Fen Wang

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.

Forced expression of hepatocyte-specific fibroblast growth factor 21 delays initiation of chemically induced hepatocarcinogenesis.

Inappropriate fibroblast growth factor (FGF) signaling is involved in most tissue‐specific pathologies including cancer. Previously we showed that inappropriate expression and chronic activity of FGF receptor (FGFR) 1 in hepatocytes accelerated diethylnitrosamine (DEN)‐initiated hepatocarcinogenesis. Here we showed that although widely expressed FGF1 and FGF2 are frequently upregulated in hepatocellular carcinoma (HCC), germline deletion of both FGF1 and FGF2 had no effect on DEN‐initiated hepatocarcinogenesis. Thus overexpression of FGF1 or FGF2 may be a consequence rather than contributor to hepatoma progression. FGF21 is the first of 22 homologues whose expression has been reported to be preferentially in the liver. We showed that similar to FGF1 and FGF2, FGF21 mRNA was upregulated in neoplastic and regenerating liver after partial hepatectomy (PH) and CCl4 administration. In situ hybridization analysis confirmed that in contrast to FGF1 and FGF2, expression of FGF21 mRNA was limited to hepatocytes. Forced overexpression of FGF21 in hepatocytes by gene targeting had no apparent impact on normal liver development and compensatory response to injury. Surprisingly, overexpression of FGF21 delayed the appearance of DEN‐induced liver tumors. At 8 and 10 mo, only 10% and 30% of transgenic mice, respectively, developed adenomas compared to 50% (all adenomas) and 80% (60% adenoma/20% HCC) in the wild‐type (WT) mice. However, the incidence and burden of HCC at 10 mo and later was equal in the FGF21 transgenic and WT mice. We propose that FGF21 may delay development of adenomas through activation of resident hepatocyte FGFR4 at early times, but counteracts the delay by acceleration of progression to HCC through interaction with ectopic FGFR1 once it appears in hepatoma cells. This indicates a dual function of FGF21 that may reflect changes in FGFR isotype during progression of differentiated hepatoma cells.

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.

CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE, FIFTH EDITION

This book is the fifth edition of Culture of Animal Cells and has a style similar to previous editions but with significant changes in the contents. The book introduces the basic concept and basic techniques of cell cuhure, as well as a list of basic reagents and equipment for general cell culture technology. The book is very useful for new researchers who want to learn tissue culturing because it describes many easy-to-follow methods for researchers seeking to use cell culture techniques in their laboratories, ranging from where to purchase reagents to how to set up a tissue culture room and operate basic tissue culture instruments. The book is also handy for veteran researchers who already have a lot of cell culture experience because the book provides a list of basic recipes of the most commonly used culture media and reagents, which are hard to memorize, even for knowledgeable researchers. I find that the book is particularly useful for tissue culture educators because of the newly added Chapter 2, Training Programs, which is a teaching manual for training new staff or students. It adds experimental and analytical elements to some of the protocols to make the learning experience more interesting, more informative, and a little more challenging. In addition, Culture of Animal Cells also describes how to culture specific cell types, tumor cells, and organ cultures, as well as a wide range of commonly used techniques related to cell cultures, including confocal microscopy, in situ molecular hybridization, somatic cell fusion, and the production of monoclonal antibodies. Overall, Culture of Animal Cells: A Manual of Basic Technique is a good and user-friendly resource. This book is easy to read with many tables and figures. The Appendix is a useful tool for locating related references. Laboratories conducting cell cultures or regular tissue cultures will find this book a useful complement to their library and a handy reference book for routing cell culture protocols.

Directional specificity of prostate stromal to epithelial cell communication via FGF7/FGFR2 is set by cell- and FGFR2 isoform-specific heparan sulfate

