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Induction of E-selectin on endothelial cell surface initiates leukocyte adhesion and subsequent migration into the subendothelium. Here, we tested the effect of hepatocyte growth factor (HGF) on inflammatory cytokine-induced expression of E-selectin and consequent leukocyte–endothelial cell interaction using human umbilical vein endothelial cells (HUVEC). Prior treatment of HUVEC with HGF significantly attenuated the tumor necrosis factor (TNF)-α-induced E-selectin protein, adhesion of HL60 cells to HUVEC and E-selectin mRNA expression in a dose-dependent manner, while HGF itself did not exert any effects. The HGF effects on the mRNA expression were inhibited in the presence of N G -nitro- l -arginine methyl ester ( l -NAME), a nitric oxide synthase (NOS) inhibitor, which also abolished HGF-stimulated eNOS activity. These results suggest HGF plays cardiovascular protective functions mediated, at least in part, through nitric oxide-dependent suppression of inflammatory cytokine-induced E-selectin expression and subsequent tethering of leukocytes to endothelial cells.

Recent studies have suggested that proinsulin C-peptide improves vascular functions, possibly through nitric oxide (NO) production. To clarify the molecular mechanisms of vascular NO production induced by C-peptide, we examined the effects of C-peptide on NO production and NO synthase expression in rat aortic endothelial cells in connection with mitogen-activated protein kinase (MAPK) activation. Aortic endothelial cells were isolated from female Wistar rats, cultured to confluence, and serum-starved for 24 h before treatment with C-peptide. Nitric oxide production was measured by the DAF-2 fluorescence dye method and relative amounts of endothelial nitric oxide synthase (eNOS) protein and its mRNA were semi-quantified by western blot and RT-PCR analyses respectively. Activation of MAPK was estimated by western blot detection of activity-related phosphorylation and in vitro kinase assay. Stimulation of cells with C-peptide for 3 h doubled NO production, which was suppressed by the NO synthase inhibitor, N G -nitro-L-arginine methyl ester (L-NAME). Stimulation also increased mRNA and protein contents of eNOS in a manner sensitive to the transcription inhibitor actinomycin D. It did not affect inducible NO synthase mRNA. C-peptide also induced rapid phosphorylation and activation of extracellular signal-regulated kinase (ERK, also known as p44/42MAPK), but not of p38MAPK. In cells pretreated with the ERK inhibitor PD98059 the C-peptide-elicited increase of NO production and eNOS was abrogated in a dose-dependent manner; suppression of ERK phosphorylation induced by C-peptide also occurred. Our results show that C-peptide increases NO production by increasing eNOS protein contents through ERK-dependent up-regulation of eNOS gene transcription. This could explain some actions of C-peptide on the vasculature, indicating a pivotal role for C-peptide in vascular homeostasis.

Non-invasive immobilization stress causes an increase in the plasma interleukin (IL)-6 level accompanied by increased IL-6 mRNA expression and IL-6 immunoactivity in the liver [Biochem. Biophys. Res. Commun. (1997) 238, 707-711]. In the present study, using rat primary cultured hepatocytes and non-parenchymal liver cells, the effect of norepinephrine (NE) on IL-6 mRNA expression was determined. IL-6 mRNA expression in hepatocytes, but not in non-parenchymal liver cells, increased when the cells were treated with NE. The stimulatory effect of NE was inhibited by the combined use of alpha- and beta-adrenergic antagonists. IL-6 mRNA expression in hepatocytes also increased on incubation with the culture medium of non-parenchymal liver cells treated with NE. The effect of the medium was blocked by an IL-1 receptor antagonist. Moreover, exogenous IL-1beta stimulated IL-6 mRNA expression in hepatocytes. IL-1beta was present in the medium of non-parenchymal liver cells and increased with NE-treatment. These results suggest that NE released from sympathetic nerve terminals during stress can directly increase IL-6 mRNA expression in hepatocytes and indirectly through IL-1beta production from non-parenchymal liver cells.

