H(2) has no known toxic effects on the human body. Thus, the results suggest that H(2)-loaded eye drops are a highly useful neuroprotective and antioxidative therapeutic treatment for acute retinal I/R injury.

Hepatocyte growth factor (HGF), 1 originally identified and cloned as a mitogenic protein for hepatocytes (1-3), evokes multiple cellular responses, including mitogenesis, morphogenesis, migration, and anti-apoptosis (4 -6). These biological activities of HGF are triggered by tyrosine phosphorylation of c-Met, a specific receptor tyrosine kinase for HGF (7). Biological activities of HGF support tissue organization during development and regeneration of organs, including the liver, kidney, placenta, and skeletal muscle (4 -6), but unregulated and/or constitutive activation of the c-Met receptor endows tumor cells with invasive and metastatic characteristics (8, 9).The c-Met receptor is a heterodimeric protein composed of extracellular ␣-chain and membrane-spanning ␤-chain, which contains an intracellular tyrosine kinase domain. Specific binding of HGF to the c-Met receptor activates tyrosine kinase activity, thereby facilitating phosphorylation of C-terminal tyrosine residues, so-called multifunctional docking sites (10, 11). In addition to the catalytic tyrosine kinase domain, the ␤-chain contains a juxtamembrane domain of 47 amino acid residues, which is highly conserved in distinct species (12). Previous studies indicated that a serine residue at position 985, which resides in the juxtamembrane domain of c-Met, is phosphorylated by treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator for protein kinase C (PKC). Most interesting, Ser-985 phosphorylation was associated with decreased tyrosine phosphorylation of the c-Met receptor (13); however, neither regulatory mechanisms nor the biological significance of the Ser-985 phosphorylation of c-Met has been elucidated.In the present study, we found that the phosphorylation status of juxtamembrane Ser-985 of the c-Met receptor is bidirectionally regulated through reverse activities of PKC␦/⑀ and protein phosphatase 2A (PP2A), a serine/threonine protein phosphatase. Likewise, oxidative stress in cells induced PKCmediated Ser-985 phosphorylation, and this was associated with inhibition of c-Met tyrosine phosphorylation and subsequent biological responses upon HGF stimulus. Our observations mean that the Ser-985 phosphorylation of c-Met mediated via PKCs and PP2A provides a unique mechanism, which confers cellular responsiveness/unresponsiveness to HGF, depending on the extracellular environment and conditions.

Inhibition of cell proliferation regulated by cell-cell contact is a fundamental characteristic of normal cells by which cellular adhesion successfully maintains formation and highly organized tissue architecture. On the other hand, the loss of contact inhibition is associated with cellular transformation, which is a precursor to malignant disorder (1-5). However, the molecular machineries involved in contact regulation are not fully understood.Although contact inhibition is a general characteristic of normal cells, the extent to which cells are inhibited by cell-cell contact differs significantly depending on cell types. Hepatocytes in primary culture provide a most appropriate experimental model in investigating contact inhibition (6 -9). Mature hepatocytes, which are terminally differentiated cells, undergo proliferation depending on exogenous growth factors. Proliferation of primary cultured hepatocytes is strictly regulated by cell-cell contact. When hepatocytes are cultured at sparse cell density, the cells undergo DNA synthesis in response to growth factors. However, when the cells are in a confluent density, DNA synthesis is mostly inhibited even in the presence of growth factors.Hepatocyte growth factor (HGF) 2 plays pivotal roles in hepatocyte proliferation through the specific receptor c-Met tyrosine kinase (10 -14). Disruption of the HGF-c-Met pathway in hepatocytes results in impaired liver regeneration (15, 16). c-Met tyrosine phosphorylation is a triggering event that forms phosphotyrosine-mediated activation cascades of the downstream signaling molecules of c-Met to elicit hepatocyte proliferation in response to HGF stimulation (14, 17). Conversely, it has been proposed that protein-tyrosine phosphatase (PTP) activities are involved in regulating the balance of tyrosine phosphorylation states of receptor tyrosine kinases, including c-Met and the possible downstream signal mediators, such as Shc, the Stat families, phospholipase C␥1, ␤-catenin, ␥-catenin, and p120 CAS in the normal liver (18). However, the precise role of PTPs in hepatocyte proliferation is not well clarified.In the present study, focusing on the activation states of c-Met, we investigated the mechanisms by which tight cell-cell contact in primary hepatocytes prevents mitogenic response to HGF. We found that LAR, a transmembrane protein-tyrosine phosphatase, plays a definitive role in inactivation, i.e. tyrosine dephosphorylation of c-Met through their physical interaction, which specifically occurs in hepatocytes under the confluent condition. Additionally, specific suppression of LAR expression prolonged tyrosine phosphorylation of c-Met and released contact inhibition. Our results indicate that regulation of the activation states of c-Met by LAR underlies contact inhibition of the mitogenic response to HGF in hepatocytes. MATERIALS AND METHODSCell Culture-Hepatocytes were prepared from 7-week-old male Sprague-Dawley rats by in situ perfusion of the liver with collagenase, as described elsewhere (6,19). The cells were cultured o...

