Milk basic protein promotes bone formation and suppresses bone resorption in healthy adult men

Milk contains several components effective for bone health. In the previous in vitro and in vivo studies, we have shown that milk whey protein, especially its basic protein fraction (milk basic protein [MBP]), promoted bone formation and suppressed bone resorption. This present study examines the effect of MBP on the biochemical markers of bone metabolism in healthy adult men. Experimental beverages containing MBP (300 mg of MBP a day) were given to 30 normal healthy adult men for 16 days. The serum osteocalcin concentration had increased significantly after 16 days of ingesting the experimental beverage containing MBP. Urinary cross-linked N-teleopeptides of type-I collagen (NTx) excretion had decreased significantly after 16 days of ingesting MBP. The urinary NTx excretion was related to the serum osteocalcin concentration after 16 days of ingestion. These results suggest that MBP promoted bone formation and suppressed bone resorption, while maintaining the balance of bone remodeling.     

Cystatin B as an intracellular modulator of bone resorption

Degradation of organic bone matrix requires proteinase activity. Cathepsin K is a major osteoclast proteinase needed for bone resorption, although osteoclasts also express a variety of other cysteine- and matrix metalloproteinases that are involved in bone remodellation. Cystatin B, an intracellular cysteine proteinase inhibitor, exhibits a lysosomal distribution preferentially in osteoclasts but it's role in osteoclast physiology has remained unknown. The current paper describes a novel regulatory function for cystatin B in bone-resorbing osteoclasts in vitro. Rat osteoclasts were cultured on bovine bone and spleen-derived cystatin B was added to the cultures. Nuclear morphology was evaluated and the number of actively resorbing osteoclasts and resorption pits was counted. Intracellular cathepsin K and tartrate-resistant acid phosphatase (TRACP) activities were monitored using fluorescent enzyme substrates and immunohistology was used to evaluate distribution of cystatin B in rat metaphyseal bone. Microscopical evaluation showed that cystatin B inactivated osteoclasts, thus resulting in impaired bone resorption. Cathepsin K and TRACP positive vesicles disappeared dose-dependently from the cystatin B-treated osteoclasts, indicating a decreased intracellular trafficking of bone degradation products. At the same time, cystatin B protected osteoclasts from experimentally induced apoptosis. These data show for the first time that, in addition to regulating cysteine proteinase activity and promoting cell survival in the nervous system, cystatin B inhibits bone resorption by down-regulating intracellular cathepsin K activity despite increased osteoclast survival.     

Release of intact and fragmented osteocalcin molecules from bone matrix during bone resorption in vitro

Osteocalcin detected from serum samples is considered a specific marker of osteoblast activity and bone formation rate. However, osteocalcin embedded in bone matrix must also be released during bone resorption. To understand the contribution of each type of bone cell in circulating osteocalcin levels, we used immunoassays detecting different molecular forms of osteocalcin to monitor bone resorption in vitro. Osteoclasts were obtained from rat long bones and cultured on bovine bone slices using osteocalcin-depleted fetal bovine serum. In addition, human osteoclasts differentiated from peripheral blood mononuclear cells were used. Both rat and human osteoclasts released osteocalcin from bovine bone into medium. The amount of osteocalcin increased in the presence of parathyroid hormone, a stimulator of resorption, and decreased in the presence of bafilomycin A1, an inhibitor of resorption. The amount of osteocalcin in the medium correlated with a well characterized marker of bone resorption, the C-terminal telopeptide of type I collagen (r > 0.9, p < 0.0001). The heterogeneity of released osteocalcin was determined using reverse phase high performance liquid chromatography, and several molecular forms of osteocalcin, including intact molecule, were identified in the culture medium. In conclusion, osteocalcin is released from the bone matrix during bone resorption as intact molecules and fragments. In addition to the conventional use as a marker of bone formation, osteocalcin can be used as a marker of bone resorption in vitro. Furthermore, bone matrix-derived osteocalcin may contribute to circulating osteocalcin levels, suggesting that serum osteocalcin should be considered as a marker of bone turnover rather than bone formation.     

Absorption of bovine angiogenin into peripheral blood of rats orally administered milk basic protein

Bovine angiogenin is a major component of the bone resorption inhibitory activity of milk basic protein (MBP). The intestinal absorption of bovine angiogenin was investigated in a rat model, where it was detected in an intact form in the peripheral blood after the oral administration of MBP. This finding demonstrates that orally administered bovine angiogenin is absorbed without being degraded.     

