Stimulated ERK Phosphorylation Research Articles

Abstract LB208: An intricate connection between canonical Wnt and MAPK/ERK signaling drives the partial hybrid EMT state involving FOXC2/p63 activation leading to cancer stemness and metastasis

Abstract The epithelial to mesenchymal transition (EMT) is a dynamic process underlying metastasis. Here we show that TCF1, a TCF7 gene product, promotes a hybrid epithelial/mesenchymal (E/M) phenotype in basal breast cancer cells that was enriched in stemness and metastasis as compared to cells residing in the M-like state. E/M cells (CD104highCD44high) were enriched in canonical Wnt signaling, thereby upregulating FOXC2 which was turn able of converting M into E/M cells. Consistent with the E/M state, FOXC2 upregulated TA and ΔNp63 isoforms which control epithelial differentiation and Wnt signaling via β4 integrin/CD104 and Frizzled 7 upregulation respectively. Importantly, Wnt3a was able of stimulating ERK phosphorylation in E/M cells leading to FOXC2 and p63 upregulation thereby preventing E/M to M differentiation. These findings were further corroborated by ATAC-sequencing pointing predominantly to FOXC2 as a master regulator of the E/M state that was suppressed by ERK inhibition. Together, these results demonstrate that TCF7 stimulates Wnt3a/ERK activation which in turn activates the FOXC2/p63 axis, thereby promoting the hybrid EMT state underlying stemness and metastasis. Citation Format: Huizhi Liang, Outhiriaradjou Benard, Zuen Ren, Kimita Suyama, Jingli Wang, Anthony Griffen, Hao Wang, Lindsay LeFave, Larry Norton, Rachel B. Hazan. An intricate connection between canonical Wnt and MAPK/ERK signaling drives the partial hybrid EMT state involving FOXC2/p63 activation leading to cancer stemness and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB208.

Preosteoclast plays a pathogenic role in syndesmophyte formation of ankylosing spondylitis through the secreted PDGFB — GRB2/ERK/RUNX2 pathway

ObjectivesAnkylosing spondylitis (AS) is a chronic inflammatory disease that mainly affects the sacroiliac joint and spine. However, the real mechanisms of immune cells acting on syndesmophyte formation in AS are not well identified. We aimed to find the key AS-associated cytokine and assess its pathogenic role in AS.MethodsA protein array with 1000 cytokines was performed in five AS patients with the first diagnosis and five age- and gender-matched healthy controls to discover the differentially expressed cytokines. The candidate differentially expressed cytokines were further quantified by multiplex protein quantitation (3 AS-associated cytokines and 3 PDGF-pathway cytokines) and ELISA (PDGFB) in independent samples (a total of 140 AS patients vs 140 healthy controls). The effects of PDGFB, the candidate cytokine, were examined by using adipose-derived stem cells (ADSCs) and human fetal osteoblast cell line (hFOB1.19) as in vitro mesenchymal cell and preosteoblast models, respectively. Furthermore, whole-transcriptome sequencing and enrichment of phosphorylated peptides were performed by using cell models to explore the underlying mechanisms of PDGFB. The xCELLigence system was applied to examine the proliferation, chemotaxis, and migration abilities of PDGFB-stimulated or PDGFB-unstimulated cells.ResultsThe PDGF pathway was observed to have abnormal expression in the protein array, and PDGFB expression was further found to be up-regulated in 140 Chinese AS patients. Importantly, PDGFB expression was significantly correlated with BASFI (Pearson coefficient/p value = 0.62/6.70E − 8) and with the variance of the mSASSS score (mSASSS 2 years − baseline, Pearson coefficient/p value = 0.76/8.75E − 10). In AS patients, preosteoclasts secreted more PDGFB than the healthy controls (p value = 1.16E − 2), which could promote ADSCs osteogenesis and enhance collagen synthesis (COLI and COLIII) of osteoblasts (hFOB 1.19). In addition, PDGFB promoted the proliferation, chemotaxis, and migration of ADSCs. Mechanismly, in ADSCs, PDGFB stimulated ERK phosphorylation by upregulating GRB2 expression and then increased the expression of RUNX2 to promote osteoblastogenesis of ADSCs.ConclusionPDGFB stimulates the GRB2/ERK/RUNX2 pathway in ADSCs, promotes osteoblastogenesis of ADSCs, and enhances the extracellular matrix of osteoblasts, which may contribute to pathological bone formation in AS.

