Osteoclast Attachment Research Articles

Tyrosine Kinase Src Is a Regulatory Factor of Bone Homeostasis.

Osteoclasts, which resorb the bone, and osteoblasts, which form the bone, are the key cells regulating bone homeostasis. Osteoporosis and other metabolic bone diseases occur when osteoclast-mediated bone resorption is increased and bone formation by osteoblasts is decreased. Analyses of tyrosine kinase Src-knockout mice revealed that Src is essential for bone resorption by osteoclasts and suppresses bone formation by osteoblasts. Src-knockout mice exhibit osteopetrosis. Therefore, Src is a potential target for osteoporosis therapy. However, Src is ubiquitously expressed in many tissues and is involved in various biological processes, such as cell proliferation, growth, and migration. Thus, it is challenging to develop effective osteoporosis therapies targeting Src. To solve this problem, it is necessary to understand the molecular mechanism of Src function in the bone. Src expression and catalytic activity are maintained at high levels in osteoclasts. The high activity of Src is essential for the attachment of osteoclasts to the bone matrix and to resorb the bone by regulating actin-related molecules. Src also inhibits the activity of Runx2, a master regulator of osteoblast differentiation, suppressing bone formation in osteoblasts. In this paper, we introduce the molecular mechanisms of Src in osteoclasts and osteoblasts to explore its potential for bone metabolic disease therapy.

The Effect of Low-Temperature Thermal Processing on Bovine Hydroxyapatite Bone Substitutes, toward Bone Cell Interaction and Differentiation.

Ideal bone grafting scaffolds are osteoinductive, osteoconductive, and encourage osteogenesis through the remodeling processes of bone resorption, new bone formation, and successful integration or replacement; however, achieving this trifecta remains challenging. Production methods of bone grafts, such as thermal processing, can have significant effects on the degree of cell-surface interactions via wide-scale changes in the material properties. Here, we investigated the effects of small incremental changes at low thermal processing temperatures on the degree of osteoclast and osteoblast attachment, proliferation, and differentiation. Bovine bone scaffolds were prepared at 100, 130, 160, 190, and 220 °C and compared with a commercial control, Bio-Oss®. Osteoclast attachment and activity were significantly higher on lower temperature processed bone and were not present ≥190 °C. The highest osteoblast proliferation and differentiation were obtained from treatments at 130 and 160 °C. Similarly, qRT2-PCR assays highlighted osteoblasts attached to bone processed at 130 and 160 °C as demonstrating the highest osteogenic gene expression. This study demonstrated the significant effects of small-scale processing changes on bone graft materials in vitro, which may translate to a tailored approach of cellular response in vivo.

Tetraspanin 7 regulates osteoclast function through association with the RANK/αvβ3 integrin complex.

Actin rings are unique structures that facilitate the attachment of osteoclasts to the bone matrix during bone resorption. Previous studies have shown that tetraspanin7 (TSPAN7) plays an important role in the reorganization of the cytoskeleton necessary for the bone-resorbing activity of osteoclasts. However, questions remain as to the mechanisms by which TSPAN7 regulates this cytoskeletal rearrangement. In this study, we investigated the roles of TSPAN7 in osteoclasts by deleting the Tm4sf2 gene in mice, which encodes TSPAN7. The Tm4sf2 global knockout model showed protective effects on pathological bone loss, but no discernible changes in bone phenotypes under physiological conditions. In vitro study revealed that ablation of Tm4sf2 caused significant defects in integrin-mediated actin ring formation, thereby leading to significantly decreased bone resorption. Additionally, we demonstrated an association between TSPAN7 and the receptor activator of nuclear factor-кB/αvβ3 integrin. Overall, our findings suggest that TSPAN7 acts as a novel modulator regulating the bone-resorbing function of osteoclasts.

