Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intraarticular injection of dexamethasone (DEX) accelerates cartilage degradation due to the suppression of chondrocyte proliferation and extracellular matrix formation. The present study first demonstrated the interaction between DEX and TGF beta, a potent growth factor for cultured rat articular chondrocytes (CRAC), and then investigated the molecular mechanism by which DEX counteracts TGF beta-induced chondrocyte proliferation and differentiation through the regulation of AP-1 activity. DEX reduced serum-deprived and TGF beta-stimulated cell growth and [(3)H]-thymidine incorporation of CRAC. DEX also inhibited the expression of (alpha)1 type II collagen with concomitant suppression of the promoter activity. Transfection studies using a reporter vector with AP-1 responsive elements showed that DEX reduced TGF beta-activated but not basal luciferase activities. Activation of 3TP-luc, another AP-1 responsive element containing reporter was also blocked by DEX. GAL4-Elk1 studies revealed that DEX suppressed TGF beta-induced ERK activation which led to c-fos gene expression followed by increase in AP-1 complex formation, whereas the Smad pathway was not involved in DEX-dependent negative regulation of AP-1 in a reporter assay that requires FAST1-Smad2 for the activation. DEX also eliminated TGF beta-induced c-fos mRNA expression and ERK activation in Northern analysis and in vitro kinase assay, respectively. Further, DNA synthesis and transactivation of type II collagen by TGF beta were inhibited by PD98059, an inhibitor of MEK. Our results indicate that DEX suppressed TGF beta-induced chondrocyte proliferation and type II collagen expression, probably through selective inhibition of ERK integrated AP-1 activation.
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PMID:Dexamethasone inhibition of TGF beta-induced cell growth and type II collagen mRNA expression through ERK-integrated AP-1 activity in cultured rat articular chondrocytes. 1096 45

Tumor necrosis factor alpha (TNFalpha) inhibits matrix synthesis by chondrocytes in rheumatoid arthritis and osteoarthritis; however, the underlying signaling pathways are poorly characterized. This study investigated the TNFalpha-activated pathways regulating expression of two key components of the cartilage matrix-link protein and type II collagen. In rat articular chondrocytes, TNFalpha decreased link protein and type II collagen mRNA to undetectable levels within 48 h. Levels of link protein mRNA recovered more readily than type II collagen mRNA following removal of the cytokine. TNFalpha-mediated reduction in mRNA of both matrix molecules occurred at the level of transcription and, for link protein, mRNA stability. Turnover of type II collagen and link protein mRNA was dependent on new protein synthesis. In both prechondrocytes and articular chondrocytes, TNFalpha induced concentration-dependent activation of MEK1/2 and NF-kappaB, but not p38 or JNK. Sustained activation of NF-kappaB was observed for up to 72 h following continuous or transient exposure to TNFalpha. Using pharmacological and molecular approaches, the MEK1/2 and NF-kappaB pathways were found to mediate inhibition of type II collagen and link protein gene expression by TNFalpha. Both prechondrocytes and articular chondrocytes are targets of TNFalpha. This study identifies pathways through which TNFalpha perturbs the synthesis and organization of articular cartilage matrix during inflammation.
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PMID:TNFalpha suppresses link protein and type II collagen expression in chondrocytes: Role of MEK1/2 and NF-kappaB signaling pathways. 1456 65

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, also known as the MEK-ERK kinase cascade, has recently been implicated in the regulation of embryonic cartilage differentiation. However, its precise role in this complex process remains controversial. To more thoroughly examine the role of the MEK-ERK kinase cascade in chondrogenesis, we analyzed the effects of two structurally different pharmacological inhibitors of MEK, the upstream kinase activator of ERK, on chondrocyte differentiation in micromass cultures of embryonic chick limb mesenchyme cells. We found that the MEK inhibitors, U0126 and PD98059, promote increased accumulation of cartilage-characteristic mRNA transcripts for type II collagen, aggrecan, and the transcription factor, Sox9. PD98059 treatment stimulated increased deposition of sulfated glycosaminoglycan into both Alcian blue-stainable cartilage matrix and the surrounding culture medium, whereas U0126 elevated glycosaminoglycan secretion into the medium fraction alone. Both MEK inhibitors increased total type II collagen protein accumulation in micromass culture and elevated the activity of a transfected type II collagen enhancer-luciferase reporter gene. Thus, pharmacological MEK inhibition induced increased expression of multiple chondrocyte differentiation markers. Conversely, transfection of limb mesenchyme cells with a constitutively active MEK1 plasmid resulted in a prominent decrease in the activity of a co-transfected type II collagen enhancer-luciferase reporter gene. Collectively, these findings support the hypothesis that signaling through the MEK-ERK kinase cascade may function as an important inhibitory regulator of embryonic cartilage differentiation.
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PMID:The MEK-ERK signaling pathway is a negative regulator of cartilage-specific gene expression in embryonic limb mesenchyme. 1461 31

