0 1992 Harwood Academic Publishers GmbH
Growth Factors, 1992, Vol. 6, pp. 93-101 Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
A Chimera between Platelet-Derived Growth Factor PReceptor and Fibroblast Growth Factor Receptor-1 Stimulates Pancreatic FCell DNA Synthesis in the Presence of PDGF-BB JAROSLAV MARES, LENA CLAESSON-WELSH* and MICHAEL WELSH
Growth Factors Downloaded from informahealthcare.com by UB der LMU Muenchen on 06/20/14 For personal use only.
Department of Medical Cell Biology, Uppsala University, Box 571, Biomedicum, S-75123 Uppsala, Sweden, and *Ludwig Institute for Cancer Research, Box 595, Biomedicum, Uppsala
(Received May 8 1991, Accepted August 26 1991)
This study was undertaken to characterize the expression of a chimeric growth factor receptor composed of the extracellular and transmembrane domains of the plateletderived growth factor (PDGF) preceptor (PDGFR-B) fused to the intracellular domain of the fibroblast growth factor receptor-1 (FGFR-I)and to assess its effect on the growth potential of pancreatic islet cells. For this purpose rat pancreatic islets or monolayers of pancreatic islet cells were transfected with recombinant DNA constructs coding for the PDGF B-chain, the PDGFR-/3, the FGFR-1 and the chimera between PDGFR-/l and FGFR-I. DNA synthesis, monitored as the percentage of labelled nuclei and [3H]thymidineincorporation, was stimulated in pancreatic islet cells cotransfected with the constructs coding for the PDGF B-chain and the PDGFR-/3 or the chimeric PDGFR-/3 /FGFR-1 as compared with that determined after transfection with control plasmid. PDGF-BB stimulated DNA synthesis when islet cells had been transfected with PDGFR/3 or PDGFR-P/FGFR-l. Cotransfection of the PDGFR-B and the chimeric PDGFR-/l /FGFR-1 constructs attenuated the stimulation of DNA synthesis in response to PDGFBB. Receptor binding studies showed binding with a & of 0.7nM to the chimeric receptor. The present findings show that when the chimeric PDGFR-/3/FGFR-l construct is expressed in pcells it is efficient in increasing DNA synthesis when stimulated with ligand. KEYWORDS islets, chimeric receptor, DNA synthesis ABBREVIATIONS PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; aFGF, acidic FGF; bFGF, basic FGF; PDGF-BB, dimer composed of two PDGF-B polypeptide chains; pHhis, recombinant DNA construct coding for PDGF-BB; pHIPDGFrec, recombinant DNA construct coding for PDGF preceptor; pPDGFR-/?/FGFR-l, chimeric construct; pFGFR-I, recombinant DNA construct coding for the FGF receptor; PCR, polymerase chain reaction; PBS, phosphate buffered saline.
increased replication (reviewed by Hellerstrom and Swenne, 1985). Transfection of P c e l l s with DNA constructs coding for certain oncogene products a n d the PDGF P r e c e p t o r increased the rates of DNA synthesis, although the effects were modest (Welsh et al., 1988; Berggren et al., 1989; Welsh et al., 1990). The results were interpreted to suggest that despite efficient transfection a n d subsequent expression of the
INTRODUCTION
with
The pancreatic /I-cell has a limited capacity for proliferation, although it responds slightly to glucose and certain polypeptide growth factors Address for correspondence: Dr Michael Welsh, Department of Medical Cell Biology, P.O. Box 571, Biomedicum, S75123, Uppsala, Sweden. Tel: 46-18 174447. Fax: 46-18556101.
93
Growth Factors Downloaded from informahealthcare.com by UB der LMU Muenchen on 06/20/14 For personal use only.
