Chondrocyte Hypertrophic Differentiation Research Articles

Mir-374-5p, mir-379-5p, and mir-503-5p Regulate Proliferation and Hypertrophic Differentiation of Growth Plate Chondrocytes in Male Rats

Growth plate chondrocytes undergo sequential differentiation to form the resting zone, the proliferative zone (PZ), and the hypertrophic zone (HZ). The important role of microRNAs (miRNAs) in the growth plate was previously revealed by cartilage-specific ablation of Dicer, an enzyme essential for biogenesis of many miRNAs. To identify specific miRNAs that regulate differentiation of PZ chondrocytes to HZ chondrocytes, we microdissected individual growth plate zones from juvenile rats and performed miRNA profiling using a solution hybridization method and miRNA sequencing. Thirty-four miRNAs were differentially expressed between the PZ and the HZ, and we hypothesized that some of the miRNAs that are preferentially expressed in the PZ may promote proliferation and inhibit hypertrophic differentiation. Consistent with this hypothesis, transfection of inhibitors for four of these miRNAs (mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p) decreased proliferation in primary epiphyseal chondrocytes. The inhibitors for three of these miRNAs (mir-374-5p, mir-379-5p, and mir-503-5p) also increased expression of multiple genes that are associated with chondrocyte hypertrophic differentiation. We next hypothesized that preferential expression of these miRNAs in the PZ is driven by the parathyroid hormone-related protein (PTHrP) concentration gradient across the growth plate. Consistent with this hypothesis, treatment of primary chondrocytes with a parathyroid hormone (PTH)/PTHrP receptor agonist, PTH1-34, increased expression of mir-374-5p, mir-379-5p, and mir-503-5p. Taken together, our findings suggest that the PTHrP concentration gradient across the growth plate induces differential expression of mir-374-5p, mir-379-5p, and mir-503-5p between the PZ and the HZ. In the PZ, the higher expression levels of these miRNAs promote proliferation and inhibit hypertrophic differentiation. In the HZ, downregulation of these miRNAs inhibits proliferation and promotes hypertrophic differentiation.

PRMT5 is necessary to form distinct cartilage identities in the knee and long bone

During skeletal development, limb progenitors become specified as chondrocytes and subsequently differentiate into specialized cartilage compartments. We previously showed that the arginine dimethyl transferase, PRMT5, is essential for regulating the specification of progenitor cells into chondrocytes within early limb buds. Here, we report that PRMT5 regulates the survival of a separate progenitor domain that gives rise to the patella. Independent of its role in knee development, PRMT5 regulates several distinct types of chondrocyte differentiation within the long bones. Chondrocytes lacking PRMT5 have a striking blockage in hypertrophic chondrocyte differentiation and are marked by abnormal gene expression. PRMT5 remains important for articular cartilage and hypertrophic cell identity during adult stages, indicating an ongoing role in homeostasis of these tissues. We conclude that PRMT5 is required for distinct steps of early and late chondrogenic specialization and is thus a critical component of multiple aspects of long bone development and maintenance.

Latexin expression correlated with mineralization of articular cartilage during progression of post-traumatic osteoarthritis in a rat model.

As latexin has been linked with chondrocyte hypertrophic differentiation it is possible that this protein may also be involved in the mineralization of cartilage in OA. Therefore, we correlated latexin expression with the mineralization marker, alkaline phosphatase and determined the mineral deposition in the articular cartilage by analyzing the Ca/P ratio and the collagen fibrils pattern, during the progression of post-traumatic OA in a rat model. OA was induced by medial meniscectomy and post-surgery exercise for 5, 10, 20 and 45 days. Protein expression in articular cartilage was evaluated by immunofluorescence, histochemistry and Western blot. Minerals and structure of collagen fibrils in the superficial zone of cartilage were analyzed by energy dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM) respectively. Protein expression analysis showed time-dependent up-regulation of latexin during OA progression. In the cartilage, latexin expression correlated with the expression and activity of alkaline phosphatase. EDX of the superficial zone of cartilage showed a Ca/P ratio closer to theoretical values for basic calcium phosphate minerals. The presence of minerals was also analyzed indirectly with AFM, as the collagen fibril pattern was less evident in the mineralized tissue. Latexin is expressed in articular cartilage from the early stages of post-traumatic OA; however, minerals were detected after latexin expression was up-regulated, indicating that its activity precedes and remains during the pathological mineralization of cartilage. Thus, our results contribute to the identification of molecules involved in the mineralization of articular chondrocytes.

