Ewa K. Stachowiak

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

FGF-2 has been implicated in the neoplastic transformation of glioma cells and in the transition of normal quiescent astrocytes to a proliferating, reactive state. In the present study we have observed that in human glial cells, levels and subcellular localization of FGF-2 are different in quiescent and proliferating cells. FGF-2 was detected in the cytoplasm of non-reactive astrocytes in human brain sections. In contrast FGF-2 was located within the cytoplasm and nuclei of reactive astrocytes in gliotic brain tissue and in neoplastic cells of glioma tumors. In vitro, FGF-2 was found predominantly in the nucleus of subconfluent proliferating astrocytes, but was detected only in the cytoplasm of density arrested quiescent astrocytes. Our results suggest that reduced cell contact stimulates nuclear accumulation of FGF-2, accompanying mitotic activation of reactive human astrocytes. FGF-2 was constitutively localized to the nucleus of continuously proliferating glioma cells independent of cell density. A role for intracellular FGF-2 was further suggested by the observation that glioma cells that are not stimulated to proliferate by extracellular FGF-2 proliferated faster when transfected with FGF-2 expressing vectors. This increased proliferation correlated with nuclear accumulation of FGF-2. Cell proliferation was attenuated by 5′-deoxy-5′-methylthioadenosine, a FGF-2 receptor tyrosine kinase inhibitor that acts within the cell, but was unaffected by myo-inositol hexakis [dihydrogen phosphate] that disrupts FGF-2 binding to plasma membrane receptors. Our results indicate that FGF-2 serves as a nuclear regulator of proliferation in astrocytic cells. In glioma cells, the constitutive presence of FGF-2 in the nucleus may promote proliferation that is insensitive to cell contact inhibition.

Integrative nuclear FGFR1 signaling (INFS) as a part of a universal “feed‐forward‐and‐gate” signaling module that controls cell growth and differentiation

A novel signaling mechanism is described through which extracellular signals and intracellular signaling pathways regulate proliferation, growth, differentiation, and other functions of cells in the nervous system. Upon cell stimulation, fibroblast growth factor receptor‐1 (FGFR1), a typically plasma membrane‐associated protein, is released from ER membranes into the cytosol and translocates to the cell nucleus by an importin‐β‐mediated transport pathway along with its ligand, FGF‐2. The nuclear accumulation of FGFR1 is activated by changes in cell contacts and by stimulation of cells with growth factors, neurotransmitters and hormones as well as by a variety of different second messengers and thus was named integrative nuclear FGFR1 signaling (INFS). In the nucleus, FGFR1 localizes specifically within nuclear matrix‐attached speckle‐domains, which are known to be sites for RNA Pol II‐mediated transcription and co‐transcriptional pre‐mRNA processing. In these domains, nuclear FGFR1 colocalizes with RNA transcription sites, splicing factors, modified histones, phosphorylated RNA Pol II, and signaling kinases. Within the nucleus, FGFR1 serves as a general transcriptional regulator, as indicated by its association with the majority of active nuclear centers of RNA synthesis and processing, by the ability of nuclear FGFR1 to activate structurally distinct genes located on different chromosomes and by its stimulation of multi‐gene programs for cell growth and differentiation. We propose that FGFR1 is part of a universal “feed‐forward‐and‐gate” signaling module in which classical signaling cascades initiated by specific membrane receptors transmit signals to sequence specific transcription factors (ssTFs), while INFS elicited by the same stimuli feeds the signal forward to the common coactivator, CREB‐binding protein (CBP). Activation of CBP by INFS, along with the activation of ssTFs by classical signaling cascades brings about coordinated responses from structurally different genes located at different genomic loci.

TC-5619: an alpha7 neuronal nicotinic receptor-selective agonist that demonstrates efficacy in animal models of the positive and negative symptoms and cognitive dysfunction of schizophrenia.

