GIT1 is a novel MEK1–ERK1/2 scaffold that localizes to focal adhesions

Cell polarity is critical for cell migration and requires localized signal transduction in subcellular domains. Recent evidence demonstrates that activation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) in focal adhesions is essential for cell migration. GIT1 (G‐protein‐coupled receptor kinase‐interacting protein 1) has been shown to bind paxillin and regulate focal‐adhesion disassembly. We have previously reported that GIT1 binds to MEK1 [MAPK (mitogen‐activated protein kinase)/ERK kinase 1] and acts as a scaffold to enhance ERK1/2 activation in response to EGF (epidermal growth factor). In the present study we show that GIT1 associates with ERK1/2 in focal adhesions and this association increases after EGF stimulation. The CC (coiled‐coil) domain of ERK1/2 is required for association with GIT1, translocation to focal adhesions, and cell spreading and migration. Immunofluorescent staining showed that, after EGF stimulation, GIT1 co‐localized with pERK1/2 (phosphorylated ERK1/2) in focal adhesions. The binding of GIT1 and ERK1/2 was functionally important, since transfecting an ERK2 mutant lacking the CC domain [ERK2(del CC)] significantly decreased pERK1/2 translocation to focal adhesions, cell spreading and migration induced by EGF. In summary, the CC domain of ERK1/2 is necessary for binding to GIT1, for ERK1/2 activation in focal adhesions, and for cell spreading and migration.     

卡铂通过抑制ERK1/2通路诱导人卵巢癌细胞S和G0/G1期阻滞

卡铂(carboplatin, CBP)是一种抗肿瘤活性较强的化疗药物, 通过诱导细胞周期阻滞抑制肿瘤细胞生长, 但其诱导细胞周期阻滞的报告不甚一致. 本研究探索卡铂对卵巢癌HO-8910细胞生长及细胞周期进程的影响. MTS结果显示, 卡铂以浓度和时间依赖方式抑制卵巢癌HO-8910细胞生长, 联合使用ERK1/2通路抑制剂PD98059可使卡铂抗卵巢癌细胞增殖作用增强. 采用Giemsa染色法观察到, 卡铂与PD98059单用或联用均能致卵巢癌细胞发生明显的形态学变化. 流式细胞术检测细胞周期发现, 随卡铂浓度的增高, S期阻滞作用增强; 抑制ERK1/2通路可拮抗卡铂对HO-8910细胞S期阻滞作用, 增加G1期阻滞作用, 而对G2/M期细胞影响不明显. Western印迹结果显示, 随卡铂浓度的增高, p-ERK1/2、Cdc2(Y15)和p Cdc2(T161)的表达逐渐升高, Cyclin E1和Cyclin B1的表达逐渐降低; 抑制ERK1/2通路可将卡铂上调,p-ERK1/2和p-Cdc2(T161)的作用反转为下调作用, 上调Cdc2(Y15)的表达受阻, 抑制Cyclin B1的下调作用, 促进Cyclin E1的下调作用. 本研究结果提示, 卡铂通过抑制ERK1/2激活, 诱导人卵巢癌HO-8910细胞S和G1期阻滞, 抑制卵巢癌细胞生长.     

Inhibition of adipocyte differentiation by phytoestrogen genistein through a potential downregulation of extracellular signal-regulated kinases 1/2 activity

In the current study, we investigated the effects of genistein on adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cell (BMSC) cultures and its potential signaling pathway. The terminal adipogenic differentiation was assessed by western-blotting analysis of adipogenic-specific proteins such as PPARgamma, C/EBPalpha, and aP2 and the formation of adipocytes. Treatment of mouse BMSC cultures with adipogenic cocktail resulted in sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are members of the mitogen-activated protein kinase (MAPK) family, at the early phase of adipogenesis (from days 3 to 9). Inhibition of ERK1/2 activation by PD98059, a specific MEK inhibitor, reversed the induced adipogenic differentiation. Genistein dose-dependently decreased the phosphorylation of ERK1/2 in mouse BMSC cultures. Genistein incubation for the entire culture period, as well as that applied during the early phase of the culture period, significantly inhibited the adipogenic differentiation of mouse BMSC cultures. While genistein was incubated at the late stage (after day 9), no inhibitory effect on adipogenic differentiation was observed. BMSC cultures treated with genistein in the presence of fibroblast growth factor-2 (FGF-2), an activator of the ERK1/2 signaling pathway, expressed normal levels of ERK1/2 activity, and, in so doing, are capable of undergoing adipogenesis. Our results suggest that activation of the ERK1/2 signaling pathway during the early phase of adipogenesis (from days 3 to 9) is essential to adipogenic differentiation of BMSC cultures, and that genistein inhibits the adipogenic differentiation through a potential downregulation of ERK1/2 activity at this early phase of adipogenesis.     

