Differential expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells

Embryonic stem (ES) cells have the potential to develop into various cell lineages including hemangioblasts (Flk1+), a common progenitor for hematopoietic and vascular endothelial cells. Previous studies indicate that Flk1+ cells, a marker for hemangioblast, can be derived from ES cell and that Flk1+ can be differentiated into hematopoietic or endothelial cells depending on culture conditions. We developed an improved in vitro system to generate Flk1+-enriched cultures from mouse ES cells and used this in vitro system to study the role of Wnt signalling in early endothelial progenitor cells. We determined the expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells. RT-PCR analyses identified significantly higher expression of non-canonical Wnt5a and Wnt11 genes in Flk1+ cells compared to Flk1- cells. In contrast, expression of canonical Wnt3a gene was reduced in Flk1+ cells. In addition, Frizzled2, Frizzled5 and Frizzled7 genes were also expressed at a higher level in Flk1+ cells. The differential expression of Wnt and Frizzled genes in Flk1+ cells provides a novel insight into the role of non-canonical Wnt signalling in vascular endothelial fate determination.     

Identification of c-Kit receptor as a regulator of adult neural stem cells in the mammalian eye: interactions with Notch signaling

Neural stem cells are present in specific regions of the adult central nervous system (CNS). Recent evidence suggests that the ciliary epithelium (CE), a CNS derivative, in the adult mammalian eye, harbors a quiescent population of neural stem cells. Here, we report the identification of c-Kit signaling as one of the regulators of adult CE neural stem cells in vitro. c-Kit receptors are expressed in proliferating adult CE neural stem cells and colocalized with neural progenitor markers. Perturbation of c-Kit signaling influences the self-renewal and differentiation of CE neural stem cells, thus demonstrating the role of c-Kit signaling in the maintenance of these cells. In addition, we observed an influence of c-Kit-mediated signaling on the expression of Notch1, another critical regulator of neural stem cells. Our observations suggest that, given the importance of preservation of a stem cell pool for generating different cell types at different times, multiple signaling pathways act in concert for the maintenance of neural stem cells.     

Matrix metalloproteinase-2, caveolins, focal adhesion kinase and c-Kit in cells of the mouse myocardium

Matrix metalloproteinase-2 (MMP-2) may play roles at intracellular and extracellular sites of the heart in ischaemia/reperfusion injury. Caveolins (Cav-1, -2 and -3) are lipid raft proteins which play roles in cell sig-nalling. This study examined, using immunohistochemistry and two photon confocal microscopy, if MMP-2 and caveolins co-localize at the plasma membrane of cardiac cells: cardiomyocytes (CM), fibroblasts (FB) and capillary endothelial cells (CEC) in the left ventricle (LV) of the Cav-1(+/+) and Cav-1(-/-) mouse heart. In Cav-1(+/+) mouse LV MMP-2 and Cav-1 co-localized at CM plasma membranes, and at multiple locations in FB and CEC. MMP-2 co-localized with Cav-2 only at CEC. MMP-2 co-localized with Cav-3 at CM plasma membranes and Z-lines, and partially at FB and CEC. In Cav-1(-/-) LV Cav-1 and MMP-2 were absent or reduced everywhere. Cav-2 appeared at CEC despite the absence of Cav-1. Cav-3 appeared at CM plasma membranes and Z-lines, FB and CEC. Also, FAK in FB and c-Kit in interstitial Cajal-like cells (ICLC) were completely absent. By transmission electron microscopy in Cav-1(+/+), regular size caveolae (Cav) were at CEC, irregular size Cav were at CM and a few were at FB. In Cav-1(-/-) there were few Cav at CM and FB and some at CEC. To conclude, MMP-2 is closely associated with caveolins at FB and CEC as well as at CM. Also, MMP-2 is closely associated with FAK at FB and c-Kit at ICLC. Thus, Cav-1 expression is not necessary for Cav-2 expression. Cav-3 or Cav-3 with Cav-2 has the capability to make Cav.     

