T Cell Alternative p38 Activation Pathway

T 细胞替代 p38 激活途径

• 批准号: 8937833
• 负责人: Jonathan Ashwell
• 金额: $ 55.94万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937833
• 关键词: Affect; Age; Animal Model; Animals; Ankylosis; Antigen Receptors; Autoimmune Diseases; Autoimmunity; B-Lymphocytes; Binding; Biological; Biopsy; CD4 Positive T Lymphocytes; Cell Cycle; Cell Lineage; Cells; Citrobacter; Clinical Trials; Collagen Arthritis; Cues; Data; Defect; Disease; Down-Regulation; Drug Targeting; Environment; Enzymes; Event; Experimental Autoimmune Encephalomyelitis; Family; GADD45; Genes; Goals; Heat-Shock Response; Human; Hypoxia; IRF4 gene; Immune; Immune Sera; Immunity; Inflammation; Inflammatory; Inflammatory Response; Injection of therapeutic agent; Interferon Type II; Interleukin-1; Interleukin-17; Joints; Knock-in Mouse; Knockout Mice; Laboratories; Lead; Lesion; Lymphocyte-Specific p56LCK Tyrosine Protein Kinase; MAP Kinase Kinase Kinase; MAP2K6 gene; MAP3K7 gene; MAPK11 gene; MAPK14 gene; Malignant neoplasm of pancreas; Mediating; Mediator of activation protein; Membrane; Mitogen-Activated Protein Kinases; Modeling; Molecular; Molecular Target; Mono-S; Mus; Mutant Strains Mice; Mutate; Mutation; Names; Osmotic Shocks; Pathway interactions; Peptides; Phosphorylation; Phosphotransferases; Phototherapy; Physiological; Play; Production; Protein Family; Protein-Serine-Threonine Kinases; Psoriasis; Receptor Signaling; Resistance; Rest; Role; SB 203580; Severity of illness; Signal Transduction; Stimulus; Stress; Substrate Specificity; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; TNF gene; Toxoplasma gondii; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Ultraviolet Rays; Wild Type Mouse; autoimmune vasculitis; cytokine; extracellular; high throughput screening; human MAPK14 protein; human TNF protein; human disease; in vivo; inhibitor/antagonist; lupus-like; mouse model; mutant; neoplastic; pancreatic neoplasm; pathogen; prevent; response; small molecule; transcription factor; tumor; tumor growth

The multistep propagation of discrete intracellular signals allows cells to respond to cues from the extracellular environment. Among the most ubiquitous and well-studied of these are the phosphorylation cascades that culminate in the activation of mitogen-activated protein kinases (MAPKs). The enzymatic activity of MAPKs is markedly influenced by extracellular events. As a rule, p38 MAPK activity is induced by environmental stress (e.g. osmotic shock, hypoxia, heat shock, ultraviolet radiation) and pro-inflammatory stimuli and cytokines such as LPS, IL-1, TGF-beta, and TNF-alpha. The most membrane-proximal enzyme activated in the classic MAPK cascade is a serine/threonine kinase known as a MAPK kinase kinase, or MAPKKK, and the MAPKKKs that lead p38 activation include TAK1, ASK1, and MTK1 (human)/MEKK4 (mouse). Growth Arrest and DNA Damage inducible 45 (Gadd45a) was initially identified as a stress-responsive gene. Our studies of Gadd45a-deficient mice found that they died at an early age of a lupus-like autoimmune disease. Because of the known ability of Gadd45-family proteins to bind and activate MTK1/MEKK4, we asked how the absence of Gadd45a might affect p38 activation. We found that, rather than being hypoactive, p38 was spontaneously active in T-lineage cells. An in-depth analysis of this initial observation led to the following findings by our laboratory: - p38 from antigen receptor-stimulated normal T cells but not B cells robustly autophosphorylates. The autophosphorylation appeared to be on the two canonical activating residues, Thr-180 and Tyr-182. - T cell p38 activation requires Lck and Zap70 but is LAT-independent. - The TCR proximal kinases Lck, Fyn, and Zap70 phosphorylate p38 on Tyr-323, which induces autophosphorylation and enhanced activity toward other substrates. Notably, even p38 lacking Tyr-182 is activated by Tyr-323 phosphorylation. - A Tyr-323 phospho-specific antiserum detects Tyr-323-phosphorylated p38 (p-Tyr-323 p38) in T but not B cells activated via the antigen receptor. - p-Tyr-323 p38 is not detected in Lck+ Zap70- Jurkat T cells, implicating Zap70 as the effector kinase in vivo. - The alternative pathway appeared to be a major mechanism of p38 activation in T cells, because (1) in Jurkat T cells, p38 containing a Y323F substitution was poorly activated in response to anti-TCR compared to wild type (WT), and (2) dual (Thr-180/Tyr-182) phosphorylation of p38 in TCR-stimulated normal resting T cells was almost completely prevented by the p38 inhibitor SB203580, indicating that it is a consequence of autophosphorylation. - p38 from Gadd45a-deficient T cells is spontaneously phosphorylated on Tyr-323. - Gadd45a specifically binds to p38 (whether phosphorylated or not) and inhibits the activity of the p-Tyr-323 form. Importantly, Gadd45a binding does not inhibit the activity of p38 