The influence of prostaglandins on steroid conversions by human gingival fibroblasts

The aim of this investigation was to study the effects of prostaglandins E1 and E2 (PGE1 and PGE2) on the accumulation, release and metabolism of C19 steroids by human gingival fibroblasts (HGF) and gingivae, due to their anabolic potential in inflammatory repair. For the accumulation studies, HGF were incubated with 14C-testosterone at timed intervals and the cell-digests analysed for label uptake. The release of 5 alpha-dihydrotestosterone (DHT) by HGF was studied by preincubating the cells with 14C-DHT and reincubating with cold steroid to quantify its release at timed intervals. For the metabolic studies, HGF/gingival tissue were incubated with 14C-testosterone and serial concentrations of PGE1 and PGE2 to study their effects on the synthesis of DHT. The incubations were terminated at 24 h and extracted metabolites were analysed and quantified. The accumulation of 14C-testosterone by human gingival fibroblasts was elevated 3-fold at 24 h by PGE1 (n = 3, p < 0.001; 1-way ANOVA). The release of 14C-DHT was enhanced nearly 2-fold by PGE1 (n = 3, p < 0.001), compared with controls. Both PGE1 and PGE2 caused 2-fold increases in DHT synthesis by HGF and 3-fold increases in 4-androstenedione formation (n = 4, p < 0.001). With the tissue incubations PGE1/PGE2 caused 3-4 fold increases in DHT synthesis (n = 5, p < 0.005; Wilcoxon signed rank statistic for paired observations). Direct stimulation of the accumulation/release and metabolism of these steroids by prostaglandins in gingivae may contribute to the anabolic potential of androgens in inflammatory periodontal disease.     

Human epidermal growth factor and the proliferation of human fibroblasts

The majority of the mRNA molecules in HeLa cells contain 1-2 residue(s) of m6Ap and one blocked, methylated 5' terminal "cap" structure. The hnRNA, which is longer than mRNA, contains both m6Ap and caps but 4-6 times as many m6Ap residues per chain. In addition, nuclear molecules contain T2 RNA ase-resistant, methyl-labeled oligonucleotides ("di-" and "tri-" nucleotides) which are not found in mRNA. Some of the dinucleotides may be precursors to the 2'-0-methylated nucleotides in the cap structures. These results are compatible with internal methylation of hnRNA molecules (both m6Ap and 2'-0-methyl) followed by hnRNA cleavage and the addition of the cap structure to generate at least some of the HeLa cell mRNA. It also appears that some hnRNA molecules, which are longer than most mRNA molecules, contain cap structures suggesting the derivation of some mRNA molecules from the 5' regions of hnRNA.     

125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts

125I-labeled human epidermal growth factor (hEGF) binds in a specific and saturable manner to human fibroblasts. At 37 degrees C, the cell-bound 125I-hEGF initially may be recovered in a native form by acid extraction; upon subsequent incubation, the cell-bound 125I-hEGF is degraded very rapidly, with the appearance in the medium of 125I-monoiodotyrosine. At 0 degrees C, cell-bound 125I-hEGF is not degraded but slowly dissociates from the cell. The data are consistent with a mechanism in which 125I-hEGF initially is bound to the cell surface and subsequently is internlized before degradation. The degradation is blocked by inhibitors of metabolic energy production (azide, cyanide, dinitrophenol), some protease inhibitors (Tos-Lys-CH2Cl, benzyl guanidobenzoate), a lysosomotropic agent (chloroquine) various local anesthetics (cocaine, lidocaine, procaine), and ammonium chloride. After the binding and degradation of 125I-hEGF the fibroblasts are no longer able to rebind fresh hormone. The binding capacity of these cells is restored by incubation in a serum-containing medium; this restoration is inhibited by cycloheximide or actinomycin D.     

