Mobility Group Nucleosomal Binding Domain Research Articles

Pan-cancer Multi-omics Analysis Reveals HMGN1 as a Potential Prognostic and Immune Infiltration-associated Biomarker.

The High Mobility Group Nucleosomal Binding Domain 1 Gene (HMGN1) is crucial for epigenetic regulation. However, the specific function of HMGN1 in cancer development is unclear. Raw data on HMGN1 expression were procured from Genotype-Tissue Expression (GTEx), the University of Alabama- Birmingham CANcer data analysis Portal (UALCAN), and The Cancer Genome Atlas (TCGA). Thereafter, the pan-cancer analysis was implemented to understand the HMGN1 expression patterns, prognostic value, and immunological features. Furthermore, the Gene Set Enrichment Analysis (GSEA) was executed via R language. In addition, the relationship between HMGN1 and the sensitivity of antitumor drugs was also determined. Finally, real-time PCR (RT-PCR) experiments were carried out. Pan-cancer analysis revealed that HMGN1 was upregulated in several solid tumors and was associated with pathological staging and poor prognosis. In addition, HMGN1 was found to be involved in regulating the tumor microenvironment. The GSEA enrichment analysis indicated that HMGN1 assisted in the regulation of oncogenic processes, especially metabolic and immune pathways. Furthermore, HMGN1 expression was linked to microsatellite instability (MSI) and tumor mutational burden (TMB) across diverse tumor types. RT-PCR assays indicated that HMGN1 was overexpressed in the gastric and breast cancer cell lines and tissues. This study highlighted the potential of HMGN1 as a biomarker for pan- - cancer analysis.

Chromatin binding protein HMGN1 promotes HBV cccDNA transcription and replication by regulating the phosphorylation of histone 3

Background and aimsDirect elimination of cccDNA remains a formidable obstacle due to the persistent and stable presence of cccDNA in hepatocyte nuclei. The silencing of cccDNA transcription enduringly is one of alternative strategies in the treatment of hepatitis B. Protein binding to cccDNA plays an important role in its transcriptional regulation; thus, the identification of key factors involved in this process is of great importance. Approaches and resultsIn the present study, high mobility group nucleosome binding domain 1 (HMGN1) was screened out based on our biotin-avidin enrichment system. First, chromatin immunoprecipitation and fluorescent in situ hybridization assays confirmed the binding of HMGN1 with cccDNA in the nucleus. Second, functional experiments in HBV-infected cells showed that the promoting effect of HMGN1 on HBV transcription and replication depended on the functional region of the nucleosomal binding domain, while transfection of the HMGN1 mutant showed no influence on HBV compared with the vector. Third, further mechanistic exploration revealed that the silencing of HMGN1 increased the level of phosphorylase CLK2 and promoted H3 phosphorylation causing the reduced accessibility of cccDNA. Moreover, silenced HMGN1 was mimicked in HBV (r) cccDNA mouse model of HBV infection in vivo. The results showed that silencing HMGN1 inhibited HBV replication in vivo. ConclusionsIn summary, our study identified that a host protein can bind to cccDNA and promote its transcription, providing a candidate strategy for anti-HBV targeting to interfere with the transcriptional activity of cccDNA microchromosomes.

OR27-06 Identification And Functional Analyses Of Therapeutic Targets For Testicular Germ Cell Tumors

