Type IX collagen is a potent inducer of PGE2 and interleukin 1 production by human monocyte macrophages

Type IX collagen and its collagenous fragments are potent stimulatory agents on human blood mononuclear cells for the production of prostaglandin E2 and interleukin 1/mononuclear cell factor. Type IX collagen is 2-4-fold more potent that type I and II and 1 alpha-, 2 alpha- and 3 alpha-collagens. This property may be important in the destructive process of cartilage in inflammatory diseases.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.     

Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor-1

The coagulation protease thrombin plays a critical role in hemostasis and exerts pro-inflammatory and pro-fibrotic effects via proteolytic activation of the major thrombin receptor, protease-activated receptor-1 (PAR-1). Connective tissue growth factor (CTGF) is a novel fibroblast mitogen and also promotes extracellular matrix protein production. It is selectively induced by transforming growth factor beta (TGF-beta) and is thought to be the autocrine agent responsible for mediating its pro-fibrotic effects. CTGF is up-regulated during tissue repair and in fibrotic conditions associated with activation of the coagulation cascade. We therefore hypothesized that coagulation proteases promote the production of CTGF by cells at sites of tissue injury. To begin to address this hypothesis, we assessed the effect of coagulation proteases on fibroblast CTGF expression in vitro, and we show that thrombin, at physiological concentrations, up-regulated CTGF mRNA levels 5-fold relative to base line (p < 0.01) in fetal fibroblasts and 7-fold in primary adult fibroblasts (p < 0.01). These effects were cycloheximide-insensitive and were not blocked with a pan-specific TGF-beta-neutralizing antibody. They were further paralleled by a concomitant increase in CTGF protein production and could be mimicked with selective PAR-1 agonists. In addition, fibroblasts derived from PAR-1 knockout mice were unresponsive to thrombin but responded normally to TGF-beta(1). Finally, factor Xa, which is responsible for activating prothrombin during blood coagulation, exerted similar stimulatory effects. We propose that coagulation proteases and PAR-1 may play a role in promoting connective tissue formation during normal tissue repair and the development of fibrosis by up-regulating fibroblast CTGF expression.     

BID-D59A is a potent inducer of apoptosis in primary embryonic fibroblasts

The proapoptotic activity of BID seems to solely depend upon its cleavage to truncated tBID. Here we demonstrate that expression of a caspase-8 non-cleavable (nc) BID-D59A mutant or expression of wild type (wt) BID induces apoptosis in Bid -/-, caspase-8 -/-, and wt primary MEFs. Western blot analysis indicated that no cleavage products appeared in cells expressing ncBID. ncBID was as effective as wtBID in inducing cytochrome c release, caspase activation, and apoptosis. ncBID and wtBID (nc/wtBID) were much less effective than tBID in localizing to mitochondria and in inducing cytochrome c release, but only slightly less effective in inducing apoptosis. Studies with Apaf-1- and caspase-9-deficient primary MEFs indicated that both proteins were essential for nc/wtBID and for tBID-induced apoptosis. Most importantly, expression of non-apoptotic levels of either ncBID or wtBID in Bid -/- MEFs induced a similar and significant enhancement in apoptosis in response to a variety of death signals, which was accompanied by enhanced localization of BID to mitochondria and cytochrome c release. Thus, these results implicate full-length BID as an active player in the mitochondria during apoptosis.     

Ethylene as a potent inducer of sexual development

A novel and critical function of ethylene, a potent plant hormone, has been well documented in Dictyostelium, because it leads cells to the sexual development (macrocyst formation) by inducing zygote formation. Zygote formation (sexual cell fusion) and the subsequent nuclear fusion are the characteristic events occurring during macrocyst formation. A novel gene, zyg1 was found to be predominantly expressed during the sexual development, and its enforced expression actually induces zygote formation. As expected, the zygote inducer, ethylene enhances the expression of zyg1. Thus the function of ethylene has been verified at all of individual (macrocyst formation), cellular (zygote formation), and molecular levels (zyg1 expression). Based on our recent studies concerning the behavior and function of the zyg1 product (ZYG1 protein), the signal transduction pathways involved in zygote formation are proposed in this review.     

