Exendin-4 promotes proliferation of adipose-derived stem cells through ERK and JNK signaling pathways.

The expression of ERK and JNK in patients with an endemic osteochondropathy, Kashin-Beck disease

A Feedback Loop between Androgen Receptor and ERK Signaling in Estrogen Receptor-Negative Breast Cancer

BackgroundHepatocellular carcinoma (HCC) is now the second leading cause of cancer death worldwide and lacks effectual therapy due to its high rate of tumor recurrence and metastasis. The aim of this study was to investigate the effects of L antigen family member 3 (LAGE3, a member of the LAGE gene family involved in positive transcription) on the progression of HCC.MethodsThe expression of LAGE3 was detected by quantitative real-time polymerase chain reaction. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation assay, EdU, and cell cycle analysis assay were employed to evaluate the proliferation of HCC cells. Annexin V-FITC/PI and TUNEL assay were used to assess the apoptosis rate of HCC cells. Wound healing and transwell assay were used to investigate the migration and invasion of HCC cells. A xenograft model of HCC was established with 2 × 106 Hep3B or SK-HEP1 cells to investigate the in vivo effects of LAGE3. Then, the protein levels of LAGE3, p-p38, p-38, c-Jun N-terminal kinase (JNK),p-JNK, extracellular signal-regulated kinase (ERK), and p-ERK were detected by western blot.ResultsWe found that LAGE3 was upregulated in HCC tissues compared to adjacent tissues, and its high expression was correlated with poor overall survival by bioinformatics analysis. Next, we manually regulated the expression of LAGE3 in HCC cells. The knockdown of LAGE3 inhibited the proliferation of HCC cells by arresting the cell cycle in G1 phase. Also the downregulation of LAGE3 inhibited cell migration and invasion and induced apoptosis of HCC cells, while overexpression of LAGE3 promoted the malignant phenotypes of HCC. These results were further confirmed by the in vivo growth of HCC xenografts and the inhibition of apoptosis of HCC tumor cells. Furthermore, we found that LAGE3 exerted cancer-promoting effects by potentiating the JNK and ERK signaling pathway. An ERK inhibitor (10 μM SCH772984) or JNK inhibitor (25 μM SP600125) repressed the upregulated LAGE3-induced proliferation, migration, and invasion of HCC cells.ConclusionsLAGE3 enhanced the malignant phenotypes of HCC by promoting the JNK and ERK signaling pathway.

Many natural and synthetic cancer chemopreventive compounds are potent inducers of phase II detoxifying and antioxidant stress responsive genes. The phase II/antioxidant gene expression plays critical role in chemoprevention of carcinogenesis. The antioxidant responsive element (ARE), located on many phase II/antioxidant genes, binds with the transcription factor Nrf2, and is required for the activation of these phase II/antioxidant gene expression induced by many natural and synthetic cancer chemopreventive compounds. In this study, we investigated the potential roles of extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) in the regulation of butylated hydroxyanisole (BHA)-induced and Nrf2-dependent ARE transcriptional activity and ARE-mediated endogenous heme oxygenase-1 (HO-1) protein expression in HepG2 cells. ARE transcriptional activity and HO-1 protein expression were increased dose dependently after treatment with BHA in HepG2 cells. Dose-response and time-course experiments showed that BHA increased the accumulation of Nrf2, and concomitantly decreased the protein level of Keap1. We next examined the phosphorylation of the MAPKs, and found that BHA significantly increased the phosphorylation levels of ERK1/2 and JNK1/2. Importantly BHA-induced ARE transcriptional activity was attenuated by the inhibition of ERK and JNK signaling using biochemical inhibitors and their dominant-negative mutants. Using confocal microscopy technique, treatment with BHA showed the release of Nrf2 sequestered by Keap1 in the cytosol, and that Nrf2 translocated into the nucleus. Importantly, cDNA transfections of ERK and JNK signaling pathways similarly released Nrf2 from Keap1 cytosolic sequestration and translocating Nrf2 into the nucleus. Taken together, these results strongly suggested that ERK and JNK signaling pathways played important and positive roles in BHA-induced and Nrf2-dependent regulation of ARE-mediated gene expression, as well as the nuclear translocation of Nrf2 in HepG2 cells.

