Altered Cell Differentiation Research Articles

Epigenetics and Axial Spondyloarthritis - DNA is not Destiny

Epigenetics is defined as heritable changes in gene expression without changing the base-sequence of the genome, ultimately altering cell differentiation, phenotype, and function. Epigenetic modifications can take place in chromosomal deoxyribonucleic acid (DNA) or in the proteins linked with the chromosomal DNA such as histones, and have been grouped into 4 main categories: DNA methylation, histone modification, small and non-coding RNAs, and chromatin remodeling. In recent years, many epigenetic proteins have been experimentally and clinically investigated, whereas strategies for modifying the epigenetic ‘machinery’ have been the frontier for discovering a ‘cure’ for chronic ailments. Gene-environment interaction is the most plausible answer.

Deregulated Genes in Hematopoietic Stem Cells Isolated from Spleen of Patients with Myelofibrosis

An important issue in myeloproliferative diseases research is to test the hypothesis that in the neo-angiogenesis, which is observed in the spleen and bone marrow of patients, involves endothelial cells derived from the neoplastic clone. In this study we analyzed the gene expression profile of 138 genes in CD34+ hematopoietic cells from the spleen and peripheral blood of patients with myelofibrosis (MF) and healthy individuals (HI). These genes have been selected among those that characterize the neoangiogenic gene signature or belonging to deregulated expression pathways in MF, and are listed in Table1.By using quantitative reverse transcription-PCR (RT-qPCR), we measured the expression levels of 141 targets and 5 reference genes in CD34+ cells isolated by immunomagnetic method from the spleen and peripheral blood of 4 patients and 3 HI. For calculation of the statistical significance of the differences we used the t-test for unpaired samples. A p<0.05 was considered to be statistically significant. All data analyses were performed with GenEx (version 6.1, MultiD).To characterize the differentially expressed genes obtained in each comparison, the lists of genes were analyzed through several tools, i.e. String, Enrichr and GSEA analysis. The comparison between spleen CD34+ cells and circulating CD34+ cells reveal a group of common genes in both patients and heathy donors. Those genes are significantly overexpressed in spleen. Some of the common genes are involved in the regulation of cell migration and in the development of blood vessels were significantly overexpressed in splenic CD34+ cells compared to circulating CD34+ cells. However, we observed that many of the genes implicated in the development of the vessels were under-expressed in CD34+ cells isolated from the spleen of patients compared to those of HI. The most significant upregulated genes in CD34+ splenic cells from patients included transcripts (GATA1, HBB, TAL1, GATA2, PTGS1) belonging to the molecular signature of CD34+ cells isolated from the bone marrow (BM) of patients with chronic myeloid leukemia (Diaz-Blanco E. et al., Leukemia 2007). Another group of upregulated genes (GATA1, TAL1, ITGB3, GATA2, PF4) are also identified in a study designed to characterize the genes essential to the development of megakaryocytes (Tenedini et al. Blood 2004). These two groups of genes are part of an expression pattern characteristic for immature stem cells as well as megakaryocyte-erythrocyte progenitor cells. Another group of genes overexpressed in patients spleens (CD34, ANGPT1, PF4, GATA2, PTGS1), which includes some of the above mentioned genes, has been observed in a comparison between circulating CD34+ cells from patients with myelofibrosis, and CD34 + cells isolated from the bone marrow of HI (Guglielmelli P. et al. Stem Cells 2007). Among the underexpressed genes in the spleen of patients we mention CXCR4, in keeping with previous observations. Interestingly, among the aforementioned genes, GATA1 and GATA2 genes are overexpressed in granulocytes of patients with MPNs regardless of the JAK2/CALR mutational status whereas PTGS1 is overexpressed in the JAK2-V617F homozygous and CALR-mutated granulocytes (Rampal et al. Blood 2014).We focused our attention on PTGS1 because its well-known activity in regulating angiogenesis, which can be inhibited in vitro by treatment with aspirin (Tsujii, M. et al. Cell 1998). To explain the mechanism of action of low-doses aspirin in this context, it has been proposed a model that involves permanent inactivation of PTGS1 in platelets. Indeed, PTGS1 is the only cyclooxygenase isoenzyme present in platelets. Moreover, Dixon and colleagues (Dixon, D.A. et al. JCI 2006), demonstrated that activated platelets induced the expression of PTGS2 in monocytes. It is believed that the synthesis of prostaglandin E2 by PTGS2 in tissues, is linked to an increment of angiogenesis and cell proliferation, and to a reduction of apoptosis. According to this concept, our data point toward a model in which, in the spleen of patients with MF, an altered hematopoietic stem cell differentiation could induce an inflammation-mediated angiogenesis through the overexpression of PTGS1 and a consequent induction of PTGS2 in cells of the splenic microenvironment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Metformin suppresses adipogenesis through both AMP-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms

People with Type 2 diabetes mellitus (T2DM) have reduced bone mineral density and an increased risk of fractures due to altered mesenchymal stem cell (MSC) differentiation in the bone marrow. This leads to a shift in the balance of differentiation away from bone formation (osteogenesis) in favour of fat cell development (adipogenesis). The commonly used anti-diabetic drug, metformin, activates the osteogenic transcription factor Runt-related transcription factor 2 (Runx2), which may suppress adipogenesis, leading to improved bone health. Here we investigate the involvement of the metabolic enzyme, AMP-activated protein kinase (AMPK), in these protective actions of metformin. The anti-adipogenic actions of metformin were observed in multipotent C3H10T1/2 MSCs, in which metformin exerted reciprocal control over the activities of Runx2 and the adipogenic transcription factor, PPARγ, leading to suppression of adipogenesis. These effects appeared to be independent of AMPK activation but rather through the suppression of the mTOR/p70S6K signalling pathway. Basal AMPK and mTOR/p70S6K activity did appear to be required for adipogenesis, as demonstrated by the use of the AMPK inhibitor, compound C. This observation was further supported by using AMPK knockout mouse embryo fibroblasts (MEFs) where adipogenesis, as assessed by reduced lipid accumulation and expression of the adipogeneic transcription factor, C/EBPβ, was found to display an absolute requirement for AMPK. Further activation of AMPK in wild type MEFS, with either metformin or the AMPK-specific activator, A769662, was also associated with suppression of adipogenesis. It appears, therefore, that basal AMPK activity is required for adipogenesis and that metformin can inhibit adipogenesis through AMPK-dependent or -independent mechanisms, depending on the cellular context.

Regulatory roles of epigenetic modulators, modifiers and mediators in lung cancer.

Lung cancer as the leading cause of cancer-related deaths can be initiated and progressed by the interaction between dynamically genetic and epigenetic elements, although mechanisms mediating lung cancer development and progression remain unclear. Tumor progenitor genes may contribute to lung carcinogenesis and cancer progression, are epigenetically disrupted at the early stages of malignancies even before mutations, and alter cell differentiation throughout tumor evolution. The present review explores potential roles and mechanisms of epigenetic modulators, modifiers and mediators in the development of lung cancer. We also overviewed potential mechanisms by which epigenetic modulators, modifiers and mediators control and regulate 3D nuclear architectures, and discussed translational efforts to epigenetic modifications for treatment of lung cancer. Deep understanding of epigenetic modulators, modifiers and mediators will benefit the discovery and development of new diagnostics and therapies for lung cancer.

Repeated whole cigarette smoke exposure alters cell differentiation and augments secretion of inflammatory mediators in air-liquid interface three-dimensional co-culture model of human bronchial tissue

In vitro models of human bronchial epithelium are useful for toxicological testing because of their resemblance to in vivo tissue. We constructed a model of human bronchial tissue which has a fibroblast layer embedded in a collagen matrix directly below a fully-differentiated epithelial cell layer. The model was applied to whole cigarette smoke (CS) exposure repeatedly from an air-liquid interface culture while bronchial epithelial cells were differentiating. The effects of CS exposure on differentiation were determined by histological and gene expression analyses on culture day 21. We found a decrease in ciliated cells and perturbation of goblet cell differentiation. We also analyzed the effects of CS exposure on the inflammatory response, and observed a significant increase in secretion of IL-8, GRO-α, IL-1β, and GM-CSF. Interestingly, secretion of these mediators was augmented with repetition of whole CS exposure. Our data demonstrate the usefulness of our bronchial tissue model for in vitro testing and the importance of exposure repetition in perturbing the differentiation and inflammation processes.