Choline dehydrogenase interacts with SQSTM1/p62 to recruit LC3 and stimulate mitophagy

CHDH (choline dehydrogenase) is an enzyme catalyzing the dehydrogenation of choline to betaine aldehyde in mitochondria. Apart from this well-known activity, we report here a pivotal role of CHDH in mitophagy. Knockdown of CHDH expression impairs CCCP-induced mitophagy and PARK2/parkin-mediated clearance of mitochondria in mammalian cells, including HeLa cells and SN4741 dopaminergic neuronal cells. Conversely, overexpression of CHDH accelerates PARK2-mediated mitophagy. CHDH is found on both the outer and inner membranes of mitochondria in resting cells. Interestingly, upon induction of mitophagy, CHDH accumulates on the outer membrane in a mitochondrial potential-dependent manner. We found that CHDH is not a substrate of PARK2 but interacts with SQSTM1 independently of PARK2 to recruit SQSTM1 into depolarized mitochondria. The FB1 domain of CHDH is exposed to the cytosol and is required for the interaction with SQSTM1, and overexpression of the FB1 domain only in cytosol reduces CCCP-induced mitochondrial degradation via competitive interaction with SQSTM1. In addition, CHDH, but not the CHDH FB1 deletion mutant, forms a ternary protein complex with SQSTM1 and MAP1LC3 (LC3), leading to loading of LC3 onto the damaged mitochondria via SQSTM1. Further, CHDH is crucial to the mitophagy induced by MPP+ in SN4741 cells. Overall, our results suggest that CHDH is required for PARK2-mediated mitophagy for the recruitment of SQSTM1 and LC3 onto the mitochondria for cargo recognition.     

Inhibition of extracellular signal-regulated kinase potentiates the apoptotic and antimetastatic effects of cyclin-dependent kinase inhibitors on metastatic DU145 and PC3 prostate cancer cells

Purvalanol and roscovitine are specific cyclin-dependent kinase (CDK) inhibitors, which have antiproliferative and apoptotic effects on various types of cancer. Although, the apoptotic accomplishment of purvalanol and roscovitine was elucidated at the molecular level, the underlying exact of drug-induced apoptosis through mitogen-activated protein kinase (MAPK) signaling still speculative. In addition, the role of CDK inhibitors in the downregulation of extracellular signal–regulated kinase 1/2 (ERK1/2)-mediated epithelial-mesenchymal transition (EMT) remains unclear. Here, we investigated the potential effect of each CDK inhibitors on cell proliferation, migration, and generation of reactive oxygen species due to the inhibition of MAPKs in metastatic DU145 and PC3 prostate cancer cells. We reported that purvalanol and roscovitine induced mitochondria membrane potential loss–dependent apoptotic cell death, which was also characterized by activation of several caspases, cleavage of poly (ADP-ribose) polymerase-1 in DU145 and PC3 cells. Cotreatment of either purvalanol or roscovitine with ERK1/2 inhibitor, U0126, synergistically suppressed cell proliferation, and induced apoptotic action. Also, ERK1/2 inhibition potentiated the effect of each CDK inhibitor on the downregulation of EMT processes via increasing the epithelial marker and decreasing mesenchymal markers through reduction of Wnt signaling regulators in DU145 cells. This study provides biological evidence about purvalanol and roscovitine have apoptotic and antimetastatic effects via MAPK signaling on prostate cancer cell by activation of GSK3β signaling and inhibition of phosphoinositide-3-kinase/AKT (PI3K/AKT) pathways involved in the EMT process.     