Lipid phosphatase SHIP-1 regulates chondrocyte hypertrophy and skeletal development.

SH2‐containing inositol‐5′‐phosphatase‐1 (SHIP‐1) controls the phosphatidylinositol‐3′‐kinase (PI3K) initiated signaling pathway by limiting cell membrane recruitment and activation of Akt. Despite the fact that many of the growth factors important to cartilage development and functions are able to activate the PI3K signal transduction pathway, little is known about the role of PI3K signaling in chondrocyte biology and its contribution to mammalian skeletogenesis. Here, we report that the lipid phosphatase SHIP‐1 regulates chondrocyte hypertrophy and skeletal development through its expression in osteochondroprogenitor cells. Global SHIP‐1 knockout led to accelerated chondrocyte hypertrophy and premature formation of the secondary ossification center in the bones of postnatal mice. Drastically higher vascularization and greater number of c‐kit + progenitors associated with sinusoids in the bone marrow also indicated more advanced chondrocyte hypertrophic differentiation in SHIP‐1 knockout mice than in wild‐type mice. In corroboration with the in vivo phenotype, SHIP‐1 deficient PDGFRα + Sca‐1 + osteochondroprogenitor cells exhibited rapid differentiation into hypertrophic chondrocytes under chondrogenic culture conditions in vitro. Furthermore, SHIP‐1 deficiency inhibited hypoxia‐induced cellular activation of Akt and extracellular‐signal‐regulated kinase (Erk) and suppressed hypoxia‐induced cell proliferation. These results suggest that SHIP‐1 is required for hypoxia‐induced growth signaling under physiological hypoxia in the bone marrow. In conclusion, the lipid phosphatase SHIP‐1 regulates skeletal development by modulating chondrogenesis and the hypoxia response of the osteochondroprogenitors during endochondral bone formation.

Inducible knockout of CHUK/IKK\u03b1 in adult chondrocytes reduces progression of cartilage degradation in a surgical model of osteoarthritis

CHUK/IKKα contributes to collagenase-driven extracellular matrix remodeling and chondrocyte hypertrophic differentiation in vitro, in a kinase-independent manner. These processes contribute to osteoarthritis (OA), where chondrocytes experience a phenotypic shift towards hypertrophy concomitant with abnormal matrix remodeling. Here we investigated the contribution of IKKα to OA in vivo. To this end, we induced specific IKKα knockout in adult chondrocytes in AcanCreERT2/+; IKKαf/f mice treated with tamoxifen (cKO). Vehicle-treated littermates were used as wild type controls (WT). At 12 weeks of age, WT and cKO mice were subjected to the destabilization of medial meniscus (DMM) model of post-traumatic OA. The cKO mice showed reduced cartilage degradation and collagenase activity and fewer hypertrophy-like features at 12 weeks after DMM. Interestingly, in spite of the protection from structural articular cartilage damage, the postnatal growth plates of IKKα cKO mice after DMM displayed abnormal architecture and composition associated with increased chondrocyte apoptosis, which were not as evident in the articular chondrocytes of the same animals. Together, our results provide evidence of a novel in vivo functional role for IKKα in cartilage degradation in post-traumatic OA, and also suggest intrinsic, cell-autonomous effects of IKKα in chondrocytes that control chondrocyte phenotype and impact on cell survival, matrix homeostasis, and remodeling.