The tyrosine phosphatase SHP-1 inhibits proliferation of activated hepatic stellate cells by impairing PDGF receptor signaling

The dimerization and auto-transphosphorylation of platelet-derived growth factor receptor (PDGFR) upon engagement by platelet-derived growth factor (PDGF) activates signals promoting the mitogenic response of hepatic stellate cells (HSCs) due to liver injury, thus contributing to the development of hepatic fibrosis. We demonstrate that the tyrosine phosphatases Src homology 2 domain-containing phosphatase 1 and 2 (SHP-1 and SHP-2) act as crucial regulators of a complex signaling network orchestrated by PDGFR activation in a spatio-temporal manner with diverse and opposing functions in HSCs. In fact, silencing of either phosphatase shows that SHP-2 is committed to PDGFR-mediated cell proliferation, whereas SHP-1 dephosphorylates PDGFR hence abrogating the downstream signaling pathways that result in HSC activation. In this regard, SHP-1 as an off-switch of PDGFR signaling appears to emerge as a valuable molecular target to trigger as to prevent HSC proliferation and the fibrogenic effects of HSC activation. We show that boswellic acid, a multitarget compound with potent anti-inflammatory action, exerts an anti-proliferative effect on HSCs, as in other cell models, by upregulating SHP-1 with subsequent dephosphorylation of PDGFR-β and downregulation of PDGF-dependent signaling after PDGF stimulation. Moreover, the synergism resulting from the combined use of boswellic acid and imatinib, which directly inhibits PDGFR-β activity, on activated HSCs offers new perspectives for the development of therapeutic strategies that could implement molecules affecting diverse players of this molecular circuit, thus paving the way to multi-drug low-dose regimens for liver fibrosis.     

Musashi 1‐positive cells derived from mouse embryonic stem cells can differentiate into neural and intestinal epithelial‐like cells in vivo

Msi1 (Musashi 1) is regarded as a marker for neural and intestinal epithelial stem cells. However, it is still unclear whether Msi1‐positive cells derived from mouse embryonic stem cells have the ability to differentiate into neural or intestinal epithelial cells. A pMsi1–GFP (green fluorescent protein) reporter plasmid was constructed in order to sort Msi1‐positive cells out of the differentiated cell population. The GFP‐positive cells (i.e. Msi1‐positive cells) were sorted by FACS and were hypodermically engrafted into the backs of NOD/SCID (non‐obese diabetic/severe combined immunodeficient) mice. The presence of neural and intestinal epithelial cells in the grafts was detected. Msi1 was highly expressed in the GFP‐positive cells, but not in the GFP‐negative cells. The markers for neural cells (Nestin and Tubulin β III) and intestinal epithelial cells [FABP2 (fatty acid binding protein 2), Lyz (lysozyme) and ChA (chromogranin A)] were more highly expressed in the grafts from Msi1‐positive cells than those from Msi1‐negative cells (P<0.05). The grafts from the Msi1‐negative cells contained more mesodermal‐like tissues than those from the Msi1‐positive cells. The pMsi1–GFP vector can be used to sort Msi1‐positive cells from a cell population derived from mouse embryonic stem cells. The Msi1‐positive cells can differentiate into neural and intestinal epithelial‐like cells in vivo.     

p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75^NTR), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75^NTR and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75^NTR and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75^NTR/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75^NTR or TrkA. Interestingly, immunoreactivity to anti-p75^NTR antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75^NTR, when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75^NTR is turned on.     

Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies

Retinoid signaling has been implicated in embryonic stem cell differentiation. Here we present a systematic analysis of gene expression changes in mouse embryonic stem cells (mESCs), during their spontaneous differentiation into embryoid bodies and the effect of all-trans retinoic acid (ATRA) on this process. We show that retinoic acid is present in the serum and is sufficient to activate retinoid signaling at a basal level in undifferentiated mESCs. This signal disappears during embryoid body formation. However exogenously added ATRA resets the spontaneous differentiation programs in embryoid bodies and initiates a distinct genetic program. These data suggest that retinoid signaling not only promotes a particular pathway but also acts as a context dependent general coordinator of the differentiation states in embryonic stem cells.     

