NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo.

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

Cell adhesion molecule KIRREL1 is a feedback regulator of Hippo signaling recruiting SAV1 to cell-cell contact sites.

The Hippo/YAP pathway controls cell proliferation through sensing physical and spatial organization of cells. How cell-cell contact is sensed by Hippo signaling is poorly understood. Here, we identified the cell adhesion molecule KIRREL1 as an upstream positive regulator of the mammalian Hippo pathway. KIRREL1 physically interacts with SAV1 and recruits SAV1 to cell-cell contact sites. Consistent with the hypothesis that KIRREL1-mediated cell adhesion suppresses YAP activity, knockout of KIRREL1 increases YAP activity in neighboring cells. Analyzing pan-cancer CRISPR proliferation screen data reveals KIRREL1 as the top plasma membrane protein showing strong correlation with known Hippo regulators, highlighting a critical role of KIRREL1 in regulating Hippo signaling and cell proliferation. During liver regeneration in mice, KIRREL1 is upregulated, and its genetic ablation enhances hepatic YAP activity, hepatocyte reprogramming and biliary epithelial cell proliferation. Our data suggest that KIRREL1 functions as a feedback regulator of the mammalian Hippo pathway through sensing cell-cell interaction and recruiting SAV1 to cell-cell contact sites.

The RSPO-LGR4/5-ZNRF3/RNF43 module in liver homeostasis, regeneration, and disease.

WNT/ß-catenin signaling plays pivotal roles during liver development, homeostasis, and regeneration. Likewise, its deregulation disturbs metabolic liver zonation and is responsible for the development of a large number of hepatic tumors. Liver fibrosis, which has become a major health burden for society and a hallmark of NASH, can also be promoted by WNT/ß-catenin signaling. Upstream regulatory mechanisms controlling hepatic WNT/ß-catenin activity may constitute targets for the development of novel therapies addressing these life-threatening conditions. The R-spondin (RSPO)-leucine-rich repeat-containing G protein-coupled receptor (LGR) 4/5-zinc and ring finger (ZNRF) 3/ring finger 43 (RNF43) module is fine-tuning WNT/ß-catenin signaling in several tissues and is essential for hepatic WNT/ß-catenin activity. In this review article, we recapitulate the role of the RSPO-LGR4/5-ZNRF3/RNF43 module during liver development, homeostasis, metabolic zonation, regeneration, and disease. We further discuss the controversy around LGR5 as a liver stem cell marker.

ZNRF3 and RNF43 cooperate to safeguard metabolic liver zonation and hepatocyte proliferation.

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/ß-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/ß-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of ß-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/ß-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/ß-Catenin activity, balancing metabolic function and hepatocyte proliferation.

Experimental study on dust removal optimization of shearer external spray in air velocity.

In order to clarify the influence of air velocity on the atomization effect of shearer external spray and optimize dust suppression performance, the dust removal experiment was carried out by utilizing the self-designed external spray experiment platform. The effect of three kinds of air velocity on the atomization effect of main spray parameters was investigated to clarify the influence of air velocity on the atomization effect of shearer external spray and optimize dust suppression performance. The results showed that the influence of air velocity on droplet size and distribution width of the droplet size was slightly less than the spray pressure. The average diameter of the droplet was 54.211 µm, and the particle size distribution of the droplet was more uniform and concentrated when air velocity was 2 m/s, the pressure was 5 MPa and nozzle diameter was 1.0 mm. When the air velocity was less than 2 m/s and the spray pressure was 3-5 MPa, the atomization effect was better by using 1.0 or 1.2 mm diameter nozzle. Through on-site applications and optimization, the leeward total dust removal efficiency of the shearer can reach 79.43%, and the maximum increase range can reach 18.51 percentage points. The respiratory dust removal efficiency of the shearer can reach 87.45%, and the maximum increase range can reach 11.87 percentage points.

AXIN2+ Pericentral Hepatocytes Have Limited Contributions to Liver Homeostasis and Regeneration.

The existence of specialized liver stem cell populations, including AXIN2+ pericentral hepatocytes, that safeguard homeostasis and repair has been controversial. Here, using AXIN2 lineage tracing in BAC-transgenic mice, we confirm the regenerative potential of intestinal stem cells (ISCs) but find limited roles for pericentral hepatocytes in liver parenchyma homeostasis. Liver regrowth following partial hepatectomy is enabled by proliferation of hepatocytes throughout the liver, rather than by a pericentral population. Periportal hepatocyte injury triggers local repair as well as auxiliary proliferation in all liver zones. DTA-mediated ablation of AXIN2+ pericentral hepatocytes transiently disrupts this zone, which is reestablished by conversion of pericentral vein-juxtaposed glutamine synthetase (GS)- hepatocytes into GS+ hepatocytes and by compensatory proliferation of hepatocytes across liver zones. These findings show hepatocytes throughout the liver can upregulate AXIN2 and LGR5 after injury and contribute to liver regeneration on demand, without zonal dominance by a putative pericentral stem cell population.

YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.

TAK1 regulates endothelial cell necroptosis and tumor metastasis.

Formation of metastases is the major cause of death in patients diagnosed with cancer. It is a complex multistep process, including tumor cell migration, intravasation, survival in the circulation, and extravasation. Previously it was shown that tumor cell-induced endothelial necroptosis promotes tumor cell extravasation and metastasis. Here, we identified endothelial TGF-ß-activated kinase 1 (TAK1) as a critical regulator of endothelial necroptosis and metastasis. Human and murine endothelial cells lacking TAK1 exhibit higher levels of necroptosis both in vitro and in vivo, and mice with endothelial cell-specific loss of TAK1 are more prone to form metastases. Endothelial RIPK3, a key component of the necroptotic machinery, was upregulated in mice with endothelial TAK1-deficiency, and endothelial knockout of RIPK3 reverted the effects of TAK1-deficiency. Moreover, altered expression levels of TAK1 and RIPK3 in pulmonary endothelial cells of mice bearing primary tumors correlated with increased endothelial necroptosis and metastasis. Together, our data suggest an important protective role for endothelial TAK1 in tumor progression by keeping endothelial necroptosis in check.

