WNT/RYK signaling functions as an antiinflammatory modulator in the lung mesenchyme.

A number of inflammatory lung diseases, including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and pneumonia, are modulated by WNT/ß-catenin signaling. However, the underlying molecular mechanisms remain unclear. Here, starting with a forward genetic screen in mouse, we identify the WNT coreceptor Related to receptor tyrosine kinase (RYK) acting in mesenchymal tissues as a cell survival and antiinflammatory modulator. Ryk mutant mice exhibit lung hypoplasia and inflammation as well as alveolar simplification due to defective secondary septation, and deletion of Ryk specifically in mesenchymal cells also leads to these phenotypes. By analyzing the transcriptome of wild-type and mutant lungs, we observed the up-regulation of proapoptotic and inflammatory genes whose expression can be repressed by WNT/RYK signaling in vitro. Moreover, mesenchymal Ryk deletion at postnatal and adult stages can also lead to lung inflammation, thus indicating a continued role for WNT/RYK signaling in homeostasis. Our results indicate that RYK signaling through ß-catenin and Nuclear Factor kappa B (NF-κB) is part of a safeguard mechanism against mesenchymal cell death, excessive inflammatory cytokine production, and inflammatory cell recruitment and accumulation. Notably, RYK expression is down-regulated in the stromal cells of pneumonitis patient lungs. Altogether, our data reveal that RYK signaling plays critical roles as an antiinflammatory modulator during lung development and homeostasis and provide an animal model to further investigate the etiology of, and therapeutic approaches to, inflammatory lung diseases.

WNT/RYK signaling restricts goblet cell differentiation during lung development and repair.

Goblet cell metaplasia and mucus hypersecretion are observed in many pulmonary diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. However, the regulation of goblet cell differentiation remains unclear. Here, we identify a regulator of this process in an N-ethyl-N-nitrosourea (ENU) screen for modulators of postnatal lung development; Ryk mutant mice exhibit lung inflammation, goblet cell hyperplasia, and mucus hypersecretion. RYK functions as a WNT coreceptor, and, in the developing lung, we observed high RYK expression in airway epithelial cells and moderate expression in mesenchymal cells as well as in alveolar epithelial cells. From transcriptomic analyses and follow-up studies, we found decreased WNT/ß-catenin signaling activity in the mutant lung epithelium. Epithelial-specific Ryk deletion causes goblet cell hyperplasia and mucus hypersecretion but not inflammation, while club cell-specific Ryk deletion in adult stages leads to goblet cell hyperplasia and mucus hypersecretion during regeneration. We also found that the airway epithelium of COPD patients often displays goblet cell metaplastic foci, as well as reduced RYK expression. Altogether, our findings reveal that RYK plays important roles in maintaining the balance between airway epithelial cell populations during development and repair, and that defects in RYK expression or function may contribute to the pathogenesis of human lung diseases.

Osteoclast differentiation by RANKL and OPG signaling pathways.

INTRODUCTION: In bone tissue, bone resorption by osteoclasts and bone formation by osteoblasts are repeated continuously. Osteoclasts are multinucleated cells that derive from monocyte-/macrophage-lineage cells and resorb bone. In contrast, osteoblasts mediate osteoclastogenesis by expressing receptor activator of nuclear factor-kappa B ligand (RANKL), which is expressed as a membrane-associated cytokine. Osteoprotegerin (OPG) is a soluble RANKL decoy receptor that is predominantly produced by osteoblasts and which prevents osteoclast formation and osteoclastic bone resorption by inhibiting the RANKL-RANKL receptor interaction. MATERIALS AND METHODS: In this review, we would like to summarize our experimental results on signal transduction that regulates the expression of RANKL and OPG. RESULTS: Using OPG gene-deficient mice, we have demonstrated that OPG and sclerostin produced by osteocytes play an important role in the maintenance of cortical and alveolar bone. In addition, it was shown that osteoclast-derived leukemia inhibitory factor (LIF) reduces the expression of sclerostin in osteocytes and promotes bone formation. WP9QY (W9) is a peptide that was designed to be structurally similar to one of the cysteine-rich TNF-receptortype-I domains. Addition of the W9 peptide to bone marrow culture simultaneously inhibited osteoclast differentiation and stimulated osteoblastic cell proliferation. An anti-sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) antibody inhibited multinucleated osteoclast formation induced by RANKL and macrophage colony-stimulating factor (M-CSF). Pit-forming activity of osteoclasts was also inhibited by the anti-Siglec-15 antibody. In addition, anti-Siglec-15 antibody treatment stimulated the appearance of osteoblasts in cultures of mouse bone marrow cells in the presence of RANKL and M-CSF. CONCLUSIONS: Bone mass loss depends on the RANK-RANKL-OPG system, which is a major regulatory system of osteoclast differentiation induction, activation, and survival.

