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The proliferative effects of retinoic acid on primary cultures of adult rat type II pneumocytes depend upon cell density

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Abstract

Retinoic acid (RA) is important for maintaining integrity of alveolar epithelial cells, but the mechanism has not been defined. We cultured type II pneumocytes at confluent, high cell density (104 cells/mm2) and found that RA (10−6 M) inhibited thymidine incorporation to 60% of control, despite a dose-dependent increase in epidermal growth factor receptor (EGFR) levels. However, at lower, subconfluent density (102 cells/mm2), RA stimulated thymidine incorporation to 280% of control. EGF increased thymidine incorporation at concentrations as low as 0.1 ng/mL, but no further increase was observed at higher concentrations up to 100 ng/mL. In subconfluent cells co-treated with EGF (100 ng/mL) and increasing concentrations of RA (10−8 M–10−5 M RA), thymidine incorporation was significantly greater at all concentrations than RA alone, with greatest increases observed at 10−7 (422% of control) and 10−6 (470% of control) M RA. In summary, the effects of RA on thymidine incorporation are sensitive to changes in cell density. RA inhibits thymidine incorporation at high cell density and stimulates thymidine incorporation at low density. RA increases EGFRs in cultured type II pneumocytes, and EGF stimulates thymidine incorporation independent of the cultured cell density. These data may help to explain how RA mediates lung repair in vivo.

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Abbreviations

B-EGF:

Biotin-labeled EGF

BSA:

Bovine serum albumin

DMEM:

Dulbecco’s modified Eagle’s medium

EGF:

Epidermal growth factor

EGFR:

Epidermal growth factor receptor

ELISA:

Enzyme-linked immunosorbent assay

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

Mab:

Monoclonal antibody

PBS:

Phosphate-buffered saline

RA:

Retinoic acid

[3H]thymidine:

Tritiated thymidine

References

  • Adamson I. Y. R.; Bowden D. H. The type II cell as progenitor of alveolar epithelial regeneration: a cytodynamic study in mice after exposure to oxygen. Lab. Invest. 30: 35–42; 1974.

    CAS  PubMed  Google Scholar 

  • Baybutt R. C.; Aziz S. M.; Fagerland J. A.; Olson J. W.; Gillespie M. N. Monocrotaline alters type II morphology and polyamine regulation. Toxicol. Appl. Pharmacol. 129: 188–195; 1994.

    Article  CAS  PubMed  Google Scholar 

  • Baybutt R. C.; Hu L.; Molteni A. Vitamin A deficiency injures lung and liver parenchyma and impairs function of rat type II pneumocytes. J. Nutr. 130: 1159–1165; 2000.

    CAS  PubMed  Google Scholar 

  • Bordier C. Phase separation of integral membrane proteins in triton X-114 solution. J. Biol. Chem. 256: 1604–1607; 1981.

    CAS  PubMed  Google Scholar 

  • Choi J. H.; Oh J. Y.; Ryu S. K.; Kim S. J.; Lee N. Y.; Kim Y. S.; Yi S. Y.; Shim K. S.; Han W. S. Detection of epidermal growth factor receptor in the serum of gastric carcinoma patients. Cancer 79: 1879–1883; 1997.

    Article  CAS  PubMed  Google Scholar 

  • Chytil F. The lungs and vitamin A. Am. J. Physiol. 262: L517–L527; 1992.

    CAS  PubMed  Google Scholar 

  • Corti M.; Brody A. R.; Harrison J. H. Isolation and primary culture of murine alveolar type II cells. Am. J. Respir. Cell Mol. Biol. 14: 309–315; 1996.

    CAS  PubMed  Google Scholar 

  • Diglio C. A.; Kikkawa Y. The type II epithelial cells of the lung, IV: adaptation and behavior of isolated type II cells in culture. Lab. Invest. 37: 622–631; 1977.

    CAS  PubMed  Google Scholar 

  • Dobbs L. G.; Gonzalez R.; Williams M. C. An improved method for isolating type II cells in high yield and purity. Am. Rev. Respir. Dis. 134: 141–145; 1986.

    CAS  PubMed  Google Scholar 

  • Dobbs L. G.; Mason R. G.; Williams M. C.; Benson B. J.; Sueishi K. Secretion of surfactant by primary cultures of alveolar type II cells isolated from rats. Biochim. Biophys. Acta 713: 118–127; 1982.