94
MARES, CLAESSON-WELSH and WELSH
active (onco)gene products (Welsh et al., 1988; Berggren et al., 1989; Welsh et al., 1990), most pancreatic pcells are permanently suppressed in their ability to replicate. To overcome such a SUPpression of &cell proliferation by expressing other potent growth promoting genes may provide new possibilities for diabetes therapy. Furthermore, examination of mechanisms for the regulation of growth in the slowly replicating p cell may have general interest. The PDGF a- and preceptors are protein tyrosine kinases which are activated upon ligand binding (Heldin and Westermark, 1990). Furthermore, since the ligands are homo- or heterodimers (PDGF-AA, -AB and -BB), ligand binding to the receptors induces dimerization of the receptors (Bishayee et al., 1989; Heldin et al., 1989; Kanakaraj et al., 1991), a step which is thought to be of importance for receptor activation. Four distinct but highly related FGF receptors have been characterized which are protein tyrosine kinases (Partanen et al., 1991). Unlike PDGF, the members of the FGF ligand family are monomers (Gospodarowicz, 1987) and are thus believed to induce receptor dimerization (S. Wennstrom, personal communication) through a different mechanism than that of PDGF. Furthermore, acidic FGF (aFGF) and basic FGF (bFGF) are synthesized without signal sequences (Abraham et al., 1986). When a signal sequence is fused to the bFGF polypeptide by recombinant DNA technology, the recombinant gene induces efficient transformation of cells, suggesting a high transforming potential of activated FGF receptors (Rogelj et al., 1988). A hybrid receptor gene was constructed consisting of the extracellular and transmembrane domains from the PDGF P-receptor and the intracellular region from the type 1 FGF receptor in order to elucidate the effects of activation by binding of a dimeric ligand on Pcell replication. The rates of DNA synthesis of pancreatic pcells were determined after transfection of the hybrid receptor construct.
MATERIALS AND METHODS Materials [methyl-3H1Thymidine (50 Ci/mmol, 1Ci= 37 GRq), I3*P1dCTP, multiprime DNA labelling
kit, T4 DNA ligase, DNAse I and restriction nucleases were from Amersham, Bucks, UK; LipofectinTM was from BRL, MD; RPMI 1640, fetal bovine and donor calf serum were from Flow, Irvine, UK; IEI-PDGF-BB and recombinant PDGF-BB were kindly provided by Dr C.-H. Heldin, Ludwig Institute for Cancer Research, Uppsala Branch, and baculovirus produced aFGF was kindly provided by Dr Ralf Pettersson, Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute; Soluene and Instagel were from Packard, IL; Falcon Primaria multiwell tissue culture plates (3847) were from Becton Dickinson, NJ; the GeneAmpTMkit was from Perkin Elmer Cetus, CT; RibocloneTM cDNA synthesis system was from Promega Biotech, WI; the eucaryotic expression vector pcDNA-1 was from Invitrogen, CA.
Construction of pPDGFR-PIFGFR-1 The 2.1 kb Acc I fragment of the FGF receptor-1 cDNA (Wennstrom et al., 1991) was ligated into the Acc I site of the bluescript plasmid vector. A Hind I11 site was created at the positions coding for Lys-559 and Pro-560 immediately beyond the transmembrane region in the wild type PDGF Preceptor (Claesson-Welsh et al., 1988). The 1.8 kb Eco RI-Hind 111 fragment from this mutated PDGF preceptor was ligated into the corresponding sites in the above bluescript-FGF receptor construct. After digestion with Hind I11 and Bal I, a synthetic oligonucleotide was ligated into the digested bluescript vector containing the PDGFR-P and FGFR-1 sequences. The synthetic oligonucleotide was composed of 55 nucleotides of FGFR-1 sequence upstream from the Bal I site plus a 5'Hind 111 site, thus generating an inframe linkage between the PDGF Preceptor extracellular and transmembrane regions and the FGF receptor-1 intracellular regions. Subsequent sequence analysis of the generated hybrid clones revealed point mutations in the stretch derived from the synthetic oligonucleotide sequences. In clone 3, the sequence corresponding to Ala-411 had been replaced by an aspartic acid codon and in clone 61, the Ser-408 was replaced by an arginine codon. The chimeric receptor constructs were digested with Eco RI+Xho I and transferred into the expression vector pcDNA I. 1
.
