Proteolytic Histone Modification by Mast Cell Tryptase,a Serglycin Proteoglycan-dependent Secretory Granule Protease

A hallmark feature of mast cells is their high content of cytoplasmic secretory granules filled with various preformed compounds, including proteases of tryptase-, chymase-, and carboxypeptidase A3 type that are electrostatically bound to serglycin proteoglycan. Apart from participating in extracellular processes, serglycin proteoglycan and one of its associated proteases, tryptase, are known to regulate cell death by promoting apoptosis over necrosis. Here we sought to outline the underlying mechanism and identify core histones as primary proteolytic targets for the serglycin-tryptase axis. During the cell death process, tryptase was found to relocalize from granules into the cytosol and nucleus, and it was found that the absence of tryptase was associated with a pronounced accumulation of core histones both in the cytosol and in the nucleus. Intriguingly, tryptase deficiency resulted in defective proteolytic modification of core histones even at baseline conditions, i.e. in the absence of cytotoxic agent, suggesting that tryptase has a homeostatic impact on nuclear events. Indeed, tryptase was found in the nucleus of viable cells and was shown to cleave core histones in their N-terminal tail. Moreover, it was shown that the absence of the serglycin-tryptase axis resulted in altered chromatin composition. Together, these findings implicate histone proteolysis through a secretory granule-derived serglycin-tryptase axis as a novel principle for histone modification, during both cell homeostasis and cell death.     

Serglycin proteoglycan promotes apoptotic versus necrotic cell death in mast cells

The mechanisms that govern whether a cell dies by apoptosis or necrosis are not fully understood. Here we show that serglycin, a secretory granule proteoglycan of hematopoietic cells, can have a major impact on this decision. Wild type and serglycin(-/-) mast cells were equally sensitive to a range of cell death-inducing regimens. However, whereas wild type mast cells underwent apoptotic cell death, serglycin(-/-) cells died predominantly by necrosis. Investigations of the underlying mechanism revealed that cell death was accompanied by leakage of secretory granule compounds into the cytosol and that the necrotic phenotype of serglycin(-/-) mast cells was linked to defective degradation of poly(ADP-ribose) polymerase-1. Cells lacking mouse mast cell protease 6, a major serglycin-associated protease, exhibited similar defects in apoptosis as observed in serglycin(-/-) cells, indicating that the pro-apoptotic function of serglycin is due to downstream effects of proteases that are complex-bound to serglycin. Together, these findings implicate serglycin in promoting apoptotic versus necrotic cell death.     

A role for serglycin proteoglycan in granular retention and processing of mast cell secretory granule components

In the absence of serglycin proteoglycans, connective tissue-type mast cells fail to assemble mature metachromatic secretory granules, and this is accompanied by a markedly reduced ability to store neutral proteases. However, the mechanisms behind these phenomena are not known. In this study, we addressed these issues by studying the functionality and morphology of secretory granules as well as the fate of the secretory granule proteases in bone marrow-derived mast cells from serglycin(+/+) and serglycin(-/-) mice. We show that functional secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defective in serglycin(-/-) mast cell granules. The low levels of mast cell proteases present in serglycin(-/-) cells had a granular location, as judged by immunohistochemistry, and were released following exposure to calcium ionophore, indicating that they were correctly targeted into secretory granules even in the absence of serglycin. In the absence of serglycin, the fates of the serglycin-dependent proteases differed, including preferential degradation, exocytosis or defective intracellular processing. In contrast, beta-hexosaminidase storage and release was not dependent on serglycin. Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive pathway of secretion, but rather that serglycin may be involved in the retention of the proteases after their entry into secretory vesicles.     

