LPS-mediated Apoptosis Research Articles

Inflammatory Signaling via PEIZO1 Engages and Enhances the LPS-Mediated Apoptosis during Clinical Mastitis.

Bovine clinical mastitis is characterized by inflammation and immune responses, with apoptosis of mammary epithelial cells as a cellular reaction to infection. PIEZO1, identified as a mechanotransduction effector channel in nonruminant animals and sensitive to both mechanical stimuli or inflammatory signals like lipopolysaccharide (LPS). However, its role in inflammatory processes in cattle has not been well-documented. The aim of this study was to elucidate the in situ expression of PIEZO1 in bovine mammary gland and its potential involvement in clinical mastitis. We observed widespread distribution and upregulation of PIEZO1 in mammary epithelial cells in clinical mastitis cows and LPS-induced mouse models, indicating a conserved role across species. In vitro studies using mammary epithelial cells (MAC-T) revealed that LPS upregulates PIEZO1. Notably, the effects of PIEZO1 artificial activator Yoda1 increased apoptosis and NLRP3 expression, effects mitigated by PIEZO1 silencing or NLRP3 inhibition. In conclusion, the activation of the PIEZO1-NLRP3 pathway induces abnormal apoptosis in mammary epithelial cells, potentially serving as a regulatory mechanism to combat inflammatory responses to abnormal stimuli.

Chrysophanol exerts a protective effect against sepsis-induced acute myocardial injury through modulating the microRNA-27b-3p/Peroxisomal proliferating-activated receptor gamma axis

ABSTRACT Sepsis, a leading contributor to the death of inpatients, results in severe organ dysfunction as complications. The heart is one of the major organs attacked by sepsis, and the effective control of the inflammatory cascade reaction in sepsis is of great significance in alleviating sepsis-associated acute myocardial injury (S-AMI). Chrysophanol, a natural anthraquinone, has been discovered to carry anti-inflammatory effects. The aim of this paper is to probe the impact of Chrysophanol on S-AMI. An S-AMI model was engineered in rats via CLP. Pathological alterations in the myocardial tissues of rats were monitored. qRT-PCR, ELISA, and western blot measured the profiles of miR-27b-3p, Peroxisomal proliferating-activated receptor gamma (PPARG), inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8), and inflammatory response proteins (NF-κB-p65, MAPK-p38, JNK1/2). Besides, miR-27b-3p mimics were transfected into cardiomyocytes, and the proliferation and apoptosis of cardiomyocytes were examined through MTT and flow cytometry. As evidenced by the experimental outcomes, chrysophanol suppressed sepsis-mediated acute myocardial injury and LPS-mediated apoptosis in myocardial cells and lessened the release of pro-inflammatory cytokines and inflammatory response proteins. Moreover, chrysophanol cramped miR-27b-3p expression and heightened PPARG expression. miR-27b-3p targeted PPARG and restrained its expression. On the other hand, the PPARG agonist (RGZ) partially eliminated the apoptosis and pro-inflammatory responses of myocardial cells elicited by LPS. Therefore, this study revealed that Chrysophanol guarded against sepsis-mediated acute myocardial injury through dampening inflammation and apoptosis via the miR-27b-3p-PPARG axis, adding to the references for treating sepsis-AMI.

Hypaphorine exerts anti-inflammatory effects in sepsis induced acute lung injury via modulating DUSP1/p38/JNK pathway.

