ATDC5 Chondrocytes Research Articles

Chitosan/collagen based biomimetic osteochondral tissue constructs: A growth factor-free approach

Tissue engineering approach offers alternative strategies to develop multi-layered/multi-component osteochondral mimetic constructs to meet the requirements of the heterogeneous and layered structure of native osteochondral tissue. Herein, an iterative overlaying process to fabricate a multi-layered scaffold with a gradient composition and layer specific structure have been developed by combining the natural extracellular matrix (ECM) components-chitosan, type I collagen, type II collagen, nanohydroxyapatite- of the osteochondral tissue in biomimetic compositions. Subchondral bone layer was prepared by using freeze-drying method to obtain 3D porous scaffolds. The calcified cartilage and cartilage layers were prepared by thermal gelation method in the hydrogel form. Osteochondral scaffolds fabricated by iterative overlaying of each distinct layer exhibited a porous, continuous gradient structure and supported cell proliferation in a co-culture of MC3T3-E1 preosteoblasts and ATDC5 chondrocytes. Histology and biochemical analysis showed enhanced extracellular matrix production and demonstrated collagen and glycosaminoglycan deposition. Expression of genes specific for bone, calcified cartilage and cartilage were improved in the osteochondral scaffold. Overall, these findings suggest that iterative overlaying of freeze-dried scaffolds and hydrogel matrices prepared by using ECM components in biomimetic ratios to fabricate gradient, multi-layered structures can be a promising strategy without the need for growth factors.

Laquinimod Mitigated IL-1β-Induced Impairment of the Cartilage Extracellular Matrix in Human ATDC5 Chondrocytes.

To date, a safe and reliable treatment of osteoarthritis (OA) has not yet been announced. Inflammatory response and degradation of the articular extracellular matrix (ECM) induced by IL-1β are important pathological characteristics of OA. Laquinimod is a quinoline-3-carboxamide and a novel oral immunomodulatory compound in clinical use. However, whether laquinimod has a beneficial effect in OA is not known. In our research, we found that laquinimod could ameliorate IL-1β-induced generation of ROS and improve mitochondrial function by increasing mitochondrial membrane potential (ΔΨm). Furthermore, treatment with laquinimod suppressed IL-1β-induced production of TNF-α and IL-6. Notably, laquinimod prevented the degradation of type II collagen by inhibiting MMP-3 and MMP-13. Meanwhile, the presence of laquinimod attenuated the reduction in aggrecan by mediating ADAMTS-4 and ADAMTS-5. Mechanistically, laquinimod ameliorated IL-1β-induced inflammation and degeneration of ECM by suppressing the activation of NF-κB. Taken together, our findings reveal that laquinimod possesses a beneficial effect against IL-1β insults in human chondrocytes, implying an important role of laquinimod in OA.

Fully physically crosslinked pectin-based hydrogel with high stretchability and toughness for biomedical application

Hydrogels derived from natural polymers have been extensively investigated in the biomedical field, while inherent brittleness and poor stability limit their applications. In this study, a tough pectin-Fe3+/poly (acrylamide-co-stearyl methacrylate) (P(AAm-co-SMA)) double physical crosslinking (DPC) network hydrogel is prepared using a three-step method. The first HPAAm network is formed via hydrophobic associations among the PSMA segment in P(AAm-co-SMA), and trivalent ions (Fe3+) crosslinked pectin network as the second network. Due to the reversibility of dual physical cross-linking structures, the pectin-Fe3+/HPAAm hydrogel exhibit excellent toughness (1.04–11.20 MJ m−3). In addition, the pectin-Fe3+/HPAAm DPC hydrogels have tunable mechanical properties (tensile strength: 0.97–1.61 MPa, elongation: 133–1346%, elastic modulus: 0.30–2.20 MPa) via adjusting the ratio of pectin network and HPAAm network. To explore their potential application in tissue engineering, ATDC5 chondrocytes were seeded on the prepared DPC hydrogels. Results suggest that the pectin-Fe3+/HPAAm DPC hydrogels can support the adhesion and proliferation of ATDC5, moreover, the ATDC5 cells can penetrate into the hydrogel. It is concluded that the prepared hydrogels exhibit potential application in the load-bearing tissue repair field.

LncRNA‑NR024118 overexpression reverses LPS‑induced inflammatory injury and apoptosis via NF‑κB/Nrf2 signaling in ATDC5 chondrocytes.

