The kinase IKKα inhibits activation of the transcription factor NF-κB by phosphorylating the regulatory molecule TAX1BP1.

In response to stimulation with proinflammatory cytokines, the deubiquitinase A20 inducibly interacts with the regulatory molecules TAX1BP1, Itch and RNF11 to form the A20 ubiquitin-editing complex. However, the molecular signal that coordinates the assembly of this complex has remained elusive. Here we demonstrate that TAX1BP1 was inducibly phosphorylated on Ser593 and Ser624 in response to proinflammatory stimuli. The kinase IKKα, but not IKKß, was required for phosphorylation of TAX1BP1 and directly phosphorylated TAX1BP1 in response to stimulation with tumor necrosis factor (TNF) or interleukin 1 (IL-1). TAX1BP1 phosphorylation was pivotal for cytokine-dependent interactions among TAX1BP1, A20, Itch and RNF11 and downregulation of signaling by the transcription factor NF-κB. IKKα therefore serves a key role in the negative feedback of NF-κB canonical signaling by orchestrating assembly of the A20 ubiquitin-editing complex to limit inflammatory gene activation.

The E3 ligase Itch negatively regulates inflammatory signaling pathways by controlling the function of the ubiquitin-editing enzyme A20.

The ubiquitin-editing enzyme A20 is a critical negative regulator of inflammation and cytokine-mediated activation of the transcription factor NF-kappaB; however, little is known about the mechanisms of A20-mediated inactivation of signaling intermediates such as RIP1. Here we demonstrate that the regulatory molecule TAX1BP1 recruited the E3 ligase Itch to A20 via two 'PPXY' motifs. Itch was essential for the termination of tumor necrosis factor receptor signaling by controlling A20-mediated recruitment and inactivation of RIP1. Furthermore, the Tax oncoprotein of human T cell leukemia virus type I targeted this complex for inactivation by disrupting the interaction among TAX1BP1, A20 and Itch. Thus, our studies show a previously unappreciated complexity of A20 substrate recognition and inactivation whereby TAX1BP1 and Itch function as essential subunits of an A20 ubiquitin-editing complex.

The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-kappaB signalling.

The RING domain protein RNF11 is overexpressed in breast cancers and promotes tumour growth factor-beta (TGF-beta) signalling. RNF11 has been proposed to regulate TGF-beta signalling by interacting with HECT- and SCF-type E3 ligases; however, the role of RNF11 in other signalling pathways is poorly understood. Here, we demonstrate a novel function of RNF11 as a negative regulator of NF-kappaB and jun N-terminal kinase (JNK) signalling pathways. Knockdown of RNF11 with siRNA resulted in persistent tumour necrosis factor (TNF)- and lipopolysaccharide (LPS)-mediated NF-kappaB and JNK signalling. RNF11 interacted with the NF-kappaB inhibitor A20 and its regulatory protein TAX1BP1 in a stimulus-dependent manner. RNF11 negatively regulated RIP1 and TRAF6 ubiquitination upon stimulation with TNF and LPS, respectively. Furthermore, RNF11 was required for A20 to interact with and inactivate RIP1 to inhibit TNF-mediated NF-kappaB activation. Our studies reveal that RNF11, together with TAX1BP1 and Itch, is an essential component of an A20 ubiquitin-editing protein complex that ensures transient activation of inflammatory signalling pathways.

Human T cell leukemia virus type 1 Tax inhibits innate antiviral signaling via NF-kappaB-dependent induction of SOCS1.

Human T cell leukemia virus type 1 (HTLV-1) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-1 Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-1-transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-I-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-1-transformed cell line MT-2 resulted in increased IFN-ß expression accompanied by reduced HTLV-1 replication and p19(Gag) levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein.

The human T-cell leukemia virus type 1 Tax oncoprotein requires the ubiquitin-conjugating enzyme Ubc13 for NF-kappaB activation.