Choline dehydrogenase interacts with SQSTM1/p62 to recruit LC3 and stimulate mitophagy

CHDH (choline dehydrogenase) is an enzyme catalyzing the dehydrogenation of choline to betaine aldehyde in mitochondria. Apart from this well-known activity, we report here a pivotal role of CHDH in mitophagy. Knockdown of CHDH expression impairs CCCP-induced mitophagy and PARK2/parkin-mediated clearance of mitochondria in mammalian cells, including HeLa cells and SN4741 dopaminergic neuronal cells. Conversely, overexpression of CHDH accelerates PARK2-mediated mitophagy. CHDH is found on both the outer and inner membranes of mitochondria in resting cells. Interestingly, upon induction of mitophagy, CHDH accumulates on the outer membrane in a mitochondrial potential-dependent manner. We found that CHDH is not a substrate of PARK2 but interacts with SQSTM1 independently of PARK2 to recruit SQSTM1 into depolarized mitochondria. The FB1 domain of CHDH is exposed to the cytosol and is required for the interaction with SQSTM1, and overexpression of the FB1 domain only in cytosol reduces CCCP-induced mitochondrial degradation via competitive interaction with SQSTM1. In addition, CHDH, but not the CHDH FB1 deletion mutant, forms a ternary protein complex with SQSTM1 and MAP1LC3 (LC3), leading to loading of LC3 onto the damaged mitochondria via SQSTM1. Further, CHDH is crucial to the mitophagy induced by MPP+ in SN4741 cells. Overall, our results suggest that CHDH is required for PARK2-mediated mitophagy for the recruitment of SQSTM1 and LC3 onto the mitochondria for cargo recognition.     

Mitogen-activated protein kinase activity may not be necessary for the neuropathology of Niemann-Pick type C mice

Hyperphosphorylation of neurofilament and tau, and formation of cytoskeletal lesions, are notable features of several human neurodegenerative diseases, including Niemann-Pick Disease Type C (NPC). Previous studies suggested that the MAPKs, extracellular signal regulated kinase 1 and 2 (ERK1/2) may play a significant role in this aspect of NPC. To test this idea, we treated npc mice with PD98059, a specific and potent inhibitor of MAPK activation. Although activity of ERK1/2 was inhibited by 40%, a 2-week intracerebroventricular infusion of PD98059 just prior to onset of cytoskeletal pathology and symptoms in npc mice did not delay or inhibit prominent hallmarks of NPC. Unexpectedly, ERK1/2 inhibition led to aggravation of tau hyperphosphorylation, particularly in oligodendroctyes, in a manner similar to that of certain human tauopathies. Our results suggest that ERK1/2 does not play a major role in NPC neuropathology, and therefore, that MAPK inhibition is unlikely to be a useful strategy for managing the disease.     

Interaction between RING1 (R1) and the Ubiquitin-like (UBL) Domains Is Critical for the Regulation of Parkin Activity

Parkin is an E3 ligase that contains a ubiquitin-like (UBL) domain in the N terminus and an R1-in-between-ring-RING2 motif in the C terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at the Ser-65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at Ser-65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on phosphorylation at Ser-65, and interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, are suppressed by binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations associated with Parkinson disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by m-chlorophenyl hydrazone treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1 and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis.     

