3-Aminobenzamide Prevents Concanavalin A-Induced Acute Hepatitis by an Anti-inflammatory and Anti-oxidative Mechanism.

BACKGROUND AND AIMS: Concanavalin A is known to activate T cells and to cause liver injury and hepatitis, mediated in part by secretion of TNFα from macrophages. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been shown to prevent tissue damage in various animal models of inflammation. The objectives of this study were to evaluate the efficacy and mechanism of the PARP-1 inhibitor 3-aminobenzamide (3-AB) in preventing concanavalin A-induced liver damage. METHODS: We tested the in vivo effects of 3-AB on concanavalin A-treated mice, its effects on lipopolysaccharide (LPS)-stimulated macrophages in culture, and its ability to act as a scavenger in in vitro assays. RESULTS: 3-AB markedly reduced inflammation, oxidative stress, and liver tissue damage in concanavalin A-treated mice. In LPS-stimulated RAW264.7 macrophages, 3-AB inhibited NFκB transcriptional activity and subsequent expression of TNFα and iNOS and blocked NO production. In vitro, 3-AB acted as a hydrogen peroxide scavenger. The ROS scavenger N-acetylcysteine (NAC) and the ROS formation inhibitor diphenyleneiodonium (DPI) also inhibited TNFα expression in stimulated macrophages, but unlike 3-AB, NAC and DPI were unable to abolish NFκB activity. PARP-1 knockout failed to affect NFκB and TNFα suppression by 3-AB in stimulated macrophages. CONCLUSIONS: Our results suggest that 3-AB has a therapeutic effect on concanavalin A-induced liver injury by inhibiting expression of the key pro-inflammatory cytokine TNFα, via PARP-1-independent NFκB suppression and via an NFκB-independent anti-oxidative mechanism.

Enhanced phosphatidylinositol 3-kinase (PI3K)/Akt signaling has pleiotropic targets in hippocampal neurons exposed to iron-induced oxidative stress.

The PI3K/Akt pathway is a key component in synaptic plasticity and neuronal survival. The aim of this work was to investigate the participation of the PI3K/Akt pathway and its outcome on different molecular targets such as glycogen synthase kinase 3ß (GSK3ß) and Forkhead box-O (FoxO) transcription factors during mild oxidative stress triggered by iron overload. The exposure of mouse hippocampal neurons (HT22) to different concentrations of Fe(2+) (25-200 µm) for 24 h led us to define a mild oxidative injury status (50 µm Fe(2+)) in which cell morphology showed changes typical of neuronal damage with increased lipid peroxidation and cellular oxidant levels but no alteration of cellular viability. There was a simultaneous increase in both Akt and GSK3ß phosphorylation. Levels of phospho-FoxO3a (inactive form) increased in the cytosolic fraction of cells treated with iron in a PI3K-dependent manner. Moreover, PI3K and Akt translocated to the nucleus in response to oxidative stress. Iron-overloaded cells harboring a constitutively active form of Akt showed decreased oxidants levels. Indeed, GSH synthesis under oxidative stress conditions was regulated by activated Akt. Our results show that activation of the PI3K/Akt pathway during iron-induced neurotoxicity regulates multiple targets such as GSK3ß, FoxO transcriptional activity, and glutathione metabolism, thus modulating the neuronal response to oxidative stress.

Interaction of purinergic receptors with GPCRs, ion channels, tyrosine kinase and steroid hormone receptors orchestrates cell function.

Extracellular purines and pyrimidines have emerged as key regulators of a wide range of physiological and pathophysiological cellular processes acting through P1 and P2 cell surface receptors. Increasing evidence suggests that purinergic receptors can interact with and/or modulate the activity of other classes of receptors and ion channels. This review will focus on the interactions of purinergic receptors with other GPCRs, ion channels, receptor tyrosine kinases, and steroid hormone receptors. Also, the signal transduction pathways regulated by these complexes and their new functional properties are discussed.

Activation of the PI3K/Akt signaling pathway through P2Y2 receptors by extracellular ATP is involved in osteoblastic cell proliferation.

