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1.
Nutrition ; 115: 112117, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37531790

ABSTRACT

OBJECTIVES: During musculoskeletal development, the vitamin D endocrine system is crucial, because vitamin D-dependent calcium absorption is a major regulator of bone growth. Because exercise regimens depend on bone mass, the direct action of active vitamin D (1,25-dihydroxyvitamin D3 [1,25(OH)2D3]) on musculoskeletal performance should be determined. METHODS: To evaluate the effect of 1,25(OH)2D3 on muscle tissue, the vitamin D receptor (Vdr) gene was genetically inactivated in mouse skeletal muscle and the role of 1,25(OH)2D3-VDR signaling on locomotor function was assessed. The direct action of 1,25(OH)2D3 on muscle development was determined using cultured C2C12 cells with myogenic differentiation. RESULTS: The lack of Vdr activity in skeletal muscle decreased spontaneous locomotor activity, suggesting that the skeletal muscle performance depended on 1,25(OH)2D3-VDR signaling. Bone phenotypes, reduced femoral bone mineral density, and accelerated osteoclast bone resorption were confirmed in mice lacking skeletal muscle Vdr activity. In vitro study revealed that the treatment with 1,25(OH)2D3 decreased the cellular adenosine triphosphate (ATP)-to-adenosine monophosphate ratio without reducing ATP production. Remarkably, protein expressions of connexin 43, an ATP releaser to extracellular space, and ATP metabolizing enzyme ectonucleotide pyrophosphatase phosphodiesterase 1 were increased responding to 1,25(OH)2D3 treatment. Furthermore, the concentration of pyrophosphate in the culture medium, which inhibits tissue calcification, was increased with 1,25(OH)2D3 treatment. In the presence of 1,25(OH)2D3-VDR signaling, calcium accumulation was suppressed in both muscle samples isolated from mice and in cultured C2C12 cells. CONCLUSIONS: This study dissected the physiological functions of 1,25(OH)2D3-VDR signaling in muscle and revealed that regulation of ATP dynamics is involved in sustaining locomotor function.

2.
Nutrition ; 91-92: 111409, 2021.
Article in English | MEDLINE | ID: mdl-34388585

ABSTRACT

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.


Subject(s)
Bone Density , Calcium , Milk Proteins , Animals , Homeostasis , Mice , Mice, Knockout , Milk , Receptors, Calcitriol
3.
Genes Cells ; 25(11): 707-717, 2020 Nov.
Article in English | MEDLINE | ID: mdl-32916757

ABSTRACT

Osteoclasts are multinucleated cells responsible for bone resorption. Src homology 3 (SH3) domain-containing protein-2 (SH3P2)/osteoclast-stimulating factor-1 regulates osteoclast differentiation, but its exact role remains elusive. Here, we show that SH3P2 suppresses osteoclast differentiation. SH3P2 knockout (KO) mice displayed decreased femoral trabecular bone mass and enhanced localization of osteoclasts on the tibial trabecular bone surface, suggesting that SH3P2 suppresses bone resorption by osteoclasts. Osteoclast differentiation based on cellular multinuclearity induced by macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL) was enhanced in bone marrow-derived macrophages lacking SH3P2. RANKL induced SH3P2 dephosphorylation, which increased the association of actin-dependent motor protein myosin 1E (Myo1E) with SH3P2 and thereby prevented Myo1E localization to the plasma membrane. Consistent with this, Myo1E in the membrane fraction increased in SH3P2-KO cells. Together with the attenuated osteoclast differentiation in Myo1E knocked down cells, SH3P2 may suppress osteoclast differentiation by preventing their cell-to-cell fusion depending on Myo1E membrane localization.


Subject(s)
Muscle Proteins/metabolism , Myosin Type I/metabolism , Osteoclasts/metabolism , Animals , Bone Marrow Cells/metabolism , Bone Resorption/metabolism , Bone Resorption/prevention & control , Carrier Proteins/metabolism , Cell Differentiation/genetics , Cell Survival/drug effects , Cells, Cultured , Femur/metabolism , Hematopoiesis/drug effects , Macrophage Colony-Stimulating Factor/metabolism , Macrophages/metabolism , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Proteins/physiology , Myosin Type I/physiology , Myosins/metabolism , Osteoclasts/physiology , RANK Ligand/metabolism , Signal Transduction/drug effects
4.
Clin Calcium ; 28(10): 1365-1371, 2018.
Article in Japanese | MEDLINE | ID: mdl-30269119

ABSTRACT

Animal studies using mice model such as vitamin D deficiency and global and conditional VDR knock out(KO)mice have disclosed that the physiological role of vitamin D strongly depends on the calcium balance. Vitamin D stimulates active intestinal calcium transport mechanism, thereby maintains normocalcemia that has priority over skeletal integrity. Besides the biological significance of vitamin D extensively studied, its precise function in non-classical target needs further investigation.


