Role In Chondrocyte Differentiation Research Articles

Autophagy Is a Crucial Path in Chondrogenesis of Adipose-Derived Mesenchymal Stromal Cells Laden in Hydrogel.

Autophagy is a cellular process that contributes to the maintenance of cell homeostasis through the activation of a specific path, by providing the necessary factors in stressful and physiological situations. Autophagy plays a specific role in chondrocyte differentiation; therefore, we aimed to analyze this process in adipose-derived mesenchymal stromal cells (ASCs) laden in three-dimensional (3D) hydrogel. We analyzed chondrogenic and autophagic markers using molecular biology, immunohistochemistry, and electron microscopy. We demonstrated that ASCs embedded in 3D hydrogel showed an increase expression of typical autophagic markers Beclin 1, LC3, and p62, associated with clear evidence of autophagic vacuoles in the cytoplasm. During ASCs chondrogenic differentiation, we showed that autophagic markers declined their expression and autophagic vesicles were rare, while typical chondrogenic markers collagen type 2, and aggrecan were significantly increased. In line with developmental animal models of cartilage, our data showed that in a 3D hydrogel, ASCs increased their autophagic features. This path is the fundamental prerequisite for the initial phase of differentiation that contributes to fueling the cells with energy and factors necessary for chondrogenic differentiation.

Sox9 deletion causes severe intervertebral disc degeneration characterized by apoptosis, matrix remodeling, and compartment-specific transcriptomic changes

SOX9 plays an important role in chondrocyte differentiation and, in the developing axial skeleton, maintains the notochord and the demarcation of intervertebral disc compartments. Diminished expression is linked to campomelic dysplasia, resulting in severe scoliosis and progressive disc degeneration. However, the specific functions of SOX9 in the adult spinal column and disc are largely unknown. Accordingly, employing a strategy to conditionally delete Sox9 in Acan-expressing cells (AcanCreERT2Sox9fl/fl), we delineated these functions in the adult intervertebral disc. AcanCreERT2Sox9fl/fl mice (Sox9cKO) showed extensive and progressive remodeling of the extracellular matrix in nucleus pulposus (NP) and annulus fibrosus (AF), consistent with human disc degeneration. Progressive degeneration of the cartilaginous endplates (EP) was also evident in Sox9cKO mice, and it preceded morphological changes seen in the NP and AF compartments. Fate mapping using tdTomato reporter, EdU chase, and quantitative immunohistological studies demonstrated that SOX9 is crucial for disc cell survival and phenotype maintenance. Microarray analysis showed that Sox9 regulated distinct compartment-specific transcriptomic landscapes, with prominent contributions to the ECM, cytoskeleton-related, and metabolic pathways in the NP and ion transport, the cell cycle, and signaling pathways in the AF. In summary, our work provides new insights into disc degeneration in Sox9cKO mice at the cellular, molecular, and transcriptional levels, underscoring tissue-specific roles of this transcription factor. Our findings may direct future cell therapies targeting SOX9 to mitigate disc degeneration.

MiR-23a-3p regulated by LncRNA SNHG5 suppresses the chondrogenic differentiation of human adipose-derived stem cells via targeting SOX6/SOX5.

Cartilage generation and degradation are controlled by miRNAs. Our previous study showed miR-23a-3p was downregulated during chondrogenic differentiation in chondrogenic human adipose-derived mesenchymal stem cells (hADSCs). In the present study, we explored the function of miR-23a-3p in chondrogenesis differentiation. The role of miR-23a-3p in chondrogenic differentiation potential of hADSCs was assessed by Alcian blue staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. We show that miR-23a-3p suppressed the chondrogenic differentiation of hADSCs. LncRNA SNHG5 interacted with miR-23a-3p, and suppression or overexpression of SNHG5 correlates with inhibition and promotion of hADSC chondrogenic differentiation, respectively. We have determined that SNHG5 can sponge miR-23a-3p to regulate the expression of SOX6/SOX5, transcription factors that play essential roles in chondrocyte differentiation. Furthermore, the overexpression of SNHG5 activates the JNK/MAPK/ERK pathway. In conclusion, miR-23a-3p regulated by lncRNA SNHG5 suppresses the chondrogenic differentiation of human adipose-derived stem cells via targeting SOX6/SOX5.