Abstract Disclosure: K. Horie: None. S. Kitayama: None. K. Ikeda: None. T. Suzuki: None. S. Kawakami: None. S. Inoue: None. Testicular germ cell tumor (TGCT) is the most common cancer among adolescent and young adult males. While cisplatin is a key drug that efficiently reduces TGCT growth, the acquisition of drug resistance is often life-threatening. To elucidate molecular mechanisms underlying the pathophysiology of TGCT, we established patient-derived TGCT spheroid cultures from nonseminomatous tumors, in which cancer stem cell population can be preferentially enriched. The TGCT spheroid cultures can be applied to the generation of in vivo xenograft models, which successfully recapitulated the pathological features of patient tumors with microenvironment. Transcriptomic analysis revealed that hypoxia-related genes were upregulated in TGCT spheroid cultures, and HIF1α inhibitor 2-methoxyestradiol could substantially repress the in vivo tumor formation. We further generated cisplatin-resistant TGCT models from embryonic cancer patient-derived spheroids and cell line, and found that transcription-related gene high mobility group nucleosome binding domain 5 (HMGN5) and meiosis-related gene testis-expressed 11 (TEX11) were remarkably upregulated in cisplatin-resistant TGCT cells compared with their parental cells. Either HMGN5 or TEX11 knockdown could reduce the viability of cisplatin-resistant TGCT cells in the presence of cisplatin. Of note, TEX11 knockdown increased cleaved PARP1 protein expression and the percentage of double-strand break marker γH2AX-positive cells in cisplatin-resistant TGCT cells. We demonstrated that siRNA-mediated TEX11 inhibition is a promising therapeutic option for cisplatin-resistant TGCT as we showed the efficacy in in vivo xenograft models generated from cisplatin-resistant patient-derived TGCT spheroids. Furthermore, chromatin immunoprecipitation sequencing for histone H3K27 acetylation status in the TGCT spheroid cultures revealed that the enrichment of “super-enhancers”, identified by the Rank Ordering of Super-enhancers algorithm [http://younglab.wi.mit.edu/super_enhancer_code.html], was well observed at chromosome 12p, where copy number gains are often observed in the tumors, and genes in the vicinity of super-enhancers identified in patient-derived TGCT models can be potential diagnostic and therapeutic targets for the advanced disease. Overall, the present study provides useful insights into the biology and chemoresistance of TGCT. Presentation: Saturday, June 17, 2023

Development of a novel modified vaccine (TheraVacM) for curative treatment of mouse solid tumors

Monotherapy with immune checkpoint blockade (ICB) antibodies (anti-CTLA4 and anti-PD1/PDL-1) is only effective for 20% to 30% of patients with certain cancers. Patients with cancers harboring few effector T cells (Teffs) are insensitive to ICB therapy. The lack of tumor-specific Teffs is predominantly caused by the paralysis of tumor-infiltrating dendritic cells (TiDCs) resulting from immunosuppression in the tumor microenvironment. We have identified a potent combination of high mobility group nucleosome binding domain 1 (HMGN1, N1) and fibroblast stimulating lipopeptide-1 (FSL-1) that can synergistically trigger maturation of both mouse and human DCs. Accordingly, we designed a combinational anti-cancer immunotherapy with two arms: an immune-activating arm consisting of N1 and FSL-1 to stimulate the generation of Teffs by triggering full maturation of TiDCs, and an ICB arm using anti-PDL-1 or anti-CTLA4 to prevent Teffs from being silenced in the tumor tissue. This combinational immunotherapeutic vaccination regimen dubbed modified TheraVac (TheraVacM) has proved particularly effective as it cured 100% of mice bearing established ectopic CT26 colon and RENCA kidney tumors. The resultant tumor-free mice were resistant to subsequent re-challenge with the same tumors, indicating the generation of long-term tumor specific protective immunity. Since the immune-activating arm also induces full maturation of human DCs, and anti-PDL-1 or anti-CTLA4 have been FDA-approved, this combinational immunotherapy has the potential to be an effective clinical therapy for patients with solid tumors.

WTAP dysregulation-mediated HMGN3-m6A modification inhibited trophoblast invasion in early-onset preeclampsia.

