Gérard Bouche

Fibroblast growth factor-2 binds to the regulatory beta subunit of CK2 and directly stimulates CK2 activity toward nucleolin.

The presence of fibroblast growth factor-2 (FGF-2) in the nucleus has now been reported both in vitro and in vivo, but its nuclear functions are unknown. Here, we show that FGF-2 added to nuclear extract binds to protein kinase CK2 and nucleolin, a CK2 natural substrate. Added to baculovirus-infected cell extracts overexpressing CK2 or its isolated subunits, FGF-2 binds to the enzyme through its regulatory β subunit. Using purified proteins, FGF-2 is shown to directly interact with CK2 and to stimulate CK2 activity toward nucleolin. Furthermore, a mitogenic-deficient FGF-2 mutant protein has an impaired ability to interact with CK2 and to stimulate CK2 activity using nucleolin as substrate. We propose that in growing cells, one function of nuclear FGF-2 is to modulate CK2 activity through binding to its regulatory β subunit.

Fibroblast growth factor-2 (FGF-2) in the nucleus : translocation process and targets

FGF-2 (basic fibroblast growth factor) was recently detected in the nucleus of a variety of cell types. The large isoforms contain a functional nuclear localization signal that allows their nuclear accumulation in producing cells, while a small amount of FGF-2 added exogenously to target cells is translocated to the nucleus in phase G1 of the cell cycle according to an unknown process. We report here using Chinese hamster ovary cell mutants bearing deficiency in heparan sulfate proteoglycans (HSPGs) synthesis that HSPGs are required for transport of exogenous FGF-2 to the nucleus. Furthermore a co-transport was suggested since an active complex containing FGF-2 and HSPGs was isolated from nuclei of treated cells. Several FGF-2 nuclear targets were described. In vivo as in vitro, it activates rDNA transcription and it binds to a specific DNA sequence that is present in the non-transcribed spacer of ribosomal genes. In vitro, FGF-2 has a strong affinity for histone H1 and it activates the protein kinase CKII. In the nucleus FGF-2 could regulate gene expression through modulation of chromatin structure.

Uncoupling of cell proliferation and differentiation activities of basic fibroblast growth factor

FGF‐2 exerts its pleiotropic effects on cell growth and differentiation by interacting with specific cell surface receptors. In addition, exogenously added FGF‐2 is translocated from outside the cell to the nucleus during G1‐S transition. In this study, we show that a single point mutation in FGF‐2 (substitution of residue serine 117 by alanine) is sufficient to drastically reduce its mitogenic activity without affecting its differentiation properties. The FGF‐2(S117A) mutant binds to and activates tyrosine kinase receptors and induces MAPK and p70S6K activation as strongly as the wild‐type FGF‐2. We demonstrate that this mutant enters NIH3T3 cells, is translocated to the nucleus, and is phosphorylated similar to the wild‐type growth factor. This suggests that FGF‐2 mitogenic activity may require, in addition to signaling through cell surface receptors and nuclear translocation, activation of nuclear targets. We have previously shown that, in vitro, FGF‐2 directly stimulates the activity of the casein kinase 2 (CK2), a ubiquitous serine/threonine kinase involved in the control of cell proliferation. We report that, in vivo, FGF‐2(WT) transiently interacts with CK2 and stimulates its activity in the nucleus during G1‐S transition in NIH3T3 cells. In contrast, the FGF‐2(S117A) mutant fails to interact with CK2. Thus, our results show that FGF‐2 mitogenic and differentiation activities can be dissociated by a single point mutation and that CK2 may be a new nuclear effector involved in FGF‐2 mitogenic activity. Bailly, K., Soulet, F., Leroy, D., Amalric, F., Bouche, G. Uncoupling of cell proliferation and differentiation activities of basic fibroblast growth factor (FGF‐2). FASEB J. 14, 333—344 (2000)

Cancer Cells Regulate Lymphocyte Recruitment and Leukocyte-Endothelium Interactions in the Tumor-Draining Lymph Node