Biocomputational Analysis of ARAP1, a novel GTPase‐Activating‐Protein Involved in Osteoclast‐Mediated Bone Digestion

RhoGTPases are important regulators of many cellular activities including cell proliferation, trafficking of proteins, DNA transcription, and apoptosis. RhoGTPases are also involved in bone remodeling, the continuous process of bone digestion, and formation that occurs during one's lifespan. Any abnormalities in these Rho Proteins can lead to physiological effects such as osteoporosis, characterized by porous bone, or osteopetrosis, characterized by highly-dense bone tissue. RhoA-GTP is responsible for disrupting sealing zone dynamics and promoting cell motility in osteoclasts. On the other hand, Cdc42-GTP and Rac1-GTP maintain osteoclast attachment to the bone surface, thereby allowing it to continue enzymatic degradation of bone. To employ RhoGTPases as potential drug targets, we need to further understand the complex physiology and control mechanisms for these G-proteins. ARAP1 has been shown to localize to the osteoclast sealing zones upon the release of phosphoinositide (3,4,5) trisphosphate, which binds to its first and third PH domains. ARAP1 belongs to the RhoGAP family of proteins and has a RhoGAP domain responsible for catalyzing the intrinsic GTPase activity of G proteins. However, it is still unknown which GTPase (i.e. RhoA, Cdc42, Rac1) preferentially binds to the ARAP1 catalytic domain. Delineating the substrate specificity for the RhoGAP domain is crucial to its characterization as a pro-migration or pro-digestion protein, allowing us to specifically target its function in managing osteoporosis or osteopetrosis-related illnesses. In this computational study, we have modeled the ARAP1 RhoGAP domain using homology modeling and ab initio techniques, characterized its biophysical features, and used docking analysis to scrutinize its interactions with potential substrates. The docking results suggest that ARAP1 shows a preferential substrate specificity for RhoA. This indicates that ARAP1-RhoGAP may behave in a similar manner as ARAP3-RhoGAP, which has been shown to prefer RhoA-targeting in an experimental setup. Lastly, we report an in silico mutagenesis analysis that induces RhoA-like mutations in Cdc42/Rac1 and provides structural data that supports our hypothesis of an ARAP1-RhoA preferential attraction. Overall, this study reveals new insights into the structure-function relations of the RhoGAP domain, specifically pertaining to its role in osteoclast-mediated bone digestion, using an integrated and comprehensive strategy of sequence and structure analyses. The novel findings from our study regarding the molecular mechanism of GTPases in the osteoclast lays the groundwork towards developing therapies that improve the quality of life for those suffering from chronic bone pathologies.

Role of CaMKK2 in Osteoclast Development and Function

Background and Hypothesis: In bone remodeling there are two opposing forces, osteoclast resorption and osteoblast synthesis of bone. When there is an imbalance, osteoporosis is likely found. We look to determine the role that Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) has on osteoclast development and attachment. Previous studies show that Camkk2-/- mice have fewer mature osteoclasts that display poor attachment to the bone. These results suggest that there is a defect in facilitating osteoclast differentiation, or a defect in the attachment itself. Thus, we hypothesize that CaMKK2 plays an integral role in the development and function of the osteoclast cytoskeleton.
 Experimental Design or Project Methods: We plated different concentrations of bone marrow cells isolated from male and female, wildtype and Camkk2-/- mice to investigate size and number of mature osteoclasts present. We examined the size of the resorption pits made by these osteoclasts, when plated on calcium phosphate-coated surfaces. We analyzed the RNA and protein and stained the cells immunohistochemically for proteins associated with cell attachment.
 Results: There were greater numbers of mature osteoclasts in the Camkk2-/- males at each density than the wildtype males. The female mice followed the same trend, but with fewer osteoclasts overall.
 The average area per resorbed pit was two-times larger in the WT males than the Camkk2-/- males. But, for the females the opposite trend is shown, the pits from the Camkk2-/- females were twice as large as those formed from the wildtype females.
 To gauge efficiency, we calculated the average area resorbed per osteoclast. For the males, significantly greater efficiency from the wildtype osteoclasts was found than the Camkk2-/- osteoclasts. For the females, there was no difference in efficiency at the lower plating densities, but the osteoclasts from the Camkk2-/- females had greater efficiency at the greater densities.
 Remaining results are to be finalized. 
 Conclusion and Potential Impact: With the decrease in efficiency of Camkk2-/- mice osteoclasts, we believe that CaMKK2 is necessary for the attachment of OCs. CaMKK2 could be a target to slow the pathologic breakdown of bone.

