Giovanni Ragnotti

Endogenous interleukin 1 alpha must be transported to the nucleus to exert its activity in human endothelial cells.

We have previously shown that the signal peptideless cytokine interleukin 1 alpha (IL-1 alpha) 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 249:1570-1574, 1990). To investigate the potential intracellular function of IL-1 alpha, transformed endothelial cells were transfected with the human cDNAs that code for the two forms of IL-1 alpha, the precursor molecule IL-1(1-271) and the mature protein IL-1(113-271). 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-1 alpha gene and the beta-galactosidase open reading frames. The IL-1(113-271) protein was cytoplasmic, while IL-1(1-271) was nuclear. The basic cluster at the NH2 terminus of IL-1, KVLKKRR, has been shown to mediate IL-1 alpha nuclear targeting. Moreover, nuclear localization of IL-1 alpha correlates with impaired cell growth and expression of some IL-1 alpha-inducible genes. These results suggest that transport of endogenous IL-1(1-271) into the nucleus is required for it to modulate endothelial cell function.

A six-amino acid deletion in basic fibroblast growth factor dissociates its mitogenic activity from its plasminogen activator-inducing capacity.

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.

Nuclear localization of endogenous basic fibroblast growth factor in cultured endothelial cells

Indirect immunofluorescence using anti-human placental bFGF antibodies demonstrates the presence of bFGF-like reactivity in the cytoplasm and in the nucleus of adult bovine aortic endothelial cells and of normal and transformed fetal bovine aortic endothelial AG 7680 and GM 7372 cells. Biologically active immunoreactive Mr 18,000 bFGF can be isolated by heparin-Sepharose affinity chromatography from the extract of GM 7372 cell nuclei. Quantitation of bFGF content by biological and immunological methods indicates that 100,000 bFGF molecules per nucleus are present in GM 7372 cells, with nuclear bFGF corresponding to 25-30% of total cellular bFGF. Immunoprecipitation experiments demonstrate that the nuclear localization of newly synthesized bFGF occurs when GM 7372 cells are biosynthetically labeled both in the absence and in the presence of suramin, a molecule that inhibits the binding of bFGF to its plasma membrane receptor. Thus the data indicate that endogenous bFGF undergoes an intracellular sorting to the nucleus of the endothelial cell.

Purification of basic fibroblast growth factor from rat brain: Identification of a Mr 22,000 immunoreactive form

A 18,000-dalton protein which stimulates plasminogen activator (PA) activity in endothelial GM 7373 cells has been purified from rat brain by using heparin affinity chromatography and ion-exchange chromatography. The purified molecule stimulates PA activity in a dose-dependent manner between 1 and 30 ng/ml. It also stimulates proliferation of GM 7373 cells and DNA synthesis in NIH 3T3 cells in a similar concentration range. The molecule has been identified as a bFGF-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with anti-human bFGF antibodies. In the final preparation of the rat brain bFGF, trace amounts (less than 5%) of a contaminant were detectable. This contaminant has a molecular weight of 22,000 and cross reacts with several anti-human placental bFGF antibodies. On the basis of its affinity for heparin-Sepharose and its immunological characteristics, this protein appears to be an high molecular weight form of bFGF.

Basic fibroblast growth factor in ovulatory cycle and postmenopausal human endometrium.

Biopsies of human endometrium were studied for the presence of basic fibroblast growth factor (bFGF). An immunoreactive Mr 18,000 bFGF-like molecule was detected at high levels both in ovulatory cycle and postmenopausal endometrium. This molecule was identified as bFGF on the basis of its molecular weight, its affinity for heparin, its capacity to induce plasminogen activator production and cell proliferation in endothelial GM 7373 cells, and its cross-reactivity with various anti-bFGF antibodies. The levels of endometrial bFGF do not change during the menstrual cycle but they increase significantly after menopause, as evaluated both by biological and immunological assays. Lower levels of an acidic FGF-like activity were also evident in ovulatory cycle endometrium but, at variance with bFGF, no significant increase of this activity was observed in postmenopausal endometrium. These data represent the first characterization of a polypeptide growth factor present in human endometrium.

