神经干细胞向少突胶质前体细胞的定向分化诱导

本研究采用神经胶质瘤细胞株(B104 neuroblatoma cells,B104 cells)培养上清(B104CM)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),将冷冻复苏的大鼠胚胎脊髓神经干细胞(neural stem cells,NSCs)定向诱导为少突胶质前体细胞(oligodendrocyte precusor cells,OPCs)。形态学和免疫组化的结果显示,诱导后95％以上的细胞具有双极或多极突起的典型OPCs形态,并表达A285和血小板源生长因子受体-α(platelet derived growth factor receptor-α,PDGFR-α等0PCs标志,所有PDGFR-α阳性的OPCs均不表达β-Tublin Ⅲ,其中仅少量细胞表达胶质原纤维酸性蛋白(glia fibrillary acidic protein,GFAP)。在B104CM和bFGF共存的培养条件下,悬浮培养的OPCs可大量增殖形成少突胶质细胞球,该细胞球可通过传代继续扩增,且扩增的OPCs仍能维持其特有的形态和自我增殖的特性。撤去bFGF和B104CM后,OPCs能进一步分化为成熟的少突胶质细胞(oligodendrocytes,OLs)或Ⅱ型星形胶质细胞。实验表明,诱导NSCs产生的OPCs在形态、增殖以及分化格局等方面均与已报道的存在于胚胎脑区的O-2A前体细胞相类似。该培养系统可为实验性细胞移植的研究提供丰富的细胞来源。     

Generation of an environmental niche for neural stem cell development by the extracellular matrix molecule tenascin C

Stem cells in the embryonic mammalian CNS are initially responsive to fibroblast growth factor 2 (FGF2). They then undergo a developmental programme in which they acquire epidermal growth factor (EGF) responsiveness, switch from the production of neuronal to glial precursors and become localized in specialized germinal zones such as the subventricular zone (SVZ). Here we show that extracellular matrix molecules act as regulators of this programme. Tenascin C is highly expressed in the SVZ, and transgenic mice lacking tenascin C show delayed acquisition of the EGF receptor. This results from alterations in the response of the stem cells to the growth factors FGF2 and bone morphogenic protein 4 (BMP4), which normally promote and inhibit acquisition of the EGF receptor, respectively. Tenascin C-deficient mice also have altered numbers of CNS stem cells and these stem cells have an increased probability of generating neurones when grown in cell culture. We conclude that tenascin C contributes to the generation of a stem cell 'niche' within the SVZ, acting to orchestrate growth factor signalling so as to accelerate neural stem cell development.     

Expression of several growth factors and their receptor genes in human colon carcinomas

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-alpha, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-alpha mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-alpha protein by DLD-1 and CoLo320DM cells was confirmed by TGF-alpha specific monoclonal antibody binding assay. The spontaneous 3H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-alpha monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-alpha produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.     

Expression of EGF receptor and FGF receptor isoforms during neuroepithelial stem cell differentiation

To characterize the role of epidermal growth factor (EGF) and fibroblast growth factor (FGF) in regulating neuroepithelial stem cells differentiation, we have examined the expression of FGF, EGF, and their receptors by neuroepithelial (NEP) cells and their derivatives. Our results indicate that undifferentiated NEP cells express a subset of FGF receptor (FGFR) isoforms, but do not express platelet-derived growth factor receptors (PDGFRs) or epidermal growth factor receptor (EGFR). The FGFR pattern of expression by differentiated neuron and glial cells differs from that found on NEP stem cells. FGFR-4 is uniquely expressed on NEP cells, while FGFR-1 is expressed by both NEP cells and neurons, and FGFR-2 is down-regulated during neuronal differentiation. FGFRs present on astrocytes and oligodendrocytes also represent a subset of those present on NEP cells. Expression of FGF and EGF by NEP cells and their progeny was also examined. NEP cells synthesize detectable levels of both FGF-1 and FGF-2, and EGF. FGF-1 and FGF-2 synthesis is likely to be biologically relevant, as cells grown at high density do not require exogenous FGF for their survival and cells grown in the presence of neutralizing antibodies to FGF show a reduction in cell survival and division. Thus, neuroepithelial cells synthesize and respond to FGF, but not to EGF, and are therefore distinct from other neural stem cells (neurospheres). The unique pattern of expression of FGF isoforms may serve to distinguish NEP cells from their more differentiated progeny.     

