TET3-mediated DNA demethylation modification activates SHP2 expression to promote endometrial cancer progression through the EGFR/ERK pathway.

OBJECTIVE: Src homology phosphotyrosin phosphatase 2 (SHP2) has been implicated in the progression of several cancer types. However, its function in endometrial cancer (EC) remains unclear. Here, we report that the ten-eleven translocation 3 (TET3)-mediated DNA demethylation modification is responsible for the oncogenic role of SHP2 in EC and explore the detailed mechanism. METHODS: The transcriptomic differences between EC tissues and control tissues were analyzed using bioinformatics tools, followed by protein-protein interaction network establishment. EC cells were treated with shRNA targeting SHP2 alone or in combination with isoprocurcumenol, an epidermal growth factor receptor (EGFR) signaling activator. The cell biological behavior was examined using cell counting kit-8, colony formation, flow cytometry, scratch assay, and transwell assays, and the median inhibition concentration values to medroxyprogesterone acetate/gefitinib were calculated. The binding of TET3 to the SHP2 promoter was verified. EC cells with TET3 knockdown and combined with SHP2 overexpression were selected to construct tumor xenografts in mice. RESULTS: TET3 and SHP2 were overexpressed in EC cells. TET3 bound to the SHP2 promoter, thereby increasing the DNA hydroxymethylation modification and activating SHP2 to induce the EGFR/extracellular signal-regulated kinase (ERK) pathway. Knockdown of TET3 or SHP2 inhibited EC cell malignant aggressiveness and impaired the EGFR/ERK pathway. Silencing of TET3 inhibited the tumorigenic capacity of EC cells, and ectopic expression of SHP2 or isoprocurcumenol reversed the inhibitory effect of TET3 knockdown on the biological activity of EC cells. CONCLUSION: TET3 promoted the DNA demethylation modification in the SHP2 promoter and activated SHP2, thus activating the EGFR/ERK pathway and leading to EC progression.

Long Non-Coding RNA TUG1 Attenuates Insulin Resistance in Mice with Gestational Diabetes Mellitus via Regulation of the MicroRNA-328-3p/SREBP-2/ERK Axis.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been illustrated to contribute to the development of gestational diabetes mellitus (GDM). In the present study, we aimed to elucidate how lncRNA taurine upregulated gene 1 (TUG1) influences insulin resistance (IR) in a high-fat diet (HFD)-induced mouse model of GDM. METHODS: We initially developed a mouse model of HFD-induced GDM, from which islet tissues were collected for RNA and protein extraction. Interactions among lncRNA TUG1/microRNA (miR)-328-3p/sterol regulatory element binding protein 2 (SREBP-2) were assessed by dual-luciferase reporter assay. Fasting blood glucose (FBG), fasting insulin (FINS), homeostasis model assessment of insulin resistance (HOMA-IR), HOMA pancreatic ß-cell function (HOMA-ß), insulin sensitivity index for oral glucose tolerance tests (ISOGTT) and insulinogenic index (IGI) levels in mouse serum were measured through conducting gain- and loss-of-function experiments. RESULTS: Abundant expression of miR-328 and deficient expression of lncRNA TUG1 and SREBP-2 were characterized in the islet tissues of mice with HFD-induced GDM. LncRNA TUG1 competitively bound to miR-328-3p, which specifically targeted SREBP-2. Either depletion of miR-328-3p or restoration of lncRNA TUG1 and SREBP-2 reduced the FBG, FINS, HOMA-ß, and HOMA-IR levels while increasing ISOGTT and IGI levels, promoting the expression of the extracellular signal-regulated kinase (ERK) signaling pathway-related genes, and inhibiting apoptosis of islet cells in GDM mice. Upregulation miR-328-3p reversed the alleviative effects of SREBP-2 and lncRNA TUG1 on IR. CONCLUSION: Our study provides evidence that the lncRNA TUG1 may prevent IR following GDM through competitively binding to miR-328-3p and promoting the SREBP-2-mediated ERK signaling pathway inactivation.

Central role for BRAF in cardiac hypertrophy: rethinking the pathological-physiological divide.

The RAF/MEK/ERK1/2 signaling cascade has been implicated in pathological cardiac hypertrophy downstream of some Gq-coupled receptors. The RAF family of kinases consists of three isoforms (ARAF, BRAF, and CRAF) and until recently most studies on this signaling pathway in the heart have focused on RAF1 (CRAF). In a recent issue of Clinical Science, Alharbi et al. utilized an inducible cardiac myocyte targeted knockout mouse model to define the role of BRAF in pathological versus physiological hypertrophy using angiotensin II and phenylephrine (PE) infusion, respectively. They reported that loss of BRAF attenuated both pathological cardiac hypertrophy and interstitial fibrosis. BRAF knockout decreased cardiac function with PE in male mice and enhanced both interstitial and perivascular cardiac fibrosis but had no effect on hypertrophy. In contrast, loss of BRAF attenuated physiological hypertrophy in female mice but had no effect on fibrosis or contractility. These observations extend those previously made by this group assessing the consequences of expressing an inducible activating mutant of BRAF in the heart and the benefit of enhancing RAF/MEK/ERK1/2 signaling by exploiting the 'RAF paradox'. Additional studies are needed to better define the role of BRAF under conditions reflective of chronic stress on the heart due to the biomechanical stimulation exerted by hypertension. In addition, the role of BRAF and its activation in overt heart failure remains to be established. Nevertheless, the new findings highlight the potential importance of additional signaling events, perhaps related to RAF1 or ERK1/2 activation, in shaping BRAF signaling in a sex- and context-dependent manner.

