Influence of the interaction between Ac­SDKP and Ang II on the pathogenesis and development of silicotic fibrosis.

N­acetyl­seryl­aspartyl­lysyl­proline (Ac­SDKP) is a natural tetrapeptide that is released from thymosin ß4 by prolyl oligopeptides. It is hydrolyzed by the key enzyme of the renin­angiotensin system, angiotensin­converting enzyme (ACE). The aim of the present study was to investigate the alterations in Ac­SDKP and the ACE/angiotensin II (Ang II)/angiotensin II type 1 (AT1) receptor axis and its impact on the pathogenesis and development of silicotic fibrosis. For in vivo studies, a HOPE MED 8050 exposure control apparatus was used to establish different stages of silicosis in a rat model treated with Ac­SDKP. For in vitro studies, cultured primary lung fibroblasts were induced to differentiate into myofibroblasts by Ang II, and were pretreated with Ac­SDKP and valsartan. The results of the present study revealed that, during silicosis development, ACE/Ang II/AT1 expression in local lung tissues increased, whereas that of Ac­SDKP decreased. Ac­SDKP and the ACE/AT1/Ang II axis were inversely altered in the development of silicotic fibrosis. Ac­SDKP treatment had an anti­fibrotic effect in vivo. Compared with the silicosis group, the expression of α­smooth muscle actin (α­SMA), Collagen (Col) I, Fibronectin (Fn) and AT1 were significantly downregulated, whereas matrix metalloproteinase­1 (MMP­1) expression and the MMP­1/tissue inhibitor of metalloproteinases­1 (TIMP­1) ratio was increased in the Ac­SDKP treatment group. In vitro, pre­treatment with Ac­SDKP or valsartan attenuated the expression of α­SMA, Col I, Fn and AT1 in Ang II­induced fibroblasts. In addition, MMP­1 expression and the MMP­1/TIMP­1 ratio were significantly higher in Ac­SDKP and valsartan pre­treatment groups compared with the Ang II group. In conclusion, the results of the present study suggest that an imbalance between Ac­SDKP and ACE/Ang II/AT1 molecules promotes the development of silicosis and that Ac­SDKP protects against silicotic fibrosis by inhibiting Ang II­induced myofibroblast differentiation and extracellular matrix production.

Effect of liposome­mediated HSP27 transfection on collagen synthesis in alveolar type II epithelial cells.

To investigate the effect of liposome Lipofectamine® 2000­mediated HSP27 plasmid transfection in A549 human alveolar type II epithelial cell line on collagen synthesis during transforming growth factor­ß1 (TGF­ß1)­induced type II epithelial cell transition to myofibroblasts. Cells were transfected with varying ratios of the Lipofectamine® 2000­mediated heat shock protein 27 (HSP27) plasmid and the transfection efficiency was determined using flow cytometry. The maximum transfection efficacy was confirmed by laser confocal microscopy. HSP gene expression and the most efficient HSP27 plasmid were determined using reverse transcription­quantitative polymerase chain reaction. Western blot analysis was used to examine HSP27 and collagen expression levels. With a transfection efficiency of 83%, the 8 µg:20 µl ratio of liposome: Plasmid had the highest transfection levels. Among the four different interference sequences in the HSP27 plasmid, the D sequence had the highest interference effect with 70% silencing of the HSP27 gene. The expression of type I and III collagen in TGF­ß1­induced transition of A549 human alveolar type II epithelial cell line to myofibroblasts was significantly downregulated by the successful transfection with HSP27­interfering plasmid. The expression of type I and III collagen in the TGF­ß1­induced transition of A549 cells to myofibroblasts was significantly downregulated by transfection of A549 cells with HSP27 plasmid D­interfering sequence and optimal ratio of Lipofectamine® 2000 and HSP27 plasmid.

Protective effects of oleanolic acid on oxidative stress and the expression of cytokines and collagen by the AKT/NF­κB pathway in silicotic rats.