Altered expression of sphingosine kinase 1 and sphingosine-1-phosphate receptor 1 in mouse hippocampus after kainic acid treatment

Kainic acid (KA) induces hippocampal cell death and astrocyte proliferation. There are reports that sphingosine kinase (SPHK)1 and sphingosine-1- phosphate (S1P) receptor 1 (S1P₁) signaling axis controls astrocyte proliferation. Here we examined the temporal changes of SPHK1/S1P₁ in mouse hippocampus during KA-induced hippocampal cell death. Mice were killed at 2, 6, 24, or 48 h after KA (30 mg/kg) injection. There was an increase in Fluoro-Jade B-positive cells in the hippocampus of KA-treated mice with temporal changes of glial fibrillary acidic protein (GFAP) expression. The lowest level of SPHK1 protein expression was found 2 h after KA treatment. Six hours after KA treatment, the expression of SPHK1 and S1P₁ proteins steadily increased in the hippocampus. In immunohistochemical analysis, SPHK1 and S1P₁ are more immunoreactive in astrocytes within the hippocampus of KA-treated mice than in hippocampus of control mice. These results indicate that SPHK1/S1P₁ signaling axis may play an important role in astrocytes proliferation during KA-induced excitotoxicity.     

Involvement of neuropeptide Y and its Y1 and Y5 receptors in maintaining self‐renewal and proliferation of human embryonic stem cells

Neuropeptide Y (NPY) and NPY receptors are widely expressed in various organs and cell types and have been shown to have pleiotropic functions. However, their presence or role in human embryonic stem cells (hESCs) remains unknown. We now show that undifferentiated hESCs primarily express NPY and its Y1 and Y5 receptors. Inhibition of NPY signalling using either the selective NPY Y1 or Y5 receptor antagonist reduces the maintenance of self‐renewal and proliferation of undifferentiated hESCs. We also provide compelling evidence that exogenous NPY supports the long‐term growth of undifferentiated hESCs in the absence of feeder cell factors using only knockout serum replacement media. Further, NPY facilitates the use of chemically defined medium made up of N2/B27 supplement and basic fibroblast growth factor (bFGF) for hESC feeder‐free culture. Our results indicate that both Y1 and Y5 receptors appear to be involved in the NPY‐mediated activation of AKT/protein kinase B and extracellular signal‐regulated kinase 1/2 (ERK1/2) in hESCs. Notably, only Y1 receptor, but not Y5 receptor, is responsible for the NPY‐induced activation of cAMP‐response element binding (CREB) in hESCs. These results provide the first evidence that NPY and its Y1 and Y5 receptors have potential role in maintaining hESC self‐renewal and pluripotency. We demonstrate the underlying importance of NPY signalling and its usefulness in the development of a defined and xeno‐free culture condition for the large‐scale propagation of undifferentiated hESCs.     

Differential effects of imatinib on PDGF-induced proliferation and PDGF receptor signaling in human arterial and venous smooth muscle cells

