argos

EVOLUTIONARY HOMOLOGS

Development of the C. elegans egg-laying systems requires the formation of a connection between the uterine lumen and the developing vulva lumen, thus allowing a passage of eggs and sperm. Development of the connection requires that cells in both tissues become specialized to participate in the connection, and that the specialized cells are brought in register. A single cell, the anchor cell, acts to induce and organize specialization of the epidermal and uterine epithelia, and registrates these tissues. The anchor cell induces the vulva from ventral epithelial cells via the LIN-3 growth factor, an Epidermal growth factor homolog. LIN-3 acts through LET-23, the C. elegans homolog of EGF-R. It then induces surrounding uterine intermediate precursors via the receptor LIN-12, a member of the Notch family of receptors. LET-23 acts via the Ras pathway, which targets LIN-1, an ETS-domain class transcription factor homologous to Drosophila Yan and Pointed, and LIN-31, an HNF3/forkhead transcription factor, both of which act to prevent vulval differentiation (Newman, 1996 and references).

Stimulation of metastatic MTLn3 cells with EGF causes the rapid extension of lamellipods, whichcontain a zone of F-actin at the leading edge. In order to establish the mechanism for accumulation ofF-actin at the leading edge and its relationship to lamellipod extension in response to EGF, the kinetics and location of EGF-induced actin nucleation activity were studied in MTLn3 cells; the actin dynamics were characterized at the leading edge by measuring the changes at the pointed andbarbed ends of actin filaments upon EGF stimulation of MTLn3 cells. The major result of this study isthat stimulation of MTLn3 cells with EGF causes a transient increase in actin nucleation activityresulting from the appearance of free barbed ends very close to the leading edge of extendinglamellipods. In addition, cytochalasin D causes a significant decrease in the total F-actin content inEGF-stimulated cells, indicating that both actin polymerization and depolymerization are stimulated byEGF. Pointed end incorporation of rhodamine-labeled actin by the EGF stimulated cells ismore than two times higher than that of control cells. Since EGF stimulation causes an increase inboth barbed and pointed end incorporation of rhodamine-labeled actin in the same location, theEGF-stimulated nucleation sites are more likely due either to severing of pre-existing filaments or denovo nucleation of filaments at the leading edge, thereby creating new barbed and pointed ends. Thetiming and location of EGF-induced actin nucleation activity in MTLn3 cells can account for theobserved accumulation of F-actin at the leading edge and demonstrate that this F-actin rich zone is theprimary actin polymerization zone after stimulation (Chan, 1998).

EGF induces the quantitative formation of sEGFR dimers that contain two EGF molecules. The data suggest a model in which one EGF monomer binds to one sEGFR monomer, and that receptor dimerization involves subsequent association of two monomeric (1:1) EGF-sEGFR complexes. Dimerization may result from bivalent binding of both EGF molecules in the dimer and/or receptor-receptor interactions. The requirement for two (possibly bivalent) EGF monomers distinguishes EGF-induced sEGFR dimerization from the growth hormone and interferon-[gamma] receptors, where multivalent binding of a single ligand species (either monomeric or dimeric) drives receptor oligomerization. The proposed model of EGF-induced sEGFR dimerization suggests possible mechanisms for both ligand-induced homo- and heterodimerization of the EGFR (or erbB) family of receptors (Lemmon, 1997).

Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferatingsmooth muscle cells (SMCs) and epidermal growth factor (Egf). Cultured SMCs requireTN-C for Egf-dependent growth on type I collagen. In this study, the regulation andfunction of TN-C was explored in SMCs. A matix metalloproteinase (MMP) inhibitor (GM6001)suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-Cexpression in a beta3 integrin- dependent manner, independent of MMPs. Floating type I collagen gelalso suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presenceof Egf. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001,restores the Egf growth response and "rescues" cells from apoptosis. The mechanism by which TN-Cfacilitates Egf-dependent survival and growth was then investigated. TN-C interactionswith alphavbeta3 integrins modify SMC shape, and Egf-dependent growth. These features areassociated with redistribution of filamentous actin to focal adhesion complexes, which colocalize withclusters of Egf-rs, tyrosine-phosphorylated proteins, and increased activation of Egf-rs afteraddition of Egf. Cross-linking SMC beta3 integrins replicates the effect of TN-C on Egf-r clusteringand tyrosine phosphorylation. Together, these studies represent a functional paradigm forECM-dependent cell survival whereby MMPs upregulate TN-C by generating beta3 integrin ligands intype I collagen. In turn, alphavbeta3 interactions with TN-C alter SMC shape and increase Egf-rclustering and Egf-dependent growth. Conversely, suppression of MMPs downregulates TN-C andinduces apoptosis (Jones, 1997).