Oleanolic acid (OA), a natural pentacyclic triterpenoid, has been reported to have several benefits and medicinal properties. However, its protective effects against silica­induced lung injury and fibrosis remain to be elucidated. The aim of the present study was to investigate the effects of OA on oxidative stress, and the expression of cytokines and collagen in silicotic rats. Male rats were induced by intratracheal instillation of silicosis (250 mg/kg), with the exception of the control group (NS). The rats in the OA group were intragastrically administered with OA (60 mg/kg/d). The rats in the solvent control group were gavaged daily with 0.6% sodium carboxymethyl cellulose (10 ml/kg) solution for 56 consecutive days. The data showed that OA significantly attenuated the extent of silicosis fibrosis by histopathologic analysis of the lung tissues. In addition, oxidative stress activated by silica exposure, as evidenced by increasing of malondialdehyde content, and activities of superoxide dismutase and glutathione peroxidase in the lung, was regulated by treatment with OA. Furthermore, enzyme­linked immunosorbent assay analysis showed that OA significantly decreased the levels of tumor necrosis factor­α and transforming growth factor­ß1. Immunohistochemistry analysis showed that OA significantly decreased collagen types I and III. In investigating the mechanisms underlying the action of OA, it was found that OA decreased the level of phosphorylated AKT1, which in turn inactivated the transcriptional of nuclear factor (NF)­κB in the development and progress of silicosis. In conclusion, these results suggested that the protective effects of OA were due, at least in part, to its anti­oxidant activity and its ability to decrease the expression of cytokines and collagen by modulating the AKT/NF­κB pathway.

ELF3 modulates type II collagen gene (COL2A1) transcription in chondrocytes by inhibiting SOX9-CBP/p300-driven histone acetyltransferase activity.

AIM: We showed previously that E74-like factor 3 (ELF3) protein levels are increased in osteoarthritic (OA) cartilage, that ELF3 accounts for inflammatory cytokine-driven MMP13 gene expression, and that, upon induction by interleukin-1ß, ELF3 binds to the COL2A1 promoter and suppresses its activity in chondrocytes. Here, we aimed to further investigate the mechanism/s by which ELF3 represses COL2A1 transcription in chondrocytes. METHODS AND RESULTS: We report that ELF3 inhibits Sox9-driven COL2A1 promoter activity by interfering with the activator functions of CBP/300 and Sox9. Co-transfection of the pGL2B-COL2A1 (-577/+3428 bp) reporter construct with Sox9 and with Sox5 and/or Sox6 increased COL2A1 promoter activity, and ELF3 overexpression significantly reduced the promoter transactivation. Co-transfection of ELF3 with the pLuc 4x48 enhancer construct, containing the 89-bp COL2A1 promoter and lacking the previously defined ELF3 binding sites, decreased both basal and Sox9-driven promoter activity. Co-transfection of ELF3 with a Gal4 reporter construct also inhibited Gal4-Sox9-driven transactivation, suggesting that ELF3 directly interacts with Sox9. Using truncated Sox9 fragments, we found that ELF3 interacts directly with the HMG domain of Sox9. Importantly, overexpression of ELF3 significantly decreased Sox9/CBP-dependent HAT activity. Finally, we show evidence that increased ELF3 mRNA expression in OA chondrocytes correlates with hypermethylation of the proximal promoter, suggesting that ELF3 transcription is subjected to epigenetic control in OA disease. CONCLUSION: Our results highlight the contribution of ELF3 to transcriptional regulation of COL2A1 and its potential role in OA disease, and uncover epigenetic mechanisms at play in the regulation of ELF3 and its downstream targets in articular chondrocytes.

Chemical vapor deposition of thin crystals of layered semiconductor SnS2 for fast photodetection application.

Layered two-dimensional (2D) semiconductors, such as MoS(2) and SnS(2), have been receiving intensive attention due to their technological importance for the next-generation electronic/photonic applications. We report a novel approach to the controlled synthesis of thin crystal arrays of SnS(2) at predefined locations on chip by chemical vapor deposition with seed engineering and have demonstrated their application as fast photodetectors with photocurrent response time ∼ 5 µs. This opens a pathway for the large-scale production of layered 2D semiconductor devices, important for applications in integrated nanoelectronic/photonic systems.

[Regulation of ursolic acid on TNF-alpha and collagen in silicotic rats].