Dual targets for mouse mast cell protease-4 in mediating tissue damage in experimental bullous pemphigoid

Mouse mast cell protease-4 (mMCP-4) has been linked to autoimmune and inflammatory diseases, although the exact mechanisms underlying its role in these pathological conditions remain unclear. Here, we have found that mMCP-4 is critical in a mouse model of the autoimmune skin blistering disease bullous pemphigoid (BP). Mice lacking mMCP-4 were resistant to experimental BP. Complement activation, mast cell (MC) degranulation, and the early phase of neutrophil (PMN) recruitment occurred comparably in mMCP-4(-/-) and WT mice. However, without mMCP-4, activation of matrix metalloproteinase (MMP)-9 was impaired in cultured mMCP-4(-/-) MCs and in the skin of pathogenic IgG-injected mMCP-4(-/-) mice. MMP-9 activation was not fully restored by local reconstitution with WT or mMCP-4(-/-) PMNs. Local reconstitution with mMCP-4(+/+) MCs, but not with mMCP-4(-/-) MCs, restored blistering, MMP-9 activation, and PMN recruitment in mMCP-4(-/-) mice. mMCP-4 also degraded the hemidesmosomal transmembrane protein BP180 both in the skin and in vitro. These results demonstrate that mMCP-4 plays two different roles in the pathogenesis of experimental BP, by both activating MMP-9 and by cleaving BP180, leading to injury of the hemidesmosomes and extracellular matrix of the basement membrane zone.     

A role for the interleukin-1 receptor in the pathway linking static mechanical compression to decreased proteoglycan synthesis in surface articular cartilage

Loading of articular cartilage during weight bearing is essential for the maintenance of cartilage function. Although certain cyclic loading protocols stimulate extracellular matrix synthesis, constant or static compression decreases proteoglycan and collagen synthesis in cartilage explants. The goal of this study was to determine whether the compression-induced decrease in proteoglycan synthesis involves an interleukin-1 (IL-1) signaling pathway. Cartilage explants were compressed 50% in the presence of IL-1 receptor antagonist (IL-1ra), and the incorporation of [35S]sulfate into macromolecules was measured. IL-1ra increased sulfate incorporation in compressed cartilage but not in cartilage maintained at the in situ thickness (0% compression). IL-1alpha and IL-1beta mRNAs were detected in cartilage compressed 50% for at least 3h, while nitric oxide synthase II mRNA was only detected in cartilage compressed 50% for 6h. The data support a role for the IL-1 receptor in the pathway linking static compression to reduced proteoglycan synthesis.     

BH3-only activator proteins Bid and Bim are dispensable for Bak/Bax-dependent thrombocyte apoptosis induced by Bcl-xL deficiency: molecular requisites for the mitochondrial pathway to apoptosis in platelets

A pivotal step in the mitochondrial pathway of apoptosis is activation of Bak and Bax, although the molecular mechanism remains controversial. To examine whether mitochondrial apoptosis can be induced by just a lack of antiapoptotic Bcl-2-like proteins or requires direct activators of the BH3-only proteins including Bid and Bim, we studied the molecular requisites for platelet apoptosis induced by Bcl-xL deficiency. Severe thrombocytopenia induced by thrombocyte-specific Bcl-xL knock-out was fully rescued in a Bak and Bax double knock-out background but not with single knock-out of either one. In sharp contrast, deficiency of either Bid, Bim, or both did not alleviate thrombocytopenia in Bcl-xL knock-out mice. An in vitro study revealed that ABT-737, a Bad mimetic, induced platelet apoptosis in association with a conformational change of the amino terminus, translocation from the cytosol to mitochondria, and homo-oligomerization of Bax. ABT-737-induced Bax activation and apoptosis were also observed in Bid/Bim-deficient platelets. Human platelets, upon storage, underwent spontaneous apoptosis with a gradual decline of Bcl-xL expression despite a decrease in Bid and Bim expression. Apoptosis was attenuated in Bak/Bax-deficient or Bcl-xL-overexpressing platelets but not in Bid/Bim-deficient platelets upon storage. In conclusion, platelet lifespan is regulated by a fine balance between anti- and proapoptotic multidomain Bcl-2 family proteins. Despite residing in platelets, BH3-only activator proteins Bid and Bim are dispensable for Bax activation and mitochondrial apoptosis.     

