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Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of proliferation, differentiation, apoptosis and neuroprotection in the central nervous system. With regard to dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc), which degenerate in Parkinson's disease, FGF-2 improves survival of mature DA neurons in vivo and regulates expansion of DA progenitors in vitro. To address the physiological role of FGF-2 in SNpc development, embryonic (E14.5), newborn (P0) and juvenile (P28) FGF-2-deficient mice were investigated. Stereological quantification of DA neurons identified normal numbers in the ventral tegmental area, whereas the SNpc of FGF-2-deficient mice displayed a 35% increase of DA neurons at P0 and P28, but not at earlier stage E14.5. Examination of DA marker gene expression by quantitative RT-PCR and in situ hybridization revealed a normal patterning of embryonic ventral mesencephalon. However, an increase of proliferating Lmx1a DA progenitors in the subventricular zone of the ventral mesencephalon of FGF-2-deficient embryos indicated altered cell cycle progression of neuronal progenitors. Increased levels of nuclear FgfR1 in E14.5 FGF-2-deficient mice suggest alterations of integrative nuclear FgfR1 signaling (INFS). In summary, FGF-2 restricts SNpc DA neurogenesis in vivo during late stages of embryonic development., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Developing and mature midbrain dopamine (DA) neurons express fibroblast growth factor (FGF) receptor-1 (FGFR1). To determine the role of FGFR1 signaling in the development of DA neurons, we generated transgenic mice expressing a dominant negative mutant [FGFR1(TK-)] from the catecholaminergic, neuron-specific tyrosine hydroxylase (TH) gene promoter. In homozygous th(tk-)/th(tk-) mice, significant reductions in the size of TH-immunoreactive neurons were found in the substantia nigra compacta (SNc) and the ventral tegmental area (VTA) at postnatal days 0 and 360. Newborn th(tk-)/th(tk-) mice had a reduced density of DA neurons in both SNc and VTA, and the changes in SNc were maintained into adulthood. The reduced density of DA transporter in the striatum further demonstrated an impaired development of the nigro-striatal DA system. Paradoxically, the th(tk-)/th(tk-) mice had increased levels of DA, homovanilic acid and 3-methoxytyramine in the striatum, indicative of excessive DA transmission. These structural and biochemical changes in DA neurons are similar to those reported in human patients with schizophrenia and, furthermore, these th(tk-)/th(tk-) mice displayed an impaired prepulse inhibition that was reversed by a DA receptor antagonist. Thus, this study establishes a new developmental model for a schizophrenia-like disorder in which the inhibition of FGF signaling leads to alterations in DA neurons and DA-mediated behavior.

Activation of cAMP signaling pathway and its transcriptional factor cyclic AMP response element binding protein (CREB) and coactivator are key determinants of neuronal differentiation and plasticity. We show that nuclear fibroblast growth factor receptor-1 (FGFR1) mediates cAMP-induced neuronal differentiation and regulates CREB and CREB binding protein (CBP) function in alpha-internexin-expressing human neuronal progenitor cells (HNPC). In proliferating HNPC, FGFR1 was associated with the cytoplasm and plasma membrane. Treatment with dB-cAMP induced nuclear accumulation of FGFR1 and caused neuronal differentiation, accompanied by outgrowth of neurites expressing MAP2 and neuron-specific neurofilament-L protein and enolase. HNPC transfected with nuclear/cytoplasmic FGFR1 or non-membrane FGFR1(SP-/NLS), engineered to accumulate exclusively in the cell nucleus, underwent neuronal differentiation in the absence of cAMP stimulation. In contrast, FGFR1/R4, with highly hydrophobic transmembrane domain of FGFR4, was membrane associated, did not enter the nucleus and failed to induce neuronal differentiation. Transfection of tyrosine kinase-deleted dominant negative receptor mutants, cytoplasmic/nuclear FGFR1(TK-) or nuclear FGFR1(SP-/NLS)(TK-), prevented cAMP-induced neurite outgrowth. Nuclear FGFR1 localized in speckle-like domains rich in phosphorylated histone 3 and splicing factors, regions known for active RNA transcription and processing, and activated the neurofilament-L gene promoter. FGFR1(SP-/NLS) transactivated CRE, up-regulated phosphorylation and transcriptional activity of CREB and stimulated the activity of CBP several-fold. Thus, cAMP-induced nuclear accumulation of FGFR1 provides a signal that triggers molecular events leading to neuronal differentiation.

