Exosome Isolation Using Chitosan Oligosaccharide Lactate-1-Pyrenecarboxylic Acid-Based Self-Assembled Magnetic Nanoclusters.

Exosomes are small extracellular vesicles that play a crucial role in intercellular communication and offer significant potential for a wide range of biomedical applications. However, conventional methods for exosome isolation have limitations in terms of purity, scalability, and preservation of exosome structural integrity. To address these challenges, an exosome isolation platform using chitosan oligosaccharide lactate conjugated 1-pyrenecarboxylic acid (COL-Py) based self-assembled magnetic nanoclusters (CMNCs), is presented. CMNCs are characterized to optimize their size, stability, and interaction dynamics with exosomes. The efficiency of CMNCs in isolating exosomes is systematically evaluated using various analytical methods to demonstrate their ability to capture exosomes based on amphiphilic lipid bilayers. CMNC-based exosome isolation consistently yields exosomes with structural integrity and purity similar to those obtained using traditional methods. The reusability of CMNCs over multiple exosome isolation cycles underscores their scalability and offers an efficient solution for biomedical applications. These results are supported by western blot analysis, which demonstrated the superiority of CMNC-based isolation in terms of purity compared to conventional methods. By providing a scalable and efficient exosome isolation process that preserves both structural integrity and purity, CMNCs can constitute a new platform that can contribute to the field of exosome studies.

Antibody-Based Targeting of Cell Surface GRP94 Specifically Inhibits Cetuximab-Resistant Colorectal Cancer Growth.

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. Cetuximab, a human/mouse chimeric monoclonal antibody, is effective in a limited number of CRC patients because of cetuximab resistance. This study aimed to identify novel therapeutic targets in cetuximab-resistant CRC in order to improve clinical outcomes. Through phage display technology, we isolated a fully human antibody strongly binding to the cetuximab-resistant HCT116 cell surface and identified the target antigen as glucose-regulated protein 94 (GRP94) using proteomic analysis. Short interfering RNA-mediated GRP94 knockdown showed that GRP94 plays a key role in HCT116 cell growth. In vitro functional studies revealed that the GRP94-blocking antibody we developed strongly inhibits the growth of various cetuximab-resistant CRC cell lines. We also demonstrated that GRP94 immunoglobulin G monotherapy significantly reduces HCT116 cell growth more potently compared to cetuximab, without severe toxicity in vivo. Therefore, cell surface GRP94 might be a potential novel therapeutic target in cetuximab-resistant CRC, and antibody-based targeting of GRP94 might be an effective strategy to suppress GRP94-expressing cetuximab-resistant CRC.

TSPYL5-mediated inhibition of p53 promotes human endothelial cell function.

Testis-specific protein, Y-encoded like (TSPYL) family proteins (TSPYL1-6), which are members of the nucleosome assembly protein superfamily, have been determined to be involved in the regulation of various cellular functions. However, the potential role of TSPYL family proteins in endothelial cells (ECs) has not been determined. Here, we demonstrated that the expression of TSPYL5 is highly enriched in human ECs such as human umbilical vein endothelial cells (HUVECs) and human pluripotent stem cell-differentiated ECs (hPSC-ECs). Importantly, TSPYL5 overexpression was shown to promote EC proliferation and functions, such as migration and tube formation, by downregulating p53 expression. Adriamycin-induced senescence was markedly blocked by TSPYL5 overexpression. In addition, the TSPYL5 depletion-mediated loss of EC functions was blocked by p53 inhibition. Significantly, TSPYL5 overexpression promoted angiogenesis in Matrigel plug and wound repair in a mouse skin wound healing model in vivo. Our results suggest that TSPYL5, a novel angiogenic regulator, plays a key role in maintaining endothelial integrity and function. These findings extend the understanding of TSPYL5-dependent mechanisms underlying the regulation of p53-related functions in ECs.

Necroptosis Is a Mechanism of Death in Mouse Induced Hepatocyte-Like Cells Reprogrammed from Mouse Embryonic Fibroblasts.