Extracellular calcium regulates the adhesion and migration of osteoclasts via integrin αv β 3 /Rho A/Cytoskeleton signaling.

Integrin αv β3 is a transmembrane integrin, which can initiate osteoclasts' attachment on bones, leading to downward signaling pathways and subsequent bone resorption. Different calcium concentrations have been reported to have an influence on the activation of integrin αv β3 . To elucidate the regulatory mechanism of extracellular calcium concentrations on osteoclasts, a controlled micro flow plate (M04S) was utilized in the ONIX flow control system to observe the osteoclasts' adhesion and migration in different calcium concentration media. Fluorescent staining is conducted to show the distribution of integrin αv β3 and cytoskeleton reorganization. In addition, western blots were performed to detect the expression of integrin αv β3 and its downstream signaling pathways related to bone resorption. Also, real-time reverse-transcription polymerase chain reaction data of transcription co-activator (YAP/TAZ) and hydrolytic enzymes (the matrix metalloproteinase 9 and cathepsin K) areevaluated. Our findings suggest that osteoclasts' migration and adhesion is better promoted at 0.5 mM than 1.2 mM, which can be partly explained by the induced cytoskeleton organization via integrin αv β3 /Rho GTPase. But the activation and nuclear localization of YAP/TAZ, and the secretion of hydrolytic enzymes were upregulated when the calcium concentration is at a higher level (1.2 mM). According to our study, there is a highpossibility that the migration and attachment of osteoclasts and subsequent osteoclastic bone resorption are regulated over a specific range of extracellular calcium concentration.

Identification of regulatory elements recapitulating early expression of L-plastin in the zebrafish enveloping layer and embryonic periderm

We have cloned and characterized an intronic fragment of zebrafish lymphocyte cytosolic protein 1 (lcp1, also called L-plastin) that drives expression to the zebrafish enveloping layer (EVL). L-plastin is a calcium-dependent actin-bundling protein belonging to the plastin/fimbrin family of proteins, and is necessary for the proper migration and attachment of several adult cell types, including leukocytes and osteoclasts. However, in zebrafish lcp1 is abundantly expressed much earlier, during differentiation of the EVL. The cells of this epithelial layer migrate collectively, spreading vegetally over the yolk. L-plastin expression persists into the larval periderm, a transient epithelial tissue that forms the first larval skin. This finding establishes that L-plastin is activated in two different embryonic waves, with a distinct regulatory switch between the early EVL and the later leukocyte. To better study L-plastin expressing cells we attempted CRISPR/Cas9 homology-driven recombination (HDR) to insert a self-cleaving peptide (Cre-P2A-EGFP-CAAX) downstream of the native lcp1 promoter. This produced a stable zebrafish line expressing Cre recombinase in EVL nuclei and green fluorescence in EVL cell membranes. In vivo tracking of these labeled cells provided enhanced views of EVL migration behavior, membrane extensions, and mitotic events. Finally, we experimentally dissected key elements of the targeted lcp1 locus, discovering a ∼300 bp intronic sequence sufficient to drive EVL expression. The lcp1: Cre-P2A-EGFP-CAAX zebrafish should be useful for studying enveloping layer specification, gastrulation movements and periderm development in this widely used vertebrate model. In addition, the conserved regulatory sequences we have isolated predict that L-plastin orthologs may have a similar early expression pattern in other vertebrate embryos.

Osteoclasts secrete osteopontin into resorption lacunae during bone resorption

Osteopontin (OPN) is a non-collagenous extracellular sialylated glycoprotein located in bone. It is believed to be one of the key components in osteoclast attachment to bone during resorption. In this study, we characterized OPN and other glycoproteins found in the resorption lacunae to confirm the role of osteoclasts in OPN secretion using electron microscopy and mass spectrometry. Additionally, we examined the glycan epitopes of resorption pits and the effects of different glycan epitopes on the differentiation and function of osteoclasts. Osteoarthritic femoral heads were examined by immunohistochemistry to reveal the presence of OPN in areas of increased bone metabolism in vivo. Our results demonstrate that human osteoclasts secrete OPN into resorption lacunae on native human bone and on carbonated hydroxyapatite devoid of natural OPN. OPN is associated with an elevated bone turnover in osteoarthritic bone under experimental conditions. Our data further confirm that osteoclasts secrete OPN into the resorption pit where it may function as a chemokine for subsequent bone formation. We show that α2,3- and α2,6-linked sialic acids have a role in the process of osteoclast differentiation. OPN is one of the proteins that has both of the above sialic residues, hence we propose that de-sialylation can effect osteoclast differentiation in bone.