Spleen serves as a reservoir of osteoclast precursors through vitamin D-induced IL-34 expression in osteopetrotic op/op mice.

Osteoclasts are generated from monocyte/macrophage-lineage precursors in response to colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). CSF-1-mutated CSF-1(op/op) mice as well as RANKL(-/-) mice exhibit osteopetrosis (OP) caused by osteoclast deficiency. We previously identified RANKL receptor (RANK)/CSF-1 receptor (CSF-1R) double-positive cells as osteoclast precursors (OCPs), which existed in bone in RANKL(-/-) mice. Here we show that OCPs do not exist in bone but in spleen in CSF-1(op/op) mice, and spleen acts as their reservoir. IL-34, a newly discovered CSF-1R ligand, was highly expressed in vascular endothelial cells in spleen in CSF-1(op/op) mice. Vascular endothelial cells in bone also expressed IL-34, but its expression level was much lower than in spleen, suggesting a role of IL-34 in the splenic generation of OCPs. Splenectomy (SPX) blocked CSF-1-induced osteoclastogenesis in CSF-1(op/op) mice. Osteoclasts appeared in aged CSF-1(op/op) mice with up-regulation of IL-34 expression in spleen and bone. Splenectomy blocked the age-associated appearance of osteoclasts. The injection of 2-methylene-19-nor-(20S)-1α,25(OH)(2)D(3) (2MD), a potent analog of 1α,25-dihidroxyvitamin D(3), into CSF-1(op/op) mice induced both hypercalcemia and osteoclastogenesis. Administration of 2MD enhanced IL-34 expression not only in spleen but also in bone through a vitamin D receptor-mediated mechanism. Either splenectomy or siRNA-mediated knockdown of IL-34 suppressed 2MD-induced osteoclastogenesis. These results suggest that IL-34 plays a pivotal role in maintaining the splenic reservoir of OCPs, which are transferred to bone in response to diverse stimuli, in CSF-1(op/op) mice. The present study also suggests that the IL-34 gene in vascular endothelial cells is a unique target of vitamin D.

[Current Topics on Vitamin D. The role of active forms of vitamin D in regulation of bone remodeling].

Active forms of vitamin D3 have been widely used in the treatment of rickets, osteomalacia, and osteoporosis in order to stimulate bone mineralization and prevent bone loss. Meanwhile, the active form of vitamin D3, 1α, 25-dihydroxyvitamin D3 [1α, 25 (OH) 2D3], upregulates expression of receptor activator of nuclear factor-κB ligand (RANKL) in osteoblastic cells. Osteoclast formation requires RANKL and macrophage colony-stimulating factor (M-CSF), which are expressed in osteoblastic cells. Recently, IL-34 was discovered as a cytokine functionally overlapping M-CSF. We found that administration of 1α, 25 (OH) 2D3 into mice enhanced IL-34 expression in spleen and bone. These results suggest that 1α, 25 (OH) 2D3 is a bone resorption-stimulating factor. However, daily administration of an active vitamin D3 analog (eldecalcitol, an osteoporotic therapeutic drug) for a month suppressed RANKL expression in osteoblasts, resulting in suppression of bone resorption. Here we discuss new findings and hypotheses to explain this contradictory effect of active vitamin D3 on bone remodeling.