Odonto/osteogenic differentiation of stem cells from the apical papilla (SCAPs) is a key process in tooth root formation and development. However, the molecular mechanisms underlying this process remain largely unknown. In the present study, it was identified that guanine and nucleotide binding protein 3 (GNAI3) was at least in part responsible for the odonto/osteogenic differentiation of SCAPs. GNAI3 was markedly induced in mouse tooth root development in vivo and in human SCAPs mineralization in vitro. Notably, knockdown of GNAI3 by lentiviral vectors expressing short-hairpin RNAs against GNAI3 significantly inhibited the proliferation, cell cycle progression and migration of SCAPs, as well as odonto/osteogenic differentiation of SCAPs in vitro, suggesting that GNAI3 may play an essential role in tooth root development. The promotive role of GNAI3 in odonto/osteogenic differentiation was further confirmed by downregulation of odonto/osteogenic makers in GNAI3-deficient SCAPs. In addition, knockdown of GNAI3 effectively suppressed activity of c-Jun N-terminal kinase (JNK) and extracellular-signal regulated kinase (ERK) signaling pathways that was induced during SCAPs differentiation, suggesting that GNAI3 promotes SCAPs mineralization at least partially via JNK/ERK signaling. Taken together, the present results implicate GNAI3 as a critical regulator of odonto/osteogenic differentiation of SCAPs in tooth root development, and suggest a possible role of GNAI3 in regeneration processes in dentin or other tissues.

BackgroundThe extracellular signal-regulated kinase (ERK) signaling pathway is considered to be associated with the pathogenesis and treatment of depression. Acupuncture has been demonstrated to ameliorate depression-related behavior and promote neurogenesis. In this study, we explored the role of the ERK signaling pathway in the antidepressant-like effects of acupuncture in rats exposed to chronic unpredictable mild stress (CUMS).MethodsEighty male Sprague–Dawley rats were randomly divided into eight groups: control group, model group, model + Acupuncture group (Acu group), model + fluoxetine group (FLX group), model + DMSO group (DMSO group), model + PD98059 group (PD group), model + Acupuncture + PD98059 group (Acu + PD group) and model + fluoxetine + PD98059 group (FLX + PD group). Except for the control group, all rats were subjected to 3 weeks of CUMS protocols to induce depression. Acupuncture was carried out for 10 min at acupoints of Baihui (GV-20) and Yintang (GV-29) each day during the experimental procedure. The ERK signaling pathway was inhibited using PD98059 through intracerebroventricular injection. The depression-like behaviors were evaluated using the sucrose intake and open-field tests. The protein levels of ERK1/2, phosphor (p)-ERK1/2, cAMP response element-binding protein (CREB), p-CREB and brain-derived neurotrophic factor (BDNF) in the hippocampus were examined using western blot.ResultsAcupuncture ameliorated the depression-like behaviors and dysfunction of the ERK signaling pathway in the hippocampus of CUMS rats. PD98059 pretreatment inhibited the improvements brought about by acupuncture on the ERK signaling pathway.ConclusionsTaken together, our results indicated that acupuncture had a significant antidepressant-like effect on CUMS-induced depression model rats, and the ERK signaling pathway was implicated in this effect.

