Expression and regulation of the osteoarthritis-associated protein asporin

Asporin (ASPN) is a small leucine-rich proteoglycan that is involved in pathological processes of osteoarthritis. Previously, we showed that asporin can inhibit transforming growth factor-beta1 (TGF-beta1)-mediated expression of cartilage matrix genes and chondrogenesis in vitro (Kizawa, H., Kou, I., Iida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., and Ikegawa, S. (2005) Nat. Genet. 37, 138-144). However, details about regulation of asporin itself are not yet known. Here, we examined ASPN expression in skeletal tissue and potential regulation of ASPN by TGF-beta. In situ hybridization revealed the presence of ASPN mRNA in the perichondrium/periosteum of long bones, but its absence in articular cartilage and growth plates. Immunohistochemical analysis also showed ASPN protein expression predominantly in the perichondrium/periosteum. TGF-beta1 induced endogenous ASPN mRNA expression over time in vitro, and this induction was suppressed by the TGF-beta type I receptor kinase inhibitor SB431542. Inhibition of Smad3 significantly reduced TGF-beta1-induced ASPN expression, whereas overexpression of Smad3 augmented the induction. Characterization of the human ASPN promoter region revealed a region from -126 to -82 that is sufficient for full promoter activity; however, TGF-beta1 failed to increase activity through the ASPN promoter. Our findings indicate that TGF-beta1 induces ASPN through Smad3 but that this induction is indirect.     

Cytokine-like 1 knock-out mice (Cytl1-/-) show normal cartilage and bone development but exhibit augmented osteoarthritic cartilage destruction

We have shown that cytokine-like 1 (Cytl1) is a novel autocrine regulatory factor that regulates chondrogenesis of mouse mesenchymal cells (Kim, J. S., Ryoo, Z. Y., and Chun, J. S. (2007) J. Biol. Chem. 282, 29359-29367). In this previous work, we found that Cytl1 expression was very low in mesenchymal cells, increased dramatically during chondrogenesis, and decreased during hypertrophic maturation, both in vivo and in vitro. Moreover, exogenous addition or ectopic expression of Cytl1 caused chondrogenic differentiation of mouse limb bud mesenchymal cells. In the current study, we generated a Cytl1 knock-out (Cytl1(-/-)) mouse to investigate the in vivo role of Cytl1. Deletion of the Cytl1 gene did not affect chondrogenesis or cartilage development. Cytl1(-/-) mice also showed normal endochondral ossification and long bone development. Additionally, ultrastructural features of articular cartilage, such as matrix organization and chondrocyte morphology, were similar in wild-type and Cytl1(-/-) mice. However, Cytl1(-/-) mice were more sensitive to osteoarthritic (OA) cartilage destruction. Compared with wild-type littermates, Cytl1(-/-) mice showed more severe OA cartilage destruction upon destabilization of the medial meniscus of mouse knee joints. In addition, expression levels of Cytl1 were markedly decreased in OA cartilage of humans and experimental mice. Taken together, our results suggest that, rather than regulating cartilage and bone development, Cytl1 is required for the maintenance of cartilage homeostasis, and loss of Cytl1 function is associated with experimental OA cartilage destruction in mice.     

Lack of association of a variable number of aspartic acid residues in the asporin gene with osteoarthritis susceptibility: case-control studies in Spanish Caucasians

