Platelet-derived Growth Factor and Growth-related Genes in Rat Lung. I. Developmental Expression Shilpa Buch, Colin Jones, Neil Sweezey, Keith Tanswell, and Martin Post Neonatal Division, Department of Paediatrics, and the Department of Nephrology, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada
The autocrine, paracrine, or systemic growth factors responsible for fetal lung cell growth are not completely defined. The progression-type insulin-like growth factors and epidermal growth factor, or transforming growth factor-a acting through the epidermal growth factor receptor, appear to act on the developing lung epithelium. The competence factors that facilitate the actions of progression factors during lung growth are unknown. Fetal rat lung cells in vitro synthesize a platelet-derived growth factor (PDGF)-like polypeptide, which we have hypothesized may play a paracrine role in normal lung development. Slot blot and Northern blot analyses of fetal rat lung mRNA have been used to determine if there is a relationship between expression of message for PDGF-A or PDGF-B chains, or their cognate receptors, and periods of maximal growth during late fetal rat lung development. Whole lung mRNA was extracted on 18, 19, 20, 21, and 22 days of gestation (term = 22 days). The peak of DNA synthesis, as assessed by expression of message for DNA polymerase a, histone 3, and the proto-oncogenes c-fos and c-myc, which are stimulated by binding of growth factors including PDGF, occurred during the canalicular stage of lung development on days 19 and 20 of gestation. Expression of message for PDGF-A and PDGF-B chains was low during the pseudoglandular stage on day 18, peaked during the canalicular stage on days 19 and 20, then fell again during the saccular stage at days 21 and 22 of gestation. Message for PDGF receptors was not apparently related to the stage of lung development but did decline dramatically immediately prior to delivery.
Growth of the mammalian fetal lung, in preparation for air breathing after birth, occurs in three well-described stages. These are, in order, the pseudoglandular stage of airway development, the canalicular stage during which the respiratory portion of the lung is developed and there is increasing vascularization of the mesenchyme, and the saccular stage of increased differentiation of the respiratory region (1). The control of proliferation of most eukaryotic cells is brought about by growth factors that eventually regulate the expression of specific growth-related genes. Cell division is believed to require the action of two types of growth factors. Progression-type growth factors result in cell division provided the cell has first been primed to respond by a competence-type growth factor (2). There is evidence that epidermal growth factor (EGF) plays a role in growth of airway epithelium (3), while insulin-like growth factors (IGF) (Received in original form October 30, 1990 and in final form March 19, 1991) Address correspondence to: Dr. M. Post, Division of Neonatology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G lX8, Canada. Abbreviations: epidermal growth factor, EGF; insulin-like growth factor, IGF; platelet-derived growth factor, PDGF; sodium dodecyl sulfate, SDS. Am. J. Respir. Cell Mol. BioI. Vol. 5. pp. 371-376, 1991
can be localized to the respiratory epithelium (4, 5). In contrast to these progression factors, very little is known about the appearance of competence-type growth factors during lung growth. Platelet-derived growth factor (PDGF), a competencetype growth factor for cells of mesenchymal origin (6), consists of a disulfide-linked Jilmer of two related polypeptide chains, A and B, that can be assembled together to give various PDGF isoforms (i.e., PDGF-AA, PDGF-AB, PDGFBB). Whereas the PDGF-AB and PDGF-BB isoforms are consistently mitogenic, the mitogenicity of the PDGF-AA isoform appears to vary between species (7). Autocrine production of PDGF in neoplastic transformation and tumor development has been well documented (6, 8, 9). Besides malignant transformation, autocrine or paracrine secretion of PDGF is believed to play an important role during normal growth and development in a variety of systems (6). For example, the c-sis gene (encoding the PDGF-B chain) is expressed throughout mouse embryonic development (10). Arterial endothelial cells (11), activated macrophages (12), and arterial smooth muscle cells from the rat (13) are also known to synthesize and secrete PDGF-like mitogens. Embryonal carcinoma cells and cytotrophoblasts from the human placenta transcribe the c-sis gene and produce the protein during the first trimester (14).
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We have previously reported (15) the secretion of a competence-like factor by purified populations of fetal lung fibroblasts. Upon partial purification of this factor, it became evident that this factor shared characteristics with that of PDGF. It was therefore of interest to us to investigate the expression of the genes for PDGF and its two receptors during fetal lung development because PDGF production has been reported to be developmentally regulated in other cell types (13). In the studies reported here, we investigated the mRNA abundance of the four growth-related genes, DNA polymerase a, histone 3, c-fos, and c-myc, in relation to the mRNA abundance of PDGF and its receptors during fetal rat (term = 22 days) lung development. Tissue was studied at the pseudoglandular stage (day 18), the canalicular stage (days 19 and 20), and the saccular stage (days 21 and 22).
