Hepatocyte Growth Factor/scatter factor-induces phosphorylation of cortactin in A431 cells in a Src kinase-independent manner.

The Hepatocyte Growth Factor receptor transduces proliferating and scattering signals in epithelial and endothelial cells. We have explored potential interactions of the HGF/SF receptor beta-subunit (p145(beta MET)) with F-actin binding partners aiming to identify novel downstream effectors implicated in HGF/SF pluripotent signalling. Cortactin, a p80/85 F-actin binding protein, was found phosphorylated on tyrosine in response to HGF-SF in A431 human epidermoid carcinoma cells, expressing the HGF/SF receptor (c-MET). The HGF/SF receptor was enriched in the detergent-insoluble fraction and was found to co-precipitate with cortactin and to associate in vitro with cortactin. The Grb2 small adapter protein known to associate via its Src homology 2 domain (SH2) with the MET C-terminus, was also associated with cortactin. Transient transfection of A431 cells with dominant-negative Grb2 constructs has revealed that the Grb2-C-SH3 domain possesses a central role in cortactin phosphorylation in response to HGF/SF. Finally, tyrosine phosphorylation of cortactin was found uncoupled of endogenous c-Src kinase activity, thus further supporting the hypothesis that cortactin is a direct target of the MET kinase. We propose that cortactin may constitute a docking site for MET-derived signals within the cytoskeleton.

The beta-subunit of the hepatocyte growth factor/scatter factor (HGF/SF) receptor phosphorylates and associates with CrkII: expression of CrkII enhances HGF/SF-induced mitogenesis.

CrkII, a 40 kDa adaptor possessing a Src homology (SH)2 domain followed by two SH3 domains, although not endowed with catalytic activity, participates in intracellular signalling, presumably by activating the Ras pathway. CrkII was found to be phosphorylated in response to hepatocyte growth factor/scatter factor (HGF/SF) and to associate with the beta-subunit of the HGF receptor (MET). CrkII associated with p(145betaMET) via its SH2 domain. Growth-factor-receptor-bound protein 2 (Grb2) co-immunoprecipitated with CrkII species. By transient transfection of A431 human epidermoid carcinoma cells with wild-type and dominant-negative Grb2 expression constructs lacking either the SH2 or SH3 domains, we have concluded that Grb2 does not contribute to the 'presentation' of CrkII to p(145betaMET). Overexpression of wild-type CrkII in A431 cells enhanced HGF/SF-induced proliferation, while a CrkII dominant-negative mutant lacking the SH2 domain prevented a similar proliferating response to HGF/SF. The effect of CrkII on HGF/SF-induced proliferation was also abolished in cells co-expressing CrkII and Son-of-sevenless lacking the guanine exchange domain, suggesting that CrkII is likely to induce cell proliferation partly via the Ras/mitogen-activated protein kinase route.

Hepatocyte growth factor/scatter factor-induced intracellular signalling.

Hepatocyte growth factor (HGF) identical to scatter factor (SF) is a glycoprotein involved in the development of a number of cellular phenotypes, including proliferation, mitogenesis, formation of branching tubules and, in the case of tumour cells, invasion and metastasis. This fascinating cytokine transduces its activities via its receptor encoded by the c-met oncogene, coupled to a number of transducers integrating the HGF/SF signal to the cytosol and the nucleus. The downstream transducers coupled to HGF/MET, most of which participate in overlapping pathways, determine the development of the cell's phenotype, which in most cell types is dual.

Cytosolic phospholipase A2 is activated by the hepatocyte growth factor receptor-kinase in Madin Darby canine kidney cells.