A reverse signaling pathway downstream of Sema4A controls cell migration via Scrib.

Semaphorins comprise a large family of ligands that regulate key cellular functions through their receptors, plexins. In this study, we show that the transmembrane semaphorin 4A (Sema4A) can also function as a receptor, rather than a ligand, and transduce signals triggered by the binding of Plexin-B1 through reverse signaling. Functionally, reverse Sema4A signaling regulates the migration of various cancer cells as well as dendritic cells. By combining mass spectrometry analysis with small interfering RNA screening, we identify the polarity protein Scrib as a downstream effector of Sema4A. We further show that binding of Plexin-B1 to Sema4A promotes the interaction of Sema4A with Scrib, thereby removing Scrib from its complex with the Rac/Cdc42 exchange factor ßPIX and decreasing the activity of the small guanosine triphosphatase Rac1 and Cdc42. Our data unravel a role for Plexin-B1 as a ligand and Sema4A as a receptor and characterize a reverse signaling pathway downstream of Sema4A, which controls cell migration.

Quantitative analysis of the TNF-α-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKKß substrate.

The inhibitor of the nuclear factor-κB (IκB) kinase (IKK) complex is a key regulator of the canonical NF-κB signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-κB activation; however, there is increasing evidence for NF-κB pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-α-IKKß-induced phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which ∼1% are regulated up on TNF-α stimulation. We identify various potential novel IKKß substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKKß on serine 298. We provide evidence that IKKß-mediated AEG-1 phosphorylation is essential for IκBα degradation as well as NF-κB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo.

Semaphorin-Plexin Signaling Controls Mitotic Spindle Orientation during Epithelial Morphogenesis and Repair.

Morphogenesis, homeostasis, and regeneration of epithelial tissues rely on the accurate orientation of cell divisions, which is specified by the mitotic spindle axis. To remain in the epithelial plane, symmetrically dividing epithelial cells align their mitotic spindle axis with the plane. Here, we show that this alignment depends on epithelial cell-cell communication via semaphorin-plexin signaling. During kidney morphogenesis and repair, renal tubular epithelial cells lacking the transmembrane receptor Plexin-B2 or its semaphorin ligands fail to correctly orient the mitotic spindle, leading to severe defects in epithelial architecture and function. Analyses of a series of transgenic and knockout mice indicate that Plexin-B2 controls the cell division axis by signaling through its GTPase-activating protein (GAP) domain and Cdc42. Our data uncover semaphorin-plexin signaling as a central regulatory mechanism of mitotic spindle orientation necessary for the alignment of epithelial cell divisions with the epithelial plane.

Grb2 mediates semaphorin-4D-dependent RhoA inactivation.

Signaling through the semaphorin 4D (Sema4D) receptor plexin-B1 is modulated by its interaction with tyrosine kinases ErbB-2 and Met. In cells expressing the plexin-B1-ErbB-2 receptor complex, ligand stimulation results in the activation of small GTPase RhoA and stimulation of cellular migration. By contrast, in cells expressing plexin-B1 and Met, ligand stimulation results in an association with the RhoGTPase-activating protein p190 RhoGAP and subsequent RhoA inactivation--a process that involves the tyrosine phosphorylation of plexin-B1 by Met. Inactivation of RhoA is necessary for Sema4D-mediated inhibition of cellular migration. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGAP interaction and activity. Here we show that the activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation by Met creates a docking site for the SH2 domain of growth factor receptor bound-2 (Grb2). Grb2 is thereby recruited into the plexin-B1 receptor complex and, through its SH3 domain, interacts with p190 RhoGAP and mediates RhoA deactivation. Phosphorylation of plexin-B1 by Met and the recruitment of Grb2 have no effect on the R-RasGAP activity of plexin-B1, but are required for Sema4D-induced, RhoA-dependent antimigratory effects of Sema4D on breast cancer cells. These data show Grb2 as a direct link between plexin and p190-RhoGAP-mediated downstream signaling.

PHF8 is a histone H3K9me2 demethylase regulating rRNA synthesis.

Dimethylation of histone H3 lysine 9 (H3K9me2) is an important epigenetic mark associated with transcription repression. Here, we identified PHF8, a JmjC-domain-containing protein, as a histone demethylase specific for this repressing mark. Recombinant full-length wild type protein could remove methylation from H3K9me2, but mutation of a conserved histidine to alanine H247A abolished the demethylase activity. Overexpressed exogenous PHF8 was colocalized with B23 staining. Endogenous PHF8 was also colocalized with B23 and fibrillarin, two well-established nucleolus proteins, suggesting that PHF8 is localized in the nucleolus and may regulate rRNA transcription. Indeed, PHF8 bound to the promoter region of the rDNA gene. Knockdown of PHF8 reduced the expression of rRNA, and overexpression of the gene resulted in upregulation of rRNA transcript. Concomitantly, H3K9me2 level was elevated in the promoter region of the rDNA gene in PHF8 knockdown cells and reduced significantly when the wild type but not the catalytically inactive H247A mutant PHF8 was overexpressed. Thus, our study identified a histone demethylase for H3K9me2 that regulates rRNA transcription.