Enhanced Extracellular Signal-regulated Kinase Research Articles

Epidermal growth factor receptor signaling protects epithelia from morphogenetic instability and tissue damage in Drosophila.

Dying cells in the epithelia communicate with neighboring cells to initiate coordinated cell removal to maintain epithelial integrity. Naturally occurring apoptotic cells are mostly extruded basally and engulfed by macrophages. Here, we have investigated the role of Epidermal growth factor (EGF) receptor (EGFR) signaling in the maintenance of epithelial homeostasis. In Drosophila embryos, epithelial tissues undergoing groove formation preferentially enhanced extracellular signal-regulated kinase (ERK) signaling. In EGFR mutant embryos at stage 11, sporadic apical cell extrusion in the head initiates a cascade of apical extrusions of apoptotic and non-apoptotic cells that sweeps the entire ventral body wall. Here, we show that this process is apoptosis dependent, and clustered apoptosis, groove formation, and wounding sensitize EGFR mutant epithelia to initiate massive tissue disintegration. We further show that tissue detachment from the vitelline membrane, which frequently occurs during morphogenetic processes, is a key trigger for the EGFR mutant phenotype. These findings indicate that, in addition to cell survival, EGFR plays a role in maintaining epithelial integrity, which is essential for protecting tissues from transient instability caused by morphogenetic movement and damage.

Canagliflozin inhibits inflammasome activation in diabetic endothelial cells – Revealing a novel calcium-dependent anti-inflammatory effect of canagliflozin on human diabetic endothelial cells

BackgroundCanagliflozin (CANA) shows anti-inflammatory and anti-oxidative effects on endothelial cells (ECs). In diabetes mellitus (DM), excessive reactive oxygen species (ROS) generation, increased intracellular calcium (Ca2+) and enhanced extracellular signal regulated kinase (ERK) 1/2 phosphorylation are crucial precursors for inflammasome activation. We hypothesized that: (1) CANA prevents the TNF-α triggered ROS generation in ECs from diabetic donors and in turn suppresses the inflammasome activation; and (2) the anti-inflammatory effect of CANA is mediated via intracellular Ca2+ and ERK1/2. MethodsHuman coronary artery endothelial cells from donors with DM (D-HCAECs) were pre-incubated with either CANA or vehicle for 2 h before exposure to 50 ng/ml TNF-α for 2–48 h. NAC was applied to scavenge ROS, BAPTA-AM to chelate intracellular Ca2+, and PD 98059 to inhibit the activation of ERK1/2. Live cell imaging was performed at 6 h to measure ROS and intracellular Ca2+. At 48 h, ELISA and infra-red western blot were applied to detect IL-1β, NLRP3, pro-caspase-1 and ASC. Results10 µM CANA significantly reduced TNF-α related ROS generation, IL-1β production and NLRP3 expression (P all <0.05), but NAC did not alter the inflammasome activation (P > 0.05). CANA and BAPTA both prevented intracellular Ca2+ increase in cells exposed to TNF-α (P both <0.05). Moreover, BAPTA and PD 98059 significantly reduced the TNF-α triggered IL-1β production as well as NLRP3 and pro-caspase-1 expression (P all <0.05). ConclusionCANA suppresses inflammasome activation by inhibition of (1) intracellular Ca2+ and (2) ERK1/2 phosphorylation, but not by ROS reduction.

SH2B1 Tunes Hippocampal ERK Signaling to Influence Fluid Intelligence in Humans and Mice.

Fluid intelligence is a cognitive domain that encompasses general reasoning, pattern recognition, and problem-solving abilities independent of task-specific experience. Understanding its genetic and neural underpinnings is critical yet challenging for predicting human development, lifelong health, and well-being. One approach to address this challenge is to map the network of correlations between intelligence and other constructs. In the current study, we performed a genome-wide association study using fluid intelligence quotient scores from the UK Biobank to explore the genetic architecture of the associations between obesity risk and fluid intelligence. Our results revealed novel common genetic loci (SH2B1, TUFM, ATP2A1, and FOXO3) underlying the association between fluid intelligence and body metabolism. Surprisingly, we demonstrated that SH2B1 variation influenced fluid intelligence independently of its effects on metabolism but partially mediated its association with bilateral hippocampal volume. Consistently, selective genetic ablation of Sh2b1 in the mouse hippocampus, particularly in inhibitory neurons, but not in excitatory neurons, significantly impaired working memory, short-term novel object recognition memory, and behavioral flexibility, but not spatial learning and memory, mirroring the human intellectual performance. Single-cell genetic profiling of Sh2B1-regulated molecular pathways revealed that Sh2b1 deletion resulted in aberrantly enhanced extracellular signal-regulated kinase (ERK) signaling, whereas pharmacological inhibition of ERK signaling reversed the associated behavioral impairment. Our cross-species study thus provides unprecedented insight into the role of SH2B1 in fluid intelligence and has implications for understanding the genetic and neural underpinnings of lifelong mental health and well-being.

