Paracrine TGF-ß1 from breast cancer contributes to chemoresistance in cancer associated fibroblasts via upregulation of the p44/42 MAPK signaling pathway.

Conventionally, Cancer-associated fibroblasts (CAFs) are considered as an inducer of chemoresistance in cancer cells. However, the underlying mechanism by which carcinomas induce chemoresistance in CAFs through tumor-stroma cross-talk is largely unknown. Henceforth, we uncovered a network of paracrine signals between carcinoma and CAFs that drives chemoresistance in CAFs. Acquired tamoxifen and 5-Fu resistant cell lines MCF-7 and MDA-MB-468 respectively showed higher apoptotic resistance compared to the parental cell. Besides, chemoresistant breast cancer cells showed overexpression of TGF-ß1 and have the higher potential to induce CAF phenotype in the normal dermal fibroblasts in a paracrine manner through the TGF-ß1 cytokine, compared to their parental cell. Moreover, the chemoresistant cancer cells augmented the EMT markers with a reduction of E-cadherin in the CAFs. Importantly we found out that the TGF- ß1 enriched conditioned media from both of the resistant cells triggered chemoresistance in the CAFs by p44/42 MAPK signaling axis. Mechanistically, pharmacological and genetic blockade of TGF-ß1 inhibits p44/42 MAPK activation with the subsequent restoration of chemosensitivity in the CAFs. Altogether we ascertained that chemoresistant cancer cells have tremendous potential to modulate the CAFs compared to the parental counterpart. Targeting TGF-ß1 and p44/42 MAPK signaling in the future may help to abrogate the chemoresistance in the CAFs.

Altered gene expression profile of Wolbachia pipientis wAlbB strain following transinfection from its native host Aedes albopictus to Aedes aegypti cells.

Wolbachia is an obligate intracellular bacterial symbiont prevalent among arthropods and nematodes. To survive and reproduce, Wolbachia interacts with and modifies host subcellular structures, while sensing and responding to changes within the cellular environment. In mutualistic associations, Wolbachia may provision the host with metabolites, or help to maintain the chemical homeostasis of the host cell. Some strains can rapidly invade insect populations by manipulating host reproductive biology, while also preventing viral replication, allowing their use in vector control of arthropod-borne viruses. The Aedes albopictus-derived strain wAlbB is promising in this regard. When transinfected into the Yellow fever mosquito, Aedes aegypti, wAlbB reaches high frequencies within wild populations, and strongly inhibits viral transmission. Despite its obvious potential, much is still unknown about the molecular interactions between Wolbachia and host that enable its use in vector control. Furthermore, most Wolbachia transinfection research to date has focused on host effects. In the current study, we used a cell line model to explore the effect of transinfection of wAlbB from Ae. albopictus to Ae. aegypti. Using RNA sequencing, we show that several genes associated with host-symbiont interactions were downregulated by transinfection, with the greatest downregulation exhibited by prophage-associated genes.

Expression and possible roles of extracellular signal-related kinases 1-2 (ERK1-2) in mouse primordial germ cell development.

In the present work, we described the expression and activity of extracellular signal-related kinases 1-2 (ERK1-2) in mouse primordial germ cells (PGCs) from 8.5-14.5 days post coitum (dpc) and investigated whether these kinases play a role in regulating the various processes of PGC development. Using immunofluorescence and immunoblotting to detect the active phosphorylated form of ERK1-2 (p-ERK1-2), we found that the kinases were present in most proliferating 8.5-10.5 dpc PGCs, low in 11.5 dpc PGCs, and progressively increasing between 12.5-14.5 dpc both in female and male PGCs. In vitro culture experiments showed that inhibiting activation of ERK1-2 with the MEK-specific inhibitor U0126 significantly reduced the growth of 8.5 dpc PGCs in culture but had little effect on 11.5-12.5 dpc PGCs. Moreover, we found that the inhibitor did not affect the adhesion of 11.5 dpc PGCs, but it significantly reduced their motility features onto a cell monolayer. Further, while the ability of female PGCs to begin meiosis was not significantly affected by U0126, their progression through meiotic prophase I was slowed down. Notably, the activity of ERK1-2 was necessary for maintaining the correct expression of oocyte-specific genes crucial for germ cells survival and the formation of primordial follicles.

