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Oncogenic RAS impacts communication between cancer cells and their microenvironment, but it is unclear how this process influences cellular interactions with extracellular vesicles (EVs). This is important as intercellular EV trafficking plays a key role in cancer invasion and metastasis. Here we report that overexpression of mutant RAS drives the EV internalization switch from endocytosis (in non-transformed cells) to macropinocytosis (in cancer cells) resulting in enhanced EV uptake. This process depends on the surface proteoglycan, fibronectin and EV engulfment mechanism regulated by CRAF. Both mutant RAS and activated CRAF expression is associated with formation of membrane ruffles to which they colocalize along with actin, sodium-hydrogen exchangers (NHEs) and phosphorylated myosin phosphatase (pMYPT). RAS-transformed cells internalize EVs in the vicinity of ruffled structures followed by apparent trafficking to lysosome and degradation. NHE inhibitor (EIPA) suppresses RAS-driven EV uptake, along with adhesion-independent clonal growth and experimental metastasis in mice. Thus, EV uptake may represent a targetable step in progression of RAS-driven cancers., Competing Interests: The authors report no conflicts of interest., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Chronic pain caused by spinal cord injury (SCI) is notoriously resistant to treatment, particularly by opioids. After SCI, DRG neurons show hyperactivity and chronic depolarization of resting membrane potential (RMP) that is maintained by cAMP signaling through PKA and EPAC. Importantly, SCI also reduces the negative regulation by Gαi of adenylyl cyclase and its production of cAMP, independent of alterations in G protein-coupled receptors and/or G proteins. Opioid reduction of pain depends on coupling of opioid receptors to Gαi/o family members. Combining high-content imaging and cluster analysis, we show that in male rats SCI decreases opioid responsiveness in vitro within a specific subset of small-diameter nociceptors that bind isolectin B4. This SCI effect is mimicked in nociceptors from naive animals by a modest 5 min depolarization of RMP (15 mm K + ; -45 mV), reducing inhibition of cAMP signaling by μ-opioid receptor agonists DAMGO and morphine. Disinhibition and activation of C-Raf by depolarization-dependent phosphorylation are central to these effects. Expression of an activated C-Raf reduces sensitivity of adenylyl cyclase to opioids in nonexcitable HEK293 cells, whereas inhibition of C-Raf or treatment with the hyperpolarizing drug retigabine restores opioid responsiveness and blocks spontaneous activity of nociceptors after SCI. Inhibition of ERK downstream of C-Raf also blocks SCI-induced hyperexcitability and depolarization, without direct effects on opioid responsiveness. Thus, depolarization-dependent C-Raf and downstream ERK activity maintain a depolarized RMP and nociceptor hyperactivity after SCI, providing a self-reinforcing mechanism to persistently promote nociceptor hyperexcitability and limit the therapeutic effectiveness of opioids. SIGNIFICANCE STATEMENT Chronic pain induced by spinal cord injury (SCI) is often permanent and debilitating, and usually refractory to treatment with analgesics, including opioids. SCI-induced pain in a rat model has been shown to depend on persistent hyperactivity in primary nociceptors (injury-detecting sensory neurons), associated with a decrease in the sensitivity of adenylyl cyclase production of cAMP to inhibitory Gαi proteins in DRGs. This study shows that SCI and one consequence of SCI (chronic depolarization of resting membrane potential) decrease sensitivity to opioid-mediated inhibition of cAMP and promote hyperactivity of nociceptors by enhancing C-Raf activity. ERK activation downstream of C-Raf is necessary for maintaining ongoing depolarization and hyperactivity, demonstrating an unexpected positive feedback loop to persistently promote pain., (Copyright © 2020 the authors.)

Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. Here we find that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Protein modifications by phosphorylation or ubiquitylation have been selected throughout evolution as efficient regulatory mechanisms of cellular processes. Cell migration is a complex, highly coordinated process where these mechanisms must participate in an integrated manner to transmit signaling during migration. In this study, we show that the ubiquitin ligase HERC1 regulates the p38 signaling pathway, and that this regulation is mediated by the MAPK kinase MKK3. Moreover, we demonstrate a crosstalk between RAF and MKK3/p38 pathways where RAF acts upstream of MKK3. Mechanistically, HERC1 regulates the protein levels of C-RAF and MKK3. Thus, HERC1 ubiquitylates C-RAF, targeting it for proteasomal degradation, and RAF proteins regulate MKK3 mRNA levels. Accordingly, HERC1 knockdown induces C-RAF stabilization and activation of RAF proteins; in turn, this activation increases MKK3, which phosphorylates and activates p38. The importance of these observations is demonstrated by HERC1 regulation of cell migration through regulation of p38 signaling via a RAF-dependent mechanism. Thus, HERC1 plays an essential role as a regulator of crosstalk between RAF/MKK3/p38 signaling pathways during cell migration.

Since Drosophila melanogaster has proven to be a useful model system to study phenotypes of oncogenic mutations and to identify new anti-cancer drugs, we generated human BRAF V600E homologous dRaf mutant (dRaf A572E ) Drosophila melanogaster strains to use these for characterisation of mutant phenotypes and exploit these phenotypes for drug testing. For mutant gene expression, the GAL4/UAS expression system was used. dRaf A572E was expressed tissue-specific in the eye, epidermis, heart, wings, secretory glands and in the whole animal. Expression of dRaf A572E under the control of an eye-specific driver led to semi-lethality and a rough eye phenotype. The vast majority of other tissue-specific and ubiquitous drivers led to a lethal phenotype only. The rough eye phenotype was used to test BRAF inhibitor vemurafenib and MEK1/2 inhibitor cobimetinib. There was no phenotype rescue by this treatment. However, a significant rescue of the lethal phenotype was observed under a gut-specific driver. Here, MEK1/2 inhibitor cobimetinib rescued Drosophila larvae to reach pupal stage in 37% of cases as compared to 1% in control experiments. Taken together, the BRAF V600E homolog dRaf A572E exerts mostly lethal effects in Drosophila. Gut-specific dRaf A572E expression might in future be developed further for drug testing., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Platinum-based chemotherapeutics represent a mainstay of cancer therapy, but resistance limits their curative potential. Through a kinome RNAi screen, we identified microtubule-associated serine/threonine kinase 1 (MAST1) as a main driver of cisplatin resistance in human cancers. Mechanistically, cisplatin but no other DNA-damaging agents inhibit the MAPK pathway by dissociating cRaf from MEK1, while MAST1 replaces cRaf to reactivate the MAPK pathway in a cRaf-independent manner. We show clinical evidence that expression of MAST1, both initial and cisplatin-induced, contributes to platinum resistance and worse clinical outcome. Targeting MAST1 with lestaurtinib, a recently identified MAST1 inhibitor, restores cisplatin sensitivity, leading to the synergistic attenuation of cancer cell proliferation and tumor growth in human cancer cells and patient-derived xenograft models., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Interleukin-33 (IL-33) is a potent contributor to antiviral immune responses and antitumor immunity. We recently discovered that IL-33 is overexpressed in dectin-1-activated dendritic cells (DCs). However, mechanisms of dectin-1-induced IL-33 expression in DCs remain elusive. Curdlan, an agonist of dectin-1, was used to mature DCs in this study. We found that dectin-1-induced IL-33 expression in DCs relies on Syk and Raf-1 pathways. By using nuclear factor (NF)-κB inhibitors, we also found that dectin-1-induced IL-33 expression relies on NF-κB signaling. Furthermore, through Syk/Raf-1-NF-κB pathway, dectin-1 signaling stimulates DCs to overexpress interferon regulatory factor 4 (IRF4), which directly upregulates the expression of IL-33 in dectin-1-activated DCs. Thus, our study provides new insights into the mechanisms of dectin-1-induced IL-33 expression in DCs and may provide new targets for improving DC-based cancer immunotherapy.