Chronic inflammatory pain alters alcohol-regulated frontocortical signaling and associations between alcohol drinking and thermal sensitivity

Alcohol use disorder (AUD) is a chronic, relapsing psychiatric disorder that is characterized by the emergence of negative affective states. The transition from recreational, limited intake to uncontrolled, escalated intake is proposed to involve a transition from positive to negative reinforcement mechanisms for seeking alcohol. Past work has identified the emergence of significant hyperalgesia/allodynia in alcohol-dependent animals, which may serve as a key negative reinforcement mechanism. Chronic pain has been associated with enhanced extracellular signal-regulated kinase (ERK) activity in cortical and subcortical nociceptive areas. Additionally, both pain and AUD have been associated with increased activity of the glucocorticoid receptor (GR), a key mediator of stress responsiveness. The objectives of the current study were to first determine relationships between thermal nociceptive sensitivity and alcohol drinking in male Wistar rats. While inflammatory pain induced by complete Freund’s adjuvant (CFA) administration did not modify escalation of home cage drinking in animals over four weeks, the relationship between drinking levels and hyperalgesia symptoms reversed between acute (1 week) and chronic (3–4 week) periods post-CFA administration, suggesting that either the motivational or analgesic effects of alcohol may be altered over the time course of chronic pain. We next examined ERK and GR phosphorylation in pain-related brain areas (including the central amygdala and prefrontal cortex subregions) in animals experiencing acute withdrawal from binge alcohol administration (2 g/kg, 6 h withdrawal) and CFA administration (four weeks) to model the neurobiological consequences of binge alcohol exposure in the context of pain. We observed a significant interaction between alcohol and pain state, whereby alcohol withdrawal increased ERK phosphorylation across all four frontocortical areas examined, although this effect was absent in animals experiencing chronic inflammatory pain. Alcohol withdrawal also increased GR phosphorylation across all four frontocortical areas, but these changes were not altered by CFA. Interestingly, we observed significant inter-brain regional correlations in GR phosphorylation between the insula and other regions investigated only in animals exposed to both alcohol and CFA, suggesting coordinated activity in insula circuitry and glucocorticoid signaling in this context. The results of these studies provide a greater understanding of the neurobiology of AUD and will contribute to the development of effective treatment strategies for comorbid AUD and pain.

Astragaloside VI and cycloastragenol-6-O-beta-D-glucoside promote wound healing in vitro and in vivo

BackgroundAstragalus genus includes most of the common, historical herbal medicines that have various applications in Asian countries. However, clinical data and mechanistic insights into their actions are still lacking. PurposeIn this study, we aimed to examine the effects of astragalosides on wound healing in vitro and in vivo, as well as the underlying mechanisms of these actions. MethodsThe wound healing activity of astragalosides was investigated in human HaCaT keratinocytes, human dermal fibroblast (HDF) cells, and murine models of wound healing. ResultsAll eight astragalosides studied enhanced epidermal growth factor receptor (EGFR) activity in HaCaT cells. Among them, astragaloside VI (AS-VI) showed the strongest EGFR activation. Consistently, AS-VI and cycloastragenol-6-O-beta-D-glucoside (CMG), which is the major metabolite of astragalosides, enhanced extracellular signal-regulated kinase (ERK) activity in a concentration-dependent manner. In agreement, both compounds induced EGFR-dependent cell proliferation and migration in HaCaT and HDF cells. In addition, we showed that AS-VI and CMG accelerated the healing of both sterile and infected wounds in vivo. These effects were associated with increased angiogenesis in the scar tissue. ConclusionAS-VI and CMG increased the proliferation and migration of skin cells via activation of the EGFR/ERK signalling pathway, resulting in the improvement of wound healing in vitro and in vivo. These findings indicate the therapeutic potential of AS-VI and CMG to accelerate wound healing; additionally, they suggest the mechanistic basis of this activity.

