Osmotic stress blocks NF-kappaB-dependent inflammatory responses by inhibiting ubiquitination of IkappaB

The inhibitory effects of hypertonic conditions on immune responses have been described in clinical studies; however, the molecular mechanism underlying this phenomenon has yet to be defined. Here we investigate osmotic stress-mediated modification of the NF-kappaB pathway, a central signaling pathway in inflammation. We unexpectedly found that osmotic stress could activate IkappaBalpha kinase but did not activate NF-kappaB. Osmotic stress-induced phosphorylated IkappaBalpha was not ubiquitinated, and osmotic stress inhibited interleukin 1-induced ubiquitination of IkappaBalpha and ultimately blocked expression of cytokine/chemokines. Thus, blockage of IkappaBalpha ubiquitination is likely to be a major mechanism for inhibition of inflammation by hypertonic conditions.     

Alendronate suppresses tumor angiogenesis by inhibiting Rho activation of endothelial cells

We previously reported that alendronate inhibits intraperitoneal dissemination in an in vivo ovarian cancer model. Recently, nitrogen-containing bisphosphonates have been reported to have antiangiogenic activities. In this study, alendronate inhibited human umbilical vein endothelial cell (HUVEC) migration and capillary-like structure formation in vitro. These inhibitory effects were associated with reduced Rho activation and suppression of the formation of actin stress fibers and focal adhesions in HUVECs. Furthermore, the inhibition by alendronate was reversed by geranylgeraniol, which abrogated the inhibition of Rho geranylgeranylation. Next, we examined the effect of alendronate on angiogenesis in disseminated ovarian tumors of athymic immunodeficient mice. Alendronate treatment reduced the intra-tumor neoangiogenesis compared with that in the non-treated mice, although tumor-derived VEGF expression was not altered. In conclusion, the in vivo anti-tumor effect of alendronate might be derived, at least in part, from its direct antiangiogenic effects on intra-tumor endothelial cells by inhibiting Rho geranylgeranylation.     

Heparan sulfate expression is affected by inflammatory stimuli in primary human endothelial cells

In diabetes the endothelium is either chronically or transiently exposed to hyperglycemic conditions. In addition, endothelial dysfunction in diabetes is related to changes in the inflammatory response and the turnover of extracellular matrix. This study was undertaken to study the effects of inflammatory stimuli on one particular matrix component, the heparan sulfate (HS) proteoglycans (PGs) synthesized by primary human umbilical cord vein endothelial cells (HUVEC). Such cells were cultured in vitro in 5 mM and 25 mM glucose. The latter concentration was used to mimic hyperglycemic conditions in short-term experiments. HUVEC were also cultured in the presence of the inflammatory agents tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), interleukin 1β (IL-1β) and transforming growth factor β (TGF-β). The cells were labeled with ³⁵S-sulfate and ³⁵S-PGs were recovered for further analyses. The major part of the ³⁵S-PGs was secreted to the medium, irrespective of type of stimuli. Secreted ³⁵S-PGs were therefore isolated and subjected to further analyses. TNF-α and IL-1α slightly increased the release of ³⁵S-PGs to the culture medium, whereas IL-1β treatment gave a significant increase. The different treatments neither changed the ratio of ³⁵S-HS and ³⁵S-chondroitin sulfate (CS) nor the macromolecular properties of the ³⁵S-PGs. However, the ³⁵S-HS chains were slightly increased in size after TNF-α treatment, and slightly decreased after TGF-β treatment, but not affected by the other treatments. Compositional analysis of labeled disaccharides showed changes in the amount of 6-O-sulfated glucosamine residues after treatment with TNF-α, IL-1α and IL-1β. Western immunoblotting showed that major HSPGs recovered from these cells were collagen XVIII, perlecan and agrin, and that secretion of these distinct PGs was increased after IL-1β stimulation. Hence, short term inflammatory stimuli increased the release of HSPGs in HUVEC and affected both the size and sulfation pattern of HS, depending on type of stimuli.     