The hepatocyte growth factor/scatter factor (HGF/SF) receptor which is a transmembrane protein encoded by the Met oncogene, possesses intrinsic tyrosine kinase activity which transduces the mitogenic, morphogenic and the scattering effect of HGF/SF. The pluripotent signal of HGF/SF is transduced through association of the Met receptor with various intracellular adaptors. Phosphorylation of cytosolic phospholipase A2 (cPLA2) is associated with activation of this molecule which in turn leads to arachidonic acid production followed by release of prostaglandins and related compounds exerting their roles onto cell proliferation, chemotaxis and vascular motility. Arachidonic acid and its metabolites were shown to be involved in processes like liver regeneration where growth factor receptors possessing tyrosine kinase activity are implicated. In this study we examined whether stimulation of the HGF/SF-receptor's tyrosine kinase activity would involve changes in the phosphorylation state and the activity of cPLA2 in MDCK cells, where HGF/SF is known to induce scattering responses rather than mitogenesis. The activated p145betaMET was shown to associate with and to phosphorylate cPLA2 on tyrosine residues, this leading to subsequent release of arachidonic acid. cPLA2 was also phosphorylated in serine residues and such a role has been so far assigned to the mitogen activated protein (MAP) kinase. Our data have also shown that MAP kinase is associated and phosphorylated on tyrosine by the activated p145betaMET. Immunodepletion of MAP kinase via electroporation of an anti-MAP kinase antibody, did not significantly decrease arachidonic acid release in HGF/SF-stimulated MDCK cells. It is therefore emerging that phosphorylation of cPLA2 on tyrosine by the HGF/SF receptor kinase is capable of triggering arachidonic acid release and that MAP kinase is contributing to full, but does not drive, the activity of cPLA2. The release of arachidonic acid by MDCK cells following HGF/SF stimulation is establishing this fatty acid and its metabolites as major components involved in the transduction of MET-driven signals and at the same time in the amplification of such signals.

Inhibition of bacterial growth by synthetic SP-B1-78 peptides.

Residues 12-34 of mature human pulmonary surfactant protein B (SP-B1-78) are 68% homologous to residues 48-72 of the frog peptide antibiotic dermaseptin b I. We examined the effects of SP-B1-78 on the growth of Escherichia coli in order to find whether full length SP-B1-78 might act as a peptide antibiotic. We found that SP-B1-78 peptide inhibited growth of E. coli (MIC = 210 micrograms.ml-1), but that the SP-B variant [R/K-->S]SP-B1-78 was less potent (MIC = 500 micrograms.ml-1).

The hepatocyte growth factor receptor kinase-mediated phosphorylation of lipocortin-1 transduces the proliferating signal of the hepatocyte growth factor.

Hepatocyte growth factor (HGF), which is identical to scatter factor (SF) through coupling to its receptor the product of c-met oncogene, was found to induce proliferation of A549 lung carcinoma cell line, accompanied by release of prostaglandin E2 (PGE2). This activity was sensitive to 0.1-100 microM indomethacin and to 5-50 nM of verapamil. Lipocortin-1, a dexamethasone-inducible inhibitor of phospholipase A2, was shown to be phosphorylated on tyrosine 10 min upon addition of HGF and to translocate to the membrane fraction for up to 6 h upon ligand stimulation. Lipocortin-1 was found to associate in vivo with the HGF receptor species, and this association was independent of the phosphorylation state of the beta-subunit of the HGF receptor (p145betaMET. Immobilized HGF receptor kinase species associated and phosphorylated in vitro lipocortin-1, thus providing evidence that lipocortin-1 is directly phosphorylated by the p145betaMET. Incubation of A549 cells with antisense 21-mer lipocortin-1 oligonucleotides reduced the synthesis and the HGF-stimulated phosphorylation of lipocortin-1 as well as the HGF-stimulated cell proliferation. In processes where the HGF receptor tyrosine kinase is activated, phosphorylation of lipocortin-1 may function as a "signal amplifier" promoting the release of intercellular messengers (PGE2) with pluripotent roles in cell proliferation, chemotaxis, and vascular remodeling.

Parathyroid hormone co-stimulation of hepatocyte proliferation is sensitive to protein kinase A and calcium channel inhibitors.

Parathyroid hormone (PTH) mobilises calcium in the hepatocyte, an effect which is abolished by verapamil and staurosporine. In our study parathyroid hormone was shown to act additively to dHGF in inducing hepatocyte DNA synthesis. It is also shown that PTH induced the production of inositol 1,4,5 trisphosphate (IP3) and c-fos expression at early times in culture. Co-incubation of PTH and dHGF with a c-fos antisense oligodeoxynucleotide inhibited hepatocyte DNA synthesis, indicating that the additive effect of PTH is correlated with the induction of c-fos. H-89, a PKA specific inhibitor, inhibited the PTH effect on IP3 production as well as the PTH effect on hepatocyte DNA synthesis. Verapamil and staurosporine also inhibited the PTH effect in dHGF-induced DNA synthesis. Therefore it is suggested that PKA mediated at a great extent the co-stimulatory effects of PTH on hepatocyte proliferation via IP3 production.

