Relationship between the MAPK/ERK pathway and neurocyte apoptosis after cerebral infarction in rats.

OBJECTIVE: The aim of this study was to explore the relationship between the mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinase (ERK) pathway and neurocyte apoptosis after cerebral infarction in rats. MATERIALS AND METHODS: Neural stem cells were isolated from rats by establishing the cerebral infarction model and sham model. Isolated cells were cultured in complete culture medium in vitro. Real-time quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was used to detect the messenger ribonucleic acid (mRNA) expression of ERK1 and ERK2 in the MARK pathway. Western blotting was applied to examine the activation of the MAPK/ERK pathway and neuron-specific markers. The expression of neuron-specific enolase (NSE) was detected via immunofluorescence. Cell activity and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. RESULTS: The mRNA expressions of ERK1 and ERK2 in neural stem cells increased in a time-dependent manner after cerebral infarction in rats. The expressions of ERK1, ERK2, cyclin D1, Nestin, NSE and glial fibrillary acidic-protein (GFAP) in neural stem cells were significantly decreased after being treated with SCH772984. Cell activity, proliferation and differentiation were markedly inhibited. However, cleaved-caspase 3 protein and apoptosis rate were remarkably increased. CONCLUSIONS: The MAPK/ERK pathway seriously affects neurocyte apoptosis after cerebral infarction in rats. When the MAPK/ERK pathway is inhibited, neurocyte apoptosis is remarkably increased after cerebral infarction in rats.

Selenium-isotopic signature toward mass spectrometric identification and enzyme activity assay.

The unraveling of enzymatic reactions, especially identification of enzymatic substrates or products, is important to elucidate biological processes. Here a selenium-isotopic signature for mass spectrometric identification of enzymatic-related species is demonstrated by using selenium-containing peptides (SePeps) as substrates. Thus a strategy is proposed for rapid and precise assay of multiple enzyme activity. These SePeps can be synthesized by introduction of one selenomethionine residue in the sequence and simply identified in the full-scan mode with the feature of distinctive selenium-isotopic distribution without MS/MS verifications, which proposes a novel solution to the specific identification of enzyme-related species, allows to exclude the interferences of species with tiny mass differences in bio-samples, and meanwhile can offer a judgement on data accuracy for the analysis of enzyme activities. As a proof-of-concept, a method for multiple analysis of two representative enzymes in MCF-7 cell lysate has been developed with the isotopic peak areas of either SePep substrates or enzymatic products with the top intensities. These results could be the foundation to extend the method for more complicated enzyme systems. The selenium-isotopic signature provides a powerful protocol for high-throughput assays of peptide-metabolizing enzymes with enhanced confidence and can be extended to screen enzymatic reaction-related substrates.

Human G protein-coupled receptor 30 is N-glycosylated and N-terminal domain asparagine 44 is required for receptor structure and activity.

G protein-coupled receptor 30 (GPR30), or G protein-coupled estrogen receptor (GPER), is a G protein-coupled receptor (GPCR) that is currently attracting considerable attention in breast cancer and cardiometabolic regulation. The receptor was reported to be a novel membrane estrogen receptor mediating rapid non-genomic responses. However, questions remain about both the cognate ligand and the subcellular localization of receptor activity. Here, we used human embryonic kidney (HEK) 293 (HEK293) cells ectopically expressing N-terminally FLAG-tagged human GPR30 and three unique antibodies (Ab) specifically targetting the receptor N-terminal domain (N-domain) to investigate the role of N-glycosylation in receptor maturation and activity, the latter assayed by constitutive receptor-stimulated extracellular-regulated protein kinase (ERK) 1/2 (ERK1/2) activity. GPR30 expression was complex with receptor species spanning from approximately 40 kDa to higher molecular masses and localized in the endoplasmatic reticulum (ER), the plasma membrane (PM), and endocytic vesicles. The receptor contains three conserved asparagines, Asn25, Asn32, and Asn44, in consensus N-glycosylation motifs, all in the N-domain, and PNGase F treatment showed that at least one of them is N-glycosylated. Mutating Asn44 to isoleucine inactivated the receptor, yielding a unique receptor species at approximately 20 kDa that was recognized by Ab only in a denatured state. On the other hand, mutating Asn25 or Asn32 either individually or in combination, or truncating successively N-domain residues 1-42, had no significant effect either on receptor structure, maturation, or activity. Thus, Asn44 in the GPR30 N-domain is required for receptor structure and activity, whereas N-domain residues 1-42, including specifically Asn25 and Asn32, do not play any major structural or functional role(s).

