Identification of RELT homologues that associate with RELT and are phosphorylated by OSR1

RELL1 and RELL2 are two newly identified RELT homologues that bind to the TNF receptor family member RELT. The expression of RELL1 at the mRNA level is ubiquitous, whereas expression of RELL2 mRNA is more restricted to particular tissues. RELT, RELL1, and RELL2 co-localized with one another at the plasma membrane. The three proteins interacted with one another as demonstrated by in vitro co-immunoprecipitation experiments. We propose that RELL1 and RELL2 be considered RELT family members based on their similar amino acid sequences and on their ability to physically interact with one another. OSR1 was identified through a yeast two-hybrid screen utilizing the intracellular portion of RELL1 as bait, and OSR1 was shown to interact with the three RELT family members by in vitro co-immunoprecipitation experiments. Additionally, OSR1 phosphorylated the RELT family members in an in vitro kinase assay. These results report two novel homologues of RELT that interact with RELT and are phosphorylated by the OSR1 kinase.     

Expression and purification of recombinant tristetraprolin that can bind to tumor necrosis factor-alpha mRNA and serve as a substrate for mitogen-activated protein kinases

Tristetraprolin (TTP) is an mRNA-binding protein, but studies of this interaction have been difficult due to problems with the purification of recombinant TTP. In the present study, we expressed human and mouse TTP as glutathione S-transferase and maltose-binding protein (MBP) fusion proteins in Escherichia coli, and purified them by affinity resins and Mono Q chromatography. TTP cleaved from the fusion protein was identified by immunoblotting, MALDI-MS, and protein sequencing, and was further purified to homogeneity by continuous-elution SDS-gel electrophoresis. Purified recombinant TTP bound to the AU-rich element of tumor necrosis factor-alpha (TNFalpha) mRNA and this binding was dependent on Zn(2+). Results from sizing columns suggested that the active species might be in the form of an oligomer of MBP-TTP. Recombinant TTP was phosphorylated by three members of the mitogen-activated protein (MAP) kinase family, p42, p38, and JNK, with half-maximal phosphorylation occurring at approximately 0.5, 0.25, and 0.25 microM protein, respectively. Phosphorylation by these kinases did not appear to affect the ability of TTP to bind to TNFalpha mRNA under the assay conditions. This study describes a procedure for purifying nonfusion protein TTP to homogeneity, demonstrates that TTP's RNA-binding activity is zinc dependent, and that TTP can be phosphorylated by JNK as well as by the other members of the greater MAP kinase family.     

APRIL-deficient mice have normal immune system development

APRIL (a proliferation-inducing ligand) is a member of the tumor necrosis factor (TNF) superfamily. APRIL mRNA shows high levels of expression in tumors of different origin and a low level of expression in normal cells. APRIL shares two TNF receptor family members, TACI and BCMA, with another TNF homolog, BLyS/BAFF. BLyS is involved in regulation of B-cell activation and survival and also binds to a third receptor, BR3/BAFF-R, which is not shared with APRIL. Recombinant APRIL and BLyS induce accumulation of B cells in mice, while BLyS deficiency results in severe B-cell dysfunction. To investigate the physiological role of APRIL, we generated mice that are deficient in its encoding gene. APRIL(-/-) mice were viable and fertile and lacked any gross abnormality. Detailed histological analysis did not reveal any defects in major tissues and organs, including the primary and secondary immune organs. T- and B-cell development and in vitro function were normal as well, as were T-cell-dependent and -independent in vivo humoral responses to antigenic challenge. These data indicate that APRIL is dispensable in the mouse for proper development. Thus, BLyS may be capable of fulfilling APRIL's main functions.     

Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA

Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor alpha (TNFalpha) release as a consequence of increased stability of TNFalpha mRNA. TTP was then shown to destabilize TNFalpha mRNA after binding directly to the AU-rich region (ARE) of the 3'-untranslated region of the TNFalpha mRNA. In mammals and in Xenopus, TTP is the prototype of a small family of three known zinc finger proteins containing two CCCH zinc fingers spaced 18 amino acids apart; a fourth more distantly related family member has been identified in Xenopus and fish. We show here that representatives of all four family members were able to bind to the TNFalpha ARE in a cell-free system and, in most cases, promote the breakdown of TNFalpha mRNA in intact cells. Because the primary sequences of these CCCH proteins are most closely related in their tandem zinc finger domains, we tested whether various fragments of TTP that contained both zinc fingers resembled the intact protein in these assays. We found that amino- and carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid fragment that contained both zinc fingers, could bind to the TNFalpha ARE in cell-free cross-linking and gel shift assays. In addition, these truncated forms of TTP could also stimulate the apparent deadenylation and/or breakdown of TNFalpha mRNA in intact cells. Alignments of the tandem zinc finger domains from all four groups of homologous proteins have identified invariant residues as well as group-specific signature amino acids that presumably contribute to ARE binding and protein-specific activities, respectively.     

Insulin increases tristetraprolin and decreases VEGF gene expression in mouse 3T3-L1 adipocytes

Objectives: Tristetraprolin (TTP) family proteins (TTP/ZFP36; ZFP36L1, ZFP36L2, ZFP36L3) destabilize adenylate uridylate‐rich element‐containing mRNAs encoding cytokines, such as tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF). Little is known about the expression and insulin regulation of TTP and related genes in adipocytes. We analyzed the relative abundance of TTP family mRNAs in 3T3‐L1 adipocytes compared to RAW264.7 macrophages and investigated insulin effects on the expression of 43 genes in 3T3‐L1 adipocytes. Methods and Procedures: Insulin was added to mouse 3T3‐L1 adipocytes. Relative abundance of mRNA levels was determined by quantitative real‐time PCR. TTP and ZFP36L1 proteins were detected by immunoblotting. Results: Zfp36l1 and Zfp36l2 genes were expressed at eight‐ to tenfold higher than Ttp in adipocytes. Zfp36l3 mRNA was detected at ～1% of Ttp mRNA levels in adipocytes and its low level expression was confirmed in RAW cells. Insulin at 10 and 100 nmol/l increased Ttp mRNA levels by five‐ to sevenfold, but decreased those of Zfp36l3 by 40% in adipocytes after a 30‐min treatment. Immunoblotting showed that insulin induced TTP but did not affect ZFP36L1 protein levels in adipocytes. Insulin decreased mRNA levels of Vegf and a number of other genes in adipocytes. Discussion: Insulin induced Ttp mRNA and protein expression and decreased Vegf mRNA levels in adipocytes. Zfp36l3 mRNA was detected, for the first time, in cells other than mouse placenta and extraembryonic tissues. This study established a basis for the investigation of TTP and VEGF genes in the regulation of obesity and suggested that Vegf mRNA may be a target of TTP in fat cells.     

Differential expression and functional analysis of the tristetraprolin family during early differentiation of 3T3-L1 preadipocytes

The tristetraprolin (TTP) family comprises zinc finger-containing AU-rich element (ARE)-binding proteins consisting of three major members: TTP, ZFP36L1, and ZFP36L2. The present study generated specific antibodies against each TTP member to evaluate its expression during differentiation of 3T3-L1 preadipocytes. In contrast to the inducible expression of TTP, results indicated constitutive expression of ZFP36L1 and ZFP36L2 in 3T3-L1 preadipocytes and their phosphorylation in response to differentiation signals. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3' untranslated region (UTR) of MAPK phosphatase-1 (MKP-1) mRNA and downregulated Mkp-1 3'UTR-mediated luciferase activity. Mkp-1 is an immediate early gene for which the mRNA is transiently expressed in response to differentiation signals. The half-life of Mkp-1 mRNA was longer at 30 min of induction than at 1 h and 2 h of induction. Knockdown of TTP or ZFP36L2 increased the Mkp-1 mRNA half-life at 1 h of induction. Knockdown of ZFP36L1, but not ZFP36L2, increased Mkp-1 mRNA basal levels via mRNA stabilization and downregulated ERK activation. Differentiation induced phosphorylation of ZFP36L1 through ERK and AKT signals. Phosphorylated ZFP36L1 then interacted with 14-3-3, which might decrease its mRNA destabilizing activity. Inhibition of adipogenesis also occurred in ZFP36L1 and TTP knockdown cells. The findings indicate that the differential expression of TTP family members regulates immediate early gene expression and modulates adipogenesis.     