Early-onset preeclampsia (ePE) originates from abnormal implantation and placentation that involves trophoblast invasion, but its pathophysiology is not entirely understood. N6-methyladenosine (m6A) regulators mediate the progression of various cancers. The invasiveness of trophoblast cells is similar to that of tumor cells. However, little is known regarding the potential role of m6A modification in ePE and the underlying mechanism. This study aimed to explore the m6A level in placental tissue samples collected from ePE patients and to investigate whether m6A modification was an essential part of PE pathogenesis. The m6A level in placental tissue samples of 80 PE participants was examined. MeRIP-microarray, RNA-Seq, luciferase reporter assay, and RNA immunoprecipitation chip (RIP) assay were performed. The m6A level in the ePE group was significantly reduced compared with the control group. Wilms' tumor 1-associating protein (WTAP) regulated trophoblast cell migration and invasion. Mechanistically, the high mobility group nucleosomal binding domain 3 (HMGN3) gene was a target gene of WTAP in trophoblast (p < .05). WTAP enhanced the stability of HMGN3 mRNA through binding with its 3'-UTR m6A site(+485A, +522A). HMGN3 was recognized by m6A recognition protein insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which was inhibited when knocking down WTAP. Both m6A and WTAP levels were downregulated in ePE. The m6A modification mediated by WTAP/IGF2BP1/HMGN3 axis might contribute to abnormal trophoblast invasion. Our work provided a foundation for further exploration of RNA epigenetic regulatory patterns in ePE, and indicated a new treatment strategy for ePE.

Camostat mesilate inhibits pro-inflammatory cytokine secretion and improves cell viability by regulating MFGE8 and HMGN1 in lipopolysaccharide-stimulated DF-1 chicken embryo fibroblasts.

Camostat mesilate (CM) possesses potential anti-viral and anti-inflammatory activities. However, it remains unknown whether CM is involved in lipopolysaccharide (LPS)-mediated inflammatory responses and cell injury. In this project, differentially expressed proteins (DEPs, fold change ≥ 1.2 or ≤ 0.83 and Q value ≤ 0.05) in response to LPS stimulation alone or in combination with CM were identified through tandem mass tags (TMT)/mass spectrometry (MS)-based proteomics analysis in DF-1 chicken embryo fibroblasts. The mRNA expression levels of filtered genes were determined by RT-qPCR assay. The results showed that CM alleviated the detrimental effect of LPS on cell viability and inhibited LPS-induced TNF-α and IL-6 secretions in DF-1 chicken embryo fibroblasts. A total of 141 DEPs that might be involved in mediating functions of both LPS and CM were identified by proteomics analysis in DF-1 chicken embryo fibroblasts. LPS inhibited milk fat globule EGF and factor V/VIII domain containing (MFGE8) expression and induced high mobility group nucleosome binding domain 1 (HMGN1) expression, while these effects were abrogated by CM in DF-1 chicken embryo fibroblasts. MFGE8 knockdown facilitated TNF-α and IL-6 secretions , reduced cell viability, stimulated cell apoptosis in DF-1 chicken embryo fibroblasts co-treated with LPS and CM. HMGN1 loss did not influence TNF-α and IL-6 secretions, cell viability, and cell apoptosis in DF-1 chicken embryo fibroblasts co-treated with LPS and CM. In conclusion, CM exerted anti-inflammatory and pro-survival activities by regulating MFGE8 in LPS-stimulated DF-1 chicken embryo fibroblasts, deepening our understanding of the roles and molecular basis of CM in protecting against Gram-negative bacteria.

The regulatory effect of acetylation of HMGN2 and H3K27 on pyocyanin-induced autophagy in macrophages by affecting Ulk1 transcription.

Pyocyanin (PYO) is a major virulence factor secreted by Pseudomonas aeruginosa, and autophagy is a crucial homeostatic mechanism for the interaction between the pathogens and the host. It remains unknown whether PYO leads to autophagy in macrophages by regulating histone acetylation. The high mobility group nucleosomal binding domain 2 (HMGN2) has been reported to regulate the PYO‐induced autophagy and oxidative stress in the epithelial cells; however, the underlying molecular mechanism has not been fully elucidated. In this study, PYO was found to induce autophagy in macrophages, and the mechanism might be correlated with the up‐regulation of HMGN2 acetylation (HMGN2ac) and the down‐regulation of H3K27 acetylation (H3K27ac) by modulation of the activities of acetyltransferases and deacetylases. Moreover, we further demonstrated that the up‐regulated HMGN2ac enhances its recruitment to the Ulk1 promoter, while the down‐regulation of H3K27ac reduces its recruitment to the Ulk1 promoter, thereby promoting or inhibiting the transcription of Ulk1. In conclusion, HMGN2ac and H3K27ac play regulatory roles in the PYO‐induced autophagy in macrophages.

