Pascale Van Vooren

A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility

The kidney has an important role in the regulation of acid–base homeostasis. Renal ammonium production and excretion are essential for net acid excretion under basal conditions and during metabolic acidosis. Ammonium is secreted into the urine by the collecting duct, a distal nephron segment where ammonium transport is believed to occur by non-ionic NH3 diffusion coupled to H+ secretion. Here we show that this process is largely dependent on the Rhesus factor Rhcg. Mice lacking Rhcg have abnormal urinary acidification due to impaired ammonium excretion on acid loading—a feature of distal renal tubular acidosis. In vitro microperfused collecting ducts of Rhcg-/- acid-loaded mice show reduced apical permeability to NH3 and impaired transepithelial NH3 transport. Furthermore, Rhcg is localized in epididymal epithelial cells and is required for normal fertility and epididymal fluid pH. We anticipate a critical role for Rhcg in ammonium handling and pH homeostasis both in the kidney and the male reproductive tract.

Alpha-fetoprotein controls female fertility and prenatal development of the gonadotropin-releasing hormone pathway through an antiestrogenic action

ABSTRACT It has been shown previously that female mice homozygous for an alpha-fetoprotein (AFP) null allele are sterile as a result of anovulation, probably due to a defect in the hypothalamic-pituitary axis. Here we show that these female mice exhibit specific anomalies in the expression of numerous genes in the pituitary, including genes involved in the gonadotropin-releasing hormone pathway, which are underexpressed. In the hypothalamus, the gonadotropin-releasing hormone gene, Gnrh1, was also found to be down-regulated. However, pituitary gene expression could be normalized and fertility could be rescued by blocking prenatal estrogen synthesis using an aromatase inhibitor. These results show that AFP protects the developing female brain from the adverse effects of prenatal estrogen exposure and clarify a long-running debate on the role of this fetal protein in brain sexual differentiation.

Genes encoding atrial and brain natriuretic peptides as candidates for sensitivity to brain ischemia in stroke-prone hypertensive rats.

-Previous studies suggested that atrial natriuretic peptide gene (Anp) and brain natriuretic peptide gene (Bnp) are plausible candidate genes for susceptibility to stroke and for sensitivity to brain ischemia in the stroke-prone spontaneously hypertensive rat (SHRSP). We performed structural and functional analyses of these 2 genes in SHRSP from Glasgow colonies (SHRSPGla) and Wistar-Kyoto rats from Glasgow colonies (WKYGla) and developed a radiation hybrid map of the relevant region of rat chromosome 5. Sequencing of the coding regions of the Anp and Bnp genes revealed no difference between the 2 strains. Expression studies in brain tissue showed no differences at baseline and at 24 hours after middle cerebral artery occlusion. Plasma concentrations of atrial natriuretic peptide (ANP) did not differ between the SHRSPGla and WKYGla, whereas concentrations of brain natriuretic peptide were significantly higher in the SHRSPGla as compared with the WKYGla (n=11 to 14; 163+/-21 pg/mL and 78+/-14 pg/mL; 95% confidence interval 31 to 138, P=0.003). We did not detect any attenuation of endothelium-dependent relaxations to bradykinin or ANP in middle cerebral arteries from the SHRSPGla; indeed the sensitivity to ANP was significantly increased in arteries harvested from this strain (WKYGla: n=8; pD2=7. 3+/-0.2 and SHRSPGla: n=8; pD2=8.2+/-0.15; P<0.01). Moreover, radiation hybrid mapping and fluorescence in situ hybridization allowed us to map the Anf marker in the telomeric position of rat chromosome 5 in close proximity to D5Rat48, D5Rat47, D5Mgh15, and D5Mgh16. These results exclude Anp and Bnp as candidate genes for the sensitivity to brain ischemia and pave the way to further congenic and physical mapping strategies.

The TORC1 effector kinase Npr1 fine tunes the inherent activity of the Mep2 ammonium transport protein

The TORC1 complex controls cell growth upon integrating nutritional signals including amino-acid availability. TORC1 notably adapts the plasma membrane protein content by regulating arrestin-mediated endocytosis of amino-acid transporters. Here we demonstrate that TORC1 further fine tunes the inherent activity of the ammonium transport protein, Mep2, a yeast homologue of mammalian Rhesus factors, independently of arrestin-mediated endocytosis. The TORC1 effector kinase Npr1 and the upstream TORC1 regulator Npr2 control Mep2 transport activity by phospho-silencing a carboxy-terminal autoinhibitory domain. Under poor nitrogen supply, Npr1 enables Mep2 S457 phosphorylation and thus ammonium transport activity. Supplementation of the preferred nitrogen source glutamine leads to Mep2 inactivation and instant S457 dephosphorylation via plasma membrane Psr1 and Psr2 redundant phosphatases. This study underscores that TORC1 also adjusts nutrient permeability to regulate cell growth in a fast and flexible response to environmental perturbation, establishing a hierarchy in the transporters to be degraded, inactivated or maintained active at the plasma membrane.

The development of several organs and appendages is impaired in mice lacking Sp6.

