ERK5 signalling pathway is a novel target of sorafenib: Implication in EGF biology.

Sorafenib is a multikinase inhibitor widely used in cancer therapy with an antitumour effect related to biological processes as proliferation, migration or invasion, among others. Initially designed as a Raf inhibitor, Sorafenib was later shown to also block key molecules in tumour progression such as VEGFR and PDGFR. In addition, sorafenib has been connected with key signalling pathways in cancer such as EGFR/EGF. However, no definitive clue about the molecular mechanism linking sorafenib and EGF signalling pathway has been established so far. Our data in HeLa, U2OS, A549 and HEK293T cells, based on in silico, chemical and genetic approaches demonstrate that the MEK5/ERK5 signalling pathway is a novel target of sorafenib. In addition, our data show how sorafenib is able to block MEK5-dependent phosphorylation of ERK5 in the Ser218/Tyr220, affecting the transcriptional activation associated with ERK5. Moreover, we demonstrate that some of the effects of this kinase inhibitor onto EGF biological responses, such as progression through cell cycle or migration, are mediated through the effect exerted onto ERK5 signalling pathway. Therefore, our observations describe a novel target of sorafenib, the ERK5 signalling pathway, and establish new mechanistic insights for the antitumour effect of this multikinase inhibitor.

p38ß and Cancer: The Beginning of the Road.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway is implicated in cancer biology and has been widely studied over the past two decades as a potential therapeutic target. Most of the biological and pathological implications of p38MAPK signaling are often associated with p38α (MAPK14). Recently, several members of the p38 family, including p38γ and p38δ, have been shown to play a crucial role in several pathologies including cancer. However, the specific role of p38ß (MAPK11) in cancer is still elusive, and further investigation is needed. Here, we summarize what is currently known about the role of p38ß in different types of tumors and its putative implication in cancer therapy. All evidence suggests that p38ß might be a key player in cancer development, and could be an important therapeutic target in several pathologies, including cancer.

The Tetraspanin TSPAN33 Controls TLR-Triggered Macrophage Activation through Modulation of NOTCH Signaling.

The involvement of NOTCH signaling in macrophage activation by Toll receptors has been clearly established, but the factors and pathways controlling NOTCH signaling during this process have not been completely delineated yet. We have characterized the role of TSPAN33, a tetraspanin implicated in a disintegrin and metalloproteinase (ADAM) 10 maturation, during macrophage proinflammatory activation. Tspan33 expression increases in response to TLR signaling, including responses triggered by TLR4, TLR3, and TLR2 activation, and it is enhanced by IFN-γ. In this study, we report that induction of Tspan33 expression by TLR and IFN-γ is largely dependent on NOTCH signaling, as its expression is clearly diminished in macrophages lacking Notch1 and Notch2 expression, but it is enhanced after overexpression of a constitutively active intracellular domain of NOTCH1. TSPAN33 is the member of the TspanC8 tetraspanin subgroup more intensely induced during macrophage activation, and its overexpression increases ADAM10, but not ADAM17, maturation. TSPAN33 favors NOTCH processing at the membrane by modulating ADAM10 and/or Presenilin1 activity, thus increasing NOTCH signaling in activated macrophages. Moreover, TSPAN33 modulates TLR-induced proinflammatory gene expression, at least in part, by increasing NF-κB-dependent transcriptional activity. Our results suggest that TSPAN33 represents a new control element in the development of inflammation by macrophages that could constitute a potential therapeutic target.

DLK1 is a novel inflammatory inhibitor which interferes with NOTCH1 signaling in TLR-activated murine macrophages.