Materials and Methods Materials Radioisotopes and nylon membrane were purchased from Amersham (Arlington Heights, IL), and restriction enzymes and dextran sulfate from Pharmacia (Baie D'Urfe, Quebec, Canada). Bovine serum albumin type V, Ficoll 400, polyvinylpyrolidone, guanidinium thiocyanate, cesium chloride, and salmon sperm DNA were purchased from Sigma Chemical Co. (St. Louis, MO), and oligo(dT)-cellulose was obtained from Boehringer Mannheim (Mannheim, Germany). Organic solvents were of high performance liquid chromatography grade. Animals were obtained from our own breeding colony of Wistar rats, originally obtained from Charles River Laboratories (Wilmington, MA). The l.3-kb human PDGF-A cDNA fragment (clone 13.1) and the 750-bp cDNA fragment of the human PDGF A-type receptor were generous gifts from Dr. C. Betsholtz (Ludwig Institute for Cancer Research, Uppsala, Sweden). The mouse PDGF B-type receptor cDNA fragment (1.16 kb, clone pGR102) was provided by Dr. 1. A. Escobedo (University of California, San Francisco, CAl, the 700-bp cDNA fragment of human pcD-KB polymerase a was obtained from Dr. T. Wong (Stanford University, Palo Alto, CAl, and the human histone 3 cDNA fragment (2.1 kb, clone pF0535) was a gift from Dr. G. S. Stein. (Massachusetts Medical School, Worcester, MA). The 2.6-kb PDGF-B cDNA fragment (clone pSM-l), the 4.8-kb mouse c-myc cDNA fragment (clone pSVcmyc1), and the 6.8-kb mouse c-fos cDNA fragment (clone pc-fos [mouse]-3) were purchased from the American Type Culture Collection (Rockville, MD). Isolation of Lung RNA Pregnant rats at known gestation (days 18 to 22) were killed by decapitation. The fetuses were immediately removed from the uterus. The fetal thoracic contents were removed en bloc, and the lungs dissected away from vessels and large airways to be flash-frozen in liquid nitrogen. Adult rats were killed with diethylether, and their lungs were isolated and flash-frozen in liquid nitrogen. Total (nuclear and cytoplasmic) RNA was isolated by lysing the tissue in 4 M guanidinium thiocyanate followed by centrifugation on a 5.7-M cesium chloride cushion to pellet RNA (16). After extraction with phenol:chloroform (1:1, vol:vol), the RNA was
ethanol-precipitated and collected by centrifugation. This RNA was lyophilized and dissolved in water. RNA integrity was confirmed by fractionation on 1.2 % (wt.vol) agaroseformaldehyde gels and staining the ribosomal RNA bands with ethidium bromide. PolytA)"-enriched mRNA was isolated directly from 19-day fetal lung tissue lysates by a modification of an oligo(dT)-cellulose affinity chromatography procedure (17). Slot Blot and Northern Blot Analysis For slot blot analysis, 0.6- to 5-J.tg aliquots of RNA were applied in a final200-J.tl solution of 6.15 M formaldehyde and iox SSC (1.5 M sodium chloride, 0.15 M sodium citrate [pH 7.0]). The RNA was denatured at 65° C for 15 min and immediately immobilized on nylon membrane under slow vacuum filtration. After the solution had filtered through, the wells were rinsed with 500 J.tllOx SSC, and RNA was then fixed by baking at 80° C for 2 h. Included in the slot blot analysis as negative control was 5S RNA. For Northern blot hybridization, 10 J.tg poly(A)+ RNA was fractionated on 1.2% (wt:vol) agarose gels containing 6% (vol:vol) formaldehyde and transferred to a nylon membrane, which was fixed by baking at 80° C for 2 h. All probes were labeled with deoxycytidine 5'-[a-32P]triphosphate by a random primed labeling system (Amersham), with specific activities of 0.5 to 2.9 x 109 cpm/ug DNA. Prehybridization (5 h to overnight) and hybridization were performed in 50% (vol:vol) formamide, 750 mM NaCl, 75 mM sodium citrate, 5x Denhardt's solution (0.4% [wt:vol] each of bovine serum albumin Ficoll, and polyvinylpyrrolidone), 10% (wt:vol) dextran sulfate, and 100 J.tg/ml denatured salmon sperm DNA for 24 to 48 h at 42° C. Washes varied with the probe used and were as follows. Blots hybridized with the histone 3 probe were washed in 5 X SSC, 0.1% (wt:vol) sodium dodecyl sulfate (SDS) at 37° C for 20 min. Blots hybridized with the DNA polymerase a probe were washed in 5x SSC, 0.1% (wt:vol) SDS at 42° C for 15 min, followed by 2x SSC, 0.1% (wt:vol) SDS at 42° C for 30 min. Blots hybridized with the c-fos and c-myc probes were washed as previously described, followed by 0.2x SSC, 0.1% (wt:vol) SDS at room temperature for 15 min. PDGF-A chain, PDGF-B chain, and PDGF-A receptor blots were washed very stringently, with a final wash of 0.1x SSC, 0.1% (wt:vol) SDS at 42° C for 30 min. Under low stringency conditions, PDGF-A receptor blots had a final wash of2x SSC, 0.1% (wt:vol) SDS at42° C for 10 min. The final wash for the PDGF-B receptor blots was 2x SSC, 0.1% (wt.vol) SDS at 42° C for 10 min. The blots were exposed for 24 to 48 h to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) using Dupont Cronex Intensifying screens (E. I. Dupont, Clifton, NJ). The films were quantified by an Ultroscan XL laser densitometer (LKB, Bromma, Sweden). Data Presentation Values are presented as the mean ± SEM of arbitrary densitometric units for three individual experiments, with RNA for each set of experiments isolated from at least two litters. Statistical significance (P < 0.05) was determined by ANOVA followed by assessment of differences using Dunnet's twosided test (18) or Duncan's multiple range test (19).