We studied the PI3K/Akt signaling pathway modulation and its involvement in the stimulation of ROS 17/2.8 osteoblast-like cell proliferation by extracellular ATP. A dose- and time-dependent increase in Akt-Ser 473 phosphorylation (p-Akt) was observed. p-Akt was increased by ATPγS and UTP, but not by ADPßS. Akt activation was abolished by PI3K inhibitors and reduced by inhibitors of PI-PLC, Src, calmodulin (CaM) but not of CaMK. p-Akt was diminished by cell incubation in a Ca²âº-free medium but not by the use of L-type calcium channel blockers. The rise in intracellular Ca²âº induced by ATP was potentiated in the presence of Ro318220, a PKC inhibitor, and attenuated by the TPA, a known activator of PKC. ATP-dependent p-Akt was diminished by TPA and augmented by Ro318220 treatment in a Ca²âº-containing but not in a Ca²âº-free medium. ATP stimulated the proliferation of both ROS 17/2.8 cells and rat osteoblasts through PI3K/Akt. In the primary osteoblasts, ATP induces alkaline phosphatase activity via PI3K, suggesting that the nucleotide promotes osteoblast differentiation. These results suggest that ATP stimulates osteoblast proliferation through PI-PLC linked-P2Y2 receptors and PI3K/Akt pathway activation involving Ca²âº, CaM and Src. PKC seems to regulate Akt activation through Src and the Ca²âº influx/CaM pathway.

Protein phosphatases: possible bisphosphonate binding sites mediating stimulation of osteoblast proliferation.

We investigated the existence of a bisphosphonate (BP) target site in osteoblasts. Binding assays using [³H]-olpadronate ([³H]OPD) in whole cells showed the presence of specific, saturable and high affinity binding for OPD (K(d)=1.39 ± 0.33 µM) in osteoblasts. [³H]OPD was displaced from its binding site by micromolar concentrations of lidadronate, alendronate and etidronate (K(d)=1.42 ± 0.15 µM, 2.00 ± 0.2 µM and 2.4 ± 0.4 µM, respectively), and by millimolar concentrations of the non-permeant protein phosphatase (PP) substrates p-nitrophenylphosphate and α-naphtylphosphate. PP inhibitors orthovanadate, NaF or vpb(bipy) did not displace [³H]OPD. As expected, specific OPD binding was detected in the plasma membrane of ROS 17/2.8 cells, although significant BP binding was also found intracellularly. Moreover, OPD increased DNA synthesis in these cells with a temporal profile similar to the protein tyrosine phosphatase (PTP) inhibitors, Na3VO4 and vpb(bipy); but different from a general PP inhibitor (NaF). The stimulatory effect of OPD and PTP inhibitors on osteoblast proliferation was inhibited by the protein tyrosine kinase inhibitors genistein and geldanamycin. These results provide new evidence on the existence of a BP target in osteoblastic cells, presumably a PTP, which may be involved in the stimulatory action of BPs on osteoblast proliferation.

Purinergic (ATP) signaling stimulates JNK1 but not JNK2 MAPK in osteoblast-like cells: contribution of intracellular Ca2+ release, stress activated and L-voltage-dependent calcium influx, PKC and Src kinases.

This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPgammaS and UTP, but not with ADPbetaS. RT-PCR studies supported the expression of P2Y(2) receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP(3) receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca(2+)-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y(2) receptors. These results show that P2Y(2) receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP(3)/intracellular Ca(2+) release and Ca(2+) influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases.

Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress.

Oxidative stress leads to the degeneration of retinal pigment epithelial (RPE) and photoreceptor cells. We evaluated the potential of adipose-derived mesenchymal stem cells (ASCs) as a therapeutic tool by studying the migration capacity of ASCs in vitro and their protective effect against RPE cell death under oxidative stress in vitro and in vivo. ASCs exhibited enhanced migration when exposed to conditioned medium of oxidative stressed RPE cells obtained by hydrogen peroxide. Migration-related axis SDF-1/CXCR4 was studied, and upregulation of SDF-1 in stressed RPE and of CXCR4 in ASCs was detected. Moreover, ASCs' conditioned medium prevented H2O2-induced cell death of RPE cells. Early passage ASCs had high expression level of HGF, low VEGF levels, and unmodulated IL-1ß levels, compared to late passage ASCs. Thus, early passage ASCs show the potential to migrate towards damaged RPE cells and protect them in a paracrine manner from cell death induced by oxidative stress. In vivo, mice received systemic injection of NaIO3, and 72 h later, ASCs were transplanted in the subretinal space. Seven days after ASC transplantation, the eyes were enucleated fixed and frozen for immunohistochemical analysis. Under such conditions, ASC-treated mice showed preservation of nuclear layers in the outer nuclear layer and stronger staining of RPE and photoreceptor layer, compared to PBS-treated mice. Taken together, our results indicate that ASCs are able to home in on damaged RPE cells and protect against damage to the RPE and PR layers caused by oxidative stress. These data imply the potential that ASCs have in regenerating RPE under oxidative stress, providing the basis for a therapeutic approach to retinal degeneration diseases related to oxidative stress that could help save the eyesight of millions of people worldwide.