Environmental signaling: from environmental estrogens to endocrine-disrupting chemicals and beyond.

The landmark report (Herbst et al. 1971) linking prenatal treatment with a synthetic estrogen, diethylstilbestrol (DES), to cancer at puberty in women whose mothers took the drug while pregnant ushered in an era of research on delayed effects of such exposures on functional outcomes in offspring. An animal model developed in our laboratory at the National Institute of Environmental Health Sciences confirmed that DES was the carcinogen and exposure to DES caused, as well, functional alterations in the reproductive, endocrine, and immune systems of male and female mice treated in utero. DES was also being used in agriculture and we discovered, at the first meeting on Estrogens in the Environment in 1979 (Estrogens in the Environment, 1980), that many environmental contaminants were also estrogenic. Many laboratories sought to discern the basis for estrogenicity in environmental chemicals and to discover other hormonally active xenobiotics. Our laboratory elucidated how DES and other estrogenic compounds worked by altering differentiation through epigenetic gene imprinting, helping explain the transgenerational effects found in mice and humans. At the Wingspread Conference on the Human-Wildlife Connection in 1991 (Advances in Modern Environmental Toxicology, 1992), we learned that environmental disruption of the endocrine system occurred in many species and phyla, and the term endocrine disruption was introduced. Further findings of transgenerational effects of environmental agents that mimicked or blocked various reproductive hormones and the ubiquity of environmental signals, such as bisphenol A increased concern for human and ecological health. Scientists began to look at other endocrine system aspects, such as cardiovascular and immune function, and other nuclear receptors, with important observations regarding obesity and metabolism. Laboratories, such as ours, are now using stem cells to try to understand the mechanisms by which various environmental signals alter cell differentiation. Since 2010, research has shown that trauma and other behavioral inputs can function as 'environmental signals,' can be encoded in gene regulation networks in a variety of cells and organs, and can be passed on to subsequent generations. So now we come full circle: environmental chemicals mimic hormones or other metabolic signaling molecules and now behavioral experience can be transduced into chemical signals that also modify gene expression.

Morc3 mutant mice exhibit reduced cortical area and thickness, accompanied by altered haematopoietic stem cells niche and bone cell differentiation.

Morc3, a member of a highly conserved nuclear matrix protein super-family plays an important part in chromatin remodeling, DNA repair, epigenetic regulation and cellular senescence. However, its role in bone homeostasis is not known. In the present study, a phenotype-driven ENU mouse mutagenesis screen revealed that Morc3mut +/− mice exhibit reduced cortical area and thickness with increased cortical porosity. Morc3mut +/− mice displayed reduced osteoclast numbers and surface per bone surface as well as osteocyte numbers, concomitant with altered gene expressions such as Rankl/Opg and Sost in ex vivo long bones. In vitro experiments revealed a significant increase in the number of Sca-1+/c-kit+ haematopoietic stem cells (HSCs), and a significant reduction in senescence associated β-galactosidase activity in bone marrow macrophages (BMMs). In addition, we observed a decrease in osteoclastogenesis and bone resorption accompanied by upregulation of STAT1 expression in osteoclast lineage cells. Strikingly, Morc3 protein localization within the nuclear membrane was shifted to the cytoplasm in Morc3mut +/− osteoclasts. Further, Morc3mut +/− mice displayed increased osteoblast differentiation and altered gene expression. Collectively, our data show that Morc3 is a previously unreported regulator of cortical bone homeostasis and haematopoietic stem cells niche, accompanied by altered bone cell differentiation.

Stromal β-catenin overexpression contributes to the pathogenesis of renal dysplasia.