PTH decreases in vitro human cartilage regeneration without affecting hypertrophic differentiation.

Regenerated cartilage formed after Autologous Chondrocyte Implantation may be of suboptimal quality due to postulated hypertrophic changes. Parathyroid hormone-related peptide, containing the parathyroid hormone sequence (PTHrP 1–34), enhances cartilage growth during development and inhibits hypertrophic differentiation of mesenchymal stromal cells (MSCs) and growth plate chondrocytes. This study aims to determine the possible anabolic and/or hypertrophic effect of PTH on human articular chondrocytes. Healthy human articular cartilage-derived chondrocytes (n = 6 donors) were cultured on type II collagen-coated transwells with/without 0.1 or 1.0 μM PTH from day 0, 9, or 21 until the end of culture (day 28). Extracellular matrix production, (pre)hypertrophy and PTH signaling were assessed by RT-qPCR and/or immunohistochemistry for collagen type I, II, X, RUNX2, MMP13, PTHR1 and IHH and by determining glycosaminoglycan production and DNA content. The Bern score assessed cartilage quality by histology. Regardless of the concentration and initiation of supplementation, PTH treatment significantly decreased DNA and glycosaminoglycan content and reduced the Bern score compared with controls. Type I collagen deposition was increased, whereas PTHR1 expression and type II collagen deposition were decreased by PTH supplementation. Expression of the (pre)hypertrophic markers MMP13, RUNX2, IHH and type X collagen were not affected by PTH. In conclusion, PTH supplementation to healthy human articular chondrocytes did not affect hypertrophic differentiation, but negatively influenced cartilage quality, the tissues’ extracellular matrix and cell content. Although PTH may be an effective inhibitor of hypertrophic differentiation in MSC-based cartilage repair, care may be warranted in applying accessory PTH treatment due to its effects on articular chondrocytes.

Changes in SOX9 and RUNX2 protein activity correlate to the health state of human primary chondrocytes

