beta-Integrin-collagen interaction reduces chondrocyte apoptosis.

We have observed that the spent culture media in suspended chondrocyte cultures is essential for the survival of the cells, since complete change of the spent media induces severe programmed cell death (apoptosis). Moreover, we showed that extracellular matrix (ECM) molecules in the culture media provide vital chondrocyte-matrix interactions; when media are changed, cells are deprived of matrix molecules and undergo apoptosis. In this paper we report that interaction with collagen, a ubiquitous extracellular matrix molecule, is essential for chondrocyte survival. Such an interaction causes chondrocyte aggregation and reduces the level of chondrocyte apoptosis. Hyaluronan, an abundant ECM molecule, can influence the effects of collagen by preventing chondrocyte aggregation. Degradation of hyaluronan with hyaluronidase results in chondrocyte aggregation, and this reduces the level of chondrocyte apoptosis. Experiments with an antibody to integrin beta1 suggest that the collagen-chondrocyte interactions are mediated through integrin beta1, and these interactions may protect chondrocytes from apoptosis. We hypothesize that hyaluronan binds aggrecan and link protein, forming stable ternary complexes, which interact with the chondrocyte surface, perhaps via CD44, and thus maintains a stable chondrocyte-matrix network.     

Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor

We examined the effect of fibroblast growth factor (FGF) on proteoglycan synthesis by rabbit costal chondrocyte cultures maintained on plastic tissue culture dishes. Low density rabbit costal chondrocyte cultures grown in the absence of FGF gave rise at confluency to a heterogeneous cell population composed of fibroblastic cells and poorly differentiated chondrocytes. When similar cultures were grown in the presence of FGF, the confluent cultures organized into a homogenous cartilage-like tissue composed of rounded cells surrounded by a refractile matrix. The cell ultrastructure and that of the pericellular matrix were similar to those seen in vivo. The expression of the cartilage phenotype in confluent chondrocyte cultures grown from the sparse stage in the presence vs. absence of FGF was reflected by a fivefold increase in the rate of incorporation of [35S]sulfate into proteoglycans. These FGF effects were only observed when FGF was present during the cell logarithmic growth phase, but not when it was added after chondrocyte cultures became confluent. High molecular weight, chondroitin sulfate proteoglycans synthesized by confluent chondrocyte cultures grown in the presence of FGF were slightly larger in size than that produced by confluent cultures grown in the absence of FGF. The major sulfated glycosaminoglycans associated with low molecular weight proteoglycan in FGF-exposed cultures were chondroitin sulfate, while in cultures not exposed to FGF they were chondroitin sulfate and dermatan sulfate. Regardless of whether or not cells were grown in the presence or absence of FGF, the 6S/4S disaccharide ratio of chondroitin sulfate chains associated with high and low molecular weight proteoglycans synthesized by confluent cultures was the same. These results provide evidence that when low density chondrocyte cultures maintained on plastic tissue culture dishes are grown in the presence of FGF, it results in a stimulation of the expression and stabilization of the chondrocyte phenotype once cultures become confluent.     

Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

Chondrocyte hypertrophy is an essential process required for endochondral bone formation. Proper regulation of chondrocyte hypertrophy is also required in postnatal cartilage homeostasis. Indian hedgehog (Ihh) and PTHrP signaling play crucial roles in regulating the onset of chondrocyte hypertrophy by forming a negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy by controlling PTHrP expression. To understand whether there is a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy, we have both activated and inactivated Ihh signaling in the absence of PTHrP during endochondral skeletal development. We found that upregulating Ihh signaling in the developing cartilage by treating PTHrP(-/-) limb explants with sonic hedgehog (Shh) protein in vitro, or overexpressing Ihh in the cartilage of PTHrP(-/-) embryos or inactivating patched 1 (Ptch1), a negative regulator of hedgehog (Hh) signaling, accelerated chondrocyte hypertrophy in the PTHrP(-/-) embryos. Conversely, when Hh signaling was blocked by cyclopamine or by removing Smoothened (Smo), a positive regulator of Hh signaling, chondrocyte hypertrophy was delayed in the PTHrP(-/-) embryo. Furthermore, we show that upregulated Hh signaling in the postnatal cartilage led to accelerated chondrocyte hypertrophy during secondary ossification, which in turn caused reduction of joint cartilage. Our results revealed a novel role of Ihh signaling in promoting chondrocyte hypertrophy independently of PTHrP, which is particularly important in postnatal cartilage development and homeostasis. In addition, we found that bone morphogenetic protein (Bmp) and Wnt/beta-catenin signaling in the cartilage may both mediate the effect of upregulated Ihh signaling in promoting chondrocyte hypertrophy.     