Chenodeoxycholate is a potent inducer of the permeability transition pore in rat liver mitochondria

Several reports support the concept that bile acids may be cytotoxic during cholestatic disease process by causing mitochondrial dysfunction. Here we report additional data and findings aimed at a better understanding of the involvement of the permeability transition pore (PTP) opening in bile acids toxicity. The mitochondrial PTP is implicated as a mediator of cell injury and death in many situations. In the presence of calcium and phosphate, chenodeoxycholic acid (CDCA) induced a permeability transition in freshly isolated rat liver mitochondria, characterized by membrane depolarization, release of matrix calcium, and osmotic swelling. All these events were blocked by cyclosporine A (CyA) and the calcium uniporter inhibitor ruthenium red (RR). The results suggest that CDCA increases the sensitivity of isolated mitochondria in vitro to the calcium-dependent induction of the PTP.     

Hedgehog signaling is a principal inducer of Myosin-II-driven cell ingression in Drosophila epithelia

Cell constriction promotes epithelial sheet invagination during embryogenesis across phyla. However, how this cell response is linked to global patterning information during organogenesis remains unclear. To address this issue, we have used the Drosophila eye and studied the formation of the morphogenetic furrow (MF), which is characterized by cells undergoing a synchronous apical constriction and apicobasal contraction. We show that this cell response relies on microtubules and F-actin enrichment within the apical domain of the constricting cell as well as on the activation of nonmuscle myosin. In the MF, Hedgehog (Hh) signaling is required to promote cell constriction downstream of cubitus interruptus (ci), and, in this context, Ci155 functions redundantly with mad, the main effector of dpp/BMP signaling. Furthermore, ectopically activating Hh signaling in fly epithelia reveals a direct relationship between the duration of exposure to this signaling pathway, the accumulation of activated Myosin II, and the degree of tissue invagination.     

The combination of apoptotic U937 cells and lupus IgG is a potent IFN-alpha inducer

Patients with active systemic lupus erythematosus (SLE) have signs of an ongoing IFN-alpha production, that may be of pathogenic significance in the disease. We previously showed that SLE patients have an IFN-alpha-inducing factor in blood, probably consisting of complexes containing anti-DNA Abs and immunostimulatory DNA. The DNA component could be derived from apoptotic cells, because SLE patients have been reported to have both increased apoptosis and reduced clearance of apoptotic cell material. In the present study, we therefore investigated whether apoptotic cells, together with IgG from SLE patients, could act as an IFN-alpha inducer in normal PBMC in vitro. We found that apoptotic cells of the myeloid leukemia cell line U937 as well as four other cell lines (MonoMac6, H9, Jurkat, U266) could induce IFN-alpha production in PBMC when combined with IgG from SLE patients. The IFN-alpha production by PBMC was much enhanced when PBMC were costimulated by IFN-alpha2b. The ability of IgG from different SLE patients to promote IFN-alpha induction by apoptotic U937 cells was associated with the presence of anti-ribonucleoprotein Abs, but not clearly with occurrence of anti-DNA Abs. These results suggest that apoptotic cells in the presence of autoantibodies can cause production of a clearly immunostimulatory cytokine, which is IFN-alpha. This mechanism for induction of IFN-alpha production could well be operative also in vivo, explain the IFN-alpha production seen in SLE patients, and be important in the pathogenesis of SLE.     

