Michael J. Hayman

Signal transduction and invasion of epithelial cells by S. typhimurium

Invasion of host cells is essential for the pathogenicity of Salmonella. We have recently shown that invasion of cultured epithelial cells by S. typhimurium is accompanied by activation of the epidermal growth factor receptor. In this report we show that S. typhimurium invasion stimulated a rapid increase in the levels of free intracellular calcium ([Ca2+]i) in cultured epithelial cells. Mutants defective in invasion were unable to induce these calcium fluxes, and addition of calcium antagonists blocked wild-type S. typhimurium entry. These results indicate that [Ca2+]i increase is required for bacterial entry. Further analysis demonstrated that phospholipase A2 and 5-lipoxygenase activities resulting in production of leukotrienes are required for bacterial entry. Addition of the leukotriene D4 to Henle-407 cells caused both an increase in [Ca2+]i and the internalization of an invasion-defective mutant of S. typhimurium. Furthermore, S. typhimurium caused the activation of mitogen-activated protein (also known as extracellular signal-regulated protein) kinase in infected cells.

Molecular Mechanism for a Role of SHP2 in Epidermal Growth Factor Receptor Signaling

ABSTRACT The Src homology 2-containing phosphotyrosine phosphatase (SHP2) is primarily a positive effector of receptor tyrosine kinase signaling. However, the molecular mechanism by which SHP2 effects its biological function is unknown. In this report, we provide evidence that defines the molecular mechanism and site of action of SHP2 in the epidermal growth factor-induced mitogenic pathway. We demonstrate that SHP2 acts upstream of Ras and functions by increasing the half-life of activated Ras (GTP-Ras) in the cell by interfering with the process of Ras inactivation catalyzed by Ras GTPase-activating protein (RasGAP). It does so by inhibition of tyrosine phosphorylation-dependent translocation of RasGAP to the plasma membrane, to its substrate (GTP-Ras) microdomain. Inhibition is achieved through the dephosphorylation of RasGAP binding sites at the level of the plasma membrane. We have identified Tyr992 of the epidermal growth factor receptor (EGFR) to be one such site, since its mutation to Phe renders the EGFR refractory to the effect of dominant-negative SHP2. To our knowledge, this is the first report to outline the site and molecular mechanism of action of SHP2 in EGFR signaling, which may also serve as a model to describe its role in other receptor tyrosine kinase signaling pathways.

Therapeutic implications of a human neutralizing antibody to the macrophage-stimulating protein receptor tyrosine kinase (RON), a c-MET family member.

RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in approximately 100 cancer cell lines and approximately 300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers.

Leukemic transformation of normal murine erythroid progenitors: v- and c-ErbB act through signaling pathways activated by the EpoR and c-Kit in stress erythropoiesis.

Primary erythroid progenitors can be expanded by the synergistic action of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids. While Epo is required for erythropoiesis in general, glucocorticoids and SCF mainly contribute to stress erythropoiesis in hypoxic mice. This ability of normal erythroid progenitors to undergo expansion under stress conditions is targeted by the avian erythroblastosis virus (AEV), harboring the oncogenes v-ErbB and v-ErbA. We investigated the signaling pathways required for progenitor expansion under stress conditions and in leukemic transformation. Immortal strains of erythroid progenitors, able to undergo normal, terminal differentiation under appropriate conditions, were established from fetal livers of p53−/− mice. Expression and activation of the EGF-receptor (HER-1/c-ErbB) or its mutated oncogenic version (v-ErbB) in these cells abrogated the requirement for Epo and SCF in expansion of these progenitors and blocked terminal differentiation. Upon inhibition of ErbB function, differentiation into erythrocytes occurred. Signal transducing molecules important for renewal induction, i.e. Stat5- and phosphoinositide 3-kinase (PI3K), are utilized by both EpoR/c-Kit and v/c-ErbB. However, while v-ErbB transformed cells and normal progenitors depended on PI3K signaling for renewal, c-ErbB also induces progenitor expansion by PI3K-independent mechanisms.

