The histone methyltransferase ASH1L protects against bone loss by inhibiting osteoclastogenesis.

Absent, small, or homeotic1-like (ASH1L) is a histone lysine methyltransferase that generally functions as a transcriptional activator in controlling cell fate. So far, its physiological relevance in bone homeostasis and osteoclast differentiation remains elusive. Here, by conditional deleting Ash1l in osteoclast progenitors of mice, we found ASH1L deficiency resulted in osteoporosis and potentiation of osteoclastogenesis in vivo and in vitro. Mechanistically, ASH1L binds the promoter of the Src homology 3 and cysteine-rich domain 2 (Stac2) and increases the gene's transcription via histone 3 lysine 4 (H3K4) trimethylation modification, thus augmenting the STAC2's protection against receptor activator of nuclear factor kB ligand (RANKL)-initiated inflammation during osteoclast formation. Collectively, we demonstrate the first piece of evidence to prove ASH1L as a critical checkpoint during osteoclastogenesis. The work sheds new light on our understanding about the biological function of ASH1L in bone homeostasis, therefore providing a valuable therapeutic target for the treatment of osteoporosis or inflammatory bone diseases.

WSB1/2 target chromatin-bound lysine-methylated RelA for proteasomal degradation and NF-κB termination.

Proteasome-mediated degradation of chromatin-bound NF-κB is critical in terminating the transcription of pro-inflammatory genes and can be triggered by Set9-mediated lysine methylation of the RelA subunit. However, the E3 ligase targeting methylated RelA remains unknown. Here, we find that two structurally similar substrate-recognizing components of Cullin-RING E3 ligases, WSB1 and WSB2, can recognize chromatin-bound methylated RelA for polyubiquitination and proteasomal degradation. We showed that WSB1/2 negatively regulated a subset of NF-κB target genes via associating with chromatin where they targeted methylated RelA for ubiquitination, facilitating the termination of NF-κB-dependent transcription. WSB1/2 specifically interacted with methylated lysines (K) 314 and 315 of RelA via their N-terminal WD-40 repeat (WDR) domains, thereby promoting ubiquitination of RelA. Computational modeling further revealed that a conserved aspartic acid (D) at position 158 within the WDR domain of WSB2 coordinates K314/K315 of RelA, with a higher affinity when either of the lysines is methylated. Mutation of D158 abolished WSB2's ability to bind to and promote ubiquitination of methylated RelA. Together, our study identifies a novel function and the underlying mechanism for WSB1/2 in degrading chromatin-bound methylated RelA and preventing sustained NF-κB activation, providing potential new targets for therapeutic intervention of NF-κB-mediated inflammatory diseases.

BRD4 Regulates Glycolysis-Dependent Nos2 Expression in Macrophages Upon H pylori Infection.

BACKGROUND & AIMS: Metabolic reprogramming is essential for the activation and functions of macrophages, including bacterial killing and cytokine production. Bromodomain-containing protein 4 (BRD4) has emerged as a critical regulator of innate immune response. However, the potential role of BRD4 in the metabolic reprogramming of macrophage activation upon Helicobacter pylori infection remains unclear. METHODS: Bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Brd4-myeloid deletion conditional knockout (Brd4-CKO) mice were infected with H pylori. RNA sequencing was performed to evaluate the differential gene expression between WT and Brd4-deficient BMDMs upon infection. An in vivo model of H pylori infection using WT and Brd4-CKO mice was used to confirm the role of BRD4 in innate immune response to infection. RESULTS: Depletion of Brd4 in BMDMs showed impaired H pylori-induced glycolysis. In addition, H pylori-induced expression of glycolytic genes, including Slc2a1 and Hk2, was decreased in Brd4-deficient BMDMs. BRD4 was recruited to the promoters of Slc2a1 and Hk2 via hypoxia-inducible factor-1α, facilitating their expression. BRD4-mediated glycolysis stabilized H pylori-induced nitric oxide synthase (Nos2) messenger RNA to produce nitric oxide. The NO-mediated killing of H pylori decreased in Brd4-deficient BMDMs, which was rescued by pyruvate. Furthermore, Brd4-CKO mice infected with H pylori showed reduced gastric inflammation and increased H pylori colonization with reduced inducible NO synthase expression in gastric macrophages. CONCLUSIONS: Our study identified BRD4 as a key regulator of hypoxia-inducible factor-1α-dependent glycolysis and macrophage activation. Furthermore, we show a novel regulatory role of BRD4 in innate immunity through glycolysis to stabilize Nos2 messenger RNA for NO production to eliminate H pylori infection.

