Multiple controlling mechanisms of FGF1 gene expression through multiple tissue-specific promoters.

We now know that fibroblast growth factor-1 (FGF1) transcription is controlled by at least four distinct promoters in a tissue-specific manner. Thus, promoter 1.A is active in the kidney, 1.B in the brain, and 1.C and 1.D in a variety of cultured cells including vascular smooth muscle cells. These promoters are separated from each other by up to 70 kbp. Multiple FGF1 transcripts arise from alternate promoter usage and alternative splicing of different 5'-untranslated exons. The 1.A and 1.B promoters are constitutively active in their respective cell types. In contrast, different biological response modifiers, including serum and transforming growth factor beta, can induce the 1.C and 1.D promoters. The 540-bp sequence upstream of the 1B transcription initiation site is sufficient to drive the expression of a heterologous luciferase reporter in cultured cells, and an 18-bp sequence within this region is important for the regulation of brain-specific gene expression. Furthermore, regulation occurs through the binding of the 18-bp sequence to a brain-specific 37-kDa protein and a ubiquitous basic helix-loop-helix protein, E2-2. We have produced transgenic mice bearing the brain-specific promoter of the human FGF1 gene joined to the SV40 immediate-early gene, which encodes the large T antigen. The resulting mice developed brain tumors that originated in the pontine gray, just rostral to the fourth ventricle. We have also identified a serum response element, comprising a CarG box and an Ets-binding site, in the 1.D promoter. Continued characterization of the mechanistic events that control the tissue-specific activation of FGF1 promoters will help us to understand the role of FGF1 in cancer, atherosclerosis, and neural development.

The small GTPases Ras, Rac, and Cdc42 transcriptionally regulate expression of human fibroblast growth factor 1.

Four distinct promoters (1A, 1B, 1C, and 1D) of fibroblast growth factor 1 (FGF1), spaced up to 70 kilobase pairs apart, direct the expression of alternatively spliced transcript variants (FGF1.A, -1. B, -1.C, and -1.D) that encode FGF1. These FGF1 transcripts can be detected in cultured cells as well as in normal and diseased tissues. These transcripts are differentially regulated in a cell-specific manner. To further delineate the biological function of multiple promoter usage by a single gene, we investigated the transcriptional regulation of these promoters by defined signaling pathways associated with cell proliferation and cell survival. Here we show a specific association of two of the FGF1 promoters, 1C and 1D, with signaling cascades of the Ras superfamily of GTPases. A serum-response element, comprised of the Ets and CArG motifs, present in promoter 1D was shown to be the target of distinct signaling cascades; the Ets motif target of Ras, Rac1, and Cdc42 regulation; and the CArG motif target of de novo protein synthesis-independent cascade. Ras and Rac1 also activated the FGF2 promoter. Further, the transcription factor Ets2 synergistically activated FGF1 gene, but not FGF2, in a Ras- and Rac1-dependent signaling pathway. In support of these conclusions high levels of intracellular FGF1 were detected in cells undergoing cytokinesis. Altogether, our results suggest that FGF1 may play a fundamental role in cell division, spreading, and migration, in addition to cell proliferation.

Tumorigenesis in transgenic mice in which the SV40 T antigen is driven by the brain-specific FGF1 promoter.

Gene expression can be manipulated by the introduction of a hybrid gene formed by linking a highly tissue-specific regulatory element to a gene whose expression might be expected to alter cellular function. Previously, we have shown that the human FGF1 gene contains four distinct tissue-specific promoters. In an effort to perturb the programming of proliferation and differentiation in a subset of neural cells, we have produced transgenic mice bearing the brain-specific promoter of the human FGF1 gene joined to the SV40 immediate early gene, which encodes the large T antigen. The resulting mice, and offspring from four individual lines, developed brain tumors that originated in the pontine gray, just rostral to the fourth ventricle. Tumors were moderately vascularized, as demonstrated by staining with both hematoxylin and eosin and antibodies to three different endothelial cell markers, but vessels were histologically normal. Scattered tumor foci were present as early as postnatal day 26; and affected animals died between 5 - 8 months of age. In mature animals, tumors lacked terminal differentiation markers for astrocytes (glial fibrillary acidic protein) or neurons (synaptophysin and neuron-specific enolase). However, they expressed high levels of proliferating cell nuclear antigen and vimentin, markers for proliferating cells. This immunophenotype is consistent with the tumor being at an early stage of differentiation. Therefore, these mice may provide a valuable tool for the study of tumorigenesis, replenishment and differentiation of neural stem cells.

Increased densities of AMPA GluR1 subunit proteins and presynaptic mossy fiber sprouting in the fascia dentata of human hippocampal epilepsy.

In human hippocampal epilepsy, there is a consistent pathology of cell loss and reactive synaptic reorganization of 'excitatory' mossy fibers (MF) into the inner molecular layer (IML) of the fascia dentata (FD). In this study, neo-Timm's histochemistry of MFs and immunocytochemistry of GluR1 were used to determine, in patients with or without hippocampal sclerosis (HS), if there was a correlation between aberrant supragranular (IML) mossy fiber sprouting and increased densities of AMPA GluR1 subunit proteins in the IML of the FD. Computerized quantified densitometric grey values of Timm and GluR1 densities were corrected for the densities of granule cell losses using cell counts. In the IML of the HS group, despite the losses of granule cells, mossy fiber sprouting was significantly greater (P<0.000001) and GluR1 protein densities were significantly higher (P<0.0005) than those of the non-HS group. Unlike supragranular mossy fiber sprouting, which was limited to the IML, the increased GluR1 stainings were distributed throughout the whole molecular layer. For all cases, MF synaptic reorganization in the supragranular ML was correlated with GluR1 subunit protein densities in the IML (R=0.784, P<0.0093). These data demonstrate that in the human epileptic fascia dentata, there are significantly increased AMPA GluR1 subunit proteins associated with aberrant MF synaptic reorganizations. This suggests that the hyperexcitability of sclerotic hippocampus occurs, at least in part, from the associated changes of both presynaptic mossy fiber glutamatergic neoinnervation and increased GluR1 subunit proteins in the dendritic domains of the FD.

Induced expression of NMDAR2 proteins and differential expression of NMDAR1 splice variants in dysplastic neurons of human epileptic neocortex.

Immunocytochemistry was used to study the expressions of glutamate receptor subunit proteins for NMDAR2A/B, NMDAR1 splice variants, and AMPA Glu-R2/3 in human brain resected for intractable epilepsy associated with cortical dysplasia. NMDAR2A/B intensely labeled dysplastic neurons showing staining in both the cell bodies and dendritic profiles. However, nondysplastic neurons were not immunoreactive to NMDAR2A/B. The antibody selective to NMDAR1 splice variants of NR1-1a. -1b, -2a, and -2b labeled dysplastic neurons, but few nondysplastic neurons. In contrast, the antibody to splice variants of NR1-1a, -1b, 2a, -2b, -3a, -3b, -4a, and -4b labeled both dysplastic and nondysplastic neurons. The different labeling patterns by these two antibodies indicate that variants of NMDAR1-3a, -3b, -4a, and -4b are present in nondysplastic neurons. Both dysplastic neurons and nondysplastic neurons were immunoreactive to AMPA GluR2/3, but denser immunoreactivity was observed in dysplastic neurons. We also found that the locations of dysplastic neurons labeled by NMDAR2A/B were related to focal epileptic EEG seizure onsets or spiking and to focal behavioral seizure types. Our results suggest that there is hyperexcitability of dysplastic cortical regions, at least in part, from the presence of NMDAR2 subunits and selectively expressed NMDAR1 splice variants in dysplastic neurons.