Cardiac β‐adrenergic receptor activation mediates distinct and cell type‐dependent changes in the expression and distribution of connexin 43

Activation of the sympatho‐β‐adrenergic receptors (β‐ARs) system is a hallmark of heart failure, leading to fibrosis and arrhythmias. Connexin 43 (Cx43) is the most abundant gap junctional protein in the myocardium. Current knowledge is limited regarding Cx43 remodelling in diverse cell types in the diseased myocardium and the underlying mechanism. We studied cell type‐dependent changes in Cx43 remodelling due to β‐AR overactivation and molecular mechanisms involved. Mouse models of isoproterenol stimulation or transgenic cardiomyocyte overexpression of β₂‐AR were used, which exhibited cardiac fibrosis and up‐regulated total Cx43 abundance. In both models, whereas Cx43 expression in cardiomyocytes was reduced and more laterally distributed, fibroblasts exhibited elevated Cx43 expression and enhanced gap junction communication. Mechanistically, activation of β₂‐AR in fibroblasts in vitro elevated Cx43 expression, which was abolished by the β₂‐antagonist ICI‐118551 or protein kinase A inhibitor H‐89, but simulated by the adenylyl cyclase activator forskolin. Our in vitro and in vivo data showed that β‐AR activation‐induced production of IL‐18 sequentially stimulated Cx43 expression in fibroblasts in a paracrine fashion. In summary, our findings demonstrate a pivotal role of β‐AR in mediating distinct and cell type‐dependent changes in the expression and distribution of Cx43, leading to pathological gap junction remodelling in the myocardium.     

IGF binding protein‐3 mediates stress‐induced apoptosis in non‐transformed mammary epithelial cells

Mammary epithelial cell (MEC) number is an important determinant of milk production in lactating dairy cows. IGF‐I increases IGF binding protein‐3 (IGFBP‐3) production in these cells, which plays a role in its ability to enhance proliferation. In the present study, we show that the apoptotic factor anisomycin (ANS) also increases IGFBP‐3 mRNA and protein in a dose‐ and concentration‐dependent manner that mirrors activation of caspase‐3 and ‐7, with significant increases in both IGFBP‐3 protein and caspase activation observed by 3 h. Knock‐down of IGFBP‐3 with small interfering (si) RNA attenuated the ability of ANS to induce apoptosis, while knock‐down of IGFBP‐2, the other major IGFBP made by bovine MEC, had no effect. Reducing IGFBP‐3 also decreased the ability of ANS to induce mitochondrial cytochrome c release, indicating its involvement in the intrinsic apoptotic pathway. In contrast, transfection with IGFBP‐3 in the absence of ANS failed to induce apoptosis. Since both the mitogen IGF‐I and the apoptotic inducer ANS increase IGFBP‐3 production in MEC, we proposed that cellular localization might determine IGFBP‐3 action. While both IGF‐I and ANS stimulated the release of IGFBP‐3 into conditioned media, only ANS induced nuclear localization of IGFBP‐3. A pan‐caspase inhibitor had no effect on ANS‐induced nuclear localization of IGFBP‐3, indicating that nuclear entry of IGFBP‐3 precedes caspase activation. Treatment with IGF‐I had no effect on ANS‐induced nuclear localization, but did block ANS‐induced apoptosis. In summary, our data indicate that IGFBP‐3 plays a role in stress‐induced apoptosis that may require nuclear localization in non‐transformed MEC. J. Cell. Physiol. 228: 734–742, 2013. © 2012 Wiley Periodicals, Inc.     

Serum concentrations of insulin‐like growth factor‐i and insulin‐like growth factor binding protein‐2 and ‐3 in eight hoofstock species

