Role of polyamines in gastroprotection induced by epidermal growth factor.

Polyamines have been shown to stimulate cellular growth and differentiation, though their role in the prevention of acute gastric lesion induced by various noxious agents has been little studied. Epidermal growth factor (EGF) exhibits gastroprotective and ulcer healing properties due to its potent mitogenic and growth promoting action. This study was designed to compare the gastroprotective effects of spermine and EGF against gastric damage induced by absolute ethanol, acidified aspirin and stress and to determine the role of endogenous polyamines in EGF-induced gastroprotection. Spermine and EGF significantly reduced the lesions induced by all three ulcerogens. Oral administration of spermine or subcutaneous infusion of EGF in 24 h fasted rats with chronic gastric fistula resulted in similar inhibition of gastric acid and pepsin secretion. Pretreatment with difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, did not affect ethanol lesions, but reversed the protective effect EGF but not spermine against ethanol. This finding indicates that polyamines mediate, at least in part, EGF-induced gastroprotection. In tests with oral administration of aminoguanidine that is known to suppress the activity of diamino-oxidase (DAO) and to inhibit the degradation of polyamines, EGF showed a markedly enhanced gastroprotective activity against ethanol damage. Since indomethacin failed to affect the gastroprotective effects of spermine and EGF and neither of these agents influenced the mucosal generation of PGE2 in intact or injured gastric mucosa, we conclude that prostaglandins are not the major factors in spermine- and EGF-induced gastroprotection. This study demonstrates that polyamines are highly effective against gastric lesions induced by various ulcerogens and that they act as primary mediators of EGF-induced gastroprotection.     

Trypanosoma brucei AP endonuclease 1 has a major role in the repair of abasic sites and protection against DNA-damaging agents

DNA repair mechanisms guarantee the maintenance of genome integrity, which is critical for cell viability and proliferation in all organisms. As part of the cellular defenses to DNA damage, apurinic/apyrimidinic (AP) endonucleases repair the abasic sites produced by spontaneous hydrolysis, oxidative or alkylation base damage and during base excision repair (BER). Trypanosoma brucei, the protozoan pathogen responsible of human sleeping sickness, has a class II AP endonuclease (TBAPE1) with a high degree of homology to human APE1 and bacterial exonuclease III. The purified recombinant enzyme cleaves AP sites and removes 3'-phosphoglycolate groups from 3'-ends. To study its cellular function, we have established TBAPE1-deficient cell lines derived from bloodstream stage trypanosomes, thus confirming that the AP endonuclease is not essential for viability in this cell type under in vitro culture conditions. The role of TBAPE1 in the removal of AP sites is supported by the inverse correlation between the level of AP endonuclease in the cell and the number of endogenously generated abasic sites in its genomic DNA. Furthermore, depletion of TBAPE1 renders cells hypersensitive to AP site and strand break-inducing agents such as methotrexate and phleomycin respectively but not to alkylating agents. Finally, the increased susceptibility that TBAPE1-depleted cells show to nitric oxide suggests an essential role for this DNA repair enzyme in protection against the immune defenses of the mammalian host.     

Involvement of tumor necrosis factor receptor-associated protein 1 (TRAP1) in apoptosis induced by beta-hydroxyisovalerylshikonin

beta-Hydroxyisovalerylshikonin (beta-HIVS), a compound isolated from the traditional oriental medicinal herb Lithospermum radix, is an ATP non-competitive inhibitor of protein-tyrosine kinases, such as v-Src and EGFR, and it induces apoptosis in various lines of human tumor cells. However, the way in which beta-HIVS induces apoptosis remains to be clarified. In this study, we performed cDNA array analysis and found that beta-HIVS suppressed the expression of the gene for tumor necrosis factor receptor-associated protein 1 (TRAP1), which is a member of the heat-shock family of proteins. When human leukemia HL60 cells and human lung cancer DMS114 cells were treated with beta-HIVS, the amount of TRAP1 in mitochondria decreased in a time-dependent manner during apoptosis. A similar reduction in the level of TRAP1 was also observed upon exposure of cells to VP16. Treatment of DMS114 cells with TRAP1-specific siRNA sensitized the cells to beta-HIVS-induced apoptosis. Moreover, the reduction in the level of expression of TRAP1 by TRAP1-specific siRNA enhanced the release of cytochrome c from mitochondria when DMS114 cells were treated with either beta-HIVS or VP16. The suppression of the level of TRAP1 by either beta-HIVS or VP16 was blocked by N-acetyl-cysteine, indicating the involvement of reactive oxygen species (ROS) in the regulation of the expression of TRAP1. These results suggest that suppression of the expression of TRAP1 in mitochondria might play an important role in the induction of apoptosis caused via formation of ROS.     

