Reactive oxygen species induced by Streptococcus pyogenes invasion trigger apoptotic cell death in infected epithelial cells

Streptococcus pyogenes (group A streptococcus, GAS), one of the most common pathogens of humans, attaches and invades into human pharyngeal or skin epithelial cells. We have previously reported that induction of apoptosis is associated with GAS invasion, which induces mitochondrial dysfunction and apoptotic cell death. We demonstrate here that GAS‐induced apoptosis is mediated by reactive oxygen species (ROS) production. Both the induction of apoptosis and ROS production markedly increased upon invasion of wild‐type GAS strain JRS4 into HeLa cells; however, the apoptotic response was not observed in fibronectin‐binding protein F1‐disrupted mutant SAM1‐infected cells. In Bcl‐2‐overexpressing HeLa cells (HBD98‐2‐4), the induction of apoptosis, ROS production and mitochondrial dysfunction were significantly suppressed, whereas the numbers of invaded GAS was not different between HeLa (mock cells) and the HeLa HBD98‐2‐4 cells. Whereas Rac1 activation occurred during GAS invasion, ROS production in GAS‐infected cells was clearly inhibited by transfection with the Rac1 mutants (L37 or V12L37), but not by the dominant active mutant (V12L61) or by the dominant negative mutant (N17). These observations indicate that GAS invasion triggers ROS production through Rac1 activation and generated ROS induced mitochondrial dysfunction leading to cellular apoptosis.     

Expression profiles of apoptosis genes in mammary epithelial cells

To investigate apoptosis in HC11 mammary epithelial cells, we compared the gene expression profiles of actively growing and serum-starved apoptotic cells using a mouse apoptosis gene array and 33P-labeled cDNA prepared from the RNA of the two cultures. Analysis of the arrays showed that expression of several genes such as clusterin, secreted frizzled related protein mRNA (sFRP-1), CREB-binding protein (CBP), and others was higher in the apoptotic cells whereas expression of certain genes including survivin, cell division cycle 2 homolog A (CDC2), and cyclin A was lower. These expression patterns were confirmed by RT-PCR and/or Northern analyses. We compared the expression of some of these genes in the mouse mammary gland under various physiological conditions. The expression levels of genes (clusterin, CBP, and M6P-R) up-regulated in apoptotic conditions were higher at involution than during lactation. On the other hand, genes (Pin, CDC2) downregulated in apoptotic conditions were relatively highly expressed in virgin and pregnant mice. We conclude that certain genes such as clusterin, sFRP-1, GAS1 and CBP are induced in apoptotic mammary epithelial cells, and others are repressed. Moreover, the apoptosis array is an efficient technique for comparing gene expression profiles in different states of the same cell type.     

Hypoxia induces p53-dependent transactivation and Fas/CD95-dependent apoptosis

p53 triggers apoptosis in response to cellular stress. We analyzed p53-dependent gene and protein expression in response to hypoxia using wild-type p53-carrying or p53 null HCT116 colon carcinoma cells. Hypoxia induced p53 protein levels and p53-dependent apoptosis in these cells. cDNA microarray analysis revealed that only a limited number of genes were regulated by p53 upon hypoxia. Most classical p53 target genes were not upregulated. However, we found that Fas/CD95 was significantly induced in response to hypoxia in a p53-dependent manner, along with several novel p53 target genes including ANXA1, DDIT3/GADD153 (CHOP), SEL1L and SMURF1. Disruption of Fas/CD95 signalling using anti-Fas-blocking antibody or a caspase 8 inhibitor abrogated p53-induced apoptosis in response to hypoxia. We conclude that hypoxia triggers a p53-dependent gene expression pattern distinct from that induced by other stress agents and that Fas/CD95 is a critical regulator of p53-dependent apoptosis upon hypoxia.     