Osteoarthritis (OA) is one of the most prevalent types of chronic joint diseases. Chondrocytes survival is closely associated with the destruction of joints in patients with OA. Long noncoding RNAs (lncRNAs) serve a critical role in OA. However, to the best of our knowledge, the role of lncRNAs NR024118 in OA has not been examined. In the present study, the expression levels of NR024118 in lipopolysaccharide (LPS)-induced chondrocytes was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the apoptosis levels of cells was determined using flow cytometry. The levels of scavenged reactive oxygen species and expression levels of the antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and heme oxygenase-1 (HO-1) were measured using specialized detection kits. The expression of interleukin (IL)-1β, IL-6 and IL-18 were measured using ELISA. Expression of the cell apoptosis markers Bcl-2, Bax, Bcl-2-like protein 11, NF-κB, phosphorylated (p)-NF-κB inhibitor β (IκBβ), IκBβ, p-transcription factor p65 (p65) and p65, and nuclear factor erythroid-2 related factor 2 (Nrf2) signaling pathways-associated proteins, Nrf2, HO-1 and quinone oxidoreductase-1 were detected by western blot analysis and RT-qPCR. The results indicated that in ATDC5 cells, apoptosis, oxidative stress and inflammation were significantly increased and the expression level of NR024118 was significantly decreased by LPS-mediated induction. NR024118 overexpression significantly reversed the effects of LPS treatment in the ATDC5 cell line. In addition, the overexpression of NR024118 decreased NF-κB expression levels and activated the Nrf2 signaling pathways in LPS-induced ATDC5 cells. The present study demonstrated that NR024118 attenuated the effects of LPS-induction on ATDC5 cells. These results suggest that NR024118 may be a potential target for diagnosis and treatment of OA.

RETRACTED: Salidroside protects ATDC5 cells against lipopolysaccharide-induced injury through up-regulation of microRNA-145 in osteoarthritis

Osteoarthritis (OA) is a kind of degenerative disease characterized by the degeneration of the articular cartilage. Salidroside (SAL) is an active component of Rhodiola rosea L., which exhibits diverse pharmacological effects in different diseases. However, the effects of SAL on OA remain largely unclear. The study aimed to investigate the roles of SAL in lipopolysaccharides (LPS)-induced inflammatory injury in murine ATDC5 chondrocyte cells. LPS induced ATDC5 cell injury model was constructed by determining cell viability, apoptosis, apoptosis-associated factors as well as inflammatory cytokines expressions and concentrations. Then, the various concentrations of SAL were used to treat ATDC5 cells, and the effect of SAL on LPS-induce inflammatory injury was detected. After treatment with SAL, the expression level of miR-145 was measured by qRT-PCR. Subsequently, miR-145 inhibitor and corresponding control were transfected into ATDC5 cells to explore the influences of miR-145 in LPS-induce inflammatory injury. Besides, the key signaling pathways of NF-κB and p38MAPK were analyzed by using western blot. LPS inhibited cell viability, induced apoptosis, activated cleaved-caspase-3/-9 expression, as well as increased IL-6, MCP-1 and TNF-α expressions and secretions in ATDC5 cells. SAL significantly alleviated LPS-induced inflammatory injury. Meanwhile, the expression of miR-145 was up-regulated by SAL. The protective effect of SAL on LPS-induced injury was obviously reversed by miR-145 inhibition. Furthermore, SAL inactivated NF-κB and p38MAPK signaling pathways by regulating miR-145. These findings suggested that SAL could protect ATDC5 cells against LPS-induced injury via up-regulation of miR-145 in ATDC5 chondrocyte cells.

RETRACTED: LncRNA PMS2L2 protects ATDC5 chondrocytes against lipopolysaccharide-induced inflammatory injury by sponging miR-203

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Given the comments of Dr Elisabeth Bik regarding this article “… the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated”, the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Release of CXCL12 From Apoptotic Skeletal Cells Contributes to Bone Growth Defects Following Dexamethasone Therapy in Rats.

