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MMP-13 Expression (mmp-13 + expression)
Selected AbstractsGene expression profiling during rat mammary carcinogenesis induced by 7,12-dimethylbenz[a]anthraceneINTERNATIONAL JOURNAL OF CANCER, Issue 6 2009Masakazu Souda Abstract 7,12-Dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma is a well-recognized model; however, the genetic alterations during its carcinogenesis have yet to be determined. We used laser capture microdissection to specifically isolate cells from terminal end buds (TEBs), the origin of carcinoma, at 2 weeks after sesame oil treatment (control) or DMBA treatment (DMBA-TEBs), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (MC). Using an oligonucleotide microarray representing 20,600 rat probe sequences, we analyzed gene expression profiles and validated mRNA and protein levels of genes of interest byreal-time quantitative PCR and immunohistochemistry. The number of differentially expressed genes dramatically increased from DMBA-TEBs (63) to DCIS (798) and MC (981). Only the expression of PEP-19, an anti-apoptotic gene, showed significant increases in DMBA-TEBs (4-fold), DCIS (10-fold) and MC (16-fold). MMP-13 expression was increased markedly in DCIS (19-fold) and MC (61-fold) while OPN expression was increased 6-fold in DCIS and 8-fold in MC. MMP-7 expression was increased 4-fold in MC. Nidogen-1; a participant in the assembly of basement membranes, TSP-2; an inhibitor of angiogenesis and COUP-TFI; a transcription repressor showed significant decreases in DCIS (4-, 9- and 17-fold, respectively) and MC (10-, 37- and 100-fold). Network analyses with IPA software revealed that the most significant network included Akt groups in DCIS and ERK groups in MC. The present findings provide us with a better understanding of the molecular alteration that occur during mammary carcinogenesis and suggest the importance of PEP-19 overexpression in the very early stage of mammary carcinogenesis. © 2009 UICC [source] Improved bioengineered cartilage tissue formation following cyclic compression is dependent on upregulation of MT1-MMPJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 7 2010J. N. Amrith De Croos Abstract The generation of bioengineered cartilage tissue suitable for transplantation is a potential therapy to treat damaged cartilage. We have shown previously that the physical and biomechanical properties of bioengineered cartilage can be improved by the application of 30,min of cyclic compression by a mechanism involving sequential upregulation of gene and protein levels of membrane type-1 matrix metalloproteinase (MT1-MMP) and MMP-13. In the current study, we demonstrated that MT1-MMP is critical to this response, as blocking the upregulation of MT1-MMP prevented the improvement in tissue formation. MT1-MMP seems to act by inducing tissue remodeling as evidenced by the presence of aggrecan degradation products by Western blot analysis and increased release of matrix molecules into the media. Release of these molecules was diminished when MT1-MMP upregulation was prevented. This matrix degradation was likely due to MT1-MMP, as under conditions where MMP-13 expression is maintained (stimulation in the presence of MT1-MMP siRNA) the release of these matrix molecules into the media was still prevented. It also appears that MT1-MMP does not regulate MMP-13 gene expression, as MT1-MMP-siRNA pretreatment had no effect on MMP-13 expression following mechanical stimulation. Further analysis of the anabolic genes and proteins involved in mechanically stimulated cartilage will lead to better understanding of the mechanism(s) underlying tissue formation yielding improved bioengineered cartilage. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:921,927, 2010 [source] Matrix metalloproteinase 13 loss associated with impaired extracellular matrix remodeling disrupts chondrocyte differentiation by concerted effects on multiple regulatory factorsARTHRITIS & RHEUMATISM, Issue 8 2010Rosa Maria Borzí Objective To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. Methods MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and ,-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage. Results Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, ,-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular localization. SOX9 was largely excluded from the nuclei of chondrocytes with MMP-13,remodeled or ,degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. Conclusion MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors. [source] Estradiol inhibits chondrogenic differentiation of mesenchymal stem cells via nonclassic signalingARTHRITIS & RHEUMATISM, Issue 4 2010Zsuzsa Jenei-Lanzl Objective We undertook this study to examine the effects of estradiol on chondrogenesis of human bone marrow,derived mesenchymal stem cells (MSCs), with consideration of sex-dependent differences in cartilage repair. Methods Bone marrow was obtained from the iliac crest of young men. Density-gradient centrifugation,separated human MSCs proliferated as a monolayer in serum-containing medium. After confluence was achieved, aggregates were created and cultured in a serum-free differentiation medium. We added different concentrations of 17,-estradiol (E2) with or without the specific estrogen receptor inhibitor ICI 182.780, membrane-impermeable E2,bovine serum albumin (E2-BSA), ICI 182.780 alone, G-1 (an agonist of G protein,coupled receptor 30 [GPR-30]), and G15 (a GPR-30 antagonist). After 21 days, the aggregates were analyzed histologically and immunohistochemically; we quantified synthesized type II collagen, DNA content, sulfated glycosaminoglycan (sGAG) concentrations, and type X collagen and matrix metalloproteinase 13 (MMP-13) expression. Results The existence of intracellular and membrane-associated E2 receptors was shown at various stages of chondrogenesis. Smaller aggregates and significantly lower type II collagen and sGAG content were detected after treatment with E2 and E2-BSA in a dose-dependent manner. Furthermore, E2 enhanced type X collagen and MMP-13 expression. Compared with estradiol alone, the coincubation of ICI 182.780 with estradiol enhanced suppression of chondrogenesis. Treatment with specific GPR-30 agonists alone (G-1 and ICI 182.780) resulted in a considerable inhibition of chondrogenesis. In addition, we found an enhancement of hypertrophy by G-1. Furthermore, the specific GPR-30 antagonist G15 reversed the GPR-30,mediated inhibition of chondrogenesis and up-regulation of hypertrophic gene expression. Conclusion The experiments revealed a suppression of chondrogenesis by estradiol via membrane receptors (GPR-30). The study opens new perspectives for influencing chondrogenesis on the basis of classic and nonclassic estradiol signaling. [source] Role of Wnt-5A in interleukin-1,,induced matrix metalloproteinase expression in rabbit temporomandibular joint condylar chondrocytesARTHRITIS & RHEUMATISM, Issue 9 2009Xianpeng Ge Objective To determine the possible involvement and regulatory mechanisms of Wnt-5A signaling in interleukin-1, (IL-1,),induced increase in matrix metalloproteinase 1 (MMP-1), MMP-3, MMP-9, and MMP-13 expression in temporomandibular joint (TMJ) condylar chondrocytes. Methods Primary rabbit condylar chondrocytes were treated with IL-1,, purified Wnt-5A protein, or both and transfected with Wnt-5A expression vector. Expression of Wnt-5A, MMP-1, MMP-3, MMP-9, MMP-13, and type II collagen, as well as cell morphologic changes, were examined. To explore the mechanisms of action of Wnt-5A, the accumulation and nuclear translocation of ,-catenin, the transcription activity of the ,-catenin,Tcf/Lef complex, phosphorylated JNK, phosphorylated ERK, and phosphorylated p38 were analyzed. SP600125, a JNK inhibitor, was used to investigate the role of the JNK pathway in Wnt-5A induction of MMP-1, MMP-3, MMP-9, and MMP-13. Results Treatment of rabbit condylar chondrocytes with IL-1, up-regulated Wnt-5A expression. Purified Wnt-5A protein and transfection with Wnt-5A expression vector promoted the expression of MMP-1, MMP-3, MMP-9, and MMP-13. Wnt-5A did not cause accumulation and nuclear