\u201cRuffled border\u201d formation on a CaP-free substrate: A first step towards osteoclast-recruiting bone-grafts materials able to re-establish bone turn-over

Osteoclasts are large multinucleated giant cells that actively resorb bone during the physiological bone turnover (BTO), which is the continuous cycle of bone resorption (by osteoclasts) followed by new bone formation (by osteoblasts). Osteoclasts secrete chemotactic signals to recruit cells for regeneration of vasculature and bone. We hypothesize that a biomaterial that attracts osteoclasts and re-establishes BTO will induce a better healing response than currently used bone graft materials. While the majority of bone regeneration efforts have focused on maximizing bone deposition, the novelty in this approach is the focus on stimulating osteoclastic resorption as the starter for BTO and its concurrent new vascularized bone formation. A biodegradable tyrosine-derived polycarbonate, E1001(1k), was chosen as the polymer base due to its ability to support bone regeneration in vivo. The polymer was functionalized with a RGD peptide or collagen I, or blended with β-tricalcium phosphate. Osteoclast attachment and early stages of active resorption were observed on all substrates. The transparency of E1001(1k) in combination with high resolution confocal imaging enabled visualization of morphological features of osteoclast activation such as the formation of the “actin ring” and the “ruffled border”, which previously required destructive forms of imaging such as transmission electron microscopy. The significance of these results is twofold: (1) E1001(1k) is suitable for osteoclast attachment and supports osteoclast maturation, making it a base polymer that can be further modified to optimize stimulation of BTO and (2) the transparency of this polymer makes it a suitable analytical tool for studying osteoclast behavior.

Single-nucleotide polymorphisms of MMP2 in MMP/TIMP pathways associated with the risk of alcohol-induced osteonecrosis of the femoral head in Chinese males: A case-control study.

The proportion of alcohol-induced osteonecrosis of the femoral head (ONFH) in all ONFH patients was 30.7%, with males prevailing among the ONFH patients in mainland China (70.1%). Matrix metalloproteinase 2 (MMP2), a member of the MMP gene family, encodes the enzyme MMP2, which can promote osteoclast migration, attachment, and bone matrix degradation. In this case–control study, we aimed to investigate the association between MMP2 and the alcohol-induced ONFH in Chinese males.In total, 299 patients with alcohol-induced ONFH and 396 healthy controls were recruited for a case–control association study. Five single-nucleotide polymorphisms within the MMP2 locus were genotyped and examined for their correlation with the risk of alcohol-induced ONFH and treatment response using Pearson χ2 test and unconditional logistic regression analysis. We identified 3 risk alleles for carriers: the allele “T” of rs243849 increased the risk of alcohol-induced ONFH in the allele model, the log-additive model without adjustment, and the log-additive model with adjustment for age. Conversely, the genotypes “CC” in rs7201 and “CC” in rs243832 decreased the risk of alcohol-induced ONFH, as revealed by the recessive model. After the Bonferroni multiple adjustment, no significant association was found. Furthermore, the haplotype analysis showed that the “TT” haplotype of MMP2 was more frequent among patients with alcohol-induced ONFH by unconditional logistic regression analysis adjusted for age.In conclusion, there may be an association between MMP2 and the risk of alcohol-induced ONFH in North-Chinese males. However, studies on larger populations are needed to confirm this hypothesis; these data may provide a theoretical foundation for future studies.

Chondrocytic ephrin B2 promotes cartilage destruction by osteoclasts in endochondral ossification.

