Phosphorylation Of Elk-1 Research Articles

Basic Fibroblast Growth Factor Induces Matrix Metalloproteinase-13 via ERK MAP Kinase-Altered Phosphorylation and Sumoylation of Elk-1 in Human Adult Articular Chondrocytes [Expression of Concern

Basic Fibroblast Growth Factor Induces Matrix Metalloproteinase-13 via ERK MAP Kinase-Altered Phosphorylation and Sumoylation of Elk-1 in Human Adult Articular Chondrocytes [Expression of Concern

Mitotic Kinases Aurora-A, Plk1, and Cdk1 Interact with Elk-1 Transcription Factor through the N-Terminal Domain.

Elk-1 is a member of the ETS domain transcription factor superfamily that is phosphorylated upon mitogen-activated protein kinase (MAPK) pathway activation, which in turn regulated its interaction with partner protein serum response factor (SRF), leading to formation of a ternary complex with DNA. It has previously been reported that Elk-1 interacts with a mitotic kinase Aurora-A, although the mechanisms or the relevance of this interaction was unclear. Elk-1 was also reported to be phosphorylated by CDK5 on Thr417 residue. In this study, we show for the first time that this transcription factor interacts not only with Aurora-A but also with other mitotic kinases Aurora-B, Plk1, and Cdk1, and we define the interaction domain on Elk-1 to the first N-terminal 205 amino acids. We also describe putative phosphorylation sites of these mitotic kinases on Elk-1 and show that Elk-1 peptides containing these residues get phosphorylated by the mitotic kinases in in vitro kinase assays. We also perform bioinformatic analysis of mitotic phosphoproteomes and determine potential interaction partners for Elk-1 in Plk or Aurora phosphoproteomes. We propose that understanding the dynamic phosphorylation of Elk-1 by mitotic kinases is important and that it can present a novel target for anticancer strategies.

An allosteric potentiator of metabotropic glutamate (mGlu) 2 receptors reduces the cocaine-stimulated ERK1/2 phosphorylation in the mouse striatum

Metabotropic glutamate (mGlu) receptors are involved in the experience-dependent neuroplasticity in the mesolimbic reward circuit. A Gαi/o-coupled mGlu2 subtype is distributed presynaptically in the striatum. These autoreceptors may have a significant influence over striatal neurons in their intracellular signaling pathways in response to a psychostimulant. Here we explored the effect of pharmacological potentiation of mGlu2 receptors on cocaine-stimulated phosphorylation (activation) of extracellular signal-regulated kinases (ERK) in the mouse striatum in vivo. We found that an mGlu2 selective positive allosteric modulator (PAM) LY487379 after a systemic injection did not alter basal phosphorylation of ERK1/2 or c-Jun N-terminal kinases in the striatum. However, pretreatment with LY487379 blocked the ERK1/2 phosphorylation induced by cocaine in the two subdivisions of the striatum, i.e., the caudate putamen and nucleus accumbens. LY487379 also blocked the cocaine-induced phosphorylation of Elk-1, a transcription factor downstream to the ERK pathway. Additionally, LY487379 reduced locomotor behavioral responses to cocaine. These results demonstrate that the mGlu2 PAM LY487379 possesses the ability to attenuate the activation of the ERK1/2 pathway in striatal neurons and reduce locomotor activity in response to cocaine in vivo.

Comparative analysis of protein expression systems and PTM landscape in the study of transcription factor ELK-1

Post-translational modifications (PTMs) are important for protein folding and activity, and the ability to recreate physiologically relevant PTM profiles on recombinantly-expressed proteins is vital for meaningful functional analysis. The ETS transcription factor ELK-1 serves as a paradigm for cellular responses to mitogens and can synergise with androgen receptor to promote prostate cancer progression, although in vitro protein function analyses to date have largely overlooked its complex PTM landscapes. We expressed and purified human ELK-1 using mammalian (HEK293T), insect (Sf9) and bacterial (E. coli) systems in parallel and compared PTMs imparted upon purified proteins, along with their performance in DNA and protein interaction assays. Phosphorylation of ELK-1 within its transactivation domain, known to promote DNA binding, was most apparent in protein isolated from human cells and accordingly conferred the strongest DNA binding in vitro, while protein expressed in insect cells bound most efficiently to the androgen receptor. We observed lysine acetylation, a hitherto unreported PTM of ELK-1, which appeared highest in insect cell-derived ELK-1 but was also present in HEK293T-derived ELK-1. Acetylation of ELK-1 was enhanced in HEK293T cells following starvation and mitogen stimulation, and modified lysines showed overlap with previously identified regulatory SUMOylation and ubiquitination sites. Our data demonstrate that the choice of recombinant expression system can be tailored to suit biochemical application rather than to maximise soluble protein production and suggest the potential for crosstalk and antagonism between different PTMs of ELK-1.