Fos plays an essential role in the upregulation of RANK expression in osteoclast precursors within the bone microenvironment.

Fos plays essential roles in the osteoclastic differentiation of precursor cells generated by colony-stimulating factor 1 (CSF-1) and receptor activator of NF-κB ligand (RANKL; also known as tumor necrosis factor ligand superfamily member 11, Tnsf11). RANKL-deficient (RANKL(-/-)) mice and Fos(-/-) mice exhibit osteopetrosis due to an osteoclast deficiency. We previously reported that RANK-positive osteoclast precursors are present in bone of RANKL(-/-) mice but not Fos(-/-) mice. Here we report the role of Fos in RANK expression in osteoclast precursors. Medullary thymic epithelial cells and intestinal antigen-sampling microfold cells have been shown to express RANK. High expression of RANK was observed in some epithelial cells in the thymic medulla and intestine but not in osteoclast precursors in Fos(-/-) mice. RANK mRNA and protein levels in bone were lower in Fos(-/-) mice than RANKL(-/-) mice, suggesting that Fos-regulated RANK expression is tissue specific. When wild-type bone marrow cells were inoculated into Fos(-/-) mice, RANK-positive cells appeared along bones. RANK expression in wild-type macrophages was upregulated by coculturing with RANKL(-/-) osteoblasts as well as wild-type osteoblasts, suggesting that cytokines other than RANKL expressed by osteoblasts upregulate RANK expression in osteoclast precursors. CSF-1 receptor-positive cells were detected near CSF-1-expressing osteoblastic cells in bone in Fos(-/-) mice. CSF-1 upregulated RANK expression in wild-type macrophages but not Fos(-/-) macrophages. Overexpression of Fos in Fos(-/-) macrophages resulted in the upregulation of RANK expression. Overexpression of RANK in Fos(-/-) macrophages caused RANKL-induced signals, but failed to recover the RANKL-induced osteoclastogenesis. These results suggest that Fos plays essential roles in the upregulation of RANK expression in osteoclast precursors within the bone environment.

Roles of cathelicidins in inflammation and bone loss.

Body surface tissues, such as the oral cavity, contact directly with the external environment and are continuously exposed to microbial insults. Cathelicidins are a family of antimicrobial peptides that are found in mammalian species. Humans and mice have only one cathelicidin. Cathelicidins are expressed in a variety of surface tissues. In addition, they are abundantly expressed in bone and bone marrow. Infectious stimuli upregulate the expression of cathelicidins, which play sentinel roles in allowing the tissues to fight against microbial challenges. Cathelicidins disrupt membranes of microorganisms and kill them. They also neutralize microbe-derived pathogens, such as lipopolysaccharide (LPS) and flagellin. Besides their antimicrobial functions, cathelicidins can also control actions of host cells, such as chemotaxis, proliferation, and cytokine production, through binding to the receptors expressed on them. LPS and flagellin induce osteoclastogenesis and the production of cathelicidins, which can in turn inhibit osteoclastogenesis. Thus, cathelicidins contribute to maintaining microbiota-host homeostasis and promoting repair responses to inflammatory insults. In this review, we describe recent findings on the multiple roles of cathelicidins in host defense. We also discuss the significance of the human cathelicidin, LL-37, as a pharmaceutical target for the treatment of inflammation and bone loss in infectious diseases, such as periodontitis.

Lack of vitamin D signalling in mesenchymal progenitors causes fatty infiltration in muscle.