RECEPTOR CHIMERA AND PCELL GROWTH
donor calf serum immediately after transfection, 24 hr in 10% donor calf serum and 24 hr in 1 or 0.1% donor calf serum, after which the media Rat pancreatic islets were isolated from female were changed to those containing [3H]thymidine Sprague-Dawley rats belonging to a local colony. (0.5 pCi/ml). At the end of the 24 hr labelling A detailed account of the islet isolation pro- period, the cell monolayers were washed with cedure has been published elsewhere (Howell PBS (154mM Na+, 4mM K+, 10mM HPOZ-, and Taylor, 1968; Lernmark et al., 1976). Groups 140 mM C1-, pH 7.4) and sonicated for 10 sec in of 150 islets were kept free floating in the RPMI 300 pl of HzO; aliquots were then taken for DNA 1640 medium containing 11.1mM glucose, 10% content analysis (Kissane and Robins, 1958). The fetal bovine serum, 2 mM L-glutamine, 100 U/ml rest of the sonicated sample was precipitated, benzpenicillin, and 0.1 mg/ml streptomycin at subsequently washed with ice-cold 10% trichlor37°C in a gas phase of 95% humidified air/5% acetic acid and dissolved in 50 pl of Soluene. The COz. On day 2, the standard medium was radioactivity incorporated was determined by exchanged. On day 4, the islets were dispersed scintillation counting after addition of 1 ml of by trypsin/EDTA treatment (Lernmark et al., Instagel. 1976) to wells of Multiwellm tissue culture plate. Pancreatic islet cell monolayers were cultured Light Microscopy and Determination of 24 hr as described above and then transfected Labelling Indices with the DNA constructs using the LipofectinTM method (Felgner et al., 1987). The transfection For estimates of autoradiographic labelling indimixture contained 30 pl of Lipofectin suspension ces, 0.5 pCi/ml of I3H1thymidine was added to and 10 p g of DNA in total volume of 100 pl. After the culture in 1%fetal bovine serum during the 15 min incubation at room temperature, the Lipo- last 2 hr of the culture period. Then the islets fectin reagent-DNA complex suspension was were rinsed by PBS, fixed in Bouin's solution for diluted by 500p1 of serum-free RPMI medium 24 hr and embedded in paraffin. Sections, 7 p m and used for transfection procedure in four wells. thick, were processed for autoradiography and After a 3 hr incubation, the transfection was stained immunocytochemically for insulin stopped by adding 1 ml of standard medium con- (Korsgren et al., 1988; Swenne et al., 1980). For taining 1% donor calf serum and monolayers each experiment 2,000 cells were evaluated with regard to immunocytochemical staining and were incubated 18 hr as described above. Since the determination of labelling indices autoradiographic labelling in order to determine requires intact islet morphology, adult rat islets the frequency of insulin-positive cells and their were collected free from exocrine tissue in corresponding labelling index. serum-free standard medium and transfected with DNA constructs immediately after isolation. Isolation of RNA Lipofectin reagent-DNA complex suspension (100 pl) was added dropwise to the islets in 500 p 1,000 adult rat islets after transfection and culture 1 of serum-free standard medium and incubated were picked carefully, rinsed with PBS, centri3 hr as described above. The transfection was fuged shortly and frozen at -70°C. Intact FWA stopped by addition of 5 ml of standard medium has been prepared from these islets by disrupting supplemented with 10% fetal bovine serum and the cells in guanidium lysis solution (Chirgwin et the islets were maintained 48hr in standard al., 1979; Sambrook et al., 1989a). Selection medium containing 10% fetal bovine serum and of poly(A') FWA was performed by batch 24 h r in 1% fetal bovine serum. For pratical affinity chromatography on oligo(dT)-cellulose reasons, the above proceudre was also used for (Sambrook et al., 1989b). transfection of islets for RNA isolation, which were washed with PBS and frozen at -70 "C after Polymerase Chain Reaction (PCR) the culture period. Estimation of pcell DNA synthesis was Isolated rat pancreatic islets were transfected slightly modified from that described by Welsh with either the coding sequences of the chimeric et al. (1988). The cells were cultured 18 hr in 1% receptor in bluescript (control) or with pPDGFXIslet Isolation, Transfection Procedures and Estimation of DNA Synthesis
Growth Factors Downloaded from informahealthcare.com by UB der LMU Muenchen on 06/20/14 For personal use only.
95
Growth Factors Downloaded from informahealthcare.com by UB der LMU Muenchen on 06/20/14 For personal use only.
