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The interplay between apoptotic cancer cells and the tumor microenvironment modulates cancer progression and metastasis. Cancer-associated fibroblasts (CAFs) play a crucial role in promoting these events through paracrine communication. Here, we demonstrate that conditioned medium (CM) from lung CAFs exposed to apoptotic cancer cells suppresses TGF-β1-induced migration and invasion of cancer cells and CAFs. Direct exposure of CAFs to apoptotic 344SQ cells (ApoSQ) inhibited CAF migration and invasion and the expression of CAF activation markers. Enhanced secretion of Wnt‐induced signaling protein 1 (WISP-1) by CAFs exposed to ApoSQ was required for these antimigratory and anti-invasive effects. Pharmacological inhibition of Notch1 activation or siRNA-mediated Notch1 silencing prevented WISP-1 production by CAFs and reversed the antimigratory and anti-invasive effects. Enhanced expression of the Notch ligand delta-like protein 1 on the surface of ultraviolet-irradiated apoptotic lung cancer cells triggered Notch1-WISP-1 signaling. Phosphatidylserine receptor brain-specific angiogenesis inhibitor 1 (BAI1)-Rac1 signaling, which facilitated efferocytosis by CAFs, participated in crosstalk with Notch1 signaling for optimal production of WISP-1. In addition, a single injection of ApoSQ enhanced WISP-1 production, suppressed the expression of CAF activation markers in isolated Thy1+ CAFs, and inhibited lung metastasis in syngeneic immunocompetent mice via Notch1 signaling. Treatment with CM from CAFs exposed to ApoSQ suppressed tumor growth and lung metastasis, whereas treatment with WISP-1-immunodepleted CM from CAFs exposed to ApoSQ reversed the antitumorigenic and antimetastatic effects. Therefore, treatment with CM from CAFs exposed to apoptotic lung cancer cells could be therapeutically applied to suppress CAF activation, thereby preventing cancer progression and metastasis.

Lung-resident neutrophils need to be tightly regulated to avoid degranulation- and cytokine-associated damage to fragile alveolar structures that can lead to fatal outcomes. Here we show that lung neutrophils (LNs) express distinct surface proteins and genes that distinguish LNs from bone marrow and blood neutrophils. Functionally, LNs show impaired migratory activity toward chemoattractants and produce high levels of interleukin-6 (IL-6) at steady state and low levels of tumor necrosis factor-α in response to lipopolysaccharide (LPS) challenge. Treating bone marrow neutrophils with bronchoalveolar lavage fluid or prostaglandin E2 induces LN-associated characteristics, including the expression of transglutaminase 2 (Tgm2) and reduced production of inflammatory cytokines upon LPS challenge. Neutrophils from Tgm2−/− mice release high levels of inflammatory cytokines in response to LPS. Lung damage is significantly exacerbated in Tgm2−/− mice in an LPS-induced acute respiratory distress syndrome model. Collectively, we demonstrate that prostaglandin E2 is a key factor for the generation of LNs with unique immune suppressive characteristics, acting through protein kinase A and Tgm2, and LNs play essential roles in protection of the lungs against pathogenic inflammation.

To improve the poor survival rate of lung cancer patients, we investigated the role of HDGF‐related protein 3 (HRP‐3) as a potential biomarker for lung cancer. The expression of endogenous HRP‐3 in human lung cancer tissues and xenograft tumor models is indicative of its clinical relevance in lung cancer. Additionally, we demonstrated that HRP‐3 directly binds to the E2F1 promoter on chromatin. Interestingly, HRP‐3 depletion in A549 cells impedes the binding of HRP‐3 to the E2F1 promoter; this in turn hampers the interaction between Histone H3/H4 and HDAC1/2 on the E2F1 promoter, while concomitantly inducing Histone H3/H4 acetylation around the E2F1 promoter. The enhanced Histone H3/H4 acetylation on the E2F1 promoter through HRP‐3 depletion increases the transcription level of E2F1. Furthermore, the increased E2F1 transcription levels lead to the enhanced transcription of Cyclin E, known as the E2F1‐responsive gene, thus inducing S‐phase accumulation. Therefore, our study provides evidence for the utility of HRP‐3 as a biomarker for the prognosis and treatment of lung cancer. Furthermore, we delineated the capacity of HRP‐3 to regulate the E2F1 transcription level via histone deacetylation., HRP‐3 depletion–mediated S‐phase accumulation augments E2F1 transcription. HRP‐3 depletion induces H3/H4 acetylation and obstructs HRP‐3 binding to the E2F1 promoter. The reduction of HDAC 1/2 recruitment in the E2F1 promoter by HRP‐3 depletion increases H3/H4 acetylation in the E2F1 promoter.

Lysophosphatidic acid (LPA), an intercellular lipid mediator, is increased in the bronchoalveolar fluids of patients with asthma after allergen exposure. LPA administration exaggerates allergic responses, and the type 2 LPA receptor (LPA

Signal transducer and activator of transcription 6 (STAT6) promotes an anti-inflammatory process by inducing the development of M2 macrophages. We investigated whether modulating STAT6 activity in macrophages using AS1517499, the specific STAT6 inhibitor, affects the restoration of homeostasis after an inflammatory insult by regulating PPARγ expression and activity. Administration of AS1517499 suppressed the enhanced STAT6 phosphorylation and nuclear translocation observed in peritoneal macrophages after zymosan injection. In addition, AS1517499 delayed resolution of acute inflammation as evidenced by enhanced secretion of pro-inflammatory cytokines, reduced secretion of anti-inflammatory cytokines in PLF and supernatants from peritoneal macrophages, and exaggerated neutrophil numbers and total protein levels in PLF. We demonstrate temporal increases in annexin A1 (AnxA1) protein and mRNA levels in peritoneal lavage fluid (PLF), peritoneal macrophages, and spleen in a murine model of zymosan-induced acute peritonitis. In vitro priming of mouse bone marrow-derived macrophages (BMDM) and peritoneal macrophages with AnxA1 induced STAT6 activation with enhanced PPARγ expression and activity. Using AS1517499, we demonstrate that inhibition of STAT6 activation delayed recovery of PPARγ expression and activity, as well as impaired efferocytosis. Taken together, these results suggest that activation of the STAT6 signaling pathway mediates PPARγ expression and activation in macrophages to resolve acute inflammation.

Retinal prostheses use periodic repetition of electrical stimuli to form artificial vision. To enhance the reliability of evoked visual percepts, repeating stimuli need to evoke consistent spiking activity in individual retinal ganglion cells (RGCs). However, it is not well known whether outer retinal degeneration alters the consistency of RGC responses. Hence, here we systematically investigated the trial-to-trial variability in network-mediated responses as a function of the degeneration level. We patch-clamp recorded spikes in ON and OFF types of alpha RGCs from rd10 mice at four different postnatal days (P15, P19, P31, and P60), representing distinct stages of degeneration. To assess the consistency of responses, we analyzed variances in spike count and timing across repeats of the same stimulus delivered multiple times. We found the trial-to-trial variability of network-mediated responses increased considerably as the disease progressed. Compared to responses taken before degeneration onset, those of degenerate retinas showed up to ~70% higher variability (Fano Factor) in spike counts (p < 0.001) and ~95% lower correlation level in spike timing (p < 0.001). These results indicate consistency weakens significantly in electrically-evoked network-mediated responses and therefore raise concerns about the ability of microelectronic retinal implants to elicit consistent visual percepts at advanced stages of retinal degeneration.

