Differentiation Of ATDC5 Research Articles

3D Bioprinting-Based Dopamine-Coupled Flexible Material for Nasal Cartilage Repair.

Since 3D printing can be used to design implants according to the specific conditions of patients, it has become an emerging technology in tissue engineering and regenerative medicine. How to improve the mechanical, elastic and adhesion properties of 3D-printed photocrosslinked hydrogels is the focus of cartilage tissue repair and reconstruction research. We established a strategy for toughening hydrogels by mixing GelMA-DOPA (GD), which is prepared by coupling dopamine (DA) with GelMA, with HAMA, bacterial cellulose (BC) to produce composite hydrogels (HB-GD). HB-GD hydrogel scaffolds were characterized in vitro by scanning electron microscopy (SEM), Young's modulus, swelling property and rheological property tests. And biocompatibility and chondrogenic ability were tested by live/dead staining, DNA quantitative analysis and immunofluorescence staining. Combined with 3D bioprinting technology, mouse chondrocytes (ADTC5) were added to form a biological chain to construct an in vitro model, and the feasibility of the model for nasal cartilage regeneration was verified by cytology evaluation. With the increase of GD concentration, the toughness of the composite hydrogel increased (47.0 ± 2.7 kPa (HB-5GD)-158 ± 3.2 kPa (HB-20GD)), and it had excellent swelling properties, rheological properties and printing properties. The HB-GD composite hydrogel promoted the proliferation and differentiation of ATDC5. Cells in 3D printed scaffolds had higher survival rates (> 95%) and better protein expression than the encapsulated cultures. The HB-10GD hydrogel can be made into a porous scaffold with precise shape, good internal pore structure, high mechanical strength and good swelling rate through extrusion 3D printing. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Ascorbic acid enhances chondrocyte differentiation of ATDC5 by accelerating insulin receptor signaling.

Chondrogenesis is strictly regulated by several factors, including cytokines, hormones, and extracellular matrix proteins. Mouse teratocarcinoma-derived lineage cells, differentiate into chondrocytes in the presence of insulin. Although ascorbic acid promotes chondrogenic differentiation, the detailed regulative mechanisms underlying its role in chondrogenesis remain unclear. Therefore, in this study, we evaluated the effects of ascorbic acid on insulin-induced chondrogenic differentiation of ATDC5 cells and the underlying intracellular signaling. The results revealed that insulin-stimulated collagen deposition, matrix formation, calcification, and expression of chondrogenic differentiation marker genes in ATDC5 cells. This enhancement by insulin was amplified with the addition of ascorbic acid. Molecular analysis revealed that the activation of insulin-induced phosphoinositide 3-kinase (PI3K)/Akt signaling was enhanced in the presence of ascorbic acid. In contrast, Wnt/β-catenin signaling was suppressed during chondrocyte differentiation via upregulation of the Wnt agonist, secreted Frizzled-related protein 1 (sFRP-1) and 3 (sFRP-3). Notably, ascorbic acid upregulated the expression of insulin receptors and their substrates (IRS-1 and IRS-2). Furthermore, ascorbic acid reversed the suppression of IRS-1 and IRS-2 protein by insulin. These results indicate that ascorbic acid positively regulates the chondrogenic differentiation of ATDC5 cells via enhancement of insulin signaling. Our findings provide a substantial basis for further elucidation of the regulatory mechanisms of chondrocyte differentiation and the pathophysiology of OA, thus aiding in development of effective treatment strategies.

Galactose Enhances Chondrogenic Differentiation of ATDC5 and Cartilage Matrix Formation by Chondrocytes.

Galactose, an important carbohydrate nutrient, is involved in several types of cellular metabolism, participating in physiological activities such as glycosaminoglycan (GAG) synthesis, glycosylation, and intercellular recognition. The regulatory effects of galactose on osteoarthritis have attracted increased attention. In this study, in vitro cell models of ATDC5 and chondrocytes were prepared and cultured with different concentrations of galactose to evaluate its capacity on chondrogenesis and cartilage matrix formation. The cell proliferation assay demonstrated that galactose was nontoxic to both ATDC5 cells and chondrocytes. RT-PCR and immunofluorescence staining indicated that the gene expressions of cartilage matrix type II collagen and aggrecan were significantly upregulated with increasing galactose concentration and the expression and accumulation of the extracellular matrix (ECM) protein. Overall, these results indicated that a galactose concentration below 8 mM exhibited the best effect on promoting chondrogenesis, which entitles galactose as having considerable potential for cartilage repair and regeneration.