Ginsenoside Rb3 inhibits osteoclastogenesis via ERK/NF-κB signaling pathway in vitro and in vivo.

OBJECTIVE: The objective of the study was to determine the anti-osteoclastogenic potential of ginsenoside Rb3 for the treatment of periodontitis. METHODS: The anti-osteoclastogenic effect was determined using RANKL-induced RAW264.7 cells and murine bone marrow-derived macrophages followed by TRAP and phalloidin staining. Expression of osteoclastogenesis-related genes and proteins were examined by qPCR and WB. Activation of signaling pathways was detected by WB and IHC techniques. Experimental periodontitis rat model was built up by gingival injections of P. gingivalis LPS. After 21 days of Rb3 treatment, rats were sacrificed for micro-CT, IHC, H&E, and TRAP staining analyses. RESULTS: Rb3 dramatically inhibits RANKL-induced osteoclastogenesis. Nfatc1, Mmp9, Ctsk, Acp5 mRNA, and MMP9, CTSK proteins were dose-dependently downregulated by Rb3 pretreatment. WB results revealed that Rb3 suppressed activations of p38 MAPK, ERK, and p65 NF-κB, and the inhibition of ERK was most pronounced. Consistently, IHC analysis revealed that p-ERK was highly expressed in alveolar bone surface, blood vessels, odontoblasts, and gingival epithelia, which were notably suppressed by Rb3 treatment. H&E staining and micro-CT analyses showed that Rb3 significantly attenuated gingivitis and alveolar bone resorption in rats. CONCLUSION: Rb3 inhibits RANKL-induced osteoclastogenesis and attenuates P. gingivalis LPS-induced gingivitis and alveolar bone resorption in rats via ERK/NF-κB signaling pathway.

Effect of edaravone on ERK-CREB signaling pathway in hippocampus of aged rats with postoperative cognitive dysfunction / 中华麻醉学杂志

Objective:To evaluate the effect of edaravone on the extracellular signal-regulated kinase (ERK)-cAMP responsive element binding protein (CREB) signaling pathway in the hippocampus of aged rats with postoperative cognitive dysfunction (POCD).Methods:Sixty SPF healthy male Sprague-Dawley rats, aged 20 months, weighing 650-700 g, were divided into 4 groups ( n=15 each) using a random number table method: control group (group C), POCD group (group P), edaravone group (group E) and ERK inhibitor group (group I). The rats received laparotomy under 3% sevoflurane anesthesia to prepare POCD model in P, E and I groups. Edaravone 3 mg/kg was intraperitoneally injected at 30 min before operation in E and I groups, ERK inhibitor PD98059 0.3 mg/kg was injected via the tail vein in group I. The open field test was performed at 3 days after operation to evaluate the spontaneous activity of rats, then Morris water maze test was performed to evaluate the cognitive function of rats on 3-7 days after operation. The rats were sacrificed after the end of Morris water maze test, and hippocampal tissues were obtained for determination of the expression of phosphorylated ERK (p-ERK), phosphorylated CREB (p-CREB), synaptophysin and postsynaptic density protein 95 (PSD-95) (by Western blot) and dendrite length and density of dendrites in hippocampal CA1 area (using Golgi staining). Results:Compared with group C, the escape latency was significantly prolonged after operation, the number of crossing the original platform was reduced, the expression of p-ERK, p-CREB, synaptophysin and PSD-95 was down-regulated, and the dendritic length and density of hippocampal neurons were reduced in group P ( P<0.05). Compared with group P, the escape latency was significantly shortened, the number of crossing the original platform was increased, the expression of p-ERK, p-CREB, synaptophysin and PSD-95 was up-regulated, and the dendritic length and density of hippocampal neurons were increased in group E ( P<0.05). Compared with group E, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the expression of p-ERK, p-CREB, synaptophysin and PSD-95 was down-regulated, the dendritic length of hippocampal neurons was shortened, and the density of hippocampal neurons was decreased in group I( P<0.05). Conclusions:The mechanism by which edaravone improves POCD may be related to activating ERK/CREB signaling pathway and changing synaptic plasticity in hippocampal CA1 region in aged rats.

Thyroxine promotes the progression of integrin α vβ 3-positive differentiated thyroid cancer through the ERK1/2 pathway / 中华核医学与分子影像杂志