Controlled trial of the effects of milk basic protein (MBP) supplementation on bone metabolism in healthy adult women

Milk has more beneficial effects on bone health compared to other food sources. Recent in vitro and in vivo studies showed that milk whey protein, especially its basic protein fraction, contains several components capable of both promoting bone formation and inhibiting bone resorption. However, the effects of milk basic protein (MBP) on bone metabolism of humans are not known. The object of this study was to examine the effects of MBP on bone metabolism of healthy adult women. Thirty-three normal healthy women were randomly assigned to treatment with either placebo or MBP (40 mg per day) for six months. The bone mineral density (BMD) of the left calcaneus of each subject was measured at the beginning of the study and after six months of treatment, by dual-energy x-ray absorptiometry. Serum and urine indices of bone metabolism were measured at the base line, three-month intervals, and the end of the study. Daily intake of nutrients was monitored by a three-day food record made at three and six months. The mean (+/- SD) rate of left calcaneus BMD gain of women in the MBP group (3.42 +/- 2.05%) was significantly higher than that of women in the placebo group (2.01 +/- 1.75%, P = 0.042). As compared with the placebo group, urinary cross-linked N-teleopeptides of type-I collagen/creatinine and deoxypyridinoline/creatinine were significantly decreased in the MBP group (p < 0.05), while no significant differences between the two groups were observed in serum osteocalcin and bone-specific alkaline phosphatase concentrations. A daily MBP supplementation of 40 mg in healthy adult women can significantly increase their BMD independent of dietary intake of minerals and vitamins. This increase in BMD might be primarily mediated through inhibition of osteoclast-mediated bone resorption by the MBP supplementation.     

Degradation of myelin basic protein in myelin by protease in cerebrospinal fluid and effects of protease inhibitors

Neutral protease is shown to be present in cell-free human cerebrospinal fluid. Incubation of heated human myelin with CSF at 25°C resulted in a marked reduction of myelin basic protein (MBP) with time. Degradation products appeared at apparent mol wt 14 KDa and 12 KDa on polyacrylamide gel electrophoresis. Optimal pH of the protease was 7.0. This protease was activated by calcium ion. Degradation of MBP was inhibited by FOY305 (camostat mesilate), Trasylol^®, and Leupeptin, but not a specific calcium-activated neutral protease inhibitor, E-64-a. FOY305, which is a synthesized specific serine protease inhibitor, was the strongest inhibitor of all. The role of this protease in CSF has not been elucidated. In may be related to the physiological turnover of MBP, and may affect myelin maintenance in pathological conditions such as demyelination.     

Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages

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 anti-resorptive 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 approximately 22-26 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 Rab-dependent intracellular membrane trafficking in osteoclasts.     

Purification and biochemical characterization of a fibroblast growth factor-binding protein (FGF-BP) from the lactoferrin fraction of bovine milk

By means of gel filtration on a TSK-gel HPLC column in the presence of 8 M urea, a 37-kDa polypeptide (p37) was completely separated from lactoferrin (LF) in the heparin HII fraction of the partially purified LF fraction prepared from bovine milk. Purified p37 was identified as a fibroblast growth factor-binding protein (FGF-BP), since its N-terminal 14 amino acid residues (KKEGRNRRGSKASA) were 100% identical to the corresponding sequence of bovine FGF-BP. It was found, in vitro, that (i) p37 had a higher binding affinity with bFGF than bLF; (ii) p37 functioned as a phosphate acceptor for at least three protein kinases (PKA, CK1 and CK2); (iii) bLF stimulated about 3-fold the PKA-mediated phosphorylation of p37, but suppressed its phosphorylation by CK1; and (iv) galloyl pedunculagin was an effective inhibitor for the phosphorylation of p37 by PKA and CK1. Furthermore, the physiological correlation between p37 and bLF may be regulated through specific phosphorylation of p37 by PKA, since p37 fully phosphorylated by PKA did not bind to bLF in vitro. The sulfatide-induced conformational changes in p37 enabled the phosphorylation of p37 by CK1 and also reduced its ability to bind with bLF in vitro. From these results presented here, it is concluded that (i) p37 (FGF-BP) may be tightly associated with bLF in bovine milk; and (ii) the physiological correlation between p37 and bLF may be regulated by the PKA-mediated full phosphorylation of p37 or by the direct binding of sulfatide to p37 in vivo.     

