Modulation of oncogene expression by epidermal growth factor and gamma-interferon in A431 squamous carcinoma cells

Epidermal growth factor (EGF) acts, in a dose dependent manner, as both a mitogen and an inhibitor of growth of the A431 squamous carcinoma cell line. gamma-interferon (IFN) also inhibits A431 cell growth. The dual effects of EGF on A431 growth and expression of the oncogenes, EGF receptor (EGFR) and Ha-ras, were evaluated with or without gamma-IFN. A mitogenic level (10pM) of EGF had no effect on expression of EGFR 10 kb mRNA or protein. gamma-IFN combined with 10pM EGF caused an initial drop in EGFR mRNA not reflected at the protein level; at 72 hours, the level of EGFR 10kb mRNA rose and inhibition of cell growth was observed. Treatment with a cytostatic amount (10nM) of EGF resulted in decreased expression of EGFR 10kb mRNA and protein within 24 hours; combined treatment with gamma-IFN caused rapid cell death. Expression of Ha-ras mRNA paralleled that of EGFR mRNA upon treatment with 10pM EGF and/or gamma-IFN, but differed with 10nM EGF.     

Epidermal growth factor enhances expression of connexin 43 protein in cultured porcine preantral follicles

Connexin 43 (Cx43) and gap junctional coupling appear to play a critical role in early follicular development because absence of Cx43 disrupts progression of follicles beyond primary stages in transgenic mouse ovaries. Two experimental culture systems were used to determine whether epidermal growth factor (EGF) stimulates expression of Cx43 in early porcine follicular development. Ovarian explants were collected from 32- to 40-day-old gilts and cultured for 6 days on membrane inserts in Waymouth MB 752/1 medium supplemented with 0, 50, or 500 ng/ml mouse EGF. Western blot analysis demonstrated significant increases (P < 0.05) in relative amounts of Cx43 protein (both phosphorylated and nonphosphorylated) with 50 and 500 ng/ml of EGF as compared with control cultures. Preantral follicles were enzymatically isolated from 70- to 86-day-old gilts and cultured for 8 days in collagen matrices. Medium and EGF treatments were the same as previously described. Western blot analysis demonstrated a significant increase (P < 0.05) in relative amounts of Cx43 protein with 50 and 500 ng/ml of EGF as compared with control cultures. EGF increased expression of Cx43 protein in secondary preantral follicles in a dose-dependent manner, which suggests that EGF or similar growth factor molecules may modulate early folliculogenesis by stimulating expression of Cx43 gap junctions.     

Regulation of VL30 gene expression by activators of protein kinase C

The mouse genome contains a retrovirus-like sequence, designated VL30, which is expressed at high levels in transformed cells and which can be induced by exogenously supplied epidermal growth factor (EGF). Binding of EGF to the EGF receptor produces changes in intracellular calcium levels and phospholipase activity which indirectly lead to activation of protein kinase C. We treated AKR-2B cells, Swiss 3T3 cells, and the 3T3 variants NR6 (EGF receptorless) and TNR9 (phorbol ester nonresponsive) with various phorbol ester tumor promoters and with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. Tumor-promoting phorbol esters (e.g. 12-O-tetradecanoyl phorbol acetate (TPA] increased the level of VL30 expression. Stimulation with either TPA or EGF produced a similar time course of VL30 expression. TPA induced VL30 expression in the EGF-receptorless NR6 cell line, indicating that neither EGF ligand-receptor binding nor phosphorylation of the EGF receptor was required for induction of VL30 expression. Protein synthesis was not required for the TPA-mediated increase in VL30 expression, as pretreatment with cycloheximide did not block or reduce the TPA effect. VL30 expression was also stimulated by treatment with sn-1,2-dioctanoylglycerol, an analog of a probable endogenous activator of protein kinase C. These results suggest that activation of protein kinase C plays a direct role in regulating VL30 expression.     

Differential control of cellular gene expression by diffusible and non-diffusible EGF

Cell gene expression is affected by both the kind and mode of growth factor stimulation (diffusive vs. non-diffusive). Epidermal growth factor (EGF) was pattern-immobilized on a polystyrene plate. Although the growth of the rat phaeochromocytoma cell line PC12 is stimulated by diffusible EGF, and differentiation is stimulated by diffusible nerve growth factor (NGF), immobilized (non-diffusible) EGF stimulated PC12 differentiation. The immobilized EGF caused a long-lasting stimulation of the intracellular signal protein mitogen-associated protein MAP kinase (MAPK, also known as ERK) and p38 (a subfamily of the MAPK superfamily) in cells, as did diffusible NGF. The switching between growth stimulation and differentiation is considered to be due to the duration of the stimulus. The function of the biosignal conjugate was regulated using conjugation methodology.     

