The Protective Effects of Eicosapentaenoic Acid for Stress-induced Accelerated Senescence in Vascular Endothelial Cells.

Background: Eicosapentaenoic acid (EPA) is an omega-3 fatty acid that protects against cardiovascular diseases in patients with hypertriglyceridemia and may have pleotropic effects beyond lowering triglycerides. Many degenerative diseases, such as atherosclerosis and diabetes, are related to cellular senescence as a pathophysiological mechanism. We aimed to examine whether EPA could protect vascular endothelial cells under stress conditions against stress-induced accelerated senescence (SIAS). Methods: Cultured human umbilical vein endothelial cells (HUVECs) were exposed to H2O2 as oxidative stress and a high glucose concentration with palmitate as a glucolipotoxic condition. Changes in cell viability, apoptosis, lactate dehydrogenase release, and cell cycle analysis were measured by cell counting kit-8 assay, annexin V/ propidium iodide staining, and enzyme-linked immunosorbent assay, respectively. EPA was applied in stress conditions. The degree of senescence was measured by senescence-associated beta-galactosidase staining and p16 staining using immunofluorescence. Apoptosis and cellular senescence-related proteins were measured by Western blotting. Results: Cultured HUVECs under oxidative and glucolipotoxic stresses revealed accelerated senescence and increased apoptosis. These changes were markedly reversed by EPA administration, and the expressions of apoptosis and cellular senescence-related proteins were reversed by EPA treatment. Conclusion: EPA effectively protects HUVECs against SIAS, which may be one of its pleotrophic effects.

Characterization of a flavin-containing monooxygenase from Corynebacterium glutamicum and its application to production of indigo and indirubin.

OBJECTIVE: To examine the role of a gene encoding flavin-containing monooxygenase (cFMO) from Corynebacterium glutamicum ATCC13032 when cloned and expressed in Escherichia coli for the production of indigo pigments. RESULTS: The blue pigments produced by recombinant E. coli were identified as indigo and indirubin. The cFMO was purified as a fused form with maltose-binding protein (MBP). The enzyme was optimal at 25 °C and pH 8. From absorption spectrum analysis, the cFMO was classified as a flavoprotein. FMO activity was strongly inhibited by 1 mM Cu(2+) and recovered by adding 1-10 mM EDTA. The enzyme catalyzed the oxidation of TMA, thiourea, and cysteamine, but not glutathione or cysteine. MBP-cFMO had an indole oxygenase activity through oxygenation of indole to indoxyl. The recombinant E. coli produced 685 mg indigo l(-1) and 103 mg indirubin l(-1) from 2.5 g L-tryptophan l(-1). CONCLUSION: The results suggest the cFMO can be used for the microbial production of both indigo and indirubin.

Testosterone Protects Pancreatic ß-cells from Apoptosis and Stress-Induced Accelerated Senescence.

PURPOSE: Androgens are steroid hormones that are very important in the sexual development and the maintenance of male reproductive system, and also have diverse actions in non-reproductive tissues, including potent antioxidant capacity. Type 2 diabetes mellitus is caused by tissue insulin resistance and insufficient insulin secretion from the pancreatic ß-cells. The progressive decline of pancreatic ß-cells in diabetes is closely related with the severity of disease. We wanted to know whether dihydrotestosterone (DHT) can protect insulin secreting pancreatic ß-cells from apoptosis and accelerated senescence induced by oxidative stress. MATERIALS AND METHODS: Cultured INS-1 cells were used. Various concentrations of H2O2 were applied to exert oxidative stresses. The degrees of apoptosis, accelerated senescence, and the changes of the expressions of related signaling molecules after the application of DHT were analyzed by CCK-8, p16 expression, SA-ß-Gal staining, reverse transcription polymerase chain reactions and Western blots. RESULTS: The application of H2O2 significantly increased (p<0.05) the degree of senescence and apoptosis of cultured INS-1 ß-cells. DHT not only showed anti-oxidant protective capacity, but also significantly reduced (p<0.05) the degree of accelerated senescence. CONCLUSIONS: DHT effectively protects pancreatic islet INS-1 ß-cells from H2O2 induced oxidative stress.

Effects of anagliptin on the stress induced accelerated senescence of human umbilical vein endothelial cells.

