Perry J. Blackshear

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 α (TNFα) release as a consequence of increased stability of TNFα mRNA. TTP was then shown to destabilize TNFα mRNA after binding directly to the AU-rich region (ARE) of the 3′-untranslated region of the TNFα 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 inXenopus and fish. We show here that representatives of all four family members were able to bind to the TNFα ARE in a cell-free system and, in most cases, promote the breakdown of TNFα 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 TNFα 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 TNFα 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.

Interleukin-10 targets p38 MAPK to modulate ARE-dependent TNF mRNA translation and limit intestinal pathology.

Interleukin‐10 (IL‐10) is a key inhibitory signal of inflammatory responses that regulates the production of potentially pathogenic cytokines like tumor necrosis factor (TNF). We show here that the development of chronic intestinal inflammation in IL‐10‐deficient mice requires the function of TNF, indicating that the IL‐10/TNF axis regulates mucosal immunity. We further show that IL‐10 targets the 3′ AU‐rich elements (ARE) of TNF mRNA to inhibit its translation. Moreover, IL‐10 does not alter TNF mRNA stability, and its action does not require the presence of the stability‐regulating ARE binding factor tristetraprolin, indicating a differential assembly of stability and translation determinants on the TNF ARE. Inhibition of TNF translation by IL‐10 is exerted mainly by inhibition of the activating p38/MAPK‐activated protein kinase‐2 pathway. These results demonstrate a physiologically significant cross‐talk between the IL‐10 receptor and the stress‐activated protein kinase modules targeting TNF mRNA translation. This cross‐talk is necessary for optimal TNF production and for the maintenance of immune homeostasis in the gut.

Protein kinase C regulates the nuclear localization of diacylglycerol kinase-ζ

Diacylglycerol kinases (DGKs) terminate signalling from diacylglycerol by converting it to phosphatidic acid. Diacylglycerol regulates cell growth and differentiation, and its transient accumulation in the nucleus may be particularly important in this regulation,. Here we show that a fraction of DGK-ζ is found inthe nucleus, where it regulates the amount of nuclear diacylglycerol. Reducing nuclear diacylglycerol levels by conditional expression of DGK-ζ attenuates cell growth. The nuclear-localization signal of DGK-ζ is located in a region that is homologous to the phosphorylation-site domain of the MARCKS protein. This is, to our knowledge, the first evidence that this domain, which is amajor target for protein kinase C, can localize a protein to thenucleus. Two isoforms of protein kinase C, but not others, regulate the localization of DGK-ζ. Our results define a cycle in which diacylglycerol activates protein kinase C, which then regulates the metabolism of diacylglycerol by alternating the intracellular location of DGK-ζ. This may be a general mechanism to control mitogenic signals that depend on nuclear diacylglycerol.

Genome-wide Analysis Identifies Interleukin-10 mRNA as Target of Tristetraprolin

Tristetraprolin (TTP) is an RNA-binding protein required for the rapid degradation of mRNAs containing AU-rich elements. Targets regulated by TTP include the mRNAs encoding tumor necrosis factor-α, granulocyte-macrophage colony-stimulating factor, interleukin-2 (IL-2), and immediate early response 3. To identify novel target mRNAs of TTP in macrophages, we used a genome-wide approach that combines RNA immunoprecipitation and microarray analysis. A list was compiled of 137 mRNAs that are associated with TTP with an estimated accuracy on the order of 90%. Sequence analysis revealed a highly significant enrichment of AU-rich element motifs, with AUUUA pentamers present in 96% and UUAUUUAUU nonamers present in 44% of TTP-associated mRNAs. We further show that IL-10 is a novel target regulated by TTP. IL-10 mRNA levels were found to be elevated because of a reduced decay rate in primary macrophages from TTP-/- mice. Our study demonstrates the importance of experimental approaches for identifying targets of RNA-binding proteins.

Tristetraprolin down-regulates IL-2 gene expression through AU-rich element-mediated mRNA decay.