BACKGROUND: Recent studies have indicated the importance of muscle quality in addition to muscle quantity in sarcopenia pathophysiology. Intramuscular adipose tissue (IMAT), which originates from mesenchymal progenitors (MPs) in adult skeletal muscle, is a key factor affecting muscle quality in older adults, suggesting that controlling IMAT formation is a promising therapeutic strategy for sarcopenia. However, the molecular mechanism underlying IMAT formation in older adults has not been clarified. We recently found that the vitamin D receptor (VDR) is highly expressed in MPs in comparison to myotubes (P = 0.028, N = 3), indicating a potential role of vitamin D signalling in MPs. In this study, we aimed to clarify the role of vitamin D signalling in MP kinetics, with a focus on adipogenesis. METHODS: MPs isolated from mouse skeletal muscles were subjected to adipogenic differentiation conditions with or without vitamin D (1α,25(OH)2D3, 100 nM) for 7 days, and adipogenicity was evaluated based on adipogenic marker expression. For in vivo analysis, tamoxifen-inducible MP-specific VDR-deficient (VdrMPcKO) mice were newly developed to investigate whether lack of vitamin D signalling in MPs is involved in IMAT formation. To induce muscle atrophy, VdrMPcKO male mice were subjected to tenotomy of the gastrocnemius muscle, and then muscle weight, myofibre cross-sectional area, adipogenic marker expression, and fatty infiltration into the muscle were evaluated at 3 weeks after operation (N = 3-4). In addition, a vitamin D-deficient diet was provided to wild-type male mice (3 and 20 months of age, N = 5) for 3 months to investigate whether vitamin D deficiency causes IMAT formation. RESULTS: Vitamin D treatment nearly completely inhibited adipogenesis of MPs through Runx1-mediated transcriptional modifications of early adipogenic factors such as PPARγ (P = 0.0031) and C/EBPα (P = 0.0027), whereas VDR-deficient MPs derived from VdrMPcKO mice differentiated into adipocytes even in the presence of vitamin D (P = 0.0044, Oil-Red O+ area). In consistency with in-vitro findings, VdrMPcKO mice and mice fed a vitamin D-deficient diet exhibited fat deposition in atrophied (P = 0.0311) and aged (P = 0.0216) skeletal muscle, respectively. CONCLUSIONS: Vitamin D signalling is important to prevent fate decision of MPs towards the adipogenic lineage. As vitamin D levels decline with age, our data indicate that decreased vitamin D levels may be one of the causes of IMAT formation in older adults, and vitamin D signalling may be a novel therapeutic target for sarcopenia.

Roles of cathelicidin-related antimicrobial peptide in murine osteoclastogenesis.

Cathelicidin-related antimicrobial peptide (CRAMP) not only kills bacteria but also binds to lipopolysaccharide (LPS) to neutralize its activity. CRAMP is highly expressed in bone marrow and its expression is reported to be up-regulated by inflammatory and infectious stimuli. Here, we examined the role of CRAMP in murine osteoclastogenesis. Osteoclasts were formed in co-cultures of osteoblasts and bone marrow cells in response to 1α,25-dihydroxyvitamin D3 [1α,25(OH)2 D3 ], prostaglandin E2 (PGE2 ), and Toll-like receptor (TLR) ligands such as LPS and flagellin through the induction of receptor activator of nuclear factor-κB ligand (RANKL) expression in osteoblasts. CRAMP inhibited the osteoclastogenesis in co-cultures treated with LPS and flagellin, but not in those treated with 1α,25(OH)2 D3 or PGE2 . Although bone marrow macrophages (BMMs) highly expressed formyl peptide receptor 2 (a receptor of CRAMP), CRAMP showed no inhibitory effect on osteoclastogenesis in BMM cultures treated with RANKL. CRAMP suppressed both LPS- and flagellin-induced RANKL expression in osteoblasts and tumour necrosis factor-α (TNF-α) expression in BMMs, suggesting that CRAMP neutralizes the actions of LPS and flagellin. LPS and flagellin enhanced the expression of CRAMP mRNA in osteoblasts. Extracellularly added CRAMP suppressed LPS- and flagellin-induced CRAMP expression. These results suggest that the production of CRAMP promoted by LPS and flagellin is inhibited by CRAMP released by osteoblasts through a feedback regulation. Even though CRAMP itself has no effect on osteoclastogenesis in mice, we propose that CRAMP is an osteoblast-derived protector in bacterial infection-induced osteoclastic bone resorption.