Activation of the MAPKs ERK1/2 by cell swelling in turbot hepatocytes

Background information. Activation of MAPKs (mitogen‐activated protein kinases), in particular ERK1/2 (extracellular‐signal‐regulated kinase 1/2), has been reported to take place in a large variety of cell types after hypo‐osmotic cell swelling. Depending on cell type, ERK1/2 phosphorylation can then serve or not the RVD (regulatory volume decrease) process. The present study investigates ERK1/2 activation after aniso‐osmotic stimulations in turbot hepatocytes and the potential link between phosphorylation of these proteins and RVD. Results. In turbot hepatocytes, Western‐blot analysis shows that a hypo‐osmotic shock from 320 to 240 mOsm·kg^?1 induced a rapid increase in ERK1/2 phosphorylation, whereas a hyper‐osmotic shock from 320 to 400 mOsm·kg^?1 induced no significant change in the phosphorylation of these proteins. The hypo‐osmotic‐induced ERK1/2 phosphorylation was significantly prevented when hypo‐osmotic shock was performed in the presence of the specific MEK (MAPK/ERK kinase) inhibitor PD98059 (100 μM). In these conditions, the RVD process was not altered, suggesting that ERK1/2 did not participate in this process in turbot hepatocytes. Moreover, the hypo‐osmotic‐induced activation of ERK1/2 was significantly prevented by breakdown of extracellular ATP by apyrase (10 units·ml^?1), by inhibition of purinergic P₂ receptors by suramin (100 μM) or by calcium depletion using EGTA (1 mM) and thapsigargin (1 μM). Conclusions. In turbot hepatocytes, hypo‐osmotic swelling but not hyper‐osmotic shrinkage induced the activation of ERK1/2. However, these proteins do not seem to be involved in the RVD process. Their hypo‐osmotic‐induced activation is partially due to cascades of signalling events triggered by the binding of released ATP on purinergic P₂ receptors and requires the presence of calcium.     

Myostatin regulates preadipocyte differentiation and lipid metabolism of adipocyte via ERK1/2

Myostatin is known as an inhibitor of muscle development, but its role in adipogenesis and lipid metabolism is still unclear, especially the underlying mechanisms. Here, we demonstrated that myostatin inhibited 3T3-L1 preadipocyte differentiation into adipocyte by suppressing C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome-proliferator-activated receptor γ), also activated ERK1/2 (extracellular-signal-regulated kinase 1/2). Furthermore, myostatin enhanced the phosphorylation of HSL (hormone-sensitive lipase) and ACC (acetyl-CoA carboxylase) in fully differentiated adipocytes, as well as ERK1/2. Besides, we noted that myostatin markedly raised the levels of leptin and adiponectin release and mRNA expression during preadipocyte differentiation, but the levels were inhibited by myostatin treatments in fully differentiated adipocytes. These results suggested that myostatin suppressed 3T3-L1 preadipocyte differentiation and regulated lipid metabolism of mature adipocyte, in part, via activation of ERK1/2 signalling pathway.     

Fibronectin promotes proplatelet formation in the human megakaryocytic cell line UT-7/TPO

We investigated PPF (proplatelet formation) in the human megakaryocytic cell line UT-7/TPO in vitro and signal transduction pathways responsible for PPF. The megakaryocytic cell lines are useful for studying megakaryocyte biology, although PPF is induced only in the presence of phorbol ester. TPO (thrombopoietin) stimulates megakaryocyte proliferation and differentiation; however, no PPF occurred in the megakaryocytic cell lines, even after the addition of TPO. Therefore, factors other than TPO may play an important role in the process of PPF. As PPF occurs in the bone marrow in vivo, we noted extracellular matrix proteins and found that soluble FN (fibronectin) induced potent PPF in UT-7/TPO without phorbol ester. A Western blot analysis showed that the expression of integrins was not increased by FN treatment. Anti-β1 antibody and the RGD (arginine-glycine-aspartate) peptide inhibited FN-induced PPF. This result indicates that the signal originated from integrin β1, which is essential to inducing PPF in UT-7/TPO. Results of the experiments using several inhibitors suggest that activation of the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]-ERK and PI3K (phosphoinositide 3-kinase) pathways are necessary for PPF. The phosphorylation of ERK gradually increased for 2 h after the addition of soluble FN, which suggests that activation of ERK is essential for the initial induction of FN-induced PPF in UT-7/TPO. UT-7/TPO is a useful cell line that enables us to study the signals of PPF without effects of chemical compounds.     

Ⅲ型纤连蛋白组件包含蛋白5通过抑制ERK1/2磷酸化抑制C3H10T1/2细胞成脂分化

旨在探究Ⅲ型纤连蛋白组件包含蛋白5(type Ⅲ domain-containing protein5,FNDC5)对C3H10T1/2细胞成脂分化的调控作用.利用qRT-PCR和Western印迹检测FNDC5在C3H10T1/2细胞成脂分化过程中的时序性表达规律;构建慢病毒包被的过表达/干扰FNDC5载体,转染C3...     

