Loss of Endometrial Plasticity in Recurrent Pregnancy Loss.

Menstruation drives cyclic activation of endometrial progenitor cells, tissue regeneration, and maturation of stromal cells, which differentiate into specialized decidual cells prior to and during pregnancy. Aberrant responsiveness of human endometrial stromal cells (HESCs) to deciduogenic cues is strongly associated with recurrent pregnancy loss (RPL), suggesting a defect in cellular maturation. MeDIP-seq analysis of HESCs did not reveal gross perturbations in CpG methylation in RPL cultures, although quantitative differences were observed in or near genes that are frequently deregulated in vivo. However, RPL was associated with a marked reduction in methylation of defined CA-rich motifs located throughout the genome but enriched near telomeres. Non-CpG methylation is a hallmark of cellular multipotency. Congruently, we demonstrate that RPL is associated with a deficiency in endometrial clonogenic cell populations. Loss of epigenetic stemness features also correlated with intragenic CpG hypomethylation and reduced expression of HMGB2, coding high mobility group protein 2. We show that knockdown of this sequence-independent chromatin protein in HESCs promotes senescence and impairs decidualization, exemplified by blunted time-dependent secretome changes. Our findings indicate that stem cell deficiency and accelerated stromal senescence limit the differentiation capacity of the endometrium and predispose for pregnancy failure.

The combination of a nuclear HMGB1-positive and HMGB2-negative expression is potentially associated with a shortened survival in patients with pancreatic ductal adenocarcinoma.

High-mobility group box (HMGB) proteins are ubiquitous, abundant nuclear non-histone chromosomal proteins that play a critical role in binding to distorted DNA structures and subsequently regulating DNA transcription, replication, repair, and recombination. Both HMGB1 and HMGB2 exhibit a high expression in several human cancers and are closely associated with tumor progression and a poor prognosis. However, the expression patterns of these molecules in pancreatic ductal adenocarcinoma (PDAC) remain to be elucidated. As most cases of postoperative relapse of PDAC occur within the first 2 years, the clinical significance of accurate biomarkers is needed. Therefore, we investigated the correlation between the immunohistochemical HMGB1 and HMGB2 expression and the clinicopathological characteristics and prognosis using 62 paraffin-embedded tumor samples obtained from patients with surgically resected PDAC. The HMGB1/2 expression was considered to be positive when 10 % or more of the cancer cells showed positive nuclear, not merely cytoplasmic, staining. Consequently, the expression of HMGB1/2 was observed in 54 (87.1 %) and 31 (50.0 %) patients, respectively. Unexpectedly, a positive HMGB1 expression was found to have a significantly close relationship with a negative HMGB2 expression. The univariate and multivariate analyses demonstrated that the patients with a HMGB1+ and HMGB2- status had markedly lower disease-specific survival rates, especially within the first 2 years postoperatively, whereas those with a HMGB1+ status alone did not. Therefore, the combination of a HMGB1+ and HMGB2- expression potentially predicts a poor prognosis in patients with PDAC, and these new biomarkers may be useful parameters for clinical management in the early postoperative phase.

Expression patterns and function of chromatin protein HMGB2 during mesenchymal stem cell differentiation.

The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis (OA). The expression of chromatin protein HMGB2 is restricted to the SZ, which contains cells expressing mesenchymal stem cell (MSC) markers. Age-related loss of HMGB2 and gene deletion are associated with reduced SZ cellularity and early onset OA. This study addressed HMGB2 expression patterns in MSC and its role during differentiation. HMGB2 was detected at higher levels in human MSC as compared with human articular chondrocytes, and its expression declined during chondrogenic differentiation of MSC. Lentiviral HMGB2 transduction of MSC suppressed chondrogenesis as reflected by an inhibition of Col2a1 and Col10a1 expression. Conversely, in bone marrow MSC from Hmgb2(-/-) mice, Col10a1 was more strongly expressed than in wild-type MSC. This is consistent with in vivo results from mouse growth plates showing that Hmgb2 is expressed in proliferating and prehypertrophic zones but not in hypertrophic cartilage where Col10a1 is strongly expressed. Osteogenesis was also accelerated in Hmgb2(-/-) MSC. The expression of Runx2, which plays a major role in late stage chondrocyte differentiation, was enhanced in Hmgb2(-/-) MSC, and HMGB2 negatively regulated the stimulatory effect of Wnt/ß-catenin signaling on the Runx2 proximal promoter. These results demonstrate that HMGB2 expression is inversely correlated with the differentiation status of MSC and that HMGB2 suppresses chondrogenic differentiation. The age-related loss of HMGB2 in articular cartilage may represent a mechanism responsible for the decline in adult cartilage stem cell populations.