Delta-like protein 1 (DLK1) is a noncanonical ligand that inhibits NOTCH1 receptor activity and regulates multiple differentiation processes. In macrophages, NOTCH signaling increases TLR-induced expression of key pro-inflammatory mediators. We have investigated the role of DLK1 in macrophage activation and inflammation using Dlk1-deficient mice and Raw 264.7 cells overexpressing Dlk1. In the absence of Dlk1, NOTCH1 expression is increased and the activation of macrophages with TLR3 or TLR4 agonists leads to higher production of IFN-ß and other pro-inflammatory cytokines, including TNF-α, IL-12, and IL-23. The expression of key proteins involved in IFN-ß signaling, such as IRF3, IRF7, IRF1, or STAT1, as well as cRel, or RelB, which are responsible for the generation of IL-12 and IL-23, is enhanced in Dlk1 KO macrophages. Consistently, Dlk1 KO mice are more sensitive to LPS-induced endotoxic shock. These effects seem to be mediated through the modulation of NOTCH1 signaling. TLR4 activation reduces DLK1 expression, whereas increases NOTCH1 levels. In addition, DLK1 expression diminishes during differentiation of human U937 cells to macrophages. Overall, these results reveal a novel role for DLK1 as a regulator of NOTCH-mediated, pro-inflammatory macrophage activation, which could help to ensure a baseline level preventing constant tissue inflammation.

P38 MAPK and Radiotherapy: Foes or Friends?

Over the last 30 years, the study of the cellular response to ionizing radiation (IR) has increased exponentially. Among the various signaling pathways affected by IR, p38 MAPK has been shown to be activated both in vitro and in vivo, with involvement in key processes triggered by IR-mediated genotoxic insult, such as the cell cycle, apoptosis or senescence. However, we do not yet have a definitive clue about the role of p38 MAPK in terms of radioresistance/sensitivity and its potential use to improve current radiotherapy. In this review, we summarize the current knowledge on this family of MAPKs in response to IR as well as in different aspects related to radiotherapy, such as their role in the control of REDOX, fibrosis, and in the radiosensitizing effect of several compounds.

The EGF-like proteins DLK1 and DLK2 function as inhibitory non-canonical ligands of NOTCH1 receptor that modulate each other's activities.

The protein DLK2, highly homologous to DLK1, belongs to the EGF-like family of membrane proteins, which includes NOTCH receptors and their DSL-ligands. The molecular mechanisms by which DLK proteins regulate cell differentiation and proliferation processes are not fully established yet. In previous reports, we demonstrated that DLK1 interacts with itself and with specific EGF-like repeats of the NOTCH1 extracellular region involved in the binding to NOTCH1 canonical ligands. Moreover, the interaction of DLK1 with NOTCH1 caused an inhibition of basal NOTCH signaling in preadipocytes and mesenchymal multipotent cells. In this work, we demonstrate, for the first time, that DLK2 interacts with itself, with DLK1, and with the same NOTCH1 receptor region as DLK1 does. We demonstrate also that the interaction of DLK2 with NOTCH1 similarly results in an inhibition of NOTCH signaling in preadipocytes and Mouse Embryo fibloblasts. In addition, we demonstrate that a membrane DLK1 variant, lacking the sequence recognized by the protease TACE, also inhibits NOTCH signaling. Furthermore, both DLK1 and DLK2 are able to decrease NOTCH activity also when triggered by specific NOTCH ligands. However, the decrease in NOTCH signaling induced by overexpression of Dlk2 is reversed by the overexpression of Dlk1, and viceversa. We conclude that DLK1 and DLK2 act as inhibitory non-canonical protein ligands for the NOTCH1 receptor that modulate NOTCH signaling.

ERK5 Is a Major Determinant of Chemical Sarcomagenesis: Implications in Human Pathology.

Sarcomas are a heterogeneous group of tumors in which the role of ERK5 is poorly studied. To clarify the role of this MAPK in sarcomatous pathology, we used a murine 3-methyl-cholanthrene (3MC)-induced sarcoma model. Our data show that 3MC induces pleomorphic sarcomas with muscle differentiation, showing an increased expression of ERK5. Indeed, this upregulation was also observed in human sarcomas of muscular origin, such as leiomyosarcoma or rhabdomyosarcoma. Moreover, in cell lines derived from these 3MC-induced tumors, abrogation of Mapk7 expression by using specific shRNAs decreased in vitro growth and colony-forming capacity and led to a marked loss of tumor growth in vivo. In fact, transcriptomic profiling in ERK5 abrogated cell lines by RNAseq showed a deregulated gene expression pattern for key biological processes such as angiogenesis, migration, motility, etc., correlating with a better prognostic in human pathology. Finally, among the various differentially expressed genes, Klf2 is a key mediator of the biological effects of ERK5 as indicated by its specific interference, demonstrating that the ERK5-KLF2 axis is an important determinant of sarcoma biology that should be further studied in human pathology.

