Epidermal growth factor and phorbol myristate acetate increase expression of the mRNA for cytosolic phospholipase A2 in glomerular mesangial cells

We have previously shown that phospholipase A2 (PLA2) activity is rapidly activated by epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) in renal mesangial cells and other cell systems in a manner that suggests a covalent modification of the PLA2 enzyme(s). This PLA2 activity is cytosolic (cPLA2) and is distinct from secretory forms of PLA2, which are also stimulated in mesangial cells in response to cytokines and other agonists. However, longer-term regulation of cPLA2 in renal cells may also occur at the level of gene expression. Cultured rat mesangial cells were used as a model system to test the effects of EGF and PMA on the regulation of cPLA2 gene expression. EGF and PMA both produced sustained increases in cPLA2 mRNA levels, with a parallel increase in enzyme activity over time. Inhibition of protein synthesis by cycloheximide increased basal cPLA2 mRNA accumulation in serum-starved mesangial cells, and the combination of EGF and cycloheximide resulted in super-induction of cPLA2 gene expression compared with EGF alone. Actinomycin D treatment entirely abrogated the effect of EGF on cPLA2 mRNA accumulation. These findings suggest that regulation of cPLA2 is achieved by factors controlling gene transcription and possibly mRNA stability, in addition to previously characterized posttranslational modifications.

Epidermal growth factor-stimulated extravillous cytotrophoblast motility is mediated by the activation of PI3-K, Akt and both p38 and p42/44 mitogen-activated protein kinases

BACKGROUND: Trophoblast invasion is a temporally and spatially regulated scheme of events that can dictate pregnancy outcome. Evidence suggests that the potent mitogen epidermal growth factor (EGF) regulates cytotrophoblast (CTB) differentiation and invasion during early pregnancy. METHODS AND RESULTS: In the present study, the first trimester extravillous CTB cell line SGHPL-4 was used to investigate the signalling pathways involved in the motile component of EGF-mediated CTB migration/invasion. EGF induced the phosphorylation of the phosphatidylinositol 3-kinase (PI3-K)-dependent proteins, Akt and GSK-3β as well as both p42/44 MAPK and p38 mitogen-activated protein kinases (MAPK). EGF-stimulated motility was significantly reduced following the inhibition of PI3-K (P < 0.001), Akt (P < 0.01) and both p42/44 MAPK (P < 0.001) and p38 MAPKs (P < 0.001) but not the inhibition of GSK-3β. Further analysis indicated that the p38 MAPK inhibitor SB 203580 inhibited EGF-stimulated phosphorylation of Akt on serine 473, which may be responsible for the effect SB 203580 has on CTB motility. Although Akt activation leads to GSK-3β phosphorylation and the subsequent expression of β-catenin, activation of this pathway by 1-azakenpaullone was insufficient to stimulate the motile phenotype. CONCLUSION: We demonstrate a role for PI3-K, p42/44 MAPK and p38 MAPK in the stimulation of CTB cell motility by EGF, however activation of β-catenin alone was insufficient to stimulate cell motility.

Expression of the disintegrin metalloprotease ADAM-10 in prostate cancer and its regulation by dihydrotestosterone, insulin like growth factor -1 and epidermal growth factor in the prostate cell model LNCaP

Purpose: The disintegrin metalloprotease ADAM-10 is a multidomain metalloprotease that is potentially significant in tumor progression due to its extracellular matrix-degrading properties. Previously, ADAM-10 mRNA was detected in prostate cancer (PCa) cell lines; however, the presence of ADAM-10 protein and its cellular localization, regulation, and role have yet to be described. We hypothesized that ADAM-10 mRNA and protein may be regulated by growth factors such as 5α-dihydrotestosterone, insulin-like growth factor I, and epidermal growth factor, known modulators of PCa cell growth and invasion.

Experimental Design: ADAM-10 expression was analyzed by in situ hybridization and immunohistochemistry in prostate tissues obtained from 23 patients with prostate disease. ADAM-10 regulation was assessed using quantitative reverse transcription-PCR and Western blot analysis in the PCa cell line LNCaP.