Buch, Jones, Sweezey et al.: PDGF and Growth-related Genes in Rat Fetal Lung
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profiles for expression of PDGF-A and PDGF-B chains closely follow those observed with polymerase cc, histone 3, c-fos, and c-myc, with a low level of expression during the pseudoglandular stage, a peak of activity at days 19 and 20 of gestation during the canalicular stage, and then a decline during the fetal saccular stage. The expression of PDGF-A in adult rat lung was slightly greater than that of day 22 fetal lung. In contrast, PDGF receptor mRNA levels do not appear to be related to the stage of lung maturation and are essentially unchanged until there is an acute decline on the day of delivery. Based on the above data, lung tissue from day 19 fetuses was chosen for isolation of poly(A)+ RNA for Northern analysis to confirm the fact that each of the probes was picking up the correct transcript. RNA transfer blot analysis illustrated in Figure 8, lane 1, shows three hybridizing bands of2.9, 2.3, and 1.7kb for the PDGF-A chain. Transcripts 1.7 and 2.3 kb, respectively, constituted a major abundance in the day 19 fetal lung, whereas the larger 2.9-kb transcript represented a small proportion of the total. A discrete band of 3.5 kb was present in the day 19 fetal lung upon hybridization with the PDGF-B chain eDNA (Figure 8, lane 2). Under low-stringency hybridization conditions, PDGF-A-type receptor eDNA detected two transcript sizes (approximately 5.3 and 3.0kb) (Figure 9, lane 1). The 5.3-kb transcript probably represents the B-type receptor transcript. Under highstringency conditions, however,only a single transcript of 3.0 kb was detected (Figure 9, lane 2). Using the B-type receptor eDNA, a 5.3-kb transcript was detected (Figure 9, lane 3).
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gestation. Northern blot analysis of poly(A)+ RNA from day 19 fetal lung revealed transcripts of 0.9,2.2, and 2.3 kb for histone 3, c-fos, and c-myc probes, respectively (Figure 4). Expression of the Genes for PDGF-A and PDGF-B Chains, and Their Receptor Genes Densitometric analysis of slot blots showing developmental changes in expression of each of the PDGF chains and the receptor genes is presented in Figures 5 through 7. The
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Buch, Jones, Sweezey et al.: PDGF and Growth-related Genes in Rat Fetal Lung
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Discussion Prenatal lung morphogenesis and growth involves various preparatory events to facilitate efficient gas exchange upon birth. These changes comprise an increase in respiratory gas exchange surface area by cell proliferation, and differentiation of cells to carry out highly specialized functions. We used mRNA abundance of the growth-coupled genes DNA polymerase ex, histone 3, c-fos, and c-myc as markers of peak proliferation. The DNA polymerase ex gene plays a major role in eukaryotic DNA replication (20), with its expression being constitutive throughout the cell cycle (21), and its regulation being exerted at the transcriptional level. DNA polymerase ex steady-state mRNA levels, and subsequent translation as assayed by enzymatic activities, positively correlate with cell proliferation (22). Histone 3 gene expression is an essential component of cell proliferation, because of the stringent requirement for histone proteins to package newly replicated DNA into nucleosomes (23, 24). We observed maximal steady-state levels of the mRNAs from these growth-coupled genes in the rat fetal iung at 19 to 20 days of gestation, the canalicular stage of lung development. The specificity of the probes used for these studies was confirmed by Northern blot analysis. Similar sized bands to those observed here, although of differing intensities, have been
reported for rat myogenic cells (25). Gestation-dependent profiles for DNA synthesis have also been observed in autoradiographic studies of developing lung (26). Expression of PDGF-A and PDGF-B chain mRNAs is maximal during this same period, suggesting a role for this competence factor during this stage of lung growth. The specificity of the probes used to measure PDGF chain mRNA expression was confirmed by Northern blot analysis. Similar transcript sizes to those observed in this study have been reported earlier in different systems by other investigators (27). The human PDGF A-type receptor cDNA, however, detects a single transcript of 3.0 kb in fetal rat lung, which differs in size from the human (28) and the rat carotid artery (29) transcript of 6.5 kb. Under conditions of lower stringency, the PDGF-A receptor cDNA detects two messages of 5.3 and 3.0 kb in fetal rat lung, most likely due to cross hybridization of the A-type receptor cDNA to the larger B-type receptor transcript. Recently, a smaller transcript of 4.8 kb for the A-type receptor has been reported in regions of monkey brain tissue (30). Similar findings of smaller sized mRNA for the A-type receptor have also been observed in other cells (30). The significance and nature of such a smaller message size detected by the A-type receptor cDNA in fetal rat lung remains to be elucidated. A truncated
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Figure 8. RNA transfer blots illustrating PDGF-A chain (lane 1) and PDGF-B chain (lane 2) transcripts in fetal rat lung at 19 days of gestation. The amount of poly(At RNA applied was 10 ug. Exposure time was 48 h.
Figure 9. Northern blots of the PDGF A-type (lanes 1 and 2) and the B-type (lane 3) receptor transcripts in fetal rat lung at 19 days of gestation. The amount of RNA applied in lane 1 was 30 p.g total RNA and that in lanes 2 and 3 was 10 ug poly/A):' RNA. Exposure time was 48 h.