Purinergic P2Y2 receptors modulate endothelial sprouting.

Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.

Ethanol Withdrawal Alters the Oxidative State of the Heart Through AT1-Dependent Mechanisms.

AIMS: We investigated the cardiac effects of ethanol withdrawal and the possible role of AT1 receptors in such response. METHODS: Male Wistar rats were treated with increasing doses of ethanol (3 to 9%, vol./vol.) for 21 days. The cardiac effects of ethanol withdrawal were investigated 48 h after abrupt discontinuation of ethanol. Some animals were orally treated with losartan (10 mg/kg/day), a selective AT1 receptor antagonist. RESULTS: Ethanol withdrawal did not affect serum levels of creatine kinase (CK)-MB. Losartan prevented ethanol withdrawal-induced increase in superoxide anion (O2•-) production in the left ventricle (LV). However, ethanol withdrawal did no alter the levels of thiobarbituric acid reactive substances (TBARS) or the expression of Nox1, Nox2 or Nox4 were found in the LV. Ethanol withdrawal reduced the concentration of hydrogen peroxide (H2O2) in the LV and this response was prevented by losartan. Ethanol withdrawal increased catalase activity in the LV and losartan attenuated this response. No changes on superoxide dismutase (SOD) activity or expression were detected in the LV during ethanol withdrawal. The expression of AT1, AT2 or angiotensin converting enzyme (ACE) was not affected by ethanol withdrawal. Similarly, no changes on the expression of ERK1/2, SAPK/JNK, COX-1 or COX-2 were found in the LV during ethanol withdrawal. CONCLUSIONS: Ethanol withdrawal altered the cardiac oxidative state through AT1-dependent mechanisms. Our findings showed a role for angiotensin II/AT1 receptors in the initial steps of the cardiac effects induced by ethanol withdrawal.

Downregulation of miR-21 promotes tibial fracture healing in rabbits through activating ERK pathway.

OBJECTIVE: The aim of this study was to investigate the effect of micro ribonucleic acid (miR)-21 on tibial fracture healing in rabbits by regulating the extracellular signal-regulated kinase (ERK) signaling pathway, and to explore its possible underlying mechanism. MATERIALS AND METHODS: A total of 15 healthy male rabbits were randomly divided into three groups, including: model group A (fracture group, n=5), model group B (fracture treatment group, n=5), and model group C (miR-21 siRNA + treatment group, n=5). Fracture healing was observed by imaging. The content of the serum collagen I and collagen II in rabbits was detected via enzyme-linked immunosorbent assay (ELISA). The morphology of bone tissues was observed via staining. Moreover, the expressions of ERK, transforming growth factor-ß1 (TGF-ß1), and Smad in osteoblasts of tibia were observed via Western blotting and Reverse Transcription-Polymerase Chain Reaction (RT-PCR), respectively. RESULTS: There was bony callus formation in group B and C when compared with group A. Compared with group B, bony callus formation was significantly accelerated in group C, while healing cycle was shortened. Hematoxylin-eosin (HE) staining and Masson staining indicated that compared with group A, group C had more fibrous calluses, new capillaries, and fibroblasts in tissues. Meanwhile, group C exerted better maturity of collagen tissues and higher osteoid content at 20 d after modeling. Compared with group C, there were more osteoid tissues with poor maturity in group B. Meanwhile, intramembranous bone formation was deformed, and collagen content was remarkably lower in group B. The content of serum collagen I and collagen II remarkably increased in group B compared with group A (p<0.05). However, it was significantly upregulated in group C compared with group B, showing statistically significant differences (p<0.05). According to the results of Western blotting, the protein expressions of TGF-ß1, Smad, and ERK in osteoblasts were significantly upregulated in group B when compared with those in group A (p<0.05). However, they increased remarkably in group C when compared with group B (p<0.05). Besides, RT-PCR results revealed that the messenger RNA (mRNA) expressions of TGF-ß1, Smad, and ERK in osteoblasts were significantly higher in group B than those in group A (p<0.05). However, they were markedly raised in group C in comparison with group B (p<0.05). CONCLUSIONS: Down-regulation of miR-21 promotes tibial fracture healing in rabbits by activating the ERK signaling pathway.

MiR-129 reduces CDDP resistance in gastric cancer cells by inhibiting MAPK3.

