Hepatocyte Growth Factor-Induced Expression of Ornithine Decarboxylase, c-met,and c-mycIs Differently Affected by Protein Kinase Inhibitors in Human Hepatoma Cells HepG2

Binding of hepatocyte growth factor (HGF) to its receptor Met induces autophosphorylation and activation of the tyrosine kinase activity. In HGF-treated HepG2 cells, we studied: (i) the expression patterns of early(c-myc,c-jun,and c-fos) and delayed-early (ornithinedecarboxylase and c-met) response genes and (ii) thepossible involvement of protein kinase transducersin the control of the expression of c-metand of other genes eventually induced downstream. c-metand c-mycmRNAs peaked 1–2 h after HGF, while c-junandc-fosmRNAs slightly increased at 1 h. Ornithinedecarboxylase activity was induced earlier (4 h) thanthe mRNA (8–10 h). The transducers involved in HGF-triggered gene inductions were investigated using different protein kinase inhibitors: genistein for the receptor tyrosine kinase, herbimycin A for the nonreceptor tyrosine kinase (pp60^c-src), wortmannin for phosphatidylinositol 3-kinase (PI3K) and H7 for protein kinase C (PKC). The similarity of responses to PKC inhibition led to suppose that c-mycand ornithinedecarboxylase mRNAs were induced sequentially along the same transduction pathway triggered by HGF. Ornithine decarboxylase activity seemed to be largely regulated by phosphorylation(s). The mRNA expression of c-junwas likely to undergo a negative regulation through a mechanism involving PI3K, while that ofc-metseemed to be almost independent from various protein kinases (PI3K, pp60^c-src, and PKC).     

Reversal of the growth inhibitory effect of α-difluoromethylornithine by putrescine but not by other divalent cations

Summary Treatment with -difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC), depletes the putrescine and spermidine content, and reduces the growth rate of Ehrlich ascites tumor cells.The addition of putrescine, which is the immediate precursor of spermidine, promptly replenished the intracellular putrescine and spermidine pools and completely reversed the antiproliferative effect of DFMO. A sequential accumulation of spermine, spermidine and putrescine was observed.1,3-diaminopropane, a lower homolog of putrescine, did not reverse the antiproliferative effect of DFMO, despite its structural similarity and identical positive charge. By inhibiting remaining ODC activity, resistant to 5 mM DFMO, and possibly by inhibiting spermine synthase activity, 1,3-diaminopropane produced a further decrease in total polyamine content by reducing the spermine content.Mg²⁺, which can replace putrescine in many in vitro reactions, completely lacked the capacity to reverse the antiproliferative effect of putrescine and spermidine deficiency.Abbreviations DFMO -difluoromethylornithine - ODC ornithine decarbxylase     

Expression and function of gingival fibroblast C1q receptors are upregulated by interleukin-1β and transforming growth factor-β

In injury and inflammation, complement (C) component C1q, in addition to its central role in initiation of classical pathway of complement activation, modulates diverse cellular functions by binding to specific cell surface receptors. Interaction of substrate-bound C1q with receptors for the collagen-like domain of C1q (C1qRC) of human gingival fibroblasts (HGF) promotes cell attachment. We investigated modulation of the adhesive function and expression of C1qRC by interleukin-1β (IL-1β) and transforming growth factor-β (TGF-β). Confluent fibroblast monolayers were incubated under standard culture conditions with or without cytokines. C1qRC function was measured by attachment assays. IL-1β and TGF-β increased fibroblast adhesion to C1q to 146% and 131% of controls, respectively. Cytokine enhancement of HGF adhesion was concentration-dependent, saturable (20 ng/ml IL-1β; 1 ng/ml TGF-β) and time-dependent (IL-1β 12-hr peak; TGF-β 24-hr peak). Effect of IL-1β and TGF-β on C1qRC expression was assessed by flow cytometry measurements of fluorescence intensity of cells stained with C1q and FITC anti-C1q antibody, and by binding studies with ¹²⁵l-C1q. Cells treated with cytokines displayed a two- to four-fold increased fluorescence of cell-bound C1q compared to controls. Binding studies indicated the increased fluorescence correlated with increase in number of C1qRC in both IL-1β (4.7 × 10⁶/cell) and TGF-β (3.9 × 10⁶/cell)-treated cells, compared to control (3.0 × 10⁶/cell), but had no effect on binding affinity. Rates of internalization of receptor-bound C1q were similar in cytokine-treated cells and controls. We propose from these data that IL-1β and TGF-β have the ability to upregulate C1qRC expression, and this effect contributes to increased adhesion of HGF to substrate-bound C1q. © 1993 Wiley-Liss, Inc.     