The somatotropic axis, which includes growth hormone, insulin‐like growth factor (IGF)‐I, and IGF binding proteins (IGFBP), is involved in the regulation of growth and metabolism. Measures of the somatotropic axis can be predictive of nutritional status and growth rate that can be utilized to identify nutritional status of individual animals. Before the somatotropic axis can be a predictive tool, concentrations of hormones of the somatotropic axis need to be established in healthy individuals. To begin to establish these data, we quantified IGF‐I, IGFBP‐2, and IGFBP‐3 in males and females of eight threatened hoofstock species at various ages. Opportunistic blood samples were collected from Bos javanicus (Java banteng), Tragelaphus eurycerus isaaci (bongo), Gazella dama ruficollis (addra gazelle), Taurotragus derbianus gigas (giant eland), Kobus megaceros (Nile lechwe), Hippotragus equines cottoni (roan antelope), Ceratotherium simum simum (white rhinoceros), and Elephas maximus (Asian elephant). Serum IGF‐I and IGFBPs were determined by radioimmunoassay and ligand blot, respectively. Generally, IGF‐I and IGFBP‐3 were greater in males, and IGFBP‐2 was greater in females. In banteng (P = 0.08) and male Nile lechwe (P<0.05), IGF‐I increased with age, but decreased in rhinoceros (P = 0.07) and female Nile lechwe (P<0.05). In banteng, IGFBP‐3 was greater (P<0.01) in males. In elephants (P<0.05) and antelope (P = 0.08), IGFBP‐2 were greater in females. Determination of concentrations of hormones in the somatotropic axis in healthy animals makes it possible to develop models that can identify the nutritional status of these threatened hoofstock species. Zoo Biol 30:275–284, 2011. © 2010 Wiley‐Liss, Inc.     

Regulation of insulin resistance by targeting the insulin‐like growth factor 1 receptor with microRNA‐122‐5p in hepatic cells

Insulin resistance (IR) is a common etiology of type 2 diabetes (T2D) defined by a state of decreased reactivity to insulin in multiple organs, such as the liver. This study aims to investigate how microRNA‐122‐5p (miR‐122) regulates the hepatic IR in vitro. We first found that the miR‐122 level was upregulated in the liver of rats fed with a high‐fat diet and injected with streptozotocin (T2D rats), while the expression level of insulin‐like growth factor 1 receptor (IGF‐1R), a potential target of miR‐122, was downregulated in the diabetic liver. In vitro, glucosamine‐induced IR was introduced in HepG2 hepatic cells, and the levels of miR‐122 and IGF‐1R were further assessed. An increase of miR‐122 level and a decrease of IGF‐IR level were observed in IR hepatic cells, which was the same as that in the diabetic liver. Results of the luciferase reporter assay validated IGF‐1R as a direct target of miR‐122. Moreover, in IR HepG2 cells, antagonizing miR‐122 with its specific inhibitor enhanced glucose uptake and suppressed the expression of glucose 6‐phosphatase and phosphoenolpyruvate carboxykinase, two key enzymes in regulating gluconeogenesis. Such alterations induced by the miR‐122 inhibitor in IR hepatic cells were impaired when IGF‐1R was simultaneously knocked down. In addition, the PI3K/Akt pathway was deactivated in IR cells, and then reactivated with miR‐122 inhibitor transfection. In conclusion, our study demonstrates that miR‐122 is able to regulate IR in hepatic cells by targeting IGF‐1R.     

Preparation of connexin43‐integrated giant Liposomes by a baculovirus expression–liposome fusion method

Connexin‐43 (Cx43) containing giant liposomes (GL) were prepared by a baculovirus expression–liposome fusion method. Recombinant budded viruses expressing Cx43 were prepared and then fused with GLs containing DOPG/DOPC at pH 4.5. Connexon formation on the GL membrane was observed by transmission electron microscope. Hydrophilic fluorescent dye transfers were observed through a Cx43‐mediated pathway not only between Sf9 (Spodoptera frugiperda) cells with Cx43 but also from giant Cx43 liposomes to Cx43‐expressing U2OS cells (human osteosarcoma cell). The functional connexin‐containing liposome is expected to be useful for cellular cytosolic delivery systems. The original orientation and function of Cx43 was maintained after integration into the liposomes. The liposome fusion method will create new opportunities as a tool for analysis of channel membrane proteins. Biotechnol. Bioeng. 2010;107: 836–843. © 2010 Wiley Periodicals, Inc.     