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form of the mouse B-type receptor has been observed in a mouse embryonal carcinoma and embryonic stem cells (31) and has been found to function independently of the ligand during early mouse embryogenesis. Whether the 3.0-kb transcript in fetal rat lung is a truncated A-type receptor form remains to be established. Progression type growth factors such as IOF-I, IGF-II (4, 5), and EGF or transforming growth factor-a binding to the EGF receptor (3) have been localized to the respiratory and airway epithelium during lung development. The mRNAs for PDGF are known to increase following lung injury (32) in adult animals. The data presented here document the in vivo expression of PDGF-A chain, PDGF-B chain, and the PDGF B-type receptor genes in the development of the rat fetal lung. The peaks of steady-state mRNA for PDGF-A chain and PDGF-B chain during the canalicular stage of lung development suggest a specific role for PDGF at this stage, during which the saccular epithelium becomes recognized and vascularization occurs (1). Unlike the situation in the adult lung following injury (32), the expression of PDGF mRNA in the fetal lung does not precede peak DNA synthesis by 2 to 3 days. The source and site of action of PDGF within the fetal rat lung remains to be determined. Not only is the expression of PDGF developmentally regulated (13), but there are apparent species differences in sensitivity (7). Despite the usual assumption that PDGF acts only on cells of mesenchymal origin (6), our previous observations in vitro (15) suggest that at some stages of fetal lung development pulmonary epithelial cells are capable of responding to PDGF. Its role during the canalicular stage of lung maturation may therefore relate to the appearance of the respiratory epithelium. It is also possible that PDGF controls the pulmonary vascularization characteristic of this stage of lung development, because small vessel endothelial cells have PDGF receptors (33). Definitive answers must await localization by immunocytochemical and in situ hybridization techniques. Acknowledgments: We thank Stuart Rae for expert technical assistance. These studies were supported by Grants PG-42 and MT7867 from the Medical Research Council of Canada and by Grant HL-43416 from the National Heart, Lung and Blood Institute. Dr. Jones is a Fellow of the Kidney Foundation of Canada.
References 1. Meyrick, B., and L. M. Reid. 1977. Ultrastructure of alveolar lining and its development. In Development of the Lung. W. A. Hodson, editor. Marcel Dekker, New York. 135-214. 2. Stiles, C. D., G. T. Capne, C. D. Scher, H. M. Antoniades, J. J. Van Wyk, and W. J. Pledger. 1979. Dual control of cell growth by somatomedins and platelet derived growth factor. Proc. Natl. Acad. Sci. USA 76: 1279-1283. 3. Stahlman, M. T., D. N. Orth, and M. E. Gray. 1989. Immunocytochemical localization of epidermal growth factor in the developing human respiratory system and in acute and chronic lung disease in the neonate. Lab. Invest. 60:539-547. 4. Han, V. K. M, D. J. Hill, A. J. Strain et al. 1987. Identification of somatomedin/insulin-Iike growth factor immunoreactive cells in the human fetus. Pediatr. Res. 22:245-249. 5. Beck, F., N. J. Samani, J. D. Penschow, B. Thorley, G. W. Tregear, and J. P. Coghlan. 1987. Histochemical localization ofIGF-I and -II mRNA in the developing rat embryo. Development 101:175-184. 6. Ross, R., E. W. Raines, and D. F. Bowen-Pope. 1986. The biology of platelet-derived growth factor. Cell 46:155-169. 7. Sturani, E., R. Brambilla, L. Morello, M. G. Cattaneo, R. Zippel, and L. Alberghina. 1989. Effect of the different dimeric forms of the platelet-
derived growth factor on cellular responses in mouse Swiss 3T3 fibroblasts. FEBS Leu. 255:191-195. 8. Deuel, T. F. 1987. Polypeptide growth factors: roles in normal and abnormal cell growth. Annu. Rev. Cell Biol. 3:443-492. 9. Betsholtz, C., B. Westermark, B. Ek, and C.-H. Heldin. 1984. Coexpression of a PDGF-like factor and PDGF receptors in human osteosarcoma cell line: implications for autocrine receptor activation. Cell 39:447-457. 10. Slamon, D. J., and M. J. Cline. 1984. Expression of cellular oncogenes during embryonic and fetal development of the mouse. Proc. Natl. Acad. Sci. USA 81:7141-7145. 11. DiCorleto, P. E., and D. F. Bowen-Pope. 1983. Cultured endothelial cells produce a platelet-derived growth factor-like protein. Proc. Natl. Acad. Sci. USA 80:1919-1923. 12. Rappolee, D. A., and Z. Werb. 1990. mRNA phenotyping for studying gene expression in small numbers of cells: platelet-derived growth factor and other growth factors in wound-derived macrophages. Am. J. Respir. Cell Mol. Bioi. 2:3-10. 13. Seifert, R. A., S. M. Schwartz, and D. F. Bowen-Pope. 1984. Developmentally regulated production of platelet-derived growth factor-like molecules. Nature 311:669-671. 14. Goustin, A. S., C. Betsholtz, S. Pfeifer-Ohlsson etal. 1985. Co-expression of the sis and myc proto-oncogenes in human placenta suggests autocrine control of trophoblast growth. Cell 41:301-312. 