96
MARES, CLAESSON-WELSH and WELSH
p/FGFR-1. Three days later RNA was isolated and treated with DNAse I (20 U/25 pl) for 30 rnin at 37 "C. The RNA was then phenol extracted and ethanol precipitated before first strand cDNA synthesis. First strand cDNA was synthesized using the Riboclone cDNA synthesis system using l p g poly(A') RNA. RNA was then hydrolyzed in 0.5 M NaOH at 55 "C for 15 rnin after which DNA was ethanol precipitated. The cDNAs were dissolved in loop1 water and samples of 3-6 p1 were used for the amplification reactions using 0.2 pM primers but otherwise the components of the PCR reactions provided by the GeneAmp kit were used. Two cycles of 1min at 94 "C, 3 min at 37 "C and 1 min at 72 "C were run before 25 cycles of 1 min at 94 "C, 2 min at 55 "C and 1 min at 72 "C. The oligonucleotide primers were 18-mers derived from amino acid 510-516 of the PDGF Preceptor and nucleotides 1330-1312 of the FGF receptor-1, thus only amplifying chimeric sequences. Subsequent to PCR the samples were subjected to Southern blot analysis (Southern, 1975) using labelled pPDGFR-P /FGFR-l as a probe.
not amplify any sequences hybridizing to pPDGFR-P/FGFR-l (Fig. 1, lane 1).
Scatchard Analysis COS cells at a density of 2x1O5/we1l in Costar 12well dishes were transfected with pPDGFR-P /FGFR-1 using Lipofectin as above. After three days, Scatchard analysis was performed using '251-PDGF-BB and different concentrations of unlabeled PDGF-BB (1-251 ng/ml) as described (Claesson-Welsh et al., 1989).
FIGURE 1. Expression of the chimeric PDGFR-PIFGFR-1 receptor construct in islet cells using PCR. Freshly isolated rat pancreatic islets were transfected with the PDGFR-B/FGFR-l coding sequences in bluescript plasmid (control; lane 1) or with pPDGFR-BIFGFR-1 (lane 2) and cultured according to the same protocol as in the experiments for the determination of labelling indices. The PCR products were separated by electrophoresis on a 1.2%agarose gel. The DNA was transferred to nitrocellulose by standard procedure, and the blots were hybridized with I3'P1labelled pPDGFR-P/FGFR-1 as a probe.
Statistical Analysis MeanskS.E.M. were calculated, and comparisons between control and the other groups were performed by Student's paired f-test.
RESULTS Expression of the Transfected Constructs Islets transfected with pPDGFR-P/FGFR-l amplified a product of 0.3 kb which hybridized to pPDGFR-P/FGFR-l sequences (Fig. 1, lane 2). Islets transfected with the sequences coding for the chimeric receptor but not inserted into an expression vector (control transfected islets) did
Scatchard Analysis To assess the binding characteristics of the chimeric receptor construct, COS-I cells were transfected using Lipofectin with pPDGFR-P/FGFR-l (3), and binding of lz5I-PDGF-BBwas then studied. Figure 2 shows the fraction bound/free ligand plotted against bound ligand. The Kd for binding of PDGF-BB was estimated using the RBINDING program (van Zoelen, 1989), and was found to be 0.7 nM which is very similar to that obtained for the wildtype PDGF P-receptor expressed endogenously in human foreskin fibroblasts (Kd=0.5 nM) (Ostman et al., 1989).
RECEPTOR CHIMERA AND /3-CELL GROWTH
97
0,12
0,lO
W
W K
0,08
FIGURE 2. Binding of lz5I-PDGF-
t n z
3
0
0,06
m
Growth Factors Downloaded from informahealthcare.com by UB der LMU Muenchen on 06/20/14 For personal use only.
0,04
0,02
0
2
4
BOUND (ng)
Effect of the Cotransfection with pHIsis and pPDGFR-/3 FGFR-1 on Islet Cell DNA Synthesis
6
BB to COS cells expressing pPDGFR-B/FGFR-l. The pPDGFR-B /FGFR-1 Lipofectin transfected COS cells were cultured for three days and then incubated with 1ng/ml of iodinated PDGF-BB in the presence of 0, 1.2, 2.5, 5, 10, 25, 100 or 250 ng/ml unlabelled PDGF-BB and the cell bound radioactivity was measured. The fraction bound/free ligand was plotted against bound ligand. Non specific binding (about 10%) was estimated by parallel 8 determination with 100-fold excess of unlabelled ligand. The I
Growth Factors, 1992, Vol. 6, pp. 93-101 Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
A Chimera between Platelet-Derived Growth Factor PReceptor and Fibroblast Growth Factor Receptor-1 Stimulates Pancreatic FCell DNA Synthesis in the Presence of PDGF-BB JAROSLAV MARES, LENA CLAESSON-WELSH* and MICHAEL WELSH
Growth Factors Downloaded from informahealthcare.com by UB der LMU Muenchen on 06/20/14 For personal use only.