Sepsis is a systemic inflammatory response syndrome attributed to infection, while sepsis-induced acute lung injury (ALI) has high morbidity and mortality. Here, we aimed to explore the specific mechanism of hypaphorine's anti-inflammatory effects in ALI. Lipopolysaccharide (LPS) was adopted to construct ALI model both in vivo and in vitro. BEAS-2B cell viability and apoptosis was testified by the MTT assay and flow cytometry. Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed to examine the expression of proinflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-18), and Western blot was adopted to examine the expression of the apoptosis-related proteins (Bax, Bcl2, and Caspase3) and the DUSP1/p38/JNK signaling pathway. At the same time, lung injury score, lactate dehydrogenase (LDH) and myeloperoxidase (MPO) activity were monitored. The dry/wet weight method was used to examine lung edema, and the total protein content in BALF was determined to test pulmonary vascular permeability. As the data suggested, hypaphorine inhibited the LPS-mediated apoptosis of alveolar epithelial cells. What is more, hypaphorine attenuated the expression of inflammatory factors (IL-1β, IL-6, TNF-α, and IL-18) and inactivated the p38/JNK signaling pathway through upregulating DUSP1 in a dose-dependent manner. Meanwhile, DUSP1 knockdown weakened the anti-inflammatory effect of hypaphorine on LPS-mediated lung injury. Furthermore, hypaphorine also relieved LPS induced ALI in rats with anti-inflammatory effects. Taken together, hypaphorine prevented LPS-mediated ALI and proinflammatory response via inactivating the p38/JNK signaling pathway by upregulating DUSP1.

Long non-coding RNA CDKN2B-AS1 enhances LPS-induced apoptotic and inflammatory damages in human lung epithelial cells via regulating the miR-140-5p/TGFBR2/Smad3 signal network

BackgroundSepsis is a complicated disease with systemic inflammation or organ dysfunction, and it is the leading cause of acute lung injury (ALI). Long non-coding RNAs (lncRNAs) have played important roles in the pathogenesis of sepsis. This study was designed to explore the biological function and regulatory mechanism of cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in lipopolysaccharide (LPS)-induced lung injury.MethodsALI model was established after human lung epithelial cell line BEAS-2B was exposed to LPS. CDKN2B-AS1, microRNA-140-5p (miR-140-5p) and transforming Growth Factor Beta Receptor II (TGFBR2) levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was measured using Cell Counting Kit-8 (CCK-8). Cell apoptosis was assessed by caspase3 activity and flow cytometry. Inflammatory cytokines were examined via enzyme-linked immunosorbent assay (ELISA). Protein analysis was performed through western blot. Dual-luciferase reporter, RNA immunoprecipitation (RIP) and pull-down assays were applied to validate the interaction between targets.ResultsCDKN2B-AS1 and TGFBR2 were abnormally upregulated in sepsis patients. Functionally, CDKN2B-AS1 or TGFBR2 knockdown promoted cell growth but inhibited cell apoptosis and inflammatory response in LPS-treated BEAS-2B cells. Moreover, the regulation of CDKN2B-AS1 in LPS-induced cell injury was achieved by increasing the TGFBR2 expression. CDKN2B-AS1 was identified as a miR-140-5p sponge and TGFBR2 was a target of miR-140-5p. Furthermore, CDKN2B-AS1 could regulate the TGFBR2/Smad3 pathway by sponging miR-140-5p.ConclusionsThese results suggested that CDKN2B-AS1 contributed to the LPS-mediated apoptosis and inflammation in BEAS-2B cells via the miR-140-5p/TGFBR2/Smad3 axis.

Rhein protects 5/6 nephrectomized rat against renal injury by reducing inflammation via NF-κB signaling.

Inflammation is well known to play a pivotal role in renal injury. Rhein is a major component of the medicinal Rhubarb. The aim of this study was to investigate whether Rhein protects against renal injury and explore its underlying mechanism. 5/6 nephrectomization (5/6 Nx) was operated on Sprague-Dawley rats. Human kidney tubular epithelial (HK-2) cells were treated with lipopolysaccharide (LPS). The level of blood urea nitrogen (BUN) and serum creatinine (SCr) was examined. Kidney tissues were stained with hematoxylin and eosin to check the morphology. The cell viability was examined. The levels of cytokines and chemokines were measured by ELISA kit. The protein expression was determined by western blot. Rhein significantly decreased SCr and BUN levels in 5/6 Nx rat. The morphologic findings indicated noteworthy amelioration of the damaged renal tissue in Rhein-treated rats. Rhein significantly protects HK-2 cells from LPS-mediated apoptosis. The productions of inflammatory signaling molecules (TNF-α, IL-6 and MCP-1) were inhibited by Rhein. LPS-induced NF-κB activation was also attenuated by Rhein via blocking its nuclear translocation by inhibiting phosphorylation of IκBα. These findings provide evidence that Rhein protect against renal injury, and NF-κB signaling pathway is involved in this protective effect.

