Tumor necrosis factor alpha-induced glucose transporter (GLUT-1) mRNA stabilization in 3T3-L1 preadipocytes. Regulation by the adenosine-uridine binding factor.

Tumor necrosis factor alpha (TNF alpha), 12-O-tetradecanoylphorbol-13-acetate and cAMP stimulate hexose transport in quiescent 3T3-L1 preadipocytes by stabilizing the relatively labile mRNA coding for the basal glucose transporter, GLUT-1. The 3'-UTR of GLUT-1 mRNA contains a single copy of the destabilizing AUUUA motif in the context of an AU-rich region. The adenosine-uridine binding factor (AUBF) is a cytosolic protein which interacts with similar AU-rich regions in a variety of labile cytokine and oncogene mRNAs. Here, we demonstrate that AUBF complexes in vitro with GLUT-1 mRNA through the AU-rich portion of the 3'-UTR. AUBF activity is very low in quiescent preadipocytes, but can be up-regulated by agonists such as TPA, TNF alpha, cAMP, and okadaic acid, all of which stabilize GLUT-1 mRNA. The time courses of TNF alpha- and TPA-mediated AUBF up-regulation and GLUT-1 mRNA stabilization are coincident, suggesting a cause and effect relationship.     

Ras participates in the activation of p38 MAPK by interleukin-1 by associating with IRAK, IRAK2, TRAF6, and TAK-1.

Interleukin-1 (IL-1) activates p38 MAP kinase via the small G protein Ras, and this activity can be down-regulated by another small G protein Rap. Here we have further investigated the role of Ras and Rap in p38 MAPK activation by IL-1. Transient transfection of cells with constitutively active forms of the known IL-1 signaling components MyD88, IRAK, and TRAF-6, or the upstream kinases MKK6 and MKK3, activated p38 MAPK. Dominant negative forms of these were found to inhibit activation of p38 MAPK by IL-1. Dominant negative RasN17 blocked the effect of the active forms of all but MKK3 and MKK6, indicating that Ras lies downstream of TRAF-6 but upstream of MKK3 and MKK6 on the pathway. Furthermore, the activation of p38 MAPK caused by overexpressing active RasVHa could not be inhibited using dominant negative mutants of MyD88, IRAK, or IRAK-2, or TRAF6, but could be inhibited by dominant negative MKK3 or MKK6. In the same manner, the inhibitory effect of Rap on the activation of p38 by IL-1 occurred at a point downstream of MyD88, IRAK, and TRAF6, since the activation of p38 MAPK by these components was inhibited by overexpressing active Rap1AV12, while neither MKK3 nor MKK6 were affected. Active RasVHa associated with IRAK, IRAK2, and TRAF6, but not MyD88. In addition we found a role for TAK-1 in the activation of p38 MAPK by IL-1, with TAK-1 also associating with active Ras. Our study suggests that upon activation Ras becomes associated with IRAK, Traf-6, and TAK-1, possibly aiding the assembly of this multiprotein signaling complex required for p38 MAPK activation by IL-1.     

Interleukin 1 alpha mRNA in virus-transformed T and B cells

IL-1 alpha cDNA clone was isolated from a T cell line infected by the human T lymphotropic retrovirus type-I (HTLV-I/ATLV). We found significant amounts of mRNA hybridizing to IL-1 alpha cDNA not only in HTLV-I-transformed T cells but also in Epstein-Barr Virus-transformed B cells. A part of IL-2 receptor inducing activity in Adult T cell leukemia (ATL) cell line seems to be due to IL-1 alpha.     

Supplementation of N-acetylcysteine inhibits NFkappaB activation and protects against alloxan-induced diabetes in CD-1 mice.

Reactive oxygen species (ROS) are involved in the destruction of pancreatic beta cells and the development of insulin-dependent diabetes mellitus (IDDM). However, the cellular mechanism responsible for beta cell death is still unclear. We hypothesize that activation of NFkappaB by ROS is the key cellular signal in initiating a cascade of events leading to beta cell death. Thus, enhancement of pancreatic GSH, a known antioxidant and key regulator of NF-kappaB, should protect against IDDM. Weanling CD1 mice (n=5) were injected with alloxan (50 mg/kg i.v.) to induce IDDM. Using EPR spin trapping techniques, we demonstrated that alloxan generated ROS in the pancreas 15 min after administration. Activation of NFkappaB in pancreatic nuclear extracts was observed 30 min after alloxan injection, as assessed by an electrophoretic mobility shift assay. Fasting blood glucose levels were monitored for 14 days. Supplementation with N-acetylcysteine (NAC, 500 mg/kg), a GSH precursor, inhibited alloxan-induced NFkappaB activation and reduced hyperglycemia. Thus, NFkappaB activation by ROS may initiate a sequence of events leading to IDDM. Inhibition of NF-kappaB activation by NAC attenuated the severity of IDDM. This research will contribute to the understanding of the etiology of IDDM and may lead to the development of better strategies for disease prevention.     