Hydrogel with chemical modification has been used for 3D printing in the biomedical field of cell and tissue-based regeneration because it provides a good cellular microenvironment and mechanical supportive ability. As a scaffold and a matrix, hydrogel itself has to be modified chemically and physically to form a &beta, sheet crosslinking structure for the strength of the biomaterials. These chemical modifications could affect the biological damage done to encapsulated cells or surrounding tissues due to unreacted chemical residues. Biological assessment, including assessment of the cytocompatibility of hydrogel in clinical trials, must involve testing with cytotoxicity, irritation, and sensitization. Here, we modified silk fibroin and glycidyl methacrylate (Silk-GMA) and evaluated the physical characterizations, residual chemical detection, and the biological effect of residual GMA depending on dialysis periods. Silk-GMA depending on each dialysis period had a typical &beta, sheet structure in the characterization analysis and residual GMA decreased from dialysis day 1. Moreover, cell proliferation and viability rate gradually increased, additionally, necrotic and apoptotic cells decreased from dialysis day 2. These results indicate that the dialysis periods during chemical modification of natural polymer are important for removing unreacted chemical residues and for the potential application of the manufacturing standardization for chemically modified hydrogel for the clinical transplantation for tissue engineering and biomedical applications.

The adverse effects of radiation are proportional to the total dose and dose rate. We aimed to investigate the effects of radiation dose rate on different organs in mice. The mice were subjected to low dose rate (LDR, ~3.4 mGy/h) and high dose rate (HDR, ~51 Gy/h) radiation. LDR radiation caused severe tissue toxicity, as observed in the histological analysis of testis. It adversely influenced sperm production, including sperm count and motility, and induced greater sperm abnormalities. The expression of markers of early stage spermatogonial stem cells, such as Plzf, c-Kit, and Oct4, decreased significantly after LDR irradiation, compared to that following exposure of HDR radiation, in qPCR analysis. The compositional ratios of all stages of spermatogonia and meiotic cells, except round spermatid, were considerably reduced by LDR in FACS analysis. Therefore, LDR radiation caused more adverse testicular damage than that by HDR radiation, contrary to the response observed in other organs. Therefore, the dose rate of radiation may have differential effects, depending on the organ; it is necessary to evaluate the effect of radiation in terms of radiation dose, dose rate, organ type, and other conditions.

SAAving vitamin A–mediated immunity The vitamin A metabolite retinol is critical for B and T cell development and homing to the gut. Intestinal myeloid cells such as dendritic cells and macrophages take up retinol and process it into retinoic acid (RA), which in turn initiates RA-dependent gene expression programs in lymphocytes. Bang et al . identified LDL receptor-related protein 1 (LRP1) as a myeloid cell surface receptor for retinol. LRP1 binds retinol chaperoned by serum amyloid A (SAA) proteins, and SAA–retinol complexes are then endocytosed and metabolized by myeloid cells. Mice lacking either Saa or myeloid-specific Lrp1 exhibited profound impairments in vitamin A–mediated immunity. B and T cell trafficking to the intestine, immunoglobulin A production by B cells, and protection from enteric Salmonella Typhimurium infection were all diminished when either of these crucial players was missing. —STS

Adiponectin (encoded by Adipoq), a fat-derived hormone, alleviates risk factors associated with metabolic disorders. Although many transcription factors are known to control adiponectin expression, the mechanism underlying its fluctuation with regard to metabolic status remains unclear. Here, we show that ribosomal protein S6 kinase 1 (S6K1) controls adiponectin expression by inducing a transcriptional switch between two transcriptional machineries, BMAL1 and EZH2. Active S6K1 induced a suppressive histone code cascade, H2BS36p-EZH2-H3K27me3, leading to suppression of adiponectin expression. Moreover, active S6K1 phosphorylated BMAL1, an important transcription factor regulating the circadian clock system, at serine 42, which led to its dissociation from the Adipoq promoter region. This response resulted in EZH2 recruitment and subsequent H3K27me3 modification of the Adipoq promoter. Upon fasting, inactivation of S6K1 induced the opposite transcriptional switch, EZH2-to-BMAL1, promoting adiponectin expression. Consistently, S6K1-depleted mice exhibited lower H3K27me3 levels and elevated adiponectin expression. These findings identify a novel epigenetic switch system by which S6K1 controls the production of adiponectin, which displays beneficial effects on metabolism.

Significance Vitamin A is an essential nutrient for immune system development and function. After absorption from the diet, vitamin A is converted to retinol, which is delivered to cells and tissues by retinol-binding proteins. Serum amyloid A (SAA) proteins are retinol-binding proteins that transport retinol specifically during an infection. In this study, we illuminate the molecular details of how SAA proteins bind to retinol. We present the cocrystal structure of mouse SAA3 bound to retinol, which reveals that 3 molecules of SAA3 assemble to form a deep binding pocket that protects retinol, a fat-soluble molecule, from the aqueous environment. Our findings thus provide structural insight into how retinol is transported throughout the body during infection., Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization. In infected mice, SAA proteins circulate in association with the vitamin A derivative retinol, suggesting that SAAs transport retinol during infection. Here we illuminate a structural basis for the retinol–SAA interaction. In the bloodstream of infected mice, most SAA is complexed with high-density lipoprotein (HDL). However, we found that the majority of the circulating retinol was associated with the small fraction of SAA proteins that circulate without binding to HDL, thus identifying free SAA as the predominant retinol-binding form in vivo. We then determined the crystal structure of retinol-bound mouse SAA3 at a resolution of 2.2 Å. Retinol-bound SAA3 formed a novel asymmetric trimeric assembly that was generated by the hydrophobic packing of the conserved amphipathic helices α1 and α3. This hydrophobic packing created a retinol-binding pocket in the center of the trimer, which was confirmed by mutagenesis studies. Together, these findings illuminate the molecular basis for retinol transport by SAA proteins during infection.

Fms-like tyrosine kinase 3 (FLT3) is a type III kinase that is highly expressed in seminal plasma of infertile men. FLT3 activation can be blocked by inhibition of its phosphorylation using the nontoxic and selective inhibitor, quizartinib. We investigated the function of FLT3 and the corresponding effects of quizartinib in mouse spermatozoa. Spermatozoa were treated with different concentrations (0.1, 1, 10, 20, and 30 μM) of quizartinib for 90 min at 37 °C in 5% CO2 in air. FLT3 was detected in capacitated and non-capacitated spermatozoa. While the level of FLT3 was unaffected, the levels of phospho-FLT3 were significantly altered in spermatozoa by quizartinib. Exposure of spermatozoa to higher concentrations of quizartinib significantly altered sperm viability, motility, motion kinematics, levels of intracellular ATP, and capacitation status. Fertilization and early embryonic development were suppressed by quizartinib. This may have occurred as a consequence of decreased protein kinase A (PKA) activity and tyrosine phosphorylation. The inhibition of FLT3 by quizartinib may affect the fertilization and embryonic development by reducing tyrosine phosphorylation through a PKA-dependent pathway. Our data implicate FLT3 as a biomarker for diagnosis and prognosis of male fertility. In addition, quizartinib has potential for development as a new contraceptive agent.

