Hypoxia and TNF-α Synergistically Induce Expression of IL-6 and IL-8 in Human Fibroblast-like Synoviocytes via Enhancing TAK1/NF-κB/HIF-1α Signaling.

Hypoxia and increased levels of inflammatory cytokines in the joints are characteristics of rheumatoid arthritis (RA). However, the effects of hypoxia and tumor necrosis factor-α (TNF-α) on interleukin (IL)-6 and IL-8 production on fibroblast-like synoviocytes (FLSs) remain to be clarified. This study aimed to explore how hypoxia and TNF-α affect the expression of IL-6 and IL-8 in human FLSs isolated from RA patients. Hypoxia or TNF-α treatment alone significantly increased the expression and promoter activity of IL-6, IL-8, and hypoxia-inducible factor-1α (HIF-1α). Treatment of hypoxic FLSs with TNF-α further significantly elevated the expression of these cytokines and enhanced promoter activity of HIF-1α, which was abrogated by treatment with the HIF-1α inhibitor YC-1. Similarly, TNF-α alone elevated the phosphorylation and promoter activity of nuclear factor-κBp65 (NF-κBp65) in the FLSs. These effects were further enhanced by the combined treatment of hypoxia and TNFα but were attenuated by the NF-κB inhibitor BAY11-7082. NF-κB-p65 inhibition decreased the effect of TNF-α on HIF-1α upregulation in the FLSs in response to hypoxia. The combination of hypoxia and TNF-α also significantly upregulated transforming growth factor-ß-activated kinase 1 (TAK1) expression, and silencing TAK1 dramatically decreased NF-κB-p65, HIF-1α, IL-6, and IL-8 expression under the same conditions. Our results indicate that hypoxia and TNF-α synergistically increase IL-6 and IL-8 expression in human FLSs via enhancing TAK1/NF-κB/HIF-1α signaling.

The pathogenesis and regulatory role of HIF-1 in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a prevalent autoimmune disease that involves the overgrowth and inflammation of synovial tissue, leading to the degeneration and impairment of joints. In recent years, numerous studies have shown a close relationship between the hypoxic microenvironment in joints and the occurrence and progression of RA. The main cause of the pathological changes in RA is widely believed to be the abnormal expression of hypoxia-inducible factor-1 (HIF-1) in joints. This paper describes and illustrates the structure and primary functions of HIF-1 and explains the main regulatory methods of HIF-1, including the PHDs/HIF-1 α/pVHL pathway, factor-inhibiting HIF (FIH), regulation of inflammatory cytokines, and the NF-κB pathway. Furthermore, this paper discusses the mechanism of HIF-1 and its impact on inflammation, angiogenesis, and cartilage destruction in greater detail. We summarize previous research findings on the mechanism of HIF-1 and propose new potential treatments for RA based on the pathogenesis of HIF-1 in RA.

Krüppel-like factor 4 regulates the expression of inducible nitric oxide synthase induced by TNF-α in human fibroblast-like synoviocyte MH7A cells.

Krüppel-like factor 4 (KLF4), a zinc finger transcription factor, has been implicated in the inflammation mediated by macrophages and endothelial cells by regulating the expression of inflammatory mediators. Here, we investigated whether KLF4 affects the expression of inducible nitric oxide synthase (iNOS), an important inflammatory mediator, in the human RA fibroblast-like synovial cell line MH7A. A pcDNA3.1-KLF4 plasmid or short interfering RNA KLF4 was transfected into MH7A cells, and the iNOS expression and nitric oxide (NO) production were analyzed by quantitative PCR, immunoblotting, and nitrite measurement. The iNOS promoter activity was determined by luciferase assay. The results showed overexpression of KLF4 increased iNOS expression and NO production in the presence or absence of TNF-α. Conversely, KLF4 knockdown markedly reduced iNOS expression and NO production induced by TNF-α. KLF4 activated the transcription activity of iNOS promoter in MH7A cells stimulated by TNF-α. This study indicates that KLF4 is important for regulating the expression of iNOS by TNF-α in human synoviocytes.

The roles of reactive oxygen species (ROS) and autophagy in the survival and death of leukemia cells.

