Bone mass protective potential mediated by bovine milk basic protein requires normal calcium homeostasis in mice.

OBJECTIVES: Milk provide protective effects against bone loss caused by an impaired calcium balance. Although the effects of some elements have previously been confirmed, the involvement of milk basic protein (MBP) in bone mineral metabolism remains poorly characterized. Moreover, the importance of mineral nutrition sufficiency to establish the effect of MBP must be evaluated. METHODS: First, to evaluate the physiological conditions required for MBP activity, we examined the bone and mineral phenotypes of mice that suffer from insufficient calcium absorption due to a lack of intestinal vitamin D signaling. Second, to determine whether vitamin D signaling affects the effect of MBP on bone resorption, in vitro osteoclastogenesis were assessed using bone marrow cells. RESULTS: In mice with systemic vitamin D receptor (Vdr) inactivation, dietary MBP supplementation was unable to normalize hypercalcemia and hyperparathyroidism and failed to rescue bone mineralization impairments. In contrast, calcium and bone homeostasis responded to MBP supplementation when Vdr inactivation was restricted to the intestines. Hyperparathyroidism in intestine-specific Vdr knockout mice was also improved by MBP supplementation, along with a decrease in bone resorption in response to the level of serum tartrate-resistant acid phosphatase 5b. These results corresponded with a reduction in tartrate-resistant acid phosphatase-stained osteoclast numbers and the eroded surface on the tibia. MBP treatment dose-dependently suppressed osteoclastogenesis in cultured bone marrow macrophages regardless of vitamin D activity. These effects of MBP were blunted when parathyroid hormone was added to the culture medium, which is in line with the in vivo phenotype observed with systemic Vdr inactivation and suggests that severe hyperparathyroidism limits MBP activity in the bone. CONCLUSIONS: Therefore, adaptive calcium homeostasis is an essential requirement when MBP exerts protective effects through the inhibition of bone resorption.

Effector memory CD4+T cells in mesenteric lymph nodes mediate bone loss in food-allergic enteropathy model mice, creating IL-4 dominance.

Intestinal inflammation can be accompanied by osteoporosis, but their relationship, mediated by immune responses, remains unclear. Here, we investigated a non-IgE-mediated food-allergic enteropathy model of ovalbumin (OVA) 23-3 mice expressing OVA-specific T-cell-receptor transgenes. Mesenteric lymph nodes (MLNs) and their pathogenic CD4+T cells were important to enteropathy occurrence and exacerbation when the mice were fed an egg-white (EW) diet. EW-fed OVA23-3 mice also developed bone loss and increased CD44hiCD62LloCD4+T cells in the MLNs and bone marrow (BM); these changes were attenuated by MLN, but not spleen, resection. We fed an EW diet to F1 cross offspring from OVA23-3 mice and a mouse line expressing the photoconvertible protein KikGR to track MLN CD4+T cells. Photoconverted MLN CD44hiCD62LloCD4+T cells migrated predominantly to the BM; pit formation assay proved their ability to promote bone damage via osteoclasts. Significantly greater expression of IL-4 mRNA in MLN CD44hiCD62LloCD4+T cells and bone was observed in EW-fed OVA23-3 mice. Anti-IL-4 monoclonal antibody injection canceled bone loss in the primary inflammation phase in EW-fed mice, but less so in the chronic phase. This novel report shows the specific inflammatory relationship, via Th2-dominant-OVA-specific T cells and IL-4 production, between MLNs and bone, a distant organ, in food-allergic enteropathy.

Short communication: Milk basic protein promotes proliferation and inhibits differentiation of mouse chondrogenic ATDC5 cells.