phosphorylated by MKK6 (on Thr-180/Tyr-182). - p38 phosphorylated on Tyr-323 is able to phosphorylate itself in trans; that is, one p38 molecule binds and phosphorylates another. - Auto-trans-phosphorylation involves just Thr-180 and not the canonical Tyr-182 found in the MAPK cascade. - The substrate specificity of mono-phosphorylated p38 is different from the di-phosphorylated form. This may explain why this alternative pathway has been evolutionarily conserved in T cells, because the biological effects of these two phosphorylated species would be expected to differ in vivo. - We generated p38 "knock-in" mice in which Tyr-323 is replaced with a Phe (p38YF). Proving the physiologic importance of the alternative pathway, TCR-mediated activation is completely incapable of activating p38 in T cells from these mice. - T cells from p38YF knockin mice are slow to transit from G0 to G1 in the cell cycle upon stimulation via the TCR. Moreover, they make much less interferon-gamma when immunized with Toxoplasma gondii. Therefore, the alternative p38 activation pathway is important for normal T cell proliferation and immune/inflammatory responses. We have established two animal models that allow us to explore the importance of the alternative pathway in normal and pathophysiologic conditions. The findings include: - The p38alpha YF knockin mice have been crossed with the GADD45a knockout mice, and we found that the T cell hyperproliferation and autoimmunity was prevented. This establishes TCR-induced p38 activation as having a key role in autoimmune vasculitis. - We have made p38beta YF knockin mice and have crossed them to the p38alpha YF knockin mice (p38abYF). Quantitation of activated p38 in the various mutant animals allowed us to determine that p38alpha contributes about 70%, and p38beta about 30%, to total p38 activation via TCR signaling. The double knockin mice have more pronounced defects in T cell proliferation than mice with single mutations. Moreover, Th1 skewing is much reduced in cells from these animals, consistent with a decrease in sustained Tbet expression. These observations prove that p38alpha and p38beta are partly redundant, and that both need to be mutated to quantitate the importance of TCR-induced p38 activity in biological responses. - The p38abYF mice are significantly more resistant to two models of autoimmunity and inflammation: collagen-induced arthritis and experimental autoimmune encephalomyelitis. The former, in particular, is remarkably milder in the mutant mice, which had no evidence of severe joint involvement, whereas 75% or more of wild type mice developed ankylosis. - T cells from p38abYF mice file to upregulate critical transcription factors involved in Th17 skewing (NFATc1, IRF4) and, as a consequence, IL-17. - p38abYF mice mount a weak IL-17 response to Citrobacter and fail to clear the pathogen from the gut. - Activation of the classic MAPK pathway results in p38 activation that antagonizes the alternative pathway, inhibition T cell functional responses. - The molecular mechanism of the antagonism is MAPK-activated p38 phosphorylation and inactivation of NFATc1. - Biopsies of human psoriatic lesions before and after UV treatment have shown that MAPK UV-activated p38 inactivates NFATc1, leading to downregulation of IRF4 and IL-17. This is the first evidence that phototherapy acts on pathogenic T cells. - Membrane-permeable Gadd45a, but not an internal deletion mutant that can't bind p38, inhibits T cell proliferation and cytokine production by WT T cells. - Intratumor injection of this compound into Panc02 pancreatic tumors halts tumor growth and inhibits proinflammatory cytokine production by tumor infiltrating CD4+ T cells (TIL). - I.V. injection of the peptide into KPC mice (a mouse model of human pancreatic cancer) inhibits the progression of pre-neoplastic to neoplastic lesions. Just as with Panc02, proinflammatory cytokine production by TIL is reduced. - Examination of 193 human pancreatic cancers revealed that all have CD4+ TIL with alternatively activated p38. Furthermore, the was a highly statistically significant correlation between the percent of TIL with alternatively activated p38 and disease severity. These T cells secreted TNF and IL-17, just like their muring counterparts. - High throughput screens have identified a family of small molecules that are cell permeable, non-toxic, and inhibit the alternative p38 activation pathway. Studies in animal models of inflammation will be performed. Our observations establish the alternative p38 pathway as the only mechanism for the activation of this important kinase upon TCR-mediated stimulation, and show that TCR-activated p38 plays a critical role in several mouse models of human disease, and is highly active in human pancreatic cancer. The data support the importance of the alternative p38 activation pathway as a molecular target.