Subject(s)
Calcium/physiology , Disease Models, Animal , Vitamin D Deficiency/pathology , Vitamin D/physiology , Animals , Mice , Mice, Knockout , Osteomalacia , Rickets
5.
FASEB J ; 32(4): 1903-1915, 2018 04.
Article in English | MEDLINE | ID: mdl-29282249

ABSTRACT

Extracellular low phosphate strongly enhances intestinal calcium absorption independently of active vitamin D [1,25(OH)2D3] signaling, but the underlying mechanisms remain poorly characterized. To elucidate the phosphate-dependent regulation of calcium transport, we investigated part of the enteral environment that is involved in 1,25(OH)2D3-independent calcium absorption, which responds to dietary phosphate levels in mice that lack intestinal vitamin D receptor ( Vdr) activity. Impaired calcium absorption in intestinal Vdr-null mice was improved by dietary phosphate restriction. Accordingly, calcium transport in cultured intestinal epithelial cells was increased when the apical side was exposed to low phosphate levels (0.5 mM) compared with normal or high phosphate levels (1.0 or 5.0 mM, respectively). Mechanistically, low phosphate increased ATP in the apical side medium and allowed calcium entry into epithelial cells via the P2X7 purinoreceptor, which results in increased calcium transport. We found that luminal ATP was regulated by the release and degradation of ATP at the epithelium, and phosphate restriction increased ATP release from epithelial cells via connexin-43 hemichannels. Furthermore, ATP degradation by ectonucleotide pyrophosphatase-1 was reduced, which was caused by the reduction of the MAPK cascade. These findings indicate that luminal ATP metabolism regulates transcellular calcium transport in the intestine by an 1,25(OH)2D3-independent mechanism in response to dietary phosphate levels.-Uekawa, A., Yamanaka, H., Lieben, L., Kimira, Y., Uehara, M., Yamamoto, Y., Kato, S., Ito, K., Carmeliet, G., Masuyama, R. Phosphate-dependent luminal ATP metabolism regulates transcellular calcium transport in intestinal epithelial cells.


Subject(s)
Adenosine Triphosphate/metabolism , Calcium/metabolism , Intestinal Mucosa/metabolism , Transcytosis , Animals , Cells, Cultured , Connexin 43/metabolism , Female , Intestinal Absorption , MAP Kinase Signaling System , Mice , Phosphates/metabolism , Pyrophosphatases/metabolism , Vitamin D/metabolism
6.
Clin Calcium ; 27(11): 1525-1532, 2017.
Article in Japanese | MEDLINE | ID: mdl-29074824

ABSTRACT

Vitamin D endocrine system is required for bone and mineral homeostasis through the active form of vitamin D[1α,25(OH)2D3]transported to the target organs, where the vitamin D receptor(VDR)is present. The biological significance of 1α,25(OH)2D3-VDR signalling is regarded not only in classical target of vitamin D involved in calcium and phosphate homeostasis, such as intestine, bone, kidney and parathyroid glands, but also in many other non-classical target cells of vitamin D including skin keratinocytes, pancreatic ß cells, cardiomyocytes, T-lymphocytes, bone marrow macrophages, among others. Although 1α,25(OH)2D3-VDR signalling in classical target organs of vitamin D has been extensively studied, its precise function in these target organs still needs further investigation.