Liver transplantation is recommended for patients with liver failure, but liver donors are limited. This necessitates the development of artificial livers, and hepatocytes are necessary to develop such artificial livers. Although induced hepatocyte-like cells are used in artificial livers, the characteristics of mouse induced hepatocyte-like cells (miHeps) reprogrammed with embryonic fibroblasts have not yet been clarified. Therefore, this study investigated the mechanisms underlying the survival, function, and death of miHeps. miHeps showed decreased cell viability, increased cytotoxicity, decreased hepatic function, and albumin and urea secretion at passage 14. Addition of necrostatin-1 (NEC-1) to miHeps inhibited necrosome formation and reactive oxygen species generation and increased cell survival. However, NEC-1 did not affect the hepatic function of miHeps. These results provide a basis for development of artificial livers using hepatocytes.

Inhibition of VEGF-dependent angiogenesis and tumor angiogenesis by an optimized antibody targeting CLEC14a.

The C-type lectin-like domain of CLEC14a (CLEC14a-C-type lectin-like domain [CTLD]) is a key domain that mediates endothelial cell-cell contacts in angiogenesis. However, the role of CLEC14a-CTLD in pathological angiogenesis has not yet been clearly elucidated. In this study, through complementarity-determining region grafting, consecutive deglycosylation, and functional isolation, we generated a novel anti-angiogenic human monoclonal antibody that specifically targets CLEC14a-CTLD and that shows improved stability and homogeneity relative to the parental antibody. We found that this antibody directly inhibits CLEC14a-CTLD-mediated endothelial cell-cell contact and simultaneously downregulates expression of CLEC14a on the surface of endothelial cells. Using various in vitro and in vivo functional assays, we demonstrated that this antibody effectively suppresses vascular endothelial growth factor (VEGF)-dependent angiogenesis and tumor angiogenesis of SNU182 human hepatocellular carcinoma, CFPAC-1 human pancreatic cancer, and U87 human glioma cells. Furthermore, we also found that this antibody significantly inhibits tumor angiogenesis of HCT116 and bevacizumab-adapted HCT116 human colorectal cancer cells. These findings suggest that antibody targeting of CLEC14a-CTLD has the potential to suppress VEGF-dependent angiogenesis and tumor angiogenesis and that CLEC14a-CTLD may be a novel anti-angiogenic target for VEGF-dependent angiogenesis and tumor angiogenesis.

A Review of Anti-Angiogenic Targets for Monoclonal Antibody Cancer Therapy.

Tumor angiogenesis is a key event that governs tumor progression and metastasis. It is controlled by the complicated and coordinated actions of pro-angiogenic factors and their receptors that become upregulated during tumorigenesis. Over the past several decades, vascular endothelial growth factor (VEGF) signaling has been identified as a central axis in tumor angiogenesis. The remarkable advent of recombinant antibody technology has led to the development of bevacizumab, a humanized antibody that targets VEGF and is a leading clinical therapy to suppress tumor angiogenesis. However, despite the clinical efficacy of bevacizumab, its significant side effects and drug resistance have raised concerns necessitating the identification of novel drug targets and development of novel therapeutics to combat tumor angiogenesis. This review will highlight the role and relevance of VEGF and other potential therapeutic targets and their receptors in angiogenesis. Simultaneously, we will also cover the current status of monoclonal antibodies being developed to target these candidates for cancer therapy.

Ig-like domain 6 of VCAM-1 is a potential therapeutic target in TNFα-induced angiogenesis.

Tumor necrosis factor alpha (TNFα)-induced angiogenesis plays important roles in the progression of various diseases, including cancer, wet age-related macular degeneration, and rheumatoid arthritis. However, the relevance and role of vascular cell adhesion molecule-1 (VCAM-1) in angiogenesis have not yet been clearly elucidated. In this study, VCAM-1 knockdown shows VCAM-1 involvement in TNFα-induced angiogenesis. Through competitive blocking experiments with VCAM-1 Ig-like domain 6 (VCAM-1-D6) protein, we identified VCAM-1-D6 as a key domain regulating TNFα-induced vascular tube formation. We demonstrated that a monoclonal antibody specific to VCAM-1-D6 suppressed TNFα-induced endothelial cell migration and tube formation and TNFα-induced vessel sprouting in rat aortas. We also found that the antibody insignificantly affected endothelial cell viability, morphology and activation. Finally, the antibody specifically blocked VCAM-1-mediated cell-cell contacts by directly inhibiting VCAM-1-D6-mediated interaction between VCAM-1 molecules. These findings suggest that VCAM-1-D6 may be a potential novel therapeutic target in TNFα-induced angiogenesis and that antibody-based modulation of VCAM-1-D6 may be an effective strategy to suppress TNFα-induced angiogenesis.