RUNX3 Meets the Ubiquitin-Proteasome System in Cancer.

RUNX3 is a transcription factor with regulatory roles in cell proliferation and development. While largely characterized as a tumor suppressor, RUNX3 can also be oncogenic in certain cancers. Many factors account for the tumor suppressor function of RUNX3, which is reflected by its ability to suppress cancer cell proliferation after expression-restoration, and its inactivation in cancer cells. Ubiquitination and proteasomal degradation represent a major mechanism for the inactivation of RUNX3 and the suppression of cancer cell proliferation. On the one hand, RUNX3 has been shown to facilitate the ubiquitination and proteasomal degradation of oncogenic proteins. On the other hand, RUNX3 can be inactivated through the ubiquitin-proteasome system. This review encapsulates two facets of RUNX3 in cancer: how RUNX3 suppresses cell proliferation by facilitating the ubiquitination and proteasomal degradation of oncogenic proteins, and how RUNX3 is degraded itself through interacting RNA-, protein-, and pathogen-mediated ubiquitination and proteasomal degradation.

Protocol for measuring NLRC4 inflammasome activation and pyroptosis in murine bone-marrow-derived macrophages.

NLR family CARD domain containing protein 4 (NLRC4) inflammasome activation and the associated pyroptosis are critical for protection against infection by bacterial pathogens. This protocol presents a detailed procedure to activate and measure NLRC4 inflammasome activation and pyroptosis upon Salmonella Typhimurium infection. The techniques can be adapted to monitoring the activation of other types of inflammasomes and pathogenic stimuli. For comprehensive details on the use and execution of this protocol, please refer to Dong et al. (2021).

Measuring NF-κB Phosphorylation and Acetylation.

Posttranslational modifications of NF-κB, including phosphorylation, acetylation, and methylation, have emerged as important regulatory mechanisms to control the transcriptional outcomes of this important transcription factor. These modifications work independently, sequentially or in combination to modulate the diverse biological functions of NF-κB in cancer and inflammatory response. Here, we describe some experimental methods to detect the in vitro and in vivo phosphorylation and acetylation of NF-κB, specifically focusing on the RelA subunit of NF-κB. These methods include labeling the phospho- or acetyl- groups with radioisotopes in vitro and immunoblotting with site-specific anti-phospho-serine or acetyl-lysine antibodies in culture cells and tissue samples.

Brd4 modulates diet-induced obesity via PPARγ-dependent Gdf3 expression in adipose tissue macrophages.

Macrophage-mediated inflammatory response has been implicated in the pathogenesis of obesity and insulin resistance. Brd4 has emerged as a key regulator in the innate immune response. However, the role of Brd4 in obesity-associated inflammation and insulin resistance remains uncharacterized. Here, we demonstrated that myeloid lineage-specific Brd4 knockout (Brd4-CKO) mice were protected from high-fat diet-induced (HFD-induced) obesity with less fat accumulation, higher energy expenditure, and increased lipolysis in adipose tissue. Brd4-CKO mice fed a HFD also displayed reduced local and systemic inflammation with improved insulin sensitivity. RNA-Seq of adipose tissue macrophages (ATMs) from HFD-fed WT and Brd4-CKO mice revealed that expression of antilipolytic factor Gdf3 was significantly decreased in ATMs of Brd4-CKO mice. We also found that Brd4 bound to the promoter and enhancers of Gdf3 to facilitate PPARγ-dependent Gdf3 expression in macrophages. Furthermore, Brd4-mediated expression of Gdf3 acted as a paracrine signal targeting adipocytes to suppress the expression of lipases and the associated lipolysis in cultured cells and mice. Controlling the expression of Gdf3 in ATMs could be one of the mechanisms by which Brd4 modulates lipid metabolism and diet-induced obesity. This study suggests that Brd4 could be a potential therapeutic target for obesity and insulin resistance.

