Metabolic programming determines the lineage-differentiation fate of murine bone marrow stromal progenitor cells

Enhanced bone marrow adipogenesis and impaired osteoblastogenesis have been observed in obesity, suggesting that the metabolic microenvironment regulates bone marrow adipocyte and osteoblast progenitor differentiation fate. To determine the molecular mechanisms, we studied two immortalized murine cell lines of adipocyte or osteoblast progenitors (BMSC

Diabetic β-Cells Can Achieve Self-Protection against Oxidative Stress through an Adaptive Up-Regulation of Their Antioxidant Defenses

Background Oxidative stress (OS), through excessive and/or chronic reactive oxygen species (ROS), is a mediator of diabetes-related damages in various tissues including pancreatic β-cells. Here, we have evaluated islet OS status and β-cell response to ROS using the GK/Par rat as a model of type 2 diabetes. Methodology/Principal Findings Localization of OS markers was performed on whole pancreases. Using islets isolated from 7-day-old or 2.5-month-old male GK/Par and Wistar control rats, 1) gene expression was analyzed by qRT-PCR; 2) insulin secretion rate was measured; 3) ROS accumulation and mitochondrial polarization were assessed by fluorescence methods; 4) antioxidant contents were quantified by HPLC. After diabetes onset, OS markers targeted mostly peri-islet vascular and inflammatory areas, and not islet cells. GK/Par islets revealed in fact protected against OS, because they maintained basal ROS accumulation similar or even lower than Wistar islets. Remarkably, GK/Par insulin secretion also exhibited strong resistance to the toxic effect of exogenous H2O2 or endogenous ROS exposure. Such adaptation was associated to both high glutathione content and overexpression (mRNA and/or protein levels) of a large set of genes encoding antioxidant proteins as well as UCP2. Finally, we showed that such a phenotype was not innate but spontaneously acquired after diabetes onset, as the result of an adaptive response to the diabetic environment. Conclusions The GK/Par model illustrates the effectiveness of adaptive response to OS by beta-cells to achieve self-tolerance. It remains to be determined to what extend such islet antioxidant defenses upregulation might contribute to GK/Par beta-cell secretory dysfunction

Implication de la voie de signalisation Wnt-/b-caténine dans la régulation de la croissance et la régénération des cellules b pancréatiques

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Mechanisms of KGF mediated signaling in pancreatic duct cell proliferation and differentiation.

BACKGROUND: Keratinocyte growth factor (KGF; palifermin) is a growth factor with a high degree of specificity for epithelial cells. KGF is an important effector of epithelial growth and tissue homeostasis in various organs including the pancreas. Here we investigated the intracellular signaling pathways involved in the mediation of pancreatic ductal cell proliferation and differentiation induced by exogenous KGF during beta-cell regeneration in diabetic rat. METHODOLOGY AND RESULTS: In vitro and in vivo duct cell proliferation was measured by BrdU incorporation assay. The implication of MAPK-ERK1/2 in the mediation of KGF-induced cell proliferation was determined by inactivation of this pathway, using the pharmacological inhibitor or antisense morpholino-oligonucleotides against MEK1. In vivo KGF-induced duct cell differentiation was assessed by the immunolocalization of PDX1 and Glut2 in ductal cells and the implication of PI3K/AKT in this process was investigated. We showed that KGF exerted a potent mitogenic effect on ductal cells. Both in vitro and in vivo, its effect on cell proliferation was mediated through the activation of ERK1/2 as evidenced by the abolition of duct cell proliferation in the context of MEK/ERK inactivation. In vivo, KGF treatment triggered ductal cell differentiation as revealed by the expression of PDX1 and Glut2 in a subpopulation of ductal cells via a PI3K-dependent mechanism. CONCLUSION: Here we show that KGF promotes beta-cell regeneration by stimulating duct cell proliferation in vivo. Moreover, we demonstrated for the first time that KGF directly induces the expression of PDX1 in some ductal cells thus inducing beta-cell neogenesis. We further explored the molecular mechanisms involved in these processes and showed that the effects of KGF on duct cell proliferation are mediated by the MEK-ERK1/2 pathway, while the KGF-induced cell differentiation is mediated by the PI3K/AKT pathway. These findings might have important implications for the in vivo induction of duct-to-beta cell neogenesis in patients with beta-cell deficiency

Coculture between hMADS and Mouse Adult CM

Heart failure occurring after acute myocardial infarction (MI) is among the main causes of death in western countries. Cell therapies, particularly those based on mesenchymal stem cells (MSC), represent one of the most promising approaches to repair damaged heart tissues. Several reports have provided evidences that injection of mesenchymal stem cells improved heart function following myocardial infarction (Shake et al., 2002; Zimmet and Hare, 2005; Zeng et al., 2007). Nevertheless, the mechanism(s) by which MSC exert their therapeutic action is far from being understood, and further knowledges in this field are required especially to optimize efficiency of current cardiac cell therapies. To assess the regenerative mechanisms developed by MSC in vitro, we developed the method described above which is expected to mimic the micro-environment typical of an infarcted heart. This method consists in a species mismatch coculture between mouse terminally differentiated cardiomyocytes in a distressed state and human Multipotent Adipose Derived Stem cells (hMADS cells) used herein as an MSC model

Inactivation of ERK1/2 pathway by MEK1-AS abolishes KGF-induced duct cell proliferation.

