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Yap/Taz negatively regulate bone angiogenesis and osteogenesis

ABSTRACT: In long bone, the metaphyseal region is less hypoxic than the bone marrow (bone shaft). To study the regional differences of vasculatures, we isolated endothelial cells (ECs) expressing membrane-anchored mTomato protein and nuclear H2B-GFP from Cdh5-mT/nG transgenic reporter femoral metaphysis and diaphyseal bone marrow cavity by fluorescence-activated cell sorting (FACS) at high purity for RNA sequencing (RNA-seq). Next, to study the Hippo signaling in bone ECs, we performed RNA-seq analysis of freshly isolated bone ECs from Cdh5Cre-ERT2-positive Yap1/Taz double floxed or Cdh5Cre-ERT2-positive Lats2 floxed allele and their Cre-negative corresponding littermate controls.

ORGANISM(S): Mus musculus

PROVIDER: GSE102181 | GEO | 2020/01/15

REPOSITORIES: GEO

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Project description:Vitamin A is the only known compound that produces spontaneous fractures in rats. In an effort to resolve the molecular mechanism behind this effect, we fed young rats high doses of vitamin A and performed a global transcriptional analysis of diaphyseal bone after one week, i.e. just before the first fractures appeared. Microarray gene expression analysis revealed that 68 transcripts were differentially expressed in hypervitaminotic cortical bone and 118 transcripts were found when the bone marrow was also included. 98% of the differentially expressed genes in the bone marrow sample were up-regulated. In contrast, hypervitaminotic cortical bone without marrow showed reduced expression of 37% of differentially expressed genes. Gene Ontology (GO) analysis revealed that only samples containing bone marrow were associated to a GO term, which principally represented extracellular matrix (ECM). This is consistent with the histological findings of increased endosteal bone formation. Four of the genes in this ECM cluster and four other genes, including Cyp26b1 which is known to be up-regulated by vitamin A, were selected and verified by real-time PCR. In addition, immunohistochemical staining of bone sections confirmed that the bone-specific molecule, osteoadherin (Omd) was up-regulated. Further analysis of the major gene expression changes revealed distinct differences between cortical bone and bone marrow, e.g. there appeared to be augmented Wnt signaling in the bone marrow but reduced Wnt signaling in cortical bone. Moreover, induced expression of hypoxia-associated genes was only found in samples containing bone marrow. Together, these results corroborate our previous observations of compartment-specific effects of vitamin A, with reduced periosteal but increased endosteal bone formation, and suggest important roles for Wnt signaling and hypoxia in the processes leading to spontaneous fractures. Rat diaphyseal bones from hypervitaminosis A and controls (8week old) were extracted for RNA and hybridized on Affymetrix microarrays. Humeri were isolated, and all connective tissue, including periosteum, was completely removed as were both epiphyses, including growth plates. Samples of cortical bone including marrow were snap-frozen in liquid nitrogen and quickly crushed into a fine powder using a mortar and pestle followed by total RNA extraction with TRI Reagent® (Sigma-Aldrich). For cortical bone without marrow, diaphyseal bones were cut into pieces, vortexed in ice-cold PBS three times for 10 s to remove marrow cells followed by snap-freezing in liquid nitrogen, crushing into a fine powder and RNA extraction. Isolated RNA was quantified using spectrophotometry by measuring the absorbance at 260 nm, and the 260/280 nm ratio was calculated. RNA was kept only when this ratio was 1.9-2.0 to ensure the absence of protein contamination and limited RNA degradation. The integrity of sample RNAs was confirmed by capillary electrophoresis separating 18 S and 28 S ribosomal RNA on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Notably, the metaphyses and growth plates were not included in theses samples. Importantly the amount of RNA extracted from cortical bone including marrow was approximately four times the amount of RNA extracted from the pure cortical bone and was therefore considered herein to mainly represent bone marrow cells.

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Yap/Taz negatively regulate bone angiogenesis and osteogenesis

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