Contribution of spermidine to stimulation by hepatocyte growth factor in repair after damage of rabbit gastric mucosal cells in primary culture

Wound-healing of the gastric mucosa is suggested to be stimulated by hepatocyte growth factor (HGF). Polyamines are shown to contribute to repair after damage in the gastric mucosa. The present study was designed to elucidate whether HGF can stimulate wound-healing of the gastric mucosa via polyamine production, using rabbit gastric mucosal cells in primary culture. A wound was made as a round cell-free area in the cell sheet of confluent cultured cells. When HGF was added to the culture medium, such denuded area was significantly reduced in size compared with the control, but the reduction was inhibited by addition of D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of a rate-limiting enzyme (ornithine decarboxylase) of polyamine biosynthesis, to the culture medium. However, the inhibitory effect by DFMO was reversed by pretreatment with spermidine, but not with putrescine. Intracellular levels of polyamines in the whole confluent cells including the cells around the denuded area were not changed by addition of HGF, but putrescine and spermidine levels were decreased by further addition of DFMO. We conclude that spermidine may be involved in stimulation by HGF in the repair after damage of gastric mucosal cells.     

Docosahexaenoic acid-containing phosphatidylethanolamine enhances HL-60 cell differentiation by regulation of c-jun and c-myc expression

18:1/docosahexaenoic acid (DHA)-containing phosphatidylethanolamine (PE) enhanced cell differentiation and growth inhibition of HL-60 induced by dibutyryl cAMP (dbcAMP) in a dose-dependent manner. The combined treatment of 200 μM dbcAMP and 50 μM 18:1/DHA-PE increased the NBT reducing activity, which is as an indicator of cell differentiation, to more than 75% from 40% of cells treated with 200 μM dbcAMP alone. In HL-60 cells treated with 50 μM 18:1/DHA-PE and 200 μM dbcAMP for 24 h, the expression level of c-jun mRNA and c-Jun protein were remarkably elevated compared to cells treated with dbcAMP alone. In contrast, there was no difference in the expression levels of c-fos mRNA and c-Fos protein between the combination of 18:1/DHA-PE + dbcAMP or dbcAMP alone. On the other hand, the combine treatment of 18:1/DHA-PE and dbcAMP markedly reduced the expression level of c-myc oncogene during 48 h incubation. The decreases of c-myc mRNA by 18:1/DHA-PE and/or dbcAMP was correlated with growth inhibition effect. Thus, 18:1/DHA-PE might enhance dbcAMP-induced HL-60 cell differentiation and growth inhibition by regulation of c-jun and c-myc mRNA and their products.     