Effect of insulin on farnesyltransferase. Specificity of insulin action and potentiation of nuclear effects of insulin-like growth factor-1, epidermal growth factor, and platelet-derived growth factor

We have previously demonstrated that insulin activates farnesyltransferase (FTase) and augments the amounts of farnesylated p21 (Goalstone, M. L., and Draznin, B. (1996) J. Biol. Chem. 271, 27585-27589). We postulated that this aspect of insulin action might explain the "priming effect" of insulin on the cellular response to other growth factors. In the present study, we show the specificity of the effect of insulin on FTase. Insulin, but not insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), or platelet-derived growth factor (PDGF), stimulated the phosphorylation of the alpha-subunit of FTase and the amounts of farnesylated p21. Even though all four growth factors utilized the Ras pathway to stimulate DNA synthesis, only insulin used this pathway to influence FTase. Insulin failed to stimulate FTase in cells expressing the chimeric insulin/IGF-1 receptor and in cells derived from the insulin receptor knock-out animals. Insulin potentiated the effects of IGF-1, EGF, and PDGF on DNA synthesis in cells expressing the wild type insulin receptor, but this potentiation was inhibited in the presence of the FTase inhibitor, alpha-hydroxyfarnesylphosphonic acid. We conclude that the effect of insulin on FTase is specific, requires the presence of an intact insulin receptor, and serves as a conduit for the "priming" influence of insulin on the nuclear effects of other growth factors.     

Immunocytochemical localization of transforming growth factor alpha, epidermal growth factor and epidermal growth factor receptor in the cat endometrium and placenta

Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5-15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell-cell communication, cell fate, and differentiation of conducting airway epithelia.     

Overexpression of cellular Src in fibroblasts enhances endocytic internalization of epidermal growth factor receptor

Previous studies have demonstrated a requirement for the nonreceptor tyrosine kinase, cellular Src (c-Src), in epidermal growth factor (EGF)-induced mitogenesis and a synergistic interaction between c-Src and EGF receptor (EGFR) in tumorigenesis. Although endocytic internalization of EGFR may be thought to attenuate EGF-stimulated signaling, recent evidence suggests that signaling through Ras can be amplified by repeated encounters of endosome-localized, receptor. Shc.Grb2.Sos complexes with the plasma membrane, where Ras resides almost exclusively. Based on these reports, we examined EGFR trafficking behavior in a set of single and double c-Src/EGFR C3H10T1/2 overexpressors to determine if c-Src affects basal receptor half-life, ligand-induced internalization, and/or recycling. Our results show that overexpression of c-Src causes no change in EGFR half-life but does produce an increase in the internalization rate constant of EGF.EGFR complexes when the endocytic apparatus is not stoichiometrically saturated; this effect of c-Src on EGFR endocytosis is negligible at high receptor occupancy in cells overexpressing the receptor. In neither case are EGFR recycling rate constants affected by c-Src. These data indicate a functional role for c-Src in receptor internalization, which in turn could alter some aspects of EGFR signaling related to mitogenesis and tumorigenesis.     

Epidermal growth factor, transforming growth factor-alpha, and epidermal growth factor receptor localization in the baboon (Papio anubis) oviduct during steroid treatment and the menstrual cycle