The majority of the skeleton arises by endochondral ossification, whereby cartilaginous templates expand and are resorbed by osteoclasts then replaced by osteoblastic bone formation. Ephrin B2 is a receptor tyrosine kinase expressed by osteoblasts and growth plate chondrocytes that promotes osteoblast differentiation and inhibits osteoclast formation. We investigated the role of ephrin B2 in endochondral ossification using Osx1Cre-targeted gene deletion. Neonatal Osx1Cre.Efnb2(Δ/Δ) mice exhibited a transient osteopetrosis demonstrated by increased trabecular bone volume with a high content of growth plate cartilage remnants and increased cortical thickness, but normal osteoclast numbers within the primary spongiosa. Osteoclasts at the growth plate had an abnormal morphology and expressed low levels of tartrate-resistant acid phosphatase; this was not observed in more mature bone. Electron microscopy revealed a lack of sealing zones and poor attachment of Osx1Cre.Efnb2(Δ/Δ) osteoclasts to growth plate cartilage. Osteoblasts at the growth plate were also poorly attached and impaired in their ability to deposit osteoid. By 6 months of age, trabecular bone mass, osteoclast morphology and osteoid deposition by Osx1Cre.Efnb2(Δ/Δ) osteoblasts were normal. Cultured chondrocytes from Osx1Cre.Efnb2(Δ/Δ) neonates showed impaired support of osteoclastogenesis but no significant change in Rankl (Tnfsf11) levels, whereas Adamts4 levels were significantly reduced. A population of ADAMTS4(+) early hypertrophic chondrocytes seen in controls was absent from Osx1Cre.Efnb2(Δ/Δ) neonates. This suggests that Osx1Cre-expressing cells, including hypertrophic chondrocytes, are dependent on ephrin B2 for their production of cartilage-degrading enzymes, including ADAMTS4, and this might be required for attachment of osteoclasts and osteoblasts to the cartilage surface during endochondral ossification.

A Model for Osteonecrosis of the Jaw with Zoledronate Treatment following Repeated Major Trauma.

This study aims to develop a reproducible rat model for post-traumatic bisphosphonate-related osteonecrosis of the jaw (BRONJ). In our previous studies using dental extraction as an inducing factor, only 30% - 60% of zoledronate-treated animals fulfilled the definition of clinical BRONJ. We modified the zoledronate regimen and introduced repeated surgical extraction to illicit quantifiable BRONJ in all animals. Eighty retired-breeder female Sprague-Dawley rats were divided between the treatment (IV zoledronate; 80 μg/kg/week for 13 weeks) and control (saline) groups. On week 13, the left mandibular first molar was surgically extracted, followed by the second molar a week later. Animals were euthanized at 1-week, 2-weeks, and 8-weeks following extraction. The occurrence and severity of BRONJ were scored in each animal based on gross and MicroCT analysis. Parameters of bone formation and osteoclast functions at the extraction site were compared between groups. All zoledronate-treated animals developed a severe case of BRONJ that fulfilled the clinical definition of the condition in humans. Osteoclast attachment continued to be defective eight weeks after stopping the treatment. There were no signs of kidney or liver toxicity. Our data confirmed that repeated surgical extraction (major trauma) by itself consistently precipitated massive bone necrosis in ZA-treated animals, eliminating the need to induce pre-existing infection or comorbidity. These results will be the basis for further studies examining the in-vivo pathogenesis and prevention of BRONJ.

Osteoprotegerin induces podosome disassembly in osteoclasts through calcium, ERK, and p38 MAPK signaling pathways

