The Deubiquitylase Otub1 Regulates the Chemotactic Response of Splenic B Cells by Modulating the Stability of the γ-Subunit Gng2.

The ubiquitin proteasome system performs the covalent attachment of lysine 48-linked polyubiquitin chains to substrate proteins, thereby targeting them for degradation, while deubiquitylating enzymes (DUBs) reverse this process. This posttranslational modification regulates key features both of innate and adaptative immunity, including antigen presentation, protein homeostasis and signal transduction. Here we show that loss of one of the most highly expressed DUBs, Otub1, results in changes in murine splenic B cell subsets, leading to a significant increase in marginal zone and transitional B cells and a concomitant decrease in follicular B cells. We demonstrate that Otub1 interacts with the γ-subunit of the heterotrimeric G protein, Gng2, and modulates its ubiquitylation status, thereby controlling Gng2 stability. Proximal mapping of Gng2 revealed an enrichment in partners associated with chemokine signaling, actin cytoskeleton and cell migration. In line with these findings, we show that Otub1-deficient B cells exhibit greater Ca2+ mobilization, F-actin polymerization and chemotactic responsiveness to Cxcl12, Cxcl13 and S1P in vitro, which manifests in vivo as altered localization of B cells within the spleen. Together, our data establishes Otub1 as a novel regulator of G-protein coupled receptor signaling in B cells, regulating their differentiation and positioning in the spleen.

Systematic proximal mapping of the classical RAD51 paralogs unravel functionally and clinically relevant interactors for genome stability.

Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as a core component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability that could be used as biomarkers for the prognosis of BC.

Common clonal origin of chronic myelomonocytic leukemia and B-cell acute lymphoblastic leukemia in a patient with a germline CHEK2 variant.

Hematological malignancies are broadly divided into myeloid and lymphoid neoplasms, reflecting the two major cellular lineages of the hematopoietic system. It is generally rare for hematological malignancies to spontaneously progress with a switch from myeloid to lymphoid lineage. We describe the exceptional case of a patient who sequentially developed myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), and B-cell acute lymphoblastic leukemia (B-ALL), as well as our investigation into the underlying pathogenesis. Using whole-exome sequencing (WES) performed on sorted CMML and B-ALL cell fractions, we identified both common and unique potential driver mutations, suggesting a branching clonal evolution giving rise to both diseases. Interestingly, we also identified a germline variant in the cancer susceptibility gene CHEK2 We validated that this variant (c.475T > C; p.Y159H), located in the forkhead-associated (FHA) domain, impairs its capacity to bind BRCA1 in cellulo. This unique case provides novel insight into the genetics of complex hematological diseases and highlights the possibility that such patients may carry inherited predispositions.

Protein kinase B/AKT mediates insulin-like growth factor 1-induced phosphorylation and nuclear export of histone deacetylase 5 via NADPH oxidase 4 activation in vascular smooth muscle cells.

Insulin-like growth factor 1 (IGF-1) mediates the generation of reactive oxygen species (ROS) and the activation of growth promoting signaling pathways. Histone deacetylases (HDACs) regulate gene transcription by deacetylating lysine residues in histone and nonhistone proteins and a heightened HDAC activation, notably of HDAC5, is associated with vascular disorders, such as atherosclerosis. Although the contribution of IGF-1 in these pathologies is well documented, its role in HDAC phosphorylation and activation remains unexplored. Here, we examined the effect of IGF-1 on HDAC5 phosphorylation in vascular smooth muscle cells (VSMCs) and identified the signaling pathways involved in controlling HDAC5 phosphorylation and nuclear export. Treatment of A10 VSMCs with IGF-1 enhanced HDAC5 phosphorylation. Blockade of the IGF-1 receptor tyrosine kinase (TK) activity with the specific pharmacological inhibitor, AG1024, significantly inhibited IGF-1-induced HDAC5 phosphorylation, whereas the epidermal growth factor receptor (EGFR) TK antagonist, AG1478, had no effect. Inhibition of the mitogen-activated protein kinase pathway with U0126, SP600125, or SB203580, did not affect HDAC5 phosphorylation, whereas two inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathways, wortmannin and SC66, almost completely attenuated IGF-1-induced responses as confirmed by immunoblotting of phospho-HDAC5 and by small interfering RNA (siRNA)-induced AKT silencing. Moreover, the NAD(P)H oxidase (Nox) inhibitor, diphenyleneiodonium (DPI), and Nox4 siRNA, attenuated IGF-1-induced phosphorylation of HDAC5 and AKT. The HDAC5 phosphorylation resulted in its nuclear export, which was reversed by SC66 and DPI. Our results indicate that IGF-1-induced phosphorylation and nuclear export of HDAC5 involve Nox4-dependent ROS generation and PI3K/AKT signaling pathways.

SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break repair pathway choice.

DNA double-strand breaks (DSBs) can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)-based approach, we identify 11 high-confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ-mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1-, RIF1-, and REV7-dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision-making process during DSB repair.

cAMP attenuates angiotensin-II-induced Egr-1 expression via PKA-dependent signaling pathway in vascular smooth muscle cells.

cAMP has been shown to inhibit vascular smooth muscle cell proliferation and exerts a vasculoprotective effect. An upregulation of the early growth response protein-1 (Egr-1) expression has been linked with the development of atherosclerosis and intimal hyperplasia. We have recently demonstrated that angiotensin-II (Ang-II) stimulates Egr-1 expression via Ca2+/ERK-mediated cAMP-response element binding protein (CREB) activation. However, whether Ang-II-induced signaling leading to Egr-1 expression is modulated by cAMP remains unexplored. Therefore, in the present studies, we have examined the effect of cAMP on Ang-II-induced expression of Egr-1 and associated signaling pathways. Isoproterenol (ISO) and forskolin (FSK) attenuated Ang-II-induced Egr-1 expression in a dose-dependent fashion. In addition, dibutyryl-cAMP and benzoyl-cAMP, as well as isobutylmethylxanthine, attenuated Ang-II-induced Egr-1 expression. Moreover, inhibition of Ang-II-induced Egr-1 expression was accompanied by an increase in the phosphorylation of the vasodilator-activated phosphoprotein (VASP), and this was associated with a concomitant decrease in ERK phosphorylation. Blockade of PKA using H89 decreased VASP phosphorylation, restored Ang-II-induced ERK phosphorylation, and abolished ISO- and FSK-mediated inhibition of Ang-II-induced Egr-1 expression. In summary, these results suggest that PKA-mediated suppression of Ang-II-induced Egr-1 expression and phosphorylation of ERK may be among the mechanisms by which cAMP exerts its vasculoprotective effects.

STIM-1 and ORAI-1 channel mediate angiotensin-II-induced expression of Egr-1 in vascular smooth muscle cells.

An upregulation of Egr-1 expression has been reported in models of atherosclerosis and intimal hyperplasia and, various vasoactive peptides and growth promoting stimuli have been shown to induce the expression of Egr-1 in vascular smooth muscle cells (VSMC). Angiotensin-II (Ang-II) is a key vasoactive peptide that has been implicated in the pathogenesis of vascular diseases. Ang-II elevates intracellular Ca2+ through activation of the store-operated calcium entry (SOCE) involving an inositol-3-phosphate receptor (IP3R)-coupled depletion of endoplasmic reticular Ca2+ and a subsequent activation of the stromal interaction molecule 1 (STIM-1)/Orai-1 complex. However, the involvement of IP3R/STIM-1/Orai-1-Ca2+ -dependent signaling in Egr-1 expression in VSMC remains unexplored. Therefore, in the present studies, we have examined the role of Ca2+ signaling in Ang-II-induced Egr-1 expression in VSMC and investigated the contribution of STIM-1 or Orai-1 in mediating this response. 2-aminoethoxydiphenyl borate (2-APB), a dual non-competitive antagonist of IP3R and inhibitor of SOCE, decreased Ang-II-induced Ca2+ release and attenuated Ang-II-induced enhanced expression of Egr-1 protein and mRNA levels. Egr-1 upregulation was also suppressed following blockade of calmodulin and CaMKII. Furthermore, RNA interference-mediated depletion of STIM-1 or Orai-1 attenuated Ang-II-induced Egr-1 expression as well as Ang-II-induced phosphorylation of ERK1/2 and CREB. In addition, siRNA-induced silencing of CREB resulted in a reduction in the expression of Egr-1 stimulated by Ang-II. In summary, our data demonstrate that Ang-II-induced Egr-1 expression is mediated by STIM-1/Orai-1/Ca2+ -dependent signaling pathways in A-10 VSMC.