OBJECTIVE: To explore the regulation of ursolic acid on the expressions of TNF-alpha and collagen on Silicotic Rats. METHODS: Seventy-five Wistar rats (class SPF) were divided into there groups according to the randomized block design, namely, control, model, ursolic acid groups with twenty-five rats in each group. SiO2 powders (250 mg/kg) were douched in the trachea of rat to make the silicotic model in model and ursolic acid groups. Ursolic acid (40 mg/kg) was injected into stomach cavity in ursolic acid group from the second day of SiO2, while the rats in control group were given sodium chloride in the same condition for 28 consecutive days. All rats were put to death on the 7th,14th and 28th day. TNF-alpha contents in serum were detected by ELISA. Total collagen contents in lung tissue were determined by hydroxyproline kits. Collagen I and III in lung tissue were investigated by western blot technique. RESULTS: After four weeks of intervention, the contents of TNF-alpha in serum of the model group had rised, showing statistically significant difference in each time compared to those of the control group (P < 0.05). Ursolic acid had depressant effect on the contents of TNF-alpha (P < 0.05). Compared with control group, the content of total collagen was significantly improved in model group (P < 0.05). However, ursolic acid depressed the expression of total collagen protein compared to those of the model group (P < 0.05). The expression levels of collagen I and III in ursolic acid and model groups were similar with the total collagen. CONCLUSIONS: Ursolic acid could decrease the expressions of TNF-alpha and collagen in the process of silicosis fibrosis.

Mice deleted for GPAT3 have reduced GPAT activity in white adipose tissue and altered energy and cholesterol homeostasis in diet-induced obesity.

Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first step in the synthesis of glycerolipids and glycerophospholipids. Microsomal GPAT, the major GPAT activity, is encoded by at least two closely related genes, GPAT3 and GPAT4. To investigate the in vivo functions of GPAT3, we generated Gpat3-deficient (Gpat3(-/-)) mice. Total GPAT activity in white adipose tissue of Gpat3(-/-) mice was reduced by 80%, suggesting that GPAT3 is the predominant GPAT in this tissue. In liver, GPAT3 deletion had no impact on total GPAT activity but resulted in a 30% reduction in N-ethylmaleimide-sensitive GPAT activity. The Gpat3(-/-) mice were viable and fertile and exhibited no obvious metabolic abnormalities on standard laboratory chow. However, when fed a high-fat diet, female Gpat3(-/-) mice showed decreased body weight gain and adiposity and increased energy expenditure. Increased energy expenditure was also observed in male Gpat3(-/-) mice, although it was not accompanied by a significant change in body weight. GPAT3 deficiency lowered fed, but not fasted, glucose levels and tended to improve glucose tolerance in diet-induced obese male and female mice. On a high-fat diet, Gpat3(-/-) mice had enlarged livers and displayed a dysregulation in cholesterol metabolism. These data establish GPAT3 as the primary GPAT in white adipose tissue and reveal an important role of the enzyme in regulating energy, glucose, and lipid homeostasis.

[Effect of quercetin on the expression of TGF-beta1 in human embryonic lung fibroblasts activated by the silicotic alveolar macrophages].

OBJECTIVE: To explore the effect s of quercetin (Que) intraperitoneal pulmonary fibrosis. METHODS: The silicotic alveolar macrophages were collected by bronchoalveolar lavage andincubated in vitro in the DMEM medium containing SiO2 (50 microg/ml) and DMEM medium without SiO2 for 18h. Then the AM supernatant incubated for 18h was collected and with quercetins (10, 20 and 40 micromol/L) as stimulus supernatant. HELFs were isolated by organize paste block method, and incubated with stimulus supernatants. The proliferation in the HELF was detected with MTT method. The expressions of TGF-beta1 in the HELF are detected with the immunocytochemistry method. RESULTS: (1) Three groups of Que could inhibit the proliferation of HELF, and more concentration, inhibition is stronger. Three groups of Que compared with SiO2 + AM group (0.620 +/- 0.096), Que20 group (0.406 +/- 0.070) and Que40 group (0.334 +/- 0.050) compared with AM group (0.490 +/- 0.064), differences were significant (P < 0.01). (2) Three groups of Que could reduce the expression of TGF-beta1 in HELF, and compared with SiO2 + AM group (0.542 +/- 0.095) differences were significant (P < 0.05). Three groups of Que compared with AM group (0.394 +/- 0.109) and blank control group (0.314 +/- 0. 066) differences were not significant. CONCLUSIONS: Quercetin could prevent and treat pulmonary fibrosis stimulated by silicon dioxide.