C-myc is required for the G0/G1-S transition of primary hepatocytes stimulated with a deleted form of hepatocyte growth factor.

Primary rat hepatocytes stimulated in vitro with the addition of a deleted form of hepatocyte growth factor (dHGF) enter the S-phase 48 h after addition of the growth factor. The c-myc gene is believed to play a role in a variety of cellular stages, such as proliferation, differentiation and cell death. In primary hepatocytes c-myc was expressed constitutively at both mRNA and protein levels, independently of the growth conditions. dHGF induced significant c-myc expression at times correlated with the long-lasting pre-S phase, and no induction was observed at the G0/G1 traverse compared with the unstimulated hepatocytes. An antisense construct coding for all three exons of c-myc was imported into hepatocytes by using the transferrin receptor-mediated endocytosis methodology (transferrinfection). Expression of the antisense construct inhibited the biosynthesis of the c-Myc protein, however it did not interfere with the expression of c-met, encoding the receptor for HGF/dHGF. Continuous expression of the antisense construct inhibited entry of the hepatocytes into the S-phase. Regulated induction of the antisense c-myc by dexamethasone for up to 6 h in culture, did not interfere with the entry of hepatocytes into the S-phase. c-myc expression was shown to be required between 6 and 12 h in dHGF-stimulated hepatocytes, and inhibition of its expression during this time by the antisense myc construct did not allow these cells to enter the S-phase. Inhibition of c-myc biosynthesis between 24 and 48 h hours slightly affected the DNA synthetic response. It is proposed that the expression of c-Myc protein interferes with the "priming' of hepatocytes to become responsive to growth-factor stimuli, or in the absence of such stimuli it interferes with the maintenance of a non-proliferating phenotype and subsequent in vitro de-differentiation.

c-Myc and Max interactions in quiescent and mitogen-stimulated primary hepatocytes.

The c-myc oncogene has been linked with cell proliferation, apoptosis, and differentiation, and when its expression is deregulated also with malignant transformation. In primary hepatocytes c-myc expression is constitutive and in part regulated by hepatocyte-specific growth factors (HGF, TGFalpha, and EGF) in a delayed early response manner. Max expression in these cells was found to be constitutive throughout the in vitro lifetime and mRNA transcript levels were increased at 12 h after induction with growth factors. Max was found to be associated in vivo with hepatocyte Myc species, with this association being independent of growth conditions and of the endogenous Myc or Max levels. Inhibition of endogenous hepatocyte Max levels via expression of an antisense max construct driven by the MMTV promoter did not affect the DNA synthetic response in the presence of dHGF (a variant of HGF). The unusually long half-life of the endogenous Myc species was found to be independent of their association with the widely accepted as "stable" partner, Max. We suggest that Myc and Max in hepatocytes are involved in the growth (proliferation, cell death) and differentiation program of these cells, acting independently or as a complex.

Hepatocyte growth factor-induced proliferation of primary hepatocytes is mediated by activation of phosphatidylinositol 3-kinase.

Primary hepatocytes respond to the proliferating signals of Hepatocyte Growth Factor (HGF) through activation of the tyrosine kinase activity of the met (p145) receptor. Addition of dHGF in hepatocyte cultures resulted in receptor phosphorylation which co-precipitated with a phosphorylated protein of 85 kDa. This protein was identified as the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). Co-precipitation of the PI 3-kinase regulatory subunit with the met receptor was observed only with the phosphorylated receptor. Wortmanin, which specifically inhibits PI 3-kinase, was found to abolish the hepatocyte DNA synthetic response due to stimulation with dHGF. It is suggested that the D-3-phosphorylated inositol phospholipids participate as major regulators in the growth and differentiation factor-initiated cascades, this not being restricted to primary hepatocytes.

Phospholipase A2 is activated by tumor necrosis factor-alpha in primary hepatocytes stimulated by a deleted form of hepatocyte growth factor.

