Biology subjects

Kikuchi, H.

Publications and source records attributed to Kikuchi, H..

12 recordsLinked to original sources

bioRxiv2025

Vasopressin-Dependent β-Catenin Phosphorylation at Ser552 and Branching Structure of Mouse Collecting Duct System

BackgroundPhosphoproteomics studies in both cultured and native collecting duct (CD) cells showed that vasopressin strongly increases PKA-dependent phosphorylation of {beta}-catenin at Ser552. Relatively little is known about the role of Ser552 phosphorylation. MethodsTo address the role of {beta}-catenin Ser552 phosphorylation in the mature renal CD, we have inserted a Ser552Ala mutation in mice using CRISPR-Cas9. ResultsThe mutation did not affect the renal abundance of the vasopressin-regulated water channel aquaporin-2 (AQP2) or urinary osmolality. However, the structure of the CD system was altered. Specifically, the cortical branching ratio (the number of nephrons that merge to form one cortical CD) was reduced from 6.18 {+/-} 0.66 in control mice to 3.33 {+/-} 0.82 in Ser552Ala mutant mice. This was associated with a greater number of cortical and medullary CDs with smaller average diameter. The total number of nephrons (glomerular counts) was not different between control and Ser552Ala mutant mice (both [~]13,500 per kidney). RNA-seq in microdissected cortical CDs of the mice revealed a highly significant enrichment of genes involved in regulation of mitosis and the cell cycle, along with decreases in mRNAs coding for two CDK inhibitor proteins, Cdkn1b and Cdkn1c. At the same time, there were no changes in abundances of major transporter mRNAs, indicative of sustained CD differentiation. A subset of cortical CD cells showed an increase in DNA content, consistent with G2/M cell-cycle arrest. ConclusionsThe observed structural changes in the collecting duct system of adult mice point to a role of vasopressin-mediated post-translational modification of {beta}-catenin at Ser552 in collecting duct development, presumably PKA-mediated Ser552 phosphorylation. We speculate that vasopressin may act to slow or halt branching morphogenesis perinatally and may affect the collecting duct elongation process that normally produces the unbranched region of the CD system in the cortex and outer medulla. Key pointsO_LIVasopressin regulates collecting duct (CD) transport by triggering phosphorylation of multiple proteins including {beta}-catenin at Ser552. C_LIO_LIMutating Ser552 to a non-phosphorylable amino acid in mice resulted in altered CD branching without loss of differentiation in adult CDs. C_LIO_LIThe findings point to a role for {beta}-catenin Ser552 phosphorylation in CD branching and sub-segmental CD elongation. C_LI

physiology↗

bioRxiv2024

The lysosomal membrane protein LAMP2B mediates microlipophagy to target obesity-related disorders

Lifestyle diseases, such as obesity, diabetes, and metabolic syndrome, are leading health problems, most of which are related to abnormal lipid metabolism. Lysosomes can degrade lipid droplets (LDs) via microautophagy. Here, we report the molecular mechanism and pathophysiological roles of microlipophagy, regulated by the lysosomal membrane protein LAMP2B. Our study revealed that LAMP2B interacts with phosphatidic acid, facilitating lysosomal-LD interactions and enhancing lipid hydrolysis via microlipophagy depending on endosomal sorting complexes required for transport. Correlative light and electron microscopy demonstrated direct LDs uptake into lysosomes at contact sites. Moreover, LAMP2B overexpression in mice prevents high-fat diet-induced obesity, insulin resistance, and adipose tissue inflammation; liver lipidomics analysis suggested enhanced triacylglycerol hydrolysis. Overall, the findings of this study elucidated the mechanism of microlipophagy, which could be promising for the treatment of obesity and related disorders.

cell biology↗

bioRxiv2023

Combined blockade of CXCR4 and PD-1 enhances intratumoral dendritic cell activation and immune responses against HCC

