Takeshi Minashima

Progressive Ankylosis Protein (ANK) in Osteoblasts and Osteoclasts Controls Bone Formation and Bone Remodeling

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.

Annexin A6 Interacts With p65 and Stimulates NF‐κB Activity and Catabolic Events in Articular Chondrocytes

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.

Lithium Protects Against Cartilage Degradation in Osteoarthritis

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.

Intracellular Modulation of Signaling Pathways by Annexin A6 Regulates Terminal Differentiation of Chondrocytes

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. Annexin A6 (AnxA6) is highly expressed in hypertrophic and terminally differentiated growth plate chondrocytes. Rib chondrocytes isolated from newborn AnxA6−/− mice showed delayed terminal differentiation as indicated by reduced terminal differentiation markers, including alkaline phosphatase, matrix metalloproteases-13, osteocalcin, and runx2, and reduced mineralization. Lack of AnxA6 in chondrocytes led to a decreased intracellular Ca2+ concentration and protein kinase C α (PKCα) activity, ultimately resulting in reduced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activities. The 45 C-terminal amino acids of AnxA6 (AnxA6(1–627)) were responsible for the direct binding of AnxA6 to PKCα. Consequently, transfection of AnxA6−/− chondrocytes with full-length AnxA6 rescued the reduced expression of terminal differentiation markers, whereas transfection of AnxA6−/− chondrocytes with AnxA6(1–627) did not or only partially rescued the decreased mRNA levels of terminal differentiation markers. In addition, lack of AnxA6 in matrix vesicles, which initiate the mineralization process in growth plate cartilage, resulted in reduced alkaline phosphatase activity and Ca2+ and inorganic phosphate (Pi) content and the inability to form hydroxyapatite-like crystals in vitro. Histological analysis of femoral, tibial, and rib growth plates from newborn mice revealed that the hypertrophic zone of growth plates from newborn AnxA6−/− mice was reduced in size. In addition, reduced mineralization was evident in the hypertrophic zone of AnxA6−/− growth plate cartilage, although apoptosis was not altered compared with wild type growth plates. In conclusion, AnxA6 via its stimulatory actions on PKCα and its role in mediating Ca2+ flux across membranes regulates terminal differentiation and mineralization events of chondrocytes.

The role of ANK interactions with MYBBP1a and SPHK1 in catabolic events of articular chondrocytes

OBJECTIVE To determine the role of progressive ankylosis protein (ANK)/Myb-binding protein 1a (MYBBP1a) and sphingosine kinase 1 (SPHK1) interactions in catabolic events of articular chondrocytes. METHOD ANK/MYBBP1a and SPHK1 interactions were identified using yeast two-hybrid screening and co-immunoprecipitation. To determine the role of these interactions in catabolic events of articular chondrocytes, ank/ank and wild type (WT) mouse chondrocytes transfected with full-length or mutant ank expression vectors (EVs) or femoral heads were treated with interleukin-1beta (IL-1β) in the absence or presence of SPHK inhibitor. Catabolic marker mRNA levels were analyzed by real time PCR; proteoglycan loss using safranin O staining and MMP-13 immunostaining were determined in femoral head explants; NF-κB activity was determined by transfecting chondrocytes with an NF-κB-specific luciferase reporter and analyzing nuclear translocation of p65 by immunoblotting; MYBBP1a nuclear or cytoplasmic amounts were determined by immunohistochemistry and immunoblotting. RESULTS The ANK N-terminal region interacted with SPHK1, whereas a cytoplasmic C-terminal loop interacted with MYBBP1a. Lack of ANK/MYBBP1a and SPHK1 interactions in ank/ank chondrocytes resulted in increased MYBBP1a nuclear amounts and decreased SPHK1 activity, and consequently decreased NF-κB activity, catabolic marker mRNA levels, proteoglycan loss, and MMP-13 immunostaining in IL-1β-treated articular chondrocytes or femoral heads. Transfection with full-length ank EV reduced nuclear MYBBP1a amounts and fully restored SPHK and NF-κB activities in IL-1β-treated ank/ank chondrocytes, whereas transfection with P5L or F376del mutant ank reduced nuclear MYBBP1a or increased SPHK activity, respectively, and consequently either transfection only partially restored NF-κB activity. CONCLUSION ANK/MYBBP1a and SPHK1 interactions stimulate catabolic events in IL-1β-mediated cartilage degradation.