Expression of several growth factors and their receptor genes in human colon carcinomas

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-α, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-α mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-α protein by DLD-1 and CoLo320DM cells was confirmed by TGF-α specific monoclonal antibody binding assay. The spontaneous³H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-α monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-α produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.     

Brain injury expands the numbers of neural stem cells and progenitors in the SVZ by enhancing their responsiveness to EGF

There is an increase in the numbers of neural precursors in the SVZ (subventricular zone) after moderate ischaemic injuries, but the extent of stem cell expansion and the resultant cell regeneration is modest. Therefore our studies have focused on understanding the signals that regulate these processes towards achieving a more robust amplification of the stem/progenitor cell pool. The goal of the present study was to evaluate the role of the EGFR [EGF (epidermal growth factor) receptor] in the regenerative response of the neonatal SVZ to hypoxic/ischaemic injury. We show that injury recruits quiescent cells in the SVZ to proliferate, that they divide more rapidly and that there is increased EGFR expression on both putative stem cells and progenitors. With the amplification of the precursors in the SVZ after injury there is enhanced sensitivity to EGF, but not to FGF (fibroblast growth factor)-2. EGF-dependent SVZ precursor expansion, as measured using the neurosphere assay, is lost when the EGFR is pharmacologically inhibited, and forced expression of a constitutively active EGFR is sufficient to recapitulate the exaggerated proliferation of the neural stem/progenitors that is induced by hypoxic/ischaemic brain injury. Cumulatively, our results reveal that increased EGFR signalling precedes that increase in the abundance of the putative neural stem cells and our studies implicate the EGFR as a key regulator of the expansion of SVZ precursors in response to brain injury. Thus modulating EGFR signalling represents a potential target for therapies to enhance brain repair from endogenous neural precursors following hypoxic/ischaemic and other brain injuries.     

Hepatocyte growth factor induces proliferation and differentiation of multipotent and erythroid hemopoietic progenitors

Hepatocyte growth factor (HGF) is a mesenchymal derived growth factor known to induce proliferation and "scattering" of epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c- MET protooncogene. Here we show that highly purified recombinant HGF stimulates hemopoietic progenitors to form colonies in vitro. In the presence of erythropoietin, picomolar concentrations of HGF induced the formation of erythroid burst-forming unit colonies from CD34-positive cells purified from human bone marrow, peripheral blood, or umbilical cord blood. The growth stimulatory activity was restricted to the erythroid lineage. HGF also stimulated the formation of multipotent CFU- GEMM colonies. This effect is synergized by stem cell factor, the ligand of the tyrosine kinase receptor encoded by the c-KIT protooncogene, which is active on early hemopoietic progenitors. By flow cytometry analysis, the receptor for HGF was found to be expressed on the cell surface in a fraction of CD34+ progenitors. Moreover, in situ hybridization experiments showed that HGF receptor mRNA is highly expressed in embryonic erythroid cells (megaloblasts). HGF mRNA was also found to be produced in the embryonal liver. These data show that HGF plays a direct role in the control of proliferation and differentiation of erythroid progenitors, and they suggest that it may be one of the long-sought mediators of paracrine interactions between stromal and hemopoietic cells within the hemopoietic microenvironment.     