Asprosin inhibits macrophage lipid accumulation and reduces atherosclerotic burden by up-regulating ABCA1 and ABCG1 expression via the p38/Elk-1 pathway

BackgroundAsprosin, a newly discovered adipokine, is a C-terminal cleavage product of profibrillin. Asprosin has been reported to participate in lipid metabolism and cardiovascular disease, but its role in atherogenesis remains elusive.MethodsAsprosin was overexpressed in THP-1 macrophage-derived foam cells and apoE−/− mice using the lentiviral vector. The expression of relevant molecules was determined by qRT-PCR and/or western blot. The intracellular lipid accumulation was evaluated by high-performance liquid chromatography and Oil red O staining. HE and Oil red O staining was employed to assess plaque burden in vivo. Reverse cholesterol transport (RCT) efficiency was measured using [3H]-labeled cholesterol.ResultsExposure of THP-1 macrophages to oxidized low-density lipoprotein down-regulated asprosin expression. Lentivirus-mediated overexpression of asprosin promoted cholesterol efflux and inhibited lipid accumulation in THP-1 macrophage-derived foam cells. Mechanistic analysis revealed that asprosin overexpression activated p38 and stimulated the phosphorylation of ETS-like transcription factor (Elk-1) at Ser383, leading to Elk-1 nuclear translocation and the transcriptional activation of ATP binding cassette transporters A1 (ABCA1) and ABCG1. Injection of lentiviral vector expressing asprosin diminished atherosclerotic lesion area, increased plaque stability, improved plasma lipid profiles and facilitated RCT in apoE−/− mice. Asprosin overexpression also increased the phosphorylation of p38 and Elk-1 as well as up-regulated the expression of ABCA1 and ABCG1 in the aortas.ConclusionAsprosin inhibits lipid accumulation in macrophages and decreases atherosclerotic burden in apoE−/− mice by up-regulating ABCA1 and ABCG1 expression via activation of the p38/Elk-1 signaling pathway.

EGF-Dependent Activation of ELK1 Contributes to the Induction of CLDND1 Expression Involved in Tight Junction Formation.

Claudin proteins are intercellular adhesion molecules. Increased claudin domain-containing 1 (CLDND1) expression is associated with the malignant transformation of estrogen receptor-negative breast cancer cells with low sensitivity to hormone therapy. Abnormal CLDND1 expression is also implicated in vascular diseases. Previously, we investigated the regulatory mechanism underlying CLDND1 expression and identified a strong enhancer region near the promoter. In silico analysis of the sequence showed high homology to the ETS domain-containing protein-1 (ELK1)-binding sequence which is involved in cell growth, differentiation, angiogenesis, and cancer. Transcriptional ELK1 activation is associated with the mitogen-activated protein kinase (MAPK) signaling cascade originating from the epidermal growth factor receptor (EGFR). Here, we evaluated the effect of gefitinib, an EGFR tyrosine kinase inhibitor, on the suppression of CLDND1 expression using ELK1 overexpression in luciferase reporter and chromatin immunoprecipitation assays. ELK1 was found to be an activator of the enhancer region, and its transient expression increased that of CLDND1 at the mRNA and protein levels. CLDND1 expression was increased following EGF-induced ELK1 phosphorylation. Furthermore, this increase in CLDND1 was significantly suppressed by gefitinib. Therefore, EGF-dependent activation of ELK1 contributes to the induction of CLDND1 expression. These findings open avenues for the development of new anticancer agents targeting CLDND1.

Critical Protein-Protein Interactions Determine the Biological Activity of Elk-1, a Master Regulator of Stimulus-Induced Gene Transcription.