Osteoclasts are critical for bone resorption and use podosomes to attach to bone matrix. Osteoprotegerin (OPG) is a negative regulator of osteoclast function that can affect the formation and function of podosomes. However, the signaling pathways that link OPG to podosome function have not been well characterized. Therefore, this study examined the roles of intracellular calcium and MAPKs in OPG-induced podosome disassembly in osteoclasts. We assessed the effects of the intracellular calcium chelator Bapta-AM, ERK inhibitor U0126, and p38 inhibitor SB202190 on OPG-treated osteoclast differentiation, adhesion structures, intracellular free Ca2+ concentration and the phosphorylation state of podosome associated proteins (Pyk2 and Src). Mouse monocytic RAW 264.7 cells were differentiated to osteoclasts using RANKL (30ng/mL) and M-CSF (25ng/mL). The cells were pretreated with Bapta-AM (5μM), U0126 (5μM), or SB202190 (10μM) for 30min, followed by 40ng/mL OPG for 3h. Osteoclastogenesis, adhesion structure, viability and morphology, intracellular free Ca2+ concentration and the phosphorylation state of Pyk2 and Src were measured by TRAP staining, scanning electron microscopy, real-time cell analyzer, flow cytometry and western blotting, respectively. OPG significantly inhibited osteoclastogenesis, the formation of adhesion structures, and reduced the amount of phosphorylated Pyk2 and Src-pY527, but increased phosphorylation of Src-pY416. Bapta-AM, U0126, and SB202190 partially restored osteoclast differentiation and adhesion structures. Both Bapta-AM and U0126, but not SB202190, restored the levels of intracellular free Ca2+ concentration, phosphorylated Pyk2 and Src-pY527. All three inhibitors blocked OPG-induced phosphorylation at Src-pY416. These results suggest OPG disrupts the attachment structures of osteoclasts and activates Src as an adaptor protein that competes for the reduced amount of phosphorylated Pyk2 through calcium- and ERK-dependent signaling pathways. p38 MAPK signaling may have a different role in OPG-induced osteoclast retraction. Our findings potentially offer novel insights into the signaling mechanisms downstream of OPG that affect osteoclast attachment to the extracellular matrix.

Histological assessment of tissue from large human bone defects repaired with β-tricalcium phosphate

This report describes the histological characteristics of large human bone defects that were implanted with β-tricalcium phosphate (β-TCP). Samples were obtained longer after the primary operation than in the earlier studies. We assessed a total of nine biopsies taken 33-208 weeks after implantation. The tissue sections were stained with hematoxylin-eosin for general observation, with Gomori stain to visualize the reticulin fibers, and with an antibody against tartrate-resistant alkaline phosphatase (TRAP) to characterize the cells. Ongoing bone remodeling was observed even 208 weeks after implantation as determined by the presence of osteoclasts and active osteoblasts and new woven and lamellar bone. We observed multinuclear giant cells phagocytosing the biomaterial and the attachment of osteoclasts to the β-TCP. The osteoclasts showed intense TRAP positivity, while the giant cells showed variable TRAP positivity. There was a zonal pattern in the original defects: The central regions showed granules and fibrous septa, while peripheral areas showed a layer of new bone formation. These data demonstrate ongoing bone remodeling long after implantation in the peripheral regions of the original defects as well as fibrous changes in the central regions and phagocytosis of biomaterial by multinuclear giant cells.

P130Cas, Crk-Associated Substrate, Plays Important Roles in Osteoclastic Bone Resorption

p130Cas, Crk-associated substrate (Cas), is an adaptor/scaffold protein that plays a central role in actin cytoskeletal reorganization. We previously reported that p130Cas is not tyrosine-phosphorylated in osteoclasts derived from Src-deficient mice, which are congenitally osteopetrotic, suggesting that p130Cas serves as a downstream molecule of c-Src and is involved in osteoclastic bone resorption. However, the physiological role of p130Cas in osteoclasts has not yet been confirmed because the p130Cas-deficient mice displayed embryonic lethality. Osteoclast-specific p130Cas conditional knockout (p130Cas(ΔOCL-) ) mice exhibit a high bone mass phenotype caused by defect in multinucleation and cytoskeleton organization causing bone resorption deficiency. Bone marrow cells from p130Cas(ΔOCL-) mice were able to differentiate into osteoclasts and wild-type cells in vitro. However, osteoclasts from p130Cas(ΔOCL-) mice failed to form actin rings and resorb pits on dentine slices. Although the initial events of osteoclast attachment, such as β3-integrin or Src phosphorylation, were intact, the Rac1 activity that organizes the actin cytoskeleton was reduced, and its distribution was disrupted in p130Cas(ΔOCL-) osteoclasts. Dedicator of cytokinesis 5 (Dock5), a Rho family guanine nucleotide exchanger, failed to associate with Src or Pyk2 in osteoclasts in the absence of p130Cas. These results strongly indicate that p130Cas plays pivotal roles in osteoclastic bone resorption.

