Session Title: Sjögrenʼs Syndrome – Basic & Clinical Science Poster I
Session Type: Poster Session (Tuesday)
Session Time: 9:00AM-11:00AM
Background/Purpose: Despite the strong association of maternal anti-Ro60 autoantibodies in the development of SS, Neonatal Lupus (NL) and scLE, understanding causality is challenging given that the intracellular location of the target RNP antigen only becomes accessible to extracellular autoantibodies during cell death. While binding Y RNA is a property of Ro60 in dying cells, Ro60 contributes to events essential to proliferation including the degradation of non-coding RNAs. To define potentially targetable molecular events associated with Ro60 accessibility and their effects on proliferation, we evaluated two candidate chaperone Ro60 binding proteins, ZBP1 (also known as IGF2BP1, which has two RNA recognition domains and plays a role in nuclear export of protein) and PTBP1 (which binds RNA in heterogeneous nuclear complexes).
Methods: Short hairpin (sh)RNA knockdown (KD) of both ZPB1 and PTBP1 was accomplished by using MISSION pLKO.1 derived lentiviral vehicles for targeted shRNA to yield each of the specific depletions in human fetal fibroblasts. Affinity purified anti-Ro60 antibodies (AP60) isolated from the serum of 2 mothers of children with cardiac NL and control IgG isolated from a healthy donor were used to evaluate Ro60 surface translocation in intact and apoptotic cells by flow cytometry. Fibroblast proliferation was determined using 5-ethynyl-2′-deoxyuridine (EdU) incorporation.
Results: KD of targeted transcripts were confirmed by qPCR and western blot. Permeabilized KD and wildtype fibroblasts demonstrated equivalent intracellular expression of Ro60. As expected, flow cytometry with AP60 revealed no surface staining of non-permeabilized wildtype or KD fibroblasts. The next set of experiments addressed binding of AP60 to polyHEMA-apoptotic fibroblasts. Staining with annexin V (a proxy of apoptosis) was uniformly consistent between the wildtype and KD cells. As expected, compared to non-apoptotic fibroblasts, AP60 but not control donor IgG readily bound the surface of wildtype apoptotic fibroblasts, supporting that Ro60 was indeed translocated during apoptosis (MFI of 97 + 5 vs 211 + 14, respectively; P< 0.05; N=3). In contrast, binding of AP60 was significantly attenuated in the ZPB1 KD fibroblasts (81.5 + 7; N = 3) vs anti-Ro binding of apoptotic wildtype fibroblasts (P< 0.05). However, the binding of AP60 was equivalent in the apoptotic PTBP1 KD fibroblasts (MFI of 203 + 6, N= 3, P=NS) compared to apoptotic wildtype fibroblasts. With regard to cell proliferation, the percent positive EdU cells of wildtype fetal fibroblasts, ZBP1 KD fibroblasts, and PTBP1 KD fibroblasts were 56%, 2% and 45%, respectively, a result suggesting that the loss of ZBP1 restrains cell cycle progression.
Conclusion: ZBP1 represents a novel and required chaperone for the translocation of Ro60 to the cell surface during apoptosis in addition to contributing to cell proliferation. Given that Ro60 antigen accessibility is essential, not only to the generation of anti-Ro60 responses but also to the formation of surface immune complexes and subsequent tissue injury, ZBP1 may represent a newly targetable candidate to forestall both the initiation and sequelae of autoimmunity.
To cite this abstract in AMA style:DiDonato F, Buyon J, Clancy R. Zipcode-Binding Protein 1 (ZBP1) Facilitates Ro60 Surface Translocation, Cellular Growth and Autoimmune Sequelae [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/zipcode-binding-protein-1-zbp1-facilitates-ro60-surface-translocation-cellular-growth-and-autoimmune-sequelae/. Accessed October 21, 2020.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/zipcode-binding-protein-1-zbp1-facilitates-ro60-surface-translocation-cellular-growth-and-autoimmune-sequelae/