Utility of Combined EZH2, p-ERK1/2, p-STAT, and MYC Expression in the Differential Diagnosis of EZH2-positive Hodgkin Lymphomas and Related Large B-Cell Lymphomas.

EZH2 is a methyltransferase that plays an important tumorigenic role in various neoplasms. We previously found that EZH2 is expressed in a range of aggressive B-cell lymphomas (ABCLs), T-cell lymphomas, and histiocytic neoplasms, with differential expression of intracellular signaling molecules p-ERK, MYC, and p-STAT3, potential regulators of EZH2 expression. We studied EZH2 expression in nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), classic Hodgkin lymphoma (cHL), T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL), and B-cell Lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphomas and classic Hodgkin lymphoma (BCLu-DLBCL/cHL), as well as the coexpression of p-ERK, MYC, and p-STAT3 in these neoplasms. The neoplastic LP cells of NLPHL and Hodgkin/Reed-Sternberg cells of cHL were strongly positive for EZH2, as were the neoplastic cells in THRLBCL and BCLu-DLBCL/cHL. EZH2 expression correlated with proliferation rate, as assessed by Ki-67 staining. LP cells in NLPHL and Hodgkin/Reed-Sternberg cells in cHL were strongly positive for p-ERK, p-STAT3, and MYC, as were the neoplastic cells in THRLBCL and BCLu-DLBCL/cHL, in contrast to the differential expression of these molecules seen in ABCLs. These findings suggest that combined expression of p-ERK, MYC, and p-STAT3 is a useful immunohistochemical pattern for the diagnosis of EZH2-positive Hodgkin lymphomas and related lymphomas, in contrast to ABCLs. Furthermore, the overexpression of EZH2, in association with coexpression of tumorigenic signaling molecules, suggests an oncogenic role for this molecule in the development of Hodgkin lymphomas and related lymphomas. THRLBCL and BCLu-DLBCL/cHL appear to have a mechanism for the regulation of EZH2 expression that is similar to NLPHL and cHL and different from that of ABCLs. In addition, EZH2 and associated signaling cascades may serve as therapeutic targets for the treatment of Hodgkin lymphomas and related lymphomas.

Biomarker and Drug Target Discovery Using Quantitative Proteomics Post-Intracerebral Hemorrhage Stroke in the Rat Brain.

The pathological mechanisms of acute intracerebral hemorrhage (ICH) remain unknown and unverified. In the present study, we used quantitative proteomics to elucidate the pathological mechanisms and to identify novel biomarker and therapeutic target candidates via tissue proteome in a rat model of acute ICH. Rats were experimentally induced with ICH (n = 6) or Sham (n = 6), and their brain tissue was obtained by 24 h. The TMT-LC-MS/MS-based proteomics approach was used to quantify the differential proteomes across brain tissue, and the results were further analyzed by ingenuity pathway analysis to explore canonical pathways and the relationship involved in the uploaded data. Upon quantification, we found that 96 secreted proteins that were identified in the ICH 24-h group were significantly different those in the control group (P < 0.05); among these proteins, 57 increased and 39 decreased in abundance. Bioinformatic analyses of differentially expressed proteins demonstrated that the protein localization and ERK1 and ERK2 cascade were the top two biological processes with the highest concentrations of differentially proteins. The top protein-protein action network with high confidence levels of protein was the albumin and ERK signaling pathways. Albumin, ERK, and p-ERK were assessed in brain tissue by western blot analysis, and higher expression levels of albumin and p-ERK were observed in the ICH group. Our proteomic results highlight important change in the biological processes of ERK1 and ERK2 cascade, which are possible targets for future interventions of ICH. To our knowledge, this study provides in-depth analysis of ICH in brain tissue, and we propose 96 new biomarker candidates for ICH, including albumin and ERK.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells.

We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

Protective effect of cyanidin-3-O-glucoside on neonatal porcine islets.