BACKGROUND: Dipeptidyl peptidase 4 (DPP-4) inhibitors have been used to treat type 2 diabetes mellitus (T2DM) via inhibition of the enzymatic activity of DPP-4 in degrading active circulating glucagon-like peptide-1. In addition to their glucose-lowering effect, DPP-4 inhibitors have pleiotropic effects. Cellular senescence regarded as important pathophysiological mechanism underlying many degenerative diseases, including atherosclerosis. This study was performed to examine whether the DPP-4 inhibitor, anagliptin, can directly protect against stress-induced accelerated senescence (SIAS) of vascular endothelial cells, regardless of changes in ambient glucose level. METHODS: Cultured human umbilical vein endothelial cells (HUVECs) were exposed to various concentrations of H2O2, and a fixed high concentration of glucose (25 mM) with varying concentrations of palmitate. Changes in cell viability, senescence-associated beta-galactosidase (SA-ß-Gal), p16 protein, markers of endoplasmic reticulum (ER) stress, NOX4, NLRP inflammasome, lactate dehydrogenase (LDH) release and interleukin (IL) 1ß levels were measured by Cell Counting Kit-8 assay, immunofluorescent staining, Western blotting, and enzyme-linked immunosorbent assay, respectively before and after application of anagliptin. RESULTS: The application of oxidative and glucolipotoxic stresses markedly increased the degree of SIAS of HUVECs, represented by increased SA-ß-Gal immunopositivity and p16 protein expression. Aggravation of ER stress and inflammatory response were also observed through increased levels of ATF4, CHOP, peIF2α, NOX4, NLRP inflammasome, LDH, and IL1ß. These changes were markedly reversed by the administration of anagliptin. CONCLUSIONS: The DPP-4 inhibitor anagliptin effectively protects HUVECs against SIAS, suggesting its potential use in the development of new treatment strategies for aging.

Melatonin protects INS-1 pancreatic ß-cells from apoptosis and senescence induced by glucotoxicity and glucolipotoxicity.

INTRODUCTION: Melatonin is a hormone known as having very strong anti-oxidant property. Senescence is a biological state characterized by the loss of cell replication and the changes consisting of a pro-inflammatory phenotype, leading to Senescence Associated Secretory Phenotype (SASP) which is now regarded as one of the fundamental processes of many degenerative diseases. Increased cell division count induces cell senescence via DNA damage in response to elevated Reactive Oxygen Species (ROS). We wanted to test whether melatonin could reduce apoptosis and stress induced premature pancreatic ß-cell senescence induced by glucotoxicity and glucolipotoxicity. MATERIALS AND METHOD: Cultured rodent pancreatic ß-cell line (INS-1 cell) was used. Glucotoxicity (HG: hyperglycemia) and glucolipotoxicity (HGP: hyperglycemia with palmitate) were induced by hyperglycemia and the addition of palmitate. The degrees of the senescence were measured by SA-ß-Gal and P16lnk4A staining along with the changes of cell viabilities, cell cycle-related protein and gene expressions, endogenous anti-oxidant defense enzymes, and Glucose Stimulated Insulin Secretion (GSIS), before and after melatonin treatment. RESULTS: Cultured INS-1 cells in HG and HGP conditions revealed accelerated senescence, increased apoptosis, cell cycle arrest, compromised endogenous anti-oxidant defense, and impaired glucose-stimulated insulin secretion. Melatonin decreased apoptosis and expressions of proteins related to senescence, increase the endogenous anti-oxidant defense, and improved glucose-stimulated insulin secretion. CONCLUSION: Melatonin protected pancreatic ß-cell from apoptosis, decreased expressions of the markers related to the accelerated senescence, and improved the biological deteriorations induced by glucotoxicity and glucolipotoxicity.

The direct effect of lobeglitazone, a new thiazolidinedione, on pancreatic beta cells: A comparison with other thiazolidinediones.

AIMS: The direct effects of thiazolidinediones (TZDs) on pancreatic beta cells have been controversial. The aim of this study was to find out whether a novel TZD, lobeglitazone, has beneficial effects on pancreatic beta cells and db/db mice compared to those of other TZDs. METHODS: INS-1 cells were incubated at a high-glucose concentration with various concentrations of troglitazone, rosiglitazone, pioglitazone, and lobeglitazone. Apoptosis and proliferation of beta cells, markers for ER stress and glucose-stimulated insulin secretion (GSIS) were assessed. In addition, C57BL/6 db/db mice were treated with pioglitazone or lobeglitazone for 4 weeks, and metabolic parameters and the configuration of pancreatic islets were also examined. RESULTS: Lobeglitazone and other TZDs decreased INS-1 cell apoptosis in high-glucose conditions. Lobeglitazone and other TZDs significantly decreased hyperglycemia-induced increases in ER stress markers and increased GSIS. Metabolic parameters showed greater improvement in db/db mice treated with pioglitazone and lobeglitazone than in control mice. Islet size, cell proliferation, and beta cell mass were increased, and collagen surrounding the islets was decreased in treated mice. CONCLUSIONS: Lobeglitazone showed beneficial effects on beta cell survival and function against hyperglycemia. The prosurvival and profunction effects of lobeglitazone were comparable to those of other TZDs.