SP6 belongs to the SP/KLF family of transcription factors, characterized by a DNA‐binding domain composed of three zinc fingers of the C2H2 type. The Sp6 gene generates two different transcripts, termed Sp6 and epiprofin, which differ in the first exon and encode the same SP6 protein. These transcripts are mainly expressed in the skin, the teeth, and the limb buds of embryos and also in the adult lungs. To gain insight into the biological function of the SP6 protein, we knocked out the gene by eliminating the full coding region. The resulting Sp6 null mice are nude, lack functional teeth, and present limb and lung malformations. We also showed that the identified abnormalities are associated with apoptotic misregulations. In conclusion, this work indicates that Sp6 plays a critical role in the development of several epithelium‐containing organs or appendages, possibly by regulating apoptosis. Developmental Dynamics 237:883–892, 2008.

Analysis of candidate genes included in the mammary cancer susceptibility 1 (Mcs1) region.

Abstract. The rat strain COP is resistant to spontaneous and carcinogen-induced mammary cancer, whereas the strain WF is susceptible. Using genetic linkage analysis of (WF × COP) F1× WF backcrosses, LC Hsu, LA Shepel and co-workers showed that a region at the centromeric end of Chromosome (Chr) 2 (2q1) segregates with the sensitivity to mammary cancer development. The responsible locus was named Mcs1 (for mammary cancer susceptibility 1). We have developed the chromosome map of the 2q1 region by localizing 18 genes, 4 ESTs, and several anonymous markers, using radiation hybrids and fluorescence in situ hybridization. The region containing Mcs1 was delineated to 2q12–q14. Five of the genes (Mef2c, Map1b, Ccnh, Rasa, Rasgrf2) were assigned to this region and were shown to be expressed in the rat mammary glands, while three possible functional candidate genes, Pi3kr1, Rad17, and Naip, were excluded from the critical region. Since cyclin H, encoded by Ccnh, plays an important role in the control of the cell cycle and since the proteins encoded by Rasa and Rasgrf2 control the activity of the RAS oncoprotein, the corresponding genes appeared as both functional and positional Mcs1 candidates. RT-PCR experiments on RNA extracted from mammary glands of the two rat strains (COP, WF) was done. No amino acid sequence difference was found between the two strains. These results argue against the hypothesis that any of these three genes is Mcs1.

Localization of 54 rat genes, and definition of new synteny groups conserved in the human and the rat

In order to improve the rat gene map and comparative mapping with the human and the mouse, we determined the chromosome localization of 54 rat genes. Most genes encode transcription factors or other regulatory proteins of cancer relevance. The human homologs of four genes were also assigned to their respective chromosome. These data generated anchor points between the recently established radiation hybrid maps and the genetic and cytogenetic maps. They improve comparative mapping between the rat, the mouse, and the human gene maps, and in particular they disclose four new synteny groups conserved in the rat and the human. These new localizations should also be useful for the identification of genes involved in the control of quantitative traits such as cancer susceptibility or diabetes.

Rat Chromosome 2: assignment of the genes encoding cyclin B1, interleukin 6 signal transducer, and proprotein convertase 1 to the Mcs1-containing region and identification of new microsatellite markers

Abstract. The rat Chromosome (Chr) 2 harbors several genes controlling tumor growth or development, blood pressure, and non-insulin-dependent diabetes mellitus. We report that the region (2q1) containing the mammary susceptibility cancer gene Mcs1 also harbors the genes encoding cyclin B1, interleukin 6 signal transducer (gp130), and proprotein convertase 1. We also generated 13 new anonymous microsatellite markers from Chr 2-sorted DNA. These markers, as well as a microsatellite marker in the cyclin B1 gene, were genetically mapped in combination with known markers. A cyclin B1-related gene was also cytogenetically assigned to rat Chr 11q22-q23.

SNPs Altering Ammonium Transport Activity of Human Rhesus Factors Characterized by a Yeast-Based Functional Assay

Proteins of the conserved Mep-Amt-Rh family, including mammalian Rhesus factors, mediate transmembrane ammonium transport. Ammonium is an important nitrogen source for the biosynthesis of amino acids but is also a metabolic waste product. Its disposal in urine plays a critical role in the regulation of the acid/base homeostasis, especially with an acid diet, a trait of Western countries. Ammonium accumulation above a certain concentration is however pathologic, the cytotoxicity causing fatal cerebral paralysis in acute cases. Alteration in ammonium transport via human Rh proteins could have clinical outcomes. We used a yeast-based expression assay to characterize human Rh variants resulting from non synonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinical phenotypes and assessed their ammonium transport efficiency, protein level, localization and potential trans-dominant impact. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditary stomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsic bidirectional ammonium transport. Moreover, this study reveals that the R202C variant of HsRhCG, the orthologue of mouse MmRhcg required for optimal urinary ammonium excretion and blood pH control, shows an impaired inherent ammonium transport activity. Urinary ammonium excretion was RHcg gene-dose dependent in mouse, highlighting MmRhcg as a limiting factor. HsRhCGR202C may confer susceptibility to disorders leading to metabolic acidosis for instance. Finally, the analogous R211C mutation in the yeast ScMep2 homologue also impaired intrinsic activity consistent with a conserved functional role of the preserved arginine residue. The yeast expression assay used here constitutes an inexpensive, fast and easy tool to screen nsSNPs reported by high throughput sequencing or individual cases for functional alterations in Rh factors revealing potential causal variants.