Notch1 upregulates LPS-induced macrophage activation by increasing NF-kappaB activity.

Macrophages present different Notch receptors and ligands on their surface. Following macrophage activation by LPS or other TLR ligands, Notch1 expression is upregulated. We report here that Notch signaling increases both basal and LPS-induced NF-kappaB activation, favoring the expression of genes implicated in the inflammatory response, such as the cytokines TNF-alpha and IL-6, or enzymes, such as iNOS. Delta4 seems to be the most effective ligand to induce Notch activation and increasing NF-kappaB transcriptional activity in macrophages. We show that Notch1 signaling promotes NF-kappaB translocation to the nucleus and DNA binding by increasing both phosphorylation of the IkappaB kinase alpha/beta complex and the expression of some NF-kappaB family members. Treatment of macrophages with the gamma-secretase inhibitor DAPT, which prevents the cleavage and activation of Notch receptors, inhibits all these processes, diminishing NF-kappaB activity following LPS stimulation. Additionally, we show that the active intracellular Notch fragment can directly interact with TNF-alpha and iNOS promoters. Our results suggest that Notch signaling results in an amplification of the macrophage-dependent inflammatory response by enhancing NF-kappaB signaling.

Predictors of acquired lipodystrophy in juvenile-onset dermatomyositis and a gradient of severity.

We describe the clinical features of 28 patients with juvenile dermatomyositis (JDM) and 1 patient with adult-onset dermatomyositis (DM), all of whom developed lipodystrophy (LD) that could be categorized into 1 of 3 phenotypes, generalized, partial, or focal, based on the pattern of fat loss distribution. LD onset was often delayed, beginning a median of 4.6 years after diagnosis of DM. Calcinosis, muscle atrophy, joint contractures, and facial rash were DM disease features found to be associated with LD. Panniculitis was associated with focal lipoatrophy while the anti-p155 autoantibody, a newly described myositis-associated autoantibody, was more associated with generalized LD. Specific LD features such as acanthosis nigricans, hirsutism, fat redistribution, and steatosis/nonalcoholic steatohepatitis were frequent in patients with LD, in a gradient of frequency and severity among the 3 sub-phenotypes. Metabolic studies frequently revealed insulin resistance and hypertriglyceridemia in patients with generalized and partial LD. Regional fat loss from the thighs, with relative sparing of fat loss from the medial thighs, was more frequent in generalized than in partial LD and absent from DM patients without LD. Cytokine polymorphisms, the C3 nephritic factor, insulin receptor antibodies, and lamin mutations did not appear to play a pathogenic role in the development of LD in our patients. LD is an under-recognized sequela of JDM, and certain DM patients with a severe, prolonged clinical course and a high frequency of calcinosis appear to be at greater risk for the development of this complication. High-risk JDM patients should be screened for metabolic abnormalities, which are common in generalized and partial LD and result in much of the LD-associated morbidity. Further study is warranted to investigate the pathogenesis of acquired LD in patients with DM.

Dlk1 influences differentiation and function of B lymphocytes.

The Dlk1 (delta-like-1) gene is a member of the epidermal growth factor (EGF)-like homeotic gene family. It influences cell-cell interactions between stromal cells and pro-B cells in vitro. To define the in vivo role of the dlk protein in B cell development, we established a Dlk1-/- mouse model. In spleens of Dlk1-/- mice, transitional B cell numbers were increased and the ratio between transitional B cell subsets was altered. Numbers of follicular B cells decreased, while the number of marginal zone B cells and the size of the marginal zone were increased. Loss of dlk resulted in increased immunoglobulin G1 (IgG1) and IgG3 in preimmune sera. Furthermore, there was an exaggerated primary T-dependent antigen-specific humoral immune response. In bone marrow, the lack of dlk led to increased numbers of the earliest B lineage cells in young mice without affecting numbers of later B lineage cells. In vitro experiments showed that lack of dlk on either stromal cells or pro-B cells caused changes in differentiation and proliferation of pro-B cells, suggesting that lack of dlk leads to changes in cell-cell interactions in the bone marrow microenvironment. These results show that dlk expression is essential for normal B cell development.