MiR-599 regulates LPS-mediated apoptosis and inflammatory responses through the JAK2/STAT3 signalling pathway via targeting ROCK1 in human umbilical vein endothelial cells.

MicroRNA plays an integral role in the development of atherosclerosis. Our study aimed to investigate the roles of miR-599 in lipopolysaccharide (LPS)-induced endothelial damage in human umbilical vein endothelial cells (HUVECs). HUVECs were transfected with a miR-599 mimic and negative control, and then exposed to LPS. The expression of miR-599 was detected by quantitative real time-polymerase chain reaction (RT-qPCR). Cell viability was analyzed by CCK-8 assay and trypan blue exclusion assay; the formation of DNA fragments was tested by Cell Death Detection ELISA Plus kit; the incidence of apoptosis was detected by flow cytometry; the expression of p53 and cleaved-caspase 3 (c-caspase 3) was evaluated by western blot. Moreover, the mRNA levels and concentrations of tumour necrosis factor (TNF)-α, interleukin (IL)-6, ICAM-1 and VCAM-1 were assayed by RT-qPCR and ELISA. The results showed that overexpression of miR-599 increased cell viability, reduced DNA fragments, the incidence of apoptosis, as well as the protein levels of p53 and c-caspase 3 in the presence of LPS. TNF-α, IL-6, ICAM-1 and VCAM-1 mRNA levels and concentrations were also decreased upon miR-599 upregulation. In addition, the dual luciferase reporter assay demonstrated that ROCK1 is a direct target of miR-599. MiR-599 overexpression inhibited ROCK1 expression. Induced expression of ROCK1 reversed the roles of miR-599 in apoptosis and inflammation. The gain function of miR-599 function inhibited activation of the JAK2/STAT3 signalling pathway, which was abrogated by overexpression of ROCK1. Taken together, our results indicate that miR-599 attenuates LPS-caused cell apoptosis and inflammatory responses through the JAK2/STAT3 signalling pathway via targeting ROCK1.

JNK Signaling Pathway Suppresses LPS-Mediated Apoptosis of HK-2 Cells by Upregulating NGAL.

Objective To explore the role of the c-Jun N-terminal kinase (JNK) signaling pathway in upregulated NGAL expression and its antiapoptotic mechanism in lipopolysaccharide (LPS)-mediated renal tubular epithelial cell injury. Methods In vitro, HK-2 cells were divided into five groups (Con, LPS 1 h, LPS 3 h, LPS 6 h, and LPS 12 h groups) based on the time of LPS (10 μM) treatment. NGAL and caspase-3 gene expression levels were detected by RT-PCR to assess dynamic changes. HK-2 cells were pretreated with SP600125 (20 μM) for 2 hours, followed by LPS (10 μM) stimulation for 3 hours. NGAL and caspase-3 gene expression levels were then determined. Results NGAL mRNA was increased significantly within 6 hours, and caspase-3 mRNA was increased within 3 hours after treatment (P < 0.05). Correlation analysis showed a high correlation between their expression (r = 0.448, P < 0.05). After pretreatment with SP600125, mRNA expression of NGAL in the LPS group was inhibited, while that of caspase-3 was increased significantly. The NGAL mRNA expression level in the SB + LPS group was decreased significantly compared with that in the LPS group, but it was slightly higher than that in the SP group (∼1.5 times of that in the Con group). However, caspase-3 mRNA expression was increased significantly in the SB + LPS group (P < 0.001) (3.5 times of that in the Con group). It also showed a significant increase compared with SP and LPS groups (P < 0.001 vs. SB group; P < 0.05 vs. LPS group). We also found that NGAL and caspase 3 proteins were increased significantly in LPS and SP + LPS groups, but SP600125 decreased the NGAL level by almost 35% and increased the caspase 3 level by 50% in the SP + LPS group compared with the LPS group (P < 0.05). Conclusions The JNK signaling pathway inhibits LPS-mediated apoptosis of renal tubular epithelial cells by upregulating NGAL.

Ginsenoside Rb2 Ameliorates LPS-Induced Inflammation and ER Stress in HUVECs and THP-1 Cells via the AMPK-Mediated Pathway.