High on/off ratio field effect transistors based on exfoliated crystalline SnS2 nano-membranes.

We report the implementation of field effect transistors based on exfoliated nano-membranes of a layered two-dimensional semiconductor SnS(2), which exhibit an on/off ratio exceeding 2 × 10(6) and a carrier mobility of ∼1 cm(2) V(-1) s(-1). The results demonstrate the great potential of SnS(2), a layered semiconductor with finite band gap, as the building block for future nanoelectronic applications complementary to graphene-based materials with zero or small band gaps.

Observation of multiple superconducting gaps in Fe1+yTe1-xSex via a nanoscale approach to point-contact spectroscopy.

We report a novel experimental approach to point-contact Andreev reflection spectroscopy with diagnostic capability via a unique design for nanoscale normal metal/superconductor devices with excellent thermomechanical stability, and have employed this method to unveil the existence of two superconducting energy gaps in iron chalcogenide Fe(1+y)Te(1-x)Se(x), which is crucial for understanding its pairing mechanism. This work opens up new opportunities to study gap structures in superconductors and elemental excitations in solids.

AB-stacked multilayer graphene synthesized via chemical vapor deposition: a characterization by hot carrier transport.

We report the synthesis of AB-stacked multilayer graphene via ambient pressure chemical vapor deposition on Cu foils and demonstrate a method to construct suspended multilayer graphene devices. In four-terminal geometry, such devices were characterized by hot carrier transport at temperatures down to 240 mK and in magnetic fields up to 14 T. The differential conductance (dI/dV) shows a characteristic dip at longitudinal voltage bias V = 0 at low temperatures, indicating the presence of hot electron effect due to a weak electron-phonon coupling. Under magnetic fields, the magnitude of the dI/dV dip diminishes through the enhanced intra-Landau level cyclotron phonon scattering. Our results provide new perspectives in obtaining and understanding AB-stacked multilayer graphene, important for future graphene-based applications.

E74-like factor 3 (ELF3) impacts on matrix metalloproteinase 13 (MMP13) transcriptional control in articular chondrocytes under proinflammatory stress.

Matrix metalloproteinase (MMP)-13 has a pivotal, rate-limiting function in cartilage remodeling and degradation due to its specificity for cleaving type II collagen. The proximal MMP13 promoter contains evolutionarily conserved E26 transformation-specific sequence binding sites that are closely flanked by AP-1 and Runx2 binding motifs, and interplay among these and other factors has been implicated in regulation by stress and inflammatory signals. Here we report that ELF3 directly controls MMP13 promoter activity by targeting an E26 transformation-specific sequence binding site at position -78 bp and by cooperating with AP-1. In addition, ELF3 binding to the proximal MMP13 promoter is enhanced by IL-1ß stimulation in chondrocytes, and the IL-1ß-induced MMP13 expression is inhibited in primary human chondrocytes by siRNA-ELF3 knockdown and in chondrocytes from Elf3(-/-) mice. Further, we found that MEK/ERK signaling enhances ELF3-driven MMP13 transactivation and is required for IL-1ß-induced ELF3 binding to the MMP13 promoter, as assessed by chromatin immunoprecipitation. Finally, we show that enhanced levels of ELF3 co-localize with MMP13 protein and activity in human osteoarthritic cartilage. These studies define a novel role for ELF3 as a procatabolic factor that may contribute to cartilage remodeling and degradation by regulating MMP13 gene transcription.

Targeting Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) with small molecule inhibitors for the treatment of metabolic diseases.

