Ninjurin1 deficiency aggravates colitis development by promoting M1 macrophage polarization and inducing microbial imbalance.

Disruption of colonic homeostasis caused by aberrant M1/M2 macrophage polarization and dysbiosis contributes to inflammatory bowel disease (IBD) pathogenesis. However, the molecular factors mediating colonic homeostasis are not well characterized. Here, we found that Ninjurin1 (Ninj1) limits colon inflammation by regulating macrophage polarization and microbiota composition under homeostatic conditions and during colitis development. Ninj1 deletion in mice induced hypersusceptibility to colitis, with increased prevalence of colitogenic Prevotellaceae strains and decreased immunoregulatory Lachnospiraceae strains. Upon co-housing (CoH) with WT mice, Ninj1-/- mice showed increased Lachnospiraceae and decreased Prevotellaceae abundance, with subsequent improvement of colitis. Under homeostatic conditions, M1 macrophage frequency was higher in the Ninj1-/- mouse colons than wild-type (WT) mouse colons, which may contribute to increased basal colonic inflammation and microbial imbalance. Following colitis induction, Ninj1 expression was increased in macrophages; meanwhile Ninj1-/- mice showed severe colitis development and impaired recovery, associated with decreased M2 macrophages and escalated microbial imbalance. In vitro, Ninj1 knockdown in mouse and human macrophages activated M1 polarization and restricted M2 polarization. Finally, the transfer of WT macrophages ameliorated severe colitis in Ninj1-/- mice. These findings suggest that Ninj1 mediates colonic homeostasis by modulating M1/M2 macrophage balance and preventing extensive dysbiosis, with implications for IBD prevention and therapy.

Neuropilin 1 balances ß8 integrin-activated TGFß signaling to control sprouting angiogenesis in the brain.

Angiogenesis in the developing central nervous system (CNS) is regulated by neuroepithelial cells, although the genes and pathways that couple these cells to blood vessels remain largely uncharacterized. Here, we have used biochemical, cell biological and molecular genetic approaches to demonstrate that ß8 integrin (Itgb8) and neuropilin 1 (Nrp1) cooperatively promote CNS angiogenesis by mediating adhesion and signaling events between neuroepithelial cells and vascular endothelial cells. ß8 integrin in the neuroepithelium promotes the activation of extracellular matrix (ECM)-bound latent transforming growth factor ß (TGFß) ligands and stimulates TGFß receptor signaling in endothelial cells. Nrp1 in endothelial cells suppresses TGFß activation and signaling by forming intercellular protein complexes with ß8 integrin. Cell type-specific ablation of ß8 integrin, Nrp1, or canonical TGFß receptors results in pathological angiogenesis caused by defective neuroepithelial cell-endothelial cell adhesion and imbalances in canonical TGFß signaling. Collectively, these data identify a paracrine signaling pathway that links the neuroepithelium to blood vessels and precisely balances TGFß signaling during cerebral angiogenesis.

Ninjurin1 deficiency attenuates susceptibility of experimental autoimmune encephalomyelitis in mice.

Ninjurin1 is a homotypic adhesion molecule that contributes to leukocyte trafficking in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, in vivo gene deficiency animal studies have not yet been done. Here, we constructed Ninjurin1 knock-out (KO) mice and investigated the role of Ninjurin1 on leukocyte trafficking under inflammation conditions such as EAE and endotoxin-induced uveitis. Ninjurin1 KO mice attenuated EAE susceptibility by reducing leukocyte recruitment into the injury regions of the spinal cord and showed less adhesion of leukocytes on inflamed retinal vessels in endotoxin-induced uveitis mice. Moreover, the administration of a custom-made antibody (Ab26-37) targeting the Ninjurin1 binding domain ameliorated the EAE symptoms, showing the contribution of its adhesion activity to leukocyte trafficking. In addition, we addressed the transendothelial migration (TEM) activity of bone marrow-derived macrophages and Raw264.7 cells according to the expression level of Ninjurin1. TEM activity was decreased in Ninjurin1 KO bone marrow-derived macrophages and siNinj1 Raw264.7 cells. Consistent with this, GFP-tagged mNinj1-overexpressing Raw264.7 cells increased their TEM activity. Taken together, we have clarified the contribution of Ninjurin1 to leukocyte trafficking in vivo and delineated its direct functions to TEM, emphasizing Ninjurin1 as a beneficial therapeutic target against inflammatory diseases such as multiple sclerosis.