Characterization of a Mr 25,000 basic fibroblast growth factor form in adult, regenerating, and fetal rat liver

A heparin-binding Mr 25,000 immunoreactive bFGF-like protein (ir-bFGF) is recognized in adult rat liver extract by affinity-purified polyclonal anti-human placental bFGF antibodies. Hepatic levels of this protein increase 4-fold in regenerating rat liver during the first 48 h after partial hepatectomy. Also, they appear to be higher in embryonic than in newborn or in adult rat liver. Mr 25,000 ir-bFGF from regenerating rat liver, partially purified by heparin-affinity chromatography, induces plasminogen activator activity and cell proliferation in transformed fetal bovine aortic endothelial GM 7373 cells and competes with Mr 18,000 [125I]bFGF for the binding to high affinity bFGF receptors. The data indicate the presence in rat liver of a high molecular weight form of bFGF whose expression is modulated during embryonic development and liver regeneration.

High molecular weight immunoreactive basic fibroblast growth factor-like proteins in rat pituitary and brain

Four proteins immunologically related to basic fibroblast growth factor (bFGF) have been detected by Western blot analysis in the extract from rat anterior pituitary. Their apparent molecular weights are 29, 27, 18, and 14 kDa, respectively. A similar immunoreactive pattern has been observed in the rat tumor pituitary GH3 cell line. In the extracts from rat neurohypophysis, hypothalamus, hippocampus, striatum, olfactory tubercles, cerebellum, and cortex only the 29 kDa form is detectable in a significant amount.

Structural and functional changes in polysomes from ischemic livers

Abstract Some aspects of the state of aggregation and functioning of polysomes have been investigated in preparations obtained from rat liver lobes subjected to ischemia. The incroporation of amino acid into protein is reduced in postmitochondrial supernatant fractions, microsomes, and C-ribosomes from ischemic livers. In the presence of cell sap from ischemic livers, normal C-ribosomes, but not normal microsomes, show a reduced incorporating capacity, which is not affected by a fivefold increase of the amount of cell sap in the incubation mixture. The analysis of the polysomal patterns shows that ischemia causes a progressive disaggregation of polyribosomes, with a reduction in the number of heavy aggregates and an increase of dimers and monomers. This disaggregation is best seen in the ribosomes recovered from the fraction bound to the membranes of the endoplasmic reticulum. The determination of RNA -used as a reference basis of the various subcellular fractions—shows that ribosomes are also reduced in number in preparations from ischemic livers. Bound ribosomes are more affected than the free forms. On the whole, ribosomes obtained from ischemic livers seem to be reduced in number, less aggregated, and less efficient in carrying out protein synthesis.

State and function of liver polysomes during recovery from ischemia

Abstract The reestablishment of blood supply to the liver lobes which have suffered ischemia is followed by an increase in protein synthesis only if ischemia did not last longer than 60 minutes. The increase is accompanied by the recovery of a normal polyribosomal pattern. Treatment of the rats with actinomycin D does not prevent this restoration. In the early phase of the reestablishment of circulation there is a sudden formation of monomers. These monosomes respond well to polyuridylic acid (poly-U) in vitro . The monosome formation can be prevented by treatment of the animal with cycloheximide. Treatment with cycloheximide also prevents the disappearance of the polysomal shoulder during ischemia. The results support the hypothesis that the lowering of the polysomal shoulder and the formation of monosomes are not the effect of ribonuclease action, but the consequence of an interference with recycling of ribosomes on mRNA. The recovery of a normal polysomal pattern after ischemia does not depend on the synthesis of new mRNA.

Decrease of the activity of the mixed function oxidase system in regenerating rat liver: An alternative explanation

Abstract Partial hepatectomy and sham-operation reduce to a similar extent the activity of two enzymes of rat liver mixed function oxidase system, aniline hydroxylase and aminopyrine demethylase. On this basis it is proposed that the decreased functional specialization of regenerating liver depends on events associated to the stress of intra-abdominal surgery and not, as previously hypotized, to cellular multiplication. This view is confirmed by the fact that regenerating liver retains its ability to respond to phenobarbitone administration with an increased activity of the two enzymes.