Platelet-derived growth factor (PDGF) has been implicated in smooth muscle cell (SMC) proliferation, a key event in the development of myointimal hyperplasia in vascular grafts. Recent evidence suggests that the PDGF receptor (PDGFR) tyrosine kinase inhibitor, imatinib, can prevent arterial proliferative diseases. Because hyperplasia is far more common at the venous anastomosis than the arterial anastomosis in vascular grafts, we investigated whether imatinib also inhibited venous SMC (VSMC) proliferation, and examined possible differences in its mechanism of action between VSMC and arterial SMC (ASMC). Human ASMC and VSMC were stimulated with PDGF-AB, in the presence or absence of imatinib (0.1-10 microM). Proliferation was assayed using the 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, while PDGFR, Akt and ERK1/2-mitogen activated protein kinase (MAPK) signaling pathways were investigated by immunoblotting. The proliferative response to PDGF at 50 and 100 ng/ml was 32 and 43% greater, respectively, in VSMC than in ASMC. Similarly, PDGF-stimulated proliferation was more sensitive to inhibition by imatinib in VSMC than ASMC (IC(50) = 0.05 microM vs. 0.4 microM; P < 0.01). Imatinib also more effectively inhibited PDGF-induced phosphorylation of PDGFRbeta and Akt in VSMC, compared to ASMC. These data highlight inherent pharmacodynamic differences between VSMC and ASMC in receptor and cell signaling functions and suggest that imatinib therapy may be useful for the prevention of venous stenosis in vascular grafts.     

小鼠胚胎干细胞分化心肌细胞血管紧张素Ⅱ 1型和2型受体的表达特征

胚胎干细胞(ESC)具有无限增殖和分化为体内3个胚层来源的各种类型组织细胞的潜能,经过体外诱导能够分化为心肌细胞,亦称为胚胎干细胞分化心肌细胞(ESCM).本研究探讨了ESC诱导分化心肌细胞过程中血管紧张素Ⅱ受体(ATR)的亚型AT1R和AT2R的表达特征.10-4mol/L维生素C体外诱导小鼠R1胚胎干细胞分化为自发搏动的心肌细胞,用免疫荧光法检测分化后的细胞表达心肌细胞特异性标志物α辅肌动蛋白.小鼠胚胎干细胞在诱导分化为心肌细胞以后,逆转录聚合酶链反应(RT-PCR)和实时定量RT-PCR(Real-timeRT-PCR)方法检测到ESCM表达AT1R,并且呈时间依赖性逐渐增加的特点,在第14d达到高峰.Western印记法检测AT1R表达特征与RT-PCR结果相符.Western印记法的结果显示,血管紧张素Ⅱ(10-6mol/L)可作为AT1R激动剂激活AT1R,并使其下游的细胞外信号调节激酶(ERK1/2)磷酸化水平上调,预孵育AT1R抑制剂Losartan(10-6mol/L),此作用被抑制.RT-PCR方法显示,与新生小鼠心室肌细胞相比,ESCM的AT2R表达水平较低.     

Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid

Cultured mouse D3 embryonic stem (ES) cells differentiating into embryoid bodies (EBs) expressed several Wnt isoforms, nearly all isotypes of the Wnt receptor Frizzled and the Wnt/Dickkopf (Dkk) co-receptor low-density lipoprotein receptor-related protein (LRP) type 5. A 4-day treatment with retinoic acid (RA), which promoted neural differentiation of EBs, substantially increased the expression of the Wnt antagonist Dkk-1, and induced the synthesis of the Wnt/Dkk-1 co-receptor LRP6. Recombinant Dkk-1 applied to EBs behaved like RA in inducing the expression of the neural markers nestin and distal-less homeobox gene (Dlx-2). Recombinant Dkk-1 was able to inhibit the Wnt pathway, as shown by a reduction in nuclear beta-catenin levels. Remarkably, the antisense- or small interfering RNA-induced knockdown of Dkk-1 largely reduced the expression of Dlx-2, and the neuronal marker beta-III tubulin in EBs exposed to RA. These data suggest that induction of Dkk-1 and the ensuing inhibition of the canonical Wnt pathway is required for neural differentiation of ES cells.     