    CAS  PubMed  Google Scholar 

  • Everett M. M.; King R. J.; Jones M. B.; Martin H. M. Lung fibroblasts from animals breathing 100% oxygen produce growth factors for alveolar type II cells. Am. J. Physiol. 259: L247–L254; 1990.

    CAS  PubMed  Google Scholar 

  • Fraslon C.; Bourbon J. R. Comparison of effects of epidermal and insulin-like growth factors, gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro. Biochim. Biophys. Acta 1123: 65–75; 1992.

    CAS  PubMed  Google Scholar 

  • Frosolono M. F.; Kress Y.; Wittner M.; Rosenbaum R. M. Culture characteristics of cells derived from type II pneumocyte enriched fractions from rabbit and rat. In Vitro 12: 708–717; 1976.

    Article  CAS  PubMed  Google Scholar 

  • Gharaee-Kermani M.; Gyetko M. R.; Hu B.; Phan S. H. New insights into the pathogenesis and treatment of idiopathic pulmonary fibrosis: a potential role for stem cells in the lung parenchyma and implications for therapy. Pharm. Res. 24: 819–841; 2007.

    Article  CAS  PubMed  Google Scholar 

  • Heger R. J.; Baybutt R. C. Regulation of polyamine synthesis and transport by retinoic acid and epidermal growth factor in cultured rat type II pneumocytes. J. Nutr. Biochem. 10: 518–524; 1999.

    Article  CAS  PubMed  Google Scholar 

  • Jetten A. M. Retinoids specifically enhance the number of epidermal growth factor receptors. Nature 284: 626–629; 1980.

    Article  CAS  PubMed  Google Scholar 

  • Kawaguchi M.; Kawashima J.; Ohshima K.; Kawaguchi S.; Wada H. Retinoic acid enhances the number of epidermal growth factor receptors in rat glomerular epithelial cells in vitro. Nephron 68: 97–103; 1994.

    Article  CAS  PubMed  Google Scholar 

  • King I. C.; Catino J. J. Nonradioactive ligand binding assay for epidermal growth factor receptor. Anal. Biochem. 188: 97–100; 1990.

    Article  CAS  PubMed  Google Scholar 

  • Klein J. M.; Blayne L. F.; McCarthy T. A.; Wohlford-Lenane C. L.; Snyder J. M. Localization of epidermal growth factor receptor in alveolar epithelium during human fetal lung development in vitro. Exp. Lung Res. 21: 917–939; 1995.

    Article  CAS  PubMed  Google Scholar 

  • Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685; 1970.

    Article  CAS  PubMed  Google Scholar 

  • Lavranos T. C.; Raymond J. R. An assay of tritiated thymidine incorporation into DNA by cells cultured under anchorage-independent conditions. Anal. Biochem. 223: 325–327; 1994.

    Article  CAS  PubMed  Google Scholar 

  • Lee R. P.; Doughty S. W.; Ashman K.; Walker J. Purification of hydrophobic integral membrane proteins from Mycoplasma hyopneumoniae by reversed-phase high-performance liquid chromatography. J. Chromatogr. 737: 273–279; 1996.

    Article  CAS  Google Scholar 

  • Leslie C. C.; McCormic-Shannon K.; Robinson P. C.; Mason R. J. Stimulation of DNA synthesis in cultured rat alveolar type II cells. Exp. Lung Res. 8: 53–66; 1985.

    Article  CAS  PubMed  Google Scholar 

  • Li C. Y.; Suardet L.; Little J. B. Potential role of WAF1/Cip1/p21 as a mediator of TGF-beta cytoinhibitory effect. J. Biol. Chem. 270: 4971–4974; 1995.

    Article  CAS  PubMed  Google Scholar 

  • Massaro G. D.; Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am. J. Physiol. 270: L305–L310; 1996.

    CAS  PubMed  Google Scholar 

  • Massaro G. D.; Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nat. Med. 3: 675–677; 1997.

    Article  CAS  PubMed  Google Scholar 

  • Nabeyrat E.; Corroyer S.; Besnard V.; Cazals-Laville V.; Bourbon J.; Clement A. Retinoic acid protects against hyperoxia-mediated cell-cycle arrest of lung alveolar epithelial cells by preserving late G1 cyclin activities. Am. J. Respir. Cell Mol. Biol. 25: 507–514; 2001.