Objective:To explore whether thyroxine (T 4) could promote differentiated thyroid cancer (DTC) progression by binding to integrin α vβ 3in vitro and its downstream mechanism. Methods:Papillary thyroid cancer cell lines TPC-1, K1 and follicular thyroid cancer (FTC) cell line FTC133 were cultured in vitro, and the expressions of integrin α vβ 3 in those 3 DTC cell lines were determined with immunofluorescence and flow cytometry analysis. After the treatment of T 4, tetraiodo thyroacetic acid (Tetrac) and Arg-Gly-Asp (RGD) peptide alone or in combination, the proliferation and metastatic potential of DTC cell lines were detected by cell counting kit-8 (CCK-8), Transwell migration and invasion assays. The small interfering RNA (siRNA) transfection was used to verify whether integrin α v or β 3 subunit knockdown could reverse the effect of T 4 on DTC cells. The expression levels of downstream signaling proteins phosphorylated extracellular signal-regulated kinase (p-ERK)1/2 and total extracellular signal-regulated kinase (ERK)1/2 were detected by Western blot. The effects of mitogen-activated protein kinase kinase (MEK)1/2 inhibitor (GSK1120212) on the proliferation, migration and invasion of T 4-treated cells were detected. One-way analysis of variance and Tukey test were used for data analysis. Results:The integrin α vβ 3 expressions in TPC-1, K1 and FTC133 cells were all positive, with the relative mean fluorescence intensity (MFI) of 61.93±18.61, 16.89±2.43 and 32.36±0.83, and the percentages of positive cells of (94.38±1.30)%, (74.11±3.87)% and (50.67±1.78)%, respectively ( F values: 13.36 and 217.30, P=0.006 and P<0.001). Compared with control group, the proliferation, migration and invasion in the three DTC cell lines treated with T 4 were significantly enhanced (96 h, F values: 62.67-297.50, q values: 13.15-20.73, all P<0.001). T 4-induced cell proliferation, migration and invasion were markedly reversed by Tetrac or RGD (96 h, q values: 8.61-17.54, all P<0.001). T 4-induced cell proliferation, migration and invasion were also significantly inhibited by the knockdown of integrin α v or β 3 subunit (72 h, F values: 7.75-70.98, q values: 4.77-15.21, all P<0.05). Western blot results showed that the phosphorylation levels of ERK1/2 in DTC cells were significantly increased by T 4 treatment, and the T 4-induced activation of ERK1/2 signaling pathway could be blocked by Tetrac, RGD, integrin α v or β 3 subunit knockdown. T 4-induced cell proliferation, migration and invasion were significantly reversed by GSK1120212 (96 h, F values: 47.53-151.40, q values: 10.32-16.65, all P<0.001). Conclusion:T 4 can promote cell proliferation and metastasis of DTC cells by binding to integrin α vβ 3 and activating the ERK1/2 pathway.

The Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Pathway in Osteoblasts.

Extracellular signal-regulated kinases (ERKs) are evolutionarily ancient signal transducers of the mitogen-activated protein kinase (MAPK) family that have long been linked to the regulation of osteoblast differentiation and bone formation. Here, we review the physiological functions, biochemistry, upstream activators, and downstream substrates of the ERK pathway. ERK is activated in skeletal progenitors and regulates osteoblast differentiation and skeletal mineralization, with ERK serving as a key regulator of Runt-related transcription factor 2, a critical transcription factor for osteoblast differentiation. However, new evidence highlights context-dependent changes in ERK MAPK pathway wiring and function, indicating a broader set of physiological roles associated with changes in ERK pathway components or substrates. Consistent with this importance, several human skeletal dysplasias are associated with dysregulation of the ERK MAPK pathway, including neurofibromatosis type 1 and Noonan syndrome. The continually broadening array of drugs targeting the ERK pathway for the treatment of cancer and other disorders makes it increasingly important to understand how interference with this pathway impacts bone metabolism, highlighting the importance of mouse studies to model the role of the ERK MAPK pathway in bone formation.

Preliminary study on the expression of MIF in HCC tissues and its relationship with ERK1/2 signaling pathway / 中华内科杂志

Objective:To investigate the expression of Macrophage migration-inhibitory factors (MIF) in hepatocellular carcinoma (HCC) tissues and its interaction with ERK1/2 signaling pathway, so as to establish a theoretical basis for further studying the molecular mechanism of MIF promoting HCC.Methods:From February 2020 to August 2021, 52 cases of hepatocellular carcinoma (HCC) tissues based on hepatitis B cirrhosis (HBV-LC) and 52 cases of adjacent tissues in Tianjin Medical University Cancer Hospital and 940th Hospital of Joint Logistic Support Force of PLA were collected as the experimental group, including 39 males and 13 females, aged 35-65 years. And 20 cases of normal liver tissue were selected as the control group. Immunohistochemistry was used to detect the expression of MIF, ERK1/2 and p-ERK1/2 proteins in liver tissues of the two groups, and in situ hybridization was used to detect the expression of ERK1/2 nucleic acid in liver tissues of the two groups.HepG2 HCC cells and L-02 normal hepatocytes were co-cultured with different concentrations of rMIF, the expression and phosphorylation levels of ERK1/2 and JNK1 proteins in the two kinds of liver cells were detected by Western-blot, and the expression levels of ERK1/2 nucleic acids in the two kinds of liver cells were detected by RT-PCR. One-way ANOVA was used for measurement data and χ 2 test was used for counting data. Results:The expressions of MIF, ERK1/2, p-ERK1/2 and ERK1/2 mRNA were significantly increased in HCC and para-cancer tissues (the expression of MIF in HCC group was 78.8%, and that in adjacent group was 75.0%; ERK1/2 80.8% in HCC group and ERK1/2 71.8% in paracancerous group. The expression of p-ERK1/2 75.0 % in HCC group and 46.2% in paracancerous group were respectively detected. ERK1/2 mRNA was expressed in HCC group 76.9%, ERK1/2 mRNA expression in paracancerous group 78.8%), and the differences were statistically significant compared with normal liver tissues ( P<0.05), but there was no significant difference between HCC and para-cancer tissues ( P>0.05). The expressions of ERK1/2, p-ERK1/2 and ERK1/2 mRNA in HepG2 HCC cells were significantly increased with the increase of rMIF concentration, and the increase was most obvious when rMIF concentration was 200 ng/ml, and the difference was statistically significant compared with L-02 normal hepatocytes ( P<0.05). Conclusion:MIF, ERK1/2 and p-ERK1/2 are highly expressed in HCC tissues and HepG2 HCC cells, suggesting that MIF promotes the occurrence and development of hepatocellular carcinoma through ERK1/2 signaling pathway.