Department of Medical Cell Biology, Uppsala University, Box 571, Biomedicum, S-75123 Uppsala, Sweden, and *Ludwig Institute for Cancer Research, Box 595, Biomedicum, Uppsala
(Received May 8 1991, Accepted August 26 1991)
This study was undertaken to characterize the expression of a chimeric growth factor receptor composed of the extracellular and transmembrane domains of the plateletderived growth factor (PDGF) preceptor (PDGFR-B) fused to the intracellular domain of the fibroblast growth factor receptor-1 (FGFR-I)and to assess its effect on the growth potential of pancreatic islet cells. For this purpose rat pancreatic islets or monolayers of pancreatic islet cells were transfected with recombinant DNA constructs coding for the PDGF B-chain, the PDGFR-/3, the FGFR-1 and the chimera between PDGFR-/l and FGFR-I. DNA synthesis, monitored as the percentage of labelled nuclei and [3H]thymidineincorporation, was stimulated in pancreatic islet cells cotransfected with the constructs coding for the PDGF B-chain and the PDGFR-/3 or the chimeric PDGFR-/3 /FGFR-1 as compared with that determined after transfection with control plasmid. PDGF-BB stimulated DNA synthesis when islet cells had been transfected with PDGFR/3 or PDGFR-P/FGFR-l. Cotransfection of the PDGFR-B and the chimeric PDGFR-/l /FGFR-1 constructs attenuated the stimulation of DNA synthesis in response to PDGFBB. Receptor binding studies showed binding with a & of 0.7nM to the chimeric receptor. The present findings show that when the chimeric PDGFR-/3/FGFR-l construct is expressed in pcells it is efficient in increasing DNA synthesis when stimulated with ligand. KEYWORDS islets, chimeric receptor, DNA synthesis ABBREVIATIONS PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; aFGF, acidic FGF; bFGF, basic FGF; PDGF-BB, dimer composed of two PDGF-B polypeptide chains; pHhis, recombinant DNA construct coding for PDGF-BB; pHIPDGFrec, recombinant DNA construct coding for PDGF preceptor; pPDGFR-/?/FGFR-l, chimeric construct; pFGFR-I, recombinant DNA construct coding for the FGF receptor; PCR, polymerase chain reaction; PBS, phosphate buffered saline.
increased replication (reviewed by Hellerstrom and Swenne, 1985). Transfection of P c e l l s with DNA constructs coding for certain oncogene products a n d the PDGF P r e c e p t o r increased the rates of DNA synthesis, although the effects were modest (Welsh et al., 1988; Berggren et al., 1989; Welsh et al., 1990). The results were interpreted to suggest that despite efficient transfection a n d subsequent expression of the
INTRODUCTION
with
The pancreatic /I-cell has a limited capacity for proliferation, although it responds slightly to glucose and certain polypeptide growth factors Address for correspondence: Dr Michael Welsh, Department of Medical Cell Biology, P.O. Box 571, Biomedicum, S75123, Uppsala, Sweden. Tel: 46-18 174447. Fax: 46-18556101.
93
Growth Factors Downloaded from informahealthcare.com by UB der LMU Muenchen on 06/20/14 For personal use only.