The physiologic function of the secondary lymphoid organs to recruit large numbers of naïve lymphocytes increases the probability that antigens encounter their rare, sometimes unique, specific T lymphocytes and initiate a specific immune response. In peripheral lymph nodes (LNs), this recruitment is a multistep process, initiated predominantly within the high endothelial venules (HEVs), beginning with rolling and chemokine-dependent firm adhesion of the lymphocytes on the venular endothelium surface. We report here that, in C57BL/6 mice, the recruitment of naïve lymphocytes is impaired in LNs draining a B16 melanoma tumor. Intravital microscopy analysis of the tumor-draining LNs revealed that this effect is associated with an important defect in lymphocyte adhesion in the HEVs and a progressive decrease in the expression of the LN chemokine CCL21. In parallel with these effects, the tumor up-regulated, essentially through a P-selectin-dependent mechanism, the rolling and sticking of circulating polymorphonuclear cells within the LN low-order venules where few rolling and sticking events are usually observed. These effects of the tumor were independent of the presence of metastasis into the LN and occurred as long as the tumor developed. Together, these results indicate that the tumor proximity disturbs the LN physiology by modifying the molecular, spatial, and cellular rules that usually control leukocyte-endothelium interactions into the peripheral LNs. In addition, they emphasize a new role for the low-order venules of the peripheral LNs, which compared with the HEVs, seem to be the preferential port of entry for cells linked to inflammatory processes.

Effects of heat shock on gene expression and subcellular protein distribution in Chinese hamster ovary cells

Incubation of Chinese Hamster Ovary (CHO) cells for one hour at 43 degrees C results in several obvious changes in protein distribution and protein synthesis. One major protein of the cytoplasm (molecular weight 45,000 daltions), also present as a minor component in the nucleus, rapidly disappeared while several proteins, especially high molecular weight peptides, were induced by heat shock. Localization of the proteins in the cytoplasm, extra-nucleolar chromatin and nucleolar bodies has been carried out. Different sets of induced proteins appear in each subcellular compartment. Four hours after restoration of the normal temperature, the normal pattern of protein synthesis was observed. The 45,000 dalton protein reappeared first. Relations between structural and functional alterations and changes in protein distribution are suggested.

Glucocorticoids repress ribosome biosynthesis in lymphosarcoma cells by affecting gene expression at the level of transcription, posttranscription and translation

Growth arrest of P1798 murine lymphosarcoma cells by glucocorticoids is accompanied by a remarkable decrease in transcription of rRNA and translation of mRNAs encoding basic ribosomal proteins (rps). Here we report that the expression of other genes involved in ribosome biogenesis is repressed in dexamethasone-treated P1798 cells. These include posttranscriptionally regulated decline in the abundance of the mRNA and primary transcript of nucleolin; abrupt drop in the transcription rate of U3 small nucleolar RNA; and inhibition of translation of mRNAs coding for P2 and L5, acidic and basic rps, respectively. Normal expression of these genes is resumed upon hormonal withdrawal.

Unusual processing of nucleolar RNA synthesized during a heat shock in CHO cells.

Maturation of pre-rRNA has been investigated through heat shock experiments in which pre-rRNA synthesis is successively turned off and turned on. After one hour at 43°C high molecular weight RNA is no longer synthesized and both the methylation and the maturation of pre-rRNA synthesized before heat shock are blocked. After two hours recovery at 37°C, methylation and simultaneous maturation, of pre-existing RNA occur while pre-rRNA synthesis is reinitiated only after 7 hours at 37°C. During the first 30 min. at 43°C, a residual synthesis of high molecular weight RNA is observed in the nucleolus with an average molecular weight slightly higher than pre-rRNA (4.6 106). During the recovery period at 37°C, RNA synthesized at 43°C is slowly processed into unusual species (39S, 35S, 29S). No new ribosomal RNA appeared in the cytoplasm. This unusual maturation pathway could be a minor pathway of nucleolar RNA processing in exponentially growing cells.