C-RAF was the first member of the RAF kinase family to be discovered. Since its discovery, C-RAF has been found to regulate many fundamental cell processes, such as cell proliferation, cell death, and metabolism. However, the majority of these functions are achieved through interactions with different proteins; the genes regulated by C-RAF in its active or inactive state remain unclear. In the work, we used RNA-seq analysis to study the global transcriptomes of C-RAF bearing or C-RAF knockout cells in quiescent or EGF activated states. We identified 3353 genes that are promoted or suppressed by C-RAF. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these genes are involved in drug addiction, cardiomyopathy, autoimmunity, and regulation of cell metabolism. Our results provide a panoramic view of C-RAF function, including known and novel functions, and have revealed potential targets for elucidating the role of C-RAF., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study, we elucidated the immunomodulatory activity of docosahexaenoic acid (DHA) on protein expression in RAW264.7 cells and its molecular mechanism. The results showed that the proliferation index of RAW264.7 cells at 48 h was about 173.03 ± 7.82% after the treatment of 2.4 μM DHA. DHA could activate RAW264.7 cells by the G-protein coupled cell membrane receptor GPR120-C-Raf- mitogen-activated protein kinases (MAPKs)-nuclear factor κB (NF-κB) p65 pathway. In addition, 2.4 μM of DHA could significantly increase (P < 0.01) the mRNA and protein expression of inducible nitric oxide synthase (iNOS), which is consistent with the result of the NO release. ELISA results revealed that DHA could enhance the protein expression of cytokines IL-1β, IL-6, IL-10, IL-12, TNF-α, IFN-γ, and TGF-β. These results indicated that the immunomodulatory mechanism of RAW264.7 cells by DHA was associated with the release of NO and cytokines by stimulating the GPR120, C-Raf, and MAPKs to the NF-κB p65 pathway.

Histone deacetylase (HDAC) inhibitors, which are approved for the treatment of cutaneous T-cell lymphoma and multiple myeloma, are undergoing evaluation in other lymphoid neoplasms. How they kill susceptible cells is incompletely understood. Here, we show that trichostatin A, romidepsin and panobinostat induce apoptosis across a panel of malignant B cell lines, including lines that are intrinsically resistant to bortezomib, etoposide, cytarabine and BH3 mimetics. Further analysis traces the pro-apoptotic effects of HDAC inhibitors to increased acetylation of the chaperone heat shock protein 90 (HSP90), causing release and degradation of the HSP90 client proteins RASGRP1 and CRAF, which in turn leads to downregulation of mitogen-activated protein kinase pathway signaling and upregulation of the pro-apoptotic BCL2 family member BIM in vitro and in vivo. Importantly, these pro-apoptotic effects are mimicked by RASGRP1 small interfering RNA (siRNA) or HSP90 inhibition and reversed by overexpression of constitutively active MEK1 or siRNA-mediated downregulation of BIM. Collectively, these observations not only identify a new HSP90 client protein, RASGRP1, but also delineate a complete signaling pathway from HSP90 acetylation through RASGRP1 and CRAF degradation to BIM upregulation that contributes to selective cytotoxicity of HDAC inhibitors in lymphoid malignancies.

Raf-1 has an important role in cellular antiapoptosis. So far, there is no solid evidence that shows that Raf-1 mutation is associated with cancer development. In the course of further study of Raf-1 signaling, we have reported that Raf-1 hyperphosphorylation inhibits its kinase activity toward its downstream mitogen-activated protein kinase kinase 1/2 (MEK1/2) and proposed a model for negative feedback regulation of Raf-1. Here, we show that there is no hyperphosphorylation in some cancer cells, which results in increased kinase activity and enhances the antiapoptotic ability. Inhibition of either Raf-1 or ALG-2 (apoptosis-linked gene 2) expression results in apoptosis signal-regulating kinase 1/c-Jun N-terminal kinase (ASK1/JNK) signaling activation, and cell sensitivity to chemotherapeutic reagents, indicating that inhibition of ASK1/JNK apoptotic signaling by Raf-1 is mediated by ALG-2. A previous report indicated that extracellular signal-regulated kinase 1/2 (ERK1/2) were responsible for Raf-1 hyperphosphorylation. However, our evidence shows that when ERK1/2 are activated and the Raf-1 gene is not mutated, Raf-1 is not hyperphosphorylated in these cells, indicating that ERK1/2 are not responsible for the Raf-1 hyperphosphorylation in these cancer cell lines. Surprisingly, we also found that Raf-1 is not a necessary kinase for MEK1/2 activation under normal tissue culture conditions, but is required for MEK1/2 activation under apoptosis-inducing conditions. Our research demonstrates that although Raf-1 gene is not mutated, an abnormality of Raf-1 kinase feedback regulation enhances its antiapoptotic function, and Raf-1 can still be a pharmaceutical target to increase chemotherapy or radiotherapy sensitivity in these cancer cells.

Purpose: SOX5 plays important roles in various kinds of cancers. However, the expression and roles of SOX5 in osteosarcoma (OS) have not been investigated well. In the present study we aimed to investigate the mechanism of SOX5 in OS., Methods: OS and adjacent non-cancerous specimens were obtained from patients with OS. PCR was applied to detect SOX5 mRNA. Then human OS cell lines (U2OS, SoSP-M, SoSP-9607, and MG-63) and one immortalized normal osteoblast hFOB1.19 were investigated. SOX5 knocking with shRNA in U2OS and SOX5 upregulation with recombinant plasmid in MG-63 were applied. Real-time cell monitoring system and invasion assay were used, and Western blot assay was performed to detect the protein level of E-cadherin, N-cadherin, Vimentin and Snail, where Glyceraldehyde3- phosphate dehydrogenase (GAPDH) was presented as control. P<0.05 was considered as statistically significant., Results: Significant upregulation of SOX5 in OS tissues and cell lines was identified. The gain- and loss-of-function studies suggested that OS cell migration and invasion were promoted significantly by SOX5. Additionally, SOX5 promoted epithelial-mesenchymal transition (EMT) by regulation of Snail., Conclusion: SOX5 is a novel regulator of EMT in OS, and is a potential target for OS.