OBJECTIVES: Polypeptide growth factors may modulate the actions of estrogen (E2) and progesterone (P) in reproductive tissues in an autocrine/paracrine manner. The objective of this study was to determine whether the baboon oviduct contains epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and EGF receptor (EGF-R) and whether changes in their expression are correlated with various hormonal states. METHODS: Oviductal tissue was obtained from adult female baboons (Papio anubis) after oophorectomy and steroid treatment, and during the menstrual cycle. Ampullary regions were fixed in Bouin's fixative and embedded in paraffin for immunocytochemistry using rabbit polyclonal antibodies against EGF and EGF-R, and mouse monoclonal antibody against TGF alpha. RESULTS: Both EGF and EGF-R were present in all tissue compartments (most strongly in the epithelium, followed by smooth muscle and stroma) at all reproductive stages and showed similar staining patterns. However, the most intense immunoreactive product was found in the tissue obtained from the E2-treated and late follicular phase animals. At this time, intense staining was present in the apical regions of the mature ciliated cells, whereas the stain was dispersed uniformly over the cytoplasm of all other cell types. Immunoreactive TGF alpha was limited primarily to the nonciliated epithelial cells, and staining was most intense in the E2-treated and late follicular phase tissues. Transforming growth factor-alpha formed intense perinuclear deposits in the mature secretory cells, an area that corresponds to the Golgi region. No immunoreactive product was observed for any of these proteins when preimmune serum was substituted for the primary antibody or when the primary antibody was preabsorbed with antigen. CONCLUSION: In summary, EGF, TGF alpha, and EGF-R are present in the ampulla of the baboon oviduct. Moreover, the localization and intensity of immunoreactive product are dependent on cell type and hormonal state. These data are consistent with the concept that EGF, TGF alpha, and EGF-R may be regulated by E2 and P and thus may play a role in cell differentiation and function. In addition, the specific localization of TGF alpha suggests that this growth factor may be synthesized for release from the secretory cells and thus may also function as a modulator of gamete/embryo viability and development.     

Transforming growth factor alpha, epidermal growth factor, and epidermal growth factor receptor expression in normal and diseased human adrenal cortex by immunohistochemistry and in situ hybridization

Because epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGFR) have been implicated in the regulation of adrenocortical function, we used immunohistochemistry and in situ hybridization of EGF and TGF-alpha to study 41 specimens of human adrenal cortex, including 10 normal specimens, 15 aldosteronomas, five Cushing's adenomas, six adrenocortical incidentalomas, and five carcinomas to determine what role these growth factors play in controlling human adrenocortical function. Neither immunoreactivity nor mRNA hybridization signals to EGF was detected in any specimens, and EGF therefore may exert its effects on adrenal function as an endocrine hormone. TGF-alpha expression was detected at both protein and mRNA levels in normal and neoplastic adrenal cortex, demonstrating that TGF-alpha is synthesized locally in human adrenal cortex. TGF-alpha expression was observed in the cells with increased steroidogenesis, including compact tumor cells and zona fasciculata cells with lipid depletion, but did not necessarily correlate with production sites of any specific steroid hormone. EGFR immunoreactivity was more widely distributed than TGF-alpha immunoreactivity. Both TGF-alpha and EGFR expression were markedly elevated in adrenocortical carcinomas. TGF-alpha and EGFR thus appear to be involved in biological function in both normal and neoplastic human adrenal cortex. In addition, TGF-alpha and EGFR may play important roles in some biological features of adrenocortical malignancy.     

Chronic pancreatitis is associated with increased concentrations of epidermal growth factor receptor, transforming growth factor alpha, and phospholipase C gamma

The epidermal growth factor (EGF) receptor is a transmembrane protein that binds EGF and transforming growth factor alpha (TGF alpha), and that stimulates phospholipase C gamma 1 (PLC gamma 1) activity. In this study the role of the EGF receptor in chronic pancreatitis was studied. By immunohistochemistry, the EGF receptor, TGF alpha, and PLC gamma 1 were found to be expressed at high concentrations in pancreatic ductal and acinar cells from chronic pancreatitis patients. Northern blot analysis showed that, by comparison with normal controls, 19 of 27 chronic pancreatitis tissues exhibited a 5.7-fold increase in EGF receptor mRNA concentrations, and 20 of 27 chronic pancreatitis tissues exhibited a sixfold increase in TGF alpha mRNA concentrations. In situ hybridisation confirmed that overexpression occurred in ductal and acinar cells, and showed that both mRNA moieties colocalised with their respective proteins. These findings suggest that TGF alpha may act through autocrine and paracrine mechanisms to excessively activate the overexpressed EGF receptor in the two major cell types of the exocrine pancreas, thereby contributing to the pathobiology of this disorder.     

