GFAP-expressing cells in the adult hypothalamus can generate multiple neural cell lineages in vitro.

Adult neural stem cells (NSCs) located in the two canonical neurogenic niches, the subventricular zone (SVZ) and the subgranular zone (SGZ), express the glial fibrillary acidic protein (GFAP). Recently, proliferative activity has been described in the hypothalamus although the characterization of hypothalamic neural stem/progenitor cells (NSPCs) is still uncertain. We therefore investigated whether hypothalamic GFAP-positive cells, as in the SVZ and SGZ, also have neurogenic potential. We used a transgenic mouse line expressing green fluorescent protein (GFP) under the control of the GFAP promoter. GFAP-GFP expressing cells are localized in the ependymal layer as well as in the parenchyma of the mediobasal hypothalamus (MBH) and express Sox2, a marker for NSCs. Interestingly, no sexual dimorphism was observed in the numbers of GFP + and GFP-Sox2 + cells. After cells sorting, these cells were able to generate neurospheres in vitro and give rise to neurons, astrocytes and oligodendrocytes. Taken together, these results show that hypothalamic GFAP-expressing cells form a population of NSPCs.

Multiparametric MR Evaluation of the Photoperiodic Regulation of Hypothalamic Structures in Sheep.

Most organisms on earth, humans included, have developed strategies to cope with environmental day-night and seasonal cycles to survive. For most of them, their physiological and behavioral functions, including the reproductive function, are synchronized with the annual changes of day length, to ensure winter survival and subsequent reproductive success in the following spring. Sheep are sensitive to photoperiod, which also regulates natural adult neurogenesis in their hypothalamus. We postulate that the ovine model represents a good alternative to study the functional and metabolic changes occurring in response to photoperiodic changes in hypothalamic structures of the brain. Here, the impact of the photoperiod on the neurovascular coupling and the metabolism of the hypothalamic structures was investigated at 3T using BOLD fMRI, perfusion-MRI and proton magnetic resonance spectroscopy (1H-MRS). A longitudinal study involving 8 ewes was conducted during long days (LD) and short days (SD) revealing significant BOLD, rCBV and metabolic changes in hypothalamic structures of the ewe brain between LD and SD. More specifically, the transition between LD and SD revealed negative BOLD responses to hypercapnia at the beginning of SD period followed by significant increases in BOLD, rCBV, Glx and tNAA concentrations towards the end of the SD period. These observations suggest longitudinal mechanisms promoting the proliferation and differentiation of neural stem cells within the hypothalamic niche of breeding ewes. We conclude that multiparametric MRI studies including 1H-MRS could be promising non-invasive translational techniques to investigate the existence of natural adult neurogenesis in-vivo in gyrencephalic brains.

Seasonal remodeling of the progenitor pool and its distribution in the ewe mediobasal hypothalamus.

Recent studies have reported the presence of adult neurogenesis in the arcuate nucleus periventricular space (pvARH) and in the median eminence (ME), two structures involved in reproductive function. In sheep, a seasonal mammal, decreasing daylight in autumn induces a higher neurogenic activity in these two structures. However, the different types of neural stem and progenitor cells (NSCs/NPCs) that populate the arcuate nucleus and median eminence, as well as their location, have not been evaluated. Here, using semi-automatic image analyzing processes, we identified and quantified the different populations of NSCs/NPCs, showing that, during short days, higher densities of [SOX2 +] cells are found in pvARH and ME. In the pvARH, higher densities of astrocytic and oligodendrocitic progenitors mainly contribute to these variations. The different populations of NSCs/NPCs were mapped according to their position relative to the third ventricle and their proximity to the vasculature. We showed that [SOX2 +] cells extended deeper into the hypothalamic parenchyma during short days. Similarly, [SOX2 +] cells were found further from the vasculature in the pvARH and the ME, at this time of year, indicating the existence of migratory signals. The expression levels of neuregulin transcripts (NRGs), whose proteins are known to stimulate proliferation and adult neurogenesis and to regulate progenitor migration, as well as the expression levels of ERBB mRNAs, cognate receptors for NRGs, were assessed. We showed that mRNA expression changed seasonally in pvARH and ME, suggesting that the ErbB-NRG system is potentially involved in the photoperiodic regulation of neurogenesis in seasonal adult mammals.

Selective Depletion of Adult GFAP-Expressing Tanycytes Leads to Hypogonadotropic Hypogonadism in Males.

