Wallace L. McKeehan

Specificity for Fibroblast Growth Factors Determined by Heparan Sulfate in a Binary Complex with the Receptor Kinase

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the FGF receptor kinase (FGFR) restricts FGF-independenttrans-phosphorylation and supports the binding of activating FGF to self-associated FGFR. Here we show that in contrast to heparin, cellular heparan sulfate forms a binary complex with FGFR that discriminates between FGF-1 and FGF-2. FGFR type 4 (FGFR4) in liver parenchymal cells binds only FGF-1, whereas FGFR1 binds FGF-1 and FGF-2 equally. Cell-free complexes of heparin and recombinant FGFR4 bound FGF-1 and FGF-2 equally. However, in contrast to FGFR1, when recombinant FGFR4 was expressed back in epithelial cells by transfection, it failed to bind FGF-2 unless heparan sulfate was depressed by chlorate or heparinase treatment. Isolated heparan sulfate proteoglycan (HSPG) from liver cells in cell-free complexes with FGFR4 restored the specificity for FGF-1 and supported the binding of both FGF-1 and FGF-2 when complexed with FGFR1. In contrast, FGF-2 bound equally well to complexes of both FGFR1 and FGFR4 formed with endothelial cell-derived HSPG, but the endothelial HSPG was deficient for the binding of FGF-1 to both FGFR complexes. These data suggest that a heparan sulfate subunit is a cell type- and FGFR-specific determinant of the selectivity of the FGFR signaling complex for FGF. In a physiological context, the heparan sulfate subunit may limit the redundancy among the current 18 FGF polypeptides for the 4 known FGFR.

Role of Fibroblast Growth Factor Type 1 and 2 in Carbon Tetrachloride-Induced Hepatic Injury and Fibrogenesis

Genomic ablation of hepatocyte-specific fibroblast growth factor receptor (FGFR)4 in mice revealed a role of FGF signaling in cholesterol and bile acid metabolism and hepatolobular restoration in response to injury without effect on liver development or hepatocyte proliferation. Although the potential role of all 23 FGF polypeptides in the liver is still unclear, the most widely studied prototypes, FGF1 and FGF2, are present and have been implicated in liver cell growth and function in vitro. To determine whether FGF1 and FGF2 play a role in response to injury and fibrosis, we examined the impact of both acute and chronic exposure to carbon tetrachloride (CCl(4)) in the livers of FGF1- and FGF2-deficient mice. After acute CCl(4) exposure, FGF1(-/-)FGF2(-/-) mice exhibited an accelerated release of serum alanine aminotransferase similar to FGFR4 deficiency, but no effect on overall hepatolobular restoration or bile acid metabolism. FGF1(-/-)FGF2(-/-) mice exhibited a normal increase in alpha-smooth muscle actin and desmin associated with activation and migration of hepatic stellate cells to damage, but a reduced level of hepatic stellate cell-derived matrix collagen alpha1(I) synthesis. Liver fibrosis resulting from chronic CCl(4) exposure was markedly decreased in the livers of FGF1/FGF2-deficient mice. These results suggest an agonist role for FGF1 and FGF2 in specifically insult-induced liver matrix deposition and hepatic fibrogenesis and a potential target for the prevention of hepatic fibrosis.

Increased Carbon Tetrachloride-Induced Liver Injury and Fibrosis in FGFR4-Deficient Mice

Carbon tetrachloride (CCl(4)) intoxification in rodents is a commonly used model of both acute and chronic liver injury. Recently, we showed that mice in which FGFR4 was ablated from the germline exhibited elevated cholesterol metabolism and bile acid synthesis coincident with unrepressed levels of cytochrome P450 7A (CYP7A), the rate-limiting enzyme in cholesterol disposal. Of the four fibroblast growth factor (FGF) receptor genes expressed in adult liver, FGFR4 is expressed specifically in mature hepatocytes. To determine whether FGFR4 plays a broader role in liver-specific metabolic functions, we examined the impact of both acute and chronic exposure to CCl(4) in FGFR4-deficient mice. Following acute CCl(4) exposure, the FGFR4-deficient mice exhibited accelerated liver injury, a significant increase in liver mass and delayed hepatolobular repair. Chronic CCl(4) exposure resulted in severe fibrosis in livers of FGFR4-deficient mice compared to normal mice. Analysis at both mRNA and protein levels indicated an 8-hour delay in FGFR4-deficient mice in the down-regulation of cytochrome P450 2E1 (CYP2E1) protein, the major enzyme whose products underlie CCl(4)-induced injury. These results show that hepatocyte FGFR4 protects against acute and chronic insult to the liver and prevents accompanying fibrosis. The results show that FGFR4 acts by promotion of processes that restore hepatolobular architecture rather than cellularity while limiting damage due to prolonged CYP2E1 activity.