MEK/ERK signal pathway was required for the differentiation of granulocytes, megakaryocytes and erythrocytes. Recently, MEK/ERK cascade was reported to be involved in all-trans retinoic acid (ATRA) induced differentiation in acute promyelocytic leukemia (APL) cells. However, the upstream and downstream molecules of MEK/ERK signal pathway in this cell model remains to be elucidated. In this work, we showed that RAF-1 was activated and the blockade of RAF-1 activation attenuated MEK/ERK activation as well as ATRA-induced differentiation. ATRA-enhanced protein levels of C/EBPβ, C/EBPε and PU.1, which were required for differentiation in APL cells, were suppressed by the specific inhibitor of MEK. However, MEK inhibition had no effect on the degradation of PML-RARα fusion protein or the restoration of PML nuclear bodies by ATRA treatment. Taken together, our study suggested that RAF-1/MEK/ERK cascade was involved in ATRA-induced differentiation in APL cells through enhancing the protein level of C/EBPβ, C/EBPε and PU.1., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

B-Raf inhibitors have been used for the treatment of some B-Raf-mutated cancers. They effectively inhibit B-Raf/MEK/ERK signaling in cancers harboring mutant B-Raf, but paradoxically activates MEK/ERK in Ras-mutated cancers. Death receptor 5 (DR5), a cell surface pro-apoptotic protein, triggers apoptosis upon ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or aggregation. This study focused on determining the effects of B-Raf inhibition on DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. Using chemical and genetic approaches, we have demonstrated that the B-Raf inhibitor PLX4032 induces DR5 upregulation exclusively in Ras-mutant cancer cells; this effect is dependent on Ras/c-Raf/MEK/ERK signaling activation. PLX4032 induces DR5 expression at transcriptional levels, largely due to enhancing CHOP/Elk1-mediated DR5 transcription. Pre-exposure of Ras-mutated cancer cells to PLX4032 sensitizes them to TRAIL-induced apoptosis; this is also a c-Raf/MEK/ERK-dependent event. Collectively, our findings highlight a previously undiscovered effect of B-Raf inhibition on the induction of DR5 expression and the enhancement of DR5 activation-induced apoptosis in Ras-mutant cancer cells and hence may suggest a novel therapeutic strategy against Ras-mutated cancer cells by driving their death due to DR5-dependent apoptosis through B-Raf inhibition.

The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K(+) channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf (+/-) mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection.

Purpose of Review: The modalities of Ras mutation detection, its role as a predictive biomarker, mechanisms of wild-type Ras activation, and the role of Ras-directed targeted therapies will be discussed mainly in colorectal cancer., Recent Findings: RAS genotype is generally considered to be highly concordant between primary colorectal tumours and metastases. However, recent data show significant discordance between primary tumours and specific metastatic sites, but also heterogeneity within primary tumours. Moreover, the mechanisms of Ras activation expand far beyond mutations through altered expression or function of physiological Ras activators and inhibitors. Accordingly, genomic signatures of Ras or epidermal growth factor receptor (EGFR) activation are being developed and are potential predictive biomarkers of response to anti-EGFR antibodies. Finally, several recent clinical trials targeting Ras or its downstream signalling with statins or Raf inhibitors have shown promising activity in chemorefractory metastatic colorectal cancer., Summary: RAS mutation remains an important biomarker predicting response to anti-EGFR therapies and perhaps clinical outcomes after surgery for metastatic colorectal cancer, but new techniques including genomic signatures need to be validated to take into account the complexity of Ras activation. The importance of Ras signalling as a therapeutic target has recently been outlined by successful clinical trials with Raf inhibitors.

Cutaneous melanoma is a malignant tumor with a high metastatic potential. If an early treatment is associated with a favorable outcome, the prognosis of metastatic melanoma remains poor. Advances in molecular characterization of cancers, notably the discovery of BRAF gene mutations in metastatic melanoma, allowed to the recent development of targeted therapies against mutated BRAF protein. Despite high tumor response rates observed in clinical trials, these new drugs are associated with frequent secondary tumor resistance occurrence and paradoxical carcinogenic side effects. The cellular and molecular mechanisms of these carcinogenic side effects and secondary resistance are not yet fully elucidated and are actually intensely studied. This review of the literature focus on the mechanisms of these carcinogenic side effects and on the tumor resistance associated with anti-BRAF targeted therapies., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

A number of inhibitors have been used to dissect the functional relevance of Jak2 in endothelial homeostasis, with disparate results. Given that Jak2 deficiency leads to embryonic lethality, the exact role of Jak2 in the regulation of postnatal endothelial function is yet to be fully elucidated. We generated a model in which Jak2 deficiency can be induced by tamoxifen in adult mice. Loss of Jak2 significantly impaired endothelium-dependent response capacity for vasodilators. Matrigel plug assays indicated a notable decrease in endothelial angiogenic function in Jak2-deficient mice. Studies in a hindlimb ischemic model indicated that Jak2 activity is likely to be a prerequisite for prompt perfusion recovery, based on the concordance of temporal changes in Jak2 expression during the course of ischemic injury and perfusion recovery. A remarkable delay in perfusion recovery, along with reduced capillary and arteriole formation, was observed in Jak2-deficient mice. Antibody array studies indicated that loss of Jak2 led to repressed eNOS expression. In mechanistic studies, Jak2 deficiency attenuated Raf-1/MEK1 signaling, which then reduced activity of Sp-1, an essential transcription factor responsible for eNOS expression. These data are important not only for understanding the exact role that Jak2 plays in endothelial homeostasis, but also for assessing Jak2-based therapeutic strategies in a variety of clinical settings., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

ATP competitive inhibitors of the BRAF(V600E) oncogene paradoxically activate downstream signaling in cells bearing wild-type BRAF (BRAF(WT)). In this study, we investigate the biochemical mechanism of wild-type RAF (RAF(WT)) activation by multiple catalytic inhibitors using kinetic analysis of purified BRAF(V600E) and RAF(WT) enzymes. We show that activation of RAF(WT) is ATP dependent and directly linked to RAF kinase activity. These data support a mechanism involving inhibitory autophosphorylation of RAF's phosphate-binding loop that, when disrupted either through pharmacologic or genetic alterations, results in activation of RAF and the mitogen-activated protein kinase (MAPK) pathway. This mechanism accounts not only for compound-mediated activation of the MAPK pathway in BRAF(WT) cells but also offers a biochemical mechanism for BRAF oncogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Arterial morphogenesis is one of the most critical events during embryonic vascular development. Although arterial fate specification is mainly controlled by the Notch signaling pathway, arterial-venous patterning is modulated by a number of guidance factors. How these pathways are regulated is still largely unknown. Here, we demonstrate that endothelial activation of RAF1/extracellular signal-regulated kinase (ERK) pathway regulates arterial morphogenesis and arterial-venous patterning via Δ/Notch and semaphorin signaling. Introduction of a single amino acid RAF1 mutant (RAF1 Ser259Ala), which renders it resistant to inhibition by phosphorylation, into endothelial cells in vitro induced expression of virtually the entire embryonic arteriogenic program and activated semaphorin 6A-dependent endothelial cell-cell repulsion. In vivo, endothelial-specific expression of RAF1(S259A) during development induced extensive arterial morphogenesis both in the yolk sac and the embryo proper and disrupted arterial-venous patterning. Our results suggest that endothelial ERK signaling is critical for both arteriogenesis and arterial-venous patterning and that RAF1 Ser(259) phosphorylation plays a critical role in preventing unopposed ERK activation.