In adult mammals, neural stem cells are localized in three neurogenic regions, the subventricular zone of the lateral ventricle (SVZ), the subgranular zone of the dentate gyrus of the hippocampus (SGZ) and the hypothalamus. In the SVZ and the SGZ, neural stem/progenitor cells (NSPCs) express the glial fibrillary acidic protein (GFAP) and selective depletion of these NSPCs drastically decreases cell proliferation in vitro and in vivo. In the hypothalamus, GFAP is expressed by α-tanycytes, which are specialized radial glia-like cells in the wall of the third ventricle also recognized as NSPCs. To explore the role of these hypothalamic GFAP-positive tanycytes, we used transgenic mice expressing herpes simplex virus thymidine kinase (HSV-Tk) under the control of the mouse Gfap promoter and a 4-week intracerebroventricular infusion of the antiviral agent ganciclovir (GCV) which kills dividing cells expressing Tk. While GCV significantly reduced the number and growth of hypothalamus-derived neurospheres from adult transgenic mice in vitro, it causes hypogonadotropic hypogonadism in vivo. The selective death of dividing tanycytes expressing GFAP indeed results in a marked decrease in testosterone levels and testicular weight, as well as vacuolization of the seminiferous tubules and loss of spermatogenesis. Additionally, GCV-treated GFAP-Tk mice show impaired sexual behavior, but no alteration in food intake or body weight. Our results also show that the selective depletion of GFAP-expressing tanycytes leads to a sharp decrease in the number of gonadotropin-releasing hormone (GnRH)-immunoreactive neurons and a blunted LH secretion. Overall, our data show that GFAP-expressing tanycytes play a central role in the regulation of male reproductive function.

Seasonal vascular plasticity in the mediobasal hypothalamus of the adult ewe.

Sheep, like most seasonal mammals, exhibit a cyclic adaptive reproductive physiology that allows ewes to give birth to their progeny during the spring when environmental conditions are favorable to their survival. This process relies on the detection of day length (or photoperiod) and is associated with profound changes in cellular plasticity and gene expression in the hypothalamic-pituitary-gonadal axis, mechanisms that are suggested to participate in the seasonal adaptation of neuroendocrine circuits. Recently, pituitary vascular growth has been proposed as a seasonally regulated process in which the vascular endothelial growth factor A (VEGFA), a well-known angiogenic cytokine, is suspected to play a crucial role. However, whether this mechanism is restricted to the pituitary gland or also occurs in the mediobasal hypothalamus (MBH), a crucial contributor to the control of the reproductive function, remains unexplored. Using newly developed image analysis tools, we showed that the arcuate nucleus (ARH) of the MBH exhibits an enhanced vascular density during the long photoperiod or non-breeding season, associated with higher expression of VEGFA. In the median eminence (ME), a structure connecting the MBH to the pituitary gland, higher VEGFA, kinase insert domain receptor (KDR/VEGFR2) and plasmalemma vesicle-associated protein (PLVAP) gene expressions were detected during the long photoperiod. We also found that VEGFA and its receptor, VEGFR2, are expressed by neurons and tanycytes in both the ARH and ME. Altogether, these data show variations in the MBH vasculature according to seasons potentially through a VEGFA-dependent pathway, paving the way for future studies aiming to decipher the role of these changes in the hypothalamic control of seasonal reproduction.

When pharmaceutical drugs become environmental pollutants: Potential neural effects and underlying mechanisms.

Pharmaceutical drugs have become consumer products, with a daily use for some of them. The volume of production and consumption of drugs is such that they have become environmental pollutants. Their transfer to wastewater through urine, feces or rinsing in case of skin use, associated with partial elimination by wastewater treatment plants generalize pollution in the hydrosphere, including drinking water, sediments, soils, the food chain and plants. Here, we review the potential effects of environmental exposure to three classes of pharmaceutical drugs, i.e. antibiotics, antidepressants and non-steroidal anti-inflammatory drugs, on neurodevelopment. Experimental studies analyzing their underlying modes of action including those related to endocrine disruption, and molecular mechanisms including epigenetic modifications are presented. In addition, the contribution of brain imaging to the assessment of adverse effects of these three classes of pharmaceuticals is approached.

Rostro-caudal maturation of glial cells in the accessory olfactory system during development: involvement in outgrowth of GnRH neurites.

