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 2

ROCK2 deficiency leads to impaired GC responses.

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ROCK2 deficiency leads to impaired GC responses. (A–C) ROCK1 and ROCK2 I...
(A–C) ROCK1 and ROCK2 IVK assays (A) were performed on extracts from CD23+ cultures from Rock2fl/fl (WT) or CD23-Cre Rock2fl/fl (CD23-Rock2) mice as in Figure 1, A and B. Kinase activity was quantified for ROCK1 (B) and ROCK2 (C) as the ratio of p-MYPT1 to input ROCK protein. n = 3. Data represent the mean ± SEM. P values were determined by 1-way ANOVA followed by Dunnett’s test for multiple comparisons. Lanes in A were run on the same gel but were noncontiguous. (D–G) WT and CD23-Rock2 mice were immunized with 100 μg NP-CGG for 7 to 10 days. Representative FACS plots (D) and pooled quantifications of total GC B cells (E) (B220+Fas+GL7+), total NP-specific B cells (F) (B220+IgM–IgD–Gr1–NP+IgG1+), and NP-specific GC B cells (G) (B220+IgM–IgD–Gr1–NP+IgG1+CD38lo) from WT (solid blue circles) and CD23-Rock2 (open blue circles) mice. (H–K) WT and Cγ1-Cre Rock2fl/fl (Cγ1-Rock2) mice were immunized as in D–G. Representative FACS plots (H) and pooled quantifications of total GC B cells (I), total NP-specific B cells (J), and NP-specific GC B cells (K) from WT (solid blue) or Cγ1-Rock2 (open blue) mice. n > 6. Data are from at least 2 independent experiments per time point and represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by unpaired 2-tailed t test.