Chelerythrine is a potent and specific inhibitor of protein kinase C

The benzophenanthridine alkaloid chelerythrine is a potent, selective antagonist of the Ca++/phospholopid-dependent protein kinase (Protein kinase C: PKC) from the rat brain. Half-maximal inhibition of the kinase occurs at 0.66 microM. Chelerythrine interacted with the catalytic domain of PKC, was a competitive inhibitor with respect to the phosphate acceptor (histone IIIS) (Ki = 0.7 microM) and a non-competitive inhibitor with respect to ATP. This effect was further evidenced by the fact that chelerythrine inhibited native PKC and its catalytic fragment identically and did not affect [3H]- phorbol 12,13 dibutyrate binding to PKC. Chelerythrine selectively inhibited PKC compared to tyrosine protein kinase, cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase. The potent antitumoral activity of celerythrine measured in vitro might be due at least in part to inhibition of PKC and thus suggests that PKC may be a model for rational design of antitumor drugs.     

Musk xylene is a novel specific inducer of cytochrome P-450IA2.

The effect of musk xylene on contents of both cytochrome P-450IA1 and cytochrome P-450IA2 in rat liver was investigated using Western blotting analysis. Rats were treated i.p. for five consecutive days with either 50, 100 or 200 mg musk xylene/kg body weight. Musk xylene increased both total cytochrome P-450 and cytochrome b5 contents in rat liver microsomes. Musk xylene induced cytochrome P-450IA2 (384 pmol/mg protein) strongly and preferentially and the ratio of cytochrome P450IA2/P-450IA1 was about 12 at the lowest dose tested. Musk xylene also induced the cytochrome P-450IA1 dose-dependently, but these extents were very small (32-174 pmol/mg protein). These results suggest that musk xylene may be a more specific inducer for cytochrome P-450IA2 than any other inducers reported.     

The saposin-like domain of the plant aspartic proteinase precursor is a potent inducer of vesicle leakage

A unique feature of plant aspartic proteinase precursors is the presence of an internal domain, known as plant-specific insert, whose function is not completely understood. The three-dimensional structure of the plant-specific insert resembles that of saposin-like proteins, a group of lipid-binding proteins involved in a variety of physiological processes. Here we show that recombinant plant-specific insert is able to interact with phospholipid vesicles and to induce leakage of their contents in a pH- and lipid-dependent manner. The leakage activity is higher at pH 4.5 and requires the presence of acidic phospholipids such as phosphatidylserine. To determine whether the same effect could be observed when the plant-specific insert is part of the precursor form, procardosin A and a mutant form lacking this specific domain were produced and characterized. Procardosin A displays a similar activity profile, whereas the mutant without the plant-specific insert shows only residual activity. These findings indicate that the plant-specific insert domain of plant aspartic proteinases mediates an interaction of their precursors with phospholipid membranes and induces membrane permeabilization. It is therefore possible that the plant-specific insert, alone or in conjunction with the proteolytic activity of plant aspartic proteinases, may function either as a defensive weapon against pathogens or in late autolysis of plant cells.     

Histamine is a potent inducer of IL-18 and IFN-gamma in human peripheral blood mononuclear cells

Histamine (10-7 to 10-4 M) concentration-dependently stimulated the production of IL-18 and IFN-gamma and inhibited the production of IL-2 and IL-10 in human PBMCs. Histamine in the same concentration range did not induce the production of IL-12 at all. The stimulatory or inhibitory effects of histamine on cytokine production were all antagonized by H2 receptor antagonists ranitidine and famotidine in a concentration-dependent manner, but not by H1 and H3 receptor antagonists. Selective H2 receptor agonists, 4-methylhistamine and dimaprit, mimicked the effects of histamine on five kinds of cytokine production. The EC50 values of histamine, 4-methylhistamine, and dimaprit for the production of IL-18 were 1.5, 1.0, and 3.8 microM, respectively. These findings indicated that histamine caused cytokine responses through the stimulation of H2 receptors. All effects of histamine on cytokine responses were also abolished by the presence of either anti-IL-18 Ab or IL-1beta-converting enzyme/caspase-1 inhibitor, indicating that the histamine action is dependent on mature IL-18 secretion and that IL-18 production is located upstream of the cytokine cascade activated by histamine. The addition of recombinant human IL-18 to the culture concentration-dependently stimulated IL-12 and IFN-gamma production and inhibited the IL-2 and IL-10 production. IFN-gamma production induced by IL-18 was inhibited by anti-IL-12 Ab, showing the marked contrast of the effect of histamine. Thus histamine is a very important modulator of Th1 cytokine production in PBMCs and is quite unique in triggering IL-18-initiating cytokine cascade without inducing IL-12 production.     