A role for mixed lineage kinases in granule cell apoptosis induced by cytoskeletal disruption

Microtubule disruption by colchicine induces apoptosis in selected neuronal populations. However, little is known about the upstream death signalling events mediating the neurotoxicity. We investigated first whether colchicine-induced granule cell apoptosis activates the c-Jun N-terminal kinase (JNK) pathway. Cultured murine cerebellar granule cells were exposed to 1 microm colchicine for 24 h. Activation of the JNK pathway was detected by western blotting as well as immunocytochemistry using antibodies against phospho-c-Jun (p-c-Jun). Next, adult male rats were injected intracerebroventricularly with colchicine (10 microg), and JNK pathway activation in dentate granule cells (DGCs) was detected by antibodies against p-c-Jun. The second part of the study tested the involvement of mixed lineage kinases (MLK) as upstream activators of the JNK pathway in colchicine toxicity, using CEP-1347, a potent MLK inhibitor. In vitro, significant inhibition of the JNK pathway, activated by colchicine, was achieved by 100-300 nm CEP-1347, which blocked both activation of cell death proteases and apoptosis. Moreover, CEP-1347 markedly delayed neurite fragmentation and cell degeneration. In vivo, CEP-1347 (1 mg/kg) significantly prevented p-c-jun increase following injection of colchicine, and enhanced survival of DGCs. We conclude that colchicine-induced neuronal apoptosis involves the JNK/MLK pathway, and that protection of granule cells can be achieved by MLK inhibition.     

Pathway analysis identifies perturbation of genetic networks induced by butyrate in a bovine kidney epithelial cell line

Ruminant species have evolved to metabolize the short-chain volatile fatty acids (VFA), acetate, propionate, and butyrate, to fulfill up to 70% of their nutrient energy requirements. The inherent VFA dependence of ruminant cells was exploited to add a level of increased sensitivity to the study of the role of butyrate gene-response elements in regulatory biochemical pathways. Global gene expression profiles of the bovine kidney epithelial cells regulated by sodium butyrate were investigated with high-density oligonucleotide microarrays. The detailed mechanisms by which butyrate induces cell growth arrest and apoptosis were analyzed using the Ingenuity Pathways Knowledge Base. The functional category and pathway analyses of the microarray data revealed that four canonical pathways (Cell cycles: G2/M DNA damage checkpoint, and pyrimidine metabolism; G1/S checkpoint regulation and purine metabolism) were significantly perturbed. The biologically relevant networks and pathways of these genes were also identified. IGF2, TGFB1, TP53, E2F4, and CDC2 were established as being centered in these genomic networks. The present findings provide a basis for understanding the full range of the biological roles and the molecular mechanisms that butyrate may play in animal cell growth, proliferation, and energy metabolisms. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Mention of trade names or commercial products in this publication is solely for providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

Erybraedin C and bitucarpin A, two structurally related pterocarpans purified from Bituminaria bituminosa, induced apoptosis in human colon adenocarcinoma cell lines MMR- and p53-proficient and -deficient in a dose-, time-, and structure-dependent fashion

Pterocarpans, the second group of natural isoflavonoids, have received considerable interest on account of their medicinal properties. These drugs are employed as antitoxins, but display antifungal, antiviral and antibacterial properties as well. Erybraedin C and bitucarpin A are two new structurally related pterocarpans recently purified and characterized. Bitucarpin A differs from erybraedin C for the absence of a prenyl group in 5' position and the presence of a methoxylate hydroxyl group in 7, 4' positions. These compounds proved not to be clastogens in human lymphocytes per se but displayed anticlastogenic activity against mytomicin C and bleomycin C. Here we extended the study of their antiproliferative and apoptosis-inducing mechanism on human cell lines. Two human adenocarcinoma cell lines, LoVo and HT29, as examples of slow-growing solid tumors, proficient and deficient in mismatch repair system (MMR), p53 and Bcl-2, were used to evaluate the cytotoxicity of the drugs and their effects on the cell cycle, measured by flow cytometry. Erybraedin C similarly affects the survival of HT29 (MMR +/+, p53 -/- and Bcl-2 +/+) and LoVo (MMR -/-, p53 +/+ and Bcl-2 -/-) cells (LD(50): 1.94 and 1.73 microg/ml, respectively). By contrast, bitucarpin A exhibits a differential cytotoxicity in the cell lines (LD(50): 6.00 microg/ml, HT29, and 1.84 microg/ml, LoVo). The cell cycle distributions of the LoVo and HT29 cells treated with erybraedin C lacked a specific checkpoint arrest, whereas they underwent a characteristic sub-G(1) peak, time- and drug-concentration dependent. So that apoptotic process induced by erybraedin C in both adenocarcinoma cell lines is independent of cell cycle arrest and of phenotypic status of the cells as well. By contrast, bitucarpin A affects cell cycle progression on both cell lines, inducing a transient block in G(0)/G(1) along 24-96 h, and induces apoptosis with a cell density and treatment time dependency. Similar results were obtained with the positive control drug etoposide. The programmed cellular death on human adenocarcinoma cell lines may be efficiently activated, via a topoisomerase II poison pattern, by erybraedin C, the drug containing regio-specific hydroxyl and prenyl groups. The apoptotic effect induced by the methoxylated bitucarpin A proved to be conditioned by cell density and required higher dose (5-fold-LD(50)) and longer treatment time. The present study provides evidences that erybraedin C may act as a potent growth inhibitory compound, at low and high cell density, comparable to other clinically important antineoplastic natural drugs including etoposide, on human colon adenocarcinoma cells. Bitucarpin A proved less active because it was conditioned by cell density effect, but this finding may represent a clinical advantage against early micrometastatic diseases.     

