Spontaneous recombinase activity of Cre-ERT2 in vivo.

Inducible Cre-ERT recombinase technology is widely used for gene targeting studies. The second generation of inducible Cre-ERT recombinase, hemizygous B6.129S-Tg(UBC-cre/ERT2)1Ejb/J (hereafter abbreviated as Cre-ERT2), a fusion of a mutated estrogen receptor and Cre recombinase, was engineered to be more efficient and specific than the original Cre-ERT. The putative mechanism of selective Cre-mediated recombination is Cre sequestration in the cytoplasm in the basal state with translocation to the nucleus only in the presence of tamoxifen. We utilized both a reporter mouse (B6.129 (Cg)-Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J) and endothelin converting enzyme-1 floxed transgenic mouse line to evaluate Cre-ERT2 activity. We observed spontaneous Cre activity in both settings. Unintended Cre activity is a confounding factor that has a potentially large impact on data interpretation. Thus, it is important to consider background Cre activity in experimental design.

Big endothelin changes the cellular miRNA environment in TMOb osteoblasts and increases mineralization.

INTRODUCTION: Endothelin (ET1) promotes the growth of osteoblastic breast and prostate cancer metastases. Conversion of big ET1 to mature ET1, catalyzed primarily by endothelin converting enzyme 1 (ECE1), is necessary for ET1's biological activity. We previously identified the Ece1, locus as a positional candidate gene for a pleiotropic quantitative trait locus affecting femoral size, shape, mineralization, and biomechanical performance. METHODS: We exposed TMOb osteoblasts continuously to 25 ng/ml big ET1. Cells were grown for 6 days in growth medium and then switched to mineralization medium for an additional 15 days with or without big ET1, by which time the TMOb cells form mineralized nodules. We quantified mineralization by alizarin red staining and analyzed levels of miRNAs known to affect osteogenesis. Micro RNA 126-3p was identified by search as a potential regulator of sclerostin (SOST) translation. RESULTS: TMOb cells exposed to big ET1 showed greater mineralization than control cells. Big ET1 repressed miRNAs targeting transcripts of osteogenic proteins. Big ET1 increased expression of miRNAs that target transcripts of proteins that inhibit osteogenesis. Big ET1 increased expression of 126-3p 121-fold versus control. To begin to assess the effect of big ET1 on SOST production we analyzed both SOST transcription and protein production with and without the presence of big ET1 demonstrating that transcription and translation were uncoupled. CONCLUSION: Our data show that big ET1 signaling promotes mineralization. Moreover, the results suggest that big ET1's osteogenic effects are potentially mediated through changes in miRNA expression, a previously unrecognized big ET1 osteogenic mechanism.

Blood pressure, artery size, and artery compliance parallel bone size and strength in mice with differing ece1 expression.

The recombinant congenic mouse strains HcB-8 and HcB-23 differ in femoral shape, size, and strength, with HcB-8 femora being more gracile, more cylindrical, weaker, and having higher Young's modulus. In previous work, we mapped a robust, pleiotropic quantitative trait locus for these bone traits. Ece1, encoding endothelin converting enzyme 1, is a positional candidate gene for this locus, and was less expressed in HcB-8 bone. We hypothesized that the same genetic factors would impose analogous developmental trajectories on arteries to those in bones. Cardiovascular hemodynamics and biomechanics of carotids were measured in adult HcB-8 and HcB-23 mice. Biological differences in heart and arteries were examined at mRNA and protein levels. As in bone, Ece1 expression was higher in HcB-23 heart and arteries (p < 0.05), and its expression was correlated with that of the endothelin B type receptor target Nos3, encoding endothelial nitric oxide synthase. HcB-8 mice had higher ambulatory blood pressure (p < 0.005) than HcB-23 mice. Ex vivo, at identical pressures, HcB-8 carotid arteries had smaller diameters and lower compliance (p < 0.05), but the same elastic modulus compared to HcB-23 carotid arteries. HcB-8 hearts were heavier than HcB-23 hearts (p < 0.01). HcB-8 has both small, stiff bones and small, stiff arteries, lower expression of Ece1 and Nos3, associated in each case with less favorable function. These findings suggest that endothelin signaling could serve as a nexus for the convergence of skeletal and vascular modeling, providing a potential mechanism for the epidemiologic association between skeletal fragility and atherosclerosis.

Placental endothelin-converting enzyme-1 is decreased in preeclampsia.

Endothelin-converting enzyme-1(ECE-1) is a key regulatory enzyme in the processing of endothelin-1 (ET-1). We quantified and localized ECE-1 in normal and preeclamptic placentas. Normal (n=6) and preeclamptic (n=6) placentas were serially sectioned for immunofluorescence (IF). Cell type specific markers identified endothelial, trophoblast, macrophage, smooth muscle, and fibroblast cells. Quantitative analyses were performed by western blot and ELISA. IF identified ECE-1 expression within the stroma and villous space. Cellular localization of ECE-1 was limited to endothelial membranes. There was significantly less ECE-1 in preeclamptic placentas, suggesting ECE-1 is important for proper regulation of ET-1 within the placenta.

Endothelin Signaling in Bone.