Ubiquitination of the human T-cell leukemia virus 1 Tax oncoprotein provides an important regulatory mechanism that promotes the Tax-mediated activation of NF-kappaB. However, the type of polyubiquitin chain linkages and the host factors that are required for Tax ubiquitination have not been identified. Here, we demonstrate that Tax polyubiquitin chains are composed predominantly of lysine 63-linked chains. Furthermore, the ubiquitination of Tax is critically dependent on the E2 ubiquitin-conjugating enzyme Ubc13. Tax interacts with Ubc13, and small interfering RNA-mediated knockdown of Ubc13 expression abrogates Tax ubiquitination and the activation of NF-kappaB. Mouse fibroblasts lacking Ubc13 exhibit impaired Tax activation of NF-kappaB despite normal tumor necrosis factor- and interleukin-1-mediated NF-kappaB activation. Finally, the interaction of Tax with NEMO is disrupted in the absence of Tax ubiquitination and Ubc13 expression, suggesting that Tax ubiquitination is critical for NEMO binding. Collectively, our results reveal that Ubc13 is essential for Tax ubiquitination, its interaction with NEMO, and Tax-mediated NF-kappaB activation.

VEGF activation of protein kinase C stimulates occludin phosphorylation and contributes to endothelial permeability.

PURPOSE: VEGF is a potent permeabilizing factor that contributes to the pathogenesis of diabetic retinopathy and brain tumors. VEGF-induced vascular permeability in vivo and in cell culture requires PKC activity, but the mechanism by which PKC regulates barrier properties remains unknown. This study was conducted to examine how VEGF and diabetes alter occludin phosphorylation and endothelial cell permeability. METHODS: Chemical PKC inhibitors and activators were used to treat primary retinal endothelial cells in culture. In vitro kinase assays and Western blot analysis of two-dimensional (2D) and one-dimensional (1D) gel retardation assays were used to analyze occludin phosphorylation. Endothelial cell permeability was determined by measuring the flux of 70-kDa dextran through a cell monolayer in culture. Exogenous expression of a dominant negative PKCbetaII mutant (S217A) was used to assess the PKC dependence of VEGF-induced occludin phosphorylation and endothelial permeability. Occludin phosphorylation was also determined in retinas of streptozotocin-induced diabetic rats. RESULTS: VEGF stimulated the phosphorylation of occludin in primary retinal endothelial cells. Chemical inhibitors of PKC activity blocked the VEGF-induced increase in occludin phosphorylation, as assessed by 2D gel and gel retardation in Western blot analysis, and blocked part of the VEGF-induced monolayer permeability to 70-kDa dextran. Expression of a dominant negative PKCbetaII mutant blocked VEGF-induced occludin phosphorylation and endothelial permeability. Finally, elevated occludin phosphorylation was observed in the retina of diabetic animals. CONCLUSIONS: These results strongly suggest that VEGF-induced endothelial permeability requires PKC-dependent phosphorylation of occludin. Regulation of PKC activity and tight junction protein modifications may have therapeutic implications for treatment of diabetic retinopathy and brain tumors.

Regulation of tight junctions and loss of barrier function in pathophysiology.

The mechanism by which epithelial and endothelial cells interact to form polarized tissue is of fundamental importance to multicellular organisms. Dysregulation of these barriers occurs in a variety of diseases, destroying the normal cellular environments and leading to organ failure. Increased levels of growth factors are a common characteristic of diseases exhibiting tissue permeability, suggesting that growth factors play a direct role in elevating permeability. Of particular concern for this laboratory, increased expression of vascular endothelial growth factor may enhance vascular permeability in diabetic retinopathy, leading to vision impairment and blindness. However, the mechanism by which growth factors increase permeability is unclear. Polarized cells form strong barriers through the development of tight junctions, which are specialized regions of the junctional complex. Tight junctions are composed of three types of transmembrane proteins, a number of peripheral membrane structural proteins, and are associated with a variety of regulatory proteins. Recent data suggest that growth factor-stimulated alterations in tight junctions contribute to permeability in a variety of disease states. The goal of this review was to elucidate potential mechanisms by which elevated growth factors elicit deregulated paracellular permeability via altered regulation of tight junctions, with particular emphasis on the tight junction proteins occludin and ZO-1, protein kinase C signaling, and endocytosis of junctional proteins. Understanding the molecular mechanisms underlying growth factor-mediated regulation of tight junctions will facilitate the development of novel treatments for diseases such as brain tumors, diabetic retinopathy and other diseases with compromised tight junction barriers.

Activation of NF-kappa B by the human T cell leukemia virus type I Tax oncoprotein is associated with ubiquitin-dependent relocalization of I kappa B kinase.