Renal dysplasia, the leading cause of renal failure in children, is characterized by disrupted branching of the collecting ducts and primitive tubules, with an expansion of the stroma, yet a role for the renal stroma in the genesis of renal dysplasia is not known. Here, we demonstrate that expression of β-catenin, a key transcriptional co-activator in renal development, is markedly increased in the expanded stroma in human dysplastic tissue. To understand its contribution to the genesis of renal dysplasia, we generated a mouse model that overexpresses β-catenin specifically in stromal progenitors, termed β-cat(GOF-S) . Histopathological analysis of β-cat(GOF) (-S) mice revealed a marked expansion of fibroblast cells surrounding primitive ducts and tubules, similar to defects observed in human dysplastic kidneys. Characterization of the renal stroma in β-cat(GOF) (-S) mice revealed altered stromal cell differentiation in the expanded renal stroma demonstrating that this is not renal stroma but instead a population of stroma-like cells. These cells overexpress ectopic Wnt4 and Bmp4, factors necessary for endothelial cell migration and blood vessel formation. Characterization of the renal vasculature demonstrated disrupted endothelial cell migration, organization, and vascular morphogenesis in β-cat(GOF) (-S) mice. Analysis of human dysplastic tissue demonstrated a remarkably similar phenotype to that observed in our mouse model, including altered stromal cell differentiation, ectopic Wnt4 expression in the stroma-like cells, and disrupted endothelial cell migration and vessel formation. Our findings demonstrate that the overexpression of β-catenin in stromal cells is sufficient to cause renal dysplasia. Further, the pathogenesis of renal dysplasia is one of disrupted stromal differentiation and vascular morphogenesis. Taken together, this study demonstrates for the first time the contribution of stromal β-catenin overexpression to the genesis of renal dysplasia. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Toxic effects of prenatal exposure to alcohol, tobacco and other drugs.

Tobacco, alcohol, cannabis and cocaine are the most consumed psychoactive drugs throughout the population. Prenatal exposure to these drugs could alter normal foetal development and could threaten future welfare. The main changes observed in prenatal exposure to tobacco are caused by nicotine and carbon monoxide, which can impede nutrient and oxygen exchange between mother and foetus, restricting foetal growth. Memory, learning processes, hearing and behaviour can also be affected. Alcohol may cause physical and cognitive alterations in prenatally exposed infants, fundamentally caused by altered NMDAR and GABAR activity. Tetrahydrocannabinol, the psychoactive compound of cannabis, is capable of activating CB1R, inducing connectivity deficits during the foetal brain development. This fact could be linked to behavioural and cognitive deficits. Many of the effects from prenatal cocaine exposure are caused by altered cell proliferation, migration, differentiation and dendritic growth processes. Cocaine causes long term behavioural and cognitive alterations and also affects the uteroplacental unit.

Genomic aberrations in spitzoid melanocytic tumours and their implications for diagnosis, prognosis and therapy

Histopathological evaluation of melanocytic tumours usually allows reliable distinction of benign melanocytic naevi from melanoma. More difficult is the histopathological classification of Spitz tumours, a heterogeneous group of tumours composed of large epithelioid or spindle-shaped melanocytes. Spitz tumours are biologically distinct from conventional melanocytic naevi and melanoma, as exemplified by their distinct patterns of genetic aberrations. Whereas common acquired naevi and melanoma often harbour BRAF mutations, NRAS mutations, or inactivation of NF1, Spitz tumours show HRAS mutations, inactivation of BAP1 (often combined with BRAF mutations), or genomic rearrangements involving the kinases ALK, ROS1, NTRK1, BRAF, RET, and MET. In Spitz naevi, which lack significant histological atypia, all of these mitogenic driver aberrations trigger rapid cell proliferation, but after an initial growth phase, various tumour suppressive mechanisms stably block further growth. In some tumours, additional genomic aberrations may abrogate various tumour suppressive mechanisms, such as cell-cycle arrest, telomere shortening, or DNA damage response. The melanocytes then start to grow in a less organised fashion and may spread to regional lymph nodes, and are termed atypical Spitz tumours. Upon acquisition of even more aberrations, which often activate additional oncogenic pathways or alter cell differentiation, the neoplastic cells become entirely malignant and may colonise and take over distant organs (spitzoid melanoma). The sequential acquisition of genomic aberrations suggests that Spitz tumours represent a continuous biological spectrum, rather than a dichotomy of benign versus malignant, and that tumours with ambiguous histological features (atypical Spitz tumours) might be best classified as low-grade melanocytic tumours. The number of genetic aberrations usually correlates with the degree of histological atypia and explains why existing ancillary genetic techniques, such as array comparative genomic hybridisation (CGH) or fluorescence in situ hybridisation (FISH), are usually capable of accurately classifying histologically benign and malignant Spitz tumours, but are not very helpful in the diagnosis of ambiguous melanocytic lesions. Nevertheless, we expect that progress in our understanding of tumour progression will refine the classification of spitzoid melanocytic tumours in the near future. By integrating clinical, pathological, and genetic criteria, distinct tumour subsets will be defined within the heterogeneous group of Spitz tumours, which will eventually lead to improvements in diagnosis, prognosis and therapy.