Role of putrescine in interleukin 1 beta production in human histiocytic lymphoma cell line U937

Treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and incubation with lipopolysaccharide (LPS) induces interleukin 1 beta (IL-1 beta) production in the histiocytic lymphoma cell line U937. Here we investigated the effect of treatment with both TPA and 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) on LPS-induced IL-1 beta production in U937 cells. To clarify the mechanism of IL-1 beta production, the possible role of polyamines in this process was examined. Combined treatment with TPA and 1,25(OH)2D3 for 72 h followed by incubation with LPS for 24 h caused synergistic induction of both IL-1 beta release and mRNA expression. On the other hand, TPA increased the numbers of vitamin D3 receptors, which may be one mechanism of this synergistic induction. Ornithine decarboxylase (ODC), a rate-limiting enzyme for polyamine biosynthesis, was also induced by these compounds biphasically: the first peak of ODC activity was observed at 4 h of the incubation with the two compounds and the second peak was at 4 h after the addition of LPS. To find whether these peaks were related to IL-1 beta production, DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, was added together with TPA and 1,25(OH)2D3. DFMO decreased the cellular levels of putrescine and spermidine and suppressed IL-1 beta release and IL-1 beta mRNA expression by 65%. Exogenous putrescine, but not spermidine, abrogated these kinds of inhibition. Similar results were obtained with DFMO and the polyamines during the differentiation of the cells up to the monocyte or macrophage stage. These results thus suggest that changes in either of these intracellular polyamines, especially putrescine, help to regulate the differentiation of U937 cells, resulting in partial control of the regulation of IL-1 beta production.     

Downregulation of T cell growth factor production by ornithine decarboxylase and its product putrescine: -α-Difluoromethylornithine suppresses general protein synthesis but augments simultaneously the production of interleukin-2

Treatment of EL-4 lymphoma cells with tetradecanoylphorbol-acetate (TPA), a well-known activator of protein kinase C, induces the production of the T cell growth factor interleukin-2 (IL-2) and the expression of IL-2-specific mRNA within 4–8 h. This system is an ideal model for studies on the induction of a differentiated function in a homogeneous lymphoid cell population by a defined signal. TPA induces also an increase of ornithine decarboxylase (ODC) activity and elevates the intracellular concentrations of putrescine and polyamines within 4–8 h. A similar increase of intracellular putrescine and polyamine concentrations can be achieved by administration of 2 mM putrescine to the culture medium. However, putrescine cannot induce the production of IL-2 in the absence of TPA and cannot reconstitute the IL-2 production in cultures with PGE₂ or cyclosporine A, i.e., two well-known immunosuppressive substances which inhibit ODC activity. Putrescine has rather a counter-regulatory effect as concluded from the observation that the TPA-induced TCGF production and IL-2-specific mRNA expression are augmented (superinduced) by the ODC inhibitor -α-difluoromethylornithine (DFMO) and again suppressed after the administration of putrescine or polyamines to DFMO-treated cultures. The glycolytic activity, general protein synthesis ([³H]leucine incorporation), and the cell cycle progression from G₂/M to G₁, in contrast, are inhibited by DFMO and reconstituted by putrescine. This demonstrates that the cells are able to sacrifice to a large extent several vital functions including their general protein synthesis and to devote themselves at the same time to a fulminant production of their functionally most relevant protein IL-2. This process is downregulated by ODC and its product putrescine. A correlation between increased IL-2 production and accumulation of cells in the G₂/M phase was also observed in cultures treated with hydroxyurea or with a combination of amethopterin and adenosine.     

Expression of c-fos and jun protooncogenes in ovine trophoblasts in relation to interferon-tau expression and early implantation process