Posttranscriptional regulation of IL-2 gene expression at the level of mRNA decay is mediated by an AU-rich element (ARE) found in the 3′-untranslated region. We hypothesized that the ARE-binding protein tristetraprolin (TTP) regulates T lymphocyte IL-2 mRNA decay by interacting with the IL-2 ARE and targeting the transcript for decay. rTTP protein expressed in HeLa cells bound specifically to the IL-2 ARE with high affinity in a gel shift assay. In primary human T lymphocytes, TTP mRNA and protein expression were induced by TCR and CD28 coreceptor stimulation. Using a gel shift assay, we identified a cytoplasmic RNA-binding activity that was induced by TCR and CD28 coreceptor stimulation and bound specifically to the IL-2 ARE sequence. Using anti-TTP Abs, we showed by supershift that this inducible activity contained TTP. We also showed that insertion of the IL-2 ARE sequence into the 3′-untranslated region of a β-globin reporter construct conferred TTP-dependent mRNA destabilization on the β-globin reporter. To determine whether TTP also regulates IL-2 gene expression in vivo, we examined IL-2 expression in primary cells from wild-type and TTP knockout mice. Compared with their wild-type counterparts, TCR- and CD28-activated splenocytes and T cells from TTP knockout mice overexpressed IL-2 mRNA and protein. Also, IL-2 mRNA was more stable in activated splenocytes from TTP knockout mice compared with wild-type mice. Taken together, these data suggest that TTP functions to down-regulate IL-2 gene expression through ARE-mediated mRNA decay.

Chorioallantoic Fusion Defects and Embryonic Lethality Resulting from Disruption of Zfp36L1, a Gene Encoding a CCCH Tandem Zinc Finger Protein of the Tristetraprolin Family

ABSTRACT 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.

Load-Dependent and -Independent Regulation of Proinflammatory Cytokine and Cytokine Receptor Gene Expression in the Adult Mammalian Heart

Background—Although previous studies have examined the effects of acute hemodynamic pressure overload on proinflammatory cytokine gene expression, the effects of sustained hemodynamic overloading have not been examined. Methods and Results—Sustained hemodynamic pressure overloading was produced in mice by transverse constriction of the aorta. Proinflammatory cytokine and cytokine receptor gene expression were determined by ribonuclease protection assays (RPA) at 6 hours and at 3, 7, 14 and 35 days after banding. M-mode echocardiography was used to assess left ventricular structure and function at identical time points. RPA showed that tumor necrosis factor (TNF), interleukin (IL)-1&bgr;, and IL-6 mRNA levels were maximal at 6 hours and returned to baseline levels within 72 hours. There was a significant increase in IL-1RII and IL-6R&agr; receptor mRNA levels after overloading but no significant increase in TNFR1, TNFR2, IL-1RI, or gp130 mRNA levels. The transient increase in expression of proinflammatory cytokine gene expression was not explained by changes in left ventricular loading conditions, left ventricular wall stress, desensitization of proinflammatory genes, or decreased nuclear factor-&kgr;B activation. It is interesting that transverse constriction of the aorta provoked an increase in the expression of tristetraprolin, a homeostatic zinc finger protein that is known to destabilize TNF mRNA. Conclusion—Sustained hemodynamic overloading provokes a transient increase in proinflammatory cytokine and cytokine receptor gene expression; however, the decrease in proinflammatory cytokine gene expression occurred in the absence of changes in loading conditions, suggesting that the expression of proinflammatory cytokines in the heart is regulated, at least in part, by load-dependent and load-independent mechanisms.

Immunogenetic risk and protective factors for the idiopathic inflammatory myopathies: distinct HLA-A, -B, -Cw, -DRB1, and -DQA1 allelic profiles distinguish European American patients with different myositis autoantibodies.

Abstract: The idiopathic inflammatory myopathies (IIM) are systemic connective tissue diseases defined by chronic muscle inflammation and weakness associated with autoimmunity. We have performed low to high resolution molecular typing to assess the genetic variability of major histocompatibility complex loci (HLA-A, -B, -Cw, -DRB1, and -DQA1) in a large population of European American patients with IIM (n = 571) representing the major myositis autoantibody groups. We established that alleles of the 8.1 ancestral haplotype (8.1 AH) are important risk factors for the development of IIM in patients producing anti-synthetase/anti-Jo-1, -La, -PM/Scl, and -Ro autoantibodies. Moreover, a random forests classification analysis suggested that 8.1 AH-associated alleles B*0801 and DRB1*0301 are the principal HLA risk markers. In addition, we have identified several novel HLA susceptibility factors associated distinctively with particular myositis-specific (MSA) and myositis-associated autoantibody (MAA) groups of the IIM. IIM patients with anti-PL-7 (anti-threonyl-tRNA synthetase) autoantibodies have a unique HLA Class I risk allele, Cw*0304 (pcorr = 0.046), and lack the 8.1 AH markers associated with other anti-synthetase autoantibodies (for example, anti-Jo-1 and anti-PL-12). In addition, HLA-B*5001 and DQA1*0104 are novel potential risk factors among anti-signal recognition particle autoantibody-positive IIM patients (pcorr = 0.024 and p = 0.010, respectively). Among those patients with MAA, HLA DRB1*11 and DQA1*06 alleles were identified as risk factors for myositis patients with anti-Ku (pcorr = 0.041) and anti-La (pcorr = 0.023) autoantibodies, respectively. Amino acid sequence analysis of the HLA DRB1 third hypervariable region identified a consensus motif, 70D (hydrophilic)/71R (basic)/74A (hydrophobic), conferring protection among patients producing anti-synthetase/anti-Jo-1 and -PM/Scl autoantibodies. Together, these data demonstrate that HLA signatures, comprising both risk and protective alleles or motifs, distinguish IIM patients with different myositis autoantibodies and may have diagnostic and pathogenic implications. Variations in associated polymorphisms for these immune response genes may reflect divergent pathogenic mechanisms and/or responses to unique environmental triggers in different groups of subjects resulting in the heterogeneous syndromes of the IIM. Abbreviations: AH = ancestral haplotype, DM = dermatomyositis, EA = European Americans, HVR3 = third hypervariable region, IBM = inclusion body myositis, IIM = idiopathic inflammatory myopathies, MAA = myositis-associated autoantibodies, MHC = major histocompatibility complex, MSA = myositis-specific autoantibodies, PM = polymyositis, RF = random forests, RSP = restrictive supertype patterns, SRP = signal recognition particle.