Among various bioreactors used in the field of tissue engineering and regenerative medicine, a magnetic bioreactor is more capable of providing steady force to the cells while avoiding direct manipulation of the materials. However, most of them are complex and difficult to fabricate, with drawbacks in terms of consistency and biocompatibility. In this study, a magnetic bioreactor system and a magnetic hydrogel were manufactured by single-stage three-dimensional (3D) printing with digital light processing (DLP) technique for differentiation of myoblast cells. The hydrogel was composed of a magnetic part containing iron oxide and glycidyl-methacrylated silk fibroin, and a cellular part printed by adding mouse myoblast cell (C2C12) to gelatin glycidyl methacrylate, that was placed in the magnetic bioreactor system to stimulate the cells in the hydrogel. The composite hydrogel was steadily printed by a one-stage layering technique using a DLP printer. The magnetic bioreactor offered mechanical stretching of the cells in the hydrogel in 3D ways, so that the cellular differentiation could be executed in three dimensions just like the human environment. Cell viability, as well as gene expression using quantitative reverse transcription-polymerase chain reaction, were assessed after magneto-mechanical stimulation of the myoblast cell-embedded hydrogel in the magnetic bioreactor system. Comparison with the control group revealed that the magnetic bioreactor system accelerated differentiation of mouse myoblast cells in the hydrogel and increased myotube diameter and length in vitro. The DLP-printed magnetic bioreactor and the hydrogel were simply manufactured and easy-to-use, providing an efficient environment for applying noninvasive mechanical force via FDA-approved silk fibroin and iron oxide biocomposite hydrogel, to stimulate cells without any evidence of cytotoxicity, demonstrating the potential for application in muscle tissue engineering.

Alzheimer&rsquo, s disease (AD) is a progressive neurodegenerative disease. In this study, to investigate the effect of microglial elimination on AD progression, we administered PLX3397, a selective colony-stimulating factor 1 receptor inhibitor, to the mouse model of AD (5xFAD mice). Amyloid-beta (A&beta, ) deposition and amyloid precursor protein (APP), carboxyl-terminal fragment &beta, ionized calcium-binding adaptor molecule 1, synaptophysin, and postsynaptic density (PSD)-95 levels were evaluated in the cortex and hippocampus. In addition, the receptor density changes in dopamine D2 receptor (D2R) and metabotropic glutamate receptor 5 were evaluated using positron emission tomography (PET). D2R, tyrosine hydroxylase (TH), and dopamine transporter (DAT) levels were analyzed in the brains of Tg (5xFAD) mice using immunohistochemistry. PLX3397 administration significantly decreased A&beta, deposition following microglial depletion in the cortex and hippocampus of Tg mice. In the neuro-PET studies, the binding values for D2R in the Tg mice were lower than those in the wild type mice, however, after PLX3397 treatment, the binding dramatically increased. PLX3397 administration also reversed the changes in synaptophysin and PSD-95 expression in the brain. Furthermore, the D2R and TH expression in the brains of Tg mice was significantly lower than that in the wild type, however, after PLX3397 administration, the D2R and TH levels were significantly higher than those in untreated Tg mice. Thus, our findings show that administering PLX3397 to aged 5xFAD mice could prevent amyloid pathology, concomitant with the rescue of dopaminergic signaling, suggesting that targeting microglia may serve as a useful therapeutic option for neurodegenerative diseases, including AD.

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder and the leading cause of dementia, but therapeutic treatment options are limited. Taurine has been reported to have neuroprotective properties against dementia, including AD. The present study aimed to investigate the treatment effect of taurine in AD mice by functional molecular imaging. To elucidate glutamate alterations by taurine, taurine was administered to 5xFAD transgenic mice from 2 months of age, known to apear amyloid deposition. Then, we performed glutamate positron emission tomography (PET) imaging studies for three groups (wild-type, AD, and taurine-treated AD, n = 5 in each group). As a result, brain uptake in the taurine-treated AD group was 31–40% higher than that in the AD group (cortex: 40%, p p p p > 0.05) and 3–14% lower than that in the WT group (cortex: 10%, p > 0.05; striatum: 15%, p > 0.05; hippocampus: 14%, p > 0.05; thalamus: 3%, p > 0.05). However, we did not observe differences in Aβ pathology between the taurine-treated AD and AD groups in immunohistochemistry experiments. Our results reveal that although taurine treatment did not completely recover the glutamate system, it significantly increased metabolic glutamate receptor type 5 brain uptake. Therefore, taurine has therapeutic potential against AD.

Introduction Due to public concerns about deleterious biological consequences of radiofrequency electromagnetic fields (RF‐EMF), the potential effects of RF‐EMF on the central nervous system have received wide consideration. Methods Here, two groups of C57BL/6 mice, aged 2 and 12 months, were exposed to 1,950‐MHz RF‐EMF at a specific absorption rate of 5.0 W/kg for chronic periods (2 hr/day and 5 days/week for 8 months). Behavioral changes were then assessed in the mice at 10 months (sham‐ or RF‐10M) and 20 months (sham‐ or RF‐20M), on the open‐field test, the Y‐maze test, and an object recognition memory task, while biological effects were analyzed via microarray gene profiling of the hippocampus. Results Open‐field test results showed a decrease in the time duration spent at the center while there was a decrease in enhanced memory shown by the Y‐maze test and the novel object recognition test in the RF‐20M mice, compared to sham‐exposed mice, but no significant changes in the RF‐10M group. Based on a 2‐fold change cutoff, the microarray data revealed that 15 genes, which are listed as being involved in neurogenesis on Gene Ontology, were altered in both groups. Quantitative real‐time PCR for validation showed increased expression of Epha8 and Wnt6 in the hippocampi of RF‐20M group mice, although 13 additional genes showed no significant changes following RF‐EMF exposure. Conclusion Therefore, cognitive enhancement following chronic exposure for 8 months to RF‐EMF from middle age may be associated with increases in neurogenesis‐related signals in the hippocampus of C57BL/6 mice., Young‐ and middle‐aged C57BL/6 mice, aged 2 and 12 months, were exposed to 1,950‐MHz RF‐EMF at an absorption rate of 5.0 W/kg for 2 hr/day and 5 days/week for 8 months. We found an association between chronic RF‐EMF exposure‐induced behavioral improvement and hippocampal gene signature in middle‐aged mice.

Problem Maternal obesity induces elevated saturated fatty acid palmitate levels in the blood and causes pregnancy complications such as gestational diabetes, preeclampsia, fetal growth abnormalities, and stillbirth. Sestrin2, a highly conserved stress-inducible protein, is involved in the cellular responses of various stress conditions and homeostatic regulation. However, the effects of Sestrin2 on trophoblast cells have not yet been investigated. Here, we investigated the role of Sestrin2 in palmitate-induced lipotoxicity and its underlying mechanisms in human first-trimester trophoblast cells (Sw.71). Method of study Mouse placental tissues were obtained from low-fat diet-fed mice (n = 14) and high-fat diet-fed mice (n = 14) at gestation day 17.5. Sw.71 cells were treated with palmitate or bovine serum albumin as vehicle controls. The role of Sestrin2 in palmitate-induced lipotoxicity was examined by immunocytochemistry, immunoblot analysis, quantitative real-time PCR, and invasion assay. Results Expression of placental Sestrin2 was elevated in high-fat diet-fed dams compared to that of low-fat diet-fed dams. Prolonged treatment of Sw.71 cells with palmitate-induced endoplasmic reticulum (ER) stress-dependent expressions of Sestrin2 protein and mRNA, and the treatment also triggered apoptosis. Knockdown of Sestrin2 increased palmitate-mediated ER stress, inflammatory signaling, and apoptosis. Furthermore, Sestrin2 suppressed impaired trophoblast invasion caused by palmitate and attenuated palmitate-induced ER stress and inflammation via AMPK/mTORC1 pathways. Conclusion Our study provides the relationship between Sestrin2, AMPK/mTORC1 pathway, and trophoblast function, suggesting that Sestrin2 may be a novel potential therapeutic target for the prevention of pregnancy complications.

