GRAIL gene knockout mice protect against aging-related and noise-induced hearing loss.

The ubiquitin E3 ligase gene related to anergy in lymphocytes (GRAIL) (Rnf128) is a type 1 transmembrane protein that induces T cell anergy through the ubiquitination activity of its cytosolic RING finger. GRAIL also contains an equally large luminal region consisting primarily of an uncharacterized protease-associated (PA) domain. Using two-hybrid technology to screen for proteins that bound the PA domain we identified CD151, a member of the tetraspanin family of membrane proteins. GRAIL bound to the luminal/extracellular portion of both CD151 and the related tetraspanin CD81 using its PA domain, which promoted ubiquitination of cytosolic lysine residues. GRAIL exhibited specificity for lysines only within the tetraspanin amino terminus even in the presence of other cytosolic lysine residues in the substrate. GRAIL-mediated ubiquitination promoted proteasomal degradation and cell surface down-regulation of tetraspanins via Lys-48 linkages. As a result, the juxtaposition of PA and RING finger domains across a lipid bilayer facilitates the capture of transmembrane substrates for subsequent ubiquitination. These findings identify for the first time a single subunit E3 ligase containing a substrate-binding domain spatially restricted by a membrane from its E2 recruitment domain as well as an E3 ligase for members of the tetraspanin family.

GRAIL (gene related to anergy in lymphocytes), is an E3 ubiquitin ligase with increased expression in anergic CD4+ T cells. The expression of GRAIL has been shown to be both necessary and sufficient for the induction of T cell (T) anergy. To date, several subsets of anergic T cells have demonstrated altered interactions with antigen-presenting cells (APC) and perturbed TCR-mediated signaling. The role of GRAIL in mediating these aspects of T cell anergy remains unclear. We used flow cytometry and confocal microscopy to examine T/APC interactions in GRAIL-expressing T cells. Increased GRAIL expression resulted in reduced T/APC conjugation efficiency as assessed by flow cytometry. Examination of single T/APC conjugates by confocal microscopy revealed altered polarization of polymerized actin and LFA-1 to the T/APC interface. When GRAIL expression was knocked down, actin polarization to the T/APC interface was restored, demonstrating that GRAIL is necessary for alteration of actin cytoskeletal rearrangement under anergizing conditions. Interestingly, proximal TCR signaling including calcium flux and phosphorylation of Vav were not disrupted by expression of GRAIL in CD4+ T cells. In contrast, interrogation of distal signaling events demonstrated significantly decreased JNK phosphorylation in GRAIL-expressing T cells. In sum, GRAIL expression in CD4+ T cells mediates alterations in the actin cytoskeleton during T/APC interactions. Moreover, in this model, our data dissociates proximal T cell signaling events from functional unresponsiveness. These data demonstrate a novel role for GRAIL in modulating T/APC interactions and provide further insight into the cell biology of anergic T cells.

Skeletal muscle atrophy occurs under a variety of conditions and can result from alterations in both protein synthesis and protein degradation. The muscle-specific E3 ubiquitin ligases, MuRF1 and MAFbx, are excellent markers of muscle atrophy and increase under divergent atrophy-inducing conditions such as denervation and glucocorticoid treatment. While deletion of MuRF1 or MAFbx has been reported to spare muscle mass following 14 days of denervation, their role in other atrophy-inducing conditions is unclear. The goal of this study was to determine whether deletion of MuRF1 or MAFbx attenuates muscle atrophy after 2 weeks of treatment with the synthetic glucocorticoid dexamethasone (DEX). The response of the triceps surae (TS) and tibialis anterior (TA) muscles to 14 days of DEX treatment (3 mg kg(-1) day(-1)) was examined in 4 month-old male and female wild type (WT) and MuRF1 or MAFbx knock out (KO) mice. Following 14 days of DEX treatment, muscle wet weight was significantly decreased in the TS and TA of WT mice. Comparison of WT and KO mice following DEX treatment revealed significant sparing of mass in both sexes of the MuRF1 KO mice, but no muscle sparing in MAFbx KO mice. Further analysis of the MuRF1 KO mice showed significant sparing of fibre cross-sectional area and tension output in the gastrocnemius (GA) after DEX treatment. Muscle sparing in the MuRF1 KO mice was related to maintenance of protein synthesis, with no observed increases in protein degradation in either WT or MuRF1 KO mice. These results demonstrate that MuRF1 and MAFbx do not function similarly under all atrophy models, and that the primary role of MuRF1 may extend beyond controlling protein degradation via the ubiquitin proteasome system.