Multi-Omics Analysis to Characterize Cigarette Smoke Induced Molecular Alterations in Esophageal Cells.

Though smoking remains one of the established risk factors of esophageal squamous cell carcinoma, there is limited data on molecular alterations associated with cigarette smoke exposure in esophageal cells. To investigate molecular alterations associated with chronic exposure to cigarette smoke, non-neoplastic human esophageal epithelial cells were treated with cigarette smoke condensate (CSC) for up to 8 months. Chronic treatment with CSC increased cell proliferation and invasive ability of non-neoplastic esophageal cells. Whole exome sequence analysis of CSC treated cells revealed several mutations and copy number variations. This included loss of high mobility group nucleosomal binding domain 2 (HMGN2) and a missense variant in mediator complex subunit 1 (MED1). Both these genes play an important role in DNA repair. Global proteomic and phosphoproteomic profiling of CSC treated cells lead to the identification of 38 differentially expressed and 171 differentially phosphorylated proteins. Bioinformatics analysis of differentially expressed proteins and phosphoproteins revealed that most of these proteins are associated with DNA damage response pathway. Proteomics data revealed decreased expression of HMGN2 and hypophosphorylation of MED1. Exogenous expression of HMGN2 and MED1 lead to decreased proliferative and invasive ability of smoke exposed cells. Immunohistochemical labeling of HMGN2 in primary ESCC tumor tissue sections (from smokers) showed no detectable expression while strong to moderate staining of HMGN2 was observed in normal esophageal tissues. Our data suggests that cigarette smoke perturbs expression of proteins associated with DNA damage response pathways which might play a vital role in development of ESCC.

High mobility group nucleosomal binding 2 reduces integrin α5/β1‐mediated adhesion of Klebsiella pneumoniae on human pulmonary epithelial cells via nuclear factor I

It has been reported that high mobility group nucleosomal binding domain 2 (HMGN2) is a nucleus-related protein that regulates gene transcription and plays a critical role in bacterial clearance. An elevated level of HMGN2 reduced integrin α5/β1 expression of human pulmonary epithelial A549 cells was demonstrated during Klebsiella pneumoniae infection, thus weakening bacterial adhesion and invasion. However, the mechanism by which HMGN2 regulates integrin expression remains unclear. This study found that a transcription factor-nuclear factor I (NFI), which serves as the potential target of HMGN2 regulated integrin expression. The results showed that HMGN2 was able to promote NFIA and NFIB expression by increasing H3K27 acetylation of NFIA/B promoter regions. The integrin α5/β1 expression was significantly enhanced by knockdown of NFIA/B via a siRNA approach. Meanwhile, NFIA/B silence could also compromise the inhibition effect of HMGN2 on the integrin α5/β1 expression. Mechanistically, it was demonstrated that HMGN2 facilitated the recruitment of NFI on the promoter regions of integrin α5/β1 according to the chromatin immunoprecipitation assay. In addition, it was further demonstrated that the knockdown of NFIA/B induced more adhesion of Klebsiella pneumoniae on pulmonary epithelial A549 cells, which could be reversed by the application of an integrin inhibitor RGD. The results revealed a regulatory role of HMGN2 on the transcription level of integrin α5/β1, indicating a potential treatment strategy against Klebsiella pneumoniae-induced infectious lung diseases.

Transcriptomic analysis of peripheral blood mononuclear cells in head and neck squamous cell carcinoma patients.