The immune system is essential to maintain homeostasis with resident microbial populations, ensuring that the symbiotic host-microbial relationship is maintained. In parallel, commensal microbes significantly shape mammalian immunity at the host mucosal surface, as well as systemically. Candida albicans is an opportunistic pathogen that lives as a commensal on skin and mucosa of healthy individuals. Little is known about its capacity to modulate responses toward other microorganisms, such as colonizing bacteria (e.g., intestinal microorganisms). The aim of this study was to assess the cytokine production of PBMCs induced by commensal bacteria when these cells were primed by C. albicans. We show that C. albicans and β-1,3-glucan induce priming of human primary mononuclear cells and this leads to enhanced cytokine production upon in vitro stimulation with TLR ligands and bacterial commensals. This priming requires the β-1,3-glucan receptor dectin-1 and the noncanonical Raf-1 pathway. In addition, although purified mannans cannot solely mediate the priming, the presence of mannosyl residues in the cell wall of C. albicans is nevertheless required. In conclusion, C. albicans is able to modify cytokine responses to TLR ligands and colonizing bacteria, which is likely to impact the inflammatory reaction during mucosal diseases.

Alkylphosphocholines are highly active against multiple myeloma (MM) cells in vitro and are devoid of myelotoxicity. Little is known about the determinants of MM cell responsiveness or resistance to these drugs. In this study we investigated the effects of disease-relevant cytokines, such as interleukin-6 (IL-6) and osteopontin (OPN), on the in vitro antimyeloma activity of erufosine and perifosine. The role of the Raf/MEK/ERK pathway was also studied. Exogenous IL-6 reduced the cytotoxicity of erufosine against OPM-2 cells and, to a smaller extent, against U-266 cells. This was accompanied by inhibition of apoptosis in OPM-2 cells. The efficacy of perifosine was similarly affected, but to a greater extent. IL-6 slightly enhanced the sensitivity of RPMI-8226 cells to erufosine, thus emphasizing the heterogeneity of MM. Induced overexpression of OPN isoforms made OPM-2 cells less sensitive to erufosine. In all cases of IL-6- or OPN-induced resistance, the effective concentrations of erufosine were still within the clinically achievable range. Like other alkylphosphocholines, erufosine enhanced Raf/MEK/ERK signaling in MM cells but in some cases this contributed to cytotoxicity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family that is associated with the disease status and outcomes of cancers. However, the effect of CCL2 on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that CCL2 increased the migration and expression of matrix metalloproteinase (MMP)-9 in human chondrosarcoma cells. CCL2-mediated migration and MMP-9 up-regulation were attenuated by CCR2, Ras, Raf-1, and MEK inhibitor. Activation of the Ras, Raf-1, MEK, ERK, and NF-κB signaling pathway after CCL2 treatment was demonstrated, and CCL2-induced expression of MMP-9 and migration activity were inhibited by the specific inhibitor, and mutant of Ras, Raf-1, MEK, ERK, and NF-κB cascades. Taken together, our results indicated that CCL2 enhances the migration of chondrosarcoma cells by increasing MMP-9 expression through the CCR2 receptor, Ras, Raf-1, MEK, ERK, and NF-κB signal transduction pathway., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Regulation of glucose homeostasis by insulin depends on pancreatic β-cell growth, survival, and function. Raf-1 kinase is a major downstream target of several growth factors that promote proliferation and survival of many cell types, including the pancreatic β cells. We have previously reported that insulin protects β cells from apoptosis and promotes proliferation by activating Raf-1 signaling in cultured human islets, mouse islets, and MIN6 cells. As Raf-1 activity is critical for basal apoptosis and insulin secretion in vitro, we hypothesized that Raf-1 may play an important role in glucose homeostasis in vivo. To test this hypothesis, we utilized the Cre-loxP recombination system to obtain a pancreatic β-cell-specific ablation of Raf-1 kinase gene (RIPCre(+/+):Raf-1(flox/flox)) and a complete set of littermate controls (RIPCre(+/+):Raf-1(wt/wt)). RIPCre(+/+):Raf-1(flox/flox) mice were viable, and no effects on weight gain were observed. RIPCre(+/+):Raf-1(flox/flox) mice had increased fasting blood glucose levels and impaired glucose tolerance but normal insulin tolerance compared to littermate controls. Insulin secretion in vivo and in isolated islets was markedly impaired, but there was no apparent effect on the exocytosis machinery. However, islet insulin protein and insulin 2 mRNA, but not insulin 1 mRNA, were dramatically reduced in Raf-1-knockout mice. Analysis of insulin 2 knockout mice demonstrated that this reduction in mRNA was sufficient to impair in vivo insulin secretion. Our data further indicate that Raf-1 specifically and acutely regulates insulin 2 mRNA via negative action on Foxo1, which has been shown to selectively control the insulin 2 gene. This work provides the first direct evidence that Raf-1 signaling is essential for the regulation of basal insulin transcription and the supply of releasable insulin in vivo.

Genetic loss-of-function studies in murine tumor models have been essential in the analysis of downstream mediators of oncogenic transformation. Unfortunately, these studies are frequently limited by the availability of genetically modified mouse strains. Here we describe a versatile method allowing the efficient expression of an oncogene and simultaneous knockdown of targets of interest (TOI) from a single retroviral vector. Both oncogene and TOI-specific miR30-based shRNA are under the control of the strong viral long terminal repeat promoter, resulting in a single shared RNA transcript. Using this vector in a murine syngeneic BM transplantation model for BCR-ABL-induced chronic myeloid leukemia, we find that oncogene expression and target knockdown in primary hematopoietic cells with this vector is efficient both in vitro and in vivo, and demonstrate that Raf1, but not BRAF, modulates BCR-ABL-dependent ERK activation and transformation of hematopoietic cells. This expression system could facilitate genetic loss-of-function studies and allow the rapid validation of potential drug targets in a broad range of oncogene-driven murine tumor models.

Background: Recent evidence has implicated the MAP kinase (MAPK) pathway with the development of castrate-resistant prostate cancer (CRPC). We have previously reported gene amplification of critical members of this pathway with the development of castrate-resistant disease. In addition, we have shown that rising Raf-1 expression, with the development of CRPC, influences time to biochemical relapse. We therefore sought to further analyse the role of both Raf-1 and its downstream target MAPK in the molecular pathogenesis of CRPC., Methods: Protein expression of Raf-1 and MAPK, including their activation status, was analysed using immunohistochemistry in a database of 65 paired tumour specimens obtained before and after the development of CRPC and correlated with other members of the pathway., Results: Patients whose nuclear expression of MAPK rose with the development of CRPC had a significantly shorter median time to death following biochemical relapse (1.40 vs 3.00 years, P=0.0255) as well as reduced disease-specific survival when compared with those whose expression fell or remained unchanged (1.16 vs 2.62 years, P=0.0005). Significant correlations were observed between protein expression of Raf-1 and MAPK with the type 1 receptor tyrosine kinases, Her2 and epidermal growth factor receptor, as well as the transcription factor AP-1 in CRPC tumours., Conclusion: We conclude that the Her2/Raf-1/MAPK/AP-1 axis may promote the development of CRPC, leading to early relapse, and reduced disease-specific survival. In addition, members of the pathway may act as novel therapeutic and/or diagnostic targets for prostate cancer.