TULA-2, a novel histidine phosphatase, regulates bone remodeling by modulating osteoclast function

Bone is a dynamic tissue that depends on the intricate relationship between protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) for maintaining homeostasis. PTKs and PTPs act like molecular on and off switches and help modulate differentiation and the attachment of osteoclasts to bone matrix regulating bone resorption. The protein T cell ubiquitin ligand-2 (TULA-2), which is abundantly expressed in osteoclasts, is a novel histidine phosphatase. Our results show that of the two family members, only TULA-2 is expressed in osteoclasts and that its expression is sustained throughout the course of osteoclast differentiation, suggesting that TULA-2 may play a role during early as well late stages of osteoclast differentiation. Skeletal analysis of mice that do not express TULA or TULA-2 proteins (DKO mice) revealed that there was a decrease in bone volume due to increased osteoclast numbers and function. Furthermore, in vitro experiments indicated that bone marrow precursor cells from DKO mice have an increased potential to form osteoclasts. At the molecular level, the absence of TULA-2 in osteoclasts results in increased Syk phosphorylation at the Y352 and Y525/526 residues and activation of phospholipase C gamma 2 (PLCγ2) upon engagement of immune-receptor-tyrosine-based-activation-motif (ITAM)-mediated signaling. Furthermore, expression of a phosphatase-dead TULA-2 leads to increased osteoclast function. Taken together, these results suggest that TULA-2 negatively regulates osteoclast differentiation and function.

Abstract A92: Oral bisphosphonate use and colorectal cancer incidence in the Women's Health Initiative

Abstract Introduction: Oral bisphosphonates are widely-prescribed medications used to increase bone density in postmenopausal women with osteopenia or osteoporosis. Bisphosphonates prevent the attachment of osteoclasts to bone, and have been shown to have numerous anticancer properties. Several studies, including the Women's Health Initiative (WHI), have found that use of oral bisphosphonates is associated with reduced risk of developing breast cancer, but less is known about potential associations with other hormonal malignancies such as colorectal cancer (CRC). A few case-control and retrospective cohort studies have reported decreased risk of CRC among oral bisphosphonate users. In contrast, a large prospective cohort study found no association. Methods: We evaluated the association between oral bisphosphonate use and CRC incidence in postmenopausal women, between 50 and 79 years of age, who participated in the WHI clinical trials and observational study. At baseline, all women completed an in-person interview covering demographic and general health information, and personal and family medical history. Oral bisphosphonate use was ascertained from an interviewer-administered inventory of regularly-used medications and supplements taken at baseline and over follow-up. Women who reported a personal history of CRC, ulcerative colitis, or colectomy at baseline were excluded from our analyses. Relative risks (RRs) were estimated by hazard ratios with 95% confidence intervals (CI) using Cox proportional hazards regression. Estimates were adjusted for WHI study component, hormone randomization assignment, age, a composite 5-year fracture probability, colorectal endoscopy, mammography, body mass index, race, education, family history of CRC, smoking status, alcohol consumption, physical activity, estrogen-only use, estrogen-progestin use, nonsteroidal anti-inflammatory drug use, aspirin use, total calcium intake, and total vitamin D intake. Results: Among 156,826 eligible women at baseline, 1,931 were diagnosed with incident invasive CRC during a median follow-up of 11.9 years. The prevalence of oral bisphosphonate use increased over study follow-up from about 2% at baseline to about 10% six years after baseline. Comparing oral bisphosphonate users at baseline to non-users at baseline, we observed no association with CRC risk (RR, 0.80; CI, 0.55-1.17, P = 0.24). Updating baseline use in regression models that treated oral bisphosphonate use as a time-varying covariate resulted in more precise estimates, but the RR did not achieve statistical significance at the 0.05 level (RR, 0.88; CI, 0.72-1.07, P = 0.20). These results were not substantially different by tumor site or stage at diagnosis. Conclusions: Use of oral bisphosphonates was not associated with the risk of developing CRC in the postmenopausal women who participated in WHI. Citation Format: Michael N. Passarelli, Polly A. Newcomb, Andrea Z. LaCroix, Dorothy S. Lane, Gloria Y.F. Ho, Rowan T. Chlebowski. Oral bisphosphonate use and colorectal cancer incidence in the Women's Health Initiative. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A92.