Defective mitochondrial fission augments NLRP3 inflammasome activation.

Despite the fact that deregulated NLRP3 inflammasome activation contributes to the pathogenesis of chronic inflammatory or metabolic disorders, the underlying mechanism by which NLRP3 inflammasome signaling is initiated or potentiated remains poorly understood. Much attention is being paid to mitochondria as a regulator of NLRP3 inflammasome activation, but little is known about the role of mitochondrial dynamics for the inflammasome pathway. Here, we present evidence that aberrant mitochondrial elongation caused by the knockdown of dynamin-related protein 1 (Drp1) lead to a marked increase in NLRP3-dependent caspase-1 activation and interleukin-1-beta secretion in mouse bone marrow-derived macrophages. Conversely, carbonyl cyanide m-chlorophenyl hydrazone, a chemical inducer of mitochondrial fission, clearly attenuated NLRP3 inflammasome assembly and activation. Augmented activation of NLRP3 inflammasome by mitochondrial elongation is not resulted from the increased mitochondrial damages of Drp1-knockdown cells. Notably, enhanced extracellular signal-regulated kinase (ERK) signaling in Drp1-knockdown macrophages is implicated in the potentiation of NLRP3 inflammasome activation, possibly via mediating mitochondrial localization of NLRP3 to facilitate the assembly of NLRP3 inflammasome. Taken together, our results provide a molecular insight into the importance of mitochondrial dynamics in potentiating NLRP3 inflammasome activation, leading to aberrant inflammation.

DUSP4 regulates neuronal differentiation and calcium homeostasis by modulating ERK1/2 phosphorylation.

Protein tyrosine phosphatases have been recognized as critical components of multiple signaling regulators of fundamental cellular processes, including differentiation, cell death, and migration. In this study, we show that dual specificity phosphatase 4 (DUSP4) is crucial for neuronal differentiation and functions in the neurogenesis of embryonic stem cells (ESCs). The endogenous mRNA and protein expression levels of DUSP4 gradually increased during mouse development from ESCs to postnatal stages. Neurite outgrowth and the expression of neuron-specific markers were markedly reduced by genetic ablation of DUSP4 in differentiated neurons, and these effects were rescued by the reintroduction of DUSP4. In addition, DUSP4 knockdown dramatically enhanced extracellular signal-regulated kinase (ERK) activation during neuronal differentiation. Furthermore, the DUSP4-ERK pathway functioned to balance calcium signaling, not only by regulating Ca(2+)/calmodulin-dependent kinase I phosphorylation, but also by facilitating Cav1.2 expression and plasma membrane localization. These data are the first to suggest a molecular link between the MAPK-ERK cascade and calcium signaling, which provides insight into the mechanism by which DUSP4 modulates neuronal differentiation.

Effects of Arsenic on Osteoblast Differentiation in Vitro and on Bone Mineral Density and Microstructure in Rats