The effect of epidermal growth factor and transforming growth factor beta 1 on proliferation and differentiation of urothelial cells in urinary bladder explant culture

The presence of the virulence markers K1 capsule, serum resistance, aerobactin, S and P/PR fimbriae were examined in a total of 395 E. coli strains from different extraintestinal infections and in 81 faecal isolates of healthy volunteers using specific DNA probes and classical phenotypic methods. All markers were more frequently detected when genotypic assays were applied. The simultaneous occurrence of 3-4 virulence determinants was typical for isolates derived from patients with septicaemia or meningitis. Isolates from blood cultures and cerebrospinal fluid were expressing the virulence phenotypes to a greater extent than isolates from urine or faeces. The use of colony hybridization with specific oligonucleotide and polynucleotide probes for the detection of virulence determinants has been proven to be more specific and reliable than phenotypic approaches.     

Effects and interactions of tumour necrosis factor alpha and bradykinin on interleukin-1 production in gingival fibroblasts

We examined the association of serum albumin concentration with diabetes mellitus and other cardiovascular risk factors, prevalent cardiovascular disease, and ultrasonographically assessed carotid artery intima-media thickness using data from 45- to 64-year-old adults in the Atherosclerosis Risk in Communities (ARIC) Study. The mean albumin concentration was 0.04 to 0.12 g/L lower in participants with diabetes and 0.02 to 0.06 g/L lower in those with cardiovascular disease, compared to participants without these conditions. However, lower serum albumin level was also correlated with most traditional risk factors and hemostatic variables. On adjustment for these, there was essentially no association between serum albumin and prevalent cardiovascular disease. Likewise, there was no association between albumin and carotid intima-media thickness (a marker of atherosclerosis). While hypoalbuminemia may be a marker for chronic disease and perhaps renal loss of albumin, it seems unlikely that it is an important cause of atherosclerosis.     

Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor

Past studies have shown that epidermal growth factor (EGF) is able to mimic the uterotropic effects of estrogen in the rodent. These studies have suggested a "cross-talk" model in which EGF receptor (EGF-R) signaling results in activation of nuclear estrogen receptor (ER) and its target genes in an estrogen-independent manner. Furthermore, in vitro studies have indicated the requirement for ER in this mechanism. To verify the requirement for ER in an in vivo system, EGF effects were studied in the uteri of ER knockout (ERKO) mice, which lack functional ER. The EGF-R levels, autophosphorylation, and c-fos induction were observed at equivalent levels in both genotypes indicating that removal of ER did not disrupt the EGF responses. Induction of DNA synthesis and the progesterone receptor gene in the uterus were measured after EGF treatment of both ERKO and wild-type animals. Wild-type mice showed increases of 4.3-fold in DNA synthesis, as well as an increase in PR mRNA after EGF treatment. However, these responses were absent in ERKO mice, confirming that the estrogen-like effects of EGF in the mouse uterus do indeed require the ER. These data conclusively demonstrate the coupling of EGF and ER signaling pathways in the rodent reproductive tract.     