Overexpression of Spry1 in chondrocytes causes attenuated FGFR ubiquitination and sustained ERK activation resulting in chondrodysplasia

The FGF signaling pathway plays essential roles in endochondral ossification by regulating osteoblast proliferation and differentiation, chondrocyte proliferation, hypertrophy, and apoptosis. FGF signaling is controlled by the complementary action of both positive and negative regulators of the signal transduction pathway. The Spry proteins are crucial regulators of receptor tyrosine kinase-mediated MAPK signaling activity. Sprys are expressed in close proximity to FGF signaling centers and regulate FGFR-ERK-mediated organogenesis. During endochondral ossification, Spry genes are expressed in prehypertrophic and hypertrophic chondrocytes. Using a conditional transgenic approach in chondrocytes in vivo, the forced expression of Spry1 resulted in neonatal lethality with accompanying skeletal abnormalities resembling thanatophoric dysplasia II, including increased apoptosis and decreased chondrocyte proliferation in the presumptive reserve and proliferating zones. In vitro chondrocyte cultures recapitulated the inhibitory effect of Spry1 on chondrocyte proliferation. In addition, overexpression of Spry1 resulted in sustained ERK activation and increased expression of p21 and STAT1. Immunoprecipitation experiments revealed that Spry1 expression in chondrocyte cultures resulted in decreased FGFR2 ubiquitination and increased FGFR2 stability. These results suggest that constitutive expression of Spry1 in chondrocytes results in attenuated FGFR2 degradation, sustained ERK activation, and up-regulation of p21Cip and STAT1 causing dysregulated chondrocyte proliferation and terminal differentiation.     

Effects of autophagy on apoptosis of articular chondrocytes in adjuvant arthritis rats

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease that eventually leads to joint deformities and loss of joint function. Previous studies have demonstrated a close relationship between autophagy and the development of RA. Although autophagy and apoptosis are two different forms of programmed death, the relationship between them in relation to RA remains unclear. In this study, we explored the effect of autophagy on apoptosis of articular chondrocytes in vivo and in vitro. Adjuvant arthritis (AA) and acid‐induced primary articular chondrocyte apoptosis were used as in vivo and in vitro models, respectively. Articular chondrocyte autophagy and apoptosis were both observed dynamically in AA rat articular cartilage at different stages (15 days, 25 days and 35 days). Moreover, chondrocyte apoptosis and articular cartilage injury in AA rats were increased by the autophagy inhibitor 3‐methyladenine (3‐MA) and decreased by the autophagy activator rapamycin. In addition, pre‐treatment with 3‐MA increased acid‐induced chondrocyte apoptosis, while pre‐treatment with rapamycin reduced acid‐induced chondrocyte apoptosis in vitro. These results suggest that autophagy might be a potential target for the treatment of RA.     

Identification and characterization of arginase II as a chondrocyte phenotype-specific gene

We have previously shown that activation of extracellular signal-regulated protein kinase-1 and -2 (ERK1/2) causes chondrocyte dedifferentiation, which contributes to the destruction of arthritic cartilage. In the present study, we identified genes involved in the ERK1/2 regulation of chondrocyte dedifferentiation. Several genes were identified by subtractive hybridization, and, of these, arginase II was selected for further functional characterization. Similar to the pattern of type II collagen expression, which is a hallmark of chondrocyte differentiation, arginase II expression was increased during chondrogenesis of mesenchymal cells. The high expression level of arginase II was decreased during dedifferentiation of chondrocytes, whereas its expression was restored during redifferentiation of the dedifferentiated chondrocytes. Inhibition of ERK1/2 signaling in chondrocytes enhanced type II collagen expression with a concomitant increase in expression and activity of arginase II. However, ectopic expression of arginase II or inhibition of its activity did not affect chondrocyte differentiation. The results collectively indicate that expression of arginase II is specific to the chondrocyte phenotype, although the expression of arginase II alone is not sufficient for articular chondrocytes to maintain a differentiated phenotype.     