Inflammation and endoplasmic reticulum (ER) stress have been documented to contribute to the development of atherosclerosis. Ginsenoside Rb2 has been reported to exhibit antidiabetic effects. However, the effects of Rb2 on atherosclerotic responses such as inflammation and ER stress in endothelial cells and monocytes remain unclear. In this study, the expression of inflammation and ER stress markers was determined using a Western blotting method. Concentrations of tumor necrosis factor alpha (TNF[Formula: see text]) and monocyte chemoattractant protein-1 (MCP-1) in culture media were assessed by enzyme-linked immunosorbent assay (ELISA) and apoptosis was evaluated by a cell viability assay and a caspase-3 activity measurement kit. We found that exposure of HUVECs and THP-1 monocytes to Rb2 attenuated inflammation and ER stress, resulting in amelioration of apoptosis and THP-1 cell adhesion to HUVECs under lipopolysaccharide (LPS) condition. Increased AMPK phosphorylation and heme oxygenase (HO)-1 expression, including GPR120 expression were observed in Rb2-treated HUVECs and THP-1 monocytes. Downregulation of both, AMPK phosphorylation and HO-1expression rescued these observed changes. Furthermore, GPR120 siRNA mitigated Rb2-induced AMPK phosphorylation. These results suggest that Rb2 inhibits LPS-mediated apoptosis and THP-1 cell adhesion to HUVECs by GPR120/AMPK/HO-1-associated attenuating inflammation and ER stress. Therefore, Rb2 can be used as a potential therapeutic molecule for treatment of atherosclerosis.

Melatonin alleviates inflammation-induced apoptosis in human umbilical vein endothelial cells via suppression of Ca2+-XO-ROS-Drp1-mitochondrial fission axis by activation of AMPK/SERCA2a pathway

Endothelia inflammation damage is vital to the development and progression of chronic venous disease. In the present study, we explored the protective effect of melatonin on endothelia apoptosis induced by LPS, particularly focusing on the mitochondrial fission. We demonstrated that human umbilical vein endothelial cells (HUVEC) subjected to LPS for 12h exhibited a higher apoptotic rate. However, melatonin (1-20μM) treatment 12h before LPS had the ability to protect HUVEC cell against LPS-mediated apoptosis in a dose-dependent manner. Furthermore, LPS induced the cytoplasmic calcium overload which was responsible for the upregulation of calcium-dependent xanthine oxidase (XO). Higher XO expression was associated with reactive oxygen species (ROS) overproduction, leading to the Drp1 phosphorylation at the Ser616 site and migration on the surface of mitochondria. Furthermore, phosphorylated Drp1 initiated the mitochondrial fission contributing to the caspase9-dependent mitochondrial apoptosis as evidenced by lower membrane potential, more cyt-c leakage into the nuclear, and higher expression of proapoptotic proteins. However, melatonin treatment could trigger the AMPK pathway, which was followed by the increased SERCA2a expression. Activation of AMPK/SERCA2a by melatonin inhibited the calcium overload, XO-mediated ROS outburst, Drp1-required mitochondrial fission, and final mitochondrial apoptosis. In summary, this study confirmed that LPS induced HUVEC apoptosis through Ca2+-XO-ROS-Drp1-mitochondrial fission axis and that melatonin reduced the apoptosis of HUVEC through activation of the AMPK/SERCA2a pathway.

Estradiol Suppresses TLR4-triggered Apoptosis of Decidual Stromal Cells and Drives an Anti-inflammatory TH2 Shift by Activating SGK1.