Suppression of G-protein-coupled receptor kinase 3 expression is a feature of classical GBM that is required for maximal growth

G-protein-coupled receptor kinases (GRK) regulate the function of G-protein-coupled receptors (GPCR). Previously, we found that GPCR (CXCR4)-mediated astrocytoma growth was dependent upon abnormally sustained CXCR4 signaling and was correlated with decreased GRK-mediated receptor phosphorylation. As CXCR4 has also been implicated in the stimulation of high-grade glioma growth, we sought to determine whether dysregulation of GRK expression and/or function might also be present in high-grade gliomas. In an analysis of data from The Cancer Genome Atlas, we found that GRK3 expression is frequently decreased in glioblastoma (GBM) of the classical subtype, which possesses signature amplification or mutational activation of the epidermal growth factor (EGF) receptor. We tested the correlation between GRK3 expression and GBM subtypes, as well as the relationship between the activation of the EGF and other growth factor receptor pathways and GRK expression. In analyses of primary GBM tissue and RNA specimens, we found that GRK3 expression is correlated with established criteria for GBM subtyping including expression of EGF receptor, platelet-derived growth factor receptor (PDGFR)α, NF1, PTEN, CDKN2A, and neurofilament. We also found that established drivers of gliomagenesis, the EGF, PDGF, and TGF-β pathways, all regulate GRK expression. Coculture experiments, designed to mimic critical interactions between tumor and brain microvascular endothelial cells, showed that specifically increasing GRK3 expression reduced the trophic effect of endothelial cells on tumor cells. Together, these experiments show that GRK3 is a negative regulator of cell growth whose expression is preferentially reduced in GBM of the classical subtype as a consequence of activity in primary gliomagenic pathways.     

Egr-1 and serum response factor are involved in growth factors- and serum-mediated induction of E2-EPF UCP expression that regulates the VHL-HIF pathway

E2-EPF ubiquitin carrier protein (UCP) has been shown to be highly expressed in common human cancers and target von Hippel-Lindau (VHL) for proteosomal degradation in cells, thereby stabilizing hypoxia-inducible factor (HIF)-1alpha. Here, we investigated cellular factors that regulate the expression of UCP gene. Promoter deletion assay identified binding sites for early growth response-1 (Egr-1) and serum response factor (SRF) in the UCP promoter. Hepatocyte or epidermal growth factor (EGF), or phorbol 12-myristate 13-acetate induced UCP expression following early induction of Egr-1 expression in HeLa cells. Serum increased mRNA and protein levels of SRF and UCP in the cell. By electrophoretic mobility shift and chromatin immunoprecipitation assays, sequence-specific DNA-binding of Egr-1 and SRF to the UCP promoter was detected in nuclear extracts from HeLa cells treated with EGF and serum, respectively. Overexpression of Egr-1 or SRF increased UCP expression. RNA interference-mediated depletion of endogenous Egr-1 or SRF impaired EGF- or serum-mediated induction of UCP expression, which was required for cancer cell proliferation. Systemic delivery of EGF into mice also increased UCP expression following early induction of Egr-1 expression in mouse liver. The induced UCP expression by the growth factors or serum increased HIF-1alpha protein level under non-hypoxic conditions, suggesting that the Egr-1/SRF-UCP-VHL pathway is in part responsible for the increased HIF-1alpha protein level in vitro and in vivo. Thus, growth factors and serum induce expression of Egr-1 and SRF, respectively, which in turn induces UCP expression that positively regulates cancer cell growth.     

The epidermal growth factor-induced cell migration and expression of the 47,000 Mr secreted glycoprotein EIP-1 of rat liver epithelial cells are down-modulated by cyclic AMP

The migration of rat liver epithelial cells induced by epidermal growth factor (EGF) was inhibited by cyclic AMP (cAMP) and cholera toxin, but not by cGMP, cAMP and cholera toxin also inhibited the expression of the EGF/transforming growth factor (TGF) alpha-inducible protein EIP-1 (Mr 47,000), but not that of other proteins induced by the growth factor. cAMP therefore specifically and selectively represses the EGF-induced expression of this protein, which by synthesis in the presence of tunicamycin and by enzymatic treatments was shown to be N-glycosylated and sialylated. The close correlation of the expression of EIP-1 with the growth factor-induced migration suggests that this glycoprotein is involved in the cellular translocation process. Modulation of cell migration and of EIP-1 expression through increased intracellular concentrations of cAMP indicate that factors operating through this signal system can modulate the phenotypic and gene expression changes mediated by the EGF-receptor. Identification of the ligand(s) that can cause the cAMP-mediated effects might be an important step towards understanding the regulation of liver cell migration in vivo.     

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Heterodimerizing peptides, such as the de novo designed E5/K5 peptide pair, have several applications including as tags for protein purification or immobilization. Recently, we demonstrated that E5-tagged epidermal growth factor (EGF), when bound to a K4 expressing adenovirus, promotes retargeting of the adenovirus to EGFR expressing target cells. In this study, we present the Escherichia coli expression, refolding and purification of human EGF fused with the E5-coil (E5-coil-EGF) or with the K5-coil (K5-coil-EGF). EGF receptor phosphorylation and cell proliferation assays demonstrated that the biological activity of the coil-tagged EGF versions was comparable to that of non-tagged EGF. Additionally, analysis of the binding of E5/K5-coil-EGF to cell surface EGFR or to soluble EGFR ectodomain, as measured by cell-based binding competition assays and by SPR-based biosensor experiments, indicated that the coil-tagged EGF versions bound to EGFR with affinities similar to that of non-tagged EGF. Finally, we show that E-coil-tagged EGF, but not non-tagged EGF, can retarget a K-coil containing adenovirus to EGF receptor expressing glioblastoma tumor cells. Overall these results indicate that E. coli expression offers a practical platform for the reproducible production of fully biologically active E5/K5-coil-tagged EGF, and support applications of heterodimerizing coil-tagged ligands, e.g. the targeting of viruses or other entities such as nanoparticles to tumor cells, or growth factor immobilization on cell culture scaffolds for tissue engineering.     