Results: ADAM-10 expression was localized to the secretory cells of prostate glands, with additional basal cell expression in benign glands. ADAM-10 protein was predominantly membrane bound in benign glands but showed marked nuclear localization in cancer glands. By Western blot, the 100-kDa proform and the 60-kDa active form of ADAM-10 were synergistically up-regulated in LNCaP cells treated with insulin-like growth factor I plus 5α-dihydrotestosterone. Epidermal growth factor also up-regulated both ADAM-10 mRNA and protein.

Conclusions: This study describes for the first time the expression, regulation, and cellular localization of ADAM-10 protein in PCa. The regulation and membrane localization of ADAM-10 support our hypothesis that ADAM-10 has a role in extracellular matrix maintenance and cell invasion, although the potential role of nuclear ADAM-10 is not yet known.

The control of epidermal growth factor grafted on mesoporous silica nanoparticles for targeted delivery

The performance of biomaterials in a biological environment is largely influenced by the surface properties of the biomaterials. In particular, grafted targeting ligands significantly impact the subsequent cellular interactions. The utilisation of a grafted epidermal growth factor (EGF) is effective for targeted delivery of drugs to tumours, but the amount of these biological attachments cannot be easily quantified as most characterization methods could not detect the extremely low amount of EGF ligands grafted on the surface of nanoparticles. In this study, hollow mesoporous silica nanoparticles (HMSNs) were functionalized with amine groups to conjugate with EGFs via carbodiimide chemistry. Time of flight secondary ion mass spectrometry (ToF-SIMS), a very surface specific technique (penetration depth <1.5 nm), was employed to study the binding efficiency of the EGF to the nanoparticles. Principal component analysis (PCA) was implemented to track the relative surface concentrations of EGFs on HMSNs. It was found that ToF-SIMS combined with the PCA technique is an effective method to evaluate the immobilization efficiency of EGFs. Based on this useful technique, the quantity and density of the EGF attachments that grafted on nanoparticles can be effectively controlled by varying the EGF concentration at grafting stages. Cell experiments demonstrated that the targeting performance of EGFR positive cells was affected by the number of EGFs attached on HMSNs.

Expression of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B, in the bovine corpus luteum

There is evidence that several fibroblast growth factors (FGFs) are involved in growth and development of the corpus luteum (CL), but many FGFs have not been investigated in this tissue, including FGF10. The objective of this study was to determine if FGF10 and its receptor (FGFR2B) are expressed in the CL. Bovine CL were collected from an abattoir and classed as corpus hemorrhagica (stage 1), developing (stage 11), developed (stage 111), and regressed (stage IV) CL. Expression of FGF10 and FGFR2B mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR). Both genes were expressed in bovine CL, and FGF10 expression did not differ between stages of CL development. FGF10 protein was localized to large and small luteal cells by immunohistochemistry. FGFR2B expression was approximately threefold higher in regressed compared to developing and developed CL (P < 0.05). To determine if FGF10 and FGFR2B expression is regulated during functional luteolysis, cattle were injected with PGF2 alpha and CL collected at 0, 0.5, 2, 4, 12, 24, 48, and 64 hr thereafter (n = 5 CL/time point), and mRNA abundance was measured by real-time RT-PCR. FGF10 mRNA expression did not change during functional luteolysis, whereas FGFR2B mRNA abundance decreased significantly at 2, 4, and 12 hr after PGF2a, and returned to pretreatment levels for the period 24-64 hr post-PGF2 alpha. These data suggest a potential role for FGFR2B signaling during structural luteolysis in bovine CL.

Expression and function of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 213, in bovine follicles

Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.