The novel gene EGFL9/Dlk2, highly homologous to Dlk1, functions as a modulator of adipogenesis.

The Dlk1 gene appears to function as a regulator of adipogenesis. Adult Dlk1-deficient mice are obese, but adipose tissue still develops in transgenic mice overexpressing an Fc-dlk1 fusion protein, and neither type of genetically modified mice displays serious abnormalities. It was therefore possible that one yet unidentified gene might either compensate or antagonize for the absence or for overexpression, respectively, of Dlk1 in those animals. In database searches, we found a novel gene, EGFL9, encoding for a protein whose structural features are virtually identical to those of dlk1, suggesting it may function in a similar way. As dlk1 does, the protein encoded by EGFL9/Dlk2 affects adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells; however, it does so in an opposite way to that of dlk1. In addition, expression levels of both genes appear to be inversely correlated in both cell lines. Moreover, enforced changes in the expression of one gene affect the expression levels of the other. Our data suggest that adipogenesis may be modulated by the coordinated expression of Dlk1 and EGFL9/Dlk2.

Autophagic cell death associated to Sorafenib in renal cell carcinoma is mediated through Akt inhibition in an ERK1/2 independent fashion.

OBJECTIVES: To fully clarify the role of Mitogen Activated Protein Kinase in the therapeutic response to Sorafenib in Renal Cell Carcinoma as well as the cell death mechanism associated to this kinase inhibitor, we have evaluated the implication of several Mitogen Activated Protein Kinases in Renal Cell Carcinoma-derived cell lines. MATERIALS AND METHODS: An experimental model of Renal Cell Carcinoma-derived cell lines (ACHN and 786-O cells) was evaluated in terms of viability by MTT assay, induction of apoptosis by caspase 3/7 activity, autophagy induction by LC3 lipidation, and p62 degradation and kinase activity using phospho-targeted antibodies. Knock down of ATG5 and ERK5 was performed using lentiviral vector coding specific shRNA. RESULTS: Our data discard Extracellular Regulated Kinase 1/2 and 5 as well as p38 Mitogen Activated Protein Kinase pathways as mediators of Sorafenib toxic effect but instead indicate that the inhibitory effect is exerted through the PI3K/Akt signalling pathway. Furthermore, we demonstrate that inhibition of Akt mediates cell death associated to Sorafenib without caspase activation, and this is consistent with the induction of autophagy, as indicated by the use of pharmacological and genetic approaches. CONCLUSION: The present report demonstrates that Sorafenib exerts its toxic effect through the induction of autophagy in an Akt-dependent fashion without the implication of Mitogen Activated Protein Kinase. Therefore, our data discard the use of inhibitors of the RAF-MEK-ERK1/2 signalling pathway in RCC and support the use of pro-autophagic compounds, opening new therapeutic opportunities for Renal Cell Carcinoma.

E1a is an exogenous in vivo tumour suppressor.

The E1a gene from adenovirus has become a major tool in cancer research. Since the discovery of E1a, it has been proposed to be an oncogene, becoming a key element in the model of cooperation between oncogenes. However, E1a's in vivo behaviour is consistent with a tumour suppressor gene, due to the block/delay observed in different xenograft models. To clarify this interesting controversy, we have evaluated the effect of the E1a 13s isoform from adenovirus 5 in vivo. Initially, a conventional xenograft approach was performed using previously unreported HCT116 and B16-F10 cells, showing a clear anti-tumour effect regardless of the mouse's immunological background (immunosuppressed/immunocompetent). Next, we engineered a transgenic mouse model in which inducible E1a 13s expression was under the control of cytokeratin 5 to avoid side effects during embryonic development. Our results show that E1a is able to block chemical skin carcinogenesis, showing an anti-tumour effect. The present report demonstrates the in vivo anti-tumour effect of E1a, showing that the in vitro oncogenic role of E1a cannot be extrapolated in vivo, supporting its future use in gene therapy approaches.