HTLV-1 Tax-induced NFkappaB activation is independent of Lys-63-linked-type polyubiquitination

Human T-cell leukemia virus type 1 (HTLV-1) Tax-induced activation of nuclear factor-kappaB (NFkappaB) is thought to play a critical role in T-cell transformation and onset of adult T-cell leukemia. However, the molecular mechanism of the Tax-induced NFkappaB activation remains unknown. One of the mitogen-activated protein kinase kinase kinses (MAP3Ks) members, TAK1, plays a critical role in cytokine-induced activation of NFkappaB, which involves lysine 63-linked (K63) polyubiquitination of NEMO, a noncatalytic subunit of the IkappaB kinase complex. Here we show that Tax induces K63 polyubiquitination of NEMO. However, TAK1 is dispensable for Tax-induced NFkappaB activation, and deubiquitination of the K63 polyubiquitin chain failed to block Tax-induced NFkappaB activation. In addition, silencing of other MAP3Ks, including MEKK1, MEKK3, NIK, and TPL-2, did not affect Tax-induced NFkappaB activation. These results strongly suggest that unlike cytokine signaling, Tax-induced NFkappaB activation does not involve K63 polyubiquitination-mediated MAP3K activation.     

Interleukin-1 (IL-1)-induced TAK1-dependent Versus MEKK3-dependent NFkappaB activation pathways bifurcate at IL-1 receptor-associated kinase modification

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is phosphorylated after it is recruited to the receptor, subsequently ubiquitinated, and eventually degraded upon IL-1 stimulation. Although a point mutation changing lysine 134 to arginine (K134R) in IRAK abolished IL-1-induced IRAK ubiquitination and degradation, mutations of serines and threonines adjacent to lysine 134 to alanines ((S/T)A (131-144)) reduced IL-1-induced IRAK phosphorylation and abolished IRAK ubiquitination. Through the study of these IRAK modification mutants, we uncovered two parallel IL-1-mediated signaling pathways for NFkappaB activation, TAK1-dependent and MEKK3-dependent, respectively. These two pathways bifurcate at the level of IRAK modification. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classical NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through IkappaBalpha phosphorylation and subsequent dissociation from NFkappaB but without IkappaBalpha degradation. These results provide significant insight to our further understanding of NFkappaB activation pathways.     

Interleukin-1alpha-induced chemokines in mouse granulosa cells: impact on keratinocyte chemoattractant chemokine, a CXC subfamily

IL-1 is well known to be involved in the immune system and have a role in ovarian inflammation as well as exhibiting inhibitory effects on steroidogenesis and folliculogenesis. Because multiple aspects of ovarian function have also been shown to involve cytokine/chemokine networks, IL-1alpha-induced chemokine gene expression in mouse granulosa cells was investigated. Granulosa cells from immature mice at 28 d of age were cultured with IL-1alpha (10 ng/ml). IL-1alpha induced abundantly and specifically keratinocyte chemoattractant (KC) chemokine, a CXC subfamily. KC chemokine mRNA and protein were increased 1-2 h after IL-1alpha and then gradually decreased. The KC promoter (-701/+30) containing three nuclear factor (NF)-kappaB sites was fully responsive to IL-1alpha, whereas deletions and mutants of the NF-kappaB sites lowered the responsiveness to IL-1alpha. The proximal NF-kappaB site (-69/-59) played a critical role in regulating IL-1alpha-induced KC chemokine promoter activity. Overexpression of the inhibitor of NF-kappaB (IkappaB) blocked KC promoter activity induced by IL-1alpha, whereas overexpression of p65, a component of NF-kappaB, increased promoter activity and mRNA of KC chemokine. In addition, FSH did not affect NF-kappaB signaling or IL-1alpha-induced KC chemokine promoter activity. Within 1-3 h after ip injection of lipopolysaccharide (100 mug/mouse), a product known to stimulate release of IL-1, KC chemokine was localized in the ovary to granulosa cells as well as the thecal-interstitial layer. The results of this study indicate that KC gene is a chemokine induced acutely by IL-1alpha via NF-kappaB signaling in mouse granulosa cells.     

Distinct differences between TNF receptor 1- and TNF receptor 2-mediated activation of NFkappaB

Tumor necrosis factor (TNF) signaling is mediated via two distinct receptors, TNFR2 and TNFR1, which shows partially overlapping signaling mechanisms and biological roles. In the present study, TNFR2 and TNFR1 signal transduction mechanisms involved in activation of NFkappaB and CMV promoter-enhancer were compared with respect to their susceptibility towards inhibitors of intracellular signaling. For this, we used SW480 cells, where we have shown that TNF-signaling can occur independently through each of the two receptors. The TNFR1 response was inhibited by D609, bromophenacyl bromide (BPB), nordihydroguararetic acid (NDGA), and by sodium salicylate, while TNFR2-mediated activation of NFkappaB and CMV promoter-enhancer was resistant to these compounds. The signaling mechanisms known to be affected by these inhibitors include phospholipases as well as redox- and pH-sensitive intracellular components. Our results imply that TNFR2 signaling involved in NFkappaB activation proceeds independently of these inhibitor-sensitive signaling components, indicating distinct signaling pathways not shared with TNFR1.