Effect of rat cardiac fibroblasts on expression of Cx43 in H9c2 cells during hypothermic hypoxia/reoxygenation / 中华麻醉学杂志

Objective:To evaluate the effect of rat cardiac fibroblasts (RCF) on the expression of connexin43 (Cx43) in H9c2 cells during hypothermic hypoxia/reoxygenation.Methods:H9c2 cells cultured in vitro were divided into 4 groups ( n=12 each) using the random number table method: control group (group C), hypothermic hypoxia/reoxygenation group (group HHR), RCF co-culture group (group Co) and RCF co-culture plus hypothermic hypoxia/reoxygenation group (group Co+ HHR). Group C was incubated at 37℃ in 5% CO 2 + 95% air for 5 h. Group HHR was incubated at 4 ℃ in 5% CO 2 + 95% N 2 for 1 h and then at 37 ℃ in 5% CO 2 + 95% air for 4 h. In group Co and group Co+ HHR, H9c2 cells 0.3×10 5 cells/well were inoculated in the lower chamber and RCF 0.6×10 5 cells/well in the the upper chamber of a transwell ? culture dish.Group Co was incubated at 37 ℃ in 5% CO 2 + 95% air for 5 h. Group Co+ HHR was incubated at 4℃ in 95% N 2 + 5% CO 2 for 1 h, and then incubated at 37 ℃ in 5% CO 2 + 95% air for 4 h. The mortality rate of H9c2 cells was measured by trypan blue staining, the expression of Cx43 and extracellular signal-regulated protein kinases 1/2 (ERK1/2) by immunofluorescence, and the expression of Cx43, phosphorylated Cx43, ERK1/2 and phosphorylated ERK1/2 by Western blot. Results:Compared with group C, the mortality rate of H9c2 cells was significantly increased, the expression and phosphorylation of Cx43 were decreased, and the expression and phosphorylation of ERK1/2 were increased in group HHR ( P<0.05), and no significant change was found in the mortality rate of H9c2 cells or expression and phosphorylation of Cx43 and ERK1/2 in group Co ( P>0.05). Compared with group Co, the mortality rate of H9c2 cells was significantly increased, and the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group Co+ HHR ( P<0.05). Compared with group HHR, the mortality rate of H9c2 cells was significantly increased, and the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group Co+ HHR ( P<0.05). Conclusions:RCFs can decrease the expression and activity of Cx43 in H9c2 cells during hypothermic hypoxia/reoxygenation, and the mechanism may be related to the down-regulation of ERK1/2 expression and inhibition of ERK1/2 activity.

CD38 deficiency up-regulated IL-1ß and MCP-1 through TLR4/ERK/NF-κB pathway in sepsis pulmonary injury.

As a disease with high mortality, many cytokines and signaling pathways are associated with sepsis. The pro-inflammatory cytokines and chemokines are participating in the pathogenesis of sepsis, especially in early stage. Moreover, the releases and expressions of cytokines are regulated by numerous signaling pathways, including TLR4/ERK pathway. But despite many studies have expounded the pathogenesis of sepsis and the regulation of cytokines in sepsis, how CD38 influence the expressions of related molecules in sepsis are still unknown. The aim of this study is illuminating the alteration of cytokines and signaling pathways in CD38-/- mice injected with Escherichia coli. Compared with WT mice, E. coli infection results in more severe pulmonary injuries and higher mRNA expressions of cytokines. Compared with E. coli infected WT mice, CD38 knockout leads to aggravated pulmonary injury, increased phosphorylated ERK1/2, p38 and NF-κB p65, and enhanced levels of IL-1ß, iNOS and MCP-1. While compared with E. coli infected CD38-/- mice, TLR4 mutation results in alleviated pulmonary injury, down-regulated phosphorylated ERK1/2 and NF-κB p65, and decreased expressions of IL-1ß and MCP-1. CD38 deficiency increased the expressions of IL-1ß andMCP-1 and aggravated pulmonary injury through TLR4/ERK/NF-κB pathway in sepsis.

The proper concentrations of dextrose and lidocaine in regenerative injection therapy: in vitro study.

BACKGROUND: Prolotherapy is a proliferation therapy as an alternative medicine. A combination of dextrose solution and lidocaine is usually used in prolotherapy. The concentrations of dextrose and lidocaine used in the clinical field are very high (dextrose 10%-25%, lidocaine 0.075%-1%). Several studies show about 1% dextrose and more than 0.2% lidocaine induced cell death in various cell types. We investigated the effects of low concentrations of dextrose and lidocaine in fibroblasts and suggest the optimal range of concentrations of dextrose and lidocaine in prolotherapy. METHODS: Various concentrations of dextrose and lidocaine were treated in NIH-3T3. Viability was examined with trypan blue exclusion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Migration assay was performed for measuring the motile activity. Extracellular signal-regulated kinase (Erk) activation and protein expression of collagen I and α-smooth muscle actin (α-SMA) were determined with western blot analysis. RESULTS: The cell viability was decreased in concentrations of more than 5% dextrose and 0.1% lidocaine. However, in the concentrations 1% dextrose (D1) and 0.01% lidocaine (L0.01), fibroblasts proliferated mildly. The ability of migration in fibroblast was increased in the D1, L0.01, and D1 + L0.01 groups sequentially. D1 and L0.01 increased Erk activation and the expression of collagen I and α-SMA and D1 + L0.01 further increased. The inhibition of Erk activation suppressed fibroblast proliferation and the synthesis of collagen I. CONCLUSIONS: D1, L0.01, and the combination of D1 and L0.01 induced fibroblast proliferation and increased collagen I synthesis via Erk activation.