Hypertrophic cardiomyopathy (HCM) is a leading cause of sudden death in children and young adults. Abnormalities in several signaling pathways are implicated in the pathogenesis of HCM, but the role of the RAS-RAF-MEK-ERK MAPK pathway has been controversial. Noonan syndrome (NS) is one of several autosomal-dominant conditions known as RASopathies, which are caused by mutations in different components of this pathway. Germline mutations in RAF1 (which encodes the serine-threonine kinase RAF1) account for approximately 3%-5% of cases of NS. Unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with HCM. To explore the pathogenesis of such mutations, we generated knockin mice expressing the NS-associated Raf1(L613V) mutation. Like NS patients, mice heterozygous for this mutation (referred to herein as L613V/+ mice) had short stature, craniofacial dysmorphia, and hematologic abnormalities. Valvuloseptal development was normal, but L613V/+ mice exhibited eccentric cardiac hypertrophy and aberrant cardiac fetal gene expression, and decompensated following pressure overload. Agonist-evoked MEK-ERK activation was enhanced in multiple cell types, and postnatal MEK inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice. These data show that different NS genes have intrinsically distinct pathological effects, demonstrate that enhanced MEK-ERK activity is critical for causing HCM and other RAF1-mutant NS phenotypes, and suggest a mutation-specific approach to the treatment of RASopathies.

Background: There is evidence that peroxynitrite is generated in pulmonary hypertension and we have therefore investigated whether peroxynitrite can cause proliferation of pulmonary artery cells., Methods: Bovine pulmonary artery endothelial (PAEC) and smooth muscle cells (PASMC) were exposed to peroxynitrite solution or to the peroxynitrite generating compound, 3-morpholinosydnonimine (SIN-1). Vascular cell proliferation was determined by cell count and (3)H-thymidine incorporation. Protein biochemistry was by western blot analysis., Results: Transient exposure to peroxynitrite stimulated the proliferation of PASMC (peroxynitrite 0.2 nM-2 μM) and PAEC (peroxynitrite 0.2 μM). Peroxynitrite 0.2 μM stimulated DNA synthesis in PASMC cell by 200 ± 22% and in PAEC by 137 ± 4%. DNA synthesis in PAEC and PASMC was also stimulated by the peroxynitrite generator SIN-1 2 μM. Cell proliferation was accompanied by activation of ERK, which peaked at 15 min and remained elevated for 12 h in PASMC. However peroxynitrite at the concentrations used in this study did not activate the stress pathways p38 mitogen activated protein kinase (MAPK) or Jun N-terminal kinase (JNK). Peroxynitrite-induced proliferation and ERK phosphorylation in PASMC were abolished by the peroxynitrite scavenger ebselen 5 μM. Peroxynitrite-induced proliferation and extracellular signal-regulated kinase (ERK) phosphorylation in PASMC was prevented by selective inhibitors of MAP kinase kinase (MEK) (U0126 5 μM, PD98059 50 μM), Raf-1 (Raf-1 kinase inhibitor 10 μM), Ras (FPT II and FPT III 10 μM) and protein kinase C (PKC) (GF109203X 10 μM). Inhibition of EGF or PDGF receptor signaling using AG-1296, AG-1478 or imatinib prevented peroxynitrite-induced cell proliferation and ERK phosphorylation in PASMC., Conclusion: Peroxynitrite can stimulate proliferation of pulmonary artery cells, involving ERK, PKC and EGF or PDGF receptors., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Some 25 years ago, Raf was discovered as the transforming principle shared by a murine sarcoma and an avian carcinoma virus. Thus, Raf and tumorigenesis have been connected from the very beginning. Ten years later, the work of many groups instated Raf as the link between Ras, the oncogene most frequently mutated in human cancers, and the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK/ERK) module, which with its manifold substrates can contribute to different aspects of carcinogenesis. Finally, the discovery of activating B-Raf mutations in a subset of human cancers, notably melanomas, conclusively established Raf as a major player in tumor development. Recent studies in animal models now show that endogenous C-Raf is essential for the development and maintenance of Ras-induced epidermal tumors. Surprisingly, the role of C-Raf in this case is not that of an mitogen-activated protein kinase activator, but rather that of an endogenous inhibitor of Rho signaling, expanding the range of tumor-related Raf targets. This review focuses on old and new targets of Raf in tumorigenesis.

Translocator protein TSPO is an 18 kDa protein implicated in numerous cell functions and is highly expressed in secretory and glandular tissues, especially in steroidogenic cells. TSPO expression is altered in pathological conditions such as certain cancers and neurological diseases. In search of the factors regulating Tspo expression, we recently showed that high levels of TSPO in steroidogenic cells may be due to high constitutive expression of protein kinase Cepsilon (PKCepsilon), while phorbol 12-myristate 13-acetate (PMA) activation of PKCepsilon drives inducible TSPO expression in nonsteroidogenic cells, likely through activator protein 1 (AP1). In this study, we aimed to identify the signal transduction pathway through which PKCepsilon regulates Tspo gene expression. The MEK1/2 specific inhibitor U0126, but not NFkappaB inhibitors, reduced basal Tspo promoter activity in TSPO-rich steroidogenic cells (MA-10 Leydig), as well as basal and PMA-induced Tspo promoter levels in TSPO-poor nonsteroidogenic cells (NIH-3T3 fibroblasts). AP1 and signal transducer and activation of transcription 3 (STAT3) have binding sites in the Tspo promoter and are downstream targets of PKCepsilon and MAPK (Raf-1-ERK1/2) pathways. PKCepsilon overexpression induced STAT3 phosphorylation in NIH-3T3 cells, while PKCepsilon knockdown reduced STAT3 and c-Jun phosphorylation in Leydig cells. MEK1/2, ERK2, c-Jun, and STAT3 knockdown reduced Tspo mRNA and protein levels in Leydig cells. Additionally, Raf-1 reduced Tspo mRNA levels in the same cells. MEK1/2, c-Jun, and STAT3 knockdown also reduced basal as well as PMA-induced Tspo mRNA levels in NIH-3T3 cells. Together, these results demonstrate that PKCepsilon regulates Tspo gene expression through a MAPK (Raf-1-MEK1/2-ERK1/2) signal transduction pathway, acting at least in part through c-Jun and STAT3 transcription factors.