Requirement for anticoagulant heparan sulfate in the fibroblast growth factor receptor complex

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the fibroblast growth factor (FGF) receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation between self-associated FGFR and determines specificity for and mediates binding of activating FGF. Here we show that only the fraction of commercial heparin or rat liver heparan sulfate which binds to immobilized antithrombin formed an FGF-binding binary complex with the ectodomain of the FGFR kinase. Conversely, only the fraction of heparin that binds to immobilized FGFR inhibited Factor Xa in the presence of antithrombin. Only the antithrombin-bound fraction of heparin competed with 3H-heparin bound to FGFR in absence of FGF, whereas both antithrombin-bound and unretained fractions competed with radiolabeled heparin bound independently to FGF-1 and FGF-2. The antithrombin-bound fraction of heparin was required to support the heparin-dependent stimulation of DNA synthesis of endothelial cells by FGF-1. The requirement for divalent cations and the antithrombin-binding motif distinguish the role of heparan sulfate as an integral subunit of the FGFR complex from the wider range of effects of heparan sulfates and homologues on FGF signaling through FGFR-independent interactions with FGF.

Lycopene and lutein inhibit proliferation in rat prostate carcinoma cells.

Abstract Consumption of lycopene, a carotenoid without provitamin A activity, has been associated with a lower risk of prostate and breast cancer. Lutein is another carotenoid that may be associated with a reduced risk of age-related macular degeneration, the leading cause of blindness in adults 65 years of age and older. Bioactive compounds such as lycopene and lutein, derived from natural plant sources, have been shown to act at low substrate levels through the action of intrinsic cytokines and growth factors and their receptors within tissues, particularly those of the fibroblast growth factor and transforming growth factor β families. The effects of grapefruit-derived and commercial lycopene and lutein preparations on androgen independent cultured malignant type II tumor cells [Dunning R3327AT3 or AT3 cells (androgen-responsive, slow-growing tumor cells with well developed epithelium and stroma)] were compared to their benign parent type I tumor epithelial cells (DTE). Results demonstrated that both lycopene, in an α -cyclodextrin water soluble carrier, and lutein inhibited malignant AT3 cells in a concentration and time-dependent manner. No such effect was observed when benign DTE cells were examined, demonstrating selective inhibition of extremely malignant AT3 prostate cancer cells relative to their benign parent. Lutein demonstrated a similar but slightly diminished response as lycopene. When cells were treated with cocktails of lycopene and lutein, no synergistic or additive effect occurred. These studies are consistent with epidemiological studies that show inverse relationships of these carotenoids with prostate cancer.

NOVEL COMPLEX INTEGRATING MITOCHONDRIA AND THE MICROTUBULAR CYTOSKELETON WITH CHROMOSOME REMODELING AND TUMOR SUPPRESSOR RASSF1 DEDUCED BY IN SILICO HOMOLOGY ANALYSIS, INTERACTION CLONING IN YEAST, AND COLOCALIZATION IN CULTURED CELLS

SummaryAvailability of the complete sequence of the human genome and sequence homology analysis has accelerated new protein discovery and clues to protein function. Protein-protein interaction cloning suggests multisubunit complexes and pathways. Here, we combine, these molecular approaches with cultured cell colocalization analysis to suggest a novel complex and a pathway that integrate the mitochondrial location and the microtubular cytoskeleton, with chromosome remodeling apoptosis, and tumor suppression based on a novel leucine-rich pentatricopeptide repeat-motif-containing protein (LRPPRC) that copurified with the fibroblast growth factor receptor complex. One round of interaction cloning and sequence homology analysis defined a primary LRPPRC complex with novel subunits cat eye syndrome chromosome region candidate 2 (CECR2), ubiquitously expressed transcript (UXT), and chromosome 19 open reading frames 5 (C19ORF5) but still of unknown function. Immuno, deoxyribonucleic acid (DNA), and green fluorescent protein (GFP) tag colocalization analyses revealed that LRPPRC appears in both cytosol and nuclei of cultured cells, colocalizes with mitochondria and β-tubulin rather than with α-actin in the cytosol of interphase cells, and exhibits phase-dependent organization around separating chromosomes in mitotic cells. GFP-tagged CECR2B was strictly nuclear and colocalized with condensed DNA in apoptotic cells. GFP-tagged UXT and GFP-tagged C19ORF5 appeared in both cytosol and nuclei and colocalized with LRPPRC and β-tubulin. Cells, exhibiting nuclear C19ORF5 were apoptotic. Screening for interactive substrates with the primary LRPPRC substrates in the human liver complementary DNA library revealed that CECR2B interacted with chromatin-associated TFIID-associated protein TAFII30 and ribonucleic acid splicing factor SRP40, UXT bridged to CBP/p300-binding factor CITED2 and kinetochore-associated factor BUB3, and C19ORF5 complexed with mitochondria-associated NADH dehydrogenase I and cytochrome c oxidase I. C19ORF5 also interacted with RASSF1, providing a bridge to apoptosis and tumor suppression.

