High-mobility group box 1 fragment ameliorates chronic pancreatitis induced by caerulein in mice.

BACKGROUND: Chronic pancreatitis (CP) is a progressive disease characterized by pancreatic fibrosis for which effective treatment options are lacking. Mesenchymal stem cells (MSCs) have shown potential for fibrosis treatment but face limitations in clinical application. The high-mobility group box 1 (HMGB1) fragment mobilizes MSCs from bone marrow into the blood and has emerged as a promising therapeutic agent for tissue regeneration in various pathological conditions. The aim of this study was to investigate the potential therapeutic effects of systemic administration of the HMGB1 fragment in a mouse model of CP. METHODS: A caerulein-induced CP mouse model was used, and the HMGB1 fragment was administered by tail vein injection. Parameters such as body weight, pancreatic tissue damage, fibrosis, inflammatory cytokine expression, and collagen-related gene expression were evaluated using various assays, including immunohistochemistry, real-time PCR, serum analysis, and single-cell transcriptome analysis. And the migration of MSCs to the pancreas was evaluated using the parabiosis model. RESULTS: Administration of the HMGB1 fragment was associated with significant improvements in pancreatic tissue damage and fibrosis. It suppressed the expression of inflammatory cytokines and activated platelet-derived growth factor receptor-α+ MSCs, leading to their accumulation in the pancreas. The HMGB1 fragment also shifted gene expression patterns associated with pancreatic fibrosis toward those of the normal pancreas. Systemic administration of the HMGB1 fragment demonstrated therapeutic efficacy in attenuating pancreatic tissue damage and fibrosis in a CP mouse model. CONCLUSION: These findings highlight the potential of the HMGB1 fragment as a therapeutic target for the treatment of CP.

Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.

Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here, we determine the roles of CHD4 in enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility. Its depletion leads to redistribution of transcription factors to previously unoccupied sites. During cellular reprogramming induced by the pioneer factor GATA3, CHD4 activity is necessary to prevent inappropriate chromatin opening. Mechanistically, CHD4 promotes nucleosome positioning over GATA3 binding motifs to compete with transcription factor-DNA interaction. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents unnecessary gene expression by editing chromatin binding activities of transcription factors.

High-mobility group box-1 peptide ameliorates bronchopulmonary dysplasia by suppressing inflammation and fibrosis in a mouse model.

BACKGROUND: This study aimed to examine the effect of the HMGB1 peptide on Bronchopulmonary dysplasia (BPD)-related lung injury in a mouse model. RESULTS: HMGB1 peptide ameliorates lung injury by suppressing the release of inflammatory cytokines and decreasing soluble collagen levels in the lungs. Single-cell RNA sequencing showed that the peptide suppressed the hyperoxia-induced inflammatory signature in macrophages and the fibrotic signature in fibroblasts. These changes in the transcriptome were confirmed using protein assays. CONCLUSION: Systemic administration of HMGB1 peptide exerts anti-inflammatory and anti-fibrotic effects in a mouse model of BPD. This study provides a foundation for the development of new and effective therapies for BPD.

Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.

Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here we determine the roles of CHD4 to enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility at transcription factor binding sites; its depletion leads to altered motif scanning and redistribution of transcription factors to sites not previously occupied. During GATA3-mediated cellular reprogramming, CHD4 activity is necessary to prevent inappropriate chromatin opening and enhancer licensing. Mechanistically, CHD4 competes with transcription factor-DNA interaction by promoting nucleosome positioning over binding motifs. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents inappropriate gene expression by editing binding site selection by transcription factors.

Synthesized HMGB1 peptide prevents the progression of inflammation, steatosis, fibrosis, and tumor occurrence in a non-alcoholic steatohepatitis mouse model.

AIM: Non-alcoholic steatohepatitis (NASH) with fibrosis eventually leads to cirrhosis and hepatocellular carcinoma. Thus, the development of therapies other than dietary restriction and exercise, particularly those that suppress steatosis and fibrosis of the liver and have a long-term beneficial effect, is necessary. We aimed to evaluate the therapeutic effects of the HMGB1 peptide synthesized from box A using the melanocortin-4 receptor-deficient (Mc4r-KO) NASH model mouse. METHODS: We performed short- and long-term administration of this peptide and evaluated the effects on steatosis, fibrosis, and carcinogenesis using Mc4r-KO mice. We also analyzed the direct effect of this peptide on macrophages and hepatic stellate cells in vitro and performed lipidomics and metabolomics techniques to evaluate the effect. RESULTS: Although this peptide did not show direct effects on macrophages and hepatic stellate cells in vitro, in the short-term administration model, we could confirm the reduction of liver damage, steatosis, and fibrosis progression. The results of lipidomics and metabolomics suggested that the peptide might ameliorate NASH by promoting lipolysis via the activation of fatty acid ß-oxidation and improving insulin resistance. In the long-term administration model, this peptide prevented progression to cirrhosis but retained the steatosis state, that is, the peptide prevents the progression to "burnt-out NASH." This peptide inhibited carcinogenesis by about one-third. CONCLUSION: This HMGB1 peptide can reduce liver damage, improve fibrosis and steatosis, and inhibit carcinogenesis, suggesting that the peptide would be a new treatment candidate for NASH and can contribute to the long-term prognosis for patients with NASH.