Adaptation of the protein translational apparatus during ATDC5 chondrogenic differentiation.

IntroductionRibosome biogenesis is integrated with many cellular processes including proliferation, differentiation and oncogenic events. Chondrogenic proliferation and differentiation require a high cellular translational capacity to facilitate cartilaginous extracellular matrix production. We here investigated the expression dynamics of factors involved in ribosome biogenesis during in vitro chondrogenic differentiation and determined whether protein translation capacity adapts to different phases of chondrogenic differentiation.MaterialsSnoRNA expression during ATDC5 differentiation was analyzed by RNA sequencing of samples acquired from day 0 (progenitor stage), 7 (chondrogenic stage) and day 14 (hypertrophic stage). RT-qPCR was used to determine expression of fibrillarin, dyskerin, UBF-1, Sox9, Col2a1, Runx2, Col10a1 mRNAs and 18S, 5.8S and 28S rRNAs. Protein expression of fibrillarin, dyskerin and UBF-1 was determined by immunoblotting. Ribosomal RNA content per cell was determined by calculating rRNA RT-qPCR signals relative to DNA content (SYBR Green assay). Total protein translational activity was evaluated with a puromycilation assay and polysome profiling.ResultsAs a result of initiation of chondrogenic differentiation (Δt0-t7), 21 snoRNAs were differentially expressed (DE). Hypertrophic differentiation caused DE of 23 snoRNAs (Δt7-t14) and 43 when t0 was compared to t14. DE snoRNAs, amongst others, target nucleotide modifications in the 28S rRNA peptidyl transferase center and the 18S rRNA decoding center. UBF-1, fibrillarin and dyskerin expression increased as function of differentiation and displayed highest fold induction at day 5–6 in differentiation. Ribosomal RNA content per cell was significantly increased at day 7, but not at day 14 in differentiation. Similar dynamics in translational capacity and monosomal ribosome fraction were observed during differentiation.ConclusionThe expression of a great number of ribosome biogenesis factors is altered during chondrogenic differentiation of ATDC5 cells, which is accompanied by significant changes in cellular translational activity. This elucidation of ribosome biogenesis dynamics in chondrogenic differentiation models enables the further understanding of the role of ribosome biogenesis and activity during chondrocyte cell commitment and their roles in human skeletal development diseases.

Regulation of cell differentiation via synergistic self-powered stimulation and degradation behavior of a biodegradable composite piezoelectric scaffold for cartilage tissue

The articular cartilage disorder at the junction mainly results from constantly repeated dynamic tension/compression effects with ageing and lack of intrinsic defect repairability. Therefore, the degradable piezoelectric scaffolds are very essential, which mimics the dynamic mechanical loading and optimizes the chondrocyte differentiation during the degradation. Here, a degradable aligned electrospun poly-L-lactic acid (PLLA) modified with graphene (rGO) and polydopamine (PDA) fibrous scaffolds with different orientations (0°, 90°) and surface morphologies (wrinkled and porous) was developed as a biocompatible and degradable piezoelectric scaffold with the self-powered tunable piezoelectricity to modulate cell behaviour and cell differentiation by tuning the degradation effect. The results show that the electrical output and mechanical properties of the composite fibrous scaffold can be improved by adding rGO and applying mechanical force along with the 90° orientation. With changing the degradation behavior, dynamic mechanical loading on the porous PLLA/rGO/PDA fibrous scaffold exhibits a significant increase in cell proliferation and secretion of extracellular matrix (ECM). More surprisingly, as extending the degradation periods to 21 days, a higher glycosaminoglycans (GAGs) synthesis was detected in prechondrogenic ATDC5 cells cultured on the degraded porous scaffold compared with that after 7 days’ culture. This indicated that long-term degradation favoured promoting cell differentiation of ATDC5 towards a chondrogenic phenotype due to dynamic mechanical loading, low-intensity electrical stimulation, and interconnected porous structural morphology. In contrast, on the wrinkled PLLA/rGO/PDA fibre with a high-intensity electrical stimulation, the ALP activity significantly increased after 21 days, inducing mineralization with the differentiation of ATDC5 into osteocytes. The modulation of the degraded environment and electrical stimulation of the piezoelectric scaffold offers an effective alternative to influence cell functions, significantly improving the ECM secretion and cell differentiation.