Several factors are released in the liver microenvironment immediately after injury. Among these factors TNF alpha is implicated as a regulator of hepatocyte proliferation. Hepatocytes in the intact liver are mostly in the G0 phase of the cell cycle and after injury (including collagenase perfusion) display a constitutive expression of the growth-regulated c-myc oncogene. TNF alpha co-added with dHGF in hepatocyte cultures, superinduced the DNA synthetic response observed at all time points. In parallel, TNF alpha/dHGF-treated hepatocytes have shown increased expression of the c-myc oncogene at times corresponding to the in vitro G1 phase of the cell cycle. TNF alpha activated PLA2 in hepatocytes and it is believed that the subsequent production of PGE2 plays a role in the "priming" process in these cells and at the same time amplifies the proliferating signals induced by hepatocyte-specific growth factors.

Transforming growth factor-alpha-induced DNA synthesis and c-myc expression in primary rat hepatocyte cultures is modulated by indomethacin.

Primary hepatocytes stimulated with epidermal growth factor (EGF) secrete prostaglandins into the culture medium as soon as 1 h after the addition of the EGF. Transforming growth factor-alpha (TGF alpha), a potent hepatocyte mitogen, shares the same receptor with EGF, and its expression is increased after partial hepatectomy. TGF alpha is also secreted in culture. We have observed that TGF alpha induced hepatocyte DNA synthesis (30 h after addition) and at the same time stimulated the production of prostaglandins E2 and F2 alpha by the cultured hepatocytes. Indomethacin at 20-100 microM inhibited the TGF alpha-induced hepatocyte DNA synthesis, and this effect was specifically due to the inhibition of prostaglandin formation. Indomethacin also inhibited a TGF-alpha-induced increase in hepatocyte c-myc expression, indicating that prostaglandins mediate this increase, as previously shown for EGF. TGF alpha increased the expression of the EGF receptor gene, and this was prevented by the presence of an antibody against TGF alpha in the culture medium. We therefore suggest that TGF alpha induces hepatocyte proliferation either through coupling with its receptor (i.e. the EGF receptor) or by subsequent phosphorylation of lipocortin I. This leads to activation of phospholipase. A2, which seems to regulate the metabolism of arachidonic acid and the formation of prostaglandins. Thus hepatocyte proliferation in vitro appears to be controlled by a self-regulatory autocrine pathway involving activation of phospholipase A2 and secretion of prostaglandins and TGF alpha.

Expression of exogenous c-myc oncogene does not initiate DNA synthesis in primary rat hepatocyte cultures.

Cultured hepatocytes from adult rats stimulated with combinations of growth factors enter into S phase but do not undergo multiple rounds of DNA synthesis nor mitosis. We have examined the potential of an introduced oncogene to induce alterations in the DNA synthetic activity of the cultured hepatocytes in response to epidermal growth factor (EGF). Overexpression of c-myc did not initiate significant DNA synthesis in rat hepatocyte cultures alone, although it cooperated with added EGF to super-induce thymidine incorporation into DNA. From our results, it is suggested that EGF is also necessary to initiate hepatocyte DNA synthesis probably by inducing a battery of cell cycle-related genes if incubated with c-myc transfected cultures for only 5 hours. Hepatocyte polypeptides reacting with anti-MYC antisera were found to migrate between 55-67 KDa in SDS-PAGE; only the 64-67 KDa species were found to be phosphorylated, and the observed size heterogeneity may be due to proteolytic degradation or may reflect presently unknown posttranslational modifications.

Hepatocyte conditioned medium modulates the response of primary rat hepatocyte cultures to epidermal growth factor.

Primary hepatocytes may produce autocrine growth trigger(s) with or without a mitogenic stimulus. We explored the potential of hepatocyte conditioned medium--from untreated quiescent cultures--to modulate the DNA synthetic responses induced by EGF. The EGF-induced responses were similar when EGF was continuously or transiently (3 h) present. Conditioned medium (CM) from 48 h hepatocyte culture was the most effective in eliciting thymidine incorporation into hepatocyte DNA. At the same time the conditioned medium from hepatocyte cultures stimulated lymphocyte DNA synthesis at levels much lower than those observed using PHA, a specific lymphocyte inducer. The maximal EGF-binding by intact hepatocytes was also significantly increased in the presence of conditioned medium (48 h). We therefore suggest that hepatocytes produce autocrine growth trigger(s) which might be in part responsible for the regulation of the in vitro and/or in vivo hepatocyte proliferation.