94
MARES, CLAESSON-WELSH and WELSH
active (onco)gene products (Welsh et al., 1988; Berggren et al., 1989; Welsh et al., 1990), most pancreatic pcells are permanently suppressed in their ability to replicate. To overcome such a SUPpression of &cell proliferation by expressing other potent growth promoting genes may provide new possibilities for diabetes therapy. Furthermore, examination of mechanisms for the regulation of growth in the slowly replicating p cell may have general interest. The PDGF a- and preceptors are protein tyrosine kinases which are activated upon ligand binding (Heldin and Westermark, 1990). Furthermore, since the ligands are homo- or heterodimers (PDGF-AA, -AB and -BB), ligand binding to the receptors induces dimerization of the receptors (Bishayee et al., 1989; Heldin et al., 1989; Kanakaraj et al., 1991), a step which is thought to be of importance for receptor activation. Four distinct but highly related FGF receptors have been characterized which are protein tyrosine kinases (Partanen et al., 1991). Unlike PDGF, the members of the FGF ligand family are monomers (Gospodarowicz, 1987) and are thus believed to induce receptor dimerization (S. Wennstrom, personal communication) through a different mechanism than that of PDGF. Furthermore, acidic FGF (aFGF) and basic FGF (bFGF) are synthesized without signal sequences (Abraham et al., 1986). When a signal sequence is fused to the bFGF polypeptide by recombinant DNA technology, the recombinant gene induces efficient transformation of cells, suggesting a high transforming potential of activated FGF receptors (Rogelj et al., 1988). A hybrid receptor gene was constructed consisting of the extracellular and transmembrane domains from the PDGF P-receptor and the intracellular region from the type 1 FGF receptor in order to elucidate the effects of activation by binding of a dimeric ligand on Pcell replication. The rates of DNA synthesis of pancreatic pcells were determined after transfection of the hybrid receptor construct.
MATERIALS AND METHODS Materials [methyl-3H1Thymidine (50 Ci/mmol, 1Ci= 37 GRq), I3*P1dCTP, multiprime DNA labelling
kit, T4 DNA ligase, DNAse I and restriction nucleases were from Amersham, Bucks, UK; LipofectinTM was from BRL, MD; RPMI 1640, fetal bovine and donor calf serum were from Flow, Irvine, UK; IEI-PDGF-BB and recombinant PDGF-BB were kindly provided by Dr C.-H. Heldin, Ludwig Institute for Cancer Research, Uppsala Branch, and baculovirus produced aFGF was kindly provided by Dr Ralf Pettersson, Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute; Soluene and Instagel were from Packard, IL; Falcon Primaria multiwell tissue culture plates (3847) were from Becton Dickinson, NJ; the GeneAmpTMkit was from Perkin Elmer Cetus, CT; RibocloneTM cDNA synthesis system was from Promega Biotech, WI; the eucaryotic expression vector pcDNA-1 was from Invitrogen, CA.
Construction of pPDGFR-PIFGFR-1 The 2.1 kb Acc I fragment of the FGF receptor-1 cDNA (Wennstrom et al., 1991) was ligated into the Acc I site of the bluescript plasmid vector. A Hind I11 site was created at the positions coding for Lys-559 and Pro-560 immediately beyond the transmembrane region in the wild type PDGF Preceptor (Claesson-Welsh et al., 1988). The 1.8 kb Eco RI-Hind 111 fragment from this mutated PDGF preceptor was ligated into the corresponding sites in the above bluescript-FGF receptor construct. After digestion with Hind I11 and Bal I, a synthetic oligonucleotide was ligated into the digested bluescript vector containing the PDGFR-P and FGFR-1 sequences. The synthetic oligonucleotide was composed of 55 nucleotides of FGFR-1 sequence upstream from the Bal I site plus a 5'Hind 111 site, thus generating an inframe linkage between the PDGF Preceptor extracellular and transmembrane regions and the FGF receptor-1 intracellular regions. Subsequent sequence analysis of the generated hybrid clones revealed point mutations in the stretch derived from the synthetic oligonucleotide sequences. In clone 3, the sequence corresponding to Ala-411 had been replaced by an aspartic acid codon and in clone 61, the Ser-408 was replaced by an arginine codon. The chimeric receptor constructs were digested with Eco RI+Xho I and transferred into the expression vector pcDNA I. 1
.