Ninjurin1 enhances the basal motility and transendothelial migration of immune cells by inducing protrusive membrane dynamics.

Ninjurin1 is involved in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, by mediating leukocyte extravasation, a process that depends on homotypic binding. However, the precise regulatory mechanisms of Ninjurin1 during inflammation are largely undefined. We therefore examined the pro-migratory function of Ninjurin1 and its regulatory mechanisms in macrophages. Interestingly, Ninjurin1-deficient bone marrow-derived macrophages exhibited reduced membrane protrusion formation and dynamics, resulting in the impairment of cell motility. Furthermore, exogenous Ninjurin1 was distributed at the membrane of filopodial structures in Raw264.7 macrophage cells. In Raw264.7 cells, RNA interference of Ninjurin1 reduced the number of filopodial projections, whereas overexpression of Ninjurin1 facilitated their formation and thus promoted cell motility. Ninjurin1-induced filopodial protrusion formation required the activation of Rac1. In Raw264.7 cells penetrating an MBEC4 endothelial cell monolayer, Ninjurin1 was localized to the membrane of protrusions and promoted their formation, suggesting that Ninjurin1-induced protrusive activity contributed to transendothelial migration. Taking these data together, we conclude that Ninjurin1 enhances macrophage motility and consequent extravasation of immune cells through the regulation of protrusive membrane dynamics. We expect these findings to provide insight into the understanding of immune responses mediated by Ninjurin1.

The astrocyte-expressed integrin αvß8 governs blood vessel sprouting in the developing retina.

The mouse retina is vascularized after birth when angiogenic blood vessels grow and sprout along a pre-formed latticework of astrocytes. How astrocyte-derived cues control patterns of blood vessel growth and sprouting, however, remains enigmatic. Here, we have used molecular genetic strategies in mice to demonstrate that αvß8 integrin expressed in astrocytes is essential for neovascularization of the developing retina. Selective ablation of αv or ß8 integrin gene expression in astrocytes leads to impaired blood vessel sprouting and intraretinal hemorrhage, particularly during formation of the secondary vascular plexus. These pathologies correlate, in part, with diminished αvß8 integrin-mediated activation of extracellular matrix-bound latent transforming growth factor ßs (TGFßs) and defective TGFß signaling in vascular endothelial cells, but not astrocytes. Collectively, our data demonstrate that αvß8 integrin is a component of a paracrine signaling axis that links astrocytes to blood vessels and is essential for proper regulation of retinal angiogenesis.

Meteorin promotes the formation of GFAP-positive glia via activation of the Jak-STAT3 pathway.

Meteorin is an orphan ligand which has been previously reported to control neuritogenesis and angiogenesis, as well as gliogenesis. However, the precise function of this factor in CNS development and the underlying molecular mechanisms are poorly understood. Here, we demonstrate that meteorin is involved in GFAP-positive glial differentiation through activation of the Jak-STAT3 pathway, by using neurosphere and retinal explant culture systems. During embryonic brain development, meteorin is highly expressed in neural stem and radial glia cells of the ventricular zone and immature neurons outside the ventricular zone but its expression disappears spontaneously as development proceeds except in GFAP-positive astrocytes. In cultured neurospheres, meteorin activates STAT3, and in turn increases the transcriptional activity of GFAP by enhancing the binding of STAT3 to the promoter. By meteorin stimulation, differentiating neurospheres show increased numbers of GFAP-positive cells, but the effect is abrogated by a blockade of the Jak-STAT3 pathway using either a Jak inhibitor or STAT3 siRNA. Furthermore, we expand our findings to the retina, and show that meteorin increases GFAP expression in Müller glia. Together, our results suggest that meteorin promotes GFAP-positive glia formation by mediating the Jak-STAT3 signaling pathway during both cortical stem cell differentiation and retinal glia development.

The N-terminal ectodomain of Ninjurin1 liberated by MMP9 has chemotactic activity.

Ninjurin1 is known as an adhesion molecule promoting leukocyte trafficking under inflammatory conditions. However, the posttranslational modifications of Ninjurin1 are poorly understood. Herein, we defined the proteolytic cleavage of Ninjurin1 and its functions. HEK293T cells overexpressing the C- or N-terminus tagging mouse Ninjurin1 plasmid produced additional cleaved forms of Ninjurin1 in the lysates or conditioned media (CM). Two custom-made anti-Ninjurin1 antibodies, Ab(1-15) or Ab(139-152), specific to the N- or C-terminal regions of Ninjurin1 revealed the presence of its shedding fragments in the mouse liver and kidney lysates. Furthermore, Matrix Metalloproteinase (MMP) 9 was responsible for Ninjurin1 cleavage between Leu(56) and Leu(57). Interestingly, the soluble N-terminal Ninjurin1 fragment has structural similarity with well-known chemokines. Indeed, the CM from HEK293T cells overexpressing the GFP-mNinj1 plasmid was able to attract Raw264.7 cells in trans-well assay. Collectively, we suggest that the N-terminal ectodomain of mouse Ninjurin1, which may act as a chemoattractant, is cleaved by MMP9.