It is well documented that Ras functions as a molecular switch for reentry into the cell cycle at the border between G0 and G1by transducing extracellular growth stimuli into early G1 mitogenic signals. The role of Ras was investigatedduring the late stage of the G1 phase by using NIH 3T3 (M17) fibroblasts in which the expression of a dominant negative Rasmutant [Ha-Ras(Asn17)] was induced in response to dexamethasone treatment. Delaying the expression ofRas(Asn17) until late in the G1 phase by introducing dexamethasone 3 h after the addition of epidermal growth factor (EGF)abolishes the downregulation of the p27kip1 cyclin-dependent kinase (CDK) inhibitor that normally occurs during thisperiod, with resultant suppression of cyclin Ds/CDK4 and cyclin E/CDK2 and G1 arrest. The immunodepletion of p27kip1completely eliminates the CDK inhibitor activity from EGF-stimulated, dexamethasone-treated cell lysate. The failure ofp27kip1 downregulation and G1 arrest is also observed in cells in which Ras(Asn17) is induced after growth stimulationwith either a phorbol ester or alpha-thrombin and is mimicked by the addition of inhibitors forphosphatidylinositol-3-kinase late in the G1 phase. Ras-mediated downregulation of p27kip1 involves both the suppression of synthesis and thestimulation of the degradation of the protein. Unlike the earlier expression of Ras(Asn17) at the border between G0 and G1,its delayed expression does not compromise the EGF-stimulated transient activation of extracellular signal-regulated kinases orinhibit the stimulated expression of a principal D-type cyclin, cyclin D1, until close to the border between G1 and S. It isconcluded that Ras plays temporally distinct, phase-specific roles throughout the G1 phase and that Ras function late in G1 isrequired for p27kip1 downregulation and passage through the restriction point, a prerequisite for entry into the S phase (Takuwa, 1997).

Protein tyrosine kinases activate the STAT (signal transducer and activator of transcription) signaling pathway, which can play essential roles in cell differentiation, cell cycle control, and development. However, the potential role of the STAT signalingpathway in the induction of apoptosis remains unexplored. Gamma interferon (IFN-gamma) activatesSTAT1 and induces apoptosis in both A431 and HeLa cells, whereas epidermal growth factor (EGF) activates STATproteins and induces apoptosis in A431 but not in HeLa cells. EGF receptor autophosphorylation and mitogen-activatedprotein kinase activation in response to EGF are similar in both cell lines. The breast cancer cell line MDA-MB-468exhibits a similar response to A431 cells, i.e., STAT activation and apoptosis correlatively results from EGF or IFN-gammatreatment. In addition, in a mutant A431 cell line in which STAT activation is abolished, no apoptosis is induced by eitherEGF or IFN-gamma. Both EGF and IFN-gamma induce caspase 1 (interleukin-1betaconverting enzyme [ICE]) gene expression in a STAT-dependent manner. IFN-gamma is unable to induce ICE geneexpression and apoptosis in either JAK1-deficient HeLa cells (E2A4) or STAT1-deficient cells (U3A). However, ICE geneexpression and apoptosis are induced by IFN-gamma in U3A cells into which STAT1 had been reintroduced. Moreover,both EGF-induced apoptosis and IFN-gamma-induced apoptosis are effectively blocked byZ-Val-Ala-Asp-fluoromethylketone (ZVAD) in all the cells tested; studies from ICE-deficient cells indicate that ICEgene expression is necessary for IFN-gamma-induced apoptosis. It is concluded that activation of the STAT signalingpathway can induce apoptosis through the induction of ICE gene expression (Chin, 1997).