Protein kinase Cβ is a modulator of the dopamine D2 autoreceptor‐activated trafficking of the dopamine transporter

The strength and duration of extracellular dopamine concentrations are regulated by the presynaptic dopamine transporter (DAT) and dopamine D2 autoreceptors (D2autoRs). There is a functional interaction between these two proteins. Activation of D2autoRs increases DAT trafficking to the surface whereas disruption of this interaction compromises activities of both proteins and alters dopaminergic transmission. Previously we reported that DAT expression and activity are subject to modulation by protein kinase Cβ (PKCβ). Here, we further demonstrate that PKCβ is integral for the interaction between DAT and D2autoR. Inhibition or absence of PKCβ abolished the communication between DAT and D2autoR. In mouse striatal synaptosomes and transfected N2A cells, the D2autoR‐stimulated membrane insertion of DAT was abolished by PKCβ inhibition. Moreover, D2autoR‐stimulated DAT trafficking is mediated by a PKCβ‐extracellular signal‐regulated kinase signaling cascade where PKCβ is upstream of extracellular signal‐regulated kinase. The increased surface DAT expression upon D2autoR activation resulted from enhanced DAT recycling as opposed to reduced internalization. Further, PKCβ promoted accelerated DAT recycling. Our study demonstrates that PKCβ critically regulates D2autoR‐activated DAT trafficking and dopaminergic signaling. PKCβ is a potential drug target for correcting abnormal extracellular dopamine levels in diseases such as drug addiction and schizophrenia.     

Hippocalcin increases phospholipase D2 expression through extracellular signal-regulated kinase activation and lysophosphatidic acid potentiates the hippocalcin-induced phospholipase D2 expression

We have previously isolated a 22 kDa protein from a rat brain which was found to be involved in activating phospholipsae D (PLD), and identified the protein as hippocalcin through sequence analysis. Nevertheless, the function of hippocalcin for PLD activation still remains to be resolved. Here, we proposed that hippocalcin was involved in extracellular signal-regulated kinase (ERK)-mediated PLD2 expression. To elucidate a role of hippocalcin, we made hippocalcin transfected NIH3T3 cells and showed that the expression of PLD2 and basal PLD activity were increased in hippocalcin transfected cells. We performed PLD assay with dominant negative PLD2 (DN-PLD2) and hippocalcin co-transfected cells. DN-PLD2 suppressed increase of basal PLD activity in hippocalcin transfected cells, suggesting that increased basal PLD activity is due to PLD2 over-expression. Hippocalcin is a Ca2+-binding protein, which is expressed mainly in the hippocampus. Since it is known that lysophosphatidic acid (LPA) increases intracellular Ca2+, we investigated the possible role of hippocalcin in the LPA-induced elevation of intracellular Ca2+. When the intracellular Ca2+ level was increased by LPA, hippocalcin was translocated to the membrane after LPA treatment in hippocalcin transfected cells. In addition, treatment with LPA in hippocalcin transfected cells markedly potentiated PLD2 expression and showed morphological changes of cell shape suggesting that increased PLD2 expression acts as one of the major factors to cause change of cell shape by making altered membrane lipid composition. Hippocalcin-induced PLD2 expression potentiated by LPA in hippocalcin transfected cells was inhibited by a PI-PLC inhibitor, U73122 and a chelator of intracellular Ca2+, BAPTA-AM suggesting that activation of hippocalcin caused by increased intracellular Ca2+ is important to induce over-expression of PLD2. However, downregulation of PKC and treatment of a chelator of extracellular Ca2+, EGTA had little or no effect on the inhibition of hippocalcin-induced PLD2 expression potentiated by LPA in the hippocalcin transfected cells. Interestingly, when we over-express hippocalcin, ERK was activated, and treatment with LPA in hippocalcin transfected cells significantly potentiated ERK activation. Specific inhibition of ERK dramatically abolished hippocalcin-induced PLD2 expression. Taken together, these results suggest for the first time that hippocalcin can induce PLD2 expression and LPA potentiates hippocalcin-induced PLD2 expression, which is mediated by ERK activation.     