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is one of two known DGAT enzymes that catalyze the final step in triglyceride synthesis. Findings from genetically modified mice as well as pharmacological studies suggest that inhibition of DGAT1 is a promising strategy for the treatment of obesity and type 2 diabetes. Here we characterize a tool DGAT1 inhibitor compound, T863. We found that T863 is a potent inhibitor for both human and mouse DGAT1 in vitro, which acts on the acyl-CoA binding site of DGAT1 and inhibits DGAT1-mediated triacylglycerol formation in cells. In an acute lipid challenge model, oral administration of T863 significantly delayed fat absorption and resulted in lipid accumulation in the distal small intestine of mice, mimicking the effects of genetic ablation of DGAT1. In diet-induced obese mice, oral administration of T863 for 2 weeks caused weight loss, reduction in serum and liver triglycerides, and improved insulin sensitivity. In addition to the expected triglyceride-lowering activity, T863 also lowered serum cholesterol. Hepatic IRS2 protein was dramatically up-regulated in mice treated with T863, possibly contributing to improved insulin sensitivity. In differentiated 3T3-L1 adipocytes, T863 enhanced insulin-stimulated glucose uptake, suggesting a possible role for adipocytes to improve insulin sensitivity upon DGAT1 inhibition. These results reveal novel mechanistic insights into the insulin-sensitizing effects of DGAT1 inhibition in mouse models. Taken together, our study provides a comprehensive evaluation of a small molecule inhibitor for DGAT1 and suggests that pharmacological inhibition of DGAT1 holds promise in treating diverse metabolic disorders.

[The effect of extracellular signal-regulated kinase 1/2 signaling pathway on the expression of transforming growth factor-beta1 in lung fibroblast activated by silicon dioxide].

OBJECTIVE: To investigate the effect and regulation of extracellular signal-regulated kinase (ERK1/2) signaling pathway on the expression of transforming growth factor-beta1 (TGF-beta1) in human embryonic lung fibroblasts induced by SiO2. METHODS: Human alveolar macrophages were collected from a silicotic patient by bronchoalveolar lavage and in the presence or absence of SiO2 (50 ug/ml) exposition for 18h, and then the conditioned supernatants were used to incubate HELF. The expressions of TGF-beta1, of the HELF acted with the conditioned AM supernatant fluid were detected with the immunocytochemistry method after treatment with PD98059 of inhibitor of ERK. RESULTS: The expression of TGF-beta1 in HELF of the SiO2 treatment group (OD value is 0.322 7 +/- 0.023 8) exceed blank group (OD value is 0.163 7 +/- 0.019 6) and AM control group (OD value is 0.240 6 +/- 0.022 5) by the immunocytochemistry method. But the expression of TGF-beta1 had reduction in some extent in the PD98059 intervention group (OD value is 0.271 1 +/- 0.022 9). The values were statistically different (P < 0.05). CONCLUSION: ERK inhibitor PD98059 have inhibition effect on the expression of transforming growth factor-beta1 and expression of cytokine of human embryonic lung fibroblasts stimulated by SiO2. The study indicate that the proliferation and collagen production of HELF activated by SiO2 are mediated by ERK/MAPK signal pathway in some extent. PD98059 may antagonizes silica-induced lung fibrosis by inhibiting the expression of transforming growth factor-beta1.

[Effect of ERK1/2 signal pathway on the proliferation of lung fibroblast activated by SiO2].