Intestinal Epithelial Toll-Like Receptor 4 Signaling Affects Epithelial Function and Colonic Microbiota and Promotes a Risk for Transmissible Colitis.

Evidence obtained from gene knockout studies supports the role of Toll-like receptor 4 (TLR4) in intestinal inflammation and microbiota recognition. Increased epithelial TLR4 expression is observed in patients with inflammatory bowel disease. However, little is known of the effect of increased TLR4 signaling on intestinal homeostasis. Here, we examined the effect of increased TLR4 signaling on epithelial function and microbiota by using transgenic villin-TLR4 mice that overexpress TLR4 in the intestinal epithelium. Our results revealed that villin-TLR4 mice are characterized by increases in the density of mucosa-associated bacteria and bacterial translocation. Furthermore, increased epithelial TLR4 signaling was associated with an impaired epithelial barrier, altered expression of antimicrobial peptide genes, and altered epithelial cell differentiation. The composition of the colonic luminal and mucosa-associated microbiota differed between villin-TLR4 and wild-type (WT) littermates. Interestingly, WT mice cohoused with villin-TLR4 mice displayed greater susceptibility to acute colitis than singly housed WT mice did. The results of this study suggest that epithelial TLR4 expression shapes the microbiota and affects the functional properties of the epithelium. The changes in the microbiota induced by increased epithelial TLR4 signaling are transmissible and exacerbate dextran sodium sulfate-induced colitis. Together, our findings imply that host innate immune signaling can modulate intestinal bacteria and ultimately the host's susceptibility to colitis.

The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

Drosophila eye developmental defect caused by elevation of the activity of the LIM-homeodomain protein, Lmx1a, requires its association with the Co-activator Chip

The LIM-homeodomain (LIM-HD) family member Lmx1a has been successfully used to induce dopaminergic neurons from other cell types, thus showing significant implications in replacement therapies of Parkinson's disease, but the underlying mechanism remains elusive. In this study, we used Drosophila eye as a model system to investigate how forced expression of dLmx1a, the fly homolog of human Lmx1a, alters cell identify. We found that ectopic expression of dLmx1a suppresses the formation of Drosophila eye tissue and identified the LIM and HD as two essential domains. dLmx1a requires and physically binds to Chip, a well-known cofactor of LIM-HD proteins. Chip connects two dLmx1a proteins to form a functional tetrameric complex. In addition, we provide evidence showing that dLmx1a expression results in the suppression of two retina determination gene eyes absent (eya) and string (stg). Taken together, our findings identified Chip as a novel partner of dLmx1a to alter cell differentiation in Drosophila eye through repressing eya and stg expression, and provide an animal model for further understanding the molecular mechanism whereby Lmx1a determines cell fate.

Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection.

Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2-responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART.

Epithelial inactivation of Yy1 abrogates lung branching morphogenesis.

Yin Yang 1 (YY1) is a multifunctional zinc-finger-containing transcription factor that plays crucial roles in numerous biological processes by selectively activating or repressing transcription, depending upon promoter contextual differences and specific protein interactions. In mice, Yy1 null mutants die early in gestation whereas Yy1 hypomorphs die at birth from lung defects. We studied how the epithelial-specific inactivation of Yy1 impacts on lung development. The Yy1 mutation in lung epithelium resulted in neonatal death due to respiratory failure. It impaired tracheal cartilage formation, altered cell differentiation, abrogated lung branching and caused airway dilation similar to that seen in human congenital cystic lung diseases. The cystic lung phenotype in Yy1 mutants can be partly explained by the reduced expression of Shh, a transcriptional target of YY1, in lung endoderm, and the subsequent derepression of mesenchymal Fgf10 expression. Accordingly, SHH supplementation partially rescued the lung phenotype in vitro. Analysis of human lung tissues revealed decreased YY1 expression in children with pleuropulmonary blastoma (PPB), a rare pediatric lung tumor arising during fetal development and associated with DICER1 mutations. No evidence for a potential genetic interplay between murine Dicer and Yy1 genes during lung morphogenesis was observed. However, the cystic lung phenotype resulting from the epithelial inactivation of Dicer function mimics the Yy1 lung malformations with similar changes in Shh and Fgf10 expression. Together, our data demonstrate the crucial requirement for YY1 in lung morphogenesis and identify Yy1 mutant mice as a potential model for studying the genetic basis of PPB.