Dear Editor: Communication between epithelial and stromal compartments of parenchymal organs is mediated, in part, by members of the fibroblast growth factor (FGF) family in which different isotypes of the FGF signal and the FGF tyrosine kinase receptor are partitioned between compartments (McKeehan et al., 1998). Similar to other parenchymal organs with both epithelial and stromal compartments, prostate epithelial cells express the Rib splice variant of the type 2 FGF receptor (FGFR2), whereas stromal cells express the IIlc variant as well as FGFRIIIIc, which is not expressed in the epithelial cells (Yan et al., 1993; Feng et al., 1997). Expression of FGF7 and FGF10 within the current family of 23 FGF homologs occurs exclusively in the stromal ceils (Yan et al., 1992; Lu et al., 1999). This partitioning of FGF7/FGF10 and FGFR2 isoforms is thought to underlie directionally specific signaling from stromal to epithelial cells. Epithelial cells respond only to FGF7/FGF10, but not to FGF2, whereas stromal ceils do not respond to FGF7/FGFIO, but respond to FGF2 (Yan et al., 1992; Lu et al., 1999). Recent reports suggest that the specificity of the FGFR isofo~cns for the FGF isoforms is mammalian cell-context dependent (Kan et al., 1999; Uematsu et al., 2000). In liver cells, FGFR4 recognizes specifically FGF1 whereas cell-free recombinant FGFR4 recognizes FGF1 and FGF2 equally. Conversely, FGFR4 recognizes only FGF2 in endothelial cells similar to the resident FGFR1 (Kan et al., 1999). The apparent loss of specificity of cell-free FGFR4 was traced to the use of heparin instead of cell-derived heparan sulfate in binary complexes with FGFR4 used to assess the binding of FGF1 and FGF2 to cell-free FGFR4 (Kan et al., 1999). Cell-type specificity for FGF1 or FGF2 was restored to the cell-free FGFR4 by substitution of the heparin with heparan sulfate from liver or endothelial cells, respectively (Kan et al., 1999). More surprising was the demonstration that cell-free binary complexes of heparin and FGFR2IIIb and FGFR2IIIc failed to exhibit selectivity for FGF7 or FGF2, respectively, in marked contrast to their profiles in epithelial and stromal cells (Uematsu et al., 2000). Here we show that, similar to FGFR4, the cell type selectivity of FGFR2IIIb and FGFR2IIIc for FGF is dependent on the cell membrane context and is determined by cell type-specific heparan sulfate. Constructs coding for the two Ig module 13 isoforms of the ectodomains, transmembrane domains and part of the intracellular juxtamembrane domains of FGFR2IIIb, FGFR2IIIc, and FGFRIIIIc fused to glutathione-S-transferase on the intracellular domain, were expressed into the membranes of baculoviral-infected Sf9 insect cells as described (Uematsu et al., 2000). After extraction with detergent and immobilization on Glutathione (GSH)-agarose beads, binary complexes were prepared with heparin or cell-derived heparan sulfate, and the binding of radiolabeled FGF1, FGF2, and FGF7 was compared (Uematsu et al., 2000). Active cellular heparan sulfate proteoglycan (HSPG) was harvested from 40 plastic 75-cm 2 culture flasks of subcontinent Dunning R3327PAP tumor prostate epithelial cells (DTE) or stroreal cells (DTS) by trypsin treatment as previously described (Kan et al., 1999). The HSPGs were partially purified by high-performance liquid chromatography (HPLC) using ion exchange (TSKDEAE-5 PW, 75 • 7.5 mm; Bio-Rad, Richmond, CA), followed by molecular fltration (Bio-Sil SEC-400, 300 • 7.8 ram; BioRad), and traced and qnantitated as described (Kan et al., 1999; McKeehan et al., 1999; Wu et al., 2001). The indicated isoforms of immobilized FGFR were incubated with HSPG preparations from either DTE or DTS cultures to allow the FGFR to affinity select FGFR-specific fractions of HSPG by formation of a binary complex prior to introduction of radiolabeled FGF. Affinity purification of the DTE cell HSPG that interacts with Ig module II of FGFR1 to homogeneity, with respect to core protein sequence, indicates that structural domains within the ectodomain of FGFR interact with a rare species of cellular HSPG with respect to the heparan sulfate chain (Wu et al., 2001). Moreover, the presence of soluble,free hep in or HSPG in binding assays can have dramatic effect on the apparent specificity of FGF binding to FGFR by formation of heparin-FGF complexes that cannot interact with FGFR (Uematsu et al., 2000). Therefore, care was taken to remove unbound heparin or HSPG by washing the binary complexes extensively prior to introduction of radiolabeled FGF1, FGF2, or FGF7. High affinity binding to the binary complexes was assessed by covalent affinity crosslinking of the FGF to the FGFR part of the complex and autoradiography of radiolabeled complexes (Fig. 1). As demonstrated previously (Uematsu et al., 2000), cell-free binary complexes of heparin (H) and FGFR2IIIb or FGFR2IIIc exhibited little specificity for FGF1, FGF2, or FGF7, whereas hepar in-FGFRlIIIc complexes failed to bind FGF7 (Fig. 1). Both HSPGs derived from DTS and DTE cells supported binding of FGF1 to FGFR2IIIb, the resident isoform in epithelial cells. In contrast, HSPG from both DTS and DTE cells failed to support the binding of FGF2 to FGFR2IIIb. The HSPG from both cell types supported the binding of FGF7 to FGFR2IIIb. This suggests that the specificity of epithelial cells for FGF7, relative to FGF2, lies in FGFR2IIIb-specific HSPG that is not cell-specific. FGFR2IIIb is expressed ectopically in inesenchymal cells in two rare cases of Aperts syndrome which is thought to result from the abnormal autocrine activity resulting from activation by FGF7 or FGFIO in the stromal cells (Oldridge et al., 1999). Our results with DTS cells