As a clonal disease of hematopoietic stem cells (HSCs), the etiology and pathogenesis of leukemia is not fully understood. Recent studies suggest that cellular homeostasis plays an essential role in maintaining the function of HSCs because dysregulation of cellular homeostasis is one of the major factors underlying the malignant transformation of HSCs. Reactive oxygen species (ROS) and autophagy, key factors regulating cellular homeostasis, are commonly observed in the human body. Autophagy can be induced by ROS through a variety of signaling pathways, and conversely inhibits ROS-induced damage to cells and tissues. ROS and autophagy coordinate to maintain cellular homeostasis. Previous studies have demonstrated that both of ROS and autophagy play important roles in the development of leukemia and are closely involved in drug resistance in leukemia. Interference with cellular homeostasis by promoting programmed leukemia cell death via ROS and autophagy has been verified to be an efficient technique in the treatment of leukemia. However, the critical roles of ROS and autophagy in the development of leukemia are largely unknown. In this review, we summarize the roles of ROS and autophagy in the pathogenesis of leukemia, which may allow the identification of novel targets and drugs for the treatment of leukemia based on the regulation of HSCs homeostasis through ROS and autophagy.

Adenovirus-Mediated Small Interfering RNA Targeting TAK1 Ameliorates Joint Inflammation with Collagen-Induced Arthritis in Mice.

Transforming growth factor ß-activated kinase-1 (TAK1) is a key upstream kinase in cell signaling during inflammation, which regulates the expression of inflammatory mediators. Small interfering RNA (siRNA) against TAK1 offers promise as a potential therapeutic strategy in immune-mediated inflammatory disorder including rheumatoid arthritis. Here, we are to evaluate the therapeutic effects of intra-articular administration of adenoviral-mediated siRNA against TAK1 (ad-siRNA-TAK1) on collagen-induced arthritis (CIA) in mice. Ad-siRNA-TAK1 was constructed. The murine RAW 264.7 macrophages were infected with ad-siRNA-TAK1, and the silencing specificity of TAK1 was assessed by quantitative polymerase chain reaction (PCR) and western blot. DBA/1 mice were injected intra-articularly with ad-siRNA-TAK1. Development and severity of arthritis was assessed histologically. Synovial inflammation and bone destruction were determined by hematoxylin and eosin (HE) staining. Articular and serum concentrations of tumor necrosis factor-α, interleukin-1, and interleukin-6 were determined using enzyme-linked immunosorbent assay. Levels of phosphorylated p38, c-Jun N-terminal kinase (JNK), and extracellular-signal-regulated kinase (ERK) were detected by western blot. In vitro, ad--siRNA-TAK1 efficiently inhibited the expression of TAK1 at both mRNA and protein levels. In vivo, intra-articular injection of ad-siRNA-TAK1 efficiently alleviated joint inflammation, decreased the expression of pro-inflammatory mediators, and suppressed JNK pathways. Our results demonstrate the efficiency of ad--siRNA-TAK1 in controlling joint inflammation of CIA, which is associated with the suppression of the expression of pro-inflammatory cytokines and JNK activation.

[Effects of TAK gene silencing on the expressions of IL-6 and IL-8 induced by TNF-α in fibroblast-like synoviocytes].

OBJECTIVE: To investigate the effects of silencing transforming growth factor-ß activating kinase 1 (TAK1)on the expressions of IL-6 and IL-8 induced by TNF-α in fibroblast-like synoviocytes, and to explore the role of TAK1 in rheumatoid arthritis (RA). METHODS: The synthesized TAK1 siRNA and scrambled siRNA (ScRNA) were transferred into cultured RA fibroblast-like synoviocyte line MH7A by lipofectamine. The expressions of the pro-inflammatory mediator IL-6 and IL-8 and the levels of phospho-P38(p-P38), phospho-C-Jun NH2-terminal kinase(p-JNK), phospho-extracellular signal-regulated kinase(p-ERK), phospho-p65(p-p65) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha(IκBa) were examined. RESULTS: Silencing of TAK was demonstrated in synoviocytes transfected by TAK siRNA. TAK1 silencing markedly attenuated the expression of IL-6 and IL-8 in the presence of TNF-α. TAK1 silencing inhibited the activation of p38 and JNK MAPK. TAK1 silencing also inhibited activation of nuclear factor-κB (NF-κB). CONCLUSIONS: TAK1 silencing attenuated the expression of IL-6 and IL-8 in synoviocytes induced by TNF-α via inhibiting the activation of p38, JNK MAPK and NF-κB.