[Effects of uncertainty and spousal support on infertility-related quality of life in women undergoing assisted reproductive technologies]. / ë³´ì¡°ìƒì‹ìˆ ì„ 받는 여성의 불확실성과 배우자 지지가 난임 ê´€ë ¨ 삶의 질에 미치는 영향.

Purpose: The purpose of this study was to investigate the effects of uncertainty and spousal support on infertility-related quality of life (QoL) in women undergoing assisted reproductive technologies. Methods: In this correlational survey study, 172 infertile women undergoing assisted reproductive technologies for infertility treatment at M hospital in Seoul participated. Data collection took place at the outpatient department of M hospital using a self-report questionnaire from July to August 2019. Data were analyzed using SPSS for Windows version 28.0. Results: The mean scores for uncertainty, spousal support, and infertility-related quality of life (QoL) were 28.35 (out of 50), 86.67 (out of 115), and 57.98 (out of 100), respectively. Infertility-related quality of life (QoL) was positively correlated with spousal support and negatively correlated with uncertainty. According to the regression analysis, infertility-related quality of life (QoL) was significantly affected by uncertainty, total number of assisted reproductive technology treatments, marriage duration, subjective health status, the financial burden of infertility testing, and the presence of a burdensome person. These variables had an explanatory power of 35.0% for infertility-related quality of life (QoL). Conclusion: Uncertainty was an important factor influencing infertility-related quality of life (QoL) among women undergoing assisted reproductive technologies. It is necessary to develop and implement a nursing intervention program focused on reducing various forms of uncertainty during assisted reproductive procedures and to consider other factors affecting infertility-related quality of life (QoL) in the clinical setting.

Ninjurin1 positively regulates osteoclast development by enhancing the survival of prefusion osteoclasts.

Osteoclasts (OCs) are bone-resorbing cells that originate from hematopoietic stem cells and develop through the fusion of mononuclear myeloid precursors. Dysregulation of OC development causes bone disorders such as osteopetrosis, osteoporosis, and rheumatoid arthritis. Although the molecular mechanisms underlying osteoclastogenesis have been well established, the means by which OCs maintain their survival during OC development remain unknown. We found that Ninjurin1 (Ninj1) expression is dynamically regulated during osteoclastogenesis and that Ninj1-/- mice exhibit increased trabecular bone volume owing to impaired OC development. Ninj1 deficiency did not alter OC differentiation, transmigration, fusion, or actin ring formation but increased Caspase-9-dependent intrinsic apoptosis in prefusion OCs (preOCs). Overexpression of Ninj1 enhanced the survival of mouse macrophage/preOC RAW264.7 cells in osteoclastogenic culture, suggesting that Ninj1 is important for the survival of preOCs. Finally, analysis of publicly available microarray data sets revealed a potent correlation between high NINJ1 expression and destructive bone disorders in humans. Our data indicate that Ninj1 plays an important role in bone homeostasis by enhancing the survival of preOCs.

Altered AKAP12 expression in portal fibroblasts and liver sinusoids mediates transition from hepatic fibrogenesis to fibrosis resolution.