Effects of Combined Exposure to Lead and High-Fat Diet on Bone Quality in Juvenile Male Mice

BackgroundLead (Pb) exposure and obesity are co-occurring risk factors for decreased bone mass in the young, particularly in low socioeconomic communities.ObjectivesThe goal of this study was to determine whether the comorbidities of Pb exposure and high-fat diet–induced obesity amplify skeletal deficits independently associated with each of these risk factors, and to explore associated mechanisms of the observed deficiencies.MethodsFive-week-old male C57BL/6J mice were placed on low-fat (10% kcal, LFD) or high-fat (60% kcal, HFD) diets for 12 weeks. Mice were exposed to lifetime Pb (50 ppm) through drinking water.ResultsHFD was associated with increased body mass and glucose intolerance. Both HFD and Pb increased fasting glucose and serum leptin levels. Pb and HFD each reduced trabecular bone quality and together had a further detrimental effect on these bone parameters. Mechanical bone properties of strength were depressed in Pb-exposed bones, but HFD had no significant effect. Both Pb and HFD altered progenitor cell differentiation, promoting osteoclastogenesis and increasing adipogenesis while suppressing osteoblastogenesis. In support of this lineage shift being mediated through altered Wnt signaling, Pb and non-esterified fatty acids in MC3T3 cells increased in vitro PPAR-γ activity and inhibited β-catenin activity. Combining Pb and non-esterified fatty acids enhanced these effects.ConclusionsPb and HFD produced selective deficits in bone accrual that were associated with alterations in progenitor cell activity that may involve reduced Wnt signaling. This study emphasizes the need to assess toxicants together with other risk factors relevant to human health and disease.CitationBeier EE, Inzana JA, Sheu TJ, Shu L, Puzas JE, Mooney RA. 2015. Effects of combined exposure to lead and high-fat diet on bone quality in juvenile male mice. Environ Health Perspect 123:935–943; http://dx.doi.org/10.1289/ehp.1408581

Immunotherapy in upper GI malignancies.

A comprehensive analysis of the interactions that can occur amongst three complex systems-the tumor cell, tissue microenvironment, and the immune response, is required if we are to realize the potential of immunotherapeutics in gastroesophageal cancer. For many years, epithelial cancers were believed to originate due to the transformation of tissue stem cells. Recently however, it has suggested that bone marrow-derived cells (BMDCs), which are frequently recruited to sites of tissue injury and inflammation, might also represent a potential source of malignancy. The link between infection, chronic inflammation, and cancer has long been recognized in gastric cancer with its close association with Helicobacter pylori as a class I carcinogen. The long-term consequences of recruiting pluripotent cells to areas of chronic inflammation which can result in altered cell signaling and differentiation needs to be defined, but this work provides a fascinating insight into the pivotal role played by the immune system in the development of upper GI tumors. Here, we discuss many of the immunotherapeutic strategies that have been assessed in gastroesophageal cancer in the last two decades. At the time of writing, the use of checkpoint inhibitors represents the most exciting approach and displays the greatest potential for widespread adoption in the clinic. Preliminary data suggest that programmed death ligand-1 (PD-L1) expression ranges from approximately 18 to 42% in gastroesophageal cancer. Phase II and phase III clinical trials involving either single agent PD-1/PD-L1 inhibition or combined with CTLA-4 inhibitors are currently enrolling, and it is expected that checkpoint inhibition will become a new standard of care in the management of advanced disease in the near future.

