Calmodulin Regulates Intracellular Trafficking of Epidermal Growth Factor Receptor and the MAPK Signaling Pathway

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13¿mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.

Immunoelectron microscopic localization of transforming growth factor alpha in rat colon

Transforming growth factor alpha (TGF alpha) is a polypeptide, which binds to the epidermal growth factor receptor to carry out its function related to cell proliferation and differentiation. The ultrastructural localisation of TGF alpha was studied in both the proximal and the distal colon. The columnar cells, lining the surface epithelium of the proximal colon, showed a strong immunoreactivity in the polyribosomes and in the interdigitations of the lateral membrane. The columnar cells of the crypts and the goblet cells in both the proximal and the distal colon showed the immunostaining in the cis and trans cisternae of the Golgi apparatus. TGF alpha seems to be processed differently in the surface columnar cells and in the crypt columnar cells and goblet cells. Moreover, it probably has different roles in proliferation and differentiation.

Immunoelectron microscopic localization of transforming growth factor alpha in rat colon

Transforming growth factor alpha (TGF alpha) is a polypeptide, which binds to the epidermal growth factor receptor to carry out its function related to cell proliferation and differentiation. The ultrastructural localisation of TGF alpha was studied in both the proximal and the distal colon. The columnar cells, lining the surface epithelium of the proximal colon, showed a strong immunoreactivity in the polyribosomes and in the interdigitations of the lateral membrane. The columnar cells of the crypts and the goblet cells in both the proximal and the distal colon showed the immunostaining in the cis and trans cisternae of the Golgi apparatus. TGF alpha seems to be processed differently in the surface columnar cells and in the crypt columnar cells and goblet cells. Moreover, it probably has different roles in proliferation and differentiation.

The antilipolytic effect of insulin and epidermal growth factor in rat adipocytes are mediated by different mechanisms

Epidermal growth factor (EGF) and insulin induced similar effects in isolated rat adipocytes. To determine whether EGF and insulin produced similar effects through the same mechanisms, we focused on lipolysis. Insulin inhibited the lipolysis stimulated by isoproterenol, glucagon (either alone or in combination with adenosine deaminase), adenosine deaminase itself, or forskolin. In contrast, EGF did not inhibit the lipolysis stimulated by forskolin or by hormones when the cells were also incubated with adenosine deaminase. The effect of insulin, but not that of EGF, on isoproterenol-stimulated lipolysis disappeared when adipocytes were incubated with 1 microM wortmannin. These results indicate that EGF and insulin affected lipolysis through different mechanisms. We observed that EGF, but not insulin, increased cytosolic Ca2+. The effect of EGF, but not that of insulin, disappeared when the cells were incubated in a Ca2+-free medium. We suggest that EGF, but not insulin, mediate its antilipolytic effect through a Ca2+-dependent mechanism which, however, do not involve Ca2+-activated protein kinase C isoforms. This is based on the following: 1) phorbol 12-myristate 13-acetate affected lipolysis in an opposite way to that of EGF; and 2) the protein kinase C inhibitor bisindolylmaleimide GF 109203X did not affect the antilipolytic action of EGF. Our results indicate that the antilipolytic effect of EGF resembles more that of vasopressin than that of insulin.

Development of an epidermal growth factor derivative with EGFR blocking activity.

The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR.

Epidermal growth factor secreted from submandibular salivary glands interferes with the lipolytic effect of adrenaline in mice