OBJECTIVE: Abnormal expression of mitogen-activated protein kinase 3 (MAPK3) is related to invasion, metastasis, and drug resistance of multiple tumor cells. MiR-129 expression is associated with gastric cancer. Bioinformatics analysis showed a targeting relation between miR-129 and MAPK3. This study investigated whether miR-129 plays a role in regulating MAPK3 expression, affecting proliferation, apoptosis, and cisplatin (CDDP) resistance of gastric cancer cells. MATERIALS AND METHODS: The dual-luciferase reporter gene assay was used to assess the targeted regulation between miR-129 and MAPK3. The expression of miR-129 and MAPK3 in CDDP-resistant cell line MGC-803/CDDP and the parental MGC-803 cells was measured. MGC-803/CDDP cells were cultured in vitro and divided into miR-NC group and miR-129 mimic group. The expression of MAPK3 and p-MAPK3 protein were detected by Western blot and the effect of CDDP treatment on cell apoptosis and proliferation was detected by flow cytometry. RESULTS: There was a targeted regulation relation between miR-129 and MAPK3 mRNA. MiR-129 expression in MGC-803/CDDP cells was significantly lower than that in MGC-803 cells and the expression of MAPK3 mRNA and protein was significantly higher than that in MGC-803 cells. Compared with miR-NC group, the expression of MAPK3 and p-MAPK3 in MHC-803/CDDP cells in miR-129 mimic transfection group was significantly decreased, with increased cell apoptosis and reduced cell proliferation. CONCLUSIONS: The decreased expression of miR-129 and the up-regulation of MAPK3 are associated with CDDP resistance in gastric cancer cells. Overexpression of miR-129 inhibits MAPK3 expression and cell proliferation, it induces cell apoptosis and reduces CDDP resistance.

Nicotine is neuroprotective to neonatal neurons of sympathetic ganglion in rat.

Sympathetic neurons of SCG are dependent on availability of nerve growth factor (NGF) for their survival. SCG neurons express nicotinic receptors (nAChR) whose expression levels are modulated by nicotine. Nicotine exerts multiple effects on neurons, including neuroprotection, through nAChR binding. Although sympathetic neurons express robust levels of nAChR, a possible neuroprotective role for nicotine in these neurons is not well-understood. Therefore we determined the effect of nicotine exposure on survival of SCG neurons during NGF withdrawal in a well-established cell culture system. NGF was withdrawn in rat neonatal SCG neuron cultures which were then treated with either 10 µM nicotine alone or with nAChR antagonists 0.1 µM α-bungarotoxin (antagonist for α7 subunit bearing nAChR) and 10 µM mecamylamine (non-specific antagonist for ganglionic nAChR) for 48 h. Apoptotic death was determined by TUNEL staining. Cell survival was also determined by MTS assay. Western blot analysis of ERK1/2 was also performed. Our results showed that exposure to 10 µM nicotine significantly reduced apoptotic cell death in SCG neurons resulting from NGF withdrawal as shown by fewer TUNEL positive cells. The MTS assay results also revealed that 10 µM nicotine concentration significantly increased cell survival thus indicating neuroprotective effect of nicotine against cell death resulting from NGF withdrawal. Nicotinic receptor antagonists (bungarotoxin & mecamylamine) attenuated the effect of nicotine's action of neuroprotection. Western blot analysis showed an increased expression of ERK1/2 in nicotine treated cultures suggesting nicotine provided neuroprotection in SCG neurons by increasing the expression of ERK1/2 through nicotinic receptor dependent mechanisms.

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.

Molecular analysis of biocompatibility of anodized titanium with deposited silver nanodendrites.

Titanium (>99.6% purity) and its anodically oxidized modifications, with and without deposited silver nanodendrites regarding its biocompatibility were evaluated. In human gingival fibroblasts and osteoblast cell lines grown on tested samples, the level of expression of genes encoding αV (ITGAV) and ß1 (ITGB1) integrin subunits also genes encoding focal adhesion (FAK) and extracellular-signal regulated (ERK) kinases was assessed. For this purpose, the qualitative and quantitative PCR technique was used. The expression of studied genes was dependent on the origin of cell lines and the type of evaluated material. The high expression of PBGD and ITGAV genes in fibroblasts grown on the surface of anodically modified titanium with deposited silver nanodendrites indicates potentially high biocompatibility of these samples for soft tissue cells. The high expression of the ITGB1 and ERK1 genes and the enhanced expression of the FAK gene in osteoblasts cells grown on the tested material was also observed. Summarizing, the nanocrystalline Ti modified with silver deposits showed higher biocompatibility in comparison with the conventional pure Ti samples.

