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. 1995 Dec 1;312(Pt 2):425–431. doi: 10.1042/bj3120425

Stimulation of phosphatidylcholine biosynthesis in mouse MLE-12 type-II cells by conditioned medium from cortisol-treated rat fetal lung fibroblasts.

J I MacDonald 1, F Possmayer 1
PMCID: PMC1136279  PMID: 8526851

Abstract

Treatment of murine adult MLE-12 type-II and fetal-rat type-II cells with fetal-rat-fibroblast-conditioned medium (FFCM) resulted in a 2-fold stimulation of [14C]choline incorporation into phosphatidylcholine. Soluble CTP:phosphocholine cytidylyltransferase (CT) activity was increased approx. 3-fold in FFCM-treated fetal-rat type-II cells but was not changed in MLE-12 cells. Neither choline kinase nor cholinephosphotransferase activities were affected by treatment of MLE-12 cells with FFCM. Long-term labelling of MLE-12 cells with [14C]choline, followed by a 14-18 h chase with FFCM, resulted in a 2.5-fold decrease in [14C]phosphocholine levels relative to controls, suggesting that CT was being activated. In contrast, oleate treatment increased CT activity in the particulate fraction in both cells. Western blots indicate that soluble CT undergoes dephosphorylation in response to FFCM, but no translocation to the particulate fraction was noted. Treatment with oleate stimulated a marked translocation. Tryptic phosphopeptide maps from FFCM-treated cells revealed only minor alterations in the phosphorylation pattern. It is concluded that FFCM and oleate activate CT through different mechanisms. The results are consistent with FFCM activating CT in MLE-12 as well as fetal type-II cells. However, the reason why this activation cannot be detected in vitro is not known.

