Rab7 and estrogen-ER as B cell-intrinsic mediators of auto/antibody responses

描述（由申请人提供）：Rab7和雌激素- er作为B细胞自身/抗体反应的内在介质。我们对R01 AI079705头四年的研究表明，雌激素促进了类转换和突变抗体/自身抗体的产生，如系统性狼疮中的抗dsdna IgG；雌激素通过上调HoxC4同源结构域转录因子增强AID的诱导（对CSR和SHM至关重要）；雌激素受体（ER）通过结合HoxC4启动子中发现的三个保守的ER响应元件（EREs）诱导HoxC4；HoxC4直接结合AICDA/ AICDA启动子诱导AID表达；HoxC4和AID在狼疮B细胞中高表达；他们的KO在正常小鼠中钝化了类切换/突变抗体，在狼疮易感小鼠中钝化了自身抗体/自身免疫。因此，我们关于雌激素在CSR/SHM中B细胞内在作用的重要发现与女性抗体/自身抗体反应增强和自身免疫（尤其是狼疮）发生率升高高度相关。这种竞争性更新将验证我们的新假设，即Rab7介导抗体/自身抗体反应，并在此功能中由雌激素（ß-estradiol）-ER （E2-ER）调节。在B细胞中诱导后，Rab7小GTPase激活NF-κB（增加HoxC4/Aicda转录），下调microrna（解除HoxC4和Aicda转录物的沉默），从而诱导HoxC4/AID和CSR/SHM。这种依赖rab7的途径会被E2-ER增强，进一步上调CSR/SHM。我们的假设得到了令人信服的初步数据的支持，包括：证明Rab7在t非依赖性和t依赖性抗体反应中在诱导AID和CSR中发挥B细胞内在作用；Rab7降低Dicer的证据，这对microRNA的生物发生至关重要；Rab7启动子中多个EREs的鉴定；雌激素对正常B细胞中Rab7的诱导上调及其在狼疮B细胞中的表达。目的1将通过Rab7抑制剂（CID 1067700）、er拮抗剂（氟维酯）和MRL/Faslpr/lpr小鼠在“诱导”B细胞中删除Rab7 [Tg(Aicda-cre)Rab7fl/fl]或ERα [Tg(Aicda-cre)ERαfl/fl]来研究Rab7和E2-ER在自身抗体反应和狼疮免疫病理中的作用。Aim 2将探讨Rab7在HoxC4/Aicda诱导、CSR/SHM和抗体反应中的B细胞内在功能、潜在机制（NF-κB的诱导）和E2-ER的增强，方法是使用B细胞删除Rab7，通过在这些B细胞中表达组成型活性IKKß突变体来增强NF-κB的激活，并使用B细胞删除ERα。Aim 3将探讨Rab7和雌激素对miR-23b、miR-26、miR-214（沉默HoxC4 mRNA）、miR-181b、miR-155和miR-361（沉默Aicda mRNA）的下调，并确定Rab7在这一过程中的作用：可能通过促进自噬（样）过程作为Dicer降解的介质。通过将自噬、雌激素和microrna三个研究领域结合起来，共同研究自身抗体产生的调控，我们的建议为理解狼疮的复杂问题提供了一个综合的方法，并有助于确定新的狼疮治疗靶点。

DESCRIPTION (provided by applicant): Rab7 and estrogen-ER as B cell-intrinsic mediators of auto/antibody responses. Our work of the first four years of R01 AI079705 has shown that estrogen promotes production of class- switched and mutated antibodies/autoantibodies, such as anti-dsDNA IgG in systemic lupus; estrogen potentiates induction of AID (critical for CSR and SHM) through upregulation of the HoxC4 homeodomain transcription factor; estrogen receptors (ERs) induce HoxC4 by binding to three conserved ER responsive elements (EREs) we identified in the HoxC4 promoter; HoxC4 directly binds to the AICDA/Aicda promoter to induce AID expression; and, HoxC4 and AID are highly expressed in lupus B cells; their KO blunts class- switched/mutated antibodies in normal mice, and autoantibodies/autoimmunity in lupus-prone mice. Thus, our significant findings on the B cell-intrinsic role of estrogen in CSR/SHM are highly relevant to heightened antibody/autoantibody responses and higher incidence of autoimmunity, particularly lupus, in females. This competitive renewal will test our novel hypothesis that Rab7 mediates antibody/autoantibody responses and is modulated in this function by estrogen (ß-estradiol)-ER (E2-ER). Upon induction in B cells, the Rab7 small GTPase would activate NF-κB (to increase HoxC4/Aicda transcription) and downregulate microRNAs (to relieve HoxC4 and Aicda transcripts from silencing), thereby inducing HoxC4/AID and CSR/SHM. This Rab7-dependent pathway would be enhanced by E2-ER, further upregulating CSR/SHM. Our hypotheses are supported by compelling preliminary data, including: demonstration that Rab7 plays a B cell-intrinsic role in inducing AID and CSR in T-independent and T-dependent antibody responses; evidence that Rab7 decreases Dicer, which is crucial to microRNA biogenesis; identification of multiple EREs in Rab7 promoter; upregulation of Rab7 induction by estrogen in normal B cells and its expression in lupus B cells. Aim 1 will address the roles of Rab7 and E2-ER in autoantibody responses and lupus immunopathology by using a Rab7-inhibitor (CID 1067700), an ER-antagonist (fulvestrant), and MRL/Faslpr/lpr mice deleting Rab7 [Tg(Aicda-cre)Rab7fl/fl] or ERα [Tg(Aicda-cre)ERαfl/fl] in "induced" B cells. Aim 2 will address the B cell- intrinsic functions of Rab7 in HoxC4/Aicda induction, CSR/SHM and antibody responses, underlying mechanisms (induction of NF-κB) and potentiation by E2-ER, by using B cells deleting Rab7, enforcing NF-κB activation through expression of a constitutively active IKKß mutant in these B cells, and by using B cells deleting ERα. Aim 3 will address Rab7 and estrogen downregulation of miR-23b, miR-26, miR-214 (silencing HoxC4 mRNA), miR-181b, miR-155 and miR-361 (silencing Aicda mRNA), and define the role of Rab7 in this process: possibly as a mediator of Dicer degradation through promotion of autophagy (-like) processes. By bringing the three research fields of autophagy, estrogen and microRNAs together to address the regulation of autoantibody production, our proposal provides an integrated approach to understand the complex problem of lupus and contributes to the definition of new lupus therapeutic targets.