We have previously shown that the signal peptideless cytokine interleukin 1a (IL-la) may play a role as an intracellular regulator of human endothelial cell senescence (J. A. M. Maier, P. Voulalas, D. Roeder, and T. Maciag, Science 249: [1570][1571][1572][1573][1574] 1990). To investigate the potential intracellular function of IL-lot, transformed endothelial cells were transfected with the human cDNAs that code for the two forms of IL-la, the precursor molecule IL-11-27, and the mature protein IL-11.3-271* The subcellular localization of the two different polypeptides was investigated directly or by using chimeric genes constructed by fusion of different fragments of the IL-la gene and the P-galactosidase open reading frames. The IL-1 113-271 protein was cytoplasmic, while IL-11-271 was nuclear. The basic cluster at the NH2 terminus of IL-1, KVLKKRR, has been shown to mediate IL-lot nuclear targeting. Moreover, nuclear localization of IL-la correlates with impaired cell growth and expression of some IL-la-inducible genes. These results suggest that transport of endogenous intracellular IL-lat has been highlighted by the discovery of an intracellular IL-1 receptor antagonist which is expressed in keratinocytes (17). This protein could function as an intracellular competitor of either internalized or endogenously produced IL-la.To determine whether IL-lat represents an intracellular regulatory molecule in endothelial cells, we transfected transformed endothelial cells (tEC) (46) with plasmids containing the cDNAs that code for IL-11-271 and IL-1 113-271 The subcellular localization of the two forms was investigated directly or by using chimeric genes constructed by fusion of the IL-lot and ,B-galactosidase (P-Gal) open reading frames. IL-11_271 was nuclear, while IL-11,3-271 was cytoplasmic. The sequence responsible for the nuclear targeting of IL-1 is contained within seven amino acid residues at positions 79 to 85. We also determined whether or not the activity of endogenous IL-la correlates with its ability to localize to the nucleus. The following data suggest that transport of IL-la into the nucleus is required for it to modulate endothelial cell properties. MATERIALS AND METHODSPlasmid construction. Most plasmids were constructed by PCR amplification of the IL-la cDNA with primers bearing unique sequence and restriction enzyme sites. Plasmid Cfla, containing the full-length cDNA for IL-lat, was kindly provided by R. Gayle (Immunex, Seattle, Wash.).The primers used to clone IL-lot in pMEXneo (29) were IL-11-271 sense (5'-CCG TCG ACC CAC CAT GGC-3'),

Abstract. Basic fibroblast growth factor (bFGF) induces cell proliferation and plasminogen activator (PA) activity in transformed fetal bovine aortic endothelial (FBAE) GM 7373 cells. A similar response is observed after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In these cells, bFGF and TPA cause activation of protein kinase C (PKC), as demonstrated by the induction of the phosphorylation of an 87-kD PKC substrate in intact cells and by the increase in membrane-associated PKC activity. Activation of PKC by bFGF or TPA is inhibited in cells made PKCdeficient by pretreatment with high concentrations of TPA. The mitogenic activity of bFGF or of TPA is completely inhibited in PKC-deficient cells or in naive cells treated with the PKC inhibitor H-7. However, these cells proliferate in response to serum, epidermal growth factor, and dibutyryl cyclic AMP. Similar results are obtained in normal FBAE AG 7680 cells. These data indicate that activation of PKC is responsible for the mitogenic activity of bFGF in FBAE cells. On the contrary, the PA-inducing activity of bFGF is unaffected by down-regulation of PKC or by treatment with the PKC inhibitor H-7 in both transformed GM 7373 and normal AG 7680 cells, bFGF induces a rapid 45Ca influx in naive and in PKC-deprived GM 7373 cells. In these cells, addition of EGTA to the incubation medium prevents both the 45Ca influx and the increase in PA activity induced by bFGF, without affecting its mitogenic activity. Even though the involvement of PKC in the increase of cell-associated PA activity induced by bFGF can not be completely dismissed, the present results suggest a role of calcium entry in the modulation of the PA-inducing activity of bFGE p ROTEI~ kinase C (PKC) ~ is a calcium and phospholipid-dependent enzyme that is activated by diacylglycerol (Nishizuka, 1984). Several lines of evidence indicate that PKC is involved in mediating the intracellular signaling that triggers various biological processes, including those elicited by certain polypeptide growth factors (Nishizuka, 1986).Basic fibroblast growth factor (bFGF) is an 18-kD mitogen characterized by its affinity for heparin. In vitro, it induces cell proliferation and a characteristic set of responses, including protease production, depending upon the cell type under investigation. In vivo, bFGF has been shown to induce neovascularization (for a recent review on bFGF see Gospodarowicz et al., 1987). The role exerted by PKC in the transduction signaling mediating bFGF activity is still unclear. bFGF has been shown to activate PKC in Swiss 3T3 and in 3T3-L1 fibroblasts (Blackshear et al., 1985;Tsuda et al.,

Basic fibroblast growth factor (bFGF) binds to heparin-like molecules present in the extracellular matrix (ECM) of transformed fetal bovine aortic endothelial GM 7373 cells. Binding of bFGF to ECM can be competed by heparin or heparan sulfate, and ECM-bound bFGF can be released by treating the cells with heparinase or heparatinase. After binding to ECM, bFGF is slowly released into the medium in a biologically active form, as shown by its capacity to induce an increase of cell-associated plasminogen activator activity and cell proliferation. The increase is prevented upon removal of ECM-bound bFGF by a neutral 2 M NaCl wash. Soluble heparin and heparan sulfate reduce the amount of ECM-bound bFGF released into the medium, possibly competing with ECM polysaccharides for heparinase-like enzymes produced by endothelial cells, suggesting that these enzymes are involved in the mobilization of ECM-bound bFGF.

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