Dexamethasone (Dex) is known to cause significant bone growth impairment in childhood. Although previous studies have suggested roles of osteocyte apoptosis in the enhanced osteoclastic recruitment and local bone loss, whether it is so in the growing bone following Dex treatment requires to be established. The current study addressed the potential roles of chemokine CXCL12 in chondroclast/osteoclast recruitment and bone defects following Dex treatment. Significant apoptosis was observed in cultured mature ATDC5 chondrocytes and IDG-SW3 osteocytes after 48 hours of 10-6 M Dex treatment, and CXCL12 was identified to exhibit the most prominent induction in Dex-treated cells. Conditioned medium from the treated chondrocytes/osteocytes enhanced migration of RAW264.7 osteoclast precursor cells, which was significantly inhibited by the presence of the anti-CXCL12 neutralizing antibody. To investigate the roles of the induced CXCL12 in bone defects caused by Dex treatment, young rats were orally gavaged daily with saline or Dex at 1 mg/kg/day for 2 weeks, and received an intraperitoneal injection of anti-CXCL12 antibody or control IgG (1 mg/kg, three times per week). Aside from oxidative stress induction systemically, Dex treatment caused reductions in growth plate thickness, primary spongiosa height, and metaphysis trabecular bone volume, which are associated with induced chondrocyte/osteocyte apoptosis and enhanced chondroclast/osteoclast recruitment and osteoclastogenic differentiation potential. CXCL12 was induced in apoptotic growth plate chondrocytes and metaphyseal bone osteocytes. Anti-CXCL12 antibody supplementation considerably attenuated Dex-induced chondroclast/osteoclast recruitment and loss of growth plate cartilage and trabecular bone. CXCL12 neutralization did not affect bone marrow osteogenic potential, adiposity, and microvasculature. Thus, CXCL12 was identified as a potential molecular linker between Dex-induced skeletal cell apoptosis and chondroclastic/osteoclastic recruitment, as well as growth plate cartilage/bone loss, revealing a therapeutic potential of CXCL12 functional blockade in preventing bone growth defects during/after Dex treatment. © 2018 American Society for Bone and Mineral Research.

Comparison of botulinum toxin type A and Rho-inhibitor on ATDC5 chondrocyte

Knee osteoarthritis (OA) has an incidence rate of 25% per year and its responses to treatment are variable. The analgesic effect of intra-articular injection of botulinum toxin type A (BTX-A) has been observed in patients with knee OA. BTX-A might inhibit Rho GTPase by ADP-ribosylation of amino acid asparagine-41. Rho activity has a role in mediation of chondrocyte catabolic signaling pathways, and Rho/ROCK (Rho-associated coiled-coil containing kinase) activation induces articular cartilage degradation. In our unpublished data, chondroprotection of BTX-A was observed based on pathological findings in rat knee OA. ROCK I & II were found to be altered in chondrocytes treated with BTXA, with dose-dependent effect. This study aimed to compare the effect on chondrocyte of BTX-A and Rho-inhibitor. ATDC5 chondrocytes were compared based on cell phenotype and markers for chondrocyte differentiation and growth after administration of BTX-A, Rho-inhibitor (Y27632), and normal saline (control) to clarify the downstream signaling pathway. Reverse transcription-PCR (RT-PCR) showed that in ATDC5 chondrocyte cell culture, Y27632 and BTX-A elevated aggrecan on culture day 4 and day 7. On day 21, the level of collagen type X and MMP13 were lowered in Y27632 and BTX-A. ROCK I and II level were decreased on day 4 and 7 in all three groups, but were remarkably higher on day 14 and 21 in Y27632. On day 8, in ATDC5 cells not pretreated with insulin, decreased cell density, small and flat chondrocyte morphology were observed after addition of Y27632 and BTX-A, in comparison to control. BTX-A and Rho-inhibitor decreased ROCK I & II and increased aggrecan level during early stage, and decreased collagen type X and MMP13 during late stage. Potential dedifferentiation effect in BTX-A and Rho-inhibitor were observed as well. Rho-signaling pathway might play different roles during early and late stage in chondrocyte growth.

Mechanical Properties of Graphene Foam and Graphene Foam - Tissue Composites.

Graphene foam (GF), a 3-dimensional derivative of graphene, has received much attention recently for applications in tissue engineering due to its unique mechanical, electrical, and thermal properties. Although GF is an appealing material for cartilage tissue engineering, the mechanical properties of GF - tissue composites under dynamic compressive loads have not yet been reported. The objective of this study was to measure the elastic and viscoelastic properties of GF and GF-tissue composites under unconfined compression when quasi-static and dynamic loads are applied at strain magnitudes below 20%. The mechanical tests demonstrate a 46% increase in the elastic modulus and a 29% increase in the equilibrium modulus after 28-days of cell culture as compared to GF soaked in tissue culture medium for 24h. There was no significant difference in the amount of stress relaxation, however, the phase shift demonstrated a significant increase between pure GF and GF that had been soaked in tissue culture medium for 24h. Furthermore, we have shown that ATDC5 chondrocyte progenitor cells are viable on graphene foam and have identified the cellular contribution to the mechanical strength and viscoelastic properties of GF - tissue composites, with important implications for cartilage tissue engineering.