Immune checkpoint inhibitors (ICIs) have transformed systemic therapy for unresectable hepatocellular carcinoma (HCC). Nevertheless, their efficacy is limited to a small percentage of patients, leaving an opportunity for enhancement through synergistic combination therapies. We tested here the combined blockade of programmed death receptor 1 (PD-1) and CXCR4, a receptor for CXCL12 and a key mediator of immunosuppression in the tumor microenvironment in orthotopic grafted and autochthonous models of HCC. We evaluated tumor growth and survival outcomes and examined the underlying mechanisms using immunofluorescence, flow cytometry, RNA-sequencing, and transgenic mice experiments. Combined anti-CXCR4/PD-1 therapy had a robust impact on tumor growth and significantly prolonged survival in all murine preclinical models. The combination treatment successfully reprogrammed antigen-presenting cells, revealing the role of conventional type 1 dendritic cells (cDC1s) in the tumor microenvironment. Moreover, DC reprogramming enhanced anti-cancer immunity by facilitating CD8 T-cell accumulation and activation in the HCC tissue. The effectiveness of the anti-CXCR4 antibody/ICI combination treatment was compromised entirely in Batf3-KO mice deficient in cDC1 cells. Thus, combined ICI therapy with an anti-CXCR4 antibody has the potential to augment the anti-cancer effects and improve survival outcomes in HCC via reprogramming intra-tumoral cDC1 cells.

cancer biology↗

bioRxiv2023

Preventing NK cell activation in the damaged liver induced by cabozantinib/PD-1 blockade increases survival in hepatocellular carcinoma models

Combining multikinase inhibitors with immune checkpoint blockade improves tumor control in hepatocellular carcinoma (HCC), but survival benefits remain inconsistent. To define this discordance, we integrated COSMIC-312 with orthotopic and autochthonous murine HCC models with or without liver fibrosis. In patients treated with cabozantinib plus atezolizumab, baseline liver function stratified overall survival but not progression-free survival, indicating uncoupling of tumor control from survival. In contrast, sorafenib outcomes tracked with both endpoints, supporting a treatment-specific effect rather than a purely prognostic effect of liver status. In murine HCC models, cabozantinib plus PD-1 blockade induced comparable tumor regression regardless of liver condition, but improved survival only in mice with preserved liver function, whereas fibrotic hosts developed hepatotoxicity. Immune profiling revealed compartment-specific remodeling, with enhanced cytotoxic T-cell programs in tumors but expansion of NK-lineage innate lymphocytes with ILC1-like features in fibrotic liver. Depletion of NK1.1-positive cells reduced liver injury and restored survival without compromising antitumor efficacy, whereas CD4-positive or CD8-positive T-cell depletion did not protect from hepatotoxicity. Transcriptomic, single-cell, adoptive-transfer, and human in vitro studies supported a model in which the fibrotic liver niche promotes NK-to-ILC1-like reprogramming, hepatocyte stress signaling, and TNF/TRAIL-associated epithelial injury. Consistently, cabozantinib and nivolumab showed liver-predominant remodeling of CD56-positive innate lymphocyte-enriched populations in human HCC samples, and ex vivo TGF-beta induced ILC1-like phenotypic changes in human NK cells. These findings identify liver fibrosis as a host determinant that can limit the survival benefit of multikinase inhibitor immunotherapy by promoting innate immune-mediated hepatotoxicity despite preserved tumor control in HCC. One Sentence SummaryLiver fibrosis limits the survival benefit of multikinase inhibitor immunotherapy in hepatocellular carcinoma by promoting innate immune-mediated hepatotoxicity despite preserved tumor control.

cancer biology↗

bioRxiv2023

ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

AMP-activated protein kinase (AMPK) is a central kinase involved in energy homeostasis. Increased intracellular adenosine monophosphate (AMP) levels result in AMPK activation through the binding of AMP to the {gamma}-subunit of AMPK. Recently, we reported that AMP-induced AMPK activation is impaired in the kidneys in chronic kidney disease (CKD) despite an increase in the AMP/ATP ratio. However, the mechanisms by which AMP sensing is disrupted in CKD are unclear. In this study, we identified mechanisms of energy homeostasis in which Unc-51-like kinase 1 (ULK1)-dependent phosphorylation of AMPK{gamma}1 at Ser260/Thr262 promotes AMP sensitivity of AMPK. AMPK activation by AMP was impaired in Ulk1-/- mice despite an increased AMP/ATP ratio. We also demonstrated that MK8722, an allosteric AMPK activator, activates AMPK in the kidneys of a CKD mouse model via a pathway that is independent of AMP sensing. MK8722 treatment significantly attenuates the deterioration of renal function in CKD and is a potential therapeutic option in CKD therapeutics.

molecular biology↗

bioRxiv2023

DNA methylation signature in NSD2 loss-of-function variants appeared similar to that in Wolf-Hirschhorn syndrome