Chlorin e6 (Ce6), with its high phototoxic potential, has wide applications in photodynamic therapy (PDT) for many human diseases. However, poor cancer cell localization of Ce6 has limited its direct application for PDT. Here, we developed cancer-targeting peptide p 18-4/chlorin e6 (Ce6)-conjugated polyhedral oligomeric silsesquioxane (PPC) nanoparticles for improving the targeting ability of Ce6 to breast cancer cells, thereby enhancing PDT efficacy. The synthesized PPC nanoparticles exhibited a spherical shape with an average diameter of 127.2 ± 11.3 nm in aqueous solution. Compared with free Ce6, the immobilization of p 18-4 enhanced the in vitro cellular uptake and targeting ability of PPC nanoparticles in breast cancer cell line MDA-MB-231. In addition, the intracellular uptake of PPC nanoparticles in MDA-MB-231 cells was dramatically increased compared with other cancer cells, indicating an obvious targeting ability of PPC nanoparticles on breast cancer cells. Upon light irradiation, PPC nanoparticles revealed significantly improved phototoxicity to MDA-MB-231 cells, mainly due to apoptotic cell death. In vivo PDT study suggested that PPC nanoparticles exhibited increased retention in tumor tissues and effectively inhibited the growth of MDA-MB-231 tumors in a target-specific manner. Overall, these results indicate that PPC nanoparticles are highly effective PDT agents for breast cancer therapy.

Given the increased public concern about the deleterious biological consequences of radiofrequency electromagnetic fields (RF-EMFs), the involvement of RF-EMFs in neurodegenerative diseases, especially Alzheimer’s disease (AD), has received increased consideration. To investigate the effect of long-term RF-EMF exposure on AD progression, we exposed 5xFAD mice to 1950 MHz RF-EMF at a specific absorption rate of 5.0 W/kg for 2 h/day and 5 days/week for 8 months. Behavioral changes were assessed by an open field test and an object recognition memory task after RF exposure was terminated. In addition, cerebral glucose metabolism was analyzed in the brains of the 5xFAD mice using 18F-deoxyglucose positron emission tomography. The hyperactivity-like and anxiolytic behaviors of the 5xFAD mice in open field tests were rescued by RF exposure. Furthermore, long-term RF-EMF exposure improved the cognitive deficits of 5xFAD mice that were observed in the object recognition memory test. Consistent with the behavioral changes, glucose metabolism in the hippocampus and amygdala regions of the brains of 5xFAD mice following RF exposure was significantly increased compared to glucose metabolism in the brains of sham-exposed mice. These data suggest that long-term exposure to RF-EMF might exert beneficial effects on AD in 5xFAD mice.

Background/Aims: The signal transducer and activator of transcription 6 (STAT6) transcription factor mediates PPARγ-regulated gene expression in macrophages. However, it remains largely unknown how proximal membrane signaling events initiated by apoptotic cell recognition upregulate PPARγ expression and activate the lung homeostatic program. Methods: The STAT6 inhibitor AS1517499 was used to determine the role of STAT6 in mediating PPARγ activity, anti-inflammatory effects, and anti-fibrotic effects induced by apoptotic cell instillation after bleomycin treatment into C57BL/6 mice. Bronchoalveolar lavage fluid, alveolar macrophages and lungs were harvested at days 2, 7, and 14 and then analyzed by real-time PCR, immunoblotting, ELISA, immunocytochemistry and immunohistochemistry assays. Results: Our data demonstrate that apoptotic cell instillation after bleomycin results in prolonged enhancement of STAT6 phosphorylation in alveolar macrophages and lung. Co-administration of the STAT6 inhibitor, AS1517499, reversed the enhanced PPARγ expression and activity induced by apoptotic cell instillation after bleomycin treatment. By reducing the expression of PPARγ target genes, including CD36, macrophage mannose receptor, and arginase 1, AS1517499 inhibited efferocytosis and restored pro-inflammatory cytokine expression, neutrophil recruitment, protein levels, hydroxyproline content, and expression of fibrosis markers, including type 1 collagen α2, fibronectin, and α-smooth muscle actin. STAT6 inhibition reversed the expression profile of hepatocyte growth factor and interleukin-10. Conclusion: These results indicate that prolonged STAT6 activation following one-time apoptotic cell instillation facilitates continuous PPARγ activation, resulting in the resolution of bleomycin-induced lung inflammation and fibrosis.

Liver damage upon exposure to ionizing radiation, whether accidental or because of therapy can contribute to liver dysfunction. Currently, radiation therapy is used for various cancers including hepatocellular carcinoma, however, the treatment dose is limited by poor liver tolerance to radiation. Furthermore, reliable biomarkers to predict liver damage and associated side-effects are unavailable. Here, we investigated fibrinogen-like 1 (FGL1)-expression in the liver and plasma after radiation exposure. We found that 30 Gy of liver irradiation (IR) induced cell death including apoptosis, necrosis, and autophagy, with fibrotic changes in the liver occurring during the acute and subacute phase in mice. Moreover, FGL1 expression pattern in the liver following IR was associated with liver damage represented by injury-related proteins and oxidative stress markers. We confirmed the association between FGL1 expression and hepatocellular injury by exposing human hepatocytes to radiation. To determine its suitability, as a potential biomarker for radiation-induced liver injury, we measured FGL1 in the liver tissue and the plasma of mice following total body irradiation (TBI) or liver IR. In TBI, FGL1 showed the highest elevation in the liver compared to other major internal organs including the heart, lung, kidney, and intestine. Notably, plasma FGL1 showed good correlation with radiation dose by liver IR. Our data revealed that FGL1 upregulation indicates hepatocellular injury in response to IR. These results suggest that plasma FGL1 may represent a potential biomarker for acute and subacute radiation exposure to the liver.

This study aims to investigate the genetic and epigenetic mechanisms involved in the pathogenesis of subacute stage of spinal cord injury (SCI). Gene-expression datasets associated with SCI were downloaded from the Gene Expression Omnibus (GEO) database, and differential expression analyses were performed in order to identify differentially expressed genes (DEGs). Multiple network types were constructed and analyzed, including protein-protein-interaction (PPI) network, miRNA-target network, lncRNA-associated competing endogenous RNA (ceRNA) network, and miRNA-transcription factor (TF)-target network. Cluster analyses were performed to identify significant modules. To verify the prediction accuracy of the in-silico identified molecules, qRT-PCR experiments were conducted. The results depicted the Ywhae gene as the hub gene with the highest degree in the PPI network. The ceRNA network identified specific genes (Flna, ID3, and HK2), miRNAs (miR-16-5p, miR-1958, and miR-185-5p), and lncRNAs (Neat1, Xist, and Malat1) as playing critical regulating roles in the pathological mechanisms of SCI. The miRNA-TF-gene interaction network identified four important TFs (Sp1, Trp53, Jun, and Rela). The miRNA-gene-TF interaction loops from the significant modules indicated that miR-325-3p can interact with the Asah1 gene and TF-Sp1 by forming a closed loop. The qRT-PCR experiments verified four selected genes (Flna, ID3, HK2, and Ywhae) and two selected TFs (Jun, and Sp1) as significantly up-regulated following SCI. The results indicated that four genes (Flna, ID3, HK2, and Ywhae), four transcription factors (Sp1, Trp53, Jun, and RelA), two miRNAs (miR-16-5p and miR-325-3p), and three lncRNAs (Neat1, Xist, and Malat1) are likely to be involved in the molecular mechanisms underlying the subacute stage of SCI. These findings uncover putative pathogenic mechanisms involved in SCI and might bear translation significance for future research towards therapeutic development.