Src tyrosine kinase mediates endothelin-1-induced early growth response protein-1 expression via MAP kinase-dependent pathways in vascular smooth muscle cells.

We have previously demonstrated that the non-receptor protein tyrosine kinase (NR-PTK) c-Src is an upstream regulator of endothelin-1 (ET-1) and angiotensin II-induced activation of protein kinase B (PKB) signaling in vascular smooth muscle cells (VSMCs). We have also demonstrated that ET-1 potently induces the expression of the early growth response protein-1 (Egr-1), a zinc finger transcription factor that is overexpressed in models of vascular diseases, such as atherosclerosis. However, the involvement of c-Src in ET-1­induced Egr-1 expression has not yet been investigated and its role in mitogen-activated protein kinase (MAPK) signaling remains controversial. Therefore, the aim of the present study was to examine the role of c-Src in the ET-1-induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 MAPK, 3 key members of the MAPK family and in the regulation of Egr-1 expression in rat aortic A10 VSMCs. ET-1 rapidly induced the phosphorylation of MAPKs, as well as the expression of Egr-1; however, treatment of the VSMCs with PP2, a specific pharmacological inhibitor of c-Src, dose-dependently reduced the phosphorylation of the 3 MAPKs and the expression of Egr-1 induced by ET-1. Furthermore, in mouse embryonic fibroblasts (MEFs) deficient in c-Src (SYF), the ET-1-induced Egr-1 expression and MAPK phosphorylation were significantly suppressed, as compared to MEFs expressing normal Src levels. These results suggest that c-Src plays a critical role in mediating ET-1-induced MAPK phosphorylation and Egr-1 expression in VSMCs.

ET-1-induced growth promoting responses involving ERK1/2 and PKB signaling and Egr-1 expression are mediated by Ca2+/CaM-dependent protein kinase-II in vascular smooth muscle cells.

Endothelin-1 (ET-1), a potent vasoactive peptide with a pathogenic role in vascular diseases, has been shown to induce the activation of ERK1/2, PKB and the expression of a transcriptional regulator, the early growth response 1 (Egr-1), key mediators of hypertrophic and proliferative responses in vascular smooth muscle cells (VSMC). We have demonstrated earlier that ET-1 requires H2O2 generation to activate these signaling pathways and Ca2+, calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII), play a critical role to trigger H2O2-induced effects in VSMC. However, an involvement of CaMKII in mediating ET-1-induced responses in VSMC remains unknown. Therefore, by utilizing pharmacological inhibitors of CaM, CaMKII, a CaMKII inhibitor peptide and CaMKII knockdown techniques, we have investigated the contribution of CaM and CaMKII in ET-1-induced ERK1/2 and PKB signaling, Egr-1 expression and hypertrophic and proliferative responses in VSMC. W-7 and calmidazolium, antagonists of CaM, as well as KN-93, an inhibitor of CaMKII activity, attenuated ET-1-induced ERK1/2 and PKB phosphorylation. In addition, transfection of VSMC with a CaMKII inhibitory peptide suppressed ET-1-evoked ERK1/2 and PKB phosphorylation. Similarly, siRNA-mediated CaMKII silencing reduced ET-1-produced ERK1/2 and PKB phosphorylation. CaM and CaMKII blockade also significantly lowered the ET-1-induced protein and DNA synthesis as well as Egr-1 expression. These findings demonstrate that CaMKII plays a critical role in ET-1-induced growth promoting signaling pathways as well as hypertrophic and proliferative responses in VSMC.