cDNA microarray analysis reveals chop-10 plays a key role in Sertoli cell injury induced by bisphenol A

We examined the time course of changes in gene expression in detail using cDNA microarray analysis of mouse Sertoli TTE3 cells treated with bisphenol A (BPA). A subtoxic dose of BPA (200 microM) transiently increased intracellular Ca(2+) concentration and time-dependently induced an increase in mRNA level of 78-kDa glucose-regulated protein, indicating that BPA induces endoplasmic reticulum stress. Of the 865 genes analyzed, 31 genes showed increased levels of expression. TaqMan analysis confirmed that the mRNA levels of chop-10, fra-2, c-myc, and ornithine decarboxylase were increased, and showed that chop-10 is the most sensitive gene. The expression level of chop-10 protein and cell injury induced by BPA were significantly reduced in stable TTE3 cells overexpressing full-length chop-10 antisense RNA. We conclude that chop-10 plays a key role in Sertoli cell injury induced by BPA.     

A PKA survival pathway inhibited by DPT-PKI, a new specific cell permeable PKA inhibitor, is induced by T. annulata in parasitized B-lymphocytes

T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI_5–24 and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.     

Different levels of p53 induced either apoptosis or cell cycle arrest in a doxycycline-regulated hepatocellular carcinoma cell line <Emphasis Type="Italic">in vitro</Emphasis>

Induction of p53 gene expression in cancer cells can lead to both cell cycle arrest and apoptosis. To clarify whether the level of p53 expression determines the apoptotic response of hepatocellullar carcinoma (HCC) cells, we assessed the effect of various levels of expression of p53 gene on a p53-deficient HCC cell line, Hep3B, utilizing a doxycycline (Dox)-regulated inducible p53 expression system. Our results showed that apoptosis was induced in HCC cells with high levels of p53 expression. However, lower level of p53 expression induced only cell cycle arrest but not apoptosis. Bax expression was up-regulated following high levels of p53 expression, while bcl-2 expression was not altered by the level of p53 expression. Moreover, p21 expression was observed in both high and low expression of p53. These results suggest the level of p53 expression could determine if the HCC cells would go into cell cycle arrest or apoptosis. Bax may participate, at least in part, in inducing p53-dependent apoptosis and the induction of p21 alone was able to cause cell cycle arrest but not apoptosis.     

Role of Ca2+-activated Cl- channels in the mechanism of apoptosis induced by cyclosporin A in a human hepatoma cell line