A-raf and B-raf are dispensable for normal endochondral bone development, and parathyroid hormone-related peptide suppresses extracellular signal-regulated kinase activation in hypertrophic chondrocytes

Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone-PTHrP receptor increase chondrocyte proliferation and delay chondrocyte maturation in endochondral bone development at least partly through cyclic AMP (cAMP)-dependent signaling pathways. Because data suggest that the ability of cAMP to stimulate cell proliferation involves the mitogen-activated protein kinase kinase kinase B-Raf, we hypothesized that B-Raf might mediate the proliferative action of PTHrP in chondrocytes. Though B-Raf is expressed in proliferative chondrocytes, its conditional removal from cartilage did not affect chondrocyte proliferation and maturation or PTHrP-induced chondrocyte proliferation and PTHrP-delayed maturation. Similar results were obtained by conditionally removing B-Raf from osteoblasts. Because A-raf and B-raf are expressed similarly in cartilage, we speculated that they may fulfill redundant functions in this tissue. Surprisingly, mice with chondrocytes deficient in both A-Raf and B-Raf exhibited normal endochondral bone development. Activated extracellular signal-regulated kinase (ERK) was detected primarily in hypertrophic chondrocytes, where C-raf is expressed, and the suppression of ERK activation in these cells by PTHrP or a MEK inhibitor coincided with a delay in chondrocyte maturation. Taken together, these results demonstrate that B-Raf and A-Raf are dispensable for endochondral bone development and they indicate that the main role of ERK in cartilage is to stimulate not cell proliferation, but rather chondrocyte maturation.     

IL-6通过MAPK/ERK信号通路调节BMSCs的软骨定向分化

白细胞介素-6（IL-6）是参与骨髓间充质干细胞（BMSCs）软骨定向分化的重要调节因子. MAPK/ERK信号通路可介导骨关节炎软骨损伤. 然而,IL-6调节BMSCs定向分化为软骨细胞的分子机制尚不清楚. IL-6通过激活MAPK/ERK信号途径,抑制BMSCs的成软骨分化. 本文发现,BMSCs在体外向软骨细胞分化时, Il-6基因表达水平显著下调,同时分泌到培养基中的IL-6蛋白水平亦明显降低. 重组IL-6可抑制BMSCs向软骨细胞分化,软骨分化标志蛋白Runx2和Sox9的诱导表达亦相应下调. IL-6可诱导MAPK/ERK信号通路活化,加入ERK特异性阻断剂后,Runx2和Sox9的诱导表达恢复正常.结果提示,IL-6通过激活MAPK/ERK信号通路抑制BMSCs的软骨细胞分化.炎症因子IL-6对软骨细胞的再生具有不利的影响,该研究为软骨组织工程研究和骨关节炎等软骨疾病的治疗提供有价值的参考.     

Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification

The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes. The expansion in the extent of hypertrophic maturation detectable histologically is accompanied by expanded and upregulated domains of expression of molecular markers of chondrocyte maturation, particularly type X collagen and osteopontin, and by expansion of mineralized cartilage matrix, which is characteristic of terminal hypertrophic differentiation. Furthermore, Dlx5 misexpression markedly reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together, these results indicate that Dlx5 is a positive regulator of chondrocyte maturation and suggest that it regulates the process at least in part by promoting conversion of immature proliferating chondrocytes into hypertrophying chondrocytes. Retroviral misexpression of Dlx5 also enhances formation of periosteal bone, which is derived from the Dlx5-expressing perichondrium that surrounds the diaphyses of the cartilage models. This suggests that Dlx5 may be involved in regulating osteoblast differentiation, as well as chondrocyte maturation, during endochondral ossification.     