Subject(s)
Vitamin D/metabolism , Animals , Bone Density , Bone and Bones/physiology , Calcification, Physiologic , Calcium/metabolism , Humans , Receptors, Calcitriol/metabolism
7.
PLoS One ; 12(5): e0177375, 2017.
Article in English | MEDLINE | ID: mdl-28494010

ABSTRACT

Gamma-glutamyl carboxylase (GGCX) gene mutation causes GGCX syndrome (OMIM: 137167), which is characterized by pseudoxanthoma elasticum (PXE)-like symptoms and coagulation impairment. Here, we present a 55-year-old male with a novel homozygous deletion mutation, c.2,221delT, p.S741LfsX100, in the GGCX gene. Histopathological examination revealed calcium deposits in elastic fibers and vessel walls, and collagen accumulation in the mid-dermis. Studies of dermal fibroblasts from the patient (GGCX dermal fibroblasts) demonstrated that the mutated GGCX protein was larger, but its expression level and intracellular distribution were indistinguishable from those of the wild-type GGCX protein. Immunostaining and an enzyme-linked immunosorbent assay showed an increase in undercarboxylated matrix gamma-carboxyglutamic acid protein (ucMGP), a representative substrate of GGCX and a potent calcification inhibitor, indicating that mutated GGCX was enzymatically inactive. Under osteogenic conditions, calcium deposition was exclusively observed in GGCX dermal fibroblasts. Furthermore, GGCX dermal fibroblast cultures contained 23- and 7.7-fold more alkaline phosphatase (ALP)-positive cells than normal dermal fibroblast cultures (n = 3), without and with osteogenic induction, respectively. Expression and activity of ALP were higher in GGCX dermal fibroblasts than in normal dermal fibroblasts upon osteogenic induction. mRNA levels of other osteogenic markers were also higher in GGCX dermal fibroblasts than in normal dermal fibroblasts, which including bone morphogenetic protein 6, runt-related transcription factor 2, and periostin (POSTN) without osteogenic induction; and osterix, collagen type I alpha 2, and POSTN with osteogenic induction. Together, these data indicate that GGCX dermal fibroblasts trans-differentiate into the osteogenic lineage. This study proposes another mechanism underlying aberrant calcification in patients with GGCX syndrome.


Subject(s)
Calcinosis/genetics , Carbon-Carbon Ligases/genetics , Dermis/pathology , Fibroblasts/pathology , Osteogenesis/genetics , Up-Regulation/genetics , Alkaline Phosphatase/metabolism , Biomarkers/metabolism , Bone Morphogenetic Proteins/metabolism , Calcinosis/pathology , Calcium-Binding Proteins/metabolism , Cell Transdifferentiation , Extracellular Matrix Proteins/metabolism , Gene Deletion , Homozygote , Humans , Intracellular Space/metabolism , Male , Middle Aged , Protein Transport , Pseudoxanthoma Elasticum/enzymology , Pseudoxanthoma Elasticum/pathology , Signal Transduction , Syndrome , Matrix Gla Protein
8.
Front Immunol ; 8: 1958, 2017.
Article in English | MEDLINE | ID: mdl-29375576

ABSTRACT

OBJECTIVES: We investigated the relationships among M1 monocytes, M2 monocytes, osteoclast (OC) differentiation ability, and clinical characteristics in patients with rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from RA patients and healthy donors, and we then investigated the number of M1 monocytes or M2 monocytes by fluorescence-activated cell sorting. We also obtained and cultured CD14-positive cells from PBMCs from RA patients and healthy donors to investigate OC differentiation in vitro. RESULTS: Forty RA patients and 20 healthy donors were included. Twenty-two patients (55%) were anticitrullinated protein antibody (ACPA) positive. The median M1/M2 ratio was 0.59 (0.31-1.11, interquartile range). There were no significant differences between the RA patients and healthy donors. There was a positive correlation between the M1/M2 ratio and the differentiated OC number in vitro in RA patients (ρ = 0.81, p < 0.001). The ACPA-positive patients had significantly higher M1/M2 ratios in vivo (p = 0.028) and significantly greater numbers of OCs in vitro (p = 0.005) than the ACPA-negative patients. Multivariable regression analysis revealed that the M1/M2 ratio was the sole significant contribution factor to in vitro osteoclastogenesis. RA patients with M1/M2 ratios >1 (having relatively more M1 monocytes) had higher C-reactive protein and erythrocyte sedimentation rates than RA patients with M1/M2 ratios ≤1. M1-dominant monocytes in vitro produced higher concentrations of interleukin-6 upon stimulation with lipopolysaccharide than M2 monocytes. CONCLUSION: M1/M2 monocytes imbalance strongly contributes to osteoclastogenesis of RA patients. Our findings cast M1 and M2 monocyte subsets in a new light as a new target of treatments for RA to prevent progression of osteoclastic bone destruction.