The role of TGF-beta/bone morphogenetic protein signaling in the chondrogenic differentiation of human synovial fibroblasts (SFs) was examined with the adenovirus vector-mediated gene transduction system. Expression of constitutively active activin receptor-like kinase 3 (ALK3CA) induced chondrocyte-specific gene expression in SFs cultured in pellets or in SF pellets transplanted into nude mice, in which both the Smad and p38 pathways are essential. To analyze downstream cascades of ALK3 signaling, we utilized adenovirus vectors carrying either Smad1 to stimulate Smad pathways or constitutively active MKK6 (MKK6CA) to activate p38 pathways. Smad1 expression had a synergistic effect on ALK3CA, while activation of p38 MAP kinase pathways alone by transduction of MKK6CA accelerated terminal chondrocytic differentiation, leading to type X collagen expression and enhanced mineralization. Overexpression of Smad1 prevented MKK6CA-induced type X collagen expression and maintained type II collagen expression. In a mouse model of osteoarthritis, activated p38 expression as well as type X collagen staining was detected in osteochondrophytes and marginal synovial cells. These results suggest that SFs can be differentiated into chondrocytes via ALK3 activation and that stimulating Smad pathways and controlling p38 activation at the proper level can be a good therapeutic strategy for maintaining the healthy joint homeostasis and treating degenerative joint disorders.
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PMID:Distinct roles of Smad pathways and p38 pathways in cartilage-specific gene expression in synovial fibroblasts. 1499 Oct 70

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21(Cip-1/SDI-1/WAF-1), is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory effects of U0126 or stimulatory effects of SP600125 on insulin-induced chondrogenic differentiation were observed when these inhibitors exist in the early phase of differentiation, suggesting that MEK/ERK and JNK act on early phase differentiation. SB202580, however, is necessary not only for early phase but also for late phase differentiation, indicating that p38 MAP kinase stimulates differentiation by acting during the entire period of cultivation. These results for the first time demonstrate that up-regulation of p21 expression by ERK1/2 and p38 MAP kinase is required for chondrogenesis, and that JNK acts as a suppressor of chondrogenesis by down-regulating p21 expression.
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PMID:p21(Cip-1/SDI-1/WAF-1) expression via the mitogen-activated protein kinase signaling pathway in insulin-induced chondrogenic differentiation of ATDC5 cells. 1524 98

Tissue engineering has the potential to provide cartilaginous constructs capable of restoring the normal function of native articular cartilage following joint injury or degradation. One approach to functional tissue engineering of cartilage involves the in vitro cultivation of tissue constructs by using: (i) chondrogenic cells that can be selected, expanded, and transfected to overexpress the genes of interest, (ii) scaffolds that provide a defined three-dimensional structure for tissue development and biodegrade at a controlled rate. Understanding the functional potential of the cells and the signaling mechanisms underlying their differentiation should lead to innovative protocols for clinical orthopaedic interventions. A large number of growth factors and hormones have been implicated in the regulation of chondrocyte biology, relatively little is known about the intracellular signaling pathways involved. We have tried to define the roles of specific TGF- dependent signaling pathways involved in the regulation of chondrogenesis from human mesenchymal stem cells. Chondrogenesis induced by TGF-beta3 in alginate bead system was confirmed by examining cartilage specific type II collagen expression and aggrecan, whereas type I collagen expression was not affected by TGF-beta3. Type II collagen mRNA expression was expressed strongly during chondrogenesis and MEK inhibition (U0126) resulted in complete down-regulation of type II collagen. In contrast, aggrecan expression was detected in same level by treatment of U0126. These results strongly suggest that the ERK signaling cascade is involved in TGF-beta3 induced-chondrogenesis signaling pathways and a role of its pathway is necessary over a longer period to promote type II collagen expression. However, their end product properties in vivo have not been well known. In this study, an articular cartilage from chondrogenic MSCs with PLGA scaffolds (75:25 and 65:35) were made and analyzed its biochemical, histological and mechanical properties in vitro and in vivo. And also, we evaluated the cartilage formation in vivo through the injection of cell-thermosensitive gel complex, a newly developed injectable material. At 12 weeks after PLGA scaffolds containing chondrogenic MSCs transplantation, the separated rabbit distal femur showed a good gross articular cartilage appearance in the transplanted site. In indentation test, compare to the native articular cartilage, the engineered cartilage from two types of (75:25 and 65:35) achieved up to 30-60% in mechanical stiffness. And also, a new model for cartilage formation in bladder, at 14 weeks after injection, we could find out mass formation in the submucosal area grossly. H&E staining, alcian blue staining and other special staining confirmed the chondrogenic differentiation in the mass. These cell therapy technologies can provide the possibility of clinical applications for vesicoureteral reflux and reflux esophagitis, and urinary incontinence as well as articular cartilage regeneration.
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PMID:Chondrogenic differentiation of mesenchymal stem cells and its clinical applications. 1525 49