Exploiting the potential of autophagy in cisplatin therapy: A new strategy to overcome resistance.

Resistance to cisplatin is a major challenge in the current cancer therapy. In order to explore new therapeutic strategies to cisplatin resistance, we evaluated, in a model of lung cancer (H1299 and H460 cell lines), the nature of the pathways leading to cell death. We observed that H1299 displayed a natural resistance to cisplatin due to an inability to trigger an apoptotic response that correlates with the induction of autophagy. However, pharmacological and genetic approaches showed how autophagy was a mechanism associated to cell death rather than to resistance. Indeed, pro-autophagic stimuli such as mTOR or Akt inhibition mediate cell death in both cell lines to a similar extent. We next evaluated the response to a novel platinum compound, monoplatin, able to promote cell death in an exclusive autophagy-dependent manner. In this case, no differences were observed between both cell lines. Furthermore, in response to monoplatin, two molecular hallmarks of cisplatin response (p53 and MAPKs) were not implicated, indicating the ability of this pro-autophagic compound to overcome cisplatin resistance. In summary, our data highlight how induction of autophagy could be used in cisplatin resistant tumours and an alternative treatment for p53 mutated patient in a synthetic lethally approach.

The non-canonical NOTCH ligand DLK1 exhibits a novel vascular role as a strong inhibitor of angiogenesis.

AIMS: The epidermal growth factor-like protein Delta-like 1 (DLK1) regulates multiple differentiation processes. It resembles NOTCH ligands structurally and is considered a non-canonical ligand. Given the crucial role of the NOTCH pathway in angiogenesis, we hypothesized that DLK1 could regulate angiogenesis by interfering with NOTCH. We therefore investigated the expression and function of DLK1 in the vascular endothelium and its role in the regulation of angiogenesis. METHODS AND RESULTS: We report DLK1 expression in the endothelium of different species, including human, cow, pig, and mouse. Angiogenesis was studied by using in vitro and in vivo models of angiotube formation in endothelial cells, retinal phenotypes in Dlk1-null mice, and vessel development in zebrafish. DLK1 overexpression strongly inhibited angiotube formation, whereas lung endothelial cells from Dlk1-null mice were highly angiogenic. In vivo studies demonstrated DLK1-mediated inhibition of neovessel formation and revealed an altered pattern of angiogenesis in the retinas of Dlk1-null mice. The expression of human DLK1 in zebrafish embryos severely altered the formation of intersegmental vessels, while knockdown of the orthologous gene was associated with ectopic and increased tumour-induced angiogenesis. NOTCH-dependent signalling as determined by gene expression reporters was inhibited by the presence of DLK1 in vascular endothelial cells. In contrast, Dlk1-null mice showed increased levels of NOTCH downstream targets, such as Snail and Slug. CONCLUSION: Our results unveil a novel inhibitory role for DLK1 in the regulation of angiogenesis, mediated by antagonism of the NOTCH pathway, and establish the basis for investigating its action in pathological settings.

DLK1 is a novel regulator of bone mass that mediates estrogen deficiency-induced bone loss in mice.

Delta-like 1/fetal antigen 1 (DLK1/FA-1) is a transmembrane protein belonging to the Notch/Delta family that acts as a membrane-associated or a soluble protein to regulate regeneration of a number of adult tissues. Here we examined the role of DLK1/FA-1 in bone biology using osteoblast-specific Dlk1-overexpressing mice (Col1-Dlk1). Col1-Dlk1 mice displayed growth retardation and significantly reduced total body weight and bone mineral density (BMD). Micro-computed tomographis (µCT) scanning revealed a reduced trabecular and cortical bone volume fraction. Tissue-level histomorphometric analysis demonstrated decreased bone-formation rate and enhanced bone resorption in Col1-Dlk1 mice compared with wild-type mice. At a cellular level, Dlk1 markedly reduced the total number of bone marrow (BM)-derived colony-forming units fibroblasts (CFU-Fs), as well as their osteogenic capacity. In a number of in vitro culture systems, Dlk1 stimulated osteoclastogenesis indirectly through osteoblast-dependent increased production of proinflammatory bone-resorbing cytokines (eg, Il7, Tnfa, and Ccl3). We found that ovariectomy (ovx)-induced bone loss was associated with increased production of Dlk1 in the bone marrow by activated T cells. Interestingly, Dlk1(-/-) mice were significantly protected from ovx-induced bone loss compared with wild-type mice. Thus we identified Dlk1 as a novel regulator of bone mass that functions to inhibit bone formation and to stimulate bone resorption. Increasing DLK1 production by T cells under estrogen deficiency suggests its possible use as a therapeutic target for preventing postmenopausal bone loss.