An orally administered multitarget tyrosine kinase inhibitor, SU11248, is a novel potent inhibitor of thyroid oncogenic RET/papillary thyroid cancer kinases.

CONTEXT: The oncogenic RET/PTC tyrosine kinase causes papillary thyroid cancer (PTC). The use of inhibitors specific for RET/PTC may be useful for targeted therapy of PTC. OBJECTIVE: The objective of the study was to evaluate the efficacies of the recently developed kinase inhibitors SU11248, SU5416, and SU6668 in inhibition of RET/PTC. DESIGN: SU11248, SU5416, and SU6668 were synthesized, and their inhibitory potencies were evaluated using an in vitro RET/PTC kinase assay. The inhibitory effects of the compounds on RET/PTC were evaluated by quantifying the autophosphorylation of RET/PTC, signal transducer and activator of transcription (STAT)-3 activation, and the morphological reversal of RET/PTC-transformed cells. RESULTS: An in vitro kinase assay revealed that SU5416, SU6668, and SU11248 inhibited phosphorylation of the synthetic tyrosine kinase substrate peptide E4Y by RET/PTC3 in a dose-dependent manner with IC(50) of approximately 944 nm for SU5416, 562 nm for SU6668, and 224 nm for SU11248. Thus, SU11248 effectively inhibits the kinase activity of RET/PTC3. RET/PTC-mediated Y705 phosphorylation of STAT3 was inhibited by addition of SU11248, and the inhibitory effects of SU11248 on the tyrosine phosphorylation and transcriptional activation of STAT3 were very closely correlated with decreased autophosphorylation of RET/PTC. SU11248 caused a complete morphological reversion of transformed NIH-RET/PTC3 cells and inhibited the growth of TPC-1 cells that have an endogenous RET/PTC1. CONCLUSION: SU11248 is a highly effective tyrosine kinase inhibitor of the RET/PTC oncogenic kinase.

Regulation of protein kinase B tyrosine phosphorylation by thyroid-specific oncogenic RET/PTC kinases.

Papillary thyroid carcinoma (PTC) is a heterogenous disorder characterized by unique gene rearrangements and gene mutations that activate signaling pathways responsible for cellular transformation, survival, and antiapoptosis. Activation of protein kinase B (PKB) and its downstream signaling pathways appears to be an important event in thyroid tumorigenesis. In this study, we found that the thyroid-specific oncogenic RET/PTC tyrosine kinase is able to phosphorylate PKB in vitro and in vivo. RET/PTC-transfected cells showed tyrosine phosphorylation of endogenous and exogenous PKB, which was independent of phosphorylation of T308 and S473 regulated by the upstream kinases phosphoinositide-dependent kinase-1 and -2, respectively. The PKB Y315 residue, which is known to be phosphorylated by Src tyrosine kinase, was also a major site of phosphorylation by RET/PTC. RET/PTC-mediated tyrosine phosphorylation results in the activation of PKB kinase activity. The activation of PKB by RET/PTC blocked the activity of the forkhead transcription factor, FKHRL1, but a Y315F mutant of PKB failed to inhibit FKHRL1 activity. In summary, these observations suggest that RET/PTC is able to phosphorylate the Y315 residue of PKB, an event that results in maximal activation of PKB for RET/PTC-induced thyroid tumorigenesis.

CR6-interacting factor 1 interacts with orphan nuclear receptor Nur77 and inhibits its transactivation.