Single-cell transcriptome analysis of fractional CO2 laser efficiency in treating a mouse model of alopecia.

OBJECTIVES: Hair loss, including alopecia, is a common dermatological issue worldwide. At present, the application of fractional carbon dioxide (CO2 ) laser in the treatment of alopecia has been documented; however, the results vary between reports. These varying results may be due to the limited knowledge of cellular action in laser-irradiated skin. The objective of this study was to investigate the molecular and cellular mechanisms of laser treatment under effective conditions for hair cycle initiation. METHODS: A fractional CO2 laser was applied and optimized to initiate the hair cycle in a mouse model of alopecia. Several cellular markers were analyzed in the irradiated skin using immunofluorescence staining. Cellular populations and their comprehensive gene expression were analyzed using single-cell RNA sequencing and bioinformatics. RESULTS: The effective irradiation condition for initiating the hair cycle was found to be 15 mJ energy/spot, which generates approximately 500 µm depth columns, but does not penetrate the dermis, only reaching approximately 1 spot/mm2 . The proportion of macrophage clusters significantly increased upon irradiation, whereas the proportion of fibroblast clusters decreased. The macrophages strongly expressed C-C chemokine receptor type 2 (Ccr2), which is known to be a key signal for injury-induced hair growth. CONCLUSIONS: We found that fractional CO2 laser irradiation recruited Ccr2 positive macrophages, and induced hair regrowth in a mouse alopecia model. These findings may contribute to the development of stable and effective fractional laser irradiation conditions for human alopecia treatment.

Longitudinal Single-Cell Transcriptomics Reveals a Role for Serpina3n-Mediated Resolution of Inflammation in a Mouse Colitis Model.

BACKGROUND & AIMS: Proper resolution of inflammation is essential to maintaining homeostasis, which is important as a dysregulated inflammatory response has adverse consequences, even being regarded as a hallmark of cancer. However, our picture of dynamic changes during inflammation remains far from comprehensive. METHODS: Here we used single-cell transcriptomics to elucidate changes in distinct cell types and their interactions in a mouse model of chemically induced colitis. RESULTS: Our analysis highlights the stromal cell population of the colon functions as a hub with dynamically changing roles over time. Importantly, we found that Serpina3n, a serine protease inhibitor, is specifically expressed in stromal cell clusters as inflammation resolves, interacting with a potential target, elastase. Indeed, genetic ablation of the Serpina3n gene delays resolution of induced inflammation. Furthermore, systemic Serpina3n administration promoted the resolution of inflammation, ameliorating colitis symptoms. CONCLUSIONS: This study provides a comprehensive, single-cell understanding of cell-cell interactions during colorectal inflammation and reveals a potential therapeutic target that leverages inflammation resolution.

Controlled induction of immune tolerance by mesenchymal stem cells transferred by maternal microchimerism.

Feto-maternal immune tolerance is established during pregnancy; however, its mechanism and maintenance remain underexplored. Here, we investigated whether mesenchymal stem/stromal cells (MSCs) as non-inherited maternal antigens (NIMAs) transferred by maternal microchimerism could induce immune tolerance. We showed that MSCs had a potential equivalent to hematopoietic stem and progenitor cells (HSPCs) to induce immune tolerance and that MSCs were essential to induce tolerance to MSC-specific antigens. Furthermore, we demonstrated that MSCs as NIMAs transferred by maternal microchimerism could induce robust immune tolerance that can be further enhanced using a drug. Our data shed light on induction of immune tolerance and serve as a foundation to develop new therapies using maternally derived cells for autoimmune or genetic diseases.

Contribution of PDGFRα lineage cells in adult mouse hematopoiesis.