Interaction of cytokines with their membrane receptors induces the proliferation and differentiation of a specific lineage of haematopoietic progenitors. The molecular mechanism of cytokine receptor-mediated signal transduction is unclear because these receptors do not have tyrosine kinase activity. Interleukin-3 and erythropoietin, however, induce transient tyrosine phosphorylation of a common set of proteins as a growth signal, and interleukin-2 induces phosphorylation of an overlapping but distinct set of proteins. Here we show that chimaeric receptors consisting of the extracellular domains of the erythropoietin receptor and the cytoplasmic domains of the interleukin-2 (or interleukin-3) receptor induce an erythropoietin-dependent tyrosine phosphorylation in interleukin-3-dependent Ba/F3 cells; however, chimaeric receptors composed of the extracellular domains of the interleukin-2 receptor and the cytoplasmic domains of the erythropoietin (or interleukin-3) receptor apparently transmit an interleukin-2-dependent signal. Our results indicate that these cytokines transmit distinct signals for activation of specific tyrosine kinases through the extracellular rather than cytoplasmic domains of the receptors.

Hepatocyte growth factor/scatter factor (HGF) was recently reported to function as a neurotrophic factor in the CNS. To investigate the intracellular signal pathways afteractivation of the HGF receptor c-Met in primary cultured rat neocortical cells, in vitro kinase assays were performed. HGF stimulation enhances the phosphorylation of endogenous 80-and 45-kDa substrates. Studies with protein kinase inhibitors and phorbol 12-myristate 13-acetate showed that protein kinase C (PKC) is activated intracellularly. The 80-kDa protein was identified to be the major PKC substrate MARCKS. Although four PKC subspecies, PKCa, PKCc, PKCy, and PKCX, were expressed in the cells, only PKCa, PKCe, and PKCy were selectively translocated in the plasma membrane after HGF stimulation. As expected from these three PKC subspecies, phosphorylation of phospholipase Cy 1 (PLCy1) but not phosphatidylinositol 3-kinase was enhanced, although the stimulation of brain-derived neurotrophic factor induced phosphorylation of phosphatidylinositol 3-kinase. In contrast to the neocortical cells, HGF did not enhance phosphorylation of PLCy1 in primary astrocytes. We also found that activated PKC(s) served as a major mitogen-activated protein kinase activator in this pathway. These findings suggest that HGF exerts neurotrophic effects through selective phosphorylation of PLCy1 and activation of distinct PKC subspecies in neocortical cells, most likely neurons. Key Words: Hepatocyte growth factor/scatter factor-c-Met-Protein kinase C subspecies-Distinct signal pathway-Neocortical cells.