YAP1 regulates chondrogenic differentiation of ATDC5 promoted by temporary TNF-α stimulation through AMPK signaling pathway.

Local injection of tumor necrosis factor-alpha (TNF-α) at bone fracture sites during the early stage of the inflammatory response is reported to improve fracture repair in a murine model. However, the underlying mechanism is unclear. Endochondral bone formation, a process that is highly related to fracture repair, requires a certain amount of chondrocyte hypertrophy. This study aimed to investigate the effect of TNF-α on the differentiation of murine chondrogenic ATDC5 cells and the underlying mechanism. In this study, improved chondrogenic differentiation of ATDC5 cells was achieved by brief TNF-α stimulation. Moreover, the expression of Yes-associated protein 1 (YAP1) was suppressed after brief TNF-α stimulation. The expressions of inflammatory mediators and chondrogenic and hypertrophic-associated genes in ATDC5 cells triggered by TNF-α were suppressed in the YAP1 overexpression group but enhanced in the YAP1 knockdown group. Mechanistically, TNF-α-induced activation of the 5' AMP-activated protein kinase (AMPK) signaling pathway was regulated by YAP1, as revealed by the phosphorylated-AMPK/AMPK change ratios in the YAP1 overexpression and knockdown groups, respectively. Moreover, the potential for TNF-α to enhance chondrogenic differentiation could be partially reversed with an AMPK inhibitor. Taken together, we demonstrate, for the first time, that YAP1 modulates the ability of TNF-α to enhance chondrocyte differentiation partly through AMPK signaling.

TET1 Directs Chondrogenic Differentiation by Regulating SOX9 Dependent Activation of Col2a1 and Acan In Vitro.

ABSTRACTSkeletal development is a tightly orchestrated process in which cartilage and bone differentiation are intricately intertwined. Recent studies have highlighted the contribution of epigenetic modifications and their writers to skeletal development. Methylated cytosine (5mC) can be oxidized to 5‐hydroxymethylcytosine (5hmC) by the Ten‐eleven‐translocation (TET) enzymes leading to demethylation. We have previously demonstrated that 5hmC is stably accumulated on lineage‐specific genes that are activated during in vitro chondrogenesis in the ATDC5 chondroprogenitors. Knockdown (KD) of Tet1 via short‐hairpin RNAs blocked ATDC5 chondrogenic differentiation. Here, we aimed to provide the mechanistic basis for TET1 function during ATDC5 differentiation. Transcriptomic analysis of Tet1 KD cells demonstrated that 54% of downregulated genes were SOX9 targets, suggesting a role for TET1 in mediating activation of a subset of the SOX9 target genes. Using genome‐wide mapping of 5hmC during ATDC5 differentiation, we found that 5hmC is preferentially accumulated at chondrocyte‐specific class II binding sites for SOX9, as compared with the tissue‐agnostic class I sites. Specifically, we find that SOX9 is unable to bind to Col2a1 and Acan after Tet1 KD, despite no changes in SOX9 levels. Finally, we compared this KD scenario with the genetic loss of TET1 in the growth plate using Tet1 −/− embryos, which are approximately 10% smaller than their WT counterparts. In E17.5 Tet1 −/− embryos, loss of SOX9 target gene expression is more modest than upon Tet1 KD in vitro. Overall, our data suggest a role for TET1‐mediated 5hmC deposition in partly shaping an epigenome conducive for SOX9 function. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

The effect of celecoxib in traumatic heterotopic ossification around temporomandibular joint in mice