OBJECTIVE: To observe the effect of ERK1/2 signal pathway activated by SiO2 in the proliferation of human embryonic lung fibroblast mediated by silicotic alveolar macrophages. METHOD: The alveolar macrophages (AM) harvested from silicotic sufferers by bronchoalveolar lavage (BAL) were interacted with SiO2 suspension once more. HELF, pretreated with the inhibitor PD98059 (50 µmol/L) for 1 hour, were stimulated by conditional supernatant fluid of silicotic sufferers. The experimentation have been classificated four group: blank group, AM control group, SiO2 treatment group, PD98059 intervention group. The proliferation activity and expressions of Phospho-ERK1/2 of lung fibroblast activated by AM supernatant fluids of silicotic are detected with the MTT assay, flow cytometry and Western blot method after being pretreatmented with PD98059. RESULT: The A values of cell proliferation in SiO2 treatment group and AM control group are 2.6 and 2.0 times that of blank group, in which the difference was statistically significant (P < 0.05). Comparing with SiO2 treatment group, the A values of every concentrations of PD98059 intervention group decreased with a dose-response relationship, after 10, 25 and 50 µmol/L PD98059 intervention. The 25 and 50 µmol/L PD98059 intervention group were 72.1% and 48.5% of SiO2 treatment group, which the difference is statistic (P < 0.05). The expression of phospho-ERK1/2 in SiO2 treatment group was up, which appeared in 15 min and apparent activated in 30 min (A value is 0.4653 ± 0.0265), and then still in the higher state afterwards declined after 60 min. In addition to 15 min, the expression of phospho-ERK1/2 protein in SiO2 treatment group at each time point are 1.25, 1.23, 1.25 times over the same period AM control group respectively, the differences were statistically significant (P < 0.05). CONCLUSION: The silicotic supernatant of alveolar macrophages have promote proliferation of HELF and activation of ERK1/2, which may involve in the development of silicosis pathogenesis by ERK1/2 signal pathway.

[Effect of p38MAPK on proliferation in human embryonic lung fibroblasts in vitro].

OBJECTIVE: To study the proliferation effect of the AM supernatant incubated activation of p38 mitogen activated protein kinases (p38MAPK) signal transduction pathway in human embryonic lung fibroblasts, and to participate in the development of fibrosis in silicosis. METHODS: The silicotic alveolar macrophages were collected by bronchoalveolar lavage and incubated in vitro in the DMEM medium containing SiO2 (50 µg/ml) and DMEM medium without SiO2 for 18 h. Then the AM supernatant incubated for 18 h was collected. HELFs were isolated by organize paste block method, and incubated with AM supernatants. HELFs were divided into four groups: blank control groups, AM groups, SiO2 + AM groups, SB203580 + SiO2 + AM groups. The proliferation in the HELF was detected with MTT method and Flow cytometry. RESULTS: The proliferation in the HELF acted with the conditioned AM supernatant fluid were more than blank control groups, AM groups and SB203580 + SiO2 + AM groups [average optical density: (0.48 ± 0.03) vs (0.29 ± 0.01), (0.38 ± 0.02), (0.33 ± 0.03)], the values with MTT method were statistically different (P < 0.05); Proliferous index with flow cytometry in SiO2 + AM groups (18.12 ± 0.82) was bigger than blank control groups (9.24 ± 0.48), AM groups (14.76 ± 0.43) and SB203580 + SiO2 + AM groups (11.71 ± 0.70) and the values were statistically different(P < 0.05). CONCLUSIONS: The AM supernatant stimulated by silicon dioxide can accelerate the proliferation in the HELF by activation of p38MAPK signal transduction pathway.

Differential expression of GADD45beta in normal and osteoarthritic cartilage: potential role in homeostasis of articular chondrocytes.

OBJECTIVE: Our previous study suggested that growth arrest and DNA damage-inducible protein 45beta (GADD45beta) prolonged the survival of hypertrophic chondrocytes in the developing mouse embryo. This study was undertaken, therefore, to investigate whether GADD45beta plays a role in adult articular cartilage. METHODS: Gene expression profiles of cartilage from patients with late-stage osteoarthritis (OA) were compared with those from patients with early OA and normal controls in 2 separate microarray analyses. Histologic features of cartilage were graded using the Mankin scale, and GADD45beta was localized by immunohistochemistry. Human chondrocytes were transduced with small interfering RNA (siRNA)-GADD45beta or GADD45beta-FLAG. GADD45beta and COL2A1 messenger RNA (mRNA) levels were analyzed by real-time reverse transcriptase-polymerase chain reaction, and promoter activities were analyzed by transient transfection. Cell death was detected by Hoechst 33342 staining of condensed chromatin. RESULTS: GADD45beta was expressed at higher levels in cartilage from normal donors and patients with early OA than in cartilage from patients with late-stage OA. All chondrocyte nuclei in normal cartilage immunostained for GADD45beta. In early OA cartilage, GADD45beta was distributed variably in chondrocyte clusters, in middle and deep zone cells, and in osteophytes. In contrast, COL2A1, other collagen genes, and factors associated with skeletal development were up-regulated in late OA, compared with early OA or normal cartilage. In overexpression and knockdown experiments, GADD45beta down-regulated COL2A1 mRNA and promoter activity. NF-kappaB overexpression increased GADD45beta promoter activity, and siRNA-GADD45beta decreased cell survival per se and enhanced tumor necrosis factor alpha-induced cell death in human articular chondrocytes. CONCLUSION: These observations suggest that GADD45beta might play an important role in regulating chondrocyte homeostasis by modulating collagen gene expression and promoting cell survival in normal adult cartilage and in early OA.

