PAX2 suppresses apoptosis in renal collecting duct cells.

PAX2 is a transcription factor belonging to the evolutionarily conserved paired box family and is required during development of the central nervous system and genitourinary axis. Mutations in the PAX2 gene cause a rare autosomal dominant renal-coloboma syndrome, characterized by optic nerve colobomas and renal hypoplasia. Recent analysis of a spontaneous PAX2 mutant mouse model (1Neu) revealed that the major cause of renal hypoplasia is reduced branching of the ureteric bud (UB) and fewer nephrons. We have observed that this abnormality is associated with a striking increase in the number of UB cells undergoing programmed cell death during nephrogenesis. To ascertain whether apoptosis is directly linked to the level of PAX2 expression, we have studied the role of PAX2 in cultured renal cells. We show that mIMCD-3 cells, a murine collecting duct cell line with high endogenous PAX2 expression, undergo apoptosis when transfected with anti-sense PAX2. In contrast, HEK293 cells expressing exogenous PAX2 are protected against apoptotic death induced by caspase-2. PAX2 has no effect on proliferation of embryonic kidney or in cultured kidney cells. Our observations imply a direct role for PAX2 in survival of ureteric bud cells.

Primary renal hypoplasia in humans and mice with PAX2 mutations: evidence of increased apoptosis in fetal kidneys of Pax2(1Neu) +/- mutant mice.

PAX2 mutations cause renal-coloboma syndrome (RCS), a rare multi-system developmental abnormality involving optic nerve colobomas and renal abnormalities. End-stage renal failure is common in RCS, but the mechanism by which PAX2 mutations lead to renal failure is unknown. PAX2 is a member of a family of developmental genes containing a highly conserved 'paired box' DNA-binding domain, and encodes a transcription factor expressed primarily during fetal development in the central nervous system, eye, ear and urogenital tract. Presently, the role of PAX2 during kidney development is poorly understood. To gain insight into the cause of renal abnormalities in patients with PAX2 mutations, kidney anomalies were analyzed in patients with RCS, including a large Brazilian kindred in whom a new PAX2 mutation was identified. In a total of 29 patients, renal hypoplasia was the most common congenital renal abnormality. To determine the direct effects of PAX2 mutations on kidney development fetal kidneys of mice carrying a Pax2 (1Neu)mutation were examined. At E15, heterozygous mutant kidneys were approximately 60% of the size of wild-type littermates, and the number of nephrons was strikingly reduced. Heterozygous 1Neu mice showed increased apoptotic cell death during fetal kidney development, but the increased apoptosis was not associated with random stochastic inactivation of Pax2 expression in mutant kidneys; Pax2 was shown to be biallelically expressed during kidney development. These findings support the notion that heterozygous mutations of PAX2 are associated with increased apoptosis and reduced branching of the ureteric bud, due to reduced PAX2 dosage during a critical window in kidney development.

The Wilms' tumor suppressor gene (wt1) product regulates Dax-1 gene expression during gonadal differentiation.

Gonadal differentiation is dependent upon a molecular cascade responsible for ovarian or testicular development from the bipotential gonadal ridge. Genetic analysis has implicated a number of gene products essential for this process, which include Sry, WT1, SF-1, and DAX-1. We have sought to better define the role of WT1 in this process by identifying downstream targets of WT1 during normal gonadal development. We have noticed that in the developing murine gonadal ridge, wt1 expression precedes expression of Dax-1, a nuclear receptor gene. We document here that the spatial distribution profiles of both proteins in the developing gonad overlap. We also demonstrate that WT1 can activate the Dax-1 promoter. Footprinting analysis, transient transfections, promoter mutagenesis, and mobility shift assays suggest that WT1 regulates Dax-1 via GC-rich binding sites found upstream of the Dax-1 TATA box. We show that two WT1-interacting proteins, the product of a Denys-Drash syndrome allele of wt1 and prostate apoptosis response-4 protein, inhibit WT1-mediated transactivation of Dax-1. In addition, we demonstrate that WT1 can activate the endogenous Dax-1 promoter. Our results indicate that the WT1-DAX-1 pathway is an early event in the process of mammalian sex determination.

What PAX genes do in the kidney.

Of the nine known PAX genes, only two (PAX2 and PAX8) are expressed in the developing kidney. Genetic evidence in mice and humans indicates that PAX2 plays a critical role in normal renal development and may sit atop a molecular cascade which unfolds during the transition from undifferentiated mesenchyme to the early stages of nephrogenesis. Less is known about the role of PAX8 in kidney development; although PAX8 is expressed in the S-shaped body and early proximal tubule, preliminary data suggest that renal morphogenesis is unaffected by its absence. In this review, we discuss the basic aspects of PAX gene structure and function and how mutations of PAX2 might interfere with structure of the developing nephron.

Effects of PAX2 expression in a human fetal kidney (HEK293) cell line.