The neurotrophic action and signalling of epidermal growth factor

Epidermal growth factor (EGF) is a conventional mitogenic factor that stimulates the proliferation of various types of cells including epithelial cells and fibroblasts. EGF binds to and activates the EGF receptor (EGFR), which initiates intracellular signalling and subsequent effects. The EGFR is expressed in neurons of the cerebral cortex, cerebellum, and hippocampus in addition to other regions of the central nervous system (CNS). In addition, EGF is also expressed in various regions of the CNS. Therefore, EGF acts not only on mitotic cells, but also on postmitotic neurons. In fact, many studies have indicated that EGF has neurotrophic or neuromodulatory effects on various types of neurons in the CNS. For example, EGF acts directly on cultured cerebral cortical and cerebellar neurons, enhancing neurite outgrowth and survival. On the other hand, EGF also acts on other cell types, including septal cholinergic and mesencephalic dopaminergic neurons, indirectly through glial cells. Evidence of the effects of EGF on neurons in the CNS is accumulating, but the mechanisms of action remain essentially unknown. EGF-induced signalling in mitotic cells is better understood than that in postmitotic neurons. Studies of cloned pheochromocytoma PC12 cells and cultured cerebral cortical neurons have suggested that the EGF-induced neurotrophic actions are mediated by sustained activation of the EGFR and mitogen-activated protein kinase (MAPK) in response to EGF. The sustained intracellular signalling correlates with the decreased rate of EGFR down-regulation, which might determine the response of neuronal cells to EGF. It is likely that EGF is a multi-potent growth factor that acts upon various types of cells including mitotic cells and postmitotic neurons.     

Bacterial steroidogenesis by periodontal pathogens and the effect of bacterial enzymes on steroid conversions by human gingival fibroblasts in culture

Studies were performed to investigate the effect of microbial culture supernatants of periodontal pathogens on the metabolism of radiolabelled testosterone in the presence or absence of human gingival fibroblasts. Subgingival plaque samples were obtained on paper points from 3 sites with probing depth values of 6-8 mm. Samples were incubated with 14C-testosterone for 24 h in brain heart infusion (BHI) broth. Similar incubations were also carried out with strains of A. actinomycetemcomitans, P. Intermedius and P. gingivalis to study the metabolism of radiolabelled testosterone by these periodontal pathogens. At the end of a 24 h incubation period with fibroblasts and supernatants or sonicates, the radioactive metabolites were extracted with ethyl acetate, evaporated and subjected to thin layer chromatography. The separated metabolites were quantified by scanning the radioactive plates using a Berthold linear analyser. When three sub-gingival plaque samples were incubated with radiolabelled testosterone there were 50-fold, 10-12-fold and 15-17-fold increases in 5 alpha-dihydrotestosterone (DHT) synthesis over 4-androstenedione production in these mixed microbial cultures. The two strains of P. intermedius produced 3- and 20-fold increases in 4-androstenedione production and DHT synthesis respectively. Both strains of A. actinomycetemcomitans and P. gingivalis showed 3-4-fold and 12-28-fold increases respectively in 4-androstenedione synthesis over that of DHT. Culture supernatants of P. intermedius and P. gingivalis caused 3-fold and 2-fold increases in DHT synthesis by fibroblasts over controls. There was little change in the case of the third pathogen. Since DHT has implications on matrix synthesis by fibroblasts in the environment of plaque associated inflammatory periodontal disease, bacterial metabolism and the effect of bacterial supernatants on human gingival fibroblasts can influence the degree of inflammatory repair.     

Comparison of the effects of interleukin-1 alpha, interleukin-1 beta and interferon-gamma-inducing factor on the production of interferon-gamma by natural killer

Interferon-gamma inducing factor (IGIF) is a recently identified cytokine which stimulates the production of interferon-gamma (IFN-gamma) by T cells and enhances natural killer (NK) cell cytolytic activity. Protein fold recognition, structure prediction and comparative modeling have revealed that IGIF is a member of the interleukin (IL)-1 cytokine family and has prompted the designation IL-1 gamma. Here we report functional similarities between members of the IL-1 family by comparing the effects of IL-1 alpha, IL-1 beta and IGIF on NK cell production of IFN-gamma. All three IL-1 types enhanced NK cell production of IFN-gamma when induced by IL-2 or IL-12, although at high concentrations (> 10 ng/ml), IGIF was five- to tenfold more potent than IL-1 alpha or IL-1 beta. This effect correlated with enhanced levels of mRNA for IFN-gamma when NK cells were stimulated with IGIF plus IL-12. In contrast to IL-12 and IL-2, the ability of IGIF to stimulate NK cell production of IFN-gamma was not increased by IL-1 alpha or IL-1 beta. The ability of IGIF to enhance IFN-gamma production was independent of the type I and type II IL-1 receptors or the IL-1R accessory protein. Together, these results identify IGIF as a potent stimulator of NK cell production of IFN-gamma and demonstrate that the effect of IGIF on NK cell production of IFN-gamma is similar to that of IL-1 alpha and IL-1 beta but distinct from that of IL-12.     