FGF2 positively regulates osteoclastogenesis via activating the ERK-CREB pathway

Fibroblast growth factor 2 (FGF2) plays crucial roles in the growth and development of several tissues. However, its function in bone homeostasis remains controversial. Here, we found that exogenous FGF2 supplementation inhibited the mineralization of bone marrow stromal cells (BMSCs), at least partially, via up-regulating the gene expression of osteoclastogenesis. The FGF receptor (FGFR) allosteric antagonist SSR128129E modestly, whereas the FGFR tyrosine kinase inhibitor AZD4547 significantly antagonized the effects of FGF2. Mechanistically, FGF2 stimulated ERK phosphorylation, and the ERK signaling inhibitor PD325901 strongly blocked FGF2 enhancement of osteoclastogenesis. Moreover, the phosphorylation of CREB was also activated in response to FGF2, thereby potentiating the interaction of p-CREB with the promoter region of Rankl gene. Notably, FGF2-deficient BMSCs exhibited higher mineralization capability and lower osteoclastogenic gene expression. Correspondingly, FGF2-knockout mice showed increased bone mass and attenuated expression of osteoclast-related markers, which were associated with moderate inhibition of the ERK signaling. In conclusion, FGF2 positively regulates osteoclastogenesis via stimulating the ERK-CREB pathway. These findings establish the importance of FGF2 in bone homeostasis, hinting the potential use of FGF2/ERK/CREB specific inhibitors to fight against bone-related disorders, such as osteoporosis.

Stimulated phosphorylation of ERK in mouse kidney mesangial cells is dependent upon expression of Cav3.1

BackgroundT-type calcium channels (TTCC) are low voltage activated channels that are widely expressed in the heart, smooth muscle and neurons. They are known to impact on cell cycle progression in cancer and smooth muscle cells and more recently, have been implicated in rat and human mesangial cell proliferation. The aim of this study was to investigate the roles of the different isoforms of TTCC in mouse mesangial cells to establish which may be the best therapeutic target for treating mesangioproliferative kidney diseases. MethodsIn this study, we generated single and double knockout (SKO and DKO) clones of the TTCC isoforms CaV3.1 and CaV3.2 in mouse mesangial cells using CRISPR-cas9 gene editing. The downstream signals linked to this channel activity were studied by ERK1/2 phosphorylation assays in serum, PDGF and TGF-β1 stimulated cells. We also examined their proliferative responses in the presence of the TTCC inhibitors mibefradil and TH1177.ResultsWe demonstrate a complete loss of ERK1/2 phosphorylation in response to multiple stimuli (serum, PDGF, TGF-β1) in CaV3.1 SKO clone, whereas the CaV3.2 SKO clone retained these phospho-ERK1/2 responses. Stimulated cell proliferation was not profoundly impacted in either SKO clone and both clones remained sensitive to non-selective TTCC blockers, suggesting a role for more than one TTCC isoform in cell cycle progression. Deletion of both the isoforms resulted in cell death.ConclusionThis study confirms that TTCC are expressed in mouse mesangial cells and that they play a role in cell proliferation. Whereas the CaV3.1 isoform is required for stimulated phosphorylation of ERK1/2, the Ca V3.2 isoform is not. Our data also suggest that neither isoform is necessary for cell proliferation and that the anti-proliferative effects of mibefradil and TH1177 are not isoform-specific. These findings are consistent with data from in vivo rat mesangial proliferation Thy1 models and support the future use of genetic mouse models to test the therapeutic actions of TTCC inhibitors.

ACAGT-007a, an ERK MAPK Signaling Modulator, in Combination with AKT Signaling Inhibition Induces Apoptosis in KRAS Mutant Pancreatic Cancer T3M4 and MIA-Pa-Ca-2 Cells.

