Loss of receptor activity-modifying protein 2 in mice causes placental dysfunction and alters PTH1R regulation

Receptor activity-modifying protein 2 (Ramp2) is a single-pass transmembrane protein that heterodimerizes with several family B G-protein coupled receptors to alter their function. Ramp2 has been primarily characterized in association with calcitonin receptor-like receptor (Calcrl, CLR), forming the canonical receptor complex for the endocrine peptide adrenomedullin (Adm, AM). However, we previously demonstrated that Ramp2+/- female mice display a constellation of endocrine-related phenotypes that are distinct from those of Adm+/- and Calcrl+/- mice, implying that RAMP2 has physiological functions beyond its canonical complex. Here, we localize Ramp2 expression in the mouse placenta, finding that Ramp2 is robustly expressed in the fetal labyrinth layer, and then characterize the effects of loss of Ramp2 on placental development. Consistent with the expression pattern of Ramp2 in the placenta, Ramp2-/- placentas have a thinner labyrinth layer with significantly fewer trophoblast cells secondary to a reduction in trophoblast proliferation. We also find that absence of Ramp2 leads to failed spiral artery remodeling unaccompanied by changes in the uterine natural killer cell population. Furthermore, we assess changes in gene expression of other RAMP2-associated G-protein coupled receptors (GPCRs), concluding that Ramp2 loss decreases parathyroid hormone 1 receptor (Pthr1) expression and causes a blunted response to systemic parathyroid hormone (PTH) administration in mice. Ultimately, these studies provide in vivo evidence of a role for RAMP2 in placental development distinct from the RAMP2-CLR/AM signaling paradigm and identify additional pathways underlying the endocrine and fertility defects of the previously characterized Ramp2 heterozygous adult females

Взаимосвязь понятий «стиль» и «имидж» как эстетическая проблема

<div><p>The intestinal microbiota influences the development and function of myeloid lineages such as neutrophils, but the underlying molecular mechanisms are unresolved. Using gnotobiotic zebrafish, we identified the immune effector Serum amyloid A (Saa) as one of the most highly induced transcripts in digestive tissues following microbiota colonization. Saa is a conserved secreted protein produced in the intestine and liver with described effects on neutrophils <i>in vitro</i>, however its <i>in vivo</i> functions remain poorly defined. We engineered <i>saa</i> mutant zebrafish to test requirements for Saa on innate immunity <i>in vivo</i>. Zebrafish mutant for <i>saa</i> displayed impaired neutrophil responses to wounding but augmented clearance of pathogenic bacteria. At baseline, <i>saa</i> mutants exhibited moderate neutrophilia and altered neutrophil tissue distribution. Molecular and functional analyses of isolated neutrophils revealed that Saa suppresses expression of pro-inflammatory markers and bactericidal activity. Saa’s effects on neutrophils depended on microbiota colonization, suggesting this protein mediates the microbiota’s effects on host innate immunity. To test tissue-specific roles of Saa on neutrophil function, we over-expressed <i>saa</i> in the intestine or liver and found that sufficient to partially complement neutrophil phenotypes observed in <i>saa</i> mutants. These results indicate Saa produced by the intestine in response to microbiota serves as a systemic signal to neutrophils to restrict aberrant activation, decreasing inflammatory tone and bacterial killing potential while simultaneously enhancing their ability to migrate to wounds.</p></div

Loss of receptor activity-modifying protein 2 in mice causes placental dysfunction and alters PTH1R regulation.

Receptor activity-modifying protein 2 (Ramp2) is a single-pass transmembrane protein that heterodimerizes with several family B G-protein coupled receptors to alter their function. Ramp2 has been primarily characterized in association with calcitonin receptor-like receptor (Calcrl, CLR), forming the canonical receptor complex for the endocrine peptide adrenomedullin (Adm, AM). However, we previously demonstrated that Ramp2+/- female mice display a constellation of endocrine-related phenotypes that are distinct from those of Adm+/- and Calcrl+/- mice, implying that RAMP2 has physiological functions beyond its canonical complex. Here, we localize Ramp2 expression in the mouse placenta, finding that Ramp2 is robustly expressed in the fetal labyrinth layer, and then characterize the effects of loss of Ramp2 on placental development. Consistent with the expression pattern of Ramp2 in the placenta, Ramp2-/- placentas have a thinner labyrinth layer with significantly fewer trophoblast cells secondary to a reduction in trophoblast proliferation. We also find that absence of Ramp2 leads to failed spiral artery remodeling unaccompanied by changes in the uterine natural killer cell population. Furthermore, we assess changes in gene expression of other RAMP2-associated G-protein coupled receptors (GPCRs), concluding that Ramp2 loss decreases parathyroid hormone 1 receptor (Pthr1) expression and causes a blunted response to systemic parathyroid hormone (PTH) administration in mice. Ultimately, these studies provide in vivo evidence of a role for RAMP2 in placental development distinct from the RAMP2-CLR/AM signaling paradigm and identify additional pathways underlying the endocrine and fertility defects of the previously characterized Ramp2 heterozygous adult females