Growth factor-independent proliferation of normal human neonatal keratinocytes: production of autocrine- and paracrine-acting mitogenic factors

When normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 x 10(3)/cells cm2, the cells proliferated continuously and the addition of IGF-I, EGF, TGF alpha, bFGF, or aFGF did not significantly alter growth rate. Heparin sulfate, TGF beta, or suramin inhibited keratinocyte growth factor-independent proliferation. The addition of EGF, TGF alpha, or aFGF reversed heparin-induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte-derived conditioned medium (CM) indicated the presence of TGF alpha peptide at a concentration of approximately 235 pg/ml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte-derived CM replaced EGF in stimulating keratinocyte clonal growth, and an anti-EGF receptor mAb inhibited CM-induced keratinocyte clonal growth. In addition to its effect on keratinocytes, keratinocyte-derived CM stimulated the incorporation of [3H]thymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR-2B cells, and EGF-receptorless mouse NR6 cells. CM-stimulated [3H]thymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti-EGF receptor mAb. Heparin sulfate partially inhibited CM-induced keratinocyte clonal growth and [3H]thymidine incorporation into quiescent AKR-2B cells. We hypothesize from these data that autocrine and paracrine-acting factors produced by keratinocytes mediated their effect through the activation of both EGF receptor-dependent and EGF receptor-independent mitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin.     

Two independent mechanisms for escaping epidermal growth factor- mediated growth inhibition in epidermal growth factor receptor- hyperproducing human tumor cells

Human squamous cell carcinoma cell lines often possess increased levels of epidermal growth factor (EGF) receptor. The growth of these EGF receptor-hyperproducing cells is usually inhibited by EGF. To investigate the mechanism of EGF-mediated inhibition of cell growth, variants displaying alternate responses to EGF were isolated from two squamous cell carcinoma lines, NA and Ca9-22; these cell lines possess high numbers of the EGF receptor and an amplified EGF receptor (EGFR) gene. The variants were isolated from NA cells after several cycles of EGF treatment and they have acquired EGF-dependent growth. Scatchard plot analysis revealed a decreased level of EGF receptor in these ER variants as compared with parental NA cells. Southern blot analysis and RNA dot blot analysis demonstrated that the ER variants had lost the amplified EGFR gene. One variant isolated from Ca9-22 cells, CER-1, grew without being affected by EGF. CER-1 cells had higher numbers of EGF receptor than parental Ca9-22 but similar EGFR gene copy number. Flow cytometric analysis indicated an increase in ploidy and cell volume which may give rise to the increase in receptor number per cell. The EGF receptors on both Ca9-22 and CER-1 cells were autophosphorylated upon EGF exposure in a similar manner suggesting no obvious alteration in receptor tyrosine kinase. However, very efficient down-regulation of the EGF receptor occurred in CER-1 cells. These data suggest two independent mechanisms by which EGF receptor-hyperproducing cells escape EGF-mediated growth inhibition: one mechanism is common and involves the loss of the amplified EGFR genes, and another is novel and involves the efficient down-regulation of the cell-surface receptor.     

Epidermal growth factor and transforming growth factor alpha mimic the effects of insulin in human fat cells and augment downstream signaling in insulin resistance

The ability of the growth factors epidermal growth factor (EGF), transforming growth factor alpha, and platelet-derived growth factor to exert insulin-like effects on glucose transport and lipolysis were examined in human and rat fat cells. No effects were found in rat fat cells, whereas EGF (EC(50) for glucose transport approximately 0.02 nm) and transforming growth factor alpha (EC(50) approximately 0.2 nm), but not platelet-derived growth factor, mimicked the effects of insulin (EC(50) approximately 0.2 nm) on both pathways. EGF receptors, but not EGF, were abundantly expressed in human fat cells as well as in human skeletal muscle. EGF increased the tyrosine phosphorylation of several proteins (the EGF receptor, insulin receptor substrate (IRS)-1, IRS-2, and Grb2-associated binder 1), whereas Shc and Gab2 were only weakly and inconsistently phosphorylated. p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), was also found to associate with all of these docking molecules, showing that EGF activated PI 3-kinase pools that were additional to those of insulin. EGF and/or insulin increased protein kinase B/Akt serine phosphorylation to a similar extent, whereas mitogen-activated protein kinase phosphorylation was more pronounced for EGF than for insulin. The impaired insulin-stimulated downstream signaling, measured as protein kinase B/Akt serine phosphorylation, in insulin-resistant cells (Type 2 diabetes) was improved by the addition of EGF. Thus, EGF receptors, but not EGF, are abundantly expressed in human fat cells and skeletal muscle. EGF mimics the effects of insulin on both the metabolic and mitogenic pathways but utilize in part different signaling pathways. Both insulin and EGF increase the tyrosine phosphorylation and activation of IRS-1 and IRS-2, whereas EGF is also capable of activating additional PI 3-kinase pools and, thus, can augment the downstream signaling of insulin in insulin-resistant states like Type 2 diabetes.     