To investigate the gene expression profile of peripheral blood mononuclear cells (PBMCs) from head and neck squamous cell carcinoma (HNSCC), including oral cancer (OC) and oropharyngeal cancer (OPC) patients, and compare them with healthy controls (HC). Transcriptomic analysis of PBMCs was performed by RNA-sequencing. The upregulated candidate genes were selected for validation by quantitative real-time polymerase chain reaction (qPCR). In addition, related plasma protein levels were determined by enzyme-linked immunosorbent assay (ELISA). Three significantly upregulated genes, including high mobility group nucleosomal binding domain 2 (HMGN2), folate receptor gamma (FOLR3), and amphiregulin (AREG), were selected. In the first cohort, the results showed that only HMGN2 expression was significantly increased in OC patients. In the larger sample size, the overall results demonstrated that HMGN2 expression had a tendency to increase in both OC and OPC patients compared with HC. Interestingly, the plasma HMGN2 (HMG-17) protein level exhibited the same trend as that observed at the transcriptional level. HMGN2 expression and plasma HMG-17 (HMGN2 protein) were increased in both cancer patients compared with HC. This gene may be important for further functional studies in the PBMCs of HNSCC patients.

Construction and dissection of the ceRNA‑ceRNA network reveals critical modules in depression.

The prevalence of mental health disorders such as depression is high. Depression is a multifactorial disorder and its underlying mechanisms remain unclear. Competing endogenous RNA (ceRNA) regulation has been reported to serve important roles in human disease. In the present study, ceRNA networks for depression and the corresponding normal physiological states were constructed. Further analysis of the ceRNA networks revealed that ceRNA regulation may be important for depression. Hub ceRNAs including high mobility group nucleosomal binding domain 3, peroxisome proliferator-activated receptor-γ coactivator 1β and leukemia inhibitory factor receptor-α were associated with depression. A common core ceRNA network was identified by comparison analysis. Functional analysis suggested that these ceRNAs may be implicated in depression. Differential expression analysis revealed that ceRNAs in the obtained ceRNA interaction networks were significantly enriched with significantly differentially expressed genes. A total of 8 key functional modules for depression were identified, and small target molecules were screened. ceRNA protocadherin-α subfamily C2 in module 1 and ceRNA Cyclin-dependent kinase 6 in module 3 were reported to be implicated in the occurrence and development of depressive disorders. Thus, the present analysis may provide insight into the pathogenesis of depression and improve its treatment.

Abstract B11: HDAC6 as a therapeutic target in human breast cancer

Abstract Stat5 is a transcription factor utilized by several cytokine/hormone receptor signaling pathways that promotes transcription of genes associated with proliferation, differentiation, and survival of cancer cells. However, there are currently no clinically approved therapies that target Stat5, despite ample evidence that it contributes to breast cancer pathogenesis. Previous research in our lab has shown that the high mobility group nucleosome binding domain 2 (HGMN2) protein serves as a Stat5 co-activator. The activity of HMGN2 has been previously shown to be regulated by acetylation. Here, we show that deacetylation of HMGN2 on lysine residue K2 by histone deacetylase 6 (HDAC6) promotes Stat5-mediated transcription and breast cancer growth. Conversely, in vitro HDAC6 inhibition by pharmacologic and knockdown approaches enhanced HMGN2 acetylation with a concomitant reduction of in vitro Stat5-mediated signaling and global gene expression, and breast cancer growth. These data, combined with global in silico ChIP-Seq analysis, indicate that HDAC6 serves as a regulator of the pioneer function of HMGN2 for Stat5 transcriptional function. In vitro and in vivo treatment of traditional and patient-derived xenograft models with HDAC6 inhibitors resulted in a highly significant reduction of tumor growth. Translationally, it was also found that high levels of acetylated K2 were present in normal human breast tissue, which was lost in primary breast cancers and lymph node metastases. This suggests that blockade of HMGN2 deacetylation as described above is a novel treatment for breast cancer, given that existing HDAC6 inhibitors have a favorable toxicity profile in phase I trials of other tumor types. Altogether, these results reveal a novel mechanism through which HDAC6 regulates the transcription of Stat5 target genes and demonstrates the utility of HDAC6 inhibition as a potential breast cancer therapeutic. Citation Format: Justin M. Craig, Suzanne M. Schauwecker, Charles V. Clevenger. HDAC6 as a therapeutic target in human breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B11.