Reply to Scott, Sale, Greeves, and Fraser

to the editor: We thank Scott et al.([1][1]) for their interest in our recent study ([2][2]) that examined the acute response of bone turnover markers to a single bout of resistance training (RT) or plyometrics (PLY). We appreciate the acknowledgement of the strengths of our study design: we

Cathepsin K‐sensitive poly(ethylene glycol) hydrogels for degradation in response to bone resorption

We propose a new strategy of biomaterial design to achieve selective cellular degradation by the incorporation of cathepsin K-degradable peptide sequences into a scaffold structure so that scaffold biodegradation can be induced at the end of the bone formation process. Poly(ethylene glycol) diacrylate (PEGDA) hydrogels were used as a model biomaterial system in this study. A cathepsin K-sensitive peptide, GGGMGPSGPWGGK (GPSG), was synthesized and modified with acryloyl-PEG-succinimidyl carbonate to produce a cross-linkable cathepsin K-sensitive polymer that can be used to form a hydrogel. Specificity of degradation of the GPSG hydrogels was tested with cathepsin K and proteinase K as a positive control, with both resulting in significant degradation compared to incubation with nonspecific collagenases over a 24-h time period. No degradation was observed when the hydrogels were incubated with plasmin or control buffers. Cell-induced degradation was evaluated by seeding differentiated MC3T3-E1 osteoblasts and RAW264.7 osteoclasts on GPSG hydrogels that were also modified with the cell adhesion peptide RGDS. Resulting surface features and resorption pits were analyzed by differential interference contrast (DIC) and fluorescent images obtained with confocal microscopy. Results from both analyses demonstrated that GPSG hydrogels can be degraded specifically in response to osteoclast attachment but not in response to osteoblasts. In summary, we have demonstrated that by incorporating a cathepsin K-sensitive peptide into a synthetic polymer structure, we can generate biomaterials that specifically respond to cues from the natural process of bone remodeling.

Functional osteoclast attachment requires inositol-1,4,5-trisphosphate receptor-associated cGMP-dependent kinase substrate

Osteoclast activity is central to balanced bone turnover to maintain normal bone mass. A specialized osteoclast attachment to bone localizes acid secretion to remove bone mineral; in some cases, attachment is functionally impaired despite normal attachment proteins. The inositol-1,4,5-trisphosphate receptor-1 (IP3R1) is an intracellular calcium channel required for regulation of reversible osteoclast attachment by nitric oxide (NO), an important regulator of both normal and pathological bone degradation. In studies using human osteoclasts produced in vitro, we found that IP3R1 binds an endosomal isoform of the IP3R-associated cGMP-dependent kinase substrate (IRAG). IRAG is a substrate of cGMP-dependent kinase-1 (PKG1) and binds the PKG1 isoform PKG1β, which was the predominant form of PKG1 in human osteoclasts. Western blots of IRAG were consistent with NO-dependent serine phosphorylation of IRAG. An additional effect of PKG1β activity in osteoclasts was disassociation of IP3R1–IRAG complexes, as shown by analysis of IP3R1 complexes and by localization of the proteins within cells. IP3R1–IRAG complexes were stabilized by PKG or Src antagonists, Src activity being a requirement for IP3R1 calcium release downstream of PKG. IP3R1-mediated calcium release regulates cellular detachment in part through the calcium-dependent proteinase μ-calpain. In osteoclasts with IRAG suppressed by siRNA, activity of μ-calpain was increased relative to cells with normal IRAG, and regulation of μ-calpain by NO was lost. Furthermore, cells deficient in IRAG detached easily from substrate and had smaller attached diameters and randomly distributed podosomes, although IRAG knockdown did not affect cell viability. Our results indicate that IRAG is required for PKG1β-regulated cyclic calcium release during motility, and that disruption of the IP3R1–IRAG calcium regulation system is a novel cause of dysfunctional osteoclasts unrelated to defects in attachment proteins or acid secretion.