The Ski oncoprotein interacts with Skip, the human homolog of Drosophila Bx42

The v-Ski avian retroviral oncogene is postulated to act as a transcription factor. Since protein–protein interactions have been shown to play an important role in the transcription process, we attempted to identify Ski protein partners with the yeast two-hybrid system. Using v-Ski sequence as bait, the human gene skip (Ski Interacting Protein) was identified as encoding a protein which interacts with both the cellular and viral forms of Ski in the two-hybrid system. Skip is highly homologous to the Drosophila melanogaster protein Bx42 which is found associated with chromatin in transcriptionally active puffs of salivary glands. The Ski-Skip interaction is potentially important in Skis transforming activity since Skip was demonstrated to interact with a highly conserved region of Ski required for transforming activity. Like Ski, Skip is expressed in multiple tissue types and is localized to the cell nucleus.

The phosphotyrosine phosphatase SHP2 is a critical mediator of transformation induced by the oncogenic fibroblast growth factor receptor 3.

Receptor tyrosine kinases (RTKs) such as the fibroblast growth factor receptor (FGFR) and the epidermal growth factor receptor are overexpressed in a variety of cancers. In addition to overexpression, the FGFRs are found mutated in some cancers. The Src homology 2 domain-containing phosphotyrosine phosphatase (SHP2) is a critical mediator of RTK signaling, but its role in oncogenic RTK-induced cell transformation and cancer development is largely unknown. In the current report, we demonstrate that constitutively activated FGFR3 (K/E-FR3) transforms NIH-3T3 cells, and that SHP2 is a critical mediator of this transformation. Infection of K/E-FR3-transformed 3T3 cells with a retrovirus carrying a dominant-negative mutant of SHP2 (C/S-SHP2) retarded cell growth, reversed the transformation phenotype and inhibited focus-forming ability. Furthermore, treatment of K/E-FR3-transformed NIH-3T3 cells with PD98059 or LY294002, specific inhibitors of MEK and PI3K, respectively, inhibited focus formation. Biochemical analysis showed that K/E-FR3 activates the Ras-ERK and the PI3K signaling pathways, and that the C/S SHP2 mutant suppressed this effect via competitive displacement of interaction of the endogenous SHP2 with FRS2. However, the C/S SHP2 protein did not show any effect on receptor autophosphorylation, FRS2 tyrosine phosphorylation or interaction of Grb2 with K/E-FR3 or FRS2. Together, the results show that K/E-FR3 is transforming and that the Ras-ERK and the PI3K-Akt signaling pathways, which are positively regulated by SHP2, are important for K/E-FR3-induced transformation.

Self-renewal and differentiation of normal avian erythroid progenitor cells: Regulatory roles of the TGFα c -ErbB and SCF c -Kit receptors

The c-kit proto-oncogene product is a major regulator of early hematopoiesis in mice. We show here that the avian c-Kit protein, together with the c-erbB protooncogene product, regulates self-renewal and differentiation in two types of normal chick erythroid progenitors. A relatively frequent progenitor expressing only c-Kit transiently proliferated in response to avian c-Kit ligand (stem cell factor [SCF]). A second, rare progenitor coexpressed c-Kit and c-ErbB and was induced to long-term self-renewal by SCF or transforming growth factor alpha (TGF alpha), a c-ErbB ligand. In the absence of SFC or TGF alpha, both progenitors underwent erythropoietin (Epo)-dependent terminal differentiation with indistinguishable kinetics. Interestingly, Epo induced differentiation in the SCF progenitors even when SCF was present. In contrast, the c-ErbB-expressing, TGF alpha-induced progenitors continued to self-renew when treated with Epo plus the growth factors SCF, TGF alpha, or both. Expression of c-ErbB thus may be a dominant determinant for the sustained self-renewal of committed erythroid progenitors.

Temperature-sensitive mutants of avian erythroblastosis virus: Surface expression of the erbB product correlates with transformation

The v-erbB gene of avian erythroblastosis virus (AEV) codes for an integral plasma membrane glycoprotein, gp74erbB. Expression of gp74erbB and its intracellular precursors, gp66erbB and gp68erbB, has been studied in cells transformed by two temperature-sensitive mutants of AEV. After shift to 42 degrees C, the processing of gp68erbB is blocked in tsAEV-transformed, but not in wtAEV-transformed, erythroblasts and fibroblasts. In addition, gp74erbB disappears from the surface of tsAEV cells within 12 hr after shift. Thus tsAEV mutants probably bear a lesion in v-erbB that affects the maturation and subcellular localization of gp74erbB. The tsAEV erythroblasts, when committed to differentiation by a pulse-shift to 42 degrees C, reexpress gp74erbB during terminal differentiation at 36 degrees C. This suggests that tsAEV erythroblasts become insensitive to the transforming functions of gp74erbB at a certain stage of differentiation.