Cell- and receptor isotype-specific phosphorylation of SNT1 by fibroblast growth factor receptor tyrosine kinases.

A partnership between the ectodomain of the fibroblast growth factor receptor (FGFR) isotypes and the chains of pericellular matrix heparan sulfate determines the fibroblast growth factor (FGF) and cell-type specificitives of the FGFR signaling complex. The contribution of the FGFR intracellular tyrosine kinase domains to the specificity of FGFR signaling is unclear. This report shows that the quantiy and quality of phosphorylation of the FGFR kinase substrate SNT1 (also called FGFR substrate 2, FRS2) is both FGFR isotype and cell-type specific in prostate tumor epithelial cells at different stages of malignancy. Epithelial cell-resident FGFR2 that promotes homeostasis yields a low level of phosphorylated 65-kDa SNT1. Phosphorylation by ectopic FGFR1 that promotes malignancy was much more intense and yielded a phosphorylated 85-kDaSNT1. The amount of the 85-kDa SNT1 increased by 20-fold during proliferative aging of FGFR1-expressing cell populations that is required for FGFR1-stimulated mitogenesis and the malignant phenotype. In addition, the receptor-specific differential phosphorylation of SNT1 by FGFR isotypes, both of which are normally anchored to the cell membrane, occurred only in intact cells. Therefore, similar to kinase subunits within the heparan sulfate-FGFR complex, cell membrane and cytoskeletal context likely determine FGFR isotype- and cell-type-specific conformational relationships between FGFR kinases and external substrates. This determines the quantity and quality of SNT1 phosphorylation and differential signaling.

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.

CANCER CELL CULTURE—METHODS AND PROTOCOLS

Since the publication of the first human cancer cell line (Hela cells) about five decades ago, cell culture has become a widely used technology. Our present understanding of the cell and molecular biology of cancers has been derived mainly from the use of cultured cancer cells. Thousands of ceil lines representing a wide spectrum of human cancers have been developed so far. The book, Cancer Cell Culture--Methods and Protocols, introduces the basic concept of cancer cell culture, as well as general cell culture technology, and describes many easy to follow methods for researchers seeking to use cell culture techniques in their laboratories. The book also describes in detail how to characterize and authenticate widely used cell lines by deoxyribonucleic acid fingerprint and cytogenetic methods, how to isolate and develop cell lines from cancers from different tissue origins, and how to eoculture different cell types. In addition, Cancer Cell Culture--Methods and Protocols also introduces how to prevent and recover cell lines from contamination, including bacteria and yeast, and how to detect and eliminate mycoplasma infection. A wide range of procedures encompassing many of the key functional features of cancer cells are also described in the book, including assays to evaluate elonogenicity, cell proliferation, apoptosis, adhesion, migration, invasion, senescence, angiogenesis, and cell cycle parameters, and methods to modify cancer cells, including protocols for transfection, development of drug resistance, immortalization, and establishing xenografts using Matrigels. Overall, Cancer Cell Culture Methods and Protocols is a good and user-friendly resource. Laboratories conducting cancer cell culture or regular tissue cultures will find this book a useful complement to their library and a handy reference book for routing cell culture protocols. Beginners in cell cultures will find this book easy to follow and a source for many needed basic cell culture techniques.