IL-34 and CSF-1: similarities and differences.

Colony-stimulating factor-1 (CSF-1) is widely expressed and considered to regulate the development, maintenance, and function of mononuclear phagocyte lineage cells such as monocytes, macrophages, dendritic cells (DCs), Langerhans cells (LCs), microglia, and osteoclasts. Interleukin-34 (IL-34) was recently identified as an alternative ligand for the CSF-1 receptor (CSF-1R) through functional proteomics experiments. It is well established that the phenotype of CSF-1R-deficient (CSF-1R⁻/⁻) mice is more severe than that of mice bearing a spontaneous null mutation in CSF-1 (CSF-1(op/op)). CSF-1R⁻/⁻ mice are severely depleted of macrophages and completely lack LCs, microglia, and osteoclasts during their lifetime. In contrast, CSF-1(op/op) mice exhibit late-onset macrophage development and osteoclastogenesis, whereas they show modestly reduced numbers of microglia and a relatively normal LC development. In contrast, IL-34-deficient (IL-34⁻/⁻) mice show a marked reduction of LCs and a decrease in microglia. IL-34 and CSF-1 display different spatiotemporal expression patterns and have distinct biological functions. In this review, we focus on the functional similarities and differences between IL-34 and CSF-1 in vivo.

The vitamin D receptor in osteoblastic cells but not secreted parathyroid hormone is crucial for soft tissue calcification induced by the proresorptive activity of 1,25(OH)2D3.

The vitamin D receptor (VDR) is expressed most abundantly in osteoblasts and osteocytes (osteoblastic cells) in bone tissues and regulates bone resorption and calcium (Ca) and phosphate (P) homeostasis in association with parathyroid hormone (PTH). We previously reported that near-physiological doses of vitamin D compounds suppressed bone resorption through VDR in osteoblastic cells. We also found that supra-physiological doses of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced bone resorption and hypercalcemia via VDR in osteoblastic cells. Here, we report that the latter, a proresorptive dose of 1,25(OH)2D3, causes soft tissue calcification through VDR in osteoblastic cells. High concentrations of vitamin D affect multiple organs and cause soft tissue calcification, with increases in bone resorption and serum Ca levels. Such a variety of symptoms is known as hypervitaminosis D, which is caused by not only high doses of vitamin D but also impaired vitamin D metabolism and diseases that produce 1,25(OH)2D3 ectopically. To clarify the biological process hierarchy in hypervitaminosis D, a proresorptive dose of 1,25(OH)2D3 was administered to wild-type mice in which bone resorption had been suppressed by neutralizing anti-receptor activator of NF-κB ligand (RANKL) antibody. 1,25(OH)2D3 upregulated the serum Ca x P product, concomitantly induced calcification of the aorta, lungs, and kidneys, and downregulated serum PTH levels in control IgG-pretreated wild-type mice. Pretreatment of wild-type mice with anti-RANKL antibody did not affect the down-regulation of PTH levels by 1,25(OH)2D3, but inhibited the increase of the serum Ca x P product and soft tissue calcification induced by 1,25(OH)2D3. Consistent with the effects of anti-RANKL antibody, VDR ablation in osteoblastic cells also did not affect the down-regulation of PTH levels by 1,25(OH)2D3, but suppressed the 1,25(OH)2D3-induced increase of the serum Ca x P product and calcification of soft tissues. Taken together with our previous results, these findings suggest that bone resorption induced by VDR signaling in osteoblastic cells is critical for the pathogenesis of hypervitaminosis D, but PTH is not involved in hypervitaminosis D.

Influence of vitamin D on sarcopenia pathophysiology: A longitudinal study in humans and basic research in knockout mice.