A recent genetic association study has identified a microsatellite in the coding sequence of the asporin gene as a susceptibility factor for osteoarthritis (OA). Alleles of this microsatellite determine the variable number of aspartic acid residues in the amino-terminal end of the asporin protein. Asporin binds directly to the growth factor transforming growth factor beta and inhibits its anabolic effects in cartilage, which include stimulation of collagen and aggrecan synthesis. The OA-associated allele, with 14 aspartic acid residues, inhibits the anabolic effects of transforming growth factor beta more strongly than other asporin alleles, leading to increased OA liability. We have explored whether the association found in several cohorts of Japanese hip OA and knee OA patients was also present in Spanish Caucasians. We studied patients that had undergone total joint replacement for primary OA in the hip (n = 303) or the knee (n = 188) and patients with hand OA (n = 233), and we compared their results with controls (n = 294) lacking overt OA clinical symptoms. No significant differences were observed in any of the multiple comparisons performed, which included global tests of allele frequency distributions and specific comparisons as well as stratification by affected joint and by sex. Our results, together with reports from the United Kingdom and Greece, indicate that the stretch of aspartic acid residues in asporin is not an important factor in OA susceptibility among European Caucasians. It remains possible that lifestyle, environmental or genetic differences allow for an important effect of asporin variants in other ethnic groups as has been reported in the Japanese, but this should be supported by additional studies.     

Identification and characterization of asporin. a novel member of the leucine-rich repeat protein family closely related to decorin and biglycan

Asporin, a novel member of the leucine-rich repeat family of proteins, was partially purified from human articular cartilage and meniscus. Cloning of human and mouse asporin cDNAs revealed that the protein is closely related to decorin and biglycan. It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to decorin and biglycan, asporin is not a proteoglycan. Instead, asporin contains a unique stretch of aspartic acid residues in its amino-terminal region. A polymorphism was identified in that the number of consecutive aspartate residues varied from 11 to 15. The 8 exons of the human asporin gene span 26 kilobases on chromosome 9q31.1-32, and the putative promoter region lacks TATA consensus sequences. The asporin mRNA is expressed in a variety of human tissues with higher levels in osteoarthritic articular cartilage, aorta, uterus, heart, and liver. The deduced amino acid sequence of asporin was confirmed by mass spectrometry of the isolated protein resulting in 84% sequence coverage. The protein contains an N-glycosylation site at Asn(281) with a heterogeneous oligosaccharide structure and a potential O-glycosylation site at Ser(54). The name asporin reflects the aspartate-rich amino terminus and the overall similarity to decorin.     

Berberine ameliorates cartilage degeneration in interleukin‐1β‐stimulated rat chondrocytes and in a rat model of osteoarthritis via Akt signalling

Berberine, a plant alkaloid used in Chinese medicine, has broad cell‐protective functions in a variety of cell lines. Chondrocyte apoptosis contributes to the pathogenesis of cartilage degeneration in osteoarthritis (OA). However, little is known about the effect and underlying mechanism of berberine on OA chondrocytes. Here, we assessed the effects of berberine on cartilage degeneration in interleukin‐1β (IL‐1β)‐stimulated rat chondrocytes and in a rat model of OA. The results of an MTT assay and western blotting analysis showed that berberine attenuated the inhibitory effect of IL‐1β on the cell viability and proliferating cell nuclear antigen expression in rat chondrocytes. Furthermore, berberine activated Akt, which triggered p70S6K/S6 pathway and up‐regulated the levels of aggrecan and Col II expression in IL‐1β‐stimulated rat chondrocytes. In addition, berberine increased the level of proteoglycans in cartilage matrix and the thickness of articular cartilage, with the elevated levels of Col II, p‐Akt and p‐S6 expression in a rat OA model, as demonstrated by histopathological and immunohistochemistry techniques. The data thus strongly suggest that berberine may ameliorate cartilage degeneration from OA by promoting cell survival and matrix production of chondrocytes, which was partly attributed to the activation of Akt in IL‐1β‐stimulated articular chondrocytes and in a rat OA model. The resultant chondroprotective effects indicate that berberine merits consideration as a therapeutic agent in OA.     