Runx2 silencing promotes adipogenesis via down-regulation of DLK1 in chondrogenic differentiating MSCs.

For cartilage regeneration, stem cells are a promising cell source; however, even the advances made in the differentiation of stem cells into precursor-differentiated cartilage cells have not been successful with respect to reprograming these cells to achieve complete differentiation and fully functioning cells until now. Previous findings suggest that Runx2 plays a major role in chondrocyte differentiation and maturation. Although targeting Runx2 has enhanced some chondrocyte properties, the adipogenic lineage shift has eventually occurred in these cells. The present study mainly aimed to reveal the mechanism of this adipogenesis. To create inducible artificial shRNA-miR expressing vectors, the designed short hairpin RNAs (shRNAs) were inserted into the pri-mir-30 backbone, cloned into lentiviral pLVET-Tet-on, and transducted into mesenchymal stem cells (MSCs). Runx2 gene was silenced in MSCs either for 1 week or 4 weeks and cultured in the chondrogenic medium. At days 7, 14 and 28, cells were harvested, and chondrogenesis, adipogenesis and hypertrophic states were examined using histochemical staining and a real-time polymerase chain reaction assay. The results showed that the designed shRNA-miR effectively targeted Runx2 in mRNA and protein levels. Chondrogenic markers were up-regulated in constantly silenced Runx2 group; however, adipogenic markers and fat droplets appeared gradually. DLK1 gene was also significantly down-regulated in this group, and overexpression of DLK1 abrogated adipogenesis in the Runx2 targeted group. Based on these results, it can be concluded that DLK1 is responsible for the lineage shift in Runx2 targeted chondrogenic differentiating MSCs.

Inhibition of Rac1 activity by NSC23766 prevents cartilage endplate degeneration via Wnt/β-catenin pathway.

Cartilage endplate (CEP) degeneration has been considered as one of important factors related to intervertebral disc degeneration (IVDD). Previous researches have showed that Rac1 played a pivotal role in chondrocyte differentiation. However, the effect of Rac1 during the process of CEP degeneration remains unclear. Herein, we explored the effect of Rac1 on CEP degeneration and elucidated the underlying molecular mechanism. We found expression of Rac1‐GTP increased in human‐degenerated CEP tissue and IL‐1β‐stimulated rat endplate chondrocytes (EPCs). Our study revealed that Rac1 inhibitor NSC23766 treatment promoted the expression of collagen II, aggrecan and Sox‐9, and decreased the expression of ADTAMTS5 and MMP13 in IL‐1β‐stimulated rat EPCs. Moreover, we also found that NSC23766 could suppress the activation of Wnt/β‐catenin pathway, suggesting that the beneficial effects of Rac1 inhibition in EPCs are mediated through the Wnt/β‐catenin signalling. Besides, puncture‐induced rats models showed that NSC23766 played a protective role on CEP and disc degeneration. Collectively, these findings demonstrated that Rac1 inhibition delayed the EPCs degeneration and its potential mechanism may be associated with Wnt/β‐catenin pathway regulation, which may help us better understand the association between Rac1 and CEP degeneration and provide a promising strategy for delaying the progression of IVDD.

Constitutive activation of the alternative NF-κB pathway disturbs endochondral ossification.