In this study, the role of inflammation in traumatic heterotopic ossification around temporomandibular joint (THO-TMJ), as well as the preventive and treatment effect of celecoxib in THO-TMJ both in vivo and in vitro were explored. A surgically-induced THO-TMJ mouse model and a co-culture model of ATDC-5 or MC3T3-E1 and RAW-264.7cells were used in this study for in vivo and in vitro research. A series of inflammatory factors, such as CD3, CD68, CD20, IL-10, IL-6 and TNF-α, were activated 48h after trauma in a THO-TMJ model. Local trauma initiated systemic inflammatory responses as well as T cell- and macrophage-mediated local inflammatory responses around TMJ. In addition, expression of COX-2 was significantly elevated. The findings also showed that local injection of celecoxib could effectively alleviate the inflammatory response around TMJ at the early stage of trauma and inhibit the formation of THO-TMJ in vivo. Meanwhile, celecoxib could inhibit chondrogenic differentiation of ATDC-5 and osteogenic differentiation of MC3T3-E1 under inflammatory condition in vitro. Furthermore, celecoxib could inhibit the expression of Bmpr1b in the injured condylar cartilage at the initiation stage of THO-TMJ, which implied that Bmpr1b expressed by the residual condylar cartilage might be related to the pathogenesis of THO-TMJ. Inflammation played a crucial role in the pathogenesis of THO-TMJ, and anti-inflammation might be a possible choice to inhibit THO-TMJ, which provided scientific clues for the mechanisms, pharmacotherapy and molecular intervention of THO-TMJ.

Inhibitory effect of retinoic acid receptor agonists on in vitro chondrogenic differentiation.

Retinoic acid (RA), an active metabolite of vitamin A, plays pivotal roles in a wide variety of biological processes, such as body patterning, organ development, and cell differentiation and proliferation. RA signaling is mediated by nuclear retinoic acid receptors, α, β, and γ (RARα, RARβ, and RARγ). RA is a well-known regulator of cartilage and skeleton formation and RARs are also essential for skeletal growth and hypertrophic chondrocyte-specific gene expression. These important roles of RA and RARs in chondrogenesis have been widely investigated using in vivo mouse models. However, few reports are available on the function of each subtype of RARs on in vitro chondrocyte differentiation. Here, we examined the effect of specific agonists of RARs on chondrogenic differentiation of ATDC5 and C3H10T1/2 cells. Subtype-specific RAR agonists as well as RA decreased the expressions of chondrogenic differentiation marker genes and inhibited chondrogenic differentiation, which was accompanied with morphological change to spindle-shaped cells. Among RAR agonists, RARα and RARγ agonists revealed a strong inhibitory effect on chondrogenic differentiation. RARα and RARγ agonists also hampered viability of ATDC5 cells. These observations suggested that RARα and RARγ are dominant receptors of RA signaling that negatively regulate chondrogenic differentiation.

Neuropeptide W regulates proliferation and differentiation of ATDC5: Possible involvement of GPR7 activation, PKA and PKC-dependent signalling cascades.

Various neuropeptides related to the energy equilibrium affect bone growth in humans and animals. Neuropeptides W (NPW) are identical in the internal ligands of the two G‐protein receptors (GPRs) included in subtypes 7 and 8. Neuropeptides W inhibits proliferation in the cultivated rat calvarial osteoblast‐like (ROB) cells. This study examines the expression of NPW and GPR7 in murine chondrocyte and their function. An immunohistochemical analysis showed that NPW and GPR7 were expressed in the proliferative chondrocytes of the growth plates in the hind limbs of mice. The NPW mRNA quickly elevated in the early differentiation (7‐14 days) of ATDC5 cells, while NPW and GPR7 mRNA were reduced during the late stage (14‐21 days) of differentiation. Neuropeptide W‐23 (NPW‐23) promoted the proliferation of ATDC5 cells, which was attenuated by inhibiting the GPR7, protein kinase A (PKA), protein kinase C (PKC) and ERK1/2 pathways. Neuropeptide W‐23 enhanced the early cell differentiation, as evaluated by collagen type II and the aggrecan gene expression, which was unaffected by inhibiting the ERK1/2 pathway, but significantly decreased by inhibiting the PKA, PKC and p38 MAPK pathways. In contrast, NPW‐23 was not involved in the terminal differentiation of the chondrocytes, as evaluated by the mineralization of the chondrocytes and the activity of the alkaline phosphatase. Neuropeptides W stimulated the PKA, PKC, p38 MAPK and ERK1/2 activities in a dose‐ and time‐dependent manner in the ATDC5 cells. These results show that NPW promotes the proliferation and early differentiation of murine chondrocyte via GPR7 activation, as well as PKA and PKC‐dependent signalling cascades, which may be involved in endochondral bone formation.