Background: Arsenic is a ubiquitous toxic element and is known to contaminate drinking water in many countries. Several epidemiological studies have shown that arsenic exposure augments the risk of bone disorders. However, the detailed effect and mechanism of inorganic arsenic on osteoblast differentiation of bone marrow stromal cells and bone loss still remain unclear.Objectives: We investigated the effects and mechanism of arsenic on osteoblast differentiation in vitro and evaluated bone mineral density (BMD) and bone microstructure in rats at doses relevant to human exposure from drinking water.Methods: We used a cell model of rat primary bone marrow stromal cells (BMSCs) and a rat model of long-term exposure with arsenic-contaminated drinking water, and determined bone microstructure and BMD in rats by microcomputed tomography (μCT).Results: We observed significant attenuation of osteoblast differentiation after exposure of BMSCs to arsenic trioxide (0.5 or 1 μM). After arsenic treatment during differentiation, expression of runt-related transcription factor-2 (Runx2), bone morphogenetic protein-2 (BMP-2), and osteocalcin in BMSCs was inhibited and phosphorylation of enhanced extracellular signal-regulated kinase (ERK) was increased. These altered differentiation-related molecules could be reversed by the ERK inhibitor PD98059. Exposure of rats to arsenic trioxide (0.05 or 0.5 ppm) in drinking water for 12 weeks altered BMD and microstructure, decreased Runx2 expression, and increased ERK phosphorylation in bones. In BMSCs isolated from arsenic-treated rats, osteoblast differentiation was inhibited.Conclusions: Our results suggest that arsenic is capable of inhibiting osteoblast differentiation of BMSCs via an ERK-dependent signaling pathway and thus increasing bone loss.Citation: Wu CT, Lu TY, Chan DC, Tsai KS, Yang RS, Liu SH. 2014. Effects of arsenic on osteoblast differentiation in vitro and on bone mineral density and microstructure in rats. Environ Health Perspect 122:559–565; http://dx.doi.org/10.1289/ehp.1307832

Hypoxia inducible factor-1α mediates iron uptake which induces inflammatory response in amoeboid microglial cells in developing periventricular white matter through MAP kinase pathway

Iron accumulation occurs in tissues such as periventricular white matter (PWM) in response to hypoxic injuries, and microglial cells sequester excess iron following hypoxic exposure. As hypoxia has a role in altering the expression of proteins involved in iron regulation, this study was aimed at examining the interaction between hypoxia inducible factor (HIF)-1α and proteins involved in iron transport in microglial cells, and evaluating the mechanistic action of deferoxamine and KC7F2 (an inhibitor of HIF-1α) in iron mediated hypoxic injury. Treating the microglial cultures with KC7F2, led to decreased expression of transferrin receptor and divalent metal transporter-1. Administration of deferoxamine or KC7F2 to hypoxic microglial cells enhanced extracellular signal-regulated kinase (ERK) phosphorylation (p-ERK), but decreased the phosphorylation of p38 (p-p38). The increased p-ERK further phosphorylated the cAMP response element-binding protein (p-CREB) which in turn may have resulted in the increased mitogen activated protein kinase (MAPK) phosphatase 1 (MKP1), known to dephosphorylate MAPKs. Consistent with the decrease in p-p38, the production of pro-inflammatory cytokines TNF-α and IL-1β was reduced in hypoxic microglia treated with deferoxamine and SB 202190, an inhibitor for p38. This suggests that the anti-inflammatory effect exhibited by deferoxamine is by inhibition of p-p38 induced inflammation through the pERK-pCREB-MKP1 pathway, whereas that of KC7F2 requires further investigation. The present results suggest that HIF-1α may mediate iron accumulation in hypoxic microglia and KC7F2, similar to deferoxamine, might provide limited protection against iron induced PWMD.

Positive regulation of osteogenesis by bile acid through FXR

Farnesoid X receptor (FXR) is a nuclear receptor that functions as a bile acid sensor controlling bile acid homeostasis. We investigated the role of FXR in regulating bone metabolism. We identified the expression of FXR in calvaria and bone marrow cells, which gradually increased during osteoblastic differentiation in vitro. In male mice, deletion of FXR (FXR(-/-) ) in vivo resulted in a significant reduction in bone mineral density by 4.3% to 6.6% in mice 8 to 20 weeks of age compared with FXR(+/+) mice. Histological analysis of the lumbar spine showed that FXR deficiency reduced the bone formation rate as well as the trabecular bone volume and thickness. Moreover, tartrate-resistant acid phosphatase (TRACP) staining of the femurs revealed that both the osteoclast number and osteoclast surface were significantly increased in FXR(-/-) mice compared with FXR(+/+) mice. At the cellular level, induction of alkaline phosphatase (ALP) activities was blunted in primary calvarial cells in FXR(-/-) mice compared with FXR(+/+) mice in concert with a significant reduction in type I collagen a1(Col1a1), ALP, and runt-related transcription factor 2 (Runx2) gene expressions. Cultures of bone marrow-derived macrophages from FXR(-/-) mice exhibited an increased number of osteoclast formations and protein expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). In female FXR(-/-) mice, although bone mineral density (BMD) was not significantly different from that in FXR(+/+) mice, bone loss was accelerated after an ovariectomy compared with FXR(+/+) mice. In vitro, activation of FXR by bile acids (chenodeoxycholic acid [CDCA] or 6-ECDCA) or FXR agonists (GW4064 or Fexaramine) significantly enhanced osteoblastic differentiation through the upregulation of Runx2 and enhanced extracellular signal-regulated kinase (ERK) and β-catenin signaling. FXR agonists also suppressed osteoclast differentiation from bone marrow macrophages. Finally, administration of a farnesol (FOH 1%) diet marginally prevented ovariectomy (OVX)-induced bone loss and enhanced bone mass gain in growing C57BL/6J mice. Taken together, these results suggest that FXR positively regulates bone metabolism through both arms of the bone remodeling pathways; ie, bone formation and resorption.

