Regulation of bFGF gene expression and subcellular distribution of bFGF protein in adrenal medullary cells.

Abstract

Abstract. Basic fibroblast growth factor (bFGF), a potent mitogenic/neurotrophic factor, controls the development and plasticity of many types of neural cells. In adrenal chromaflin cells, the appearance of bFGF protein coincided with the establishment of functional innervation, suggesting induction by trans-synaptic signals. In cultured bovine adrenal medullary cells Western blot analysis revealed 18-, 23-, and 24-kD bFGF isoforms in the cytosolic and nuclear fractions. Stimulation of acetylcholine nicotinic receptors or hormonal angiotensin II receptors or the direct stimulation of adenylate cyclase with forskolin or protein kinase C (PKC) with PMA increased the content of all bFGF isoforms. Increases in the levels of intracellular bFGF did not result in detectable presence of bFGF proteins in culture medium. Instead, bFGF proteins accumulated in the cytoplasm or the nucleus depending on whether PKC or cAMP pathways were activated.The long-term nuclear forskolin-induced accumulation of bFGF was prevented by cycloheximide or by antisense bFGF oligonucleotide and was also accompanied by an increase in bFGF mRNA. We used luciferase reporter plasmids containing the human bFGF promoter to show that the induction of bFGF resulted from transcriptional activation of the bFGF gene and was mediated by regulatory sequences located upstream from its transcription start site. Stimulation of bFGF gene expression by forskolin and PMA was synergistic and was mediated through different promoter regions. The results suggest that stimulation by cAMP and PKC is mediated through novel cis elements. The regulation of bFGF protein content also involves posttranscriptional mechanisms since changes in the levels of individual bFGF isoforms were different depending on whether cells were treated with carbachol or angiotensin II, forskolin, or PMA.The present study indicates that bFGF is an intracrine cytoplasmic-nuclear factor, whose expression is regulated by trans-synaptic and hormonal stimuli and which may act as a direct mediator of genomic responses to afferent stimulation.

Replication-defective herpes simplex virus vectors for neurotrophic factor gene transfer in vitro and in vivo

“…Three protein isoforms were identified in Western blot analysis, in accordance with previous reports. 32 All three isoform levels were increased to a similar extent after TH FGF-2 injection. A kinetics analysis was run by examining FGF-2 levels 4, 7, 15 and 30 days after vector injection: maximal increases have been observed at 4 days, while a lower, nonsignificant increase was still present at 7 days, and levels returned to baseline by 15 days (Figure 5b).…”

Nuclear accumulation of FGF-2 is associated with proliferation of human astrocytes and glioma cells

“…Heparin-sepharosebound proteins were eluted directly into SDS sample bu er and resolved by SDS ± PAGE gel electrophoresis on a 12% polyacrylamide gel. Proteins were transferred onto nitrocellulose membranes and Western analysis performed using an anti-FGF-2 monoclonal antibody (UBI, Lake Placid, NY) followed by rabbit anti-mouse IgG and 125 I-labeled protein A (Florkiewicz and Sommer, 1989;Stachowiak et al, 1994). In some experiments 10 ml of conditioned media were processed through heparin sepharose and analysed in a similar way to the cell extracts to determine whether FGF-2 was present in the media.…”

“…Nuclear presence of FGF-2 was detected in rat brain astrocytes both in vivo (Woodward et al, 1992) and in vitro (Vijayan et al, 1993) and in cultured human astrocytes and glioma cells (Mo ett et al, 1996;Stachowiak et al, 1996a). With the observation that FGF-2 remains cell associated and may not be secreted by a classical mechanism, a regulated targeting of endogenous FGF-2 to the nucleus (Puchacz et al, 1993;Stachowiak et al, 1994Stachowiak et al, , 1996bMo ett et al, 1994Mo ett et al, , 1996 suggests that this growth factor may serve as an intracrine signaling molecule.…”

“…At least four amino terminus extended isoforms of FGF-2 arise from the same gene due to initiation of translation at di erent start codons with di erent cells expressing di erent isoforms (Florkiewicz and Sommer, 1989). All FGF-2 isoforms are detected in both the cytoplasm and the nucleus of expressing cells (Powell and Klagsbrun, 1991;Florkiewicz et al, 1991;Gualandris et al, 1993;Woodward et al, 1992;Vijayan et al, 1993;Stachowiak et al, 1994Stachowiak et al, , 1996a. Nuclear presence of FGF-2 was detected in rat brain astrocytes both in vivo (Woodward et al, 1992) and in vitro (Vijayan et al, 1993) and in cultured human astrocytes and glioma cells (Mo ett et al, 1996;Stachowiak et al, 1996a).…”

Basic fibroblast growth factor transcriptional autoregulation requires EGR-1

“…Deletion analysis indicated two negative regulatory elements, one of which is in the upstream promoter region. It was reported that the bFGF promoter was induced by PMA and cAMP through cis regulatory elements (Stachowiak et al, 1994). An AP-1 element in the bFGF promoter has been shown to be functional in the transcriptional regulation of bFGF gene expression Erdos et al, 1995).…”