Stretch-activated signaling pathways responsible for early response gene expression in fetal lung epithelial cells

High-tidal volume ventilation has been shown to increase the expression of several inflammation-associated genes prior to overt physiologic lung injury. Herein, using an in vitro stretch system, we investigated the mechanotransduction pathways involved in ventilation-induced expression of these early response genes (i.e., early growth response gene (Egr)1, heat-shock protein (HSP)70, and the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6, and MIP-2). Mechanical stretch of fetal lung epithelial cells activated various signaling pathways, resulting in transient or progressive increases in gene expression of the early response genes. The transient increase in Egr1 and IL-6 expression was mediated via p44/42 mitogen-activated protein kinase (p44/42 MAPK), while nuclear factor-kappaB (NF-kappaB) was responsible for the sustained and progressive increase in expression of HSP70 and MIP-2. Blockage of Egr-1 expression did not affect the upregulation of IL-6, HSP70, MIP-2, and itself by stretch. Inhibition of calcium mobilization abolished stretch-induced p44/42 MAPK activation and NF-kappaB nuclear translocation as well as increased expression of all early response genes. Similar results were obtained with an inhibitor of Ras. These results suggest that mechanical stretch of fetal lung epithelial cells evokes a complex network of signaling molecules, which diverge downstream to regulate the temporal expression of a unique set of early response genes, but upstream converge at calcium. Thus, calcium mobilization may be a point of hierarchical integration of mechanotransduction in lung epithelial cells.     

Hepatitis B virus x gene-downregulated growth-arrest specific 5 inhibits the cell viability and invasion of hepatocellular carcinoma cell lines by activating Y-box-binding protein 1/p21 signaling

The long noncoding RNA growth-arrest specific 5 (GAS5) is a suppressor of many cancers. However, the role and mechanism of action of GAS5 in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remain unclear. Here, the expression of hepatitis B virus x gene (HBx) mRNA and GAS5 was assessed by qRT–PCR, and western blot analysis was performed to determine the protein expression levels. In addition, the cell viability and invasion of cells were confirmed using  MTT assay and Transwell assay, respectively. The DNA methylation level of GAS5 was measured by methylation-specific PCR. Moreover, RIP assay and RNA pull down assay were carried out to examine the combination of Y-box-binding protein 1 (YBX1) and GAS5. First, our data proved that HBx is increased, while GAS5 is decreased in HCC cell lines. Subsequently, we found that HBx facilitates HCC cell viability and invasion by inhibiting GAS5 expression. Then, we further clarified that HBx induces the DNA methylation of GAS5 by promoting methyltransferase expression, thereby suppressing GAS5 expression. Furthermore, GAS5 binds YBX1 and promotes YBX1 and p21 expression. Finally, the functional analysis revealed that the upregulation of GAS5 could attenuate cell viability and invasion by boosting p21 expression via binding YBX1. Overall, our results demonstrated that HBx promotes HCC progression by inducing GAS5 methylation to reduce its expression. The upregulation of GAS5 suppressed HBV-related HCC by activating YBX1/p21 signaling. Our data provide novel evidence supporting the potential of GAS5 as a treatment target in HBV-related HCC.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00645-z.     

Intradermal delivery of IL-12 naked DNA induces systemic NK cell activation and Th1 response in vivo that is independent of endogenous IL-12 production.

In this study four murine IL-12 naked DNA expression plasmids (pIL-12), containing both the p35 and p40 subunits, were shown to induce systemic biological effects in vivo after intradermal injection. Three of the four IL-12 expression vectors augmented NK activity and induced expression of the IFN-gamma and IFN-gamma-inducible Mig genes. Both IL-12 p70 heterodimer and IFN-gamma proteins were documented in the serum within 24 h after intradermal injection of the pIL-12o- plasmid, which also induced the highest level of NK activity in the spleen and liver among the IL-12 constructs. Interestingly, both p40 mRNA expression at the injection site and serum protein levels followed a biphasic pattern of expression, with peaks on days 1 and 5. Subsequent studies revealed that the ability of intradermally injected pIL-12o- to augment NK lytic activity was prevented by administration of a neutralizing anti-IL-12 mAb. Finally, injection of the pIL-12o- into BALB/c IL-12 p40-/- mice also resulted in a biphasic pattern of IL-12 p70 appearance in the serum, and induced IFN-gamma protein and activated NK lytic activity in liver and spleen. These results demonstrate that injection of delivered naked DNA encoding the IL-12 gene mediates the biphasic systemic production of IL-12-inducible genes and augments the cytotoxic function of NK cells in lymphoid and parenchymal organs as a direct result of transgene expression. The results also suggest that these naked DNA plasmids may be useful adjuvants for vaccines against infectious and neoplastic diseases.     

Caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis

Phosphorylation and dephosphorylation are important cellular events regulating major metabolic activities such as signal transduction, gene expression, cell cycle progression, and apoptosis. It is well documented that okadaic acid, a potent inhibitor of protein phosphatase-1 (PP-1) and -2A (PP-2A), can induce apoptosis in a variety of cell lines. Our recent studies have revealed that in the immortal rabbit lens epithelial cell line, N/N1003A, inhibition of PP-1, but not PP-2A, leads to rapid apoptosis of the lens epithelial cells. This induction of cell death is associated with up-regulated expression of a set of genes, including the tumor-suppressor gene, p53, and the proapoptotic gene, bax. In the present study, we demonstrate that inhibition of PP-1 by okadaic acid in the primary cultures of rat lens epithelial cells also leads to apoptotic death. Moreover, we show that the cysteine protease, caspase-3, is important in the execution of okadaic acid-induced apoptosis. Treatment of the primary cultures of rat lens epithelial cells with 100 nM okadaic acid up-regulates expression of caspase-3 at the mRNA, protein, and enzyme activity levels. Inhibition of the caspase-3 activity with a chemically synthesized inhibitor prevents okadaic acid-induced apoptosis in rat lens epithelial cells. Similar results are also observed in the immortal cell line N/N1003A. Furthermore, stable expression of the mouse gene encoding lens alphaB crystallin inhibits okadaic acid-induced apoptosis, and this inhibition is associated with repression of the okadaic acid-induced up-regulation of caspase-3 activity. Taken together, these results demonstrate that caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis.     

Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells

Epithelial cells are the initial sites of host invasion by group A Streptococcus pyogenes (GAS), and their infection of epithelial cells has been suggested to induce apoptosis. However, the mechanism responsible for bacteria–host interaction and the induction of apoptosis has not been clearly understood. We demonstrate here that human pharyngeal epithelial HEp-2 cells became apoptotic with DNA fragmentation by invasion of GAS strains JRS4 (M6⁺, F1⁺) and JRS145 (M6⁻, F1⁺ mutant of JRS4), whereas apoptotic cellular changes were not observed in SAM1 (M6⁺, F1⁻ mutant) or SAM2 (M6⁻, F1⁻ mutant) infected HEp-2 cells. Confocal microscopy revealed that Bax translocation to mitochondria and cytochrome c release occurred after 4 h of infection. Western blot analyses showed that the amounts of Bcl-2 and Bcl-x_L were decreased in the mitochondria of infected cells. In addition, we demonstrated that the release of nuclear histone from infected cells was prevented by the addition of caspase-9 inhibitor (Ac-LEHD-CHO). We conclude that the internalization of GAS in epithelial cells is necessary and sufficient for the induction of apoptosis, which is initiated by mitochondrial dysfunction, and the mechanism of GAS-induced apoptosis is clearly different from that induced by other intracellular invasive bacteria, e.g. Shigella and Salmonella species.     