The c-jun proto-oncogene encodes a transcription factor that is activated by mitogens bothtranscriptionally and as a result of phosphorylation by Jun N-terminal kinase (JNK). Thecellular signaling pathways involved in epidermal growth factor (EGF) induction of the c-jun promoter have been investigated.Two sequence elements that bind ATF1 (a leucine zipper DNA binding protein) and MEF2D transcription factors are requiredin HeLa cells, although these elements are not sufficient for maximal induction. Activated forms of Ras, RacI,Cdc42Hs, and MEKK increase expression of the c-jun promoter, while dominant negative forms ofRas, RacI, and MEK kinase (MEKK) inhibit EGF induction. These resultssuggest that EGF activates the c-jun promoter by a Ras-to-Rac-to-MEKK pathway. No change is found in protein binding to the jun ATF1 site in EGF-treated cells. A potential mechanism for regulation of ATF1 and CREB is phosphorylation (Clarke, 1997).

The pattern-forming event of kidney tubulogenesis is initiated by the inductive transition of mesenchymal cells to epithelial phenotype, a transition that is critically dependent on the regulated expression of the developmental control gene, Pax-2. Because of a defined role in in vitro renal tubulogenesis, an evaluation was made of the effects of epidermal growth factor (EGF), transforming growth factor (TGF)-beta 1and retinoic acid on Pax-2 gene expression in proximal tubule cells (PTC). Rabbit cultured PTC grown toconfluent quiescent conditions were analyzed for the effect of various factors on Pax-2 gene expression. PTC express high levels of Pax-2. A 24-hour exposure to EGF, a potent mitogen of PTC, increases this level of expression. In contrast, Pax-2 gene expression is suppressed by treating PTC with retinoic acid, a well-described differentiating factor, and with TGF-beta 1, a recognized antiproliferative agent for these cells, which suggests that Pax-2 has a role in renal cell proliferation. The mechanism of the effect of TGF-beta 1 on Pax-2 mRNA levels was further detailed. TGF-beta 1 does not affect Pax-2 transcription rates; however, in a dose-dependent manner, it diminishes the stability of Pax-2 mRNA. TGF-beta 1 reduces Pax-2mRNA stability from a control half-life of 120 min to a half-life of less than 60 min.This study demonstrates that various soluble inductive factors affectPax-2 gene expression in renal tubule cells. TGF-beta 1 downregulates Pax-2gene expression through a posttranscriptional process, an acknowledged mechanismfor modulating important growth regulatory gene products (Liu, 1997).

Sp1 nuclear levels have been shown to directly correlate with the proliferative state of the cell. Changes in the abundance of Sp1 were studied in a rat pituitary cell line (GH4) whose growth rate isregulated by epidermal growth factor (EGF). Nuclear extracts from GH4 cells treated with 10 nMEGF for at least 16 h show a 50% decrease in Sp1 binding to a GC-rich DNA sequence element present in thegastrin promoter. The decrease in binding correlates with a decrease in cell proliferation, a loss ofnuclear Sp1 protein and a 50-60% decrease in Sp1-mediated transactivation through an Sp1 enhancerelement in transfection assays. Okadaic acid, a phosphatase inhibitor, is synergistic with the effect ofEGF on Sp1 protein levels, suggesting that the loss of Sp1 is mediated by phosphorylation events.A 2-fold increase in orthophosphate-labeled Sp1 occurs with EGF treatment andokadaic acid. Cycloheximide prevents the expected loss of Sp1 mediated by EGF and okadaic acid. This suggests that the synthesis of a protease may mediate these events. This hypothesis was testeddirectly by showing that the cysteine protease inhibitor leupeptin prevents Sp1 degradation. Sp1 has a domain with a high concentration of proline, glutamic acid, serine,and threonine residues, as reported for a number of proteins with inducible rates of degradation.Collectively, these results indicate that sustained stimulation of GH4 cells by EGF initiates a cascade ofphosphorylation events that promotes Sp1 proteolysis, decreases Sp1 nuclear levels and decreasescellular proliferation (Mortensen, 1997).

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