Elk-1 is a transcription factor that binds together with a dimer of the serum response factor (SRF) to the serum-response element (SRE), a genetic element that connects cellular stimulation with gene transcription. Elk-1 plays an important role in the regulation of cellular proliferation and apoptosis, thymocyte development, glucose homeostasis and brain function. The biological function of Elk-1 relies essentially on the interaction with other proteins. Elk-1 binds to SRF and generates a functional ternary complex that is required to activate SRE-mediated gene transcription. Elk-1 is kept in an inactive state under basal conditions via binding of a SUMO-histone deacetylase complex. Phosphorylation by extracellular signal-regulated protein kinase, c-Jun N-terminal protein kinase or p38 upregulates the transcriptional activity of Elk-1, mediated by binding to the mediator of RNA polymerase II transcription (Mediator) and the transcriptional coactivator p300. Strong and extended phosphorylation of Elk-1 attenuates Mediator and p300 recruitment and allows the binding of the mSin3A-histone deacetylase corepressor complex. The subsequent dephosphorylation of Elk-1, catalyzed by the protein phosphatase calcineurin, facilitates the re-SUMOylation of Elk-1, transforming Elk-1 back to a transcriptionally inactive state. Thus, numerous protein–protein interactions control the activation cycle of Elk-1 and are essential for its biological function.

PARK7 enhances antioxidative-stress processes of BMSCs via the ERK1/2 pathway.

Oxidative stresss in the microenvironment surrounding lesions induces apoptosis of transplanted bone‐marrow‐derived mesenchymal stem cells (BMSCs). Hence, there is an urgent need for improving antioxidative‐stress processes of transplanted BMSCs to further promote their survival. The present study reports the role and mechanism of Parkinson's disease protein 7 (PARK7) in enhancing antioxidative activity in BMSCs. We used a PARK7 lentivirus to transfect BMSCs to up‐ or downregulate PARK7, and then used H2O2 to simulate oxidative stress in BMSCs in vitro. Overexpression of PARK7 effectively reduced reactive oxygen species and malondialdehyde, protected mitochondrial membrane potential, and resisted oxidative‐stress‐induced apoptosis of BMSCs, but the expression of PARK7 was downregulated, these results were reversed. At the same time, we also found that overexpression of PARK7 increased extracellular‐regulated protein kinase 1/2 (ERK1/2) phosphorylation and nuclear translocation, as well as upregulated Elk1 phosphorylation and superoxide dismutase (SOD) expression. In contrast, when U0126 was used to block the ERK1/2 pathway, ERK1/2 and Elk1 phosphorylation levels were downregulated, ERK1/2 nuclear translocation and SOD content were significantly reduced, and PARK7‐overexperssion‐induced antioxidative activity was completely blocked. Collectively, our results suggest that PARK7 overexpression increased antioxidative‐stress processes and survival of BMSCs subjected to H2O2 via activating the ERK1/2 signaling pathway. Our findings may guide the development of a PARK7‐specific strategy for improving the transplantation efficacy of BMSCs.

CXXC finger protein 4 inhibits the CDK18-ERK1/2 axis to suppress the immune escape of gastric cancer cells with involvement of ELK1/MIR100HG pathway.

Gastric cancer, is the fourth most common tumour type yet, ranks second in terms of the prevalence of cancer‐related deaths worldwide. CXXC finger protein 4 (CXXC4) has been considered as a novel cancer suppressive factor, including gastric cancer. This study attempted to investigate the possible function of CXXC4 in gastric cancer and the underlying mechanism. The binding of the ETS domain‐containing protein‐1 (ELK1) to the long non‐coding RNA MIR100HG promoter region was identified. Then, their expression patterns in gastric cancer tissues and cells (SGC7901) were detected. A CCK‐8 assay was used to detect SGC7901 cell proliferation. Subsequently, SGC7901 cells were co‐cultured with CD3+ T cells, followed by measurement of CD3+ T cell proliferation, magnitude of IFN‐γ+ T cell population and IFN‐γ secretion. A nude mouse model was subsequently developed for in vivo validation of the in vitro results. Low CXXC4 expression was found in SGC7901 cells. Nuclear entry of ELK1 can be inhibited by suppression of the extent of ELK1 phosphorylation. Furthermore, ELK1 is able to bind the MIR100HG promoter. Overexpression of CXXC4 resulted in weakened binding of ELK1 to the MIR100HG promoter, leading to a reduced proliferative potential of SGC7901 cells, and an increase in IFN‐γ secretion from CD3+ T cells. Moreover, in vivo experiments revealed that CXXC4 inhibited immune escape of gastric cancer cells through the ERK1/2 axis. Inhibition of the CXXC4/ELK1/MIR100HG pathway suppressed the immune escape of gastric cancer cells, highlighting a possible therapeutic target for the treatment of gastric cancer.