Annexins: Novel Therapeutic Targets for the Treatment of Osteoarthritis?

Osteoarthritis (OA) is the most common form of arthritis, affecting an estimated 27 million Americans. 1 The lack of current treatments to slow OA progression is due to the lack of both early detection methods and therapeutic targets for OA. Thus, there is an urgent need for the discovery of potential new therapeutic targets for the treatment of OA. Annexins are cytoplasmic proteins that, in the presence of Ca 2+ , translocate and bind to membranes. The role of annexins in disease pathology is an emerging field of investigation, with many studies highlighting the role of annexins not only as prognostic and diagnostic markers but also as being actively involved in causing diseases such as Alzheimer’s disease, autoimmunity, cancer, diabetes, and cardiovascular diseases. Specifically, their modulatory action on major signaling pathways involved in disease pathologies has made the annexins novel and exciting therapeutic targets. 2

Engineered Coiled-Coil Protein for Delivery of Inverse Agonist for Osteoarthritis

Osteoarthritis (OA) results from degenerative and abnormal function of joints, with localized biochemistry playing a critical role in its onset and progression. As high levels of all- trans retinoic acid (ATRA) in synovial fluid have been identified as a contributive factor to OA, the synthesis of de novo antagonists for retinoic acid receptors (RARs) has been exploited to interrupt the mechanism of ATRA action. BMS493, a pan-RAR inverse agonist, has been reported as an effective inhibitor of ATRA signaling pathway; however, it is unstable and rapidly degrades under physiological conditions. We employed an engineered cartilage oligomeric matrix protein coiled-coil (CccS) protein for the encapsulation, protection, and delivery of BMS493. In this study, we determine the binding affinity of CccS to BMS493 and the stimulator, ATRA, via competitive binding assay, in which ATRA exhibits approximately 5-fold superior association with CccS than BMS493. Interrogation of the structure of CccS indicates that ATRA causes about 10% loss in helicity, while BMS493 did not impact the structure. Furthermore, CccS self-assembles into nanofibers when bound to BMS493 or ATRA as expected, displaying 11-15 nm in diameter. Treatment of human articular chondrocytes in vitro reveals that CccS·BMS493 demonstrates a marked improvement in efficacy in reducing the mRNA levels of matrix metalloproteinase-13 (MMP-13), one of the main proteases responsible for the degradation of the extracellular cartilage matrix compared to BMS493 alone in the presence of ATRA, interleukin-1 beta (IL-1β), or IL-1 β together with ATRA. These results support the feasibility of utilizing coiled-coil proteins as drug delivery vehicles for compounds of relatively limited bioavailability for the potential treatment of OA.

The role of the progressive ankylosis protein (ANK) in adipogenic/osteogenic fate decision of precursor cells.

The progressive ankylosis protein (ANK) is a transmembrane protein that transports intracellular pyrophosphate (PPi) to the extracellular milieu. In this study we show increased fatty degeneration of the bone marrow of adult ank/ank mice, which lack a functional ANK protein. In addition, isolated bone marrow stromal cells (BMSCs) isolated from ank/ank mice showed a decreased proliferation rate and osteogenic differentiation potential, and an increased adipogenic differentiation potential compared to BMSCs isolated from wild type (WT) littermates. Wnt signaling pathway PCR array analysis revealed that Wnt ligands, Wnt receptors and Wnt signaling proteins that stimulate osteoblast differentiation were expressed at markedly lower levels in ank/ank BMSCs than in WT BMSCs. Lack of ANK function also resulted in impaired bone fracture healing, as indicated by a smaller callus formed and delayed bone formation in the callus site. Whereas 5weeks after fracture, the fractured bone in WT mice was further remodeled and restored to original shape, the fractured bone in ank/ank mice was not fully restored and remodeled to original shape. In conclusion, our study provides evidence that ANK plays a critical role in the adipogenic/osteogenic fate decision of adult mesenchymal precursor cells. ANK functions in precursor cells are required for osteogenic differentiation of these cells during adult bone homeostasis and repair, whereas lack of ANK functions favors adipogenic differentiation.

