Research Of The Mechanisms Of The Effects Of ER-?36, LOC147710 And Omentin-1 On The Regulation Of Bone Metabolism

Part one Estrogen Receptor-?36 Mediates a Bone-Sparing Effect of 17?-Estrodiol in Postmenopausal WomenObject Estrogen receptor-alpha36 (ER-?36) is a membrane-based protein. Unlike ER-?66?ER-?46 and ER-?which mediate nuclear effects of estrogens, ER-?36 only mediates membrane effects of estrodiol, such as activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway. This study was aimed to investigate whether ER-?36 is involved in the regulation of bone metabolism as done by ER-?66.Methods The expression of ER-?36 protein in bone sections was assessed by immunohistochemistry. Cell proliferation and apoptosis were analyzed by 3H-thymidine incorporation and ELISA, respectively. Alkaline phosphatase (ALP) activity was assayed by measurement of p-nitrophenol. Osteocalcin (OCN) was measured by radioimmunoassay (RIA). Matrix mineralization was examined with alizarin red S staining. Expression levels of ER-?36, ER-?66, ERK1/2 and p-ERK1/2 protein were detected by Western blot. Reactive oxygen species (ROS) level was measured by 2',7'-dichlorodihydrofluorescein diacetate (H2-DCFDA) probe. To investigate the involvement of ER-?36 and post-receptor ERK and ROS signaling pathways in the regulation of proliferation, differentiation or apoptosis of osteoblasts and osteoclasts by E2, ER-?36 shRNA and eukaryon expression vector were used for regulating ER-?36 expression, and, inhibitors for ERK and ROS were also used. Expression levels of OCN and tartrate-resistant acid phosphatase (TRAP) mRNA were analyzed by quantitative real-time PCR (QRT-PCR). Furthermore, we selected 154 female operative cases (premenopausal:60; postmenopausal:33 normal,31 osteopenic, and 30 osteoporotic) who underwent surgery for intervertebral disk hernia, spinal stenosis or spondylolisthesis were selected in this study. BMD was measured using a dual-energy X-ray absorptiometry (DXA). Blood samples were collected for detection of serum levels of bone formation marker OCN and bone resorption marker cross-linked N-telopeptides of type?collagen (NTX). We also performed QRT-PCR for detection of the mRNA levels of ER-?36, ER-?66, ER-a46 and ER-?in collected vertebral cancellous bone explants in these patients. Furthermore, animal experiments were performed to observe the effects of the IC162, a selective ER-?36 modulator, on the bone metabolism in ovariectomized mice.Results (1) Cell culture experiments results:ER-?36 mediated mitogenic, antiapoptotic, and antiosteogenic effects of postmenopausal low level (10 pM) E2 in osteoblasts through transient activation of the ERK, whereas ER-?36 mediated proapoptotic effect of 10 pM E2 in osteoclasts through prolonged activation of the ERK with the involvement of ROS; 10 pM E2 had no effect on the proliferation, apoptosis or differentiation of ER-?36 negative osteoblasts and osteoclasts. (2) Bone tissue culture experiments results:10 pM E2 significantly inhibited the mRNA expression of osteoblast marker OCN and osteoclast marker TRAP in bone tissues that express high levels of endogenous ER-?36, while had no significant effect on the mRNA levels of OCN and TRAP in bone tissues that express extremely low levels of endogenous ER-?36. (3) The ANOVA showed that the mRNA levels of ER-?36 in the premenopausal group were significantly lower than in the three postmenopausal groups; the mRNA levels of ER-?36 in the normal postmenopausal group were significantly higher than in the other two postmenopausal groups (osteoporotic or osteopenic); the mRNA levels of ER-?36 in the osteopenic group were significantly higher than in the osteoporotic group; there was a decrease of ER-?46, ER-?66 and ER-?levels in all postmenopausal groups compared with the premenopausal group; there was no significant difference in mRNA levels of ER-?46, ER-?66 and ER-?expression among all postmenopausal groups. (4) The correlation analysis showed that in postmenopausal women, a significant positive correlation was observed between the levels of ER-?36 mRNA in bone and BMD, while a significantly negative relationship was observed between the levels of ER-?36 mRNA in bone and serum OCN and NTX; no significant correlations were observed between the levels of ER-?36 mRNA in bone and BMD and serum OCN and NTX in premenopausal women; no significant correlations were found between the levels of ER-?46, ER-?66 and ER-?mRNA in bone with BMD and serum OCN and NTX in both pre- and post-menopausal women. (5) Animal experiment results:In ovariectomized mice, IC162 increased BMD but had no effect on the body weight and uterus weight.Conclusions ER-?36, but not ER-?66, ER-a46 or ER-?, is a critical regulator of bone metabolism in postmenopausal women�osteoblasts and osteoclasts expressing high levels of ER-?36 are hypersensitive to E2, ER-?36 mediates a bone-sparing effect of the low level of E2 in postmenopausal women. IC162, a selective ER-?36 modulator, exerts bone-sparing effect in ovariectomized mice. Part twoIdentification of a hypothetical protein LOC147710 mutation associated with autosomal recessive osteopetrosisObject Osteopetrosis is a highly heterogeneous group of inherited skeleton disorder characterized by a generalized increase in bone mineral density due to a failure of osteoclast differentiation or function. To date, loss-of-function mutations in 11 genes have been identified as being associated with human osteopetrosis. All of the heritage osteopetrosis patients were caused by single gene mutation. In this study, the homozygous genotype of 5 members of a Chinese family (including 11 members) affected by an autosomal recessive osteopetrosis was investigated with the aim of identifying novel gene mutations associated with osteopetrosis in this family.Methods BMD was measured using a dual-energy X-ray absorptiometry (DXA). Total genomic DNA was isolated from peripheral blood of all 11 family members and 180 normal controls. Genome-wide scan and fine mapping study and haplotype analysis were performed to identify the critical region. Exons of genes located in the critical interval were sequenced to identify gene mutation. Northern blot and Western blot were performed to reveal the the expression pattern of LOC147710 in human tissues and in the osteoclast precursor CD14+ peripheral blood mononuclear cells (PBMCs) which undergoing osteoclastic differentiation. Western blot was also used for detection of the subcellular localization of LOC147710 protein. TRAP staining and Toluidine blue staining were used for observing osteoclast formation and activity, respectively. LOC147710 eukaryon expression vector was constructed and transfected to LOC147710-/- PBMCs for investigating whether the osteoclastic differentiation defect of LOC147710-/- PBMCs could be restored by re-introduction of LOC147710.Results A single critical region of homozygosity on chromosome 19q13.2-ql3.3 between D19S197 and D19S545 spanning 8.36 cM with maximum LOD score of 2.907 at?=0 was identified. Sequencing of the genes in this region revealed a homozygous nonsense mutation (c.295C>T) in exon 2 of the LOC147710 gene. This mutation introduced a premature stop codon, resulting in p.R99X in the translated protein. LOC147710 mRNA is primarily detected in human bone and small intestine, but not in other tissues. In vitro, LOC147710 mRNA was expressed in human primary osteoclasts, but not in huamn osteoblasts and PBMCs. LOC147710 mRNA and protein expression in PBMCs increased progressively during the osteoclastic differentiation process induced by macrophage colony-stimulating factor (M-CSF) and receptor activator for nuclear factor?B ligand (RANKL). LOC147710-/- PBMCs from the osteopetrosis subject exhibited poor ability to differentiate into osteoclasts upon exposure to M-CSF and RANKL. Furthermore, reintroduction of LOC147710 restored the ability of LOC147710-/ PBMCs to differentiate into functional osteoclasts in the presence of M-CSF and RANKL.Conclusions This study showed that a loss-of-function homozygous mutation in LOC147710 gene caused a novel type of autosomal recessive osteopetrosis via down-regulating osteoclast differentiation. Part threeOmentin-1 exerts bone-sparing effect in ovariectomized mice and osteoprotegerin gene knockout miceObject Omentin-1 is a recently identified visceral adipose tissue-derived cytokine and is highly abundant in plasma. This study was undertaken to investigate the effect of omentin-1 on bone metabolism.Methods Osteoblast differentiation was assessed by measuring ALP activity, osteocalcin production and matrix mineralization. ELISA was used to detect receptor activator for nuclear factor?B ligand (RANKL) and osteoprotegerin (OPG) protein expression in osteoblasts. The effect of recombinant omentin-1 on osteoclasts formation was examined in the co-culture systems of osteoblasts and osteoclast precursors. The effects of omentin-1 on bone mass, bone strength and bone turnover were examined in the ovariectomized (OVX) mice or OPG gene knockout (OPG-/-) mice by injection of omentin-1 adenovirus.Results (1) In vitro, omentin-1 directely inhibited osteoblast differentiation, but had no direct effect on osteoclast differentiation; however, it indirectly reduced osteoclast formation in the co-culture systems of osteoblasts and osteoclast precursors through stimulating OPG and inhibiting RANKL production in osteoblasts. (2) In vivo, omentin-1 partially restored bone mineral density (BMD) and bone strength in OVX mice, accompanied by decreased levels of plasma osteocalcin (OCN) and tartrate-resistant acid phosphatase-5b (TRAP-5b) and a lower serum RANKL/OPG ratio. (3) Omentin-1 partially restored BMD in OPG-mice, accompanied by decreased levels of serum RANKL, OCN and TRAP-5b.Conclusions The present study suggests that omentin-1 ameliorates bone loss induced by estrogen-deficiency or OPG gene knockout via regulating RANKL and OPG expression.