Extracellular signal-regulated kinase (ERK) activation preserves cardiac function in pressure overload induced hypertrophy.

BACKGROUND: Chronic pressure overload and a variety of mediators induce concentric cardiac hypertrophy. When prolonged, cardiac hypertrophy culminates in decreased myocardial function and heart failure. Activation of the extracellular signal-regulated kinase (ERK) is consistently observed in animal models of hypertrophy and in human patients, but its role in the process is controversial. METHODS: We generated transgenic mouse lines with cardiomyocyte restricted overexpression of intrinsically active ERK1, which similar to the observations in hypertrophy is phosphorylated on both the TEY and the Thr207 motifs and is overexpressed at pathophysiological levels. RESULTS: The activated ERK1 transgenic mice developed a modest adaptive hypertrophy with increased contractile function and without fibrosis. Following induction of pressure-overload, where multiple pathways are stimulated, this activation did not further increase the degree of hypertrophy but protected the heart through a decrease in the degree of fibrosis and maintenance of ventricular contractile function. CONCLUSIONS: The ERK pathway acts to promote a compensated hypertrophic response, with enhanced contractile function and reduced fibrosis. The activation of this pathway may be a therapeutic strategy to preserve contractile function when the pressure overload cannot be easily alleviated. The inhibition of this pathway, which is increasingly being used for cancer therapy on the other hand, should be used with caution in the presence of pressure-overload.

Aspergillus fumigatus Increased PAR-2 Expression and Elevated Proinflammatory Cytokines Expression Through the Pathway of PAR-2/ERK1/2 in Cornea.

Purpose: To determine the role of protease-activated receptor-2 (PAR-2) in cornea infected by Aspergillus fumigatus. Methods: PAR-2 was tested in normal and infected corneas of C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of PAR-2 antagonist (FSLLRY-NH2). Polymorphonuclear neutrophilic leukocytes (PMNs) were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of FSLLRY-NH2. Disease severity was documented by clinical score and photographs with a slit lamp. PCR, Western blot, and ELISA tested expression of PAR-2, IL-1ß, TNF-α, IFN-γ, MIP-2, and p-ERK1/2. PMN infiltration was assessed by myeloperoxidase assay and immunofluorescent staining. Results: PAR-2 expression was significantly elevated by A. fumigatus, whereas the upregulation was significantly inhibited by FSLLRY-NH2 in mice corneas. FSLLRY-NH2 decreased disease response, PMN infiltration, and proinflammatory cytokine expression compared with infected control. In PMNs, PAR-2 expression was also significantly increased by A. fumigatus, which was significantly inhibited by FSLLRY-NH2. FSLLRY-NH2 significantly inhibited proinflammatory cytokine protein expression, as compared with that in infected control cells, which may be modified by p-ERK1/2. Conclusions: These data provide evidence that A. fumigatus increased PAR-2 expression and elevated disease, PMN infiltration, and proinflammatory cytokine expression through PAR-2, which may be modified by p-ERK1/2.

Cell cycle-dependent translocation and regulatory mechanism of CacyBP/SIP in gastric cancer cells.

