C-type lectin 2D (CLEC2D) is upregulated in clear cell renal cell carcinoma (ccRCC) tissues and predicts poor prognosis.

Clear cell renal cell carcinoma (ccRCC) is known as the most common type of renal cancer. Recently, a series of advances have been made in targeted therapy for ccRCC. To combat this highly metastatic tumor, novel therapeutic targets still need to be developed. C-type lectins (CLECs) contain a characteristic C-type lectin-like domain and affect several physiological functions. The effects of C-type lectin 2D (CLEC2D) on cancer progression have been revealed in several types of cancers; however, its expression in ccRCC tissues, and the possible effects on the progression and metastasis of ccRCC, are still unclear. Herein, we found the high mRNA and protein levels of CLEC2D in ccRCC tissues. We further found that CLEC2D expression was correlated with the prognosis of ccRCC patients and correlated with the tumor size (p = 0.019*) of patients. In addition, CLEC2D affected tumor immune infiltration, confirmed by the further analysis. CLEC2D knockdown suppressed the proliferation of ccRCC cells in vitro and restrained ccRCC tumor growth and immune infiltration in mice. Therefore, we believe that CLEC2D has the potential to serve as a promising ccRCC therapeutic target.

Endosomal trafficking of two-pore K+ efflux channel TWIK2 to plasmalemma mediates NLRP3 inflammasome activation and inflammatory injury.

Potassium efflux via the two-pore K+ channel TWIK2 is a requisite step for the activation of NLRP3 inflammasome, however, it remains unclear how K+ efflux is activated in response to select cues. Here, we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in the extrusion of K+. We showed that ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP-ligated purinergic receptor P2X7 each prevented endosomal fusion with the plasmalemma and K+ efflux as well as NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-depleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. We conclude that Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 localization and activity at the cell surface and the downstream activation of the NLRP3 inflammasome. Results show that endosomal trafficking of TWIK2 to the plasmalemma is a potential therapeutic target in acute or chronic inflammatory states.

NCAPG Promotes the Proliferation of Renal Clear Cell Carcinoma via Mediating with CDK1.

Objective: Currently, lots of scholars have proved that the expression of NCAPG is associated with the prognosis of several cancers, while the relationship between NCAPG and renal clear cell carcinoma remains unclear, so the main aim of this research is to explore the effects of NCAPG on the progression of renal clear cell carcinoma. Methods: We observed the differential expression of NCAPG in several cancers from GEPIA online database, and the expression of NCAPG in renal clear cell carcinoma and normal tissue was compared and further verified by IHC assay. CCK-8 assay and clone formation experiment were conducted to observe the change of NCAPG on the proliferation. GraphPad was used for data analysis, and t-test and χ 2 analysis were used to analyze the correlation between NCAPG/CDK1 and renal clear cell carcinoma. Results: NCAPG was upregulated in renal clear cell carcinoma compared with the normal tissue, and the expression of NCAPG was associated with the clinical prognosis of pancreatic cancer especially with tumor size (P = 0.010). Knockdown NCAPG could restrain the proliferation of renal clear cell carcinoma. CDK1 was found to be tightly related with NCAPG, and the expression of CDK1 was also associated with the prognosis. Conclusions: NCAPG was upregulated in renal clear cell carcinoma, which was related with tumor size and overall survival. NCAPG might promote the proliferation of renal clear cell carcinoma via mediating CDK1. NCAPG/CDK1 complex might provide a new treatment strategy for lots of patients with renal clear cell carcinoma.

Angiogenic factor AGGF1 acts as a tumor suppressor by modulating p53 post-transcriptional modifications and stability via MDM2.

Angiogenesis factors are widely known to promote tumor growth by increasing tumor angiogenesis in the tumor microenvironment, however, little is known whether their intracellular function is involved in tumorigenesis. Here we show that AGGF1 acts as a tumor suppressor by regulating p53 when acting inside tumor cells. AGGF1 antagonizes MDM2 function to inhibit p53 ubiquitination, increases the acetylation, phosphorylation, stability and expression levels of p53, activates transcription of p53 target genes, and regulates cell proliferation, cell cycle, and apoptosis. AGGF1 also interacts with p53 through the FHA domain. Somatic AGGF1 variants in the FHA domain in human tumors, including p.Q467H, p.Y469 N, and p.N483T, inhibit AGGF1 activity on tumor suppression. These results identify a key role for AGGF1 in an AGGF1-MDM2-p53 signaling axis with important functions in tumor suppression, and uncover a novel trans-tumor-suppression mechanism dependent on p53. This study has potential implications in diagnosis and therapies of cancer.