Purpose: SOX9 and RUNX2 are the master transcription factors involved in cartilage and bone development respectively. Altered SOX9 and RUNX2 activity is implicated in osteoarthritis (OA) pathophysiology and hypertrophic differentiation of chondrocytes. We have previously shown that SOX9 and RUNX2 protein activity can be measured by Transcription Factor - Fluorescence Recovery After Photobleaching (TF-FRAP). To decipher the underlying signaling mechanism in OA pathophysiology, we aim to use TF-FRAP to correlate SOX9 and RUNX2 mobility to the health state of the chondrocytes. We hypothesize that the mobility of SOX9 increases with OA progression, while the mobility of RUNX2 decreases in chondrocyte hypertrophy. We measured the mobility and correlated protein activity of these transcription factors at the single cell level by TF-FRAP in healthy, preserved and OA chondrocytes. Methods: Healthy human primary chondrocytes (hPCs) were purchased from Articular Engineering, USA. Human preserved and OA hPCs were isolated from patients undergoing total knee replacement therapy. hPCs were seeded on glass coverslips and transfected with either SOX9-mGFP or eGFP-RUNX2. TF-FRAP was performed in at least 40 cells per condition in two healthy donors and three donors for each preserved and OA chondrocytes. TF-FRAP measurements were done using a Nikon A1 confocal microscope (Japan). In TF-FRAP, a small circular region (Ø 25 pixels) of the nucleus was bleached using a high intensity laser and the recovery of the fluorescence in the bleach spot is recorded. For every TF-FRAP measurement, 25 pre-bleach images and 260 post-bleach images were acquired at 4 frames per second for 60 seconds, with a frame size of 256x256 pixels. Higher immobile fraction (i.e., the fraction of transcription factors bound to DNA) and higher half-time recovery (longer DNA interaction times) were associated with a higher transcriptional activity. For all donors, early passage (≤3) cells were used for TF-FRAP and qPCR measurements. To correlate protein mobility and DNA binding with the protein activity, we quantified the expression levels of SOX9 target genes (ACAN and COL2A) and RUNX2 target genes (MMP13 and COL10) by qPCR. Results: In the healthy hPCs, the SOX9 immobile fraction and half-time recovery were 60% (SD ±6%) and 14.2 sec (SD ±3.2 sec) respectively. In preserved hPCs, the SOX9 immobile fraction and half-time to recover was significantly lower, ranging from 50% - 44% (SD ±7% - ±9.7%), and 14.2 - 13.3 sec (SD ±3.9 - ±4.5 sec) respectively. In OA hPCs, the SOX9 immobile fraction and half-time recovery was significantly lower than in the healthy and preserved hPCs, ranging from 48.6% - 42.8% (SD ±7.3% - ±8.4%), and half-time to recover ranged from 13.3 - 12.2 sec (SD ±3.9 - ±5.3 sec). RUNX2 immobile fraction and half-time to recover data spread was large in healthy, preserved and OA hPCs. In healthy hPCs, immobile fraction and half-time to recover were ranging from 33.6% - 47.9% (SD 19.4% - 20.7%) and 13.97 sec - 14.46 sec (SD 7.9 - 10.1 sec) respectively. In the preserved hPCs, the immobile fraction and half-time to recover were ranging from 38.8% - 47.0 % (SD 19.7% - 22.2%) and 11.7 sec - 14.8 sec (SD 6.8 - 8.2 sec) respectively. In OA hPCs, the immobile fraction and half-time to recover ranged from 33.0% - 41.02% (SD 20.3% - 25.7%) and 11.2 sec - 12.9 sec (SD 7.4 - 9.1 sec) respectively. We have an initial non-quantitative indication for a distinct cell population that, irrespective of the health state, shows decreased RUNX2 mobility, which could be indicative of a hypertrophic cell state. We will show the quantification and segregation. RT-qPCR measurements show decreased SOX9 and increased RUNX2 target gene expression during OA progression. Conclusions: Our data show that the SOX9 and RUNX2 transcriptional activity is differentially regulated depending on the health state of hPCs. If we assume a correlation between mobility and transcription factor activity, then we can say that SOX9 protein activity decreases with increasing OA progression. RUNX2 mobility data showed large spread especially in the preserved and OA donors possibly due to cells were in the different health state in the OA joint. Our findings at the single cell level provide novel insights into SOX9 and RUNX2 protein activity with respect to OA progression.

Neural Cell Adhesion Molecule Regulates Chondrocyte Hypertrophy in Chondrogenic Differentiation and Experimental Osteoarthritis

Chondrocyte hypertrophy-like change is an important pathological process of osteoarthritis (OA), but the mechanism remains largely unknown. Neural cell adhesion molecule (NCAM) is highly expressed and participate the chondrocyte differentiation of mesenchymal stem cells (MSCs). In this study, we found that NCAM deficiency leads to hypertrophic chondrocyte differentiation in murine MSCs, in which extracellular signal-regulated kinase (ERK) signaling play an important role. Moreover, in an interleukin (IL)-1β induced OA cellular model, NCAM expression is down-regulated. Interestingly, overexpression of NCAM substantially inhibits hypertrophic differentiation in the OA cellular model. Consistently, NCAM deficiency accelerates chondrocyte hypertrophy in growth-plate and articular cartilage of OA mice. In conclusion, NCAM could inhibit hypertrophic chondrocyte differentiation of MSCs by activating ERK signaling and reduce chondrocyte hypertrophy in experimental OA model, suggesting the potential utility of NCAM as a novel therapeutic target for alleviating chondrocyte hypertrophy of OA. Funding Statement: This work was supported by the grants from the National Natural Science Foundation of China (No. 81403161, U1704186, 81402416 and U1404816), Key Scientific Research Project of Colleges and Universities in Henan Province (No. 19A310020 and 16A180015), Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine (No. XTCX-2015-ZD3) and Scientific and Technological Research Project of Henan Province (No. 182102311136). Declaration of Interests: The authors declare no competing financial interest. Ethics Approval Statement: All animal experiments were approved by the Ethics Committee of Xinxiang Medical University.