A pro-inflammatory cytokine profile at the feto-maternal interface may predispose immune maladaptation notably in early miscarriages. We investigated the involvement of estradiol (E2)-activated serum-glucocorticoid regulated kinase 1 (SGK1) in preserving the tolerogenic and pro-survival intrauterine microenvironment beneficial to gestation maintenance. Decidual SGK1 was down-regulated in early miscarriage, consistent with the lower serum E2 concentration seen in pregnancy loss. Lipopolysaccharide (LPS)/Toll-like receptors 4 (TLR4) signaling induced apoptosis and the pro-inflammatory T helper type (TH) 1 response of decidual stromal cells (DSCs) were associated with miscarriage. SGK1 activation was suppressed by LPS/TLR4 signaling and would be rescued by E2 administration via the PI3K signaling pathway in DSCs. SGK1 activation attenuated TLR4-mediated cell apoptosis, while promoting cell viability of DSCs by up-regulating the pro-survival genes BCL2 and XIAP, and enhancing the phosphorylation of FOXO1. Furthermore, E2-induced SGK1 activation reduced the secretion of pro-inflammatory TH1 cytokines, and promoted the generation of TH2 cytokines and elevated IRF4 mRNA and protein levels in LPS-incubated DSCs. Pharmacologic inhibition of SGK1 or suppression by small interfering (si) RNA increased the phosphorylation and nuclear translocation of NF-κB to reverse the pro-TH2 and anti-inflammatory effects of E2 pretreatment, leading to compromised pregnancy. These findings suggest that the E2-mediated SGK1 activation suppressed LPS-mediated apoptosis and promoted the anti-inflammatory TH2 responses in DSCs, ultimately contributing to a successful pregnancy.

RETRACTED: Modification of TAK1 by O-linked N-acetylglucosamine facilitates TAK1 activation and promotes M1 macrophage polarization

Macrophages play many different roles in tissue inflammation and immunity, and the plasticity of macrophage polarization is closely associated with acute inflammatory responses. O-GlcNAcylation is an important type of post-translational modification, which subtly modulates inflammation responses. Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is a key serine/threonine protein kinase that mediates signals transduced by pro-inflammatory cytokines such as TGF-β, tumor necrosis factor (TNF), and interleukin-1 (IL-1). It is here reported that TGFβ-activated kinase (TAK1) is modified with N-acetylglucosamine (O-GlcNAc) on S427. Both IL-1 and osmotic stress, which are known as the TAK1-signaling inducers, significantly trigger the O-GlcNAcylation of TAK1 in macrophages. By overexpressing wild-type (WT) or S427A TAK1 mutant into macrophages, it was determined that O-GlcNAcylation of TAK1 on S427 is required for T187/S192 phosphorylation and full activation of TAK1 upon stimulation with IL-1α and NaCl. Aborting O-GlcNAcylation of TAK1 on S427 was found to inhibit the downstream JNK and nuclear factor-κB activation and reduce the final amount of cytokines produced in activated macrophages to a great extent. Results also showed that overexpression of the O-GlcNAcylation-deficient mutant of TAK1 promotes LPS-mediated apoptosis in macrophages. Importantly, TAK1 O-GlcNAcylation was found to promote M1 macrophage polarization in activated macrophages. Taken together, these data demonstrate that O-GlcNAcylation of TAK1 on S427 critically regulates the pro-inflammatory activation and M1 polarization of macrophages via modulation of the TAK1/JNK/NF-κB signaling pathway.

Negative role of inducible PD-1 on survival of activated dendritic cells

PD-1 is a well-established negative regulator of T cell responses by inhibiting proliferation and cytokine production of T cells via interaction with its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), expressed on non-T cells. Recently, PD-1 was found to be expressed in innate cells, including activated DCs, and plays roles in suppressing production of inflammatory cytokines. In this study, we demonstrate that PD-1 KO DCs exhibited prolonged longevity compared with WT DCs in the dLNs after transfer of DCs into hind footpads. Interestingly, upon LPS stimulation, WT DCs increased the expression of PD-1 and started to undergo apoptosis. DCs, in spleen of LPS-injected PD-1 KO mice, were more resistant to LPS-mediated apoptosis in vivo than WT controls. Moreover, treatment of blocking anti-PD-1 mAb during DC maturation resulted in enhanced DC survival, suggesting that PD-1:PD-L interactions are involved in DC apoptosis. As a result, PD-1-deficient DCs augmented T cell responses in terms of antigen-specific IFN-γ production and proliferation of CD4 and CD8 T cells to a greater degree than WT DCs. Moreover, PD-1 KO DCs exhibited increased MAPK1 and CD40-CD40L signaling, suggesting a possible mechanism for enhanced DC survival in the absence of PD-1 expression. Taken together, our findings further extend the function of PD-1, which plays an important role in apoptosis of activated DCs and provides important implications for PD-1-mediated immune regulation.