199 THE ROLE OF THE PROGRESSIVE ANKYLOSIS PROTEIN (ANK) IN OSTEOARTHRITIS

INTRODUCTION: Currently there are no treatments available for osteoarthritis (OA). In order to establish new therapeutic strategies for the treatment of OA, a better understanding of the cellular and molecular changes during OA progression is required. The progressive ankylosis protein (ANK) is a transmembrane protein that transports intracellular pyrophosphate (PPi) to the extracellular milieu. Previous studies have shown that extracellular PPi and inorganic phosphate (Pi) resulting from PPi hydrolysis affect cartilage calcification as well as chondrocyte differentiation. Extracellular PPi has been shown to inhibit basic calcium phosphate (BCP) crystal formation. However, excessive extracellular PPi concentrations lead to the formation of calcium pyrophosphate (CPPD) crystals. Both types of crystals are found in human OA patients. In addition, several lines of evidence suggest that ANK via its cytoplasmic N-terminal and C-terminal domains interacts with other proteins and these interactions may modulate signaling pathway activities. Since ANK expression is low in normal healthy articular cartilage and its expression levels increase in OA pathology, we have determined the role of ANK in OA pathology. METHODS: Mouse articular chondrocytes isolated from articular cartilage caps of 2-month-old ank/ank mice and wild type littermates were cultured as described. Because of a spontaneous mutation leading to a premature stop codon in the ank gene, ank/ank mice express a non-functional ANK protein, which does not insert into the plasma membrane and is degraded in the cells. Femoral heads isolated from 18-week-old ank/ank mice and wild type littermates were cultured in the absence or presence of 10ng/ml interleukin-1 (IL-1). Post-traumatic OA was surgically induced in 4-week-old ank/ank and wild type mice using the transection of the medial collateral ligament and partial medial meniscetomy (PMX) joint instability model. Human articular chondrocytes isolated from leftover tissue after knee replacement surgery were transfected with ank expression vector and cultured in the absence or presence of interleukin1 (IL-1) and zolendronate (10, 10, 10M), a bisphosphonate and nonhydrolysable PPi analogue. The mRNA levels of articular chondrocyte markers (aggrecan and type II collagen), hypertrophic markers (alkaline phosphatase (APase), runx2, and type X collagen), and MMP-13 were determined by real time PCR. NF-κB activity was determined by the transfection of human chondrocytes with a NF-κB-specific luciferase reporter plasmid. The mineralization of human chondrocytes in the absence or presence of PPi and levamisole (to prevent PPi hydrolysis) was determined using alizarin red S staining. RESULTS: We first determined how the lack of ANK in ank/ank mice affects cartilage destruction and OA pathology. Proteoglycan loss and immunostaining for MMP-13 were markedly reduced in IL-1-treated ank/ank femoral head explants compared with IL-1-treated wild type explants. In addition, cartilage destruction and OA severity was markedly reduced in ank/ank mice 10 weeks after PMX surgery compared with wild type mice. These findings suggest that the loss of ANK function protects articular cartilage against destruction in OA pathology. Since previous findings suggest that possible ANK interactions with other proteins may affect the activities of signaling pathways, we used a yeast two-hybrid screen to determine possible binding partners of ANK. We found that ANK binds to MYBBP1a, a cytoplasmic factor that can shuttle into the nucleus where it inhibits NFκB. The binding of MYBPP1a to ANK was confirmed by coimmunoprecipitation experiments. To determine the role of these interactions in NF-κB activity, we determined the amount of nuclear MYBBP1a in ank/ank and wild type articular chondrocytes cultured in the absence or presence of IL-1. In the absence of IL-1, only cytoplasmic staining, no nuclear immunostaining, for MYBBP1a was detected in wild type cells, whereas ank/ank cells showed mostly nuclear staining for MYBBP1a. Consequently, the luciferase activity from an NF-κB reporter plasmid was markedly