It has been reported that the intake of milk basic protein (MBP) increases bone density by promoting bone formation and suppressing bone resorption. However, few studies have been done on MBP in cartilage, the tissue adjacent to bone. We therefore investigated the effect of MBP on a chondrocyte cell line, ATDC5. In a proliferative assay using the WST-1 method, the addition of 10, 100, and 1,000 µg/mL of MBP to ATDC5 cells significantly increased the cell number by about 1.2-, 1.5-, and 1.7-fold, respectively, compared with the control cells. The cell cycle analysis using flow-cytometry revealed that the proportion of S- and G2/M-phase cells was increased but that of G0/G1 phase was decreased in a dose-dependent manner with MBP addition. We measured the alkaline phosphatase activity of MBP-treated ATDC5 cells to examine the differentiation stage of the cells. Alkaline phosphatase activity was suppressed in a dose-dependent manner with MBP addition and was especially drastic at higher doses of MBP (100 and 1,000 µg/mL). The Alizarin Red S staining intensity, the indicator for calcification of cells, was lower in the MBP-treated (100 µg/mL) cells than in nontreated control cells. In the reverse-transcription PCR experiment, the mRNA level of SRY-box containing gene 9 (Sox9) and type II collagen (Col2) was significantly increased in the MBP-treated cells compared with the control cells. A significant decrease of the mRNA level of runt-related transcription factor 2 (Runx2) and type X collagen (Col10) was also observed in the MBP-treated cells. These results suggested that MBP promoted the proliferation of chondrocytes by suppressing their differentiation toward calcification.

Milk basic protein supplementation exerts an anti-inflammatory effect in a food-allergic enteropathy model mouse.

To examine novel functions of milk basic protein (MBP) in T-cell-related inflammatory diseases, such as autoimmune diseases and allergies, we evaluated the effects of MBP on the causative responses of ovalbumin (OVA)-specific T cells in a food-allergic enteropathy model, OVA23-3 mice, which express an OVA-specific T-cell receptor gene. The OVA-specific CD4+ T cells of the mesenteric lymph nodes (MLN) from OVA23-3 mice were cultured with CD11c+ dendritic cells of MLN from BALB/cA mice in the absence or presence of MBP following stimulation with OVA; then the levels of CD69 expression and the levels of cytokine production by CD4+ T cells were measured to evaluate activation. The effects of MBP supplementation of OVA 23-3 mice were assessed by feeding a diet containing OVA (OVA diet) with or without MBP for 28 d. Intestinal inflammation, together with activation and cytokine production of CD4+ T cells by MLN, as well as femoral bone mineral density, were measured. In in vitro culture, MBP inhibited excess activation and IL-4 production by CD4+ T cells. The supplementation of MBP to the OVA diet attenuated OVA-specific IgE production in OVA-diet-fed OVA23-3 mice and slightly resolved developing enteropathy caused by excess IL-4 production by CD4+ T cells. Feeding OVA diet to OVA23-3 mice exhibited bone loss accompanied with enteropathy, whereas MBP supplementation prevented bone loss and increased osteoprotegerin, an osteoclastogenesis inhibitory factor, in the mice. The inhibition of T-cell-activation in both MLN and bone marrow by MBP supplementation may help prevent increased IgE levels caused by excessive IL-4 production and bone loss accompanied by enteropathy. Our findings show that MBP may help attenuate both T-cell-related inflammation and bone loss.

Milk Basic Protein Facilitates Increased Bone Mass in Growing Mice.

Milk basic protein (MBP) comprises a group of basic whey proteins and is effective in preventing bone loss by promoting bone deposition (bone formation) and suppressing withdrawn (bone resorption). We previously revealed the bone protective effects of MBP during life phases involving excessive bone resorption, such as in adults and postmenopausal women, and in animal models (ovariectomized rats and mice). However, it was unclear whether MBP increases bone mass during the growth stage, when there is more bone formation than resorption. We therefore investigated the effect of MBP supplementation on bone mass in 6-wk-old mice provided water supplemented with MBP [0.01%, 0.1%, 1.0% (w/w)] or deionized water (control) ad libitum for 10 wk. Analysis by micro-computerized tomography showed that MBP significantly increased tibia cortical bone mineral density and femur trabecular bone volume to tissue volume compared with mice provided deionized water. Next, the function of MBP in bone remodeling (bone formation and resorption) was evaluated using an in vitro system and the results demonstrated that MBP directly promoted osteoblast proliferation and inhibited osteoclastogenesis. Moreover, the plasma level of insulin-like growth factor-1 was increased by MBP supplementation, suggesting that MBP indirectly promoted osteoblast proliferation/differentiation. These effects enhance bone formation and/or inhibit bone resorption, resulting in increased bone mass in growing mice.

Milk basic protein increases ghrelin secretion and bone mineral density in rodents.

OBJECTIVES: Milk basic protein (MBP), a mixture of proteins isolated from bovine milk, is known to increase bone formation. Ghrelin, a stomach-derived peptide hormone, also has been reported to stimulate osteoblast formation. The aim of this study was to determine whether MBP-induced bone formation is mediated via ghrelin. METHODS: MBP was chronically administered to mice in their drinking water for 3 wk, and body weight, water intake, and bone mineral density were measured. Additionally, plasma bone-specific alkaline phosphatase, tartrate-resistant acid phosphatase isoform 5b, and ghrelin concentrations were determined by enzyme-linked immunosorbent assay. To examine the direct effect of MBP on ghrelin secretion, gastric tissue culture and primary mucosal cells were stimulated by MBP. RESULTS: The in vivo study of young, growing mice showed that chronic MBP intake for 3 wk increased the plasma ghrelin concentration and bone mineral density of the hind limb tibia. In vitro studies using minced rat gastric mucosa tissues and primary murine isolated gastric mucosal cells revealed that MBP stimulated ghrelin release in a dose-dependent manner. Moreover, MBP-induced ghrelin secretion was partly inhibited by adrenergic blockers. CONCLUSIONS: These findings suggest a novel mechanism by which MBP directly acts on ghrelin secretion. Additionally, the elevated ghrelin level induced by MBP may act as a mediator for bone formation.

Chloroquine reduces osteoclastogenesis in murine osteoporosis by preventing TRAF3 degradation.

The cytokines RANKL and TNF activate NF-κB signaling in osteoclast precursors (OCPs) to induce osteoclast (OC) formation. Conversely, TNF can limit OC formation through NF-κB p100, which acts as an inhibitor, and TNF receptor-associated receptor 3 (TRAF3); however, a role for TRAF3 in RANKL-mediated OC formation is unknown. We found that TRAF3 limits RANKL-induced osteoclastogenesis by suppressing canonical and noncanonical NF-κB signaling. Conditional OC-specific Traf3-KO (cKO) mice had mild osteoporosis and increased OC formation. RANKL induced TRAF3 degradation via the lysosome/autophagy system. The autophagy/lysosome inhibitor chloroquine reduced RANKL-induced OC formation and function by increasing TRAF3 expression in OCPs in vitro and in vivo. Although chloroquine had no effect on basal bone resorption, it inhibited parathyroid hormone- and ovariectomy-induced OC activation in WT, but not cKO, mice. Deletion of the transcription factor gene Relb resulted in increased TRAF3 expression in OCPs, which was associated with decreased RANKL-induced TRAF3 degradation. RelB directly increased expression of BECN1, a key autophagy regulator, by binding to its promoter. These data indicate that autophagic/lysosomal degradation of TRAF3 is an important step in RANKL-induced NF-κB activation in OCPs. Furthermore, treatments that increase TRAF3 levels in OCPs, including pharmacological inhibition of its degradation with compounds such as chloroquine, may limit bone destruction in common bone diseases.

Noncanonical NF-κB signaling regulates hematopoietic stem cell self-renewal and microenvironment interactions.

RelB and nuclear factor κB (NF-κB2) are the main effectors of NF-κB noncanonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-κB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Furthermore, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-κB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment.

Identification of angiogenin as the osteoclastic bone resorption-inhibitory factor in bovine milk.

We identified, for the first time, the factor responsible for inhibiting osteoclast-mediated bone resorption in the basic protein fraction of bovine milk (milk basic protein, MBP). The protein was purified by a combination of ion and gel column chromatography from MBP, based on its activity to prevent unfractionated rabbit bone cells from forming pits on dentine slices. It was found to have a molecular weight of 15 kDa on SDS-PAGE, and the sequence of the N-terminal 25 amino acid residues was identical to that of bovine angiogenin. The purified bovine angiogenin inhibited the pit-forming activity of both unfractionated bone cells and purified osteoclasts in a dose-dependent manner, and the inhibitory activity was markedly suppressed by treatment with anti-bovine angiogenin antibody. The inhibitory activity was confirmed in mice both in vitro and in vivo. Treatment of osteoclasts with bovine angiogenin resulted in an impairment of the formation F-actin ring and a reduction in the mRNA levels of TRAP and cathepsin K, both known to be essential for the bone resorption activity of osteoclasts. These results suggest that bovine angiogenin is the substance mainly responsible for the inhibitory effect of bovine milk on osteoclast-mediated bone resorption, and that it exerts its activity by acting directly on the osteoclasts.