Xinhuang Tablets Improve Intestinal Barrier Function via Regulating Epithelial Tight Junctions in Dextran Sulfate Sodium-Induced Ulcerative Colitis Mice.

Intestinal mucosal barrier dysfunction is involved in the pathogenesis of inflammatory bowel disease, including ulcerative colitis (UC). Xinhuang tablets (XHTs) have been prescribed for several kinds of inflammatory diseases, including UC, whereas its possible underlying molecular mechanisms had never been explored. Mouse model of UC was constructed by DSS treatment and followed by XHT treatment. Disease activity index, histopathological of colonic tissue, tumor necrosis factor-alpha (TNF-α), and serum amyloid A (SAA) levels in serum were further assessed. The underlying mechanism was further explored by determination of the expression of epithelial tight junction-related protein. XHT administration ameliorated dextran sulfate sodium (DSS)-induced clinical symptoms, colonic histological injury, and decreased the circulating levels of TNF-α and SAA. Moreover, XHT treatment significantly increased the protein levels of zona occludens (ZO)-1, whereas decreased the levels of phosphorylation of Elk-1. In conclusion, this study confirmed the therapeutic effects of XHT treatment on UC in a DSS-induced mouse model, and indicated that by increasing expression of epithelial tight junctions and decreasing phosphorylation of Elk-1 might be one of the underlying mechanisms of XHT treatment on UC.

Insulin-Like Growth Factor Binding Protein-2 Promotes Proliferation and Predicts Poor Prognosis in Hepatocellular Carcinoma.

BackgroundInsulin-like growth factor binding protein-2 (IGFBP2) levels are significantly increased in the plasma of hepatocellular carcinoma (HCC) patients. However, the correlation between IGFBP2 levels and clinical parameters and the exact role of IGFBP2 in HCC are unclear. In this study, we identified the role and potential molecular mechanisms of IGFBP2 in HCC.Materials and MethodsELISA assays were used to detect plasma IGFBP2 levels in HCC patients and healthy controls, and the correlations with patients’ clinicopathological data were analyzed. The CCK8 assay was used to explore cell proliferation. Luciferase reporter, co-immunoprecipitation, and immunofluorescence assays were used to demonstrate the molecular mechanism of IGFBP2 in HCC.ResultsPlasma IGFBP2 levels were determined blindly in 37 HCC patients and 37 matched healthy controls. The mean plasma IGFBP2 concentrations in HCC patients were higher than in healthy controls, and IGFBP2 levels in HCC were positively correlated with the degree of differentiation, tumor size, metastasis, and portal venous invasion. Exogenous IGFBP2 activated integrin β1 and thus induced the combination and colocalization of activated integrin β1 and p-FAK, which promoted the phosphorylation of FAK, Erk, and Elk1, eventually inducing EGR1-mediated proliferation of the HCC cell lines HepG2 and HCCLM3. Meanwhile, neutralization of integrin β1 inhibited IGFBP2-induced FAK, Erk, Elk1, and EGR1 activation.ConclusionTaken together, these results indicated that exogenous IGFBP2 promoted the integrin β1/FAK/Erk/Elk1/EGR1 pathway, which stimulated the proliferation of HCC cells. Plasma IGFBP2 could be a novel prognostic biomarker for HCC patients.