Effect of remifentanil on MAPK signaling pathway during intestinal epithelial cell apoptosis induced by intestinal ischemia-reperfusion in rats / 中华麻醉学杂志

Objective:To evaluate the effect of remifentanil on mitogen-activated protein kinase (MAPK) signaling pathway during intestinal epithelial cell apoptosis induced by intestinal ischemia-reperfusion (I/R) in rats.Methods:Thirty-six clean grade healthy adult male Sprague-Dawley rats, weighing 200-250 g, aged 2 months, were divided into 3 groups ( n=12 each) by a random number table method: sham operation group (Sham group), intestinal I/R group (I/R group) and remifentanil group (R group). Intestinal I/R was produced by occlusion of superior mesenteric artery for 1 h followed by reperfusion in anesthetized rats.At 30 min before ischemia, 0.2 μg·kg -1·min -1 of remifentanil was infused intravenously for 5 min , followed by infusion of normal saline for 5 min, repeating for 3 cycles in group R. At 2 h of reperfusion, blood samples were collected from right ventricle to measure the concentration of diamine oxidase (DAO). The animals were then sacrificed and the intestinal tissues were obtained for examination of pathological changes and scored according to Chiu, for calculation of intestinal epithelial cell apoptosis rate (by TUNEL), for determination of the expression of phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated p38 MAPK (p-p38 MAPK), cleaved caspase-3 and nuclear factor kappa B p65 (NF-κB p65) in nucleoprotein and for calculation of p-ERK/ERK ratio, p-JNK/JNK ratio and p-p38 MAPK/p38 MAPK ratio in the intestinal tissues. Results:Compared with group Sham, Chiu′s scores, serum DAO concentration, apoptosis rate, p-ERK/ERK ratio, p-JNK/JNK ratio and p-p38 MAPK/p38 MAPK ratio and the expression of cleaved caspase-3 and NF-κB p65 in the intestinal tissues were significantly increased in group I/R, and Chiu′s scores was increased ( P<0.05), and no significant change was found in serum DAO concentration, apoptosis rate, p-ERK/ERK ratio, p-JNK/JNK ratio, p-p38 MAPK/p38 MAPK ratio and the expression of cleaved caspase-3 and NF-κB p65 in the intestinal tissues in group R ( P>0.05). Compared with group I/R, Chiu′s scores, apoptosis rate, serum DAO concentration, p-ERK/ERK ratio and expression of cleaved caspase-3 and NF-κB p65 were significantly decreased in group R ( P<0.05). Conclusion:The mechanism by which remifentanil inhibits intestinal epithelial cell apoptosis induced by intestinal I/R is related to promoting activation of ERK in rats.

Macrophage-Stimulating Protein Enhances Osteoblastic Differentiation via the Recepteur d'Origine Nantais Receptor and Extracellular Signal-Regulated Kinase Signaling Pathway.

BACKGROUND: Macrophage-stimulating protein (MSP; also known as macrophage stimulating 1 and hepatocyte growth factor-like protein) has been shown to play a crucial role in calcium homeostasis and skeletal mineralization in zebrafish. However, the precise role of MSP in osteoblasts has not been elucidated. In this study, we investigated the effect of MSP on osteoblast differentiation of pre-osteoblast cells. METHODS: Osteoblast differentiation upon MSP treatment was evaluated by analyzing the osteogenic gene expression, alkaline phosphatase (ALP) activity, and mineralized nodule formation. To assess changes in the MSP-RON signaling pathway, knockdown of Ron gene was performed using siRNA and pharmacological inhibitor treatment. RESULTS: Expression of the tyrosine kinase receptor RON, a receptor of MSP, was found to be significantly increased during osteoblast differentiation. MSP treatment significantly upregulated the expression of osteogenic marker genes and remarkably increased ALP activity and mineralized nodule formation. Conversely, knockdown of Ron significantly attenuated the expression of osteogenic marker genes and ALP activity that were induced upon MSP treatment. Mechanistically, MSP treatment significantly enhanced the phosphorylation of extracellular signal-regulated kinase (ERK); however, additional treatment with the selective ERK inhibitor PD98059 attenuated the effect of MSP on osteoblast differentiation. CONCLUSIONS: Altogether, these results indicate that the MSP-RON axis is involved in promoting osteoblast differentiation via activation of the ERK signaling pathway.

Oncogenic Ras downregulates mdr1b expression through generation of reactive oxygen species.

In the present study, we investigated the effect of oncogenic H-Ras on rat mdr1b expression in NIH3T3 cells. The constitutive expression of H-RasV12 was found to downregulate the mdr1b promoter activity and mdr1b mRNA expression. The doxorubicin-induced mdr1b promoter activity of the H-RasV12 expressing NIH3T3 cells was markedly lower than that of control NIH3T3 cells. Additionally, there is a positive correlation between the level of H-RasV12 expression and a sensitivity to doxorubicin toxicity. To examine the detailed mechanism of H-RasV12-mediated down-regulation of mdr1b expression, antioxidant N-acetylcysteine (NAC) and NADPH oxidase inhibitor diphenylene iodonium (DPI) were used. Pretreating cells with either NAC or DPI significantly enhanced the oncogenic H-Ras-mediated down-regulation of mdr1b expression and markedly prevented doxorubicin-induced cell death. Moreover, NAC and DPI treatment led to a decrease in ERK activity, and the ERK inhibitors PD98059 or U0126 enhanced the mdr1b-Luc activity of H-RasV12-NIH3T3 and reduced doxorubicin-induced apoptosis. These data suggest that RasV12 expression could downregulate mdr1b expression through intracellular reactive oxygen species (ROS) production, and ERK activation induced by ROS, is at least in part, contributed to the downregulation of mdr1b expression.

Activation of extracellular signal-regulated kinase is associated with hepatocellular carcinoma with aggressive phenotypes.