NF-kappaB-dependent induction of osteoprotegerin by Porphyromonas gingivalis in endothelial cells

Porphyromonas gingivalis is a major etiological pathogen of adult periodontitis characterized by alveolar bone resorption. Vascular endothelial cells supply many inflammatory cytokines into periodontal tissue. However, whether the cells contribute to bone metabolism in periodontitis remains unknown. In this study, we investigated the effect of P. gingivalis on osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) production, both of which are key regulators of bone metabolism, in human microvascular endothelial cells (HMVECs). We showed that P. gingivalis upregulated expression of OPG but not RANKL mRNA in HMVEC. P. gingivalis induced NF-kappaB activation, and the induction of OPG in HMVEC by the pathogen was blocked by the inhibitors of NF-kappaB. In addition, incubation of OPG with P. gingivalis supernatant resulted in loss of the protein. These results indicate that P. gingivalis-stimulated HMVEC secrete OPG via a NF-kappaB-dependent pathway, while the OPG is partly degraded by the bacteria. Thus, microvascular endothelial cells can act as a source of OPG and thereby may play an important role in regulating bone metabolism in periodontitis.     

Calbindin-D28k is expressed in osteoblastic cells and suppresses their apoptosis by inhibiting caspase-3 activity

The rate of osteoblast apoptosis is a critical determinant of the rate of bone formation. Because the calcium-binding protein calbindin-D(28k) has anti-apoptotic properties in neuronal cells and lymphocytes, we searched for the presence of this protein in osteoblastic cells and investigated whether it can modify their response to proapoptotic signals. Calbindin-D(28K) was expressed at low levels in several osteoblastic cell lines and at high levels in primary cultures of murine osteoblastic cells. Transient transfection of rat calbindin-D(28k) cDNA blocked tumor necrosis factor alpha (TNFalpha)-induced apoptosis in osteoblastic MC3T3-E1 cells, as determined by cell viability and nuclear morphology of cells cotransfected with the green fluorescent protein targeted to the nucleus, whereas transfection of the empty vector had no effect. Calbindin-D(28k) levels in several stably transfected MC3T3-E1 lines were directly related to protection from TNFalpha-induced apoptosis. Purified rat calbindin-D(28k) markedly reduced the activity of caspase-3, a critical molecule for the degradation phase of apoptosis, in a cell-free assay. In addition, cell extracts from MC3T3-E1 cells expressing high levels of calbindin-D(28k) decreased caspase-3 activity, compared with extracts from vector-transfected cells. This effect was apparently unrelated to the calcium binding properties of calbindin, as chelation of calcium by EGTA or addition of other calcium-binding proteins such as calbindin-D(9k), S100, calmodulin, and osteocalcin, did not affect caspase-3 activity. Last, calbindin-D(28k) interacts with the active form of caspase-3 as demonstrated by a GST pull-down assay. These results demonstrate that calbindin-D(28k) is a biosynthetic product of osteoblasts with a role in the regulation of apoptosis. They also reveal that the antiapoptotic properties of calbindin-D(28k) may result not only from calcium buffering but also from the ability of the protein to interact with and to inhibit caspase-3 activity, a property that is independent of its calcium binding capability.     

Curcumin suppresses the expression of extracellular matrix genes in activated hepatic stellate cells by inhibiting gene expression of connective tissue growth factor

Upon liver injury, quiescent hepatic stellate cells (HSCs), the most relevant cell type for hepatic fibrogenesis, become active and overproduce extracellular matrix (ECM). Connective tissue growth factor (CTGF) promotes ECM production. Overexpression of CTGF during hepatic fibrogenesis is induced by transforming growth factor (TGF)-beta. We recently demonstrated that curcumin reduced cell growth and inhibited ECM gene expression in activated HSCs. Curcumin induced gene expression of peroxisome proliferator-activated receptor (PPAR)-gamma and stimulated its activity in activated HSCs, which was required for curcumin to suppress ECM gene expression, including alphaI(I)-collagen. The underlying mechanisms remain largely unknown. The aim of this study was to elucidate the mechanisms by which curcumin suppresses alphaI(I)-collagen gene expression in activated HSCs. We hypothesize that inhibition of alphaI(I)-collagen gene expression in HSCs by curcumin is mediated by suppressing CTGF gene expression through attenuating oxidative stress and interrupting TGF-beta signaling. The present report demonstrated that curcumin significantly reduced the abundance of CTGF in passaged HSCs and suppressed its gene expression. Exogenous CTGF dose dependently abrogated the inhibitory effect of curcumin. Activation of PPAR-gamma by curcumin resulted in the interruption of TGF-beta signaling by suppressing gene expression of TGF-beta receptors, leading to inhibition of CTGF gene expression. The phytochemical showed its potent antioxidant property by significantly increasing the level of total glutathione (GSH) and the ratio of GSH to GSSG in activated HSCs. De novo synthesis of cellular GSH was a prerequisite for curcumin to interrupt TGF-beta signaling and inhibited gene expression of CTGF and alphaI(I)-collagen in activated HSCs. Taken together, our results demonstrate that inhibition of alphaI(I)-collagen gene expression by curcumin in activated HSCs results from suppression of CTGF gene expression through increasing cellular GSH contents and interruption of TGF-beta signaling. These results provide novel insights into the mechanisms underlying inhibition of HSC activation by curcumin.     

