Bone morphogenetic protein 15 gene disruption affects the in vitro maturation of porcine oocytes by impairing spindle assembly and organelle function.

Bone morphogenetic protein 15 (BMP15) plays a crucial role in the porcine follicular development. However, its exact functions in the in vitro maturation (IVM) of porcine oocytes remain largely unknown. Here, through cytoplasmic injection of a preassembled crRNA-tracrRNA-Cas9 ribonucleoprotein complex, we achieved BMP15 disruption in approximately 54 % of the cultured porcine oocytes. Editing BMP15 impaired the IVM of porcine oocytes, as indicated by the significantly increased abnormal spindle assembly and reduced first polar body (PB1) extrusion. The editing also impaired cytoplasmic maturation of porcine oocytes, as reflected by reduced abundant of Golgi apparatus and impaired functions of mitochondria. The impaired IVM of porcine oocytes by editing BMP15 possibly was associated with the attenuated SMAD1/5 and EGFR-ERK1/2 signaling in the cumulus granulosa cells (CGCs) and the inhibited MOS/ERK1/2 signaling in oocytes. The attenuated MOS/ERK1/2 signaling may contribute to the inactivation of maturation promoting factor (MPF) and the increased abnormal spindle assembly, leading to reduced PB1 extrusion. It also may contribute to reduced Golgi apparatus formation, and impaired functions of mitochondria. These findings expand our understanding of the regulatory role of BMP15 in the IVM of porcine oocytes and provide a basis for manipulation of porcine reproductive performance.

Molecular characterization of the follicular development of BMP15-edited pigs.

In brief: The regulatory role of BMP15 on porcine ovarian follicular development still remains unclear. This study reveals that biallelic editing of BMP15 impairs SMAD signaling and inhibits granulosa cell proliferation, resulting in porcine follicular development arrest and ovarian hypoplasia. Abstract: Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor beta (TGF-ß) superfamily, which is critical for facilitating ovarian folliculogenesis in mono-ovulatory mammalian species but is not essential in polyovulatory mice. Our previously established BMP15-edited pigs presented varied female reproductive phenotypes, suggesting the important role of BMP15 in ovarian folliculogenesis in polyovulatory pigs. To understand the regulatory mechanism underlying the effect of BMP15 on porcine ovarian follicular development, we molecularly characterized infertile biallelic-BMP15-edited gilts with ovarian hypoplasia. We found that an absence of BMP15 proteins in biallelic-BMP15-edited gilts can lead to premature activation of primordial follicles, possibly through the upregulation of KITLG-KIT-PI3K-AKT signaling pathways. However, this absence severely impaired SMAD (Sma and Mad proteins from Caenorhabditis elegans and Drosophila, respectively) signaling, causing severely reduced granulosa cell proliferation, leading to the arrest of follicular development during the preantral stage and ovarian hypoplasia, resulting in complete infertility. Our study expands the understanding of the molecular functions of BMP15 in nonrodent polyovulatory mammals.

Interaction of Zap70 and CXCR4 receptor at lamellipodia that determines the directionality during Jurkat T cells chemotaxis.

Directional migration of T-lymphocytes is a key process during immune activation and is tightly regulated both temporally and spatially. The initial cell membrane protrusion at a particular site is critical for determining the direction of cell migration. In this study, we found that ZAP-70 protein appeared not only at the margin of the spreading areas of polarized Jurkat T cells but also formed clusters near the center of the cell body on a fibronectin plate. Specifically, some pZAP-70 was located at the lamellipodia/filopodia and was closely associated with the most extended membrane contact. To visualize the dynamic distribution of ZAP-70 on migrating Jurkat T cells, we generated a fluorescent ZAP-70-EGFP fusion protein (hZAP70G). Expression of the hZAP70G in P116 cells, a ZAP-70 defective Jurkat derivative, restored its chemotactic migration toward SDF-1, adhesion to fibronectin matrix, and integrin activation. In addition, the distribution of hZAP70G protein is associated with changes in cell shape, specifically the membrane protrusion step, forming filopodia/lamellipodia and a retracting uropod. Furthermore, SDF-1 stimulated the formation of ZAP-70 and CXCR4 complex. CXCR4 was observed mainly at the leading edge of migrating cell. The localization of ZAP-70 at the very front edge of protruding lamellipodia was close to CXCR4 and a part of them were overlapped. Collectively, our data describe the critical early step of directional cell movement toward SDF-1 that ZAP-70 is recruited to the CXCR4 at the leading edge of membrane and consequently modulates lamellipodia/filopodia formation and integrin activation.

Activation of endoplasmic reticulum stress and its effect on osteogenic differentiation induced by micropit/nanotube topography / 中华口腔医学杂志

Objective@#To explore the activation of endoplasmic reticulum stress (ERS) in bone marrow mesenchymal stem cell (BMMSC) and its effect on osteogenic differentiation induced by micropit/nanotube topography (MNT), so as to provide guidance for the topography design of biomaterials. @*Methods@#Four sample groups were fabricated: polishing control group (polished titanium, PT, no treatment), thapsigargin treatment (TG, 0.1 μmol/L TG treated for 9 h), MNT5 and MNT20 (anodized at 5 V and 20 V after acid etching). Scanning electron microscope (SEM) was used to observe the topography of Ti samples. The alkaline phosphatase (ALP) production, collagen secretion and extracellular matrix (ECM) mineralization of BMMSC (osteogenic induced for 7, 14 and 21 d) on Ti samples were detected to evaluate the osteogenic differentiation. After 12 h incubation, the shape and size of ER was examined using a transmission electron microscope (TEM), and ERS-related genes including immunoglobulin heavy chain binding protein (BiP), protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 4 (ATF4) were detected by quantitative real-time PCR (qRT-PCR). @*Results@#After 7, 14 and 21 d of induction, the ALP production, collagen secretion and ECM mineralization in TG and MNT20 all significantly increased compared to PT (P<0.05). The cells grown on TG, MNT5 and MNT20 surfaces displayed gross distortions of the ER. Compared to PT, BiP, PERK, ATF4 mRNA expression in TG was respectively 1.87±0.10, 2.24±0.35, 1.85±0.14; BiP, ATF4 mRNA expression in MNT5 were respectively 1.27±0.09, 1.25±0.04; BiP, PERK, ATF4 mRNA expression in MNT20 were respectively 1.44±0.09, 2.40±0.60, 1.48±0.05 (P<0.05). @*Conclusions@#MNT triggered different degree of ERS, and the activated ERS may promote MNT-induced osteogenic differentiation.

Dependence of fibroblast infiltration in tumor stroma on type IV collagen-initiated integrin signal through induction of platelet-derived growth factor.

Cancer-associated fibroblasts play a crucial role in accelerating tumor progression, but there is a knowledge gap regarding the chemotactic signal activated in a tumor microenvironment. In this study, the expression of type IV collagen was knocked down using a lentiviral-mediated short hairpin RNA strategy. Although there was no obvious effect on cell growth in vitro, silencing the Col4-α1 gene decreased the tumorigenicity of B16F10 in C57BL/6 mice, which was accompanied by a reduction in the infiltration of alpha-smooth muscle actin-positive (α-SMA+) fibroblasts. Silencing the Col4-α1 gene or disrupting integrin engagement by blocking the antibody reduced the expression of platelet-derived growth factor A (PDGF-A), a potent chemotactic factor for fibroblasts. Furthermore, ectopic expression of the autoclustering integrin mutant significantly stimulated PDGF-A expression in murine B16F10 and human U118MG and Huh7 cells. PDGF-A-specific sh-RNA and neutralizing anti-PDGF-A antibody effectively inhibited the transwell migration of fibroblasts. Adding recombinant PDGF-A back to shCol cell-conditioned media restored the fibroblast-attraction ability indicating that PDGF-A is a major chemotactic factor for fibroblasts in the current study model. The integrin-associated PDGF-A production correlated with the activation of Src and ERK. High type IV collagen staining intensity colocalized with elevated PDGF-A expression was observed in tumor tissues obtained from hepatoma and glioma patients. The integrin signal pathway was activated by collagen engagement through Src and ERK, leading to enhanced PDGF-A production, which serves as a key regulator of fibroblast recruitment.

[The technology of apical infection control].

Root canal therapy is the most efficient way to treat pulptitis and periapical inflammation, which can clear infections of root canal systems, fill the root canal firmly, and avoid reinfection. However, the variations in root canal morphology and complexity of infection confer difficulty in thoroughly eliminating microorganisms and their by-products in the root canal system, especially in the root apex area (including the top one-third of the root canal and periapical tissue), which is described as the hardest area to clean during endodontic treatment. Infection is difficult to remove entirely because the apex area is hard to approach using dental instruments and because of the existence of special morphological structures, such as apical ramification, intercanal anastomoses, and lateral branch of root canal. This review gives a brief introduction of the characteristics and difficulties of apical infection and knowledge on how to control such infections, including root apex preparation, irrigation and disinfection, and root canal filling.

Modulation of tumor cell stiffness and migration by type IV collagen through direct activation of integrin signaling pathway.

Excessive collagen deposition plays a critical role in tumor progression and metastasis. To understand how type IV collagen affects mechanical stiffness and migration, low-collagen-IV-expressing transfectants of B16F10, U118MG, and Huh7 (denoted shCol cells) were established by the lentiviral-mediated delivery of small interfering RNA against type IV-α1 collagen (Col4A1). Although having similar growth rates, shCol cells showed a flatter morphology compared to that of the corresponding controls. Notably, knocking down the Col4A1 gene conferred the cells with higher levels of elasticity and lower motility. Exposure to blocking antibodies against human ß1 integrin or α2ß1 integrin or the pharmacological inhibition of Src and ERK activity by PP1 and U0126, respectively, effectively reduced cell motility and raised cell stiffness. Reduced Src and ERK activities in shCol cells indicate the involvement of a collagen IV/integrin signaling pathway. The forced expression of ß1 integrin significantly stimulated Src and ERK phosphorylation, reduced cell stiffness, and accelerated cell motility. In an experimental metastasis assay using C57BL/6 mice, B16F10 shCol cells formed significantly fewer and smaller lung nodules, confirming the contribution of collagen to metastasis. In summary, the integrin signaling pathway activated in a tumor environment with collagen deposition is responsible for low cell elasticity and high metastatic ability.

Expression of Th17-related genes in PHA/IL-2-activated human T cells by Fas signaling via caspase-1- and Stat3-dependent pathway.

T helper 17 (Th17) cells, which produce interleukin 17 (IL-17), are involved in the pathogenesis of autoimmune diseases and inflammatory conditions. Th17 cells have been detected in many Fas ligand-positive tumors. This study investigates the expression of Th17-related genes in PHA/IL-2-activated human T cells upon Fas ligation. Activated T cells transiently express RORγt, IL-17A, and IL-17F. A subsequent Fas receptor stimulation or contact with FasL-expressing glioma cells significantly prolongs the induction of RORγt and Th17-related cytokines. Treatments with inhibitors of caspase-1 and Stat3 reduce the Fas-signal-associated induction of RORγt, IL-17A, and IL-17F, as well as the phosphorylation of Stat3. Although the ligation of Fas results in caspase-8 cleavage and ERK1/2 phosphorylation, inhibitors for caspase-8 and MEK have no effect on the expressions of RORγt, IL-17A, and IL-17F. The results suggest that the Fas signal favors the Th17-phenotypic features of human T cells through the caspase-1/Stat3 signaling pathway.

Fas ligand enhances malignant behavior of tumor cells through interaction with Met, hepatocyte growth factor receptor, in lipid rafts.

Many late-stage cancer cells express Fas ligand (FasL) and show high malignancy with metastatic potential. We report here a novel signaling mechanism for FasL that hijacks the Met signal pathway to promote tumor metastasis. FasL-expressing human tumor cells express a significant amount of phosphorylated Met. The down-regulation of FasL in these cells led to decreased Met activity and reduced cell motility. Ectopic expression of human FasL in NIH3T3 cells significantly stimulated their migration and invasion. The inhibition of Met and Stat3 activities reverted the FasL-associated phenotype. Notably, FasL variants activated the Met pathway, even though most of their intracellular domain or Fas binding sites were deleted. FasL interacted with Met through the FasL(105-130) extracellular region in lipid rafts, which consequently led to Met activation. Knocking down Met gene expression by RNAi technology reverted the FasL-associated motility to basal levels. Furthermore, treatment with synthetic peptides corresponding to FasL(117-126) significantly reduced the FasL/Met interaction, Met phosphorylation, and cell motility of FasL(+) transfectants and tumor cells. Finally, the transfectants of truncated FasL showed strong anchorage-independent growth and lung metastasis potential in null mice. Collectively, our results establish the FasL-Met-Stat3 signaling pathway and explains the metastatic phenotype of FasL-expressing tumors.

Catenarin Prevents Type 1 Diabetes in Nonobese Diabetic Mice via Inhibition of Leukocyte Migration Involving the MEK6/p38 and MEK7/JNK Pathways.

Inflammation contributes to leukocyte migration, termed insulitis, and ß-cell loss in type 1 diabetes (T1D). Naturally occurring anthraquinones are claimed as anti-inflammatory compounds; however, their actions are not clear. This study aimed to investigate the effect and mechanism of catenarin on the inflammatory disease, T1D. Catenarin and/or its anthraquinone analogs dose-dependently suppressed C-X-C chemokine receptor type 4 (CXCR4)- and C-C chemokine receptor type 5 (CCR5)-implicated chemotaxis in leukocytes. Catenarin, the most potent anthraquinone tested in the study, prevented T1D in nonobese diabetic mice. Mechanistic study showed that catenarin did not act on the expression of CCR5 and CXCR4. On the contrary, catenarin inhibited CCR5- and CXCR4-mediated chemotaxis via the reduction of the phosphorylation of mitogen-activated protein kinases (p38 and JNK) and their upstream kinases (MKK6 and MKK7), and calcium mobilization. Overall, the data demonstrate the preventive effect and molecular mechanism of action of catenarin on T1D, suggesting its novel use as a prophylactic agent in T1D.

Extracellular matrix of glioblastoma inhibits polarization and transmigration of T cells: the role of tenascin-C in immune suppression.

Dense accumulations of T cells are often found in peritumoral areas, which reduce the efficiency of contact-dependent lysis of tumor cells. We demonstrate in this study that the extracellular matrix (ECM) produced by tumors can directly regulate T cell migration. The transmigration rate of several T cells including peripheral blood primary T cell, Jurkat, and Molt-4 measured for glioma cells or glioma ECM was consistently low. Jurkat cells showed reduced amoeba-like shape formation and delayed ERK activation when they were in contact with monolayers or ECM of glioma cells as compared with those in contact with HepG2 and MCF-7 cells. Phospho-ERK was located at the leading edge of migrating Jurkat cells. Glioma cells, but not MCF-7 and HepG2 cells, expressed tenascin-C. Knocking down the tenascin-C gene using the short hairpin RNA strategy converted glioma cells to a transmigration-permissive phenotype for Jurkat cells regarding ERK activation, transmigration, and amoeba-like shape formation. In addition, exogenous tenascin-C protein reduced the amoeba-like shape formation and transmigration of Jurkat cells through MCF-7 and HepG2 cell monolayers. A high level of tenascin-C was visualized immunohistochemically in glioma tumor tissues. CD3(+) T cells were detected in the boundary tumor area and stained strongly positive for tenascin-C. In summary, glioma cells can actively paralyze T cell migration by the expression of tenascin-C, representing a novel immune suppressive mechanism achieved through tumor ECM.

Zap70 controls the interaction of talin with integrin to regulate the chemotactic directionality of T-cell migration.

Aberrant lymphocyte infiltration is crucial for many disorders such as tumor immune escape and autoimmunity. In this study, we have investigated T-cell migration in a three-dimensional collagen matrix containing tumor spheroids and by using micro-Slide chemotaxis and found that Zap70 regulates directionality during cell chemotaxis. Jurkat cells actively migrated toward SDF-1, nutrition, and spheroids of MCF-7 breast carcinoma cells embedded in collagen matrix. Inhibition of Zap70 activity impaired transmigration and mu-Slide chemotaxis but not the random movement of T cells in the collagen/fibronectin matrix. P116 cells, a Zap70 deficient variant of Jurkat, showed active random movement but failed to migrate against chemoattractants. P116 cells exhibited a reduced polarization of cell morphology, showing less lamellipodia formation accompanied with a fast pseudopod turnover rate. Instead of direct interacting with F-actin, Zap70 formed a complex with talin which is an integrin scaffold for F-actin. SDF-1 enhanced Zap70 phosphorylation and also stimulated binding of talin and beta1 integrin activation. P116 cells showed reduced complex of talin and beta1 integrin in parallel with impaired integrin activation. Collectively, Zap70 modulates integrin activation by interacting with talin, which contributes to directionality of T-cell migration, severing as a potential target for anti-inflammation therapy.

Aberrant integrin activation induces p38 MAPK phosphorylation resulting in suppressed Fas-mediated apoptosis in T cells: implications for rheumatoid arthritis.

Delayed Fas-mediated apoptosis in T cells is associated with inflammatory diseases including rheumatoid arthritis (RA). CD3(+) T cells in RA synovia expressed high amounts of phospho-p38 MAPK. Exposure to RA synovial fluid or soluble collagen, a degradation product of extracellular matrix abundant in RA synovium, induced the phosphorylation of p38 MAPK in Jurkat T cells accompanied by resistance against Fas-mediated apoptosis. Blocking beta1 integrin by antibody diminished this effect. In addition, ectopic expression of auto-activated beta1 integrin variant in T cells profoundly induced the phosphorylation of p38 MAPK. Suppression of p38 MAPK sensitized T cells to Fas-mediated apoptosis and increased caspase-8 and caspase-3 cleavage. A physical interaction of p38 MAPK and caspase-8 was demonstrated by using confocal microscopic imaging and co-immunoprecipitation assay. RA synovial fluid markedly increased the formation of phospho-p38 MAPK/caspase-8 complex in Jurkat T cells. In conclusion, abnormal activation of p38 MAPK to prevent Fas-mediated apoptosis may represent a common survival mechanism of RA synovial T cells contributing to the persistent inflammation of affected synovium.

Chronic and repeated Chlamydophila pneumoniae lung infection can result in increasing IL-4 gene expression and thickness of airway subepithelial basement membrane in mice.

BACKGROUND/PURPOSE: Chlamydophila pneumoniae infection has been associated with several pulmonary and cardiac diseases. However, it has not been explored for its ability to activate the same immunopathologic mechanisms of asthma, namely, a predominant Th2 immune response and structural changes that are termed airway remodeling. This study evaluated immune responses in the lung and airway pathology of BALB/c mice with chronic and repeated C. pneumoniae infections. METHODS: Mice were inoculated intranasally with 5 x 10(6) inclusion-forming units of C. pneumoniae TWAR strain, and re-inoculated at 14 and 42 days after the primary inoculation. Cytokine gene expression in bronchoalveolar lavage (BAL) cells was analyzed by RT-PCR on day 70. Airway pathology was also evaluated by morphometric measurements. RESULTS: A significant increase of interleukin (IL)-4 mRNA was detected in BAL cells in infected mice, and a significant increase in subepithelial basement membrane thickness of the airways was also noted in infected mice as compared with control mice (8.95 +/- 0.28 microm vs. 5.54 +/- 0.22 microm, p < 0.0001). We further analyzed the correlation between IL-4 cytokine expression and the increased subepithelial basement membrane thickness of airways in infected mice. We found that mice with increased IL-4 mRNA expression had significant increases in the thickness of subepithelial basement membrane as compared with mice without increased IL-4 mRNA expression (9.87 +/- 0.51 microm vs. 6.49 +/- 0.52 microm, p < 0.0001). CONCLUSION: It is believed that our results demonstrated for the first time that chronic and repeated infections with C. pneumoniae increased IL-4 gene expression and thickness of airway subepithelial basement membrane in mice.

Preliminary evaluation of the deep inferior epigastric artery perforator with CT angiography / 中华放射学杂志

Objective To evaluate the clinical value of multislice-CT angiography (MSCTA)in planning for the patients undergoing deep inferior epigastric artery perforator (DIEAP) flap operations. Methods Eighteen patients were performed with a 16-slice CT scanner to evaluate the deep inferior epigastric artery perforator prior to DIEAP flap operations. Axial, multiplanar reconstruction( MPR), maximum intensity projection(MIP) and volume rendered (VR) images were analysed and the origins, calibers, courses and anatomic relationships of the deep inferior epigastric artery perforator were evaluated. The anastomosis between the superficial inferior epigastric artery and the main perforator was observed as well. The images were classified into three grades based on the vessels'appearance. A + indicated the vessel appeared clear,continuous and thick. A- indicated the vessel appeared foggy,discontinuous and thin or the vessel partly showed. B indicated no related vessel can be seen. Other 18 patients undergoing conventional abdomen-pelvis CT scans for other reasons were used for control group to compare their CT findings of the deep inferior epigastric artery perforator. Results MSCTA well showed the course of the deep inferior epigastric artery (DIEA). Of the 18 cases, 17 cases appeared as A +, another one A -. It precisely displayed the origins, subcutaneous and intramuscular courses, relations of the main perforators on all cases of showing A +. The exact points where the chosen perforator vessels emerged from the rectus abdominis muscle fascia were located precisely. The superficial inferior epigastric arteries were mostly displayed and the connection between the arteries and the largest-caliber perforator from the deep system could also be shown clearly. Strict concordance with operative findings was found in CTA. Conclusion MSCTA can precisely locate the chosen perforator vessels emerging from the rectus abdominis muscle fascia and it may be a feasible, fast, safe and effective method for preoperative evaluation of DIEAP.