15. Stiles, A. D., B. T. Smith, and M. Post. 1986. Reciprocal autocrine and paracrine regulation of growth of mesenchymal and alveolar epithelial cells from fetal lung. Exp. Lung Res. 11:165-177. 16. Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. 1. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299. 17. Badley, J. E., G. A. Bishop, T. St. John, and J. A. Frelinger. 1988. A simple, rapid method for the purification of poly A+ RNA. Biotechniques 6:114-116. 18. Snedecor, G. W., and W. G. Cochran. 1980. Statistical Methods. Iowa State University Press, Ames, IA. 19. Steel, R. G. D., and J. H. Torrie. 1970. Principles and Procedures of Statistics. McGraw Hill, New York. 20. Fry, M., and L. A. Loeb. 1986. Animal Cell DNA Polymerases. 1986. CRC Press, Boca Raton, FL. 13-60. 21. Wahl, A. F., A. M. Geis, B. H. Spain, S. W. Wong, D. Korn, and T. S.-F. Wang. 1988. Gene expression of human DNA polymerase a during cell proliferation and the cell cycle. Mol. Cell. Bioi. 8:5016-5025. 22. Kaczmarek, L., M. R. Miller, R. A. Hammond, and W. E. Mercer. 1986. A microinjected monoclonal antibody against human DNA polymerase-a inhibits DNA replication in human, hamster, and mouse cell-lines. 1 Bioi. Chem. 261:10802-10807. 23. McGhee,1. D., and G. Felsenfeld. 1980. Nucleosome structure. Annu. Rev. Biochem. 49:1115-1156. 24. Weisbrod, S. 1982. Active chromatin. Nature 297:289-295. 25. Sejersen, T., C. Betsholtz, M. Sjolund, C.-H. Heldin, B. Westermark, and 1. Thyberg. 1986. Rat skeletal myoblasts and arterial smooth muscle cells express the gene for the A-chain but not the gene for the B-chain (c-sis) of platelet derived growth factor (PDGF) and produce a PDGF-Iike protein. Proc. Natl. Acad. Sci. USA 83:6844-6848. 26. Adamson, I. Y. R., and G. M. King. 1984. Sex differences in development offetal rat lung. I. Autoradiographic and biochemical studies. Lab. Invest. 50:456-460. 27. Betsholtz, C., A. Johnsson, C.-H. Heldin et al. 1986. cDNA sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumour cell lines. Nature 320: 695-699. 28. Claesson-Welsh, L., A. Eriksson, B. Westermark, and C.-H. Heldin. 1989. cDNA cloning and expression of the human A-type platelet-derived growth factor (PDGF) receptor establishes structural similarity to the B-type PDGF receptor. Proc. Natl. Acad. Sci. USA 86:4917-4921. 29. Mayesky,M. W., M. A. Reidy, D. F. Bowen-Pope, C. E. Hart, J. N. Wilcox, and S. M. Schwartz. 1990. PDGF ligand and receptor gene expressionduring repair of arterial injury. J. Cell Bioi. 111:2149-2158. 30. Sasahara, M., 1. W. U. Fries, E. W. Raines et al. 1991. PDGF-B chain in neurons of the central nervous system, posterior pituitary, and in a transgenic model. Cell 64:217-227. 31. Vu, T. H., G. R. Martin, P. Lee, D. Mark, A. Wang, and L. T. Williams. 1989. Developmentally regulated use of alternative promoters creates a novel platelet-derived growth factor receptor transcript in mouse teratocarcinoma and embryonic stem cells. Mol. Cell. Bioi. 9:4563-4467. 32. Fabisiak, 1. P., 1. N. Evans, and 1. Kelley. 1989. Increased expression of PDGF-B (c-sis) mRNA in rat lung precedes DNA synthesis and tissue repair during chronic hyperoxia. Am. J. Respir. Cell Mol. Bioi. 1:181-189. 33. Bar, R. S., M. Boes, B. A. Booth, B. L. Dake, S. Henley, and M. N. Hart. 1989. The effects of platelet-derived growth factor in cultured microvessel endothelial cells. Endocrinology 124:1841-1848.
The autocrine, paracrine, or systemic growth factors responsible for fetal lung cell growth are not completely defined. The progression-type insulin-like growth factors and epidermal growth factor, or transforming growth factor-a acting through the epidermal growth factor receptor, appear to act on the developing lung epithelium. The competence factors that facilitate the actions of progression factors during lung growth are unknown. Fetal rat lung cells in vitro synthesize a platelet-derived growth factor (PDGF)-like polypeptide, which we have hypothesized may play a paracrine role in normal lung development. Slot blot and Northern blot analyses of fetal rat lung mRNA have been used to determine if there is a relationship between expression of message for PDGF-A or PDGF-B chains, or their cognate receptors, and periods of maximal growth during late fetal rat lung development. Whole lung mRNA was extracted on 18, 19, 20, 21, and 22 days of gestation (term = 22 days). The peak of DNA synthesis, as assessed by expression of message for DNA polymerase a, histone 3, and the proto-oncogenes c-fos and c-myc, which are stimulated by binding of growth factors including PDGF, occurred during the canalicular stage of lung development on days 19 and 20 of gestation. Expression of message for PDGF-A and PDGF-B chains was low during the pseudoglandular stage on day 18, peaked during the canalicular stage on days 19 and 20, then fell again during the saccular stage at days 21 and 22 of gestation. Message for PDGF receptors was not apparently related to the stage of lung development but did decline dramatically immediately prior to delivery.