Phosphorylated proteins involved in the regulation of rRNA synthesis in CHO cells recovering from heat shock

Various hypothesis have been advanced to explain the regulation of rRNA synthesis. Transcription of ribosomal genes may be dependent on the presence of rapidly turning over proteins, on the concentration of amino acids [l-5], or under the control of intracellular ATP and GTP pool sizes [6]. Also, there may be a role for protein phosphoxylation in the regulation of gene expression [7-l 01, Finally, interrelations between these various control mechanisms must also be considered. We have investigated the role of a number of factors postulated to be involved in the regulation of transcription in a heat shock system using CHO cells. Incubation of cells for 1 h at 43°C induces reversible inhibition of rRNA synthesis [ 1 1 121. Following heat shock, cells incubated at 37°C recover a normal level of RNA synthesis by 7 h. Results indicate that in our experimental system, RNA polymerase activities ATP and GTP pool concentrations and overall proteins transport were not directly related to rRNA synthesis. On the other hand, heat shock induced the phosphorylation of two nuclear proteins, one of which is specific to the nucleolus (95 000 M,). An apparent correlation between the initiation of RNA synthesis and the dephosphorylation of the 95 000 MI protein was observed.

Effect of heat shock on RNA polymerase activities in Chinese hamster ovary cells

Abstract Incubation of Chinese hamster ovary cells at supranormal temperature (1 h at 43°C) results in an inhibition of high molecular weight RNA (rRNA and hnRNA) synthesis while the low molecular weight RNAs transcribed by RNA polymerase C(III) are still synthesized. In “in vitro” assays, the three RNA polymerase activities are detectable in extracts of heat shocked cells but are present at lower specific activities than in control cells (A (I):50 %; B (II): 81 %; C (III): 76 %). Correlations have been established respectively between the “in vivo” RNA polymerase A and B activities and the level of phosphorylation of two proteins, one specific of the nucleolus (95,000 Mr) and one specific of the non nucleolar fraction of the nucleus (54,000 Mr). In their phosphorylated form, these two proteins could be specific “in vivo” inhibitors of the initiation of transcription by RNA polymerase I and II.

The Basic Fibroblast Growth Factor Isoforms: Endogenous and Exogenous Behavior

The fibroblast growth factors (FGFs) constitute a family of at least seven structurally related polypeptides sharing 35-55% sequence homology. The acidic FGF (FGF-1) and basic FGF (FGF-2) are the prototypes. Other members of this family include int-2 (FGF-3), K-fgf (FGF4), FGF-5, FGF-6 and KGF (FGF-7). They are potent modulators of cell function proliferation, motility, differentiation and survival. Furthermore, they play an important role in normal physiological processes such as embryonic development, angiogenesis, nervous cell system differentiation and wound-repair (Rifkin and Moscatelli, 1989; Burgess and Maciag, 1989). The FGFs exert their biological activity through a complex interaction with high and low affinity receptors. To date, four human genes have been identified each encoding a distinct high affinity receptor (Kd of l0-11). Each of these genes encodes multiple proteins derived from alternative mRNA splicing (Houssaint et al., 1990; Johnson et al., 1990; Partanen et al., 1991). Despite their structural similarities, these high affinity receptors may differ in their ability to bind various members of FGF family. The heparin sulfate proteoglycans (HSPGs) are the low affinity receptors (Kd of 10-9) which are essential for bFGF activity and high affinity binding (Moscatelli, 1987; Yayon et al., 1991; Rapraeger et al., 1991). Recently, syndecan, an integral membrane HSPG, has been identified as a low affinity binding site for bFGF (Kiefer et al., 1990). Basic FGF has several unique properties that differentiate it from other growth factors. One of these is the lack of a signal peptide normally required for vectorial transfer into the endoplasmic reticulum and for secretion (Abraham et al., 1986a). A second property of bFGF, shared with FGF-3, is the use of CUG codons for the initiation of translation of High Molecular Weight (HMW) isoforms, in addition to the canonic AUG codon used to generate the Low Molecular Weight bFGF (LMWbFGF) form (Florkiewicz and Sommer, 1989; Prats et al., 1989). In this paper, we will review some developments in the biology of bFGF(FGF-2), which have recently been achieved by ourselves and other authors.