The epithelial-mesenchymal transition (EMT) is important in organ fibrosis. We hypothesized that growth arrest-specific protein 6 (Gas6) and its underlying mechanisms play roles in the prevention of EMT in alveolar epithelial cells (ECs). In this study, to determine whether Gas6 prevents TGF-&beta, 1-induced EMT in LA-4 and primary alveolar type II ECs, real-time PCR and immunoblotting in cell lysates and ELISA in culture supernatants were performed. Migration and invasion assays were performed using Transwell chambers. Pretreatment of ECs with Gas6 inhibited TGF-&beta, 1-induced EMT based on cell morphology, changes in EMT marker expression, and induction of EMT-activating transcription factors. Gas6 enhanced the levels of cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) and PGD2 as well as of their receptors. COX-2 inhibitors and antagonists of PGE2 and PGD2 receptors reversed the inhibition of TGF-&beta, 1-induced EMT, migration, and invasion by Gas6. Moreover, knockdown of Axl or Mer reversed the enhancement of PGE2 and PGD2 and suppression of EMT, migration and invasion by Gas6. Our data suggest Gas6-Axl or -Mer signalling events may reprogram ECs to resist EMT via the production of PGE2, PGD2, and their receptors.

Increasing antibiotic resistance has led to the development of new strategies to combat bacterial infection. Anti-virulence strategies that impair virulence of bacterial pathogens are one of the novel approaches with less selective pressure for developing resistance than traditional strategies that impede viability. In this study, a small molecule CM14 [N-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)-3-phenylprop-2-ynamide] that inhibits the activity of HlyU, a transcriptional regulator essential for the virulence of the fulminating human pathogen Vibrio vulnificus, has been identified. Without affecting bacterial growth or triggering the host cell death, CM14 reduces HlyU-dependent expression of virulence genes in V. vulnificus. In addition to the decreased hemolysis of human erythrocytes, CM14 impedes host cell rounding and lysis caused by V. vulnificus. Notably, CM14 significantly enhances survival of mice infected with V. vulnificus by alleviating hepatic and renal dysfunction and systemic inflammation. Biochemical, mass spectrometric, and mutational analyses revealed that CM14 inhibits HlyU from binding to target DNA by covalently modifying Cys30. Remarkably, CM14 decreases the expression of various virulence genes of other Vibrio species and thus attenuates their virulence phenotypes. Together, this molecule could be an anti-virulence agent against HlyU-harboring Vibrio species with a low selective pressure for the emergence of resistance.

Significance Vitamin A is a nutrient that is essential for the development of intestinal immunity. It is absorbed by gut epithelial cells, which convert it to retinol and retinoic acid. Here we show that the transcription factor retinoic acid receptor β (RARβ) allows epithelial cells to sense vitamin A in the diet and regulate vitamin A-dependent immunity in the intestine. We find that epithelial RARβ regulates several intestinal immune responses, including production of the immunomodulatory protein serum amyloid A, T-cell homing to the intestine, and B-cell production of immunoglobulin A. Our findings provide insight into how epithelial cells sense vitamin A to regulate intestinal immunity, and highlight why vitamin A is so important for immunity to infection., Vitamin A is a dietary component that is essential for the development of intestinal immunity. Vitamin A is absorbed and converted to its bioactive derivatives retinol and retinoic acid by the intestinal epithelium, yet little is known about how epithelial cells regulate vitamin A-dependent intestinal immunity. Here we show that epithelial cell expression of the transcription factor retinoic acid receptor β (RARβ) is essential for vitamin A-dependent intestinal immunity. Epithelial RARβ activated vitamin A-dependent expression of serum amyloid A (SAA) proteins by binding directly to Saa promoters. In accordance with the known role of SAAs in regulating Th17 cell effector function, epithelial RARβ promoted IL-17 production by intestinal Th17 cells. More broadly, epithelial RARβ was required for the development of key vitamin A-dependent adaptive immune responses, including CD4+ T-cell homing to the intestine and the development of IgA-producing intestinal B cells. Our findings provide insight into how the intestinal epithelium senses dietary vitamin A status to regulate adaptive immunity, and highlight the role of epithelial cells in regulating intestinal immunity in response to diet.

The increased use of mobile phones has generated public concern about the impact of radiofrequency electromagnetic fields (RF-EMF) on health. In the present study, we investigated whether RF-EMFs induce molecular changes in amyloid precursor protein (APP) processing and amyloid beta (Aβ)-related memory impairment in the 5xFAD mouse, which is a widely used amyloid animal model. The 5xFAD mice at the age of 1.5 months were assigned to two groups (RF-EMF- and sham-exposed groups, eight mice per group). The RF-EMF group was placed in a reverberation chamber and exposed to 1950 MHz electromagnetic fields for 3 months (SAR 5 W/kg, 2 h/day, 5 days/week). The Y-maze, Morris water maze, and novel object recognition memory test were used to evaluate spatial and non-spatial memory following 3-month RF-EMF exposure. Furthermore, Aβ deposition and APP and carboxyl-terminal fragment β (CTFβ) levels were evaluated in the hippocampus and cortex of 5xFAD mice, and plasma levels of Aβ peptides were also investigated. In behavioral tests, mice that were exposed to RF-EMF for 3 months did not exhibit differences in spatial and non-spatial memory compared to the sham-exposed group, and no apparent change was evident in locomotor activity. Consistent with behavioral data, RF-EMF did not alter APP and CTFβ levels or Aβ deposition in the brains of the 5xFAD mice. These findings indicate that 3-month RF-EMF exposure did not affect Aβ-related memory impairment or Aβ accumulation in the 5xFAD Alzheimer's disease model. Bioelectromagnetics. 37:391-399, 2016. © 2016 The Authors Bioelectromagnetics published by Wiley Periodicals, Inc. on behalf of Bioelectromagnetics Society.

S6K1 has been implicated in a number of key metabolic responses, which contribute to obesity. Critical among these is the control of a transcriptional program required for the commitment of mesenchymal stem cells to the adipocytic lineage. However, in contrast to its role in the cytosol, the functions and targets of nuclear S6K1 are unknown. Here, we show that adipogenic stimuli trigger nuclear translocation of S6K1, leading to H2BS36 phosphorylation and recruitment of EZH2 to H3, which mediates H3K27 trimethylation. This blocks Wnt gene expression, inducing the upregulation of PPARγ and Cebpa and driving increased adipogenesis. Consistent with this finding, white adipose tissue from S6K1-deficient mice exhibits no detectable H2BS36 phosphorylation or H3K27 trimethylation, whereas both responses are highly elevated in obese humans or in mice fed a high-fat diet. These findings define an S6K1-dependent mechanism in early adipogenesis, contributing to the promotion of obesity.

Ethnopharmacological relevance Salvia miltiorrhiza is a traditional oriental medicine widely used for preventing and treating disorders of the liver, menstrual, and blood circulation systems. Osteoporosis, loss of bone with age and/or estrogen deficiency, is an important causal factor of fracture. S. miltiorrhiza extract has been used to alleviate dysmenorrhea and painful osteoarthritis. Aim of the study This study was performed to investigate the anti-osteoporosis activity of the Salvia miltiorrhiza ethanol extract (SME) in osteoporosis-prone conditions: ovariectomized (OVX) and naturally menopaused (NM) ICR mice. Materials and methods Anti-osteoporotic potentials of SME (50–200 mg/kg) were evaluated based on bone mineral density using microCT analysis, biochemical parameters, and changes in the gene expressions involved in bone resorption. Results SME ameliorated the loss of trabecular bone both in OVX and NM mice. SME was effective in correcting aberrant levels of RANKL, osteocalcin, and BALP, which are critically involved in bone resorption. In addition, SME suppressed the expression of TRAF6 and NFATc1, which play a role in osteoclast differentiation. Conclusions SME suppressed the loss of trabecular bone via suppressing bone resorption and osteoclast differentiation both in OVX and NM mice. SME is likely to be developed as a therapeutic agent for osteoporosis.