HMGN5 promotes proliferation and invasion via the activation of Wnt/β-catenin signaling pathway in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive carcinoma with a poor prognosis. A recent study has demonstrated that high mobility group nucleosome binding domain 5 (HMGN5) was involved in tumorigenesis and progression of multiple types of human cancers. However, the role of HMGN5 in PDAC is unknown. The objective of the present study was to analyze the function and novel mechanism of HMGN5 involved in PDAC cell progression. It was firstly revealed that the expression of HMGN5 was significantly upregulated in PDAC cell lines and tissues, when compared with that in normal pancreatic duct epithelial cells and adjacent normal pancreatic tissues. In vitro assay revealed that HMGN5 silencing impaired PDAC cell viability, proliferation, migration and invasion. Similarly, tumor growth rate was also decreased in vivo following HMGN5 silencing. Furthermore, it was demonstrated that HMGN5 silencing significantly inhibited epithelial-mesenchymal transition in vitro. Notably, HMGN5-medicated Wnt/β-catenin signaling pathway activation was observed to be one of the critical signal transduction pathways that associates HMGN5 with EMT activation. Collectively, the results indicated the important role of HMGN5 in PDAC cell proliferation and metastasis, and provide a promising target against the transcriptional program of PDAC.

HMGN5 (High Mobility Group Nucleosome binding domain 5)

HMGN5 (High Mobility Group Nucleosome binding domain 5)

Abstract P3-06-08: HDAC6 deacetylates HMGN2 to regulate Stat5a activity and breast cancer growth

Abstract Stat5 is a transcription factor utilized by several cytokine/hormone receptor signaling pathways that promotes transcription of genes associated with proliferation, differentiation, and survival of cancer cells. However, there are currently no clinically approved therapies that target Stat5, despite ample evidence that it contributes to breast cancer pathogenesis. Previous research in our lab has shown that the high mobility group nucleosome binding domain 2 (HGMN2) protein serves as a Stat5 co-activator. The activity of HMGN2 has been previously shown to be regulated by acetylation. Here, we show that deacetylation of HMGN2 on lysine residue K2 by histone deacetylase 6 (HDAC6) promotes Stat5-mediated transcription and breast cancer growth. Conversely, in vitro HDAC6 inhibition by pharmacologic and knock-down approaches enhanced HMGN2 acetylation with a concomitant reduction of in vitro Stat5-mediated signaling and global gene expression, and breast cancer growth. In vitro and in vivo treatment of traditional and patient-derived xenograft models with HDAC6 inhibitors resulted in a highly significant reduction of tumor growth. Translationally, it was also found that high levels of acetylated K2 were present in normal human breast tissue, which was lost in primary breast cancers and lymph node metastases. This suggests that blockade of HMGN2 deacetylation as described above is a novel treatment for breast cancer, given that existing HDAC6 inhibitors have a favorable toxicity profile in Phase I trials of other tumor types. Altogether, these results reveal a novel mechanism through which HDAC6 regulates the transcription of Stat5 target genes and demonstrates the utility of HDAC6 inhibition as a potential breast cancer therapeutic. Citation Format: Clevenger CV, Craig JM, Fiorillo AA, Feeney YB, Harrell JC, Medler TR. HDAC6 deacetylates HMGN2 to regulate Stat5a activity and breast cancer growth [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-06-08.

