Abrogation of Cbl–PI3K Interaction Increases Bone Formation and Osteoblast Proliferation

Cbl is an adaptor protein and E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and phosphorylated by Src family kinases. Phosphorylated CblY737 creates a binding site for the p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) that also plays an important role in the regulation of bone homeostasis. To investigate the role of Cbl–PI3K interaction in bone homeostasis, we examined knock-in mice in which the PI3K binding site on Cbl was ablated due to the substitution of tyrosine 737 to phenylalanine (CblYF/YF, YF mice). We previously reported that bone volume in these mice is increased due to decreased osteoclast function (Adapala et al., J Biol Chem 285:36745–36758, 19). Here, we report that YF mice also have increased bone formation and osteoblast numbers. In ex vivo cultures bone marrow-derived YF osteoblasts showed increased Col1A expression and their proliferation was also significantly augmented. Moreover, proliferation of MC3T3-E1 cells was increased after treatment with conditioned medium generated by culturing YF bone marrow stromal cells. Expression of stromal derived factor-1 (SDF-1) was increased in YF bone marrow stromal cells compared to wild type. Increased immunostaining of SDF-1 and CXCR4 was observed in YF bone marrow stromal cells compared to wild type. Treatment of YF condition medium with neutralizing anti-SDF-1 and anti-CXCR4 antibodies attenuated MC3T3-E1 cell proliferation. Cumulatively, these results show that abrogation of Cbl–PI3K interaction perturbs bone homeostasis, affecting both osteoclast function and osteoblast proliferation

Hypoxia Inhibits Osteogenesis in Human Mesenchymal Stem Cells through Direct Regulation of RUNX2 by TWIST

Bone loss induced by hypoxia is associated with various pathophysiological conditions, however, little is known about the effects of hypoxia and related signaling pathways on osteoblast differentiation and bone formation. Because bone marrow-derived mesenchymal stem cells (MSCs) survive under hypoxic conditions and readily differentiate into osteoblasts by standard induction protocols, they are a good in vitro model to study the effects of hypoxia on osteoblast differentiation.Using human MSCs, we discovered TWIST, a downstream target of HIF-1α, was induced under hypoxia and acted as a transcription repressor of RUNX2 through binding to the E-box located on the promoter of type 1 RUNX2. Suppression of type 1 RUNX2 by TWIST under hypoxia further inhibited the expression of BMP2, type 2 RUNX2 and downstream targets of RUNX2 in MSCs.Our findings point to the important role of hypoxia-mediated signalling in osteogenic differentiation in MSCs through direct regulation of RUNX2 by TWIST, and provide a method for modifying MSC osteogenesis upon application of these cells in fracture healing and bone reconstruction

Growth factor concentrations and their placental mRNA expression are modulated in gestational diabetes mellitus: possible interactions with macrosomia

<p>Abstract</p> <p>Background</p> <p>Gestational diabetes mellitus (GDM) is a form of diabetes that occurs during pregnancy. GDM is a well known risk factor for foetal overgrowth, termed macrosomia which is influenced by maternal hypergycemia and endocrine status through placental circulation. The study was undertaken to investigate the implication of growth factors and their receptors in GDM and macrosomia, and to discuss the role of the materno-foeto-placental axis in the <it>in-utero </it>regulation of foetal growth.</p> <p>Methods</p> <p>30 women with GDM and their 30 macrosomic babies (4.75 ± 0.15 kg), and 30 healthy age-matched pregnant women and their 30 newborns (3.50 ± 0.10 kg) were recruited in the present study. Serum concentrations of GH and growth factors, <it>i.e</it>., IGF-I, IGF-BP3, FGF-2, EGF and PDGF-B were determined by ELISA. The expression of mRNA encoding for GH, IGF-I, IGF-BP3, FGF-2, PDGF-B and EGF, and their receptors, <it>i.e</it>., GHR, IGF-IR, FGF-2R, EGFR and PDGFR-β were quantified by using RT-qPCR.</p> <p>Results</p> <p>The serum concentrations of IGF-I, IGF-BP3, EGF, FGF-2 and PDGF-B were higher in GDM women and their macrosomic babies as compared to their respective controls. The placental mRNA expression of the growth factors was either upregulated (FGF-2 or PDGF-B) or remained unaltered (IGF-I and EGF) in the placenta of GDM women. The mRNA expression of three growth factor receptors, <it>i.e</it>., IGF-IR, EGFR and PDGFR-β, was upregulated in the placenta of GDM women. Interestingly, serum concentrations of GH were downregulated in the GDM women and their macrosomic offspring. Besides, the expression of mRNAs encoding for GHR was higher, but that encoding for GH was lower, in the placenta of GDM women than control women.</p> <p>Conclusions</p> <p>Our results demonstrate that growth factors might be implicated in GDM and, in part, in the pathology of macrosomia via materno-foeto-placental axis.</p

Present state and future perspectives of using pluripotent stem cells in toxicology research

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed

Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration

International audienceDistinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches

Human embryonic stem-cell derivatives for full reconstruction of the pluristratified epidermis: a preclinical study

Background: Cell therapy for large burns is dependent upon autologous epidermis reconstructed in vitro. However, the effectiveness of current procedures is limited by the delay needed to culture the patient's own keratinocytes. To assess whether the keratinocyte progeny of human embryonic stem cells (hESCs) could be used to form a temporary skin substitute for use in patients awaiting autologous grafts, we investigated the cells' capability of constructing a pluristratified epidermis. Methods: hESCs from lines H9 and SA01 were seeded at least in triplicate on fibroblast feeder cells for 40 days in a medium supplemented with bone morphogenetic protein 4 and ascorbic acid. Molecular characterisation of cell differentiation was done throughout the process by quantitative PCR, fluorescence-activated cell sorting, and immunocytochemical techniques. Keratinocyte molecular differentiation and functional capacity to construct a human epidermis were assessed in vitro and in vivo. Findings: From hESCs, we generated a homogeneous population of cells that showed phenotypic characteristics of basal keratinocytes. Expression levels of genes encoding keratin 14, keratin 5, integrin alpha6, integrin beta4, collagen VII, and laminin 5 in these cells were similar to those in basal keratinocytes. After seeding on an artificial matrix, keratinocytes derived from hESCs (K-hESCs) formed a pluristratified epidermis. Keratin-14 immunostaining was seen in the basal compartment, with keratin 10 present in layers overlying the basal layer. Involucrin and filaggrin, late markers of epidermal differentiation, were detected in the uppermost layers only. 12 weeks after grafting onto five immunodeficient mice, epidermis derived from K-hESCs had a structure consistent with that of mature human skin. Human involucrin was appropriately located in spinous and granular layers and few Ki67-positive cells were detected in the basal layer. Interpretation: hESCs can be differentiated into basal keratinocytes that are fully functional--ie, able to construct a pluristratified epidermis. This resource could be developed to provide temporary skin substitutes for patients awaiting autologous grafts. Funding: Institut National de la Santé et de la Recherche Médicale, University Evry Val d'Essonne, Association Française contre les Myopathies, Fondation René Touraine, and Genopole