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 (-estradiol)-ER (E2-ER). Upon induction in B cells, the Rab7 small GTPase would activate NF-?B (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 ER? [Tg(Aicda-cre)ER?fl/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-?B) and potentiation by E2-ER, by using B cells deleting Rab7, enforcing NF-?B activation through expression of a constitutively active IKK mutant in these B cells, and by using B cells deleting ER?.
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.
This proposal will identify the Rab7 small GTPase as a critical element in the production of class-switched and somatically mutated antibodies and autoantibodies, and will address the molecular mechanisms by which estrogen promotes the function of Rab7 in this process. Class switch DNA recombination (CSR) and somatic hypermutation (SHM) play a central role in the generation of highly specific and advantageous antibody responses to microbial pathogens and tumors by changing antibodies from IgM to IgG, IgA and/or IgE and increasing their affinity for antigens. However, CSR and SHM can also lead to the generation of autoantibodies that can damage tissues and organs, such as those in systemic lupus (pathogenic autoantibodies). As a result of estrogen-mediated enhancement of antibody and autoantibody responses, females display a stronger antibody response to vaccines and infections, as well as a predominance of autoimmunity involving pathogenic autoantibodies. By addressing the mechanisms underlying CSR and SHM, particularly the role of Rab7 and how it is regulated by estrogen, these studies will help determine how specific pathogenic autoantibodies are generated in the human body, and will eventually provide direction for the definition for new targets of lupus therapeutics.
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