17β-Estradiol on the Expression of G-Protein Coupled Estrogen Receptor (GPER/GPR30) Mitophagy, and the PI3K/Akt Signaling Pathway in ATDC5 Chondrocytes In Vitro.

BackgroundOsteoarthritis is a progressive inflammatory joint disease resulting in damage to articular cartilage. G-protein coupled estrogen receptor (GPER/GPR30) activates cell signaling in response to 17β-estradiol, which can be blocked by the GPR30 agonist, G15, an analog of G-1. The aims of this study were to investigate the effects of 17β-estradiol on the expression of G-protein coupled estrogen receptor (GPER/GPR30) on mitophagy and the PI3K/Akt signaling pathway in ATDC5 chondrocytes in vitro.Material/MethodsCultured ATDC5 chondrocytes were treated with increasing concentrations of 17β-estradiol with and without G15, p38 inhibitor (SB203580), JNK inhibitor (SP600125), PI3K inhibitor (LY294002, S1737), and mTOR inhibitor (S1842). Expression of GPER/GPR30 and components of the PI3K/Akt pathway in cultured ATDC5 chondrocytes were detected by immunofluorescence (IF) staining, Western blot, and real-time polymerase chain reaction (RT-PCR). Transmission electron microscopy (TEM) and IF were used to detect mitophagosomes. Expression of LC-3, LAMP2, TOM20, Hsp60, p-Akt, p-mTOR, p-p38, and p-JNK was investigated by Western blot. Proliferation and viability of the ATDC5 chondrocytes were determined using BrdU and MTT assays.ResultsIn 17β-estradiol-treated ATDC5 chondrocytes, increased expression of GPER/GPR30 was found, but fewer mitophagosomes were observed, and decreased numbers of TOM20-positive granules were co-localized with decreased LAMP2 and increased expression levels of TOM20, Hsp60, p-Akt, and p-mTOR, and reduced expression of LC3-II, were found. In 17β-estradiol-treated ATDC5 chondrocytes, the proliferation and viability of the 17β-estradiol-treated ATDC5 chondrocytes were significantly elevated.ConclusionsTreatment with 17β-estradiol protected ATDC5 chondrocytes against mitophagy via the GPER/GPR30 and the PI3K/Akt signaling pathway.

Evidences for a New Role of miR-214 in Chondrogenesis

miR-214 is known to play a role in mammalian skeletal development through inhibition of osteogenesis and stimulation of osteoclastogenesis, but data regarding other vertebrates, as well as a possible role in chondrogenesis, remain unknown. Here, we show that miR-214 expression is detected in bone and cartilage of zebrafish skeleton, and is downregulated during murine ATDC5 chondrocyte differentiation. Additionally, we observed a conservation of the transcriptional regulation of miR-214 primary transcript Dnm3os in vertebrates, being regulated by Ets1 in ATDC5 chondrogenic cells. Moreover, overexpression of miR-214 in vitro and in vivo mitigated chondrocyte differentiation probably by targeting activating transcription factor 4 (Atf4). Indeed, miR-214 overexpression in vivo hampered cranial cartilage formation of zebrafish and coincided with downregulation of atf4 and of the key chondrogenic players sox9 and col2a1. We show that miR-214 overexpression exerts a negative role in chondrogenesis by impacting on chondrocyte differentiation possibly through conserved mechanisms.

LncRNA GAS5 Overexpression Reverses LPS-Induced Inflammatory Injury and Apoptosis Through Up-Regulating KLF2 Expression in ATDC5 Chondrocytes

Background/Aims: Osteoarthritis (OA) is the most frequently occurring joint disease and characterized by degeneration of cartilage. As the unique cell type in cartilage, chondrocytes play a crucial role during OA. Our study explored the influence of long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) on lipopolysaccharides (LPS)-induced injury in ATDC5 cells. Methods: Cell viability, apoptosis and expression of inflammatory cytokines were all assessed to evaluate LPS-induce inflammatory injury. Expression of GAS5 in LPS-induced cells was evaluated by qRT-PCR. After cell transfection, effect of abnormally expressed GAS5 on LPS-induced inflammatory injury was determined. Then, the possible target of GAS5 was screened by bioinformatics and verified by qRT-PCR and luciferase activity assay. Together, whether aberrant expression of target gene affected the modulation of GAS5 in LPS-induced inflammatory injury was also assessed. Finally, the influences of aberrant expressed Kruppel-like factor 2 (KLF2) on nuclear factor κB (NF-κB) and Notch pathways were detected by Western blot analysis. Results: LPS reduced cell viability and promoted cell apoptosis and secretion of inflammatory cytokines, along with down-regulation of GAS5. LPS-induced injury was alleviated by GAS5 overexpression while was exacerbated by GAS5 silence. KLF2 was predicted and verified as a target of GAS5, and GAS5 functioned through regulating expression of KLF2. Besides, aberrant expression of KLF2 regulated expressions of key kinases involved in the NF-κB and Notch pathways. Conclusion: GAS5 might ameliorate LPS-induced inflammatory injury in ATDC5 chondrocytes by inhibiting the NF-κB and Notch signaling pathways.