Acute behavioral effects of triazolam and caffeine, alone and in combination, in humans.

The acute behavioral effects of triazolam (0, 0.375, and 0.75 mg/70 kg) and caffeine (0, 250, and 500 mg/70 kg), alone and in combination, were assessed in 9 male volunteers. Ss received all possible dose combinations according to a Latin square design. Triazolam administered alone dose dependently disrupted learning and performance on the Repeated Acquisition and Performance procedure and the Digit Symbol Substitution Test and increased S ratings of sedation. Caffeine administered alone did not significantly affect learning or performance measures, but it did dose dependently increase S ratings of drug strength. Caffeine significantly attenuated triazolam-induced decrements in learning and performance. Consistent with effects on learning and performance, caffeine offset triazolam-induced increases in S ratings of sedation. Combining caffeine and triazolam did not significantly alter increases in S ratings of drug strength observed with caffeine alone. These effects are qualitatively similar to those observed with other benzodiazepines (e.g., lorazepam) and document a high degree of consistency in the behavioral pharmacology of benzodiazepine-caffeine combinations in humans. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Antitumor effects of the combination immunotherapy with interleukin-12 and tumor necrosis factor alpha in mice

Nickel hypersensitivity is an increasing problem in adolescents, especially in girls, with a prevalence of up to 30%. The presence of nickel in orthodontic appliances and the possibility of causing nickel hypersensitivity has been discussed in case reports. A review of the literature concerning nickel hypersensitivity in relation to orthodontic appliances has shown that the risk is very low for patients who are not nickel hypersensitive at the start of the treatment. A patient who is already nickel hypersensitive at the start of orthodontic treatment may in rare cases show adverse reactions induced by the appliance. The slow long-term release of nickel from orthodontic appliances may induce tolerance to nickel in individuals who are not hypersensitive at the start of orthodontic treatment.     

Expression of epidermal growth factors and epidermal growth factor receptor in normal cycling human ovaries

Immunolocalization of transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), cripto-1, amphiregulin and epidermal growth factor receptor (EGFR) was studied in 51 premenopausal human ovaries at various phases of the menstrual cycle. Localization of mRNA for TGF alpha and EGF was also studied by in-situ hybridization. Immunoreactive TGF alpha was observed predominantly in theca cells in 12 of 33 antral follicles in the follicular phase (6/14 dominant follicles, and 6/19 non-dominant) but not in any of the 18 follicles in the luteal phase or in primordial and pre-antral follicles. TGF alpha immunoreactivity was present predominantly in the luteinized granulosa cells in 13 of 15 corpora lutea in the luteal phase, which are considered to be active in steroidogenesis, but not in any of the regressed corpora lutea. Accumulation of TGF alpha mRNA hybridization signal was observed only in the theca cells in the follicles and luteinized theca cells in the ovaries that were immunohistochemically positive for TGF alpha. EGFR immunoreactivity was detected in 24 of 33 antral follicles in the follicular phase and in two of 18 follicles in the luteal phase but not in any of the corpora lutea. Immunoreactive EGF, cripto-1 and amphiregulin or EGF mRNA was not detected in any follicles, corpora lutea, or the stroma cells examined. These results indicate that, of the epidermal growth factors examined in this study, TGF alpha is locally synthesized in normal cycling human ovaries and TGF alpha may be synthesized in theca cells and act on the granulosa cells in a paracrine fashion through the EGFR in ovarian follicles.