The mitogen-activated protein kinase (MAPK)/ERK and phosphatidylinositol-3 kinase (PI3K)/AKT pathways are dysregulated in various human cancers, including pancreatic ductal adenocarcinoma (PDAC), which has a very poor prognosis due to its lack of efficient therapies. We have previously identified ACAGT-007a (GT-7), an anti-cancer compound that kills ERK-active melanoma cells by inducing ERK-dependent apoptosis. Here, we investigated the apoptosis-inducing effect of GT-7 on three PDAC cell lines and its relevance with the MAPK/ERK and PI3K/AKT signaling pathways. GT-7 induced apoptosis in PDAC cells with different KRAS mutations (MIA-Pa-Ca-2 (KRAS G12C), T3M4 (KRAS Q61H), and PANC-1 (KRAS G12D)), being T3M4 most susceptible, followed by MIA-Pa-Ca-2, and PANC-1 was most resistant to apoptosis induction by GT-7. GT-7 stimulated ERK phosphorylation in the three PDAC cells, but only T3M4 displayed ERK-activation-dependent apoptosis. Furthermore, GT-7 induced a marked down-regulation of AKT phosphorylation after a transient peak in T3M4, whereas PANC-1 displayed the strongest and most sustained AKT activation, followed by MIA-Pa-Ca-2, suggesting that sustained AKT phosphorylation as a determinant for the resistance to GT-7-mediated apoptosis. Consistently, a PI3K inhibitor, Wortmannin, abolished AKT phosphorylation and enhanced GT-7-mediated apoptosis in T3M4 and MIA-Pa-Ca-2, but not in PANC-1, which showed residual AKT phosphorylation. This is the first report that ERK stimulation alone or in combination with AKT signaling inhibition can effectively induce apoptosis in PDAC and provides a rationale for a novel concurrent targeting of the PI3K/AKT and ERK pathways.

Sema4C modulates the migration of primary tumor-associated lymphatic endothelial cells via an ERK-mediated pathway.

Although lymphatic endothelial cells (LECs) serve a positive role in tumor lymphatic metastasis, the regulation of LECs undergoing migration similar to that of tumor cells remains poorly understood. A previous study revealed that semaphorin 4C (Sema4C) could be a marker of LECs in cervical cancer. Thus, the present study aimed to understand the mechanism via which Sema4C could promote the development of tumor-associated characteristics in LECs in cervical cancer. Primary tumor-associated LECs (TLECs) were distinguished from cervical cancer by flow cytometry. The promigratory ability was assessed using the Transwell assay. Lentivirus infection was used to alter the expression of Sema4C in TLECs. Confocal laser scanning was used to determine the infection efficiency of lentivirus infection. Sema4C/ERK/E-cadherin pathway was measured by reverse transcription-quantitative PCR and western blotting. The co-localization of Sema4C and the lymphatic marker lymphatic vessel endothelial hyaluronan receptor 1 was verified. Primary tumor-associated LECs (TLECs) were isolated from a mouse xenograft cervical tumor model. It was revealed that overexpressing Sema4C stimulated the migratory ability of TLECs, downregulated E-cadherin expression and stimulated ERK phosphorylation, whereas knocking down Sema4C had the opposite effects. The treatment of PD98059 (ERK inhibitor) blocked the pro-migratory ability of TLECs, which indicated a dependence on the ERK signaling pathway. It was identified that the Sema4C/ERK/E-cadherin pathway may be critical for the migration of TLECs, which may promote lymph node metastasis. Therefore, Sema4C could be a promising target for the treatment of cervical cancer with lymphatic metastasis.

Down-regulation of dual-specificity phosphatase 6, a negative regulator of oncogenic ERK signaling, by ACA-28 induces apoptosis in NIH/3T3 cells overexpressing HER2/ErbB2.

Dual-specificity phosphatase 6 (DUSP6) is a key negative feedback regulator of the member of the RAS-ERK MAPK signaling pathway that is associated with cellular proliferation and differentiation. Deterioration of DUSP6 expression could therefore result in deregulated growth activity. We have previously discovered ACA-28, a novel anticancer compound with a unique property to stimulate ERK phosphorylation and induce apoptosis in ERK-active melanoma cells. However, the mechanism of cancer cell-specific-apoptosis by ACA-28 remains obscure. Here, we investigated the involvement of DUSP6 in the mechanisms of the ACA-28-mediated apoptosis by using the NIH/3T3 cells overexpressing HER2/ErbB2 (A4-15 cells), as A4-15 exhibited higher ERK phosphorylation and are more susceptible to ACA-28 than NIH/3T3. We showed that A4-15 exhibited high DUSP6 protein levels, which require ERK activation. Notably, the silencing of the DUDSP6 gene by siRNA inhibited proliferation and induced apoptosis in A4-15, but not in NIH/3T3, indicating that A4-15 requires high DUSP6 expression for growth. Importantly, ACA-28 preferentially down-regulated the DUSP6 protein and proliferation in A4-15 via the proteasome, while it stimulated ERK phosphorylation. Collectively, the up-regulation of DUSP6 may exert a growth-promoting role in cancer cells overexpressing HER2. DUSP6 down-regulation in ERK-active cancer cells might have the potential as a novel cancer measure.