The angiocrine Rspondin3 instructs interstitial macrophage transition via metabolic-epigenetic reprogramming and resolves inflammatory injury.

Macrophages demonstrate remarkable plasticity that is essential for host defense and tissue repair. The tissue niche imprints macrophage identity, phenotype and function. The role of vascular endothelial signals in tailoring the phenotype and function of tissue macrophages remains unknown. The lung is a highly vascularized organ and replete with a large population of resident macrophages. We found that, in response to inflammatory injury, lung endothelial cells release the Wnt signaling modulator Rspondin3, which activates ß-catenin signaling in lung interstitial macrophages and increases mitochondrial respiration by glutaminolysis. The generated tricarboxylic acid cycle intermediate α-ketoglutarate, in turn, serves as the cofactor for the epigenetic regulator TET2 to catalyze DNA hydroxymethylation. Notably, endothelial-specific deletion of Rspondin3 prevented the formation of anti-inflammatory interstitial macrophages in endotoxemic mice and induced unchecked severe inflammatory injury. Thus, the angiocrine-metabolic-epigenetic signaling axis specified by the endothelium is essential for reprogramming interstitial macrophages and dampening inflammatory injury.

CircPCNXL2 sponges miR-153 to promote the proliferation and invasion of renal cancer cells through upregulating ZEB2.

Increasing evidence showed that circular RNAs (circRNAs) play critical roles in tumorigenesis. However, the roles and underlying mechanisms of circRNAs in clear cell renal cell carcinoma (ccRCC) remain unclear. In the present study, we identified a novel circRNA circPCNXL2, which was significantly upregulated in ccRCC by circular RNA microarray. Further analysis revealed that circPCNXL2 was significantly increased and correlated with poor overall survival of ccRCC patients. Function assays revealed that circPCNXL2 knockdown reduced RCC cells proliferation, invasion in vitro, and decreased tumor growth in vivo. In mechanism study, we showed that circPCNXL2 could be bind to miR-153 as a miRNA sponge to regulate ZEB2 expression in RCC progression. In addition, our data reported that the effects of circPCNXL2 inhibition on RCC cells proliferation and invasion could be abolished by miR-153 inhibitors. Altogether, we demonstrated that circPCNXL2 could regulate RCC cells proliferation and invasion by miR-153/ZEB2 axis, suggesting circPCNXL2 might serve as a potential therapeutic target for ccRCC treatment.

Angiogenic Factor With G Patch and FHA Domains 1 Is a Novel Regulator of Vascular Injury.

OBJECTIVE: Phenotypic modulation of vascular smooth muscle cells represents a hallmark event in vascular injury. The underlying mechanism is not completely sorted out. We investigated the involvement of angiogenic factor with G patch and FHA domains 1 (Aggf1) in vascular injury focusing on the transcriptional regulation of vascular smooth muscle cell signature genes. APPROACH AND RESULTS: We report here that Aggf1 expression was downregulated in several different cell models of phenotypic modulation in vitro and in the vessels after carotid artery ligation in mice. Adenovirus-mediated Aggf1 overexpression dampened vascular injury and normalized vascular smooth muscle cell signature gene expression. Mechanistically, Aggf1 interacted with myocardin and was imperative for the formation of a serum response factor-myocardin complex on gene promoters. In response to injurious stimuli, kruppel-like factor 4 was recruited to the Aggf1 promoter and enlisted histone deacetylase 11 to repress Aggf1 transcription. In accordance, depletion of kruppel-like factor 4 or histone deacetylase 11 restored Aggf1 expression and abrogated vascular smooth muscle cell phenotypic modulation. Finally, treatment of a histone deacetylase 11 inhibitor attenuated vascular injury in mice. CONCLUSIONS: Therefore, we have unveiled a previously unrecognized role for Aggf1 in regulating vascular injury.

HIC1 epigenetically represses CIITA transcription in B lymphocytes.