Platelet-derived growth factor receptor alpha (PDGFRα) is a dominant marker of mesodermal mesenchymal cells in mice. Previous studies demonstrated that PDGFRα-positive (PDGFRα+) mesodermal cells develop not only into mesenchymal cells but also into a subset of total hematopoietic cells (HCs) in the limited period during mouse embryogenesis. However, the precise characteristics of the PDGFRα lineage positive (PDGFRα Lin+) HCs in adult mouse hematopoiesis are largely unknown. In this study, we systematically evaluated the characteristics of PDGFRα Lin+ HCs in the bone marrow and peripheral blood using PDGFRα-CRE; ROSAtdTomato mice. Flow cytometry analysis revealed that PDGFRα Lin+ HCs accounted for approximately 20% of total HCs in both the bone marrow and peripheral blood in adult mice. Compositions of myeloid and lymphoid subpopulations among CD45+ mononuclear cells were almost identical in both PDGFRα Lin+ and PDGFRα Lin- cells. Single-cell RNA-sequencing analysis also demonstrated that the transcriptomic signatures of the PDGFRα Lin+ HCs in the peripheral blood largely overlapped with those of the PDGFRα Lin- HCs, suggesting equivalent functions of the PDGFRα Lin+ and PDGFRα Lin- HCs. Although pathophysiological activities of the PDGFRα Lin + HCs were not evaluated, our data clearly demonstrate a significant role of the PDGFRα Lin + HCs in physiological hematopoiesis in adult mice.

Transcriptionally distinct mesenchymal stem/stromal cells circulate in fetus.

Umbilical cord blood contains mesenchymal stem/stromal cells (MSCs) in addition to hematopoietic stem cells, serving as an attractive tool for regenerative medicine. As umbilical cord blood originates from fetus, abundant MSCs are expected to circulate in fetus. However, the properties of circulating MSCs in fetus have not been fully examined. In the present study, we aimed to analyze circulating MSCs, marked by the expression of platelet-derived growth factor receptor α (PDGFRα), during fetal development. Using PDGFRα GFP knock-in mice, we quantified the number of circulating PDGFRα positive MSCs during development. We further performed whole transcriptome analysis of circulating MSCs at single cell levels. We found that abundant PDGFRα positive cells circulate in embryo and diminish immediately after birth. In addition, single cell RNA-sequencing revealed transcriptional heterogeneity of MSCs in fetal circulation. These data lay a foundation to analyze the function of circulating MSCs during development.

Chromatin accessibility identifies diversity in mesenchymal stem cells from different tissue origins.

Mesenchymal stem cells (MSCs), which can differentiate into tri-lineage (osteoblast, adipocyte, and chondrocyte) and suppress inflammation, are promising tools for regenerative medicine. MSCs are phenotypically diverse based on their tissue origins. However, the mechanisms underlying cell-type-specific gene expression patterns are not fully understood due to the lack of suitable strategy to identify the diversity. In this study, we investigated gene expression programs and chromatin accessibilities of MSCs by whole-transcriptome RNA-seq analysis and an assay for transposase-accessible chromatin using sequencing (ATAC-seq). We isolated MSCs from four tissues (femoral and vertebral bone marrow, adipose tissue, and lung) and analysed their molecular signatures. RNA-seq identified the expression of MSC markers and both RNA-seq and ATAC-seq successfully clustered the MSCs based on their tissue origins. Interestingly, clustering based on tissue origin was more accurate with chromatin accessibility signatures than with transcriptome profiles. Furthermore, we identified transcription factors potentially involved in establishing cell-type specific chromatin structures. Thus, epigenome analysis is useful to analyse MSC identity and can be utilized to characterize these cells for clinical use.

DNA Methylation Changes in Tbx3 in a Mouse Model Exposed to Polybrominated Diphenyl Ethers.

DE-71, a commercial mixture of polybrominated diphenyl ethers widely used in flame retardants, is a pervasive environmental contaminant due to its continuing release from waste material and its long half-life in humans. Although the genotoxic potential of DE-71 appears to be low based on bacterial mutagenicity, it remains a public health concern due to its reported involvement in tumor development. Molecular mechanisms by which DE-71 influences tumor incidence or progression remain understudied. We used liver carcinoma tissue from mice exposed to DE-71 to test the hypothesis that epigenetic alterations consistent with tumor development, specifically DNA methylation, result from long-term DE-71 exposure. We profiled DNA methylation status using the methylated-CpG island recovery assay coupled with microarray analysis of hepatocellular carcinoma DNA from animals exposed to DE-71. DE-71 exposure had little impact on global DNA methylation. However, we detected gene body-specific hypomethylation within the Tbx3 locus, a transcription factor important in liver tumorigenesis and in embryonic and cancer stem cell proliferation. This nonpromoter hypomethylation was accompanied by upregulation of Tbx3 mRNA and protein and by alterations in downstream cell cycle-associated marker expression. Thus, exposure to DE-71 may facilitate tumor development by inducing epigenetic programs that favor expansion of progenitor cell populations.