Augmented Cardiac Hypertrophy in Response to Pressure Overload in Mice Lacking ELTD1

BackgroundEpidermal growth factor (EGF), latrophilin and seven transmembrane domain-containing protein 1 (ELTD1) is developmentally upregulated in the heart. Little is known about the relationship between ELTD1 and cardiac diseases. Therefore, we aimed to clarify the role of ELTD1 in pressure overload–induced cardiac hypertrophy.Methods and ResultsC57BL/6J wild-type (WT) mice and ELTD1-knockout (KO) mice were subjected to left ventricular pressure overload by descending aortic banding (AB). KO mice exhibited more unfavorable cardiac remodeling than WT mice 28 days post AB; this remodeling was characterized by aggravated cardiomyocyte hypertrophy, thickening of the ventricular walls, dilated chambers, increased fibrosis, and blunted systolic and diastolic cardiac function. Analysis of signaling pathways revealed enhanced extracellular signal-regulated kinase (ERK) and the c-Jun amino-terminal kinase (JNK) phosphorylation in response to ELTD1 deletion.ConclusionsELTD1 deficiency exacerbates cardiac hypertrophy and cardiac function induced by AB-induced pressure overload by promoting both cardiomyocyte hypertrophy and cardiac fibrosis. These effects are suggested to originate from the activation of the ERK and JNK pathways, suggesting that ELTD1 is a potential target for therapies that prevent the development of cardiac disease.

Role of G-protein Coupled Receptor Kinases GRK2 and GRK3 in C3a Receptor Signaling in Human Mast Cells (133.12)

Abstract Studies with animal models have shown that complement components C3a and C5a play an important role in the effector phase of allergic asthma. We have previously shown that C3a activates human CD34+-derived mast cells to induce degranulation. To determine the role of GRKs on C3a receptor (C3aR) signaling in human mast cells, we used shRNA to knockdown GRK2 and GRK3 a human mast cell line, HMC-1 and CD34+-derived cultures of primary mast cells. We found that GRK2 and GKR3 knockdown inhibited C3aR desensitization as measured by enhanced C3a-induced Ca2+ mobilization in HMC-1 cells and greater degranulation in CD34+ mast cells but had no effect on receptor internalization. In addition, GRK3-knockdown in HMC-1 cells showed increased expression of cell surface C3aR and this was correlated with enhanced extracellular-signal-regulated kinase (ERK) activation and a four-fold increase in C3a-induced chemotaxis. Surprisingly, there was no enhancement of these responses in GRK2 knockdown mast cells. These findings suggest that while GRK2 and GRK3 are required for C3aR sensitization, presumably by promoting receptor phosphorylation, they play distinct roles in modulating downstream signaling such as ERK phosphorylation and chemotaxis.

A functional role for the p62–ERK1 axis in the control of energy homeostasis and adipogenesis

In vivo genetic inactivation of the signalling adapter p62 leads to mature-onset obesity and insulin resistance, which correlate with reduced energy expenditure (EE) and increased adipogenesis, without alterations in feeding or locomotor functions. Enhanced extracellular signal-regulated kinase (ERK) activity in adipose tissue from p62-knockout (p62(-/-)) mice, and differentiating fibroblasts, suggested an important role for this kinase in the metabolic alterations of p62(-/-) mice. Here, we show that genetic inactivation of ERK1 in p62(-/-) mice reverses their increased adiposity and adipogenesis, lower EE and insulin resistance. These results establish genetically that p62 is a crucial regulator of ERK1 in metabolism.