BACKGROUND: Vitamin D is an essential nutrient in musculoskeletal function; however, its relationship to sarcopenia remains ambiguous, and the mechanisms and targets of vitamin D activity have not been elucidated. This study aimed to clarify the role of vitamin D in mature skeletal muscle and its relationship with sarcopenia. METHODS: This epidemiological study included 1653 community residents who participated in both the fifth and seventh waves of the National Institute for Longevity Sciences, Longitudinal Study of Aging and had complete background data. Participants were classified into two groups: vitamin D-deficient (serum 25-hydroxyvitamin D < 20 ng/mL) and non-deficient (serum 25-hydroxyvitamin D ≥ 20 ng/mL); they underwent propensity-score matching for background factors (age, sex, height, weight, comorbidities, smoker, alcohol intake, energy intake, vitamin D intake, steps, activity, season and sarcopenia). Changes in muscle strength and mass over the 4-year period were compared. For basic analysis, we generated Myf6CreERT2 Vitamin D Receptor (VDR)-floxed (VdrmcKO ) mice with mature muscle fibre-specific vitamin D receptor knockout, injected tamoxifen into 8-week-old mice and analysed various phenotypes at 16 weeks of age. RESULTS: Grip strength reduction was significantly greater in the deficient group (-1.55 ± 2.47 kg) than in the non-deficient group (-1.13 ± 2.47 kg; P = 0.019). Appendicular skeletal muscle mass reduction did not differ significantly between deficient (-0.05 ± 0.79 kg) and non-deficient (-0.01 ± 0.74 kg) groups (P = 0.423). The incidence of new cases of sarcopenia was significantly higher in the deficient group (15 vs. 5 cases; P = 0.039). Skeletal muscle phenotyping of VdrmcKO mice showed no significant differences in muscle weight, myofibre percentage or myofibre cross-sectional area; however, both forelimb and four-limb muscle strength were significantly lower in VdrmcKO mice (males: forelimb, P = 0.048; four-limb, P = 0.029; females: forelimb, P < 0.001; four-limb, P < 0.001). Expression profiling revealed a significant decrease in expression of sarcoendoplasmic reticulum Ca2+ -ATPase (SERCA) 1 (P = 0.019) and SERCA2a (P = 0.049) genes in the VdrmcKO mice. In contrast, expression of non-muscle SERCA2b and myoregulin genes showed no changes. CONCLUSIONS: Vitamin D deficiency affects muscle strength and may contribute to the onset of sarcopenia. Vitamin D-VDR signalling has minimal influence on the regulation of muscle mass in mature myofibres but has a significant influence on muscle strength.

Angiotensin II Induces Aortic Rupture and Dissection in Osteoprotegerin-Deficient Mice.

Background The biological mechanism of action for osteoprotegerin, a soluble decoy receptor for the receptor activator of nuclear factor-kappa B ligand in the vascular structure, has not been elucidated. The study aim was to determine if osteoprotegerin affects aortic structural integrity in angiotensin II (Ang II)-induced hypertension. Methods and Results Mortality was higher (P<0.0001 by log-rank test) in 8-week-old male homozygotes of osteoprotegerin gene-knockout mice given subcutaneous administration of Ang II for 28 days, with an incidence of 21% fatal aortic rupture and 23% aortic dissection, than in age-matched wild-type mice. Ang II-infused aorta of wild-type mice showed that osteoprotegerin immunoreactivity was present with proteoglycan. The absence of osteoprotegerin was associated with decreased medial and adventitial thickness and increased numbers of elastin breaks as well as with increased periostin expression and soluble receptor activator of nuclear factor-kappa B ligand concentrations. PEGylated human recombinant osteoprotegerin administration decreased all-cause mortality (P<0.001 by log-rank test), the incidence of fatal aortic rupture (P=0.08), and aortic dissection (P<0.001) with decreasing numbers of elastin breaks, periostin expressions, and soluble receptor activator of nuclear factor-kappa B ligand concentrations in Ang II-infused osteoprotegerin gene-knockout mice. Conclusions These data suggest that osteoprotegerin protects against aortic rupture and dissection in Ang II-induced hypertension by inhibiting receptor activator of nuclear factor-kappa B ligand activity and periostin expression.