A novel glucosamine derivative with low cytotoxicity enhances chondrogenic differentiation of ATDC5.

Glucosamine (GlcN) is a component of native cartilage extracellular matrix and useful in cartilage repair, but it was limited by toxicity in high concentrations. With the aim of altering bioactive properties of GlcN to reduce the toxicity and to facilitate chondrogenesis for hyaline cartilage formation, we introduced an amino-group modification with double bond into GlcN to produce N-acryloyl-glucosamine (AGA). The cell ATDC5 was chosen to evaluate its cytotoxicity and chondrogenesis capability. Cell proliferation and cytotoxicity assay showed that AGA had significantly reduced the cytotoxicity compared to GlcN, and promoted ATDC5 proliferation. Alcian blue staining and biochemical analysis indicated that AGA enhanced extracellular matrix deposition. Both the mRNA and protein levels of articular cartilage markers, like Collagen II and Aggrecan were up-regulated, as shown by quantitative real-time PCR and immunofluorescence staining. Moreover, the level of fibrocartilage marker Collagen I and hypertrophic marker Collagen Χ weren't significantly changed. Overall, these results demonstrated that the AGA achieved the functional double-bond, reduction in toxicity and enhancement in chondrogenesis could be more potential in cartilage repair.

Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells.

ObjectivesFibroblast Growth Factor 23 (FGF23) is well documented as a crucial player in the systemic regulation of phosphate homeostasis. Moreover, loss-of-function experiments have revealed that FGF23 also has a phosphate-independent and local impact on skeletogenesis. Here, we used ATDC5 cell line to investigate the expression of FGF23 and the role it may play locally during the differentiation of these cells.MethodsATDC5 cells were differentiated in the presence of insulin, and treated with recombinant FGF23 (rFGF23), inorganic phosphate (Pi) and/or PD173074, an inhibitor of FGF receptors (FGFRs). The mRNA expressions of FGF23, FGFRs and markers of hypertophy, Col X and MMP13, were determined by qPCR analysis and FGF23 production was assessed by ELISA. FGFR activation was determined by immunoprecipitation and immunoblotting.ResultsFGF23 mRNA expression and production were increased during ATDC5 differentiation. At D28 in particular, rFGF23 stimulation increased hypertrophic markers expression, as Col X and MMP13, and mineralization. A synergic effect of Pi and rFGF23 stimulation was observed on these markers and on the mineralization process. The use of PD173074, a pan-FGFR inhibitor, decreased terminal differentiation of ATDC5 by preventing rFGF23 pro-hypertrophic effects.ConclusionsAltogether, our results provide evidence that FGF23 plays an important role during differentiation of ATDC5 cell line, by promoting both hypertrophy and mineralization.

Effect of mechanical stimuli on chondrogenic differentiation of ATDC5

Effect of mechanical stimuli on chondrogenic differentiation of ATDC5

Oleuropein, quercetin and curcumin prevent cartilage breakdown in bovine chondrocytes

Oleuropein, quercetin and curcumin prevent cartilage breakdown in bovine chondrocytes

The Effect of Growth Differentiation Factor-5, 6, 7 in Chondrogenic Cell Differentiation of ATDC-5

The proteins known as growth differentiation factors (GDFs) are members of the BMP family; GDF-5, -6, and -7 play important roles in skeletogenesis, especially with regard to chondrogenesis. The functional differences among these GDFs in chondrogenesis and chondrocyte cell differentiation remains unclear. Here, we attempt to assay cell proliferation, the production of chondrogenic matrices indicated by alcian blue intensity, and the profile of a chondrogenic cell differentiation marker gene in a cell culture of the chondrogenic cell line ATDC5 with or without recombinant mouse GDF-5, -6, and -7. After 24h culture, the ATDC5 cell number was significantly decreased in the exogenous presence of each GDF compared with a control culture absent all GDFs. On the other hand, alcian blue staining of cell cultures after 2w culture showed significantly increased intensity compared with that of the control. The expression levels of the chondrogenic cell differentiation marker genes Sox9 and aggrecan were increased after 24h and 48h culture by all GDFs, but were significantly increased in the presence of GDF-5 compared with the presence of GDF-6 or -7. These findings suggest that GDF-5, -6, and -7 could all promote chondrogenic cell differentiation of ATDC5, but GDF-5 may induce chondrogenic genes more potently than GDF-6 and -7 do.