Oxidative stress is a major cause of islet injury and dysfunction during isolation and transplantation procedures. Cyanidin-3-O-glucoside (C3G), which is present in various fruits and vegetables especially in Chinese bayberry, shows a potent antioxidant property. In this study, we determined whether C3G could protect neonatal porcine islets (NPI) from reactive oxygen species (H2O2)-induced injury in vitro and promote the function of NPI in diabetic mice. We found that C3G had no deleterious effect on NPI and that C3G protected NPI from damage induced by H2O2 Significantly higher hemeoxygenase-1 (HO1) gene expression was detected in C3G-treated NPI compared to untreated islets before and after transplantation (P < 0.05). Western blot analysis showed a significant increase in the levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K/Akt) proteins in C3G-treated NPI compared to untreated islets. C3G induced the nuclear translocation of nuclear erythroid 2-related factor 2 (NRF2) and the significant elevation of HO1 protein. Recipients of C3G-treated NPI with or without C3G-supplemented drinking water achieved normoglycemia earlier compared to recipients of untreated islets. Mice that received C3G-treated islets with or without C3G-supplemented water displayed significantly lower blood glucose levels at 5-10 weeks post-transplantation compared to mice that received untreated islets. Mice that received C3G-treated NPI and C3G-supplemented drinking water had significantly (P < 0.05) lower blood glucose levels at 7 and 8 weeks post-transplantation compared to mice that received C3G-treated islets. These findings suggest that C3G has a beneficial effect on NPI through the activation of ERK1/2- and PI3K/AKT-induced NRF2-mediated HO1 signaling pathway.

Biological screening of cucurbitacin inspired estrone analogs targeting mitogen-activated protein kinase (MAPK) pathway.

Assembly of cucurbitacin inspired estrone analogs has been previously synthesized and screened against melanoma cell lines. Further synthetic optimization was executed via installation of Azide polar functional moiety across 23, 24 α, ß-unsaturated ketone side chain using Michael addition reaction. This was followed by biological screening against melanoma cell lines employing MTT assay, in-cell-based ELISA assay, and Western blot analysis to monitor the potential of the synthesized analogs to inhibit the phosphorylated ERK levels. This resulted in evolution of MH-4 possessing IC50 of 3.59 µm with significant decrease in the p-ERK and targeting MAPK pathway.

Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM.

Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM.

A tissue microarray study of toll-like receptor 4, decoy receptor 3, and external signal regulated kinase 1/2 expressions in astrocytoma.

INTRODUCTION: Decoy receptor 3 (DcR3) functions as a death decoy inhibiting apoptosis mediated by the tumor necrosis factor receptor family. It is highly expressed in many tumors and its expression can be regulated by the MAPK/ERK signaling pathway and ERK is a vital member of this pathway. Toll-like receptor 4 (TLR4) is expressed on immune cells. Increased TLR4 expression has been associated with various types of cancers. MATERIAL AND METHODS: The study was conducted to investigate the expression of DcR3, ERK1/2, and TLR4 in astrocytomas and evaluate if they are validating markers for discriminating glioblastoma from anaplastic astrocytoma in limited surgical specimen. Expression of DcR3, ERK1/2, and TLR4 was determined by immunohistochemical staining of tissue microarray from 48 paraffin-embedded tissues. A binary logistic regression method was used to generate functions that discriminate between anaplastic astrocytomas and glioblastomas. RESULTS: The expression of TLR4 and DcR3 was significantly higher in glioblastomas than in anaplastic astrocytomas. DcR3 could discriminate anaplastic astrocytomas from glioblastomas with high sensitivity (93.8%), specificity (90%), and accuracy (92.3%). CONCLUSION: Our results suggest that DcR3 may be a useful marker for discriminating anaplastic astrocytomas from glioblastomas.

Differential expression of enhancer of zeste homolog 2 (EZH2) protein in small cell and aggressive B-cell non-Hodgkin lymphomas and differential regulation of EZH2 expression by p-ERK1/2 and MYC in aggressive B-cell lymphomas.

EZH2, a member of the polycomb protein group, is an important methyltransferase that is overexpressed in various neoplasms. We found that in small cell B-cell lymphomas, EZH2 is expressed in <40% of neoplastic cells, with heterogenous signal intensity. In aggressive B-cell lymphomas, 70-100% of tumor cells were positive for EZH2 expression with high signal intensity, which correlated with a high proliferation rate. We investigated the potential signaling molecules that regulate EZH2 overexpression in aggressive B-cell lymphomas and found that 80% of cases of EZH2-positive diffuse large B-cell lymphoma show high p-ERK1/2 expression (average ~57% tumor cell positivity). In contrast, only a small percentage of tumor cells (~10%) show p-ERK1/2 expression in Burkitt lymphoma and double hit lymphoma. On average, 91 and 76% of neoplastic cells were positive for MYC expression in Burkitt lymphoma and double hit lymphoma, respectively, while only 20% neoplastic cells were positive for MYC expression in diffuse large B-cell lymphoma. None of the aggressive B-cell lymphomas showed significant p-STAT3 expression in EZH2-overexpressed cases. The correlation of EZH2 expression with aggressive behavior and proliferation rate in B-cell neoplasms suggests that this molecule may function as an oncogenic protein in these neoplasms, with possible regulation by different signaling cascades in different types of aggressive B-cell lymphomas: p-ERK-related signaling in diffuse large B-cell lymphoma, and MYC-related signaling in Burkitt lymphoma and double hit lymphoma. Furthermore, EZH2 and associated signaling cascades may serve as therapeutic targets for the treatment of aggressive B-cell lymphomas.