translocation of ,-catenin or activation of the ,-catenin-Tcf/Lef transcription complex. Instead, Wnt-5A activated JNK, and an inhibitor of JNK blocked the Wnt-5A,induced up-regulated expression of MMPs. Conclusion These findings indicate that IL-1, up-regulates Wnt-5A, and the activation of Wnt-5A signaling induces the expression of MMP-1, MMP-3, MMP-9, and MMP-13 via the JNK signaling pathway in rabbit TMJ condylar chondrocytes. Blockage of JNK signaling impairs the Wnt-5A,induced up-regulation of MMPs. Thus, Wnt-5A may be associated with cartilage destruction by promoting the expression of MMPs. [source] Accelerated development of aging-associated and instability-induced osteoarthritis in osteopontin-deficient miceARTHRITIS & RHEUMATISM, Issue 8 2009Yuichiro Matsui Objective To investigate the role of osteopontin (OPN) in the development of osteoarthritis (OA) under in vivo and in vitro conditions. Methods Both instability-induced and aging-associated OA models were generated using OPN-deficient (OPN,/,) and control wild-type (WT) mice. An in vitro cartilage degradation model was also used, to evaluate the effect of OPN on proteoglycan loss from joint cartilage. Results OPN deficiency exacerbated both aging-associated and instability-induced OA. Both structural changes and an increased loss of proteoglycan from cartilage tissue were augmented in the absence of OPN. OPN deficiency also led to the induction of matrix metalloproteinase 13 (MMP-13), which degrades a major component of the cartilage matrix protein type II collagen. Both the loss of proteoglycan and the induction of the collagen-degrading enzyme MMP-13 facilitated the development of OA. Conclusion OPN plays a pivotal role in the progression of both instability-induced and aging-associated spontaneous OA. OPN is a critical intrinsic regulator of cartilage degradation via its effects on MMP-13 expression and proteoglycan loss. [source] Expression of MicroRNA-146a in osteoarthritis cartilageARTHRITIS & RHEUMATISM, Issue 4 2009Keiichiro Yamasaki Objective A role of microRNA, which are ,22-nucleotide noncoding RNAs, has recently been recognized in human diseases. The objective of this study was to identify the expression pattern of microRNA-146a (miR-146a) in cartilage from patients with osteoarthritis (OA). Methods The expression of miR-146a in cartilage from 15 patients with OA was analyzed by quantitative reverse transcription,polymerase chain reaction (RT-PCR) and by in situ hybridization. Induction of the expression of miR-146a by cultures of normal human articular chondrocytes following stimulation with interleukin-1, (IL-1,) was examined by quantitative RT-PCR. Results All cartilage samples were divided into 3 groups according to a modification of the Mankin score (grade I = mild OA scored 0,5, grade II = moderate OA scored 6,10, and grade III = severe OA scored 11,14). In grade I OA cartilage samples, the expression of miR-146a and COL2A1 was significantly higher than that in the other groups (P < 0.05). In grades II and III OA cartilage, the expression of miR-146a and COL2A1 was decreased, whereas the expression of matrix metalloproteinase 13 (MMP-13) was elevated in grade II OA cartilage. These data showed that miR-146a is expressed intensely in cartilage with a low Mankin grade and that miR-146a expression decreases in parallel with the level of MMP-13 expression. Tissue section in situ hybridization of primary miR-146a (pri-miR-146a) revealed that pri-miR-146a was expressed in chondrocytes residing in all tissue layers, especially in the superficial layer, where it was intensely expressed. The expression of miR-146 was markedly elevated by IL-1, stimulation in human chondrocytes in vitro. Conclusion This study shows that miR-146 is intensely expressed in low-grade OA cartilage and that its expression is induced by stimulation of IL-1,. Thus, miR-146 might play a role in OA cartilage pathogenesis. [source] CCAAT/ENHANCER binding protein , mediates expression of matrix metalloproteinase 13 in human articular chondrocytes in inflammatory arthritisARTHRITIS & RHEUMATISM, Issue 