Osteoclastic Bone Degradation Research Articles

The Src family kinase inhibitor dasatinib delays pain-related behaviour and conserves bone in a rat model of cancer-induced bone pain

Pain is a severe and debilitating complication of metastatic bone cancer. Current analgesics do not provide sufficient pain relief for all patients, creating a great need for new treatment options. The Src kinase, a non-receptor protein tyrosine kinase, is implicated in processes involved in cancer-induced bone pain, including cancer growth, osteoclastic bone degradation and nociceptive signalling. Here we investigate the role of dasatinib, an oral Src kinase family and Bcr-Abl tyrosine kinase inhibitor, in an animal model of cancer-induced bone pain. Daily administration of dasatinib (15 mg/kg, p.o.) from day 7 after inoculation of MRMT-1 mammary carcinoma cells significantly attenuated movement-evoked and non-evoked pain behaviour in cancer-bearing rats. Radiographic - and microcomputed tomographic analyses showed significantly higher relative bone density and considerably preserved bone micro-architecture in the dasatinib treated groups, suggesting a bone-preserving effect. This was supported by a significant reduction of serum TRACP 5b levels in cancer-bearing rats treated with 15 mg/kg dasatinib. Furthermore, immunoblotting of lumbar spinal segments showed an increased activation of Src but not the NMDA receptor subunit 2B. These findings support a role of dasatinib as a disease modifying drug in pain pathologies characterized by increased osteoclast activity, such as bone metastases.

Small changes in bone structure of female α7 nicotinic acetylcholine receptor knockout mice.

BackgroundRecently, analysis of bone from knockout mice identified muscarinic acetylcholine receptor subtype M3 (mAChR M3) and nicotinic acetylcholine receptor (nAChR) subunit α2 as positive regulator of bone mass accrual whereas of male mice deficient for α7-nAChR (α7KO) did not reveal impact in regulation of bone remodeling. Since female sex hormones are involved in fair coordination of osteoblast bone formation and osteoclast bone degradation we assigned the current study to analyze bone strength, composition and microarchitecture of female α7KO compared to their corresponding wild-type mice (α7WT).MethodsVertebrae and long bones of female 16-week-old α7KO (n = 10) and α7WT (n = 8) were extracted and analyzed by means of histological, radiological, biomechanical, cell- and molecular methods as well as time of flight secondary ion mass spectrometry (ToF-SIMS) and transmission electron microscopy (TEM).ResultsBone of female α7KO revealed a significant increase in bending stiffness (p < 0.05) and cortical thickness (p < 0.05) compared to α7WT, whereas gene expression of osteoclast marker cathepsin K was declined. ToF-SIMS analysis detected a decrease in trabecular calcium content and an increase in C4H6N+ (p < 0.05) and C4H8N+ (p < 0.001) collagen fragments whereas a loss of osteoid was found by means of TEM.ConclusionsOur results on female α7KO bone identified differences in bone strength and composition. In addition, we could demonstrate that α7-nAChRs are involved in regulation of bone remodelling. In contrast to mAChR M3 and nAChR subunit α2 the α7-nAChR favours reduction of bone strength thereby showing similar effects as α7β2-nAChR in male mice. nAChR are able to form heteropentameric receptors containing α- and β-subunits as well as the subunits α7 can be arranged as homopentameric cation channel. The different effects of homopentameric and heteropentameric α7-nAChR on bone need to be analysed in future studies as well as gender effects of cholinergic receptors on bone homeostasis.