    CAS  PubMed  Google Scholar 

  • Nesterenko M. V.; Tilley M.; Upton S. J. A simple modification of Blum’s silver stain method allows for 30 min detection of proteins in polyacrylamide gels. J. Biochem. Biophys. Methods 28: 239–242; 1994.

    Article  CAS  PubMed  Google Scholar 

  • Nici L.; Medina M.; Frackelton A. R. The epidermal growth factor receptor network in type II pneumocytes exposed to hyperoxia in vitro. Am. J. Physiol. 270: L242–L250; 1996.

    CAS  PubMed  Google Scholar 

  • Oberg K. C.; Carpenter G. Dexamethasone and retinoic acid regulate the expression of epidermal growth factor receptor mRNA by distinct mechanisms. J. Cell. Physiol. 149: 244–251; 1991.

    Article  CAS  PubMed  Google Scholar 

  • Oberg K. C.; Soderquist A. M.; Carpenter G. Accumulation of epidermal growth factor receptors in retinoic acid-treated fetal rat cells is due to enhanced receptor synthesis. Mol. Endocrinol. 2: 959–965; 1988.

    Article  CAS  PubMed  Google Scholar 

  • Oh M. J.; Choi J. H.; Kim I. H.; Lee Y. H.; Huh J. Y.; Park Y. K.; Lee K. W.; Chough S. Y.; Joo K. S.; Ku B. S.; Saw H. S. Detection of epidermal growth factor receptor in the serum of patients with cervical carcinoma. Clin. Cancer Res. 6: 4760–4763; 2000.

    CAS  PubMed  Google Scholar 

  • Olson J. A. In situ enzyme-linked immunosorbent assay to quantitate in vitro development of Eimeria tenella. Antimicrob. Agents Chemother. 34: 1435–1439; 1990.

    CAS  PubMed  Google Scholar 

  • Pegg A. E. Recent advances in the biochemistry of polyamines in eukaryotes. Biochem. J. 234: 249–262; 1986.

    CAS  PubMed  Google Scholar 

  • Raaberg L.; Nexo E.; Buckley S.; Luo W.; Snead M. L.; Warburton D. Epidermal growth factor transcription, translation, and signal transduction by rat type II pneumocytes in culture. Am. J. Respir. Cell Mol. Biol. 6: 44–49; 1992.

    CAS  PubMed  Google Scholar 

  • Raaberg L.; Poulsen S. S.; Nexo E. Epidermal growth factor in the lung. Histochemistry 95: 471–475; 1991.

    Article  CAS  PubMed  Google Scholar 

  • Raymond V. W.; Grisham J. W.; Earp H. S. Characterization of epidermal growth factor receptor induction by retinoic acid in a chemically transformed rat liver cell line. Cell Growth Differ. 1: 393–399; 1990.

    CAS  PubMed  Google Scholar 

  • Rice A. B.; Moomaw C. R.; Morgan D. L.; Bonner J. C. Specific inhibitors of platelet-derived growth factor or epidermal growth factor receptor tyrosine kinase reduce pulmonary fibrosis in rats. Am. J. Pathol. 155: 213–221; 1999.

    CAS  PubMed  Google Scholar 

  • Rouzina I.; Bloomfield V. A. DNA bending by small, mobile multivalent cations. Biophys. J. 74: 3152–3164; 1998.

    Article  CAS  PubMed  Google Scholar 

  • Saminathan M.; Antony T.; Shirahata A.; Sigal L. H.; Thomas T.; Thomas T. J. Ionic and structural specificity effects of natural and synthetic polyamines on the aggregation and resolubilization of single-, double-, and triple-stranded DNA. Biochemistry 38: 3821–3830; 1999.

    Article  CAS  PubMed  Google Scholar 

  • Schooler K.; Wiley H. S. Ratiometric assay of epidermal growth factor receptor tyrosine kinase activation. Anal. Biochem. 277: 135–142; 2000.

    Article  CAS  PubMed  Google Scholar 

  • Stahlman M. T.; Gray M. E.; Chytil F.; Sundell H. Effect of retinol on fetal lamb tracheal epithelium, with and without epidermal growth factor. A model for the effect of retinol on the healing lung of human preterm infants. Lab. Invest. 59: 25–35; 1988.

