The Wnt/-catenin pathway has been implicated in bone cell response to their mechanical environment. This response is the origin of the mechanism by which bone cells adjust bone architecture to maintain bone strength. Osteoporosis is the most widespread failure of this mechanism. The degree of osteoporotic bone loss in men and women is related to bio-available estrogen. Here we report that in osteoblastic ROS 17/2.8 cells and primary osteoblast cultures, a single short period of dynamic mechanical strain, as well as the glycogen synthase kinase-3 (GSK-3) inhibitor LiCl, increased nuclear accumulation of activated -catenin and stimulated TCF/LEF reporter activity. This effect was blocked by the estrogen receptor (ER) modulators ICI 182,780 and tamoxifen and was absent in primary osteoblast cultures from mice lacking ER␣. Microarray expression data for 25,000 genes from total RNA extracted from tibiae of wild-type mice within 24 h of being loaded in vivo showed differential gene regulation between loaded and contralateral non-loaded bones of 10 genes established to be involved in the Wnt pathway. Only 2 genes were involved in loaded tibiae from mice lacking ER␣ (ER␣ ؊/؊ ). Together these data suggest that Wnt/-catenin signaling contributes to bone cell early responses to mechanical strain and that its effectiveness requires ER␣. Reduced effectiveness of bone cell responses to bone loading, associated with estrogen-related decline in ER␣, may contribute to the failure to maintain structurally appropriate bone mass in osteoporosis in both men and women.
Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak antiresorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass ϳ22-26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22-26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [ 3 H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [ 14 C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rabdependent intracellular membrane trafficking in osteoclasts.
The secreted glycoprotein sclerostin has recently emerged as a key negative regulator of Wnt signaling in bone and has stimulated considerable interest as a potential target for therapeutics designed to treat conditions associated with low bone mass, such as osteoporosis. We have determined the structure of sclerostin, which resulted in the identification of a previously unknown binding site for heparin, suggestive of a functional role in localizing sclerostin to the surface of target cells. We have also mapped the interaction site for an antibody that blocks the inhibition of Wnt signaling by sclerostin. This shows minimal overlap with the heparin binding site and highlights a key role for this region of sclerostin in protein interactions associated with the inhibition of Wnt signaling. The conserved N-and C-terminal arms of sclerostin were found to be unstructured, highly flexible, and unaffected by heparin binding, which suggests a role in stabilizing interactions with target proteins.
Background: Sclerostin, an inhibitor of Wnt signaling, binds to the -propeller domain-containing Wnt co-receptors LRP6 and LRP4. Results: An NXI motif in sclerostin mediates interactions with LRP6 (but not LRP4) and blocks Wnt1 signaling. Conclusion:The sclerostin/LRP6 interaction shares features with the well characterized nidogen/laminin interaction. Significance: NXI motifs are important in mediating interactions with -propeller containing proteins.
The role of mechanical strain and estrogen status in regulating ER␣ levels in bone cells was studied in female rats. OVX is associated with decreased ER␣ protein expression/osteocyte, whereas habitual strain and artificial loading has only a small but positive effect, except on the ulna's medial surface, where artificial loading stimulates reversal of resorption to formation. Introduction: Osteoporosis is the most widespread failure of bones' ability to match their architectural strength to their habitual load bearing. In men and women, the severity of bone loss is associated with bioavailability of estrogen. This association could result from the estrogen receptor (ER) involvement in bone cells' adaptive response to loading. Materials and Methods:In vivo semiquantitative analysis of the amount of ER␣ protein per osteocyte was performed in immuno-cytochemically stained sections from control and loaded rat ulna, as well as tibias of ovariectomy (OVX) and sham-operated female rats. In vitro, the effect of exogenous estrogen (10 −8 M) and mechanical strain (3400 , 1 Hz, 600 cycles) on the expression of ER␣ mRNA levels was assessed in ROS 17/2.8 cells in monolayers using real-time PCR and ER promoter activity. ER␣ translocation in response to exogenous estrogen and mechanical strain was assessed in both ROS 17/2.8 and MLO-Y4 cells. Results: More than 90 percent of tibial osteocytes express ER␣, the level/osteocyte being higher in cortical than cancellous bone. OVX is associated with decreased ER␣ protein expression/osteocyte, whereas in the ulna habitual strain and that caused by artificial loading had only a small but positive effect, except on the medial surface, where loading stimulates reversal of resorption to formation. In unstimulated osteocytes and osteoblasts in situ, and osteocyte-like and osteoblast-like cells in vitro, ER␣ is predominantly cytoplasmic. In vitro, both strain and estrogen stimulate transient ER␣ translocation to the nucleus and transient changes in ER␣ mRNA. Strain but not estrogen also induces discrete membrane localization of ER␣. Conclusions: Bone cells' responses to both strain and estrogen involve ER␣, but only estrogen regulates its cellular concentration. This is consistent with the hypothesis that bone loss associated with estrogen deficiency is a consequence of reduction in ER␣ number/activity associated with lower estrogen concentration reducing the effectiveness of bone cells' anabolic response to strain.
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