Apoptotic cell clearance by phagocytes is essential in tissue homeostasis. We demonstrated that conditioned medium (CM) from macrophages exposed to apoptotic cancer cells inhibits the TGFβ1-induced epithelial–mesenchymal transition (EMT), migration, and invasion of cancer cells. Apoptotic 344SQ (ApoSQ) cell-induced PPARγ activity in macrophages increased the levels of PTEN, which was secreted in exosomes. Exosomal PTEN was taken up by recipient lung cancer cells. ApoSQ-exposed CM from PTEN knockdown cells failed to enhance PTEN in 344SQ cells, restore cellular polarity, or exert anti-EMT and anti-invasive effects. The CM that was deficient in PPARγ ligands, including 15-HETE, lipoxin A4, and 15d-PGJ2, could not reverse the suppression of PPARγ activity or the PTEN increase in 344SQ cells and consequently failed to prevent the EMT process. Moreover, a single injection of ApoSQ cells inhibited lung metastasis in syngeneic immunocompetent mice with enhanced PPARγ/PTEN signaling both in tumor-associated macrophages and in tumor cells. PPARγ antagonist GW9662 reversed the signaling by PPARγ/PTEN; the reduction in EMT-activating transcription factors, such as Snai1 and Zeb1; and the antimetastatic effect of the ApoSQ injection. Thus, the injection of apoptotic lung cancer cells may offer a new strategy for the prevention of lung metastasis.

We tested whether GV1001 has any ototoxic side effects at different doses and whether it protects hearing in an aminoglycoside-induced ototoxicity mouse model.GV1001, a novel peptide vaccine currently being examined in a Phase 3 clinical trial to treat pancreatic cancer, also has anti-inflammatory and antioxidant effects.In the first experiment, C57/BL6 mice were injected with GV1001 preparations at concentrations of 0.1 to 100 mg/kg for 7 days to evaluate the toxicity of GV1001 on the inner ear and kidneys. In the second experiment, the protective effect of GV1001 was tested in an ototoxicity mouse model that was generated by injecting 800 mg/kg kanamycin (KM) for 2 weeks. The hearing threshold and hair cell loss were compared between the KM + GV1001 group (treated with 10 mg/kg GV1001 for 2 wk) and the KM + saline group. The hearing threshold was measured before, and 7, 14, and 21 days after the initial treatment. The blood urea nitrogen level was measured.No ototoxicity or renal toxicity was found following treatment with different doses of GV1001 (0.1-100 mg/kg). The KM + saline group showed impaired auditory function and markedly disoriented and missing cochlear hair cells, while the KM + GV1001 group showed significant hearing and hair cell preservation in comparison (p 0.05).GV1001 itself did not have any detrimental effects on the inner ear or kidney. In the KM induced ototoxicity model, concomitant administration of GV1001 protected against cochlear hair cell damage and preserve hearing.

Previously, we demonstrated that growth arrest-specific protein 6 (Gas6)/Axl or Mer signaling inhibited the transforming growth factor (TGF)-&beta, 1-induced epithelial&ndash, mesenchymal transition (EMT) in lung epithelial cells. Hepatocyte growth factor (HGF) has also been shown to inhibit TGF-&beta, 1-induced changes in EMT markers. Here, we examined whether Gas6 signaling can induce the production of HGF and c-Met in lung alveolar epithelial cells to mediate the inhibition of EMT and to inhibit the migration and invasion of epithelial cells. The inhibition of the RhoA/Rho kinase pathway, using either a RhoA-targeted small interfering RNA (siRNA) or the Rho kinase pharmacologic inhibitor Y27362, prevented the inhibition of TGF-&beta, 1-induced EMT in LA-4 cells and primary alveolar type II (AT II) epithelial cells. The c-Met antagonist PHA-665752 also blocked the anti-EMT effects associated with Gas6. Moreover, treatment with Y27362 or PHA-665752 prevented the Gas6-mediated inhibition of TGF-&beta, 1-induced migration and invasion. Our data provided evidence that the RhoA-dependent production of HGF and c-Met mediated the Gas6-induced inhibition of EMT, migration and invasion in lung alveolar epithelial cells. Thus, Gas6/Axl and Mer/RhoA signaling may be necessary for the maintenance of homeostasis in the alveolar epithelium, via HGF and c-Met.

Skeletal muscle cell differentiation requires a family of proteins called myogenic regulatory factors (MRFs) to which MyoD belongs. The activity of MyoD is under epigenetic regulation, however, the molecular mechanism by which histone KMTs and KDMs regulate MyoD transcriptional activity through methylation remains to be determined. Here we provide evidence for a unique regulatory mechanism of MyoD transcriptional activity through demethylation by Jmjd2C demethylase whose level increases during muscle differentiation. G9a decreases MyoD stability via methylation-dependent MyoD ubiquitination. Jmjd2C directly associates with MyoD in vitro and in vivo to demethylate and stabilize MyoD. The hypo-methylated MyoD due to Jmjd2C is significantly more stable than hyper-methylated MyoD by G9a. Cul4/Ddb1/Dcaf1 pathway is essential for the G9a-mediated MyoD degradation in myoblasts. By the stabilization of MyoD, Jmjd2C increases myogenic conversion of mouse embryonic fibroblasts and MyoD transcriptional activity with erasing repressive H3K9me3 level at the promoter of MyoD target genes. Collectively, Jmjd2C increases MyoD transcriptional activity to facilitate skeletal muscle differentiation by increasing MyoD stability through inhibiting G9a-dependent MyoD degradation.

The ubiquitin-proteasome system (UPS) plays an important role in protein quality control, cellular signalings, and cell differentiation through the regulated turnover of key transcription factors in cardiac tissue. However, the molecular mechanism underlying Fbxo25-mediated ubiquitination of cardiac transcription factors remains elusive. We report that an Fbxo25-mediated SCF ubiquitination pathway regulates the protein levels and activities of Tbx5 and Nkx2-5 based on our studies using MG132, proteasome inhibitor, and the temperature sensitive ubiquitin system in ts20 cells. Our data indicate that Fbxo25 directly interacts with Tbx5 and Nkx2-5 in vitro and in vivo. In support of our findings, a dominant-negative mutant of Fbxo25, Fbxo251-236, prevents Tbx5 degradation and increases Tbx5 transcriptional activity in a Tbx5 responsive luciferase assay. Therefore, Fbxo25 facilitates Tbx5 degradation in an SCF-dependent manner. In addition, the silencing of endogenous Fbxo25 increases Tbx5 and Nkx2-5 mRNA levels and suppresses mESC-derived cardiomyocyte differentiation. Likewise, the exogenous expression of FBXO25 downregulates NKX2-5 level in human ESC-derived cardiomyocytes. In myocardial infarction model, Fbxo25 mRNA decreases, whereas the mRNA and protein levels of Tbx5 and Nkx2-5 increase. The protein levels of Tbx5 and Nkx2-5 are regulated negatively by Fbxo25-mediated SCF ubiquitination pathway. Thus, our findings reveal a novel mechanism for regulation of SCFFbox25-dependent Nkx2-5 and Tbx5 ubiquitination in cardiac development and provide a new insight into the regulatory mechanism of Nkx2-5 and Tbx5 transcriptional activity.