L-Theanine attenuates neointimal hyperplasia via suppression of vascular smooth muscle cell phenotypic modulation

Neointimal hyperplasia is a prominent pathological phenomenon in the process of stent restenosis. Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) play major pathological processes involved in the development of restenosis. l-Theanine, one of the major amino acid components in green tea, has been reported to improve vascular function. Here we display the effects of l-theanine on neointima formation and the underlying mechanism. In the rat carotid-artery balloon-injury model, l-theanine greatly inhibited neointima formation and prevented VSMCs from a contractile phenotype switching to a synthetic phenotype. In vitro study showed that l-theanine significantly inhibited PDGF-BB-induced VSMC proliferation and migration, which was comparable with the effect of l-theanine on AngII-induced VSMC proliferation and migration. Western blot analysis demonstrated that l-theanine suppressed PDGF-BB and AngII-induced reduction of SMA and SM22α and increment of OPN, suggesting that l-theanine inhibited the transformation of VSMCs from contractile to the synthetic phenotype. Further experiments showed that l-theanine exhibits potential preventive effects on neointimal hyperplasia and related vascular remodeling via inhibition of phosphorylation of Elk-1 and activation of MAPK1. The present study provides the new experimental evidence that l-theanine has potential clinical application as an anti-restenosis agent for the prevention of restenosis.

Mutant Huntingtin affects toll-like receptor 4 intracellular trafficking and cytokine production in mast cells

BackgroundHuntington’s disease (HD) is caused by the expression of a mutated variant of Huntingtin (mHtt), which results in the complex pathology characterized by a defective function of the nervous system and altered inflammatory responses. While the neuronal effects of mHtt expression have been extensively studied, its effects on the physiology of immune cells have not been fully described. Mast cells (MCs) are unique tissue-resident immune cells whose activation has been linked to protective responses against parasites and bacteria, but also to deleterious inflammatory allergic reactions and, recently, to neurodegenerative diseases.MethodsBone marrow-derived mast cells (BMMCs) were obtained from wild-type (WT-) and mHtt-expressing (R6/1) mice to evaluate the main activation parameters triggered by the high-affinity IgE receptor (FcεRI) and the Toll-like receptor (TLR) 4. Degranulation was assessed by measuring the secretion of β-hexosaminidase, MAP kinase activation was detected by Western blot, and cytokine production was determined by RT-PCR and ELISA. TLR-4 receptor and Htt vesicular trafficking was analyzed by confocal microscopy. In vivo, MC-deficient mice (c-KitWsh/Wsh) were intraperitonally reconstituted with WT or R6/1 BMMCs and the TLR4-induced production of the tumor necrosis factor (TNF) was determined by ELISA. A survival curve of mice treated with a sub-lethal dose of bacterial lipopolysaccharide (LPS) was constructed.ResultsR6/1 BMMCs showed normal β-hexosaminidase release levels in response to FcεRI, but lower cytokine production upon LPS stimulus. Impaired TLR4-induced TNF production was associated to the lack of intracellular dynamin-dependent TLR-4 receptor trafficking to perinuclear regions in BMMCs, a diminished ERK1/2 and ELK-1 phosphorylation, and a decrease in c-fos and TNF mRNA accumulation. R6/1 BMMCs also failed to produce TLR4-induced anti-inflammatory cytokines (like IL-10 and TGF-β). The detected defects were also observed in vivo, in a MCs-dependent model of endotoxemia. R6/1 and c-KitWsh/Wsh mice reconstituted with R6/1 BMMCs showed a decreased TLR4-induced TNF production and lower survival rates to LPS challenge than WT mice.ConclusionsOur data show that mHtt expression causes an impaired production of pro- and anti-inflammatory mediators triggered by TLR-4 receptor in MCs in vitro and in vivo, which could contribute to the aberrant immunophenotype observed in HD.

EGFR activates GDH1 transcription to promote glutamine metabolism through MEK/ERK/ELK1 pathway in glioblastoma.