Differentiation of B lymphocytes into isotope-specific plasma cells represents a hallmark event in adaptive immunity. During B cell maturation, expression of the class II transactivator (CIITA) gene is down-regulated although the underlying epigenetic mechanism is not completely defined. Here we report that hypermethylated in cancer 1 (HIC1) was up-regulated in differentiating B lymphocytes paralleling CIITA repression. Over-expression of HIC1 directly repressed endogenous CIITA transcription in B cells. Reporter assay and chromatin immunoprecipitation (ChIP) assay confirmed that HIC1 bound to the proximal CIITA type III promoter (-545/-113); mutation of a conserved HIC1 site within this region abrogated CIITA trans-repression. More important, depletion of HIC1 with small interfering RNA (siRNA) restored CIITA expression in differentiating B cells. Mechanistically, HIC1 preferentially interacted with and recruited DNMT1 and DNMT3b to the CIITA promoter to synergistically repress CIITA transcription. On the contrary, silencing of DNMT1/DNMT3b or inhibition of DNMT activity with 5-aza-dC attenuated CIITA trans-repression. Therefore, our data identify HIC1 as a novel factor involved in B cell differentiation acting as an epigenetic repressor of CIITA transcription.

HDAC4 mediates IFN-γ induced disruption of energy expenditure-related gene expression by repressing SIRT1 transcription in skeletal muscle cells.

Metabolic homeostasis is achieved through balanced energy storage and output. Impairment of energy expenditure is a hallmark event in patients with obesity and type 2 diabetes. Previously we have shown that the pro-inflammatory cytokine interferon gamma (IFN-γ) disrupts energy expenditure in skeletal muscle cells via hypermethylated in cancer 1 (HIC1)-class II transactivator (CIITA) dependent repression of SIRT1 transcription. Here we report that repression of SIRT1 transcription by IFN-γ paralleled loss of histone acetylation on the SIRT1 promoter region with simultaneous recruitment of histone deacetylase 4 (HDAC4). IFN-γ activated HDAC4 in vitro and in vivo by up-regulating its expression and stimulating its nuclear accumulation. HIC1 and CIITA recruited HDAC4 to the SIRT1 promoter and cooperated with HDAC4 to repress SIRT1 transcription. HDAC4 depletion by small interfering RNA or pharmaceutical inhibition normalized histone acetylation on the SIRT1 promoter and restored SIRT1 expression in the presence of IFN-γ. Over-expression of HDAC4 suppressed the transcription of genes involved in energy expenditure in a SIRT1-dependent manner. In contrast, HDAC4 knockdown/inhibition neutralized the effect of IFN-γ on cellular metabolism by normalizing SIRT1 expression. Therefore, our data reveal a role for HDAC4 in regulating cellular energy output and as such provide insights into rationalized design of novel anti-diabetic therapeutics.

Myocardin-related transcription factor A (MRTF-A) plays an essential role in hepatic stellate cell activation by epigenetically modulating TGF-ß signaling.

Fibrosis following injury is a common adaptive response in the liver, which can lead to irreparable and life-threatening cirrhosis and hepatocellular carcinoma without effectual intervention. The molecular mechanisms underlying fibrogenic response in the liver remains poorly understood. Here we report that mice with deficiency in myocardin-related transcription factor A (MRTF-A) showed resistance to thioacetamide (TAA)-induced liver fibrosis with significantly reduced expression of pro-fibrogenic genes when compared to wild type littermates. Over-expression of MRTF-A enhanced whereas depletion of MRTF-A alleviated pro-fibrogenic transcription induced by TGF-ß, a major pro-fibrogenic factor in hepatic stellate cells (HSCs). Mechanistically, MRTF-A silencing in HSCs impacted the chromatin structure by reducing the deposition of methylated histone H3K4 on the promoters of pro-fibrogenic genes. Further analyses revealed that MRTF-A interacted with and recruited several key epigenetic factors involved in H3K4 methylation, including ASH2, WDR5, and SET1, to the promoters of pro-fibrogenic genes in response to TGF-ß treatment. Over-expression of ASH2, WDR5, or SET1 enhanced the transactivation of pro-fibrogenic gene promoters by TGF-ß in an MRTF-A-dependent manner. In conclusion, MRTF-A regulates liver fibrosis by epigenetically tuning the TGF-ß signaling pathway in HSCs.

MKL1 is an epigenetic modulator of TGF-ß induced fibrogenesis.