Tuberous sclerosis complex (TSC) may not receive as much attention as Down syndrome or other genetic diseases that affect brain development. But as its lengthy name suggests, it is a multifaceted disorder that can have wide-ranging effects on an individual's life. TSC affects about 1 in 6,000 people, causing seizures, mental retardation, and benign tumors in the brain and other organs. Moreover, about one-third of children with TSC meet criteria for autism spectrum disorder. TSC is caused by mutations in two genes known as TSC1 and TSC2, leading to increased activity in the mammalian target of rapamycin (mTOR) signaling pathway, which regulates neuronal signaling and other important cellular processes. Whereas normal adult mice use the mTOR pathway for the generation of Long Term Depression (LTD) after stimulation of metabotropic Glutamate Receptor 5 (mGluR5), TSC2 mutant mice sidestep the tuberous sclerosis mutation by invoking the Erk signaling pathway. ... Although mTOR inhibitors and other drugs can control symptoms, such as tumors and seizures, these medications sometimes cause serious side effects. A better understanding of the underlying causes of the disorder could lead to the development of more effective treatments. As reported in this issue of PLOS Biology, Avtar Roopra of the University of Wisconsin-Madison and his team have made tremendous strides toward this goal by revealing novel signaling pathways involved in TSC. The researchers found that adult mice with a TSC2 mutation make use of unusual signaling pathways for long-term depression (LTD)—a cellular process that normally relies on the mTOR pathway and is thought to be important for learning and memory. Drug treatments that inhibited these overactive pathways fixed the molecular abnormality, reduced seizure-like activity in brain slices, and corrected an autistic-like behavioral problem in the mutant mice. These findings open up promising new avenues for the treatment of a wide range of TSC symptoms. Previous studies had shown that juvenile mice with a TSC2 mutation exhibit reduced LTD in the hippocampus—a brain region crucial for learning and memory. In the new study, Roopra and his team found that adult mice with the same mutation show a normal degree of LTD. Instead of relying on mTOR signaling, however, these adult mutant mice normalized their LTD through increased activation of the metabotropic glutamate receptor 5 (mGluR5) and extracellular signal-regulated kinases (ERK) signaling pathways. While activity in the mGluR5 and ERK pathways decreased in normal mice as they become adults, these pathways remained very active in adult mutant mice. The findings suggest that adult mice with the TSC2 mutation achieve a normal degree of LTD by activating compensatory pathways that are not normally involved in this cellular process. When the researchers inhibited ERK and mGluR5 signaling in hippocampal cells, LTD became dependent on mTOR signaling and seizure-like activity in the cells decreased. Roopra and his team next tested whether the mGluR5 pathway also underlies behavioral problems that characterize TSC. The investigators placed normal and mutant mice in a pool of water with a platform hidden just below the surface. Once the mice learned the location of the platform, the researchers moved it to another spot. Compared to normal mice, mutant mice were much more likely to swim to the original location of the hidden platform. This behavior resembles repetitive, autistic-like patterns that are common among individuals with TSC. Treatment with an mGluR5 inhibitor corrected the behavioral abnormality in mutant mice, enhancing their ability to find the hidden platform in new locations. Taken together, the findings suggest that abnormalities in ERK and mGluR5 signaling are at the root of a variety of TSC symptoms. By revealing that the signaling pathways affected by TSC change with age, the study sheds new light on the complexity of the disorder, reconciles conflicting findings from different laboratories about the role of the mTOR pathway in TSC, and highlights the importance of characterizing age-dependent effects to reach more reliable conclusions. The work also suggests that disruptions in the mGluR5 pathway may be a common mechanism underlying a range of neurodevelopmental disorders, including fragile X syndrome—the most common known cause of inherited intellectual disability. Moreover, the findings suggest that drugs targeting the mGluR5 and ERK signaling pathways could represent a novel and promising strategy to treat seizures and behavioral problems in TSC patients. Potter WB, Basu T, O'Riordan KJ, Kirchner A, Rutecki P, et al. (2013) Reduced Juvenile Long-Term Depression in Tuberous Sclerosis Complex Is Mitigated in Adults by Compensatory Recruitment of mGluR5 and Erk Signaling. doi:10.1371/journal.pbio.1001627