Our previous results showed that calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP) inhibits the proliferation and tumorigenicity of gastric cancer; however, the exact mechanism remains unclear, especially from the aspect of cell cycle. The subcellular localization of CacyBP/SIP, Siah-1, and Skp1 in SGC7901 gastric cancer cells was assessed by immunofluorescence after cell cycle synchronization. Levels of CacyBP/SIP, Siah-1, Skp1, ß-catenin, and p-ERK1/2 were analyzed by western blotting. CacyBP/SIP phosphorylation (p-CacyBP/SIP) and the combining capacity of Siah-1 and Skp1 with CacyBP/SIP in nucleoprotein were determined by immunoprecipitation. CacyBP/SIP, Siah-1, and Skp1 were mainly in the cytoplasm in the G1 phase, but translocated to the nucleus during G2. Their expression in total protein was not altered, but elevated in the G2 phase in nucleoprotein. The CacyBP/SIP nucleus translocation of cells transfected with mutant CacyBP/SIP that does not bind S100 (CacyBP-ΔS100) was significantly increased compared with wild-type CacyBP/SIP. In the G2 phase, p-CacyBP/SIP expression and the combining capacity of Siah-1 and Skp1 with CacyBP/SIP were all increased, whereas levels of ß-catenin and p-ERK1/2 reduced, compared with the G1 phase. CacyBP/SIP or CacyBP-ΔS100 overexpression was correlated with constitutively low ß-catenin expression and affected its level through cell cycle. CacyBP/SIP overexpression led to retarded proliferation, G1 arrest, and ß-catenin reduction, which could be abolished by lithium chloride, ß-catenin activator, and further enhanced by the Wnt inhibitor XAV-939. In addition, CacyBP-ΔS100 further suppressed cell proliferation and induced G1 arrest compared with CacyBP/SIP. In conclusion, CacyBP/SIP nuclear localization, dependent on S100 protein, suppresses gastric cancer tumorigenesis through ß-catenin degradation and the dephosphorylation of ERK1/2 during the G2 phase.

Maternal insulin therapy does not restore foetoplacental endothelial dysfunction in gestational diabetes mellitus.

Pregnant women diagnosed with gestational diabetes mellitus subjected to diet (GDMd) that do not reach normal glycaemia are passed to insulin therapy (GDMi). GDMd associates with increased human cationic amino acid transporter 1 (hCAT-1)-mediated transport of L-arginine and nitric oxide synthase (NOS) activity in foetoplacental vasculature, a phenomenon reversed by exogenous insulin. Whether insulin therapy results in reversal of the GDMd effect on the foetoplacental vasculature is unknown. We assayed whether insulin therapy normalizes GDMd-associated foetoplacental endothelial dysfunction. Primary cultures of human umbilical vein endothelial cells (HUVECs) from GDMi pregnancies were used to assay L-arginine transport kinetics, NOS activity, p44/42mapk and protein kinase B/Akt activation, and umbilical vein rings reactivity. HUVECs from GDMi or GDMd show increased hCAT-1 expression and maximal transport capacity, NOS activity, and eNOS, and p44/42mapk, but not Akt activator phosphorylation. Dilation in response to insulin or calcitonin-gene related peptide was impaired in umbilical vein rings from GDMi and GDMd pregnancies. Incubation of HUVECs in vitro with insulin (1 nmol/L) restored hCAT-1 and eNOS expression and activity, and eNOS and p44/42mapk activator phosphorylation. Thus, maternal insulin therapy does not seem to reverse GDMd-associated alterations in human foetoplacental vasculature.

SlMAPK3 enhances tolerance to tomato yellow leaf curl virus (TYLCV) by regulating salicylic acid and jasmonic acid signaling in tomato (Solanum lycopersicum).

Several recent studies have reported on the role of mitogen-activated protein kinase (MAPK3) in plant immune responses. However, little is known about how MAPK3 functions in tomato (Solanum lycopersicum L.) infected with tomato yellow leaf curl virus (TYLCV). There is also uncertainty about the connection between plant MAPK3 and the salicylic acid (SA) and jasmonic acid (JA) defense-signaling pathways. The results of this study indicated that SlMAPK3 participates in the antiviral response against TYLCV. Tomato seedlings were inoculated with TYLCV to investigate the possible roles of SlMAPK1, SlMAPK2, and SlMAPK3 against this virus. Inoculation with TYLCV strongly induced the expression and the activity of all three genes. Silencing of SlMAPK1, SlMAPK2, and SlMAPK3 reduced tolerance to TYLCV, increased leaf H2O2 concentrations, and attenuated expression of defense-related genes after TYLCV infection, especially in SlMAPK3-silenced plants. Exogenous SA and methyl jasmonic acid (MeJA) both significantly induced SlMAPK3 expression in tomato leaves. Over-expression of SlMAPK3 increased the transcript levels of SA/JA-mediated defense-related genes (PR1, PR1b/SlLapA, SlPI-I, and SlPI-II) and enhanced tolerance to TYLCV. After TYLCV inoculation, the leaves of SlMAPK3 over-expressed plants compared with wild type plants showed less H2O2 accumulation and greater superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) activity. Overall, the results suggested that SlMAPK3 participates in the antiviral response of tomato to TYLCV, and that this process may be through either the SA or JA defense-signaling pathways.