Endochondral ossification is important for skeletal development. Recent findings indicate that the p65 (RelA) subunit, a main subunit of the classical nuclear factor-κB (NF-κB) pathway, plays essential roles in chondrocyte differentiation. Although several groups have reported that the alternative NF-κB pathway also regulates bone homeostasis, the role of the alternative NF-κB pathway in chondrocyte development is still unclear. Here, we analyzed the in vivo function of the alternative pathway on endochondral ossification using p100-deficient (p100-/-) mice, which carry a homozygous deletion of the COOH-terminal ankyrin repeats of p100 but still express functional p52 protein. The alternative pathway was activated during the periarticular stage in wild-type mice. p100-/- mice exhibited dwarfism, and histological analysis of the growth plate revealed abnormal arrangement of chondrocyte columns and a narrowed hypertrophic zone. Consistent with these observations, the expression of hypertrophic chondrocyte markers, type X collagen (ColX) or matrix metalloproteinase 13, but not early chondrogenic markers, such as Col II or aggrecan, was suppressed in p100-/- mice. An in vivo BrdU tracing assay clearly demonstrated less proliferative activity in chondrocytes in p100-/- mice. These defects were partly rescued when the RelB gene was deleted in p100-/- mice. Taken together, the alternative NF-κB pathway may regulate chondrocyte proliferation and differentiation to maintain endochondral ossification.

ZNF606 interacts with Sox9 to regulate chondrocyte differentiation

SRY-related high-mobility-group box 9 (Sox9) gene is a transcription factor that plays an essential role in chondrocyte differentiation and cartilage formation. In this study, we identified the transcriptional factor ZNF606 as an interacting partner for Sox9 in cells. We further demonstrated that overexpression of ZNF606 inhibited the transcriptional activity of Sox9, while knockdown of ZNF606 increased Sox9-mediated transcription. Chromatin immunoprecipitation analysis revealed that ZNF606 prevents Sox9 binding to the enhancers of its target gene col2a1. Importantly, the interaction between ZNF606 and Sox9 was decreased during chondrocyte differentiation. Consistent with these findings, ZNF606 inhibited chondrocyte differentiation. Thus, our results demonstrate that ZNF606 acts as a novel Sox9 co-regulator that inhibits Sox9-mediated chondrocyte differentiation.

Compression regulates gene expression of chondrocytes through HDAC4 nuclear relocation via PP2A-dependent HDAC4 dephosphorylation

Biomechanics plays a critical role in the modulation of chondrocyte function. The mechanisms by which mechanical loading is transduced into intracellular signals that regulate chondrocyte gene expression remain largely unknown. Histone deacetylase 4 (HDAC4) is specifically expressed in chondrocytes. Mice lacking HDAC4 display chondrocyte hypertrophy, ectopic and premature ossification, and die early during the perinatal period. HDAC4 has a remarkable ability to translocate between the cell's cytoplasm and nucleus. It has been established that subcellular relocation of HDAC4 plays a critical role in chondrocyte differentiation and proliferation. However, it remains unclear whether subcellular relocation of HDAC4 in chondrocytes can be induced by mechanical loading. In this study, we first report that compressive loading induces HDAC4 relocation from the cytoplasm to the nucleus of chondrocytes via stimulation of Ser/Thr-phosphoprotein phosphatases 2A (PP2A) activity, which results in dephosphorylation of HDAC4. Dephosphorylated HDAC4 relocates to the nucleus to achieve transcriptional repression of Runx2 and regulates chondrocyte gene expression in response to compression. Our results elucidate the mechanism by which mechanical compression regulates chondrocyte gene expression through HDAC4 relocation from the cell's cytoplasm to the nucleus via PP2A-dependent HDAC4 dephosphorylation.

R-spondin 2 facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β-catenin signaling in endochondral ossification

Endochondral ossification is a crucial process for longitudinal growth of bones. Differentiating chondrocytes in growth cartilage form four sequential zones of proliferation, alignment into column, hypertrophy, and substitution of chondrocytes with osteoblasts. Wnt/β-catenin signaling is essential for differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. R-spondin 2 (Rspo2), a member of R-spondin family, is an agonist for Wnt signaling, but its role in chondrocyte differentiation remains unknown. Here we report that growth cartilage of Rspo2-knockout mice shows a decreased amount of β-catenin and increased amounts collagen type II (CII) and Sox9 in the abnormally extended proliferating zone. In contrast, expression of collagen type X (CX) in the hypertrophic zone remains unchanged. Differentiating chondrogenic ATDC5 cells, mimicking proliferating chondrocytes, upregulate Rspo2 and its putative receptor, Lgr5, in parallel. Addition of recombinant human Rspo2 to differentiating ATDC5 cells decreases expressions of Col2a1, Sox9, and Acan, as well as production of proteoglycans. In contrast, lentivirus-mediated knockdown of Rspo2 has the opposite effect. The effect of Rspo2 on chondrogenic differentiation is mediated by Wnt/β-catenin signaling, and not by Wnt/PCP or Wnt/Ca2+ signaling. We propose that Rspo2 activates Wnt/β-catenin signaling to reduce Col2a1 and Sox9 and to facilitate differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage.

