Site-1 protease function is essential for the generation of antibody secreting cells and reprogramming for secretory activity

www.nature.com/scientificreports OPEN Received: 12 February 2018 Accepted: 11 September 2018 Published: xx xx xxxx Site-1 protease function is essential for the generation of antibody secreting cells and reprogramming for secretory activity Muna Al-Maskari1, Matthew A. Care1,2, Emily Robinson & Gina M. Doody 1 1 , Mario Cocco1, Reuben M. Tooze 1 The unfolded protein response (UPR) and activation of XBP1 is necessary for high secretory efficiency and functional differentiation of antibody secreting cells (ASCs). The UPR additionally includes a branch in which membrane-bound transcription factors, exemplified by ATF6, undergo intramembraneproteolysis by the sequential action of site-1 (MBTPS1/S1P) and site-2 proteases (MBTPS2/S2P) and release of the cytoplasmic domain as an active transcription factor. Such regulation is shared with a family of CREB3-related transcription factors and sterol regulatory element-binding proteins (SREBPs). Of these, we identify that the CREB3 family member CREB3L2 is strongly induced and activated during the transition from B-cell to plasma cell state. Inhibition of site-1 protease leads to a profound reduction in plasmablast number linked to induction of autophagy. Plasmablasts generated in the presence of site-1 protease inhibitor segregated into CD38high and CD38low populations, the latter characterized by a marked reduction in the capacity to secrete IgG. Site-1 protease inhibition is accompanied by a distinctive change in gene expression associated with amino acid, steroid and fatty acid synthesis pathways. These results demonstrate that transcriptional control of metabolic programs necessary for secretory activity can be targeted via site-1 protease inhibition during ASC differentiation. During terminal differentiation of B-cells to plasma cells (PCs), specific gene expression programs are instigated to allow adaptation to the secretion of large amounts of immunoglobulin. A critical role for the transcription factor XBP1 has been identified linking differentiation, ER stress and secretory apparatus expansion1,2. The initial data describing a role for XBP1 in PC generation was consistent with the secretion of immunoglobulin triggering an unfolded protein response (UPR)3. Later reports suggested that XBP1 could be expressed in cells that did not secrete immunoglobulin, challenging the idea that a UPR is required4,5. Furthermore, results from a B-cell conditional knockout demonstrated that XBP1 was not required for the early stages of PC differentiation, but was required for efficient immunoglobulin secretion6. These data were additionally corroborated in another model of B-cell specific deletion of XBP1, linking XBP1 to the regulation of ER remodelling required for high rates of secretion7. Unlike the proposed requirement for XBP1, available data suggest that both the PERK and ATF6 axes of the UPR may be dispensable for the formation of PCs8,9. Collectively, the available evidence suggests that B-cells utilize the UPR in an unconventional fashion10, and other components of the ER stress response may provide partially redundant regulation of the secretory apparatus during PC differentiation. Amongst these the CREB family has not been explored in the context of B-cell differentiation. CREB3L2 is one of 5 members of the CREB3 (CREB-cAMP response element binding protein) family11. This is a group of bZIP transcription factor proteins that are synthesized as latent ER resident transmembrane proteins and require protease cleavage in the Golgi to release the active transcription factor component12. The CREB3 family members are implicated as evolutionarily conserved regulators of the secretory apparatus and potentially of the UPR. CREB3L2 and other CREB3 family members share their mechanism of activation with ATF6 and sterol regulatory element binding protein, SREBP, a major transcriptional regulator of sterol and lipid synthesis. 1 Section of Experimental Haematology, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, LS9 7TF, United Kingdom. 2Bioinformatics Group, School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom. Correspondence and requests for materials should be addressed to G.M.D. (email: ) SCientifiC Reports | (2018) 8:14338 | DOI:10.1038/s41598-018-32705-7 1 www.nature.com/scientificreports/ A B-cell Activated B-cell Plasmablast Plasma cell Day 0 Day 3 Day 6 Day 13 CD19 +CD20 ++ CD38 loCD138 - CD19 ++CD20 ++ CD38 loCD138 - CD19 +CD20 CD38 +CD138 - CD19 +/-CD20 CD38 ++CD138 + Relative expression log2 B 14 12 CREB3L2 CREB3L2 IRF4 IRF4 IRF8 IRF8 MBTPS1 MBTPS1 MBTPS2 MBTPS2 10 8 6 0 10 20 30 40 50 Days C d3 d7 d9 d11 d13 CREB3L2 Full length Cleaved ACTIN Figure 1. Expression of CREB3L2 in differentiating primary human B-cells. (A) Schematic of in vitro B-cell differentiation with key stages and phenotypes. (B) Relative mRNA expression levels for IRF4, IRF8, CREB3L2, MBTPS1 and MBTPS2 during in vitro generation of PCs derived from microarray data. (C) Protein expression of CREB3L2 during in vitro differentiation of primary human B-cells. The day of culture is indicated at the top. Intact and dashed arrows show migration of full-length protein and cleaved C-terminus, respectively. All of these factors are released from the ER, following appropriate stimulation, and migrate to the Golgi where they are cleaved by the sequential action of S1P and S2P11,13. This event releases the cytosolic transcription factor component that migrates to the nucleus and binds to DNA, regulating gene expression. The sequential process of intramembrane proteolysis controlled by S1P and S2P provides a potential avenue for therapeutic intervention targeting the group of transcription factors sharing this process of regulation. Evaluation of the pathway was originally performed in relation to control of the SREBP in the context of potential control of hepatic lipid synthesis14. This led to the development of a selective inhibitor and tool compound for selective dissection of the pathway in cell biology. Here, we describe the progressive accumulation of CREB3L2 during PC differentiation and utilize the selective S1P inhibitor PF-429242 to establish that S1P-regulated events are essential for efficient ASC differentiation and regulation of genes involved in the metabolic pathways necessary for adaptation to antibody secretion. This pathway reinforces the direct link between the secretory apparatus and the establishment of ASC state. Results CREB3L2 is induced and processed to the active form during PC differentiation. After appropriate stimulation B-cells undergo a step-wise reprogramming for dedicated antibody secretion. Recent developments of in vitro models of human PC differentiation provide the opportunity to dissect the regulatory networks that govern this transition (Fig. 1A)15–17. In a detailed characterization of gene expression spanning multiple stages o (...truncated)