A novel way to induce erythroid progenitor self renewal: cooperation of c-Kit with the erythropoietin receptor

Red blood cells are of vital importance for oxygen transport in vertebrates. Thus, their formation during development and homeostasis requires tight control of both progenitor proliferation and terminal red cell differentiation. Self renewal (i.e. long-term proliferation without differentiation) of committed erythroid progenitors has recently been shown to contribute to this regulation. Avian erythroid progenitors expressing the EGF receptor/c-ErbB (SCF/TGFalpha progenitors) can be induced to long-term proliferation by the c-ErbB ligand transforming growth factor alpha and the steroids estradiol and dexamethasone. These progenitors have not yet been described in mammals and their factor requirements are untypical for adult erythroid progenitors. Here we describe a second, distinct type of erythroid progenitor (EpoR progenitors) which can be established from freshly isolated bone marrow and is induced to self renew by ligands relevant for erythropoiesis, i.e. erythropoietin, stem cell factor, the ligand for c-Kit and the glucocorticoid receptor ligand dexamethasone. Limiting dilution cloning indicates that these EpoR progenitors are derived from normal BFU-E/CFU-E. For a detailed study, mEpoR progenitors were generated by retroviral expression of the murine Epo receptor in bone marrow erythroblasts. These progenitors carry out the normal erythroid differentiation program in recombinant differentiation factors only. We show that mEpoR progenitors are more mature than SCF/TGFalpha progenitors and also do no longer respond to transforming growth factor alpha and estradiol. In contrast they are now highly sensitive to low levels of thyroid hormone, facilitating their terminal maturation into erythrocytes.     

Amiloride directly inhibits growth factor receptor tyrosine kinase activity

Addition of amiloride to A431 human epidermoid carcinoma cell membranes inhibited autophosphorylation of the epidermal growth factor (EGF) receptor. The tyrosine phosphorylation of histone H2B catalyzed by an affinity-purified preparation of EGF receptor was also inhibited by amiloride. The inhibition was noncompetitive with respect to histone but competitive with ATP, suggesting that amiloride may act as an ATP analogue which causes the formation of nonproductive enzyme-substrate complexes. The tyrosine phosphorylation of histone H2B catalyzed by the purified EGF receptor was inhibited by amiloride at concentrations identical to those previously reported to block EGF action on cell proliferation (Ki = 350 microM). Amiloride similarly inhibited the tyrosine phosphorylation of the human placental insulin receptor and the platelet-derived growth factor receptor of Swiss 3T3 cells. Immunoprecipitation of the EGF receptor from A431 cells labeled for 24 h with [32P]phosphate demonstrated that amiloride decreased the phosphorylation of the EGF receptor on serine and threonine residues and blocked the effect of EGF to cause phosphorylation of the receptor on tyrosine residues. Phosphoamino acid analysis of total cell proteins indicated that amiloride inhibited the increase in phosphotyrosine levels caused by EGF. We conclude that amiloride directly inhibits the tyrosine kinase activity of the receptors for EGF, insulin, and platelet-derived growth factor in in vitro and can mediate such actions in vivo. This effect of amiloride demonstrates that it is unsuitable as a drug to test the hypothesis that the stimulation of the Na+/H+ antiporter is essential for mitogenic signaling by growth factor receptors.     

Down-regulation of cellular platelet-derived growth factor receptors induced by an activated neu receptor tyrosine kinase.