Role of phospholipase Cgamma-induced activation of protein kinase Cepsilon (PKCepsilon) and PKCbetaI in epidermal growth factor-mediated protection of tight junctions from acetaldehyde in Caco-2 cell monolayers

Epidermal growth factor (EGF) protects the intestinal epithelial tight junctions from acetaldehyde-induced insult. The role of phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) isoforms in the mechanism of EGF-mediated protection of tight junction from acetaldehyde was evaluated in Caco-2 cell monolayers. EGF-mediated prevention of acetaldehyde-induced decrease in transepithelial electrical resistance and an increase in inulin permeability, and subcellular redistribution of occludin and ZO-1 was attenuated by reduced expression of PLCgamma1 by short hairpin RNA. EGF induced a rapid activation of PLCgamma1 and PLC-dependent membrane translocation of PKCepsilon and PKCbetaI. Inhibition of PKC activity or selective interference of membrane translocation of PKCepsilon and PKCbetaI by RACK interference peptides attenuated EGF-mediated prevention of acetaldehyde-induced increase in inulin permeability and redistribution of occludin and ZO-1. BAPTA-AM and thapsigargin blocked EGF-induced membrane translocation of PKCbetaI and attenuated EGF-mediated prevention of acetaldehyde-induced disruption of tight junctions. EGF-induced translocation of PKCepsilon and PKCbetaI was associated with organization of F-actin near the perijunctional region. This study shows that PLCgamma-mediated activation of PKCepsilon and PKCbetaI and intracellular calcium is involved in EGF-mediated protection of tight junctions from acetaldehyde-induced insult.     

Role of vascular endothelial growth factor (VEGF) in endothelial cell protection against cytotoxic agents

Autocrine expression of VEGF has been detected in endothelial cells under hypoxia or oxidative stress. However, the functional significance of this VEGF autocrine expression remains undefined. To analyze the role of autocrine VEGF in the endothelial response against injury, cultured bovine aorta endothelial cells (BAEC) were challenged with potentially cytotoxic substances with different chemical structure and pharmacologic properties, namely cytochalasin D (CyD), hydrogen peroxide (H2O2) and cyclosporine A (CsA). Our results revealed that: i. In particular conditions, exposure to potentially cytotoxic agents as CyD, H2O2 or CsA results in significant BAEC cytoprotection rather than injury. ii. The response to the 3 agents is shifted to a cell damaging pattern in the presence of a specific anti VEGF monoclonal antibody (mAb). iii. CyD and H2O2 markedly stimulate the autocrine expression of VEGF mRNA and VEGF protein. In conclusion, the present study reveals a protective mechanism of endothelial cells against injury involving autocrine VEGF production. Moreover, the occurrence of a significant increase in VEGF expression accompanying this defensive mechanism is further disclosed.     

The extracellular matrix protein Del1 induces apoptosis via its epidermal growth factor motif

Mouse Del1 is an extracellular matrix protein mainly expressed in the developing embryo. Del1 has three EGF motifs and two discoidin domains. The second EGF motif reportedly contains an RGD sequence that binds to integrin receptors. Here, we provide evidence that Del1 protein induces cell death in vitro. Chromatin condensation and DNA laddering were observed, suggestive of apoptosis. The results of analysis using the TUNEL method and annexin V staining were also consistent with apoptosis. The apoptosis-inducing activity of Del1 could be mapped to the third EGF motif, which fitted the consensus sequence CX(D/N)XXXX(F/Y)XCXC, wherein the aspartic acid residue (D) could be β-hydroxylated. As little as twenty-five picomolar of recombinant E3 could induce apoptosis.     

Induction of apoptosis in porcine thyroid follicles by transforming growth factor beta1 and epidermal growth factor.

For thyroid cells in culture DNA fragmentation and morphological changes related to apoptosis were first described in dog thyroid cells after deprivation of serum, epidermal growth factor or thyrotropin. With intact porcine thyroid follicles in three-dimensional culture, the effect of deprivation of growth factors and of incubation with transforming growth factor beta1 (TGF-beta1), epidermal growth factor (EGF), thyrotropin (TSH) or insulin-like growth factor I (IGF-I) on the incidence of apoptosis was studied. Thyroid follicles were embedded in growth factor-depleted Matrigel and cultured in serum-free medium with or without growth factors for 7 days followed by incubation for 4, 24 and 72 h with TGF-beta1 (2 or 5 ng/mL). The percentage of apoptotic cells was determined by direct counting in electron-microscopy. Approximately 1% of apoptotic bodies could be detected in unstimulated follicles. This was unchanged in the presence of TSH (1 mU/mL) or IGF (10 ng/mL) but significantly increased up to 3.99 +/- 1.24% with 2 ng/mL of EGF. After incubation with TGF-beta apoptosis increased dose-dependently to 4.05 +/- 0.67% with 2 ng/mL TGF-beta1 and 5.16 +/- 1.75% with 5 ng/mL TGF-beta1. The incidence of necrotic cells remained constant at about 1 to 2%. Preincubation of follicles with 2 ng/mL of EGF followed by incubation with 5 ng/mL TGF-beta1 increased the rate of apoptic bodies up to 13.19 +/- 1.9%. We conclude that growth factor depletion in thyroid follicles in three-dimensional culture does not lead to apoptosis. TGF-beta1, however, induces apoptosis even in quiescent thyroid follicular cells and is significantly more pronounced in growing thyroid cells. EGF, which is a dedifferentiating growth factor for thyroid cells, also induces apoptosis. As EGF enhances TGF-beta1 mRNA and protein in thyroid follicular cells, the induction of apoptosis by EGF might also be due to TGF-beta1.     

Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells

Rat pheochromocytoma cells (clone PC12) respond to nerve growth factor (NGF) by the acquirement of a phenotype resembling neuronal cells. In an earlier study we showed that NGF causes an increase in Na+,K+ pump activity, as monitored by ouabain-sensitive Rb+ influx. Here we show that addition of epidermal growth factor (EGF) to PC12 cells resulted in a stimulation of Na+,K+ pump activity as well. The increase of Na+,K+ pump activity by NGF or EGF was due to increased Na+ influx. This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+,H+ exchange. Furthermore, no changes in membrane potential were observed upon addition of NGF or EGF. Amiloride-sensitive Na+,H+ exchange in PC12 cells was demonstrated by H+ efflux measurements and the effects of weak acids on Na+ influx. These observations suggest that both NGF and EGF activate an amiloride-sensitive, electroneutral Na+,H+ exchange mechanism in PC12 cells. These findings were surprising in view of the opposite ultimate biological effects of NGF and EGF, e.g., growth arrest vs. growth stimulation. However, within 24 h after addition, NGF was found to stimulate growth of PC12 cells, comparable to EGF. In the presence of amiloride, this stimulated growth by NGF and EGF was abolished. In contrast, amiloride did not affect NGF-induced neurite outgrowth of PC12 cells. From these observations it is concluded that in PC12 cells: (a) NGF has an initial growth stimulating effect; (b) neurite outgrowth is independent of increased amiloride-sensitive Na+ influx; and (c) growth stimulation by NGF and EGF is associated with increased amiloride-sensitive Na+ influx.     

Suppression of protein tyrosine kinase activity of the epidermal growth factor receptor by epidermal growth factor

Epidermal growth factor (EGF) receptor protein kinase activity, estimated by the use of peptide substrates, was reduced by as much as 70% after the treatment of intact A431 human carcinoma cells with EGF. The apparent decrease in protein kinase activity was observed after immunoprecipitation of the receptor or after purification of the receptor by lectin chromatography. By the use of [35S]methionine, it was determined that the total amount of receptor obtained was the same whether or not cells were treated with EGF. EGF stimulated the purified receptor protein kinase activity in vitro; however, the EGF-stimulated activity of receptor from EGF-treated cells continued to be reduced by as much at 70% compared to the EGF-stimulated activity from untreated cells. The reduction in receptor protein kinase activity induced by EGF may represent a feedback mechanism by which responsiveness to the growth factor is regulated.     

Caspase-dependent apoptosis and -independent poly(ADP-ribose) polymerase cleavage induced by transforming growth factor beta1

Apoptosis is an important cell suicide program which involves the caspases activation and is implicated in physiological and pathological processes. Poly(ADP-ribose) polymerase (PARP) cleavage is often associated with apoptosis and has been served as one hallmark of apoptosis and caspase activation. In this study, we aimed to determine TGF-beta1-induced apoptosis and to examine the involvement of caspases and its relationship with PARP cleavage. TGF-beta1 induces strong apoptosis of AML-12 cells which can be detected by DNA fragmentation, FACS, and morphological assays. Z-VAD-fmk, a selective caspase inhibitor, partially inhibits the TGF-beta1-induced apoptosis; but has no effect on TGF-beta1-induced DNA fragmentation and PARP cleavage. However, BD-fmk, a broad-spectrum caspase inhibitor, completely suppresses TGF-beta1-induced apoptosis, but unexpectedly does not inhibit TGF-beta1-induced PARP cleavage. Furthermore, Z-VAD-fmk treatment is able to completely inhibit the daunorubicin-induced apoptosis in A-431 cells, but only slightly blocks the daunorubicin-induced PARP cleavage, whereas BD-fmk can inhibit both daunorubicin-induced apoptosis and PARP cleavage completely. In addition, we observed that both TGF-beta1-induced apoptosis and PARP degradation in AML-12 cells can be completely blocked by inhibiting the protein synthesis with cycloheximide. These results demonstrate for the first time that TGF-beta1-induced caspase-dependent apoptosis is associated with caspase-independent PARP cleavage that requires the TGF-beta1-induced synthesis of new proteins. The results indicate that caspase-3 is not a major caspase involved in TGF-beta1-induced apoptosis in AML-12 cells, and is not required for apoptosis-associated DNA fragmentation. The results also suggest that PARP cleavage may occur as an independent event that can be disassociated with cell apoptosis.