PurposeWolf-Hirschhorn syndrome (WHS), a contiguous gene syndrome caused by the hemizygous deletion of the distal short arm of chromosome 4 where NSD2 is, reportedly exhibits specific DNA methylation signatures in peripheral blood cells. However, responsible genomic loci for signatures are unreported. The objective of the study is to define the loci of WHS-related DNA methylation signatures and to explore the role of NSD2 for the signatures. MethodsWe conducted genome-wide methylation analysis of individuals with WHS or NSD2 variants using array. We studied genome-edited knock in mice or induced pluripotent stem cells to explore the function of NSD2 variants which are observed in congenital anomaly cases. ResultsThree undiagnosed cases with NSD2 variants showed WHS-related DNA methylation signatures. These variants were validated to be NSD2 loss-of-function in induced pluripotent stem cells or genome-edited knock-in mice. p.Pro905Leu variant decreased Nsd2 protein levels, and changed Histone H3-Lysine 36 demethylation levels in similar way in the same genomic regions as Nsd2 knock out mice regulated. Nsd2 knock out mice exhibited common DNA methylation changes. ConclusionThese results revealed that WHS-related DNA methylation signatures are dependent on NSD2 dysfunction and are useful in diagnosing NSD2 variants of unknown significance.

genetics↗

bioRxiv2022

Early Signaling Events in Renal Compensatory Hypertrophy Revealed by Multi-Omics

Loss of a kidney results in compensatory growth of the remaining kidney, a phenomenon of considerable clinical importance. However, the mechanisms involved are largely unknown. Here, we used a multi-omic approach in a mouse unilateral nephrectomy model to identify signaling processes associated with compensatory hypertrophy of the renal proximal tubule. Morphometry applied to microdissected proximal tubules showed that growth of the proximal tubule involves a marked, rapid increase in cell volume rather than cell number. Measurements of DNA accessibility (ATAC-seq), transcriptome (RNA-seq) and proteome (quantitative protein mass spectrometry) independently identified patterns of change that are indicative of activation of the lipid-regulated transcription factor, PPAR. Activation of PPAR by fenofibrate administration increased proximal tubule cell size, while genetic deletion of PPAR in mice decreased it. The results indicate that PPAR is an important determinant of proximal tubule cell size and is a likely mediator of compensatory proximal tubule hypertrophy.

systems biology↗

bioRxiv2022

Circadian Gene Expression in Mouse Renal Proximal Tubule

Circadian variability in kidney function has long been recognized but is often ignored as a potential confounding variable in in vivo physiological experiments. To provide a guide for physiological studies on the kidney proximal tubule, we have now created a data resource consisting of expression levels for all measurable mRNA transcripts in microdissected proximal tubule segments from mice as a function of the time of day. This approach employs small-sample RNA-sequencing (RNA-seq) applied to microdissected renal proximal tubules including both S1 proximal convoluted tubules (PCTs) and S2 proximal straight tubules (PSTs). The data were analyzed using JTK-Cycle to detect periodicity. The data are provided as a user-friendly web page at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox/. In PCTs, 234 transcripts were found to vary in a circadian manner (3.7 % of total quantified). In PSTs, 334 transcripts were found to vary in a circadian manner (5.3 % of total quantified). Transcripts previously known to be associated with corticosteroid action and transcripts associated with increased flow were found to be overrepresented among circadian transcripts peaking during the "dark" portion of the day (Zeitgeber 14-22), corresponding to the peak levels of corticosterone and glomerular filtration rate in mice. BlurbCircadian variation in gene expression can be an important determinant in the regulation of kidney function. The authors used RNA-seq in microdissected proximal S1 and S2 segments to identify transcripts that vary in a circadian manner. The data were used to construct a user-friendly web resource.

systems biology↗

bioRxiv2022

IFNA pathway drives the more aggressive phenotype of KRASG12D-mutant pancreatic ductal adenocarcinomas via IFNAR1/STAT3 activation

Activating mutations of KRAS play critical roles in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC). Accumulating evidence indicates that distinct KRAS alleles associate with different prognoses, but the underlying mechanisms are not known. We established isogenic KRAS mutants (KRASG12D, KRASG12V, and KRASWT) using a KRASG12R patient-derived PDAC cell line by CRISPR/Cas9 knock-in. We used these isogenic cell lines, a collection of characterized human PDAC patient-derived cell lines, and murine PDAC models to study the role of these KRAS alleles in vitro and in vivo. We verified that the growth of KRASG12D cells is more aggressive compared to KRASG12V isogenic cells in vitro and in vivo using orthotopic mouse models. Signal transducer and activator of transcription (STAT) activation was the most significant difference between KRASG12D and KRASG12V isogenic PDACs. Furthermore, activation of interferon-alpha (IFNA)/IFNA receptor (IFNAR)1/STAT3 signaling in the cancer cells mediated the more aggressive phenotype of KRASG12D PDACs. Conversely, inhibition of IFNAR1 in patient-derived PDAC cells suppressed tumor growth. Finally, IFNAR1 blockade was also effective in murine PDAC models and induced a significant increase in survival when combined with immune checkpoint blockade therapy. We conclude that the IFNA pathway and IFNAR1/STAT3 axis contribute to a more aggressive tumor progression in human KRASG12D PDACs and that IFNAR1 inhibition is a potential therapeutic target for overcoming resistance to immunotherapy in PDAC. One Sentence SummaryIFNA pathway drives the more aggressive phenotype of KRASG12D-mutant pancreatic ductal adenocarcinomas via IFNAR1/STAT3 activation.