Krüppel-Like Factor 4 Is a Regulator of Proinflammatory Signaling in Fibroblast-Like Synoviocytes through Increased IL-6 Expression.

Human fibroblast-like synoviocytes play a vital role in joint synovial inflammation in rheumatoid arthritis (RA). Proinflammatory cytokines induce fibroblast-like synoviocyte activation and dysfunction. The inflammatory mediator Krüppel-like factor 4 is upregulated during inflammation and plays an important role in endothelial and macrophage activation during inflammation. However, the role of Krüppel-like factor 4 in fibroblast-like synoviocyte activation and RA inflammation remains to be defined. In this study, we identify the notion that Krüppel-like factor 4 is higher expressed in synovial tissues and fibroblast-like synoviocytes from RA patients than those from osteoarthritis patients. In vitro, the expression of Krüppel-like factor 4 in RA fibroblast-like synoviocytes is induced by proinflammatory cytokine tumor necrosis factor-α. Overexpression of Krüppel-like factor 4 in RA fibroblast-like synoviocytes robustly induced interleukin-6 production in the presence or absence of tumor necrosis factor-α. Conversely, knockdown of Krüppel-like factor 4 markedly attenuated interleukin-6 production in the presence or absence of tumor necrosis factor-α. Krüppel-like factor 4 not only can bind to and activate the interleukin-6 promoter, but also may interact directly with nuclear factor-kappa B. These results suggest that Krüppel-like factor 4 may act as a transcription factor mediating the activation of fibroblast-like synoviocytes in RA by inducing interleukin-6 expression in response to tumor necrosis factor-α.

TNF-α-induced programmed cell death in the pathogenesis of acquired aplastic anemia.

The mechanism of acquired aplastic anemia (AA), a bone marrow hematopoiesis failure disease, has not been fully understood. TNF-α is a pleiotropic cytokine involved in cell proliferation, differentiation and death, and inflammation through binding to specific receptors on cell membranes. Aberrant secretion of TNF-α contributes to a number of human diseases, including tumor development and inflammation. TNF-α is also an important negative regulator of hematopoiesis. Over-expression of TNF-α not only directly inhibits the proliferation and differentiation of hematopoietic cells, but also initiates the intracellular death pathway to induce hematopoietic cell death, leading to bone marrow hematopoiesis failure. In this review, we summarize the mechanisms underlying extrinsic apoptosis and necroptosis of hematopoietic cells induced by TNF-α, and discuss the role of TNF-α-induced programmed cell death in the pathogenesis of acquired AA.

The role of RIP1 and RIP3 in the development of aplastic anemia induced by cyclophosphamide and busulphan in mice.