9.
J Bone Miner Metab ; 34(5): 526-31, 2016 Sep.
Article in English | MEDLINE | ID: mdl-26202855

ABSTRACT

The increasing number of osteoporosis patients is a pressing issue worldwide. Osteoporosis frequently causes fragility fractures, limiting activities of daily life and increasing mortality. Many osteoporosis patients take numerous medicines due to other health issues; thus, it would be preferable if a single medicine could ameliorate osteoporosis and other conditions. Here, we screened 96 randomly selected drugs targeting various diseases for their ability to inhibit differentiation of osteoclasts, which play a pivotal role in development of osteoporosis, and identified methotrexate (MTX), as a potential inhibitor. MTX is currently used to treat sarcomas or leukemic malignancies or auto-inflammatory diseases such as rheumatoid arthritis (RA) through its anti-proliferative and immunosuppressive activities; however, a direct effect on osteoclast differentiation has not been shown. Here, we report that osteoclast formation and expression of osteoclastic genes such as NFATc1 and DC-STAMP, which are induced by the cytokine RANKL, are significantly inhibited by MTX. We found that RANKL-dependent calcium (Ca) influx into osteoclast progenitors was significantly inhibited by MTX. RA patients often develop osteoporosis, and osteoclasts are reportedly required for joint destruction; thus, MTX treatment could have a beneficial effect on RA patients exhibiting high osteoclast activity by preventing both osteoporosis and joint destruction.


Subject(s)
Calcium/metabolism , Methotrexate/pharmacology , Osteoclasts/drug effects , Osteogenesis/drug effects , Stem Cells/drug effects , Animals , Cells, Cultured , Mice, Inbred C57BL , Osteoclasts/metabolism , RANK Ligand/pharmacology , Stem Cells/metabolism
10.
Bone ; 81: 502-512, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26319498

ABSTRACT

The active form of vitamin D, 1,25(OH)2D, is a crucial regulator of calcium homeostasis, especially through stimulation of intestinal calcium transport. Lack of intestinal vitamin D receptor (VDR) signaling does however not result in hypocalcemia, because the increased 1,25(OH)2D levels stimulate calcium handling in extra-intestinal tissues. Systemic VDR deficiency, on the other hand, results in hypocalcemia because calcium handling is impaired not only in the intestine, but also in kidney and bone. It remains however unclear whether low intestinal VDR activity, as observed during aging, is sufficient for intestinal calcium transport and for mineral and bone homeostasis. To this end, we generated mice that expressed the Vdr exclusively in the gut, but at reduced levels. We found that ~15% of intestinal VDR expression greatly prevented the Vdr null phenotype in young-adult mice, including the severe hypocalcemia. Serum calcium levels were, however, in the low-normal range, which may be due to the suboptimal intestinal calcium absorption, renal calcium loss, insufficient increase in bone resorption and normal calcium incorporation in the bone matrix. In conclusion, our results indicate that low intestinal VDR levels improve intestinal calcium absorption compared to Vdr null mice, but also show that 1,25(OH)2D-mediated fine-tuning of renal calcium reabsorption and bone mineralization and resorption is required to maintain fully normal serum calcium levels.


Subject(s)
Calcium/blood , Calcium/metabolism , Intestinal Absorption/physiology , Aging/blood , Aging/metabolism , Animals , Biological Transport, Active , Bone Remodeling/physiology , Calcitriol/metabolism , Calcitriol/pharmacology , Calcium, Dietary/metabolism , Cells, Cultured , Gene Expression/drug effects , Homeostasis , Intestinal Mucosa/metabolism , Kidney/metabolism , Mice , Mice, Knockout , Organ Specificity , Osteoblasts/drug effects , Osteoblasts/metabolism , Parathyroid Hormone/pharmacology , RANK Ligand/genetics , Receptors, Calcitriol/deficiency , Receptors, Calcitriol/genetics , Receptors, Calcitriol/metabolism
11.
Clin Calcium ; 25(7): 1023-8, 2015 Jul.
Article in Japanese | MEDLINE | ID: mdl-26119315