High-quality ChIP-seq analysis of MBD3 in human breast cancer cells.

Chromatin accessibility is tightly regulated by multiple factors/mechanisms to establish different cell type-specific gene expression programs from a single genome. Dysregulation of this process can lead to diseases including cancer. The Mi-2/nucleosome remodeling and deacetylase (NuRD) complex is thought to orchestrate chromatin structure using its intrinsic nucleosome remodeling and histone deacetylase activities. However, the detailed mechanisms by which the NuRD complex regulates chromatin structure in vivo are not yet known. To explore the regulatory mechanisms of the NuRD complex, we mapped genome-wide localization of MBD3, a structural component of NuRD, in a human breast cancer cell line (MDA-MB-231) using a modified ChIP-seq protocol. Our data showed high quality localization information (i.e., high mapping efficiency and low PCR duplication rate) and excellent consistency between biological replicates. The data are deposited in the Gene Expression Omnibus (GSE76116).

GATA3-dependent cellular reprogramming requires activation-domain dependent recruitment of a chromatin remodeler.

BACKGROUND: Transcription factor-dependent cellular reprogramming is integral to normal development and is central to production of induced pluripotent stem cells. This process typically requires pioneer transcription factors (TFs) to induce de novo formation of enhancers at previously closed chromatin. Mechanistic information on this process is currently sparse. RESULTS: Here we explore the mechanistic basis by which GATA3 functions as a pioneer TF in a cellular reprogramming event relevant to breast cancer, the mesenchymal to epithelial transition (MET). In some instances, GATA3 binds previously inaccessible chromatin, characterized by stable, positioned nucleosomes where it induces nucleosome eviction, alters local histone modifications, and remodels local chromatin architecture. At other loci, GATA3 binding induces nucleosome sliding without concomitant generation of accessible chromatin. Deletion of the transactivation domain retains the chromatin binding ability of GATA3 but cripples chromatin reprogramming ability, resulting in failure to induce MET. CONCLUSIONS: These data provide mechanistic insights into GATA3-mediated chromatin reprogramming during MET, and suggest unexpected complexity to TF pioneering. Successful reprogramming requires stable binding to a nucleosomal site; activation domain-dependent recruitment of co-factors including BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex; and appropriate genomic context. The resulting model provides a new conceptual framework for de novo enhancer establishment by a pioneer TF.

Genome-wide binding of MBD2 reveals strong preference for highly methylated loci.

MBD2 is a subunit of the NuRD complex that is postulated to mediate gene repression via recruitment of the complex to methylated DNA. In this study we adopted an MBD2 tagging-approach to study its genome wide binding characteristics. We show that in vivo MBD2 is mainly recruited to CpG island promoters that are highly methylated. Interestingly, MBD2 binds around 1 kb downstream of the transcription start site of a subset of ∼ 400 CpG island promoters that are characterized by the presence of active histone marks, RNA polymerase II (Pol2) and low to medium gene expression levels and H3K36me3 deposition. These tagged-MBD2 binding sites in MCF-7 show increased methylation in a cohort of primary breast cancers but not in normal breast samples, suggesting a putative role for MBD2 in breast cancer.

13-Cis retinoic acid can enhance the antitumor activity of non-replicating Sendai virus particle against neuroblastoma.

Hemagglutinating virus of Japan-envelope (HVJ-E) is a drug delivery vector based on inactivated Sendai virus. Recently, antitumor activities were found for HVJ-E itself and clinical trials of HVJ-E for some malignant tumors are now ongoing. We investigated the in vitro and in vivo antitumor effects of HVJ-E against neuroblastoma, which is one of the most common malignant solid tumors in childhood. The sensitivity of human neuroblastoma cell lines to HVJ-E correlated with the expression level of gangliosides, Sialylparagloboside (SPG) and GD1a, receptors for HVJ. Among the cell lines, SK-N-SH was the most sensitive to HVJ-E in vitro and total SPG and GD1a expression was the highest. Complete eradication of subcutaneous tumors derived from SK-N-SH cells was achieved by intratumoral injection of HVJ-E in SCID mice and no recurrence was observed for more than 300 days after HVJ-E inoculation. In contrast, NB1 cells expressed the lowest amount of GD1a and SPG and were resistant to HVJ-E in vitro. The expression of GD1a increased by 13-cis retinoic acid (13cRA), which is a therapeutic drug for high risk neuroblastoma, thus leading to an improved sensitivity to HVJ-E in vitro. Only growth inhibition of the subcutaneous tumors derived from NB1 cells was achieved by HVJ-E in the SCID mice, but the combination of 13cRA and HVJ-E could achieve partial eradication of the xenograft and also lead to an improved prognosis. In conclusion, HVJ-E is a promising therapeutic modality for neuroblastoma and 13cRA can be used as an adjuvant to HVJ-E.