Presence and Importance of Connexin43 During Myogenesis

We analyzed the expression of connexin(Cx)43 in proliferating and differentiating C₂C₁₂cells and in myoblasts obtained from newborn mice. Cx43 was present in both cell types and under both conditions. The functional role of gap junctional communication (GJC) during terminal differentiation was evaluated in C₂C₁₂myoblasts in the presence or absence of the gap junction blocker 18β-glycyrrhetinic acid (β-GA). Differentiation was temporally analyzed through myogenin expression, activity of creatine kinase (CK), and yield of multinucleated cells. In cells treated with β-GA, the CK activity and myotube formation were reversibly blocked. While in control cultures positive myogenin expression was seen in cell clusters, in β-GA treated cultures the myogenin immunoreactivity was detected in few, preferentially sparse cells. The role of Cx43 during terminal differentiation was evaluated in cultures of myoblasts obtained from Cx43^Cre-ER(T)/fltransgenic mice. Inducible deletion of Cx43 was obtained upon activation of Cre-ER(T) via 4-OH-tamoxifen applications. Cx43 deletion led to a drastic decrease in myogenin expression at 24 h of differentiation as compared to myoblasts from control mice. Our results indicate that Cx43-containing gap junctions are required for normal skeletal muscle terminal differentiation. These channels might provide a pathway for the intercellular transfer of signals involved in myogenesis.     

Efficiency of gene transfection into donor cells for nuclear transfer of bovine embryos

The production of transgenic (TG) animals by somatic cell nuclear transfer (SCNT) has proven to be a more efficient method than other methods, such as gene injection or sperm mediation. The present study was intended to evaluate the efficiency of gene transfection by Effectene (Qiagen, Inc.), a lipid-based reagent compared to electroporation in fetal-derived fibroblast cells (FFC), cumulus-derived fibroblast cells (CFC), and adult ear skin-derived fibroblast cells (AEFC). Parameters compared were factors such as chromosome abnormality, gene expression, and the incidence of apoptosis. Further, the TG embryos with transfected donor cells generated by electroporation or Effectene were compared to IVF and SCNT embryos in terms of rates of cleavage, blastocyst formation, and blastocyst cell number. Most of the cells (>80%) at confluence were at G0/G1 and considered to be suitable nuclear donors for cloning. Transfection with a plasmid containing the enhanced green fluorescent protein (pEGFP-N1) gene into FFC did not increase the incidence of chromosomal abnormalities. The rates of apoptosis in different cell types transfected with pEGFP-N1 were 3.3%-5.0%, and the values did not differ among groups. In addition, the rates of apoptosis in various cells between 5-7 and 20-22 cell passages did not differ. However, the efficiency of gene transfecton into FFC by Effectene reagent (14.2 +/- 1.7) was significantly (P < 0.05) higher than that obtained by electroporation (5.1 +/- 1.0). Among various cell types, the efficiency of gene transfection by Effectene and eletroporation of FFC (14.2 +/- 1.7 and 5.1 +/- 1.0, respectively) was significantly (P < 0.05) higher than transfection of CFC and AEFC by either method (9.4 +/- 1.5 and 3.3 +/- 0.8, 8.8 +/- 0.7, and 2.1 +/- 0.4, respectively). In TG embryos produced by SCNT with electroporation and Effectene, the rates of cleavage and blastocyst formation were significantly lower (P < 0.05) than those of IVF controls, but rates did not differ between SCNT and TG embryos. Similarly, significantly higher (P < 0.05) total cell numbers in day-8 blastocysts were observed in IVF controls than those in SCNT and TG embryos, but did not differ between SCNT and TG (136 vs. approximately 110, respectively). The results demonstrated that, though there were no difference in the rates of chromosomal aneuploidy and the incidence of apoptosis among various cell types, transfected with or without pEGFP-N1, FFC were the cell type most effectively transfected and Effectene was a suitable agent for transfection.     

Endogenous IGFBP‐3 is required for both growth factor‐stimulated cell proliferation and cytokine‐induced apoptosis in mammary epithelial cells

