Faculty Bibliography

The latent transforming growth factor-beta-binding protein-1 (LTBP-1) belongs to a family of extracellular glycoproteins that includes three additional isoforms (LTBP-2, -3, and -4) and the matrix proteins fibrillin-1 and -2. Originally described as a TGF-beta-masking protein, LTBP-1 is involved both in the sequestration of latent TGF-beta in the extracellular matrix and the regulation of its activation in the extracellular environment. Whereas the expression of LTBP-1 has been analyzed in normal and malignant cells and rodent and human tissues, little is known about LTBP-1 in embryonic development. To address this question, we used murine embryonic stem (ES) cells to analyze the appearance and role of LTBP-1 during ES cell differentiation. In vitro, ES cells aggregate to form embryoid bodies (EBs), which differentiate into multiple cell lineages. We analyzed LTBP-1 gene expression and LTBP-1 fiber appearance with respect to the emergence and distribution of cell types in differentiating EBs. LTBP-1 expression increased during the first 12 d in culture, appeared to remain constant between d 12 and 24, and declined thereafter. By immunostaining, fibrillar LTBP-1 was observed in those regions of the culture containing endothelial, smooth muscle, and epithelial cells. We found that inclusion of a polyclonal antibody to LTBP-1 during EB differentiation suppressed the expression of the endothelial specific genes ICAM-2 and von Willebrand factor and delayed the organization of differentiated endothelial cells into cord-like structures within the growing EBs. The same effect was observed when cultures were treated with either antibodies to TGF-beta or the latency associated peptide, which neutralize TGF-beta. Conversely, the organization of endothelial cells was enhanced by incubation with TGF-beta 1. These results suggest that during differentiation of ES cells LTBP-1 facilitates endothelial cell organization via a TGF-beta-dependent mechanism

Covalent association of LTBP-1 (latent TGF-beta binding protein-1) to latent TGF-beta is mediated by the third eight-cysteine (also referred to as TB) module of LTBP-1, a domain designated as CR3. Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3, and we show here that another lepidopteran cell line, Trichoplusia niTN-5B1-4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3. CR3 contains an N-glycosylation site, which is conserved in all forms of LTBP known to date. When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells. Sf9 cells mainly processed the fucosylated paucomannosidic structure (GlcNAc)(2)(Mannose)(3)Fucose, although hybrid and complex N-glycosylations were also detected. In High-Five cells, the peptide was found to be modified with a wide variety of hybrid and complex sugars in addition to paucomanosidic oligosaccharides. Most glycans had one or two fucose residues bound through alpha1,3 and alpha1,6 linkages to the innermost GlcNAc. On the basis of these results and on the structure of an eight-cysteine domain from fibrillin-1, we present a model of glycosylated CR3 and discuss the role of glycosylation in eight-cysteine domain protein-protein interactions

The post-translational methylation of the N-terminally extended or high molecular weight (HMW) forms of fibroblast growth factor-2 (FGF-2) has been shown to affect the nuclear accumulation of the growth factor. In this study, we determined the extent and position of methyl groups in HMW FGF-2. Using mass spectrometry and amino acid sequence analysis, we have shown that the 22- and 22.5-kDa forms of HMW FGF-2 contain five dimethylated arginines located at positions -22, -24, -26, -36, and -38 using the methionine residue normally used to initiate the 18-kDa form as position 0. The 24-kDa form of HMW FGF-2 contains seven to eight dimethylated arginines located at positions -48, -50, and -52, in addition to positions -22, -24, -26, -36, and -38. In vitro methylation reactions demonstrate that the N-terminal extension of HMW FGF-2 acts as a specific substrate for yeast Hmt1p and human HRMT1L2 arginine methyltransferases. These findings indicate that HMW FGF-2, with the presence of five or more dimethylated Gly-Arg-Gly repeats, contains an RGG box-like domain, which may be important for protein-protein and/or protein-RNA interactions