Multiplexed Western Blotting Using Microchip Electrophoresis.

Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 µg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 µm deep × 50 µm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps.

Localization of active, dually phosphorylated extracellular signal-regulated kinase 1 and 2 in colorectal cancer with or without activating BRAF and KRAS mutations.

Colorectal cancers (CRC) often show activating mutations of the KRAS or BRAF genes, which stimulate the extracellular signal-regulated kinase (ERK) pathway, thus increasing cell proliferation and inhibiting apoptosis. However, immunohistochemical results on ERK activation in such tumors differ greatly. Recently, using a highly optimized immunohistochemical method, we obtained evidence that high levels of ERK activation in rectal adenocarcinomas were associated with resistance to radiochemotherapy. In order to determine whether KRAS and/or BRAF mutations correlate to immunohistochemically detectable increases in phosphorylation of ERK (pERK), we stained biopsies from 36 CRC patients with activating mutations in the BRAF gene (BRAFV600E: BRAF(m)), the KRAS gene (KRAS(m)) or in neither (BRAF/KRAS(n)) with this optimized method. Staining was scored in blind-coded specimens by two observers. Staining of stromal cells was used as a positive control. BRAF(m) or KRAS(m) tumors did not show higher staining scores than BRAF/KRAS(n) tumors. Although BRAFV600E staining occurred in over 90% of cancer cells in all 9 BRAF(m) tumors, 3 only showed staining for pERK in less than 10% of cancer cell nuclei. The same applied to 4 of the 14 KRAS(m) tumors. A phophorylation-insensitive antibody demonstrated that lack of pERK staining did not reflect defect expression of ERK1/2 protein. Thus, increased staining for pERK does not correlate to BRAF or KRAS mutations even with a highly optimized procedure. Further studies are required to determine whether this reflects differences in expression of counterregulatory molecules, including ERK phosphatases.

Possible Mechanisms of Di(2-ethylhexyl) Phthalate-Induced MMP-2 and MMP-9 Expression in A7r5 Rat Vascular Smooth Muscle Cells.

Proliferation and migration of vascular smooth muscle cells (VSMC) are important in the development and/or progression of many cardiovascular diseases, including atherosclerosis. Evidence shows that matrix metalloproteinase (MMP)-2 and MMP-9 are related to the pathogenesis of atherosclerosis. The expressions of MMP-2 and MMP-9 in atherosclerosis are regulated via various pathways, such as p38 mitogen activated protein kinase (MAPK), extracellular signal regulated kinase 1 and 2 (ERK1/2), Akt, and nuclear factor kappa (NF-κB). Di(2-ethylhexyl) phthalate (DEHP) has been shown to induce atherosclerosis by increasing tumor necrosis factor (TNF)-α, interleukin (IL)-6, and intercellular adhesion molecule (ICAM) productions. However, whether DEHP poses any effects on MMP-2 or MMP-9 expression in VSMC has not yet been answered. In our studies, rat aorta VSMC was treated with DEHP (between 2 and 17.5 ppm) and p38 MAPK, ERK1/2, Akt, NF-κB, and MMP-2 and MMP-9 proteins and activities were measured. Results showed that the presence of DEHP can induce higher MMP-2 and MMP-9 expression than the controls. Similar results on MMP-regulating proteins, i.e., p38 MAPK, ERK1/2, Akt, and NF-κB, were also observed. In summary, our current results have showed that DEHP can be a potent inducer of atherosclerosis by increasing MMP-2 and MMP-9 expression at least through the regulations of p38 MAPK, ERK1/2, Akt, and NF-κB.

Extracardiac rhabdomyomas--presentation of two cases with analysis of AKT/mTOR and ERK1/2 signaling.

Extracardiac rhabdomyomas (RM) are very rare benign tumors with a poorly understood pathogenesis. In this report we describe two RM cases--a sublingual adult type tumor and a genital type tumor involving the uterine cervix. The patho-clinical characteristics, as well as the pioneer immunohistochemical analysis of ERK1/2 and AKT/mTOR pathway status is included. The expression of key proteins involved in above signaling gives new insight into the biology of extracardiac RM.