Biomarker Discovery by Modeling Behçet's Disease with Patient-Specific Human Induced Pluripotent Stem Cells.

Behçet's disease (BD) is a chronic inflammatory and multisystemic autoimmune disease of unknown etiology. Due to the lack of a specific test for BD, its diagnosis is very difficult and therapeutic options are limited. Induced pluripotent stem cell (iPSC) technology, which provides inaccessible disease-relevant cell types, opens a new era for disease treatment. In this study, we generated BD iPSCs from patient somatic cells and differentiated them into hematopoietic precursor cells (BD iPSC-HPCs) as BD model cells. Based on comparative transcriptome analysis using our BD model cells, we identified eight novel BD-specific genes, AGTR2, CA9, CD44, CXCL1, HTN3, IL-2, PTGER4, and TSLP, which were differentially expressed in BD patients compared with healthy controls or patients with other immune diseases. The use of CXCL1 as a BD biomarker was further validated at the protein level using both a BD iPSC-HPC-based assay system and BD patient serum samples. Furthermore, we show that our BD iPSC-HPC-based drug screening system is highly effective for testing CXCL1 BD biomarkers, as determined by monitoring the efficacy of existing anti-inflammatory drugs. Our results shed new light on the usefulness of patient-specific iPSC technology in the development of a benchmarking platform for disease-specific biomarkers, phenotype- or target-driven drug discovery, and patient-tailored therapies.

Heat shock protein 70-1A is a novel angiogenic regulator.

Heat shock protein 70-1A (HSP70-1A) is a stress-inducible protein that provides an essential intracellular molecular chaperone function; however, the mechanism of HSP70-1A in angiogenesis has not been clarified. Herein, HSP70-1A gene silencing implicated this protein in angiogenesis. Additionally, recombinant human HSP70-1A (rhHSP70-1A) was able to stimulate human umbilical vein endothelial cell (HUVEC) migration and tube formation in vitro and microvessel formation in vivo similarly to recombinant human vascular endothelial growth factor (rhVEGF). Furthermore, rhHSP70-1A was tightly bound to the surface of HUVECs and participated in extracellular signal-related kinase (ERK)-dependent angiogenesis. Together, these results implicate HSP70-1A as a novel angiogenic regulator.

Extension of the in vivo half-life of endostatin and its improved anti-tumor activities upon fusion to a humanized antibody against tumor-associated glycoprotein 72 in a mouse model of human colorectal carcinoma.

Endostatin is an endogenous angiogenesis inhibitor that exhibits potential anti-tumor efficacy in various preclinical animal models. However, its relatively short in vivo half-life and the long-term, frequent administration of high doses limit its widespread clinical use. In this study, we evaluated whether a fusion protein of murine endostatin (mEndo) to a humanized antibody against tumor-associated glycoprotein 72 (TAG-72), which is highly expressed in several human tumor tissues including colon cancer, can extend the serum half-life and improve the anti-tumor efficacy of endostatin by targeted delivery to the tumor mass. The fusion protein (3E8-mEndo) and mEndo showed improved anti-angiogenic activity in vitro and in vivo, predominantly by interfering with pro-angiogenic signaling triggered by vascular endothelial growth factor (VEGF). Moreover, in mice treated with 3E8-mEndo, we observed a markedly prolonged serum half-life and significantly inhibited tumor growth. The improved anti-tumor activity of 3E8-mEndo can be partially explained by increased local concentration in the tumor mass due to targeted delivery of 3E8-mEndo to implanted colon tumors. Collectively, our data clearly indicate that tumor-targeting antibody fusions to endostatin are a powerful strategy that improves the poor pharmacokinetic profile and anti-tumor efficacy of endostatin.