The Src Inhibitor AZD0530 Reversibly Inhibits the Formation and Activity of Human Osteoclasts

Tumor cells in the bone microenvironment are able to initiate a vicious cycle of bone degradation by mobilizing osteoclasts, multinucleated cells specialized in bone degradation. c-Src is highly expressed both in tumors and in osteoclasts. Therefore, drugs like AZD0530, designed to inhibit Src activity, could selectively interfere with both tumor and osteoclast activity. Here we explored the effects of AZD0530 on human osteoclast differentiation and activity. The effect on osteoclasts formed in vivo was assessed in mouse fetal calvarial explants and in isolated rabbit osteoclasts, where it dose-dependently inhibited osteoclast activity. Its effect on formation and activity of human osteoclasts in vitro was determined in cocultures of human osteoblasts and peripheral blood mononuclear cells. AZD0530 was most effective in inhibiting osteoclast-like cell formation when present at the onset of osteoclastogenesis, suggesting that Src activity is important during the initial phase of osteoclast formation. Formation of active phosphorylated c-Src, which was highly present in osteoclast-like cells in cocultures and in peripheral blood mononuclear cell monocultures, was significantly reduced by AZD0530. Furthermore, it reversibly prevented osteoclast precursor migration from the osteoblast layer to the bone surface and subsequent formation of actin rings and resorption pits. These data suggest that Src is pivotal for the formation and activity of human osteoclasts, probably through its effect on the distribution of the actin microfilament system. The reversible effect of AZD0530 on osteoclast formation and activity makes it a promising candidate to temper osteoclastic bone degradation in bone diseases with enhanced osteoclast activity such as osteolytic metastatic bone disease.

Plasminogen activators are involved in the degradation of bone by osteoclasts

Osteoclastic bone degradation depends on the activity of several proteolytic enzymes, in particular to those belonging to the classes of cysteine proteinases and matrix metalloproteinases (MMPs). Yet, several findings suggest that the two types of plasminogen activators (PA), the tissue- and urokinase-type PA (tPA and uPA, respectively) are also involved in this process. To investigate the involvement of these enzymes in osteoclast-mediated bone matrix digestion, we analyzed bone explants of mice that were deficient for both tPA and uPA and compared them to wild type mice. The number of osteoclasts as well as their ultrastructural appearance was similar for both genotypes. Next, calvarial and metatarsal bone explants were cultured for 6 or 24 h in the presence of selective inhibitors of cysteine proteinases or MMPs and the effect on osteoclast-mediated bone matrix degradation was assessed. Inhibition of the activity of cysteine proteinases in explants of control mice resulted in massive areas of non-digested demineralized bone matrix adjacent to the ruffled border of osteoclasts, an effect already maximal after 6 h. However, at that time point these demineralized areas were not observed in bone explants from uPA/tPA deficient mice. After prolonged culturing (24 h), a comparable amount of demineralized bone matrix adjacent to actively resorbing osteoclasts was observed in the two genotypes, suggesting that degradation was delayed in uPA/tPA deficient bones. The activity of cysteine proteinases as assessed in bone extracts, proved to be higher in extracts from uPA/tPA −/− bones. Immunolocalization of the integrin α vβ 3 of in vitro generated osteoclasts demonstrated a more diffuse labeling of osteoclasts derived from uPA/tPA −/− mice. Taken together, our data indicate that the PAs play a hitherto unrecognized role in osteoclast-mediated bone digestion. The present findings suggest that the PAs are involved in the initial steps of bone degradation, probably by a proper integrin-dependent attachment to bone.

Osteoclastic Bone Degradation and the Role of Different Cysteine Proteinases and Matrix Metalloproteinases: Differences Between Calvaria and Long Bone