Cancer metabolism research has recently been revived and its focus expanded from glucose and the Warburg's effects on other nutrients, such as glutamine. The underlying mechanism of oncogenic alterations of glutaminolysis remains unclear. Genetic alterations of EGFR are observed in ~50% of glioblastoma (GBM) patients, and have been found to play important roles in the metabolic abnormalities of GBM. In this study, we found that glutamine metabolism was upregulated after EGFR activation in a GDH1 (glutamate dehydrogenase 1)-dependent manner. Knockdown of GDH1 significantly reduced the cell proliferation, colony formation and tumorigenesis abilities of glioblastoma cells. Furthermore, we showed that GDH1-mediated glutaminolysis was involved in EGF-promoted cell proliferation. EGFR triggered the phosphorylation of ELK1 at Ser 383 through activating MEK/ERK signaling. Phosphorylated ELK1 enriched in the promoter of GDH1 to activate the transcription of GDH1, which then promoted glutamine metabolism. In addition, EGFR activation did not accelerate glutaminolysis in ELK1 knockdown or ELK1 Ser383-mutated cells. Collectively, our findings indicate that EGFR phosphorylates ELK1 to activate GDH1 transcription and glutaminolysis through MEK/ERK pathway, providing new insight into oncogenic alterations of glutamine metabolism.

P1‐206: PIAS1 SER‐503 PHOSPHORYLATION‐MEDIATED ELK‐1 SUMOYLATION PROMOTES NEURONAL SURVIVAL IN APP/PS1 MICE

PIAS1 is phosphorylated by IKKα at Ser-90 in a PIAS1 E3 ligase activity-dependent manner. Whether PIAS1 is also phosphorylated at other residues, and what potential functional significance such additional phosphorylation events might have, are not known. The transcription factor Elk-1 remains SUMOylated under basal conditions, but the role of Elk-1 SUMOylation in the brain is unknown. Here, we examined the functional significance of PIAS1-mediated Elk-1 SUMOylation in the context of Alzheimer's disease (AD) using the APP/PS1 mouse model of AD and amyloid beta microinjection in vivo. Novel phosphorylation site(s) on PIAS1 were identified by LC-MS/MS, and MAPK/ERK-mediated phosphorylation of Elk-1 was demonstrated using in vitro kinase assays. Elk-1 SUMOylation by PIAS1 in the brain was determined using in vitro SUMOylation assays. Apoptosis in the hippocampus was assessed by measuring GADD45α expression by Western blotting, and apoptosis of neurons in the hippocampus of APP/PS1 mice was assessed by TUNEL assay. Using LC-MS/MS, we identified a novel MAPK/ERK-mediated phosphorylation site on PIAS1 at Ser-503 and showed that this phosphorylation determines PIAS1 E3 ligase activity. In the rat brain, Elk-1 is SUMOylated by PIAS1, which decreases Elk-1 phosphorylation and downregulates GADD45α expression. Moreover, lentiviral-mediated transduction of Elk-1-SUMO1 in the hippocampus reduces the number of apoptotic neurons in APP/PS1 mice. MAPK/ERK-mediated phosphorylation of PIAS1 at Ser-503 determines PIAS1 E3 ligase activity. Moreover, PIAS1 mediates SUMOylation of Elk-1, and this modification functions as an endogenous defense mechanism against Aβ toxicity in vivo. Targeting Elk-1 SUMOylation could be considered a novel therapeutic strategy against AD.

Role of integrin-linked kinase in the hypoxia-induced phenotypic transition of pulmonary artery smooth muscle cells: Implications for hypoxic pulmonary hypertension

The phenotypic transition of pulmonary artery smooth muscle cells (PASMCs) from a contractile/differentiated to synthetic/de-differentiated phenotype is an important mechanism for the occurrence and development of hypoxic pulmonary hypertension (HPH). Integrin-linked kinase (ILK) is an early hypoxic response factor whose kinase activity is significantly affected during early hypoxia. Myocardin and ETS-like protein 1 (Elk-1) are co-activators of serum response factor (SRF) and can bind to SRF to mediate the phenotypic transition of PASMCs. However, little is known about the role of ILK on the phenotypic transition of these PASMCs. Thus, in our study, we explored the role of ILK in this process. We found that the expression of ILK and myocardin decreased gradually with the increase in hypoxia exposure time in the pulmonary arteries of rats. We observed that hypoxia exposure for 1 h caused an increase in the phosphorylation of Elk-1 but did not affect the expression of ILK, myocardin, or SRF. Exposure to hypoxic treatment for 1 h decreased ILK kinase activity and caused Elk-1 to suppress myocardin binding to SRF and the smooth muscle (SM) α-actin gene promoters. In addition, hypoxia exposure for 24 h decreased the expression of ILK, myocardin, SM α-actin, and calponin but increased the expression of osteopontin. Silencing of the myocardin gene significantly decreased the expression of SM α-actin and calponin but increased the expression of osteopontin. Silencing of the ILK gene significantly decreased the expression of myocardin, SM α-actin, and calponin but increased the expression of osteopontin. ILK overexpression reversed the effects of 24 h of hypoxia on the expression of myocardin, SM α-actin, calponin, and osteopontin and reversed the decrease in binding of myocardin to the SM α-actin promoter caused by 24 h of hypoxia exposure. Thus, our results suggest that ILK initiates the phenotypic transition of PASMCs. The underlying mechanism may involve hypoxia downregulating ILK kinase activity and protein expression, causing Elk-1 to compete with myocardin for binding to the SM α-actin promoter, which downregulates the expression of the downstream target myocardin and results in the phenotypic transition of PASMCs from a contractile to a synthetic phenotype. This may be an important mechanism in the development of HPH.

