Amy S. Burnside

Effect of Fibroblast Donor Cell Age and Cell Cycle on Development of Bovine Nuclear Transfer Embryos In Vitro

Abstract The effects of cell cycle stage and the age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages, and the in vitro life span of these cell lines was analyzed. Fibroblasts from both fetuses and calves had similar in vitro life spans of approximately 30 population doublings (PDs) compared with 20 PDs in fibroblasts obtained from adult animals. When fibroblasts from both fetuses and adult animals were cultured as a population, the percentage of cells in G1 increased linearly with time, whereas the percentage of S-phase cells decreased proportionately. Furthermore, the percentage of cells in G1 at a given time was higher in adult fibroblasts than in fetal fibroblasts. To study the individual cells from a population, a shake-off method was developed to isolate cells in G1 stage of the cell cycle and evaluate the cell cycle characteristics of both fetal and adult fibroblasts from either 25% or 100% confluent cultures. Irrespective of the age, the mean cell cycle length in isolated cells was shorter (9.6–15.5 h) than that observed for cells cultured as a population. Likewise, the length of the G1 stage in these isolated cells, as indicated by 5-bromo-deoxyuridine labeling, lasted only about 2–3 h. There were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells from 25% or 100% confluent cultures of fetal or adult cell lines. This study suggests that there are substantial differences in cell cycle characteristics in cells derived from animals of different ages or cultured at different levels of confluence. However, these factors had no effect on in vitro development of nuclear transfer embryos.

Induction of Oct-3/4 expression in somatic cells by gap junction-mediated cAMP signaling from blastomeres

We report the induction of embryonic gene expression in epithelial HC-11 cells upon communication with blastomeres in compacting mouse embryos. In contrast to NIH3T3 fibroblasts, HC-11 epithelial cells form gap junctions with blastomeres after injection into cleavage-stage embryos, as shown by targeting of phosphorylated connexin43 (pCx43) to areas of cell-to-blastomere contact and dye coupling. This was accompanied by expression of the embrvo-specific transcription factor, Oct-3/4, in the HC-11 cells. Dye coupling and Oct-3/4 expression were abolished with heptanol and 18beta- glycyrrhetinic acid, two gap junction blockers. Oleamide, which blocks gap junction-mediated communication but not electrical conductance, also inhibited Oct-3/4 expression in HC-11 cells, suggesting that Oct-3/4 induction results from transfer of molecules of < 1 kDa through gap junctions. Inhibition of cAMP signaling in blastomeres abolishes Oct-3/4 expression in somatic cells despite gap junction formation. In addition, reprogramming of NIH3T3 fibroblasts in an extract of HC-11 cells enabled assembly of pCx43 and Oct-3/4 expression after contact of the reprogrammed cells with blastomeres. We propose that gap junction-mediated cAMP signaling between blastomeres and somatic cells results in changes in somatic cell gene expression.

Expression of the hypothalamic transcription factor Nhlh2 is dependent on energy availability

Mice with a deletion of the hypothalamic basic helix‐loop‐helix transcription factor Nhlh2 display adult onset obesity, implicating Nhlh2 in the neuronal circuits regulating energy availability. Nhlh2 colocalises with the hypothalamic thyrotrophin‐releasing hormone (TRH) neurones in the paraventricular nucleus (PVN) and pro‐opiomelanocortin (POMC) neurones in the arcuate nucleus. We show that Nhlh2 expression is significantly reduced in response to 24‐h food deprivation in the arcuate nucleus, PVN, lateral hypothalamus, ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH). Food intake for 2 h following deprivation stimulates Nhlh2 expression in the arcuate nucleus and the PVN, and leptin injection following deprivation results in increased Nhlh2 expression in the arcuate nucleus, PVN, lateral hypothalamus, VMH, and DMH. Hypothalamic Nhlh2 expression in response to leptin injection is maximal by 2 h. Following leptin injection, Nhlh2 mRNA colocalises in POMC neurones in the arcuate nucleus and TRH neurones in the PVN. Nhlh2 mRNA expression in POMC neurones in the arcuate nucleus and TRH neurones in the PVN is reduced with energy deprivation and is stimulated with food intake and leptin injection. Modulation of POMC expression in response to changes in energy availability is not affected in mice with a targeted deletion of Nhlh2. However, deletion of Nhlh2 does result in loss of normal TRH mRNA expression in mice exposed to food deprivation and leptin stimulation. These data implicate Nhlh2 as a regulatory target of the leptin‐mediated energy availability network of the hypothalamus, and TRH as a putative downstream target of Nhlh2.