Effect of the PTHrP(1-34) analog abaloparatide on inducing chondrogenesis involves inhibition of intracellular reactive oxygen species production

Osteoarthritis (OA) is a degenerative joint disease characterized by a progressive loss of articular cartilage. Mesenchymal stem cells transplanted to damaged tissues are promising for OA cartilage repair. However, these cells are poor survival after transplantation and acquire hypertrophic properties during chondrogenic induction. Parathyroid hormone-related protein (PTHrP) promotes chondrogenesis and suppresses chondrocyte hypertrophic differentiation. Additionally, PTHrP was reported to have anti-oxidant effects. The synthetic PTHrP(1-34) analog abaloparatide (ABL) is a newly approved drug for osteoporosis therapy. It is unknown whether ABL stimulates chondrogenesis and affects intracellular reactive oxygen species (ROS) production. By using mouse embryonic limb bud mesenchymal stem cells in micromass culture as an in vitro model of chondrogenic differentiation, we found that mesenchymal stem cells in micromass cultures spontaneously produced ROS, and N-acetyl-l-cysteine, a potent antioxidant, enhanced chondrogenesis. The effect of ABL on stimulation of chondrogenesis is involved in its inhibition of intracellular ROS generation. These novel findings support the use of ABL for the damaged cartilage regeneration.

Retinoic Receptor Signaling Regulates Hypertrophic Chondrocyte-specific Gene Expression.

Retinoid signaling is important for the maturation of growth-plate chondrocytes. The effect of retinoid receptor gamma (RARγ) signaling on the expression of genes in hypertrophic chondrocytes is unclear. This study investigated the role of RARγ signaling in regulation of hypertrophic chondrocyte-specific genes. The gene expression in mouse E17.5 tibial cartilage was examined by in situ hybridization analysis. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting were used for analysis of mRNA and phosphorylated mitogen-activated protein kinase (MAPK). mRNA expression of Rarg and connective tissue growth factor (Ccn2) was detected in maturing chondrocytes throughout the cartilaginous skeletal elements. In chondrogenic ATDC5 cells, an RARγ agonist induced the gene expression of type-X collagen (Col10A1), transglutaminase-2 (Tg2), matrix metalloproteinase-13 (Mmp13), and Ccn2 mRNA, whereas a retinoic acid pan-agonist suppressed RARγ agonist-stimulated gene expression. Phosphorylated extracellular signal regulated-kinases (pERK1/2), p-p38, and phosphorylated c-Jun N-terminal kinase (pJNK) MAPK were time-dependently increased by RARγ agonist treatment. Experimental p38 inhibition led to a severe drop in the RARγ agonist-stimulated expressions of Col10A1, Tg2, Mmp13, and Ccn2 mRNA. RARγ signaling is required for the differentiation of hypertrophic chondrocytes, with differential cooperation with p38 MAPK.

QRICH1 mutations cause a chondrodysplasia with developmental delay.

In many children with short stature, the etiology of the decreased linear growth remains unknown. We sought to identify the underlying genetic etiology in a patient with short stature, irregular growth plates of the proximal phalanges, developmental delay, and mildly dysmorphic facial features. Exome sequencing identified a de novo, heterozygous, nonsense mutation (c.1606C>T:p.R536X) in QRICH1. In vitro studies confirmed that the mutation impaired expression of the QRICH1 protein. SiRNA-mediated knockdown of Qrich1 in primary mouse epiphyseal chondrocytes caused downregulation of gene expression associated with hypertrophic differentiation. We then identified an unrelated individual with another heterozygous de novo nonsense mutation in QRICH1 who had a similar phenotype. A recently published study identified QRICH1 mutations in three patients with developmental delay, one of whom had short stature. Our findings indicate that QRICH1 mutations cause not only developmental delay but also a chondrodysplasia characterized by diminished linear growth and abnormal growth plate morphology due to impaired growth plate chondrocyte hypertrophic differentiation.