3 2009Mitsumasa Hayashida Objective To determine the function of CCAAT/enhancer binding protein , (C/EBP,) in the expression of matrix metalloproteinase 13 (MMP-13) in chondrocytes in inflammatory arthritis. Methods Cartilage obtained from patients with rheumatoid arthritis and osteoarthritis was immunostained for expression of C/EBP, or MMP-13. Interleukin-1,, or tumor necrosis factor , (TNF,),stimulated chondrocytes were subjected to Western blotting and real-time reverse transcriptase,polymerase chain reaction (RT-PCR). MMP-13 promoter assays were conducted, and the C/EBP, response element was characterized by deletion and mutation analysis. C-28/I2 cells were treated with TNF, and subjected to chromatin immunoprecipitation (ChIP) assays. Finally, C/EBP,,liver-enriched activator protein (LAP) was overexpressed in C-28/I2 cells or cartilage tissues, and MMP-13 expression was analyzed. Results C/EBP, and MMP-13 expression was colocalized in chondrocytes in arthritic cartilage. MMP-13 promoter activity was stimulated by C/EBP, overexpression in a dose-dependent manner. Luciferase assays revealed that a ,981-bp promoter had the greatest activity, while deletion to ,936 bp strongly diminished promoter activity. Luciferase activity was repressed to basal levels by mutations in potential C/EBP binding sites. The stimulatory effects of C/EBP, overexpression were diminished by mutation. ChIP assays revealed that TNF, treatment enhanced the binding of C/EBP, to the MMP-13 promoter. When C/EBP,-LAP was overexpressed in C-28/I2 cells, endogenous MMP-13 expression was stimulated up to 32-fold as detected by real-time RT-PCR. Furthermore, following adenoviral overexpression of C/EBP,-LAP in organ culture of articular cartilage, stimulation of MMP-13 was also detected by immunohistochemistry. Conclusion C/EBP, directly binds to the MMP-13 promoter region and stimulates the expression of MMP-13 in chondrocytes in inflammatory arthritis. [source] Inhibition of interleukin-1,,induced matrix metalloproteinases 1 and 13 production in human osteoarthritic chondrocytes by prostaglandin D2ARTHRITIS & RHEUMATISM, Issue 11 2008Nadia Zayed Objective To investigate the effects of prostaglandin D2 (PGD2) on interleukin-1, (IL-1,),induced matrix metalloproteinase 1 (MMP-1) and MMP-13 expression in human chondrocytes and the signaling pathways involved in these effects. Methods Chondrocytes were stimulated with IL-1 in the presence or absence of PGD2, and expression of MMP-1 and MMP-13 proteins was evaluated by enzyme-linked immunosorbent assay. Messenger RNA (mRNA) expression and promoter activity were analyzed by real-time reverse transcription,polymerase chain reaction and transient transfections, respectively. The role of the PGD2 receptors D prostanoid receptor 1 (DP1) and chemoattractant receptor,like molecule expressed on Th2 cells (CRTH2) was evaluated using specific agonists and antibody-blocking experiments. The contribution of the cAMP/protein kinase A (PKA) pathway was determined using cAMP-elevating agents and PKA inhibitors. Results PGD2 decreased in a dose-dependent manner IL-1,induced MMP-1 and MMP-13 protein and mRNA expression as well as their promoter activation. DP1 and CRTH2 were expressed and functional in chondrocytes. The effect of PGD2 was mimicked by BW245C, a selective agonist of DP1, but not by 13,14-dihydro-15-keto-PGD2, a selective agonist of CRTH2. Furthermore, treatment with an anti-DP1 antibody reversed the effect of PGD2, indicating that the inhibitory effect of PGD2 is mediated by DP1. The cAMP-elevating agents 8-Br-cAMP and forskolin suppressed IL-1,induced MMP-1 and MMP-13 expression, and the PKA inhibitors KT5720 and H89 reversed the inhibitory effect of PGD2, suggesting that the effect of PGD2 is mediated by the cAMP/PKA pathway. Conclusion PGD2 inhibits IL-1,induced production of MMP-1 and MMP-13 by chondrocytes through the DP1/cAMP/PKA signaling pathway. These data also suggest that modulation of PGD2 levels in the joint may have therapeutic potential in the prevention of cartilage degradation. [source] | ||||||||||