The progressive ankylosis gene (ank) encodes a transmembrane protein that transports intracellular inorganic pyrophosphate (PPi) to the extracellular milieu. ank/ank mice, which express a truncated nonfunctional ANK, showed a markedly reduced bone mass, bone-formation rate, and number of tartrate-resistant acid phosphatase–positive (TRAP+) multinucleated osteoclasts. ANK function deficiency suppressed osteoblastic differentiation of ank/ank bone marrow stromal cells, as indicated by the decrease in the expression of bone marker genes, including osterix, reduced alkaline phosphatase activity, and mineralization. Runx2 gene expression levels were not altered. Conversely, overexpression of ANK in the preosteoblastic cell line MC3T3-E1 resulted in increased expression of bone marker genes, including osterix. Whereas runx2 expression was not altered in ANK-overexpressing MC3T3-E1 cells, runx2 transcriptional activity was increased. Extracellular PPi or Pi stimulated osteoblastogenic differentiation of MC3T3-E1 cells or partially rescued delayed osteoblastogenic differentiation of ank/ank bone marrow stromal cells. A loss of PPi transport function ANK mutation also stimulated osteoblastogenic differentiation of MC3T3-E1 cells. Furthermore, ANK function deficiency suppressed the formation of multinucleated osteoclasts from ank/ank bone marrow cells cultured in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand. In conclusion, ANK is a positive regulator of osteoblastic and osteoclastic differentiation events toward a mature osteoblastic and osteoclastic phenotype. © 2010 American Society for Bone and Mineral Research.

Objective. ANXA6, the gene for annexin A6, is highly expressed in osteoarthritic (OA) articular chondrocytes but not in healthy articular chondrocytes. This study was undertaken to determine whether annexin A6 affects catabolic events in these cells.Methods. Articular chondrocytes were isolated from Anxa6-knockout mice, wild-type (WT) mice, and human articular cartilage in which ANXA6 was overexpressed. Cells were treated with interleukin-1␤ (IL-1␤) or tumor necrosis factor ␣ (TNF␣), and expression of catabolic genes and activation of NF-B were determined by real-time polymerase chain reaction and luciferase reporter assay. Anxa6؊/؊ and WT mouse knee joints were injected with IL-1␤ or the medial collateral ligament was transected and partial resection of the medial meniscus was performed to determine the role of Anxa6 in IL-1␤-mediated cartilage destruction and OA progression. The mechanism by which Anxa6 stimulates NF-B activity was determined by coimmunoprecipitation and immunoblot analysis of nuclear and cytoplasmic fractions of IL-1␤-treated Anxa6 ؊/؊ and WT mouse chondrocytes for p65 and Anxa6.Results. Loss of Anxa6 resulted in decreased NF-B activation and catabolic marker messenger RNA (mRNA) levels in IL-1␤-or TNF␣-treated articular chondrocytes, whereas overexpression of ANXA6 resulted in increased NF-B activity and catabolic marker mRNA levels. Annexin A6 interacted with p65, and loss of Anxa6 caused decreased nuclear translocation and retention of the active p50/p65 NF-B complex. Cartilage destruction in Anxa6 ؊/؊ mouse knee joints after IL-1␤ injection or partial medial meniscectomy was reduced as compared to that in WT mouse joints.Conclusion. Our data define a role of annexin A6 in the modulation of NF-B activity and in the stimulation of catabolic events in articular chondrocytes.Annexins, cytoplasmic proteins that, in the presence of Ca 2ϩ

Objective. To determine the actions of lithium chloride (LiCl) on catabolic events in human articular chondrocytes, and the effects of LiCl on the progression and severity of cartilage degradation in interleukin-1␤ (IL-1␤)-treated mouse knee joints and after surgical induction of osteoarthritis (OA) in a mouse model.Methods. Human articular chondrocytes were treated with LiCl followed by IL-1␤, and the expression levels of catabolic genes were determined by real-time polymerase chain reaction. To understand the mechanism by which LiCl affects catabolic events in articular chondrocytes after IL-1␤ treatment, the activation of NF-B was determined using luciferase reporter assays, and the activities of MAPKs and the STAT-3 signaling pathway were determined by immunoblot analysis of total cell lysates. Cultures of mouse femoral head explants treated with IL-1␤ and a mouse model of surgically induced OA were used to determine the effects of LiCl on proteoglycan loss and cartilage degradation.Results. LiCl treatment resulted in decreased catabolic marker messenger RNA levels and activation of NF-B, p38 MAPK, and STAT-3 signaling in IL-1␤-treated articular chondrocytes. Furthermore, LiCl directly inhibited IL-6-stimulated activation of STAT-3 signaling. Consequently, the loss of proteoglycan and severity of cartilage destruction in LiCl-treated mouse knee joints 8 weeks after OA induction surgery or in LiCl-treated mouse femoral head explants after IL-1␤ treatment were markedly reduced compared to that in vehicle-treated joints or explants. Conclusion.LiCl reduced catabolic events in IL-1␤-treated human articular chondrocytes and attenuated the severity of cartilage destruction in IL-1␤-treated mouse femoral head explants and in the knee joints of mice with surgically induced OA, acting via inhibition of the activities of the NF-B, p38, and STAT-3 signaling pathways.

Background: Regulation of terminal chondrocyte differentiation is important for bone development and cartilage pathology. Results: Annexin A6 regulates terminal chondrocyte differentiation by modulating key signaling pathway activities. Conclusion: Annexin A6 acts an intracellular regulator of terminal chondrocyte differentiation. Significance: The understanding of the mechanisms that regulate terminal chondrocyte differentiation is crucial for the development of novel therapeutic strategies for the treatment of cartilage diseases.

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There are two independent serotonin (5-HT) systems of organization: one in the central nervous system and the other in the periphery. 5-HT affects feeding behavior and obesity in the central nervous system. On the other hand, peripheral 5-HT also may play an important role in obesity, as it has been reported that 5-HT regulates glucose and lipid metabolism. Here we show that the intraperitoneal injection of 5-HT to mice inhibits weight gain, hyperglycemia and insulin resistance and completely prevented the enlargement of intra-abdominal adipocytes without having any effect on food intake when on a high fat diet, but not on a chow diet. 5-HT increased energy expenditure, O2 consumption and CO2 production. This novel metabolic effect of peripheral 5-HT is critically related to a shift in the profile of muscle fiber type from fast/glycolytic to slow/oxidative in soleus muscle. Additionally, 5-HT dramatically induced an increase in the mRNA expression of peroxisome proliferator-activated receptor coactivator 1α (PGC-1α)-b and PGC-1α-c in soleus muscle. The elevation of these gene mRNA expressions by 5-HT injection was inhibited by treatment with 5-HT receptor (5HTR) 2A or 7 antagonists. Our results demonstrate that peripheral 5-HT may play an important role in the relief of obesity and other metabolic disorders by accelerating energy consumption in skeletal muscle.

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Membranous (M) cells of the follicle-associated epithelium (FAE) are believed to sample antigens from the gut lumen. However, the origin, differentiation mechanism, and cell death of M cells are still a matter of controversy. Therefore, we investigated the process of M cell differentiation and determined their fate in the intestine of three-way crossbred female pigs. We used anti-cytokeratin 18 and anti-PCNA antibodies to distinguish M cells and proliferative cells and performed immunohistochemistry, enzyme histochemistry, and scanning electron microscopy on fresh ileal Peyer's patches. Cell migration and apoptotic cells were detected by BrdU labeling and the TUNEL method, respectively. The turnover of the FAE was similar to that of the villi. M cells were mostly observed from the FAE crypt to the FAE periphery, but not in the FAE apex. As proliferative M cells (cytokeratin 18(+)/PCNA(+) cells) have previously been detected in the FAE crypt, porcine M cells may be directly derived from intestinal epithelial stem cells and committed as a distinct cell lineage in the crypts. M cells from the FAE periphery were unstained or only weakly stained for alkaline phosphatase, whereas cytokeratin 18(+)/alkaline phosphatase(+) cells lying near to the FAE apex showed a columnar shape similar to that of adjacent enterocytes. These data suggest that the committed M cells differentiate to mature M cells by contact with lymphocytes at the FAE periphery, and that they trans-differentiate to enterocytes and are finally excluded near the FAE apex.