Regulation of nitric oxide production in snail (Lymnaea stagnalis) defence cells: a role for PKC and ERK signalling pathways

Background information. Nitric oxide (NO) is an important molecule in innate immune responses. In molluscs NO is produced by mobile defence cells called haemocytes; however, the molecular mechanisms that regulate NO production in these cells is poorly understood. The present study focused on the role of cell signalling pathways in NO production by primary haemocytes from the snail Lymnaea stagnalis. Results. When haemocytes were challenged with PMA (10 μM) or the β‐1,3‐glucan laminarin (10 mg/ml), an 8‐fold and 4‐fold increase in NO production were observed after 60 min respectively. Moreover, the NOS (NO synthase) inhibitors L‐NAME (N^G‐nitro‐L‐arginine methyl ester) and L‐NMMA (N^G‐monomethyl‐L‐arginine) were found to block laminarin‐ and PMA‐induced NO synthesis. Treatment of haemocytes with PMA or laminarin also increased the phosphorylation (activation) status of PKC (protein kinase C). When haemocytes were preincubated with PKC inhibitors (calphostin C or GF109203X) or inhibitors of the ERK (extracellular‐signal‐regulated kinase) pathway (PD98059 or U0126) prior to challenge, significant reductions in PKC and ERK phosphorylation and NO production were observed following exposure to laminarin or PMA. The greatest effect on NO production was seen with GF109203X and U0126, with PMA‐induced NO production inhibited by 94% and 87% and laminarin‐induced NO production by 50% and 91% respectively. Conclusions. These data suggest that ERK and PKC comprise part of the signalling machinery that regulates NOS activation and subsequent production of NO in molluscan haemocytes. To our knowledge, this is the first report that shows a role for these signalling proteins in the generation of NO in invertebrate defence cells.     

未成熟钠通道调控大鼠胚胎神经干细胞分化

钠通道在各类神经元上高表达,参与细胞多种生理功能的调节,是神经元实现功能活动的基本单位．未成熟神经元上钠/钙通道所诱发和自发的电位活动对后期的发育成熟至关重要．然而,发育中的钠通道是否参与神经干细胞(neural stem cells, NSCs)分化的调控尚不清楚．本研究证明,未成熟的钠通道参与NSCs分化调控．Western印迹结果显示,在分化第1,3,5,7 d的NSCs上钠通道和胞外信号调节激酶（ERK）的蛋白表达与分化时间正相关.免疫组化结果发现,与对照组比较,加入电压门控钠通道阻断剂TTX可明显下调NeuN、GFAP和Gal-c在NSCs中的表达（P＜0.05）,提示钠通道参与NSCs分化的调控.当采用veratridine激动钠通道后,激光共聚焦检测到细胞内Ca2+浓度明显升高,免疫组化和Western印迹结果显示细胞内Ca2+浓度明显升高,p-ERK表达量明显上调;相反,TTX可明显阻断Veratridine所引起的细胞内Ca2+浓度上调,并使p-ERK峰值明显降低和延后（P＜0.05）．研究结果表明,未成熟钠通道可通过激活ERK信号途径促进NSCs的分化．钠通道的这种作用可能是由钙离子介导的,其详尽机制有待进一步研究．     

Urotensin-2 promotes collagen synthesis via ERK1/2-dependent and ERK1/2-independent TGF-β1 in neonatal cardiac fibroblasts

U2 (urotensin-2) is the most potent vasoconstrictor in mammals which is involved in cardiac remodelling, including cardiac hypertrophy and cardiac fibrosis. Although the cellular mechanisms of the U2-induced vasoconstriction have been extensively studied, the signalling pathways involved in U2-induced TGF-β1 (transforming growth factor-β1) expression and collagen synthesis remain unclear. In this study, we show that U2 promoted collagen synthesis and ERK1/2 (extracellular signal-regulated kinase 1/2) activation in neonatal cardiac fibroblasts. The U2-induced collagen synthesis and TGF-β1 production were significantly but not completely inhibited by blocking ERK1/2. Both ERK1/2 inhibitor and TGF-β1 antibody could separately inhibit U2-induced collagen synthesis, and the synergistic inhibition effect was observed by blocking ERK1/2 and TGF-β1 simultaneously. These data suggest that U2 promotes collagen synthesis via ERK1/2-dependent and independent TGF-β1 pathway in neonatal cardiac fibroblasts.