Brd4 regulates NLRC4 inflammasome activation by facilitating IRF8-mediated transcription of Naips.

NLRC4 inflammasome activation and the subsequent maturation of IL-1ß and IL-18 are critical for protection against infection by bacterial pathogens. The epigenetic regulator Brd4 has emerged as a key player in inflammation by regulating the expression of inflammatory cytokines. However, whether Brd4 has any role in inflammasome activation remains undetermined. Here, we demonstrated that Brd4 is an important regulator of NLRC4 inflammasome activation in response to Salmonella typhimurium infection. Brd4-deficient bone marrow-derived macrophages (BMDMs) displayed impaired caspase-1 activation, ASC oligomerization, IL-1ß maturation, gasdermin-D cleavage, and pyroptosis in response to S.typhimurium infection. RNA sequencing and RT-PCR results revealed that the transcription of Naips was decreased in Brd4-deficient BMDMs. Brd4 formed a complex with IRF8/PU.1 and bound to the IRF8 and PU.1 binding motifs on the promoters of Naips to maintain the expression of Naips. Furthermore, myeloid lineage-specific Brd4 conditional knockout mice were more susceptible to S.typhimurium infection with increased mortality, bacterial loads, and tissue damage; impaired inflammasome-dependent cytokine production; and pyroptosis. Our studies identify a novel function of Brd4 in innate immunity by controlling inflammasome-mediated cytokine release and pyroptosis to effectively battle S.typhimurium infection.

H. pylori infection confers resistance to apoptosis via Brd4-dependent BIRC3 eRNA synthesis.

H. pylori infection is one of the leading causes of gastric cancer and the pathogenicity of H. pylori infection is associated with its ability to induce chronic inflammation and apoptosis resistance. While H. pylori infection-induced expression of pro-inflammatory cytokines for chronic inflammation is well studied, the molecular mechanism underlying the apoptosis resistance in infected cells is not well understood. In this study, we demonstrated that H. pylori infection-induced apoptosis resistance in gastric epithelial cells triggered by Raptinal, a drug that directly activates caspase-3. This resistance resulted from the induction of cIAP2 (encoded by BIRC3) since depletion of BIRC3 by siRNA or inhibition of cIAP2 via BV6 reversed H. pylori-suppressed caspase-3 activation. The induction of cIAP2 was regulated by H. pylori-induced BIRC3 eRNA synthesis. Depletion of BIRC3 eRNA decreased H. pylori-induced cIAP2 and reversed H. pylori-suppressed caspase-3 activation. Mechanistically, H. pylori stimulated the recruitment of bromodomain-containing factor Brd4 to the enhancer of BIRC3 and promoted BIRC3 eRNA and mRNA synthesis. Inhibition of Brd4 diminished the expression of BIRC3 eRNA and the anti-apoptotic response to H. pylori infection. Importantly, H. pylori isogenic cagA-deficient mutant failed to activate the synthesis of BIRC3 eRNA and the associated apoptosis resistance. Finally, in primary human gastric epithelial cells, H. pylori also induced resistance to Raptinal-triggered caspase-3 activation by activating the Brd4-dependent BIRC3 eRNA synthesis in a CagA-dependent manner. These results identify a novel function of Brd4 in H. pylori-mediated apoptosis resistance via activating BIRC3 eRNA synthesis, suggesting that Brd4 could be a potential therapeutic target for H. pylori-induced gastric cancer.

Pinning Down the Transcription: A Role for Peptidyl-Prolyl cis-trans Isomerase Pin1 in Gene Expression.