RECEPTOR CHIMERA AND PCELL GROWTH
donor calf serum immediately after transfection, 24 hr in 10% donor calf serum and 24 hr in 1 or 0.1% donor calf serum, after which the media Rat pancreatic islets were isolated from female were changed to those containing [3H]thymidine Sprague-Dawley rats belonging to a local colony. (0.5 pCi/ml). At the end of the 24 hr labelling A detailed account of the islet isolation pro- period, the cell monolayers were washed with cedure has been published elsewhere (Howell PBS (154mM Na+, 4mM K+, 10mM HPOZ-, and Taylor, 1968; Lernmark et al., 1976). Groups 140 mM C1-, pH 7.4) and sonicated for 10 sec in of 150 islets were kept free floating in the RPMI 300 pl of HzO; aliquots were then taken for DNA 1640 medium containing 11.1mM glucose, 10% content analysis (Kissane and Robins, 1958). The fetal bovine serum, 2 mM L-glutamine, 100 U/ml rest of the sonicated sample was precipitated, benzpenicillin, and 0.1 mg/ml streptomycin at subsequently washed with ice-cold 10% trichlor37°C in a gas phase of 95% humidified air/5% acetic acid and dissolved in 50 pl of Soluene. The COz. On day 2, the standard medium was radioactivity incorporated was determined by exchanged. On day 4, the islets were dispersed scintillation counting after addition of 1 ml of by trypsin/EDTA treatment (Lernmark et al., Instagel. 1976) to wells of Multiwellm tissue culture plate. Pancreatic islet cell monolayers were cultured Light Microscopy and Determination of 24 hr as described above and then transfected Labelling Indices with the DNA constructs using the LipofectinTM method (Felgner et al., 1987). The transfection For estimates of autoradiographic labelling indimixture contained 30 pl of Lipofectin suspension ces, 0.5 pCi/ml of I3H1thymidine was added to and 10 p g of DNA in total volume of 100 pl. After the culture in 1%fetal bovine serum during the 15 min incubation at room temperature, the Lipo- last 2 hr of the culture period. Then the islets fectin reagent-DNA complex suspension was were rinsed by PBS, fixed in Bouin's solution for diluted by 500p1 of serum-free RPMI medium 24 hr and embedded in paraffin. Sections, 7 p m and used for transfection procedure in four wells. thick, were processed for autoradiography and After a 3 hr incubation, the transfection was stained immunocytochemically for insulin stopped by adding 1 ml of standard medium con- (Korsgren et al., 1988; Swenne et al., 1980). For taining 1% donor calf serum and monolayers each experiment 2,000 cells were evaluated with regard to immunocytochemical staining and were incubated 18 hr as described above. Since the determination of labelling indices autoradiographic labelling in order to determine requires intact islet morphology, adult rat islets the frequency of insulin-positive cells and their were collected free from exocrine tissue in corresponding labelling index. serum-free standard medium and transfected with DNA constructs immediately after isolation. Isolation of RNA Lipofectin reagent-DNA complex suspension (100 pl) was added dropwise to the islets in 500 p 1,000 adult rat islets after transfection and culture 1 of serum-free standard medium and incubated were picked carefully, rinsed with PBS, centri3 hr as described above. The transfection was fuged shortly and frozen at -70°C. Intact FWA stopped by addition of 5 ml of standard medium has been prepared from these islets by disrupting supplemented with 10% fetal bovine serum and the cells in guanidium lysis solution (Chirgwin et the islets were maintained 48hr in standard al., 1979; Sambrook et al., 1989a). Selection medium containing 10% fetal bovine serum and of poly(A') FWA was performed by batch 24 h r in 1% fetal bovine serum. For pratical affinity chromatography on oligo(dT)-cellulose reasons, the above proceudre was also used for (Sambrook et al., 1989b). transfection of islets for RNA isolation, which were washed with PBS and frozen at -70 "C after Polymerase Chain Reaction (PCR) the culture period. Estimation of pcell DNA synthesis was Isolated rat pancreatic islets were transfected slightly modified from that described by Welsh with either the coding sequences of the chimeric et al. (1988). The cells were cultured 18 hr in 1% receptor in bluescript (control) or with pPDGFXIslet Isolation, Transfection Procedures and Estimation of DNA Synthesis
Growth Factors Downloaded from informahealthcare.com by UB der LMU Muenchen on 06/20/14 For personal use only.
95
Growth Factors Downloaded from informahealthcare.com by UB der LMU Muenchen on 06/20/14 For personal use only.