During mammalian embryonic development, GnRH neurones differentiate from the nasal placode and migrate through the nasal septum towards the forebrain. We previously showed that a category of glial cells, the olfactory ensheathing cells (OEC), forms the microenvironment of migrating GnRH neurones. Here, to characterize the quantitative and qualitative importance of this glial, we investigated the spatiotemporal maturation of glial cells in situ and the role of maturing glia in GnRH neurones development ex vivo. More than 90% of migrating GnRH neurones were found to be associated with glial cells. There was no change in the cellular microenvironment of GnRH neurones in the regions crossed during embryonic development as glial cells formed the main microenvironment of these neurones (53.4%). However, the phenotype of OEC associated with GnRH neurones changed across regions. The OEC progenitors immunoreactive to brain lipid binding protein formed the microenvironment of migrating GnRH neurones from the vomeronasal organ to the telencephalon and were also present in the diencephalon. However, during GnRH neurone migration, maturation of OEC to [GFAP+] state (glial fibrillary acid protein) was only observed in the nasal septum. Inducing depletion of OEC in maturation, using transgenic mice expressing herpes simplex virus thymidine kinase driven by the GFAP promoter, had no impact on neurogenesis or on triggering GnRH neurones migration in nasal explant culture. Nevertheless, depletion of [GFAP+] cells decreased GnRH neurites outgrowth by 57.4%. This study suggests that specific maturation of OEC in the nasal septum plays a role in morphological differentiation of GnRH neurones.

Yolk hormones influence in ovo chemosensory learning, growth, and feeding behavior in domestic chicks.

In this study, we assessed whether prenatal exposure to elevated yolk steroid hormones can influence in ovo chemosensory learning and the behavior of domestic chicks. We simulated a maternal environmental challenge by experimentally enhancing yolk progesterone, testosterone, and estradiol concentrations in hen eggs prior to incubation. The embryos from these hormones-treated eggs (HO) as well as sham embryos (O) that had received the vehicle-only were exposed to the odor of fish oil (menhaden) between embryonic Days 11 and 20. An additional group of control embryos (C) was not exposed to the odor. All chicks were tested following hatching for their feeding preferences between foods that were or were not odorized with the menhaden odor. In the 3-min choice tests, the behavior of O chicks differed significantly according to the type of food whereas C and HO chicks showed no preference between odorized and non-odorized food. Our result suggests weaker response in HO chicks. In addition, HO chicks showed impaired growth and reduced intake of an unfamiliar food on the 24-h time scale compared to controls. Our data suggest that embryonic exposure to increased yolk hormone levels can alter growth, chemosensory learning, and the development of feeding behaviors.

GnRH Episodic Secretion Is Altered by Pharmacological Blockade of Gap Junctions: Possible Involvement of Glial Cells.

Episodic release of GnRH is essential for reproductive function. In vitro studies have established that this episodic release is an endogenous property of GnRH neurons and that GnRH secretory pulses are associated with synchronization of GnRH neuron activity. The cellular mechanisms by which GnRH neurons synchronize remain largely unknown. There is no clear evidence of physical coupling of GnRH neurons through gap junctions to explain episodic synchronization. However, coupling of glial cells through gap junctions has been shown to regulate neuron activity in their microenvironment. The present study investigated whether glial cell communication through gap junctions plays a role in GnRH neuron activity and secretion in the mouse. Our findings show that Glial Fibrillary Acidic Protein-expressing glial cells located in the median eminence in close vicinity to GnRH fibers expressed Gja1 encoding connexin-43. To study the impact of glial-gap junction coupling on GnRH neuron activity, an in vitro model of primary cultures from mouse embryo nasal placodes was used. In this model, GnRH neurons possess a glial microenvironment and were able to release GnRH in an episodic manner. Our findings show that in vitro glial cells forming the microenvironment of GnRH neurons expressed connexin-43 and displayed functional gap junctions. Pharmacological blockade of the gap junctions with 50 µM 18-α-glycyrrhetinic acid decreased GnRH secretion by reducing pulse frequency and amplitude, suppressed neuronal synchronization and drastically reduced spontaneous electrical activity, all these effects were reversed upon 18-α-glycyrrhetinic acid washout.

Olfactory ensheathing cells form the microenvironment of migrating GnRH-1 neurons during mouse development.