Pin1 is a peptidyl-prolyl cis-trans isomerase that specifically binds to a phosphorylated serine or threonine residue preceding a proline (pSer/Thr-Pro) motif and catalyzes the cis-trans isomerization of proline imidic peptide bond, resulting in conformational change of its substrates. Pin1 regulates many biological processes and is also involved in the development of human diseases, like cancer and neurological diseases. Many Pin1 substrates are transcription factors and transcription regulators, including RNA polymerase II (RNAPII) and factors associated with transcription initiation, elongation, termination and post-transcription mRNA decay. By changing the stability, subcellular localization, protein-protein or protein-DNA/RNA interactions of these transcription related proteins, Pin1 modulates the transcription of many genes related to cell proliferation, differentiation, apoptosis and immune response. Here, we will discuss how Pin regulates the properties of these transcription relevant factors for effective gene expression and how Pin1-mediated transcription contributes to the diverse pathophysiological functions of Pin1.

[Analysis on Factors Influencing Posttransfusion Effectiveness in Plasma Transfusion of 4 423 Cases-Times].

OBJECTIVE: To investigate the related factors influencing plasma transfusion efficacy so as to improve the plasma transfusion efficiency. METHODS: According to the clinical symptoms and the laboratorial results, the patients were divided into transfusion efficient and inefficient groups. A total of13090.8 units of plasma were transfused to 4423 patients. The clinical symptoms and the hemorrhage related index per- and pro-transfusion, plasma components sorts, storage time, and the dose of plasma (kg/ml) transfusion were analyzed. RESULTS: The largest transfusion volume of plasma were in intensive care unit (ICU) accounted for 30.36%, the largest blood plasma per patient transfusion was in cardiac surgery (3.96 U). The analysis of transfusion efficiency showed that in terms of patient age, there were difference in transfusion efficiency among the patients with different ages (P＜0.001). The effective transfusion rate in the group of age ＜18 was 53%, which was higher than that in group of age 18-60(41%) and group of age ＞60 (30%); in terms of sex, the effective transfusion rate in female group was higher than that in male group (42% vs 37%) (P＜0.001); in terms of transfusion plasma volume/body weight, there were differences in transfusion efficiency (P＞0.05). The multi-factor logistic regression analysis showed that there was no significant correlation among the plasma sorts, storage time of the plasma pre-transfusion and transfusion efficiency(P＞0.05). The analysis of the non-hemolytic fever reaction caused by plasma transfusion revealed that there was no statistical difference between the plasma and the leukocyte-depleted plasma groups (P＞0.05). CONCLUSION: The plasma transfusion effectiveness relates with age and sex, but not relates with the transfusion plasma voume/body weight, plasma sorts, and the duration of storage.

[Analysis on Factors Influencing Posttransfusion Effectiveness in Plasma Transfusion of 4423 Cases-Times].

OBJECTIVE: To investigate the related factors influencing plasma transfusion efficacy so as to improve the plasma transfusion efficiency. METHODS: According to the clinical symptoms and the laboratorial results, the patients were divided into transfusion efficient and inefficient groups. A total of13090.8 units of plasma were transfused to 4423 patients. The clinical symptoms and the hemorrhage related index per- and pro-transfusion, plasma components sorts, storage time, and the dose of plasma (kg/ml) transfusion were analyzed. RESULTS: The largest transfusion volume of plasma were in intensive care unit (ICU) accounted for 30.36%, the largest blood plasma per patient transfusion was in cardiac surgery (3.96 U). The analysis of transfusion efficiency showed that in terms of patient age, there were difference in transfusion efficiency among the patients with different ages (P＜0.001). The effective transfusion rate in the group of age ＜18 was 53%, which was higher than that in group of age 18-60(41%) and group of age ＞60 (30%); in terms of sex, the effective transfusion rate in female group was higher than that in male group (42% vs 37%) (P＜0.001); in terms of transfusion plasma volume/body weight, there were differences in transfusion efficiency (P＞0.05). The multi-factor logistic regression analysis showed that there was no significant correlation among the plasma sorts, storage time of the plasma pre-transfusion and transfusion efficiency(P＞0.05). The analysis of the non-hemolytic fever reaction caused by plasma transfusion revealed that there was no statistical difference between the plasma and the leukocyte-depleted plasma groups (P＞0.05). CONCLUSION: The plasma transfusion effectiveness relates with age and sex, but not relates with the transfusion plasma voume/body weight, plasma sorts, and the duration of storage.