AIM: Sorafenib inhibits multiple kinase signaling pathways, including the rat sarcoma virus (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and is a promising therapy for hepatocellular carcinoma (HCC). However, the role of ERK activation in HCC remains unclear. This study was designed to investigate the potential link between ERK activation and aggressive HCC phenotypes. METHODS: We evaluated nuclear ERK expression by immunohistochemistry in 154 resected HCC nodules from 136 patients. We then investigated the associations of ERK expression with the clinicopathological characteristics of HCC, c-MET expression, and the molecular subclass biomarkers Ki-67, keratin 19 (KRT19, CK19, or K19), and sal-like protein 4. Multivariate Cox regression analysis was carried out to determine independent prognostic factors for overall survival and recurrence-free survival. The effects of ERK activation by hepatocyte growth factor (HGF) on eight HCC cell lines were further examined. RESULTS: High-level nuclear expression of ERK was observed in 20 (13%) of 154 nodules and was significantly associated with higher serum alpha-fetoprotein levels (P = 0.034), poorer differentiation (P = 0.003), a higher Ki-67 index (P < 0.001), high-level expression of c-MET (P = 0.008), KRT19 (P = 0.002), or sal-like protein 4 (P < 0.001), and shorter overall survival (multivariate hazard ratio 3.448; P = 0.028) and recurrence-free survival (multivariate hazard ratio 2.755; P = 0.004). HCC cells treated with hepatocyte growth factor showed enhanced cell proliferation together with ERK activation and upregulated KRT19 expression, both of which were inhibited by sorafenib. CONCLUSIONS: High-level ERK activation is associated with a KRT19-positive highly proliferative subtype of HCC with a dismal prognosis. These findings support the key role of the hepatocyte growth factor/c-MET/ERK axis in HCC progression.

Alterações moleculares no tumor marrom do hiperparatireoidismo dos maxilares / Molecular alterations in brown tumor of hyperparathyroidism of the jaws

Tumores marrons são lesões resultantes de alterações no metabolismo ósseo em decorrência do hiperparatireoidismo, uma das desordens endócrinas mais comuns no mundo. Os tumores marrons podem ocorrer no hiperparatireoidismo primário, secundário e terciário. Essas lesões ocorrem principalmente nos ossos longos, mas podem afetar os maxilares e apesar de apresentarem características clínicas e microscópicas bem estabelecidas, a patogênese molecular dos tumores marrons ainda não foi elucidada. Recentemente, mutações patogênicas nos genes TRPV4, FGFR1 e KRAS foram descritas em lesões de células gigantes dos maxilares e em fibroma não-ossificante dos ossos longos (FGFR1 e KRAS), lesões que são mímicas histológicas dos tumores marrons do hiperparatireoidismo. O objetivo do presente estudo foi investigar em tumores marrons dos maxilares a presença de mutações nesses genes. Para investigar tais mutações, foi realizado o sequenciamento de Sanger em uma amostra de conveniência composta por 13 tumores marrons dos maxilares associados ao hiperparatireoidismo primário e secundário. Sabendo-se que mutações nos genes analisados levam a ativação da via de sinalização celular MAPK/ERK, a detecção imuno-histoquímica da forma fosforilada da proteína ERK1/2 (pERK1/2) também foi avaliada nessas lesões. Mutações patogênicas no gene KRAS foram detectadas em sete casos (p.G12V n=4, p.G12D n=1, p.G13D n=1, p.A146T n=1). Variantes de significado incerto, p.A134T e p.E37K também foram detectadas. Todas as amostras exibiram sequências selvagens para os genes FGFR1 e TRPV4. A ativação da via MAPK/ERK foi demonstrada pela positividade imuno-histoquímica das células mononucleares dos tumores marrons para pERK1/2. Portanto, mutações no gene KRAS e ativação da via de sinalização celular MAPK/ERK foram detectadas em tumores marrons do hiperparatireoidismo dos maxilares. Tais resultados expandem o espectro de lesões de células gigantes cuja patogênese molecular envolve a sinalização RAS.

Brown tumors are lesions that result from abnormal bone metabolism in hyperparathyroidism, which is one of the most common endocrine disorders worldwide. Brown tumors can occur in primary, secondary and even in tertiary hyperparathyroidism. It occurs mainly in long bones, but occasionally affects the jaws and, despite its well-known clinical and microscopic features, the molecular pathogenesis of brown tumors remains unclear. Recently, pathogenic mutations in TRPV4, FGFR1 and KRAS were described in giant-cell lesions of the jaws and nonossifying fibromas of the bones (FGFR1 and KRAS), which are histologic mimics of brown tumors. The aim of this study was to investigate in brown tumors of the jaws the presence of mutations in these genes. To assess such mutations, a convenience sample of 13 brown tumors of the jaws associated with primary or secondary hyperparathyroidism was targeted by Sanger sequencing. As mutations in these genes are known to activate the MAPK/ERK signaling pathway, the immunostaining of the phosphorylated form of ERK1/2 (pERK1/2) was also assessed in these lesions. KRAS pathogenic mutations were detected in seven cases (p.G12V n=4, p.G12D n=1, p.G13D n=1, p.A146T n=1). KRAS variants of unknown significance, p.A134T and p.E37K, were also detected. All samples showed wild-type sequences for FGFR1 and TRPV4 genes. The activation of the MAPK/ERK signaling pathway was demonstrated by pERK1/2 immunohistochemical positivity of the brown tumors´ mononuclear cells. In conclusion, mutations in KRAS and activation of the MAPK/ERK signaling pathway were detected in brown tumors of hyperparathyroidism of the jaws, expanding the spectrum of giant cell lesions whose molecular pathogenesis involve RAS signaling

[Role and mechanism of nonreceptor tyrosine kinase Tec in endotoxin/lipopolysaccharide-induced interleukin-8 production in human alveolar epithelial cells A549].