Human T cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia. HTLV-1 encodes a trans-activating protein, Tax, which is largely responsible for the oncogenic properties of the virus. Tax promotes T cell transformation by deregulating the activity of various cellular factors, including the transcription factor NF-kappaB. Tax activates the IkappaB kinase (IKK) via physical interaction with the regulatory subunit, IKKgamma, although it is unknown precisely how Tax activates the IKK complex. Here we show that Tax modulates the cellular localization of the IKK complex. The IKKs relocalize from a broad distribution in the cytoplasm to concentrated perinuclear "hot spots" in both HTLV-1-transformed lines and in Tax-expressing Jurkat cells. Relocalization of IKK is not observed with Tax mutants unable to activate NF-kappaB, suggesting that only activated forms of IKK are relocalized. However, relocalization of IKK is strictly dependent on Tax expression because it does not occur in ATL cell lines that lack Tax expression or in Jurkat cells treated with phorbol 12-myristate 13-acetate and ionomycin. Furthermore, IKKgamma is required for redistribution because cells lacking IKKgamma were unable to relocalize IKKalpha upon expression of Tax. We also find that Tax ubiquitination likely regulates IKK relocalization because mutation of three critical lysine residues in Tax renders it unable to relocalize IKK and activate the canonical and noncanonical NF-kappaB pathways. Finally, we have observed that the perinuclear IKK in Tax-expressing cells colocalizes with the Golgi, and disruption of Golgi with either nocodazole or brefeldin A leads to a redistribution of IKK to the cytoplasm. Together, these results demonstrate that Tax induces relocalization of the IKK complex in a ubiquitin-dependent manner, and dynamic changes in the subcellular localization of the IKK complex may be critical for Tax function.

Essential role for TAX1BP1 in the termination of TNF-alpha-, IL-1- and LPS-mediated NF-kappaB and JNK signaling.

The NF-kappaB transcription factor is normally transiently activated by proinflammatory cytokines and bacterial lipopolysaccharide (LPS); however, persistent NF-kappaB activation is commonly observed in inflammatory disease and malignancy. The ubiquitin editing enzyme A20 serves an essential role in the termination of TNF-alpha- and LPS-mediated NF-kappaB signaling by inactivating key signaling molecules. However, little is known about how A20 is regulated and if other molecules play a role in the termination of NF-kappaB signaling. Here we demonstrate that Tax1-binding protein 1 (TAX1BP1) is essential for the termination of NF-kappaB and JNK activation in response to TNF-alpha, IL-1 and LPS stimulation. In TAX1BP1-deficient mouse fibroblasts, TNF-alpha-, IL-1- and LPS-mediated IKK and JNK activation is elevated and persistent owing to enhanced ubiquitination of RIP1 and TRAF6. Furthermore, in the absence of TAX1BP1, A20 is impaired in RIP1 binding, deubiquitination of TRAF6 and inhibition of NF-kappaB activation. Thus, TAX1BP1 is pivotal for the termination of NF-kappaB and JNK signaling by functioning as an essential regulator of A20.

Deregulated expression of CD40 ligand in HTLV-I infection: distinct mechanisms of downregulation in HTLV-I-transformed cell lines and ATL patients.

HTLV-I infection is associated with the development of adult T cell leukemia (ATL) and the neuroinflammatory disease HAM/TSP. There are quantitative and qualitative differences in the antiviral cytotoxic T cell (CTL) response in ATL and HAM/TSP although the underlying mechanisms are unclear. Here, we demonstrate that the HTLV-I Tax trans-activating protein is a transcriptional activator of CD40 ligand (CD40L), a critical regulator of dendritic cell maturation and adaptive immunity. Tax activates CD40L expression via a cyclosporin A insensitive pathway that is also independent of NF-kappaB. Although Tax upregulates CD40L gene expression, CD40L expression is absent in Tax-expressing HTLV-I-transformed cell lines via an epigenetic mechanism involving methylation. T lymphocytes cultured ex vivo from ATL patients, but not HAM/TSP or normal controls, exhibit a potent block in the induction of CD40L, but not CD69. However, the CD40L gene is not silenced by methylation in ATL patients, thus CD40L is downregulated by distinct mechanisms in HTLV-I-transformed cell lines and ATL patients.

Mechanisms of persistent NF-kappaB activation by HTLV-I tax.

Human T cell leukemia virus type I (HTLV-I) is the causative agent of a fatal malignancy known as adult T cell leukemia (ATL). The HTLV-I Tax protein is thought to play a significant role in the initiation and pathogenesis of HTLV-I-mediated disease. Tax is a potent oncogene that deregulates cellular gene expression by persistently activating signaling pathways such as NF-kappaB. Tax activation of NF-kappaB is critical for the immortalization and survival of HTLV-I-infected T cells. In this review, we describe recent insights into the mechanisms employed by Tax to activate the canonical and noncanonical NF-kappaB signaling pathways. The adaptor function of Tax appears to be a common and important mechanism for the pathological activation of both NF-kappaB pathways.