Evaluation of the upper airway measurements by multi-slice CT before and after operations in obstructive sleep apnea syndrome patients / 中华放射学杂志

Objective To evaluate the changes of the upper airway of the patients with obstructive sleep apnea syndrome (OSAS) before and after operations and to know the effects of operations by MSCT. Methods The upper airway dimensions of 26 patients with OSAS were measured on multiplanar reformatted (MPR), curved-planar reformatted (CPR), volume rendering(VR) images of 16-slice spiral CT. The measurements include the anteroposterior calibres and the areas on the reformatted axial images on the pharyngeal cavity levels, the calibres and the minimum areas in retropalatal and retroglossal regions, the areas of the soft palate and uvula on the reformatted sagittal view with maximum thickness, the maximum wall thickness of the right and left the upper airway on the coronary images, the volume of the upper airway before and after the operations. The measurements were correlated with the polysomnography (PSG) records. The data were analyzed paired-samples t-test and Pearson correlations. Results By comparison, the anteroposterior calibres and the cross-sectional areas on the reformatted axial view of the lower retropalatal region (slice 4) of the upper airway increased significantly after operations. The anteroposterior diameter increased from 5. 9 mm before operations to 12.8 mm after operations, where t = - 5.506, P < 0.05. The areas increased from 51.0 mm~2 before operations to 275.0 mm~2 after operations, where t = -5.011, P <0.05. In the higher retropalatal region (shce 2) of the upper airway, the anteroposterior diameter decresased from 14.8 mm before operations to 9.2 mm after operations, where t = 2.867, P < 0.05. The areas decreased from 241.0 mm~2 before operations to 128.0 mm~2 after operations, where t = 3.087, P < 0.05. The anteroposterior calibres of retroglossal region (slice 7) decreased from 12.7 mm before operations to 10.3 mm after operations,where t = 3.718, P <0.05. The L-R calibres and the minimum areas of of retropalatal increased significantly from 6.4 mm, 33.0 mm~2 before operation to 10.9 mm, 76. 0 mm~2 after operation, where t = -3.413, -2. 216, respectively and P < 0.05. Of the 9 cases whose apnea and hypopnea index (AHI) ≤5 events/hour after operations, the minimum areas of retropalatal region, the anterio-posterior diameter, L-R calibres increased significantly. The areas increased from 41.0 mm2 before operations to 76.0 mm~2 after operations, were t = -4. 932, P <0.05. The anteroposterior calibres increased from 4.6 mm before operations to 6.6 mm after operations, where t = - 7. 308, P < 0.05. The L-R calibres increased from 8.3 mm before operations to 13.6 mm after operations, where t = - 4.320, P < 0.05. Conclusions MPR、CPR、VR of MSCT can evaluate the not only the morphology but the function changes of the upper airways on the OSAS patients. The increasing of the minimum cross-sectional area may be one of the important indications for evaluating operations. The narrowing of the higher retropalatal region of the upper airway after operations should be an alert to the clinicians.

Caspase-3 enhances lung metastasis and cell migration in a protease-independent mechanism through the ERK pathway.

Caspase-3 is known as a cysteine protease that primarily executes the cell death program. However, some tumors express higher levels of caspase-3 in positive correlation with malignancy. Here, we showed that caspase-3 can promote tumor metastasis in a protease-independent mechanism. Ectopic expression of caspase-3 enhanced lung metastasis and cell motility of caspase-3 deficient MCF-7 cells. By contrast, caspase-3 siRNA reduced the invasiveness and metastasis ability of A549 cells that express high level of caspase-3. Moreover, caspase-3 induced ERK activation. Alteration of caspase-3 by introducing non-processable mutation at its cleavage site or treatment of caspase-3 inhibitor did not diminish the caspase-3-associated increases in ERK phosphorylation and cell migration. Confocal microscopy study showed that caspase-3 was not physically associated with ERK. Inhibiting ceramide formation by blockage of the ceramide synthase or acid sphingomyelinase activity resulted in significant reduction of ERK phosphorylation and cell migration. In summary, caspase-3 induces ERK activation through a ceramide-dependant, protease activity-independent mechanism, which represents a novel role of caspase-3 in tumor metastasis.