Cells may feel a hard substrate even on a grafted layer of soft hydrogel.

Introducing or grafting molecules onto biomaterial surfaces to regulate cell destination via biophysical cues is one of the important steps for biomaterial design in tissue engineering. Understanding how cells feel the substrate makes it easier to learn the mechanism behind cell-material interaction. In this study, on a glass substrate, we constructed poly-phenoxyethyl methacrylate (PHEMA) brushes having different lengths via a surface-induced atom transfer radical polymerization (SI-ATRP) method. FTIR-ATR and XPS tests of the formed polymer brushes indicate that these brushes have characteristic chemical structures of PHEMA; the polymer brush length revealed by the AFM tests increases linearly with reaction time. Cell lines of BMSCs, ATDC5, and human chondrocytes (HC) were cultured on these substrates to evaluate proliferation, adhesion, and differentiation. Our results demonstrated that the cells cultured on the substrates with short PHEMA brushes developed a spread morphology and organized actin fibers as compared to the cells cultured on those with long brushes. Different cell lines showed different responses depending on the PHEMA brush length. Cells cultured on long PHEMA brushes displayed a more rounded shape, higher gene expression of FAK and integrin, and lower gene expression of NCAM and N-cadherin as compared to those, especially ATDC5 cells, cultured on short PHEMA brushes. On PHEMA brushes with a long length, the cell lines express higher cartilage-specific genes including Sox9 and Col2 and GAG in ECM. The results suggest that polymer brushes having different lengths may interfere with the behavior of the cells cultured on them.

Runx2 mediated Induction of Novel Targets ST2 and Runx3 Leads to Cooperative Regulation of Hypertrophic Differentiation in ATDC5 Chondrocytes

Knowledge concerning expression and function of Suppression of Tumorigenicity 2 (ST2) in chondrocytes is at present, limited. Analysis of murine growth plates and ATDC5 chondrocytes indicated peak expression of the ST2 transmembrane receptor (ST2L) and soluble (sST2) isoforms during the hypertrophic differentiation concomitant with the expression of the hypertrophic markers Collagen X (Col X), Runx2 and MMP-13. Gain- and loss-of-function experiments in ATDC5 and primary human growth plate chondrocytes (PHCs), confirmed regulation of ST2 by the key transcription factor Runx2, indicating ST2 to be a novel Runx2 target. ST2 knock-out mice (ST2−/−) exhibited noticeable hypertrophic zone (HZ) reduction in murine growth plates, accompanied by lower expression of Col X and Osteocalcin (OSC) compared to wild-type (WT) mice. Likewise, ST2 knockdown resulted in decreased Col X expression and downregulation of OSC and Vascular Endothelial Growth Factor (VEGF) in ATDC5 cells. The ST2 suppression was also associated with upregulation of the proliferative stage markers Sox9 and Collagen II (Col II), indicating ST2 to be a new regulator of ATDC5 chondrocyte differentiation. Runx3 was, furthermore, identified as a novel Runx2 target in chondrocytes. This study suggests that Runx2 mediates ST2 and Runx3 induction to cooperatively regulate hypertrophic differentiation of ATDC5 chondrocytes.

High hydrostatic pressure induces pro-osteoarthritic changes in cartilage precursor cells: A transcriptome analysis.