Pharmacological Characterization of the Stick Insect Carausius morosus Allatostatin-C Receptor with Its Endogenous Agonist.

G protein-coupled receptors (GPCRs) play a pivotal role in regulating key physiological events in all animal species. Recent advances in collective analysis of genes and proteins revealed numerous potential neuropeptides and GPCRs from insect species, allowing for the characterization of peptide–receptor pairs. In this work, we used fluorescence resonance energy transfer (FRET)-based genetically encoded biosensors in intact mammalian cells to study the pharmacological features of the cognate GPCR of the type-C allatostatin (AST-C) peptide from the stick insect, Carausius morosus. Analysis of multiple downstream pathways revealed that AST-C can activate the human Gi2 protein, and not Gs or Gq, through AST-C receptor (AlstRC). Activated AlstRC recruits β-arrestin2 independent of the Gi protein but stimulates ERK phosphorylation in a Gi protein-dependent manner. Identification of Gαi-, arrestin-, and GRK-like transcripts from C. morosus revealed high evolutionary conservation at the G protein level, while β-arrestins and GRKs displayed less conservation. In conclusion, our study provides experimental and homology-based evidence on the functionality of vertebrate G proteins and downstream signaling biosensors to characterize early signaling steps of an insect GPCR. These results may serve as a scaffold for developing assays to characterize pharmacological and structural aspects of other insect GPCRs and can be used in deorphanization and pesticide studies.

Integrin α7 and Extracellular Matrix Laminin 211 Interaction Promotes Proliferation of Acute Myeloid Leukemia Cells and Is Associated with Granulocytic Sarcoma.

Acute myeloid leukemia (AML) with granulocytic sarcoma (GS) is characterized by poor prognosis; however, its underlying mechanism is unclear. Bone marrow samples from 64 AML patients (9 with GS and 55 without GS) together with AML cell lines PL21, THP1, HL60, Kasumi-1, and KG-1 were used to elucidate the pathology of AML with GS. RNA-Seq analyses were performed on samples from seven AML patients with or without GS. Gene set enrichment analyses revealed significantly upregulated candidates on the cell surface of the GS group. Expression of the adhesion integrin α7 (ITGA7) was significantly higher in the GS group, as seen by RT-qPCR (p = 0.00188) and immunohistochemistry of bone marrow formalin-fixed, paraffin-embedded (FFPE) specimens. Flow cytometry revealed enhanced proliferation of PL21 and THP1 cells containing surface ITGA7 in the presence of laminin 211 and stimulated ERK phosphorylation; this effect was abrogated following ITGA7 knockdown or ERK inhibition. Overall, high ITGA7 expression was associated with poor patient survival (p = 0.0477). In summary, ITGA7 is highly expressed in AML with GS, and its ligand (laminin 211) stimulates cell proliferation through ERK signaling. This is the first study demonstrating the role of integrin α7 and extracellular matrix interactions in AML cell proliferation and extramedullary disease development.

Fission Yeast as a Model System for Studying Cancer Signaling and Drug Discovery: Discovery of ACA-28 as a Novel Inducer of ERK-dependent Apoptosis Reveals a New Cancer Therapy

Mitogen-activated protein kinase (MAPK) pathways are evolutionarily conserved kinase modules that link extracellular signals to the machinery that controls fundamental cellular processes such as growth, proliferation, differentiation, and apoptosis. The Ras/Raf/MEK/ERK MAPK pathway is one of the most studied of the mammalian MAPK pathways and has attracted intense research interest because of its critical involvement in the regulation of cell proliferation. The mutational activation of upstream signaling components that constitutively activate ERK MAPKs as seen in various primary tumor samples has validated this pathway for drug discovery. The fission yeast Schizosaccharomyces pombe is an important tool for cancer research. This well-studied model organism has enabled groundbreaking, Nobel Prize-winning discoveries and has provided insights into how both normal and cancerous cells grow and divide. We performed chemical genetic screening using a fission yeast phenotypic assay and demonstrated that ACA-28, a synthetic derivative of 1'-acetoxychavicol acetate (ACA), effectively inhibited the growth of melanoma cancer cells wherein ERK MAPK signaling is hyperactivated due to mutations in the upstream activating regulators. Importantly, the growth of normal human epidermal melanocytes was less affected by ACA-28. In addition, ACA-28 specifically induced apoptosis in NIH/3T3 cells oncogenically transformed with HER2/ErbB2 but not in the parental cells. Notably, the ACA-28-induced apoptosis was abrogated when ERK activation was blocked with the specific MEK inhibitor U0126. Consistently, ACA-28 more strongly stimulated ERK phosphorylation in melanoma cells as compared with normal human epidermal melanocytes. ACA-28 might serve as a promising seed compound to combat ERK-dependent cancers by stimulating oncogenic signaling.

