|Growth factor-induced transcription of GluR1 increases functional AMPA receptor density in glial progenitor cells. |
Chew, L J, et al.
J. Neurosci., 17: 227-40 (1997)
We analyzed the effects of two growth factors that regulate oligodendrocyte progenitor (O-2A) development on the expression of glutamate receptor (GluR) subunits in cortical O-2A cells. In the absence of growth factors, GluR1 was the AMPA subunit mRNA expressed at the lowest relative level. Basic fibroblast growth factor (bFGF) caused an increase in GluR1 and GluR3 steady-state mRNA levels. Platelet-derived growth factor (PDGF) did not modify the mRNA levels for any of the AMPA subunits but selectively potentiated the effects of bFGF on GluR1 mRNA (4.5-fold increase). The kainate-preferring subunits GluR7, KA1, and KA2 mRNAs were increased by bFGF, but these effects were not modified by cotreatment with PDGF. Nuclear run-on assays demonstrated that PDGF+bFGF selectively increased the rate of GluR1 gene transcription (2.5-fold over control). Western blot analysis showed that GluR1 protein levels were increased selectively (sixfold over control) by PDGF+bFGF. Functional expression was assessed by rapid application of AMPA to cultured cells. AMPA receptor current densities (pA/pF) were increased nearly fivefold in cells treated with PDGF+bFGF, as compared with untreated cells. Further, AMPA receptor channels in cells treated with PDGF+bFGF were more sensitive to voltage-dependent block by intracellular polyamines, as expected from the robust and selective enhancement of GluR1 expression. Our combined molecular and electrophysiological findings indicate that AMPA receptor function can be regulated by growth factor-induced changes in the rate of gene transcription.
|Antisense inhibition of basic fibroblast growth factor induces apoptosis in vascular smooth muscle cells. |
Fox, J C and Shanley, J R
J. Biol. Chem., 271: 12578-84 (1996)
Basic fibroblast growth factor (bFGF), a potent mitogen for many cell types, is expressed by vascular smooth muscle cells and plays a prominent role in the proliferative response to vascular injury. Basic FGF has also been implicated as a survival factor for a variety of quiescent or terminally differentiated cells. Autocrine mechanisms could potentially mediate both proliferation and cell survival. To probe such autocrine pathways, endogenous bFGF production was inhibited in cultured rat vascular smooth muscle cells by the expression of antisense bFGF RNA. Inhibition of endogenous bFGF production induced apoptosis in these cells independent of proliferation, and apoptosis could be prevented by exogenous bFGF but not serum or epidermal growth factor. The induction of apoptosis was associated with an inappropriate entry into S phase. These data demonstrate that interruption of autocrine bFGF signaling results in apoptosis of vascular smooth muscle cells, and that the mechanism involves disruption of normal cell cycle regulation.
|Differential modulation of basic fibroblast and epidermal growth factor receptor activation by ganglioside GM3 in cultured retinal Müller glia. |
Meuillet, E, et al.
Glia, 17: 206-16 (1996)
Polypeptide growth factors and membrane-bound gangliosides are involved in cell signaling, including that observed in cells of neural origin. To analyze possible interactions between these two systems, we investigated the modulation of short- and long-term responses to basic fibroblast and epidermal growth factor (bFGF and EGF, respectively) in cultured retinal Müller glial cells following experimental modification of their ganglioside composition. These glial cells readily incorporated exogenously administered GM3 ganglioside, which was not substantially metabolized within 24 h. Such treatments significantly inhibited bFGF-induced DNA replication and cell migration, while having much less effect on analogous EGF-mediated behaviors. To explore GM3/growth factor interactions further, different aspects of glial metabolism in response to bFGF or EGF stimulation were examined: membrane fluidity, growth factor binding, global and individual changes in growth factor-induced phosphotyrosine levels, and growth factor-induced activation of mitogen-activated protein kinase. GM3 reduced the intensity of immunocytochemical labeling of phosphotyrosine-containing proteins within bFGF-stimulated cells and down-regulated FGF receptor activation and tyrosine phosphorylation of its cellular substrates, whereas similar parameters in EGF-stimulated cells were much less affected. Hence the data reveal a complex relationship in normal neural cells between polypeptide growth factors and membrane-bound gangliosides, which may participate in retinal cellular physiology in vivo.
|Hematopoietic commitment during embryonic stem cell differentiation in culture. |
Keller, G, et al.
Mol. Cell. Biol., 13: 473-86 (1993)
We report that embryonic stem cells efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiation, and by day 6, more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction profile of marker genes for primitive endoderm (collagen alpha IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-1, GATA-3, and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis, indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added growth factors and are not significantly influenced by the addition of a broad spectrum of factors, including interleukin-3 (IL-3), IL-1, IL-6, IL-11, erythropoietin, and Kit ligand. At days 10 and 14 of differentiation, EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and IL-11 to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first, approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next, and precursors of the mast cell lineage develop last. The kinetics of precursor development, as well as the growth factor responsiveness of these early cells, is similar to that found in the yolk sac and early fetal liver, indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo.
|Human basic fibroblast growth factor: nucleotide sequence and genomic organization. |
Abraham, J A, et al.
EMBO J., 5: 2523-8 (1986)