<p>(A) Rats from STZ/Std and STZ/MEK1-AS groups were sacrificed at day 2 post natal. Pancreases were removed and pancreatic protein extracts were analyzed by western blot using a specific MEK1 antibody. Representative blots and MEK1 protein quantification are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004734#pone-0004734-g006" target="_blank">Figure 6A</a>. Results are expressed as mean values ±sem. Four rats were analyzed in each experimental group. * <i>p</i><0.05. (B) BrdU (brown nuclei) and CK20 staining (red cytosol) of pancreatic sections from 2-day-old STZ/Std/KGF and STZ/MEK1-AS/KGF newborns (actual magnification ×500). (C) Ductal cell proliferation was assessed by BrdU staining of pancreatic sections of STZ/Std, STZ/MEK1-AS, STZ/Std/KGF and STZ/MEK1-AS/KGF groups. BrdU labelling index of ductal cells in all experimental groups was evaluated in 2-day-old pups and the results were expressed as the percentage of BrdU-positive ductal cells. Three to five rats were analyzed in each experimental group. ** <i>p</i><0.01.</p

Inactivation of ERK1/2 pathway by pharmacological inhibitor abolishes KGF-induced Panc-1 cell proliferation.

<p>(A) Panc-1 cells were pre-incubated for 1 h with 20 µM, 10 µM and 1 µM of PD98059, an inhibitor of MEK1. Cells were then treated for 5 minutes with 30 ng/ml KGF. Cell lysates were analyzed by western blot using specific antibodies against P-ERK1/2 and total-ERK1/2. (B) Panc-1 cells were cultured in serum-free medium. Cells were then exposed or not to KGF or PD98059 alone, or to the combination of the two agents for 24 h. Cell proliferation was assessed by BrdU staining (brown nuclei). Red arrows show BrdU-positive Panc-1 cells and blue arrows show BrdU-negative Panc-1 cells (actual magnification ×250). (C) Panc-1 cells were treated or not with 20 µM PD98059, 1 h prior to the 24 h incubation with KGF or 10% FBS. BrdU labelling index of Panc-1 cells was evaluated and expressed as the percentage of BrdU-positive Panc-1 cells. Results are presented as mean values ±sem of three independent experiments. ** <i>p</i><0.01.</p

Inactivation of PI3K/AKT pathway by wortmannin abolishes KGF-induced duct-to-beta cell differentiation but does not alter the effect of KGF on proliferation.

<p>(A) Double staining for insulin (red cytosol) and PDX1 (brown nuclei) were performed on pancreatic sections of STZ, STZ/KGF, STZ/wortmannin and STZ/wortmannin/KGF rats (wortmannin was administrated at the dose of 0.75 mg/kg body weight). Differentiated beta cells express both insulin and PDX1 (blue arrow). A subset of ductal cells express PDX1 (red arrow) while the majority of ductal cells are PDX1 negative (green arrow) (actual magnification ×500). d = duct. (B) PDX1-positive / insulin-negative ductal cells were quantified after insulin/PDX1 staining on pancreatic sections of the 2-day-old STZ, STZ/KGF, STZ/wortmannin and STZ/wortmannin/KGF rats. Results are expressed as the percentage of PDX1-positive/insulin-negative cells over total ductal cells. Four rats were analyzed in each experimental group. * <i>p</i><0.05; ** <i>p</i><0.01. (C) Ductal cell proliferation was assessed by BrdU staining of pancreatic sections of the 2-day-old STZ, STZ/KGF, STZ/wortmannin and STZ/wortmannin/KGF rats and the results were expressed as the percentage of BrdU-positive ductal cells. Four rats were analyzed in each experimental group. * <i>p</i><0.05; ** <i>p</i><0.01.</p

Inactivation of ERK1/2 pathway by antisense morpholino-oligonucleotides abolishes KGF-induced proliferation of Panc-1 cells.

<p>(A) Panc-1 cells were cultured in serum-free medium and treated for 24 h with MEK1 antisense morpholino-oligonucleotides (MEK1-AS, 10 nmol/ml) or standard morpholino-oligonucleotides (Std, 10 nmol/ml). Cell lysates were analyzed by western blot using specific antibody against MEK1. Representative blots and MEK1 protein quantification are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004734#pone-0004734-g003" target="_blank">Figure 3A</a>. Results are expressed as mean values ±sem of 4 independent experiments. * <i>p</i><0.05. (B) Panc-1 cells were treated with MEK1-AS or Std during 24 h, then stimulated with KGF for 5 minutes before the end of the experiments. Cell lysates were analyzed by western blot for P-ERK1/2 and total-ERK1/2 expression. (C) Panc-1 cells were cultured in serum-free medium and pre-treated with MEK1-AS during 24 h prior the addition of KGF or 10% FBS. BrdU labelling index of Panc-1 cells was evaluated and expressed as the percentage of BrdU-positive Panc-1 cells. Results are presented as mean values ±sem of three independent experiments. * <i>p</i><0.05.</p