Histone demethylase Jmjd3 regulates osteoblast apoptosis through targeting anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim

Posttranslational modifications including histone methylation regulate gene transcription through directly affecting the structure of chromatin. Trimethylation of histone H3K27 (H3K27me3) contributes to gene silencing and the histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes the methylation of H3K27me3, followed by the activation of gene expression. In the present study, we explored the roles of Jmjd3 in regulating osteoblast apoptosis. Knockdown of Jmjd3 promoted osteoblast apoptosis induced by serum deprivation with decreased mitochondrial membrane potential and increased levels of caspase-3 activation, PARP cleavage, and DNA fragmentation. B cell lymphoma-2 (Bcl-2), an anti-apoptotic protein, was down-regulated by knockdown of Jmjd3 through retaining H3K27me3 on its promoter region. Knockdown of Jmjd3 increased the pro-apoptotic activity of Bim through inhibiting ERK-dependent phosphorylation of Bim. Protein kinase D1 (PKD1), which stimulates ERK phosphorylation, decreased in the Jmjd3-knockdown cells and introduction of PKD1 relieved osteoblast apoptosis in the Jmjd3-knockdown cells through increasing ERK-regulated Bim phosphorylation. These results suggest that Jmjd3 regulates osteoblast apoptosis through targeting Bcl-2 expression and Bim phosphorylation.

RF9 Acts as a KISS1R Agonist In Vivo and In Vitro.

RF9, a reported antagonist of the mammalian gonadotropin-inhibitory hormone receptor, stimulates gonadotropin secretion in mammals. Recent studies have suggested that the stimulatory effect of RF9 on gonadotropin secretion relies on intact kisspeptin receptor (KISS1R) signaling, but the underlying mechanisms remain to be elucidated. Using Chinese Hamster Ovary cells stably transfected with KISS1R, we show that RF9 binds specifically to KISS1R, with a Kd of 1.6 × 10(-5)M, and stimulates an increase in intracellular calcium and inositol phosphate accumulation in a KISS1R-dependent manner, with EC50 values of 3.0 × 10(-6)M and 1.6 × 10(-7)M, respectively. RF9 also stimulated ERK phosphorylation, with a time course similar to that of kisspeptin-10. RFRP-3, the putative endogenous ligand for NPFFR1, did not stimulate inositol phosphate accumulation or pERK, nor did it alter responses to of kisspeptin-10 or RF9. In agreement with these in vitro data, we found that RF9 stimulated a robust LH increase in Npffr1(-/-) mice, similar to that in wild-type littermates, whereas the stimulatory effect of RF9 was markedly reduced in Kiss1r(-/-) and double Kiss1r(-/-)/Npfrr1(-/-) mice. The stimulatory effect of RF9 on LH secretion was restored by the selective rescue of Kiss1r expression in GnRH neurons, in Kiss1r(-/-T) mice. Taken together, our study demonstrates that RF9 acts primarily as a KISS1R agonist, but not as an allosteric modulator, to stimulate LH secretion. Our findings raise questions regarding the utility of RF9 for assessing NPFF1R function and de-emphasize a predominant role of this signaling system in central regulation of reproduction.

Hematopoietic stem/progenitor cells directly contribute to arteriosclerotic progression via integrin β2.