ABSTRACT

Vitamin D endocrine system is required for normal calcium and bone homeostasis. Trans-epithelial calcium absorption is initiated with calcium entry into the intestinal epithelial cells from luminal fluid through calcium permeable channels, and those expressions are strongly supported by vitamin D action. On the other hands, dietary treatment, mineral supplementation or restriction, successfully improves intestinal calcium absorption in global vitamin D receptor knock-out (VDR KO) mice, though vitamin D dependent active transport pathway is lacking. Dietary rescue of intestinal calcium absorption provided a positive calcium balance in this mouse model, and suggested that the major role of vitamin D function on calcium homeostasis was considered to be intestinal active absorption. To elucidate the entire process of intestinal calcium absorption, vitamin D independent calcium transport system was characterized into either trans-cellular or para-cellular process.


Subject(s)
Calcium/metabolism , Intestinal Absorption , Intestine, Small/metabolism , Vitamin D/physiology , Animals , Bone and Bones/metabolism , Epithelial Cells/metabolism , Estrogens/physiology , Homeostasis , Humans , Intestinal Absorption/drug effects , Intestinal Mucosa/cytology , Intestinal Mucosa/metabolism , Intestine, Small/cytology , Mice , Phosphorus, Dietary/pharmacology , Progesterone/physiology , Receptors, Calcitriol/physiology , Tight Junctions/metabolism , Vitamin D/pharmacology
12.
Curr Opin Pharmacol ; 22: 87-99, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25938686

ABSTRACT

The physiological role of vitamin D depends on calcium supply and calcium balance. When the calcium balance is normal, the major target of vitamin D is intestine. Vitamin D stimulates mainly active intestinal calcium transport mechanism. During a negative calcium balance, bone effects of vitamin D become dominant. Thus, the role of vitamin D in maintaining normocalcemia appears to have priority over skeletal integrity in these situations.


Subject(s)
Calcium/metabolism , Receptors, Calcitriol/genetics , Vitamin D/physiology , Animals , Biological Transport/physiology , Bone and Bones/metabolism , Humans , Intestinal Mucosa/metabolism , Mice , Mice, Knockout
13.
Exp Anim ; 63(3): 297-304, 2014.
Article in English | MEDLINE | ID: mdl-25077759

ABSTRACT

To determine the prevalence of drug resistant bacteria colonizing laboratory mice, we isolated and characterized vancomycin-resistant Enterococcus species (VRE) from commercially available mice. A total of 24 VRE isolates were obtained from 19 of 21 mouse strains supplied by 4 commercial breeding companies. Of these, 19 isolates of E. gallinarum and 5 isolates of E. casseliflavus possessing the vanC1 and vanC2/3 genes intrinsically, exhibited intermediate resistance to vancomycin respectively. In addition, these isolates also exhibited diverse resistant patterns to erythromycin, tetracycline, and ciprofloxacin, whereas the use of antibiotics had not been undertaken in mouse strains tested in this study. Although 6 virulence-associated genes (ace, asa, cylA, efaA, esp, and gelE) and secretion of gelatinase and hemolysin were not detected in all isolates, 23 of 24 isolates including the isolates of E. casselifalvus secreted ATP into culture supernatants. Since secretion of ATP by bacteria resident in the intestinal tract modulates the local immune responses, the prevalence of ATP-secreting VRE in mice therefore needs to be considered in animal experiments that alter the gut microflora by use of antibiotics.


Subject(s)
Anti-Bacterial Agents/pharmacology , Enterococcus/drug effects , Enterococcus/isolation & purification , Mice, Inbred Strains/microbiology , Mice/microbiology , Vancomycin/pharmacology , Adenosine Triphosphate/metabolism , Animals , Bacterial Proteins , Drug Resistance, Bacterial/genetics , Enterococcus/genetics , Enterococcus/metabolism , Female , Intestines/immunology , Intestines/microbiology , Male , Peptide Synthases
14.
Bonekey Rep ; 3: 496, 2014.
Article in English | MEDLINE | ID: mdl-24605213

ABSTRACT

Calcium and phosphate regulate numerous biological processes and they are essential for bone mass and bone quality. The calcium and phosphate balance largely depends on intestinal absorption, and the dietary content of these ions determines the type of transport. High dietary intake of calcium and phosphate enables absorption by passive transport, but often the dietary content of these ions is in the low-normal range, especially for calcium. In this condition, the contribution of active intestinal calcium transport will increase to maintain normal serum levels. This adaptation is mainly regulated by the active form of vitamin D, 1,25 dihydroxyvitamin D, and requires normal concentrations of the precursor 25-hydroxyvitamin D. When intestinal calcium absorption is insufficient, hormonal adaptations will release calcium from bones to secure normocalcemia, not only by increasing bone loss but also by decreasing bone mineralization. These data underline the fact that adequate calcium intake is critical to secure skeletal integrity. Despite the insights that sufficient dietary calcium intake and normal 25-hydroxyvitamin D levels are critical for calcium and bone homeostasis, surprisingly little is known on the proteins that mediate intestinal calcium transport. Also, the interaction between the intestine and the kidney to control serum phosphate levels is still incompletely understood.

15.
J Bone Miner Metab ; 32(1): 1-9, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24213217

ABSTRACT

Mouse genetic studies have demonstrated that the 1,25-dihydroxyvitamin D [1,25(OH)2D] endocrine system is required for calcium (Ca(2+)) and bone homeostasis. These studies reported severe hypocalcemia and impaired bone mineralization associated with rickets in mutant mice. Specific phenotypes of these mice with an engineered deletion of 1,25(OH)2D cell signaling resemble the features observed in humans with the same congenital disease or severe 1,25(OH)2D deficiency. Decreased active intestinal Ca(2+) absorption because of reduced expression of epithelial Ca(2+) channels is a crucial mechanism that contributes to the major phenotypes observed in the mutant mice. The importance of intestinal Ca(2+) absorption supported by 1,25(OH)2D-mediated transport was further emphasized by the observation that Ca(2+) supplementation rescues hypocalcemia and restores bone mineralization in both patients and mice lacking 1,25(OH)2D signaling. This observation questions the direct role of 1,25(OH)2D signaling in bone tissue. Studies regarding tissue-specific manipulation of 1,25(OH)2D function have provided a consensus on this issue by demonstrating a direct action of 1,25(OH)2D on cells in bone tissue through bone metabolism and mineral homeostasis. In addition, movement of Ca(2+) from the bone as a result of osteoclastic bone resorption also provides a large Ca(2+) supply in Ca(2+) homeostasis; however, the system controlling Ca(2+) homeostasis in osteoclasts has not been fully identified. Transient receptor potential vanilloid (TRPV) 4 mediates Ca(2+) influx during the late stage of osteoclast differentiation, thereby regulating the Ca(2+) signaling essential for cellular events during osteoclast differentiation; however, the system-modifying effect of TRPV4 activity should be determined. Furthermore, it remains unknown how local Ca(2+) metabolism participates in systemic Ca(2+) homeostasis through bone remodeling. New insights are therefore required to understand this issue.


Subject(s)
Bone and Bones/metabolism , Calcium/metabolism , Homeostasis , Signal Transduction , Vitamin D/metabolism , Animals , Biological Transport , Humans
16.
Nat Genet ; 44(5): 586-92, 2012 May.
Article in English | MEDLINE | ID: mdl-22466610

ABSTRACT

UV-sensitive syndrome (UV(S)S) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UV(S)S, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UV(S)S cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS , one of whom is described for the first time here, formed a separate UV(S)S-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UV(S)S-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER­deficient disorders.


Subject(s)
Carrier Proteins/genetics , Cockayne Syndrome/genetics , DNA Damage/genetics , DNA Repair/genetics , Mutation/genetics , RNA Polymerase II/genetics , Transcription, Genetic , Ultraviolet Rays , DNA Damage/radiation effects , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Repair/radiation effects , DNA Repair Enzymes/chemistry , DNA Repair Enzymes/genetics , Exome/genetics , Humans , Poly-ADP-Ribose Binding Proteins , RNA Polymerase II/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics
17.
J Clin Invest ; 122(5): 1803-15, 2012 May.
Article in English | MEDLINE | ID: mdl-22523068