TRAIL negatively regulates VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions.

Solid tumors supply oxygen and nutrients required for angiogenesis by producing vascular endothelial growth factor (VEGF). Thus, inhibitors of VEGF signaling abrogate tumor angiogenesis, resulting in the suppression of tumor growth and metastasis. We here investigated the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on VEGF-induced angiogenesis. TRAIL inhibited VEGF-induced in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs) and in vivo neovascularization in chicken embryos and mice. TRAIL blocked VEGF-induced angiogenic signaling by inhibiting ERK, Src, FAK, paxillin, Akt, and eNOS. Further, TRAIL blocked intracellular Ca(2+) elevation and actin reorganization in HUVECs stimulated with VEGF, without inhibiting VEGF receptor-2 tyrosine phosphorylation. TRAIL increased caspase-8 activity, without inducing caspase-9/-3 activation and apoptosis. Moreover, TRAIL resulted in cleavage of FAK into FAK-related non-kinase-like fragments in VEGF-stimulated HUVECs, which was blocked by a caspase-8 inhibitor and cellular caspase-8-like inhibitory protein. Biochemical and pharmacological inhibition of caspase-8 and FAK blocked the inhibitory effects of TRAIL on VEGF-stimulated anti-angiogenic signaling and events. In addition, caspase-8 knockdown also suppressed VEGF-mediated signaling and angiogenesis, suggesting that procaspase-8 plays a role of a non-apoptotic modulator in VEGF-induced angiogenic signaling. These results suggest that TRAIL inhibits VEGF-induced angiogenesis by increasing caspase-8 activity and subsequently decreasing non-apoptotic signaling functions of procaspase-8, without inducing caspase-3 activation and endothelial cell cytotoxicity. These data indicate that caspase-8 may be used as an anti-angiogenic drug for solid tumors resistant to TRAIL and anti-tumor drugs.

TXNIP deficiency exacerbates endotoxic shock via the induction of excessive nitric oxide synthesis.

Thioredoxin-interacting protein (TXNIP) has multiple functions, including tumor suppression and involvement in cell proliferation and apoptosis. However, its role in the inflammatory process remains unclear. In this report, we demonstrate that Txnip⁻/⁻ mice are significantly more susceptible to lipopolysaccharide (LPS)-induced endotoxic shock. In response to LPS, Txnip⁻/⁻ macrophages produced significantly higher levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), and an iNOS inhibitor rescued Txnip⁻/⁻ mice from endotoxic shock-induced death, demonstrating that NO is a major factor in TXNIP-mediated endotoxic shock. This susceptibility phenotype of Txnip⁻/⁻ mice occurred despite reduced IL-1ß secretion due to increased S-nitrosylation of NLRP3 compared to wild-type controls. Taken together, these data demonstrate that TXNIP is a novel molecule that links NO synthesis and NLRP3 inflammasome activation during endotoxic shock.

TXNIP maintains the hematopoietic cell pool by switching the function of p53 under oxidative stress.

Reactive oxygen species (ROS) are critical determinants of the fate of hematopoietic stem cells (HSCs) and hematopoiesis. Thioredoxin-interacting protein (TXNIP), which is induced by oxidative stress, is a known regulator of intracellular ROS. Txnip(-/-) old mice exhibited elevated ROS levels in hematopoietic cells and showed a reduction in hematopoietic cell population. Loss of TXNIP led to a dramatic reduction of mouse survival under oxidative stress. TXNIP directly regulated p53 protein by interfering with p53- mouse double minute 2 (MDM2) interactions and increasing p53 transcriptional activity. Txnip(-/-) mice showed downregulation of the antioxidant genes induced by p53. Introduction of TXNIP or p53 into Txnip(-/-) bone marrow cells rescued the HSC frequency and greatly increased survival in mice following oxidative stress. Overall, these data indicate that TXNIP is a regulator of p53 and plays a pivotal role in the maintenance of the hematopoietic cells by regulating intracellular ROS during oxidative stress.

Endothelial progenitor cells from human dental pulp-derived iPS cells as a therapeutic target for ischemic vascular diseases.