The mechanism of apoptosis induced by cyclosporin A (CsA) in a human hepatoma cell line was investigated. CsA induced apoptosis in a dose- and time-dependent manner in HepG2 human hepatoma cells. CsA induced Cl- efflux, which was significantly blocked by niflumic acid (NA), a specific inhibitor, and flufenamic acid (FA), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), and 4,4'-diisothiocyanoto-stibene-2,2'-disulfonic acid (DIDS), non-specific inhibitors of Ca2+-activated Cl- channels (CaCCs), not by calyculin A, an inhibitor of K+,Cl- -cotransport. In addition, CsA did not alter intracellular K+ concentration. Moreover, CsA increased intracellular Ca2+ concentration, and treatment with BAPTA/AM, an intracellular Ca2+ chelator, significantly inhibited the CsA-induced Cl- efflux, indicating that CsA induced Cl- efflux through the activation of CaCCs. Treatment with these CaCC inhibitors (NA, FA, NPPB, and DIDS) markedly prevented the CsA-induced apoptosis. Taken together, these results suggest that CaCCs may mediate apoptosis induced by CsA in HepG2 cells. Furthermore, these results provide a new insight into the novel function of CaCCs in the regulation of cancer cell apoptosis associated with perturbation of intracellular Ca2+ signal.     

The role of reactive oxygen species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells

Diallyl disulfide (DADS), an oil soluble constituent of garlic (Allium sativum), has been reported to cause antimutagentic and anticarcinogenic effects in vitro and in vivo by modulating phases I and II enzyme activities. In recent years, several studies suggested that the chemopreventive effects of DADS can also be attributed to induction of cell cycle arrest and apoptosis in cancer cells. In the present study, we reported that DADS-induced cell cycle arrest at G2/M and apoptosis in human A549 lung cancer cells in a time- and dose-dependent manner. Additionally, a significant increase of intracellular reactive oxygen species (ROS) was induced in A549 cells less than 0.5 h after DADS treatment, indicating that ROS may be an early event in DADS-modulated apoptosis. Treatment of A549 cells with N-acetyl cysteine (NAC) completely abrogated DADS-induced cell cycle arrest and apoptosis. The result indicated that oxidative stress modulates cell proliferation and cell death induced by DADS.     

On the mechanism of palmitic acid-induced apoptosis: the role of a pore induced by palmitic acid and Ca2+ in mitochondria

Palmitic acid (Pal) is known to promote apoptosis (Sparagna G et al (2000) Am J Physiol Heart Circ Physiol 279: H2124–H2132) and its amount in blood and mitochondria increases under some pathological conditions. Yet, the mechanism of the proapoptotic action of Pal has not been elucidated. We present evidence for the involvement of the mitochondrial cyclosporin A-insensitive pore induced by Pal/Ca²⁺ complexes in the apoptotic process. Opening of this pore led to a fall of the mitochondrial membrane potential and the release of the proapoptotic signal cytochrome c. The addition of cytochrome c prevented these effects and recovered membrane potential, which is in contrast to the cyclosporin A-sensitive mitochondrial permeability transition pore. Oleic and linoleic acids prevented the Pal/Ca²⁺-induced pore opening in the intact mitochondria, this directly and significantly correlating with the effect of these fatty acids on Pal-induced apoptosis in cells (Hardy S et al (2003) J Biol Chem 278: 31861–31870). The specific probe for cardiolipin, 10-N-nonyl acridine orange, inhibited formation of this pore.     

A novel functional T cell hybridoma recognizes macrophage cell death induced by bacteria: a possible role for innate lymphocytes in bacterial infection

We have established a novel TCRalphabeta (TCRVbeta6)(+)CD4(-)CD8(-) T cell hybridoma designated B6HO3. When the B6HO3 cells were cocultured with bacterial-infected J774 macrophage-like cells, IFN-gamma production by B6HO3 cells was triggered through direct cell-cell contact with dying J774 cells infected with Listeria monocytogenes (LM), Shigella flexneri, or Salmonella typhimurium that expressed the type III secretion system, but not with intact J774 cells infected with heat-killed LM, nonhemolytic lysteriolysin O-deficient (Hly(-)) LM, plasmid-cured Shigella, or stationary-phase Salmonella. However, the triggering of B6HO3 cells for IFN-gamma production involved neither dying hepatoma cells infected with LM nor dying J774 cells caused by gliotoxin treatment or freeze thawing. Cycloheximide and Abs to H-2K(d), H-2D(d), Ia(d), CD1d, TCRVbeta6, and IL-12 did not inhibit the contact-dependent IFN-gamma response, indicating that this IFN-gamma response did not require de novo protein synthesis in bacterial-infected J774 cells and was TCR and IL-12 independent. Thus, in an as yet undefined way, B6HO3 hybridoma recognizes a specialized form of macrophage cell death resulting from bacterial infection and consequently produces IFN-gamma. Moreover, contact-dependent interaction of minor subsets of splenic alphabeta T cells, including NKT cells with dying LM-infected J774 and bone marrow-derived macrophage (BMM) cells, proved to provide an IFN-gamma-productive stimulus for these minor T cell populations, to which the parental T cell of the B6HO3 hybridoma appeared to belong. Unexpectedly, subsets of gammadelta T and NK cells similarly responded to dying LM-infected macrophage cells. These results propose that innate lymphocytes may possess a recognition system sensing macrophage cell "danger" resulting from bacterial infection.     