The failure of chondrocytes to replace the lost extracellular matrix contributes to the progression of degenerative disorders of cartilage. Inflammatory mediators present in the joint regulate the breakdown of the established matrix and the synthesis of new extracellular matrix molecules. In the present study, we investigated the effects of tumor necrosis factor alpha (TNF-alpha) and epidermal growth factor (EGF) on chondrocyte morphology and matrix gene expression. Chondrocytes were isolated from distal femoral condyles of neonatal rats. Cells in primary culture displayed a cobblestone appearance. EGF, but not TNF-alpha, increased the number of cells exhibiting an elongated morphology. TNF-alpha potentiated the effect of EGF on chondrocyte morphology. Individually, TNF-alpha and EGF diminished levels of aggrecan and type II collagen mRNA. In combination, the effects of TNF-alpha and EGF were additive, indicating the involvement of discrete signaling pathways. Cell viability was not compromised by TNF-alpha or by EGF, alone or in combination. EGF alone did not activate NF-kappaB or alter NF-kappaB activation by TNF-alpha. Pharmacologic studies indicated that the effects of TNF-alpha and EGF alone or in combination were independent of protein kinase C signaling, but were dependent on MEK1/2 activity. Finally, we analyzed the involvement of Sox-9 using a reporter construct of the 48 base pair minimal enhancer of type II collagen. TNF-alpha attenuated enhancer activity as expected; in contrast, EGF did not alter either the effect of TNF-alpha or basal activity. TNF-alpha and EGF, acting through distinct signaling pathways, thus have additive adverse effects on chondrocyte function. These findings provide critical insights into the control of chondrocytes through the integration of multiple extracellular signals.
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PMID:Tumor necrosis factor alpha and epidermal growth factor act additively to inhibit matrix gene expression by chondrocyte. 1564 33

Transforming growth factor (TGF)-beta, bone morphogenetic protein (BMP), and interleukin-1beta activate TGF-beta-activated kinase 1 (TAK1), which lies upstream of the p38 MAPK, JNK, and NF-kappaB pathways. Our knowledge remains incomplete of TAK1 target genes, requirement for cooperative signaling, and capacity for shared or segregated ligand-dependent responses. We show that adenoviral overexpression of TAK1a in articular chondrocytes stimulated type II collagen protein synthesis 3-6-fold and mimicked the response to TGF-beta1 and BMP2. Both factors activated endogenous TAK1 and its activating protein, TAB1, and the collagen response was inhibited by dominant-negative TAK1a. Isoform-specific antibodies to TGF-beta blocked the response to endogenous and exogenous TGF-beta but not the response to TAK1a. Expression of Smad3 did not stimulate type II collagen synthesis or enhance that caused by TGF-beta1 or TAK1a, in contrast to its effects on its endogenous targets, CTGF and plasminogen-activated inhibitor-1. TAK1a, overexpressed alone and immunoprecipitated, phosphorylated MKK6 and stimulated the plasminogen-activated inhibitor-1 promoter following transient transfection; both effects were enhanced by TAB1 coexpression, but type II collagen synthesis was not. Stimulation by TAK1a or TGF-beta did not require increased Col2a1 mRNA, and TAK1 actually reduced Col2a1 mRNA in parallel with the cartilage markers, SRY-type HMG box 9 (Sox9) and aggrecan. Thus, TAK1 increased target gene expression (Col2a1) by translational or posttranslational mechanisms as a Smad3-independent response shared by TGF-beta1 and BMP2.
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PMID:Transforming growth factor (TGF)-beta-activated kinase 1 mimics and mediates TGF-beta-induced stimulation of type II collagen synthesis in chondrocytes independent of Col2a1 transcription and Smad3 signaling. 1574 58