Chorioallantoic fusion defects and embryonic lethality resulting from disruption of Zfp36L1, a gene encoding a CCCH tandem zinc finger protein of the Tristetraprolin family

The mouse gene Zfp36L1 encodes zinc finger protein 36-like 1 (Zfp36L1), a member of the tristetraprolin (TTP) family of tandem CCCH finger proteins. TTP can bind to AU-rich elements within the 3'-untranslated regions of the mRNAs encoding tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), leading to accelerated mRNA degradation. TTP knockout mice exhibit an inflammatory phenotype that is largely due to increased TNF secretion. Zfp36L1 has activities similar to those of TTP in cellular RNA destabilization assays and in cell-free RNA binding and deadenylation assays, suggesting that it may play roles similar to those of TTP in mammalian physiology. To address this question we disrupted Zfp36L1 in mice. All knockout embryos died in utero, most by approximately embryonic day 11 (E11). Failure of chorioallantoic fusion occurred in about two-thirds of cases. Even when fusion occurred, by E10.5 the affected placentas exhibited decreased cell division and relative atrophy of the trophoblast layers. Although knockout embryos exhibited neural tube abnormalities and increased apoptosis within the neural tube and also generalized runting, these and other findings may have been due to deficient placental function. Embryonic expression of Zfp36L1 at E8.0 was greatest in the allantois, consistent with a potential role in chorioallantoic fusion. Fibroblasts derived from knockout embryos had apparently normal levels of fully polyadenylated compared to deadenylated GM-CSF mRNA and normal rates of turnover of this mRNA species, both sensitive markers of TTP deficiency in cells. We postulate that lack of Zfp36L1 expression during mid-gestation results in the abnormal stabilization of one or more mRNAs whose encoded proteins lead directly or indirectly to abnormal placentation and fetal death.     

Tristetraprolin inhibits Ras-dependent tumor vascularization by inducing vascular endothelial growth factor mRNA degradation

Vascular endothelial growth factor (VEGF) is one of the most important regulators of physiological and pathological angiogenesis. Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway and overexpression of VEGF are common denominators of tumors from different origins. We have established a new link between these two fundamental observations converging on VEGF mRNA stability. In this complex phenomenon, tristetraprolin (TTP), an adenylate and uridylate-rich element-associated protein that binds to VEGF mRNA 3′-untranslated region, plays a key role by inducing VEGF mRNA degradation, thus maintaining basal VEGF mRNA amounts in normal cells. ERKs activation results in the accumulation of TTP mRNA. However, ERKs reduce the VEGF mRNA-destabilizing effect of TTP, leading to an increase in VEGF expression that favors the angiogenic switch. Moreover, TTP decreases RasVal12-dependent VEGF expression and development of vascularized tumors in nude mice. As a consequence, TTP might represent a novel antiangiogenic and antitumor agent acting through its destabilizing activity on VEGF mRNA. Determination of TTP and ERKs status would provide useful information for the evaluation of the angiogenic potential in human tumors.     

Regulation and localization of endogenous human tristetraprolin

Tumor necrosis factor (TNF) has been implicated in the development and pathogenicity of infectious diseases and autoimmune disorders, such as septic shock and arthritis. The zinc-finger protein tristetraprolin (TTP) has been identified as a major regulator of TNF biosynthesis. To define its intracellular location and examine its regulation of TNF, a quantitive intracellular staining assay specific for TTP was developed. We establish for the first time that in peripheral blood leukocytes, expression of endogenous TTP is confined to the cytoplasm. Baseline expression of TTP was higher in monocytes than in lymphocytes or neutrophils. After in vitro incubation with lipopolysaccharide (LPS), leukocyte TTP levels increased rapidly, peaking after approximately 2 hours. Monocytes showed the greatest response to LPS stimulation and lymphocytes the least. TTP levels were also studied in leukocytes isolated from healthy volunteers infused with a bolus dose of LPS. TTP expression and initial upregulation in response to LPS infusion were consistent with the in vitro data. Neutrophil TTP levels responded first, reaching an initial peak within 1 hour, monocyte levels peaked next at 2 hours, followed by lymphocytes at 4 hours. This response paralleled plasma TNF levels, which peaked 2 hours after infusion and were no longer detectable after 12 hours. A second rise in intracellular TTP levels, which did not parallel plasma TNF levels, was observed in all leukocyte populations, starting 12 hours after infusion. These data establish the cytoplasmic location of TTP, supporting a major role for this protein in regulating TNF production, and suggest that TTP levels are not regulated solely by TNF.     