Liver fibrosis can be reversed by removing its causative injuries; however, the molecular mechanisms mediating the resolution of liver fibrogenesis are poorly understood. We investigate the role of a scaffold protein, A-Kinase Anchoring Protein 12 (AKAP12), during liver fibrosis onset, and resolution. Biliary fibrogenesis and fibrosis resolution was induced in wild-type (WT) or AKAP12-deficient C57BL/6 mice through different feeding regimens with 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing chow. AKAP12 expression in portal fibroblasts (PFs) and liver sinusoidal endothelial cells (LSECs) gradually decreased as fibrosis progressed but was restored after cessation of the fibrotic challenge. Histological analysis of human liver specimens with varying degrees of fibrosis of different etiologies revealed that AKAP12 expression diminishes in hepatic fibrosis from its early stages onward. AKAP12 KO mice displayed reduced fibrosis resolution in a DDC-induced biliary fibrosis model, which was accompanied by impaired normalization of myofibroblasts and capillarized sinusoids. RNA sequencing of the liver transcriptome revealed that genes related to ECM accumulation and vascular remodeling were mostly elevated in AKAP12 KO samples. Gene ontology (GO) and bioinformatic pathway analyses identified that the differentially expressed genes were significantly enriched in GO categories and pathways, such as the adenosine 3',5'-cyclic monophosphate (cAMP) pathway. Knockdown of the AKAP12 gene in cultured primary PFs revealed that AKAP12 inhibited PF activation in association with the adenosine 3',5'-cyclic monophosphate (cAMP) pathway. Moreover, AKAP12 knockdown in LSECs led to enhanced angiogenesis, endothelin-1 expression and alterations in laminin composition. Collectively, this study demonstrates that AKAP12-mediated regulation of PFs and LSECs has a central role in resolving hepatic fibrosis.

A-kinase anchoring protein 12 is downregulated in human hepatocellular carcinoma and its deficiency in mice aggravates thioacetamide-induced liver injury.

AKAP12 belongs to A-kinase anchoring protein (AKAP) family of scaffold proteins and is known as a tumor suppressor in several human cancer types. Its role as a tumor suppressor in hepatocellular carcinoma (HCC) was proposed due to its downregulation and epigenetic modification in human HCC; however, the effect of its deficiency on liver injuries, such as liver fibrosis and cancer has been poorly studied. By analyzing tumor and non-tumor tissues of 15 patients with HCC, it was confirmed that AKAP12 expression was downregulated in human HCC as compared with adjacent non-tumor tissues. Immunohistochemical staining of mouse liver tissue for AKAP12 revealed that its sinusoidal expression was diminished in capillarized endothelium after 8 weeks of thioacetamide (TAA) administration. AKAP12 deficiency resulted in the promotion of ductular response of biliary epithelial cells, whereas overall fibrosis and myofibroblast activation were comparable between genotypes after short-term TAA treatment. The mRNA expressions of some fibrosis-related genes such as those encoding epithelial cell adhesion molecule, collagen type 1 α1 and elastin were upregulated in liver tissues of AKAP12-knockout mice. Long-term administration of TAA for 26 weeks led to the development of liver tumors; the incidence of tumor development was higher in AKAP12-deficient mice than in wild-type littermates. Together, these results suggest that AKAP12 functions as a tumor suppressor in liver cancer and is associated with the regulation of hepatic non-parenchymal cells.

Claudin5a is required for proper inflation of Kupffer's vesicle lumen and organ laterality.

Left-right asymmetric organ development is critical to establish a proper body plan of vertebrates. In zebrafish, the Kupffer's vesicle (KV) is a fluid-filled sac which controls asymmetric organ development, and a properly inflated KV lumen by means of fluid influx is a prerequisite for the asymmetric signal transmission. However, little is known about the components that support the paracellular tightness between the KV luminal epithelial cells to sustain hydrostatic pressure during KV lumen expansion. Here, we identified that the claudin5a (cldn5a) is highly expressed at the apical surface of KV epithelial cells and tightly seals the KV lumen. Downregulation of cldn5a in zebrafish showed a failure in organ laterality that resulted from malformed KV. In addition, accelerated fluid influx into KV by combined treatment of forskolin and 3-isobutyl-1-methylxanthine failed to expand the partially-formed KV lumen in cldn5a morphants. However, malformed KV lumen and defective heart laterality in cldn5a morphants were significantly rescued by exogenous cldn5a mRNA, suggesting that the tightness between the luminal epithelial cells is important for KV lumen formation. Taken together, these findings suggest that cldn5a is required for KV lumen inflation and left-right asymmetric organ development.

Hsp70 acetylation prevents caspase-dependent/independent apoptosis and autophagic cell death in cancer cells.

Cancer cells are continuously challenged by adverse environmental factors including hypoxia, metabolite restriction, and immune reactions, and must adopt diverse strategies to survive. Heat shock protein (Hsp) 70 plays a central role in protection against stress-induced cell death by maintaining protein homeostasis and interfering with the process of programmed cell death. Recent findings have suggested that Hsp70 acetylation is a key regulatory modification required for its chaperone activity, but its relevance in the process of programmed cell death and the underlying mechanisms involved are not well understood. In this study, we sought to investigate mechanisms mediated by Hsp70 acetylation in relation to apoptotic and autophagic programmed cell death. Upon stress-induced apoptosis, Hsp70 acetylation inhibits apoptotic cell death, mediated by Hsp70 association with apoptotic protease-activating factor (Apaf)-1 and apoptosis-inducing factor (AIF), key modulators of caspase-dependent and -independent apoptotic pathways, respectively. Hsp70 acetylation also attenuated autophagic cell death associated with upregulation of autophagy-related genes and autophagosome induction. Collectively, these results suggest that the acetylation of Hsp70 plays key regulatory roles in cell death pathways as well as in its function as a chaperone, together enabling cellular protection in response to stress.