Genetic interactions indicate a role for Mdg1p and the SH3 domain protein Bem1p in linking the G-protein mediated yeast pheromone signalling pathway to regulators of cell polarity

The pheromone signal in the yeastSaccharomyces cerevisiae is transmitted by the and subunits of the mating response G-protein. TheSTE20 gene, encoding a protein kinase required for pheromone signal transduction, has recently been identified in a genetic screen for high-gene-dosage suppressors of a partly defective G mutation. The same genetic screen identifiedBEM1, which encodes an SH3 domain protein required for polarized morphogenesis in response to pheromone, and a novel gene, designatedMDG1 (multicopy suppressor ofdefectiveG-protein). TheMDG1 gene was independently isolated in a search for multicopy suppressors of abem1 mutation. TheMDG1 gene encodes a predicted hydrophilic protein of 364 amino acids with a molecular weight of 41 kDa that has no homology with known proteins. A fusion of Mdg1p with the green fluorescent protein fromAequorea victoria localizes to the plasma membrane, suggesting that Mdg1p is an extrinsically bound membrane protein. Deletion ofMDG1 causes sterility in cells in which the wild-type G has been replaced by partly defective G derivatives but does not cause any other obvious phenotypes. The mating defect of cells deleted forSTE20 is partially suppressed by multiple copies ofBEM1 andCDC42, which encodes a small GTP-binding protein that binds to Ste20p and is necessary for the development of cell polarity. Elevated levels ofSTE20 andBEM1 are capable of suppressing a temperature-sensitive mutation inCDC42. This complex network of genetic interactions points to a role for Bem1p and Mdg1p in G-protein mediated signal transduction and indicates a functional linkage between components of the pheromone signalling pathway and regulators of cell polarity during yeast mating.     

MicroRNA-567 inhibits cell proliferation and induces cell apoptosis in A549 NSCLC cells by regulating cyclin-dependent kinase 8

MicroRNA-567 (miR-567) plays a decisive role in cancers whereas its role in non-small cell lung cancer (NSCLC) is still unexplored. This study was therefore planned to explore the regulatory function of miR-567 in A549 NSCLC cells and investigate its possible molecular mechanism that may help in NSCLC treatment. In the current study, miR-567 expression was examined by quantitative real time-polymerase chain reaction (qRT-PCR) in different NSCLC cell lines in addition to normal cell line. A549 NSCLC cells were transfected by miR-567 mimic, miR-567 inhibitor, and negative control siRNA. Cell proliferation was evaluated by MTT and 5-bromo-2′deoxyuridine assays. Cell cycle distribution and apoptosis were studied by flow cytometry. Bioinformatics analysis programs were used to expect the putative target of miR-567. The expression of cyclin-dependent kinase 8 (CDK8) gene at mRNA and protein levels were evaluated by using qRT-PCR and western blotting. Our results found that miR-567 expressions decreased in all the studied NSCLC cells as compared to the normal cell line. A549 cell proliferation was suppressed by miR-567 upregulation while cell apoptosis was promoted. Also, miR-567 upregulation induced cell cycle arrest at sub-G1 and S phases. CDK8 was expected as a target gene of miR-567. MiR-567 upregulation decreased CDK8 mRNA and protein expression while the downregulation of miR-567 increased CDK8 gene expression. These findings revealed that miR-567 may be a tumor suppressor in A549 NSCLC cells through regulating CDK8 gene expression and may serve as a novel therapeutic target for NSCLC treatment.     

A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.