The endothelin (ET) system includes 3 small peptide hormones and a pair of G-protein-coupled receptors. This review first outlines the ET signaling pathway and ET metabolism. Next, it summarizes the role of ET1 signaling in craniofacial development. Then, it discusses observations relating ET signaling to osteoblastic and other osteosclerotic processes in cancer. Finally, it describes recent work in our laboratory that points to endothelin signaling as an upstream mediator of WNT signaling, promoting bone matrix synthesis and mineralization. It concludes with a statement of some remaining gaps in knowledge and proposals for future research.

Endothelin signaling regulates mineralization and posttranscriptionally regulates SOST in TMOb cells via miR 126-3p.

Previously, our laboratory identified ECE-1, encoding endothelin-converting enzyme-1 (ECE-1), as a positional candidate for a pleiotropic quantitative trait locus affecting femoral size, shape, and biomechanical performance. We hypothesized that endothelin-1 (ET-1) signaling promotes osteogenesis. Exposure of immortalized mouse osteoblast (TMOb) cells to big ET-1 increased mineralization. Following big ET-1 treatment, we measured the secretion of insulin-like-growth factor-1 (IGF1), dickkopf-homolog-1 protein 1 (DKK1), and sclerostin (SOST). In each case, big ET-1 signaling changed secretion in a manner that favored increased osteogenic activity. Treatment with ECE-1, endothelin receptor A (EDNRA), or WNT receptor antagonists inhibited the big ET-1-mediated increase in mineralization. In the presence of big ET-1, message levels of Runx2, Igf1, Dkk1, and Sost are uncoupled from protein production, suggesting posttranscriptional regulation. To evaluate the role of big ET-1 in normal bone physiology, we inhibited EDNRA signaling during mineralization in the absence of exogenous ET-1. EDNRA blockade reduced mineralization, decreased IGF1, and increased DKK1 and SOST secretion, responses opposite to those induced by exogenous big ET-1. Pharmacological and siRNA knockdown to inhibit ECE-1 reduced mineralization and IGF1 secretion with decreasing DKK1 and decreasing or stable SOST secretion, suggesting a further, unknown role of ECE-1 in osteoblast maturation. Previously we identified miR 126-3p as a potential ET-1-responsive regulator of SOST in murine cells. Overexpression of miR126-3p increased mineralization in TMOb cells and decreased SOST secretion. Osteoblasts express the ET-1 signaling pathway and ET-1 signaling is necessary for normal osteoblast differentiation and mineralization, acting through regulation of miRs that target osteogenic molecules.

Congenic Strains Confirm the Pleiotropic Effect of Chromosome 4 QTL on Mouse Femoral Geometry and Biomechanical Performance.

A pleiotropic quantitative trait locus (QTL) for bone geometry and mechanical performance in mice was mapped to distal chromosome 4 via an intercross of recombinant congenic mice HcB-8 and HcB-23. To study the QTL in isolation, we have generated C3H.B10-(rs6355453-rs13478087) (C.B.4.3) and C3H.B10-(rs6369860-D4Mit170) (C.B.4.2) congenic strains that harbor ~20 Mb and ~3 Mb, respectively, of chromosome 4 overlapping segments from C57BL/10ScSnA (B10) within the locus on a C3H/DiSnA (C3H) background. Using 3-point bend testing and standard beam equations, we phenotyped these mice for femoral mid-diaphyseal geometry and biomechanical performance. We analyzed the results via 2-way ANOVA, using sex and genotype as factors. In the C.B.4.3 strain, we found that homozygous B10/B10 male mice had smaller cross sectional area (CSA) and reduced total displacement than homozygous C3H/C3H mice. Sex by genotype interaction was also observed for maximum load and stiffness for C3H/C3H and B10/B10 mice, respectively. In C.B.4.2 strain, we found that homozygous B10/B10 mice had lower total displacement, post-yield displacement (PYD), stiffness, yield load and maximum load than mice harboring C3H allele. Sex by genotype interaction was observed in B10/B10 mice for perimeter, outer minor axis (OMA) and CSA. There were no significant differences in tissue level mechanical performance, which suggest that the QTL acts primarily on circumferential bone size. These data confirm the prior QTL mapping data and support other work demonstrating the importance of chromosome 4 QTL on bone modeling and bone responses to mechanical loading.

Role of Endothelin-1 in a Syndrome of Myelofibrosis and Osteosclerosis.

CONTEXT: Primary myelofibrosis is one of the chronic myeloproliferative disorders characterized by bone marrow fibrosis associated with extramedullary hematopoiesis and osteosclerosis. Endothelin-1 (ET1) is a potent vasoconstrictor that is also a key mediator of osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation. CASE DESCRIPTION: We report laboratory, radiographic, bone densitometry, and bone histology data of a patient presenting with newly diagnosed, biopsy-proven myelofibrosis and osteosclerosis. We were able to demonstrate abundant ET1 signaling in the bones of our patient. CONCLUSIONS: We believe that ET1 is responsible for the osteosclerosis that develops with advanced myelofibrosis and suggest that ET1 signaling may play a role in other osteosclerotic settings as well.