Involvement of ERK1/2-mediated ELK1/CHOP/DR5 pathway in 6-(methylsulfinyl)hexyl isothiocyanate-induced apoptosis of colorectal cancer cells.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a major bioactive compound in Wasabi. Although 6-MSITC is reported to have cancer chemopreventive activities in rat model, the molecular mechanism is unclear. In this study, we investigated the anticancer mechanisms using two types of human colorectal cancer cells (HCT116 p53+/+ and p53-/-). 6-MSITC caused cell cycle arrest in G2/M phase and induced apoptosis in both types of cells in the same fashion. Signaling data revealed that the activation of ERK1/2, rather than p53, is recruited for 6-MSITC-induced apoptosis. 6-MSITC stimulated ERK1/2 phosphorylation, and then activated ERK1/2 signaling including ELK1 phosphorylation, and upregulation of C/EBP homologous protein (CHOP) and death receptor 5 (DR5). The MEK1/2 inhibitor U0126 blocked all of these molecular events induced by 6-MSITC, and enhanced the cell viability in both types of cells in the same manner. These results indicated that ERK1/2-mediated ELK1/CHOP/DR5 pathway is involved in 6-MSITC-induced apoptosis in colorectal cancer cells. Abbreviations: CHOP: C/EBP homologous protein; DR5: death receptor 5; ELK1: ETS transcription factor; ERK1/2: extracellular signal-regulated kinase 1/2; JNK: Jun-N-terminal kinase; MAPK: mitogen-activated protein kinase; MEK1/2: MAP/ERK kinase 1/2; 6-MSITC: 6-(methylsulfinyl)hexyl isothiocyanate; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP: poly(ADP-ribose) polymerase.

Protein inhibitor of activated STAT1 Ser503 phosphorylation‐mediated Elk‐1 SUMOylation promotes neuronal survival in APP/PS1 mice

Protein inhibitor of activated STAT1 (PIAS1) is phosphorylated by IKKα at Ser90 in a PIAS1 E3 ligase activity-dependent manner. Whether PIAS1 is also phosphorylated at other residues and the functional significance of these additional phosphorylation events are not known. The transcription factor Elk-1 remains SUMOylated under basal conditions, but the role of Elk-1 SUMOylation in brain is unknown. Here, we examined the functional significance of PIAS1-mediated Elk-1 SUMOylation in Alzheimer's disease (AD) using the APP/PS1 mouse model of AD and amyloid β (Aβ) microinjections in vivo. Novel phosphorylation site(s) on PIAS1 were identified by LC-MS/MS, and MAPK/ERK-mediated phosphorylation of Elk-1 demonstrated using in vitro kinase assays. Elk-1 SUMOylation by PIAS1 in brain was determined using in vitro SUMOylation assays. Apoptosis in hippocampus was assessed by measuring GADD45α expression by western blotting, and apoptosis of hippocampal neurons in APP/PS1 mice was assessed by TUNEL assay. Using LC-MS/MS, we identified a novel MAPK/ERK-mediated phosphorylation site on PIAS1 at Ser503 and showed this phosphorylation determines PIAS1 E3 ligase activity. In rat brain, Elk-1 was SUMOylated by PIAS1, which decreased Elk-1 phosphorylation and down-regulated GADD45α expression. Moreover, lentiviral-mediated transduction of Elk-1-SUMO1 reduced the number of hippocampal apoptotic neurons in APP/PS1 mice. MAPK/ERK-mediated phosphorylation of PIAS1 at Ser503 determines PIAS1 E3 ligase activity. Moreover, PIAS1 mediates SUMOylation of Elk-1, which functions as an endogenous defence mechanism against Aβ toxicity in vivo. Targeting Elk-1 SUMOylation could be considered a novel therapeutic strategy against AD.