Effects of progesterone on growth factor expression in human uterine leiomyoma

It is now evident that the use of levonorgestrel-releasing intrauterine system (LNg-IUS) is effective for long-term management of menorrhagic women with uterine myomas because of a striking reduction in menorrhagia. This prompted us to characterize the effects of progesterone (P4) on the growth and apoptosis of uterine leiomyoma cells. On the other hand, we have recently noted that epidermal growth factor (EGF) and IGF-I play a crucial role in prompting uterine leiomyoma growth through stimulating the proliferative potential and inhibiting apoptosis of cultured human leiomyoma cells. In the present review, attention was paid to evaluate the effects of P4 on the expression of growth factors (EGF, IGF-I) and apoptosis-related factors (TNFalpha, Bcl-2 protein) in cultured uterine leiomyoma cells. Treatment with P4 augmented EGF and Bcl-2 protein expression, but inhibited IGF-I and TNFalpha expression in cultured leiomyoma cells. It is known that TNFalpha induces apoptosis in a variety of cell types and Bcl-2 protein is an apoptosis-inhibiting gene product. Thus, the results obtained suggest that P4 has dual actions on uterine leiomyoma growth: one is to stimulate leiomyoma cell growth and survival through up-regulating EGF and Bcl-2 protein expression as well as down-regulating TNFalpha expression in those cells, and the other is to inhibit leiomyoma cell growth through down-regulating IGF-I expression in those cells. This may explain why the size of uterine myomas during use of LNg-IUS increases in some but decreases in other instances. This may also explain why the size of uterine myomas during pregnancy does not increase despite the overwhelming increase in circulating concentrations of sex steroid hormones.     

A mutant epidermal growth factor receptor with defective protein tyrosine kinase is unable to stimulate proto-oncogene expression and DNA synthesis.

Cultured NIH-3T3 cells devoid of endogenous epidermal growth factor (EGF) receptors were transfected with cDNA expression constructs encoding either normal human EGF receptor or a receptor mutated in vitro at Lys-721, a residue that is thought to function as part of the ATP-binding site of the kinase domain. Unlike the wild-type EGF-receptor expressed in these cells, which exhibited EGF-dependent protein tyrosine kinase activity, the mutant receptor lacked protein tyrosine kinase activity and was unable to undergo autophosphorylation and to phosphorylate exogenous substrates. Despite this deficiency, the mutant receptor was normally expressed on the cell surface, and it exhibited both high- and low-affinity binding sites. The addition of EGF to cells expressing wild-type receptors caused the stimulation of various responses, including enhanced expression of proto-oncogenes c-fos and c-myc, morphological changes, and stimulation of DNA synthesis. However, in cells expressing mutant receptors, EGF was unable to stimulate these responses, suggesting that the tyrosine kinase activity is essential for EGF receptor signal transduction.     

Activation of the Ras-ERK pathway inhibits retinoic acid-induced stimulation of tissue transglutaminase expression in NIH3T3 cells

Retinoic acid (RA) is a potent activator of tissue transglutaminase (TGase) expression, and it was recently shown that phosphoinositide 3-kinase (PI3K) activity was required for RA to increase TGase protein levels. To better understand how RA-mediated TGase expression is regulated, we considered whether co-stimulation of NIH3T3 cells with RA and epidermal growth factor (EGF), a known activator of PI3K, would facilitate the induction or increase the levels of TGase expression. Instead of enhancing these parameters, EGF inhibited RA-induced TGase expression. Activation of the Ras-ERK pathway by EGF was sufficient to elicit this effect, since continuous Ras signaling mimicked the actions of EGF and inhibited RA-induced TGase expression, whereas blocking ERK activity in these same cells restored the ability of RA to up-regulate TGase expression. However, TGase activity is not antagonistic to EGF signaling. The mitogenic and anti-apoptotic effects of EGF were not compromised by TGase overexpression, and in fact, exogenous TGase expression promoted basal cell growth and resistance to serum deprivation-induced apoptosis. Moreover, analysis of TGase expression and GTP binding activity in a number of cell lines revealed high basal TGase GTP binding activity in tumor cell lines U87 and MDAMB231, indicating that constitutively active TGase may be a characteristic of certain cancer cells. These findings demonstrate that TGase may serve as a survival factor and RA-induced TGase expression requires the activation of PI3K but is antagonized by the Ras-ERK pathway.