Protease from bovine spleen with kininogenase activity]

A protease with kininogenase activity at pH 7.5 was isolated from bovine spleen extract by gel filtration and ion exchange chromatography. The protease was found in the fraction with molecular weight lower than 25.000 and was separated from the other neutral SH-dependent protease by chromatography on KM-cellulose. The kininogenase activity was inhibited by DFP and trasylol; soybean trypsin inhibitor had no effect. The protease did not split N-benzoyl-L-arginine ethyl ester and N-benzoyl-D, L-arginine p-nitroanilide.     

Caldecrin:A pancreas-derived hypocalcemic factor,regulates osteoclast formation and function

Caldecrin was originally isolated from the pancreas as afactor that reduced serum calcium levels. This secreted serine protease has chymotrypsin-like activity and is also known as chymotrypsin C; it belongs to the elastase family. Although intravenous administration of caldecrin decreases the serum calcium concentration even when its protease activity is blocked,this effect does require cleavage of caldecrin's pro-peptide by trypsin,converting it to the mature enzyme. Ectopic intramuscular expression of caldecrin prevented bone resorption in ovariectomized mice. Caldecrin inhibited parathyroid hormone-stimulated calcium release from fetal mouse long bone organ cultures. Furthermore,caldecrin suppressed the formation of osteoclasts from bone marrow cells by inhibiting the receptor activator of nuclear factor-k B ligand(RANKL)-stimulated phospholipase Cγ-calcium oscillation-calcineurinnuclear factor of activated T-cells,cytoplasmic 1 pathway. Caldecrin also suppressed the bone resorption activity of mature osteoclasts by preventing RANKL-stimulated Src activation,calcium entry,and actin ring formation. In vivo and in vitro studies have indicated that caldecrin is a unique multifunctional protease with anti-osteoclastogenic activities that are distinct from its protease activity. Caldecrin might be a potential therapeutic target for the treatment of osteolytic diseases such as osteoporosis and osteoarthritis. This mini-review describes caldecrin's historical background and its mechanisms of action.     

Purification and characterization of a cystatin from the leaves of methyl jasmonate treated tomato plants

A multidomain cystatin was purified from the leaves of mature and seedling tomato plants (Lycopersicon esculentum, cv Bonnie Best) that had been sprayed with methyl jasmonate. For seedlings, cystatin purification was accomplished using EDTA washing, KCI extraction, 70 degrees C heat treatment, ammonium sulfate fractionation and gel filtration chromatography. For mature plants, DEAE chromatography was also needed to separate a protease, hydrolysis products of cystatin and serine proteinase inhibitors from the intact cystatin. Purified tomato cystatin has a molecular weight (Mr) of 88 kDa, eight papain binding domains, is a non-competitive inhibitor of papain with K1 of 1.4 nM and is not a glycoprotein. Tryptic peptides (Mr 26, 13 kDa) and most chymotryptic peptides (Mr 33, 13 kDa) of tomato cystatin retain inhibitor activity. Amino acid analysis revealed no Cys; Asx, Glx, Gly, Ser accounted for almost half the residues and there was some homology with potato multicystatin. Activity is stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at > 60 degrees C (Ea = 92.5 kJ/mole). Extracts of mature plants treated with methyl jasmonate contain lower Mr cystatins that appear to result from the action of an endogenous 26 kDa protease on the 88 kDa inhibitor.     

miR-31 controls osteoclast formation and bone resorption by targeting RhoA

Introduction

Increased activity of osteoclasts is responsible for bone loss and joint destruction in rheumatoid arthritis. For osteoclast development and bone resorption activity, cytoskeletal organization must be properly regulated. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that suppress expression of their target genes. This study was conducted to identify crucial miRNAs to control osteoclasts.

Methods

miRNA expression in the bone marrow-derived macrophages (BMM) with or without receptor activator of nuclear factor κB ligand (RANKL) stimulation was analyzed by miRNA array. To examine the role of specific miRNAs in osteoclast formation, bone resorption activity and actin ring formation, the BMM were retrovirally transduced with miRNA antagomirs. To confirm whether the suppressive effects on osteoclastogenesis by miR-31 inhibition were mediated by targeting RhoA, osteoclast formation was analyzed in the presence of the RhoA inhibitor, exoenzyme C3.

Results

miR-31 was identified as one of the highly upregulated miRNAs during osteoclast development under RANKL stimulation. Inhibition of miR-31 by specific antagomirs suppressed the RANKL-induced formation of osteoclasts and bone resorption. Phalloidin staining of osteoclasts revealed that actin ring formation at the cell periphery was severely impaired by miR-31 inhibition, and clusters of small ringed podosomes were observed instead. In these osteoclasts, expression of RhoA, one of the miR-31 target genes, was upregulated by miR-31 inhibition in spite of the impaired osteoclastogenesis. Treatment with the RhoA inhibitor, exoenzyme C3, rescued the osteoclastogenesis impaired by miR-31 inhibition.