Yap1 Regulates Multiple Steps of Chondrocyte Differentiation during Skeletal Development and Bone Repair.

Hippo signaling controls organ size and tissue regeneration in many organs, but its roles in chondrocyte differentiation and bone repair remain elusive. Here, we demonstrate that Yap1, an effector of Hippo pathway inhibits skeletal development, postnatal growth, and bone repair. We show that Yap1 regulates chondrocyte differentiation at multiple steps in which it promotes early chondrocyte proliferation but inhibits subsequent chondrocyte maturation both in vitro and in vivo. Mechanistically, we find that Yap1 requires Teads binding for direct regulation of Sox6 expression to promote chondrocyte proliferation. In contrast, Yap1 inhibits chondrocyte maturation by suppression of Col10a1 expression through interaction with Runx2. In addition, Yap1 also governs the initiation of fracture repair by inhibition of cartilaginous callus tissue formation. Taken together, our work provides insights into the mechanism by which Yap1 regulates endochondral ossification, which may help the development of therapeutic treatment for bone regeneration.

Overexpression of heparan sulfate 6-O-sulfotransferase-2 enhances fibroblast growth factor-mediated chondrocyte growth and differentiation.

In our previous study, we reported that heparan sulfate 6-O-sulfotransferase‑2 (HS6ST2) plays an important role in the cartilage of patients with osteoarthritis and Kashin-Beck disease and that it regulates aggrecan (Acan) metabolism and the viability of chondrocytes. However, its role in chondrocyte differentiation remains poorly understood. In the present study, we aimed to investigate the role of HS6ST2 in chondrocyte differentiation in vitro using mouse prechondrocytic cells. We found that the overexpression or silencing of HS6ST2 significantly enhanced or abrogated the effects of fibroblast growth factor (FGF)‑2 on chondrocyte growth, respectively. We found that the overexpression of HS6ST2 significantly induced the expression of Acan as well as the amount of total proteoglycans in the prechondrocytic cells in the presence of FGF‑2, whereas the silencing of HS6ST2 caused the opposite effect. Furthermore, the expresssion of FGF‑2‑induced sex‑determining region Y‑type high mobility group box protein 9 (SOX9), a major transcription factor for chondrocyte proliferation and differentiation, was also enhanced or blocked by HS6ST2 overexpression or HS6ST2 knockdown, respectively. Additionally, Wnt/β‑catenin signaling, which inhibited chondrocyte proliferation and differentiation, was suppressed by HS6ST2. Taken together, these data suggest that HS6ST2 plays an important role in regulating chondrocyte growth and differentiation by modulating FGF‑2 signaling, thus indicating that it may be a potential and valuable molecular target for the treatment of skeletal dysplasias, such as dwarfism.

Prostaglandin F2α receptor (FP) signaling regulates Bmp signaling and promotes chondrocyte differentiation