Noise-induced hearing loss (NIHL) is a major unresolved public health problem. Here, we investigate pathomechanisms of sensory hair cell death and suggest a novel target for protective intervention. Cellular survival depends upon maintenance of energy homeostasis, largely by AMP-activated protein kinase (AMPK). In response to a noise exposure in CBA/J mice, the levels of phosphorylated AMPKα increased in hair cells in a noise intensity-dependent manner. Inhibition of AMPK via siRNA or the pharmacological inhibitor compound C attenuated noise-induced loss of outer hair cells (OHCs) and synaptic ribbons, and preserved auditory function. Additionally, noise exposure increased the activity of the upstream AMPK kinase liver kinase B1 (LKB1) in cochlear tissues. The inhibition of LKB1 by siRNA attenuated the noise-increased phosphorylation of AMPKα in OHCs, reduced the loss of inner hair cell synaptic ribbons and OHCs, and protected against NIHL. These results indicate that noise exposure induces hair cell death and synaptopathy by activating AMPK via LKB1-mediated pathways. Targeting these pathways may provide a novel route to prevent NIHL. Our results demonstrate for the first time that the activation of AMP-activated protein kinase (AMPK) α in sensory hair cells is noise intensity dependent and contributes to noise-induced hearing loss by mediating the loss of inner hair cell synaptic ribbons and outer hair cells. Noise induces the phosphorylation of AMPKα1 by liver kinase B1 (LKB1), triggered by changes in intracellular ATP levels. The inhibition of AMPK activation by silencing AMPK or LKB1, or with the pharmacological inhibitor compound C, reduced outer hair cell and synaptic ribbon loss as well as noise-induced hearing loss. This study provides new insights into mechanisms of noise-induced hearing loss and suggests novel interventions for the prevention of the loss of sensory hair cells and cochlear synaptopathy.

The (E3) ubiquitin ligase, GRAIL (gene related to anergy in lymphocytes), is associated with CD4 + T cell anergy, a state of T cell nonresponsiveness to antigen. MacKenzie et al . found that amounts of GRAIL mRNA and protein were greater in mouse CD4 + CD25 + Tregs than in CD4 + CD25 – naïve cells. Transfer of naïve CD4 + T cells from TCR-DO11.10/Rag2 –/– (DO11) mice to BALB/c mice, followed by injection of the mice with OVA peptide, induced tolerized CD4 + CD25 + cells detected with the DO11-specific antibody KJ1.26. This is a routinely used method for studying tolerance, because the donor cells express a T cell receptor (TCR) that responds only to OVA peptide, and the donor mice cannot produce any mature T cells because of the lack of the Rag2 gene (recombinase activating gene 2). KJ1.26 + CD25 + cells had larger amounts of GRAIL mRNA and Foxp3 mRNA and protein than did the KJ1.26 + CD25 – subset, as measured by real-time reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. A retroviral system was used to generate cell lines producing large amounts of GRAIL (DO11 GRAIL) or GFP (DO11 GFP). Co-culture of naïve CD4 + CD25 – T cells with antigen-presenting cells, OVA peptide, and DO GFP cells resulted in proliferation of naïve cells. However, when DO11 GRAIL cells were used instead of DO11 GFP cells, proliferation of naïve CD4 + CD25 – T cells was suppressed. DO11 GRAIL cells produced more transforming growth factor-β (TGF-β), a cytokine associated with Treg function, than did DO11 cells. DO11 GRAIL cells also had larger amounts of the surface Treg markers, CD25, GITR, and CTLA4. Surprisingly, DO11 GRAIL cells did not produce the cytokine, IL-10, or the transcription factor, Foxp3, both of which are associated with Treg function. However, this study demonstrates the presence of GRAIL in CD4 + CD25 + Tregs and suggests a role for GRAIL in suppressor function. D. A. MacKenzie, J. Schartner, J. Lin, A. Timmel, M. Jennens-Clough, C. G. Fathman, C. M. Seroogy, GRAIL is up-regulated in CD4 + CD25 + T regulatory cells and is sufficient for conversion of T cells to a regulatory phenotype. J. Biol. Chem. 282 , 9696-9702 (2007). [Abstract] [Full Text]

