The absence of ZO-2 promotes an increase in cell size by two mechanisms: an increase in cyclin D, which extends the time that the cells spend in the G1 phase of the cell cycle, and an accumulation of YAP at the nucleus, which promotes its transcriptional activity, triggering the activation of the mTORC1 complex and its target, S6K1.

Background: Cerebral cavernous malformations (CCMs) are neurovascular lesions caused by loss of function mutations in 1 of 3 genes, including KRIT1 ( CCM1 ), CCM2 , and PDCD10 ( CCM3 ). CCMs affect ≈1 out of 200 children and adults, and no pharmacologic therapy is available. CCM lesion count, size, and aggressiveness vary widely among patients of similar ages with the same mutation or even within members of the same family. However, what determines the transition from quiescent lesions into mature and active (aggressive) CCM lesions is unknown. Methods: We use genetic, RNA-sequencing, histology, flow cytometry, and imaging techniques to report the interaction between CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils (CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils interaction) during the pathogenesis of CCMs in the brain tissue. Results: Expression profile of astrocytes in adult mouse brains using translated mRNAs obtained from the purification of EGFP (enhanced green fluorescent protein)-tagged ribosomes ( Aldh1l1-EGFP/Rpl10a ) in the presence or absence of CCM lesions ( Slco1c1-iCreERT2;Pdcd10 fl/fl ; Pdcd10 BECKO ) identifies a novel gene signature for neuroinflammatory astrocytes. CCM-induced reactive astrocytes have a neuroinflammatory capacity by expressing genes involved in angiogenesis, chemotaxis, hypoxia signaling, and inflammation. RNA-sequencing analysis on RNA isolated from brain endothelial cells in chronic Pdcd10 BECKO mice (CCM endothelium), identified crucial genes involved in recruiting inflammatory cells and thrombus formation through chemotaxis and coagulation pathways. In addition, CCM endothelium was associated with increased expression of Nlrp3 and Il1b . Pharmacological inhibition of NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) significantly decreased inflammasome activity as assessed by quantification of a fluorescent indicator of caspase-1 activity (FAM-FLICA caspase-1) in brain endothelial cells from Pdcd10 BECKO in chronic stage. Importantly, our results support the hypothesis of the crosstalk between astrocytes and CCM endothelium that can trigger recruitment of inflammatory cells arising from brain parenchyma (microglia) and the peripheral immune system (leukocytes) into mature active CCM lesions that propagate lesion growth, immunothrombosis, and bleedings. Unexpectedly, partial or total loss of brain endothelial NF-κB (nuclear factor κB) activity (using Ikkb fl/fl mice) in chronic Pdcd10 BECKO mice does not prevent lesion genesis or neuroinflammation. Instead, this resulted in a trend increase in the number of lesions and immunothrombosis, suggesting that therapeutic approaches designed to target inflammation through endothelial NF-κB inhibition may contribute to detrimental side effects. Conclusions Our study reveals previously unknown links between neuroinflammatory astrocytes and inflamed CCM endothelium as contributors that trigger leukocyte recruitment and precipitate immunothrombosis in CCM lesions. However, therapeutic approaches targeting brain endothelial NF-κB activity may contribute to detrimental side effects.

ZO-2 is a cytoplasmic protein of tight junctions (TJs). Here, we describe ZO-2 involvement in the formation of the apical junctional complex during early development and in TJ biogenesis in epithelial cultured cells. ZO-2 acts as a scaffold for the polymerization of claudins at TJs and plays a unique role in the blood–testis barrier, as well as at TJs of the human liver and the inner ear. ZO-2 movement between the cytoplasm and nucleus is regulated by nuclear localization and exportation signals and post-translation modifications, while ZO-2 arrival at the cell border is triggered by activation of calcium sensing receptors and corresponding downstream signaling. Depending on its location, ZO-2 associates with junctional proteins and the actomyosin cytoskeleton or a variety of nuclear proteins, playing a role as a transcriptional repressor that leads to inhibition of cell proliferation and transformation. ZO-2 regulates cell architecture through modulation of Rho proteins and its absence induces hypertrophy due to inactivation of the Hippo pathway and activation of mTOR and S6K. The interaction of ZO-2 with viral oncoproteins and kinases and its silencing in diverse carcinomas reinforce the view of ZO-2 as a tumor regulator protein.

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Tight junction protein Zonula occludens 2 (ZO-2) presence at the nucleus inhibits the transcription of genes regulated by TEAD transcription factor. Here, we analyzed if the movement of ZO-2 into the nucleus modulates the nuclear concentration of TEAD. In sparse cultures of ZO-2 knock down (KD) MDCK cells, nuclear TEAD diminished, as in parental cells transfected with a ZO-2 construct without nuclear localization signals, indicating that ZO-2 facilitates the entry of TEAD into the nucleus. Inhibition of nPKCδ in parental cells triggers the interaction between ZO-2 and TEAD at the cytoplasm and facilitates TEAD/ZO-2 complex nuclear importation. Using proximity ligation, immunoprecipitation and pull-down assays TEAD/ZO-2 interaction was confirmed. Nuclear TEAD is phosphorylated and its exit in parental cells is enhanced by activation of a ZO-2 nuclear exportation signal by nPKCε, while the nuclear accumulation of ZO-2 triggered by the mutation of ZO-2 nuclear export signals, induces the no change in TEAD nuclear concentration. In summary, our results indicate that the movements of ZO-2 in and out of the nucleus modulate the intracellular traffic of TEAD through a process regulated by nPKC δ and ε, and provide a novel role of ZO-2 as a nuclear translocator of TEAD.

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