Adverse Fat Depots and Marrow Adiposity Are Associated With Skeletal Deficits and Insulin Resistance in Long-Term Survivors of Pediatric Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem-cell transplantation (alloHSCT) survivors treated with total body irradiation (TBI) exhibit bone deficits and excess adiposity, potentially related to altered mesenchymal stem cell differentiation into osteoblasts or adipocytes. We examined associations among fat distribution, bone microarchitecture, and insulin resistance in alloHSCT survivors after TBI. This was a cross-sectional observational study of 25 alloHSCT survivors (aged 12 to 25 years) a median of 9.7 (4.3 to 19.3) years after alloHSCT compared to 25 age-, race-, and sex-matched healthy controls. Vertebral MR spectroscopic imaging and tibia micro-MRI were used to quantify marrow adipose tissue (MAT) and trabecular microarchitecture. Additional measures included DXA whole-body fat mass (WB-FM), leg lean mass (Leg-LM), trunk visceral adipose tissue (VAT), and CT calf muscle density. Insulin resistance in alloHSCT survivors was estimated by HOMA-IR. AlloHSCT survivors had lower Leg-LM (p < 0.001) and greater VAT (p < 0.01), MAT (p < 0.001), and fat infiltration of muscle (p = 0.04) independent of WB-FM, versus matched controls; BMI did not differ. Survivors had lower bone volume fraction and abnormal microarchitecture including greater erosion and more rod-like structure versus controls (all p = 0.04); 14 had vertebral deformities and two had compression fractures. Greater WB-FM, VAT, MAT, and muscle fat infiltration were associated with abnormal trabecular microarchitecture (p < 0.04 for all). AlloHSCT HOMA-IR was elevated, associated with younger age at transplantation (p < 0.01), and positively correlated with WB-FM and VAT (both p < 0.01). In conclusion, the markedly increased marrow adiposity, abnormal bone microarchitecture, and abnormal fat distribution highlight the risks of long-term treatment-related morbidity and mortality in alloHSCT recipients after TBI. Trabecular deterioration was associated with marrow and visceral adiposity. Furthermore, long-term survivors demonstrated sarcopenic obesity, insulin resistance, and vertebral deformities. Future studies are needed to identify strategies to prevent and treat metabolic and skeletal complications in this growing population of childhood alloHSCT survivors.

Fibroblast growth factor 10 alters the balance between goblet and Paneth cells in the adult mouse small intestine.

Intestinal epithelial cell renewal relies on the right balance of epithelial cell migration, proliferation, differentiation, and apoptosis. Intestinal epithelial cells consist of absorptive and secretory lineage. The latter is comprised of goblet, Paneth, and enteroendocrine cells. Fibroblast growth factor 10 (FGF10) plays a central role in epithelial cell proliferation, survival, and differentiation in several organs. The expression pattern of FGF10 and its receptors in both human and mouse intestine and their role in small intestine have yet to be investigated. First, we analyzed the expression of FGF10, FGFR1, and FGFR2, in the human ileum and throughout the adult mouse small intestine. We found that FGF10, FGFR1b, and FGFR2b are expressed in the human ileum as well as in the mouse small intestine. We then used transgenic mouse models to overexpress Fgf10 and a soluble form of Fgfr2b, to study the impact of gain or loss of Fgf signaling in the adult small intestine. We demonstrated that overexpression of Fgf10 in vivo and in vitro induces goblet cell differentiation while decreasing Paneth cells. Moreover, FGF10 decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2, and Sox9. FGF10 inhibited Hes1 expression in vitro, suggesting that FGF10 induces goblet cell differentiation likely through the inhibition of Notch signaling. Interestingly, Fgf10 overexpression for 3 days in vivo and in vitro increased the number of Mmp7/Muc2 double-positive cells, suggesting that goblet cells replace Paneth cells. Further studies are needed to determine the mechanism by which Fgf10 alters cell differentiation in the small intestine.

The acinar regulator Gata6 suppresses KrasG12V-driven pancreatic tumorigenesis in mice

Background and aimsGata6 is required to complete and maintain acinar differentiation in the mouse pancreas. Pancreas-specific Gata6 ablation during development causes extensive and persistent acinar-ductal metaplasia, which is considered an...