Effect of Subclinical and Overt Form of Rat Maternal Hypothyroidism on Offspring Endochondral Bone Formation

Abstract Maternal hypothyroidism in its overt form affects skeletal development of the offspring, but these data are not available for the subclinical form which is becoming very frequent among pregnant women. We hypothesized that the subclinical form of hypothyroidism in rat dams, infl uences the process of offspring endochondral ossifi cation affecting proliferation and differentiation of chondrocytes, osteoclasts and osteoblasts in pups. Seven-day-old male pups (n=18) derived from control dams and dams treated with a low dose (1.5 mg/L) or high dose (150 mg/L) of propylthiouracil in drinking water during pregnancy and lactation were used. Histomorphometric analysis of pups’ tibia proximal growth plate, expression of mRNA, immunohistochemical and histochemical visualization of extracellular matrix components was performed. The length of the tibia was reduced in hypothyroid pups. Secretion of type 2 and 10 collagens in the subclinical and overt form were lower while the amount of glycosaminoglycans was higher when compared with controls. Down-regulated tartrate resistant acid phosphatase mRNA indicated altered osteoclasts function while lower expression of dentin matrix acid protein-1 mRNA and reduced synthesis of type 1 collagen accentuated a compromised bone formation in the overt form of hypothyroidism. The subclinical form of maternal hypothyroidism had a negative effect on the differentiation of hypertrophic chondrocytes and calcifi ed cartilage removal in 7-day-old pups. In addition, overt hypothyroidism had a negative effect on the proliferation of chondrocytes and deposition of osteoid. Both forms of hypothyroidism resulted in a decrease of tibia length due to changes in growth plate formation.

Phenotypic instability of chondrocytes in osteoarthritis: on a path to hypertrophy.

Articular chondrocytes are quiescent, fully differentiated cells responsible for the homeostasis of adult articular cartilage by maintaining cellular survival functions and the fine-tuned balance between anabolic and catabolic functions. This balance requires phenotypic stability that is lost in osteoarthritis (OA), a disease that affects and involves all joint tissues and especially impacts articular cartilage structural integrity. In OA, articular chondrocytes respond to the accumulation of injurious biochemical and biomechanical insults by shifting toward a degradative and hypertrophy-like state, involving abnormal matrix production and increased aggrecanase and collagenase activities. Hypertrophy is a necessary, transient developmental stage in growth plate chondrocytes that culminates in bone formation; in OA, however, chondrocyte hypertrophy is catastrophic and it is believed to initiate and perpetuate a cascade of events that ultimately result in permanent cartilage damage. Emphasizing changes in DNA methylation status and alterations in NF-κB signaling in OA, this review summarizes the data from the literature highlighting the loss of phenotypic stability and the hypertrophic differentiation of OA chondrocytes as central contributing factors to OA pathogenesis.

Smad4 deficiency impairs chondrocyte hypertrophy via the Runx2 transcription factor in mouse skeletal development

