The phenotypes of NIH 3T3 cells transfected with basic fibroblast growth factor (bFGF) cDNAs that express only the high molecular weight (HMW) forms of bFGF, the 1 8-kDa form, or all forms were examined. Cells producing the 18 kDa or all forms of bFGF were transformed at high levels of growth factor expression but were nontransformed at low levels. Cell producing low levels of HMW forms of bFGF were growth impaired when compared with the parental cells. These cells tended to form multinucleated giant cells, did not grow in soft agar, were nontumorigenic, had a normal bFGF receptor number, and had a nontransformed morphology. Cells expressing high levels of HMW bFGFs had a transformed morphology and were tumorigenic. These data suggest a specific functional role for HMWbFGF.

We have cloned a murine cDNA encoding a tyrosine kinase receptor with about 90% similarity to the chicken fibroblast growth factor (FGF) receptor and the humanfms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a 15-to 20-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two growth factors of the FGF family.The mitogenic effect ofgrowth factors requires binding to and activating specific cell surface receptors, many ofwhich have tyrosine kinase activity (1,2 done/0.02% Ficoll/0.02% bovine serum albumin.) The filters were then washed at 650C with 2x SSC/0.1% SDS for 30 min and with lx SSC/0.1% SDS for 45 min. The cDNAs were subcloned into pBluescript (Stratagene) and sequenced by the dideoxynucleotide chain-termination method (9).Cell Lines and Transfections. CHO-DG44 cells, which are dihydrofolate reductase-negative (DHFR-), were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum, 5 mM hypoxanthine, 2 mM thymidine, and 10 mM proline. Cells were transfected by calcium phosphate precipitation with cDNA 4A in p91023B vector (10), which carried DHFR as a selectable marker. Positive clones were selected in medium lacking hypoxanthine and thymidine.Scatchard Analysis. Recombinant bFGF was iodinated as described (11) (specific activity = 552 cpm/fmol). CHO clone 3-3 and clone 4-1 cells were incubated at 40C with DMEM containing 0.15% gelatin, 25 mM Hepes (pH 7.4), and various concentrations of '2"I-labeled bFGF from 0.3 to 7 ng/ml.After 2 hr, the medium was removed, the cells were washed once with ice-cold isotonic phosphate-buffered saline (PBS), and 1251I-labeled bFGF bound to the extracellular matrix was removed by washing twice with 2 M NaCl/20 mM Hepes, pH 7.4 (11). Radioactive bFGF bound to high-affinity receptors was released with two subsequent washes with 2 M NaCl/20 mM sodium acetate, pH 4.0. Radioactivity in the combined medium and PBS wash (free fraction) and in the combined 2 M NaCl washes buffered at pH 4 (bound fraction) was quantitated on a Beckman y scintillation counter. The cells of parallel cultures were treated with trypsin and quantitated using a Coulter particle counter.Competition Assays. To investigate the ability of K-FGF to displace 1251-labeled bFGF, CHO cells were incub...

Human PREP1, a novel homeodomain protein of the TALE super-family, forms a stable DNA-binding complex with PBX proteins in solution, a ternary complex with PBX and HOXB1 on DNA, and is able to act as a co-activator in the transcription of PBX-HOXB1 activated promoters (Berthelsen, J., Zappavigna, V., Ferretti, E., Mavilio, F., Blasi, F. , 1998b. The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperatity. EMBO J. 17, 1434-1445; Berthelsen, J., Zappavigna, V., Mavilio, F., Blasi, F., 1998c. Prep1, a novel functional partner of Pbx proteins. EMBO J. 17, 1423-1433). Here we demonstrate the presence of DNA-binding PREP1-PBX complexes also in murine cells. In vivo, PREP1 is a predominant partner of PBX proteins in various murine tissues. However, the choice of PBX family member associated with PREP1 is largely tissue-type specific. We report the cloning and expression domain of murine Prep1 gene. Murine PREP1 shares 100% identity with human PREP1 in the homeodomain and 95% similarity throughout the whole protein. In the adult mouse, PREP1 is expressed ubiquitously, with peaks in testis and thymus. We further demonstrate the presence of murine Prep1 mRNA and protein, and of different DNA-binding PREP1-PBX complexes, in mouse embryos from at least 9.5 days p.c. Moreover, we show that PREP1 is present in all embryonic tissues from at least 7.5-17.5 days p.c with a predominantly nuclear staining. PREP1 is able to super-activate the PBX-HOXB-1 autoregulated Hoxb-1 promoter, and we show that all three proteins, PREP1, PBX and HOXB-1, are present together in the mouse rhombomere 4 domain in vivo, compatible with a role of PREP1 as a regulator of PBX and HOXB-1 proteins activity during development.

During the process of embryo implantation, trophoblast cells invade deep into uterine stroma and play a key role in establishing fetomaternal exchange of molecules. We have studied the in vivo expression patterns of the molecules of the urokinase system, during the process of mouse embryo implantation and early placentation. The sites of synthesis of urokinase-type plasminogen activator (uPA), uPA-receptor (uPAR), plasminogen activator inhibitor type 1 (PAI-1) and alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) transcripts were determined by in situ hybridization. These genes were found to be expressed in a finely regulated pattern. High levels of uPA mRNA were found in invasive trophoblast cells, while the same cells did not appear to synthesize PAI-1. Starting from day 6.5, endothelial cells of newly forming vessels also transcribed uPA gene. uPAR and alpha 2MR/LRP were in all stages expressed by decidual tissue, and their expression domains overlapped in large areas. Immunohistochemistry with uPA and PAI-1 antibodies revealed areas of co-localization of these secreted proteins with the expression domains of uPAR and alpha 2MR/LRP, which is of great interest in view of the role of these two receptors in clearing uPA-PAI-1 complexes. In situ zymography demonstrated the presence of active uPA in the ectoplacental cone region at 7.5 and 8.5 days. Our studies outline the expression of a set of functionally related genes that is well coordinated between fetal and maternal tissues. This coordination may model other physiological and pathological invasive processes.