CR6-interacting factor 1 (CRIF1) was recently identified as a nuclear protein that interacts with the Gadd45 (growth arrest and DNA damage inducible 45) family of proteins and participates in the regulation of the G1/S phase of the cell cycle. However, the nuclear action of CRIF1 is largely unknown. In this study, we demonstrate that CRIF1 acts as a novel coregulator of transactivation of the orphan nuclear receptor Nur77. Both in vitro and in vivo studies show that CRIF1 interacts with Nur77 via the Nur77 AB domain and that it dramatically inhibits the AB domain-mediated transactivation of Nur77. Transient transfection assays demonstrate that CRIF1 inhibits steroid receptor coactivator-2-mediated Nur77 transactivation, and silencing of endogenous CRIF1 by small interfering RNA relieves this repression. CRIF1 possesses intrinsic repressor activities that are not affected by the histone deacetylase inhibitor Trichostatin A. In addition, overexpression of CRIF1 inhibits TSH/protein kinase A-induced Nur-responsive element promoter activity. CRIF1 inhibited Nur77-dependent induction of E2F1 promoter activity, mRNA expression, and Nur77-mediated G1/S progression in cell cycle. These results suggest that CRIF1 acts as a repressor of the orphan nuclear receptor Nur77 by inhibiting AB domain-mediated transcriptional activity.

Ginsenoside Rg3 prevents INS-1 cell death from intermittent high glucose stress.

BACKGROUND: Ginsenoside Rg3 has been proposed to mediate anti-diabetic effects, but their direct effect on pancreatic ß cell viability and mechanisms are not clearly understood. Recent studies suggest that intermittent high glucose (IHG) could be more harmful to pancreatic ß cells than sustained high glucose. There are few reports about the effect of the ginsenosideRg3 to ß cell apoptosis and proliferation against IHG. METHODS: INS-1 cells were treated with alternative glucose concentration with or without ginsenoside Rg3. Cell apoptosis and viability were detected by Annexin V staining and MTT assay. The activation of mitogen-activated protein kinases (MAPKs) was analyzed by Western blotting using specific antibodies. Quantification of secreted insulin protein was measured using rat/mouse Insulin ELISA kits. Bromodeoxyuridine (BrdU) staining and florescence in situ hybridization (FISH) analysis was performed to compare cell proliferation. RESULT: INS-1 cell viability was decreased under IHG and increased with Rg3 treatment.Rg3 significantly reduced the apoptotic INS-1 cells against IHG. The quantification of secreted insulin concentration was increased with Rg3. Rg3 increased INS-1 cell proliferation. ERK and p38 MAPK pathways reduced by IHG were activated by the ginsenoside Rg3. CONCLUSION: Ginsenoside Rg3 protected INS-1 cell death from IHG with reducing apoptosis and increasing proliferation.

Repeated Glucose Deprivation/Reperfusion Induced PC-12 Cell Death through the Involvement of FOXO Transcription Factor.

BACKGROUND: Cognitive impairment and brain damage in diabetes is suggested to be associated with hypoglycemia. The mechanisms of hypoglycemia-induced neural death and apoptosis are not clear and reperfusion injury may be involved. Recent studies show that glucose deprivation/reperfusion induced more neuronal cell death than glucose deprivation itself. The forkhead box O (FOXO) transcription factors are implicated in the regulation of cell apoptosis and survival, but their role in neuronal cells remains unclear. We examined the role of FOXO transcription factors and the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt and apoptosis-related signaling pathways in PC-12 cells exposed to repeated glucose deprivation/reperfusion. METHODS: PC-12 cells were exposed to control (Dulbecco's Modified Eagle Medium [DMEM] containing 25 mM glucose) or glucose deprivation/reperfusion (DMEM with 0 mM glucose for 6 hours and then DMEM with 25 mM glucose for 18 hours) for 5 days. MTT assay and Western blot analysis were performed for cell viability, apoptosis, and the expression of survival signaling pathways. FOXO3/4',6-diamidino-2-phenylindole staining was done to ascertain the involvement of FOXO transcription factors in glucose deprivation/reperfusion conditions. RESULTS: Compared to PC-12 cells not exposed to hypoglycemia, cells exposed to glucose deprivation/reperfusion showed a reduction of cell viability, decreased expression of phosphorylated Akt and Bcl-2, and an increase of cleaved caspase-3 expression. Of note, FOXO3 protein was localized in the nuclei of glucose deprivation/reperfusion cells but not in the control cells. CONCLUSION: Repeated glucose deprivation/reperfusion caused the neuronal cell death. Activated FOXO3 via the PI3K/Akt pathway in repeated glucose deprivation/reperfusion was involved in genes related to apoptosis.

Regulation of signal transducer and activator of transcription 1 (STAT1) and STAT1-dependent genes by RET/PTC (rearranged in transformation/papillary thyroid carcinoma) oncogenic tyrosine kinases.