BACKGROUND Streptococcus pneumoniae is the most common cause of community-acquired pneumonia worldwide. During pneumococcal pneumonia, the human airway epithelium is exposed to large amounts of H2O2 as a product of host and pathogen oxidative metabolism. Airway cells are known to be highly vulnerable to oxidant damage, but the pathophysiology of oxidative stress induced by S. pneumoniae and the role of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant systems of the host are not well characterized. METHODS For gluthation/gluthathion disulfide analysis BEAS-2B cells, primary broncho-epithelial cells (pBEC), explanted human lung tissue and mouse lungs were infected with different S. pneumoniae strains (D39, A66, R6x, H2O2/pneumolysin/LytA- deficient mutants of R6x). Cell death was proven by LDH assay and cell viability by IL-8 ELISA. The translocation of Nrf2 and the expression of catalase were shown via Western blot. The binding of Nrf2 at the catalase promoter was analyzed by ChIP. RESULTS We observed a significant induction of oxidative stress induced by S. pneumoniae in vivo, ex vivo, and in vitro. Upon stimulation, the oxidant-responsive transcription factor Nrf2 was activated, and catalase was upregulated via Nrf2. The pneumococci-induced oxidative stress was independent of S. pneumoniae-derived H2O2 and pneumolysin but depended on the pneumococcal autolysin LytA. The Nrf2 inducer resveratrol, as opposed to catalase, reversed oxidative stress in lung epithelial cells. CONCLUSIONS These observations indicate a H2O2-independent induction of oxidative stress in lung epithelial cells via the release of bacterial factors of S. pneumoniae. Resveratrol might be an option for prevention of acute lung injury and inflammatory responses observed in pneumococcal pneumonia.

Radiation-induced intestinal toxicity is common among cancer patients after radiotherapy. Endothelial cell dysfunction is believed to be a critical contributor to radiation tissue injury in the intestine. Geranylgeranylacetone (GGA) has been used to treat peptic ulcers and gastritis. However, the protective capacity of GGA against radiation-induced intestinal injury has not been addressed. Therefore, we investigated whether GGA affects intestinal damage in mice and vascular endothelial cell damage in vitro. GGA treatment significantly ameliorated intestinal injury, as evident by intestinal crypt survival, villi length and the subsequently prolonged survival time of irradiated mice. In addition, intestinal microvessels were also significantly preserved in GGA-treated mice. To clarify the effect of GGA on endothelial cell survival, we examined endothelial function by evaluating cell proliferation, tube formation, wound healing, invasion and migration in the presence or absence of GGA after irradiation. Our findings showed that GGA plays a role in maintaining vascular cell function; however, it does not protect against radiation-induced vascular cell death. GGA promoted endothelial function during radiation injury by preventing the loss of VEGF/VEGFR1/eNOS signaling and by down-regulating TNFα expression in endothelial cells. This finding indicates the potential impact of GGA as a therapeutic agent in mitigating radiation-induced intestinal damage.

The signal transducer and activator of transcription 6 (STAT6) transcription factor activates peroxisome proliferator-activated receptor gamma (PPAR-γ)-regulated gene expression in immune cells. We investigated proximal membrane signaling that was initiated in macrophages after exposure to apoptotic cells that led to enhanced PPAR-γ expression and activity, using specific siRNAs for ABCA1, STAT6, and PPAR-γ, or their antagonists. The interactions between mouse bone marrow-derived macrophages or RAW 264.7 cells and apoptotic Jurkat cells, but not viable cells, resulted in the induction of STAT6 phosphorylation as well as PPAR-γ expression and activation. Knockdown of ATP-binding cassette transporter A1 (ABCA1) after the transfection of macrophages with ABCA1-specific siRNAs reduced apoptotic cell-induced STAT6 phosphorylation as well as PPAR-γ mRNA and protein expression. ABCA1 knockdown also reduced apoptotic cell-induced liver X receptor α (LXR-α) mRNA and protein expression. Moreover, inhibition of STAT6 with specific siRNAs or the pharmacological inhibitor AS1517499AS reversed the induction of PPAR-γ, LXR-α, and ABCA1 by apoptotic Jurkat cells. PPAR-γ-specific siRNAs or the PPAR-γ antagonist GW9662 inhibited apoptotic cell-induced increases in LXR-α and ABCA1 mRNA and protein levels. Thus, these results indicate that apoptotic cells trigger the ABCA1/STAT6 pathway, leading to the activation of the PPAR-γ/LXR-α/ABCA1 pathway in macrophages.

Promoting energy metabolism is known to provide therapeutic effects for obesity and associated metabolic disorders. The present study evaluated the therapeutic effects of the newly identified bouchardatine (Bou) on obesity-associated metabolic disorders and the molecular mechanisms of these effects.The molecular mode of action of Bou for its effects on lipid metabolism was first examined in 3T3-L1 adipocytes and HepG2 cells. This was followed by an evaluation of its metabolic effects in mice fed a high-fat diet for 16 weeks with Bou being administered in the last 5 weeks. Further mechanistic investigations were conducted in pertinent organs of the mice and relevant cell models.In 3T3-L1 adipocytes, Bou reduced lipid content and increased sirtuin 1 (SIRT1) activity to facilitate liver kinase B1 (LKB1) activation of AMPK. Chronic administration of Bou (50 mg∙kgBou may have therapeutic potential for obesity-related metabolic diseases by increasing the capacity of energy expenditure in adipose tissues and liver through a mechanism involving the SIRT1-LKB1-AMPK axis.

The alveolar bone process is the thickened ridge of bone that bears the teeth and is known to have dynamic functional interactions with surrounding tissues. However, the detailed morphological changes that occur during alveolar bone process development and the underlying molecular mechanisms behind this morphogenesis have not been elucidated. In this study, we examined the detailed morphological changes of the alveolar bone process during mouse development using HE and MTC staining. In addition, we evaluated the precise localization pattern of various signaling molecules involved in blood vessel formation including CD31, α-SMA, VEGF, periostin, and TGF-β. Innervation of the alveolar bone process was examined following injection of the nerve terminal dye AM1-43. The morphological and immunohistochemical data suggested that there is an intimate relationship between alveolar bone process development and blood vessel formation. To more closely examine the role of blood vessels in alveolar bone process formation, we microinjected mice with a clinically available anti-VEGF antibody, bevacizumab, at PN5 and analyzed the effects 5 days later. Compared to the control animals, anti-VEGF treated animals showed a disruption of the integration of bony tissues to form the alveolar bone process structures, which should contain the periodontal ligaments. Based on these data, we conclude that specific morphogenesis of the alveolar bone process is closely associated with blood vessel formation.

Growth arrest-specific protein 6 (Gas6)/Mer receptor tyrosine kinase (Mer) signaling modulates cytokine secretion and helps to regulate the immune response and apoptotic cell clearance. Signaling pathways that activate an epithelial growth program in macrophages are still poorly defined. We report that Gas6/Mer/RhoA signaling can induce the production of epithelial growth factor hepatic growth factor (HGF) in macrophages, which ultimately promotes epithelial cell proliferation and wound repair. The RhoA/protein kinase B (Akt)/mitogen-activated protein (MAP) kinases, including p38 MAP kinase, extracellular signal-regulated protein kinase, and Jun NH2-terminal kinase axis in RAW 264.7 cells, was identified as Gas6/Mer downstream signaling pathway for the upregulation of HGF mRNA and protein. Conditioned medium from RAW 264.7 cells that had been exposed to Gas6 or apoptotic cells enhanced epithelial cell proliferation of the epithelial cell line LA-4 and wound closure. Cotreatment with an HGF receptor-blocking antibody or c-Met antagonist downregulated this enhancement. Inhibition of Mer with small interfering RNA (siRNA) or the RhoA/Rho kinase pathway by RhoA siRNA or Rho kinase pharmacologic inhibitor suppressed Gas6-induced HGF mRNA and protein expression in macrophages and blocked epithelial cell proliferation and wound closure induced by the conditioned medium. Our data provide evidence that macrophages can be reprogrammed by Gas6 to promote epithelial proliferation and wound repair via HGF, which is induced by the Mer/RhoA/Akt/MAP kinase pathway. Thus, defects in Gas6/Mer/RhoA signaling in macrophages may delay tissue repair after injury to the alveolar epithelium.