Decreases in labile intracellular zinc (Zni) contribute to LPS‐induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC)

After iron, zinc is the most abundant intracellular metal and Zni quota is maintained in a narrow range (100‐500 uM) by the activity of zinc importers (SLC39A1‐14) and zinc exporters (SLC30A1‐10). Although virtually all zinc is bound to protein, a chelatable pool of Zni is capable of participating in intracellular signaling and functions. We used chemical and genetic techniques to reveal a role for decreases in Zni to affect LPS mediated apoptosis in SPAEC. Early (0.5 to 4.0 hrs) after a proapototic dose (100 ng/ml) of LPS, we noted a decrease in Zni as revealed by the zinc sensitive fluorophore, FluoZin‐3, via FACS. Confirmation of this relative decrease after LPS was obtained via: a) EPI fluorescence microscopy in live cells that revealed a 60% decrease in Zni ; and b) 40% decrease of activity of zinc sensitive metal transcription element (MRE) fused to luciferase reporter that was previously transfected into SPAEC. The functional consequences of decreases in Zni were ascertained by: a) rescuing SPAEC from LPS induced increases in caspase 3/7 by addition of exogenous zinc to normalize potential LPS induced decreases in Zni; and b) mimicking effect of LPS on caspase 3/7 activity with zinc chelator, TPEN. We conclude that LPS results in a change in net activity of zinc transporters resulting in decrease in Zni and this decrease is an important intracellular signaling pathway transducing the apoptotic effects of LPS on SPAEC.

Toll-like receptor signaling in hen ovarian granulosa cells is dependent on stage of follicle maturation

The recent identification of toll-like receptor (TLR) signaling within ovarian granulosa cells has broad implications for ovarian physiology. Functions of TLRs within granulosa cells of the laying hen are of particular interest due to the method of transovarian transmission of Salmonella enteritidis, which results in egg contamination. This study utilized hen granulosa cells to evaluate the expression and function of Gallus TLR-signaling at distinct stages of follicular maturity. Data presented herein demonstrate the presence of TLR2, TLR4, and TLR15 mRNAs in undifferentiated granulosa cells from prehierarchal follicles and differentiated granulosa cells from preovulatory follicles, together with mRNAs encoding adaptor proteins and signaling components required for TLR signaling gene. Treatment with lipopolysaccharide (LPS) or LH, in vitro, led to the differential regulation of TLRs based on the stage of follicle maturation, with the largest (F1) follicle granulosa cells having the most rapid response. Furthermore, treatment with LPS resulted in attenuation of agonist-induced progesterone synthesis in undifferentiated, but not differentiated, granulosa cells. Additionally, undifferentiated granulosa cells were significantly more sensitive to LPS-induced apoptosis than differentiated granulosa cells from the F1 follicle. Together, these data provide evidence for a complete and functional TLR signaling pathway in hen granulosa cells, with effects on steroidogenesis and cell viability dependent upon stage of maturation. These differences may reflect the susceptibility of granulosa cells at early stages of maturation to undergo apoptosis in response to select pathogenic stimuli, thus attenuating transovarian transmission, whereas granulosa cells from preovulatory follicles are comparably resistant to LPS-mediated apoptosis.

Apoptosis of Dedifferentiated Hepatoma Cells is Independent of NF-κB Activation in Response to LPS