Recent studies described the association between hematopoietic stem/progenitor cell (HSPC) expansion in the bone marrow (BM), leukocytosis in the peripheral blood, and accelerated atherosclerosis. We hypothesized that circulating HSPC may home to inflamed vessels, where they might contribute to inflammation and neointima formation. We demonstrated that Lin− Sca-1+ cKit+ (LSK cells) in BM and peripheral blood of LDLr−/− mice on high fat diet expressed significantly more integrin β2, which was responsible for LSK cell adhesion and migration toward ICAM-1 in vitro, and homing to injured arteries in vivo, all of which were blocked with an anti-CD18 blocking antibody. When homed LSK cells were isolated from ligated artery and injected to irradiated recipients, they resulted in BM reconstitution. Injection of CD18+/+ LSK cells to immunodeficient Balb/C Rag2− γC−/− recipients resulted in more severe inflammation and reinforced neointima formation in the ligated carotid artery, compared to mice injected with PBS and CD18−/− LSK cells. Hypercholesterolemia stimulated ERK phosphorylation (pERK) in LSK cells of LDLr−/− mice in vivo. Blockade of pERK reduced ARF1 expression, leading to decreased integrin β2 function on HSPC. In addition, integrin β2 function could be regulated via ERK-independent LRP1 pathway. Integrin β2 expression on HSPC is regulated by hypercholesterolemia, specifically LDL, in pERK-dependent and -independent manners, leading to increased homing and localization of HSPC to injured arteries, which is highly correlated with arteriosclerosis. Stem Cells 2015;33:1230–1240

Mitogen-Activated Protein Kinase 8 (MAP3K8) Mediates the Signaling Pathway of Estradiol Stimulating Progesterone Production Through G Protein-Coupled Receptor 30 (GPR30) in Mouse Corpus Luteum.

The corpus luteum (CL) is a transient endocrine gland developed from the ovulated follicles, and the most important function is to synthesize and secrete progesterone (P(4)), a key hormone to maintain normal pregnancy and estrous cycle in most mammals. It is known that estrogen has a vital role in stimulating P(4) synthesis in CL, but it still remains unclear about the mechanism of estradiol (E(2)) regulating P(4) production in CL. Our results here first show that all of the CL cells express MAPK 8 (MAP3K8), and the MAP3K8 level is much higher at the midstage than at the early and late stages during CL development. The further functional studies show that the forced inhibition of endogenous MAP3K8 by using MAP3K8 small interfering RNA and MAP3K8 signaling inhibitor (MAP3K8i) in the luteal cells significantly block the P(4) synthesis and neutralize the enhancing effect of E(2) on P(4) production in the CL. In addition, our results here demonstrate that the stimulating effect of E(2) on P(4) synthesis relies on the estrogen no-classical protein-coupled receptor 30, and MAP3K8 is involved in mediating the protein-coupled receptor 30signaling of E(2) affecting P(4) synthesis via stimulating ERK phosphorylation. These novel findings are critical for our understanding the ovary physiology and pathological mechanism.

Bortezomib enhances cancer cell death by blocking the autophagic flux through stimulating ERK phosphorylation.

The antitumor activity of an inhibitor of 26S proteasome bortezomib (Velcade) has been observed in various malignancies, including colon cancer, prostate cancer, breast cancer, and ovarian cancer. Bortezomib has been proposed to stimulate autophagy, but scientific observations did not always support this. Interactions between ERK activity and autophagy are complex and not completely clear. Autophagy proteins have recently been shown to regulate the functions of ERK, and ERK activation has been found to induce autophagy. On the other hand, sustained activation of ERK has also been shown to inhibit the maturation step of the autophagy process. In this study, we sought to identify the mechanism of autophagy regulation in cancer cells treated with bortezomib. Our results indicate that bortezomib blocked the autophagic flux without inhibiting the fusion of the autophagosome and lysosome. In ovarian cancer, as well as endometrial cancer and hepatocellular carcinoma cells, bortezomib inhibited protein degradation in lysosomes by suppressing cathepsins, which requires the participation of ERK phosphorylation, but not JNK or p38. Our findings that ERK phosphorylation reduced cathepsins further explain how ERK phosphorylation inhibits the autophagic flux. In conclusion, bortezomib may induce ERK phosphorylation to suppress cathepsin B and inhibit the catalytic process of autophagy in ovarian cancer and other solid tumors. The inhibition of cisplatin-induced autophagy by bortezomib can enhance chemotherapy efficacy in ovarian cancer. As we also found that bortezomib blocks the autophagic flux in other cancers, the synergistic cytotoxic effect of bortezomib by abolishing chemotherapy-related autophagy may help us develop strategies of combination therapies for multiple cancers.

EBI2 regulates intracellular signaling and migration in human astrocyte.