The single-copy fibroblast growth factor 2 (FGF-2) gene encodes four coexpressed isoforms of different molecular masses. The 18-kDa FGF-2 is primarily localized in the cytoplasm, whereas the higher molecular mass isoforms (HMW FGF-2) localize to the nucleus and nucleolus. The overexpression of either 18-kDa FGF-2 or HMW FGF-2 promotes cell transformation in a dose-dependent manner. In NIH 3T3 cells, the selective overexpression of HMW FGF-2 but not of 18-kDa FGF-2 confers upon the cells the unique phenotype of growth in low serum-containing medium. Thus, the distinct intracellular localization and the level of expression of FGF-2 are pivotal requirements for the differential effects of FGF-2 isoforms on the cellular phenotype. On this basis, we established a doxycycline-regulatable FGF-2 expression system that permitted us to regulate the expression of each isoform in a time- and dose-dependent manner. We analyzed the growth properties of cells in the presence and absence of doxycycline in both normal and low serum-containing medium and in soft agar. The doxycycline-activated expression of 18-kDa FGF-2 did not allow growth in low serum medium. The growth of cells expressing HMW FGF-2 was increased by doxycycline under all three conditions, and a relationship between the level of HMW FGF-2 expression and cell growth was observed for all three conditions. This doxycycline-regulatable FGF-2 expression system provides a mechanism to analyze changes in FGF-2 targeted pathways and genes and to characterize pathways specifically activated by either the 18-kDa FGF-2 or the HMW FGF-2 isoforms.

The glycosylphosphatidylinositol (GPI)-anchored, multifunctional receptor for the serine proteinase, urokinase plasminogen activator (uPAR, CD87), regulates plasminogen activation and cell migration, adhesion, and proliferation. uPAR occurs in functionally distinct, membrane-anchored and soluble isoforms (s-uPAR) in vitro and in vivo. Recent evidence indicates that s-uPAR present in the circulation of cancer patients correlates with tumor malignancy and represents a valuable prognostic marker in certain types of cancer. We have therefore analyzed the mechanism of uPAR shedding in vitro. We present evidence that uPAR is actively released from ovarian cancer cells since the rate of receptor shedding did not correlate with uPAR expression. While s-uPAR was derived from the cell surface, it lacked the hydrophobic portion of the GPI moiety indicating anchor cleavage. We show that uPAR release is catalyzed by cellular GPI-specific phospholipase D (GPI-PLD), an enzyme cleaving the GPI anchor of the receptor. Thus, recombinant GPI-PLD expression increased receptor release up to fourfold. Conversely, a 40% reduction in GPI-PLD activity by GPI-PLD antisense mRNA expression inhibited uPAR release by more than 60%. We found that GPI-PLD also regulated uPAR expression, possibly by releasing a GPI-anchored growth factor. Our data suggest that cellular GPI-PLD might be involved in the generation of circulating prognostic markers in cancer and possibly regulate the function of GPI-anchored proteins by generating functionally distinct, soluble counterparts

Human basic fibroblast growth factor (FGF-2) occurs in four isoforms: a low molecular weight (LMW FGF-2, 18 kDa) and three high molecular weight (HMW FGF-2, 22, 22.5, and 24 kDa) forms. LMW FGF-2 is primarily cytoplasmic and functions in an autocrine manner, whereas HMW FGF-2s are nuclear and exert activities through an intracrine, perhaps nuclear, pathway. Selective overexpression of HMW FGF-2 forms in fibroblasts promotes growth in low serum, whereas overexpression of LMW FGF-2 does not. The HMW FGF-2 forms have two functional domains: an amino-terminal extension and a common 18-kDa amino acid sequence. To investigate the role of these regions in the intracrine signaling of HMW FGF-2, we produced stable transfectants of NIH 3T3 fibroblasts overexpressing either individual HMW FGF-2 forms or artificially nuclear-targeted LMW FGF-2. All of these forms of FGF-2 localize to the nucleus/nucleolus and induce growth in low serum. The nuclear forms of FGF-2 trigger a mitogenic stimulus under serum starvation conditions and do not specifically protect the cells from apoptosis. These data indicate the existence of a specific role for nuclear FGF-2 and suggest that LMW FGF-2 represents the biological messenger in both the autocrine/paracrine and intracrine FGF-2 pathways