Modulation of let-7 miRNAs controls the differentiation of effector CD8 T cells

The differentiation of naive CD8 T cells into effector cytotoxic T lymphocytes upon antigen stimulation is necessary for successful antiviral, and antitumor immune responses. Here, using a mouse model, we describe a dual role for the let-7 microRNAs in the regulation of CD8 T cell responses, where maintenance of the naive phenotype in CD8 T cells requires high levels of let-7 expression, while generation of cytotoxic T lymphocytes depends upon T cell receptor-mediated let-7 downregulation. Decrease of let-7 expression in activated T cells enhances clonal expansion and the acquisition of effector function through derepression of the let-7 targets, including Myc and Eomesodermin. Ultimately, we have identified a novel let-7-mediated mechanism, which acts as a molecular brake controlling the magnitude of CD8 T cell responses. DOI: http://dx.doi.org/10.7554/eLife.26398.001

Leptin signaling regulates hypothalamic expression of nescient helix-loop-helix 2 (Nhlh2) through signal transducer and activator 3 (Stat3).

Mice with a deletion of the hypothalamic basic helix-loop-helix transcription factor Nhlh2 display adult onset obesity. We have previously shown that Nhlh2 expression is induced by leptin. In this study, we identify a small proximal leptin-responsive promoter region in the Nhlh2 gene. This 163bp promoter contains five putative binding sites for the leptin-activated Stat3 transcription factor, and two putative binding sites for the NFκB transcription factor. Results of mutagenesis studies reveal that deletion of the NFκB sites have little effect, mutagenesis of the third Stat3 site eliminates both leptin-induced and basal expression of Nhlh2. Mutagenesis of the 4th and 5th sites eliminates leptin-induced expression, and increases basal expression above the WT promoter. Stat3 can be preferentially pulled down from leptin-treated mouse hypothalamic chromatin extracts. This study identifies leptin-induced Stat3 transcription factor as the major transcriptional regulator of Nhlh2. As Nhlh2 transcriptionally regulates genes within the melanocortin pathway, these findings have implications for human body weight control.

Activins A and B Regulate Fate-Determining Gene Expression in Islet Cell Lines and Islet Cells From Male Mice.

TGFβ superfamily ligands, receptors, and second messengers, including activins A and B, have been identified in pancreatic islets and proposed to have important roles regulating development, proliferation, and function. We previously demonstrated that Fstl3 (an antagonist of activin activity) null mice have larger islets with β-cell hyperplasia and improved glucose tolerance and insulin sensitivity in the absence of altered β-cell proliferation. This suggested the hypothesis that increased activin signaling influences β-cell expansion by destabilizing the α-cell phenotype and promoting transdifferentiation to β-cells. We tested the first part of this hypothesis by treating α- and β-cell lines and sorted mouse islet cells with activin and related ligands. Treatment of the αTC1-6 α cell line with activins A or B suppressed critical α-cell gene expression, including Arx, glucagon, and MafB while also enhancing β-cell gene expression. In INS-1E β-cells, activin A treatment induced a significant increase in Pax4 (a fate determining β-cell gene) and insulin expression. In sorted primary islet cells, α-cell gene expression was again suppressed by activin treatment in α-cells, whereas Pax4 was enhanced in β-cells. Activin treatment in both cell lines and primary cells resulted in phosphorylated mothers against decapentaplegic-2 phosphorylation. Finally, treatment of αTC1-6 cells with activins A or B significantly inhibited proliferation. These results support the hypothesis that activin signaling destabilized the α-cell phenotype while promoting a β-cell fate. Moreover, these results support a model in which the β-cell expansion observed in Fstl3 null mice may be due, at least in part, to enhanced α- to β-cell transdifferentiation.

Intracellular Delivery of Anti-pPKCθ (Thr538) via Protein Transduction Domain Mimics for Immunomodulation

Targeting cellular proteins with antibodies, to better understand cellular signaling pathways in the context of disease modulation, is a fast-growing area of investigation. Humanized antibodies are increasingly gaining attention for their therapeutic potential, but the collection of cellular targets is limited to those secreted from cells or expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as putative targets for antibody binding. Protein kinase Cθ (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic capable of efficiently delivering an antibody against phosphorylated PKCθ (Thr538) into human peripheral mononuclear blood cells and altering expression of downstream indicators of T cell activation and differentiation. We used a humanized, lymphocyte transfer model of graft-versus-host disease, to evaluate the durability of protein transduction domain mimic:Anti-pPKCθ modulation, when delivered into human peripheral mononuclear blood cells ex vivo. We demonstrate that protein transduction domain mimic:Antibody complexes can be readily introduced with high efficacy into hard-to-transfect human peripheral mononuclear blood cells, eliciting a biological response sufficient to alter disease progression. Thus, protein transduction domain mimic:Antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.