Uric acid stimulates proliferative pathways in vascular smooth muscle cells through the activation of p38 MAPK, p44/42 MAPK and PDGFRß.

Hyperuricemia and angiotensin II (Ang II) may have a pathogenetic role in the development of hypertension and atherosclerosis as well as cardiovascular disease (CVD) and its prognosis. The purpose of this study was to investigate whether uric acid can induce proliferative pathways of vascular smooth muscle cell (VSMC) that are thought to be responsible for the development of CVD. The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and platelet-derived growth factor receptor ß (PDGFRß) was measured by Elisa and Western blot techniques to determine the activation of proliferative pathways in primary cultured VSMCs from rat aorta. Results demonstrated that uric acid can stimulate p38 MAPK, p44/42 MAPK and PDGFRß phosphorylation in a time- and concentration-dependent manner. Furthermore, treatment of VSMCs with the angiotensin II type I receptor (AT1R) inhibitor losartan suppressed p38 MAPK and p44/42 MAPK induction by uric acid. The stimulatory effect of uric acid on p38 MAPK was higher compared to that of Ang II. The results of this study show for the first time that uric acid-induced PDGFRß phosphorylation plays a crucial role in the development of CVDs and that elevated uric acid levels could be a potential therapeutical target in CVD patients.

Insulin-like growth factor 1 rescues R28 retinal neurons from apoptotic death through ERK-mediated BimEL phosphorylation independent of Akt.

Insulin-like growth factor 1 (IGF-1) can provide long-term neurotrophic support by activation of Akt, inhibition of FoxO nuclear localization and suppression of Bim gene transcription in multiple neuronal systems. However, MEK/ERK activation can also promote neuron survival through phosphorylation of BimEL. We explored the contribution of the PI3K/Akt/FoxO and MEK/ERK/BimEL pathways in IGF-1 stimulated survival after serum deprivation (SD) of R28 cells differentiated to model retinal neurons. IGF-1 caused rapid activation of Akt leading to FoxO1/3-T32/T24 phosphorylation, and prevented FoxO1/3 nuclear translocation and Bim mRNA upregulation in response to SD. IGF-1 also caused MAPK/MEK pathway activation as indicated by ERK1/2-T202/Y204 and Bim-S65 phosphorylation. Overexpression of FoxO1 increased Bim mRNA expression and amplified the apoptotic response to SD without shifting the serum response curve. Inhibition of Akt activation with LY294002 or by Rictor knockdown did not block the protective effect of IGF-1, while inhibition of MEK activity with PD98059 prevented Bim phosphorylation and blocked IGF-1 protection. In addition, knockdown of Bim expression was protective during SD, while co-silencing of FoxO1 and Fox03 expression had little effect. Thus, the PI3K/Akt/FoxO pathway was not essential for protection from SD-induced apoptosis by IGF-1 in R28 cells. Instead, IGF-1 protection was dependent on activation of the MEK/ERK pathway leading to BimEL phosphorylation, which is known to prevent Bax/Bak oligomerization and activation of the intrinsic mitochondrial apoptosis pathway. These studies demonstrate the requirement of the MEK/ERK pathway in a model of retinal neuron cell survival and highlight the cell specificity for IGF-1 signaling in this response.

Reduced miR-550a-3p leads to breast cancer initiation, growth, and metastasis by increasing levels of ERK1 and 2.

Hyperactivation of the Ras/ERK pathway contributes to breast cancer initiation and progression, and recent evidence suggests aberrant signaling of miRNAs that regulate the Ras/ERK pathway play important roles during carcinogenesis and cancer progression. In this study, we demonstrate that miR-550a-3p expression is negatively correlated with levels of ERK1 and ERK2, two pivotal effectors in the Ras/ERK pathway. MiR-550a-3p gradually decreased during breast cancer initiation and progression and this reduction was a prognostic indicator of poorer overall survival (OS) and disease-free survival (DFS) among breast cancer patients. Our mechanistic studies demonstrated that miR-550a-3p exerts its tumor-suppressor role by directly repressing ERK1 and ERK2 protein expression, thereby suppressing the oncogenic ERK/RSK cascades, which reduced breast cancer cell viability, survival, migration, invasion, tumorigenesis, and metastasis. The inhibitory effects of miR-550a-3p were rescued by ectopic expression of ERK1 and/or ERK2. The novel connection between miR-550a-3p and ERK defines a new diagnostic and prognostic role for miR-550a-3p and highlights ERK inhibition as a candidate therapeutic target for breast cancers exhibiting hyperactivated Ras/ERK signaling.