96
MARES, CLAESSON-WELSH and WELSH
p/FGFR-1. Three days later RNA was isolated and treated with DNAse I (20 U/25 pl) for 30 rnin at 37 "C. The RNA was then phenol extracted and ethanol precipitated before first strand cDNA synthesis. First strand cDNA was synthesized using the Riboclone cDNA synthesis system using l p g poly(A') RNA. RNA was then hydrolyzed in 0.5 M NaOH at 55 "C for 15 rnin after which DNA was ethanol precipitated. The cDNAs were dissolved in loop1 water and samples of 3-6 p1 were used for the amplification reactions using 0.2 pM primers but otherwise the components of the PCR reactions provided by the GeneAmp kit were used. Two cycles of 1min at 94 "C, 3 min at 37 "C and 1 min at 72 "C were run before 25 cycles of 1 min at 94 "C, 2 min at 55 "C and 1 min at 72 "C. The oligonucleotide primers were 18-mers derived from amino acid 510-516 of the PDGF Preceptor and nucleotides 1330-1312 of the FGF receptor-1, thus only amplifying chimeric sequences. Subsequent to PCR the samples were subjected to Southern blot analysis (Southern, 1975) using labelled pPDGFR-P /FGFR-l as a probe.
not amplify any sequences hybridizing to pPDGFR-P/FGFR-l (Fig. 1, lane 1).
Scatchard Analysis COS cells at a density of 2x1O5/we1l in Costar 12well dishes were transfected with pPDGFR-P /FGFR-1 using Lipofectin as above. After three days, Scatchard analysis was performed using '251-PDGF-BB and different concentrations of unlabeled PDGF-BB (1-251 ng/ml) as described (Claesson-Welsh et al., 1989).
FIGURE 1. Expression of the chimeric PDGFR-PIFGFR-1 receptor construct in islet cells using PCR. Freshly isolated rat pancreatic islets were transfected with the PDGFR-B/FGFR-l coding sequences in bluescript plasmid (control; lane 1) or with pPDGFR-BIFGFR-1 (lane 2) and cultured according to the same protocol as in the experiments for the determination of labelling indices. The PCR products were separated by electrophoresis on a 1.2%agarose gel. The DNA was transferred to nitrocellulose by standard procedure, and the blots were hybridized with I3'P1labelled pPDGFR-P/FGFR-1 as a probe.
Statistical Analysis MeanskS.E.M. were calculated, and comparisons between control and the other groups were performed by Student's paired f-test.
RESULTS Expression of the Transfected Constructs Islets transfected with pPDGFR-P/FGFR-l amplified a product of 0.3 kb which hybridized to pPDGFR-P/FGFR-l sequences (Fig. 1, lane 2). Islets transfected with the sequences coding for the chimeric receptor but not inserted into an expression vector (control transfected islets) did
Scatchard Analysis To assess the binding characteristics of the chimeric receptor construct, COS-I cells were transfected using Lipofectin with pPDGFR-P/FGFR-l (3), and binding of lz5I-PDGF-BBwas then studied. Figure 2 shows the fraction bound/free ligand plotted against bound ligand. The Kd for binding of PDGF-BB was estimated using the RBINDING program (van Zoelen, 1989), and was found to be 0.7 nM which is very similar to that obtained for the wildtype PDGF P-receptor expressed endogenously in human foreskin fibroblasts (Kd=0.5 nM) (Ostman et al., 1989).
RECEPTOR CHIMERA AND /3-CELL GROWTH
97
0,12
0,lO
W
W K
0,08
FIGURE 2. Binding of lz5I-PDGF-
t n z
3
0
0,06
m
Growth Factors Downloaded from informahealthcare.com by UB der LMU Muenchen on 06/20/14 For personal use only.
0,04
0,02
0
2
4
BOUND (ng)
Effect of the Cotransfection with pHIsis and pPDGFR-/3 FGFR-1 on Islet Cell DNA Synthesis
6
BB to COS cells expressing pPDGFR-B/FGFR-l. The pPDGFR-B /FGFR-1 Lipofectin transfected COS cells were cultured for three days and then incubated with 1ng/ml of iodinated PDGF-BB in the presence of 0, 1.2, 2.5, 5, 10, 25, 100 or 250 ng/ml unlabelled PDGF-BB and the cell bound radioactivity was measured. The fraction bound/free ligand was plotted against bound ligand. Non specific binding (about 10%) was estimated by parallel 8 determination with 100-fold excess of unlabelled ligand. The I