Dedifferentiated hepatoma cells, in contrast to most other cell types including hepatoma cells, undergo apoptosis when treated with lipopolysaccharide (LPS) plus the protein synthesis inhibitor cycloheximide (CHx). We recently reported that the dedifferentiated hepatoma cells also exhibit a strong and prolonged NF-kappaB induction phenotype upon exposure to LPS, suggesting that NF-kappaB signaling may play a pro-survival role, as reported in several other cell systems. To test the role of NF-kappaB in preventing LPS-mediated apoptosis, we examined the dedifferentiated cell line M38. Results show that antioxidants strongly inhibited LPS + CHx-mediated cell death in the M38 cells, yet only modestly inhibited NF-kappaB induction. In addition, inhibition of NF-kappaB translocation by infection of the M38 cells with an adenoviral vector expressing an IkappaBalpha super-repressor did not result in LPS-mediated cell death. These results suggest that unlike TNFalpha induction, the cell survival pathway activated in response to LPS is independent of NF-kappaB translocation in the dedifferentiated cells. Addition of inhibitors of JNK, p38 and ERK pathways also failed to elicit LPS-mediated apoptosis similar to that observed when protein synthesis is prevented. Thus, cell survival pathways other than those involving NF-kappaB inducible gene expression or other well-known pathways appear to be involved in protecting the dedifferentiated hepatoma variant cells from LPS-mediated apoptosis. Importantly, this pro-apoptotic function of LPS appears to be a function of loss of hepatic gene expression, as the parental hepatoma cells resist LPS-mediated apoptosis in the presence of protein synthesis inhibitors.

LPS-induced apoptosis is dependent upon mitochondrial dysfunction.

Bacterial infection induces apoptotic cell death in human monoblastic U937 cells that have been pretreated with interferon gamma (U937IFN). Apoptosis occurs in a manner that is independent of bacterial virulence proteins. In the present study, we show that lipopolysaccharide (LPS), a membrane constituent of gram-negative bacteria, also induces apoptosis in U937IFN cells. LPS treatment led to the appearance of characteristic markers of apoptosis such as nuclear fragmentation and activation of caspases. While the caspase inhibitor Z-VAD-fmk prevented LPS-induced apoptosis as judged by its inhibition of nuclear fragmentation, it failed to inhibit cytochrome c release and loss of mitochondrial membrane potential. Transfection of peptides containing the BH4 (Bcl-2 homology 4) domain derived from the anti-apoptotic protein Bcl-XL blocked LPS-induced nuclear fragmentation and the limited digestion of PARP. These results suggest that LPS does not require caspase activation to induce mitochondrial dysfunction and that mitochondria play a crucial role in the regulation of LPS-mediated apoptosis in U937IFN cells.

Divergence of apoptosis-inducing and preventing signals in bacteria-faced macrophages through myeloid differentiation factor 88 and IL-1 receptor-associated kinase members.

The induction of apoptosis in host cells is a common strategy by which pathogenic bacteria interfere with the host immune response. The Yersinia enterocolitica outer protein P (YopP) inhibits activation of transcription factor NF-kappa B in macrophages, which suppresses NF-kappa B-dependent antiapoptotic activities. The simultaneous initiation of proapoptotic signaling by yersiniae infection or LPS treatment results in macrophage apoptosis. In this study, we used YopP as a tool to dissect survival- and death-inducing pathways in bacteria-faced macrophages. We cotransfected J774A.1 macrophages with expression plasmids for YopP and dominant-negative mutants of signal transmitters of the NF-kappa B cascade downstream from the LPS receptor complex. Dominant-negative myeloid differentiation factor 88 (MyD88) or IL-1R-associated kinase (IRAK) 2 diminished LPS-induced apoptosis in YopP-transfected macrophages, suggesting implication of MyD88 and IRAK2 in signaling cell death. In contrast, dominant-negative IRAK1 and TNFR-associated factor 6 (TRAF6) did not provide protection, but augmented LPS-mediated apoptosis in the absence of YopP, which indicates roles of IRAK1 and TRAF6 in the antiapoptotic signal relay of the NF-kappa B cascade. The distinct functions of IRAK members in macrophage survival were reflected by opposing effects of dominant-negative IRAK1 and IRAK2 on Y. enterocolitica-mediated apoptosis. Yersiniae- and LPS-dependent cell death were substantially attenuated by a specific caspase-8 inhibitory peptide or by dominant negative Fas-associated death domain protein (FADD). This suggests, that Yersinia-induced apoptosis involves a proapoptotic signal relay through MyD88 and IRAK2, which potentially targets the Fas-associated death domain protein/caspase-8 apoptotic pathway, whereas IRAK1 and TRAF6 counteract the bacteria-induced cytotoxic response by signaling macrophage survival.