Resident Hepatocyte Fibroblast Growth Factor Receptor 4 Limits Hepatocarcinogenesis

Fibroblast growth factor (FGF) family signaling mediates cell‐to‐cell communication in development and organ homeostasis in adults. Of the FGF receptor (FGFR) isotypes, FGFR4 is the sole resident isotype present in mature parenchymal hepatocytes. FGFR1 that is normally associated with activated nonparenchymal cells appears ectopically in hepatoma cells. Ectopic expression and chronic activity of FGFR1 in hepatocytes accelerates diethylnitrosamine (DEN)‐initiated hepatocarcinogenesis by driving unrestrained cell proliferation and tumor angiogenesis. Hepatocyte FGFR4 mediates livers role in systemic cholesterol/bile acid and lipid metabolism and affects proper hepatolobular restoration after damage without effect on cell proliferation. Here we ask whether FGFR4 plays a role in progression of hepatocellular carcinoma (HCC). We report that although spontaneous HCC was not detected in livers of FGFR4‐deficient mice, the ablation of FGFR4 accelerated DEN‐induced hepatocarcinogenesis. In contrast to FGFR1 that induced a strong mitogenic response and depressed rate of cell death in hepatoma cells, FGFR4 failed to induce a mitogenic response and increased the rate of cell death. FGFR1 but not FGFR4 induced cyclin D1 and repressed p27 expression. Analysis of activation of Erk, JNK, and PI3K‐related AKT signaling pathways indicated that in contrast to FGFR1, FGFR4 failed to sustain Erk activation and did not activate AKT. These differences may underlie the opposing effects of FGFR1 and FGFR4. These results suggest that in contrast to ectopic FGFR1 that is a strong promoter of hepatoma, resident FGFR4 that mediates differentiated hepatocyte metabolic functions also serves to suppress hepatoma progression.

Ligand binding properties of binary complexes of heparin and immunoglobulin-like modules of FGF receptor 2.

Epithelial cells, which express FGFR2IIIb, bind and respond to FGF-1, FGF-7 and FGF-10, but not FGF-2. Stromal cells, which bind and respond to FGF-1 and FGF-2, but not FGF-7 and FGF-10, express FGFR2IIIc or FGFR1IIIc. Here we show that when both isolated FGFR2betaIIIb and FGFR2betaIIIc or their common Ig module II are allowed to affinity select heparin from a mixture, the resultant binary complexes bound FGF-1, FGF-2, and FGF-7 with nearly equal affinity. In addition, FGF-2 and FGF-7 bound to both heparin-Ig module IIIb and IIIc complexes, but FGF-1 bound to neither Ig module III. The results show that in isolation both Ig modules II and III of FGFR2 can interact with heparin and that each exhibits a binding site for FGF. We suggest that the specificity of FGFR2IIIb and FGFR2IIIc is dependent on the cell membrane environment and heparin/heparan sulfate. Ig modules II and III cooperate both within monomers and across dimers with cellular heparan sulfates to confer cell type-dependent specificity of the FGFR complex for FGF.

Tissue culture: the unlimited potential

Lack of tissue-specific differentiated functions of cells in tissue culture, once thought to be due to “dedifferentiation”, was shown to be due to selective overgrowth of fibroblasts by a series of simple experiments that challenged the prevailing dogma. Following this insight, enrichment culture techniques (alternate animal and culture passage) were designed to give functionally differentiated tumor cells selective advantage over the fibroblasts. These experiments resulted in the derivation of a large number of functionally differentiated clonal strains of a range of cell types, providing the final point of destruction of the dogma of “dedifferentiation.” Instead, the hypothesis was proposed that cells in culture accurately represent cells in vivo, but without the complex in vivo environment. With the development of hormonally defined media and its combination with functionally differentiated clonal cell lines, this concept has been strengthened and the potential of tissue culture studies has been greatly augmented. Hormonally defined media allow the culture of cell types that cannot be grown in conventional, serum-supplemented media. These approaches demonstrate that hormonal responses and dependencies can be discovered in culture. Following this thinking and the discovery of hormonal dependencies of cancer cells has led to a new rationale for therapy. Tissue culture and cell technology continue to play an important role in solving human health problems.

CHAPTER 46 – The Fibroblast Growth Factor (FGF) Signaling Complex

The fibroblast growth factor (FGF) signaling system is a ubiquitous cellular sensor of local environmental changes and mediator of cell-to-cell communication with broad roles in development and organ homeostasis in the adult. Through the interaction of heparan sulfate (HS) with both activating FGF polypeptides and transmembrane FGF receptor (FGFR) tyrosine kinases, the system is rigorously modulated by tissue architecture. Diversity and cell and tissue specificity of signaling result from the combinatorial oligomerization of a family of 23 FGF homologs, diverse oligosaccharide motifs within HS chains of proteoglycans, and a plethora of ectodomains resulting from splice variations from four genes coding for four intracellular tyrosine kinases.