Prevalent mutations in the mitogen-activated protein kinase 1 (MAPK1)/extracellular signal-regulated kinase 2 (ERK2) pathway have been identified in cervical squamous cell carcinoma in a large-scale genome sequencing effort. Furthermore, mutations in the rat sarcoma viral oncogene homolog (RAS)/Raf/Mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway have also been revealed to have important roles in the pathogenesis of human cancer. However, whether the potential hotspot mutations in ERK2 and other components of the RAS/RAF/MEK/ERK signaling pathway also exist in Chinese patients with cervical carcinoma remains to be elucidated. In the present study, a total of 260 patients with cervical carcinoma of distinct subtypes were analyzed for the presence of potential hotspot mutations in the RAS/RAF/MEK/ERK signaling pathway. No ERK2 mutations were detected in these samples; however, Kirsten RAS (KRAS) p.G12D (c.35G>A) mutation was identified in 2/26 (7.7%) cervical adenocarcinoma cases, including 1/20 cervical mucinous adenocarcinoma and 1/6 cervical endometrioid carcinoma cases. In addition, no mutations in the ERK1, neuroblastoma RAS, Harvey RAS or B-Raf proto-oncogene serine/threonine kinase genes were detected in the present study. These results indicated that ethnic differences may be a primary reason for the discrepancy in ERK2 mutation frequencies between the current study and previous studies. Furthermore, mutation in the KRAS gene, but not other genes in the RAS/RAF/MEK/ERK signaling pathway, may have an active role in the pathogenesis of cervical carcinoma.

Nephrolithiasis plagues a great number of patients all over the world. Increasing evidence shows that the extracellular signal-regulated kinase (ERK) signaling pathway and renal tubular epithelial cell (RTEC) dysfunction and attrition are central to the pathogenesis of kidney diseases. Mucin 4 (MUC4) is reported as an activator of ERK signaling pathway in epithelial cells. In this study, using rat models of calcium oxalate (CaOx) nephrolithiasis, the present study aims to define the roles of MUC4 and ERK signaling pathway as contributors to oxidative stress and CaOx crystal formation in RTEC. Data sets of nephrolithiasis were searched using GEO database and a heat flow map was drawn. Then MUC4 function was predicted. Wistar rats were prepared for the purpose of model establishment of ethylene glycol and ammonium chloride induced CaOx nephrolithiasis. In order to assess the detailed regulatory mechanism of MUC4 silencing on the ERK signaling pathway and RTEC, we used recombinant plasmid to downregulate MUC4 expression in Wistar rat-based models. Samples from rat urine, serum and kidney tissues were reviewed to identify oxalic acid and calcium contents, BUN, Cr, Ca2+ and P3+ levels, calcium crystal formation in renal tubules and MUC4 positive expression rate. Finally, RT-qPCR, Western blot analysis, and ELISA were employed to access oxidative stress state and CaOx crystal formation in RTEC. Initially, MUC4 was found to have an influence on the process of nephrolithiasis. MUC4 was upregulated in the CaOx nephrolithiasis model rats. We proved that the silencing of MUC4 triggered the inactivation of ERK signaling pathway. Following the silencing of MUC4 or the inhibition of ERK signaling pathway, the oxalic acid and calcium contents in rat urine, BUN, Cr, Ca2+ and P3+ levels in rat serum, p-ERK1/2, MCP-1 and OPN expressions in RTEC and H2O2 and MDA levels in the cultured supernatant were downregulated, but the GSH-Px, CAT and SOD levels in the cultured supernatant were increased. Moreover, MUC4 silencing or ERK signaling pathway inactivation may decrease the formation of CaOx crystals. Taken together, silencing of MUC4 can inactivate the ERK signaling pathway and further restrain oxidative stress and CaOx crystal formation in RTEC. Thus, MUC4 represents a potential investigative focus target in nephrolithiasis.