Histone H1 and Chromosomal Protein HMGN2 Regulate Prolactin-induced STAT5 Transcription Factor Recruitment and Function in Breast Cancer Cells

The hormone prolactin (PRL) contributes to breast cancer pathogenesis through various signaling pathways, one of the most notable being the JAK2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL-induced activation of the transcription factor STAT5 results in the up-regulation of numerous genes implicated in breast cancer pathogenesis. However, the molecular mechanisms that enable STAT5 to access the promoters of these genes are not well understood. Here, we show that PRL signaling induces chromatin decompaction at promoter DNA, corresponding with STAT5 binding. The chromatin-modifying protein high mobility group nucleosomal binding domain 2 (HMGN2) specifically promotes STAT5 accessibility at promoter DNA by facilitating the dissociation of the linker histone H1 in response to PRL. Knockdown of H1 rescues the decrease in PRL-induced transcription following HMGN2 knockdown, and it does so by allowing increased STAT5 recruitment. Moreover, H1 and STAT5 are shown to function antagonistically in regulating PRL-induced transcription as well as breast cancer cell biology. While reduced STAT5 activation results in decreased PRL-induced transcription and cell proliferation, knockdown of H1 rescues both of these effects. Taken together, we elucidate a novel mechanism whereby the linker histone H1 prevents STAT5 binding at promoter DNA, and the PRL-induced dissociation of H1 mediated by HMGN2 is necessary to allow full STAT5 recruitment and promote the biological effects of PRL signaling.

Knockdown of HMGN2 increases the internalization of Klebsiellapneumoniae by respiratory epithelial cells through the regulation of α5β1 integrin expression.

Integrin receptors, a large family of adhesion receptors, are involved in the attachment of Klebsiella pneumoniae to respiratory epithelial cells, and subsequently cause the internalization of K. pneumoniae by host cells. Although a number of molecules have been reported to regulate the expression and activity of integrin receptors in respiratory epithelial cells, the specific underlying molecular mechanisms remain largely unknown. High mobility group nucleosomal binding domain 2 (HMGN2), a non-histone nuclear protein, is present in eukaryotic cells as a ubiquitous nuclear protein. Our previous studies have demonstrated that HMGN2 affects chromatin function and modulates the expression of antibacterial peptide in A549 cells exposed to lipopolysaccharide, which indicates the critical role of HMGN2 in innate immune responses. In addition, our cDNA microarray analysis suggested that HMGN2 knockdown induced the enhanced expression of α5β1 integrin in A549 cells. Therefore, we hypothesized that intercellular HMGN2 may mediate the internalization of K. pneumoniae by altering the expression of α5β1 integrin. Using the A549 cell line, we demonstrated that HMGN2 knockdown induced the increased expression of α5β1 integrin on cell membranes, which resulted in a significant increase in K. pneumoniae internalization. Further results revealed that HMGN2 silencing induced the expression of talin and the activation of α5β1 integrin, which led to actin polymerization following the phosphorylation of FAK and Src. This study suggests a possible therapeutic application for bacterial internalization by targeting HMGN2 in order to treat K. pneumoniae infection.

Release mechanism of high mobility group nucleosome binding domain 1 from lipopolysaccharide-stimulated macrophages.

Alarmins are identified as endogenous mediators that have potent immune-activating abilities. High mobility group nucleosome binding domain 1 (HMGN1), a highly conserved, non-histone chromosomal protein, which binds to the inner side of the nucleosomal DNA, regulates chromatin dynamics and transcription in cells. Furthermore, HMGN1 acts as a cytokine in the extracellular milieu by inducing the recruitment and maturation of antigen-presenting cells (dendritic cells) to enhance Th1-type antigen-specific immune responses. Thus, HMGN1 is expected to act as an alarmin, when released into the extracellular milieu. The present study investigated the release mechanism of HMGN1 from macrophages using mouse macrophage‑like RAW264.7 cells. The results indicated that HMGN1 was released from lipopolysaccharide (LPS)‑stimulated RAW264.7 cells, accompanied by cell death as assessed by the release of lactate dehydrogenase (LDH). Subsequently, the patterns of cell death involved in HMGN1 release from LPS‑stimulated RAW264.7 cells were determined using a caspase‑1 inhibitor, YVAD, and a necroptosis inhibitor, Nec‑1. YVAD and Nec‑1 did not alter LPS‑induced HMGN1 and LDH release, suggesting that pyroptosis (caspase‑1‑activated cell death) and necroptosis are not involved in the release of HMGN1 from LPS‑stimulated RAW264.7 cells. In addition, flow cytometric analysis indicated that LPS stimulation did not induce apoptosis but substantially augmented necrosis, as evidenced by staining with annexin V/propidium iodide. Together these findings suggest that HMGN1 is extracellularly released from LPS‑stimulated RAW264.7 macrophage‑like cells, accompanied by unprogrammed necrotic cell death but not pyroptosis, necroptosis or apoptosis.