Hypoxia-induced MIR155 is a potent autophagy inducer by targeting multiple players in the MTOR pathway

Hypoxia activates autophagy, an evolutionarily conserved cellular catabolic process. Dysfunction in the autophagy pathway has been implicated in an increasing number of human diseases, including cancer. Hypoxia induces upregulation of a specific set of microRNAs (miRNAs) in a variety of cell types. Here, we describe hypoxia-induced MIR155 as a potent inducer of autophagy. Enforced expression of MIR155 increases autophagic activity in human nasopharyngeal cancer and cervical cancer cells. Knocking down endogenous MIR155 inhibits hypoxia-induced autophagy. We demonstrated that MIR155 targets multiple players in MTOR signaling, including RHEB, RICTOR, and RPS6KB2. MIR155 suppresses target-gene expression by directly interacting with their 3′ untranslated regions (UTRs), mutations of the binding sites abolish their MIR155 responsiveness. Furthermore, by downregulating MTOR signaling, MIR155 also attenuates cell proliferation and induces G₁/S cell cycle arrest. Collectively, these data present a new role for MIR155 as a key regulator of autophagy via dysregulation of MTOR pathway.     

Interleukin-6 is a potent inducer of S100P, which is up-regulated in androgen-refractory and metastatic prostate cancer

Elevated circulating interleukin-6 (IL6) and up-regulated S100P in prostate cancer (PCa) specimens correlate independently with progression to androgen-independent and metastatic PCa. The cause of up-regulated S100P levels in advanced PCa remains to be determined. We investigated the possibility that IL6 is an inducer of S100P. Determination of mRNA and protein levels by real-time PCR and Western blotting revealed that IL6 is a more potent inducer of S100P than the synthetic androgen, R1881, in the LNCaP/C4-2B model of PCa progression. IL6 did not require androgen to induce S100P in these cells, which express a functional androgen receptor (AR). Like R1881, IL6 was unable to induce S100P in PC3 cells that lack a functional AR. IL6 did not strongly induce the AR-dependent genes PSA and KLK2 and, contrary to R1881, down-regulated Cyr61/CCN1, a potential marker that is down-regulated in PCa. Epidermal growth factor (EGF), which like IL6 is a non-androgen activator of the AR, did not induce S100P. The data identifies a unique gene-induction profile for IL6 and suggests that IL6 may require a functional AR for S100P induction. A link between elevated IL6 and up-regulated S100P in androgen-refractory and metastatic PCa is postulated.     

R-etodolac is a more potent Wnt signaling inhibitor than enantiomer,S-etodolac

Etodolac is an FDA-approved nonsteroidal anti-inflammatory drug (NSAID) used to treat a variety of inflammatory diseases. The drug is administered as a racemate (50/50 mixture of R- and S- enantiomers), however, studies have shown that the two enantiomers have distinct biologic and pharmacokinetic differences. Wnt signaling, which plays key roles in cell proliferation, polarity, and differentiation, has been shown to be inhibited by R-etodolac; however, comparative analyses of R- and S-etodolac in this function have not been conducted. We used in silico molecular docking and TOPflash functional biologic assays to compare R- and S-enantiomers effect on Wnt signaling inhibition. Further, we used a cultivated limbal stem epithelial cell (cLSCs) model to investigate enantiospecific changes in the colony-forming efficiency (CFE) of cLSCs. The data shows that R-etodolac is a more potent inhibitor of Wnt signaling. In addition, consistently, while both enantiomers demonstrate a dose-dependent decrease in CFE of cLSCs, R-etodolac is a more potent inhibitor.