a result of induced ÀKTS wt1 splice variants, although total wt1 alternative RNA splicing, multiple proteins are gener- mRNA levels remained unchanged. A significant decrease ated ranging in molecular weight from 49 to 62 kDa in total WT1 protein was observed by both immunocyto- (Scharnhorst et al., 1999). One alternative splicing event chemistry and Western analysis in cultures challenged results in inclusion/exclusion of exon 5 (7exon 5), a with BaP compared to controls. Comparison of meta- sequence that encodes for a stretch of 17 amino acids nephric cultures from Ahr+/+ and AhrÀ/À mice showed within the N-terminus of the four zinc fingers. The most that Ahr is involved in kidney development, and required functionally significant splicing event involves a splice for BaP-induced deficits in nephrogenesis. These results site in exon 9 resulting in the insertion of lysine– indicate that ligand activation of Ahr signaling disrupts threonine–serine between zinc fingers 3 and 4 in the nephrogenesis in vitro, and that this response involves DNA-binding domain (7KTS). These four splice modulation of wt1 alternative splicing and post-transcrip- variants are expressed in the kidney in temporal, spatial, tional control. and evolutionarily stable ratios to support different Oncogene (2003) 22, 2160–2171. doi:10.1038/sj.onc.1206238 functions (Haber et al., 1991; Renshaw et al., 1997; Aswin et al., 1998). In vitro experiments suggest distinct Keywords: Ahr; WT1 splicing; nephrogenesis molecular functions for + and À isoforms. Hetero- zygous mice with a reduction in the +KTS isoform develop glomerulosclerosis, while homozygous mutants of both strains die after birth because of kidney defects. Introduction A second translational initiation site has been identified that gives rise to WT1 proteins of higher molecular Nephrogenesis proceeds through a series of carefully weight (Bruening and Pelletier, 1996; Scharnhorst et al., integrated cell–cell and cell–matrix interactions invol- 1997). The combination of alternative splicing, alter- ving specific induction of metanephric blastema by the native translation start sites, and RNA editing leads to ampullary portion of the ureteric bud and epithelializa- the expression of at least 24 different WT1 isoforms tion (Saxon, 1987). The mechanisms of disrupted (Sharma et al., 1994). nephrogenesis are largely unknown, but interference The essential role of wt1 in kidney development was with genes that encode transcriptional regulatory or unequivocally proven by the phenotype of homozygous wt1 knockout mice which fail to develop metanephric *Correspondence: KS Ramos, Center for Environmental and Rural kidney and die in utero. In homozygous wt1 knockout Health, Texas A&M University, College Station, TX 77843-4455, mice, the ureteric bud fails to grow out of the USA; E-mail: [email protected] mesonephric duct and metanephric mesenchyme dies Received 4 June 2002; revised 8 November 2002; accepted 12 November (Kreidberg et al., 1993). wt1 is mutated in a proportion 2002 of embryonic kidney tumors or nephroblastomas, Role of Ahr signaling in WT1 splicing MH Falahatpisheh and KS Ramos 2161 tumors characterized histologically by incomplete at- Ahr dependent. Other studies have provided evidence tempts at epithelialization. Repression of Pax-2 by wt1 that Ahr participates in the development of prostate, has been demonstrated in vitro (Ryan et al., 1995), a thymus, and ovaries (Hundeiker et al., 1999). A role for response believed to be necessary for renal cell Ahr in renal development was established based on the differentiation. In the mature kidney, expression of observation that exposure of Ahr (+/+) mice to wt1 ultimately becomes confined to the podocyte layer TCDD, an Ahr agonist, induces hydronephrosis, small of the nephron (Pritchard-Jones et al., 1990). The kidneys, tortous ureters, and dilation of the renal pelvis importance of wt1 in kidney development is reflected and ureters (Peters et al., 1999). Hydronephrosis refers by mutations which lead to Denys–Drash syndrome to the dilation of the renal calyces and pelvis proximal to (DDS) and Frasier syndromes (FS). DDS patients a point of obstruction that interrupts the unidirectional exhibit defects in podocyte structure and suffer from flow of urine. However, virtually nothing is known diffuse mesangial sclerosis, urogenital abnormalities, about the pathogenesis of developmental renal disor- and high risk of development of Wilms’ tumors. ders. Glomerular nephropathy is the most consistent finding The molecular mechanisms responsible for deficits in in DDS (Habib et al., 1985). FS patients have focal renal development following inappropriate Ahr signal- segmental glomerular sclerosis attributed to shifts in the ing are unknown. Evidence is presented here that normal +KTS isoform ratio and predominance of unregulated activation of Ahr signaling by BaP inter- ÀKTS isoforms (Barbaux et al., 1997). Developmental feres with glomerulogenesis, branching morphogenesis, regulation of transcription factors by alternative splicing and podocyte differentiation in vitro. These alterations is a widespread phenomenon, with functional implica- correlate with a marked induction of ÀKTS wt1 tions for DNA binding specificities and affinities, transcripts and decreases in WT1 protein levels. activation and repression properties, and protein dimer- ization. Ahr is a member of the large basic helix–loop–helix (bHLH) and PAS homology domain family of tran- Results scription factors that includes proteins involved in myoblast differentiation, such as MyoD, the cellular Induction of metanephric mesenchyme, condensation, response to hypoxia, such as Arnt (Ahr nuclear and subsequent nephron development were monitored translocator) and hypoxia-inducible factor-1 (HIF-1), in metanephric cultures from E11.5 C57BL/6J mice. the Drosophila neurogenic protein Sim (‘single- Murine cultures underwent normal differentiation in the minded’), and the Drosophila circadian rhythm protein presence of vehicle (DMSO), as evidenced by condensa- Per (‘period’). bHLH–PAS proteins generally form tion of renal blastema, formation of comma- and S- heterodimeric transcription factors that regulate devel- shaped bodies, glomerologenesis, and ureteric bud opment and differentiation. Within this family, Ahr is formation (Figures 1–4). Ahr expression was detected the only member conditionally activated in response to in day 4 metanephric cultures by RT–PCR, and Ahr ligand binding. Although an endogenous ligand for Ahr DNA binding was activated by Ahr ligands (data not has not yet been identified, environmental hydrocarbons shown). Treatment with 3 mm BaP, a hydrocarbon ligand and dietary indole carbinols are known to activate Ahr of Ahr, decreased the rates of glomerulogenesis and signaling (Crews and Fan, 1999). Following hydrocar- compromised cellular differentiation within the glomer- bon ligand binding within the PAS domain, the cytosolic ular region (Figure 1a, panels 4–6). The blastema in the Ahr undergoes a conformational change, dissociates proliferating zone appeared less condensed, with deficits from two 90 kDa heat-shock proteins and the hepatitis B in cellular differentiation involving mostly glomerular virus X-associated protein 2 (XAP2) and translocates to mesenchymal cells. Morphometric analysis of BaP- the nucleus where it dimerizes with Arnt (Carver and treated metanephric cultures revealed decreases in Bradfield, 1997). The Ahr/Arnt heterodimer interacts comma- and S-shaped bodies and numbers of glomeruli with Ahr responsive elements (50-TNGCGTG-30) up- and tubulo-epithelial structures (Figure 1b, c, respec-