Human dental pulp cells (hDPCs) are a valuable source for the generation of patient-specific human induced pluripotent stem cells (hiPSCs). An advanced strategy for the safe and efficient reprogramming of hDPCs and subsequent lineage-specific differentiation is a critical step toward clinical application. In present research, we successfully generated hDPC-iPSCs using only two non-oncogenic factors: Oct4 and Sox2 (2F hDPC-hiPSCs) and evaluated the feasibility of hDPC-iPSCs as substrates for endothelial progenitor cells (EPCs), contributing to EPC-based therapies. Under conventional differentiation conditions, 2F hDPC-hiPSCs showed higher differentiation efficiency, compared to hiPSCs from other cell types, into multipotent CD34(+) EPCs (2F-hEPCs) capable to differentiate into functional endothelial and smooth muscle cells. The angiogenic and neovasculogenic activities of 2F-hEPCs were confirmed using a Matrigel plug assay in mice. In addition, the therapeutic effects of 2F-hEPC transplantation were confirmed in mouse models of hind-limb ischemia and myocardial infarction. Importantly, 2F-EPCs effectively integrated into newly formed vascular structures and enhanced neovascularization via likely both direct and indirect paracrine mechanisms. 2F hDPC-hiPSCs have a robust capability for the generation of angiogenic and vasculogenic EPCs, representing a strategy for patient-specific EPC therapies and disease modeling, particularly for ischemic vascular diseases.

Inhibition of MKK7-JNK by the TOR signaling pathway regulator-like protein contributes to resistance of HCC cells to TRAIL-induced apoptosis.

BACKGROUND & AIMS: The TOR signaling pathway regulator-like (TIPRL) protein, the mammalian ortholog of yeast TIP41, was identified in an expression profiling screen for factors that regulate human liver carcinogenesis. We investigated the role of human TIPRL protein in hepatocellular carcinoma (HCC). METHODS: We measured the level of TIPRL in HCC and adjacent nontumor tissues from patients. We used small interfering RNAs and zebrafish to study the function of TIPRL. We used annexin V propidium iodide staining and immunoblot analyses to measure apoptosis and activation of apoptotic signaling pathways. We used confocal microscopy, coimmunoprecipitation, and glutathione-S transferase pull-down analyses to determine interactions among mitogen-activated protein kinase kinase 7 (MKK7 or MAP2K7), TIPRL, and the protein phosphatase type 2A (PP2Ac). We studied the effects of TIPRL in tumor xenografts in mice. RESULTS: Levels of TIPRL were higher in HCC tissues and cell lines than nontumor tissues and primary hepatocytes. Knockdown of tiprl expression in zebrafish led to large amounts of apoptosis throughout the embryos. Incubation of HCC cells, but not primary human hepatocytes, with small interfering RNA against TIPRL (siTIPRL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) caused prolonged activation (phosphorylation) of MKK7 and c-Jun N-terminal kinase (JNK) and led to apoptosis, indicated by cleavage of procaspase-8,-3 and of poly-(adenosine diphosphate-ribose) polymerase. TIPRL bound to MKK7 and PP2Ac and promoted the interaction between MKK7 and PP2Ac. In mice, injection of HCC xenograft tumors with siTIPRL and TRAIL led to tumor apoptosis and regression. CONCLUSIONS: TIPRL is highly up-regulated in human HCC samples and cell lines, compared with noncancerous liver tissues. TIPRL prevents prolonged activation of MKK7 and JNK and TRAIL-induced apoptosis by mediating the interaction between MKK7 and PP2Ac.

Autophagy promotes intracellular degradation of type I collagen induced by transforming growth factor (TGF)-ß1.