cancer biology↗

bioRxiv2022

Inflammasome targeting with an NLRP3 agonist therapy is feasible but ineffective in murine hepatocellular carcinoma models with liver damage

Co-inhibition of programmed cell death receptor-1 (PD-1) and vascular endothelial growth factor receptor (VEGFR) pathway has shown efficacy in hepatocellular carcinoma (HCC). NLRP3 is a component of the inflammasome involved in the initiation, development, and progression of multiple cancers. We examined whether adding an NLRP3 agonist to dual PD-1/VEGFR inhibitors is feasible and can address treatment resistance in orthotopic HCC in mice with underlying liver damage. Mice with established tumors were treated with an NLRP3 agonist alone, combination of anti-VEGFR2 or the multikinase inhibitor regorafenib with anti-PD1 antibodies, or their combination. In all models tested, NLRP3 agonist therapy showed acceptable toxicity but no effect on tumor growth delay, disease morbidity, or survival. Pharmacodynamic analyses confirmed the effects of NLRP3 agonist therapy on inflammasome, evidenced by a significant elevation in plasma levels of pro-inflammatory cytokines such as IL-1{beta}. However, these changes were not detectable in tumor tissues, where we detected increased expression of immunosuppressive markers IL-6, KC/GRO, CCL9, and IL-18, and immune checkpoint molecules (PD1, PD-L1, and CTLA-4) after NLRP3 agonist therapy. Thus, modulation of the inflammasome with a novel NLRP3 agonist was feasible in mice with orthotopic HCC and liver damage but did not enhance efficacy when combined with anti-PD1/VEGFR therapies.

immunology↗

bioRxiv2020

Direct uptake mechanism in lysosome required for neuromuscular homeostasis

The degradation of cellular components plays an essential role in homeostasis. However, the known degradation pathways cannot account for the levels of proteolysis in cells. Here, we demonstrate that cytosolic proteins are imported into lysosomes in an ATP-dependent manner for degradation through a direct uptake mechanism distinct from any known pathway. SIDT2, a lysosomal membrane protein previously reported as an RNA transporter, translocates substrate proteins across the lysosomal membrane. Furthermore, we identify a dominant-negative mutation in SIDT2 that causes neuropathy and distal myopathy with rimmed vacuoles, a protein aggregation disease in humans. We generate Sidt2 knockout mice, recapitulating the characteristic features of this disease. Our results reveal a novel degradation pathway and illustrate its crucial role in cellular proteostasis, physiology, and pathophysiology. One Sentence SummaryDiscovery of a novel proteolytic pathway in cells, the dysfunction of which leads to protein aggregation disease in humans.

cell biology↗

bioRxiv2017

Rice blast resistance gene Pii is controlled by a pair of NBS-LRR genes Pii-1 and Pii-2

Nucleotide-binding, leucine-rich repeat receptors (NLRs) are conserved cytosolic receptors that recognize pathogen effectors and trigger immunity in plants. Recent studies indicate that NLRs function in pairs. Rice resistance gene Pii has been known to confer resistance against rice blast pathogen Magnaporthe oryzae carrying AVR-Pii. Previously we reported isolation of Pii gene from the rice cultivar Hitomebore (Takagi et al. 2013). To further understand rice components required for Pii-mediated resistance, we screened 5,600 mutant lines of Hitomebore cultivar and identified two mutants that lost Pii resistance without any changes in Pii gene sequence. Application of MutMap-Gap, the whole genome sequencing-based method of mutation identification, to the two mutants revealed that they have mutations in another NLR gene located close to Pii. The F1 plants derived from a cross of the two mutants showed pii phenotype, demonstrating that the newly identified NLR gene is indeed a component of Pii resistance. We thus designate the previously isolated Pii gene as Pii-1 and the newly isolated NLR gene as Pii-2.

genetics↗