TNF‐α and IGF‐I exert opposing effects on mammary epithelial cell (MEC) growth and survival. However, both increase IGF binding protein‐3 (IGFBP‐3) expression, a multifunctional protein that plays both IGF‐dependent as well as independent roles in these processes. We have reported that IGF‐I utilizes the PI3‐K and MAPK pathways to induce IGFBP‐3 expression in bovine MEC. Here we show that TNF‐α requires the SAPK pathway p38, but not JNK, to induce IGFBP‐3 expression. Contrary to reports in cancer cell lines, TNF‐α retained its ability to decrease DNA synthesis in cells transfected with IGFBP‐3 siRNA. It also retained its ability to inhibit IGF‐I‐stimulated DNA synthesis in these cells. In contrast, the ability of IGF‐I to increase DNA synthesis was attenuated with IGFBP‐3 knockdown. IGFBP‐3 knockdown also decreased basal DNA synthesis, indicating that a certain level of IGFBP‐3 may be required for cell proliferation. While TNF‐α alone failed to induce apoptosis, it increased cell death when added with the JNK agonist anisomycin (ANS). TNF‐α and ANS were unable to induce apoptosis when either IGFBP‐3 or JNK‐2 was knocked‐down, suggesting that both JNK and IGFBP‐3 may interact with a downstream molecule central to apoptosis. There are reports that IGFBP‐3 promotes either cell proliferation or apoptosis in different cell systems. However, this is the first report that endogenous IGFBP‐3 is required for the action of both stimulatory and inhibitory factors within the same cell line. Therefore, the actions of IGFBP‐3 are not pre‐determined, but instead governed by cellular context such as JNK activation. J. Cell. Physiol. 220: 182–188, 2009. © 2009 Wiley‐Liss, Inc.     

A novel TXNIP‐based mechanism for Cx43‐mediated regulation of oxidative drug injury

Gap junctions (GJs) play an important role in the regulation of cell response to many drugs. However, little is known about their mechanisms. Using an in vitro model of cytotoxicity induced by geneticin (G418), we explored the potential signalling mechanisms involved. Incubation of cells with G418 resulted in cell death, as indicated by the change in cell morphology, loss of cell viability and activation of caspase‐3. Before the onset of cell injury, G418 induced reactive oxygen species (ROS) generation, activated oxidative sensitive kinase P38 and caused a shift of connexin 43 (Cx43) from non‐phosphorylated form to hyperphosphorylated form. These changes were largely prevented by antioxidants, suggesting an implication of oxidative stress. Downregulation of Cx43 with inhibitors or siRNA suppressed the expression of thioredoxin‐interacting protein (TXNIP), activated Akt and protected cells against the toxicity of G418. Further analysis revealed that inhibition of TXNIP with siRNA activated Akt and reproduced the protective effect of Cx43‐inhibiting agents, whereas suppression of Akt sensitized cells to the toxicity of G418. Furthermore, interference of TXNIP/Akt also affected puromycin‐ and adriamycin‐induced cell injury. Our study thus characterized TXNIP as a presently unrecognized molecule implicated in the regulatory actions of Cx43 on oxidative drug injury. Targeting Cx43/TXNIP/Akt signalling cascade might be a promising approach to modulate cell response to drugs.     

Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non‐diabetic cells

During early wound healing (WH) events Connexin 43 (Cx43) is down‐regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down‐regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non‐diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non‐diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non‐diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non‐diabetic cells. The cause of the discrepancy between diabetic and non‐diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368‐phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.     

Cyclooxygenase‐2 over‐expression inhibits liver apoptosis induced by hyperglycemia

Increased expression of COX‐2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX‐2 protects hepatocytes from several pro‐apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX‐2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX‐2 (hCOX‐2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post‐injection, Wt diabetic animals showed a decrease in PI3K activity and P‐Akt levels, an increase of P‐JNK, P‐p38, pro‐apoptotic Bad and Bax, release of cytochrome c and activities of caspases‐3 and ‐9, leading to an increased apoptotic index. This situation was improved in diabetic COX‐2 Tg. In addition, SID COX‐2 Tg showed increased expression of anti‐apoptotic Mcl‐1 and XIAP. Pro‐apoptotic state in the liver of diabetic animals was improved by over‐expression of COX‐2. We also analyzed the roles of high glucose‐induced apoptosis and hCOX‐2 in vitro. Non‐transfected and hCOX‐2‐transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non‐transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX‐2‐transfected cells was suppressed by addition of DFU (COX‐2 selective inhibitor), and mimicked by addition of PGE₂ in non‐transfected cells. Taken together, these results demonstrate that hyperglycemia‐induced hepatic apoptosis is protected by hCOX‐2 expression. J. Cell. Biochem. 114: 669–680, 2013. © 2012 Wiley Periodicals, Inc.     