The p38/MK2-Driven Exchange between Tristetraprolin and HuR Regulates AU–Rich Element–Dependent Translation

TNF expression of macrophages is under stringent translational control that depends on the p38 MAPK/MK2 pathway and the AU–rich element (ARE) in the TNF mRNA. Here, we elucidate the molecular mechanism of phosphorylation-regulated translation of TNF. We demonstrate that translation of the TNF-precursor at the ER requires expression of the ARE–binding and -stabilizing factor human antigen R (HuR) together with either activity of the p38 MAPK/MK2 pathway or the absence of the ARE-binding and -destabilizing factor tristetraprolin (TTP). We show that phosphorylation of TTP by MK2 decreases its affinity to the ARE, inhibits its ability to replace HuR, and permits HuR-mediated initiation of translation of TNF mRNA. Since translation of TTP''s own mRNA is also regulated by this mechanism, an intrinsic feedback control of the inflammatory response is ensured. The phosphorylation-regulated TTP/HuR exchange at target mRNAs provides a reversible switch between unstable/non-translatable and stable/efficiently translated mRNAs.     

Novel Antibodies Reactive with Sialyl Lewis X in Both Humans and Mice Define Its Critical Role in Leukocyte Trafficking and Contact Hypersensitivity Responses

Sialyl Lewis X (sLe^x) antigen functions as a common carbohydrate determinant recognized by all three members of the selectin family. However, its expression and function in mice remain undefined due to the poor reactivity of conventional anti-sLe^x monoclonal antibodies (mAbs) with mouse tissues. Here, we developed novel anti-sLe^x mAbs, termed F1 and F2, which react well with both human and mouse sLe^x, by immunizing fucosyltransferase (FucT)-IV and FucT-VII doubly deficient mice with 6-sulfo-sLe^x-expressing cells transiently transfected with an expression vector encoding CMP-N-acetylneuraminic acid hydroxylase. F1 and F2 specifically bound both the N-acetyl and the N-glycolyl forms of sLe^x as well as 6-sulfo-sLe^x, a major ligand for L-selectin expressed in high endothelial venules, and efficiently blocked physiological lymphocyte homing to lymph nodes in mice. Importantly, both of the mAbs inhibited contact hypersensitivity responses not only when administered in the L-selectin-dependent sensitization phase but also when administered in the elicitation phase in mice. When administered in the latter phase, F1 and F2 efficiently blocked rolling of mouse leukocytes along blood vessels expressing P- and E-selectin in the auricular skin in vivo. Consistent with these findings, the mAbs blocked P- and E-selectin-dependent leukocyte rolling in a flow chamber assay. Taken together, these results indicate that novel anti-sLe^x mAbs reactive with both human and mouse tissues, with the blocking ability against leukocyte trafficking mediated by all three selectins, have been established. These mAbs should be useful in determining the role of sLe^x antigen under physiological and pathological conditions.     

Intact p53-Dependent Responses in miR-34-Deficient Mice

MicroRNAs belonging to the miR-34 family have been proposed as critical modulators of the p53 pathway and potential tumor suppressors in human cancers. To formally test these hypotheses, we have generated mice carrying targeted deletion of all three members of this microRNA family. We show that complete inactivation of miR-34 function is compatible with normal development in mice. Surprisingly, p53 function appears to be intact in miR-34-deficient cells and tissues. Although loss of miR-34 expression leads to a slight increase in cellular proliferation in vitro, it does not impair p53-induced cell cycle arrest or apoptosis. Furthermore, in contrast to p53-deficient mice, miR-34-deficient animals do not display increased susceptibility to spontaneous, irradiation-induced, or c-Myc-initiated tumorigenesis. We also show that expression of members of the miR-34 family is particularly high in the testes, lungs, and brains of mice and that it is largely p53-independent in these tissues. These findings indicate that miR-34 plays a redundant function in the p53 pathway and suggest additional p53-independent functions for this family of miRNAs.