Prostaglandins are a group of lipid signaling molecules involved in various physiological processes. In addition, prostaglandins have been implicated in the development and progression of diseases including cancer, cardiovascular disease, and arthritis. Prostaglandins exert their effects through the activation of specific G protein-coupled receptors (GPCRs). In this report, we examined the role of prostaglandin F2α receptor (FP) signaling as a regulator of chondrocyte differentiation. We found that FP expression was dramatically induced during the differentiation of chondrocytes and was up-regulated in cartilages. Forced expression of FP in ATDC5 chondrogenic cell line resulted in the increased expression of differentiation-related genes and increased synthesis of the extracellular matrix (ECM) regardless of the presence of insulin. Similarly, PGF2α treatment induced the expression of chondrogenic marker genes. In contrast, knockdown of endogenous FP expression suppressed the expression of chondrocyte marker genes and ECM synthesis. Organ culture of cartilage rudiments revealed that PGF2α induces chondrocyte hypertrophy. Additionally, FP overexpression increased the levels of Bmp-6, phospho-Smad1/5, and Bmpr1a, while knockdown of FP reduced expression of those genes. These results demonstrate that up-regulation of FP expression plays an important role in chondrocyte differentiation and modulates Bmp signaling.

RAGE, receptor of advanced glycation endoproducts, negatively regulates chondrocytes differentiation.

RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE) demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms.

Expression of genes encoding matrilin-3 and cyclin-I during the impairment and recovery of chicken growth plate in tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is a skeletal disease characterized by the disruption ofendochondral bone formation in avian species. The aim of this study was to determine the expression of Matrilin-3 and Cyclin-I genes in chicken growth plate during impairment and recovery from TD. Gene expressions were analyzed by polymerase chain reaction, and proteins by immunohistochemistry and in situ hybridizations. Expression of genes encoding Matrilin-3 and Cyclin-I were diminished with parallel decrease in proteins during TD, with fewer signs of cartilage cell differentiation. In contrast, there was an increase in mRNA expressions and protein levels of both genes during the recovery phase. These findings suggest that the Matrilin-3 and Cyclin-I genes also play a role in chondrocyte differentiation during the impairment and recovery of growth plate in TD.

LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation

Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling.

Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

Age-related alterations in TGF beta signaling as a causal factor of cartilage degeneration in osteoarthritis.

Age is the most important risk factor for primary osteoarthritis (OA). Members of the TGF-β superfamily play a crucial role in chondrocyte differentiation and maintenance of healthy articular cartilage. We have investigated whether age-related changes in TGF-β superfamily signaling components play a role in the relationship between OA-related cartilage degradation and aging. The relationship between age, OA and TGF-β superfamily signaling was studied using murine experimental OA models, aging mice, bovine articular cartilage and human OA cartilage. The effects of TGF-β on cartilage homeostasis was studied with immunohistochemistry, Q-RT-PCR and signaling pathway analysis with Western blotting and the application of specific TGF-β inhibitors. We have found that TGF-β loses its protective effects in old cartilage. Moreover, we found that on chondrocytes, TGF-β not only signals via the canonical type I receptor ALK5 (TGFBR1) but also via the ALK1 (ACVRL1) receptor. Remarkably, signaling via ALK5 (Smad2/3 route) results in protective while ALK1 signaling (Smad1/5/8 route) results in deleterious responses in articular chondrocytes. In cartilage of aging mice it was detected that the ALK1/ALK5 ratio is significantly increased, favoring TGF-β signaling via the Smad1/5/8 route, inducing changes in chondrocyte differentiation and matrix metalloproteinase-13 (MMP-13) expression. Moreover, human OA cartilage showed a significant correlation between ALK1 and MMP-13 expression. Since in mice aging and OA in often goes hand in hand, we also analyzed age-related expression of TGF-β superfamily related signaling molecules in healthy bovine cartilage in an age range from 6 months to 14 years. In this cohort of aging cartilage, we found that mainly signaling receptors determining the Smad2/3 pathway were decreased with age while Smad1/5/8-related signaling molecules did not alter, confirming our findings in aging mice. Old cartilage appears to be less protected by TGF-β and shows significant alterations in TGF-β signaling pathways. Loss of the protective Smad2/3 pathway during aging can provide an explanation for the relationship between OA and aging.