Transforming growth factor-beta modulates the expression of osteoblast and chondroblast phenotypes in vitro

Transforming growth factor beta (TGF-beta) has been shown to induce chondrogenesis by embryonic rat mesenchymal cells (Seyedin et al., J. Biol. Chem., 261: 5693, 1986). Here we report the effects of bovine TGF-beta on the phenotypic expression of differentiated primary rat osteoblastic and chondroblastic cells. Culture of rat calvarial osteoblasts with TGF-beta resulted in a dose and time-dependent decrease in alkaline phosphatase activity. Levels of alkaline phosphatase were reduced to less than 10% of control values by 0.4 nM TGF-beta. The decrease became apparent after 24 hours and reached a maximum by 72 hours. Similarly, treatment of chondroblasts with 0.4 nM TGF-beta resulted in decreased production of cartilage-specific macromolecules: type II collagen and cartilage proteoglycan. Both cell types exhibited dramatic changes in cell shape after treatment with TGF-beta. Modulation of these differentiated markers by TGF-beta could be mimicked, in part, by addition of fibronectin. Addition of dihydrocytochalasin B blocked the inhibition of phenotypic expression by TGF-beta. These results indicate that TGF-beta inhibits phenotypic expression by osteoblasts and chondroblasts in vitro and suggest that this activity of TGF-beta may be mediated through interactions between the extracellular matrix and cytoskeletal elements.     

Evidence that miR‐146a attenuates aging‐ and trauma‐induced osteoarthritis by inhibiting Notch1, IL‐6, and IL‐1 mediated catabolism

Primary osteoarthritis (OA) is associated with aging, while post‐traumatic OA (PTOA) is associated with mechanical injury and inflammation. It is not clear whether the two types of osteoarthritis share common mechanisms. We found that miR‐146a, a microRNA‐associated with inflammation, is activated by cyclic load in the physiological range but suppressed by mechanical overload in human articular chondrocytes. Furthermore, miR‐146a expression is decreased in the OA lesions of human articular cartilage. To understand the role of miR‐146a in osteoarthritis, we systemically characterized mice in which miR‐146a is either deficient in whole body or overexpressed in chondrogenic cells specifically. miR‐146a‐deficient mice develop early onset of OA characterized by cartilage degeneration, synovitis, and osteophytes. Conversely, miR‐146a chondrogenic overexpressing mice are resistant to aging‐associated OA. Loss of miR‐146a exacerbates articular cartilage degeneration during PTOA, while chondrogenic overexpression of miR‐146a inhibits PTOA. Thus, miR‐146a inhibits both OA and PTOA in mice, suggesting a common protective mechanism initiated by miR‐146a. miR‐146a suppresses IL‐1β of catabolic factors, and we provide evidence that miR‐146a directly inhibits Notch1 expression. Therefore, such inhibition of Notch1 may explain suppression of inflammatory mediators by miR‐146a. Chondrogenic overexpression of miR‐146a or intra‐articular administration of a Notch1 inhibitor alleviates IL‐1β‐induced catabolism and rescues joint degeneration in miR‐146a‐deficient mice, suggesting that miR‐146a is sufficient to protect OA pathogenesis by inhibiting Notch signaling in the joint. Thus, miR‐146a may be used to counter both aging‐associated OA and mechanical injury‐/inflammation‐induced PTOA.     

Effects of diacerein on biosynthesis activities of chondrocytes in culture

The maintenance of articular cartilage integrity requires a balance between anabolic and catabolic processes which are under the control of chondrocytes. These cells are living in an anaerobic environment and normally do not divide. They are responsible for the continuous maintenance of the cartilage extracellular matrix (ECM). Although multiple factors are involved in the dynamic homeostasis of cartilage, increases in cytokines such as interleukin-1 (IL-1) are associated with a decrease in synthesis and an increase in degradation of the proteoglycans and collagens. Conversely, growth factors such as transforming growth factor-beta (TGF-beta) stimulate chondrocyte synthesis of collagens and proteoglycans, and reduce the activity of IL-1 stimulated metalloproteases, thus opposing the inhibitory and catabolic effects of IL-1. By its capability to reduce IL-1 effects and to stimulate TGF-beta expression in cultured articular chondrocytes, diacerein could favour anabolic processes in the OA cartilage and, hence may contribute to delay the progression of the disease.     