Synthesis and phosphorylation of an extracellular polypeptide reacting with anti-MYC antisera in primary rat hepatocyte cultures.

Primary hepatocytes stimulated with appropriate growth factors enter into S phase and it is believed that c-myc and other cell cycle-related genes play an important role in the G0-G1/S phase transition. Four polypeptides reacting with anti-MYC antisera were detected in normal primary rat hepatocyte lysates, showing a pattern of 55-67 KDa on SDS-PAGE. A 67 KDa polypeptide was detected in the extracellular medium of the hepatocyte culture capable of undergoing phosphorylation. Both extracellular and intracellular polypeptides reacting with MYC-specific antisera exhibited an unusually long half life. It is believed that the intracellular MYC polypeptides may represent degradation products or species undergoing at present unknown post-transcriptional modification(s). We suggest that the extracellular MYC polypeptide(s) may be involved in the regulation of cell growth and differentiation.

Prostaglandins E2 and F2a mediate the increase in c-myc expression induced by EGF in primary rat hepatocyte cultures.

Epidermal Growth Factor (EGF) and prostaglandins (PGs) E2 and F2a, have been shown to stimulate primary hepatocyte proliferation. Verapamil (5-20 microM), a calcium channel inhibitor, inhibited hepatocyte DNA synthesis and c-myc expression, induced by EGF (50 ng/dish) and prostaglandins (1-12 micrograms/dish). Indomethacin (20-100 microM) decreased significantly the EGF-induced hepatocyte DNA synthesis and c-myc expression. Addition of PGs (1-9 micrograms) in hepatocyte cultures treated with EGF+indomethacin (100 microM) restored the capacity of EGF to increase c-myc expression and DNA synthesis. We propose that arachidonic acid derivatives and calcium channel blockers modulate c-myc expression in primary hepatocytes.

A growth factor for hepatocytes is inactivated by sub-lethal gamma-irradiation in vivo or in vitro.

Serum from partially hepatectomized rats promoted DNA synthesis in primary adult rat hepatocyte cultures. If the rats had been exposed to sub-lethal gamma-irradiation immediately following operation or if their serum, collected at 3 h, was exposed to irradiation in vitro, the growth-promoting activity was destroyed. Prostaglandin E2 also stimulated DNA synthesis in the cultures; if PGE2 was irradiated in serum from intact or partially hepatectomized rats its growth-promoting activity was markedly diminished.

Serum from partially hepatectomized rats induces primary hepatocytes to enter S phase: a role for prostaglandins?

Serum obtained from partially hepatectomized rats 1, 3 or 24 h after operation was more effective in stimulating DNA synthesis in primary adult rat hepatocytes than serum from sham-operated rats; exposure to the serum for 2 h was sufficient to promote growth. Serum from the partially hepatectomized rats contained elevated levels of PGE2 and PGF2 alpha; it promoted hepatocytes to release prostaglandins into their culture medium. Growth-promoting effects of the serum and its capacity to elicit prostaglandin release into the culture medium were inhibited by 0.1 mM-indomethacin or 1 mM-aspirin. 0.1 mM-indomethacin also prevented DNA synthesis if the inhibitor were added 4 h after growth had been initiated by serum from partially hepatectomized rats, suggesting that prostaglandins continue to be important for the maintenance of hepatocyte growth for at least 6 h.

Regulation of the proliferation of primary rat hepatocytes by eicosanoids.

DNA synthesis in primary adult rat hepatocyte cultures was promoted by epidermal growth factor (EGF), arachidonic acid, and prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha). Growth promotion by EGF was blocked by 0.1 mM indomethacin and 1 mM aspirin, without affecting cell viability. If verapamil was present in the medium when EGF was added, the growth response was inhibited. Hepatocytes stimulated by EGF or arachidonic acid released PGE2 and PGF2 alpha into the culture medium. This was diminished if 0.1 mM indomethacin was also in the medium. The importance of autocrine regulation of hepatocyte growth by prostaglandins is discussed.