Detection and Differentiation of Threonine- and Tyrosine-Monophosphorylated Forms of ERK1/2 by Capillary Isoelectric Focusing-Immunoassay.

The extracellular signal regulated kinases ERK1/2 play important roles in the regulation of diverse cellular functions and have been implicated in several human diseases. In addition to the fully activated, diphosphorylated ERK1/2 protein, monophosphorylated forms of ERK1/2 have been observed, which may have distinct biological functions. We report here on the highly sensitive detection and differentiation of unphosphorylated, threonine-phosphorylated (pT), tyrosine-phosphorylated (pY) and diphosphorylated ERK1 and ERK2 by capillary isoelectric focusing followed by immunological detection (CIEF-immunoassay). Eight different phosphorylated and unphosphorylated forms of ERK1/2 were resolved according to charge. The unequivocal identification and differentiation of ERK1 and ERK2 forms monophosphorylated at either threonine or tyrosine was achieved by competitive blocking with specific phospho-peptides and different phosphorylation-sensitive antibodies. The suitability of the additional pT-ERK1/2 and pY-ERK1/2 differentiation for the time-resolved in-depth study of phospho-form distribution in response to specific stimuli is demonstrated in human neuroblastoma SH-SY5Y and monocytic THP-1 cell lines, and in human peripheral blood mononuclear cells.

Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics.

Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-Iα and PKG-Iß isoforms are present in preadipocytes, only PKG-Iß isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems.

Erk1/2 activation in stromal fibroblasts from sporadic basal cell carcinomas.

BACKGROUND: Constitutive activation of the Erk pathway can lead to oncogenic transformation. However, the Erk pathway is not activated in human basal cell carcinomas (BCCs); although in animal models, this seems to be important. OBJECTIVE: To help understand the role of Erk activity in BCC formation. MATERIALS AND METHODS: The authors assayed the specific levels of phosphorylated Erk by immunohistochemistry in BCCs and normal skin biopsies. They have also analyzed Erk activation by immunoblot in fibroblasts isolated from BCC. RESULTS: By immunohistochemical analysis, the authors have observed that 10 of BCCs (56%) did not show phosphor-Erk staining in tumor masses and 7 (40%) showed a gradient staining exhibiting phospho-Erk only in the epidermal side of tumor masses. Remarkably, 15 BCC samples (83%) showed phospho-Erk accumulation in stroma. Six of the 9 independent cultures of dermal fibroblasts isolated from BCC maintained Erk activation "in vitro." CONCLUSION: The authors propose that there is a specific cell-type regulation of Erk activity in BCC, and this feature may be relevant during BCC formation. Stroma region from BCCs showed Erk activation and reduced proliferation. Conversely, Erk activation is barely detectable in proliferative BCCs.

Fluid shear stress stimulates osteogenic differentiation of human periodontal ligament cells via the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling pathways.

BACKGROUND: Fluid shear stress (FSS) is a major type of mechanical stress that is loaded on human periodontal ligament cells (hPDLCs) during mastication and orthodontic tooth movement. This study aims to clarify the effect of FSS on the osteogenic differentiation of hPDLCs and to further verify the involvement of mitogen-activated protein kinase (MAPK) signaling in this process. METHODS: After isolation and characterization, hPDLCs were subjected to 2-hour FSS at 12 dynes/cm(2), and cell viability, osteogenic gene mRNA expression, alkaline phosphatase (ALP) activity, secretion of Type I collagen (COL-I), and calcium deposition were assayed. The levels of phosphorylated p38 and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) in response to FSS were detected by Western blot, and the involvement of ERK1/2 and p38 MAPK signaling pathways in hPDLC osteogenesis under FSS was investigated using the specific MAPK inhibitors U0126 (2Z,3Z)-2,3-bis[amino(2-aminophenylthio)methylene]succinonitrile,ethanol) and SB203580 (4-[4-(4-fluorophenyl)-2-(4-[methylsulfinyl]phenyl)-1H-imidazol-5-yl]pyridine). RESULTS: The application of FSS on hPDLCs induced an early morphologic change and rearrangement of filamentous actin. ALP activity, messenger RNA (mRNA) levels of osteogenic genes, COL-I, and osteoid nodules were significantly increased by FSS. Moreover, ERK1/2 and p38 were activated in different ways after FSS exposure. U0126 and SB203580 completely blocked the FSS-induced increases in ALP activity and osteogenic gene mRNA expression and osteoid nodules formation. CONCLUSIONS: FSS is an effective approach for stimulating osteogenic differentiation of hPDLCs. The ERK1/2 and p38 MAPK signaling pathways are involved in this cellular process.