ABSTRACT

Serum calcium levels are tightly controlled by an integrated hormone-controlled system that involves active vitamin D [1,25(OH)(2)D], which can elicit calcium mobilization from bone when intestinal calcium absorption is decreased. The skeletal adaptations, however, are still poorly characterized. To gain insight into these issues, we analyzed the consequences of specific vitamin D receptor (Vdr) inactivation in the intestine and in mature osteoblasts on calcium and bone homeostasis. We report here that decreased intestinal calcium absorption in intestine-specific Vdr knockout mice resulted in severely reduced skeletal calcium levels so as to ensure normal levels of calcium in the serum. Furthermore, increased 1,25(OH)(2)D levels not only stimulated bone turnover, leading to osteopenia, but also suppressed bone matrix mineralization. This resulted in extensive hyperosteoidosis, also surrounding the osteocytes, and hypomineralization of the entire bone cortex, which may have contributed to the increase in bone fractures. Mechanistically, osteoblastic VDR signaling suppressed calcium incorporation in bone by directly stimulating the transcription of genes encoding mineralization inhibitors. Ablation of skeletal Vdr signaling precluded this calcium transfer from bone to serum, leading to better preservation of bone mass and mineralization. These findings indicate that in mice, maintaining normocalcemia has priority over skeletal integrity, and that to minimize skeletal calcium storage, 1,25(OH)(2)D not only increases calcium release from bone, but also inhibits calcium incorporation in bone.


Subject(s)
Bone Density Conservation Agents/pharmacology , Calcification, Physiologic/drug effects , Calcium/blood , Receptors, Calcitriol/deficiency , Vitamin D/pharmacology , Absorption , Animals , Bone Density/drug effects , Bone Diseases, Metabolic/blood , Bone Diseases, Metabolic/metabolism , Bone and Bones/metabolism , Bone and Bones/pathology , Calcification, Physiologic/genetics , Calcium/metabolism , Cell Line , Gene Expression Profiling , Gene Expression Regulation , Homeostasis , Intestinal Mucosa/metabolism , Mice , Mice, Knockout , Osteoblasts/metabolism , Phosphate Transport Proteins/genetics , Phosphate Transport Proteins/metabolism , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Receptors, Calcitriol/genetics , Signal Transduction
18.
J Bone Miner Res ; 27(8): 1708-21, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22492541

ABSTRACT

Osteoclast differentiation is critically dependent on calcium (Ca(2+)) signaling. Transient receptor potential vanilloid 4 (TRPV4), mediates Ca(2+) influx in the late stage of osteoclast differentiation and thereby regulates Ca(2+) signaling. However, the system-modifying effect of TRPV4 activity remains to be determined. To elucidate the mechanisms underlying TRPV4 activation based on osteoclast differentiation, TRPV4 gain-of-function mutants were generated by the amino acid substitutions R616Q and V620I in TRPV4 and were introduced into osteoclast lineage in Trpv4 null mice to generate Trpv4(R616Q/V620I) transgenic mice. As expected, TRPV4 activation in osteoclasts increased the number of osteoclasts and their resorption activity, thereby resulting in bone loss. During in vitro analysis, Trpv4(R616Q/V620I) osteoclasts showed activated Ca(2+)/calmodulin signaling compared with osteoclasts lacking Trpv4. In addition, studies of Trpv4(R616Q/V620I) mice that lacked the calmodulin-binding domain indicated that bone loss due to TRPV4 activation was abrogated by loss of interactions between Ca(2+)/calmodulin signaling and TRPV4. Finally, modulators of TRPV4 interactions with the calmodulin-binding domain were investigated by proteomic analysis. Interestingly, nonmuscle myosin IIa was identified by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis, which was confirmed by immunoblotting following coimmunoprecipitation with TRPV4. Furthermore, myosin IIa gene silencing significantly reduced TRPV4 activation concomitant with impaired osteoclast maturation. These results indicate that TRPV4 activation reciprocally regulates Ca(2+)/calmodulin signaling, which involves an association of TRPV4 with myosin IIa, and promotes sufficient osteoclast function.