Activin Enhances α- to β-Cell Transdifferentiation as a Source For β-Cells In Male FSTL3 Knockout Mice

Diabetes results from inadequate β-cell number and/or function to control serum glucose concentrations so that replacement of lost β-cells could become a viable therapy for diabetes. In addition to embryonic stem cell sources for new β-cells, evidence for transdifferentiation/reprogramming of non-β-cells to functional β-cells is accumulating. In addition, de-differentiation of β-cells observed in diabetes and their subsequent conversion to α-cells raises the possibility that adult islet cell fate is malleable and controlled by local hormonal and/or environmental cues. We previously demonstrated that inactivation of the activin antagonist, follistatin-like 3 (FSTL3) resulted in β-cell expansion and improved glucose homeostasis in the absence of β-cell proliferation. We recently reported that activin directly suppressed expression of critical α-cell genes while increasing expression of β-cell genes, supporting the hypothesis that activin is one of the local hormones controlling islet cell fate and that increased activin signaling accelerates α- to β-cell transdifferentiation. We tested this hypothesis using Gluc-Cre/yellow fluorescent protein (YFP) α-cell lineage tracing technology combined with FSTL3 knockout (KO) mice to label α-cells with YFP. Flow cytometry was used to quantify unlabeled and labeled α- and β-cells. We found that Ins+/YFP+ cells were significantly increased in FSTL3 KO mice compared with wild type littermates. Labeled Ins+/YFP+ cells increased significantly with age in FSTL3 KO mice but not wild type littermates. Sorting results were substantiated by counting fluorescently labeled cells in pancreatic sections. Activin treatment of isolated islets significantly increased the number of YFP+/Ins+ cells. These results suggest that α- to β-cell transdifferentiation is influenced by activin signaling and may contribute substantially to β-cell mass.

Effects of activin A on survival, function and gene expression of pancreatic islets from non-diabetic and diabetic human donors

Emerging evidence suggests that activin with its associated receptors, second messengers, and antagonists would be excellent targets for therapeutic drug development in the treatment of diabetes. We undertook the current study to investigate the ability to extrapolate findings from rodent studies to human islets in which data thus far has been scarce. We tested the hypothesis that human islets synthesize activin and that activin participates in the regulation of islet β-cells. Human islets from 33 separate isolations were categorized based on functional status, culture status and diabetic status. Statistical comparisons were made by ANOVA with Tukey post-hoc adjustment for multiple comparisons. Experiments investigating activin utilized qPCR, FACS cell sorting, immunofluorescent antibody staining, functionality assays, viability assays and protein secretion assays. We have defined the transcript expression patterns of activin and the TGFβ superfamily in human islets. We found INHBA (the gene encoding activin A) to be the most highly expressed of the superfamily in normal, cultured islets. We elucidated a link between the islet microenvironment and activin A. We found differential ligand expression based on diabetic, culture and functional status. Further, this is also the first report that links direct effects of activin A with the ability to restore glucose-stimulated insulin secretion in human islets from type 2 diabetic donors thereby establishing the relevance of targeting activin for therapeutic drug development.

Moderate Intensity Exercise Reduces Activated and Apoptotic Endothelial Microparticles in Healthy Midlife Women

Endothelial microparticles (EMPs) are related to cardiovascular disease (CVD) risk. Risk factors for CVD increase with menopause, and greater cardiorespiratory fitness is generally expected to reduce CVD risk. The effects of habitual physical activity on endothelial health may be due in part to the effect of acute exercise on circulating EMPs. This study was performed to evaluate the effect of an acute bout of exercise on CD62E+ and CD31+/42b- EMPs in healthy fit midlife women at different menopausal stages. Healthy, active premenopausal (PRE), perimenopausal (PERI), and postmenopausal (POST) women completed a single bout of moderate-intensity treadmill exercise. Activated (CD62E+) and apoptotic (CD31+/42b-) EMPs were evaluated before and 30 min after exercise by using fluorescent activated cell sorting. In an exploratory analysis, these results were compared with data from low-fit peri- and postmenopausal women. Differences by group and time point were evaluated with repeated-measure ANOVAs. There was a reduction in the number of total microparticles ( P < 0.001), CD62E+ ( P = 0.003), and CD31+/42b- ( P < 0.001) EMPs/μl plasma following acute exercise. The percentage of CD62E+ EMPs increased with acute exercise ( P < 0.001), whereas the percentage of CD31+/42b- EMPs did not change ( P = 0.40). There was no effect of menopausal status on CD62E+or CD31+/42b- EMPs, or on total microparticles (all P > 0.05). The exploratory analysis revealed that low-fit women had similar changes in EMPs with acute exercise. We concluded that acute moderate-intensity exercise reduces CD62E+and CD31+/42b- EMPs, as well as total microparticles, in healthy midlife women. These effects occurred despite differences in menopausal status and fitness. NEW & NOTEWORTHY This study demonstrates that acute moderate-intensity exercise reduces activated and apoptotic endothelial microparticles in healthy midlife women.