Structural environment built by AKAP12+ colon mesenchymal cells drives M2 macrophages during inflammation recovery.

Macrophages exhibit phenotypic plasticity, as they have the ability to switch their functional phenotypes during inflammation and recovery. Simultaneously, the mechanical environment actively changes. However, how these dynamic alterations affect the macrophage phenotype is unknown. Here, we observed that the extracellular matrix (ECM) constructed by AKAP12+ colon mesenchymal cells (CMCs) generated M2 macrophages by regulating their shape during recovery. Notably, rounded macrophages were present in the linear and loose ECM of inflamed colons and polarized to the M1 phenotype. In contrast, ramified macrophages emerged in the contracted ECM of recovering colons and mainly expressed M2 macrophage markers. These contracted structures were not observed in the inflamed colons of AKAP12 knockout (KO) mice. Consequently, the proportion of M2 macrophages in inflamed colons was lower in AKAP12 KO mice than in WT mice. In addition, clinical symptoms and histological damage were more severe in AKAP12 KO mice than in WT mice. In experimentally remodeled collagen gels, WT CMCs drove the formation of a more compacted structure than AKAP12 KO CMCs, which promoted the polarization of macrophages toward an M2 phenotype. These results demonstrated that tissue contraction during recovery provides macrophages with the physical cues that drive M2 polarization.

ARD1-mediated aurora kinase A acetylation promotes cell proliferation and migration.

Aurora kinase A (AuA) is a prerequisite for centrosome maturation, separation, and mitotic spindle assembly, thus, it is essential for cell cycle regulation. Overexpression of AuA is implicated in poor prognosis of many types of cancer. However, the regulatory mechanisms underlying the functions of AuA are still not fully understood. Here, we report that AuA colocalizes with arrest defective protein 1 (ARD1) acetyltransferase during cell division and cell migration. Additionally, AuA is acetylated by ARD1 at lysine residues at positions 75 and 125. The double mutations at K75/K125 abolished the kinase activity of AuA. Moreover, the double mutant AuA exhibited diminished ability to promote cell proliferation and cell migration. Mechanistic studies revealed that AuA acetylation at K75/K125 promoted cell proliferation via activation of cyclin E/CDK2 and cyclin B1. In addition, AuA acetylation stimulated cell migration by activating the p38/AKT/MMP-2 pathway. Our findings indicate that ARD1-mediated acetylation of AuA enhances cell proliferation and migration, and probably contributes to cancer development.

Disruption of Ninjurin1 Leads to Repetitive and Anxiety-Like Behaviors in Mice.

Over the last few decades, molecular neurobiology has uncovered many genes whose deficiency in mice results in behavioral traits associated with human neuropsychiatric disorders such as autism, obsessive-compulsive disorder (OCD), and schizophrenia. However, the etiology of these common diseases remains enigmatic with the potential involvement of a battery of genes. Here, we report abnormal behavioral phenotypes of mice deficient in a cell adhesion molecule Ninjurin 1 (Ninj1), which are relevant to repetitive and anxiety behaviors of neuropsychiatric disorders. Ninj1 knockout (KO) mice exhibit compulsive grooming-induced hair loss and self-made lesions as well as increased anxiety-like behaviors. Histological analysis reveals that Ninj1 is predominantly expressed in cortico-thalamic circuits, and neuron-specific Ninj1 conditional KO mice manifest aberrant phenotypes similar to the global Ninj1 KO mice. Notably, the brains of Ninj1 KO mice display altered synaptic transmission in thalamic neurons as well as a reduced number of functional synapses. Moreover, the disruption of Ninj1 leads to glutamatergic abnormalities, including increased ionotropic glutamate receptors but reduced glutamate levels. Furthermore, chronic treatment with fluoxetine, a drug reportedly ameliorates compulsive behaviors in mice, prevents progression of hair loss and alleviates the compulsive grooming and anxiety-like behavior of Ninj1 KO mice. Collectively, our results suggest that Ninj1 could be involved in neuropsychiatric disorders associated with impairments of repetitive and anxiety behaviors.