This study aimed to investigate the role of RIP1 and RIP3 in the pathogenesis of aplastic anemia (AA) induced by cyclophosphamide and busulphan in mice. Animals were randomly divided into three groups: the control group, the AA group, and the Nec-1 group. Mouse AA model was established by intraperitoneal injection of cyclophosphamide (40 mg/kg/d) and busulfan (20 mg/kg/d) for 12 days. The Nec-1 group mice received intraperitoneal injection of Nec-1 (2 mg/kg/d) for 12 days prior to intraperitoneal injection of cyclophosphamide (40 mg/kg/d) and busulfan (20 mg/kg/d) for 12 days. The control mice received intraperitoneal injection of equal volume of saline. At 12 h after the last intraperitoneal injection, blood and bone marrow tissues were collected from mice. Peripheral blood cells were analyzed using hematology analyzer and the histological changes of bone marrow tissues were examined using scanning electron microscopy (SEM). The levels of RIP3 and RIP3 in bone marrow were measured using Western blot analysis and the interaction of RIP1 and RIP3 proteins was investigated on the basis of immunoprecipitation analysis. ELISA was used to measure the levels of IL-6, TNF-α, and FLT-3L in bone marrow tissue supernatant. Apoptosis and necrosis of bone marrow cells were analyzed using flow cytometry. Western blot showed that the expression of RIP1 and RIP3 was significantly increases in AA mice compared to the normal controls. Immunoprecipitation detected the pro-necrotic RIP1-RIP3 complex, suggesting that RIP1 and RIP3 mediated necroptosis may involved in the damage of bone marrow cells. Compared to the AA mice, Nec-1 group mice exhibited significantly increase of peripheral blood cells and mononuclear cells in bone marrow tissues and decrease of the apoptosis/necrosis of bone marrow cells. In addition, we observed significant decrease of IL-6, TNF-α, and FLT-3L in bone marrow tissue supernatant in the Nec-1 group mice compared to AA mice. Our results suggest that Nec-1 can prevent the development of AA by inhibiting bone marrow cells necrosis and the production of inflammatory mediators. RIP1 and RIP3-mediated necroptosis may involve in the pathogenesis of AA induced by cyclophosphamide and busulfan in mice.

[The effect of Hsp72 on IL-6, IL-8 expression and activation of NF-kappaB in synoviocytes of rheumatoid arthritis].

OBJECTIVE: To investigate the effects of heat shock protein 72 (Hsp72) on the expression of IL-6 and IL-8 and activation of NF-kappaB in synoviocytes from patients suffered from rheumatoid arthritis (RA). METHODS: IL6 and IL8 concentrations in culture supernatants were measured using enzyme-linked immunosorbent assays (ELISA). Nuclear translocation of NF-kappaB and degradation of the inhibitory protein IkappaBalpha were examined using immunohistochemistry and Western blot. RESULTS: Hsp72 down-regulated IL-6 and IL-8 production in RA synoviocytes induced by tumor necrosis factor-alpha (TNF-alpha). Hsp72 inhibited nuclear translocation of NF-kappaB and degradation of IkappaBalpha induced by TNF-alpha. CONCLUSION: Hsp72 has an anti-inflammatory effect on RA by down-regulation of IL-6 and IL-8 in synoviocytes, which is mediated through inhibiting the activation of NF-KalphaB signal pathways.

Extracellular heat shock protein 72 protects schwann cells from hydrogen peroxide-induced apoptosis.

The cytoprotective role of extracellular heat shock protein (Hsp) 72 has been demonstrated in various cell types, including neuronal cells; however, few studies have investigated the actual role of Hsp72 in the survival of Schwann cells (SCs). In the present study, we investigated the effect of exogenous Hsp72 on Schwann cell apoptotic cell death induced by H2O2. We determined that extracellular exposure to Hsp72 reduced cell death in rat SCs in a dose-dependent manner, with the protection resulting from downregulation of apoptosis induced by H2O2 (as shown by TUNEL and annexin V flow cytometry analyses). Moreover, we observed that Hsp72 suppressed caspase-3 and -9 activation induced by H2O2. This was accompanied by upregulation of the antiapoptotic protein Bcl-2. These findings indicate that extracellular Hsp72 can afford neuroprotection to peripheral nerves via its ability to inhibit Schwann cell apoptosis and diminish oxidative stress-mediated injuries.

Treatment with recombinant Hsp72 suppresses collagen-induced arthritis in mice.

Although the level of heat shock protein (Hsp72) has been shown to be enhanced in rheumatoid arthritis (RA) synovial tissues and RA synovial fluid, it remains unclear what role extracellular Hsp72 plays in the pathogenesis of RA. This study was conducted to investigate the effects of recombinant human Hsp72 on collagen-induced arthritis (CIA) when administered therapeutically and elucidate its underlying mechanism. We demonstrated that recombinant Hsp72 significantly reduced disease severity. Hsp72-treated animals displayed significantly less cartilage and bone destruction than that in the controls. Hsp72 treatment also reduced the expression of tumor necrosis factor alpha and interleukin 6 in the sera. Furthermore, Hsp72 treatment significantly inhibited activation of nuclear factor kappa B (NF-κB) in synovial tissues of CIA mice. These findings suggest that recombinant Hsp72 effectively suppressed synovial inflammation and the development and progress of CIA, which is mediated through the reduction of production of proinflammatory cytokines and the suppression of activation of NF-κB pathway.

Hydrogen peroxide induces apoptosis through the mitochondrial pathway in rat Schwann cells.

Oxidative stress is one of the several mechanisms that induces apoptosis in cells. It has been shown that hydrogen peroxide (H(2)O(2)) induces apoptosis in several kinds of cells; however, the role of H(2)O(2) in the apoptosis of Schwann cells (SCs) is currently unclear. The objective of this study was to determine whether H(2)O(2) is capable of inducing apoptosis in SCs and whether or not such an effect is associated with the activation of mitochondrial pathway. We demonstrated that H(2)O(2) induces apoptosis in SCs, and is associated with increased release of cytochrome c from mitochondria and the activation of caspase-3 and -9 by up-regulation of Bax and down-regulation of Bcl-2. These results suggest a potential role for H(2)O(2) in SC injury by triggering apoptosis via the mitochondrial pathway under oxidative stress.

Overexpression of MIP2, a novel WD-repeat protein, promotes proliferation of H9c2 cells.

WD40 repeat proteins have a wide range of diverse biological functions including signal transduction, cell cycle regulation, RNA splicing, and transcription. Myocardial ischemic preconditioning up-regulated protein 2 (MIP2) is a novel member of the WD40 repeat proteins superfamily that contains five WD40 repeats. Little is known about its biological role, and the purpose of this study was to determine the role of MIP2 in regulating cellular proliferation. Transfection and constitutive expression of MIP2 in the rat cardiomyoblast cell line H9c2 results in enhanced growth of those cells as measured by cell number and is proportional to the amount of MIP2 expressed. Overexpression of MIP2 results in a shorter cell cycle, as measured by flow cytometry. Collectively, these data suggest that MIP2 may participate in the progression of cell proliferation in H9c2 cells.

A role for forkhead box A1 in acute lung injury.

Forkhead box protein A1 (FoxA1) is an evolutionarily conserved winged helix transcription factor with diverse regulatory functions. However, little is known about the role of FoxA1 in acute lung injury (ALI) and pulmonary cell injury. In this study, an in vivo model was employed whereby rats were administered an intravenous injection of oleic acid (OA, 0.1 ml/kg), and alveolar type II epithelial cells (AT-2 cells) injury was induced by hydrogen peroxide (H(2)O(2)) in vitro. OA injection resulted in lung injury and AT-2 cells apoptosis in vivo. OA injection and H(2)O(2) upregulated FoxA1 mRNA and protein in lung tissue of the in vivo ALI model and in H(2)O(2) challenged AT-2 cells. Overexpression of FoxA1 promoted apoptosis, whereas FoxA1 deficiency, induced by antisense oligonucleotides, decreased AT-2 cells apoptosis induced by H(2)O(2), as shown by flow cytometry. These results suggest that FoxA1 may play an important role in ALI by promoting apoptosis of pulmonary epithelial cells.

Role of Foxa1 in regulation of bcl2 expression during oxidative-stress-induced apoptosis in A549 type II pneumocytes.

Forkhead box protein A1 (Foxa1) is an evolutionarily conserved winged helix transcription factor that was traditionally considered to be involved in embryonic development and cell differentiation. However, little is known about the role of Foxa1 in oxidative-stress-induced apoptosis. In this study, hydrogen peroxide (H(2)O(2))-induced apoptosis, upregulation of Foxa1, and the role of Foxa1 in the regulation of bcl2 gene expression were studied in A549 type II pneumocytes. H(2)O(2) upregulated Foxa1 mRNA and protein in a time- and dose-dependent manner. Overexpression of Foxa1 promoted apoptosis, whereas Foxa1 deficiency, induced by antisense oligonucleotides, decreased A549 cell apoptosis induced by H(2)O(2), as shown by flow cytometry. Moreover, Foxa1 overexpression decreased the expression of bcl2, while Foxa1 depletion increased the expression of bcl2. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed that Foxa1 bound to bcl2 promoter, and H(2)O(2) promoted its DNA binding activity. Luciferase reporter showed that Foxa1 also decreased the transcription activity of bcl2 promoter under normal conditions and oxidative stress. These results indicate that Foxa1 plays a pro-apoptotic role by inhibiting the expression of anti-apoptotic gene bcl2.

Release of heat shock protein 70 and the effects of extracellular heat shock protein 70 on the production of IL-10 in fibroblast-like synoviocytes.

It has recently been suggested that heat shock protein (Hsp) 70, an intracellular protein, can be released into the extracellular compartment and exert important immunomodulatory functions. Although elevated Hsp70 has been found in synovial fluid from patients with rheumatoid arthritis (RA), its sources and extracellular functions remain unclear. In this study, we explored whether stress response such as heat stress or exposure to tumor necrosis factor-alpha (TNF-alpha) could induce Hsp70 release from RA fibroblast-like synoviocytes (FLSs) and whether extracellular Hsp70 would stimulate cytokine production in RA FLSs. Cultured FLSs were obtained from patients with RA. The expression of intracellular Hsp70 was studied by Western blot. Hsp70 release and the production of interleukin (IL)-6, IL-8, and IL-10 by RA FLSs were studied by specific enzyme-linked immunosorbent assays. The levels of Toll-like receptor (TLR) 2 and 4 mRNA and protein in FLSs were analyzed using reverse transcription-polymerase chain reaction and Western blotting. Treatment with sublethal heat shock or TNF-alpha results in the up-regulation of intracellular Hsp70 in FLSs and Hsp70 release from RA FLSs. In vitro studies show that extracellular Hsp70 can induce anti-inflammatory cytokine IL-10 production in FLSs. The mRNA and protein expression of TLR2 and TLR4 was demonstrated in FLSs, and TLR4 blocking abrogated the up-regulatory effects of Hsp70 on IL-10 production. Thus, these results lend support to the hypothesis that Hsp70 is actively released from FLSs in response to heat shock or TNF-alpha and Hsp70 may be a major paracrine/autocrine inducer of IL-10 production in FLSs via TLR4.

Extracellular heat shock protein 70 inhibits tumour necrosis factor-alpha induced proinflammatory mediator production in fibroblast-like synoviocytes.

INTRODUCTION: It was recently suggested that heat shock protein (HSP)70, an intracellular protein, is a potential mediator of inflammatory disease when it is released into the extracellular compartment. Although elevated HSP70 levels have been identified in rheumatoid arthritis (RA) synovial tissues and RA synovial fluid compared with patients with osteoarthritis and healthy individuals, it remains unclear what role extracellular HSP70 plays in the pathogenesis of RA. This study was conducted to investigate the effects of extracellular HSP70 on the production of RA-associated cytokines in fibroblast-like synoviocytes from patients with RA and to elucidate the mechanisms involved. METHODS: IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 levels in culture supernatants were measured using enzyme-linked immunosorbent assays. Activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated protein kinases (ERKs), c-Jun amino-terminal kinase (JNK) and p38 MAPK, was detected using Western blotting. Nuclear translocation of nuclear factor-kappaB (NF-kappaB) and degradation of the inhibitory protein IkappaBalpha were examined using immunohistochemistry and Western blotting. RESULTS: Human HSP70 downregulated IL-6, IL-8 and MCP-1 production in RA fibroblast-like synoviocytes induced by tumour necrosis factor (TNF)-alpha in a concentration dependent manner. HSP70 inhibited the activation of ERK, JNK and p38 MAPK in fibroblast-like synoviocytes stimulated by TNF-alpha. Furthermore, HSP70 also significantly inhibited nuclear translocation of nuclear factor-kappaB and degradation of IkappaBalpha induced by TNF-alpha. CONCLUSION: Extracellular HSP70 has an anti-inflammatory effect on RA by downregulating production of IL-6, IL-8 and MCP-1 in fibroblast-like synoviocytes, which is mediated through inhibited activation of the MAPKs and NF-kappaB signal pathways.