Lipopolysaccharide activates matrix metalloproteinase-2 in endothelial cells through an NF-kappaB-dependent pathway

Vascular endothelial cells release proteinases that degrade the extracellular matrix, thus enabling cell migration during angiogenesis and vasculogenesis. Endothelial cells secrete mainly the proform of matrix metalloproteinase-2 (proMMP-2). In this report, we examined several growth factors, cytokines, and other molecules for activation of MMP-2 by human umbilical vein endothelial cells. Of these factors, we found that lipopolysaccharide (LPS) is the strongest activator of MMP-2. LPS induced MMP-2 activation in a time- and dose-dependent manner. While pretreatment with zinc chelators or nuclear factor kappaB (NF-kappaB) inhibitors suppressed LPS-induced MMP-2 activation, pretreatment with phosphatidylinositol 3'-kinase inhibitors had no effect. These results indicate that, in endothelial cells, LPS can directly enhance angiogenesis by inducing MMP-2 activation mediated through an NF-kappaB pathway.     

Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity.     

Ascochlorin suppresses oxLDL-induced MMP-9 expression by inhibiting the MEK/ERK signaling pathway in human THP-1 macrophages

The critical initiating event in atherogenesis involves the invasion of monocytes through the endothelial walls of arteries and the transformation of monocytes from macrophages into foam cells. Human THP-1 monocytic cells can be induced to differentiate into macrophages by phorbol myristate acetate (PMA) and can then be converted into foam cells by exposure to oxidized low-density lipoprotein (oxLDL). Also, during a chronic inflammatory response, monocytes/macrophages produce the 92-kDa matrix metalloproteinase-9 (MMP-9) that may contribute to the extravasation, migration, and tissue remolding capacities of the phagocytic cells. Here, we investigate the effect of ascochlorin (ASC), a prenylphenol antiviral compound from the fungus Ascochyta viciae, on oxLDL-induced MMP-9 expression and activity in human THP-1 macrophages. ASC reduced oxLDL-induced MMP-9 expression and activity in a time-dependent and dose-dependent manner. Also, an analysis of MMP-9 activity using pharmacologic inhibitors showed that ASC inhibits MMP-9 activity via the extracellular signal-regulated kinase 1 and kinase 2 pathways. Our results suggest that ASC may be useful as a potent clinical antiatherogenic agent, a topic of considerable interest in the biological chemistry of chemotherapeutic agents.     

Activation of PKC-epsilon and ERK1/2 participates in shear-induced endothelial MCP-1 expression that is repressed by nitric oxide

Vascular endothelial cells (ECs) continuously experience hemodynamic shear stress generated from blood flow. Previous studies have demonstrated that shear stress modulates monocyte chemotactic protein-1 (MCP-1) expression in ECs. This study explored the roles of protein kinase C (PKC), extracellular signal-regulated protein kinase (ERK1/2), and nitric oxide (NO) in sheared-induced MCP-1 expression in ECs. The activation of PKC-alpha and PKC-epsilon isoforms was observed in ECs exposed to shear stress. The use of an inhibitor (calphostin C) to PKC-alpha and PKC-epsilon decreased ERK1/2 activation and MCP-1 induction by shear, whereas an inhibitor (Go6976) to PKC-alpha did not affect ERK1/2 activation or MCP-1 induction. Inhibition of ERK1/2 activation by PD98059 blocked MCP-1 induction. Transfection of ECs with an antisense to PKC-epsilon abolished the shear inducibility of MCP-1 promoter. These results demonstrate that PKC-epsilon and ERK1/2 participate in shear-induced MCP-1 expression. We also examined the regulatory role of NO in MCP-1 expression. An NO donor (NOC18) suppressed shear-induced activation of PKC-epsilon and ERK1/2, and also repressed MCP-1 induction. Consistently, overexpression of endothelial nitric oxide synthase (eNOS) to enhance the endogenous generation of NO in ECs decreased the activation of PKC-epsilon and ERK1/2, and also inhibited MCP-1 expression. Taken together, these findings suggest that PKC-epsilon and ERK1/2 are critical in the signaling pathway(s) leading to the MCP-1 expression induced by shear stress. Additionally, this study indicates that NO, by repressing PKC-epsilon activity and ERK pathway activation, attenuates shear-induced MCP-1 expression.     

Sophoridine suppresses lenvatinib-resistant hepatocellular carcinoma growth by inhibiting RAS/MEK/ERK axis via decreasing VEGFR2 expression

Hepatocellular carcinoma (HCC) is one of the most lethal cancer types with insufficient approved therapies, among which lenvatinib is a newly approved multi-targeted tyrosine kinase inhibitor for frontline advanced HCC treatment. However, resistance to lenvatinib has been reported in HCC treatment recently, which limits the clinical benefits of lenvatinib. This study aims to investigate the underlying mechanism of lenvatinib resistance and explore the potential drug to improve the treatment for lenvatinib-resistant (LR) HCC. Here, we developed two human LR HCC cell lines by culturing with long-term exposure to lenvatinib. Results showed that the vascular endothelial growth factor receptors (VEGFR)2 expression and its downstream RAS/MEK/ERK signalling were obviously up-regulated in LR HCC cells, whereas the expression of VEGFR1, VEGFR3, FGFR1-4 and PDGFRα/β showed no difference. Furthermore, ETS-1 was identified to be responsible for VEGFR2 mediated lenvatinib resistance. The cell models were further used to explore the potential strategies for restoration of sensitivity of lenvatinib. Sophoridine, an alkaloid extraction, inhibited the proliferation, colony formation, cell migration and increased apoptosis of LR HCC cells. In vivo and in vitro results showed Sophoridine could further sensitize the therapeutic of lenvatinib against LR HCC. Mechanism studies revealed that Sophoridine decreased ETS-1 expression to down-regulate VEGFR2 expression along with downstream RAS/MEK/ERK axis in LR HCC cells. Hence, our study revealed that up-regulated VEGFR2 expression could be a predicator of the resistance of lenvatinib treatment against HCC and provided a potential candidate to restore the sensitivity of lenvatinib for HCC treatment.     

Shear stress-dependent expression of apoptosis-regulating genes in endothelial cells

Laminar shear stress exerts potent anti-apoptotic effects. Therefore, we analyzed the influence of laminar shear stress on the expression of apoptosis-regulating genes in human umbilical vein endothelial cells (HUVEC). Application of high levels of laminar shear stress (15 and 30 dyn/cm(2)) decreased the susceptibility of HUVEC to undergo apoptosis, whereas low shear stress (1 dyn/cm(2)) had no effect. These diminished signs of apoptosis were accompanied by a decreased mRNA expression of apoptosis-inducing Fas receptor. Furthermore, mRNA and protein expression of anti-apoptotic, soluble Fas isoform FasExo6Del and anti-apoptotic Bcl-x(L) were induced. Surprisingly, high shear stress also elevated mRNA and protein expression of pro-apoptotic Bak. The shear stress-induced up-regulation of Bcl-x(L) and Bak mRNA can be abrogated by inhibition of the endothelial NO synthase. We propose that altered expression of Bcl-x(L) and the Fas system is involved in the protective effect of laminar shear stress against apoptosis in human endothelial cells.     

ERK7 expression and kinase activity is regulated by the ubiquitin-proteosome pathway

ERK7 is a unique member of the extracellular signal-regulated kinase (ERK) subfamily of MAP kinases. Although ERK7 shares a TEY motif in the activation loop of the kinase, it displays constitutive activation, nuclear localization, and growth inhibitory properties that are regulated by its C-terminal domain. Because ERK7 is expressed at low levels compared with ERK2 and its activity is dependent upon its expression level, we investigated the mechanism by which ERK7 expression is regulated. We now show that ERK7 expression is regulated by ubiquitination and rapid proteosomal turnover. Furthermore, both the kinase domain and the C-terminal tail are independently degraded at a rate comparable with that of the intact protein. Analysis of a series of chimeras between ERK2 and ERK7 reveal that the N-terminal 20 amino acids of the kinase domain are a primary determinant of ERK7 degradation. Fusion of the N-terminal 20 amino acids is both necessary and sufficient to cause proteolytic degradation of both ERK2 and green fluorescent protein. Finally, ERK7 is stabilized by an N-terminal mutant of Cullin-1 suggesting that ERK7 is ubiquitinated by the Skip1-Cullin-F box complex. These results indicate that ERK7 is a highly regulated enzyme whose cellular expression and kinase activation level is tightly controlled by the ubiquitin-proteosome pathway.