During development, GnRH-1 neurons differentiate extracerebraly from the nasal placode and migrate from the vomeronasal organ to the forebrain along vomeronasal and terminal nerves. Numerous studies have described the influence of different molecules on the migration of GnRH-1 neurons, however, the role of microenvironment cells remains poorly understood. This study used GFAP-GFP transgenic mice to detect glial cells at early developmental stages. Using nasal explant cultures, the comigration of glial cells with GnRH-1 neurons was clearly demonstrated. This in vitro approach showed that glial cells began migrating from the explants before GnRH-1 neurons. They remained ahead of the GnRH-1 migratory front and stopped migrating after the GnRH-1 neurons. The association of these glial cells with the axons combined with gene expression analysis of GFAP-GFP sorted cells enabled them to be identified as olfactory ensheathing cells (OEC). Immunohistochemical analysis revealed the presence of multiple glial cell-type markers showing several OEC subpopulations surrounding GnRH-1 neurons. Moreover, these OEC expressed genes whose products are involved in the migration of GnRH-1 neurons, such as Nelf and Semaphorin 4. In situ data confirmed that the majority of the GnRH-1 neurons were associated with glial cells along the vomeronasal axons in nasal septum and terminal nerves in the nasal forebrain junction as early as E12.5. Overall, these data demonstrate an OEC microenvironment for migrating GnRH-1 neurons during mouse development. The fact that this glial cell type precedes GnRH-1 neurons and encodes for molecules involved in their nasal migration suggests that it participates in the GnRH-1 system ontogenesis.

Lentiviral-mediated integrin α5 expression in human adult mesenchymal stromal cells promotes bone repair in mouse cranial and long-bone defects.

Abstract Adult human mesenchymal stromal cells (hMSCs) are an important source for tissue repair in regenerative medicine. Notably, targeted gene therapy in hMSCs to promote osteogenic differentiation may help in the development of novel therapeutic approaches for bone repair. We recently showed that α5 integrin (ITGA5) promotes osteoblast differentiation in bone marrow-derived hMSCs. Here, we determined whether lentiviral (LV)-mediated expression of ITGA5 in hMSCs derived from the bone-marrow stroma of healthy individuals may promote bone repair in vivo in two relevant critical-size bone defects in the mouse. In a first series of experiments, control or LV-ITGA5-transduced hMSCs were seeded on collagen-based gelatin sponge and transplanted in a cranial critical-size defect (5 mm) in Nude-Foxn1nu mice. Microcomputed tomography and quantitative histological analyses after 8 weeks showed no or little de novo bone formation in defects implanted with collagen sponge alone or with hMSCs, respectively. In contrast, implantation of collagen sponge with LV-ITGA5-transduced hMSCs showed greater bone formation compared with control hMSCs. We also tested the bone-repair potential of LV-mediated ITGA5 expression in hMSCs in a critical-size long-bone defect (2 mm) in femur in Nude-Foxn1nu mice. Bone remnants were stabilized with external fixation, and control or LV-ITGA5-transduced hMSCs mixed with coral/hydroxyapatite particles were transplanted into the critical-size long-bone defect. Histological analysis after 8 weeks showed that LV-ITGA5-transduced hMSCs implanted with particles induced 85% bone regeneration and repair. These results demonstrate that repair of critical-size mouse cranial and long-bone defects can be induced using LV-mediated ITGA5 gene expression in hMSCs, which provides a novel gene therapy for bone regeneration.

CYR61 downregulation reduces osteosarcoma cell invasion, migration, and metastasis.

Osteosarcoma is the most common primary tumor of bone. The rapid development of metastatic lesions and resistance to chemotherapy remain major mechanisms responsible for the failure of treatments and the poor survival rate for patients. We showed previously that the HMGCoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitor statin exhibits antitumoral effects on osteosarcoma cells. Here, using microarray analysis, we identify Cyr61 as a new target of statins. Transcriptome and molecular analyses revealed that statins downregulate Cyr61 expression in human and murine osteosarcoma cells. Cyr61 silencing in osteosarcoma cell lines enhanced cell death and reduced cell migration and cell invasion compared with parental cells, whereas Cyr61 overexpression had opposite effects. Cyr61 expression was evaluated in 231 tissue cores from osteosarcoma patients. Tissue microarray analysis revealed that Cyr61 protein expression was higher in human osteosarcoma than in normal bone tissue and was further increased in metastatic tissues. Finally, tumor behavior and metastasis occurrence were analyzed by intramuscular injection of modified osteosarcoma cells into BALB/c mice. Cyr61 overexpression enhanced lung metastasis development, whereas cyr61 silencing strongly reduced lung metastases in mice. The results reveal that cyr61 expression increases with tumor grade in human osteosarcoma and demonstrate that cyr61 silencing inhibits in vitro osteosarcoma cell invasion and migration as well as in vivo lung metastases in mice. These data provide a novel molecular target for therapeutic intervention in metastatic osteosarcoma.