Chondrocyte Proliferation Regulated by Secreted Luminal Domain of ER Stress Transducer BBF2H7/CREB3L2

The endoplasmic reticulum (ER) stress transducer BBF2H7/CREB3L2 is an ER-resident transmembrane transcription factor. In response to physiological ER stress, it is processed at the transmembrane region to generate a cytoplasmic N terminus, which contains a basic leucine zipper (bZIP) domain, and luminal C terminus. The BBF2H7 N terminus functions as a transcription factor to promote the expression of ER-Golgi trafficking-related genes and plays crucial roles in chondrocyte differentiation. Here, we found that the BBF2H7 C terminus is secreted into the extracellular space as a signaling molecule for cell-to-cell communication. The secreted BBF2H7 C terminus directly binds to both Indian hedgehog and its receptor Patched-1, followed by activation of Hedgehog signaling, resulting in promoting the proliferation of neighboring chondrocytes. The dual N- and C-terminal functions of BBF2H7 triggered by physiological ER stress may allow chondrocytes to simultaneously regulate distinct cellular events for differentiation and proliferation in developing cartilage.

Downregulation of Carbonic Anhydrase IX Promotes Col10a1 Expression in Chondrocytes

Carbonic anhydrase (CA) IX is a transmembrane isozyme of CAs that catalyzes reversible hydration of CO2. While it is known that CA IX is distributed in human embryonic chondrocytes, its role in chondrocyte differentiation has not been reported. In the present study, we found that Car9 mRNA and CA IX were expressed in proliferating but not hypertrophic chondrocytes. Next, we examined the role of CA IX in the expression of marker genes of chondrocyte differentiation in vitro. Introduction of Car9 siRNA to mouse primary chondrocytes obtained from costal cartilage induced the mRNA expressions of Col10a1, the gene for type X collagen α-1 chain, and Epas1, the gene for hypoxia-responsible factor-2α (HIF-2α), both of which are known to be characteristically expressed in hypertrophic chondrocytes. On the other hand, forced expression of CA IX had no effect of the proliferation of chondrocytes or the transcription of Col10a1 and Epas1, while the transcription of Col2a1 and Acan were up-regulated. Although HIF-2α has been reported to be a potent activator of Col10a1 transcription, Epas1 siRNA did not suppress Car9 siRNA-induced increment in Col10a1 expression, indicating that down-regulation of CA IX induces the expression of Col10a1 in chondrocytes in a HIF-2α-independent manner. On the other hand, cellular cAMP content was lowered by Car9 siRNA. Furthermore, the expression of Col10a1 mRNA after Car9 silencing was augmented by an inhibitor of protein kinase A, and suppressed by an inhibitor for phosphodiesterase as well as a brominated analog of cAMP. While these results suggest a possible involvement of cAMP-dependent pathway, at least in part, in induction of Col10a1 expression by down-regulation of Car9, more detailed study is required to clarify the role of CA IX in regulation of Col10a1 expression in chondrocytes.

Arid5a cooperates with Sox9 to stimulate chondrocyte-specific transcription

SRY-box-containing gene 9 (Sox9) is an essential transcription factor in chondrocyte lineage determination and differentiation. Recent studies demonstrated that Sox9 controls the transcription of chondrocyte-specific genes in association with several other transcriptional regulators. To further understand the molecular mechanisms by which Sox9 influences transcriptional events during chondrocyte differentiation, we attempted to identify transcriptional partners of Sox9 and to examine their roles in chondrocyte differentiation. We isolated AT-rich interactive domain-containing protein 5a (Arid5a; also known as Mrf1) as an activator of the Col2a1 gene promoter from an ATDC5 cDNA library. Arid5a was highly expressed in cartilage and induced during chondrocyte differentiation. Furthermore, Arid5a physically interacted with Sox9 in nuclei and up-regulated the chondrocyte-specific action of Sox9. Overexpression of Arid5a stimulated chondrocyte differentiation in vitro and in an organ culture system. In contrast, Arid5a knockdown inhibited Col2a1 expression in chondrocytes. In addition, Arid5a binds directly to the promoter region of the Col2a1 gene and stimulates acetylation of histone 3 in the region. Our results suggest that Arid5a may directly interact with Sox9 and thereby enhance its chondrocyte-specific action.