Osteoclastic bone degradation involves the activity of cathepsin K. We found that in addition to this enzyme other, yet unknown, cysteine proteinases participate in digestion. The results support the notion that osteoclasts from different bone sites use different enzymes to degrade the collagenous bone matrix. The osteoclast resorbs bone by lowering the pH in the resorption lacuna, which is followed by secretion of proteolytic enzymes. One of the enzymes taken to be essential in resorption is the cysteine proteinase, cathepsin K. Some immunolabeling and enzyme inhibitor data, however, suggest that other cysteine proteinases and/or proteolytic enzymes belonging to the group of matrix metalloproteinases (MMPs) may participate in the degradation. In this study, we investigated whether, in addition to cathepsin K, other enzymes participate in osteoclastic bone degradation. In bones obtained from mice deficient for cathepsin K, B, or L or a combination of K and L, the bone-resorbing activity of osteoclasts was analyzed at the electron microscopic level. In addition, bone explants were cultured in the presence of different selective cysteine proteinase inhibitors and an MMP inhibitor, and the effect on resorption was assessed. Because previous studies showed differences in resorption by calvarial osteoclasts compared with those present in long bones, in all experiments, the two types of bone were compared. Finally, bone extracts were analyzed for the level of activity of cysteine proteinases and the effect of inhibitors hereupon. The analyses of the cathepsin-deficient bone explants showed that, in addition to cathepsin K, calvarial osteoclasts use other cysteine proteinases to degrade bone matrix. It was also shown that, in the absence of cathepsin K, long bone osteoclasts use MMPs for resorption. Cathepsin L proved to be involved in the MMP-mediated resorption of bone by calvarial osteoclasts; in the absence of this cathepsin, calvarial osteoclasts do not use MMPs for resorption. Selective inhibitors of cathepsin K and other cysteine proteinases showed a stronger effect on calvarial resorption than on long bone resorption. Our findings suggest that (1) cathepsin K-deficient long bone osteoclasts compensate the lack of this enzyme by using MMPs in the resorption of bone matrix; (2) cathepsin L is involved in MMP-mediated resorption by calvarial osteoclasts; (3) in addition to cathepsin K, other, yet unknown, cysteine proteinases are likely to participate in skull bone degradation; and finally, (4) the data provide strong additional support for the existence of functionally different bone-site specific osteoclasts.

Family 2 cystatins inhibit osteoclast-mediated bone resorption in calvarial bone explants

Osteoclastic bone resorption depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Biochemical studies have shown that cystatins, naturally occurring inhibitors of these enzymes, inhibit bone matrix degradation. Since the mechanism by which cystatins exert this inhibitory effect is not completely resolved yet, we studied the effect of cystatins on bone resorption microscopically and by Ca-release measurements. Calvarial bone explants were cultured in the presence or absence of family 2 cystatins and processed for light and electron microscopic analysis, and the culture media were analyzed for calcium release. Both egg white cystatin and human cystatin C decreased calcium release into the medium significantly. Microscopic analyses of the bone explants demonstrated that in the presence of either inhibitor, a high percentage of osteoclasts was associated with demineralized non-degraded bone matrix. Following a 24-h incubation in the presence of cystatin C, 41% of the cells were adjacent to areas of demineralized non-degraded bone matrix, whereas in controls, this was only 6%. If bone explants were cultured with both PTH and cystatin C, 60% of the osteoclasts were associated with demineralized non-degraded bone matrix, compared to 27% for bones treated with PTH only ( P < 0.01). Our study provides evidence that cystatins, the naturally occurring inhibitors of cysteine proteinases, reversibly inhibit bone matrix degradation in the resorption lacunae adjacent to osteoclasts. These findings suggest the involvement of cystatins in the modulation of osteoclastic bone degradation.

The bisphosphonate zoledronate prevents vertebral bone loss in mature estrogen-deficient rats as assessed by micro-computed tomography.

The effect of long-term treatment with the bisphosphonate zoledronate on vertebral bone architecture was investigated in estrogen-deficient mature rats. 4-month-old rats were ovariectomized and development of cancellous osteopenia was assessed after 1 year. The change of bone architectural parameters was determined with a microtomographic instrument of high resolution. After 1 year of estrogen-deficiency, animals lost 55% of vertebral trabecular bone in comparison to sham operated control animals. Trabecular number (Tb.N) and trabecular thickness (Tb.Th) were significantly reduced in ovariectomized animals, whereas trabecular separation (Tb.Sp), bone surface to volume fraction (BS/BV) and trabecular bone pattern factor (TBPf) were significantly increased, indicating a loss of architectural integrity throughout the vertebral body. 3 groups of animals were treated subcutaneously with zoledronate for 1 year with 0.3, 1.5 and 7.5 microgram/kg/week to inhibit osteoclastic bone degradation. Administration started immediately after ovariectomy and treatment dose-dependently prevented the architectural bone deterioration and completely suppressed the effects of estrogen deficiency at the higher doses. The results show that microtomographic determination of static morphometric parameters can be used to quantitate the effects of drugs on vertebral bone architecture in small laboratory animals and that zoledronate is highly effective in this rat model.