Effects of macrophages and CXCR2 on adipogenic differentiation of bone marrow mesenchymal stem cells.

Macrophages and many chemokines are closely associated with the adipogenic differentiation of bone marrow mesenchymal stem cells (MSCs), but their roles in adipogenesis and the underlying mechanisms are not fully understood. Here, we first investigated the influence of macrophages on the differentiation of MSCs in vitro. We found that RAW246.7 macrophages cocultured with MSCs strongly blocked the differentiation progress and inhibited the expression of C-X-C motif chemokine ligand 1 (CXCL1) during adipogenesis. Coculture with MSCs mainly induced macrophages toward M2 polarization. In addition, the expression of CXCL1 and its receptor, C-X-C chemokine receptor type 2, CXCR2 are high during adipogenic differentiation of MSCs and not in mature adipocytes. Although CXCL1 had no effect on adipogenesis, treatment with a specific CXCR2 inhibitor, SB225002, hampered the adipogenic differentiation of MSCs. Blocking CXCR2 decreased p38 and Elk1 phosphorylation but increased the extracellular signal-regulated kinase (ERK) phosphorylation at the initial stage of adipogenesis, which suppressed the phosphorylation of p38/ERK-Elk1 at the late stage. Inhibition of ERK had similar effects on adipogenesis and Elk1 phosphorylation. Our data suggest that MSCs interact with macrophages during adipogenic differentiation. CXCR2 regulates the adipogenic differentiation of MSCs by altering the activation of the p38/ERK-Elk1 signaling pathway.

Design, synthesis and biological evaluation of fused naphthofuro[3,2-c] quinoline-6,7,12-triones and pyrano[3,2-c]quinoline-6,7,8,13-tetraones derivatives as ERK inhibitors with efficacy in BRAF-mutant melanoma

Approximately 60% of human cancers exhibit enhanced activity of ERK1 and ERK2, reflecting their multiple roles in tumor initiation and progression. Acquired drug resistance, especially mechanisms associated with the reactivation of the MAPK (RAF/MEK/ERK) pathway represent a major challenge to current treatments of melanoma and several other cancers. Recently, targeting ERK has evolved as a potentially attractive strategy to overcome this resistance. Herein, we report the design and synthesis of novel series of fused naphthofuro[3,2-c]quinoline-6,7,12-triones 3a-f and pyrano[3,2-c]quinoline-6,7,8,13-tetraones 5a,b and 6, as potential ERK inhibitors. New inhibitors were synthesized and identified by different spectroscopic techniques and X-ray crystallography. They were evaluated for their ability to inhibit ERK1/2 in an in vitro radioactive kinase assay. 3b and 6 inhibited ERK1 with IC50s of 0.5 and 0.19 µM, and inhibited ERK2 with IC50s of 0.6 and 0.16 µM respectively. Kinetic mechanism studies revealed that the inhibitors are ATP-competitive inhibitors where 6 inhibited ERK2 with a Ki of 0.09 µM. Six of the new inhibitors were tested for their in vitro anticancer activity against the NCI-60 panel of tumor cell lines. Compound 3b and 6 were the most potent against most of the human tumor cell lines tested. Moreover, 3b and 6 inhibited the proliferation of the BRAF mutant A375 melanoma cells with IC50s of 3.7 and 0.13 µM, respectively. In addition, they suppressed anchorage-dependent colony formation. Treatment of the A375 cell line with 3b and 6 inhibited the phosphorylation of ERK substrates p-90RSK and ELK-1 and induced apoptosis in a dose dependent manner. Finally, a molecular docking study showed the potential binding mode of 3b and 6 within the ATP catalytic binding site of ERK2.