T-cell anergy may serve to limit autoreactive T-cell responses in vivo. Anergy induction in vitro is blocked by calcineurin inhibitors and by inhibition of protein synthesis. In order to look for a potential anergy specific gene, we examined early changes in gene expression in murine CD4+ T-cell clones after antigen-T-cell receptor signaling in the presence (activation) or absence (anergy) of B7 co-stimulation. GRAIL (Gene Related to Anergy in Lymphocytes) was a novel transcript whose expression was markedly induced in anergic T cells in vitro compared with activated or resting T cells. GRAIL is a novel murine type I transmembrane protein that localizes to the endocytic pathway and bears homology to several RING Zinc-finger proteins. GRAIL functions as an E3 ubiquitin ligase. Expression of GRAIL in retrovirally transduced T-cell hybridomas dramatically limits activation-induced IL-2 production in vitro. Substitution of histidine for asparagine at two positions in the ring finger (H2N2 GRAIL) blocks enzymatic function of GRAIL. Retroviral transduction of hematopoietic stem cells to express GRAIL reiterates the anergy phenotype in resultant CD4+ T cells, including inability to secrete IL-2 or proliferate following antigen stimulation. Expression of the enzymatically inactive (dominant-negative) form of H2N2 GRAIL blocks anergy induction in T cells in vivo. These data demonstrate that GRAIL is necessary and sufficient to induce anergy in CD4+ T cells.

Anergy is an important mechanism for the maintenance of peripheral tolerance and avoidance of autoimmunity. The up-regulation of E3 ubiqitin ligases, including GRAIL (gene related to anergy in lymphocytes), is a key event in the induction and preservation of anergy in T cells. However, the mechanisms of GRAIL-mediated anergy induction are still not completely understood. We examined which proteins serve as substrates for GRAIL in anergic T cells. Arp2/3-5 (actin-related protein 2/3 subunit 5) and coronin 1A were polyubiquitinated by GRAIL via Lys-48 and Lys-63 linkages. In anergic T cells and GRAIL-overexpressed T cells, the expression of Arp2/3-5 and coronin 1A was reduced. Furthermore, we demonstrated that GRAIL impaired lamellipodium formation and reduced the accumulation of F-actin at the immunological synapse. GRAIL functions via the ubiquitination and degradation of actin cytoskeleton-associated proteins, in particular Arp2/3-5 and coronin 1A. These data reveal that GRAIL regulates proteins involved in the actin cytoskeletal organization, thereby maintaining the unresponsive state of anergic T cells.

Gene related to anergy in lymphocytes (GRAIL) is an E3 ubiquitin ligase that regulates energy in T-lymphocytes. Whereas, the relevance of GRAIL to T lymphocyte function is well established, the role of this protein in other cell types remains unknown. Given that GRAIL is abundant in the liver, we investigated the potential function of GRAIL in nutrient metabolism by generating mice in which the expression of GRAIL is reduced specifically in the liver. Adenovirus-mediated transfer of a short hairpin RNA specific for GRAIL mRNA markedly reduced the amounts of GRAIL mRNA and protein in the liver. Blood glucose levels of the mice with hepatic GRAIL deficiency did not differ from those of control animals in the fasted or fed states. However, these mice manifested glucose intolerance in association with a normal increase in plasma insulin levels during glucose challenge. The mice also manifested an increase in the serum concentration of free fatty acids, whereas the serum levels of cholesterol and triglyceride were unchanged. The hepatic abundance of mRNAs for glucose-6-phosphatase, catalytic (a key enzyme in hepatic glucose production) and for sterol regulatory element-binding transcription factor 1 (an important transcriptional regulator of lipogenesis) was increased in the mice with hepatic GRAIL deficiency, possibly contributing to the metabolic abnormalities of these animals. Our results thus demonstrate that GRAIL in the liver is essential for maintenance of normal glucose and lipid metabolism in living animals.

It has long been appreciated that CD4 T cells are held in G1/S cell cycle arrest. How this is initially achieved, and then lost upon activation, is not well understood. Recent studies from our lab have demonstrated that the E3 ligase, GRAIL (gene related to anergy in lymphocytes, also known as RNF128), is expressed only in late stage QA2 positive thymocytes upon their exit from the thymus, and that its expression holds CD4 T cells in cell cycle arrest. GRAIL is not only expressed in late stage thymic migrants, but in resting CD4 T cells as well as in anergic CD4 T cells suggesting that GRAIL might be involved in holding CD4 T cells in cell cycle arrest. Using a novel E3 substrate screen, we identified cdc37 (a G1-specific cyclin) as a target of GRAIL mediated ubiquitination. Consistent with this idea, GRAIL expression leads to diminished cdc37 expression and maintained cell cycle arrest upon activation. Inversely, primary CD4 T cells lacking GRAIL have higher levels of cdc37 compared to wildtype T cells in resting conditions and allow full activation with TCR stimulation in the absence of costimulation. These data suggest that GRAIL ubiquinates and targets cdc37 for degradation and thus, maintains CD4 T cells in G1/S cell cycle arrest. These data also highlight a role for cdc37 in GRAIL-mediated cell cycle regulation and CD4 T cell proliferation. The activated CD4 T cells reacquire GRAIL expression and return to the resting G1/S interphase by unknown mechanisms.

CD4+ T cells are the master regulators of adaptive immune responses, and many autoimmune diseases arise due to a breakdown of self tolerance in CD4+ cells. Gene related to anergy in lymphocytes (GRAIL), the E3 ubiqutine ligase, has acknowledged as one of key molecules implicated in T cell activation and tolerance. In order to understand the physiological function of GRAIL in immune regulation, we have generated and analyzed GRAIL deficient mice. Naive T cells lacking GRAIL showed greatly enhanced proliferation and cytokine production after T cell receptor (TcR) activation. In addition, lack of GRAIL abrogated suppressive function of regulatory T (Treg) cells. We found that GRAIL deficient naive and Treg cells after TcR activation expressed substantially higher amounts of NFATc1 compared to wild-type cells, whereas the activation of other factors in AP-1 and NFκB pathways were normal. Our data also suggested that sustained TcR cell-surface expression in the absence of GRAIL led to selective NFATc1 expression in both naive T cells and Treg cells. In contrast to naïve T cells, GRAIL, through controlling NFATc1 expression, inhibits IL-21 production and upregulation of Th17-specific genes in Treg cells. Thus, the immune regulation by GRAIL in both naive and Treg cells is absolutely critical as evidenced by the failure of T cell tolerance induction and greatly increased susceptibility to autoimmune diseases of GRAIL deficient mice.

Oxidative stress plays a substantial role in the genesis of noise-induced cochlear injury that causes permanent hearing loss. We present the results of three different approaches to enhance intrinsic cochlear defense mechanisms against oxidative stress. This article explores, through the following set of hypotheses, some of the postulated causes of noise-induced cochlear oxidative stress (NICOS) and how noise-induced cochlear damage may be reduced pharmacologically. 1) NICOS is in part related to defects in mitochondrial bioenergetics and biogenesis. Therefore, NICOS can be reduced by acetyl-L carnitine (ALCAR), an endogenous mitochondrial membrane compound that helps maintain mitochondrial bioenergetics and biogenesis in the face of oxidative stress. 2) A contributing factor in NICOS injury is glutamate excitotoxicity, which can be reduced by antagonizing the action of cochlear -methyl-D-aspartate (NMDA) receptors using carbamathione, which acts as a glutamate antagonist. 3) Noise-induced hearing loss (NIHL) may be characterized as a cochlear-reduced glutathione (GSH) deficiency state; therefore, strategies to enhance cochlear GSH levels may reduce noise-induced cochlear injury. The objective of this study was to document the reduction in noise-induced hearing and hair cell loss, following application of ALCAR, carbamathione, and a GSH repletion drug D-methionine (MET), to a model of noise-induced hearing loss. This was a prospective, blinded observer study using the above-listed agents as modulators of the noise-induced cochlear injury response in the species chinchilla langier. Adult chinchilla langier had baseline-hearing thresholds determined by auditory brainstem response (ABR) recording. The animals then received injections of saline or saline plus active experimental compound starting before and continuing after a 6-hour 105 dB SPL continuous 4-kHz octave band noise exposure. ABRs were obtained immediately after noise exposure and weekly for 3 weeks. After euthanization, cochlear hair cell counts were obtained and analyzed. RESULTS ALCAR administration reduced noise-induced threshold shifts. Three weeks after noise exposure, no threshold shift at 2 to 4 kHz and <10 dB threshold shifts were seen at 6 to 8 kHz in ALCAR-treated animals compared with 30 to 35 dB in control animals. ALCAR treatment reduced both inner and outer hair cell loss. OHC loss averaged <10% for the 4- to 10-kHz region in ALCAR-treated animals and 60% in saline-injected-noise-exposed control animals. Noise-induced threshold shifts were also reduced in carbamathione-treated animals. At 3 weeks, threshold shifts averaged 15 dB or less at all frequencies in treated animals and 30 to 35 dB in control animals. Averaged OHC losses were 30% to 40% in carbamathione-treated animals and 60% in control animals. IHC losses were 5% in the 4- to 10-kHz region in treated animals and 10% to 20% in control animals. MET administration reduced noise-induced threshold shifts. ANOVA revealed a significant difference (P <.001). Mean OHC and IHC losses were also significantly reduced (P <.001). These data lend further support to the growing body of evidence that oxidative stress, generated in part by glutamate excitotoxicity, impaired mitochondrial function and GSH depletion causes cochlear injury induced by noise. Enhancing the cellular oxidative stress defense pathways in the cochlea eliminates noise-induced cochlear injury. The data also suggest strategies for therapeutic intervention to reduce NIHL clinically.

Cochlin is the most abundant protein in the inner ear. To study its function in response to noise trauma, we exposed adolescent wild-type (Coch+/+) and cochlin knock-out (Coch–/–) mice to noise (8–16 kHz, 103 dB SPL, 2 h) that causes a permanent threshold shift and hair cell loss. Two weeks after noise exposure, Coch–/– mice had substantially less elevation in noise-induced auditory thresholds and hair cell loss than Coch+/+ mice, consistent with cochlin deficiency providing protection from noise trauma. Comparison of pre-noise exposure thresholds of auditory brain stem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) in Coch–/– mice and Coch+/+ littermates revealed a small and significant elevation in thresholds of Coch–/– mice, overall consistent with a small conductive hearing loss in Coch–/– mice. We show quantitatively that the pro-inflammatory component of cochlin, LCCL, is upregulated after noise exposure in perilymph of wild-type mice compared to unexposed mice, as is the enzyme catalyzing LCCL release, aggrecanase1, encoded by Adamts4. We further show that upregulation of pro-inflammatory cytokines in perilymph and cochlear soft-tissue after noise exposure is lower in cochlin knock-out than wild-type mice. Taken together, our data demonstrate for the first time that cochlin deficiency results in conductive hearing loss that protects against physiologic and molecular effects of noise trauma.

Adenosine receptors regulate susceptibility to noise-induced neural injury in the mouse cochlea and hearing loss

NK cells (natural killer cells) being a part of the innate immune system have been shown to be involved in immunoregulation of autoimmune diseases. Previously we have shown that HINT1/Hsp70 treatment induced regulatory NK cells ameliorating experimental autoimmune encephalomyelitis (EAE) course and CD4+ T cells proliferation. NK cells were isolated from mice treated with HINT1/Hsp70 and co-cultured with proteolipid protein (PLP)-stimulated CD4+ T cells isolated from EAE mice. Cell proliferation was assessed by thymidine uptake, cytotoxicity by lactate dehydrogenase (LDH) release assay and fluorescence activated cell sorting (FACS) analysis, protein expression by Western blot, mRNA by quantitative RT-PCR. Gene related to anergy in lymphocytes (GRAIL) expression was downregulated by specific siRNA and GRAIL overexpression was induced by pcDNA-GRAIL transfection. HINT1/Hsp70 pretreatment of EAE SJL/J mice ameliorated EAE course, suppressed PLP-induced T cell proliferation by enhancing T cell expression of GRAIL as GRAIL downregulation restored T cell proliferation. HINT1/Hsp70 treatment induced immunoregulatory NK cells which inhibited PLP-stimulated T cell proliferation not depending on T cell necrosis and apoptosis. This immunoregulatory NK cell function depended on NK cell expression of GRAIL as GRAIL downregulation diminished inhibition of NK cell suppression of T cell proliferation. Similarly GRAIL overexpression in NK cells induced their regulatory function. HINT1/Hsp70 treatment generated regulatory NK cells characterized by expression of GRAIL.

A novel GRAIL E3 ubiquitin ligase promotes environmental salinity tolerance in euryhaline tilapia