Serine-threonine kinase ROCK2 regulates germinal center B cell positioning and cholesterol biosynthesis
Edd Ricker, … , James K. Liao, Alessandra B. Pernis
Edd Ricker, … , James K. Liao, Alessandra B. Pernis
Published March 31, 2020
Citation Information: J Clin Invest. 2020;130(7):3654-3670. https://doi.org/10.1172/JCI132414.
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Research Article Immunology

Serine-threonine kinase ROCK2 regulates germinal center B cell positioning and cholesterol biosynthesis

Abstract

Germinal center (GC) responses require B cells to respond to a dynamic set of intercellular and microenvironmental signals that instruct B cell positioning, differentiation, and metabolic reprogramming. RHO-associated coiled-coil–containing protein kinase 2 (ROCK2), a serine-threonine kinase that can be therapeutically targeted by ROCK inhibitors or statins, is a key downstream effector of RHOA GTPases. Although RHOA-mediated pathways are emerging as critical regulators of GC responses, the role of ROCK2 in B cells is unknown. Here, we found that ROCK2 was activated in response to key T cell signals like CD40 and IL-21 and that it regulated GC formation and maintenance. RNA-Seq analyses revealed that ROCK2 controlled a unique transcriptional program in GC B cells that promoted optimal GC polarization and cholesterol biosynthesis. ROCK2 regulated this program by restraining AKT activation and subsequently enhancing FOXO1 activity. ATAC-Seq (assay for transposase-accessible chromatin with high-throughput sequencing) and biochemical analyses revealed that the effects of ROCK2 on cholesterol biosynthesis were instead mediated via a novel mechanism. ROCK2 directly phosphorylated interferon regulatory factor 8 (IRF8), a crucial mediator of GC responses, and promoted its interaction with sterol regulatory element–binding transcription factor 2 (SREBP2) at key regulatory regions controlling the expression of cholesterol biosynthetic enzymes, resulting in optimal recruitment of SREBP2 at these sites. These findings thus uncover ROCK2 as a multifaceted and therapeutically targetable regulator of GC responses.

Authors

Edd Ricker, Yurii Chinenov, Tania Pannellini, Danny Flores-Castro, Chao Ye, Sanjay Gupta, Michela Manni, James K. Liao, Alessandra B. Pernis

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Figure 7

ROCK2 promotes cholesterol biosynthesis pathways in GC B cells by enhancing the activity of SREBP2.

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ROCK2 promotes cholesterol biosynthesis pathways in GC B cells by enhanc...
(A) Plot shows the top enriched gene sets downregulated in CD23-Rock2 GC B cells compared with WT GC B cells. Dotted line indicates significance cutoff at FDR Q = 0.05. (B) GSEA plot shows the enrichment of the GO_STEROL_BIOSYNTHETIC_PROCESS gene set in WT GC B cells. (C) Schematic shows the enzymes and intermediates involved in the cholesterol biosynthesis pathway. Enzymes highlighted in red are encoded by genes that contribute to the downregulation of the GO_STEROL_BIOSYNTHETIC_PROCESS gene set in CD23-Rock2 GC B cells (A). (D) Representative RT-qPCR analysis of the indicated genes in sorted FoBs or GC B cells from WT or CD23-Rock2 mice 7 days after immunization. n = 3. Data are based on technical triplicates and are representative of 3 independent experiments. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by unpaired, 2-tailed t test. (E) Total cholesterol in sorted FoBs and GC B cells 7 days after immunization. n = 2. Data are from 2 independent experiments and represent the mean ± SEM. **P < 0.01, by unpaired, 2-tailed t test. (F) CD23+ B cells from WT (black) or CD23-Rock2 (blue) mice were purified and stimulated for 3 days as in Figure 1, A and B. Plots show representative data for ChIP-qPCR of SREBP2 binding to the indicated loci. Data are representative of 3 independent experiments and indicate the mean ± SD.