The G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2) is activated by 7α, 25-dihydroxycholesterol (7α25HC) and plays a role in T cell-dependant antibody response and B cell migration. Aberrant EBI2 signaling is implicated in a range of autoimmune disorders however its role in the CNS remains unknown. Here we characterize the functional role of EBI2 in GLIA cells using primary human astrocytes and EBI2 knockout animals. We find human and mouse astrocytes express EBI2 and the enzymes necessary for synthesis and degradation of 7α25HC. In astrocytes, EBI2 activation stimulates ERK phosphorylation, Ca(2+) signaling and induces cellular migration. These results, for the first time, demonstrate a role for EBI2 in astrocyte function and suggest that modulation of this receptor may be beneficial in neuroinflammatory disorders.

The regulation of ERK and p-ERK expression by cisplatin and sorafenib in gastric cancer cells

Previous studies have reported strong antitumor effects of cisplatin and sorafenib. Our results indicated that cisplatin and sorafenib exhibited anti-tumor effects on gastric cancer cells. They significantly inhibited gastric cell growth and induced apoptosis. They effectively inhibited gastric cancer cell proliferation and induced G0/G1 phase arrest. Western blotting analysis indicated that it also promoted the phosphorylation extracellular signal regulated kinase (p-ERK). Moreover, cisplatin and sorafenib played a synergistic antitumor effect. These results suggested that the antitumor mechanism of cisplatin and sorafenib involved altering the cell cycle and stimulating ERK phosphorylation in the ERK signaling pathway.

Sorafenib use in hepatocellular carcinoma: More questions than answers

Sorafenib use in hepatocellular carcinoma: More questions than answers

Modulation of Epidermal Growth Factor Stimulated ERK Phosphorylation and Cell Motility by Inositol Trisphosphate Kinase.

Epidermal growth factor [EGF] mediated stimulation of its receptor in endothelial cell [EC] is accompanied by phosphorylation of the EGF-receptor [EGFR] and activation of phospholipase C-γ, resulting in the breakdown of phosphatidylinositol(4,5)-bisphosphate and generating inositol (1,4,5)-trisphosphate [IP3] and diacylglycerol. IP3 thus formed can be further converted to inositol (1,3,4,5)-tetrakisphosphate [IP4] by an enzyme called IP3-kinase [IP3K]. In this study we have investigated the effect of modulation of intracellular IP3K activity by the use of an inhibitor, 2-trifluoromethyl [6-(4-nitrobenzyl)-purine] [IP3KI] and siRNA against IP3KB on EGF-induced ERK-phosphorylation and cell motility. EGF stimulated ERK-phosphorylation that has been implicated in EGF-stimulated cell migration was inhibited by both IP3KI and siRNA against IP3KB. Inhibition of ERK-phosphorylation was accompanied by decreased cell migration in the presence of IP3KI.

Effects of IL8 and immune cells on the regulation of luteal progesterone secretion

Recent studies have suggested that chemokines may mediate the luteolytic action of prostaglandin F2α (PGF). Our objective was to identify chemokines induced by PGF in vivo and to determine the effects of interleukin 8 (IL8) on specific luteal cell types in vitro. Mid-cycle cows were injected with saline or PGF, ovaries were removed after 0.5-4 h, and expression of chemokine was analyzed by qPCR. In vitro expression of IL8 was analyzed after PGF administration and with cell signaling inhibitors to determine the mechanism of PGF-induced chemokine expression. Purified neutrophils were analyzed for migration and activation in response to IL8 and PGF. Purified luteal cell types (steroidogenic, endothelial, and fibroblast cells) were used to identify which cells respond to chemokines. Neutrophils and peripheral blood mononuclear cells (PBMCs) were cocultured with steroidogenic cells to determine their effect on progesterone production. IL8, CXCL2, CCL2, and CCL8 transcripts were rapidly increased following PGF treatment in vivo. The stimulatory action of PGF on IL8 mRNA expression in vitro was prevented by inhibition of p38 and JNK signaling. IL8, but not PGF, TNF, or TGFB1, stimulated neutrophil migration. IL8 had no apparent action in purified luteal steroidogenic, endothelial, or fibroblast cells, but stimulated ERK phosphorylation in neutrophils. In coculture experiments neither IL8 nor activated neutrophils altered basal or LH-stimulated luteal cell progesterone synthesis. In contrast, activated PBMCs inhibited LH-stimulated progesterone synthesis from cultured luteal cells. These data implicate a complex cascade of events during luteolysis, involving chemokine signaling, neutrophil recruitment, and immune cell action within the corpus luteum.