Human T cell leukemia virus type I Tax activates CD40 gene expression via the NF-kappa B pathway.

The human T cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus that is etiologically linked to the genesis of adult T cell leukemia (ATL) as well as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Emerging evidence suggests that the pathogenicity of HTLV-I involves deregulated activation of immune cells, especially T lymphocytes, although the underlying mechanism remains unclear. In this study, we demonstrate that HTLV-I Tax induces the aberrant expression of CD40, a member of the tumor necrosis factor receptor (TNFR) family that plays an important role in lymphocyte activation and differentiation. In a panel of HTLV-I-transformed T cell lines analyzed, CD40 expression was highly elevated compared to HTLV-I-negative T cells. Using Tax mutants and a genetically manipulated T cell system, we demonstrated that Tax-induced CD40 expression required the NF-kappaB signaling pathway. In addition, ligation of CD40 on T cells with recombinant CD40L elicited NF-kappaB activation, suggesting that the CD40 pathway is intact and may participate in a positive regulatory loop in T cells. CD40 ligation strongly synergized with Tax to activate NF-kappaB, suggesting that CD40 signals may costimulate Tax-mediated NF-kappaB activation, particularly when Tax is expressed at low levels. Collectively, these results indicate that CD40 is a novel Tax-regulated gene, and the regulation of CD40 by Tax may play a role in cellular activation and HTLV-I-induced disease pathogenesis.

Platelet-derived growth factor mediates tight junction redistribution and increases permeability in MDCK cells.

Increased tissue permeability is a common characteristic of a number of diseases such as pulmonary edema, inflammatory bowel disease, several kidney diseases, diabetic retinopathy, and tumors. We hypothesized that growth factors increase permeability by redistribution of tight junction proteins away from the cell border. To investigate mechanisms of growth factor-mediated permeability, we examined the effect of platelet derived growth factor (PDGF) on Madin-Darby canine kidney (MDCK) cell tight junction protein distribution and on permeability. PDGF altered the cellular distribution of occludin and ZO-1 from the cell border to the cytoplasm and increased permeability to 70 kDa dextran in a concentration-dependent manner. Treatment of MDCK cells with PDGF prior to fixation allowed binding of the lectin concanavalin A to the basement membrane of fixed cells, while binding was prevented in untreated control monolayers, implying that PDGF induced the formation of a paracellular transport pathway. Cell fractionation experiments with PDGF-treated cells revealed a novel occludin-containing low-density, detergent resistant subcellular structure, which increased in the buoyant fractions relative to occludin in the pellet in a time- and concentration-dependent manner. Immunocytochemistry revealed that a pool of internalized occludin co-labels with the early endosome marker, EEA1, suggesting that PDGF may stimulate occludin to enter an endosomal pathway. PDGF may act as a permeabilizing agent by moving tight junction proteins away from the cell border in discrete microdomains, and the effects of PDGF on permeability and tight junction protein distribution may model the regulation of epithelial and endothelial barrier properties by other peptide growth factors.

Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin.

Corticosteroids provide an effective treatment to reduce edema for conditions in which the blood-brain or blood-retinal barrier is compromised. However, little is known about the mechanism by which these hormones affect endothelial cell function. We hypothesized that hydrocortisone would reduce transport of water and solutes across bovine retinal endothelial cell (BREC) monolayers coincident with changes to the tight junction protein occludin. Treatment of BREC with 103 nm hydrocortisone for two days significantly decreased water and solute transport across cell monolayers. Immunoblot analysis of occludin extracted in SDS or urea based buffers revealed a 1.65- or 2.57-fold increase in content, respectively. A similar two-fold increase in occludin mRNA was observed by real-time PCR. Immunocytochemistry revealed hydrocortisone dramatically increased both occludin and ZO-1 staining at the cell border. Additionally, 4 h of hydrocortisone treatment significantly reduced occludin phosphorylation. To our knowledge, this is the first example of a regulated decrease in occludin phosphorylation associated with increased barrier properties. In conclusion, hydrocortisone directly affects retinal endothelial cell barrier properties coincident with changes in occludin content, phosphorylation and tight junction assembly. Localized hydrocortisone therapy may be developed as a treatment option for patients suffering from retinal edema due to diabetes.