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'),

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A recombinant deletion mutant of the 155-amino acid form of human basic fibroblast growth factor (bFGF), lacking amino acid residues 27-32 (Lys-Asp-Pro-Lys-Arg-Leu), was expressed in Escherichia coli and purified to homogeneity by heparin-Sepharose affinity chromatography. When maintained in the presence of an equimolar concentration of soluble heparin, the bFGF mutant (M1-bFGF) is as potent as bFGF in stimulating cell proliferation in normal and transformed fetal bovine aortic endothelial cells, in adult bovine aortic endothelial cells, and in NIH 3T3 fibroblasts. However, under the same experimental conditions, M1-bFGF is at least 100 times less efficient than bFGF in stimulating plasminogen activator (PA) production in endothelial cells, as assayed by chromogenic PA assay, SDS/PAGE zymography, and Northern blot analysis of urokinase-type PA mRNA. In the presence of heparin, M1-bFGF binds to bFGF plasma membrane receptors present on endothelial cells in a manner undistinguishable from bFGF. It also induces the same tyrosine phosphorylation pattern when added to NIH 3T3 cells. The data suggest that the PA-inducing activity of bFGF may depend upon a functional domain that differs from those involved in the mitogenic activity of the growth factor and that the binding of bFGF to its plasma membrane receptor may not be sufficient to induce urokinase-type PA production in endothelial cells.