离散细胞内信号的多步繁殖使细胞可以从细胞外环境中响应线索。其中最普遍和研究的是其中最终导致有丝分裂原激活蛋白激酶（MAPK）激活的磷酸化级联反应。 MAPK的酶促活性受细胞外事件的显着影响。通常，p38 MAPK活性是由环境应力（例如渗透性休克，缺氧，热休克，紫外线辐射）和促炎性刺激以及细胞因子以及LPS，IL-1，TGF-beta和TNF-Alpha等细胞因子引起的。在经典MAPK级联反应中激活的最膜型酶是一种丝氨酸/苏氨酸激酶，称为MAPK激酶激酶或MAPKKK，而引起P38激活的MAPKKK包括TAK1，ASK1，ASK1，ASK1，ASK1和MTK1（MTK1（HUMAN）/MEKKK4）。最初将生长停滞和DNA损伤诱导的45（GADD45A）鉴定为应激响应基因。我们对GADD45A缺陷小鼠的研究发现，它们死于狼疮样的自身免疫性疾病。由于GADD45家庭蛋白结合和激活MTK1/MEKK4的已知能力，我们询问GADD45A的缺失如何影响p38激活。我们发现，p38在T-linege细胞中自发活性，而不是发动不足。对这一初始观察结果的深入分析导致了我们的实验室的以下发现：-p38来自抗原受体刺激的正常T细胞，而不是B细胞可牢固地自磷酸化。自磷酸化似乎在两个规范激活残基THR-180和Tyr-182上。 -T细胞p38激活需要LCK和ZAP70，但不依赖于LAT。 - TRY-323上的TCR近端激酶LCK，FYN和ZAP70磷酸化p38，从而诱导自磷酸化并增强对其他底物的活性。值得注意的是，即使缺乏Tyr-182的p38也会被Tyr-323磷酸化激活。 - Tyr-323磷酸化特异性抗血清检测到T中Tyr-323-磷酸化的p38（p-Tyr-323 p38），但没有通过抗原受体激活的B细胞。 -P-TYR-323 p38在LCK+ ZAP70- JURKAT T细胞中未检测到，这意味着ZAP70是体内效应子激酶。 - 替代途径似乎是T细胞中p38激活的主要机制，因为（1）在Jurkat T细胞中，含有Y323F替代的p38与抗TCR相比，与野生型（wt）相比，与抗TCR（WT）相比，p38的激活较差，并且（2）双重（THR-180/Tyr-182）在TCR p38 p38中的p38 p38在TCR中的p38 p38几乎完全避免了TIMSTIM p38 p38 p38 p38 p38 p38抑制剂SB203580，表明它是自磷酸化的结果。 - 缺乏GADD45A的T细胞的p38在Tyr-323上自发地磷酸化。 -GADD45a特异性结合了p38（无论是否磷酸化），并抑制p-tyr-323形式的活性。重要的是，GADD45A结合并不能抑制MKK6磷酸化的p38活性（在THR-180/TYR-182上）。 - 在Tyr-323上磷酸化的p38能够在反式中磷酸化；也就是说，一个p38分子结合并磷酸化另一种分子。 - 自动传输磷酸化仅涉及THR-180，而不仅涉及MAPK级联中的规范Tyr-182。 - 单磷酸化p38的底物特异性与二磷酸化形式不同。这可以解释为什么这种替代途径在T细胞中在进化上保守，因为这两种磷酸化物种的生物学作用预计在体内会有所不同。 - 我们生成了p38“敲入”小鼠，其中Tyr-323被PHE取代（P38YF）。