Chondrocyte hypertrophy is the terminal step in chondrocyte differentiation and is crucial for endochondral bone formation. How signaling pathways regulate chondrocyte hypertrophic differentiation remains incompletely understood. In this study, using a Tbx18:Cre (Tbx18Cre/+) gene-deletion approach, we selectively deleted the gene for the signaling protein SMAD family member 4 (Smad4f/f ) in the limbs of mice. We found that the Smad4-deficient mice develop a prominent shortened limb, with decreased expression of chondrocyte differentiation markers, including Col2a1 and Acan, in the humerus at mid-to-late gestation. The most striking defects in these mice were the absence of stylopod elements and failure of chondrocyte hypertrophy in the humerus. Moreover, expression levels of the chondrocyte hypertrophy-related markers Col10a1 and Panx3 were significantly decreased. Of note, we also observed that the expression of runt-related transcription factor 2 (Runx2), a critical mediator of chondrocyte hypertrophy, was also down-regulated in Smad4-deficient limbs. To determine how the skeletal defects arose in the mouse mutants, we performed RNA-Seq with ChIP-Seq analyses and found that Smad4 directly binds to regulatory elements in the Runx2 promoter. Our results suggest a new mechanism whereby Smad4 controls chondrocyte hypertrophy by up-regulating Runx2 expression during skeletal development. The regulatory mechanism involving Smad4-mediated Runx2 activation uncovered here provides critical insights into bone development and pathogenesis of chondrodysplasia.

Antarctic Krill Oil improves articular cartilage degeneration via activating chondrocyte autophagy and inhibiting apoptosis in osteoarthritis mice

In this study, the in vivo effects of Antarctic krill oil (AKO) on cartilage degeneration in destabilization of the medial meniscus (DMM) mouse were investigated. Results showed that AKO improved the cartilage structure, inhibited chondrocyte hypertrophic differentiation and inhibited abnormal apoptosis in articular cartilage. Autophagy is a self-protective metabolic process required for maintaining cartilage homeostasis. We observed that expression of mTOR were reduced, which was consist with high PPAR-γ levels. The expression of key genes related to autophagy, including LC-3B, Beclin-1, ATG-5, and BNIP-3 were significantly enhanced after treatment with AKO. In addition, we observed that AKO suppressed the expression of key genes in p53-dependent mitochondrial apoptotic pathway, such as p53, Bax, Bid, cytochrome c, caspase-9, caspase-3 and enhanced the expression of anti-apoptotic genes, including Bcl-2 and Bcl-xl. These findings provided a new perspective for the effect of AKO on OA, which associated with the regulation of autophagy and apoptosis.

Cyclic compressive loading activates angiotensin II type 1 receptor in articular chondrocytes and stimulates hypertrophic differentiation through a G-protein-dependent pathway.

Angiotensin II type 1 receptor (AT1R) appears to have a mechanosensing function in a number of cell types. The purpose of this study was to examine whether AT1R expressed in articular chondrocytes is involved in osteoarthritis (OA) progression in vivo and whether cyclic compressive loading activates the AT1R and stimulates hypertrophic differentiation of chondrocytes in vitro. The relationships between the modified Mankin score for cartilage degeneration and the expression of AT1R and type X collagen (Col X) were studied in mouse knees with OA induced using the destabilization‐of‐medial‐meniscus model. Cyclic compressive loads were applied to cultured bovine articular chondrocytes in three‐dimensional agarose scaffolds. Expression of Col X and runt‐related transcription factor 2 (Runx2) was analyzed using RT‐PCR and western blotting. We dissected the downstream pathway for intracellular signal transductions of AT1R including G‐protein‐dependent and G‐protein‐independent pathways. Positive significant correlations between the Mankin score and the rate of AT1R‐immunopositive cells and between the rates of AT1R and Col X expression were noted. The expression of Col X and Runx2 was increased by compressive loading but suppressed by addition of olmesartan, an Ang II receptor blocker, to the agarose scaffolds. Compressive loading upregulated the phosphorylation of c‐Jun N‐terminal kinase (JNK), Src, and STAT1, but olmesartan significantly suppressed only JNK phosphorylation. We conclude that AT1R expressed by articular chondrocytes may be involved in OA progression in vivo. Mechanical stress can activate AT1R and stimulate hypertrophic differentiation of chondrocytes through the G‐protein‐dependent pathway. AT1R has a mechanosensing function in chondrocytes and may be a new therapeutic target in OA.