Objective: To investigate the role and mechanism of nonreceptor tyrosine kinase Tec in the production of pro-inflammatory cytokine interleukin-8 (IL-8) induced by endotoxin/lipopolysaccharide (LPS) in human alveolar epithelial cells A549. Methods: Human alveolar epithelial cells A549 were routinely cultured and passaged in Roswell Park Memorial Institute-1640 medium containing 10% fetal bovine serum. The second or third passage of cells were collected for subsequent experiments. (1) Cells were collected and divided into 6 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in simple LPS group were routinely cultured for 1 h and then stimulated by 1 µg/mL LPS for 1 h. Cells in simple LFM-A13 group were cultured with conventional culture medium adding 75 µmol/L LFM-A13 for 1 h and then cultured with replaced conventional culture medium for 1 h. Cells in 25 µmol/L LFM-A13+ LPS group, 75 µmol/L LFM-A13+ LPS group, and 100 µmol/L LFM-A13+ LPS group were cultured with conventional culture medium adding 25, 75, and 100 µmol/L LFM-A13 respectively for 1 h and then all stimulated by 1 µg/mL LPS added into the replaced conventional culture medium for 1 h. The protein expression of Tec in cells of each group was detected by Western blotting, and the content of IL-8 in cell culture supernatant of each group was determined by enzyme-linked immunosorbent assay. (2) Cells were collected and divided into 5 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in small interfering RNA (siRNA) control+ LPS group were transfected with empty lentivirus for 10 h and then stimulated by 1 µg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec mus-298 RNA interference (RNAi)+ LPS group, Tec mus-299 RNAi+ LPS group, and Tec mus-300 RNAi+ LPS group were transfected with lentivirus loaded with Tec mus-298 RNAi, Tec mus-299 RNAi, and Tec mus-300 RNAi respectively for 10 h and then stimulated by 1 µg/mL LPS added into the conventional culture medium for 2 h. The protein expression of Tec in cells of each group was detected by Western blotting to screen Tec-siRNA with the best silencing effect on Tec gene. (3) Cells were collected and divided into 4 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in virus control group were transfected with empty lentivirus for 10 h and then routinely cultured for 2 h. Cells in simple LPS group were stimulated by 1 µg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec-siRNA+ LPS group were transfected with lentivirus loaded with Tec-siRNA with the best silencing effect on Tec gene for 10 h and then stimulated by 1 µg/mL LPS added into the conventional culture medium for 2 h. The protein expressions of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) MAPK of cells in each group were detected by Western blotting. Data were processed with one-way analysis of variance and the least significant difference-t test. Results: (1) Compared with that of blank control group, the protein expression of Tec of cells in simple LPS group was obviously increased (t=9.72, P<0.05), but the protein expression of Tec of cells in simple LFM-A13 group was not obviously changed (t=4.31, P=0.05). Compared with that of simple LPS group, the protein expression of Tec of cells in 25 µmol/L LFM-A13+ LPS group, 75 µmol/L LFM-A13+ LPS group, or 100 µmol/L LFM-A13+ LPS group was obviously decreased (t=9.72, 9.07, 16.33, P<0.05 or P<0.01). Compared with (189±22) pg/mL of blank control group, the content of IL-8 in culture supernatant of cells in simple LPS group was obviously increased [(214±10) pg/mL, t=2.18, P<0.05], but the content of IL-8 in culture supernatant of cells in simple LFM-A13 group was not obviously changed [(173±43) pg/mL, t=0.64, P>0.05]. Compared with that of simple LPS group, the content of IL-8 in culture supernatant of cells in 25 µmol/L LFM-A13+ LPS group was not obviously changed [(204±38) pg/mL, t=0.54, P>0.05], but the content of IL-8 in culture supernatant of cells in 75 µmol/L LFM-A13+ LPS group and 100 µmol/L LFM-A13+ LPS group was obviously decreased [(144±44), (137±51) pg/mL, t=3.63, 2.55, P<0.05 or P<0.01]. (2) Compared with that of blank control group, the protein expression of Tec of cells in siRNA control+ LPS group was obviously increased (t=14.24, P<0.01). Compared with that of siRNA control+ LPS group, the protein expression of Tec of cells in Tec mus-298 RNAi+ LPS group or Tec mus-299 RNAi+ LPS group was obviously decreased (t=36.03, 18.23, P<0.01), but the protein expression of Tec of cells in Tec mus-300 RNAi+ LPS group was not obviously changed (t=4.08, P>0.05). The protein expression of Tec was the lowest in cells of Tec mus-298 RNAi+ LPS group, so Tec mus-298 RNAi was used in subsequent experiment. (3) Compared with 1.16±0.16 and 0.78±0.11 of blank control group, the protein expressions of p38 MAPK and ERK MAPK of cells in virus control group were not obviously changed (1.66±0.13, 0.89±0.11, t=11.09, 3.60, P>0.05), but the protein expressions of p38 MAPK and ERK MAPK of cells in simple LPS group were obviously increased (2.83±0.29, 1.86±0.37, t=9.70, 7.23, P<0.05). Compared with those of simple LPS group, the protein expression of p38 MAPK and protein expression of ERK MAPK of cells in Tec-siRNA+ LPS group were obviously decreased (0.69±0.16, 1.03±0.24, t=13.78, 4.12, P<0.05 or P<0.01). Conclusions: Tec may mediate the production and release of pro-inflammatory cytokine IL-8 from human alveolar epithelial cells A549 induced by LPS via the p38 MAPK and ERK MAPK signal pathways.