Conclusions

miR-31 controls cytoskeleton organization in osteoclasts for optimal bone resorption activity by regulating the expression of RhoA.     

A cysteine protease from maize isolated in a complex with cystatin

We recently purified a latent but SDS-activated protease complex (40, 15- or 13-kDa proteins) from maize [Yamada et al. (1998) Plant Cell Physiol. 39: 106]. Here, we revealed that the complex was composed of a cysteine protease (40 kDa) and a cystatin, cysteine protease inhibitor (15- or 13-kDa). This is the first report on the isolation of a complex consisting of a cystatin and a target cysteine protease from plants. Cloning of the cysteine protease revealed that it had low homology (25-30%) to other maize cysteine proteases cloned to date but was highly homologous to other plant cysteine proteases such as rice oryzain alpha (84%) and the homologs (50-80%). The cysteine protease expressed in Escherichia coli showed the same substrate and inhibitor specificities as the protease of the complex, demonstrating that the isolated cDNA clone exactly encodes the protease of the complex. The protease expressed in E. coli itself was active but not latent, probably because it was not bound to cystatin. It is most likely that in vitro activation of the protease complex by SDS is caused by the release of bound cystatin. The mRNA of protease was expressed in various tissues except for seeds.     

Involvement of calpain in osteoclastic bone resorption

There is increasing evidence that calpain contributes to the reorganization of the cytoskeleton in the integrin-mediated signaling pathway. Osteoclastic bone resorption requires cell-matrix contact, an event mediated by integrin alphavbeta3, and subsequent cytoskeletal reorganization to form characteristic membrane domains such as the sealing zone and ruffled border. In this study, therefore, we investigated whether calpain is involved in osteoclastic bone resorption. Membrane-permeable calpain inhibitors suppress the resorption activity of human osteoclasts, but an impermeable inhibitor does not. Upon the attachment of osteoclasts to bone, micro-calpain is translocated from the cytosolic to the cytoskeletal fraction and is autolytically activated. Both the activation of micro-calpain and the formation of actin-rings, the cytoskeletal structures essential for bone resorption, are inhibited by membrane-permeable calpain inhibitors. The activated micro-calpain in osteoclasts selectively cleaves talin, which links the matrix-recognizing integrin to the actin cytoskeleton. These findings suggest that calpain is a regulator of the bone resorption activity of osteoclasts through reorganization of the cytoskeleton related to actin-ring formation.     

Cleavage of Myelin Basic Protein by Neutral Protease Activity of Human White Matter and Myelin

Polypeptides arising from neutral in vitro proteolysis of myelin basic protein (MBP) of human brain were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At pH 7 a marked breakdown of MBP resulted in the formation of 8-12 polypeptides ranging from 6 to 17 kd in molecular weight. As neutral proteolytic activity was not eliminated by either gel filtration or cation-exchange chromatography acid-soluble protease(s) involved probably have a size and electric charge similar to that of MBP. The enzymatic nature of neutral proteolysis was ascertained by heat inactivation and inhibition by alpha 2-macroglobulin. Incomplete inhibition of proteolysis and the failure of small peptides (less than 6 kd) to show up on electrophoresis seem to suggest that MBP was degraded by exopeptic proteases as well. Acid extracts of purified myelin yielded polypeptides similar to those of MBP of delipidated white matter. The results are consistent with a sequential limited proteolysis of MBP by neutral proteases probably associated with myelin and possibly related to the in situ catabolism of MBP in man.     

Relationship between bisphosphonate concentration and osteoclast activity and viability