The diagnostic potential of MPT63‐derived HLA‐A*0201‐restricted CD8+T‐cell epitopes for active pulmonary tuberculosis

MPT63 protein is found only in Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis. Detection of MPT63‐specific IFN‐γ‐secreting T cells could be useful for the diagnosis of tuberculosis (TB) diseases. In the present study, the HLA‐A*0201 restriction of ten predicted MPT63‐derived CD8 ⁺ T‐cell epitopes was assessed on the basis of T2 cell line and HLA‐A*0201 transgenic mice. The diagnostic potential of immunogenic peptides in active pulmonary TB patients was evaluated using an IFN‐γ enzyme‐linked immunospot assay. It was found that five peptides bound to HLA‐A*0201 with high affinity, whereas the remaining peptides exhibited low affinity for HLA‐A*0201. Five immunogenic peptides (MPT63_18–26, MPT63_29–37, MPT63_20–28, MPT63_5–14 and MPT63_10–19) elicited large numbers of cytotoxic IFN‐γ‐secreting T cells in HLA‐A*0201 transgenic mice. Each of the five immunogenic peptides was recognized by peripheral blood mononuclear cells from 45% to 73% of 40 HLA‐A*0201 positive TB patients. The total diagnostic sensitivity of the five immunogenic peptides was higher than that of a T‐SPOT.TB assay (based on ESAT‐6 and CFP‐10) (93% versus 90%). It is noticeable that the diagnostic sensitivity of the combination of five immunogenic peptides and T‐SPOT.TB assay reached 100%. These MPT63‐derived HLA‐A*0201‐restricted CD8 ⁺ T‐cell epitopes would likely contribute to the immunological diagnosis of M. tuberculosis infection and may provide the components for designing an effective TB vaccine.     

Aliskiren‐attenuated myocardium apoptosis via regulation of autophagy and connexin‐43 in aged spontaneously hypertensive rats

There are controversies about the mechanism of myocardium apoptosis in hypertensive heart disease. The aim of this study was to investigate the relationship among autophagy, Cx43 and apoptosis in aged spontaneously hypertensive rats (SHRs) and establish whether Aliskiren is effective or not for the treatment of myocardium apoptosis. Twenty‐one SHRs aged 52 weeks were randomly divided into three groups, the first two receiving Aliskiren at a dose of 10 and 25 mg/kg/day respectively; the third, placebo for comparison with seven Wistar‐Kyoto (WKY) as controls. After a 2‐month treatment, systolic blood pressure (SBP), heart to bw ratios (HW/BW%) and angiotensin II (AngII) concentration were significantly enhanced in SHRs respectively. Apoptotic cardiomyocytes detected with TUNEL and immunofluorescent labelling for active caspase‐3 increased nearly fourfolds in SHRs, with a decline in the expression of survivin and AKT activation, and an increase in caspase‐3 activation and the ratio of Bax/Bcl‐2. Myocardium autophagy, detected with immunofluorescent labelling for LC3‐II, increased nearly threefolds in SHRs, with the up‐regulation of Atg5, Atg16L1, Beclin‐1 and LC3‐II. The expression of Cx43 plaque was found to be down‐regulated in SHRs. Aliskiren significantly reduced SBP, HW/BW%, AngII concentration and the expression of AT₁R. Thus, Aliskiren protects myocardium against apoptosis by decreasing autophagy, up‐regulating Cx43. These effects showed a dose‐dependent tendency, but no significance. In conclusion, the myocardium apoptosis developed during the hypertensive end‐stage of SHRs could be ameliorated by Aliskiren via the regulation of myocardium autophagy and maladaptive remodelling of Cx43.     