The functional integration of growth factor signaling occurs at several levels in target cells. One of the most proximal mechanisms is receptor transmodulation, by which one activated receptor can regulate the expression of other receptors in the same cells. Well-established transregulatory loops involve platelet-derived growth factor (PDGF) down-regulation of epidermal growth factor (EGF) receptors and beta-type transforming growth factors modulation of PDGF receptors. We have studied the relationship between neu tyrosine kinase activation and the expression of the PDGF receptors in transfected NIH/3T3 cells. Expression of the neu oncogene, but not of the neu proto-oncogene, was associated with a decrease of PDGF alpha- and beta-receptors on the cell surface, as measured by [125-I]PDGF-AA and -BB binding. These results were corroborated by metabolic labeling and immunoprecipitation of the PDGF beta-receptors. PDGF alpha- and beta-receptor mRNAs were strongly decreased in the neu oncogene-transformed cells in comparison with control cells expressing the neu proto-oncogene. Down-regulation of the PDGF receptors and their mRNAs was also observed after EGF treatment of cells expressing a chimeric EGF receptor/neu receptor, where the neu tyrosine kinase is activated by EGF binding. These results show that the neu tyrosine kinase can down-modulate PDGF receptor expression, and the effect is mediated via decreased PDGF receptor mRNA levels.     

Epidermal growth factor stimulates proliferation of mouse uterine epithelial cells in primary culture

Epidermal growth factor (EGF) is one of growth factors that are thought to mediate the stimulatory effects of estrogen on the proliferation of uterine epithelial cells. The present study was attempted to obtain direct evidence for the mitogenic effects of EGF on uterine epithelial cells, and to prove that EGF and EGF receptors are expressed in these cells. Mouse uterine epithelial cells were isolated from immature female mice and cultured with or without EGF for 5 days. EGF (1 to 100 ng/ml) significantly increased the number of uterine epithelial cells, and the maximal growth (141.9+/- 8.3% of controls) was obtained at a dose of 10 ng/ml. In addition, EGF (0.1 to 100 ng/ml) increased the number of DNA-synthesizing cells immunocytochemically detected by bromodeoxyuridine uptake to the nucleus. Northern blot analysis revealed that the uterine epithelial cells expressed both EGF mRNA (4.7 kb) and EGF receptor mRNAs (10.5, 6.6, and 2.7 kb) These results suggest that the proliferation of uterine epithelial cells is regulated by the paracrine and/or autocrine action of EGF. Our previous study demonstrated the mitogenic effect of IGF-I on uterine epithelial cells. To examine whether the EGF- and IGF-I signaling act at the same level in the regulation of the proliferation of uterine epithelial cells, the cultured cells were simultaneously treated with IGF-I and EGF. IGF-I was found to additively stimulate the mitogenic effects of EGF, suggesting that the EGF-induced growth of uterine epithelial cells is distinct from IGF-I-induced growth.     

Phorbol ester or epidermal growth factor (EGF) stimulates the concurrent accumulation of mRNA for the EGF receptor and its ligand transforming growth factor-alpha in a breast cancer cell line

We have previously reported that both 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) can stimulate the synthesis rate of EGF receptors. We now show that the MDA468 breast cancer cells express the mRNA for the EGF-like molecule, transforming growth factor-alpha (TGF-alpha), and demonstrate that TPA or EGF cause an accumulation of both EGF receptor and TGF-alpha mRNA. The levels of EGF receptor mRNA paralleled our earlier protein data, with peak accumulations of 2-3-fold with 10(-9) M EGF and 3-5-fold with 100 ng/ml TPA seen between 6 and 8 h. A 7-fold accumulation of TGF-alpha mRNA was seen following 4 h of treatment with TPA, and a 2-fold accumulation was seen after 8 h with EGF. These changes in EGF receptor and TGF-alpha mRNAs were observed in the absence of any change in the mRNA level of the alpha-subunit of hexosaminidase A (a lysosomal enzyme), demonstrating some degree of specificity. Detectable quantities of immunoreactive TGF-alpha accumulated in the cell culture medium of MDA468 cell treated with the blocking anti-EGF receptor monoclonal antibody B1D8 while no immunoreactive TGF-alpha was detected in the medium of cells with unblocked receptors. The concentration of B1D8 used was sufficient to block the binding of exogenously added 125I-EGF to undetectable levels but had only minor effects on cell growth and no effect on the expression of the TGF-alpha and EGF receptor mRNA.