Teraspanin transmembrane protein, Perp (P53 apoptosis effector related to PMP22), which is found in the plasma membrane as a component of the desmosome, is reported to be involved in the morphogenesis of the epithelium and the enamel formation of the incisor. However, its expression pattern and signaling regulation during molar development have not been elucidated in detail. We have examined the precise expression patterns of Perp in developing lower molars and employed the knock-down of Perp by antisense oligodeoxynucleotide treatment during in vitro organ cultivation at embryonic day 13 to define the precise developmental function of Perp. Perp was expressed mainly in the dental lamina and stellate reticulum regions at the bud and cap stages. After Perp knock-down, the tooth germ showed disruption of the dental lamina and stellate reticulum with altered apoptosis and proliferation. The changed expression levels of related signaling molecules from the enamel knot and desmosome were evaluated by real-time quantitative polymerase chain reaction. A renal capsule transplantation method was employed to examine the effects of Perp knock-down on molar crown development. Ultrastructural observations revealed that enamel was deposited more densely in an irregular pattern in the cusp region, and that dentin was hypo-mineralized after Perp knock-down at the cap stage. Thus, Perp might play important roles in the formation and integration of stellate reticulum, dental lamina structure and enamel formation through signaling interactions with the enamel knot and desmosome-related signaling molecules at the cap stage of lower molar development.

Recognition of apoptotic cells by macrophages is crucial for resolution of inflammation, immune tolerance, and tissue repair. Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) and hepatocyte growth factor (HGF) play important roles in the tissue repair process. We investigated the characteristics of macrophage COX-2 and PGE2expression mediated by apoptotic cells and then determined how macrophages exposed to apoptotic cellsin vitroandin vivoorchestrate the interaction between COX-2/PGE2and HGF signaling pathways. Exposure of RAW 264.7 cells and primary peritoneal macrophages to apoptotic cells resulted in induction of COX-2 and PGE2. The COX-2 inhibitor NS-398 suppressed apoptotic cell-induced PGE2production. Both NS-398 and COX-2-siRNA, as well as the PGE2receptor EP2 antagonist, blocked HGF expression in response to apoptotic cells. In addition, the HGF receptor antagonist suppressed increases in COX-2 and PGE2induction. Thein vivorelevance of the interaction between the COX-2/PGE2and HGF pathways through a positive feedback loop was shown in cultured alveolar macrophages followingin vivoexposure of bleomycin-stimulated lungs to apoptotic cells. Our results demonstrate that upregulation of the COX-2/PGE2and HGF in macrophages following exposure to apoptotic cells represents a mechanism for mediating the anti-inflammatory and antifibrotic consequences of apoptotic cell recognition.

Summary Mouse embryonic stem cells (ESCs) are maintained in serum with leukemia inhibitory factor (LIF) to maintain self-renewal and pluripotency. Recently, a 2i culture method was reported using a combination of MEK inhibition (MEKi) and GSK3 inhibition (GSK3i) with LIF to maintain ESCs in a naive ground state. How 2i maintains a ground state of ESCs remains elusive. Here we show that MEKi and GSK3i maintain the ESC ground state by downregulating global DNA methylation through two distinct mechanisms. MEK1 phosphorylates JMJD2C for ubiquitin-mediated protein degradation. Therefore, MEKi increased JMJD2C protein levels but decreased DNMT3 expression. JMJD2C promotes TET1 activity to increase 5-hydroxymethylcytosine (5hmC) levels. GSK3i suppressed DNMT3 expression, thereby decreasing DNA methylation without affecting 5hmC levels. Furthermore, 2i increased PRDM14 expression to inhibit DNMT3A/B protein expression by promoting G9a-mediated DNMT3A/B protein degradation. Collectively, 2i allows ESCs to maintain a naive ground state through JMJD2C-dependent TET1 activation and PRDM14/G9a-mediated DNMT3A/B protein degradation., Graphical Abstract, Highlights • MEKi increases JMJD2C protein levels and decreases DNMT3 expression in ESCs • JMJD2C promotes TET1 hydroxylase activity to increase global 5hmC levels • GSK3i decreases global DNA methylation without affecting 5hmC levels • 2i-induced PRDM14 expression promotes G9a-mediated DNMT3A/B protein degradation, In this article, Kim and colleagues show that MEKi and GSK3i maintain the ESC ground state by downregulating global DNA methylation through two distinct mechanisms. MEKi increased JMJD2C protein levels, thereby enhancing TET1 activity, whereas GSK3i decreased DNMT3 family expression. Therefore, they uncover that DNA demethylation pathways under 2i conditions are governed by the JMJD2C/TET1 and PRDM14/G9a/DNMT3 complexes.

Mer receptor tyrosine kinase (Mer) signaling plays a central role in the intrinsic inhibition of the inflammatory response to Toll-like receptor activation. Previously, we found that lung Mer protein expression decreased after lipopolysaccharide (LPS) treatment due to enhanced Mer cleavage. The purpose of the present study was to examine whether pharmacologically restored membrane-bound Mer expression upregulates the Mer signaling pathways and suppresses lung inflammatory responses. Pretreatment with the ADAM17 (a disintegrin and metalloproteinase-17) inhibitor TAPI-0 (tumor necrosis factor alpha protease inhibitor-0) reduced LPS-induced production of soluble Mer protein in bronchoalveolar lavage (BAL) fluid, restored membrane-bound Mer expression, and increased Mer activation in alveolar macrophages and lungs after LPS treatment. TAPI-0 also enhanced Mer downstream signaling, including phosphorylation of protein kinase b, focal adhesion kinase, and signal transducer and activator of transcription 1. As expected from enhanced Mer signaling, TAPI-0 also augmented suppressor of cytokine signaling-1 and -3 mRNA and protein levels and inhibited nuclear factor κB activation at 4 and 24 hours after LPS treatment. TAPI-0 suppressed LPS-induced inflammatory cell accumulation, total protein level elevation in BAL fluid, and production of inflammatory mediators, including tumor necrosis factor-α, interleukin-1β, and macrophage inflammatory protein-2. Additionally, the effects of TAPI-0 on the activation of Mer signaling and the production of inflammatory responses could be reversed by cotreatment with specific Mer-neutralizing antibody. Restored Mer protein expression by treatment with TAPI-0 efficiently prevents the inflammatory cascade during acute lung injury.

Hypoxia augments inflammatory responses and osteoclastogenesis by incompletely understood mechanisms. We identified COMMD1 as a cell-intrinsic negative regulator of osteoclastogenesis that is suppressed by hypoxia. In human macrophages, COMMD1 restrained induction of NF-κB signaling and a transcription factor E2F1-dependent metabolic pathway by the cytokine RANKL. Downregulation of COMMD1 protein expression by hypoxia augmented RANKL-induced expression of inflammatory and E2F1 target genes and downstream osteoclastogenesis. E2F1 targets included glycolysis and metabolic genes including CKB that enabled cells to meet metabolic demands in challenging environments, as well as inflammatory cytokine-driven target genes. Expression quantitative trait locus analysis linked increased COMMD1 expression with decreased bone erosion in rheumatoid arthritis. Myeloid deletion of Commd1 resulted in increased osteoclastogenesis in arthritis and inflammatory osteolysis models. These results identify COMMD1 and an E2F-metabolic pathway as key regulators of osteoclastogenic responses under pathological inflammatory conditions, and provide a mechanism by which hypoxia augments inflammation and bone destruction.