Transmembrane TNF protects mutant mice against intracellular bacterial infections, chronic inflammation and autoimmunity

Using targeted mutagenesis in mice, we have blocked shedding of endogenous murine TNF by deleting its cleavage site. Mutant mice produce physiologically regulated levels of transmembrane TNF (tmTNF), which suffice to support thymocyte proliferation but cannot substitute for the hepatotoxic activities of wild‐type TNF following LPS/D‐galactosamine challenge in vivo and are not sufficient to support secondary lymphoid organ structure and function. Notably, however, tmTNF is capable of exerting anti‐Listerial host defenses while remaining inadequate to mediate arthritogenic functions, as tested in the tristetraprolin‐deficient model of TNF‐dependent arthritis. Most interestingly, in the EAE model of autoimmune demyelination, tmTNF suppresses disease onset and progression and retains the autoimmune suppressive properties of wild‐type TNF. Together, these results indicate that tmTNF preserves a subset of the beneficial activities of TNF while lacking detrimental effects. These data support the hypothesis that selective targeting of soluble TNF may offer several advantages over complete blockade of TNF in the treatment of chronic inflammation and autoimmunity.

Regulation of Suppressor of Cytokine Signaling 3 (SOCS3) mRNA Stability by TNF-α Involves Activation of the MKK6/p38MAPK/MK2 Cascade

The potential of some proinflammatory mediators to inhibit gp130-dependent STAT3 activation by enhancing suppressor of cytokine signaling (SOCS) 3 expression represents an important molecular mechanism admitting the modulation of the cellular response toward gp130-mediated signals. Thus, it is necessary to understand the mechanisms involved in the regulation of SOCS3 expression by proinflammatory mediators. In this study, we investigate SOCS3 expression initiated by the proinflammatory cytokine TNF-α. In contrast to IL-6, TNF-α increases SOCS3 expression by stabilizing SOCS3 mRNA. Activation of the MAPK kinase 6 (MKK6)/p38MAPK-cascade is required for TNF-α-mediated stabilization of SOCS3 mRNA and results in enhanced SOCS3 protein expression. In fibroblasts or macrophages deficient for MAPK-activated protein kinase 2 (MK2), a downstream target of the MKK6/p38MAPK cascade, basal SOCS3-expression is strongly reduced and TNF-α-induced SOCS3-mRNA stabilization is impaired, indicating that MK2 is crucial for the control of SOCS3 expression by p38MAPK-dependent signals. As a target for SOCS3 mRNA stability-regulating signals, a region containing three copies of a pentameric AUUUA motif in close proximity to a U-rich region located between positions 2422 and 2541 of the 3′ untranslated region of SOCS3 is identified. One factor that could target this region is the zinc finger protein tristetraprolin (TTP), which is shown to be capable of destabilizing SOCS3 mRNA via this region. However, data from TTP-deficient cells suggest that TTP does not play an irreplaceable role in the regulation of SOCS3 mRNA stability by TNF-α. In summary, these data indicate that TNF-α regulates SOCS3 expression on the level of mRNA stability via activation of the MKK6/p38MAPK cascade and that the activation of MK2, a downstream target of p38MAPK, is important for the regulation of SOCS3 expression.