Perioperative safety after cytoreductive surgery plus hyperthermic intraperitoneal chemotherapy for pseudomyxoma peritonei from appendiceal origin: Experience on 254 patients from a single center.

OBJECTIVE: Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) is a standard treatment for pseudomyxoma peritonei (PMP) recommended by Peritoneal Surface Oncology Group International (PSOGI). The study is to analyze the incidence of perioperative serious adverse events (SAEs) of CRS + HIPEC to treat PMP patients, and identify the risk factors, for guiding the prevention of SAEs. METHODS: This is a retrospective study on the PMP database established at our center. The clinicopathological features, treatment details and SAEs information on the PMP patients are systematically established in this database. The incidence, organ system distribution and severity of perioperative SAEs are analyzed. Univariate and multivariate analyses are performed to identify the independent risk factors. RESULTS: Among the 272 CRS + HIPEC procedures for 254 PMP patients, there are 93 (34.2%) SAEs. Six systems are involved in the SAEs, including infections (9.6%), digestive system (8.1%), respiratory system (6.3%), cardiovascular system (5.5%), hematological system (2.9%), and urinary system (1.5%), in terms of frequency. In terms of severity, the majority is grade III SAEs (27.9%), followed by grade IV SAEs (4.8%) and grade V SAEs (1.5%). Univariate analysis reveals 4 risk factors for perioperative SAEs: HIPEC regimens (P = 0.020), PCI (P = 0.025), intraoperative red blood cell transfusion volume (P = 0.004), and intraoperative blood loss volume (P = 0.002). Multivariate and logistic regression model analysis identifies only one independent risk factor for perioperative SAEs: intraoperative blood loss volume (P = 0.001, OR = 0.344, 95%CI: 0.182-0.649). CONCLUSIONS: PMP patients treated by CRS + HIPEC at experienced centers could have acceptable safety. Improving the surgical techniques and developing the integrated hemostasis techniques are essential to reduce intraoperative blood loss and decrease SAEs rate.

PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome.

The innate immune sensor NLRP3 assembles an inflammasome complex with NEK7 and ASC to activate caspase-1 and drive the maturation of proinflammatory cytokines IL-1ß and IL-18. NLRP3 inflammasome activity must be tightly controlled, as its over-activation is involved in the pathogenesis of inflammatory diseases. Here, we show that NLRP3 inflammasome activation is suppressed by a centrosomal protein Spata2. Spata2 deficiency enhances NLRP3 inflammasome activity both in the macrophages and in an animal model of peritonitis. Mechanistically, Spata2 recruits the deubiquitinase CYLD to the centrosome for deubiquitination of polo-like kinase 4 (PLK4), the master regulator of centrosome duplication. Deubiquitination of PLK4 facilitates its binding to and phosphorylation of NEK7 at Ser204. NEK7 phosphorylation in turn attenuates NEK7 and NLRP3 interaction, which is required for NLRP3 inflammasome activation. Pharmacological or shRNA-mediated inhibition of PLK4, or mutation of the NEK7 Ser204 phosphorylation site, augments NEK7 interaction with NLRP3 and causes increased NLRP3 inflammasome activation. Our study unravels a novel centrosomal regulatory pathway of inflammasome activation and may provide new therapeutic targets for the treatment of NLRP3-associated inflammatory diseases.

T63 inhibits osteoclast differentiation through regulating MAPKs and Akt signaling pathways.