Autophagy is a highly conserved cellular process regulating turnover of cytoplasmic proteins via a lysosome-dependent pathway. Here we show that kidneys from mice deficient in autophagic protein Beclin 1 exhibited profibrotic phenotype, with increased collagen deposition. Reduced Beclin 1 expression, through genetic disruption of beclin 1 or knockdown by specific siRNA in primary mouse mesangial cells (MMC), resulted in increased protein levels of type I collagen (Col-I). Inhibition of autolysosomal protein degradation by bafilomycin A(1) also increased Col-I protein levels and colocalization of Col-I with LC3, an autophagy marker, or LAMP-1, a lysosome marker, whereas treatment with TFP, an inducer of autophagy, resulted in decreased Col-I protein levels induced by TGF-ß1, without alterations in Col-I α1 mRNA. Heterozygous deletion of beclin 1 increased accumulation of aggregated Col-I under nonstimulated conditions, and stimulation with TGF-ß1 further increased aggregated Col-I. These data indicate that Col-I and aggregated, insoluble procollagen I undergo intracellular degradation via autophagy. A cytoprotective role of autophagy is implicated in kidney injury, and we demonstrate that low-dose carbon monoxide, shown to exert cytoprotection against renal fibrosis, induces autophagy to suppress accumulation of Col-I induced by TGF-ß1. We also show that TGF-ß1 induces autophagy in MMC via TAK1-MKK3-p38 signaling pathway. The dual functions of TGF-ß1, as both an inducer of Col-I synthesis and an inducer of autophagy and Col-I degradation, underscore the multifunctional nature of TGF-ß1. Our findings suggest a novel role of autophagy as a cytoprotective mechanism to negatively regulate and prevent excess collagen accumulation in the kidney.

TGF-{beta}1 protects against mesangial cell apoptosis via induction of autophagy.

Autophagy can lead to cell death in response to stress, but it can also act as a protective mechanism for cell survival. We show that TGF-ß1 induces autophagy and protects glomerular mesangial cells from undergoing apoptosis during serum deprivation. Serum withdrawal rapidly induced autophagy within 1 h in mouse mesangial cells (MMC) as determined by increased microtubule-associated protein 1 light chain 3 (LC3) levels and punctate distribution of the autophagic vesicle-associated-form LC3-II. We demonstrate that after 1 h there was a time-dependent decrease in LC3 levels that was accompanied by induction of apoptosis, evidenced by increases in cleaved caspase 3. However, treatment with TGF-ß1 resulted in induction of the autophagy protein LC3 while suppressing caspase 3 activation. TGF-ß1 failed to rescue MMC from serum deprivation-induced apoptosis upon knockdown of LC3 by siRNA and in MMC from LC3 null (LC3(-/-)) mice. We show that TGF-ß1 induced autophagy through TAK1 and Akt activation, and inhibition of PI3K-Akt pathway by LY294002 or dominant-negative Akt suppressed LC3 levels and enhanced caspase 3 activation. TGF-ß1 also up-regulated cyclin D1 and E protein levels while down-regulating p27, thus stimulating cell cycle progression. Bafilomycin A1, but not MG132, blocked TGF-ß1 down-regulation of p27, suggesting that p27 levels were regulated through autophagy. Taken together, our data indicate that TGF-ß1 rescues MMC from serum deprivation-induced apoptosis via induction of autophagy through activation of the Akt pathway. The autophagic process may constitute an adaptive mechanism to glomerular injury by inhibiting apoptosis and promoting mesangial cell survival.

Transforming growth factor-beta (TGF-beta1) activates TAK1 via TAB1-mediated autophosphorylation, independent of TGF-beta receptor kinase activity in mesangial cells.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that signals through the interaction of type I (TbetaRI) and type II (TbetaRII) receptors to activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-beta1. However, the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated by TGF-beta1 in primary mouse mesangial cells. Under unstimulated conditions, endogenous TAK1 is stably associated with TbetaRI. TGF-beta1 stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates that the juxtamembrane region including the GS domain of TbetaRI is crucial for its interaction with TAK1. Both TbetaRI-mediated TAK1 phosphorylation and TGF-beta1-induced TAK1 phosphorylation do not require kinase activity of TbetaRI. Moreover, TbetaRI-mediated TAK1 phosphorylation correlates with the degree of its association with TbetaRI and requires kinase activity of TAK1. TAB1 does not interact with TGF-beta receptors, but TAB1 is indispensable for TGF-beta1-induced TAK1 activation. We also show that TRAF6 and TAB2 are required for the interaction of TAK1 with TbetaRI and TGF-beta1-induced TAK1 activation in mouse mesangial cells. Taken together, our data indicate that TGF-beta1-induced interaction of TbetaRI and TbetaRII triggers dissociation of TAK1 from TbetaRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation and not by the receptor kinase activity of TbetaRI.