Growth of the mammalian fetal lung, in preparation for air breathing after birth, occurs in three well-described stages. These are, in order, the pseudoglandular stage of airway development, the canalicular stage during which the respiratory portion of the lung is developed and there is increasing vascularization of the mesenchyme, and the saccular stage of increased differentiation of the respiratory region (1). The control of proliferation of most eukaryotic cells is brought about by growth factors that eventually regulate the expression of specific growth-related genes. Cell division is believed to require the action of two types of growth factors. Progression-type growth factors result in cell division provided the cell has first been primed to respond by a competence-type growth factor (2). There is evidence that epidermal growth factor (EGF) plays a role in growth of airway epithelium (3), while insulin-like growth factors (IGF) (Received in original form October 30, 1990 and in final form March 19, 1991) Address correspondence to: Dr. M. Post, Division of Neonatology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G lX8, Canada. Abbreviations: epidermal growth factor, EGF; insulin-like growth factor, IGF; platelet-derived growth factor, PDGF; sodium dodecyl sulfate, SDS. Am. J. Respir. Cell Mol. BioI. Vol. 5. pp. 371-376, 1991
can be localized to the respiratory epithelium (4, 5). In contrast to these progression factors, very little is known about the appearance of competence-type growth factors during lung growth. Platelet-derived growth factor (PDGF), a competencetype growth factor for cells of mesenchymal origin (6), consists of a disulfide-linked Jilmer of two related polypeptide chains, A and B, that can be assembled together to give various PDGF isoforms (i.e., PDGF-AA, PDGF-AB, PDGFBB). Whereas the PDGF-AB and PDGF-BB isoforms are consistently mitogenic, the mitogenicity of the PDGF-AA isoform appears to vary between species (7). Autocrine production of PDGF in neoplastic transformation and tumor development has been well documented (6, 8, 9). Besides malignant transformation, autocrine or paracrine secretion of PDGF is believed to play an important role during normal growth and development in a variety of systems (6). For example, the c-sis gene (encoding the PDGF-B chain) is expressed throughout mouse embryonic development (10). Arterial endothelial cells (11), activated macrophages (12), and arterial smooth muscle cells from the rat (13) are also known to synthesize and secrete PDGF-like mitogens. Embryonal carcinoma cells and cytotrophoblasts from the human placenta transcribe the c-sis gene and produce the protein during the first trimester (14).
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We have previously reported (15) the secretion of a competence-like factor by purified populations of fetal lung fibroblasts. Upon partial purification of this factor, it became evident that this factor shared characteristics with that of PDGF. It was therefore of interest to us to investigate the expression of the genes for PDGF and its two receptors during fetal lung development because PDGF production has been reported to be developmentally regulated in other cell types (13). In the studies reported here, we investigated the mRNA abundance of the four growth-related genes, DNA polymerase a, histone 3, c-fos, and c-myc, in relation to the mRNA abundance of PDGF and its receptors during fetal rat (term = 22 days) lung development. Tissue was studied at the pseudoglandular stage (day 18), the canalicular stage (days 19 and 20), and the saccular stage (days 21 and 22).
Materials and Methods Materials Radioisotopes and nylon membrane were purchased from Amersham (Arlington Heights, IL), and restriction enzymes and dextran sulfate from Pharmacia (Baie D'Urfe, Quebec, Canada). Bovine serum albumin type V, Ficoll 400, polyvinylpyrolidone, guanidinium thiocyanate, cesium chloride, and salmon sperm DNA were purchased from Sigma Chemical Co. (St. Louis, MO), and oligo(dT)-cellulose was obtained from Boehringer Mannheim (Mannheim, Germany). Organic solvents were of high performance liquid chromatography grade. Animals were obtained from our own breeding colony of Wistar rats, originally obtained from Charles River Laboratories (Wilmington, MA). The l.3-kb human PDGF-A cDNA fragment (clone 13.1) and the 750-bp cDNA fragment of the human PDGF A-type receptor were generous gifts from Dr. C. Betsholtz (Ludwig Institute for Cancer Research, Uppsala, Sweden). The mouse PDGF B-type receptor cDNA fragment (1.16 kb, clone pGR102) was provided by Dr. 1. A. Escobedo (University of California, San Francisco, CAl, the 700-bp cDNA fragment of human pcD-KB polymerase a was obtained from Dr. T. Wong (Stanford University, Palo Alto, CAl, and the human histone 3 cDNA fragment (2.1 kb, clone pF0535) was a gift from Dr. G. S. Stein. (Massachusetts Medical School, Worcester, MA). The 2.6-kb PDGF-B cDNA fragment (clone pSM-l), the 4.8-kb mouse c-myc cDNA fragment (clone pSVcmyc1), and the 6.8-kb mouse c-fos cDNA fragment (clone pc-fos [mouse]-3) were purchased from the American Type Culture Collection (Rockville, MD). Isolation of Lung RNA Pregnant rats at known gestation (days 18 to 22) were killed by decapitation. The fetuses were immediately removed from the uterus. The fetal thoracic contents were removed en bloc, and the lungs dissected away from vessels and large airways to be flash-frozen in liquid nitrogen. Adult rats were killed with diethylether, and their lungs were isolated and flash-frozen in liquid nitrogen. Total (nuclear and cytoplasmic) RNA was isolated by lysing the tissue in 4 M guanidinium thiocyanate followed by centrifugation on a 5.7-M cesium chloride cushion to pellet RNA (16). After extraction with phenol:chloroform (1:1, vol:vol), the RNA was
ethanol-precipitated and collected by centrifugation. This RNA was lyophilized and dissolved in water. RNA integrity was confirmed by fractionation on 1.2 % (wt.vol) agaroseformaldehyde gels and staining the ribosomal RNA bands with ethidium bromide. PolytA)"-enriched mRNA was isolated directly from 19-day fetal lung tissue lysates by a modification of an oligo(dT)-cellulose affinity chromatography procedure (17). Slot Blot and Northern Blot Analysis For slot blot analysis, 0.6- to 5-J.tg aliquots of RNA were applied in a final200-J.tl solution of 6.15 M formaldehyde and iox SSC (1.5 M sodium chloride, 0.15 M sodium citrate [pH 7.0]). The RNA was denatured at 65° C for 15 min and immediately immobilized on nylon membrane under slow vacuum filtration. After the solution had filtered through, the wells were rinsed with 500 J.tllOx SSC, and RNA was then fixed by baking at 80° C for 2 h. Included in the slot blot analysis as negative control was 5S RNA. For Northern blot hybridization, 10 J.tg poly(A)+ RNA was fractionated on 1.2% (wt:vol) agarose gels containing 6% (vol:vol) formaldehyde and transferred to a nylon membrane, which was fixed by baking at 80° C for 2 h. All probes were labeled with deoxycytidine 5'-[a-32P]triphosphate by a random primed labeling system (Amersham), with specific activities of 0.5 to 2.9 x 109 cpm/ug DNA. Prehybridization (5 h to overnight) and hybridization were performed in 50% (vol:vol) formamide, 750 mM NaCl, 75 mM sodium citrate, 5x Denhardt's solution (0.4% [wt:vol] each of bovine serum albumin Ficoll, and polyvinylpyrrolidone), 10% (wt:vol) dextran sulfate, and 100 J.tg/ml denatured salmon sperm DNA for 24 to 48 h at 42° C. Washes varied with the probe used and were as follows. Blots hybridized with the histone 3 probe were washed in 5 X SSC, 0.1% (wt:vol) sodium dodecyl sulfate (SDS) at 37° C for 20 min. Blots hybridized with the DNA polymerase a probe were washed in 5x SSC, 0.1% (wt:vol) SDS at 42° C for 15 min, followed by 2x SSC, 0.1% (wt:vol) SDS at 42° C for 30 min. Blots hybridized with the c-fos and c-myc probes were washed as previously described, followed by 0.2x SSC, 0.1% (wt:vol) SDS at room temperature for 15 min. PDGF-A chain, PDGF-B chain, and PDGF-A receptor blots were washed very stringently, with a final wash of 0.1x SSC, 0.1% (wt:vol) SDS at 42° C for 30 min. Under low stringency conditions, PDGF-A receptor blots had a final wash of2x SSC, 0.1% (wt:vol) SDS at42° C for 10 min. The final wash for the PDGF-B receptor blots was 2x SSC, 0.1% (wt.vol) SDS at 42° C for 10 min. The blots were exposed for 24 to 48 h to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) using Dupont Cronex Intensifying screens (E. I. Dupont, Clifton, NJ). The films were quantified by an Ultroscan XL laser densitometer (LKB, Bromma, Sweden). Data Presentation Values are presented as the mean ± SEM of arbitrary densitometric units for three individual experiments, with RNA for each set of experiments isolated from at least two litters. Statistical significance (P < 0.05) was determined by ANOVA followed by assessment of differences using Dunnet's twosided test (18) or Duncan's multiple range test (19).
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profiles for expression of PDGF-A and PDGF-B chains closely follow those observed with polymerase cc, histone 3, c-fos, and c-myc, with a low level of expression during the pseudoglandular stage, a peak of activity at days 19 and 20 of gestation during the canalicular stage, and then a decline during the fetal saccular stage. The expression of PDGF-A in adult rat lung was slightly greater than that of day 22 fetal lung. In contrast, PDGF receptor mRNA levels do not appear to be related to the stage of lung maturation and are essentially unchanged until there is an acute decline on the day of delivery. Based on the above data, lung tissue from day 19 fetuses was chosen for isolation of poly(A)+ RNA for Northern analysis to confirm the fact that each of the probes was picking up the correct transcript. RNA transfer blot analysis illustrated in Figure 8, lane 1, shows three hybridizing bands of2.9, 2.3, and 1.7kb for the PDGF-A chain. Transcripts 1.7 and 2.3 kb, respectively, constituted a major abundance in the day 19 fetal lung, whereas the larger 2.9-kb transcript represented a small proportion of the total. A discrete band of 3.5 kb was present in the day 19 fetal lung upon hybridization with the PDGF-B chain eDNA (Figure 8, lane 2). Under low-stringency hybridization conditions, PDGF-A-type receptor eDNA detected two transcript sizes (approximately 5.3 and 3.0kb) (Figure 9, lane 1). The 5.3-kb transcript probably represents the B-type receptor transcript. Under highstringency conditions, however,only a single transcript of 3.0 kb was detected (Figure 9, lane 2). Using the B-type receptor eDNA, a 5.3-kb transcript was detected (Figure 9, lane 3).