Differential effects of tamoxifen‐like compounds on osteoclastic bone degradation, H‐ATPase activity, calmodulin‐dependent cyclic nucleotide phosphodiesterase activity, and calmodulin binding

Differential effects of tamoxifen‐like compounds on osteoclastic bone degradation, H‐ATPase activity, calmodulin‐dependent cyclic nucleotide phosphodiesterase activity, and calmodulin binding

Differential effects of tamoxifen-like compounds on osteoclastic bone degradation, H+-ATPase activity, calmodulin-dependent cyclic nucleotide phosphodiesterase activity, and calmodulin binding

We studied effects of calmodulin antagonists on osteoclastic activity and calmodulin-dependent HCl transport. The results were compared to effects on the calmodulin-dependent phosphodiesterase and antagonist-calmodulin binding affinity. Avian osteoclast degradation of labeled bone was inhibited approximately 40% by trifluoperazine or tamoxifen with half-maximal effects at 1-3 microM. Four benzopyrans structurally resembling tamoxifen were compared: d-centchroman inhibited resorption 30%, with half-maximal effect at approximately 100 nM, cischroman and CDRI 85/287 gave 15-20% inhibition, and l-centchroman was ineffective. No benzopyran inhibited cell attachment or protein synthesis below 10 microM. However, ATP-dependent membrane vesicle acridine transport showed that H(+)-ATPase activity was abolished by all compounds with 50% effects at 0.25-1 microM. All compounds also inhibited calmodulin-dependent cyclic nucleotide phosphodiesterase at micromolar calcium. Relative potency varied with assay type, but d- and l-centchroman, surprisingly, inhibited both H(+)-ATPase and phosphodiesterase activity at similar concentrations. However, d- and l-centchroman effects in either assay diverged at nanomolar calcium. Of benzopyrans tested, only the d-centchroman effects were calcium-dependent. Interaction of compounds with calmodulin at similar concentrations were confirmed by displacement of labeled calmodulin from immobilized trifluoperazine. Thus, the compounds tested all interact with calmodulin directly to varying degrees, and the observed osteoclast inhibition is consistent with calmodulin-mediated effects. However, calmodulin antagonist activity varies between specific reactions, and free calcium regulates specificity of some interactions. Effects on whole cells probably also reflect other properties, including transport into cells.

Regulation of Osteoclastic Bone Resorption by Glucose

Osteoclasts degrade bone by pumping molar quantities of HCl to dissolve the calcium salts of bone, an energy intensive process evidently supported by abundant mitochondria. This is the first study to directly examine the ability of various metabolites to serve as potential energy sources for osteoclastic bone resorption. Glucose, and to a lesser extent lactate, supported osteoclastic bone degradation. However, fatty acids (palmitate, myristate and stearate), essential amino acids plus 20 mM alanine, or ketone bodies (acetoacetate, β-hydroxybutyrate and α-ketoglutarate) did not support bone degradation. Resorption declined to 10-30% of glucose controls when fatty acids or ketoacids were substituted for glucose. Resorption was glucose concentration dependent, with maximal activity at ∼7 mM (KM∼3 mM). Glucose transport was linear for ∼15 minutes, specific for D-glucose, and inhibited by cytochalasin B. Osteoclasts cultured on bone transported glucose at almost twice the rate of those off bone (Vmax23 versus 13 nmols/mg/min, respectively) and medium acid accumulation paralleled glucose uptake, while the KMwas unchanged. We conclude that glucose is the principal energy source required for bone degradation. Further, characteristics of glucose transport are consistent with the hypothesis that fluctuations in serum glucose concentration are an important component in regulation of osteoclastic bone degradation.