Transforming growth factor (TGF-ß) induced activation of portal fibroblast cells serves as a primary cause for liver fibrosis following cholestatic injury. The underlying epigenetic mechanism is not clear. We studied the role of a transcriptional modulator, megakaryoblastic leukemia 1 (MKL1) in this process. We report here that MKL1 deficiency ameliorated BDL-induced liver fibrosis in mice as assessed by histological stainings and expression levels of pro-fibrogenic genes. MKL1 silencing by small interfering RNA (siRNA) abrogated TGF-ß induced transactivation of pro-fibrogenic genes in portal fibroblast cells. TGF-ß stimulated the binding of MKL1 on the promoters of pro-fibrogenic genes and promoted the interaction between MKL1 and SMAD3. While SMAD3 was necessary for MKL1 occupancy on the gene promoters, MKL1 depletion impaired SMAD3 binding reciprocally. TGF-ß treatment induced the accumulation of trimethylated histone H3K4 on the gene promoters by recruiting a methyltransferase complex. Knockdown of individual members of this complex significantly weakened the binding of SMAD3 and down-regulated the activation of portal fibroblast cells. In conclusion, we have identified an epigenetic pathway that dictates TGF-ß induced pro-fibrogenic transcription in portal fibroblast thereby providing novel insights for the development of therapeutic solutions to treat liver fibrosis.

Endothelial MRTF-A mediates angiotensin II induced cardiac hypertrophy.

Angiotensin II (Ang II) stimulates endothelin (ET-1) transcription, which contributes to cardiac hypertrophy and fibrosis. We have previously reported that myocardin related transcription factor A (MRTF-A) is indispensable for ET-1 transcription in vascular endothelial cells under hypoxic conditions, indicating that MRTF-A might mediate Ang II-induced pathological hypertrophy. Here we report that Ang II augmented the expression of MRTF-A in cultured endothelial cells and in the lungs of mice with cardiac hypertrophy. Over-expression of MRTF-A enhanced, whereas depletion of MRTF-A attenuated, transcriptional activation of ET-1 gene by Ang II. MRTF-A deficiency ameliorated Ang II induced cardiac hypertrophy and fibrosis in mice paralleling diminished synthesis and release of ET-1. Mechanistically, MRTF-A was recruited to the ET-1 promoter by c-Jun/c-Fos (AP-1) in response to Ang II treatment. Once bound, MRTF-A altered the chromatin structure by modulating histone acetylation and H3K4 methylation on the ET-1 promoter. More importantly, mice with endothelial-specific MRTF-A silencing by lentiviral particles phenocopied mice with systemic MRTF-A deletion in terms of Ang II-induced pathological hypertrophy. In conclusion, we data have unveiled a MRTF-A-containing complex that links ET-1 transactivation in endothelial cells to cardiac hypertrophy and fibrosis by Ang II.

MRTF-A steers an epigenetic complex to activate endothelin-induced pro-inflammatory transcription in vascular smooth muscle cells.

Endothelin (ET-1) was initially identified as a potent vasoconstrictor contributing to the maintenance of vascular rhythm. Later studies have implicated ET-1, when aberrantly up-regulated within the vasculature, in a range of human pathologies associated with disruption of vascular homeostasis. ET-1 has been shown to invoke strong pro-inflammatory response in vascular smooth muscle cells (VSMCs); the underlying mechanism, however, remains elusive. Here, we report that the transcriptional modulator MRTF-A mediates the activation of pro-inflammatory mediators by ET-1 in VSMCs. ET-1 increased nuclear enrichment and activity of MRTF-A in cultured VSMCs. MRTF-A silencing attenuated ET-1 induced synthesis and release of pro-inflammatory mediators including IL-6, MCP-1 and IL-1 likely as a result of diminished NF-κB activity. In addition, MRTF-A was indispensible for the accumulation of active histone modifications on the gene promoters. Of intrigue, MRTF-A interacted with and recruited ASH2, a component of the mammalian histone methyltransferase complex, to transactivate pro-inflammatory genes in response to ET-1 treatment. The chromatin remodeling proteins BRG1 and BRM were also required for ET-1-dependent induction of pro-inflammatory mediators by communicating with ASH2, a process dependent on MRTF-A. In conclusion, our data have identified a novel epigenetic complex responsible for vascular inflammation inflicted by ET-1.

MiR-144 regulates hematopoiesis and vascular development by targeting meis1 during zebrafish development.