DAP12 ITAM Motif Regulates Differentiation and Apoptosis in M1 Leukemia Cells

DAP12 is an immunoreceptor tyrosine-based activation motif (ITAM)-bearing transmembrane adapter molecule that is associated with the NK-activating receptors. DAP12 is expressed not only in NK cells, but also in myeloid cells. Previously, we reported that DAP12 was likely to be involved in monocyte differentiation to macrophage. In this study, we established the mutant DAP12-M1 transfectants (Y76F-M1) that have mutation at their ITAM motifs. We observed that Y76F-M1 cells could not differentiate to macrophages by stimulation via DAP12, whereas wild type DAP12 transfectants (FDAP-M1) could. Furthermore, we demonstrated that the apoptosis signal mediated by LPS was inhibited in Y76F-M1 cells, but was augmented in FDAP-M1 cells. In contrast to the LPS-mediated apoptosis, the combination of LPS and DAP12 stimulation showed good cell viability in FDAP-M1 cells. Collectively our studies demonstrated that DAP12 has a critical role for macrophage differentiation and LPS induced apoptosis in M1 leukemia cells.

A common regulatory locus affects both HNF4/HNF1alpha pathway activation and sensitivity to LPS-mediated apoptosis in rat hepatoma cells.

Lipopolysaccharide (LPS) has been shown to protect certain cultured mammalian cells from undergoing programmed cell death (apoptosis) when exposed to tumor necrosis factor (TNF). However, LPS has also been reported to induce apoptosis in cultured endothelial cells, suggesting that apoptotic response mechanisms may be dependent upon cell type. In order to understand the influence of tissue-specific gene expression on apoptosis, we compared LPS-induced apoptosis in hepatoma cells with dedifferentiated hepatoma variant cells that have been selected for the loss of the liver-enriched HNF4/HNF1alpha transcriptional activation pathway. We report here that while human, rat and mouse hepatoma cell lines are resistant to LPS-mediated cell death, the HNF4-/HNF1alpha- rat hepatoma variant cells undergo rapid apoptosis (as determined by morphological analysis, DNA laddering and the TUNEL assay) upon exposure to LPS. Genetic rescue experiments show that restoration of the HNF4/HNF1alpha pathway via chromosome transfer render the hepatoma variant cells resistant to LPS-mediated apoptosis. However, the introduction of HNF1alpha alone failed to alter the apoptotic phenotype, suggesting that the defect(s) in the hepatoma variant cells that influence apoptotic responses lies upstream of HNF4/HNF1alpha expression. This study provides for the first time direct evidence of a common regulatory locus involved in activation of hepatic gene expression and sensitivity to LPS-mediated apoptosis.

The heat-shock response attenuates lipopolysaccharide-mediated apoptosis in cultured sheep pulmonary artery endothelial cells.

We recently reported that lipopolysaccharide (LPS) induces apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC). Information about survival signals against this and other stimuli for endothelial cell apoptosis is limited to factors in the extracellular space. In other cell types, apoptosis is also affected by intracellular gene products. The heat-shock response is a highly conserved cellular stress response affording cytoprotection against a variety of cytotoxic conditions. Accordingly, we tested the hypothesis that prior induction of the heat-shock response would affect apoptosis in cultured SPAEC. Exposure of SPAEC to either heat (43 degrees C, 90 min) or sodium arsenite (100 microM, 90 min) induced expression of heat-shock protein-70 (HSP-70). LPS (0.1 microg/ml) treatment of SPAEC induced apoptotic morphology, cell detachment, high molecular weight (> 30 kb) DNA fragmentation, and internucleosomal DNA fragmentation. Prior induction of the heat-shock response attenuated LPS-mediated apoptosis, a protective event associated with a concomitant attenuation of rapid (within minutes) LPS-stimulated superoxide anion (O2.-) generation. Subsequent experiments involving transient overexpression of HSP-70, by direct gene transfer, suggest a direct role for HSP-70 in the attenuation of LPS-mediated apoptosis. We conclude that the heat-shock response is an intracellular survival signal against LPS-mediated apoptosis, and that the protective mechanism may involve HSP-70 directly, as well as inhibition of LPS-mediated O2.- generation.