A peptidomimetic antagonist of the alpha(v)beta3 integrin inhibits bone resorption in vitro and prevents osteoporosis in vivo.

Osteoclastic bone degradation requires intimacy between the matrix and the resorptive cell. While the precise role the integrin alpha(v)beta3 plays in the process is not yet understood, occupancy of the heterodimer by soluble ligand or by blocking antibody effectively inhibits bone resorption in vitro and in vivo, suggesting that alpha(v)beta3 blockade may prevent postmenopausal osteoporosis. Thus, we identified a synthetic chemical peptide mimetic, beta-[2-[[5-[(aminoiminomethyl)amino]-1-oxopentyl]amino]-1-+ ++oxoethyl]amino-3-pyridinepropanoic acid, bistrifluoroacetate (SC56631) based upon the alpha(v)beta3 ligand, Arg-Gly-Asp (RGD), which recognizes the isolated integrin, and its relative, alpha(v)beta5, as effectively as does the natural peptide. The mimetic dampens osteoclastic bone resorption in vitro and in vivo. Most importantly, intravenous administration of the mimetic prevents the 55% loss of trabecular bone sustained by rats within 6 wk of oophorectomy. Histological examination of bones taken from SC56631-treated, oophorectomized animals also demonstrates the compound's bone sparing properties and its capacity to decrease osteoclast number. Thus, an RGD mimetic prevents the rapid bone loss that accompanies estrogen withdrawal.

Effect of nitric oxide synthase inhibitors on bone metabolism in growing rats.

We examined the effects of chronic nitric oxide (NO) blockade on bone mineral status in growing rats. Oral administration of NG-nitro-L-arginine methyl ester (L-NAME) for 4 wk caused hypertension and a significant reduction in urinary NO2- and NO3- excretion. Four-week oral aminoguanidine (AG, 400 mg/dl of drinking water) did not alter blood pressure but caused a significant decrease in urinary NO2- and NO3-. Rats treated with L-NAME at doses of 20 and 50 mg/dl had normal bone mineral mass in the lumbar spine, but the highest dose (80 mg/dl) caused a slight decrease in bone mass. Chronic AG induced a significant spine osteopenia. This effect of AG was abolished by the simultaneous administration of L-arginine (2.0 g/dl). AG-induced osteopenia was associated with a significant increase in urine excretion of collagen cross-links with normal serum osteocalcin. These findings indicate that chronic AG administration can cause an imbalance between bone resorption and formation, resulting in a decrease in bone mass in growing rats, and suggest that NO produced by inducible NO synthase plays an important role in basal osteoclast bone degradation activity in vivo.

Effect of calcitonin deficiency on bone density and bone turnover in totally thyroidectomized patients

To investigate the influence of calcitonin deficiency on bone turnover and density we studied 25 premenopausal female and 12 male patients (age 23 to 49 years) who had undergone total thyroidectomy for differentiated thyroid cancer 1 to 15 years previously. Basal and calcium stimulated extractable calcitonin, representing the monomeric, biologically active form of the hormone, was lacking or markedly decreased in all patients. There was a relative increase of urine hydroxyproline excretion (an index of osteoclastic bone degradation) in relation to serum osteocalcin (an index of osteoblastic bone formation) indicating an imbalance of bone turnover with a tendency to increased degradation in all patients. Total and trabecular bone density, measured with quantitative computed tomography at the distal forearm were significantly decreased in the male and normal in the female patients, without a relation to the duration of the calcitonin deficiency. The study indicates that patients with calcitonin deficiency, suppressive thyroid hormone treatment, or both may have a higher risk of increased bone degradation and osteopenia. Whether the effect is more due to calcitonin deficiency or thyroid hormone therapy, cannot be concluded from this study design. The fact that only the male patients had a decreased bone density may be due to a lower parathyroid activity in our female patients and the greater thyroidectomy-induced decrement of monomeric calcitonin in our male patients compared with male controls.