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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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 4

ROCK2 regulates a distinctive transcriptional program in GC B cells.

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ROCK2 regulates a distinctive transcriptional program in GC B cells. WT ...
WT or CD23-Rock2 mice were immunized with 100 μg NP-CGG for 7 days, and FoBs (B220+GL7–CD38hiCD23+) and GC B cells (B220+GL7+CD38lo) were sorted for RNA-Seq analyses. (A) Representative RT-qPCR analysis of Rock2 in sorted FoBs and GC B cells from the indicated mice. n = 4. Data are from technical triplicates and are representative of 4 independent experiments. Data represent the mean ± SD. ***P < 0.001, by unpaired, 2-tailed t test. (B) Plot shows the log-transformed FC (logFC) values for genes differentially expressed (unadjusted P < 0.01) between WT and CD23-Rock2 FoBs. (C) Volcano plot shows the genes differentially expressed (unadjusted P < 0.01) between WT and CD23-Rock2 GC B cells. 46. The unadjusted P values in B and C were determined using edgeR3.24.3 in R. (D) Plot shows the top enriched gene sets by GSEA that were upregulated in CD23-Rock2 GC B cells. Dotted line indicates the significance cutoff at a FDR of Q = 0.05. GO, Gene Ontology. (E) Heatmap of the scaled expression of genes enriching the GPCR_Activity gene set in CD23-Rock2 GC B cells. (F) Representative RT-qPCR analyses of the indicated genes in sorted GC B cells. n = 4. Data show technical triplicates and are representative of 4 independent experiments. Data represent the mean ± SD. *P < 0.05, ***P < 0.001, and ****P < 0.0001, by 2-tailed t test. Rel, relative. (G) H&E-stained images of splenic sections on day 7 after immunization. Scale bars: 200 μm. (H) Representative immunofluorescence images show the expression of B220 (blue) and PNA (red) on splenic sections from WT and CD23-Rock2 mice and pooled quantifications of the average GC count per ×10 field per mouse and the average GC size per mouse. Scale bars: 100 μm. n = 6 per genotype. Data are from 3 independent experiments and represent the mean ± SEM. **P < 0.01 and P = 0.10, by Mann-Whitney U test. DOWN, downregulated; UP, upregulated.