Objective To evaluate the role of extracellular signal-regulated kinase (ERK) signaling pathway in intrathecal dexmedetomidine-induced reduction of spinal cord ischemia-reperfusion (I/R) injury in rats. Methods Eighty clean-grade male Sprague-Dawley rats, aged 9-10 weeks, weighing 300-350 g, were divided into 4 groups (n=20 each) using a random number table method: sham operation group (group S), spinal cord I/R group (group I/R), dexmedetomidine group (group D), and dexmedetomidine plus ERK signaling pathway blocker PD98059 group (group P). Spinal cord ischemia was produced by cross-clamping of the abdominal aorta distal to the left renal artery for 25 min followed by reperfusion to establish the model of spinal cord I/R injury.Dexmedetomidine 1 μg/kg was intrathecally injected at 20 min before establishing the model in D and P groups, PD98059 2 mg/kg was given via the tail vein at the same time in group P, and the equal volume of normal saline was given instead in S and I/R groups.Five rats were selected at 6, 8, 10 and 12 h of reperfusion, and the modified Basso, Beattie, Bresnahan (BBB) scale was used to assess the hindlimb locomotor function.Five rats were sacrificed after assessing the locomotor function at 6 h of reperfusion, and the L3-5 segments of the spinal cord were taken for determination of cell apoptosis (by TUNEL) and expression of phosphorylated ERK (p-ERK) (by Western blot). The apoptosis index was calculated. Results Compared with group S, the BBB scores were significantly decreased at each time point of reperfusion, the apoptosis index was increased, and the expression of p-ERK was up-regulated in the other three groups (P 0.05). Compared with group D, the BBB scores were significantly decreased at each time point of reperfusion, the apoptosis index was increased, and the expression of p-ERK was down-regulated in group P (P<0.05). Conclusion The mechanism by which intrathecal dexmedetomidine reduces spinal cord I/R injury is related to activating ERK signaling pathway in rats. Key words: Dexmedetomidine; Reperfusion injury; Spinal cord; Extracellular signal-regulated MAP kinases

Rho-associated kinase (ROCK) is a key regulatory protein involved in inflammatory secretion in microglia in the central nervous system. Our previous studies showed that ROCK inhibition enhances phagocytic activity in microglia through the extracellular signal-regulated kinase (ERK) signaling pathway, but its effect on microglial migration was unknown. Therefore, in this study, we investigated the effects of the ROCK inhibitors Y27632 and fasudil on the migratory activity of primary cultured microglia isolated from the spinal cord, and we examined the underlying mechanisms. The microglia were treated with Y27632, fasudil and/or the ERK inhibitor U0126. Cellular morphology was observed by immunofluorescence. Transwell chambers were used to assess cell migration. ERK levels were measured by in-cell western blot assay. Y27632 and fasudil increased microglial migration, and the microglia were irregularly shaped and had many small processes. These inhibitors also upregulated the levels of phosphorylated ERK protein. The ERK inhibitor U0126 suppressed these effects of Y27632 and fasudil. These findings suggest that the ROCK inhibitors Y27632 and fasudil promote microglial migration in the spinal cord through the ERK signaling pathway.

Earlier studies have shown that intervertebral disc cells express TonEBP, a transcription factor that permits adaptation to osmotic stress and regulates aggrecan gene expression. However, the mechanism of hyperosmotic activation of TonEBP in disc cells is not known. Results of this study show that hypertonic activation of ERK signaling regulates transactivation activity of TonEBP, modulating its function. In an earlier report, we showed that tonicity enhancer binding protein (TonEBP) positively regulates aggrecan gene expression in disc cells, thereby autoregulating its osmotic environment. Although these studies indicated that the cells of the nucleus pulposus were optimally adapted to a hyperosmotic state, the mechanism by which the cells transduce the osmotic stress was not delineated. The primary goal of this study was to test the hypothesis that, in a hyperosmotic medium, the extracellular signal-regulated kinase (ERK) signaling pathway regulated TonEBP activity. Nucleus pulposus cells were maintained in isotonic or hypertonic media, and MAPK activation and TonEBP expression were analyzed. To study the role of MAPK in regulation of TonEBP function, gel shift and luciferase reporter assays were performed. ERK expression in cells was modulated by using expression plasmids or siRNA, and transactivation domain (TAD)-TonEBP activity was studied. We found that hypertonicity resulted in phosphorylation and activation of ERK1/2 proteins and concomitant activation of C terminus TAD activity of ELK-1, a downstream transcription factor. In hypertonic media, treatment with ERK and p38 inhibitors resulted in downregulation of TonE promoter activity of TauT and HSP-70 and decreased binding of TonEBP to TonE motif. Similarly, forced expression of DN-ERK and DN-p38 in nucleus pulposus cells suppressed TauT and HSP-70 reporter gene activity. Finally, we noted that ERK was needed for transactivation of TonEBP. Expression of DN-ERK significantly suppressed, whereas, WT-ERK and CA-MEK1 enhanced, TAD activity of TonEBP. Experiments performed with HeLa cells indicated that the ERK signaling pathway also served a major role in regulating the osmotic response in nondiscal cells. Together, these studies showed that adaptation of the nucleus pulposus cells to their hyperosmotic milieu is dependent on activation of the ERK and p38- MAPK pathways acting through TonEBP and its target genes.