Akt1 interacts with epidermal growth factor receptors and hedgehog signaling to increase stem/transit amplifying cells in the embryonic mouse cortex

A subset of precursors in the embryonic mouse cortex and in neurospheres expresses a higher level of the serine/threonine kinase Akt1 than neighboring precursors. We reported previously that the functional significance of high Akt1 expression was enhanced Akt1 activity, resulting in an increase in survival, proliferation, and self-renewal of multipotent stem/transit amplifying cells. Akt1 can interact with a number of signaling pathways, but the extrinsic factors that are required for specific effects of elevated Akt1 expression have not been identified. In this study we addressed the contributions of signaling via epidermal growth factor (EGF) and hedgehog (Hh) receptors. In EGF receptor-null precursors or following transient inhibition of EGF receptor tyrosine kinase activity, elevating Akt1 by retroviral transduction could still increase survival and proliferation but could not increase self-renewal. We also found that elevated Akt1 expression induced the expression of EGF receptors (EGFRs) in wild-type precursors. Several extrinsic factors, including Shh, can induce EGFR expression by cortical precursors, and we found that elevating Akt1 allowed them to respond to a subthreshold concentration of Shh to induce EGFRs. In precursors that lack the Hh receptor smoothened, however, elevating Akt1 did not increase EGFR expression or self-renewal, though it could still stimulate proliferation. These findings suggest that a subset of precursors in the embryonic cortex that express an elevated level of Akt1 can respond to lower concentrations of Shh than neighboring precursors, resulting in an increase in their expression of EGFRs. Signaling via EGFRs is required for their self-renewal.     

Chicken CD1 genes are located in the MHC: CD1 and endothelial protein C receptor genes constitute a distinct subfamily of class-I-like genes that predates the emergence of mammals

Mammals have several major histocompatibility complex (MHC) class-I-like genes. Although some of them are assumed to have originated before the emergence of mammals, the origin of class-I-like genes is poorly understood. We analyzed here the recently released chicken draft genome sequence and identified two families of class-I-like genes: CD1 and PROCR (the gene for the endothelial protein C receptor). Chickens have two CD1 genes, designated CD1.1 and CD1.2, located in tandem approximately 840 bp apart from each other. Chicken CD1.1 and CD1.2 are neither group 1- nor group 2-like, indicating that the two groups of CD1 emerged in a mammalian lineage. Although the database provides no information as to their chromosomal localization, we found that chicken CD1 genes are located adjacent to the previously characterized MHC B system contig on chromosome 16. We confirmed the linkage of CD1 to the B system by dual-color fluorescence in situ hybridization. Chickens have a single copy of PROCR. Among known class-I-like genes, PROCR is most closely related to CD1, indicating that CD1 and PROCR constitute a distinct subfamily of class-I-like genes that predates the emergence of mammals.     

A cluster of five cell wall-associated receptor kinase genes,Wak1–5, are expressed in specific organs of Arabidopsis

WAK1 (wall-associated kinase 1) is a cytoplasmic serine/threonine kinase that spans the plasma membrane and extends into the extracellular region to bind tightly to the cell wall. The Wak1 gene was mapped and found to lie in a tight cluster of five highly similar genes (Wak1–5) within a 30 kb region. All of the Wak genes encode a cytoplasmic serine/threonine protein kinase, a transmembrane domain, and an extracytoplasmic region with several epidermal growth factor (EGF) repeats. The extracellular regions also contain limited amino acid identities to the tenascin superfamily, collagen, or the neurexins. RNA blot analysis with gene-specific probes revealed that Wak1, Wak3 and Wak5 are expressed primarily in leaves and stems of Arabidopsis. Wak4 mRNA is only detected in siliques, while Wak2 mRNA is found in high levels in leaves and stems, and in lower levels in flowers and siliques. A trace amount of Wak2 can also be detected in roots. Wak1 is induced by pathogen infection and salicylic acid or its analogue INA and is involved in the plant's response, and Wak2, Wak3 and Wak5 also can be greatly induced by salicylic acid or INA. The WAK proteins have the potential to serve as both linkers of the cell wall to the plasma membrane and as signaling molecules, and since Wak expression is organ-specific and the isoforms vary significantly in the cell wall associated domain this family of proteins may be involved in cell wall-plasma membrane interactions that direct fundamental processes in angiosperms.