MicroRNA-488 regulates zinc transporter SLC39A8/ZIP8 during pathogenesis of osteoarthritis

Background

Even though osteoarthritis (OA) is the most common musculoskeletal dysfunction, there are no effective pharmacological treatments to treat OA due to lack of understanding in OA pathology. To better understand the mechanism in OA pathogenesis and investigate its effective target, we analyzed miRNA profiles during OA pathogenesis and verify the role and its functional targets of miR-488.

Results

Human articular chondrocytes were obtained from cartilage of OA patients undergoing knee replacement surgery and biopsy samples of normal cartilage and the expression profile of miRNA was analyzed. From expression profile, most potent miR was selected and its target and functional role in OA pathogenesis were investigated using target validation system and OA animal model system. Among miRNAs tested, miR-488 was significantly decreased in OA chondrocytes Furthermore, we found that exposure of IL-1β was also suppressed whereas exposure of TGF-β3 induced the induction of miR-488 in human articular chondrocytes isolated from biopsy samples of normal cartilages. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation.

Conclusions

miR-488 acts as a positive role for chondrocyte differentiation/cartilage development by inhibiting MMP-13 activity through targeting ZIP-8.     

Prostaglandin E2 exerts catabolic effects in osteoarthritis cartilage: evidence for signaling via the EP4 receptor

Elevated levels of PGE(2) have been reported in synovial fluid and cartilage from patients with osteoarthritis (OA). However, the functions of PGE(2) in cartilage metabolism have not previously been studied in detail. To do so, we cultured cartilage explants, obtained from patients undergoing knee replacement surgery for advanced OA, with PGE(2) (0.1-10 muM). PGE(2) inhibited proteoglycan synthesis in a dose-dependent manner (maximum 25% inhibition (p < 0.01)). PGE(2) also induced collagen degradation, in a manner inhibitable by the matrix metalloproteinase (MMP) inhibitor ilomastat. PGE(2) inhibited spontaneous MMP-1, but augmented MMP-13 secretion by OA cartilage explant cultures. PCR analysis of OA chondrocytes treated with PGE(2) with or without IL-1 revealed that IL-1-induced MMP-13 expression was augmented by PGE(2) and significantly inhibited by the cycolooygenase 2 selective inhibitor celecoxib. Conversely, MMP-1 expression was inhibited by PGE(2), while celecoxib enhanced both spontaneous and IL-1-induced expression. IL-1 induction of aggrecanase 5 (ADAMTS-5), but not ADAMTS-4, was also enhanced by PGE(2) (10 muM) and reversed by celecoxib (2 muM). Quantitative PCR screening of nondiseased and end-stage human knee OA articular cartilage specimens revealed that the PGE(2) receptor EP4 was up-regulated in OA cartilage. Moreover, blocking the EP4 receptor (EP4 antagonist, AH23848) mimicked celecoxib by inhibiting MMP-13, ADAMST-5 expression, and proteoglycan degradation. These results suggest that PGE(2) inhibits proteoglycan synthesis and stimulates matrix degradation in OA chondrocytes via the EP4 receptor. Targeting EP4, rather than cyclooxygenase 2, could represent a future strategy for OA disease modification.     

IL-1beta synthesis by chondrocyte analyzed by 3D microscopy and flow cytometry: effect of Rhein