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.

Bone morphogenetic protein-7 (BMP-7), a member of the transforming growth factor (TGF)-beta superfamily of signaling cytokines, induces dendritic growth in rat sympathetic neurons. In this study, we present evidence that the recently discovered integrative nuclear FGFR1 signaling (INFS) pathway is involved in dendrite outgrowth mediated by BMP-7. Immunocytochemical analysis of expressed fibroblast growth factors (FGFs) showed that little FGF-2 was detected in control neurons, but the expression of this molecule in the cytoplasm and nucleus increased within 6 h after BMP-7 treatment. In contrast, FGF-1 was constitutively present in the peripheral cytoplasm and in neurites under control conditions, and its distribution did not change with BMP-7 exposure. The high-affinity receptor FGFR1 was present in low amounts in control neurons and was associated with the cytoplasm, the plasma membrane, and the nucleus. Twenty-four hours of BMP-7 treatment elicited an increase in FGFR1 nuclear localization. Overexpressed constructs of FGFR1 that lack the tyrosine kinase domain, and have been shown to act in a dominant-negative manner on FGFR1 signaling, inhibited BMP-7 mediated initial dendrite outgrowth in transfected neurons by approximately 50%. However, targeted inhibition of extracellular FGF-2 by overexpression of a secreted receptor mutant FGFR1(TM-) lacking the transmembrane domain failed to affect BMP-7 induced dendritic growth, as did treatment with the extracellular FGFR antagonist inositol hexakisphosphate. These results suggest that the INFS, which has already been implicated in a broad range of activities in other cell types, may also be required for BMP-7 to stimulate dendritic development.

The cis elements mediating activation of the tyrosine hydroxylase gene by angiotensin II were examined by transfecting tyrosine hydroxylase promoter-luciferase constructs into cultured bone adrenal medullary cells. Angiotensin II-responsive elements are located within -54/+25-bp and -269/-55-bp promoter regions and were identified, respectively, as cyclic AMP (CRE)- and 12-O-tetradecanoylphorbol 13-acetate responsive element (TRE)-like sequences. Unlike CRE, TRE also supports basal promoter activity. Mutations of TRE or CRE that reduced angiotensin II stimulation abolished in vitro binding of nuclear proteins to those elements, suggesting that proteins forming CRE- and TRE-inducible complexes may mediate angiotensin II stimulation. The TRE is adjacent to a dyad symmetry element. Those two sites form a common regulatory unit in which the dyad symmetry element acts as a repressor of the TRE site. Isolated dyad symmetry element did not bind nuclear proteins in vitro. In supercoiled DNA it exhibited S1 nuclease sensitivity and was recognized by a DNA cruciform-specific antibody consistent with the extrusion of a cruciform structure that overlaps with the TRE. A mutation that abolished formation of the cruciform correlated with a loss of repressor activity. We propose a novel model of tyrosine hydroxylase gene regulation in which functions of the TRE are modulated via structural transition in the adjacent DNA.