Testes‐specific transgene expression in insulin‐like growth factor‐I transgenic mice

Insulin‐like growth factor‐I (IGF‐I) is a low molecular weight peptide that mediates the cell proliferating actions of growth hormone. Evidence exists indicating that IGF‐I is produced by various cell types and this growth factor has been implicated in a variety of reproductive processes. To investigate the effect of IGF‐I over‐expression on reproductive systems, we generated three independent lines of transgenic mice harbouring a human IGF‐I cDNA (hIGF‐I) under the control of a Cytomegalovirus immediate early (CMV) promoter. The CMV promoter was used in an attempt to direct expression of IGF‐I into a variety of tissues both reproductive and non‐reproductive. Yet expression of the foreign hIGF‐I gene, determined by Northern blot, was found to occur only in the testicular tissues of the male mice, apparently due to methylation of the transgene in all the tissues tested except the testes, which demonstrate transgene hypomethylation. Evaluation of the transgene expression during testicular development revealed that expression begins between 10 and 15 days of development, coinciding with the appearance of the zygotene and pachytene primary spermatocytes during early spermatogenesis, therefore indicating germ line expression of the transgene. Extensive study of the CMV‐hIGF‐I transgenic lines of mice has revealed that the effects of the transgene expression do not extend beyond the testicular tissues. No significant differences (P > 0.05) in the IGF‐I serum levels, growth rates, or testicular histology have been observed between transgenic and non‐transgenic male siblings. The ability of transgenic males to produce offspring also appears unaffected. Evaluation of the IGF binding protein (IGFBP) levels in the testicular tissues of CMV‐hIGF‐I transgenic mice by Western ligand blot revealed an increase in the concentration of testicular proteins with molecular weights corresponding to IGFBP‐2 and IGFBP‐3. These results suggest that the testicular over‐expression of IGF‐I induces increased IGFBP localization in this tissue. Inhibition of IGF activity by the IGFBPs would explain the lack of a dramatic physiological effect in the CMV‐hIGF‐I transgenic mice, despite the presence of elevated testicular IGF‐I. The observation that testis specific IGF‐I overexpression induces localization of IGFBPs in this tissue confirms the existence of a well regulated testicular IGF system and supports the convention that this growth factor plays an important role in testicular function. Mol. Reprod. Dev. 54:32–42, 1999. © 1999 Wiley‐Liss, Inc.     

Epigallocatechin‐3‐O‐gallate up‐regulates microRNA‐199a‐3p expression by down‐regulating the expression of cyclooxygenase‐2 in stimulated human osteoarthritis chondrocytes

Osteoarthritis (OA) is a most common form of arthritis worldwide leading to significant disability. MicroRNAs (miRNAs) are non‐coding RNAs involved in various aspects of cartilage development, homoeostasis and pathology. Several miRNAs have been identified which have shown to regulate expression of target genes relevant to OA pathogenesis such as matrix metalloproteinase (MMP)‐13, cyclooxygenase (COX)‐2, etc. Epigallocatechin‐3‐O‐gallate (EGCG), the most abundant and active polyphenol in green tea, has been reported to have anti‐arthritic effects, however, the role of EGCG in the regulation of miRNAs has not been investigated in OA. Here, we showed that EGCG inhibits COX‐2 mRNA/protein expression or prostaglandin E₂ (PGE₂) production via up‐regulating microRNA hsa‐miR‐199a‐3p expression in interleukin (IL)‐1β‐stimulated human OA chondrocytes. This negative co‐regulation of hsa‐miR‐199a‐3p and COX‐2 by EGCG was confirmed by transfection of OA chondrocytes with anti‐miR‐199a‐3p. Transfection of OA chondrocytes with anti‐miR‐199a‐3p significantly enhanced COX‐2 expression and PGE₂ production (P < 0.001), while EGCG treatment significantly inhibited anti‐miR‐199a‐3p transfection‐induced COX‐2 expression or PGE₂ production in a dose‐dependent manner. These results were further re‐validated by co‐treatment of these transfection OA chondrocytes with IL‐1β and EGCG. EGCG treatment consistently up‐regulated the IL‐1β‐decreased hsa‐miR‐199a‐3p expression (P < 0.05) and significantly inhibited the IL‐1β‐induced COX‐2 expression/PGE₂ production (P < 0.05) in OA chondrocytes transfected with anti‐hsa‐miR‐199a‐3p. Taken together, these results clearly indicate that EGCG inhibits COX‐2 expression/PGE₂ production via up‐regulation of hsa‐miR‐199a‐3p expression. These novel pharmacological actions of EGCG on IL‐1β‐stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG‐derived compounds inhibit cartilage breakdown or pain by up‐regulating the expression of microRNAs in human chondrocytes.