RANKL/OPG ratio regulates odontoclastogenesis in damaged dental pulp.

Bone-resorbing osteoclasts are regulated by the relative ratio of the differentiation factor, receptor activator NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). Dental tissue-localized-resorbing cells called odontoclasts have regulatory factors considered as identical to those of osteoclasts; however, it is still unclear whether the RANKL/OPG ratio is a key factor for odontoclast regulation in dental pulp. Here, we showed that odontoclast regulators, macrophage colony-stimulating factor-1, RANKL, and OPG were detectable in mouse pulp of molars, but OPG was dominantly expressed. High OPG expression was expected to have a negative regulatory effect on odontoclastogenesis; however, odontoclasts were not detected in the dental pulp of OPG-deficient (KO) mice. In contrast, damage induced odontoclast-like cells were seen in wild-type pulp tissues, with their number significantly increased in OPG-KO mice. Relative ratio of RANKL/OPG in the damaged pulp was significantly higher than in undamaged control pulp. Pulp damages enhanced hypoxia inducible factor-1α and -2α, reported to increase RANKL or decrease OPG. These results reveal that the relative ratio of RANKL/OPG is significant to pulpal odontoclastogenesis, and that OPG expression is not required for maintenance of pulp homeostasis, but protects pulp from odontoclastogenesis caused by damages.

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.

Strain-dependent embryonic lethality and exaggerated vascular remodeling in heparin cofactor II-deficient mice.

Heparin cofactor II (HCII) specifically inhibits thrombin action at sites of injured arterial wall, and patients with HCII deficiency exhibit advanced atherosclerosis. However, the in vivo effects and the molecular mechanism underlying the action of HCII during vascular remodeling remain elusive. To clarify the role of HCII in vascular remodeling, we generated HCII-deficient mice by gene targeting. In contrast to a previous report, HCII(-/-) mice were embryonically lethal. In HCII(+/-) mice, prominent intimal hyperplasia with increased cellular proliferation was observed after tube cuff and wire vascular injury. The number of protease-activated receptor-1-positive (PAR-1-positive) cells was increased in the thickened vascular wall of HCII(+/-) mice, suggesting enhanced thrombin action in this region. Cuff injury also increased the expression levels of inflammatory cytokines and chemokines in the vascular wall of HCII(+/-) mice. The intimal hyperplasia in HCII(+/-) mice with vascular injury was abrogated by human HCII supplementation. Furthermore, HCII deficiency caused acceleration of aortic plaque formation with increased PAR-1 expression and oxidative stress in apoE-KO mice. These results demonstrate that HCII protects against thrombin-induced remodeling of an injured vascular wall by inhibiting thrombin action and suggest that HCII is potentially therapeutic against atherosclerosis without causing coagulatory disturbance.

The Vitamin D Receptor in Osteoblast-Lineage Cells Is Essential for the Proresorptive Activity of 1α,25(OH)2D3 In Vivo.