IDENTIFICATION OF THE CYTOPLASMIC REGIONS OF FIBROBLAST GROWTH FACTOR (FGF) RECEPTOR 1 WHICH PLAY IMPORTANT ROLES IN INDUCTION OF NEURITE OUTGROWTH IN PC12 CELLS BY FGF-1

ABSTRACT Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. Recently, we have shown that the FGF receptor 1 (FGFR-1) is much more potent than FGFR-3 in induction of neurite outgrowth. To identify the cytoplasmic regions of FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells, we took advantage of this difference and prepared receptor chimeras containing different regions of the FGFR-1 introduced into the FGFR-3 protein. The chimeric receptors were introduced into FGF-nonresponsive variant PC12 cells (fnr-PC12 cells), and their ability to mediate FGF-stimulated neurite outgrowth of the cells was assessed. The juxtamembrane (JM) and carboxy-terminal (COOH) regions of FGFR-1 were identified as conferring robust and moderate abilities, respectively, for induction of neurite outgrowth to FGFR-3. Analysis of FGF-stimulated activation of signal transduction revealed that the JM region of FGFR-1 conferred strong and sustained tyrosine phosphorylation of several cellular proteins and activation of MAP kinase. The SNT/FRS2 protein was demonstrated to be one of the cellular substrates preferentially phosphorylated by chimeras containing the JM domain of FGFR-1. SNT/FRS2 links FGF signaling to the MAP kinase pathway. Thus, the ability of FGFR-1 JM domain chimeras to induce strong sustained phosphorylation of this protein would explain the ability of these chimeras to activate MAP kinase and hence neurite outgrowth. The role of the COOH region of FGFR-1 in induction of neurite outgrowth involved the tyrosine residue at amino acid position 764, a site required for phospholipase C gamma binding and activation, whereas the JM region functioned primarily through a non-phosphotyrosine-dependent mechanism. In contrast, assessment of the chimeras in the pre-B lymphoid cell line BaF3 for FGF-1-induced mitogenesis revealed that the JM region did not play a role in this cell type. These data indicate that FGFR signaling can be regulated at the level of intracellular interactions and that signaling pathways for neurite outgrowth and mitogenesis use different regions of the FGFR.

Primary, self-renewing erythroid progenitors develop through activation of both tyrosine kinase and steroid hormone receptors

BACKGROUNDnSelf renewal in the hematopoietic system is thought to be restricted to a class of pluripotent stem cells. The capacity of cells with the properties of committed progenitors to self renew in many leukemias is thought to be an abnormal property resulting from the mutations responsible for leukemic transformation. It is not known how cells that can self-renew differ from cells that cannot. The notion that only pluripotent stem cells self renew has recently been challenged: normal committed erythroid progenitors capable of sustained self renewal have been described. These cells, called SCF/TGF alpha progenitors, co-express the c-Kit receptor tyrosine kinase and c-ErbB, the avian receptor for epidermal growth factor and transforming growth factor (TGF) alpha, and they undergo continuous self renewal in response to TGF alpha and estradiol. In contrast, common erythroid progenitors (termed SCF progenitors) express only c-Kit and undergo a limited number of cell divisions in response to the c-Kit ligand, stem cell factor (SCF). Both types of progenitor faithfully reproduce terminal erythroid differentiation in vitro when exposed to differentiation factors. Here, we have investigated the developmental origin of these two classes of self-renewing erythroid progenitors.nnnRESULTSnWe show that SCF progenitors can develop into SCF/TGF alpha progenitors. This developmental conversion requires 10-14 days and is accompanied by a gradual up-regulation of bioactive TGF alpha receptor. Using sera depleted of endogenous growth factors, we demonstrate that the development of SCF progenitors into SCF/TGF alpha progenitors absolutely requires the simultaneous presence of SCF, TGF alpha and estradiol, and is strongly enhanced by an unknown activity in chicken serum.nnnCONCLUSIONSnSCF progenitors can be induced to develop into self-renewing SCF/TGF alpha progenitors. The development of self renewal is triggered by specific combinations of growth factors and hormones. This has important implications for understanding leukemogenesis, as the self renewal of leukemic cells may reflect the normal potential of certain committed progenitor cells and not, as has been thought, a unique abnormal property of leukemic cells.