Inhibition of excessive osteoclast differentiation and activity is a valid approach for the treatment of osteoporosis. T63 is a small-molecule compound identified from a high throughput screening based on RUNX2 transcriptional activity, and has been reported to stimulate osteoblast formation. However, whether the compound has any effect on osteoclast differentiation remains unknown. Here, we examined the in vitro effect of T63 on osteoclastogenesis. T63 was found to inhibit the number of TRAP-positive cells in an osteoblast-osteoclast co-culture system, and inhibited Rankl expression in the preosteoblast MC3T3-E1 cells. The compound also directly suppressed RANKL-induced osteoclast differentiation in both dose- and time-dependent manner, as evidenced by the decrease of TRAP activity, F-actin formation and osteoclastogenesis-related genes expression in RAW264.7 cells. Moreover, pretreatment with T63 markedly decreased the activation of mitogen-activated protein kinases and Akt, both of which are positively involved in the regulation of osteoclastogenesis. Collectively, our findings suggest T63 has a protective effect against bone loss by inhibiting bone resorption. Its regulatory effect on bone metabolism makes the compound a more promising candidate for the potential application in the treatment of osteoporosis.

BRD4 regulates cellular senescence in gastric cancer cells via E2F/miR-106b/p21 axis.

Small molecules targeting bromodomains of BET proteins possess strong anti-tumor activities and have emerged as potential therapeutics for cancer. However, the underlying mechanisms for the anti-proliferative activity of these inhibitors are still not fully characterized. In this study, we demonstrated that BET inhibitor JQ1 suppressed the proliferation and invasiveness of gastric cancer cells by inducing cellular senescence. Depletion of BRD4, which was overexpressed in gastric cancer tissues, but not other BET proteins recapitulated JQ1-induced cellular senescence with increased cellular SA-ß-Gal activity and elevated p21 levels. In addition, we showed that the levels of p21 were regulated at the post-transcriptional level by BRD4-dependent expression of miR-106b-5p, which targets the 3'-UTR of p21 mRNA. Overexpression of miR-106b-5p prevented JQ1-induced p21 expression and BRD4 inhibition-associated cellular senescence, whereas miR-106b-5p inhibitor up-regulated p21 and induced cellular senescence. Finally, we demonstrated that inhibition of E2F suppressed the binding of BRD4 to the promoter of miR-106b-5p and inhibited its transcription, leading to the increased p21 levels and cellular senescence in gastric cancer cells. Our results reveal a novel mechanism by which BRD4 regulates cancer cell proliferation by modulating the cellular senescence through E2F/miR-106b-5p/p21 axis and provide new insights into using BET inhibitors as potential anticancer drugs.

Selective killing of Helicobacter pylori with pH-responsive helix-coil conformation transitionable antimicrobial polypeptides.

Current clinical treatment of Helicobacter pylori infection, the main etiological factor in the development of gastritis, gastric ulcers, and gastric carcinoma, requires a combination of at least two antibiotics and one proton pump inhibitor. However, such triple therapy suffers from progressively decreased therapeutic efficacy due to the drug resistance and undesired killing of the commensal bacteria due to poor selectivity. Here, we report the development of antimicrobial polypeptide-based monotherapy, which can specifically kill H. pylori under acidic pH in the stomach while inducing minimal toxicity to commensal bacteria under physiological pH. Specifically, we designed a class of pH-sensitive, helix-coil conformation transitionable antimicrobial polypeptides (HCT-AMPs) (PGA)m-r-(PHLG-MHH)n, bearing randomly distributed negatively charged glutamic acid and positively charged poly(γ-6-N-(methyldihexylammonium)hexyl-l-glutamate) (PHLG-MHH) residues. The HCT-AMPs showed unappreciable toxicity at physiological pH when they adopted random coiled conformation. Under acidic condition in the stomach, they transformed to the helical structure and exhibited potent antibacterial activity against H. pylori, including clinically isolated drug-resistant strains. After oral gavage, the HCT-AMPs afforded comparable H. pylori killing efficacy to the triple-therapy approach while inducing minimal toxicity against normal tissues and commensal bacteria, in comparison with the remarkable killing of commensal bacteria by 65% and 86% in the ileal contents and feces, respectively, following triple therapy. This strategy renders an effective approach to specifically target and kill H. pylori in the stomach while not harming the commensal bacteria/normal tissues.