The non-provitamin A carotenoid, lutein, inhibits NF-kappaB-dependent gene expression through redox-based regulation of the phosphatidylinositol 3-kinase/PTEN/Akt and NF-kappaB-inducing kinase pathways: role of H(2)O(2) in NF-kappaB activation.

Reactive oxygen species (ROS) have been implicated in the regulation of NF-kappaB activation, which plays an important role in inflammation and cell survival. However, the molecular mechanisms of ROS in NF-kappaB activation remain poorly defined. We found that the non-provitamin A carotenoid, lutein, decreased intracellular H(2)O(2) accumulation by scavenging superoxide and H(2)O(2) and the NF-kappaB-regulated inflammatory genes, iNOS, TNF-alpha, IL-1beta, and cyclooxygenase-2, in lipopolysaccharide (LPS)-stimulated macrophages. Lutein inhibited LPS-induced NF-kappaB activation, which highly correlated with its inhibitory effect on LPS-induced IkappaB kinase (IKK) activation, IkappaB degradation, nuclear translocation of NF-kappaB, and binding of NF-kappaB to the kappaB motif of the iNOS promoter. This compound inhibited LPS- and H(2)O(2)-induced increases in phosphatidylinositol 3-kinase (PI3K) activity, PTEN inactivation, NF-kappaB-inducing kinase (NIK), and Akt phosphorylation, which are all upstream of IKK activation, but did not affect the interaction between Toll-like receptor 4 and MyD88 and the activation of mitogen-activated protein kinases. The NADPH oxidase inhibitor apocynin and gp91(phox) deletion reduced the LPS-induced NF-kappaB signaling pathway as lutein did. Moreover, lutein treatment and gp91(phox) deletion decreased the expressional levels of the inflammatory genes in vivo and protected mice from LPS-induced lethality. Our data suggest that H(2)O(2) modulates IKK-dependent NF-kappaB activation by promoting the redox-sensitive activation of the PI3K/PTEN/Akt and NIK/IKK pathways. These findings further provide new insights into the pathophysiological role of intracellular H(2)O(2) in the NF-kappaB signal pathway and inflammatory process.

CT20126, a novel immunosuppressant, prevents collagen-induced arthritis through the down-regulation of inflammatory gene expression by inhibiting NF-kappaB activation.

The colchicine-derived CT20126 compound has recently been shown to exert an immune regulatory effect and prolong the survival of allograft skins. In this study, we explored the anti-inflammatory and anti-arthritic effects of CT20126 in vivo and in vitro as well as investigated its underlying action mechanism. CT20126 suppressed the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha, and interleukin-1beta as well as the production of nitric oxide and prostaglandin E(2) in lipopolysaccharide (LPS)-treated macrophages as well as LPS-administered mice. This drug also inhibited the production of nitric oxide, prostaglandin E(2), and the chemokines, RANTES, GROalpha, and ENA-78, in cytokine-stimulated human synoviocytes. CT20126 suppressed NF-kappaB activation and iNOS promoter activity, which correlated with its inhibitory effect on phosphorylation-dependent IkappaB kinase activation, IkappaB phosphorylation and degradation, and NF-kappaB nuclear translocation, in LPS-stimulated macrophages. This compound also inhibited LPS-induced NF-kappaB-inducing kinase (NIK) and Akt phosphorylation, which are upstream of NF-kappaB activation. Furthermore, CT20126 significantly decreased the incidence and severity of arthritis as well as inhibited the expression of inflammatory cytokines, chemokines, iNOS, and cyclooxygenase-2 in the paws of collagen-induced arthritic mice. These findings indicate that CT20126 exerts an anti-inflammatory effect through NF-kappaB-responsive inflammatory gene expression by inhibiting the NIK- and Akt-dependent canonical NF-kappaB pathway and can be used as a therapeutic agent for rheumatoid arthritis related to chronic inflammation.