ESE-1 is a potent repressor of type II collagen gene (COL2A1) transcription in human chondrocytes.

The epithelium-specific ETS (ESE)-1 transcription factor is induced in chondrocytes by interleukin-1beta (IL-1beta). We reported previously that early activation of EGR-1 by IL-1beta results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE-1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF-kappaB-dependent inhibition by IL-1beta. Of the ETS factors tested, this response was specific to ESE-1, since ESE-3, which was also induced by IL-1beta, suppressed COL2A1 promoter activity only weakly. In contrast, overexpression of ETS-1 increased COL2A1 promoter activity and blocked the inhibition by IL-1beta. These responses to ESE-1 and ETS-1 were confirmed using siRNA-ESE1 and siRNA-ETS1. In transient cotransfections, the inhibitory responses to ESE-1 and IL-1beta colocalized in the -577/-132 bp promoter region, ESE-1 bound specifically to tandem ETS sites at -403/-381 bp, and IL-1-induced binding of ESE-1 to the COL2A1 promoter was confirmed in vivo by ChIP. Our results indicate that ESE-1 serves a potent repressor function by interacting with at least two sites in the COL2A1 promoter. However, the endogenous response may depend upon the balance of other ETS factors such as ETS-1, and other IL-1-induced factors, including EGR-1 at any given time. Intracellular ESE-1 staining in chondrocytes in cartilage from patients with osteoarthritis (OA), but not in normal cartilage, further suggests a fundamental role for ESE-1 in cartilage degeneration and suppression of repair.

Increased expression of the collagen receptor discoidin domain receptor 2 in articular cartilage as a key event in the pathogenesis of osteoarthritis.

OBJECTIVE: To investigate the role of the collagen receptor discoidin domain receptor 2 (DDR-2) in the pathogenesis of osteoarthritis (OA). METHODS: Histologic and immunohistochemical analyses were performed to characterize femoral head cartilage from 7 patients with OA and 4 patients with fracture, as well as articular cartilage from the knee joints of mice with surgically induced OA. Gene constructs encoding human Raf kinase inhibitor protein (RKIP), DDR-2 lacking the discoidin (DS) domain (DeltaDS-DDR-2) or the protein tyrosine kinase (PTK) core (DeltaPTK-DDR-2), DDR-2 containing a substitution of tyrosine for alanine at position 740 (Y740A), and luciferase driven by the matrix metalloproteinase 13 (MMP-13) promoter were transfected into human chondrocyte cell lines. Activated and neutralized alpha2beta1 integrin polyclonal antibodies, interleukin-1 receptor antagonist, and the chemical inhibitors SB203580, for p38, and SP600125, for JNKs, were used in cell cultures. Real-time polymerase chain reaction was performed to examine MMP-13 and DDR-2 messenger RNA (mRNA). RESULTS: Increased immunostaining for DDR-2, MMP-13, and MMP-derived type II collagen fragments was detected in cartilage from patients with OA and from mice with surgically induced OA. The discoidin domain and PTK core of DDR-2 were essential for signal transmission and the resulting increased expression of MMP-13 in chondrocytes. Y740A mutation of DDR-2 reduced levels of mRNA for MMP-13 and endogenous DDR-2. The overexpression of RKIP or preincubation with the p38 inhibitor reduced MMP-13 mRNA levels. DDR-2 signaling was independent of the alpha2beta1 integrin and the interleukin-1-induced signaling pathways in chondrocytes. CONCLUSION: These findings suggest that increased expression of DDR-2, resulting in the elevated expression of MMP-13, may be one of the common events in OA progression.