High Mobility Group Nucleosomal Binding Domain 2 (HMGN2) SUMOylation by the SUMO E3 Ligase PIAS1 Decreases the Binding Affinity to Nucleosome Core Particles

High mobility group nucleosomal binding domain 2 (HMGN2) is a small and unique non-histone protein that has many functions in a variety of cellular processes, including regulation of chromatin structure, transcription, and DNA repair. In addition, it may have other roles in antimicrobial activity, cell homing, and regulating cytokine release. Although the biochemical properties of HMGN2 protein are regulated by acetylation and phosphorylation, it is not yet known whether HMGN2 activity can also be regulated by SUMOylation. In this study, we demonstrated for the first time that HMGN2 is modified by covalent attachment of small ubiquitin-related modifier 1 (SUMO1) by pro-inflammatory signal and identified the major SUMOylated lysine residues that localize to the HMGN2 nucleosome-binding domain at Lys-17 and Lys-35. SENP1 can deSUMOylate SUMOylated HMGN2, and PIAS1 is the E3 ligase responsible for SUMOylation of HMGN2. Finally, using SUMO1-conjugated HMGN2 purified from a basal SUMOylation system in Escherichia coli, we demonstrated that SUMOylated HMGN2 has decreased the binding affinity to nucleosome core particles in comparison to unSUMOylated HMGN2. These observations potentially provide new perspectives for understanding the functions of HMGN2 in inflammatory reaction.

The expression and clinical significance of high mobility group nucleosome binding domain 5 in human osteosarcoma

The high mobility group nucleosome binding domain 5 (HMGN5) is a member of the high mobility group proteins family and highly expressed in multiple malignancies. Nevertheless, the role of HMGN5 in osteosarcoma remains unclear. This study aimed to investigate the expression and clinical significance of HMGN5 in human osteosarcoma, confirm the oncogenic role of HMGN5 and explore the mechanism by which HMGN5 contributes to invasion and metastasis. HMGN5 expression was detected in osteosarcoma tissues and corresponding adjacent non-cancerous tissues (ANCTs) from 52 patients by immunohistochemical (IHC) assay and the clinicopathologic characteristics of all patients were also recorded. Next, osteosarcoma cells were transfected by HMGN5 RNA interference and cell viability, apoptosis and invasion were detected by cell vitality test, flow cytometry and transwell assay, respectively. As a result, IHC assay showed strong immunized activity of HMGN5 in the nucleus in all osteosarcoma tissues compared with the ANCT (53.5 ± 4.3 % vs. 17.0 ± 3.9 %, P < 0.01). HMGN5 expression level was associated with pathologic staging and TNM staging. Knockdown of HMGN5 induced cell cycle arrest, inhibited invasion and increased sensitivity to doxorubicin-induced cell apoptosis in U2-OS and SaO2 cells. Western blot analysis demonstrated that there were increased expressions of cleaved caspase-3, cleaved PARP and decreased expressions of PCNA, PI3Kp85α, p-AKT, MMP-9, and cyclin B1 in U2-OS and SaO2 cells depleted of HMGN5. HMGN5 plays oncogenic role in osteosarcoma by promoting cell proliferation and invasion, and could be exploited as a target for therapy in osteosarcoma.