One year of treatment with bisphosphonates at 5x the dose used for post-menopausal osteoporosis significantly increases failure load and microdamage, and decreases toughness at multiple skeletal sites in intact female beagles. The goal of this study was to determine if similar changes occur with doses equivalent to those used for PMO treatment. Skeletallymature female beagles were treated daily for one year with vehicle (VEH) or one of three doses of risedronate (RIS; 0.05, 0.10, 0.50 mg/kg/day) or alendronate (ALN; 0.10, 0.20, 1.00 mg/kg/day). Doses of ALN corresponded to treatment dose for PMO, ½ that dose, and 5x that dose on a mg/kg basis; RIS was given at a dose-equivalent to ALN. Vertebral density, geometry, percent ash, static/dynamic histology, microdamage, and biomechanical parameters were quantified. Trabecular bone activation frequency (Ac.f) was dosedependently lower in RIS-treated groups (-40%,-66%,-84%, p < 0.05 vs VEH) while the three ALN groups were all similarly lower compared to VEH (-65%,-71%,-76%; p < 0.05). Crack surface density (Cr.S.Dn) was significantly higher than VEH for all doses of RIS and ALN (+2.9 to 5.4-fold vs VEH). Stiffness was significantly increased with both agents while there were no significant changes in any other structural or estimated material properties. Cr.S.Dn and Ac.f exhibited a significant non-linear correlation (r 2 =0.21; p<0.001) while there was no relationship between Cr.S.Dn and any mechanical properties. These results document that 1-year of bisphosphonate treatment at clinical doses allows significant accumulation of microdamage in the vertebra but this is offset by increases in bone volume and mineralization such that there is no significant impairment of mechanical properties. 45

Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Antiphosphotyrosine and anti-EGFr affinity chromatography, isotope-coded LC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In

In rheumatoid arthritis (RA), synovial inflammation results in focal erosion of articular bone. Despite treatment attenuating inflammation, repair of erosions with adequate formation of new bone is uncommon in RA, suggesting that bone formation may be compromised at these sites. Dynamic bone histomorphometry was used in a murine model of RA to determine the impact of inflammation on osteoblast function within eroded arthritic bone. Bone formation rates at bone surfaces adjacent to inflammation were similar to those observed in nonarthritic bone; therefore, osteoblast activity is unlikely to compensate for the increased bone resorption at these sites. Within arthritic bone, the extent of actively mineralizing surface was reduced at bone surfaces adjacent to inflammation compared with bone surfaces adjacent to normal marrow. Consistent with the reduction in mineralized bone formation, there was a notable paucity of cells expressing the mid-to late stage osteoblast lineage marker alkaline phosphatase, despite a clear presence of cells expressing the early osteoblast lineage marker Runx2. In addition, several members of the Dickkopf and secreted Frizzled-related protein families of Wnt signaling antagonists were upregulated in arthritic synovial tissues, suggesting that inhibition of Wnt signaling could be one mechanism contributing to impaired osteoblast function within arthritic bone. Together, these data indicate that the presence of inflammation within arthritic bone impairs osteoblast capacity to form adequate mineralized bone, thus contributing to the net loss of bone and failure of bone repair at sites of focal bone erosion in RA.

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Abstract2 Anti-remodeling agents produce similar reductions in vertebral fracture risk despite large 3 differences in BMD changes suggesting the mechanism of fracture risk reduction may 4 differ among these agents. Forty-eight intact (non-ovariectomized) skeletally mature 5 female beagle dogs were treated orally for 12 months with clinically-relevant doses of 6 risedronate (RIS, 0.10 mg/kg/day), alendronate (ALN, 0.2 mg/kg/day), raloxifene (RAL, 7 0.50 mg/kg/day), or saline (VEH, 1 ml/kg/day). After sacrifice, the following 8 measurements were made on vertebral bone: areal (aBMD) and volumetric (vBMD) 9 bone mineral densities, tissue mineralization by ash content, static and dynamic 10 histomorphometric parameters, microdamage, and extrinsic and intrinsic measures of 11 biomechanical strength, stiffness and energy to fracture. At these doses, RAL 12 suppressed bone turnover (-20%) significantly less than the bisphosphonates (-66 and -13 71%), and did not produce significant differences in aBMD, vBMD, BV/TV or percent 14 ash compared to VEH-treated animals. Microdamage accumulation in RAL-treated 15 animals was not significantly different than VEH; both RIS and ALN had significantly 16 higher crack surface density compared to VEH. Stiffness was significantly higher than 17 VEH in all treatment groups. Ultimate load divided by aBMD, a measure of strength 18 independent of BMD, was significantly higher only in RAL-treated animals compared to 19 VEH (+16%, p = 0.015). Based on these data, we conclude that raloxifene produces 20 improvements in bone mechanical properties in ways that do not involve increases in 21 BMD. 22 23

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The H1 histamine receptor (H1HR) is a member of the G protein-coupled receptor superfamily and regulates numerous cellular functions through its activation of the G q/11 subfamily of heterotrimeric G proteins. Although the H1HR has been shown to undergo desensitization in multiple cell types, the mechanisms underlying the regulation of H1HR signaling are poorly defined. To address this issue, we examined the effects of wild type and mutant G protein-coupled receptor kinases (GRKs) on the phosphorylation and signaling of human H1HR in HEK293 cells. Overexpression of GRK2 promoted H1HR phosphorylation in intact HEK293 cells and completely inhibited inositol phosphate production stimulated by H1HR, whereas GRK5 and GRK6 had lesser effects on H1HR phosphorylation and signaling. Interestingly, catalytically inactive GRK2 (GRK2-K220R) also significantly attenuated H1HR-mediated inositol phosphate production, as did an N-terminal fragment of GRK2 previously characterized as a regulator of G protein signaling (RGS) protein for G␣ q/11 . Disruption of this RGS function in holo-GRK2 by mutation (GRK2-D110A) partially reversed the quenching effect of GRK2, whereas deletion of both the kinase activity and RGS function (GRK2-D110A/K220R) effectively relieved the inhibition of inositol phosphate generation. To evaluate the role of endogenous GRKs on H1HR regulation, we used small interfering RNAs to selectively target GRK2 and GRK5, two of the primary GRKs expressed in HEK293 cells. A GRK2-specific small interfering RNA effectively reduced GRK2 expression and resulted in a significant increase in histamine-promoted calcium flux. In contrast, knockdown of GRK5 expression was without effect on H1HR signaling. These findings demonstrate that GRK2 is the principal kinase mediating H1 histamine receptor desensitization in HEK293 cells and suggest that rapid termination of H1HR signaling is mediated by both the kinase activity and RGS function of GRK2.

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