Summary Difluoromethylidene bisphosphonate (F₂MBP) is one of the many bisphosphonates known to inhibit bone resorption in vitro and in vivo. We have developed an analytical method, employing anion exchange and postcolumn indirect fluorescence detection, by which F₂MBP can be quantified in bone samples. The objective of this study was to relate the concentration of F₂MBP in embryonic bones treated in organ culture to the physiological effects of the compound, such as bone resorption (i.e., the amount of ⁴⁵Ca released into the medium from prelabeled bones) and viability of the osteoclast population (i.e., the incidence of abnormal osteoclasts). Osteoclasts in bones treated with F₂MBP exhibited morphological features of apoptosis, such as nuclear fragmentation. Both the number and percentage of these abnormal cells increased with dose of F₂MBP and duration of incubation. The decrease in normal osteoclasts was correlated with the decreased amount of ⁴⁵Ca released into the medium. Bones treated with F₂MBP for only the first 5 min of the 48-h incubation period had similar numbers of abnormal osteoclasts and amounts of ⁴⁵Ca released, as had bones incubated with F₂MBP continuously for 48 h. The uptake of F₂MBP into the bone was rapid. Bones treated with F₂MBP for 6 h were similar to bones treated with F₂MBP for the entire 48-h incubation period, both in F₂MBP concentration and the ⁴⁵Ca release ratios. These relationships between concentrations of F₂MBP within bone and osteoclast activity and viability implicate apoptosis in the mechanism by which this bisphosphonate inhibits bone resorption.     

Stimulation of osteoclastic bone resorption by hydrogen peroxide.

The molecular mechanisms underlying the pathophysiology of bone destruction still remain poorly understood. We have found that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is a potent stimulator of osteoclastic bone resorption and cell motility. A marked enhancement of bone resorption was noted when rat osteoclasts, cultured on devitalised bovine cortical bone, were exposed to 10 nM [H2O2]. Apart from exposing osteoclasts to a low extracellular pH, which is known to enhance osteoclastic bone resorption, we provide first evidence for a molecule that stimulates osteoclastic bone resorption in osteoclast cultures that do not respond to parathyroid hormone and 1, 25 dihydroxyvitamin D3. We envisage that both basic biological and practical clinical implications may eventually follow from these studies.     

Cystatin F is a cathepsin C-directed protease inhibitor regulated by proteolysis

Cystatins are a family of naturally occurring cysteine protease inhibitors, yet the target proteases and biological processes they regulate are poorly understood. Cystatin F is expressed selectively in immune cells and is the only cystatin to be synthesised as an inactive disulphide-linked dimeric precursor. Here, we show that a major target of cystatin F in different immune cell types is the aminopeptidase cathepsin C, which regulates the activation of effector serine proteases in T cells, natural killer cells, neutrophils and mast cells. Surprisingly, recombinant cystatin F was unable to inhibit cathepsin C in vitro even though overexpression of cystatin F suppressed cellular cathepsin C activity. We predicted, using structural models, that an N-terminal processing event would be necessary before cystatin F can engage cathepsin C and we show that the intracellular form of cystatin F indeed has a precise N-terminal truncation that creates a cathepsin C inhibitor. Thus, cystatin F is a latent protease inhibitor itself regulated by proteolysis in the endocytic pathway. By targeting cathepsin C, it may regulate diverse immune cell effector functions.     

Sclerostin is expressed in osteoclasts from aged mice and reduces osteoclast‐mediated stimulation of mineralization

Osteoclast‐mediated bone resorption precedes osteoblast‐mediated bone formation through early adulthood, but formation fails to keep pace with resorption during aging. We previously identified several factors produced by osteoclasts that promote bone formation. In this study, we determined if osteoclast‐produced factors contribute to the impaired bone formation with aging. We previously found that mice between the ages of 18 and 22 months develop age‐related bone loss. Bone marrow‐derived pre‐osteoclasts were isolated from 6‐week, 12‐month, and 18‐ to 24‐month‐old mice and differentiated into osteoclasts in vitro. Conditioned media were collected and compared for osteoblast mineralization support. Conditioned medium from osteoclasts from all ages was able to support mineralization of bone marrow stromal cells. Concentrating the conditioned medium from 6‐week‐old and 12‐month‐old mouse marrow cells‐derived osteoclasts enhanced mineralization support whereas concentrated conditioned medium from 18‐ to 24‐month‐old mouse marrow‐derived osteoclasts repressed mineralization compared to base medium. This observation suggests that an inhibitor of mineralization was secreted by aged murine osteoclasts. Gene and protein analysis revealed that the Wnt antagonist sclerostin was significantly elevated in the conditioned media from 24‐month‐old mouse cells compared to 6‐week‐old mouse cells. Antibodies directed to sclerostin neutralized the influences of the aged mouse cell concentrated conditioned media on mineralization. Sclerostin is primarily produced by osteocytes in young animals. This study demonstrates that osteoclasts from aged mice also produce sclerostin in quantities that may contribute to the age‐related impairment in bone formation. J. Cell. Biochem. 114: 1901–1907, 2013. © 2013 Wiley Periodicals, Inc.