Loss of <i>Ramp2</i> alters mRNA expression of RAMP2-associating GPCRs, including <i>Pthr1</i>.

<p>(A) mRNA expression of RAMP2-interacting GPCRs from e13.5 wild type and <i>Ramp2</i>^<i>-/-</i> placentas (n≥3 placentas per genotype). (B) PTH1R protein expression by immunoblot of e13.5 tissue lysates from wild type and <i>Ramp2</i>^<i>-/-</i> placentas. (C and D), Immunofluorescence for PTH1R in the labyrinth of wild type and <i>Ramp2</i>^<i>-/-</i> placentas. Scale bars, 100 μm. <i>Pth1r</i>, parathyroid hormone receptor 1; <i>Casr</i>, calcium-sensing receptor; <i>Ctr</i>, calcitonin receptor; <i>Calcrl</i>, calcitonin receptor-like receptor; <i>Gcgr</i>, glucagon receptor; <i>Vpac1r</i>, vasoactive intestinal peptide receptor 1; <i>Crhr1</i>, corticotrophin releasing hormone receptor 1. *p<0.01.</p

Loss of <i>Ramp2</i> leads to a proliferation defect in the labyrinth layer.

<p>(A and B) Immunofluorescence for BrdU in e13.5 placentas. Scale bars, 500<b>μ</b>m. (C and D) Immunofluorescence for Ki67 in the labyrinth of e13.5 placentas. Scale bars, 500 μm. (E) Ratio of <i>Bax</i> to <i>Bcl2</i> mRNA expression normalized to <i>Gapdh</i> as measured by qRT-PCR (n≥5 placentas per genotype). lb, labyrinth; jz, junctional zone; dec, decidua.</p

<i>Ramp2</i> is essential for proper development of the labyrinth layer of the placenta.

<p>(A and B), Hematoxylin and eosin staining of e13.5 wild type and <i>Ramp2</i>^<i>-/-</i> littermate placentas. Horizontal and vertical lines are representative of axes measured in panels (C) and (D) Scale bars, 1 mm. (C and D), Quantification of width and thickness of wild type and <i>Ramp2-/-</i> labyrinth layers (n≥6 placentas per genotype). (E and F), Immunofluorescence for cytokeratin in wild type and <i>Ramp2</i>^<i>-/-</i> placentas, showing trophoblast cells in the fetal compartment of the placenta. Scale bar, 500<b>μ</b>m. (G) Quantitation of labyrinth trophoblast cells, identified by histology (n = 3 fields per animal, n≥3 placentas per genotype). (H and I) Representative images of wild type and <i>Ramp2</i>^<i>-/-</i> placentas with pseudocolored maternal (yellow) and fetal (blue) sinuses. (J) Comparison of the total area of the non-vascular region and of the maternal sinuses in wild type and <i>Ramp2</i>^<i>-/-</i> placentas (n≥2 fields per placenta from multiple placentas per genotype). cp, chorionic plate; lb, labyrinth; jz, junctional zone; dec, dedicua; myo, myometrium. *p<0.05, **p<0.01, ***p<0.001</p

<i>In situ</i> hybridization of <i>Ramp2</i> in e13.5 placentas reveals robust expression in the labyrinth layer.

<p>(A) <i>In situ</i> hybridization in both the fetal and maternal compartments of the wild type placenta (n = 4). Scale bar, 100 μm. (B) <i>In situ</i> hybridization in the labyrinth layer. Scale bar, 50 μm. Inset images in both panels show sense control probes. CP, chorionic plate; LB, labyrinth; SP, spongiotrophoblast cells; DEC, decidua. Arrows point to giant trophoblast cells.</p