Journal of Histochemistry and Cytochemistry, Vol. 45, 1005-1020, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Differential Expression of the Fibroblast Growth Factor Receptor (FGFR) Multigene Family in Normal Human Adult Tissues

Siân E. Hughesa
a Division of Histopathology, United Medical and Dental Schools, St Thomas's Campus, London, United Kingdom

Correspondence to: Siân E. Hughes, Div. of Histopathology, UMDS, St Thomas’s Campus, Lambeth Palace Road, London SE1 7EH, UK.


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

This report describes a systematic analysis of the expression of the fibroblast growth factor receptor (FGFR) multigene family (FGFR1, FGFR2, FGFR3, and FGFR4) in archival serial sections of normal human adult tissues representing the major organ systems, using immunohistochemical techniques. Polyclonal antisera specific for FGFR1, FGFR2, FGFR3, and FGFR4 and a three-stage immunoperoxidase technique were employed to determine the cellular distribution of these receptors at the protein level. The expression profiles for the tissue-specific cellular localization of the FGFR multigene family demonstrated widespread and striking differential patterns of expression of individual receptors in the epithelia and mesenchyme of multiple tissues (stomach, salivary glands, pancreas, thymus, ureter, and cornea) and co-expression of FGFR1-4 in the same cell types of other tissues. The widespread expression of FGFR1-4 in multiple organ systems suggests an important functional role in normal tissue homeostasis. Differences in the spatial patterns of FGFR gene expression may generate functional diversity in response to FGF-1 and FGF-2, both of which bind with equally high affinity to more than one receptor subtype. In vivo, this may lead to functional differences that are crucial for the regulation of normal physiological processes and are responsible for the pathological mechanisms that orchestrate various disease processes. (J Histochem Cytochem 45:1005-1019, 1997)

Key Words: immunolocalization, human tissues, fibroblast growth factor receptor, differential expression


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The fibroblast growth factors (FGFs) constitute a family of at least nine structurally related heparin-binding polypeptide mitogens which can induce diverse cellular responses in multiple biological systems. The best-characterized family members are the prototypes FGF-1 (Jaye et al. 1986 ) and FGF-2 (Abraham et al. 1986 ), formerly known as acidic FGF and basic FGF, respectively. Their regulatory role during embryological development and in physiological processes, such as angiogenesis, wound healing, and tissue repair, has been the focus of considerable interest in recent years. Moreover, it is emerging that aberrant FGF activity is a feature of certain forms of neoplasia and other pathological conditions including Alzheimer's disease, Duchenne muscular dystrophy, diabetic retinopathy, and atherosclerosis (Baird and Bohlen 1990 ). FGF-1 and FGF-2 are widely expressed in normal adult tissues (Hughes and Hall 1993a ), whereas the other family members exhibit restricted patterns of distribution and are found predominantly in embryonic and tumor tissues (Basilico and Moscatelli 1992 ).

The FGFs mediate their biological effects by binding to high-affinity cell-surface receptors with protein tyrosine kinase activity. Four receptors have been identified in the human. These include FGFR1 (Dionne et al. 1990 ), FGFR2 (Dionne et al. 1990 ), FGFR3 gene (Partanen et al. 1990 ; Keegan et al. 1991a ), and FGFR4 gene (Partanen et al. 1990 ). These receptors share common structural features and consist of an extracellular ligand-binding domain containing three Ig-like loops and a unique acidic region, a trans-membrane domain, and the cytoplasmic region, which contains the tyrosine kinase catalytic domain and kinase insert. The FGFRs belong to Subclass IV of the receptor tyrosine kinase family of proteins. Receptor tyrosine kinases are critical to normal cell growth and differentiation, and overexpression is associated with malignant transformation (Schlessinger and Ullrich 1992 ).

FGF-FGFR interactions are extremely complex. Individual receptors show specific patterns of expression in adult tissues and during development (Partanen et al. 1991 ; Luqmani et al. 1992 ), and each receptor type has the capacity to bind multiple FGF ligands with similar affinity (Dionne et al. 1990 ; Keegan et al. 1991b ; Vainikka et al. 1992 ). The ligand-receptor interaction is further complicated by the discovery that cell-surface and extracellular matrix heparan sulfate proteoglycans are essential for binding of FGF-1 and FGF-2 to their cognate receptors (Givol and Yayon 1992 ). Ligand binding causes the FGFRs to dimerize and activate specific intracellular signaling pathways (Bellot et al. 1991 ). The extreme diversity of (and in some instances opposing) cell responses induced by FGFs appears in part to be due to the existence of multiple receptor isoforms of FGFR1 and FGFR2 (Dionne et al. 1990 ; Champion-Arnaud et al. 1991 ; Crumley et al. 1991 ; Eisemann et al. 1991 ; Hou et al. 1991 ; Miki et al. 1992 ). These receptor isoforms are the result of alternative mRNA splicing and internal polyadenylation that is specific for each tissue or cell type. Despite a limited number of genes encoding FGFRs, transcriptional controls can generate many receptor proteins with structural permutations involving the extracellular, juxtamembrane, and cytoplasmic tyrosine kinase catalytic domains (Givol and Yayon 1992 ). Thus, the potential repertoire of FGF-mediated intracellular signaling events is significantly increased. Furthermore, evidence suggests that FGFR1 and FGFR2 isoforms have different biological functions. For example, splice variants that bind distinct ligands (Champion-Arnaud et al. 1991 ; Miki et al. 1992 ) or with altered catalytic activity (Shi et al. 1993 ) have been identified, as well as intracellullar receptor isoforms that may provide intracrine growth loops (Yan et al. 1992 ). Thus, tissue-specific alternative mRNA splicing permits cells expressing a single FGFR gene to significantly diversify their biological response by generating distinct receptor isoforms that may exhibit differences in ligand specificitiy and function. Such exquisite regulation of growth factor receptor activity is likely to be essential for the regulation of complex physiological processes. Subtle variations in the cell- and tissue-specific patterns of FGFR gene expression may be pivotal to complex physiological processes, such as angiogenesis and development, as well as to the pathological mechanisms driving multiple disease processes.

Despite major progress in the characterization of the FGFR multigene family, there are limited data regarding the tissue-specific cell distribution of individual receptors in normal human adult tissues at the protein level. In the currently available reports that have specifically addressed this issue in human and murine tissues, techniques such as reverse transcriptase polymerase chain reaction (RT-PCR) (Luqmani et al. 1992 ), RNAse protection assays (Bernard et al. 1991 ; Templeton and Hauschka 1992 ; Werner et al. 1992 ), and Northern blotting (Holtrich et al. 1991 ; Korhonen et al. 1991 ; Partanen et al. 1991 ; Katoh et al. 1992 ) have been employed. Despite the superior sensitivity of the former techniques over Northern analysis, an inherent drawback is their failure to demonstrate the specific cellular localization of FGFRs. Furthermore, in the majority of these studies FGFR expression was examined in only a small number of tissues and was restricted to the analysis of FGFR1 and FGFR2 mRNA transcripts.

More extensive in situ hybridization analyses specifically evaluating the tissue-specific cell distribution of FGFR mRNA transcripts in a wider range of tissues have been confined largely to the mouse (Kornbluth et al. 1988 ; Reid et al. 1990 ; Safran et al. 1990 ; Orr-Urtreger et al. 1991 ; Peters et al. 1992 ; Stark et al. 1991 ), rat (Wanaka et al. 1991 ), and chicken embryos (Patstone et al. 1993 ). These studies have revealed distinct cell type-specific spatial and temporal patterns of receptor expression during development, consistent with a functional role for the FGF family of growth factors as morphogens during embryogenesis. Throughout the developmental period there is marked variability in the spatial and temporal patterns of FGF ligand and receptor expression. Moreover, expression of FGFRs during fetal development and embryogenesis may not necessarily correlate with adult patterns of expression. For example, expression of the FGF proto-oncogenes FGF-3 (int-2), FGF-5, and FGF-4 (hst/Kaposi FGF) is seen throughout many developmental stages but, in general, these growth factors are not expressed in the adult (Jakobovits et al. 1986 ; Yoshida et al. 1988 ; Wilkinson et al. 1989 ; Hebert et al. 1990 ; Haub and Goldfarb 1991 ). Similarly, significant differences have been reported in the patterns of FGFR localization between adult and fetal tissues, notably in the adrenal gland (Korhonen et al. 1991 ), brain (Wanaka et al. 1990 ; Wanaka et al. 1991 ), and heart (Orr-Urtreger et al. 1991 ; Peters et al. 1992 ; Engelmann et al. 1993 ). Therefore, the aim of this study was to characterize the distribution of the FGFR family members in serial sections of normal adult human tissues from the major organ systems, using immunohistochemical techniques. Analyses of the patterns of FGFR gene expression are essential if further insights are to be gained into the putative functional role of these highly complex growth factor and receptor multigene families under normal physiological conditions in vivo, and to direct future experimental strategies.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Tissue Preparation
Formalin-fixed, wax-embedded normal human tissues representing the major organ systems were selected from the surgical diagnostic files of the Department of Histopathology, St Thomas's Campus, UMDS, London. A series of fresh normal human tissues (tonsil, skin, colon, skeletal muscle) was obtained at surgical resection and snap-frozen in liquid nitrogen-cooled isopentane. These tissues were embedded in OCT before storage at -70C. Serial sections from frozen and wax-embedded tissues were cut at 5 µm and 4 µm, respectively, and mounted on silane-coated slides (Sigma; Poole, UK) before use in immunohistochemistry. In addition, the effects of different fixatives were evaluated in the panel of fresh frozen tissues. Cryostat sections were fixed in either ice-cold acetone, acetone:methanol (1:1), methanol, or formalin for 10 minutes before immunostaining.

Cell Culture
The following cell lines were grown in 150-mm dishes for the preparation of protein lysates for immunoblotting experiments: human stomach cancer cells (Kato III), human chronic myeloid leukemia cells (K-562), human umbilical vein endothelial cells (ECV304), FGFR1-transfected (L631) and parental (L6V) rat skeletal muscle myoblasts, FGFR4-transfected (F4) and parental (Neo) murine NIH3T3 fibroblasts. Kato III, L6V, and L631 skeletal muscle myoblasts, Neo and F4 NIH3T3 cells were grown in DMEM supplemented with 10% fetal calf serum (FCS) in 10% CO2/90% air. ECV304 and K-562 cells were grown in Medium 199 and RPMI supplemented with 10% FCS, respectively. ECV304 cells were maintained in 5% CO2/95% air and K-562 cells in 10% CO2/90% air at 37C. Media and FCS were purchased from Gibco (Paisley, Renfrewshire, UK). K-562 cells, FGFR1 and FGFR4 overexpressing cell lines, and Kato III cells were kindly provided by Dr. J. Knight, Department of Histopathology, St Thomas's Campus, UMDS, London; Dr. N. Lemoine, ICRF Oncology Group, Hammersmith Hospital, London; and Dr. Andrew Stubbs, Department of Gastroenterology, Guy's Campus, UMDS, London, respectively. ECV304 cells were purchased from the European Collection of Animal Cell Cultures (ECACC; Salisbury, Wiltshire, UK) No. 92091712.

Antibodies
Rabbit polyclonal antibodies raised against synthetic residues 808-822 of human flg gene product/FGFR1 (Dionne et al. 1990 ), residues 805-821 of human bek gene product/FGFR2 (Dionne et al. 1990 ; Crumley et al. 1991 ), residues 792-806 of human FGFR3, and residues 789-802 of human FGFR4 (NBS Biologicals; Hatfield, Herts, UK) were used in this study. These synthetic peptide antigens correspond to amino acid residues within the divergent carboxy terminus tail of the FGFR family members. This region is one of the least conserved between individual FGFRs (Partanen et al. 1991 ). Identical and similar synthetic peptide antigens directed against this region have been used successfully by other investigators to generate FGFR1-4 antibodies that are both highly specific and capable of distinguishing between individual receptors, despite the high overall structural homology of this multigene family (Dionne et al. 1990 ; Crumley et al. 1991 ; Keegan et al. 1991b ; Vainikka et al. 1992 ).

Western Blotting
To confirm the specificities of the anti-FGFR1 and anti-FGFR4 antibodies, immunoblot analyses using protein lysates prepared from FGFR1 and FGFR4 overexpressing cell lines were carried out. For the anti-FGFR3 and anti-FGFR2 antisera, protein lysates from the chronic myeloid leukemic cell line K-562 (Keegan et al. 1991a ) and Kato III cells (Hattori et al. 1990 ; Katoh et al. 1992 ) as well as ECV304 cells (Takahashi et al. 1990 ) were utilized, respectively. Protein samples were boiled for 5 min in EDTA sample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% ß-mercaptoethanol, 2 mM EDTA), then electrophoresed on 8% SDS-PAGE gels with rainbow molecular weight markers as standards (Amersham International; Amersham, Bucks, UK). Proteins were transferred to 0.45-µm nitrocellulose membranes (Schleicher & Schuell; Dassell, Germany). Nitrocellulose blots were blocked with 0.05% Tween-20/3% normal goat serum (Vector Laboratories; Bretton, Peterborough, UK) containing 3% bovine serum albumin (BSA) (Sigma) in Tris-buffered saline (50 mM Tris, pH 7.5, 150 mM NaCl) before incubation with the relevant antisera. Further control experiments were performed by probing duplicate blots with the corresponding anti-human FGFR antibodies. Blots were washed in 0.05% Tween-20 in Tris-buffered saline (TBS) and incubated with alkaline phosphatase-conjugated goat anti-rabbit antisera (1:1000) (Bio-Rad; Hemel Hempstead, Herts, UK) for 1 hr at 22C. After further washing of the blot in 0.05% Tween-20/TBS, antibody-reactive bands were visualized using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrate solution (Sigma).

Immunocytochemistry
Immunostaining was performed using a sensitive three-layer avidin-biotin complex (ABC) method with the rabbit IgG Vectastain Elite ABC (peroxidase) kit as outlined by the manufacturer (Vector Laboratories). Before application of normal goat serum, fixed and unfixed frozen sections were immersed in PBS, pH 7.4. Similarly, paraffin sections were dewaxed in xylene and hydrated through graded alcohols to water, then subsequently placed in PBS. To optimize immunostaining with the anti-human FGFR2 antiserum, enzymatic predigestion of paraffin sections with 0.01% protease XXIV (Sigma) was carried out for 15, 10, and 5 min at 37C, respectively. Optimal digestion of tissue sections was obtained with 0.01% protease for 10 min at 37C. Proteolytic digestion of tissue sections for the other FGFR antisera was found in earlier experiments not to improve immunostaining. Primary anti-FGFR1-4 antisera were diluted in PBS/0.3% BSA and used at predetermined optimal dilutions of 1:700 (anti-FGFR1), 1:200 (anti-FGFR2), 1:50 (anti-FGFR3), and 1:100 (anti-FGFR4). After overnight incubation at 4C, sections were rinsed in PBS and incubated for 1 hr at 22C with a biotinylated goat anti-rabbit antibody. Endogenous peroxidase was quenched by incubation in 0.3% hydrogen peroxide in methanol for 30 min; sections were rinsed in PBS and incubated for 30 min with the ABC solution. After further washes in PBS, the reaction product was visualized using diaminobenzidine (Sigma) as chromogen. Sections were counterstained with Harris's hematoxylin, dehydrated in graded alcohols, cleared in xylene, and mounted.

Controls
Control immunocytochemical experiments were performed by substitution of the primary antibody with normal rabbit serum at the same concentration as that of the respective primary antisera or by preincubation of the primary anti-FGFR1-4 antiserum with a molar excess of the corresponding synthetic peptide antigen (NBS Biologicals) used for immunization. Because of the high overall structural homology and the similar molecular weights of the FGFR family members, which limits Western analysis, additional cross-blocking peptide antigen neutralization experiments were performed. In these experiments, the respective primary anti-FGFR1-4 antisera were preincubated with a molar excess of either the corresponding or reciprocal peptide synthetic antigens in serial sections from tissues known to preferentially express individual receptors.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Specificity of Antibodies
The specificities of the the anti-human FGFR1-4 antibodies were confirmed by 8% SDS-PAGE using protein lysates prepared from cell lines known to preferentially express individual receptors and cell lines transfected with human FGFR1 and FGFR4 cDNA constructs. The FGFR1 antiserum recognized protein bands of 130 and 150 kD in lysates from rat L6 skeletal myoblasts overexpressing FGFR1 protein (Figure 1). Likewise, in lysates from FGFR4-transfected murine NIH3T3 fibroblasts, the anti-FGFR4 antibody recognized protein bands of 110 and 95 kD (Figure 2). The FGFR2 and FGFR3 antisera recognized protein bands of 135 kD and 115 kD in Kato III cell lysates and of 135, 125, and 97 kD in K-562 cell lysates, respectively (data not shown). These antibody-reactive bands were not observed in the nontransfected parental cell lines or in duplicate blots probed with irrelevant FGFR antisera or nonimmune normal rabbit serum (Hughes et al. 1994 ). The molecular weights obtained were in accordance with those previously reported for individual FGFRs (Dionne et al. 1990 ; Partanen et al. 1990 ; Crumley et al. 1991 ; Keegan et al. 1991b ; Keegan et al. 1991c ). Further confirmation of antibody specificity was provided by the lack of immunostaining seen in immunocytochemical controls, where positivity was abolished by prior incubation of the primary antisera with peptide antigen, and by the preservation of staining when the primary antisera were incubated with irrelevant synthetic peptide antigens (Hughes et al. 1994 ).



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Figure 1. Eight percent SDS-PAGE of human FGFR1 cDNA-transfected L631 rat skeletal muscle myoblast and parental L6V cell lysates, probed with the anti-human FGFR1 antibody. Lane 1, L631 cells; Lane 2, L6V cells. Antibody-reactive bands were observed at 130 kD and 150 kD (Lane 1). No antibody-reactive bands were seen in the parental L6V cell line (Lane 2) or in control experiments, where duplicate blots were probed with either normal rabbit serum or irrelevant FGFR antisera (data not shown). Molecular weight standards (kD) are indicated at left.

Figure 2. Eight percent SDS-PAGE of human FGFR4 cDNA-transfected F4 murine fibroblast and parental Neo cell lysates, probed with the anti-human FGFR4 antibody. Lane 1, F4 cells; Lane 2, Neo cells. Antibody-reactive bands were observed at 110 kD and 95 kD (Lane 1). These antibody-reactive bands were not seen in the parental Neo cell line (Lane 2) or in control experiments (data not shown). An additional protein band of 88 kD, probably representing endogenous FGFR4 protein, was also detected in both F4 and Neo cells. Molecular weight standards (kD) are indicated at right.

Moreover, other investigators have used identical synthetic peptide antigens to those employed in this study to generate specific polyclonal antisera to FGFR1 and FGFR2 (Dionne et al. 1990 ; Crumley et al. 1991 ; Engelmann et al. 1993 ). These antisera have been extensively characterized by immunoprecipitation and Western blot analyses and are specific for FGFR1 and FGFR2, respectively (Dionne et al. 1990 ; Crumley et al. 1991 ). A similar peptide antigen directed against the 16 amino acids of the carboxy terminal tail of FGFR4 has been characterized by Vainikka et al. 1992 and was shown to specifically recognize FGFR4 protein. This peptide immunogen contains two additional amino acid residues but is otherwise identical to the the immunogen used to generate the anti-FGFR4 antiserum in this study. Similarly, Keegan et al. 1991b raised specific polyclonal FGFR3 antisera using a trpE fusion protein incorporating residues 577-806 of the second kinase domain and carboxy terminal tail region of FGFR3. These data confirm the specificity and lack of crossreactivity of anti-FGFR1-4 antisera raised against synthetic peptide antigens corresponding to amino acid residues in the carboxy terminal tail region of these highly homologous molecules.

Tissue Distribution
All four receptors were found to have distinct spatial patterns of distribution in many tissues, and the results in human tissues are summarized in Table 1. The most widespread expression was observed for FGFR1 and FGFR2. High levels of immunoreactive FGFR1 were seen in the skin, cornea, lung, heart, placenta, kidney, and ureter, and moderate levels in testis and ovary. Abundant FGFR2 expression was found in the prostate and stomach. In contrast, no expression was seen in the pancreas, ovary, cornea, and placenta. FGFR3 and FGFR4 exhibited more restricted patterns of tissue distribution. Marked FGFR3 positivity was seen in the appendix, colon, liver, sublingual gland, placenta, and cervix, although the overall intensity of immunostaining for FGFR3 was found to be much lower in the majority of tissues and associated vasculature compared with that of FGFR1, FGFR2, and FGFR4. Tissues exhibiting minimal or no FGFR3 expression included the stomach, duodenum, ileum, kidney, ureter, and ovary. Prominent expression of FGFR4 was observed in the liver, sublingual gland ducts, kidney, and ureter, as well as in the media of some (but not all) arterioles and veins in most tissues. A far higher proportion of tissues lacked immunoreactive FGFR4 compared with the proportion of tissues showing nonreactivity for the other receptors. FGFR4 has been shown to bind FGF-1 with higher affinity than FGF-2 (Partanen et al. 1991 ). In an earlier study, the less widespread tissue distribution of FGF-1 compared with that of FGF-2 was reported (Hughes and Hall 1993a ). It is possible that the generally lower levels of FGF-1 in most tissues may, in part, account for this difference.


 
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Table 1. Summary of results of immunohistochemical detection of FGFR1-4 in normal human adult tissuesa

The degree of immunoreactivity, reflecting differences in the amount of protein expressed at the cellular level, varied qualitatively from intense to moderate or low in many of the tissues examined. The various degrees of cellular positivity both within and between given tissues for the same and different FGFRs are in concordance with the findings of other investigators (Partanen et al. 1991 ; Luqmani et al. 1992 ; Werner et al. 1992 ), who have reported significant variation in the levels of FGFR1 mRNA transcripts and RT-PCR products in different tissues. Patterns of immunostaining observed in freshly fixed cryostat sections were concordant with those in wax-embedded, formalin-fixed material.

Skin
The anti-human FGFR1 antibody revealed widespread strong staining for this receptor in the epidermis and appendages of the skin and in the media of dermal arterioles, veins, and microvasculature. Similarly, with the anti-human FGFR3-4 antisera, the patterns of immunostaining in the vasculature were practically concordant with those obtained for FGFR1, the principal exceptions being the lack of expression of these receptors in the epidermis. FGFR2 was expressed in the epidermis and dermal fibroblasts.

Urinary System
Distinct differences in FGFR expression were also detected in the ureter. Prominent immunoreactivity for FGFR4 was seen in both the urothelium and muscularis of the ureter (Figure 3A). This was in sharp contrast to the intense urothelial expression of FGFR1 and lack of reactivity in the muscularis (Figure 3B). In kidney, the tubule epithelium showed variable levels of FGFR expression. The strongest expression was that for FGFR4 (Figure 4), with intermediate and low levels of immunoreactive FGFR1 and FGFR2, respectively.



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Figure 3. Immunostaining of ureter (A) with anti-human FGFR4 antiserum and (B) with anti-human FGFR1 antibody. Cytoplasmic positivity was seen for FGFR4 in the the urothelium and the muscularis. Similarly, intense FGFR1 positivity was seen in the urothelium but the muscularis was nonreactive. Bars = 50 µm.

Figure 4. Immunostaining of kidney with anti-human FGFR4 antiserum. Positivity for FGFR4 was seen in the cytoplasm of the tubule epithelium of the kidney. Glomeruli were nonreactive. Immunoperoxidase reaction counterstained with hematoxylin. Bar = 100 µm.

Figure 5. Immunostaining of breast with anti-human FGFR2 antibody. Widespread immunoreactivity for FGFR2 was seen in the cytoplasm of all cell types comprising breast tissue, including the microvasculature. Immunoperoxidase reaction counterstained with hematoxylin. Bar = 100 µm.

Female and Male Reproductive Tracts
In the oviduct and ovary, spatial differences in FGFR expression were observed. Widespread expression of FGFR2 was seen in the epithelia of the oviduct and throughout the tissue vasculature, but in the ovary there was little staining for this receptor. FGFR4 was not expressed in the oviduct, but focal immunoreactivity for FGFR1 and FGFR3 was seen in the epithelia and occasional blood vessel of this tissue. In the ovary there was widespread expression of FGFR1 in stromal fibroblasts. Perhaps the most unusual receptor distribution was seen in the cervix, where widespread FGFR1 positivity was found in the endocervical epithelia. In contrast, immunoreactive FGFR1 was confined to the stratum spinosum, with total sparing of the basal epithelial cell layer in the ectocervix. Widespread immunoreactive FGFR3 was seen in the ecto- and endocervix, stromal fibroblasts, and the tissue vasculature, in contrast to the lack of FGFR2 and FGFR4 positivity in the cervix. Similarly, wide expression of FGFR1 and FGFR3 was seen in placental chorionic villi, with no FGFR2 or FGFR4 expression at this site. High levels of FGFR2 were found in the ducts, stromal fibroblasts, and vasculature of the breast (Figure 5), with little FGFR1 and FGFR3 expression and total lack of FGFR4.

Conversely, tissues constituting the male reproductive tract showed only weak positivity for FGFRs. The only tissue to exhibit wide expression was the prostate gland. In this tissue, FGFR1 and FGFR2 expression was seen in prostate epithelium and the microvasculature. In the epididymis, testis, and vas deferens there was little FGFR expression, and this was essentially confined to the media of small blood vessels and the muscularis of the vas deferens.

Heart and Respiratory System
Expression of FGFR1 and FGFR4 was especially marked in cardiac myocytes, with barely detectable staining for the other receptors. In the respiratory system, the distribution of FGFR2 was more widespread than that of the other three receptors. Intense FGFR2 positivity was detected in the respiratory epithelium, chondrocytes, muscularis, and vasculature, although FGFR2 was absent from the alveoli. In contrast, FGFR1 immunoreactivity was seen in the basal layer of respiratory epithelial cells and in many microvessels, with weak staining of chondrocytes and monocytes. Overall, there was limited expression of FGFR3 and FGFR4 in respiratory tissues. However, immunoreactive FGFR4 was seen in the smooth muscle cells underlying the bronchi and bronchioles.

Endocrine Tissues
Compared with all other tissues analyzed, the most widespread and greatest degree of immunoreactivity for all four receptors was observed in endocrine tissues. Staining for FGFRs was also frequently observed in cells of the same type. Intense immunoreactive FGFR1 and FGFR2 were seen focally in discrete populations of basophils and acidophils located at the periphery of the pituitary gland (Figure 6A). In contrast, low levels of FGFR3 and no expression of FGFR4 were seen in these cells. In general, widespread expression of FGFR family members was seen in the parathyroid gland and adrenal cortex, although immunoreactive FGFR4 was absent from the former tissue. The steroidogenic cells of the adrenal cortex and the oxyphilic and chief cells of the parathyroid gland showed intense positivity for FGFR1 (Figure 6B), FGFR2, and FGFR3, as did the tissue vasculature. In the thyroid gland, FGFR2 was seen focally in follicular epithelial cells, and expression of FGFR1 and FGFR3 was confined to the vasculature.



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Figure 6. Immunostaining of (A) pituitary gland with anti-human FGFR2 antibody. Bar = 100 µm. (B) Parathyroid gland with anti-human FGFR1 antibody. There was intense focal expression of FGFR2 in the cytoplasm of pituicytes and marked cytoplasmic expression of FGFR1 in the parenchyma of the parathyroid gland. Adipocytes were nonreactive. Bar = 50 µm. Immunoperoxidase reaction counterstained with hematoxylin.

Figure 7. Immunostaining of pancreas with (A) anti-human FGFR3 antibody. Bar = 100 µm. (B) Anti-human FGFR2 antibody. Bar = 50 µm. A striking differential pattern of FGFR3 expression was seen in the endocrine pancreas. There was intense positivity for FGFR3 in the cytoplasm of the islets of Langerhans but not in glandular epithelium or ducts. All cell types comprising the pancreas were nonreactive for FGFR2. Immunoperoxidase reaction counterstained with hematoxylin.

Figure 8. Immunostaining of sublingual salivary gland with (A) anti-human FGFR3 antibody; (B) Anti-human FGFR1 antibody. Bars = 50 µm. Differential FGFR expression was seen in the sublingual gland, where immunoreactive FGFR1 was confined to the cytoplasm of ducts but not epithelial cells. Striking deviation from this pattern of immunoreactivity was observed with the anti-FGFR3 antiserum, whereby the ducts were nonreactive but marked positivity was found in the cytoplasm of glandular epithelial cells. Immunoperoxidase reaction counterstained with hematoxylin.

Gastrointestinal System
Immunostaining with the anti-human FGFR1-4 antibodies revealed an unusual pattern of staining in the gastrointestinal tract. FGFR1 was not observed in the epithelium or vasculature of the stomach, and extremely faint staining of the muscularis was seen. A similar lack of expression in this tissue was observed for FGFR3. In contrast, marked expression of FGFR4 was seen predominantly in the muscularis, and expression of FGFR2 was seen throughout the gastric epithelium and submucosal macro- and microvasculature, with marked expression in the muscularis mucosae and muscularis. In the duodenum, positivity for FGFR1 was seen in the epithelial cells at the tips of villi. The muscularis mucosae, muscularis, and vessels of the submucosa were essentially nonreactive. Foci of FGFR1-positive microvessels in association with the outer layer of the muscularis were seen. For FGFR4, moderate focal immunoreactivity was detected in the muscularis but there was total lack of expression in the muscularis mucosae, lamina propria, and mucosa. The media of a few submucosal arterioles and veins demonstrated FGFR4 immunoreactivity, as well as the occasional capillary. In contrast, the FGFR1-positive microvessels associated with the muscularis were nonreactive for this receptor despite moderate FGFR4 positivity in microvessels at other sites. There was little expression of FGFR3 in this tissue.

Patterns of FGFR3 and FGFR1 expression in both the ileum and appendix were remarkably similar, with positivity seen primarily in the epithelia at the tips of villi. FGFR4 expression was not observed in the appendix, and faint focal staining of the muscularis of the ileum was seen. In these tissues, FGFR2 expression was more widespread than that observed for the other receptors, and the entire mucosal epithelium and muscularis of the appendix exhibited marked positivity, as did the ileal submucosal vasculature and muscularis mucosae. High levels of FGFR1 were seen in the colon, where there was marked positivity throughout the epithelium lining colonic glands, fibroblasts of the lamina propria, muscularis mucosae, and vessels of the submucosa and microvessels. A similar pattern of expression was seen for FGFR3 and FGFR2. Notably, the epithelial cells at the crypt luminal surface were nonreactive for FGFR3. Little staining for FGFR4 was seen in this tissue, although focal areas of positive smooth muscle cells in the muscularis were noted.

A striking differential pattern of FGFR expression was observed in the pancreas. There was intense positivity for FGFR3 in the islets of Langerhans (Figure 7A). Small foci of FGFR4-positive cells were also seen in the islets, but there was total lack of FGFR2 (Figure 7B) and FGFR1 reactivity at this site. Similarly, in the vasculature of this organ, the media of small arterioles and veins showed variable levels of FGFR1 and FGFR3 expression. In contrast, FGFR4 was seen predominantly in the media of arterioles, with absence of staining in the media of adjacent veins. This unusual differential pattern of FGFR4 expression in the vasculature was not apparent in other tissues examined, in which medial smooth muscle cells of both veins and arterioles tended to show positivity.

In the liver, differential patterns of FGFR expression were observed. Marked levels of expression of FGFR1, FGFR3, and FGFR4 were seen in this tissue, with variable degrees of immunostaining for all three receptors in the hepatocytes and portal tract vasculature. In addition, expression of FGFR1, FGFR3, and FGFR4 was seen in bile duct epithelium. In contrast, minimal FGFR2 expression was observed in hepatocytes and bile duct epithelium, but there was strong staining of medial smooth muscle cells of the portal tract vasculature. There was total absence of staining for all four receptors in the fibroblasts of the portal tracts.

The submandibular and sublingual salivary glands provided further examples of distinct differences in the spatial patterns of FGFR expression. In the sublingual gland, high levels of FGFR3 expression were seen in the glandular epithelium and moderate levels in the tissue vasculature (Figure 8A). The ducts of the sublingual gland were nonreactive for FGFR3, but expression of FGFR1 (Figure 8B), FGFR2, and FGFR4 was seen at this site. There was marked deviation in the submandibular gland, where FGFR3 was detected primarily in the ducts but not in the glandular epithelium. For FGFR1 and FGFR2, the patterns of expression seen in the sublingual gland were essentially maintained in the submandibular gland. No expression of FGFR1-4 was seen in connective tissue fibroblasts of either type of salivary gland.

Lymphoid Tissues
In the thymus, intense FGFR1 positivity was seen in epithelial cells, Hassl's corpuscles, and the endothelium and smooth muscle cells of blood vessels. Expression of FGFR2, FGFR3, and FGFR4 was seen predominantly in the thymic vasculature. In the lymph node and tonsil, intense expression of FGFR1 and FGFR2 was noted in high endothelial venules and in the media of all arterioles and venules. FGFR3 and FGFR4 expression was not observed in the high endothelial venules, and FGFR3 was absent from vessels in the lymph node itself, whereas the media of small blood vessels in the lymph node showed marked FGFR4 positivity. Similarly, the media of the muscular artery included in this section showed moderate expression of FGFR4, but the medial smooth muscle cells of neighboring veins showed intense immunoreactivity for this receptor. The pattern of FGFR expression described in the vasculature of the lymph node and attached connective tissue is not unique. Highly variable and complex patterns of receptor expression in the vasculature were a common feature for most tissues examined in this study.

Other Tissues
In addition to reviewing the major organ systems, the cornea and sympathetic ganglia were also examined. The anti-human FGFR1-4 antisera revealed a widespread and subtle differential distribution of the FGFRs in the cornea. For example, immunoreactive FGFR3 was found throughout the cornea, and intense positivity was seen in the corneal epithelium, endothelium, Descemet's membrane, and fibroblasts of the substantia propria. In contrast, expression of FGFR1 and FGFR4 was confined to corneal epithelial and endothelial cells, with lack of immunoreactivity in fibroblasts. FGFR2 was entirely absent in this tissue. In sympathetic ganglia, intense immunoreactive FGFR1 and FGFR3 were detected in Schwann cells, but not FGFR4 or FGFR2. Heterogeneous expression of FGFR1, FGFR2, and FGFR3 was seen in the associated vasculature, but not FGFR4.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The present study describes the tissue-specific cell localization of the FGFR multigene family in a panel of normal human adult tissues using a sensitive immunohistochemical technique and specific polyclonal FGFR1-4 antisera. The results obtained show the widespread spatial distribution of FGFR1-4 in tissues from the major organ systems and striking differential FGFR expression in multiple tissues. These data show good correlation with previous immunolocalization studies documenting the wide expression of the ligands for these receptors, FGF-1 and FGF-2, in the same panel of tissues (Hughes and Hall 1993a ). In general, the results described herein are in concordance with other studies that have examined FGFR expression in human fetal and adult tissues (Korhonen et al. 1991 ; Partanen et al. 1991 ; Katoh et al. 1992 ; Luqmani et al. 1992 ).

Luqmani et al. 1992 performed extensive reverse transcriptase-polymerase chain reaction (RT-PCR) studies in a series of normal human adult tissues and detected FGFR1 and FGFR2 RT-PCR products in almost all tissues examined (breast, lung, spinal cord, adrenal, skin, ovary, heart, thyroid, ileum, colon, stomach). High levels of FGFR1 PCR products were seen in skin, ovary, and heart, with moderate or low levels in other tissues. Likewise, FGFR2 was detected in the same series of tissues, with high levels of this receptor in the stomach and thyroid, but no FGFR2 PCR products were detected in the heart. In agreement with the findings of Luqmani et al. 1992 , the present study also revealed the broad tissue expression of FGFR1, with marked FGFR1 staining in the heart and skin but only moderate levels in the ovary. Immunoreactive FGFR2 was also detected in the same tissues. In particular, prominent FGFR2 expression was found throughout the stomach, with lower levels at other sites (ileum, colon) in the gastrointestinal tract. The high levels of FGFR2 in the normal stomach may signify a role for this receptor in carcinoma of the stomach. FGFR2 is preferentially expressed in the human stomach cancer cell line (Kato III) (Hattori et al. 1990 ), from which the FGFR2 gene was initially isolated. In contrast to this and other reports (Kornbluth et al. 1988 ; Templeton and Hauschka 1992 ; Engelmann et al. 1993 ) that failed to detect FGFR2 mRNA transcripts in the human, rat, and mouse heart, low levels of FGFR2 protein were detected in myocardium. These results are in agreement with those of other investigators (Orr-Urtreger et al. 1991 ; Peters et al. 1992 ; Patstone et al. 1993 ) who have detected FGFR2 mRNA transcripts in the developing heart.

Partanen et al. 1991 reported the widespread and differential expression of FGFR1-4 in an extensive range of tissues from the 18- and 17-week human fetus, using Northern analysis. In agreement with this report, FGFR4 was detected in adult human adrenal, lung, kidney, intestine, pancreas, skeletal muscle, spleen, and liver, and overall the expression profiles for FGFR2, FGFR1, and FGFR3 show correlation with the reported tissue-specific expression of these receptors at the mRNA level. The principal differences in the expression profiles of FGFR1-4 between the work of Partanen and co-workers (1991) and the current study were seen in the heart and adrenal gland. Partanen et al. 1991 reported very low levels of FGFR1 and FGFR2 mRNA and a total lack of FGFR3 in the fetal adrenal. In contrast, moderate to low levels of expression of all three receptors were found in these tissues. In support of the present findings, Luqmani et al. 1992 detected FGFR1 and FGFR2 PCR products in the adult adrenal, and in a later study by the same group (Korhonen et al. 1991 ), moderate levels of FGFR3, FGFR2, and FGFR4 mRNA, but not FGFR1, were found in the fetal adrenal. In the adult adrenal, only FGFR2 was expressed, indicating temporal differences in patterns of growth factor receptor expression between adult and fetal human tissues (Korhonen et al. 1991 ).

Similarly, comparison of these results with patterns of FGFR expression in other species reveals further variation. For example, the extensive studies of Peters et al. 1992 and Orr-Urtreger et al. 1991 , documenting FGFR1 and FGFR2 expression in the mouse embryo, reveal the almost exclusive localization of FGFR2 and FGFR1 to embryonic epithelia and mesenchyme, respectively. In the current report and that of Patstone et al. 1993 , this distinct pattern of localization was less marked. FGFR1 and FGFR2 were detected in both epithelial and mesenchyme-derived tissues. For example, in this analysis, co-expression of FGFR1-4 was seen in kidney tubule epithelium and wide expression of FGFRs was observed in the smooth muscle (mesenchymal origin) underlying epithelia at many sites. Furthermore, differential expression of FGFR1-4 was seen in the smooth muscle component of the gastrointestinal, respiratory, and urinary systems. For example, marked expression of FGFR2 was observed in the muscularis of the stomach, duodenum, and appendix, with barely detectable levels in the muscularis of the ileum and colon. Similarly, FGFR4 was found in the muscularis of the ureter and stomach, with little or no expression in smooth muscle at other sites. Curiously, Patstone et al. 1993 , describe similar variability in the pattern of smooth muscle FGFR expression according to anatomic site.

There are many possible reasons for the conflicting reports in FGFR1-4 tissue distribution. Evidence suggests that temporal differences in receptor expression exist between adult and fetal tissues. Indeed, caution must be exercised in extrapolating results obtained in the fetus to those seen in the adult, in view of such differences. Species-specific variations may also occur. Technical variation may also be partly responsible. For example, the decreased sensitivity of Northern blotting may give rise to misleadingly low or lack of receptor expression. Similarly, a panoply of different molecular probes has been employed to detect FGFR gene expression. Furthermore, the cellular expression of FGFR mRNA transcripts does not necessarily correlate with the expression of protein at the cell surface (Armstrong et al. 1992 ).

The cell type-specific alternate processing of FGFR1 and FGFR2 mRNA transcripts by different tissues provides yet another potential explanation. Tissue- and cell type-specific variations in patterns of FGFR1 and FGFR2 splice variant expression are well-documented (Reid et al. 1990 ; Bernard et al. 1991 ; Champion-Arnaud et al. 1991 ; Eisemann et al. 1991 ; Katoh et al. 1992 ; Templeton and Hauschka 1992 ; Werner et al. 1992 ). Using RNAse protection analyses and probes directed against the three alternate FGFR1 exons (exons IIIa, IIIb, and IIIc) encoding secreted and trans-membrane FGFR1 splice variants differing in the second half of the third immunoglobulin-like domain of the extracellular ligand binding region, Werner et al. 1992 reported the differential expression of FGFR1 mRNA transcripts in a series of murine adult tissues. Furthermore, the expression of FGFR1 splice variants encoded by exon IIIa or exon IIIb was found to exhibit restricted patterns of distribution compared to those receptor isoforms encoded by exon IIIc. Splice variants encoded by exon IIIa were found in brain, skeletal muscle, and skin, but not in the stomach or liver. FGFR1 isoforms encoded by exon IIIb were detected predominantly in skin, with lower levels in brain, kidney, muscle, and placenta, and no expression in liver, spleen, and testis, whereas exon IIIc was ubiquitously expressed in all tissues under investigation (spleen, skin, brain, skeletal muscle, kidney) apart from the liver. Splice variants encoded by the former exons show more restricted patterns of expression compared to the expression profile of FGFR1 isoforms encoded by the latter exon. Similarly, marked tissue-specific differences between FGFR1 and FGFR2 splice variants in the mouse have been reported by Templeton and Hauschka 1992 . Clearly, the use of alternate exons may lead to striking differences in the patterns of FGFR expression in the same tissue.

Further evidence to reinforce this notion is provided by an earlier analysis of the tissue distribution of human FGFR1 utilizing a polyclonal antibody directed against the acidic region of the chicken basic FGF receptor (cek-1 gene product), the chicken homologue of human FGFR1 (Hughes and Hall 1993a ). In the latter study, FGFR1 was predominantly expressed in the microvasculature of multiple tissues and was detected in the basal epithelia of a restricted number of tissues (cervix, respiratory epithelium, tonsil, breast). This is in contrast to the more widespread distribution of FGFR1 described in the present report using polyclonal antisera directed against the receptor carboxy terminus region. Overall, the anti-human FGFR1 and FGFR2 antisera employed in the present analysis do not distinguish among many of the described FGFR1 and FGFR2 splice variants, although one would not expect these antisera to recognize the truncated secreted FGFR1 (Eisemann et al. 1991 ; Hou et al. 1991 ) and FGFR2 isoforms (Crumley et al. 1991 ) lacking the carboxy terminus region or FGFR2 splice variants with altered carboxy terminal tail sequences. The expression of these splice variants in human tissues has yet to be addressed.

FGFR1, FGFR2, and FGFR3 were widely expressed in many adult human tissues. These receptors bind FGF-1 and FGF-2 with high affinity (Dionne et al. 1990 ; Keegan et al. 1991b ). In comparison, FGFR4 was found to have a more limited distribution. Interestingly, FGFR4 preferentially binds FGF-1 and FGF-2 with significantly lower affinity (Partanen et al. 1991 ). It is possible that the generally lower levels of FGF-1 in many tissues may in part account for the relatively restricted distribution of this receptor. However, the relationship between FGFR4 expression and that of its preferred ligand, FGF-1, did not always show close correlation. A study evaluating the ligand-binding profiles of FGFR4 indicated that this receptor binds FGF-4 and FGF-6 (Vainikka et al. 1992 ). Furthermore, the proto-oncogenes FGF-3 (brain, testis), FGF-4 (normal stomach mucosa), and FGF-6 have also been detected in normal adult tissues, albeit at low levels (Yoshida et al. 1988 ; Wilkinson et al. 1989 ; DeLapeyriere et al. 1990 ), and FGF-7 (KGF), a specific epithelial mitogen, has been identified in adult skin, intestine, and kidney (Finch et al. 1989 ). In general, although the other FGF-related growth factors appear to be expressed primarily in embryonic and tumur tissues, these observations raise the possibility that other FGF ligands may potentially be expressed at these sites or suggest the existence of perhaps as yet unidentified novel FGF ligands. In the light of these and other data, this could potentially explain why such highly complex patterns of FGFR expression exist in adult tissues under normal conditions.

FGF-1 and FGF-2 and their cognate receptors have been implicated in a wide range of normal physiological processes in vivo and in various disease states, including certain forms of neoplasia, atherosclerosis, diabetic retinopathy, and neurodegenerative disorders (Hughes and Hall 1993b ). The co-localization of these ligands, coupled with the broad expression of FGFR1-4 in multiple adult tissues, suggests a role for these growth factors as participants in the maintenance of normal tissue homeostasis by paracrine and/or autocrine modulation of FGF-responsive cells. However, this interpretation is, in part, tempered by the controversy surrounding the in vivo release of FGF-1 and FGF-2, which lack signal peptides necessary for cellular export. It has emerged that continuous baseline FGF release occurs in cultured fibroblasts by as yet undefined mechanisms (Mignatti et al. 1992 ). In the adult, one speculates that a similar scenario may account for FGF release in healthy tissues. Such a mechanism of release is especially pertinent to those tissues composed predominantly of terminally differentiated cell populations, such as the heart. Continuous low-level release of FGF is compatible with observations that the tissue mRNA levels of FGF-1 and FGF-2 are very low (Abraham et al. 1986 ; Jaye et al. 1986 ), yet abundant FGF accumulates in basal laminae and the extracellular matrix of essentially quiescent cell populations in adult tissues (Cordon-Cardo et al. 1990 ).

In conclusion, the present results indicate that expression of the FGFRs is regulated spatially in a cell type- and tissue-specific manner in the adult. This phenomenon is a common theme for FGFR family members during the development of various species. The functional significance of these highly complex differential patterns of receptor expression in adult tissues under normal physiological conditions in vivo remains speculative, although such differences probably serve to create functional diversity. This interpretation is consistent with the multifunctional nature of FGF ligands that induce diverse cellular responses in multiple cell types (e.g., proliferation, differentiation, chemotaxis) and the overlapping ligand-binding specificities exhibited by FGFRs and their isoforms. At the cellular level, variations in FGFR expression may in turn lead to functional differences essential for the coordinate regulation of normal tissue homeostasis and the orchestration of complex processes, such as wound healing and tissue repair, that involve more than one cell type and demand a repertoire of biological responses from individual cells. Moreover, the aberrant or inappropriate expression of FGFRs in normal human adult tissues, coupled with abundant FGF-1 and FGF-2 in tissues of endodermal, neuroectodermal, and mesenchymal origin may play a critical role in the development and/or progression of a wide range of tumors. Indeed, there is growing evidence implicating the FGF-FGFR multigene families in the pathogenesis of carcinoma of the colon (New and Yeoman 1992 ), breast (Luqmani et al. 1992 ), prostate (Yan et al. 1993 ), kidney, bladder (Chodak et al. 1988 ; Barritault et al. 1991 ), malignant melanoma (Becker et al. 1992 ), meningioma, and malignant glioma (Takahashi et al. 1991 ), as well as in tumor neovascularisation (Brem et al. 1992 ).


  Acknowledgments

Supported by grants from the British Heart Foundation and the Special Trustees for St Thomas's Hospital.

Received for publication October 7, 1996; accepted February 6, 1997.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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