Chimeric RET/PTC (rearranged in transformation/papillary thyroid carcinoma) oncoproteins are constitutively active tyrosine kinases found in thyroid papillary carcinoma and nonneoplastic Hashimoto's thyroiditis. Although several proteins have been identified to be substrates of RET/PTC kinases, the pathogenic roles played by RET/PTC in malignant and benign thyroid diseases and the molecular mechanisms that are involved are not fully understood. We found that RET/PTC expression phosphorylates the Y701 residue of STAT1, a type II interferon (IFN)-responsive protein. RET/PTC-mediated signal transducer and activator of transcription 1 (STAT1) phosphorylation requires RET/PTC kinase activity to be intact but other tyrosine kinases, such as Janus kinases or c-Src, are not involved. RET/PTC-induced STAT1 transcriptional activation was not inhibited by suppressor of cytokine signaling-1 or -3, or protein inhibitors of activated STAT3 [(protein inhibitor of activated STAT (PIAS3)], but PIAS1 strongly repressed the RET/PTC-induced transcriptional activity of STAT1. RET/PTC-induced STAT1 activation caused IFN regulatory factor-1 expression. We found that STAT1 and IFN regulatory factor-1 cooperated to significantly increase transcription from type IV IFN-gamma responsive promoters of class II transactivator genes. Significantly, cells stably expressing RET/PTC expressed class II transactivator and showed enhanced de novo membrane expression of major histocompatibility complex (MHC) class II proteins. Furthermore, RET/PTC1-bearing papillary thyroid carcinoma cells strongly expressed MHC class II (human leukocyte-associated antigen-DR alpha) genes, whereas the surrounding normal tissues did not. Thus, RET/PTC is able to phosphorylate and activate STAT1. This may lead to enhanced MHC class II expression, which may explain why the tissues surrounding RET/PTC-positive cancers are infiltrated with lymphocytes. Such immune response-promoting activity of RET/PTC may also relate to the development of Hashimoto's thyroiditis.

Co-Culture of α TC-6 Cells and ß TC-1 Cells: Morphology and Function.

BACKGROUND: In vitro experiments using only ß-cell lines instead of islets are limited because pancreatic islets are composed of four different types of endocrine cells. Several recent studies have focused on cellular interactions among these cell types, especially α- and ß-cells. Because islet isolation needs time and experience, we tested a simple co-culture system with α- and ß-cells. Their morphology and function were assessed by comparison to each single cell culture and pancreatic islets. METHODS: α TC-6 cells and ß TC-1 cells were maintained in Dulbecco's Minimal Essential Medium containing 5 mM glucose and 10% fetal bovine serum. Cells were mixed at a 1:1 ratio (5×105) in 6-well plates and cultured for 24, 48, and 72 hours. After culture, cells were used for insulin and glucagon immunoassays and tested for glucose-stimulated insulin secretion (GSIS). RESULTS: α TC-6 and ß TC-1 cells became condensed by 24 hours and were more strongly compacted after 48 hours. ß TC-1 cells showed both ß-ß and ß-α cell contacts. GSIS increased with increasing glucose concentration in co-cultured cells, which showed lower secreted insulin levels than ß TC-1 cells alone. The increase in the secreted insulin/insulin content ratio was significantly lower for co-cultured cells than for ß-cells alone (P=0.04). Compared to islets, the α-/ß-cell co-culture showed a higher ratio of GSIS to insulin content, but the difference was not statistically significant (P=0.09). CONCLUSION: α TC-6 and ß TC-1 cells in the co-culture system showed cell-to-cell contacts and a similar stimulated insulin secretion pattern to islets. The co-culture system may be used to better mimic pancreatic islets in in vitro assessments.

The Effect of Glucose Fluctuation on Apoptosis and Function of INS-1 Pancreatic Beta Cells.