Suppression of CD81 promotes bladder cancer cell invasion through increased matrix metalloproteinase expression via extracellular signal-regulated kinase phosphorylation.

Purpose: CD81 is a prognostic biomarker for high-grade bladder cancer (BC). In this study, we aimed to determine the functional mechanisms underlying the role of CD81 in BC progression. Materials and Methods: In two invasive BC cell lines (T24, J82), CD81 expression was suppressed by the transfection of lentiviral vectors including CD81-specific shRNAs, and then the migration and invasion of BC cells was analyzed. Enzymatic activity of matrix metalloproteinases (MMPs) was also analyzed by collagen-zymography. The expression of MMPs was confirmed by western blotting using culture supernatants from each cell line. Signaling pathways related to MMPs were investigated using various antibodies. Results: CD81 was successfully knocked down by shRNAs in T24 and J82 cell lines. While the migration of BC cells was not affected after the knockdown of CD81, the invasive activity was significantly increased in both cell lines. Zymography produced distinct bands using supernatants from CD81-knockdown cells, whereas only faint bands were observed with empty vector-transfected cells. We also observed an increased expression of MMPs, specifically MMP2 and 9, in the conditioned media from CD81-knockdown cells by western blotting. Mechanistically, the phosphorylation of extracellular signal-regulated kinase (ERK) was associated with the invasive activity of BC cells, while U0126 (an ERK inhibitor) reduced the invasive activity of CD81-knockdown BC cells. Conclusions: Taken together, CD81 suppression promotes the invasive property of BC cells through MMP signaling via ERK phosphorylation. Our results suggest that the regulation of CD81 expression may have some therapeutic potential in BC.

Intermittent parathyroid hormone promotes cementogenesis in a PKA- and ERK1/2-dependent manner.

BACKGROUND: Intermittent parathyroid hormone (PTH) promotes cementogenesis and provides a promising biotherapeutic to rehabilitate resorbed roots. However, the underlying mechanisms remain inconclusive. Cyclic aenosine monophosphate (AMP)-dependent protein kinases A (PKA) and extracellular signal-regulated MAP kinases 1/2 (ERK1/2) are key regulators of bone remodeling. The present study aims to investigate whether PKA and ERK1/2 are involved in the process of intermittent PTH-promoted cementogenesis. METHODS: Sprague-Dawley rats in experimental group (n = 30) received a daily subcutaneous injection of PTH and the control (n = 30) received placebo vehicle for 1, 2, 3, 4, and 5 weeks. Results were evaluated by hematoxylin and eosin and immunohistochemistry staining. In vitro, OCCM-30 cells were incubated with intermittent PTH. H89 and U0126 were used to determine the role of PKA and ERK1/2, respectively. The cementogenic results were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), western blotting, alkaline phosphatase activity assay and Alizarin Red S staining. The interaction of PKA and p-ERK1/2 was determined by co-immunoprecipitation (Co-IP). RESULTS: Intermittent PTH exerted anabolic effect on cellular cementum in developing teeth with elevated expression of osteocalcin, osteopontin, and PKA (catalytic subunit) in PTH injection group. The promoting effects of intermittent PTH on cementogenesis and osteogenic differentiation were abrogated by H89 and U0126 in vitro, respectively. Blocking of PKA pathway downregulated intermittent PTH-induced ERK1/2 phosphorylation. CONCLUSIONS: Intermittent PTH promotes cementogenesis in a PKA- and ERK1/2-dependent manner. In this process, PKA and p-ERK1/2 interact with each other. These results support the future biotherapeutic applications of PTH in cementum resorption.

Dexmedetomidine attenuates the neurotoxicity of propofol toward primary hippocampal neurons in vitro via Erk1/2/CREB/BDNF signaling pathways.

BACKGROUND: Propofol is a commonly used general anesthetic for the induction and maintenance of anesthesia and critical care sedation in children, which may add risk to poor neurodevelopmental outcome. We aimed to evaluate the effect of propofol toward primary hippocampal neurons in vitro and the possibly neuroprotective effect of dexmedetomidine pretreatment, as well as the underlying mechanism. MATERIALS AND PROCEDURES: Primary hippocampal neurons were cultured for 8 days in vitro and pretreated with or without dexmedetomidine or phosphorylation inhibitors prior to propofol exposure. Cell viability was measured using cell counting kit-8 assays. Cell apoptosis was evaluated using a transmission electron microscope and flow cytometry analyses. Levels of mRNAs encoding signaling pathway intermediates were assessed using qRT-PCR. The expression of signaling pathway intermediates and apoptosis-related proteins was determined by Western blotting. RESULTS: Propofol significantly reduced cell viability, induced neuronal apoptosis, and downregulated the expression of the BDNF mRNA and the levels of the phospho-Erk1/2 (p-Erk1/2), phospho-CREB (p-CREB), and BDNF proteins. The dexmedetomidine pretreatment increased neuronal viability and alleviated propofol-induced neuronal apoptosis and rescued the propofol-induced downregulation of both the BDNF mRNA and the levels of the p-Erk1/2, p-CREB, and BDNF proteins. However, this neuroprotective effect was abolished by PD98059, H89, and KG501, further preventing the dexmedetomidine pretreatment from rescuing the propofol-induced downregulation of the BDNF mRNA and p-Erk1/2, p-CREB, and BDNF proteins. CONCLUSION: Dexmedetomidine alleviates propofol-induced cytotoxicity toward primary hippocampal neurons in vitro, which correlated with the activation of Erk1/2/CREB/BDNF signaling pathways.