PAX2, a member of the "paired-box" family of homeotic genes, is a nuclear transcription factor expressed in the early stages of nephrogenesis by induced blastemal cells as they progress from mesenchymal condensates to the "S-shaped" stage and also by the ureteric bud. Spontaneous mutations in one copy of PAX2 in humans causes a syndrome of proteinuric renal failure and coloboma of the eye (P. Sanyanusin et al., Nat. Genet. 9 (1995) 358-363); transgenic mice with disruption of the PAX2 gene are anephric (M. Torres et al., Development 121 (1995) 4057-4067. Although PAX2 is clearly critical for normal kidney development, its direct effects on kidney cell phenotype are unknown. To address this issue, we developed stable transfectants of the HEK293 human fetal kidney epithelial cell line expressing human PAX2 protein under tetracycline-regulatable promoter. In these cells, PAX2 had no effect on the proliferative rate, but increased the expression of the Wilms' tumor gene (2-fold) and E-cadherin (7-fold). PAX2 had a strong inhibitory effect on vimentin; vimentin/GAPDH mRNA ratio was suppressed to 8% of control whereas cytokeratin-18/GAPDH mRNA ratio was unchanged. During nephrogenesis, loss of vimentin and onset of low-level WT1 and E-cadherin expression occur in mesenchymal condensates. Our observations suggest that these events may be, in part, regulated by PAX2.

F329L polymorphism in the human PAX8 gene.

We screened patients with juvenile nephronophthisis for mutations of the tightly linked PAX8 gene. No disease-associated mutations were found, but we identified the first known human PAX8 polymorphism, F329L, in 1 of 15 patients and 2 of 20 controls. This polymorphic variant involves a conservative amino acid change (phenylalanine to leucine) in the C-terminal portion of the PAX8 protein.

Regulation of renal EGF receptor expression is normal in Denys-Drash syndrome.

In patients with Denys-Drash syndrome, mutations of the Wilms' tumor suppressor gene are associated with nephroblastomas and developmental abnormalities of the genital tract and renal glomerulus. Normally, the Wilms' tumor gene product (WT1) is expressed at high levels in visceral glomerular epithelial cells (VGEC) of the emerging fetal glomerulus. We demonstrate that WT1 could normally serve to suppress EGF receptor expression in VGEC, since immunoreactive EGF receptor is strikingly absent compared to epithelial cells of the emerging proximal and distal tubule, which lack WT1. When HEK293 cells were co-transfected with plasmids containing EGFR enhancer/promoter elements linked to a CAT reporter and plasmids containing WT1 cDNA, EGFR enhancer/promoter activity was suppressed by all wild-type WT1 isoforms, but not by deletion mutants of WT1 lacking normal zinc-finger or N-terminal domains. Surprisingly, plasmids expressing a Denys-Drash WT1 mutant (R394W) retained the ability to suppress EGFR promoter activity in this system. Furthermore, we found that immunoreactive EGFR was appropriately undetectable in glomeruli from a three-year-old girl with Denys-Drash syndrome and in sections of her Wilm's tumor. These data suggest that faulty suppression of EGFR cannot account for the abnormalities of glomerulogenesis seen in Denys-Drash patients.

Repression of the retinoic acid receptor-alpha gene by the Wilms' tumor suppressor gene product, wt1.

The Wilms' tumor (WT) suppressor gene, WT1, encodes a zinc finger DNA binding protein (wt1) which functions as a transcriptional regulator. Germline WT1 mutations predispose to WTs and in many cases are associated with urogenital anomalies. Identification of wt1 downstream targets is essential to understanding regulatory processes involved in development of this system. In this study, we demonstrate that wt1 can repress transcription of the retinoic acid receptor-alpha 1 (RAR-alpha 1) promoter. Transient transfection, deletion mutagenesis, and mobility shift assays suggest that wt1 mediates repression of the human RAR-alpha 1 promoter through a GC-rich DNA binding motif (5'-GCGGGGGCG-3'), at positions -111 to -120 bp (relative to the transcription initiation site). In contrast, the murine RAR-alpha 1 promoter contains a cryptic binding motif and is not responsive to wt1. These results indicate that some wt1-regulatory pathways are not conserved across species, suggesting a molecular basis for differences in phenotypes between humans and mice harboring WT1 lesions.

Isolation and expression of a cDNA encoding the precursor for a novel member (ACADSB) of the acyl-CoA dehydrogenase gene family.

The acyl-CoA dehydrogenases (ACDs) are a family of mitochondrial enzymes that oxidize straight chain or branched chain acyl-CoAs in the metabolism of fatty acids or branched chain amino acids. Deficiencies in members of this gene family are important causes of human disease. A cDNA encoding the human precursor for a novel member (gene symbol ACADSB) of the ACD gene family has been isolated and characterized. The open reading frame of 1.3 kb encodes a precursor protein of 431 amino acids, which is processed in vitro to yield a mature protein of 399 amino acids. The cDNA has significant sequence similarity to other members of the acyl-CoA dehydrogenase family, with the greatest homology (38%) to the short chain acyl-CoA dehydrogenase. The cDNA was expressed in eukaryotic (COS) and prokaryotic (Escherichia coli) cells, producing a protein of the expected size, with activity toward the short branched chain acyl-CoA derivatives ((S)-2-methylbutyryl-CoA, isobutyryl-CoA, and 2-methylhexanoyl-CoA), as well as toward the short straight chain acyl-CoAs (butyryl-CoA and hexanoyl-CoA).