We previously reported that daily administration of a pharmacological dose of eldecalcitol, an analog of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], increased bone mass by suppressing bone resorption. These antiresorptive effects were found to be mediated by the vitamin D receptor (VDR) in osteoblast-lineage cells. Using osteoblast-lineage-specific VDR conditional knockout (Ob-VDR-cKO) mice, we examined whether proresorptive activity induced by the high-dose 1α,25(OH)2D3 was also mediated by VDR in osteoblast-lineage cells. Administration of 1α,25(OH)2D3 (5 µg/kg body weight/day) to wild-type mice for 4 days increased the number of osteoclasts in bone and serum concentrations of C-terminal crosslinked telopeptide of type I collagen (CTX-I, a bone resorption marker). The stimulation of bone resorption was concomitant with the increase in serum calcium (Ca) and fibroblast growth factor 23 (FGF23) levels, and decrease in body weight. This suggests that a toxic dose of 1α,25(OH)2D3 can induce bone resorption and hypercalcemia. In contrast, pretreatment of wild-type mice with neutralizing anti-receptor activator of NF-κB ligand (RANKL) antibody inhibited the 1α,25(OH)2D3-induced increase of osteoclast numbers in bone, and increase of CTX-I, Ca, and FGF23 levels in serum. The pretreatment with anti-RANKL antibody also inhibited the 1α,25(OH)2D3-induced decrease in body weight. Consistent with observations in mice conditioned with anti-RANKL antibody, the high-dose administration of 1α,25(OH)2D3 to Ob-VDR-cKO mice failed to significantly increase bone osteoclast numbers, serum CTX-I, Ca, or FGF23 levels, and failed to reduce the body weight. Taken together, this study demonstrated that the proresorptive, hypercalcemic, and toxic actions of high-dose 1α,25(OH)2D3 are mediated by VDR in osteoblast-lineage cells.

[Osteoclastogenesis and bone resorption].

Osteoclasts, the multinucleated cells that resorb bone, originate from monocyte-macrophage lineage cells. Various hormones, cytokines and growth factors are involved in osteoclastogenesis, via interaction with osteoblasts. Deficiency of osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of NF-kappa B ligand (RANKL), in mice induces osteoporosis caused by enhanced bone resorption but also accelerates bone formation. OPG-deficient mice exhibite high serum alkaline phosphatase activity and osteocalcin concentration, both of which are decreased to the levels of wild-type mice by the bisphosphonate injection. This suggests that bone formation is coupled with bone resorption in vivo. RANKL expressed by osteoblasts is a requirement for osteoclastogenesis, osteoblasts also play important roles in osteoclastogenesis through offering the critical microenvironment for the action of RANKL.

Blockade of the angiotensin II type 1 receptor increases bone mineral density and left ventricular contractility in a mouse model of juvenile Paget disease.

Juvenile Paget disease (JPD1), an autosomal-recessive disorder, is characterized by extremely rapid bone turnover due to osteoprotegerin deficiency. Its extra-skeletal manifestations, such as hypertension and heart failure, suggest a pathogenesis with shared skeletal and cardiovascular system components. In spite of this, the effects of anti-hypertensive drugs on bone morphometry remain unknown. We administered an angiotensin II type 1 receptor blocker, olmesartan (5 mg/kg/day) to 8-week-old male mice lacking the osteoprotegerin gene, with and without 1 µg/kg/min of angiotensin II infusion for 14 days. Olmesartan treatment decreased systolic blood pressure, and echocardiography showed increased left ventricular systolic contractility. Three-dimensional micro-computed tomography scans demonstrated that olmesartan treatment increased trabecular bone volume (sham, +176%; angiotensin II infusion, +335%), mineral density (sham, +150%; angiotensin II infusion, +313%), and trabecular number (sham, +407%; angiotensin II infusion, +622%) in the tibia. Olmesartan increased cortical mineral density (sham, +19%; angiotensin II infusion, +24%), decreased the cortical bone section area (sham, -16%; angiotensin II infusion, -18%), decreased thickness (sham, -18%; angiotensin II infusion, -31%), and decreased the lacunar area (sham, -41%; angiotensin II infusion, -27%) in the tibia. Similar trend was observed in the femur. Moreover, olmesartan decreased angiotensin II-induced increases in tartrate-resistant acid phosphatase concentrations in plasma, but it affected neither type I procollagen N-terminal propeptides, nor the receptor activator of nuclear factor kappa-B ligand. Our data suggest that blockade of the angiotensin II type 1 receptor improves bone vulnerability, and helps to maintain the heart's structural integrity in osteoprotegerin-deficient mice.

WNT Activates the AAK1 Kinase to Promote Clathrin-Mediated Endocytosis of LRP6 and Establish a Negative Feedback Loop.

ß-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity.