The progressive ankylosis gene (ank) encodes a transmembrane protein that transports intracellular inorganic pyrophosphate (PP_i) 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 PP_i or P_i stimulated osteoblastogenic differentiation of MC3T3-E1 cells or partially rescued delayed osteoblastogenic differentiation of ank/ank bone marrow stromal cells. A loss of PP_i 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

Biomineralization is a multifactorial and complex process, which results in the deposition of mineral crystals in the extracellular matrix of various tissues. Physiological mineralization is restricted to tissues, such as bones, teeth, and certain areas of cartilage. Pathological or ectopic mineralization can occur in many soft tissues, including articular cartilage, cardiovascular tissues, kidney, ligaments, and tendons, and can lead to serious problems. Therefore, the understanding of factors and mechanisms that regulate the mineralization process is essential for the development of novel therapeutic strategies to prevent or inhibit ectopic mineralization. This review will discuss some of the mechanisms and factors that regulate physiological mineralization and their potential roles in ectopic mineralization. Finally, potential therapeutic approaches for the treatment of ectopic mineralization are being discussed.

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‐1β (IL‐1β)–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‐1β, 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‐1β treatment, the activation of NF‐κB 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‐1β 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‐κB, p38 MAPK, and STAT‐3 signaling in IL‐1β–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‐1β treatment were markedly reduced compared to that in vehicle‐treated joints or explants.Conclusion: LiCl reduced catabolic events in IL‐1β–treated human articular chondrocytes and attenuated the severity of cartilage destruction in IL‐1β–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‐κB, p38, and STAT‐3 signaling pathways.

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-1β (IL-1β) or tumor necrosis factor α (TNFα), and expression of catabolic genes and activation of NF-κB were determined by real-time polymerase chain reaction and luciferase reporter assay. Anxa6^−/− and WT mouse knee joints were injected with IL-1β or the medial collateral ligament was transected and partial resection of the medial meniscus was performed to determine the role of Anxa6 in IL-1β–mediated cartilage destruction and OA progression. The mechanism by which Anxa6 stimulates NF-κB activity was determined by coimmunoprecipitation and immunoblot analysis of nuclear and cytoplasmic fractions of IL-1β–treated Anxa6^−/− and WT mouse chondrocytes for p65 and Anxa6. Results Loss of Anxa6 resulted in decreased NF-κB activation and catabolic marker messenger RNA (mRNA) levels in IL-1β– or TNFα-treated articular chondrocytes, whereas overexpression of ANXA6 resulted in increased NF-κB 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-κB complex. Cartilage destruction in Anxa6^−/− mouse knee joints after IL-1β 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-κB activity and in the stimulation of catabolic events in articular chondrocytes.

Physiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Because mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface-expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca²⁺ influx resulting in an increased intracellular Ca²⁺ concentration, [Ca²⁺]_i, and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca²⁺]_i, alkaline phosphatase activity, and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-l-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N terminus-deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca²⁺ channel activities) decreased [Ca²⁺]_i, alkaline phosphatase activity, and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Because annexin V is up-regulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization.