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Wnt signaling plays a key role in regulating bone remodeling. In vitro studies suggest that sclerostin's inhibitory action on Lrp5 is facilitated by the membrane-associated receptor Lrp4. We generated an Lrp4 R1170W knockin mouse model (Lrp4KI), based on a published mutation in patients with high bone mass (HBM). Lrp4KI mice have an HBM phenotype (assessed radiographically), including increased bone strength and formation. Overexpression of a Sost transgene had osteopenic effects in Lrp4-WT but not Lrp4KI mice. Conversely, sclerostin inhibition had blunted osteoanabolic effects in Lrp4KI mice. In a disuse-induced bone wasting model, Lrp4KI mice exhibit significantly less bone loss than wild-type (WT) mice. In summary, mice harboring the Lrp4-R1170W missense mutation recapitulate the human HBM phenotype, are less sensitive to altered sclerostin levels, and are protected from disuse-induced bone loss. Lrp4 is an attractive target for pharmacological targeting aimed at increasing bone mass and preventing bone loss due to disuse.

Mechanical stimulation is a key regulator of bone mass, maintenance, and turnover. Wnt signaling is a key regulator of mechanotransduction in bone, but the role of β-catenin-an intracellular signaling node in the canonical Wnt pathway-in disuse mechanotransduction is not defined. Using the β-catenin exon 3 flox (constitutively active [CA]) mouse model, in conjunction with a tamoxifen-inducible, osteocyte-selective Cre driver, we evaluated the effects of degradation-resistant β-catenin on bone properties during disuse. We hypothesized that if β-catenin plays an important role in Wnt-mediated osteoprotection, then artificial stabilization of β-catenin in osteocytes would protect the limbs from disuse-induced bone wasting. Two disuse models were tested: tail suspension, which models fluid shift, and botulinum-toxin (botox)-induced muscle paralysis, which models loss of muscle force. Tail suspension was associated with a significant loss of tibial bone mass and density, reduced architectural properties, and decreased bone formation indices in uninduced (control) mice, as assessed by dual-energy X-ray absorptiometry (DXA), micro-computed tomography (µCT), and histomorphometry. Activation of the βcatCA allele in tail-suspended mice resulted in little to no change in those properties; ie, these mice were protected from bone loss. Similar protective effects were observed among botox-treated mice when the βcatCA was activated. RNAseq analysis of altered gene regulation in tail-suspended mice yielded 35 genes, including Wnt11, Gli1, Nell1, Gdf5, and Pgf, which were significantly differentially regulated between tail-suspended β-catenin stabilized mice and tail-suspended nonstabilized mice. Our findings indicate that selectively targeting/blocking of β-catenin degradation in bone cells could have therapeutic implications in mechanically induced bone disease. © 2019 American Society for Bone and Mineral Research.

Maintenance of bone structural integrity depends in part on the rate of apoptosis of bone-forming osteoblasts. Because substrate adhesion is an important regulator of apoptosis, we have investigated the role of focal adhesions in regulating bone cell apoptosis. To test this, we expressed a truncated form of [alpha]-actinin (ROD-GFP) that competitively displaces endogenous [alpha]-actinin from focal adhesions, thus disrupting focal adhesions. Immunofluorescence and morphometric analysis of vinculin and tyrosine phosphorylation revealed that ROD-GFP expression dramatically disrupted focal adhesion organization and reduced tyrosine phosphorylation at focal adhesions. In addition, Bcl-2 protein levels were reduced in ROD-GFP-expressing cells, but caspase 3 cleavage, poly-(ADP-ribose) polymerase cleavage, histone H2A.X phosphorylation, and cytotoxicity were not increased due to ROD-GFP expression alone. Increases in both ERK and Akt phosphorylation were also observed in ROD-GFP-expressing cells, although inhibition of either ERK or Akt individually or together failed to induce apoptosis. However, we did find that ROD-GFP expression sensitized, whereas [alpha]-actinin-GFP expression protected, cells from TNF-[alpha]-induced apoptosis. Further investigation revealed that activation of TNF-[alpha]-induced survival signals, specifically Akt phosphorylation and NF-[kappa]B activation, was inhibited in ROD-GFP-expressing cells. The reduced expression of antiapoptotic Bcl-2 and inhibited survival signaling rendered ROD-GFP-expressing cells more susceptible to TNF-[alpha]-induced apoptosis. Thus we conclude that [alpha]-actinin plays a role in regulating cell survival through stabilization of focal adhesions and regulation of TNF-[alpha]-induced survival signaling. tumor necrosis factor-[alpha]; survival; cytoskeleton; nuclear factor-[kappa]B

An investigation of smooth muscle contraction in the canine extrathoracic trachea has provided evidence for the role of tyrosine phosphorylation of paxillin in the regulation of muscle contraction. Western blot analysis showed an increase in tyrosine phosphorylation of paxillin during contraction but not during relaxation.