A growing body of evidence suggests that the alpha7 neuronal nicotinic receptor (NNR) subtype is an important target for the development of novel therapies to treat schizophrenia, offering the possibility to address not only the positive but also the cognitive and negative symptoms associated with the disease. In order to probe the relationship of alpha7 function to relevant behavioral correlates we employed TC-5619, a novel selective agonist for the alpha7 NNR subtype. TC-5619 binds with very high affinity to the alpha7 subtype and is a potent full agonist. TC-5619 has little or no activity at other nicotinic receptors, including the alpha4beta2, ganglionic (alpha3beta4) and muscle subtypes. The transgenic th(tk-)/th(tk-) mouse model that reflects many of the developmental, anatomical, and multi-transmitter biochemical aspects of schizophrenia was used to assess the antipsychotic effects of TC-5619. In these mice TC-5619 acted both alone and synergistically with the antipsychotic clozapine to correct impaired pre-pulse inhibition (PPI) and social behavior which model positive and negative symptoms, respectively. Antipsychotic and cognitive effects of TC-5619 were also assessed in rats. Similar to the results in the transgenic mice, TC-5619 significantly reversed apomorphine-induced PPI deficits. In a novel object recognition paradigm in rats TC-5619 demonstrated long-lasting enhancement of memory over a wide dose range. These results suggest that alpha7-selective agonists such as TC-5619, either alone or in combination with antipsychotics, could offer a new approach to treating the constellation of symptoms associated with schizophrenia, including cognitive dysfunction.

Nuclear accumulation of fibroblast growth factor receptors in human glial cells : association with cell proliferation

In this study we describe the presence of high affinity FGF-2 binding sites in the nuclei of U251MG glioma cells (Kd=7 pM). Immunoprecipitation of total cell extracts with FGF receptor (FGFR) 1-4 antibodies showed that U251MG glioma cells express only FGFR1. [125I]FGF-2 cross linking to nuclear extracts followed by FGFR1 immunoprecipitation showed that FGFR1 may account for the nuclear FGF-2 binding sites. Western blot analysis demonstrated the presence of 103, 118 kDa and small amounts of 145 kDa FGFR1 isoforms in the nuclei of glioma cells. All isoforms contain both the C- and N-terminal domains. Nuclear FGFR1 retains kinase activity. Immunocytochemistry using confocal microscopy showed specific FGFR1 immunoreactivity within the nuclear interior. In continuously proliferating glioma cells, nuclear FGFR1 is constitutively expressed, independent of cell density. In contrast, in nontransformed human astrocytes, nuclear FGFR1 levels fluctuate with the proliferative state of the cell. In quiescent, confluent astrocytes nuclear FGFR1 protein was depleted. An accumulation of nuclear FGFR1 was observed following the transition to a subconfluent, proliferating state. Transfection of a pcDNA3.1-FGFR1 expression vector into glioma cells that do not express FGFR1 resulted in the nuclear accumulation of FGFR1, increased cell proliferation, and stimulated transition from the G0/G1 to the S-phase of the cell cycle. The increased proliferative rate was resistant to inhibition by the cell-impermeable FGF binding antagonist, myoinositol hexakis [dihydrogen phosphate]. Our results suggest that the constitutive nuclear presence of FGFR1 contributes to the increased proliferation of glioma cells while the transient nuclear accumulation of FGFR1 in normal astrocytes may play a role in the transition to a reactive state.

Growth factor regulation of cell growth and proliferation in the nervous system : a new intracrine nuclear mechanism

This article discusses a novel intracrine mechanism of growth-factor action in the nervous system whereby fibroblast growth factor-2 (FGF-2) and its receptor accumulate in the cell nucleus and act as mediators in the control of cell growth and proliferation. In human and rat brain the levels and subcellular localization of FGF-2 differ between quiescent and reactive astrocytes. Quiescent cells express a low level of FGF-2, which is located predominantly within the cytoplasm. In reactive astrocytes, the expression of FGF-2 increases and the proteins are found in both the cytoplasm and nucleus. In glioma tumors, FGF-2 is overexpressed in the nuclei of neoplastic cells. Similar changes in FGF-2 expression and localization are found in vitro. The nuclear accumulation of FGF-2 reflects a transient activation of the FGF-2 gene by potentially novel transactivating factors interacting with an upstream regulatory promoter region. In parallel with FGF-2, the nuclei of astrocytes contain the high-affinity FGF-2 receptor, FGFR1. Nuclear FGFR1 is full length, retains kinase activity, and is localized within the nuclear interior in association with the nuclear matrix. Transfection of either FGF-2 or FGFR1 into cells that do not normally express these proteins results in their nuclear accumulation and concomitant increases in cell proliferation. A similar regulation of nuclear FGF-2 and FGFR1 is observed in neural crest-derived adrenal medullary cells and of FGF-2 in the nuclei of cerebellar neurons. Thus, the regulation of the nuclear content of FGF-2 and FGFR1 could serve as a novel mechanism controlling growth and proliferation of glial and neuronal cells.