Molecular silencing of Twist1 enhances osteogenic differentiation of murine mesenchymal stem cells: implication of FGFR2 signaling.

The capacity of mesenchymal stem cells (MSCs) to differentiate into functional osteoblasts is tightly controlled by transcription factors that trigger osteoblast commitment and differentiation. The role of Twist1, a basic helix-loop-helix (bHLH) transcription factor, in osteogenic differentiation of MSCs remains unclear. Here we investigated the role of Twist1 in the osteogenic differentiation program of murine C3H10T1/2 mesenchymal cells. We showed that molecular silencing of Twist1 using short hairpin RNA (shRNA) expression moderately increased C3H10T1/2 cell proliferation and had no effect on cell survival. In contrast, Twist1 silencing enhanced osteoblast gene expression and matrix mineralization in vitro. Biochemical analyses revealed that Twist1 silencing increased the expression of FGFR2 protein level, which was reduced by a mutant Runx2. Consistent with this finding, Twist1 silencing increased ERK1/2 and PI3K signaling. Moreover, molecular or pharmacological inhibition of FGFR2 or of ERK1/2 and PI3K signaling partly abolished the increased osteoblast gene expression induced by Twist1 silencing in C3H10T1/2 cells. These results reveal that Twist1 silencing upregulates osteoblast differentiation of murine mesenchymal cells in part via activation of FGFR2 expression and downstream signaling pathways, which provides novel insights into the molecular signals by which this transcription factor regulates the osteogenic differentiation program in MSCs.

Autocrine fibroblast growth factor 18 mediates dexamethasone-induced osteogenic differentiation of murine mesenchymal stem cells.

The potential of mesenchymal stem cells (MSC) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors capable of promoting osteoblast differentiation in MSCs is therefore critical to enhance the osteogenic potential of MSCs. Using microarray analysis combined with biochemical and molecular approach, we found that FGF18, a member of the FGF family, is upregulated during osteoblast differentiation induced by dexamethasone in murine MSCs. We showed that overexpression of FGF18 by lentiviral (LV) infection, or treatment of MSCs with recombinant human (rh)FGF18 increased the expression of the osteoblast specific transcription factor Runx2, and enhanced osteoblast phenotypic marker gene expression and in vitro osteogenesis. Molecular silencing using lentiviral shRNA demonstrated that downregulation of FGFR1 or FGFR2 abrogated osteoblast gene expression induced by either LV-FGF18 or rhFGF18, indicating that FGF18 enhances osteoblast differentiation in MSCs via activation of FGFR1 or FGFR2 signaling. Biochemical and pharmacological analyses showed that the induction of phenotypic osteoblast markers by LV-FGF18 is mediated by activation of ERK1/2-MAPKs and PI3K signaling in MSCs. These results reveal that FGF18 is an essential autocrine positive regulator of the osteogenic differentiation program in murine MSCs and indicate that osteogenic differentiation induced by FGF18 in MSCs is triggered by FGFR1/FGFR2-mediated ERK1/2-MAPKs and PI3K signaling.

Priming integrin alpha5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis.

Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin alpha5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised.

KCa2.3 channel-dependent hyperpolarization increases melanoma cell motility.

Cell migration and invasion are required for tumour cells to spread from the primary tumour bed so as to form secondary tumours at distant sites. We report evidence of an unusual expression of KCa2.3 (SK3) protein in melanoma cell lines but not in normal melanocytes. Knockdown of the KCa2.3 channel led to plasma membrane depolarization, decreased 2D and 3D cell motility. Conversely, enforced production of KCa2.3 protein in KCa2.3 non-expressing cells led to the plasma membrane becoming hyperpolarized, and enhanced cell motility. In contrast, KCa3.1 channels had no effect on cell motility despite an active role in regulating membrane potential. Our data also suggest that membrane hyperpolarization increases melanoma cell motility and that this occurs through the KCa2.3 channel. Our findings reveal a previously unknown function of the KCa2.3 channel, and suggest that the KCa2.3 channel might be the only member of the Ca(2+)-activated K(+) channel family involved in melanoma cell motility pathways.

Chimeric hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies.

The hepatitis B virus (HBV) envelope protein (S) self-assembles into subviral particles used as commercial vaccines against hepatitis B. These particles are excellent carriers for foreign epitopes, which can be inserted into the external hydrophilic loop or at the N- or C-terminal end of the HBV S protein. We show here that the N-terminal transmembrane domain (TMD) of HBV S can be replaced by the TMDs of the hepatitis C virus (HCV) envelope proteins E1 and E2, to generate fusion proteins containing the entire HCV E1 or E2 sequence that are efficiently coassembled with the HBV S into particles. This demonstrates the remarkable tolerance of the HBV S protein to sequence substitutions conserving its subviral particle assembly properties. These findings may have implications for the design of new vaccine strategies based on the use of HBV subviral particles as carriers for various transmembrane proteins and produced using the same industrial procedures that are established for the HBV vaccine.