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gestation. Northern blot analysis of poly(A)+ RNA from day 19 fetal lung revealed transcripts of 0.9,2.2, and 2.3 kb for histone 3, c-fos, and c-myc probes, respectively (Figure 4). Expression of the Genes for PDGF-A and PDGF-B Chains, and Their Receptor Genes Densitometric analysis of slot blots showing developmental changes in expression of each of the PDGF chains and the receptor genes is presented in Figures 5 through 7. The
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Buch, Jones, Sweezey et al.: PDGF and Growth-related Genes in Rat Fetal Lung
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Discussion Prenatal lung morphogenesis and growth involves various preparatory events to facilitate efficient gas exchange upon birth. These changes comprise an increase in respiratory gas exchange surface area by cell proliferation, and differentiation of cells to carry out highly specialized functions. We used mRNA abundance of the growth-coupled genes DNA polymerase ex, histone 3, c-fos, and c-myc as markers of peak proliferation. The DNA polymerase ex gene plays a major role in eukaryotic DNA replication (20), with its expression being constitutive throughout the cell cycle (21), and its regulation being exerted at the transcriptional level. DNA polymerase ex steady-state mRNA levels, and subsequent translation as assayed by enzymatic activities, positively correlate with cell proliferation (22). Histone 3 gene expression is an essential component of cell proliferation, because of the stringent requirement for histone proteins to package newly replicated DNA into nucleosomes (23, 24). We observed maximal steady-state levels of the mRNAs from these growth-coupled genes in the rat fetal iung at 19 to 20 days of gestation, the canalicular stage of lung development. The specificity of the probes used for these studies was confirmed by Northern blot analysis. Similar sized bands to those observed here, although of differing intensities, have been
reported for rat myogenic cells (25). Gestation-dependent profiles for DNA synthesis have also been observed in autoradiographic studies of developing lung (26). Expression of PDGF-A and PDGF-B chain mRNAs is maximal during this same period, suggesting a role for this competence factor during this stage of lung growth. The specificity of the probes used to measure PDGF chain mRNA expression was confirmed by Northern blot analysis. Similar transcript sizes to those observed in this study have been reported earlier in different systems by other investigators (27). The human PDGF A-type receptor cDNA, however, detects a single transcript of 3.0 kb in fetal rat lung, which differs in size from the human (28) and the rat carotid artery (29) transcript of 6.5 kb. Under conditions of lower stringency, the PDGF-A receptor cDNA detects two messages of 5.3 and 3.0 kb in fetal rat lung, most likely due to cross hybridization of the A-type receptor cDNA to the larger B-type receptor transcript. Recently, a smaller transcript of 4.8 kb for the A-type receptor has been reported in regions of monkey brain tissue (30). Similar findings of smaller sized mRNA for the A-type receptor have also been observed in other cells (30). The significance and nature of such a smaller message size detected by the A-type receptor cDNA in fetal rat lung remains to be elucidated. A truncated
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Figure 9. Northern blots of the PDGF A-type (lanes 1 and 2) and the B-type (lane 3) receptor transcripts in fetal rat lung at 19 days of gestation. The amount of RNA applied in lane 1 was 30 p.g total RNA and that in lanes 2 and 3 was 10 ug poly/A):' RNA. Exposure time was 48 h.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991
form of the mouse B-type receptor has been observed in a mouse embryonal carcinoma and embryonic stem cells (31) and has been found to function independently of the ligand during early mouse embryogenesis. Whether the 3.0-kb transcript in fetal rat lung is a truncated A-type receptor form remains to be established. Progression type growth factors such as IOF-I, IGF-II (4, 5), and EGF or transforming growth factor-a binding to the EGF receptor (3) have been localized to the respiratory and airway epithelium during lung development. The mRNAs for PDGF are known to increase following lung injury (32) in adult animals. The data presented here document the in vivo expression of PDGF-A chain, PDGF-B chain, and the PDGF B-type receptor genes in the development of the rat fetal lung. The peaks of steady-state mRNA for PDGF-A chain and PDGF-B chain during the canalicular stage of lung development suggest a specific role for PDGF at this stage, during which the saccular epithelium becomes recognized and vascularization occurs (1). Unlike the situation in the adult lung following injury (32), the expression of PDGF mRNA in the fetal lung does not precede peak DNA synthesis by 2 to 3 days. The source and site of action of PDGF within the fetal rat lung remains to be determined. Not only is the expression of PDGF developmentally regulated (13), but there are apparent species differences in sensitivity (7). Despite the usual assumption that PDGF acts only on cells of mesenchymal origin (6), our previous observations in vitro (15) suggest that at some stages of fetal lung development pulmonary epithelial cells are capable of responding to PDGF. Its role during the canalicular stage of lung maturation may therefore relate to the appearance of the respiratory epithelium. It is also possible that PDGF controls the pulmonary vascularization characteristic of this stage of lung development, because small vessel endothelial cells have PDGF receptors (33). Definitive answers must await localization by immunocytochemical and in situ hybridization techniques. Acknowledgments: We thank Stuart Rae for expert technical assistance. These studies were supported by Grants PG-42 and MT7867 from the Medical Research Council of Canada and by Grant HL-43416 from the National Heart, Lung and Blood Institute. Dr. Jones is a Fellow of the Kidney Foundation of Canada.
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