Previously, we demonstrated that hepatocyte growth factor (HGF) potently stimulates endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production through a calcium- and Akt-mediated phosphorylation at Ser-1179 (Ser-1177 human) in bovine aortic endothelial cells. The regulation of eNOS, however, also involves interaction with chaperone proteins such as heat shock protein (HSP) 90, which can be enhanced by agonist stimulation of the enzyme. In the present work, the role of HSP90 in HGF stimulation of eNOS was examined in an endothelial cell culture system. Treatment of endothelial cells with geldanamycin, a commonly used HSP90 inhibitor, augmented HGF-stimulated eNOS phosphorylation at Ser-1179, while it did not alter eNOS phosphorylation at Thr-497. However, other HSP90 inhibitors, namely 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and radicicol, did not possess similar effects. Neither HGF nor geldanamycin treatment, independently or in combination, altered HSP90/eNOS interaction in endothelial cells. In addition, geldanamycin treatment did not enhance the HGF-induced phosphorylation of Akt, ERK1/2 and p38MAPK. Src kinase inhibition by PP2 also failed to block the geldanamycin effects. These results suggest that geldanamycin, but neither 17-AAG nor radicicol, may enhance HGF-mediated eNOS Ser-1179 phosphorylation by some as yet unknown mechanisms independently of HSP90 inhibition.

The proliferation of peripheral blood mononuclear cells (PBMC) containing both monocyte/macrophages and T lymphocytes increased after treatment with T-cell mitogen (concanavalin A: Con A). PBMC treated with either leptin alone or combination of leptin and ConA showed enhanced proliferative activity by 10-40%, compared with those treated with ConA alone. In contrast, isolated T lymphocytes treated with leptin and ConA showed lowered proliferative activity than the ConA-treated alone, indicating that leptin induced production of some cytokines from monocyte/macrophages, that subsequently resulted in enhancement of T lymphocytes proliferation in PBMC. Among the cytokines examined, monocyte/monocytes constitutively expressed interleukin (IL)-1beta, IL-12p35, IL-18 mRNA, and faintly expressed tumor necrosis factor (TNF)-alpha and IL-12p40 mRNA. Leptin treatment augmented the monocyte/macrophages mRNA expression of only TNF-alpha and IL-12p40 to comparable levels of cells treated with lipopolysaccharide (LPS). However, leptin treatment increased monocyte/macrophages production of IL-1beta as well as TNF-alpha, and induced the mRNA expression of caspase-1, which is shown to mediate the conversion of latent pro-IL-1beta and pro-IL-18 to active forms. These results suggest that leptin directly acts on monocyte/macrophages to produce factors that induce T lymphocytes proliferation such as IL-12p35/p40 complex through IL-12p40 induction and IL-1beta/IL-18 production through caspase-1 induction.

Leptin, a pleiotropic hormone regulating food intake and energy expenditure, has been shown to directly modulate human polymorphonuclear neutrophil (PMN) functions or indirectly through the action of tumor necrosis factor-alpha (TNF-alpha). Bovine PMN have considerable different characteristics from human PMN. For example, it does not respond to N-formyl-Methionyl-Leucyl-phenylalanine, a well known human PMN activator. In the present study, we tested the effects of leptin and TNF-alpha on superoxide production and degranulation of bovine peripheral PMN, in which both long isoform of leptin receptor (Ob-Rb) and TNF receptor 1 were expressed. Human leptin, human TNF-alpha, phorbol myristate acetate (PMA) and opsonized zymosan particles (OZP) did not stimulate degranulation responses, while zymosan-activated serum (ZAS) did. Neither leptin nor TNF-alpha enhanced the ZAS-induced degranulation responses. TNF-alpha, PMA, OZP and ZAS increased superoxide production in different magnitudes, whereas leptin did not. TNF-alpha, but not leptin, enhanced OZP- and ZAS-induced superoxide production, possibly, in part due to facilitating translocation of p47(phox), a component of NADPH oxidase. These results indicate that, unlike in human PMN, leptin does not have any direct effect on degranulation and superoxide production in bovine PMN, although TNF-alpha influences superoxide production.

Proinsulin C-peptide has been reported to have some biological activities and to be possibly involved in the development of diabetic microangiopathy. In the present study, we examined the effects of C-peptide on the mitogen-activated protein kinase pathway in LEII mouse lung capillary endothelial cells. Stimulation of the cells with C-peptide increased both p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK1/2) activities and activity-related site-specific phosphorylation of the respective kinases in a concentration-dependent manner, but failed to activate c-Jun N-terminal kinase. Stimulation of the cells with C-peptide also induced site-specific phosphorylation of cAMP response element (CRE)-binding protein (CREB)/activating transcription factor 1 (ATF1), and thereby binding of these transcription factors to CRE. Among three CREB kinases tested, phosphorylation of mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2) was induced after stimulation with C-peptide. The phosphorylation of CREB, ATF1 and MAPKAP-K2 were inhibited by SB203580, a p38MAPK inhibitor, but not by PD98059, an ERK kinase inhibitor. These results indicate that C-peptide activates p38MAPK followed by MAPKAP-K2 to enhance DNA—CREB/ATF1 interactions.