Interleukin (IL)-6 is known to indirectly enhance osteoclast formation by promoting receptor activator of nuclear factor kappa-B ligand (RANKL) production by osteoblastic/stromal cells. However, little is known about the direct effect of IL-6 on osteoclastogenesis. Here, we determined the direct effects of IL-6 and its soluble receptor (sIL-6R) on RANKL-induced osteoclast formation by osteoclast precursors in vitro. We found IL-6/sIL-6R significantly promoted and suppressed osteoclast differentiation induced by low- (10 ng/ml) and high-level (50 ng/ml) RANKL, respectively. Using a bone resorption pit formation assay, expression of osteoclastic marker genes and transcription factors confirmed differential regulation of RANKL-induced osteoclastogenesis by IL-6/sIL-6R. Intracellular signaling transduction analysis revealed IL-6/sIL-6R specifically upregulated and downregulated the phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), ERK (extracellular signal–regulated kinase) and JNK (c-Jun N-terminal kinase) induced by low- and high level RANKL, respectively. Taken together, our findings demonstrate that IL-6/sIL-6R differentially regulate RANKL-induced osteoclast differentiation and activity through modulation of NF-κB, ERK and JNK signaling pathways. Thus, IL-6 likely plays a dual role in osteoclastogenesis either as a pro-resorption factor or as a protector of bone, depending on the level of RANKL within the local microenvironment.

Background Activation of glial cells and the extracellular signal-regulated kinase (ERK) signaling pathway play an important role in the development and maintenance of neuropathic pain. Curcumin can alleviate the symptom of inflammatory pain by inhibiting the production and release of interleukin and tumor necrosis factor. However, whether curcumin affects neuropathic pain induced by nerve injury and the possible mechanism involved are still unknown. This study investigated the effects of tolerable doses of curcumin on the activation of astrocytes and ERK signaling in the spinal dorsal horn in rat model of neuropathic pain. Methods Adult male Sprague-Dawley rats were randomly divided into three groups: a control (sham operated) group, and chronic constriction injury groups (to induce neuropathic pain) that were either untreated or treated with curcumin. Thermal and mechanical hyperalgesia thresholds were measured. The distribution and morphological changes of astrocytes were observed by immunofluorescence. Western blotting was used to detect changes in the expression of glial fibrillary acid protein (GFAP) and phosphorylated ERK. Results Injured rats showed obvious mechanical allodynia and thermal hyperalgesia. The number of GFAP-positive astrocytes, and the fluorescence intensity of GFAP were significantly increased in the spinal dorsal horn of injured compared with control rats. The soma of astrocytes also appeared hypertrophied in injured animals. Expression of GFAP and phosphorylated ERK was also significantly increased in the spinal dorsal horn of injured compared with control rats. Curcumin reduced the injury-induced thermal and mechanical hyperalgesia, the increase in the fluorescence intensity of GFAP and the hypertrophy of astrocytic soma, activation of GFAP and phosphorylation of ERK in the spinal dorsal horn. Conclusions Curcumin can markedly alleviate nerve injury-induced neuropathic pain in rats. The analgesic effect of curcumin may be attributed to its inhibition of astrocyte hypertrophy in the spinal dorsal horn and phosphorylation of the ERK signaling pathway.

Epigallocatechin gallate inhibited IFN-β induced MUC5AC overexpression in airway epithelial cells by modulating JAK1 mediated STAT1 and ERK pathways activation.