Chondrogenic properties of collagen type XI, a component of cartilage extracellular matrix

Cartilage extracellular matrix (ECM) has been used for promoting tissue engineering. However, the exact effects of ECM on chondrogenesis and the acting mechanisms are not well understood. In this study, we investigated the chondrogenic effects of cartilage ECM on human mesenchymal stem cells (MSCs) and identified the contributing molecular components. To this end, a preparation of articular cartilage ECM was supplemented to pellets of chondrogenically differentiating MSCs, pellets of human chondrocytes, and bovine articular cartilage explants to evaluate the effects on cell proliferation and the production of cartilaginous matrix. Selective enzymatic digestion and screening of ECM components were conducted to identify matrix molecules with chondrogenic properties. Cartilage ECM promoted MSC proliferation, production of cartilaginous matrix, and maturity of chondrogenic differentiation, and inhibited the hypertrophic differentiation of MSC-derived chondrocytes. Selective digestion of ECM components revealed a contributory role of collagens in promoting chondrogenesis. The screening of various collagen subtypes revealed strong chondrogenic effect of collagen type XI. Finally, collagen XI was found to promote production and inhibit degradation of cartilage matrix in human articular chondrocyte pellets and bovine articular cartilage explants. Our results indicate that cartilage ECM promotes chondrogenesis and inhibits hypertrophic differentiation in MSCs. Collagen type XI is the ECM component that has the strongest effects on enhancing the production and inhibiting the degradation of cartilage matrix.

Ablation of Dnmt3b in chondrocytes suppresses cell maturation during embryonic development.

DNA methylation is a major mode of epigenetic regulation in the mammalian genome and is essential for embryonic development. The three catalytic DNA methyltransferases (Dnmts), Dnmt1, Dnmt3a, and Dnmt3b, catalyze the methylation of cytosine. Dnmt3b is highly expressed in chondrocytes and global knockout of Dnmt3b led to skeletal deformations and embryonic lethality, suggesting an essential role of Dnmt3b in endochondral bone formation. To further define the role of Dnmt3b in skeletal development, Dnmt3b was deleted in Col2 positive chondrocyte lineage cells. Both axial and appendicular skeletal size were reduced and bone mineralization was delayed in Col2Cre+ ;Dnmt3bf/f (Dnmt3bCol2 ) mice at E14.5 and E18.5. While Alcian Blue Hematoxylin/Orange G (ABH/OG) staining showed normal chondrocyte columns in control growth plates, the length of hypertrophic chondrocyte zone and type X collagen expression were decreased in E18.5 growth plates from Dnmt3bCol2 mice. TUNEL and PCNA staining demonstrated that the delay in chondrocyte maturation observed in the Dnmt3bCol2 growth plates was not secondary to altered chondrocyte apoptosis or proliferation. Complementary in vitro experiments were performed on primary sternal chondrocytes isolated from control and Dnmt3bCol2 mice. Gene expression studies confirmed delayed terminal maturation as Mmp13 and Col10a1 expression was down-regulated in Dnmt3bCol2 chondrocytes. In addition, alkaline phosphatase (ALP) and Alizarin Red staining confirmed that Dnmt3b deletion in chondrocytes delays in vitro chondrocyte hypertrophic differentiation and matrix mineralization. Mechanistically, Dnmt3b gene deletion resulted in decreased BMP signaling through reduction of Smad1 phosphorylation. These findings show that epigenetic factor, Dnmt3b is necessary for normal chondrocyte hypertrophic maturation and limb development.

Prehypertrophic to hypertrophic differentiation of chondrocytes induces IL-34 expression, a new cytokine with potential osteochondral junction remodeling activity in osteoarthritis

Prehypertrophic to hypertrophic differentiation of chondrocytes induces IL-34 expression, a new cytokine with potential osteochondral junction remodeling activity in osteoarthritis

BCP crystals drive chondrocyte hypertrophic differentiation via binding WNT ligands and activation of canonical WNT signaling

BCP crystals drive chondrocyte hypertrophic differentiation via binding WNT ligands and activation of canonical WNT signaling