Diurnal variation of biopyrrin excretion in random urine specimens is not corrected by creatinine.

Circulating bilirubin is thought to function as a physiological antioxidant. One of the decomposition products of this process is the biopyrrins, which include two regioisomers: biotripyrrin-a (1,14,15,17-tetrahydro-2,7,13-trimethyl-1,14-deoxy-3-vinyl-16H-tripyrrin-8,12-dipropionic acid) and biotripyrrin-b (1,14,15,17-tetrahydro-3,7,13-trimethyl-1,14-deoxy-3-vinyl-16H-tripyrrin-8,12-dipropionic acid). We measured biopyrrins in random urine specimens and investigated whether the biopyrrin values obtained were valid when expressed as a ratio of the creatinine (Cr) concentrations. All of the random urine specimens collected over 48 hr were from presumably healthy adults. We measured the biopyrrins by means of an enzyme-linked immunosorbent assay (ELISA) using an anti-bilirubin monoclonal antibody. When the values were expressed in terms of the ratio to Cr, the within-day coefficient of variation (%CV) of the excretion of biopyrrins was reduced to 27%+/-10% (P<0.05) from 59%+/-27%. However, assay values on random or spot urine specimens were unreliable because of the large %CV. The biopyrrin concentrations only in the first-morning-urine specimens in terms of both absolute amounts and ratios to Cr significantly reflected those in a 24-hr urine specimen (P<0.001). Concentrations in a random urine specimen voided at the second collection or later did not correlate with the concentration in a 24-hr urine specimen (P>0.05), even if their values were corrected by Cr. The amounts of biopyrrins excreted in 24-hr urine specimens were significantly correlated with the 24-hr cortisol excretion (P<0.001) but not to uropepsin (P>0.05).

The fragments of bovine high molecular weight kininogen promote osteoblast proliferation in vitro.

High molecular weight (HMW) kininogen is known to be a large plasma protein and cleaved by plasma proteinase kallikrein, then it generates four fragments in the blood coagulation cascade: heavy chain, bradykinin, fragment 1.2, and light chain. The fragment 1.2 has also been found in the basic protein fraction of bovine milk as a bioactive protein which promotes osteoblast proliferation. The milk basic protein has been shown to be a multi functional edible protein which promotes bone formation and inhibits bone resorption. In the present study, we purified the fragment 1.2 from bovine plasma and assessed it could promote osteoblast proliferation and posses the activity after pepsin digestion. Purified plasma HMW kininogen did not promote the proliferation, however, the kallikrein-cleaved HMW kininogen promoted the proliferation. The fragment 1.2, purified from the proteolysate, also promoted the proliferation. The pepsin digestion was performed according to the method of the assessment of allergenesity of genetically modified crops. After pepsin digestion, the fragment 1.2 generated resistant fragments and showed the promoting activity of osteoblast proliferation. These results suggest that the enzymatically-digested fragments of bovine HMW kininogen are able to be a naturally occurred active protein that promotes the bone formation by oral administration.

Cystatin C in milk basic protein (MBP) and its inhibitory effect on bone resorption in vitro.

A cystein protease inhibitor was identified in the basic fraction of bovine milk. We have reported in our previous study that the milk basic protein (MBP) fraction suppressed osteoclast-mediated bone resorption in vitro. Since osteoclasts secreted cystein protease to digest collagen in the bone matrix, we identified the cystein protease inhibitor in MBP. A 12-kDa inhibitor was purified from MBP by papain affinity gel chromatography and subsequent Hi-Load Superdex 75 gel filtration chromatography. The N-terminal sequence of the 18 amino acid residues of the inhibitor corresponded to bovine cystatin C. The 12-kDa cystein protease inhibitor in MBP therefore seemed to be cystatin C. Purified cystatin suppressed bone resorption with the use of isolated osteoclasts in vitro. Cystatin in MBP is suggested as one of the factors inhibiting bone resorption.