We had described that epidermal growth factor (EGF) interfered with the lipolytic effect of catecholamines in isolated adipocytes. Since catecholamines stimulate the release of EGF from submandibular salivary glands to blood plasma in male mice, we studied whether EGF affected also the lipolytic response to adrenaline in whole animals. We studied the effect of adrenaline in sialoadenectomized and sham-operated mice receiving or not a high dose of EGF following adrenaline injection. There was no difference in plasma EGF concentration between sham-operated and sialoadenectomized animals receiving saline. After adrenaline administration plasma EGF increased by 20-fold in sham-operated but did not increase in sialoadenectomized mice. Indeed, the increase was much higher (more than 100-fold) in mice receiving exogenous EGF. The effect of adrenaline on plasma concentration of both glycerol and nonesterified fatty acids was higher as lower was plasma EGF concentration. Isolated adipocytes obtained from sham-operated or sialoadenectomized mice had identical lipolytic response to adrenaline. The lipolytic response of adipocytes to isoproterenol was decreased by addition of EGF. To study whether the interference with the in vivo lipolytic effect of adrenaline had further metabolic consequences, we measured plasma b-hydroxybutyrate concentration in plasma. There was no difference in the response to adrenaline between sham-operated and sialoadenectomized mice in spite of the difference in plasma nonsterified fatty acid concentration. Studies in isolated hepatocytes indicated that ketogenesis run at near maximal rate in this range of substrate concentration. These results suggest that EGF in the physiological range decreases the lipolytic effect of adrenaline but does not compromise further metabolic events like the enhancement of ketogenesis.

An update of the mechanisms of resistance to EGFR-tyrosine kinase inhibitors in breast cancer: gefitinib (Iressatrade mark)-induced changes in the expression and nucleo-cytoplasmic trafficking of HER-ligands (Review)

Intrinsic resistance to the epidermal growth factor receptor (EGFR; HER1) tyrosine kinase inhibitor (TKI) gefitinib, and more generally to EGFR TKIs, is a common phenomenon in breast cancer. The availability of molecular criteria for predicting sensitivity to EGFR-TKIs is, therefore, the most relevant issue for their correct use and for planning future research. Though it appears that in non-small-cell lung cancer (NSCLC) response to gefitinib is directly related to the occurrence of specific mutations in the EGFR TK domain, breast cancer patients cannot be selected for treatment with gefitinib on the same basis as such EGFR mutations have beenreported neither in primary breast carcinomas nor in several breast cancer cell lines. Alternatively, there is a generalagreement on the hypothesis that the occurrence of molecular alterations that activate transduction pathways downstreamof EGFR (i.e., MEK1/MEK2 - ERK1/2 MAPK and PI-3'K - AKT growth/survival signaling cascades) significantly affect the response to EGFR TKIs in breast carcinomas. However,there are no studies so far addressing a role of EGF-related ligands as intrinsic breast cancer cell modulators of EGFR TKIefficacy. We recently monitored gene expression profiles andsub-cellular localization of HER-1/-2/-3/-4 related ligands (i.e., EGF, amphiregulin, transforming growth factor-α, ß-cellulin,epiregulin and neuregulins) prior to and after gefitinib treatment in a panel of human breast cancer cell lines. First, gefitinibinduced changes in the endogenous levels of EGF-related ligands correlated with the natural degree of breast cancer cellsensitivity to gefitinib. While breast cancer cells intrinsically resistant to gefitinib (IC50 ≥15 μM) markedly up-regulated(up to 600 times) the expression of genes codifying for HERspecific ligands, a significant down-regulation (up to 106 times)of HER ligand gene transcription was found in breast cancer cells intrinsically sensitive to gefitinib (IC50 ≤1 μM). Second,loss of HER1 function differentially regulated the nuclear trafficking of HER-related ligands. While gefitinib treatment induced an active import and nuclear accumulation of the HER ligand NRG in intrinsically gefitinib-resistant breastcancer cells, an active export and nuclear loss of NRG was observed in intrinsically gefitinib-sensitive breast cancer cells.In summary, through in vitro and pharmacodynamic studies we have learned that, besides mutations in the HER1 gene,oncogenic changes downstream of HER1 are the key players regulating gefitinib efficacy in breast cancer cells. It now appears that pharmacological inhibition of HER1 functionalso leads to striking changes in both the gene expression and the nucleo-cytoplasmic trafficking of HER-specific ligands,and that this response correlates with the intrinsic degree of breast cancer sensitivity to the EGFR TKI gefitinib. Therelevance of this previously unrecognized intracrine feedback to gefitinib warrants further studies as cancer cells could bypassthe antiproliferative effects of HER1-targeted therapeutics without a need for the overexpression and/or activation of other HER family members and/or the activation of HER-driven downstream signaling cascades

Growth factor- and cytokine-driven pathways governing liver stemness and differentiation.

Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/progenitor cell expansion and differentiation, and the relevance of these signals in liver development, regeneration and carcinogenesis. Tyrosine kinase receptor signaling, in particular, c-Met, epidermal growth factor receptors or fibroblast growth factor receptors, contribute to proliferation, survival and differentiation of liver stem/progenitor cells. Different evidence suggests a dual role for the transforming growth factor (TGF)-β signaling pathway in liver stemness and differentiation. On the one hand, TGF-β mediates progression of differentiation from a progenitor stage, but on the other hand, it contributes to the expansion of liver stem cells. Hedgehog family ligands are necessary to promote hepatoblast proliferation but need to be shut off to permit subsequent hepatoblast differentiation. In the same line, the Wnt family and β-catenin/T-cell factor pathway is clearly involved in the maintenance of liver stemness phenotype, and its repression is necessary for liver differentiation during development. Collectively, data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates to contribute, under adequate conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role. From a clinical perspective, this is a fundamental issue for liver stem/progenitor cell-based therapies.

egr-4, a target of EGFR signaling, is required for the formation of the brian primordial and head regeneration in planarians

During the regeneration of freshwater planarians, polarity and patterning programs play essential roles in determining whether a head or a tail regenerates at anterior or posterior-facing wounds. This decision is made very soon after amputation. The pivotal role of the Wnt/β-catenin and Hh signaling pathways in re-establishing anterior-posterior (AP) polarity has been well documented. However, the mechanisms that control the growth and differentiation of the blastema in accordance with its AP identity are less well understood. Previous studies have described a role of Smed-egfr-3, a planarian epidermal growth factor receptor, in blastema growth and differentiation. Here, we identify Smed-egr-4, a zinc-finger transcription factor belonging to the early growth response gene family, as a putative downstream target of Smed-egfr-3. Smed-egr-4 is mainly expressed in the central nervous system and its silencing inhibits anterior regeneration without affecting the regeneration of posterior regions. Single and combinatorial RNA interference to target different elements of the Wnt/β-catenin pathway, together with expression analysis of brain- and anterior-specific markers, revealed that Smed-egr-4: (1) is expressed in two phases - an early Smed-egfr-3-independent phase and a late Smed-egfr-3-dependent phase; (2) is necessary for the differentiation of the brain primordia in the early stages of regeneration; and (3) that it appears to antagonize the activity of the Wnt/β-catenin pathway to allow head regeneration. These results suggest that a conserved EGFR/egr pathway plays an important role in cell differentiation during planarian regeneration and indicate an association between early brain differentiation and the proper progression of head regeneration.

Mutant huntingtin impairs the post-Golgi trafficking of brain-derived neurotrophic factor but not its Val66Met polymorphism.

Brain-derived neurotrophic factor (BDNF) polymorphism is associated with the pathophysiology of several neurodegenerative disorders, including Huntington"s disease. In view ofthese data andthe involvement of huntingtin in intracellular trafficking, we examined the intracellular transport and release of Val66Val BDNF (Val-BDNF) and Val66Met BDNF (Met-BDNF) in transfected striatal knock-in cells expressing wild-type or mutant full-length huntingtin. Colocalization studies with specific markers for endoplasmic reticulum showed no differences between the Val-BDNF and Met-BDNF and were not modified by mutant huntingtin. However, post-Golgi trafficking was altered by mutant huntingtin dependent on the BDNF form. Thus, fluorescence recovery after photobleaching (FRAP) and inverse FRAP analysis showed retention of Met-BDNF inthe Golgi apparatus with respectto Val-BDNF in wild-type cells. Strikingly, mutant huntingtin diminished post-Golgi trafficking of Val-BDNF, whereas Met-BDNF was not modified. Accordingly, a reduction in the number of transport vesicles was only observed in mutant huntingtin cells transfected with Val-BDNF but not Met-BDNF. Moreover, mutant huntingtin severely affectedthe KCl-evoked release of Val-BDNF, although it had little effect on Met-BDNF regulated release. The constitutive release of Val-BDNF or Met-BDNF in mutant cells was only slightly reduced. Interestingly, mutant huntingtin only perturbed post-Golgi trafficking of proteins that follow the regulated secretory pathway (epidermal growth factor receptor or atrial natriuretic factor), whereas it did not change those that follow the constitutive pathway (p75 NTR ). We conclude that mutant huntingtin differently affects intracellular transport and release of Val-BDNF and Met-BDNF. In addition, our findings reveal a new role for huntingtin in the regulation of the post-Golgi trafficking of the regulated secretory pathway.

Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.

The membrane-permeable calmodulin inhibitors (W-7/W-13) bind to membranes changing the electrostatic surface potential. Dual effect of W-13 on EGFR activation

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca 2 /CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR;W-13 inhibits epidermal growth factordependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due toW-13inhibition of Ca 2 /CaM, but thelatter results could be due to binding of W-13 to the plasma membrane.

Green tea catechin inhibits fatty acid synthase without stimulating carnitine Palmitoyltransferase-1 or inducing weight loss in experimental animals

Background: The enzyme fatty acid synthase (FASN) is highly expressed in many human carcinomas and its inhibition is cytotoxic to human cancer cells. The use of FASN inhibitors has been limited until now by anorexia and weight loss, which is associated with the stimulation of fatty acid oxidation. Materials and Methods: The in vitro effect of (-)-epigallocatechin-3-gallate (EGCG) on fatty acid metabolism enzymes, on apoptosis and on cell signalling was evaluated. In vivo, the effect of EGCG on animal body weight was addressed. Results: EGCG inhibited FASN activity, induced apoptosis and caused a marked decrease of human epidermal growth factor receptor 2 (HER2), phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular (signal)-regulated kinase (ERK) 1/2 proteins, in breast cancer cells. EGCG did not induce a stimulatory effect on CPT-1 activity in vitro (84% of control), or on animal body weight in vivo (99% of control). Conclusion: EGCG is a FASN inhibitor with anticancer activity which does not exhibit cross-activation of fatty acid oxidation and does not induce weight loss, suggesting its potential use as an anticancer drug.

Vascular endothelial growth factor and angiopoietin-2 play a major role in the pathogenesis of vascular leakage in cirrhotic rats.

Background and aims: The extent and molecular mechanisms governing plasma extravasation and formation of ascites in cirrhosis are unknown. Vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Ang-2) are endogenous substances with powerful vascular permeability effects. We assessed regional blood flow, vascular leakage, mRNA and tissular expression of VEGF-A and Ang-2 and vascular permeability following VEGF receptor 2 blockade in control and cirrhotic rats to define the vascular territories showing altered vascular permeability in cirrhosis and to determine whether VEGF-A and Ang-2 are involved in this phenomenon. Methods: Arterial blood flow was analysed with the coloured microsphere method. Vascular leakage was measured and visualised with the dye Evan¿s Blue and colloidal carbon techniques, respectively. VEGF-A and Ang-2 expression were determined by real-time polymerase chain reaction (RT-PCR), immunohistochemistry and western blot. The effect on vascular permeability induced by VEGFR2 blockade was assessed by administration of the receptor inhibitor SU11248. Results: Arterial blood flow was increased in the mesentery, pancreas and small intestine but not in the kidney and spleen of cirrhotic rats as compared to controls. Increased vascular leakage was observed in the mesentery and liver, where colloidal carbon spread from microvessels to the adjacent fibrotic tracts. Increased hepatic and mesenteric expression of VEGF-A and Ang-2 was found in cirrhotic rats as compared to controls. Blockade of VEGFR2 markedly reduced hepatic and mesenteric vascular leakage in cirrhotic rats. Conclusions: Enhanced endothelial permeability is restricted to the hepatic and mesenteric vascular beds in cirrhotic rats with ascites and VEGF-A and Ang-2 are key factors in the signalling pathways regulating this dysfunction.