Hypoxia regulates ABCG2 activity through the activivation of ERK1/2/HIF-1α and contributes to chemoresistance in pancreatic cancer cells.

Pancreatic cancer is a drug resistant hypovascular tumor. Although there are many studies on the mechanism of chemoresistance in pancreatic cancers, studies on the relationship between ABCG2 and chemoresistance during hypoxia of pancreatic cancer are rare. Hypoxia-inducible factor-1 (HIF-1α) is a master regulator of the transcriptional response to oxygen deprivation in cancer cells. The aim of this study was to examine the role of ABCG2 and HIF-1α in mediating chemoresistance during hypoxia in pancreatic cancer. In this study, we detected the expression levels of ABCG2, ERK/phosphorylated-ERK (p-ERK) and HIF-1α by immunohistochemistry in fresh pancreatic cancer and paracarcinoma tissues obtained from 25 patients. The mechanism by which p-ERK1/2 and HIF-1α affect ABCG2s expression was analyzed in the hypoxic cultured human pancreatic cancer cell line Capan-2. ABCG2-mediatedregulation of gemcitabine response under hypoxic conditions in pancreatic cancer cells was observed. It was found that ABCG2, ERK/p-ERK and HIF-1α were overexpressed in cancer tissues. ABCG2, HIF-1α and p-ERK levels were demonstrated to be high during hypoxic conditions in pancreatic cancer cells. Hypoxia induced phosphorylation of ERK1/2 to activate HIF-1α and contribute the ABCG2 expression and mediated gemcitabine chemoresistance in pancreatic cancer cells. Hypoxic conditions induced HIF-1α binding to target gene sequences in the ABCG2 promoter, resulting in increased transcription in pancreatic cancer cells. We demonstrated that hypoxia-induced chemoresistance is due to the regulation of ABCG2 through the activation of ERK1/2/HIF-1α. ABCG2 could serve as a predictor of gemcitabine response and, potentially, as a chemotherapeutic target in pancreatic cancer. Inhibition of ERK1/2 and HIF-1αcould result in increased gemcitabine sensitization in pancreatic cancer with highly expressed ABCG2 cell member protein.

Dual inhibition of histone deacetylases and phosphoinositide 3-kinases: effects on Burkitt lymphoma cell growth and migration.

Burkitt lymphoma is a highly aggressive non-Hodgkin lymphoma that is characterized by MYC deregulation. Recently, the PI3K pathway has emerged as a cooperative prosurvival mechanism in Burkitt lymphoma. Despite the highly successful results of treatment that use high-dose chemotherapy regimens in pediatric Burkitt lymphoma patients, the survival rate of pediatric patients with progressive or recurrent disease is low. PI3Ks are also known to regulate cell migration, and abnormal cell migration may contribute to cancer progression and dissemination in Burkitt lymphoma. Little is known about Burkitt lymphoma cell migration, but the cooperation between MYC and PI3K in Burkitt lymphoma pathogenesis suggests that a drug combination could be used to target the different steps involved in Burkitt lymphoma cell dissemination and disease progression. The aim of this study was to investigate the effects of the histone deacetylase inhibitor suberoylanilide hydroxamic acid combined with the PI3K inhibitor LY294002 on Burkitt lymphoma cell growth and migration. The combination enhanced the cell growth inhibition and cell-cycle arrest induced by the PI3K inhibitor or histone deacetylase inhibitor individually. Moreover, histone deacetylase inhibitor/PI3K inhibitor cotreatment suppressed Burkitt lymphoma cell migration and decreased cell polarization, Akt and ERK1/2 phosphorylation, and leads to RhoB induction. In summary, the histone deacetylase inhibitor/PI3Ki combination inhibits cell proliferation and migration via alterations in PI3K signaling and histone deacetylase activity, which is involved in the acetylation of α-tubulin and the regulation of RhoB expression.