Effects of epidermal growth factor on the invasion activity of the oral cancer cell lines HSC3 and SAS

Epidermal growth factor (EGF) is excreted in a high concentration in human saliva and modulates the growth and differentiation of various cancer cells. To elucidate the molecular mechanisms by which EGF affects oral cancer growth and invasion, we analyzed the Matrigel invasion activity of the cultured oral cancer cell line. Cells grown under the influence of EGF were subjected to Matrigel invasion assays and cells grown in the absence of EGF were used as controls. Gelatin-zymography and Northern blot analyses quantified the invasiveness and tumorigenicity. Chloramphenicol acetyltransferase assay (CAT assay) determined the EGF stimulation of matrix metalloproteinase (MMP) expression. EGF increased the number of cells penetrating a Matrigel membrane. Gelatin-zymography and Northern blot analysis revealed that MMP9 and Ets1 expressions correlated with EGF but MMP2 was not changed. a transient transfection assay revealed that EGF increased the promoter activities of the MMP9 genes in HSC3 and SAS cells. These results suggest that EGF increases the invasion activity of oral cancer cells partly by increasing MMP9.

Rsk-2 activity is necessary for epidermal growth factor-induced phosphorylation of CREB protein and transcription of c-fos gene

Activation by growth factors of the Ras-dependent signaling cascade results in the induction of p90 ribosomal S6 kinases (p90rsk). These are translocated into the nucleus upon phosphorylation by mitogen-activated protein kinases, with which p90rsk are physically associated in the cytoplasm. In humans there are three isoforms of the p90rsk family, Rsk-1, Rsk-2, and Rsk-3, which are products of distinct genes. Although these isoforms are structurally very similar, little is known about their functional specificity. Recently, mutations in the Rsk-2 gene have been associated with the Coffin–Lowry syndrome (CLS). We have studied a fibroblast cell line established from a CLS patient that bears a nonfunctional Rsk-2. Here we document that in CLS fibroblasts there is a drastic attenuation in the induced Ser-133 phosphorylation of transcription factor CREB (cAMP response element-binding protein) in response to epidermal growth factor stimulation. The effect is specific, since response to serum, cAMP, and UV light is unaltered. Furthermore, epidermal growth factor-induced expression of c-fos is severely impaired in CLS fibroblasts despite normal phosphorylation of serum response factor and Elk-1. Finally, coexpression of Rsk-2 in transfected cells results in the activation of the c-fos promoter via the cAMP-responsive element. Thus, we establish a link in the transduction of a specific growth factor signal to changes in gene expression via the phosphorylation of CREB by Rsk-2.

Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase

The functional role of the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in epidermal growth factor (EGF)-induced expression of 12(S)-lipoxygenase gene in human epidermoid carcinoma A431 cells was studied. Coimmunoprecipitation experiments indicated that EGF stimulated interaction between c-Jun and Sp1 in a time-dependent manner. Overexpression of Ha-ras and c-Jun also enhanced the amount of c-Jun binding to Sp1. In addition, the c-Jun dominant negative mutant TAM-67 not only inhibited the coimmunoprecipitated c-Jun binding to Sp1 in a dose-dependent manner in cells overexpressing c-Jun but also reduced promoter activity of the 12(S)-lipoxygenase gene induced by c-Jun overexpression. Treatment of cells with EGF increased the interaction between the Sp1 oligonucleotide and nuclear c-Jun/Sp1 in a time-dependent manner. Furthermore, EGF activated the chimeric promoter consisting of 10 tandem GAL4-binding sites, which replaced the three Sp1-binding sites in the 12(S)lipoxygenase promoter only when coexpressed with GAL4-c-Jun () fusion proteins. These results indicate that the direct interaction between c-Jun and Sp1 induced by EGF cooperatively activated expression of the 12(S)-lipoxygenase gene, and that Sp1 may serve at least in part as a carrier bringing c-Jun to the promoter, thus transactivating the transcriptional activity of 12(S)-lipoxygenase gene.

The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide bonding pattern.

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thrombomodulin have been determined. In previous work, this fragment was expressed and purified to homogeneity, and its cofactor activity, as measured by Kcat for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation yielded the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2-carboxyethyl)phosphine according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is the same as that found previously in EGF and in a synthetic version of the fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found in EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bound to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.