The tyrosine hydroxylase gene is expressed specifically in catecholaminergic cells, and its activity is regulated by afferent stimuli. To characterize molecular mechanisms underlying those regulations, we have constructed chimeric genes consisting of bovine tyrosine hydroxylase gene promoters (wild-type or deletion mutants) and a luciferase reporter gene. The basal expression of these genes and their regulation by angiotensin II were examined in cultured bovine adrenal medullary cells. Luciferase activity was normalized to the amount of transfected plasmid DNA. A pTHgoodLUC plasmid containing the -428/+21-bp fragment of the tyrosine hydroxylase gene promoter expressed luciferase activity at severalfold higher levels than the promoterless pOLUC plasmid. Deletion of the -194/-54-bp promoter fragment containing POU/Oct, SP1, and other putative regulatory elements increased luciferase expression fivefold. An additional deletion further upstream (-269/-194 bp), including a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-like site, reduced promoter activity. These results indicate the presence of negatively and positively acting regions in the bovine tyrosine hydroxylase gene promoter controlling basal promoter activity in adrenal medullary cells. Angiotensin II stimulated the expression of endogenous tyrosine hydroxylase gene and pTHgood-LUC approximately threefold without affecting the expression of pOLUC. A comparable threefold stimulation was observed following the deletion of the -194/-54-bp promoter region, despite the increase in basal promoter activity. Additional deletion of the -269/-194-bp promoter fragment reduced stimulation by angiotensin II to 1.5-fold. These results indicate that the angiotensin II receptor-responsive element is located in the -269/-194-bp promoter region containing the TRE-like site. Additional angiotensin II-responsive site(s) may be present outside this region. Gel mobility shift assays demonstrated constitutive and angiotensin II-induced protein binding to the tyrosine hydroxylase gene promoter. Some DNA-protein complexes were displaced with c-Fos antibodies. The results suggest that c-Fos-related antigens support basal promoter activity and mediate activation of tyrosine hydroxylase by angiotensin II receptor.

Previous results from our laboratory suggest that long-term treatment of primary cultured bovine adrenal medullary (BAM) chromaffin cells with nicotine or phorbol 12-myristate 13-acetate, either of which directly activates protein kinase C (PKC), increases the mRNA levels encoding catecholamine-synthesizing enzymes and proenkephalin. In the present study, we have examined the effects of nicotine on BAM cell PKC activity with special emphasis on long-term effects. Nicotine increased particulate PKC activity in a concentration-dependent manner when measured using in vitro enzyme assay with histone as the substrate. This effect is mediated through nicotinic cholinergic receptors, because 1,1-dimethylphenylpiperazinium, a nicotinic agonist, had a similar effect. In addition, chlorisondamine, a specific nicotine-receptor blocking drug, antagonized the effect of nicotine. Nicotine also increased specific [3H]phorbol 12,13-dibutyrate ([3H]PdBu) binding within 1 min, the effect of which was maximal between 3 and 12 min. This effect was reversed by chlorisondamine similarly after 12 min and after 18 h of nicotine treatment, indicating that continual nicotinic-receptor occupancy is required for persistent PKC activation. Compared to PKC activation, the onset of nicotine-stimulated diacylglycerol production was slow, and it was observed after 12 min of incubation with nicotine. The diacylglycerol levels, specific [3H]PdBu binding, and PKC activity remained significantly elevated for at least 18 h with continuous nicotine incubation. Furthermore, nicotine increased the PKC immunoreactivity of a particulate protein with a molecular mass of 82 kDa in the western blot. These results suggest that nicotinic-receptor activation increases PKC activity and immunoreactivity in BAM cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies from our laboratory suggest that protein kinase C (PKC) is involved in the angiotensin II (AII)-induced increase in the expression of genes encoding proenkephalin and catecholamine biosynthesizing enzymes in primary cultured bovine adrenal medullary (BAM) cells. The purpose of this study was to examine the effects of [Sar1]-AII (S1-AII), an AII agonist, on PKC activity in BAM cells. Thirty-minute incubation with S1-AII produced a dose-dependent activation of PKC. The particulate PKC activity was significantly increased by 2 nM S1-AII after both 30 min and 12 h of incubation. A high concentration of S1-AII (200 nM) caused an increase in particulate PKC activity after 30 min of incubation and this increase was still observed after 18 h of continuous incubation. [Sar1, Thr8]-angiotensin II (S1, T8-AII) (100 microM), an AII antagonist, inhibited the effect of S1-AII (20 nM) on PKC activity, suggesting a specific AII receptor-mediated effect. An increase in BAM cell particulate PKC immunoreactivity after 18 h of S1-AII treatment was observed in Western blot analysis of PKC-immunoreactive protein (82 kDa). The persistent activation of PKC seen in this study is consistent with our hypothesis that PKC may mediate the S1-AII-induced increase in the expression of genes encoding proenkephalin and catecholamine synthesizing enzymes in BAM cells.