Background & Aims: Death receptors expressed on tumor cells can prevent metastasis formation by inducing apoptosis, but they also can promote migration and invasion. The determinants of death receptor signaling output are poorly defined. Here we investigated the role of oncogenic K-Ras in determining death receptor function and metastatic potential., Methods: Isogenic human and mouse colorectal cancer cell lines differing only in the presence or absence of the K-Ras oncogene were tested in apoptosis and invasion assays using CD95 ligand and tumor necrois factor-related apoptosis-inducing ligand (TRAIL) as stimuli. Metastatic potential was assessed by intrasplenic injections of green fluorescent protein- or luciferase-expressing tumor cells, followed by intravital fluorescence microscopy or bioluminescence imaging, and confocal microscopy and immunohistochemistry. Ras-effector pathway control of CD95 output was assessed by an RNA-interference and inhibitor-based approach., Results: CD95 ligand and TRAIL stimulated invasion of colorectal tumor cells and liver metastases in a K-Ras-dependent fashion. Loss of mutant K-Ras switched CD95 and TRAIL receptors back into apoptosis mode and abrogated metastatic potential. Raf1 was essential for the switch in CD95 function, for tumor cell survival in the liver, and for K-Ras-driven formation of liver metastases. K-Ras and Raf1 suppressed Rho kinase (ROCK)/LIM kinase-mediated phosphorylation of the actin-severing protein cofilin. Overexpression of ROCK or LIM kinase allowed CD95L to induce apoptosis in K-Ras-proficient cells and prevented metastasis formation, whereas their suppression protected K-Ras-deficient cells against apoptosis., Conclusions: Oncogenic K-Ras and its effector Raf1 convert death receptors into invasion-inducing receptors by suppressing the ROCK/LIM kinase pathway, and this is essential for K-Ras/Raf1-driven metastasis formation., (Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Skin cancer is the most frequently diagnosed cancer in the United States. Ultraviolet B (UVB) rays (wavelength: 280-320nm) play a pivotal role in the development of skin cancer by inducing the expression of inflammatory proteins such as cyclooxygenase-2 (COX-2). Cyanidin, the most plentiful of the plant pigments known as anthocyanidins, is a potent chemopreventive agent. In the present study, we examined the molecular mechanisms underlying the chemopreventive activity of cyanidin and identified its molecular targets. Cyanidin inhibited UVB-induced COX-2 expression and prostaglandin E(2) secretion in the epidermal skin cell line JB6 P+ by suppressing the transactivation of nuclear factor-kappaB and activator protein-1 which are well-known transcription factors regulated by mitogen-activated protein kinase. Cyanidin markedly inhibited the phosphorylation of JNK1/2, ERK1/2, and MEK1/2 than the of MKK4 and Raf-1, two upstream kinases of JNK1/2, ERK1/2, and MEK1/2. Cyanidin significantly suppressed the activities of MKK4, MEK1, and Raf-1 through direct binding. Transient transfection of a small interfering RNA specific for MKK4 inhibited the UVB-induced expression of COX-2 in JB6 P+ cells, as did the expression of a dominant-negative ERK2 mutant. We conclude that MKK4, MEK1, and Raf-1 are targets of cyanidin for the suppression of UVB-induced COX-2 expression., (2010. Published by Elsevier Inc. All rights reserved.)

We have recently reported that activation of the Raf-1/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2)/ERK1/2 signaling cascade in gastrointestinal carcinoid cell line (BON) alters cellular morphology and neuroendocrine phenotype. The mechanisms by which Raf-1 mediates these changes in carcinoid cells are unclear. Here, we report that activation of the Raf-1 signaling cascade in BON cells induced the expression of focal adhesion kinase (FAK) protein, suppressed the production of neuroendocrine markers, and resulted in significant decreases in cellular adhesion and migration. Importantly, inactivation of MEK1/2 by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene or abolition of FAK induction in Raf-1-activated BON cells by targeted siRNA led to reversal of the Raf-1-mediated reduction in neuroendocrine markers and cellular adhesion and migration. Phosphorylation site-specific antibodies detected the phosphorylated FAK(Tyr407), but not FAK(Tyr397), in these Raf-1-activated cells, indicating that FAK(Tyr407) may be associated with changes in the neuroendocrine phenotype. Overexpression of constitutively active FAK plasmids (wild-type FAK or FAK(Tyr397) mutant) into BON cells reduced neuroendocrine markers, whereas the FAK(Tyr407) mutant plasmid did not show any decrease in the levels of neuroendocrine markers, indicating that phosphorylation of FAK at the Tyr(407) residue may be important for these effects. Our results showed for the first time that FAK is an essential downstream effector of the Raf-1/MEK1/2/ERK1/2 signaling cascade and negatively regulated the neuroendocrine and metastatic phenotype in BON cells., ((c)2010 AACR.)

Cutaneous malignant melanoma is considered one of the most deadly human cancers, based on both its penchant for metastatic spread and its typical resistance to currently available therapy. Long known to harbor oncogenic NRAS mutations, melanomas were more recently reported to be frequent bearers of activating mutations in BRAF, one of the effectors situated downstream of wild-type NRAS. NRAS and BRAF mutations are rarely found in the same melanoma, suggesting that they may possess important overlapping oncogenic activities. Here, we compare and contrast the oncogenic roles of the three major NRas downstream effectors, Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine exchange factor (RalGEF), using genetically engineered Arf-deficient immortalized mouse melanocytes as a model system. Although no single downstream pathway could recapitulate all of the consequences of oncogenic NRas expression, our data indicate a prominent role for BRaf and PI3K in melanocyte senescence and invasiveness, respectively. More surprisingly, we discovered that constitutive RalGEF activation had a major impact on several malignant phenotypes, particularly anchorage-independent growth, indicating that this often overlooked pathway should be more carefully evaluated as a possible therapeutic target.

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on cell cycle progression, gene expression, prevention of apoptosis and sensitivity to chemotherapeutic drugs were examined in FL/ΔAkt-1:ER*(Myr(+)) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf-1 and Akt-1 activation by treatment with testosterone or tamoxifen respectively. In these cells we can compare the effects of normal cytokine vs. oncogene mediated signaling in the same cells by changing the culture conditions. Raf-1 was more effective than Akt-1 in inducing cell cycle progression and preventing apoptosis in the presence and absence of chemotherapeutic drugs. The normal cytokine for these cells, interleukin-3 induced/activated most downstream genes transiently, with the exception of p70S6K that was induced for prolonged periods of time. In contrast, most of the downstream genes induced by either the activate Raf-1 or Akt-1 oncogenes were induced for prolonged periods of time, documenting the differences between cytokine and oncogene mediated gene induction which has important therapeutic consequences. The FL/ΔAkt-1:ER*(Myr(+)) + ΔRaf-1:AR cells were sensitive to MEK and PI3K/mTOR inhibitors. Combining MEK and PI3K/mTOR inhibitors increased the induction of apoptosis. The effects of doxorubicin on the induction of apoptosis could be enhanced with MEK, PI3K and mTOR inhibitors. Targeting the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be an effective approach for therapeutic intervention in those cancers which have upstream mutations which result in activation of these pathways.

CD24 is a glycosylphosphatidylinositol-anchored membrane protein reported to be overexpressed in human tumorigenesis and progression. Our purpose was to determine the role of CD24 in the proliferation of colorectal cancer cells and the potential mechanisms in this process. Our data showed that CD24 promoted cell growth and induced activation of extracellular signal-regulated kinases, Raf-1, and p38 mitogen-activated protein kinase. Furthermore, suppression of extracellular signal-regulated kinases and p38 mitogen-activated protein kinase activity by their specific inhibitors, U0126 and SB203580, abrogated CD24-induced proliferation in vitro. By tumorigenicity assay in female BALB/c nude mice, we further demonstrated that CD24 promoted tumor growth in vivo. Immunohistochemical analysis revealed that CD24 expression occurred in 92.5% of human colorectal cancer tissue, and increased with tumor progression. More importantly, the stainings of phospho-extracellular signal-regulated kinases and phospho-p38 mitogen-activated protein kinase were strongly correlated with CD24 expression. Taken together, our data suggest that CD24-dependent extracellular signal-regulated kinases and p38 mitogen-activated protein kinase activations are required for colorectal cancer cell proliferation in vitro and in vivo. The linkage of CD24 and the mitogen-activated protein kinase pathway may unravel a novel mechanism in the regulation of colorectal cancer proliferation.