decreased in ank/ank articular chondrocytes compared with wild type chondrocytes. In the presence of IL-1, translocation of MYBBP1a occurred in wild type and ank/ank cells; however, luciferase activity from the NF-κB reporter was reduced in ank/ank cells compared with wild type cells. Over-expression of ANK in human articular chondrocytes resulted in increased luciferase activity from the NF-κB reporter and increased MMP-13 mRNA levels in the absence or presence of IL-1 compared with empty vector-transfected human cells. Since ANK is a transmembrane protein that transports intracellular PPi to the extracellular milieu, we also determined the role of extracellular PPi on articular chondrocyte function and phenotype. Since extracellular PPi is hydrolyzed into Pi by APase, we cultured human articular chondrocytes in the presence of PPi and in the presence or absence of levamisole, a specific inhibitor of APase. In the absence of levamisole, when most of extracellular PPi is hydrolyzed into Pi, MMP13 and hypertrophic marker mRNA levels were increased, while articular cartilage marker mRNA levels were decreased compared with the mRNA levels of these genes in untreated cells. In the presence of levamisole, PPi treatment resulted in increased articular chondrocyte marker mRNA levels, whereas hypertrophic marker and MMP-13 mRNA levels were decreased compared with the levels of these genes in untreated cells. However, treatment of human articular chondrocytes with PPi and levamisole resulted in CPPD crystal formation. Since bisphosphonates are non-hydrolysable PPi analogues that, similar to PPi, inhibit BCP crystal formation but do not lead to CPPD crystal formation, we determined whether the bisphosphonate zolendronate may be a novel treatment modality to slow down cartilage destruction during OA pathology. Treatment of human articular chondrocytes with zolendronate resulted in decreased MMP-13 and hypertrophic marker mRNA levels and increased articular chondrocyte marker mRNA levels in the absence or presence of IL-1. DISCUSSION: ANK expression markedly increases during OA progression. Here we demonstrate that increased ANK expression in OA cartilage acts catabolically on articular cartilage and increases cartilage destruction during OA pathology. Our findings reveal that the catabolic events of increased ANK expression in articular chondrocytes result from different mechanisms: (a) the activation of NF-κB by preventing the shuttling of MYBBP1a into the nucleus where it inhibits NF-κB and potentially other mechanisms; (b) the extracellular Pi resulting from the hydrolysis of increased extracellular PPi levels stimulates hypertrophic changes and MMP-13 expression; (c) the potential BCP and/or CPPD crystal formation resulting from increased extracellular Pi and PPi concentrations. On the other hand, our results reveal that extracellular PPi inhibited MMP-13 mRNA levels and hypertrophic changes of articular chondrocytes. Consequently, zolendronate, a non-hydrolysable analogue of PPi, inhibited MMP-13 expression and hypertrophic changes of articular chondrocytes and increased aggrecan and type II collagen expression. In conclusion, ANK stimulates hypertrophic differentiation, mineralization, NF-κB activity, and MMP-13 expression in chondrocytes during OA pathology, whereas bisphosphonates as nonhydrolysable PPi analogues protect articular cartilage function and phenotype. SIGNIFICANCE: The understanding of the mechanisms of how ANK affects articular chondrocyte function and phenotype during OA pathology may lead to the discovery of novel therapeutic targets for the treatment of OA. REFERENCES: 1. Ho AM, Johnson MD, Kingsley DM 2000 Science 289:265-270. 2. Kim HJ, Delaney JD, Kirsch T 2010 Bone 47:657-665. 3. Terkeltaub RA 2001 Am J Physiol Cell Physiol 281:C1-C11. 4. Fleisch H 1981 Metab Bone Dis Relat Res 3:279-287. 5. Halverson PB, Derfus BA 2001 Curr Opin Rheumatol 13:221-224. 6. Kim HJ et al. 2010 J Bone Miner Res. 7. Wang W, Xu J, Du B, Kirsch T 2005 Mol Cell Biol 25:312-323. 8. Johnson K, Terkeltaub R 2004 Osteoarthritis Cartilage 12:321-335. 9. Merz D et al 2003 J Immunol 171:4406-4415. 10. 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