Exogenous ceramide-1-phosphate (C1P) and phospho-ceramide analogue-1 (PCERA-1) regulate key macrophage activities via distinct receptors.

Inflammation is an ensemble of tightly regulated steps, in which macrophages play an essential role. Previous reports showed that the natural sphingolipid ceramide 1-phosphate (C1P) stimulates macrophages migration, while the synthetic C1P mimic, phospho-ceramide analogue-1 (PCERA-1), suppresses production of the key pro-inflammatory cytokine TNFα and amplifies production of the key anti-inflammatory cytokine IL-10 in LPS-stimulated macrophages, via one or more unidentified G-protein coupled receptors. We show that C1P stimulated RAW264.7 macrophages migration via the NFκB pathway and MCP-1 induction, while PCERA-1 neither mimicked nor antagonized these activities. Conversely, PCERA-1 synergistically elevated LPS-dependent IL-10 expression in RAW264.7 macrophages via the cAMP-PKA-CREB signaling pathway, while C1P neither mimicked nor antagonized these activities. Interestingly, both compounds have the capacity to additively inhibit TNFα secretion; PCERA-1, but not C1P, suppressed LPS-induced TNFα expression in macrophages in a CREB-dependent manner, while C1P, but not PCERA-1, directly inhibited recombinant TNFα converting enzyme (TACE). Finally, PCERA-1 failed to interfere with binding of C1P to either the cell surface receptor or to TACE. These results thus indicate that the natural sphingolipid C1P and its synthetic analog PCERA-1 bind and activate distinct receptors expressed in RAW264.7 macrophages. Identification of these receptors will be instrumental for elucidation of novel activities of extra-cellular sphingolipids, and may pave the way for the design of new sphingolipid mimics for the treatment of inflammatory diseases, and pathologies which depend on cell migration, as in metastatic tumors.

TRPC3-like protein and vitamin D receptor mediate 1alpha,25(OH)2D3-induced SOC influx in muscle cells.

1alpha,25-Dihydroxy-Vitamin-D3 (1alpha,25(OH)2-Vitamin D3) stimulates in skeletal muscle cells Ca2+ release from inner stores and influx through both voltage-dependent and store-operated Ca2+ (SOC, CCE) channels. We investigated the involvement of TRPC proteins and Vitamin D receptor (VDR) in CCE induced by 1alpha,25(OH)2D3 in chick muscle cells. Two fragments were amplified by RT-PCR, exhibiting approximately 80% sequence homology with mammalian TRPC3/6/7. Northern and Western blots employing a TRPC3-probe and anti-TRPC3 antibodies, respectively, confirmed endogenous expression of a TRPC3-like protein of 140 kDa. Spectrofluorimetric measurements in Fura-2 loaded cells showed reduced CCE and Mn2+ entry in response to either thapsigargin or 1alpha,25(OH)2D3 upon transfection with anti-TRPC3/6/7 antisense oligodeoxynucleotides (ODNs). Transfection with anti-VDR antisense ODNs diminished 1alpha,25(OH)2D3-dependent Ca2+ and Mn2+ influx. Co-immunoprecipitation of TRPC3-like protein and VDR under non-denaturating conditions was observed. We propose that endogenous TRPC3-like proteins and the VDR participate in the modulation of CCE by 1alpha,25(OH)2D3 in muscle cells, which could be mediated by an interaction between these proteins.