Chondrogenic differentiation of ATDC5 and hMSCs could be induced by a novel scaffold‐tricalcium phosphate‐collagen‐hyaluronan without any exogenous growth factorsin vitro

Application of chondrogenic growth factors is a routine strategy to induce chondrogenesis of hMSCs, but they have economic and safety problems in the long term. It is expected that scaffold material itself could play an important role in chondrogenesis of hMSCs. In this study we tested whether a novel tricalcium phosphate-collagen-hyaluronan scaffold (TCP-COL-HA) had inherent chondro-inductive capacity for chondrogenesis of both ATDC5 and hMSCs without any exogenous growth factors in vitro. hMSCs and ATDC5 were seeded onto TCP-COL-HA scaffolds and cultured in basal medium for 3 weeks to investigate whether the TCP-COL-HA scaffold itself had differentiation-inductive capacity in basal culture. With hMSCs-seeded scaffold in chondrogenic medium (including TGF-β1) as positive control, we then compared the chondrogenic induction of TCP-COL-HA in basal culture and in chondrogenic culture. The chondrogenic differentiation was evaluated by sulfated glycosaminoglycans (GAGs) quantification, type II collagen immunohistochemistry, and RT-PCR. Mechanical strength was evaluated by compression test and the cell death rate of hMSCs was assessed with TUNEL assay. The results showed TCP-COL-HA scaffold itself could efficiently induce chondrogenic differentiation of both ATDC5 and hMSCs after 3 weeks in basal culture. The accumulation of GAGs and the expression of chondrocyte marker genes were all significantly increased. In addition, hMSCs-seeded scaffold showed a significantly higher mechanical strength after 3 weeks in basal culture. The chondrogenic induction of TCP-COL-HA scaffolds in basal medium were almost similar to that in chondrogenic medium on hMSCs. The chondrogenesis-inducing capacity of TCP-COL-HA scaffold might help to improve cartilage tissue engineering with economic and safe benefits.

O-GlcNAc protein modification stimulates chondrogenesis in vitro and chondrocyte hypertrophy in mouse

Backgroundand objectives Chondrocyte differentiation that allows endocondral ossification sequentially includes cell proliferation, extracellular matrix synthesis, cellular hypertrophy, matrix mineralisation, vascular invasion and eventually apoptosis, allowing cartilage remodeling into bone. Most of these processes have been also associated to the development of osteoarthritis. Insulin ameliorate impaired bone growth and impaired bone healing both in vitro and in vivo, and is able to induce chondrocyte hypertrophy and growth plate chondrogenesis, although the specific molecular mechanisms are mostly unknown. Our aim was to investigate whether insulin-induced chondrocyte hypertrophy occurs through a modification in the amount of O-GlcNAc glycosylated proteins and in the expression of the key enzymes of this pathway: O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA). Furthermore, the authors studied if O-GlcNAc-accumulation per se was able to induce prehypertrophic chondrocyte differentiation both in vitro and in vivo. Methods Prechondrogenic ATDC5 cells were cultured in 5% FBS DMEM/F12. Insulin-induced differentiation was studied for 24 days. The accumulation of O-GlcNAcylated proteins was induced employing the OGA inhibitor Thiamet-G (TG). The gene expression of the differentiation markers collagen X (ColX), PTH receptor (PTHR), Indian hedgehog protein (IHH), Runx-2 and alkaline phosphatase (AP) was studied by real time-PCR. O-GlcNAcylation, OGT, OGA, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were studied by western-blot. The effect of the in vivo accumulation of O-GlcNAc proteins in the tibial growth plate was studied in 23-day old C57/bl mice that received 20mg/kg TG during 15 days. Results Insulin-induced differentiation paralleled with a gradual increase in the accumulation of O-GlcNacylated proteins that was appreciable since day 3, and with an increase in the expression of OGT and OGA. These increases occurred previously to any modification in the differentiation markers. O-GlcNAc accumulation per se induced by TG, in the absence of insulin, induced ATDC5 differentiation measured by the increase in the expression of differentiation markers. TG cells also induced an increase in the activity of MMP-1,-2 and -9, similar to that evoked by insulin. TG activated pERK, p-JNK and p-p38 in a similar extent to insulin. The administration of TG to C57/bl mice induced an accumulation of O-GlcNAcylated proteins in brain, liver, muscle and knee joint. TG induced a significant expansion in the growth plate height and in the hypertropic zone height, in comparison to untreated mice, without a significant modification of body weight. Conclusions TG induced the expression of chondrogenic markers in ATDC5 cells, and increased chondrogenic differentiation in vivo. Our results show that o-GlcNAc glycosylation has chondromodulating activity.