Involvement of HMGB1 and HMGB2 proteins in exogenous DNA integration reaction into the genome of HeLa S3 cells.

High mobility group 1 and 2 proteins (HMGB1 and HMGB2) are abundant chromosomal proteins in eukaryotic cells. We examined the involvement of HMGB1 and HMGB2 in nonhomologous illegitimate recombination. The HMGB1 or HMGB2 expression plasmid, carrying the neo(r) gene as a selection marker, was introduced into HeLa S3 cells to obtain stably-transfected cells. The number of G418-resistant colonies was about 10 times the number of colonies of control cells transfected with plasmids not carrying the HMGB genes. The copy number of the stably-integrated neo(r) gene was higher in the cells transfected with the HMGB expression plasmids than in control cells. The exogenous DNA integration was suggested to have occurred by nonhomologous illegitimate recombination. On the contrary, the introduction of the HMGB antisense RNA expression plasmid with a reporter plasmid carrying the neo(r) gene into HeLa S3 cells decreased the number of G418-resistant colonies. These results indicate that HMGB1 and HMGB2 each have a novel function as stimulators of stable integration of plasmid DNA into the host genome and that they may be important for the process of spontaneous DNA integration in living cells.

Molecular characterization of three 3-hydroxy-3-methylglutaryl-CoA reductase genes including pathogen-induced Hmg2 from pepper (Capsicum annuum).

Sesquiterpene phytoalexins, a class of plant defense metabolites, are synthesized from the cytosolic acetate/mevalonate pathway in isoprenoids biosynthetic system of plants. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the synthesis of mevalonate, which is the specific precursor of this pathway, as a multi gene family. Three kinds of cDNA clones encoding HMGR were isolated from Korean red pepper (Capsicum annuum L. cv. NocKwang) and the HMGR2 gene (Hmg2) was especially obtained from a cDNA library constructed with Phytophthora capsici-infected pepper root RNAs. The Hmg2 encoding a 604-amino-acid peptide had typical features as an elicitor-induced isoform among HMGRs on its gene structure and had a predicted amino acid sequence homology. In addition, the expression of Hmg2 was rapidly induced within 1 h in response to a fungal pathogen and continuously increased up to 48 h. Together with sesquiterpene cyclase gene that was strongly induced 24 h after pathogen-infection, the Hmg2 and farnesyl pyrophosphate synthase gene were coordinately and sequentially regulated for the biosynthesis of defense-related sesquiterpene phytoalexins in pepper.

Gene expression profiling of a clonal isolate of oxaliplatin-resistant ovarian carcinoma cell line A2780/C10.

The efficacy of platinum drugs in the treatment of cancer is often restricted by the acquisition of tumor cell resistance subsequent to treatment. To better understand mechanisms involved in this phenomenon, a clonal subline (A2780/C10B) isolated from an oxaliplatin-resistant human ovarian carcinoma cell line (A2780/C10) was developed, as reported previously. This cell line is 18-fold resistant to oxaliplatin and shows a 3-fold cross resistance to cisplatin. Here, we report on the gene expression analysis using Affymetrix HG-U95Av2 oligonucleotide arrays of cells in log phase growth from both the parental cell line and drug-resistant variant. Probe level analysis was perfomed using the model based expression index (dChip) and robust multichip average (RMA) methods. Genes that were differentially expressed between the two groups were identified using the significance analysis of microarrays (SAM) method with a minimum false discovery rate <1%. We identified 43 genes that were overexpressed, and 39 underexpressed in the drug-resistant cell line. Collagen VI (COL6A3) was overexpressed 62-fold and the most highly up-regulated gene. This finding is consistent with other published data based on serial analysis of gene expression (SAGE) profiling of cisplatin-resistant and sensitive ovarian carcinoma cells. Among the significant functional groups of overexpressed genes in our study were extracellular matrix genes (9 of 43) and those involved in signal transduction (7 of 43). Extracellular matrix genes included two matrix metalloproteinases (MMP3 and MMP12). Integrin alpha 1 (ITGA1) and WNT5A were also overexpressed. Genes that encode for extracellular matrix proteins were also among those found down-regulated in the resistant cell line. Several genes involved in the regulation of cell cycle and growth were found to be underexpressed, including the suppressor of cytokine signaling 2 (SOCS2), necdin (NDN), and glypicans (GPC3 and GPC4). The mRNA levels of six differentially expressed genes (COL6A3, MMP12, MMP3, WNT5A, NID, and HMGB2) were validated using real-time quantitative RT-PCR. The identification of these genes should aid in a better understanding of the pathways resulting in platinum drug resistance.