We investigated the potential role of Raf-1 kinase in mesenteric arterial contraction. Inhibitors of Raf-1 kinase, GW5074, L779450 and ZM 336372 reversed phenylephrine (PE)-induced mesenteric vascular contraction. Studies in vivo in rats showed that GW5074 inhibited PE-induced increase in mean arterial pressure in adult female Sprague-Dawley rats. Isometric tension studies in mesenteric arteries of rats showed that GW5074 did not change the KCl-evoked contraction but significantly inhibited the contractions to PE, 5-HT, U46619, endothelin 1, angiotensin II and phorbol 12, 13-dibutyrate (PDBu). In mesenteric vascular smooth muscle cells (VSMCs), PE stimulated increase in Raf-1 phosphorylation which was inhibited by GW5074. Measurement of [Ca(2+)](i) with Fura-2 showed that GW5074-mediated inhibition of PE-induced contraction was not associated with decreases in [Ca(2+)](i). VSMCs treated with PE exhibited higher levels of the contractile proteins, p-MYPT1 and p-MLC(20), which was inhibited by GW5074. Similarly, PDBu induced increases in phosphorylation of Raf-1, MLC(20) and MYPT1 and this was inhibited by GW5074. However, GW5074 did not have any significant effect on PE/PDBu-induced MEK/ERK activation. The results indicate that Raf-1 kinase plays an important role in the regulation of vascular contractility through regulation of calcium sensitization.

The activity of Raf-1 and Rok-alpha kinases is regulated by intramolecular binding of the regulatory region to the kinase domain. Autoinhibition is relieved upon binding to the small guanosine triphosphatases Ras and Rho. Downstream of Ras, Raf-1 promotes migration and tumorigenesis by antagonizing Rok-alpha, but the underlying mechanism is unknown. In this study, we show that Rok-alpha inhibition by Raf-1 relies on an intermolecular interaction between the Rok-alpha kinase domain and the cysteine-rich Raf-1 regulatory domain (Raf-1reg), which is similar to Rok-alpha's own autoinhibitory region. Thus, Raf-1 mediates Rok-alpha inhibition in trans, which is a new concept in kinase regulation. This mechanism is physiologically relevant because Raf-1reg is sufficient to rescue all Rok-alpha-dependent defects of Raf-1-deficient cells. Downstream of Ras and Rho, the Raf-1-Rok-alpha interaction represents a novel paradigm of pathway cross talk that contributes to tumorigenesis and cell motility.

Background: Thrombospondin-1 (TSP-1) induces vascular smooth muscle cell (VSMC) migration after arterial injury. TSP-1 up-regulates hyaluronic acid (HyA)-inducing genes in VSMCs. HyA also induces VSMC migration. Our hypothesis was that TSP-1-induced VSMC migration is dependent on the CD44 receptor, and that HyA and TSP-1 share migratory signaling pathways., Methods: VSMC migration was assessed using TSP-1, HyA, or serum-free medium as chemoattractants. VSMCs were treated with inhibitors to CD44, Ras, phosphatidylinositol-3 kinase, Raf-1 kinase, or c-SRC. TSP-1- and HyA-induced epidermal growth factor receptor (EGFR) activity was determined by enzyme-linked immunosorbent assay. Comparisons were made by the Student t test and a P value less than .05 was considered significant., Results: Inhibiting CD44 reduced TSP-1- and HyA-induced migration. Phosphatidylinositol-3 kinase and c-SRC inhibitors prevented TSP-1- and HyA-induced migration, whereas Ras and Raf-1 kinase inhibitors only affected TSP-1. TSP-1 and HyA activate the EGFR., Conclusions: TSP-1- and HYA-induced migration share some of the same signaling pathways and the EGFR/CD44 receptors may be a common link.

A key issue in stem cell biology is the differentiation of homogeneous stem cells towards different fates which are also organized into desired configurations. Little is known about the mechanisms underlying the process of periodic patterning. Feather explants offer a fundamental and testable model in which multi-potential cells are organized into hexagonally arranged primordia and the spacing between primordia. Previous work explored roles of a Turing reaction-diffusion mechanism in establishing chemical patterns. Here we show that a continuum of feather patterns, ranging from stripes to spots, can be obtained when the level of p-ERK activity is adjusted with chemical inhibitors. The patterns are dose-dependent, tissue stage-dependent, and irreversible. Analyses show that ERK activity-dependent mesenchymal cell chemotaxis is essential for converting micro-signaling centers into stable feather primordia. A mathematical model based on short-range activation, long-range inhibition, and cell chemotaxis is developed and shown to simulate observed experimental results. This generic cell behavior model can be applied to model stem cell patterning behavior at large.

The C-type lectin dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) mediates the innate immune recognition of microbial carbohydrates. We investigated the function of this molecule in the host response to pathogens in vivo, by generating mouse lines lacking the DC-SIGN homologues SIGNR1, SIGNR3, and SIGNR5. Resistance to Mycobacterium tuberculosis was impaired only in SIGNR3-deficient animals. SIGNR3 was expressed in lung phagocytes during infection, and interacted with M. tuberculosis bacilli and mycobacterial surface glycoconjugates to induce secretion of critical host defense inflammatory cytokines, including tumor necrosis factor (TNF). SIGNR3 signaling was dependent on an intracellular tyrosine-based motif and the tyrosine kinase Syk. Thus, the mouse DC-SIGN homologue SIGNR3 makes a unique contribution to protection of the host against a pulmonary bacterial pathogen.

Successful implantation necessitates modulation of the uterine environment by the embryo for a specific period of time during the menstrual cycle. Infusion of chorionic gonadotropin (CG) into the oviducts of baboons to mimic embryo transit induces a myriad of morphological, biochemical, and molecular changes in the endometrium. Endometrial epithelial cells from both baboons and humans when stimulated by CG in vitro, activates a cAMP-independent MAPK pathway leading to prostaglandin E(2) (PGE(2)) synthesis. This study shows that in the human endometrial cell line, HES, CG, acting via its G-protein coupled receptor, phosphorylates protein kinase B, c-Raf, and ERK1/2 in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Furthermore, ERK1/2 phosphorylation is independent of the signaling paradigms of Galpha(s), Galpha(I), and epidermal growth factor receptor (EGFR) transactivation, typical of gonadal cells, indicating an alternative signaling pattern in the endometrium. After phosphorylation by CG, ERK1/2 translocates to the nucleus in a time-dependent manner. Downstream of ERK1/2, CG activates the nuclear transcription factor, Elk1, also in a PI3K-MAPK-dependent manner. Lastly, we show that in HES cells, this pathway regulates the expression of the microsomal enzyme PGE(2) synthase (mPTGES), a terminal prostanoid synthase responsible for PGE(2) synthesis. CG regulates the mPTGES promoter and also induces mPTGES synthesis in HES cells via the PI3K-ERK1/2 pathway. We suggest that this alternative PI3K-ERK-Elk pathway activated by CG regulates prostaglandin production by the endometrial epithelium and serves as an early trigger to prepare the endometrium for implantation.

