ABSTRACT
Perlecan, a widespread heparan sulfate proteoglycan, functions as a bioactive reservoir for growth factors by stabilizing them against misfolding or proteolysis. These factors, chiefly members of the fibroblast growth factor (FGF) gene family, are coupled to the N-terminal heparan sulfate chains, which augment high affinity binding and receptor activation. However, rather little is known about biological partners of the protein core. The major goal of this study was to identify novel proteins that interact with the protein core of perlecan. Using the yeast two-hybrid system and domain III of perlecan as bait, we screened approximately 0.5 10(6) cDNA clones from a keratinocyte library and identified a strongly interactive clone. This cDNA corresponded to FGF-binding protein (FGF-BP), a secreted protein previously shown to bind acidic and basic FGF and to modulate their activities. Using a panel of deletion mutants, FGF-BP binding was localized to the second EGF repeat of domain III, a region very close to the binding site for FGF7. FGF-BP could be coimmunoprecipitated with an antibody against perlecan and bound in solution to recombinant domain III-alkaline phosphatase fusion protein. Immunohistochemical analyses revealed colocalization of FGF-BP and perlecan in the pericellular stroma of various squamous cell carcinomas suggesting a potential in vivo interaction. Thus, FGF-BP should be considered a novel biological ligand for perlecan, an interaction that could influence cancer growth and tissue remodeling.
Subject(s)
Carrier Proteins/metabolism , Heparan Sulfate Proteoglycans/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Carcinoma, Squamous Cell/metabolism , Carrier Proteins/biosynthesis , Carrier Proteins/chemistry , Carrier Proteins/genetics , DNA, Complementary/metabolism , Fibroblast Growth Factor 2/metabolism , Gene Library , Heparan Sulfate Proteoglycans/chemistry , Humans , Immunohistochemistry , Intercellular Signaling Peptides and Proteins , Models, Biological , Models, Genetic , Molecular Sequence Data , Plasmids/metabolism , Polymerase Chain Reaction , Precipitin Tests , Protein Binding , Protein Biosynthesis , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Transcription, Genetic , Transfection , Two-Hybrid System Techniques , Up-RegulationABSTRACT
Previous studies have demonstrated that 1,25-dihydroxyvitamin D3 (calcitriol) is a potent inhibitor of human T lymphocyte proliferation. It has been reported that only CD4+ cells are sensitive to the anti-proliferative action of calcitriol. To further evaluate this observation, we first performed cell cycle analysis of unfractionated phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMC) in the absence or presence of calcitriol. The CD4/CD8 ratio was similar between control and treated cells for each phase of cycle (G0----G1, S, G2 + M), suggesting that calcitriol did not selectively block proliferation of either T cell subpopulation. Secondly, the growth-inhibitory activity of calcitriol on PBMC selectively depleted of either CD4+ or CD8+ cells was comparable to that observed with unfractionated PBMC. Furthermore, the induction of transferrin receptors was inhibited by calcitriol to a comparable degree in each T cell subset, suggesting that equivalent inhibition of transition into late G1 was observed. Finally, calcitriol inhibited the proliferation of highly purified T cell subsets (greater than 99% pure) to equivalent degrees. These data suggest that T cell subsets defined by either the CD4 or by the CD8 antigen are both sensitive to the growth inhibitory effects of calcitriol.