Phosphatidylinositol 3-kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells.

It has recently become apparent that the microenvironment made up of the extracellular matrix may affect cell signaling. In this study, we evaluated Fas-triggered apoptosis in T cells in contact with tumor cells, which resembles the cell-to-cell interactions found in tumor regions. Jurkat cells were less susceptible to the Fas-mediated apoptosis when cocultured with U118, HeLa, A549, and Huh-7 tumor cells. This was indicated by less plasma membrane alteration, an amelioration of the loss of mitochondria membrane potential, a decrease in caspase-8 and caspase-3 activation, a decrease in DNA fragmentation factor-45/35 cleavage, and a reduction in the breakage of DNA when compared with Jurkat cells cultured alone. In contrast, the tumor cell lines MCF-7 and HepG2 produced no such protective effect. This protective event was independent of the expression of Fas ligand on the tumor cells. Interrupting the beta integrins-matrix interaction diminished the coculture effect. In Jurkat cells, cell matrix contact reduced the assembly of the Fas death-inducing signaling complex and Bcl-x(L) cleavage, but enhanced the phosphorylation of ERK1/2, p38 MAPK, and Akt. Only PI3K inhibitor, but not kinase inhibitors for MEK, ERK1/2, p38 MAPK, JNK, protein kinase C, and protein kinase A, completely abolished this tumor cell contact-associated protection and in parallel restored Fas-induced Bcl-x(L) cleavage as well as decreasing the phosphorylation of Bad at serine 136. Together, our results indicate that stimulation of the beta integrin signal of T cells by contact with tumor cells may trigger a novel protective signaling through the PI3K/Akt pathway of T cells against Fas-mediated apoptosis.

Lead increases lipopolysaccharide-induced liver-injury through tumor necrosis factor-alpha overexpression by monocytes/macrophages: role of protein kinase C and P42/44 mitogen-activated protein kinase.

Although lead and lipopolysaccharide (LPS), both important environmental pollutants, activate cells through different receptors and participate in distinct upstream signaling pathways, Pb increases the amount of LPS-induced tumor necrosis factor-alpha (TNF-alpha). We examined the cells responsible for the excess production of Pb-increased LPS-induced TNF-alpha and liver injury, and the roles of protein kinase C (PKC) and p42/44 mitogen-activated protein kinase (MAPK) in the induction of TNF-alpha. Peritoneal injection of Pb alone (100 micromol/kg) or a low dose of LPS (5 mg/kg) did not affect serum TNF-alpha or liver functions in A/J mice. In contrast, coexposure to these noneffective doses of Pb plus LPS (Pb+LPS) strongly induced TNF-alpha expression and resulted in profound liver injury. Direct inhibition of TNF-alpha or functional inactivation of monocytes/macrophages significantly decreased the level of Pb+LPS-induced serum TNF-alpha and concurrently ameliorated liver injury. Pb+LPS coexposure stimulated the phosphorylation of p42/44 MAPK and the expression of TNF-alpha in CD14+ cells of cultured mouse whole blood, peritoneal macrophages, and RAW264.7 cells. Moreover, blocking PKC or MAPK effectively reduced Pb+LPS-induced TNF-alpha expression and liver injury. In summary, monocytes/macrophages were the cells primarily responsible for producing, through the PKC/MAPK pathway, the excess Pb-increased/LPS-induced TNF-alpha that caused liver injury. alpha.

Coexposure of lead- and lipopolysaccharide-induced liver injury in rats: involvement of nitric oxide-initiated oxidative stress and TNF-alpha.

In this study, we investigated the interaction between lipopolysaccharide (LPS) and lead (Pb) and the involvement of tumor necrosis factor-alpha (TNF-alpha) and oxidative stress in Pb-plus-LPS (Pb/LPS)-induced liver damage in rats. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), TNF-alpha, nitric oxide (NO), and lipid peroxidation (LPO) were determined in rats treated with Pb and/or LPS. Pb ranging from 0 to 15 mg/kg dose dependently increased AST, ALT, NO, or LPO in LPS-treated rats. Pretreatment with iNOS inhibitor 1400W reduced NO, LPO, TNF-alpha, AST, and ALT in Pb/LPS-treated rats. Thus, Pb increased LPS-induced liver damage, which might be associated with increased NO-initiated oxidative stress and TNF-alpha in rats.