Brd4 modulates the innate immune response through Mnk2-eIF4E pathway-dependent translational control of IκBα.

Bromodomain-containing factor Brd4 has emerged as an important transcriptional regulator of NF-κB-dependent inflammatory gene expression. However, the in vivo physiological function of Brd4 in the inflammatory response remains poorly defined. We now demonstrate that mice deficient for Brd4 in myeloid-lineage cells are resistant to LPS-induced sepsis but are more susceptible to bacterial infection. Gene-expression microarray analysis of bone marrow-derived macrophages (BMDMs) reveals that deletion of Brd4 decreases the expression of a significant amount of LPS-induced inflammatory genes while reversing the expression of a small subset of LPS-suppressed genes, including MAP kinase-interacting serine/threonine-protein kinase 2 (Mknk2). Brd4-deficient BMDMs display enhanced Mnk2 expression and the corresponding eukaryotic translation initiation factor 4E (eIF4E) activation after LPS stimulation, leading to an increased translation of IκBα mRNA in polysomes. The enhanced newly synthesized IκBα reduced the binding of NF-κB to the promoters of inflammatory genes, resulting in reduced inflammatory gene expression and cytokine production. By modulating the translation of IκBα via the Mnk2-eIF4E pathway, Brd4 provides an additional layer of control for NF-κB-dependent inflammatory gene expression and inflammatory response.

Aesculin modulates bone metabolism by suppressing receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and transduction signals.

Aesculin (AES), a coumarin compound derived from Aesculus hippocasanum L, is reported to exert protective role against inflammatory diseases, gastric disease and cancer. However, direct effect of AES in bone metabolism is deficient. In this study, we examined the effects of AES on osteoclast (OC) differentiation in receptor activator of NF-κB ligand (RANKL)-induced RAW264.7 cells. AES inhibits the OC differentiation in both dose- and time-dependent manner within non-toxic concentrations, as analyzed by Tartrate Resistant Acid Phosphatase (TRAP) staining. The actin ring formation manifesting OC function is also decreased by AES. Moreover, expressions of osteoclastogenesis related genes Trap, Atp6v0d2, Cathepsin K and Mmp-9 are decreased upon AES treatment. Mechanistically, AES attenuates the activation of MAPKs and NF-κB activity upon RANKL induction, thus leading to the reduction of Nfatc1 mRNA expression. Moreover, AES inhibits Rank expression, and RANK overexpression markedly decreases AES's effect on OC differentiation and NF-κB activity. Consistently, AES protects against bone mass loss in the ovariectomized and dexamethasone treated rat osteoporosis model. Taken together, our data demonstrate that AES can modulate bone metabolism by suppressing osteoclastogenesis and related transduction signals. AES therefore could be a promising agent for the treatment of osteoporosis.

Myeloid STAT3 Promotes Lung Tumorigenesis by Transforming Tumor Immunosurveillance into Tumor-Promoting Inflammation.

One of the most fundamental and challenging questions in the cancer field is how immunity in patients with cancer is transformed from tumor immunosurveillance to tumor-promoting inflammation. Here, we identify the transcription factor STAT3 as the culprit responsible for this pathogenic event in lung cancer development. We found that antitumor type 1 CD4+ T-helper (Th1) cells and CD8+ T cells were directly counter balanced in lung cancer development with tumor-promoting myeloid-derived suppressor cells (MDSCs) and suppressive macrophages, and that activation of STAT3 in MDSCs and macrophages promoted tumorigenesis through pulmonary recruitment and increased resistance of suppressive cells to CD8+ T cells, enhancement of cytotoxicity toward CD4+ and CD8+ T cells, induction of regulatory T cell (Treg), inhibition of dendritic cells (DC), and polarization of macrophages toward the M2 phenotype. The deletion of myeloid STAT3 boosted antitumor immunity and suppressed lung tumorigenesis. These findings increase our understanding of immune programming in lung tumorigenesis and provide a mechanistic basis for developing STAT3-based immunotherapy against this and other solid tumors. Cancer Immunol Res; 5(3); 257-68. ©2017 AACR.