Expression of c-fos and jun protooncogenes was analyzed in the ovine extraembryonic trophoblast from days 14–18 of gestation, using Northern and Western blotting and immunohistochemistry. This study was carried out in relation to the early implantation process and the expression of interferon-tau, which is secreted in large amounts for a few days before implantation. Our results demonstrated that c-fos, c-jun, and junB were differently expressed in the ovine trophoblast around the time of implantation. The c-fos mRNA and protein were detected at high levels prior to attachment and decreased thereafter, following the pattern of expression of interferon-tau, whereas c-jun expression was maintained at relatively high levels during the implantation process. By contrast, the levels of junB mRNA and protein decreased prior to attachment. Immunohistochemical studies indicated that JunB, like C-Fos and interferon tau, was no longer expressed in the trophoblastic cells which had established cellular contacts with the uterine epithelium. A striking finding in this study is the temporal correlation between the accumulation of c-Fos and c-Jun proteins and the expression of the interferon-tau (days 14 and 15 of gestation). We also showed by gel-retardation assays that an AP-1-like site present in the promoter of one interferon-tau gene was functional in vitro, as judged by its ability to bind day-15 trophoblast nuclear protein extracts. Nuclear proteins binding to this site had the characteristics of AP-1, as judged by the ability to be competed efficiently by a consensus TRE (12.0-tetradecanoyl phorbol 13-acetate-responsive element)-site oligonucleotide and by antibodies to c-Fos and Jun proteins. These results suggest that Fos and Jun could form regulatory complexes of interferon-tau expression and/or are regulated by common mechanisms which are still unknown. Mol Reprod Dev 46:127–137, 1997. © 1997 Wiley-Liss, Inc.     

Effects of putrescine and inhibitors of putrescine biosynthesis on organogenesis inEuphorbia esula L.

Summary Exogenous putrescine (≤5 mM) had little effect on root or shoot formation in aseptically isolated hypocotyl segments of leafy spurge (Euphorbia esula L.) grown on full-strength B5 medium. Unexpectedly, putrescine inhibited root and shoot formation in hypocotyl segments grown on B5 medium diluted 10-fold. In the full-strength medium, root and shoot formation were inhibited by 0.5 mM concentrations ofdl-α-difluoromethylornithine (DFMO) anddl-α-difluoromethylarginine (DFMA). DFMO and DFMA are inhibitors of the ornithine decarboxylase and arginine decarboxylase pathways, respectively, of putrescine biosynthesis in plants. Exogenous putrescine (0.5 to 5 mM) did not reverse either the DFMO-or DFMA-induced inhibition of shoot formation. However, the DFMA-induced inhibition of root formation was partially reversed by exogenous putrescine. The auxin, indole-3-acetic acid (IAA), reduced the inhibitory effects of DFMO+DFMA (applied together) on both roots and shoots. In the first few days of culture, the endogenous levels of putrescine and spermidine, but not of spermine, increased in the presence of IAA. The levels of putrescine and spermidine in the tissues did not correlate well with either root or shoot production in the later stages of organ formation; especially in tissues treated with IAA. These results show that there were no obvious correlations between polyamine levels and organogenesis in leafy spurge hypocotyl segments, although residual putrescine or spermidine or both in the tissues at the time of excision may be indirectly involved in the early stages of root formation.     

Effects of polyamine biosynthesis inhibitors on the progesterone-initiated increase in intracellular free Ca2+ and acrosome reactions in human sperm

This laboratory has previously reported that progesterone can initiate a rapid transient increase in the concentration of intracellular free Ca²⁺([Ca²⁺]_i) and an increase in a Ca²⁺-requiring exocytotic event, the acrosome reaction (AR) in human sperm. Rapid increases in Ca²⁺ fluxes of some mammalian cells caused by another steroid, testosterone, require polyamine biosynthesis. Herein, we tested two polyamine biosynthesis suicide inhibitors for their effects on the progesterone-initiated increase in [Ca²⁺]_i and AR in capacitated human sperm in vitro: DL-α-(difluoromethyl)ornithine hydrochloride (DFMO), an inhibitor of putrescine synthesis by ornithine decarboxylase and (5′-{[(Z))-4-amino-2-butenyl]methylamino}-5′-deoxyadenosine (MDL 73811), an inhibitor of S-adenosylmethionine decarboxylase (required for spermidine and spermine synthesis). Sperm were capacitated in vitro and preincubated 10 min with 4.9 mM DFMO or 9.8 μM MDL 73811 with or without various polyamines (245 μM). Progesterone (3.09 μM final concentration) or progesterone solvent (ethanol, 0.1% final concentration) was then added, sperm fixed 1 min after additions and AR assayed by indirect immunofluorescence or with fluorescein-labeled Con A lectin. DFMO strongly inhibited the AR but putrescine (product of ornithine decarboxylase and precursor of spermidine and spermine) reversed that inhibition. Preincubation for 25 min with DMFO + spermidine also reversed DFMO inhibition. MDL 73811 inhibited the progesterone-initiated AR, and a 10 min preincubation with spermidine, but not putrescine or spermine, reversed that inhibition. Preincubations with putrescine alone or with spermidine alone followed by addition of the progesterone solvent did not initiate the AR, and such preincubations followed by progesterone addition did not increase the AR more than progesterone alone. MDL 73811 and DFMO partially inhibited the rapid progesterone-initiated increase in [Ca²⁺]_i (assayed with fura-2), and those inhibitions were partially reversed by putrescine and spermidine, respectively. Putrescine or spermidine alone did not increase [Ca²⁺]_i nor did preincubation with either polyamine followed by progesterone addition increase [Ca²⁺]_i more than progesterone alone. Neither inhibitor was able to inhibit the AR initiated by the calcium ionophore, ionomycin. Our results suggest that human sperm polyamine biosynthesis is necessary for the progesterone-initiated rapid increase in [Ca²⁺]_i and subsequent membrane events of the AR. © 1993 Wiley-Liss, Inc.     

Anti-inflammatory effects of hepatocyte growth factor: induction of interleukin-1 receptor antagonist

Hepatocyte growth factor (HGF) prevents liver failure in various animal models including endotoxin-induced acute liver failure. We were interested to find out whether human HGF exerts anti-inflammatory effects by modulation of cytokine synthesis. Therefore, human HepG2 cells were cultured with increasing concentrations of HGF. HGF dose-dependently upregulated the production of interleukin-1 receptor antagonist (IL-1Ra). Incubation of HepG2 cells with interleukin-1beta (IL-1beta) caused an increase in IL-1Ra levels, while interleukin-6 (IL-6) had no effect on IL-1Ra synthesis. Co-stimulation of HepG2 cells with HGF + IL-1beta resulted in a synergistic effect on IL-1Ra mRNA and protein expression. Stimulation of freshly isolated mouse hepatocytes from male C57 BL/6 mice with HGF increased IL-1Ra mRNA and protein synthesis dose-dependently. A co-stimulation with HGF and IL-1beta had a synergistic effect on IL-1Ra mRNA expression but only a partially additive effect on IL-1Ra protein synthesis. HGF-induced IL-1Ra production was significantly decreased by the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Accordingly, HGF stimulation specifically increased MAPK-dependent signalling pathway (p42/44). In contrast, in preactivated PBMC mRNA expression and protein synthesis of IL-1Ra, interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) were unaffected after stimulation with HGF. In conclusion, our data suggest that HGF exerts anti-inflammatory effects by modulating the signal transduction cascade leading to increased expression of IL-1Ra, which might explain the protective and regenerative properties of this cytokine in animal models of liver failure.     

Effects of exogenous polyamines and difluoromethylornithine on seed germination and root growth of Arabidopsis thaliana

Effects of exogenous polyamines and difluoromethylornithine (DFMO) on seed germination and seedling root growth of Arabidopsis thaliana were investigated. Root growth was stimulated by low concentrations of putrescine but was increasingly inhibited by high concentrations of putrscine. DFMO inhibited root growth and this inhibition was reversed by applying putrescine. In contrast, both spermidine and spermine had no effect on root growth but inhibited seed germination. The results suggest a possible requirement of endogeneous putrescine for normal root growth of Arabidopsis seedlings.Abbreviations DFMO difluoromethylornithine - DFMA difluoromethylarginine - ODC ornithine decarboxylase - Put Putrescine - Spd Spermidine - Spm Spermine