Hematopoiesis is a dynamic process by which peripheral blood lineages are developed. It is a process tightly regulated by many intrinsic and extrinsic factors, including transcriptional factors and signaling molecules. However, the epigenetic regulation of hematopoiesis, for example, regulation via microRNAs (miRNAs), remains incompletely understood. Here we show that miR-144 regulates hematopoiesis and vascular development in zebrafish. Overexpression of miR-144 inhibited primitive hematopoiesis as demonstrated by a reduced number of circulating blood cells, reduced o-dianisidine staining of hemoglobin, and reduced expression of hbαe1, hbße1, gata1 and pu.1. Overexpression of miR-144 also inhibited definitive hematopoiesis as shown by reduced expression of runx1 and c-myb. Mechanistically, miR-144 regulates hematopoiesis by repressing expression of meis1 involved in hematopoiesis. Both real-time RT-PCR and Western blot analyses showed that overexpression of miR-144 repressed expression of meis1. Bioinformatic analysis predicts a target binding sequence for miR-144 at the 3'-UTR of meis1. Deletion of the miR-144 target sequence eliminated the repression of meis1 expression mediated by miR-144. The miR-144-mediated abnormal phenotypes were partially rescued by co-injection of meis1 mRNA and could be almost completely rescued by injection of both meis1 and gata1 mRNA. Finally, because meis1 is involved in vascular development, we tested the effect of miR-144 on vascular development. Overexpression of miR-144 resulted in abnormal vascular development of intersegmental vessels in transgenic zebrafish with Flk1p-EGFP, and the defect was rescued by co-injection of meis1 mRNA. These findings establish miR-144 as a novel miRNA that regulates hematopoiesis and vascular development by repressing expression of meis1.

Role of microRNA-27a in down-regulation of angiogenic factor AGGF1 under hypoxia associated with high-grade bladder urothelial carcinoma.

Hypoxia stimulates angiogenesis under a variety of pathological conditions, including malignant tumors by inducing expression of angiogenic factors such as VEGFA. Surprisingly, here we report significant association between down-regulation of a new angiogenic factor AGGF1 and high-grade urothelial carcinoma. The proportion of strong AGGF1 expression cases was significantly lower in the high-grade urothelial carcinoma group than that in the low-grade urothelial carcinoma group (P=1.40×10(-5)) or than that in the normal urothelium tissue group (P=2.11×10(-4)). We hypothesized that tumor hypoxia was responsible for differential expression of the AGGF1 protein in low- and high-grade urothelial carcinomas, and therefore investigated the molecular regulatory mechanism for AGGF1 expression under hypoxia. Under hypoxic conditions, AGGF1 protein levels declined without any change in mRNA levels and protein stability. Hypoxia-induced down-regulation of AGGF1 was mediated by miR-27a. Overexpression of miR-27a suppressed AGGF1 expression through translational inhibition, but not by RNA degradation. Moreover, the hypoxia-induced decrease of AGGF1 expression disappeared after miR-27a expression was inhibited. Furthermore, down-regulation of AGGF1 reduced hypoxia-induced apoptosis in cancer cells. Taken together, the results of this study indicate that (1) hypoxia down-regulates expression of the AGGF1 protein, but not AGGF1 mRNA, by inducing expression of miR-27a; (2) Down-regulation of AGGF1 had an apparent protective role for cancer cells under hypoxia; (3) Down-regulation of the AGGF1 protein confers a significant risk of high-grade human urothelial bladder carcinoma.

Transcriptional activation of the Prox1 gene by HIF-1α and HIF-2α in response to hypoxia.

Prox1 encodes a homeobox transcription factor critical to organ development, but its regulation is poorly understood. Here, we show that Prox1 expression is induced by hypoxia, and controlled by a hypoxia-response element (HRE) at the Prox1 promoter/regulatory region and HIF-1α/HIF-2α. EMSA and ChIP assays demonstrated the direct interaction of the HRE with HIF-1α or HIF-2α. Overexpression of HIF-1α or HIF-2α increased activation of the Prox1 promoter, whereas knockdown of HIF-1α or HIF-2α inhibited the activation. These data reveal a novel molecular mechanism for regulation of Prox1 expression in response to hypoxia and provide new insights into Prox1-controlled processes such as lymphangiogenesis.

MicroRNA-503 targets FGF2 and VEGFA and inhibits tumor angiogenesis and growth.

FGF2 and VEGFA are the two most potent angiogenic factors. Here we report that miR-503 can simultaneously down-regulate FGF2 and VEGFA. The expression of miR-503 is repressed in HCC cells and primary tumors due to a potential epigenetic mechanism. Overexpression of miR-503 reduced tumor angiogenesis in vitro and in vivo. We also found that miR-503 expression was down-regulated by hypoxia through HIF1α. These results identify a miRNA that targets both FGF2 and VEGFA in cancers, demonstrate the anti-angiogenesis role of miR-503 in tumorigenesis, and provide a novel mechanism for hypoxia-induced FGF2 and VEGFA through HIF1α-mediated inhibition of miR-503.