Ras activation is common to many human cancers and promotes cell proliferation and survival by initiating multiple signaling cascades. Accordingly, Ras-transformed cells are generally considered too resourceful to become addicted to a single effector. In contrast to this tenet, we now demonstrate an absolute, cell autonomous requirement for Raf-1 in the development and maintenance of Ras-induced skin epidermis tumors. Mechanistically, Raf-1 functions as an endogenous inhibitor dimming the activity of the Rho-dependent kinase Rok-alpha in the context of a Ras-induced Raf-1:Rok-alpha complex. Raf-1-induced Rok-alpha inhibition allows the phosphorylation of STAT3 and Myc expression and promotes dedifferentiation in Ras-induced tumors. These data link the Raf-1:Rok-alpha complex to STAT3/Myc activation and delineate a pathway crucial for cell fate decision in Ras-induced tumorigenesis.

Squamous cell carcinoma (SCC) is the second most common form of nonmelanoma skin cancer. In this issue of Cancer Cell, Ehrenreiter et al. unveil a critical role for the Raf-1/Rok-alpha interaction in the pathogenesis of SCCs, thus paving the way for the development of therapeutic modalities to treat this malignancy.

Purpose: The expression and activation of the Ras/Raf-1/mitogen-activated protein kinase (MAPK) pathway plays an important role in the development and progression of cancer, and may influence response to treatments such as tamoxifen and chemotherapy. In this study we investigated whether the expression and activation of the key components of this pathway influenced clinical outcome, to test the hypothesis that activation of the MAPK pathway drives resistance to tamoxifen and chemotherapy in women with breast cancer., Experimental Design: Breast tumors from patients at the Glasgow Royal Infirmary and others treated within the BR9601 trial were analyzed for expression of the three Ras isoforms, total Raf-1, active and inactive forms of Raf-1 [pRaf(ser338) and pRaf(ser259), respectively], MAPK, and phospho-MAPK using an immunohistochemical approach. Analyses were done with respect to disease free-survival and overall survival., Results: Expression and activation of the Ras pathway was associated with loss of benefit from treatment with tamoxifen but not chemotherapy. Overexpression of pRaf(ser338) was associated with shortened disease-free and overall survival time in univariate analyses. Multivariate analysis suggested pRaf(ser338) was independent of known prognostic markers in predicting outcome following tamoxifen treatment (P=0.03)., Conclusion: This study suggests that activation of the Ras pathway predicts for poor outcome on tamoxifen but not chemotherapy, and identifies pRaf(ser338) as a potential marker of resistance to estrogen receptor-targeted therapy. In addition, it suggests that expression of pRaf(ser338) could identify patients for whom tamoxifen alone is insufficient adjuvant systemic therapy, but for whom the addition of chemotherapy may be of benefit.

Haloperidol, a classical antipsychotic drug, affects the extracellular signal-regulated kinase (ERK) pathway in the brain. However, findings are inconsistent and the mechanism by which haloperidol regulates ERK is poorly understood. Therefore, we examined the ERK pathway and the related protein phosphatase 2A (PP2A) in detail after haloperidol administration. Haloperidol (0.5 and 1 mg/kg) induced biphasic changes in the phosphorylation level of mitogen-activated protein kinase kinase (MEK), ERK, and p90 ribosomal S6 kinase (p90RSK) without changing Raf-1 phosphorylation. Fifteen minutes after haloperidol administration, MEK-ERK-p90RSK phosphorylation increased, whilst PP2A activity decreased. At 60 min, the reverse was observed and the binding of PP2A to MEK and ERK increased. Higher dosages of haloperidol (2 and 4 mg/kg), affected neither MEK-ERK-p90RSK phosphorylation nor PP2A activity. Accordingly, PP2A regulates acute dose- and time-dependent changes in MEK-ERK-p90RSK phosphorylation after haloperidol treatment. These findings suggest the involvement of a dephosphorylating mechanism in the acute action of haloperidol.

A relative decrease in beta-cell mass is key in the pathogenesis of type 1 diabetes, type 2 diabetes, and in the failure of transplanted islet grafts. It is now clear that beta-cell duplication plays a dominant role in the regulation of adult beta-cell mass. Therefore, knowledge of the endogenous regulators of beta-cell replication is critical for understanding the physiological control of beta-cell mass and for harnessing this process therapeutically. We have shown that concentrations of insulin known to exist in vivo act directly on beta-cells to promote survival. Whether insulin stimulates adult beta-cell proliferation remains unclear. We tested this hypothesis using dispersed primary mouse islet cells double labeled with 5-bromo-2-deoxyuridine and insulin antisera. Treating cells with 200-pm insulin significantly increased proliferation from a baseline rate of 0.15% per day. Elevating glucose from 5-15 mm did not significantly increase beta-cell replication. beta-Cell proliferation was inhibited by somatostatin as well as inhibitors of insulin signaling. Interestingly, inhibiting Raf-1 kinase blocked proliferation stimulated by low, but not high (superphysiological), insulin doses. Insulin-stimulated mouse insulinoma cell proliferation was dependent on both phosphatidylinositol 3-kinase/Akt and Raf-1/MAPK kinase pathways. Overexpression of Raf-1 was sufficient to increase proliferation in the absence of insulin, whereas a dominant-negative Raf-1 reduced proliferation in the presence of 200-pm insulin. Together, these results demonstrate for the first time that insulin, at levels that have been measured in vivo, can directly stimulate beta-cell proliferation and that Raf-1 kinase is involved in this process. These findings have significant implications for the understanding of the regulation of beta-cell mass in both the hyperinsulinemic and insulin-deficient states that occur in the various forms of diabetes.

Activation of the fibroblast growth factor (FGFR) and melanocyte stimulating hormone (MC1R) receptors stimulates B-Raf and C-Raf isoforms that regulate the dynamics of MAPK1,2 signaling. Network topology motifs in mammalian cells include feed-forward and feedback loops and bifans where signals from two upstream molecules integrate to modulate the activity of two downstream molecules. We computationally modeled and experimentally tested signal processing in the FGFR/MC1R/B-Raf/C-Raf/MAPK1,2 network in human melanoma cells; identifying 7 regulatory loops and a bifan motif. Signaling from FGFR leads to sustained activation of MAPK1,2, whereas signaling from MC1R results in transient activation of MAPK1,2. The dynamics of MAPK activation depends critically on the expression level and connectivity to C-Raf, which is critical for a sustained MAPK1,2 response. A partially incoherent bifan motif with a feedback loop acts as a logic gate to integrate signals and regulate duration of activation of the MAPK signaling cascade. Further reducing a 106-node ordinary differential equations network encompassing the complete network to a 6-node network encompassing rate-limiting processes sustains the feedback loops and the bifan, providing sufficient information to predict biological responses.