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, which contain a zone of F-actin at the leading edge. In order to establish the mechanism for accumulation of F-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 and barbed ends of actin filaments upon EGF stimulation of MTLn3 cells. The major result of this study is that stimulation of MTLn3 cells with EGF causes a transient increase in actin nucleation activity resulting from the appearance of free barbed ends very close to the leading edge of extending lamellipods. In addition, cytochalasin D causes a significant decrease in the total F-actin content in EGF-stimulated cells, indicating that both actin polymerization and depolymerization are stimulated by EGF. Pointed end incorporation of rhodamine-labeled actin by the EGF stimulated cells is more than two times higher than that of control cells. Since EGF stimulation causes an increase in both barbed and pointed end incorporation of rhodamine-labeled actin in the same location, the EGF-stimulated nucleation sites are more likely due either to severing of pre-existing filaments or de novo nucleation of filaments at the leading edge, thereby creating new barbed and pointed ends. The timing and location of EGF-induced actin nucleation activity in MTLn3 cells can account for the observed accumulation of F-actin at the leading edge and demonstrate that this F-actin rich zone is the primary 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 proliferating smooth muscle cells (SMCs) and epidermal growth factor (Egf). Cultured SMCs require TN-C for Egf-dependent growth on type I collagen. In this study, the regulation and function 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-C expression in a beta3 integrin- dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of 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-C facilitates Egf-dependent survival and growth was then investigated. TN-C interactions with alphavbeta3 integrins modify SMC shape, and Egf-dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of Egf-rs, tyrosine-phosphorylated proteins, and increased activation of Egf-rs after addition of Egf. Cross-linking SMC beta3 integrins replicates the effect of TN-C on Egf-r clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating beta3 integrin ligands in type I collagen. In turn, alphavbeta3 interactions with TN-C alter SMC shape and increase Egf-r clustering and Egf-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces 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 G1 by transducing extracellular growth stimuli into early G1 mitogenic signals. The role of Ras was investigated during the late stage of the G1 phase by using NIH 3T3 (M17) fibroblasts in which the expression of a dominant negative Ras mutant [Ha-Ras(Asn17)] was induced in response to dexamethasone treatment. Delaying the expression of Ras(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 this period, with resultant suppression of cyclin Ds/CDK4 and cyclin E/CDK2 and G1 arrest. The immunodepletion of p27kip1 completely eliminates the CDK inhibitor activity from EGF-stimulated, dexamethasone-treated cell lysate. The failure of p27kip1 downregulation and G1 arrest is also observed in cells in which Ras(Asn17) is induced after growth stimulation with either a phorbol ester or alpha-thrombin and is mimicked by the addition of inhibitors for phosphatidylinositol-3-kinase late in the G1 phase. Ras-mediated downregulation of p27kip1 involves both the suppression of synthesis and the stimulation 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 or inhibit the stimulated expression of a principal D-type cyclin, cyclin D1, until close to the border between G1 and S. It is concluded that Ras plays temporally distinct, phase-specific roles throughout the G1 phase and that Ras function late in G1 is required 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 signaling pathway in the induction of apoptosis remains unexplored. Gamma interferon (IFN-gamma) activates STAT1 and induces apoptosis in both A431 and HeLa cells, whereas epidermal growth factor (EGF) activates STAT proteins and induces apoptosis in A431 but not in HeLa cells. EGF receptor autophosphorylation and mitogen-activated protein kinase activation in response to EGF are similar in both cell lines. The breast cancer cell line MDA-MB-468 exhibits a similar response to A431 cells, i.e., STAT activation and apoptosis correlatively results from EGF or IFN-gamma treatment. In addition, in a mutant A431 cell line in which STAT activation is abolished, no apoptosis is induced by either EGF or IFN-gamma. Both EGF and IFN-gamma induce caspase 1 (interleukin-1beta converting enzyme [ICE]) gene expression in a STAT-dependent manner. IFN-gamma is unable to induce ICE gene expression and apoptosis in either JAK1-deficient HeLa cells (E2A4) or STAT1-deficient cells (U3A). However, ICE gene expression 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 by Z-Val-Ala-Asp-fluoromethylketone (ZVAD) in all the cells tested; studies from ICE-deficient cells indicate that ICE gene expression is necessary for IFN-gamma-induced apoptosis. It is concluded that activation of the STAT signaling pathway 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 both transcriptionally and as a result of phosphorylation by Jun N-terminal kinase (JNK). The cellular 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 required in 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 of Ras, RacI, and MEK kinase (MEKK) inhibit EGF induction. These results suggest 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 1 and retinoic acid on Pax-2 gene expression in proximal tubule cells (PTC). Rabbit cultured PTC grown to confluent 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-2 mRNA 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 affect Pax-2 gene expression in renal tubule cells. TGF-beta 1 downregulates Pax-2 gene expression through a posttranscriptional process, an acknowledged mechanism for 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 is regulated by epidermal growth factor (EGF). Nuclear extracts from GH4 cells treated with 10 nM EGF for at least 16 h show a 50% decrease in Sp1 binding to a GC-rich DNA sequence element present in the gastrin promoter. The decrease in binding correlates with a decrease in cell proliferation, a loss of nuclear Sp1 protein and a 50-60% decrease in Sp1-mediated transactivation through an Sp1 enhancer element in transfection assays. Okadaic acid, a phosphatase inhibitor, is synergistic with the effect of EGF 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 and okadaic 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 tested directly 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 of phosphorylation events that promotes Sp1 proteolysis, decreases Sp1 nuclear levels and decreases cellular proliferation (Mortensen, 1997).