MBD3 localizes at promoters, gene bodies and enhancers of active genes.

The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. Recent reports describing localization of NuRD provide new insights that question previous models on NuRD action, but are not in complete agreement. Here, we provide location analysis of endogenous MBD3, a component of NuRD complex, in two human breast cancer cell lines (MCF-7 and MDA-MB-231) using two independent genomic techniques: DNA adenine methyltransferase identification (DamID) and ChIP-seq. We observed concordance of the resulting genomic localization, suggesting that these studies are converging on a robust map for NuRD in the cancer cell genome. MBD3 preferentially associated with CpG rich promoters marked by H3K4me3 and showed cell-type specific localization across gene bodies, peaking around the transcription start site. A subset of sites bound by MBD3 was enriched in H3K27ac and was in physical proximity to promoters in three-dimensional space, suggesting function as enhancers. MBD3 enrichment was also noted at promoters modified by H3K27me3. Functional analysis of chromatin indicated that MBD3 regulates nucleosome occupancy near promoters and in gene bodies. These data suggest that MBD3, and by extension the NuRD complex, may have multiple roles in fine tuning expression for both active and silent genes, representing an important step in defining regulatory mechanisms by which NuRD complex controls chromatin structure and modification status.

TRAIL and Noxa are selectively upregulated in prostate cancer cells downstream of the RIG-I/MAVS signaling pathway by nonreplicating Sendai virus particles.

PURPOSE: The treatment of cancer with oncolytic viruses primarily depends on the selective viral replication in cancer cells. However, a replication-incompetent hemagglutinating virus of Japan (HVJ; Sendai virus) envelope (HVJ-E) suppresses the growth of human cancer cells as effectively as replication-competent live HVJ without producing toxic effects in nonmalignant cells. Here, we analyze the molecular mechanism of the oncolytic activity of HVJ-E. EXPERIMENTAL DESIGN: The molecules responsible for HVJ-E-induced cancer cell death were elucidated in prostate cancer cell lines, and the effect of HVJ-E on orthotopic prostate cancers was evaluated in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. RESULTS: The liposome-mediated transfer of viral RNA genome fragments from HVJ-E suppressed the viability of prostate cancer cells but not the viability of the noncancerous prostate epithelium. Knockdown experiments using siRNAs showed that the cancer cell-selective killing induced by HVJ-E was mediated by retinoic acid-inducible gene I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). Downstream of the RIG-I/MAVS pathway, both TNF-related apoptosis-inducing ligand (TRAIL) and Noxa were upregulated by HVJ-E in the castration-resistant prostate cancer cell line PC3 but not in the noncancerous prostate epithelial cell line PNT2. TRAIL siRNA but not Noxa siRNA significantly inhibited HVJ-E-induced cell death in PC3 cells. However, Noxa siRNA effectively suppressed HVJ-E-induced cell death in DU145 cells, another castration-resistant prostate cancer cell line, in which Noxa but not TRAIL was upregulated by HVJ-E. Furthermore, the orthotopic prostate cancers were dramatically eradicated in immunodeficient mice injected with HVJ-E. CONCLUSION: The RIG-I/MAVS signaling pathway represents an attractive target for cancer therapy.

PDGFRalpha-positive cells in bone marrow are mobilized by high mobility group box 1 (HMGB1) to regenerate injured epithelia.

The role of bone marrow cells in repairing ectodermal tissue, such as skin epidermis, is not clear. To explore this process further, this study examined a particular form of cutaneous repair, skin grafting. Grafting of full thickness wild-type mouse skin onto mice that had received a green fluorescent protein-bone marrow transplant after whole body irradiation led to an abundance of bone marrow-derived epithelial cells in follicular and interfollicular epidermis that persisted for at least 5 mo. The source of the epithelial progenitors was the nonhematopoietic, platelet-derived growth factor receptor α-positive (Lin(-)/PDGFRα(+)) bone marrow cell population. Skin grafts release high mobility group box 1 (HMGB1) in vitro and in vivo, which can mobilize the Lin(-)/PDGFRα(+) cells from bone marrow to target the engrafted skin. These data provide unique insight into how skin grafts facilitate tissue repair and identify strategies germane to regenerative medicine for skin and, perhaps, other ectodermal defects or diseases.