The deformation behavior and viscoelastic properties of chondrocytes in articular cartilage

Chondrocytes in articular cartilage utilize mechanical signals in conjunction with other environmental factors to regulate their metabolic activity. However, the sequence of biomechanical and biochemical events involved in the process of mechanical signal transduction has not been fully deciphered. A fundamental step in determining the role of various factors in regulating chondrocyte activity is to characterize accurately the biophysical environment within the tissue under physiological conditions of mechanical loading. Microscopic imaging studies have revealed that chondrocytes as well as their nuclei undergo shape and volume changes in a coordinated manner with deformation of the tissue matrix. Through micromechanical experiments, it has been shown that the chondrocyte behaves as a viscoelastic solid material with a mechanical stiffness that is several orders of magnitude lower than that of the cartilage extracellular matrix. These properties seem to be due to the structure of the chondrocyte cytoskeleton, and in part, the viscoelastic properties of the cell nucleus. The mechanical properties of the pericellular matrix that immediately surrounds the chondrocyte significantly differ from those of the chondrocyte and the extracellular matrix, suggesting that the pericellular matrix plays an important role in defining the mechanical environment of the chondrocyte. These experimentally measured values for chondrocyte and cartilage mechanical properties have been used in combination with theoretical constitutive modeling of the chondrocyte within articular cartilage to predict the non-uniform and time-varying stress-strain and fluid flow environment of the cell. The ultimate goal of these studies has been to elucidate the sequence of biomechanical and biochemical events through which mechanical stress influences chondrocyte activity in both health and in disease.     

Regulation of chondrocyte differentiation by actin-severing protein adseverin

The importance of actin organization in controlling the chondrocyte phenotype is well established, but little is known about the cytoskeletal components regulating chondrocyte differentiation. Previously, we have observed up-regulation of an actin-binding gelsolin-like protein in hypertrophic chondrocytes. We have now identified it as adseverin (scinderin). Adseverin is drastically up-regulated during chondrocyte maturation, as shown by Northern blot analysis, in situ hybridization, and real-time RT-PCR. Its expression is positively regulated by PKC and MEK signaling as shown by inhibitory analyses. Over-expression of adseverin in non-hypertrophic chondrocytes causes rearrangement of the actin cytoskeleton, a change in cell morphology, a dramatic (3.5-fold) increase in cell volume, and up-regulation of Indian hedgehog (Ihh) and of collagen type X--all indicative of chondrocyte differentiation. These changes are mediated by ERK1/2 and p38 kinase pathways. Thus, adseverin-induced rearrangements of the actin cytoskeleton may mediate the PKC-dependent activation of p38 and Erk1/2 signaling pathways necessary for chondrocyte hypertrophy, as evidenced by changes in cell morphology, increase in cell size and expression of the chondrocyte maturation markers. These results demonstrate that interdependence of cytoskeletal organization and chondrogenic gene expression is regulated, at least in part, by actin-binding proteins such as adseverin.     

Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix

Initial assembly of extracellular matrix occurs within a zone immediately adjacent to the chondrocyte cell surface termed the cell- associated or pericellular matrix. Assembly within the pericellular matrix compartment requires specific cell-matrix interactions to occur, that are mediated via membrane receptors. The focus of this study is to elucidate the mechanisms of assembly and retention of the cartilage pericellular matrix proteoglycan aggregates important for matrix organization. Assembly of newly synthesized chondrocyte pericellular matrices was inhibited by the addition to hyaluronan hexasaccharides, competitive inhibitors of the binding of hyaluronan to its cell surface receptor. Fully assembled chondrocyte pericellular matrices were displaced using hyaluronan hexasaccharides as well. When exogenous hyaluronan was added to matrix-free chondrocytes in combination with aggrecan, a pericellular matrix equivalent in size to an endogenous matrix formed within 30 min of incubation. Addition of hyaluronan and aggrecan to glutaraldehyde-fixed chondrocytes resulted in matrix assembly comparable to live chondrocytes. These matrices could be inhibited from assembling by the addition of excess hyaluronan hexasaccharides or displaced once assembled by subsequent incubation with hyaluronan hexasaccharides. The results indicate that the aggrecanrich chondrocyte pericellular matrix is not only on a scaffolding of hyaluronan, but actually anchored to the cell surface via the interaction between hyaluronan and hyaluronan receptors.