Subject(s)
Bone and Bones/metabolism , Bone and Bones/pathology , Calcium Signaling , Calmodulin/metabolism , Osteoclasts/metabolism , Osteoclasts/pathology , TRPV Cation Channels/metabolism , Animals , Bone Resorption/blood , Bone Resorption/pathology , HEK293 Cells , Humans , Ion Channel Gating , Mice , Myosins/metabolism , Organ Size , Protein Structure, Tertiary , TRPV Cation Channels/chemistry
19.
Biochem Biophys Res Commun ; 420(1): 210-5, 2012 Mar 30.
Article in English | MEDLINE | ID: mdl-22426477

ABSTRACT

Nonsteroidal anti-inflammatory drugs (NSAIDs) are valuable agents; however, their use has been limited by their association with mucosal damage in the upper gastrointestinal tract. NSAIDs inhibit cyclooxygenase and consequently block the synthesis of prostaglandins, which have cytoprotective effects in gastric mucosa; these effects on prostaglandins have been thought to be major cause of NSAID-induced ulceration. However, studies indicate that additional NSAID-related mechanisms are involved in formation of gastric lesions. Here, we used a toxicoproteomic approach to understand cellular processes that are affected by NSAIDs in mouse stomach tissue during ulcer formation. We used fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS)-which consists of fluorogenic derivatization, separation and fluorescence detection by LC, and identification by LC-tandem mass spectrometry-in this proteomic analysis of pyrolic stomach from control and diclofenac (Dic)-treated mice. FD-LC-MS/MS results were highly sensitive; 10 differentially expressed proteins were identified, and all 10 were more highly expressed in Dic-treated mice than in control mice. Specifically, expression levels of 78 kDa glucose-regulated protein (GRP78), heat shock protein beta-1 (HSP27), and gastrin were more than 3-fold higher in Dic-treated mice than in control mice. This study represents a first step to ascertain the precise actors of early NSAID-induced ulceration.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Cyclooxygenase Inhibitors/adverse effects , Gastric Mucosa/drug effects , Gastric Mucosa/metabolism , Proteomics/methods , Stomach Ulcer/chemically induced , Stomach Ulcer/metabolism , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Chromatography, Liquid/methods , Cyclooxygenase Inhibitors/administration & dosage , Diclofenac/administration & dosage , Diclofenac/adverse effects , Disease Models, Animal , Endoplasmic Reticulum Chaperone BiP , Fluorescent Dyes/analysis , Gastric Mucosa/pathology , Gastrins/analysis , Gastrins/biosynthesis , HSP27 Heat-Shock Proteins/analysis , HSP27 Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/analysis , Heat-Shock Proteins/biosynthesis , Male , Mice , Mice, Inbred C57BL , Stomach Ulcer/pathology , Tandem Mass Spectrometry/methods
20.
Bone ; 50(1): 409-19, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21803180

ABSTRACT

Disuse osteoporosis, which occurs commonly in prolonged bed rest and immobilization, is becoming a major problem in modern societies; however, the molecular mechanisms underlying unloading-driven bone loss have not been fully elucidated. The osteocyte network is considered to be an ideal mechanosensor and mechanotransduction system. We searched for the molecules responsible for disuse osteoporosis using BCL2 transgenic mice, in which the osteocyte network was disrupted. Pyruvate dehydrogenase kinase 4 (Pdk4), which inactivates pyruvate dehydrogenase complex (PDC), was upregulated in femurs and tibiae of wild-type mice but not of BCL2 transgenic mice after tail suspension. Bone in Pdk4(-/-) mice developed normally and was maintained. At unloading, however, bone mass was reduced due to enhanced osteoclastogenesis and Rankl expression in wild-type mice but not in Pdk4(-/-) mice. Osteoclast differentiation of Pdk4(-/-) bone marrow-derived monocyte/macrophage lineage cells (BMMs) in the presence of M-CSF and RANKL was suppressed, and osteoclastogenesis was impaired in the coculture of wild-type BMMs and Pdk4(-/-) osteoblasts, in which Rankl expression and promoter activity were reduced. Further, introduction of Pdk4 into Pdk4(-/-) BMMs and osteoblasts enhanced osteoclastogenesis and Rankl expression and activated Rankl promoter. These findings indicate that Pdk4 plays an important role in bone loss at unloading by promoting osteoclastogenesis.


Subject(s)
Bone Resorption/metabolism , Osteoclasts/physiology , Protein Kinases/metabolism , Animals , Cell Differentiation , Gene Expression , Hindlimb Suspension , Humans , Macrophages/cytology , Macrophages/metabolism , Male , Mechanotransduction, Cellular/physiology , Mice , Mice, Knockout , Mice, Transgenic , Microarray Analysis , Osteoblasts/cytology , Osteoblasts/metabolism , Osteoclasts/cytology , Osteoporosis/metabolism , Protein Kinases/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RANK Ligand/genetics , RANK Ligand/metabolism
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