SAMHD1 acetylation enhances its deoxynucleotide triphosphohydrolase activity and promotes cancer cell proliferation.

SAM domain and HD domain containing protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase (dNTPase) that inhibits retroviruses by depleting intracellular deoxynucleotide triphosphates (dNTPs) in non-cycling myeloid cells. Although SAMHD1 is expressed ubiquitously throughout the human body, the molecular mechanisms regulating its enzymatic activity and function in non-immune cells are relatively unexplored. Here, we demonstrate that the dNTPase activity of SAMHD1 is regulated by acetylation, which promotes cell cycle progression in cancer cells. SAMHD1 is acetylated at residue lysine 405 (K405) in vitro and in vivo by an acetylatransferase, arrest defective protein 1 (ARD1). Acetylated SAMHD1 wildtype proteins have enhanced dNTPase activity in vitro, whereas non-acetylated arginine substituted mutants (K405R) do not. K405R mutant expressing cancer cells have reduced G1/S transition and slower proliferation compared to wildtype. SAMHD1 acetylation levels are strongest during the G1 phase, indicating a role during G1 phase. Collectively, these findings suggest that SAMHD1 acetylation enhances its dNTPase activity and promotes cancer cell proliferation. Therefore, SAMHD1 acetylation may be a potent therapeutic target for cancer treatment.

AMP-activated protein kinase activator, HL156A reduces thioacetamide-induced liver fibrosis in mice and inhibits the activation of cultured hepatic stellate cells and macrophages.

Cirrhosis, the end-stage of hepatic fibrosis, is not only life-threatening by itself, but also a causative factor of liver cancer. Despite efforts to develop treatment for liver fibrosis, there are no approved agents as anti-fibrotic drugs to date. In the present study, we aimed to investigate the anti-fibrotic effect of the AMP-activated protein kinase (AMPK) activator, HL156A. A mouse model of thioacetamide (TAA)-induced liver fibrosis was used to examine the effect of HL156A in vivo. Mice received either TAA alone or a combination of TAA and HL156A intraperitoneally for a total duration of 6 weeks. Including HL156A during exposure to TAA significantly reduced extracellular matrix (ECM) deposition and production of the hepatic transforming growth factor-ß1 (TGF-ß1). Immunohistochemical analysis revealed that the activation of hepatic stellate cells and the capillarization of liver sinusoids were also diminished significantly by HL156A co-treatment. The anti-fibrotic effect of HL156A was further studied in vitro by using a rat hepatic stellate cell line, HSC-T6 cells. The induction of α-smooth muscle actin (α-SMA) by TGF-ß1 treatment was reversed by HL156A, which was likely via the activation of AMPK. Moreover, HL156A showed anti-inflammatory effects on macrophages. Treatment with HL156A diminished LPS-induced activation of both Raw264.7 macrophage cells and primary cultured mouse macrophages. Taken together, these results imply that the AMPK activator HL156A inhibits hepatic fibrosis via multiple mechanisms and could be a potentially effective agent for fibrosis treatment.

Ninjurin1 regulates lipopolysaccharide-induced inflammation through direct binding.

Ninjurin1 is a transmembrane protein involved in macrophage migration and adhesion during inflammation. It was recently reported that repression of Ninjurin1 attenuated the lipopolysaccharide (LPS)-induced inflammatory response in macrophages; however, the precise mechanism by which Ninjurin1 modulates LPS-induced inflammation remains poorly understood. In the present study, we found that the interaction between Ninjurin1 and LPS contributed to the LPS-induced inflammatory response. Notably, pull-down assays using lysates from HEK293T cells transfected with human or mouse Ninjurin1 and biotinylated LPS (LPS-biotin) showed that LPS directly bound Ninjurin1. Subsequently, LPS binding assays with various truncated forms of Ninjurin1 protein revealed that amino acids (aa) 81-100 of Ninjurin1 were required for LPS binding. In addition, knockdown experiments using Ninj1 siRNA resulted in decreased nitric oxide (NO) and tumor necrosis factor-α (TNFα) secretion upon LPS treatment in Raw264.7 cells. Collectively, our results suggest that Ninjurin1 regulates the LPS-induced inflammatory response through its direct binding to LPS, thus, identifying Ninjurin1 as a putative target for the treatment of inflammatory diseases, such as sepsis and inflammation-associated carcinogenesis.

ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation.

Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress.