Fibroblast growth factor receptor 1 in the adrenal gland and PC12 cells: developmental expression and regulation by extrinsic molecules

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 36 (1996) 70-78

Research report

Fibroblast growth factor receptor 1 in the adrenal gland and PC12 cells: developmental expression and regulation by extrinsic molecules Christof Meisinger, Alexander Hertenstein, Claudia Grothe * Institute of Anatomy, Unicersi~ of Freiburg, Albertstr. 17, D-79104 Freiburg, Germany Accepted 19 September 1995

Abstract

In the present study we have analyzed the expression of fibroblast growth factor receptor 1 (FGFR-I) mRNA in the developing and adult rat adrenal gland and in PC12 cells under different culture conditions. For this purpose a sensitive ribonuclease protection assay using 33p-labelled riboprobes was established. 33p-labelled riboprobes show a high resolution and are relatively easy to handle. FGFR-I mRNA was found to be present in the postnatal and adult adrenal gland. In the cortex high levels of FGFR-1 mRNA were detected at postnatal day (P) 1 and P8, during the third week the mRNA levels declined, and reached low levels during adulthood. PC12 cells also contained detectable amounts of FGFR-I mRNA. With the exception of NGF, however, the different treatment procedures did not affect FGFR-1 mRNA levels. The expression pattern of the FGFR-I transcript matches that of the expression of FGF-2 and of the mitotic activity in the developing and adult cortex. This supports the idea that FGF-2 might act as an autocrine mitogen for adrenocortical cells. In the medulla FGFR-1 mRNA levels were low at the first 3 postnatal weeks and increased towards the adult. In accordance with the developing expression pattern of FGF-2 in the medulla and in vitro effects of this protein on chromaffin and PC12 cells an autocrine/paracrine role as a maintenance and differentiation factor for chromaffin cells is conceivable. Keywords: Fibroblast growth factor; Fibroblast growth factor receptor; Adrenal gland; Chromaffin cell; RNase protection assay

1. Introduction

The fibroblast growth factors (FGF) are heparin binding proteins with widespread distribution in mesoderm- and neuroectoderm-derived tissues and a broad spectrum of functions including stimulation of proliferation, angiogenesis, differentiation and promotion of maintenance, chemotaxis and repair [10]. The FGF family currently comprises nine members [31]. Acidic and basic FGF (FGF-1 and FGF-2) were first to be characterized, high levels of these molecules are found in the brain [3]. FGF-2 is expressed in the developing nervous system and in different regions of the adult central (CNS) and peripheral nervous system (PNS) [5,15,21,55,59,60]. Many in vitro and in vivo studies have reported on FGF-2 effects in the nervous system like for example the stimulation of survival, neurite outgrowth, transmitter metabolism, and synapse formation of neurons [1,8,19,20,35,37,54,56,57]. FGF-2 was also shown to affect the proliferation and differentiation of glial pre-

• Corresponding author. Fax: (49) (07611 203 5091. 0169-328X/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved

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cursor cells [61]. The physiological role of FGF-2 in the developing and mature nervous system, however, is still contested. Chromaffin cells of the adrenal medulla are modified sympathetic neurons. They develop together with sympathetic neurons from common neural crest-derived precursor cells [2,39]. Chromaffin cells contain FGF-2 and its mRNA. Immunoreactivity for FGF-2 displays a distinct spatial and temporal pattern in the developing rat adrenal medulla. At postnatal day (P) 8 FGF-2 positive chromaffin cells become detectable and increase in number during the second and third week. The staining intensity of the adrenal medulla increases between P18 and adult [18]. The immunoreactivity is confined to the noradrenergic subpopulation [18]. Stimulation of neurotransmitter and hormonal receptors increases the expression of FGF-2 mRNA in cultured bovine chromaffin cells [46]. With regard to the putative physiological role of the medullary FGF-2 several possibilities have been discussed. According to the observation that exogenously applied FGF-2 is able to prevent the lesion-induced death of the preganglionic sympathetic neurons of the spinal cord after

C. Meisinger et al. /Moh'cular Brain Research 36 (1996) 70-78

adrenomedullectomy [8] it was suggested that the medullary FGF-2 could act as a neurotrophic factor for these neurons. On the other hand it was shown that FGF-2 mediates several effects on postnatal and adult chromaffin cells and on PCI2 cells. This could imply an autocrine/paracrine role of FGF-2 for chromaffin cells. FGF-2 stimulates proliferation and neurite outgrowth of embryonic chromaffin precursor cells [48], enhances chromaffin cell proliferation of neonatal and adult rats synergistically with insulinlike growth factors [13], promotes catecholamine storage and synthesis of early postnatal chromaffin cells [53], activates the expression of the tyrosine hydroxylase and proenkephalin genes of adult chromaffin cells [41], and enhances in combination with ciliary neurotrophic factor (CNTF) the ability of nerve growth factor (NGF) to induce transdifferentiation of postnatal and adult chromaffin cells into neurons [23]. PCI2 cells differentiate into sympathetic-like neurons and become electrically excitable in the presence of FGF-2 [36,40,42,44,45,52]. The mediation of the FGF signal apparently requires low-affinity and high-affinity receptors. Low-affinity receptors have been characterized as cell surface heparan sulfate proteoglycans, e.g., the integral membrane proteoglycan syndecan [7,26]. At present four members of the high-affinity FGF receptor (FGFR) family are known. These tyrosine kinase transmembrane receptors that belong to the immunoglobulin superfamily express a vast variety of different isoforms by differential splicing [14,24,32]. FGF-I and FGF-2 bind FGFR-I and FGFR-2 with similar affinity whereas FGFR-3 shows lower binding capacity for FGF-2 than for FGF-I [49]. Up to now there are no data on FGFRs in the adrenal medulla or in chromaffin and PC12 cells. This information, however, would have important physiological implications. The mRNAs for FGFR-1, FGFR-2, and FGFR-4 are found in extracts of the adrenal gland [29,38]. The FGFR-4 mRNA could be demonstrated in the adrenal cortex of the embryonic mouse by in situ hybridization [47]. In order to help elucidate the physiological function of FGF-2 for chromaffin cells more information on FGFRs in the adrenal medulla is required. We have approached this question by analyzing the expression of the FGFR-1 mRNA in the adrenal medulla and cortex during postnatal development and in PCI2 cells under different culture conditions. PCI2 cclls, a catecholamine-secreting pheochromocytoma cell line cloned from the rat adrenal medulla, have been a useful model system to study the effects and/or regulation of growth factors on neuronal differentiation. For this purpose we have established a ribonuclease protection assay using 33p-labelled riboprobes. 33p probes represent a good alternative to 3Zp probes, since they are less critical to handle while providing similar sensitivity and higher resolution. In addition to the ribonuclease protection assay, Western blotting was used to detect FGFR-1 protein in extracts of PCI2 cells.

71

2. Materials and methods 2.1. Preparation of the adrenal cortex and medulla P1, P8, P18 and adult Hanover Wistar rats were decapitated and adrenal glands were quickly removed. Cortices and medullae were carefully dissected on ice under binocular control and collected in liquid nitrogen. Tissues were stored at - 8 0 ° C until used.

2.2. Cell culture PCI2 cells (kindly provided by Dr. R. Westermann, Marburg, Germany) were grown to confluency in 75-cm 2 plastic tissue culture flasks (Falcon) in Dulbecco's modified Eagle's medium containing 5% fetal calf serum and 10% horse serum at 37°C in a humidified 6% CO~ incubator. Cell were routinely split in a 1:3 ratio, the medium was changed twice a week. For experiments, cells were seeded on 75-cm 2 flasks and after 1 day medium was changed and cells received the factors dissolved in serumcontaining (see above) or serum-free (see below) medium. Serum-free medium was supplemented with N I additives [9] and 0.25% bovine serum albumin. For low-density cultures 2500 cells/cm 2, for high-density cultures 40000 cells/cm 2 were seeded. Factors were presented at the following concentrations: 50 n g / m i NGF (R & D Systems, Bad Nauheim, Germany), 50 n g / m l FGF-2 (Progen, Heidelberg, Germany), 10 /zM dexamethasone (DEX), 0.4 mM carbachol, 5 g M forskolin. After a 36 h culture period cells were dislodged by 0.1% trypsin/20 mM EDTA for RNA isolation and by distilled water for Western blotting. Cells were frozen in liquid nitrogen and stored at - 8 0 ° C until used. The number and morphology of PCI2 cells was checked under microscopical control in sister cultures in the presence of the respective molecules by counting the cells in suspension, after gentle detachment from the flasks at the end of the experiments.

2.3. Riboprobe synthesis The plasmid pJDM508 (a gift from Dr. J. Milbrandt, Department of Pathology, St. Louis, MO) is a derivative of a bluescript plasmid (Stratagene) containing a 350-base portion (verified by sequencing) of the rat FGFR-1 eDNA [58]. EcoRI linearized plasmid was used as a template for the in vitro transcription synthesis with T3 RNA polymerase. The procedure generated a 33p-labelled 407-base probe, which includes the 350 bases of FGFR-I antisense cRNA and 57 bases of the bluescript polylinker region (Fig. 1). Hinfl linearized plasmid was used for in vitro transcription with T7 RNA polymerase generating a 150base FGFR-I sense cRNA (Fig. 1). The mouse /3-tubulin plasmid (gift from Dr. H. Koseki, Chiba University, Japan) was linearized with Hindlll. Labelling with T7 RNA polymerase by in vitro transcription generated a 33Plabelled 303-base tubulin antisense cRNA.

72

C. Meisinger et al. / Molecular Brain Research 36 (1996) 70-78 EcoR I

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Fig. 1. Schematic diagram of the cRNA probe used in the ribonuclease protection assay. The plasmid pJDM508, linearized with EcoRI. was used as a template for the T3 RNA polymerase. In vitro transcription reaction generated a 407 base cRNA probe which includes 57 bases of the plasmid polylinker region and 350 bases of FGFR-I antisense cRNA. In vitro transcription reaction with T7 RNA polymerase using Hinfl linearized plasmid generated a 216 base cRNA probe, including 66 base plasmid polylinker region and 150 base FGFR-1 sense cRNA. The ~n~ probe was generated without radioactive nucleotides. 2.4. Isolation o f RNA and ribonuclease protection assay

Total RNA was isolated according to the method of Chomczynski and Sacchi [11] and quantified by absorbance at 260 nm. 33p-labelled riboprobes (spec. act. approx. 4 - 8 × 107 c p m / / z g for FGFR-I, 1 - 2 × 107 cpm//,tg for tubulin) were prepared freshly and gel-purified immediately before use. Total RNA (40 /zg) was dissolved in 20 /J,I of hybridization solution (80% formamide, 40 mM PIPES, pH 6.4, 400 mM NaCI, 1 mM EDTA) containing 100000 cpm of 33p-labelled cRNA probe. After heating to 95°C for 10 min, the hybridization was performed at 49°C overnight. Subsequently, the solu-

tion was diluted with 300/xl of RNase digestion buffer (10 mM Tris-HCl, pH 7.4, 300 mM NaCI, 5mM EDTA) containing 0.48 /xg RNase A and 0.96 units RNase TI and incubated for 60 min at 30°C. 6 /,ti Proteinase K (20 m g / m l ) and SDS (20 /,tl of a 10% stock solution) were added and the mixture was incubated for 25 min at 35°C. After a phenol/chloroform extraction the supernatant was precipitated by 2 vol. ethanol and 2 ~1 glycogen (Boehringer). The pellet containing the RNA:RNA hybrid was dried and resuspended in 5 /zl loading buffer (80% formamide, 10 mM EDTA, 0.1% bromophenol blue). After heating for 5 min at 85°C samples were separated on a 4% polyacrylamide/urea sequencing gel. After fixation (10% acetic acid, 10% methanol) and drying of the gel thc protected fragment was visualized by autoradiography on a Kodak BioMax film. Riboprobe hybridized to 40 /,tg yeast tRNA was used as a negative control for ribonuclease protection. Riboprobe hybridized to 1 0 0 / z g of RNA of rat L6 myoblasts which lack endogenous FGFR was used as a negative tissue control (data not shown). Experiments were reproduced with 2 - 3 different sets of RNA preparations. 2.5. Densitometric evaluation

Quantification of bands was done by measuring the density of each band using image analysis (analySIS 2.0). All ribonuclease protection assay data were expressed as the ratio of FGFR-1 mRNA and tubulin mRNA. Tubulin

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C'. Meisinger et al. / Molecular Brain Research 36 (1996) 70-78

was used as an internal standard for the amount and integrity of RNA preparation [34]. In some experiments 18S was used as an additional internal standard to exclude alterations of the tubulin transcript level by treatment with various extrinsic molecules (data not shown). Different exposure times of the same gel ensured that the measurement was performed in the linear range.

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2.6. Western blot analysis

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Fig. 4. Ribonuclease protection analysis of FGFR- 1 expression in adrenal glands of P8, PI8 and adult (AD) rats. A: Protection assay was performed on 80 /zg of total RNA. B: Graphic representation of FGFR-I mRNA levels measured by densitometry. FGFR-1 mRNA levels were normalized to tubulin.

min, 140 × g ) in a crude cytoplasmic (supernatant) and a membraneous fraction (pellet). After S D S - P A G E and semi-dry blotting onto PVDF membranes (Bio-Rad) immunological detection of the FGFR-1 was performed by using anti-FGFR-1 antibody (raised against an external region of the FGFR-1; Upstate Biotechnology, Inc.) and the P A P - D A B system.

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Fig. 3. Ribonuclease protection analysis of FGFR-1 expression in adrenal glands of newborn (PI) and adult (AD) rats. A: Protection assay was performed on 30 /.tg of total RNA. Undigested tubulin probe is present in the control tRNA lane and in the sample lanes. MED, medulla; COR, cortex. B: Graphic representation of FGFR-I mRNA levels measured by densitometry. FGFR-I mRNA levels were normalized to tubulin.

3.1. Establishment of the ribonuclease protection assay using 33p riboprobes We established a protection assay using 33p-labelled F G F R - I antisense c R N A probes (Fig. I). The standard curve was performed using unlabeiled F G F R - I sense riboprobe (Fig. 1). Between 3 pg and 3 ng of sense riboprobe were tested. These amounts of RNA allowed the measure-

74

C. Meisingeret aL/ Molecular Brain Research36 (1996) 70-78 t~ I,U

normalized tubulin standard (Figs. 3 and 4). The highest amounts of FGFR-1 mRNA were found in PI and P8 adrenal cortices. In the cortex the FGFR-1 mRNA level was constantly high at P1 and P8, declined during the third week, and reached low levels during adulthood (Figs. 3 and 4). In the medulla the FGFR-1 mRNA levels were relatively low during the first 3 postnatal weeks and increased towards the adult (Figs. 3 and 4).

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3.3. FGFR-1 mRNA in PC12 cells

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Proliferation and morphology of P C I 2 cells were examined in parallel cultures treated at the same time and in the same way as those cultures which were run for the protection assay analysis. Under all culture conditions performed no significant changes in the number of P C I 2 cells were found. There were also no differences in the RNA amount isolated from the cultures which were used for the protection assays. As expected, both NGF and FGF-2 displayed neurite-promoting effects. FGFR-I mRNA in PC12 cells was detectable in serumcontaining as well as in serum-free cultures. However, the expression of FGFR-1 was found to be cell density-dependent; FGFR-I mRNA levels in high-density cultures were higher as compared to low-density cultures (Fig. 5). Neither application of growth factors in serum-free cultures (FGF-2, NGF, F G F - 2 / N G F , DEX), nor stimulation of cholinergic receptors with carbachol or of adenylate cyclase with forskolin did affect the level of FGFR-I expression (Fig. 6). In serum-containing cultures the presence of NGF led to a significant increase of the FGFR-1 transcript as compared to untreated cultures (Fig. 7). The effects of extrinsic molecules were analyzed only at high cell den-

UBULIN

Fig. 5. Ribonuclease protection analysis of FGFR-I expression in PCI2 cells at low (LD) and high (HD) cell density. Protection assay was performed on 40 /xg of total RNA. ment in the linear range (Fig. 2). In addition, at least 3 pg of RNA were detectable after 3 days exposure time (Fig. 2). Lower concentrations of RNA were not tested. 3.2. Developmental expression of FGFR-I in the postnatal and adult adrenal gland FGFR-1 m R N A was analyzed in extracts of carefully separated cortices and medullae of P1, P8, P18 and adult rat adrenals. FGFR-1 m R N A was present in all tissues examined. Their relative quantities, however, were found to differ, as measured densitometrically in relation to the

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Fig. 6. Ribonuclease protection analysis of FGFR-I expression in PC12 cells. Cells were cultured under serum-free conditions in the absence (CONTROL) or in the presence of FGF-2 + NGF, FGF-2, NGF, CARBACHOL,FORSKOLINand dexamethasone(DEX). Protection assay was performed on 50/zg of total RNA.

C. Meisinger et al. / Molecular Brain Research 36 (1996) 70-78

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Fig. 7. Ribonuclcase protection analysis of FGFR-I expression in PCI2 cells. Cells wcrc cultured under serum-containing conditions in the absence ( - ) or presence ( + ) of NGF. A: Protection assay was performed on 50 t.tg of total RNA. B: Graphic representation of FGFR-1 mRNA levels measured by densitometry. FGFR-1 mRNA levels were normalized to tubulin.

sity. We did not test the effects at low cell density because of the very high amounts of factors needed.

135 kDa, 85 kDa and 56 kDa in the membraneous but not in the cytoplasmic fraction (Fig. 8).

3.4. Detection of FGFR-1 protein Western blot analysis of PC12 cells using FGFR-I antibodies revealed three immunoreactive bands of about

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47 Fig. 8. Western blot analysis of FGFR-I protein in PC12 cells. Cell were homogenized and separated in a cytoplasmic (C) and membranous (M) fraction. Immunoreactive bands of 135 kDa, 85 kDa and 56 kDa were detected in the membranous fraction.

4. Discussion The establishment of a sensitive ribonuclease protection assay using 33P-labelled riboprobes allowed the detection of the FGFR-I transcript in the adrenal cortex and medulla and in PCI2 cells. Main advantages of the assay are a high resolution and an easier handling in comparison to 32p_ labelled riboprobes. The higher energy of 32P-labelled probes could be compensated by a longer exposure time of the autoradiographs. Using ~3p riboprobes we were able to detect 3 pg of RNA after 3 days exposure time. This is comparable to another study with 3:,p riboprobes where 1 pg of RNA was detectable after an exposure time of 12-48 h [28]. The present protocol allowed the detection of mRNAs which are known to be present only in few copies (Meisinger, unpublished observation). The present study is the first presentation of the FGFR-I mRNA in the postnatal and adult rat adrenal cortex and medulla. We could further show that the FGFR-1 transcript is present in PC12 cells under different culture conditions. NGF-treatment which induces neuronal differentiation of PC12 cells increased the level of FGFR-I mRNA. In the adrenal cortex we found high levels of FGFR-I mRNA at P1 and P8, which decreased during the third week and reached low levels in the adult. Previous in vitro studies have shown that FGF-2 stimulates proliferation and

76

C. Meisinger et al. / Molecular Brain Research 36 (1996) 70-78

delays cell senescence of adrenocortical cells [16]. This was interpreted as a direct evidence that FGF-2 plays a role as an autocrine mitogen in the adrenal cortex. The expression of FGFR-1 mRNA during postnatal development corroborates this idea. In correlation with the high mitotic activity during postnatal development in the cortex [33] high levels of FGFR-1 mRNA and FGF-2 protein [18] were found. In the adult cortex the FGFR-1 mRNA is decreased and the FGF-2 immunoreactivity is confined to the glomerulosa zone and to few cells in the fasciculata zone [17,18]. Binding studies using iodinated FGF-2 suggested that FGFR-1 is localized primarily to the capsule and glomerulosa zone of the adult cortex [4]. The glomerulosa zone is considered as a region of high mitotic activity during development and in the adult [22,27]. In the adrenal medulla we found an increase of FGFR-1 mRNA levels to adulthood. Although rat adrenal chromaffin cells proliferate in vivo throughout life [30] a principal mitogenic role of FGF-2 at least in the postnatal and adult medulla is not very likely. In the adult medulla where the mitotic activity is very low [25,50] the highest level of FGFR-1 mRNA is found and the number and intensity of FGF-2 immunoreactive chromaffin cells increase to adulthood [18]. In addition, in vitro studies revealed that FGF-2 does not stimulate proliferation of adult rat chromaffin cells [13,51]. The developmental expression pattern of FGF-2 immunoreactivity [18] and FGFR-1 mRNA as shown in the present paper suggests an autocrine/paracrine role of this factor. Several results support the idea that FGF-2 could be a physiological maintenance and differentiation factor for chromaffin cells. The in vitro effects on postnatal and adult chromaffin cells, which could be mediated by FGF-2 include maintenance, support on catecholamine storage and synthesis, and stimulation of proenkephalin expression [41,53]. Finally, FGF-2 in combination with CNTF enhances the ability of NGF to induce transdifferentiation into neurons dramatically [23]. PC12 cells which display characteristics similar to precursors of adrenal medullary cells are used as a model system for the study of the effects and the regulation of growth factors during neuronal differentiation. The synergistic effect of NGF and FGF-2 on neurite outgrowth of PCI2 cells [52] might be explained by the effect of NGF on the expression of FGFR1 mRNA. NGF could enhance the sensitivity of PC12 cells for FGF-2 by elevating the FGFRI mRNA expression. In addition to the FGFR-1 transcript we were able to detect the FGFR-I protein in PC12 cells. The sizes of the FGFR-1 immunoreactive bands, 135 kDa, 85 kDa and 56 kDa, correspond to the sizes found by cross-linking experiments using iodinated FGF-2 and by in vitro translation of FGFR cDNA clones [6,43]. The authors have suggested that the 90 kDa protein could be the result of differential glycosylation [6]. Also different splice variants are possible. Since FGFR-I mRNA is found in PC12 cells it seems very likely that the

chromaffin cells in vivo are the source of the medullary FGFR-I mRNA. Whether both the adrenaline- and the noradrenaline-storing subpopulations of chromaffin cells possess FGFR-I and respond to FGF-2 is not clear. The FGF-2 immunoreactivity, however, is restricted to the noradrenergic subpopulation [18]. In situ hybridization experiments are in progress to analyze the cellular distribution of FGFR-1 mRNA in the adrenal medulla. Expression of FGF-2 and functional FGF receptors are also found in dopaminergic neurons of the substantia nigra [5,12,21]. Thus it could be possible that FGF-2 could also mediate auto-/paracrine regulation of transmitter metabolism in dopaminergic brain neurons.

Acknowledgements We thank Dr. J. Milbrandt for kindly providing the FGFR-1 eDNA, Dr. H. Koseki for the fl-tubulin cDNA, B. R6sler for excellent technical assistance, and C. Micucci for excellent help in preparing the figures. We are very grateful to Dr. H. Kurz for the introduction and help in the densitometric analysis. We also thank Drs. S. and K. Wewetzer for critical reading of the manuscript. This work was supported by Deutsche Forschungsgemeinschafl (Gr 857/8-1).

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