BMP-2 and BMP-7: differential regulation of chondrogenic differentiation

BMP-2 and BMP-7: differential regulation of chondrogenic differentiation

O-GlcNAc protein modification stimulates chondrogenesis in vitro and chondrocyte hypertrophy in mouse

Backgroundand objectives Chondrocyte differentiation that allows endocondral ossification sequentially includes cell proliferation, extracellular matrix synthesis, cellular hypertrophy, matrix mineralisation, vascular invasion and eventually apoptosis, allowing cartilage remodeling into bone. Most of these processes have been also associated to the development of osteoarthritis. Insulin ameliorate impaired bone growth and impaired bone healing both in vitro and in vivo, and is able to induce chondrocyte hypertrophy and growth plate chondrogenesis, although the specific molecular mechanisms are mostly unknown. Our aim was to investigate whether insulin-induced chondrocyte hypertrophy occurs through a modification in the amount of O-GlcNAc glycosylated proteins and in the expression of the key enzymes of this pathway: O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA). Furthermore, the authors studied if O-GlcNAc-accumulation per se was able to induce prehypertrophic chondrocyte differentiation both in vitro and in vivo. Methods Prechondrogenic ATDC5 cells were cultured in 5% FBS DMEM/F12. Insulin-induced differentiation was studied for 24 days. The accumulation of O-GlcNAcylated proteins was induced employing the OGA inhibitor Thiamet-G (TG). The gene expression of the differentiation markers collagen X (ColX), PTH receptor (PTHR), Indian hedgehog protein (IHH), Runx-2 and alkaline phosphatase (AP) was studied by real time-PCR. O-GlcNAcylation, OGT, OGA, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were studied by western-blot. The effect of the in vivo accumulation of O-GlcNAc proteins in the tibial growth plate was studied in 23-day old C57/bl mice that received 20mg/kg TG during 15 days. Results Insulin-induced differentiation paralleled with a gradual increase in the accumulation of O-GlcNacylated proteins that was appreciable since day 3, and with an increase in the expression of OGT and OGA. These increases occurred previously to any modification in the differentiation markers. O-GlcNAc accumulation per se induced by TG, in the absence of insulin, induced ATDC5 differentiation measured by the increase in the expression of differentiation markers. TG cells also induced an increase in the activity of MMP-1,-2 and -9, similar to that evoked by insulin. TG activated pERK, p-JNK and p-p38 in a similar extent to insulin. The administration of TG to C57/bl mice induced an accumulation of O-GlcNAcylated proteins in brain, liver, muscle and knee joint. TG induced a significant expansion in the growth plate height and in the hypertropic zone height, in comparison to untreated mice, without a significant modification of body weight. Conclusions TG induced the expression of chondrogenic markers in ATDC5 cells, and increased chondrogenic differentiation in vivo. Our results show that o-GlcNAc glycosylation has chondromodulating activity.

O-Glcnac protein modification stimulates chondrogenesis in vitro and chondrocyte hypertrophy in mouse

Backgroundand objectivesChondrocyte differentiation that allows endocondral ossification sequentially includes cell proliferation, extracellular matrix synthesis, cellular hypertrophy, matrix mineralisation, vascular invasion and eventually apoptosis, allowing cartilage remodeling into bone. Most of...