证明了替代途径的生理重要性，TCR介导的激活完全无法激活这些小鼠的T细胞中的p38。 - 通过TCR刺激后，来自P38YF敲击蛋白小鼠的T细胞在细胞周期中从G0转到G1的速度慢。此外，当用弓形虫弓形虫免疫时，它们会减少干扰素伽马。因此，替代p38激活途径对于正常的T细胞增殖和免疫/炎症反应很重要。我们已经建立了两个动物模型，使我们能够在正常和病理生理状况下探索替代途径的重要性。研究结果包括： - P38Alpha YF敲除小鼠已与GADD45A敲除小鼠交叉，我们发现T细胞的过度增殖和自身免疫性得到了阻止。这确立了TCR诱导的p38激活，因为它在自身免疫性血管炎中具有关键作用。 - 我们制造了P38Beta YF敲击蛋白小鼠，并将它们越过P38Alpha YF敲击蛋白小鼠（P38abyf）。在各种突变动物中对活化的p38进行定量使我们能够确定p38Alpha通过TCR信号传导贡献了约70％，p38beta约30％，大约30％。与具有单个突变的小鼠相比，双敲蛋白小鼠在T细胞增殖中具有更明显的缺陷。此外，这些动物的细胞中Th1偏斜大大降低，与持续的TBET表达降低一致。这些观察结果证明p38alpha和p38beta是部分冗余的，并且必须突变以量化TCR诱导的p38活性在生物学反应中的重要性。 - P38ABYF小鼠对两种自身免疫性和炎症模型具有更大的抵抗力：胶原蛋白诱导的关节炎和实验性自身免疫性脑脊髓炎。特别是前者在突变小鼠中非常温和，没有严重的关节参与，而75％或更多的野生型小鼠出现了强直性强直。 - p38abyf小鼠的T细胞归档，以上调涉及Th17偏斜（NFATC1，IRF4）的关键转录因子，并因此是IL-17。 -P38ABYF小鼠对柠檬酸菌的IL-17反应较弱，无法从肠道中清除病原体。 - 经典MAPK途径的激活导致p38激活拮抗替代途径，抑制T细胞功能响应。 - 拮抗作用的分子机制是MAPK激活的p38磷酸化和NFATC1的失活。 - 紫外线治疗前后人牛皮癣病变活检表明，MAPK UV激活的p38使NFATC1失活，从而导致IRF4和IL-17的下调。这是光疗作用于致病性T细胞的第一个证据。 - 膜可渗透的GADD45A，但不能结合p38的内部缺失突变体，可以抑制WT T细胞的T细胞增殖和细胞因子的产生。 - 将这种化合物内注射到PANC02胰腺肿瘤中停止肿瘤生长，并通过浸润肿瘤浸润CD4+ T细胞（TIL）抑制促炎性细胞因子的产生。 -I.V。将肽注射到KPC小鼠（人胰腺癌的小鼠模型）中抑制了肿瘤前向肿瘤病变的进展。就像PANC02一样，TIL的促炎细胞因子的产生也减少了。 - 对193次人类胰腺癌的检查表明，所有人都有CD4+ TIL，替代活化的p38。此外，这是TIL百分比与替代激活的p38和疾病严重程度之间的高度统计学意义相关性。这些T细胞分泌TNF和IL-17，就像它们的介绍对应物一样。 - 高吞吐量筛选已经确定了一个小分子家族，这些分子是可渗透，无毒的，并抑制了替代性p38激活途径。将在炎症的动物模型中进行研究。我们的观察结果确定了替代性p38途径是在TCR介导的刺激下激活这种重要激酶的唯一机制，并表明TCR激活的p38在几种小鼠人类疾病模型中起着至关重要的作用，并且在人胰腺癌中高度活跃。数据支持替代p38激活途径作为分子靶标的重要性。