cAMP-induced differentiation of human neuronal progenitor cells is mediated by nuclear fibroblast growth factor receptor-1 (FGFR1).

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 α‐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.

Vitamin D increases expression of the tyrosine hydroxylase gene in adrenal medullary cells

We examined expression of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3] receptors in chromaffin cells of the adrenal medulla and the effects of 1,25(OH)2 D3 on expression of the tyrosine hydroxylase (TH) gene. Accumulation of 1,25(OH)2 D3 in the nuclei of adrenal medullary cells, but not in the adrenal cortex, was observed in mice intravenously injected with radioactively labeled hormone. 1,25(OH)2 D3 produced concentration-dependent increases in the TH mRNA levels in cultured bovine adrenal medullary cells (BAMC). The maximal increases (2-3-fold) occurred at 10(-8) M 1,25(OH)2 D3. Combined treatment with 1,25(OH)2 D3 and 20 microM nicotine had no additive effect on TH mRNA levels suggesting that transsynaptic (nicotinic) and vitamin D (hormonal) stimulation of TH gene expression are mediated through converging mechanisms. Induction of TH mRNA by 1,25(OH)2 D3 was not affected by calcium antagonist TMB-8. By increasing expression of the rate limiting enzyme in the catecholamine biosynthetic pathway, 1,25-(OH)2 D3 may participate in the regulation of catecholamine production in adrenal chromaffin cells. This regulation provides mechanisms through which 1,25(OH)2 D3 may control response and adaptation to stress.

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II, depolarization and protein kinase C

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.

Nuclear localization of functional FGF receptor 1 in human astrocytes suggests a novel mechanism for growth factor action

Fractionation of human astrocytes revealed the presence of 103, 118, and 145 kDa forms of FGF receptor 1 (FGFR1) in isolated nuclei. Only trace amounts of FGFR1 proteins were detected in the cell membrane or cytoplasmic fractions. Nuclear FGFR1 is found in the nucleoplasm and nuclear matrix but not in chromatin. Immuno-confocal microscopy further demonstrates the intranuclear presence of FGFR1 and its colocalization with FGF-2. Nuclear FGFR1 binds to FGF-2 and has tyrosine kinase activity. Translocation of functional growth factor receptors into the cell nucleus offers a novel mechanism for growth factor action.

Fibroblast Growth Factor Receptor-1 (FGFR1) Nuclear Dynamics Reveal a Novel Mechanism in Transcription Control

Nuclear FGFR1 acts as a developmental gene regulator in cooperation with FGF-2, RSK1, and CREB-binding protein (CBP). FRAP analysis revealed three nuclear FGFR1 populations: i) a fast mobile, ii) a slower mobile population reflecting chromatin-bound FGFR1, and iii) an immobile FGFR1 population associated with the nuclear matrix. Factors (cAMP, CBP) that induce FGFR1-mediated gene activation shifted FGFR1 from the nuclear matrix (immobile) to chromatin (slow) and reduced the movement rate of the chromatin-bound population. Transcription inhibitors accelerated FGFR1 movement; the content of the chromatin-bound slow FGFR1 decreased, whereas the fast population increased. The transcriptional activation appears to involve conversion of the immobile matrix-bound and the fast nuclear FGFR1 into a slow chromatin-binding population through FGFR1s interaction with CBP, RSK1, and the high-molecular-weight form of FGF-2. Our findings support a general mechanism in which gene activation is governed by protein movement and collisions with other proteins and nuclear structures.