CXCL12/CXCR4 promotes laryngeal and hypopharyngeal squamous cell carcinoma metastasis through MMP-13-dependent invasion via the ERK1/2/AP-1 pathway

Laryngeal and hypopharyngeal squamous cell carcinomas (LHSCCs) are common head and neck cancers with a high propensity for lymph node (LN) and lung metastasis. Here, we report that LHSCCs express high levels of functional CXCR4 receptors, native for chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). Primary tumor immunohistochemistry from LHSCC patients has revealed significant expression of CXCR4 and CXCL12. Greater expression of CXCR4 but not that of CXCL12 is correlated with LN and distant metastasis. Reverse transcription-polymerase chain reaction and western blots have demonstrated that CXCR4 messenger RNA (mRNA) and protein were expressed in LHSCC cell lines as well, but failed to detect CXCL12 mRNA expression. CXCL12 treatment enhanced extracellular signal-regulated kinase (ERK) pathway activation and the motility/invasiveness of LHSCC cell lines, which were blocked by treatment with a CXCR4 antagonist (AMD3100) and a specific MEK inhibitor (U0126). Results show that the mRNA and protein levels of matrix metalloproteinase (MMP)-13, but not MMP-2 or MMP-9, were elevated in HEp-2 cells in response to CXCL12. Again, U0126 almost inhibited the induction of MMP-13 in HEp-2 cells by stimulating CXCL12. The transcriptional factor, c-Jun, a downstream factor of ERK pathway, was found to be readily phosphorylated and translocated to the nucleus after 10 min of exposure to CXCL12. Blockage of c-Jun activity by transfection with c-jun antisense oligodeoxynucleotide significantly decreased CXCL12-induced MMP-13 expression and cell invasion. CXCL12 seems to enhance LHSCC cell invasion through paracrine-activated CXCR4, which triggers ERK/c-Jun-dependent MMP-13 upregulation.

Protein kinase C δ enhances proliferation and survival of murine mammary cells

Protein kinase C (PKC) delta, a member of the novel family of PKC serine-threonine kinases, has been implicated in negative regulation of proliferation and apoptosis in a large number of cell types, including breast cancer cell lines, and postulated as a tumor suppressor gene. In this study we show that in murine NMuMG mammary cells PKCdelta promotes a mitogenic response. Overexpression of PKCdelta in NMuMG cells leads to a significant increase in [3H]-tymidine incorporation and cell proliferation, as well as enhanced extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) activation. Activation of PKCdelta with a phorbol ester leads to elevated cyclin D1 expression and an hyperphosphorylated Rb state. Surprisingly, ectopic expression of PKCdelta conferred anchorage-independent growth capacity to NMuMG cells. PKCdelta overexpressors showed enhanced resistance to apoptotic stimuli, such as serum deprivation or doxorubicin treatment, an effect that correlates with hyperactivation of the Akt survival pathway. Our results provide evidence for a role of PKCdelta as a positive modulator of proliferative and survival signals in immortalized mammary cells. The fact that PKCdelta exerts differential responses depending on the cell context not only highlights the necessity to carefully understand the signaling events controlled by this PKC in each cell type but also suggests that we should be cautious in considering this kinase a target for cancer therapy.

Loss of Raf Kinase Inhibitor Protein Promotes Cell Proliferation and Migration of Human Hepatoma Cells

The Raf kinase inhibitor protein (RKIP) has been identified as a suppressor of the mitogen-activated protein kinase (MAPK) pathway. Loss of RKIP function promotes tumor metastasis in prostate cancer and melanoma. The insulin-like growth factor I (IGF-I)-mediated MAPK cascade is often activated in hepatocellular carcinoma (HCC), but the role of RKIP in the molecular pathogenesis of these tumors is unknown. This study was performed to evaluate the role of RKIP in the development of HCC. The levels of RKIP expression in HCC tumor and corresponding peritumoral tissues were determined by immunohistochemistry and Western blot analysis. The underlying mechanisms of RKIP were assessed with immunoblot analysis, Raf kinase activity assay, cell proliferation, and migration assays after either overexpression or knockdown of RKIP expression in HCC cell lines. RKIP expression is down-regulated in human HCC compared with adjacent peritumoral tissues. Low RKIP levels were correlated with enhanced extracellular signal-regulated-kinase (ERK)/MAPK pathway activation. Reconstitution experiments antagonized IGF-I-mediated MAPK pathway activation, resulting in reduced nuclear accumulation of phospho-ERK. In contrast, knockdown of RKIP expression using small interfering RNA induced activation of the ERK/MAPK pathway. Ectopic expression of RKIP altered HCC cell proliferation and migration. Our findings indicate that down-regulation of RKIP expression is a major factor in activation of the IGF-I/ERK/MAPK pathway during human hepatocarcinogenesis.

CAMP inhibits CSF‐1‐stimulated tyrosine phosphorylation but augments CSF‐1R‐mediated macrophage differentiation and ERK activation

Macrophage colony stimulating factor (M-CSF) or CSF-1 controls the development of the macrophage lineage through its receptor tyrosine kinase, c-Fms. cAMP has been shown to influence proliferation and differentiation in many cell types, including macrophages. In addition, modulation of cellular ERK activity often occurs when cAMP levels are raised. We have shown previously that agents that increase cellular cAMP inhibited CSF-1-dependent proliferation in murine bone marrow-derived macrophages (BMM) which was associated with an enhanced extracellular signal-regulated kinase (ERK) activity. We report here that increasing cAMP levels, by addition of either 8-bromo cAMP (8BrcAMP) or prostaglandin E(1) (PGE1), can induce macrophage differentiation in M1 myeloid cells engineered to express the CSF-1 receptor (M1/WT cells) and can potentiate CSF-1-induced differentiation in the same cells. The enhanced CSF-1-dependent differentiation induced by raising cAMP levels correlated with enhanced ERK activity. Thus, elevated cAMP can promote either CSF-1-induced differentiation or inhibit CSF-1-induced proliferation depending on the cellular context. The mitogen-activated protein kinase/extracellular signal-related protein kinase kinase (MEK) inhibitor, PD98059, inhibited both the cAMP- and the CSF-1R-dependent macrophage differentiation of M1/WT cells suggesting that ERK activity might be important for differentiation in the M1/WT cells. Surprisingly, addition of 8BrcAMP or PGE1 to either CSF-1-treated M1/WT or BMM cells suppressed the CSF-1R-dependent tyrosine phosphorylation of cellular substrates, including that of the CSF-1R itself. It appears that there are at least two CSF-1-dependent pathway(s), one MEK/ERK dependent pathway and another controlling the bulk of the tyrosine phosphorylation, and that cAMP can modulate signalling through both of these pathways.

Protective effects of endogenous adrenomedullin on cardiac hypertrophy, fibrosis, and renal damage.

Adrenomedullin (AM) is a novel vasodilating peptide thought to have important effects on cardiovascular function. The aim of this study was to assess the activity of endogenous AM in the cardiovascular system using AM knockout mice. Mice heterozygous for an AM-null mutation (AM+/-) and their wild-type littermates were subjected to aortic constriction or angiotensin II (Ang II) infusion. The resultant cardiovascular stress led to increases in heart weight/body weight ratios, left ventricular wall thickness, and perivascular fibrosis, as well as expression of genes encoding angiotensinogen, ACE, transforming growth factor-beta, collagen type I, brain natriuretic peptide, and c-fos. In addition, renal damage characterized by decreased creatinine clearance with glomerular sclerosis was noted. In all cases, the effects were significantly more pronounced in AM+/- mice. Hearts from adult mice subjected to aortic constriction showed enhanced extracellular signal-regulated kinase (ERK) activation, as did cardiac myocytes from neonates treated acutely with Ang II. Again the effect was more pronounced in AM+/- mice, which showed increases in cardiac myocyte size, protein synthesis, and fibroblast proliferation. ERK activation was suppressed by protein kinase C inhibition to a greater degree in AM+/- myocytes. In addition, treatment of cardiac myocytes with recombinant AM suppressed Ang II-induced ERK activation via a protein kinase A-dependent pathway. Endogenous AM exerts a protective effect against stress-induced cardiac hypertrophy via protein kinase C- and protein kinase A-dependent regulation of ERK activation. AM may thus represent a useful new tool for the treatment of cardiovascular disease.