FHL2 mediates dexamethasone-induced mesenchymal cell differentiation into osteoblasts by activating Wnt/beta-catenin signaling-dependent Runx2 expression.

The differentiation of bone marrow mesenchymal stem cells (MSCs) into osteoblasts is a crucial step in bone formation. However, the mechanisms involved in the early stages of osteogenic differentiation are not well understood. In this study, we identified FHL2, a member of the LIM-only subclass of the LIM protein superfamily, that is up-regulated during early osteoblast differentiation induced by dexamethasone in murine and human MSCs. Gain-of-function studies showed that FHL2 promotes the expression of the osteoblast transcription factor Runx2, alkaline phosphatase, type I collagen, as well as in vitro extracellular matrix mineralization in murine and human mesenchymal cells. Knocking down FHL2 using sh-RNA reduces basal and dexamethasone-induced osteoblast marker gene expression in MSCs. We demonstrate that FHL2 interacts with beta-catenin, a key player involved in bone formation induced by Wnt signaling. FHL2-beta-catenin interaction potentiates beta-catenin nuclear translocation and TCF/LEF transcription, resulting in increased Runx2 and alkaline phosphatase expression, which was inhibited by the Wnt inhibitor DKK1. Reduction of Runx2 transcriptional activity using a mutant Runx2 results in inhibition of FHL2-induced alkaline phosphatase expression in MSCs. These findings reveal that FHL2 acts as an endogenous activator of mesenchymal cell differentiation into osteoblasts and mediates osteogenic differentiation induced by dexamethasone in MSCs through activation of Wnt/beta-catenin signaling- dependent Runx2 expression.

Phosphatase PTEN in chicken muscle is regulated during ontogenesis.

The phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase able to inhibit significant actors of cell signaling (i.e. phosphatidylinositol-3'kinase and mitogen-activated protein kinase pathways). The aim of this study was to characterize PTEN and to investigate its regulation during ontogenesis in chicken muscle. Pectoralis major muscle was sampled on day 18 of the embryonic period (E18), at hatching (d0) and in fed chickens at 2, 7 and 43 days after hatching (d2, d7 and d43). We first cloned the totality of chicken PTEN cDNA; its translation into a putative protein showed more than 95% sequence identity with that characterized in mammals (humans, mice). PTEN was expressed under two major transcripts in the majority of tissues, including muscles where the expression of PTEN mRNA increased with age (P < 0.05). Surprisingly, the protein levels of PTEN (protein characterized with an apparent molecular weight of 55kDa) and its activity were considerably decreased between the E18 and d43 stages (approximately 8-10-fold reduction, P < 0.001). An association between these decreases and higher phosphorylation levels of two potential indirect downstream targets of phosphatase (i.e. AKT and ERK) was observed only in the early growth phases. It was concluded that phosphatase PTEN was expressed in chicken muscle and that its expression was regulated during ontogenesis.

Mechanisms involved in the nutritional regulation of mRNA translation: features of the avian model.

Insulin and amino acids are key factors in regulating protein synthesis. The mechanisms of their action have been widely studied for several years. The insulin signal is mediated by the activation of intracellular kinases such as phosphatidylinositol-3'kinase and the mammalian target of rapamycin (mTOR), affecting the phosphorylation of some major effectors involved in the regulation of translation initiation, i.e. p70 S6 kinase (p70S6K) and the translational repressor eukaryotic initiation factor 4E binding protein (4E-BP1). The amino acid-induced signalling cascade also originates from mTOR and promotes p70S6K and 4E-BP1 activation. However, the mechanisms of regulation are complex and little understood, especially in vivo. Elucidating these mechanisms is important for both fundamental physiology and nutritional applications, i.e. better control of the use of nutrients and optimisation of dietary amino acid supplies in various physiological and physiopathological situations. In comparative physiology, the chicken is an interesting model to gain better understanding of the nutritional regulation of mRNA translation because of the very high rates of muscle growth and protein synthesis, and the unusual features compared with mammals. In the present review we provide an overview of the roles of insulin and amino acids as regulators of protein synthesis in both mammals and avian species.