    CAS  PubMed  Google Scholar 

  • Steck P. A.; Hadi A.; Lotan R.; Yung W. K. Inhibition of epidermal growth factor receptor activity by retinoic acid in glioma cells. J. Cell. Biochem. 42: 83–94; 1990.

    Article  CAS  PubMed  Google Scholar 

  • Stiles A. D.; Smith B. T.; Post M. Reciprocal autocrine and paracrine regulation of growth of mesenchymal and alveolar epithelial cells from fetal lung. Exp. Lung Res. 11: 165–177; 1986.

    Article  CAS  PubMed  Google Scholar 

  • Strandjord T. P.; Clark J. G.; Madtes D. K. Expression of TGF-a, EGF, and EGF receptor in fetal rat lung. Am. J. Physiol. 267: L384–L389; 1994.

    CAS  PubMed  Google Scholar 

  • Takahashi Y.; Takashi M.; Takashi K. Vitamin A is involved in maintenance of epithelial cells on the bronchioles and cells in the alveoli of rats. J. Nutr. 123: 634–641; 1993.

    CAS  PubMed  Google Scholar 

  • Tanswell A. K.; Byrne P. J.; Han R. N.; Edelson J. D.; Han V. K. Limited division of low-density adult rat type II pneumocytes in serum-free culture. Am. J. Physiol. 260: L395–L402; 1991.

    CAS  PubMed  Google Scholar 

  • Towbin H.; Staehelin T.; Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. 76: 4350–4354; 1979.

    Article  CAS  PubMed  Google Scholar 

  • Uhal B. D. Cell cycle kinetics in the alveolar epithelium. Am. J. Physiol. 272: L1031–L1045; 1997.

    CAS  PubMed  Google Scholar 

  • Van Zoelen E. J. J.; van Oostwaard T. M. J.; de Laat S. W. Transforming growth factor-b and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor. J. Biol. Chem. 261: 5003–5009; 1986.

    PubMed  Google Scholar 

  • Wolf G. Recent progress in vitamin A research: nuclear retinoic acid receptors and their interaction with gene elements. J. Nutr. Biochem. 1: 284–289; 1990.

    Article  CAS  PubMed  Google Scholar 

  • Yongshan Y.; DeBauche D. M.; Stanley W. S. Epidermal growth factor receptor expression in a retinoic acid-treated human melanoma cell line. Cancer Genet. Cytogenet. 46: 261–269; 1990.

    Article  CAS  PubMed  Google Scholar 

  • Zheng Z.; Goldsmith L. A. Modulation of epidermal growth factor receptors by retinoic acid in ME180 cells. Cancer Res. 50: 1201–1205; 1990.

    CAS  PubMed  Google Scholar 

  • Zheng Z.; Polakowska R.; Johnson A.; Goldsmith L. A. Transcriptional control of epidermal growth factor receptor by retinoic acid. Cell Growth Differ. 3: 225–232; 1992.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Supported by the NRI Competitive Grants Program/USDA#99-35200-7602, Kansas Agricultural Experiment Station (Contribution no. 97-392-J), and the American Heart Association, Kansas Affiliate. The authors thank Dr. Michael Nesterenko for technical assistance and critical review of this manuscript. We also thank Dr. Sung Koo for his critical review and Mrs. Eileen K. Schofield for her skillful editing. The Lord is acknowledged for His sovereignty and grace. We acknowledge Wheaton College with its motto, “For Christ and His Kingdom”.

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Authors and Affiliations

  1. Applied Health Science Department, Wheaton College, 501 College Ave, Wheaton, IL, 60187, USA

    Richard C. Baybutt & Brendon W. Smith

  2. Department of Human Nutrition, College of Human Ecology, Kansas State University, 119 Justin Hall, Manhattan, KS, 66506, USA

    Elena V. Donskaya, Ling Hu, Ting Li & Weiqun Wang

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  1. Richard C. Baybutt
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  2. Brendon W. Smith
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  3. Elena V. Donskaya
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  4. Ling Hu
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  5. Ting Li
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Correspondence to Richard C. Baybutt.

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Editor: J. Denry Sato

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Baybutt, R.C., Smith, B.W., Donskaya, E.V. et al. The proliferative effects of retinoic acid on primary cultures of adult rat type II pneumocytes depend upon cell density. In Vitro Cell.Dev.Biol.-Animal 46, 20–27 (2010). https://doi.org/10.1007/s11626-009-9236-z

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