BACKGROUND: Blood glucose level continuously fluctuates within a certain range in the human body. In diabetes patients, the extent of such fluctuation is large, despite the strict control of blood glucose. Blood glucose fluctuation has been shown to mediate more adverse effects on vascular endothelial cells and diabetes complications than chronic hyperglycemia, which has been explained as due to oxidative stress. As few previous studies have reported the effects of chronic and intermittent hyperglycemia on the apoptosis and function of pancreatic beta cells, this study reported herein was performed to investigate such effects on these cells. METHODS: For chronic hyperglycemia, INS-1 cells were cultured for 5 days with changes of RPMI 1640 medium containing 33 mM glucose every 12 hours. For intermittent hyperglycemia, the medium containing 11 mM glucose was exchanged with the medium containing 33 mM glucose every 12 hours. Apoptosis was assessed by TUNEL assay Hoechst staining and cleaved caspase 3. Insulin secretory capacity was assessed, and the expression of Mn-SOD and Bcl-2 was measured by Western blotting. RESULTS: In comparison to the control group, INS-1 cells exposed to chronic hyperglycemia and intermittent hyperglycemia showed an increase in apoptosis. The apoptosis of INS-1 cells exposed to intermittent hyperglycemia increased significantly more than the apoptosis of INS-1 cells exposed to chronic hyperglycemia. In comparison to the control group, the insulin secretory capacity in the two hyperglycemic states was decreased, and more with intermittent hyperglycemia than with chronic hyperglycemia. The expression of Mn-SOD and Bcl-2 increased more with chronic hyperglycemia than with intermittent hyperglycemia. CONCLUSION: Intermittent hyperglycemia induced a higher degree of apoptosis and decreased the insulin secretory capacity more in pancreatic beta cells than chronic hyperglycemia. This activity may be mediated by the anti-oxidative enzyme Mn-SOD and the anti-apoptotic signal Bcl-2.

Antiinflammatory effects of a combined herbal preparation (RAH13) of Phellodendron amurense and Coptis chinensis in animal models of inflammation.

In an attempt to develop an antiinflammatory herbal remedy that is as potent as current synthetic medicines, the cortex of Phellodendron amurense Rupr (Rutaceae) and the rhizomes of Coptis chinensis Franch (Ranunculaceae) were combined in a 2:1 ratio. This ratio was chosen based on in vitro experiments and traditional Asian medicine prescriptions. The combined ethanol extract, named RAH13, was evaluated for antiinflammatory properties using animal models of acute inflammation such as the croton oil-induced ear edema test and an acetic acid-induced capillary permeability test. Models of chronic inflammation were also tested using the cotton pellet test and a delayed-type hypersensitivity (DTH) test. Oral administration of RAH13 at a dose of 200 mg/kg showed in vivo antiinflammatory activity as potent as the effects associated with 100 mg/mL of celecoxib or 1 mg/kg of dexamethasone. These effects were seen in both acute and chronic inflammation models, suggesting that RAH13 may be effective in controlling some inflammation-related diseases.

Taurine chloramine differentially inhibits matrix metalloproteinase 1 and 13 synthesis in interleukin-1beta stimulated fibroblast-like synoviocytes.

It has been suggested that taurine chloramine (TauCl) plays an important role in the downregulation of proinflammatory mediators. However, little is known about its effect on the expression of matrix metalloproteinases (MMPs). In this study, we investigated the effects of TauCl on synovial expression of MMPs. The effects of TauCl on MMP expression in IL-1beta stimulated fibroblast-like synoviocytes (FLSs) were studied using the following techniques. Real-time PCR and semi-quantitative PCR were employed to analyze the mRNA expression of MMPs. ELISA was used to determine protein levels of MMPs. Western blot analyses were performed to analyze the mitogen-activated protein kinase and inhibitor of nuclear factor-kappaB (IkappaB) kinase signalling pathways. Finally, electrophoretic mobility shift assay and immunohistochemistry were used to assess localization of transcription factors. IL-1beta increased the transcriptional and translational levels of MMP-1 and MMP-13 in rheumatoid arthritis FLSs, whereas the levels of MMP-2 and MMP-9 were unaffected. TauCl at a concentration of 400 to 600 micromol/l greatly inhibited the transcriptional and translational expression of MMP-13, but the expression of MMP-1 was significantly inhibited at 800 micromol/l. At a concentration of 600 micromol/l, TauCl did not significantly inhibit phosphorylation of mitogen-activated protein kinase or IkappaB degradation in IL-1beta stimulated rheumatoid arthritis FLSs. The degradation of IkappaB was significantly inhibited at a TauCl concentration of 800 micromol/l. The inhibitory effect of TauCl on IkappaB degradation was confirmed by electrophoretic mobility shift assay and immunochemical staining for localization of nuclear factor-kappaB. TauCl differentially inhibits the expression of MMP-1 and MMP-13, and inhibits expression of MMP-1 primarily through the inhibition of IkappaB degradation, whereas it inhibits expression of MMP-13 through signalling pathways other than the IkappaB pathway.