Cytosolic HMGB1 expression in human renal clear cell cancer indicates higher pathological T classifications and tumor grades.

PURPOSE: High mobility group box (HMGB) proteins are nuclear nonhistone chromosomal proteins that bend DNA, bind preferentially to distorted DNA structures, and promote the assembly of site-specific DNA binding proteins. Recent reports indicate that HMGB1 functions as a proinflammatory cytokine. Here, we studied expressions of HMGB1 and HMGB2 in human renal cancer. MATERIAL AND METHODS: Immunohistological expressions of HMGB1 and HMGB2 were assessed in 39 surgically resected human renal cancer specimens. RESULTS: HMGB1 was expressed in the nucleus in 37 out of 39 (94.9%) renal clear cell cancers,while its expression in the cytosol was noted in 19 cases (48.7%). Cytosolic HMGB1 is expressed more frequently in cancers beyond the pT1b classification than in those at the pT1a classification. Higher tumor grades (≥ G2) were also significantly linked with the cytosolic expression of HMGB1. HMGB2 was expressed in the nucleus in 35 of 39 (89.7%) renal clear cell cancers, while its expression in the cytosol was observed in only 7 case (17.9%). Linkage between cytosolic expression of HMGB2 and T classifications was weakly observed, while that between nuclear expression and T classifications was not. CONCLUSION: HMGB1 expressed in the cytoplasm may be an effective marker indicating higher T classifications and tumor grades.

Overexpression of high-mobility group box 2 is associated with tumor aggressiveness and prognosis of hepatocellular carcinoma.

PURPOSE: We investigated the expression of high-mobility group box 2 (HMGB2) in patients with hepatocellular carcinoma (HCC) and its clinical effects with underlying mechanisms. EXPERIMENTAL DESIGN: HMGB2 mRNA levels were measured in 334 HCC patients by real-time reverse transcription-PCR and HMGB2 protein levels in 173 HCC patients by immunohistochemical studies. The HMGB2 expression level was measured by Western blotting for three HCC cell lines. To clarify the precise role of HMGB2 on cell proliferation, we did in vitro analysis with expression vectors and small interfering RNAs. RESULTS: HMGB2 mRNA and protein expression were significantly higher in HCC than in noncancerous surrounding tissues (P < 0.0001) and showed a positive correlation (ρ = 0.35, P < 0.001). HMGB2 overexpression was significantly correlated with shorter overall survival time, both at mRNA (P = 0.0054) and protein level (P = 0.023). Moreover, HMGB2 mRNA level was an independent prognostic factor for overall survival in a multivariate analysis (P = 0.0037). HMGB2 knockdown by small interfering RNAs decreased cell proliferation, and overexpression of HMGB2 by expression vectors diminished cisplatin- and etoposide-induced cell death. CONCLUSIONS: Our clinical and in vitro data suggest that HMGB2 plays a significant role in tumor development and prognosis of HCC. These results can partly be explained by altered cell proliferations by HMGB2 associated with the antiapoptotic pathway.

Bacterial LPS-mediated acute inflammation-induced spermatogenic failure in rats: role of stress response proteins and mitochondrial dysfunction.

Bacterial Lipopolysaccharide (LPS) induced inflammation is implicated in the infection associated testicular tissue damage. Earlier, using a LPS induced acute endotoxemic rat model, we have shown the involvement of inflammation-induced oxidative stress in the impaired steroidogenesis and spermatogenesis. In the present study, we report a significant induction (more than 2-fold) of stress response proteins HSP-60, HMGB-1 and 2 in the testes, as early as 6 h after LPS injection with a later decrease. This induction of acute stress is closely followed by a significant reduction (74%) in Bcl2/Bax ratio along with leakage of cytochrome c (3 fold increase, p < 0.05) from mitochondria and increased caspase-3 activity levels (2.9 fold, p < 0.05) at 12 h and 24 h post LPS injection respectively. Further studies on PARP cleavage revealed a pattern similar to necrotic death during early periods (3 h to 24 h) and apoptosis at later periods (24 h to 72 h) after LPS treatment. In conclusion, the present study shows the involvement of stress response proteins and mitochondrial dysfunction in LPS-induced germ cell death in male rats.