Notch-1 up-regulation and signaling following macrophage activation modulates gene expression patterns known to affect antigen-presenting capacity and cytotoxic activity.

Notch signaling has been extensively implicated in cell-fate determination along the development of the immune system. However, a role for Notch signaling in fully differentiated immune cells has not been clearly defined. We have analyzed the expression of Notch protein family members during macrophage activation. Resting macrophages express Notch-1, -2, and -4, as well as the Notch ligands Jagged-1 and -2. After treatment with LPS and/or IFN-gamma, we observed a p38 MAPK-dependent increase in Notch-1 and Jagged-1 mRNA and protein levels. To study the role of Notch signaling in macrophage activation, we forced the transient expression of truncated, active intracellular Notch-1 (Notch-IC) proteins in Raw 264.7 cells and analyzed their effects on the activity of transcription factors involved in macrophage activation. Notch-IC increased STAT-1-dependent transcription. Furthermore, Raw 264.7 Notch-IC stable transfectants increased STAT1-dependent transcription in response to IFN-gamma, leading to higher expression of IFN regulatory factor-1, suppressor of cytokine signaling-1, ICAM-1, and MHC class II proteins. This effect was independent from an increase of STAT1 Tyr or Ser phosphorylation. However, inducible NO synthase expression and NO production decreased under the same conditions. Our results show that Notch up-regulation and subsequent signaling following macrophage activation modulate gene expression patterns known to affect the function of mature macrophages.

dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats.

The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis.

The EGF-like homeotic protein dlk affects cell growth and interacts with growth-modulating molecules in the yeast two-hybrid system.

Levels of dlk, an EGF-like homeotic protein, are critical for several differentiation processes. Because growth and differentiation are, in general, exclusive of each other, and increasing evidence indicates that Dlk1 expression changes in tumorigenic processes, we studied whether dlk could also affect cell growth. We found that, in response to glucocorticoids, Balb/c 3T3 cells with diminished levels of dlk expression develop foci-like cells that have lost contact inhibition, display altered morphology, and grow faster than control cell lines. Balb/c 3T3 cells spontaneously growing more rapidly are also dlk-negative cells. Moreover, screening by the yeast two-hybrid system, using Dlk1 constructs as baits, resulted in the isolation of GAS1 and acrogranin cDNAs. Interestingly, these proteins are cysteine-rich molecules involved in the control of cell growth. Taken together, these observations suggest that dlk may participate in a network of interactions controlling how the cells respond to growth or differentiation signals.

dlk modulates mitogen-activated protein kinase signaling to allow or prevent differentiation.

The EGF-like membrane protein dlk plays a crucial role in the control of cell differentiation. Overexpression of the protein prevents, whereas inhibition of its expression increases, adipocyte differentiation of 3T3-L1 cells in response to Insulin-like Growth Factor I (IGF-1) or insulin. We have investigated whether dlk modulates the signaling pathways known to control this process. We found that the levels of dlk expression modulated signaling through the IGF-1 receptor, causing changes in the activation levels and kinetics of Extracellular-Regulated Kinase/Mitogen-Activated Protein Kinase (ERK/MAPK) that correlated with differentiation outcome. These changes occurred in response to IGF-1 or insulin but not in response to Epidermal Growth Factor. However, the levels of expression of IGF-1 receptor, or the activation of Insulin Receptor Substrate-1 in response to IGF-1, were not affected by the levels of dlk expression. Therefore, dlk appears to modulate ERK/MAPK signaling in response to specific differentiation signals.