Immunoelectronic microscopy indicates that the cytoplasmic domain of leukocyte adhesion molecule L-selectin interacts with the cytoskeletal proteins via alpha-actinin. These interactions are not required for localization of L-selectin to the microvilli. Vinculin and Talin are the two cytoskeletal proteins involved in the L-selectin-cytoskeleton interaction.

The study of phosphorylation of paxillin and dense-plaque proteins talin reveals that anchoring of contractivle filaments to the cell membrane results in the pharmacological activation of airway smooth muscles. The stimulation of membranes with Ach results in the phosphorylation of the tyrosine residues efficiently.

Disruption of microfilaments in human umbilical vein endothelial cells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA) resulted in a 3.3- to 5.7-fold increase in total synthesis of prostaglandin [E.sub.2] ([PGE.sub.2]) and a 3.4- to 6.5-fold increase in prostacyclin ([PGI.sub.2]) compared with control cells. Disruption of the microtubule network with nocodazole or colchicine increased synthesis of [PGE.sub.2] 1.7- to 1.9-fold and [PGI.sub.2] 1.9- to 2.0-fold compared with control cells. Interestingly, however, increased release of [PGE.sub.2] and [PGI.sub.2] from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold more [PGE.sub.2] and 3.8-fold more [PGI.sub.2] released from HUVEC compared with control cells; latA treatment resulted in 17.7-fold more [PGE.sub.2] and 11.2-fold more [PGI.sub.2] released compared with control cells. Both increased synthesis and release of prostaglandins in response to all drug treatments were completely inhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2). Disruption of either microfilaments using cytD or latA or of microtubules using nocodazole or colchicine resulted in a significant increase in COX-2 protein levels, suggesting that the increased synthesis of prostaglandins in response to drug treatments may result from increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest that the cytoskeleton plays an important role in maintenance of endothelial barrier function by regulating prostaglandin synthesis and release from HUVEC. cyclooxygenase; microfilaments; microtubules; vasoprotection; prostaglandin [E.sub.2]; prostacyclin

[Ca.sup.2+] regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts. Am J Physiol Cell Physiol 278: C989-C997, 2000.--Osteoblasts subjected to fluid shear increase the expression of the early response gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]][.sub.i]) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/[cm.sup.2]). Before application of shear, cells were treated with two [Ca.sup.2+] channel inhibitors or various blockers of intracellular [Ca.sup.2+] release for 0.5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [[Ca.sup.2+]][.sub.i] response, neither gadolinium nor nifedipine, an L-type channel [Ca.sup.2+] channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intracellular [Ca.sup.2+] chelator, or thapsigargin, which empties intracellular [Ca.sup.2+] stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate ([IP.sub.3])-induced intracellular [Ca.sup.2+] release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that [IP.sub.3]-mediated intracellular [Ca.sup.2+] release is required for modulating flow-induced responses in MC3T3-E1 cells. actin cytoskeleton; intracellular calcium, c-fos; cyclooxygenase-2; phospholipase C

Recent studies suggest that megakaryocytes (MKs) may play a significant role in skeletal homeostasis, as evident by the occurrence of osteosclerosis in multiple MK related diseases (Lennert et al., 1975; Thiele et al., 1999; Chagraoui et al., 2006). We previously reported a novel interaction whereby MKs enhanced proliferation of osteoblast lineage/osteoprogenitor cells (OBs) by a mechanism requiring direct cell-cell contact. However, the signal transduction pathways and the downstream effector molecules involved in this process have not been characterized. Here we show that MKs contact with OBs, via beta1 integrin, activate the p38/MAPKAPK2/p90RSK kinase cascade in the bone cells, which causes Mdm2 to neutralizes p53/Rb-mediated check point and allows progression through the G1/S. Interestingly, activation of MAPK (ERK1/2) and AKT, collateral pathways that regulate the cell cycle, remained unchanged with MK stimulation of OBs. The MK-to-OB signaling ultimately results in significant increases in the expression of c-fos and cyclin A, necessary for sustaining the OB proliferation. Overall, our findings show that OBs respond to the presence of MKs, in part, via an integrin-mediated signaling mechanism, activating a novel response axis that de-represses cell cycle activity. Understanding the mechanisms by which MKs enhance OB proliferation will facilitate the development of novel anabolic therapies to treat bone loss associated with osteoporosis and other bone-related diseases. J. Cell. Physiol. 230: 578-586, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company [ABSTRACT FROM AUTHOR]