Due to the high water content of cartilage, hydrostatic pressure is likely one of the main physical stimuli sensed by chondrocytes. Whereas, in the physiological range (0 to around 10 MPa), hydrostatic pressure exerts mostly pro-chondrogenic effects in chondrocyte models, excessive pressures have been reported to induce detrimental effects on cartilage, such as increased apoptosis and inflammation, and decreased cartilage marker expression. Though some genes modulated by high pressure have been identified, the effects of high pressure on the global gene expression pattern have still not been investigated. In this study, using microarray technology and real-time PCR validation, we analyzed the transcriptome of ATDC5 chondrocyte progenitors submitted to a continuous pressure of 25 MPa for up to 24 h. Several hundreds of genes were found to be modulated by pressure, including some not previously known to be mechano-sensitive. High pressure markedly increased the expression of stress-related genes, apoptosis-related genes and decreased that of cartilage matrix genes. Furthermore, a large set of genes involved in the progression of osteoarthritis were also induced by high pressure, suggesting that hydrostatic pressure could partly mimic in vitro some of the genetic alterations occurring in osteoarthritis.

Effect of Various Ratios of Co-Cultured ATDC5 Cells and Chondrocytes on the Expression of Cartilaginous Phenotype in Microcavitary Alginate Hydrogel.

The present study introduced a direct co-culture of mouse ATDC5 cells and primary porcine chondrocytes into a microcavitary hydrogel, which possessed advantages in promoting the growth of chondrocytes and retaining the phenotype. These two types of cells were encapsulated with gelatin microspheres in alginate hydrogels in either of the three ratios (3:1, 1:1, or 1:3 of ATDC5 cells to chondrocytes) and cultured in chondrogenic medium for 28 days. Simultaneously, the single encapsulation of ATDC5 cells or chondrocytes was set as a control. Cell Counting Kit-8 (CCK-8), real-time PCR, and immunohistochemistry staining were used to evaluate the effect of various ratios of co-cultured ATDC5 cells and chondrocytes on the expression of the cartilaginous phenotype. The CCK-8 data indicated that the ratio of 3:1 group had an outstanding ability of cell growth. The other results demonstrated that higher the ATDC5 ratios and longer the culture duration, greater the expression of cartilage-specific genes (including type II collagen and aggrecan) and more the synthesized cartilaginous extracellular matrix. Also, the Western blot analysis suggested that p44/42 MAP Kinase was involved in cell proliferation. However, due to the direct co-culture of the two cell types, the underlying mechanism necessitates further investigation. Overall, the co-culture system in microcavitary hydrogel improved the effect of chondrogenesis and exhibited promising strategy for cartilage tissue engineering therapies. J. Cell. Biochem. 118: 3607-3615, 2017. © 2017 Wiley Periodicals, Inc.

Hypoxia inducible factor-1[alpha] directly induces the expression of receptor activator of nuclear factor-[kappa]B ligand in ATDC5 chondrocyte

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

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.

Oxygen Tension Affects Lubricin Expression in Chondrocytes

We assessed the effects of oxygen tension on lubricin expression in bovine chondrocytes and cartilage explants and a role for hypoxia-inducible transcription factor (HIF)-1α in regulating lubricin expression was investigated using a murine chondroprogenitor cell line, ATDC5, and bovine chondrocytes isolated from superficial and middle/deep zones of femoral cartilage. ATDC5 cells and bovine chondrocytes were cultured in micromass under different oxygen tensions (21%, 5%, and 1%). ATDC5 cells and middle/deep zone chondrocytes that initially had low lubricin expression levels were also cultured with or without transforming growth factor (TGF)-β1. Quantitative reverse transcription (RT)-PCR was used to determine lubricin and chondrogenic marker gene mRNA levels and immunohistochemistry was used to assess lubricin protein expression. Explant cartilage plugs cultured under different oxygen tensions were also subjected to immunohistological analysis for lubricin. HIF-1α gene silencing was achieved by electroporatic transfer into ATDC5 cells. A low oxygen tension reduced lubricin gene expression levels in bovine superficial chondrocytes, TGF-β1-treated middle/deep zone chondrocytes, and TGF-β1-treated ATDC5 cells. Lubricin expression in explant cartilage was also suppressed under hypoxia. HIF-1α gene silencing in ATDC5 cells attenuated the lubricin expression response to the oxygen tension. These results corroborate with previous studies that the oxygen tension regulates lubricin gene expression and suggest that HIF-1α plays an important role in this regulation. The normal distribution of lubricin in articular cartilage may be due to the hypoxic oxygen environment of cartilage as it is an avascular tissue. An oxygen tension gradient may be a key factor for engineering cartilage tissue with a layered morphology.

Human Immunodeficiency Virus Type 1 Enhancer-binding Protein 3 Is Essential for the Expression of Asparagine-linked Glycosylation 2 in the Regulation of Osteoblast and Chondrocyte Differentiation

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.