Several factors are known to be involved in the destruction of the articular cartilage. Interleukin-1 (IL-1) plays an important role in the pathogenesis of osteoarthritis (OA) either directly or through the stimulation of catabolic factors. The action of IL-1 on articular cartilage is multifaceted and it most likely plays an important role in the mechanism of cartilage destruction. IL-1 suppresses the synthesis of the cartilage matrix components and promotes the degradation of cartilage matrix macromolecules. Diacerein is an anthraquinone molecule that has been shown to reduce the severity of OA, both in man and in animal models. The present study was designed to evaluate in vitro effects of diacerein on IL-1beta expression in LPS or IL-1alpha stimulated chondrocytes. Intracellular IL-1beta production was analysed in articular chondrocytes cultured in monolayer or in alginate 3D-biosystems in the presence of lipopolysaccharide (LPS) or IL-1alpha, with or without diacerein. The results show that LPS and IL-1alpha increase intracellular IL-1beta and Diacerein inhibited LPS-induced and IL-1alpha induced IL-1beta production by articular chondrocytes. Moreover, the effect of mechanical stimulation was analysed. An inhibitory effect of DAR at therapeutic concentrations on IL-1beta production in articular chondrocytes is suggested.     

The proton-activated G protein-coupled receptor GPR4 regulates the development of osteoarthritis via modulating CXCL12/CXCR7 signaling

Inflammatory diseases decrease the extracellular environmental pH. However, whether proton-activated G protein-coupled receptors (GPCRs) can regulate the development of osteoarthritis (OA) is largely unknown. In this study, we report that proton-activated GPR4 is essential for OA development. We found a marked increase in expression of the proton-activated GPR4 in human and mouse OA cartilage. Lentivirus-mediated overexpression of GPR4 in mouse joints accelerated the development of OA, including promotion of articular cartilage damage, synovial hyperplasia, and osteophyte formation, while Gpr4 knockout effectively attenuated the development of posttraumatic and aging-associated OA in mice. We also found that inhibition of GPR4 with the antagonist NE52-QQ57 ameliorated OA progression in mice, promoted extracellular matrix (ECM) production, and protected cartilage from degradation in human articular cartilage explants. Moreover, GPR4 overexpression upregulated matrix-degrading enzymes’ expression and inflammation factors under pro-inflammatory and slightly acidic conditions. Mechanistically, GPR4 suppressed chondrocyte differentiation and upregulated cartilage homeostasis through NF-κB/MAPK signaling activation by regulating CXCR7/CXCL12 expression. Together, our results take the lead to illustrate that proton-activated GPCR acts as a key regulator for OA pathogenesis in vivo, and support that GPR4 could be a promising therapeutic target for OA treatment.Subject terms: Cartilage development, Osteoarthritis     

Expression analysis of the novel gene collagen triple helix repeat containing-1 (Cthrc1)

We recently identified collagen triple helix repeat containing-1 (Cthrc1) as a novel gene induced in adventitial fibroblasts after arterial injury. Cthrc1 is a 30 kDa secreted protein that has the ability to inhibit collagen matrix synthesis. Cthrc1 is also glycosylated and retains a signal sequence consistent with the presence of Cthrc1 in the extracellular space. In injured arteries and skin wounds, we have found Cthrc1 expression to be associated with myofibroblasts and sites of collagen matrix deposition. Furthermore, we demonstrated that Cthrc1 inhibits collagen matrix deposition in vitro. Using in situ hybridization and immunohistochemistry, we characterized the expression domains of Cthrc1 during murine embryonic development and in postnatal tissues. In mouse embryos, Cthrc1 was expressed in the visceral endoderm, notochord, neural tube, developing kidney, and heart. Abundant expression of Cthrc1 was observed in the developing skeleton, i.e., in cartilage primordia, in growth plate cartilage with exclusion of the hypertrophic zone, in the bone matrix and periostium. Bones from adults showed expression of Cthrc1 only in the bone matrix and periostium while the articular cartilage lacked expression. Cthrc1 is typically expressed at epithelial-mesenchymal interfaces that include the epidermis and dermis, basal corneal epithelium, airway epithelium, esophagus epithelium, choroid plexus epithelium, and meninges. In the adult kidney, collecting ducts and distal tubuli expressed Cthrc1. Collectively, the sites of Cthrc1 expression overlap considerably with those reported for TGF-beta family members and interstitial collagens. The present study provides useful information towards the understanding of potential Cthrc1 functions.