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Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death.

ABSTRACT: Rho-associated kinase (ROCK), including the ROCK-I and ROCK-II isoforms, is a protein kinase involved in signaling from Rho to actin cytoskeleton. However, in vivo functions of each ROCK isoform remain largely unknown. We generated mice deficient in ROCK-II by gene targeting. ROCK-II(-/-) embryos were found at the expected Mendelian frequency until 13.5 days postcoitum, but approximately 90% died thereafter in utero. ROCK-II(-/-) mice of both genders that survived were born runts, subsequently developed without gross abnormality, and were fertile. Whole-mount staining for a knocked-in lacZ reporter gene revealed that ROCK-II was highly expressed in the labyrinth layer of the placenta. Disruption of architecture and extensive thrombus formation were found in the labyrinth layer of ROCK-II(-/-) mice. While no obvious alteration in actin filament structures was found in the labyrinth layer of ROCK-II(-/-) placenta and stress fibers were formed in cultured ROCK-II(-/-) trophoblasts, elevated expression of plasminogen activator inhibitor 1 was found in ROCK-II(-/-) placenta. These results suggest that ROCK-II is essential in inhibiting blood coagulation and maintaining blood flow in the endothelium-free labyrinth layer and that loss of ROCK-II leads to thrombus formation, placental dysfunction, intrauterine growth retardation, and fetal death.

SUBMITTER: Thumkeo D

PROVIDER: S-EPMC162229 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death.

Thumkeo Dean D Keel Jeongsin J Ishizaki Toshimasa T Hirose Masaya M Nonomura Kimiko K Oshima Hiroko H Oshima Masanobu M Taketo Makoto M MM Narumiya Shuh S

Molecular and cellular biology 20030701 14

Rho-associated kinase (ROCK), including the ROCK-I and ROCK-II isoforms, is a protein kinase involved in signaling from Rho to actin cytoskeleton. However, in vivo functions of each ROCK isoform remain largely unknown. We generated mice deficient in ROCK-II by gene targeting. ROCK-II(-/-) embryos were found at the expected Mendelian frequency until 13.5 days postcoitum, but approximately 90% died thereafter in utero. ROCK-II(-/-) mice of both genders that survived were born runts, subsequently d ...[more]

PMID: 12832488

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Project description:Intrauterine fetal death or stillbirth occurs in approximately 1 out of every 160 pregnancies and accounts for 50% of all perinatal deaths. Postmortem evaluation fails to elucidate an underlying cause in many cases. Long QT syndrome (LQTS) may contribute to this problem.To determine the spectrum and prevalence of mutations in the 3 most common LQTS susceptible genes (KCNQ1, KCNH2, and SCN5A) for a cohort of unexplained cases.In this case series, retrospective postmortem genetic testing was conducted on a convenience sample of 91 unexplained intrauterine fetal deaths (mean [SD] estimated gestational age at fetal death, 26.3 [8.7] weeks) that were collected from 2006-2012 by the Mayo Clinic, Rochester, Minnesota, or the Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. More than 1300 ostensibly healthy individuals served as controls. In addition, publicly available exome databases were assessed for the general population frequency of identified genetic variants.Comprehensive mutational analyses of KCNQ1 (KV7.1, LQTS type 1), KCNH2 (HERG/KV11.1, LQTS type 2), and SCN5A (NaV1.5, LQTS type 3) were performed using denaturing high-performance liquid chromatography and direct DNA sequencing on genomic DNA extracted from decedent tissue. Functional analyses of novel mutations were performed using heterologous expression and patch-clamp recording.The 3 putative LQTS susceptibility missense mutations (KCNQ1, p.A283T; KCNQ1, p.R397W; and KCNH2 [1b], p.R25W), with a heterozygous frequency of less than 0.05% in more than 10 000 publicly available exomes and absent in more than 1000 ethnically similar control patients, were discovered in 3 intrauterine fetal deaths (3.3% [95% CI, 0.68%-9.3%]). Both KV7.1-A283T (16-week male) and KV7.1-R397W (16-week female) mutations were associated with marked KV7.1 loss-of-function consistent with in utero LQTS type 1, whereas the HERG1b-R25W mutation (33.2-week male) exhibited a loss of function consistent with in utero LQTS type 2. In addition, 5 intrauterine fetal deaths hosted SCN5A rare nonsynonymous genetic variants (p.T220I, p.R1193Q, involving 2 cases, and p.P2006A, involving 2 cases) that conferred in vitro electrophysiological characteristics consistent with potentially proarrhythmic phenotypes.In this molecular genetic evaluation of 91 cases of intrauterine fetal death, missense mutations associated with LQTS susceptibility were discovered in 3 cases (3.3%) and overall, genetic variants leading to dysfunctional LQTS-associated ion channels in vitro were discovered in 8 cases (8.8%). These preliminary findings may provide insights into mechanisms of some cases of stillbirth.

Project description:BackgroundIntrauterine growth retardation (IUGR) affects approximately 10% to 15% of all pregnancies worldwide. IUGR is not only associated with stillbirth and newborn death, but also the delay of cognition in childhood and the promotion of metabolic and vascular disorders in adulthood. Figuring out the mechanism of IUGR is rather meaningful and valuable.MethodsDatasets related to IUGR were searched in the Gene Expression Omnibus website. Principal component analysis (PCA) was used for normalization. Differential expressed genes (DEGs) were screened out using the ggpot2 tool. DEGs were used to conduct Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analyses, and protein-protein interaction (PPI) analysis. IUGR related genes were searched in the OMIM website to look for the intersection with the DEGs. The DEGs were analyzed for tissue-specific expression by the online resource BioGPS. The results were displayed through volcano map, Venn map, box plot, heat map, and GSEA enrichment plots drawn by R language packages.ResultsEleven DEGs were screened out of 2 datasets. One hundred ninety-five genes related to IUGR in OMIM were retrieved. EGR2 was the only intersection gene that was found in both groups. Genes associated with placental tissue expression include COL17A1, HSD11B1, and LGALS14. Molecular functions of the DEGs are related to the oxidoreductase activity. The following 4 signaling pathways, reactome signaling by interleukins, reactome collagen degradation, Naba secreted factors, and PID NFAT tfpathway, were enriched by GSEA. Two critical modules comprising 5 up-regulated genes (LEP, PRL, TAC3, MMP14, and ADAMTS4) and 4 down-regulated genes (TIMP4, FOS, CCK, and KISS1) were identified by PPI analysis. Finally, we identified 6 genes (PRL, LGALS14, EGR2, TAC3, LEP, and KISS1) that are potentially relevant to the pathophysiology of IUGR.ConclusionThe candidate down-regulated genes LGALS14 and KISS1, as well as the up-regulated genes PRL, EGR2, TAC3, and LEP, were found to be closely related to IUGR by bioinformatics analysis. These hub genes are related to hypoxia and oxidoreductase activities in placental development. We provide useful and novel information to explore the potential mechanism of IUGR and make efforts to the prevention of IUGR.

Dataset Information

Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death.

Publications

Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death.

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