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, also known as the MEK-ERK cascade, has been shown to regulate cartilage differentiation in embryonic limb mesoderm and several chondrogenic cell lines. In the present study, we employed the micromass culture system to define the roles of MEK-ERK signaling in the chondrogenic differentiation of neural crest-derived ectomesenchyme cells of the embryonic chick facial primordia. In cultures of frontonasal mesenchyme isolated from stage 24/25 embryos, treatment with the MEK inhibitor U0126 increased type II collagen and glycosaminoglycan deposition into cartilage matrix, elevated mRNA levels for three chondrogenic marker genes (col2a1, aggrecan, and sox9), and increased expression of a Sox9-responsive collagen II enhancer-luciferase reporter gene. Transfection of frontonasal mesenchyme cells with dominant negative ERK increased collagen II enhancer activation, whereas transfection of constitutively active MEK decreased its activity. Thus, MEK-ERK signaling inhibits chondrogenesis in stage 24/25 frontonasal mesenchyme. Conversely, MEK-ERK signaling enhanced chondrogenic differentiation in mesenchyme of the stage 24/25 mandibular arch. In mandibular mesenchyme cultures, pharmacological MEK inhibition decreased cartilage matrix deposition, cartilage-specific RNA levels, and collagen II enhancer activity. Expression of constitutively active MEK increased collagen II enhancer activation in mandibular mesenchyme, while dominant negative ERK had the opposite effect. Interestingly, MEK-ERK modulation had no significant effects on cultures of maxillary or hyoid process mesenchyme cells. Moreover, we observed a striking shift in the response of frontonasal mesenchyme to MEK-ERK modulation by stage 28/29 of development.
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PMID:MEK-ERK signaling plays diverse roles in the regulation of facial chondrogenesis. 1645 13

The ability of insulin-like growth factor I (IGF-I) to stimulate cartilage matrix synthesis is reduced in aged and osteoarthritic cartilage. Aging and osteoarthritis are associated with an increase in reactive oxygen species, which we hypothesized would interfere with normal IGF-I signaling. We compared IGF-I signaling in normal and osteoarthritic human articular chondrocytes and investigated the effects of oxidative stress induced by tert-butylhydroperoxide (tBHP). In normal human chondrocytes, IGF-I initiated a strong and sustained phosphorylation of IRS-1 (Tyr-612) and Akt (Ser-473) and transient ERK phosphorylation. In contrast, in osteoarthritic chondrocytes, which possessed elevated basal IRS-1 (Ser-312) and ERK phosphorylation, IGF-I failed to stimulate IRS-1 (Tyr-612) or Akt phosphorylation. In normal human chondrocytes, tBHP triggered strong IRS-1 (Ser-312 and Ser-616) and ERK phosphorylation and inhibited IGF-I-induced IRS-1 (Tyr-612) and Akt phosphorylation. Lentivirus-mediated overexpression of constitutively active (CA) Akt significantly enhanced proteoglycan synthesis, whereas both dominant negative Akt and CA MEK inhibited proteoglycan synthesis. CA Akt also promoted type II collagen and Sox9 expression, whereas tBHP treatment and CA MEK inhibited aggrecan, collagen II, and Sox9 mRNA expression. In osteoarthritic chondrocytes, the antioxidants Mn(III) tetrakis(4-benzoic acid)porphyrin and N-acetylcysteine increased the ratio of Akt to ERK phosphorylation and promoted IGF-I-mediated proteoglycan synthesis. Chemical inhibition of ERK significantly enhanced IGF-I phosphorylation of Akt and alleviated tBHP inhibition of Akt phosphorylation. These results demonstrate opposing roles for phosphatidylinositol 3-kinase-Akt and MEK-ERK in cartilage matrix synthesis and suggest that elevated levels of reactive oxygen species cause chondrocyte IGF-I resistance by altering the balance of Akt to ERK activity.
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PMID:Oxidative stress inhibits insulin-like growth factor-I induction of chondrocyte proteoglycan synthesis through differential regulation of phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK signaling pathways. 1976 15


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