Differential role for p38 mitogen-activated protein kinase in regulating CD40-induced gene expression in dendritic cells and B cells

We investigated whether human monocyte-derived dendritic cells (DCs) differed from tonsillar B cells in the set of cell fate genes they express constitutively and in the way these genes are affected after CD40 ligation. In particular, Bcl-2, TNF receptor-associated factor-2 (TRAF2), and TRAF4 were clearly inducible via CD40 in B cells but not in DCs. DCs, unlike B cells, were induced to increase expression of IL-1beta, IL-1Ra, IL-8, IL-12 p40, RANTES, macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1 after CD40 ligation. We next tested whether CD40-induced signaling pathways were different in DCs vs B cells. In DCs, as in B cells, CD40 ligation activated p38 mitogen-activated protein kinase (MAPK), its downstream target, MAPKAPK-2, and the c-Jun N-terminal kinase. The p38 MAPK-specific inhibitor, SB203580, blocked CD40-induced MAPKAPK-2 activation, but did not affect activation of c-Jun N-terminal kinase. Furthermore, unlike in B cells, extracellular signal-regulated kinase-1 and -2 were activated after CD40 ligation in DCs. SB203580 strongly blocked CD40-induced IL-12 p40 production in DCs at both mRNA and protein levels, while having minimal effect on CD40-induced expression of the chemokine RANTES. In contrast, no detectable IL-12 p40 protein was secreted in CD40-stimulated B cells. Furthermore, CD40-induced mRNA expression of cellular inhibitor of apoptosis protein-2 was also dependent on the p38 MAPK pathway in DCs and differed compared with that in B cells. In conclusion, CD40 induces distinct programs in DCs and B cells, and the set of p38 MAPK-dependent genes in DCs (IL-12 p40 and cellular inhibitor of apoptosis protein-2) is different from that in B cells (IL-10 and IL-1beta).     

高毒力A组链球菌致病机制研究进展

A组链球菌(Group A Streptococcus,GAS)常导致咽炎和皮肤感染,也能引起严重侵袭性感染.根据其表面M蛋白编码基因emm可将GAS分为200多型,严重侵袭性感染多由高毒力株引起,以emm1、emm3、emm12、emm28和emm89型常见.研究发现高毒力GAS株中CovRS基因突变可导致细菌逃逸固...     

Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms

Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration, and matrix synthesis. However, SMCs within atherosclerotic plaques can also express a number of proinflammatory genes, and in cultured SMCs the inflammatory cytokine IL-1β represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1β modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1β- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1β distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1β exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1β-induced upregulation of proinflammatory genes as well as repression of SMC differentiation marker genes. Interestingly, PDGF-DD-induced SMC marker gene repression was not NF-κB dependent. Finally, immunofluorescent staining of mouse atherosclerotic lesions revealed the presence of cells positive for the marker of an IL-1β-stimulated inflammatory SMC, chemokine (C-C motif) ligand 20 (CCL20), but not the PDGF-DD-induced gene, regulator of G protein signaling 17 (RGS17). Results demonstrate that IL-1β- but not PDGF-DD-induced phenotypic modulation of SMC is characterized by NF-κB-dependent activation of proinflammatory genes, suggesting the existence of a distinct inflammatory SMC phenotype. In addition, studies provide evidence for the possible utility of CCL20 and RGS17 as markers of inflammatory and proliferative state SMCs within atherosclerotic plaques in vivo.     

GAS6 is an estrogen-inducible gene in mammary epithelial cells

To identify estrogen-responsive genes in mammary glands, microarray assays were performed. Twenty genes were found to be up-regulated while 16 genes were repressed in the 9h estrogen treated glands. The induction of GAS6, one of the genes up-regulated by estrogen, was confirmed by RNase protection assay. Furthermore, GAS6 was also demonstrated to be induced by estrogen in ER positive breast cancer cells. Analysis of GAS6 promoter revealed that GAS6 promoter was regulated by estrogen. An estrogen response element (ERE) was identified in the GAS6 promoter. Electrophoretic mobility shift assay revealed that ERalpha interacted with the ERE in the GAS6 promoter. Chromatin immunoprecipitation demonstrated that ERalpha was recruited to the GAS6 promoter upon estrogen stimulation. These results suggested that GAS6 is an estrogen target gene in mammary epithelial cells.