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Selected References

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  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Burkhardt R., Von Wichert P., Batenburg J. J., Van Golde L. M. Fatty acids stimulate phosphatidylcholine synthesis and CTP:choline-phosphate cytidylyltransferase in type II pneumocytes isolated from adult rat lung. Biochem J. 1988 Sep 1;254(2):495–500. doi: 10.1042/bj2540495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caniggia I., Tseu I., Han R. N., Smith B. T., Tanswell K., Post M. Spatial and temporal differences in fibroblast behavior in fetal rat lung. Am J Physiol. 1991 Dec;261(6 Pt 1):L424–L433. doi: 10.1152/ajplung.1991.261.6.L424. [DOI] [PubMed] [Google Scholar]
  5. Choy P. C., Vance D. E. Lipid requirements for activation of CTP: phosphocholine cytidylyltransferase from rat liver. J Biol Chem. 1978 Jul 25;253(14):5163–5167. [PubMed] [Google Scholar]
  6. Cornell R. B. Regulation of CTP:phosphocholine cytidylyltransferase by lipids. 1. Negative surface charge dependence for activation. Biochemistry. 1991 Jun 18;30(24):5873–5880. doi: 10.1021/bi00238a010. [DOI] [PubMed] [Google Scholar]
  7. Cornell R. B. Regulation of CTP:phosphocholine cytidylyltransferase by lipids. 2. Surface curvature, acyl chain length, and lipid-phase dependence for activation. Biochemistry. 1991 Jun 18;30(24):5881–5888. doi: 10.1021/bi00238a011. [DOI] [PubMed] [Google Scholar]
  8. Feldman D. A., Brubaker P. G., Weinhold P. A. Activation of CTP : phosphocholine cytidylyltransferase in rat lung by fatty acids. Biochim Biophys Acta. 1981 Jul 24;665(1):53–59. doi: 10.1016/0005-2760(81)90231-9. [DOI] [PubMed] [Google Scholar]
  9. Feldman D. A., Kovac C. R., Dranginis P. L., Weinhold P. A. The role of phosphatidylglycerol in the activation of CTP:phosphocholine cytidylyltransferase from rat lung. J Biol Chem. 1978 Jul 25;253(14):4980–4986. [PubMed] [Google Scholar]
  10. Feldman D. A., Rounsifer M. E., Charles L., Weinhold P. A. CTP:phosphocholine cytidylyltransferase in rat lung: relationship between cytosolic and membrane forms. Biochim Biophys Acta. 1990 Jun 28;1045(1):49–57. doi: 10.1016/0005-2760(90)90202-9. [DOI] [PubMed] [Google Scholar]
  11. Feldman D. A., Weinhold P. A. CTP:phosphorylcholine cytidylyltransferase from rat liver. Isolation and characterization of the catalytic subunit. J Biol Chem. 1987 Jul 5;262(19):9075–9081. [PubMed] [Google Scholar]
  12. Fraslon C., Batenburg J. J. Pre-translational regulation of lipid synthesizing enzymes and surfactant proteins in fetal rat lung in explant culture. FEBS Lett. 1993 Jul 5;325(3):285–290. doi: 10.1016/0014-5793(93)81090-m. [DOI] [PubMed] [Google Scholar]
  13. Hatch G. M., Jamil H., Utal A. K., Vance D. E. On the mechanism of the okadaic acid-induced inhibition of phosphatidylcholine biosynthesis in isolated rat hepatocytes. J Biol Chem. 1992 Aug 5;267(22):15751–15758. [PubMed] [Google Scholar]
  14. Hogan M., Zimmermann L. J., Wang J., Kuliszewski M., Liu J., Post M. Increased expression of CTP:phosphocholine cytidylyltransferase in maturing type II cells. Am J Physiol. 1994 Jul;267(1 Pt 1):L25–L32. doi: 10.1152/ajplung.1994.267.1.L25. [DOI] [PubMed] [Google Scholar]
  15. Houweling M., Jamil H., Hatch G. M., Vance D. E. Dephosphorylation of CTP-phosphocholine cytidylyltransferase is not required for binding to membranes. J Biol Chem. 1994 Mar 11;269(10):7544–7551. [PubMed] [Google Scholar]
  16. Kent C. Regulation of phosphatidylcholine biosynthesis. Prog Lipid Res. 1990;29(2):87–105. doi: 10.1016/0163-7827(90)90010-i. [DOI] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lim P., Cornell R., Vance D. E. The supply of both CDP-choline and diacylglycerol can regulate the rate of phosphatidylcholine synthesis in HeLa cells. Biochem Cell Biol. 1986 Jul;64(7):692–698. doi: 10.1139/o86-095. [DOI] [PubMed] [Google Scholar]
  19. MacDonald J. I., Kent C. Baculovirus-mediated expression of rat liver CTP:phosphocholine cytidylyltransferase. Protein Expr Purif. 1993 Feb;4(1):1–7. doi: 10.1006/prep.1993.1001. [DOI] [PubMed] [Google Scholar]
  20. MacDonald J. I., Kent C. Identification of phosphorylation sites in rat liver CTP: phosphocholine cytidylyltransferase. J Biol Chem. 1994 Apr 8;269(14):10529–10537. [PubMed] [Google Scholar]
  21. Mallampalli R. K., Hunninghake G. W. Expression of immunoreactive cytidine 5'-triphosphate: cholinephosphate cytidylyltransferase in developing rat lung. Pediatr Res. 1993 Oct;34(4):502–511. doi: 10.1203/00006450-199310000-00024. [DOI] [PubMed] [Google Scholar]
  22. Oka T., Perry J. W. Glucocorticoid stimulation of choline kinase activity during the development of mouse mammary gland. Dev Biol. 1979 Jan;68(1):311–318. doi: 10.1016/0012-1606(79)90264-1. [DOI] [PubMed] [Google Scholar]
  23. Pelech S. L., Cook H. W., Paddon H. B., Vance D. E. Membrane-bound CTP:phosphocholine cytidylyltransferase regulates the rate of phosphatidylcholine synthesis in HeLa cells treated with unsaturated fatty acids. Biochim Biophys Acta. 1984 Oct 4;795(3):433–440. doi: 10.1016/0005-2760(84)90169-3. [DOI] [PubMed] [Google Scholar]
  24. Pelech S. L., Pritchard P. H., Brindley D. N., Vance D. E. Fatty acids promote translocation of CTP:phosphocholine cytidylyltransferase to the endoplasmic reticulum and stimulate rat hepatic phosphatidylcholine synthesis. J Biol Chem. 1983 Jun 10;258(11):6782–6788. [PubMed] [Google Scholar]
  25. Possmayer F., Casola P. G., Chan F., MacDonald P., Ormseth M. A., Wong T., Harding P. G., Tokmakjian S. Hormonal induction of pulmonary maturation in the rabbit fetus: effects of maternal treatment with estradiol-17 beta on th endogenous levels of cholinephosphate, CDP-choline and phosphatidylcholine. Biochim Biophys Acta. 1981 Apr 23;664(1):10–21. doi: 10.1016/0005-2760(81)90024-2. [DOI] [PubMed] [Google Scholar]
  26. Possmayer F., Casola P., Chan F., Hill S., Metcalfe I. L., Stewart-DeHaan P. J., Wong T., las Heras J., Gammal E. B., Harding P. G. Glucocorticoid induction of pulmonary maturation in the rabbit fetus. The effect of maternal injection of betamethasone on the activity of enzymes in fetal lung. Biochim Biophys Acta. 1979 Aug 30;574(2):197–211. doi: 10.1016/0005-2760(79)90002-x. [DOI] [PubMed] [Google Scholar]
  27. Post M., Barsoumian A., Smith B. T. The cellular mechanism of glucocorticoid acceleration of fetal lung maturation. Fibroblast-pneumonocyte factor stimulates choline-phosphate cytidylyltransferase activity. J Biol Chem. 1986 Feb 15;261(5):2179–2184. [PubMed] [Google Scholar]
  28. Post M., Batenburg J. J., Van Golde L. M., Smith B. T. The rate-limiting reaction in phosphatidylcholine synthesis by alveolar type II cells isolated from fetal rat lung. Biochim Biophys Acta. 1984 Oct 4;795(3):558–563. doi: 10.1016/0005-2760(84)90186-3. [DOI] [PubMed] [Google Scholar]
  29. Post M., van Golde L. M. Metabolic and developmental aspects of the pulmonary surfactant system. Biochim Biophys Acta. 1988 Jun 9;947(2):249–286. doi: 10.1016/0304-4157(88)90011-1. [DOI] [PubMed] [Google Scholar]
  30. Rooney S. A., Smart D. A., Weinhold P. A., Feldman D. A. Dexamethasone increases the activity but not the amount of choline-phosphate cytidylyltransferase in fetal rat lung. Biochim Biophys Acta. 1990 Jun 14;1044(3):385–389. doi: 10.1016/0005-2760(90)90085-c. [DOI] [PubMed] [Google Scholar]
  31. Rooney S. A. The surfactant system and lung phospholipid biochemistry. Am Rev Respir Dis. 1985 Mar;131(3):439–460. doi: 10.1164/arrd.1985.131.3.439. [DOI] [PubMed] [Google Scholar]
  32. Teegarden D., Taparowsky E. J., Kent C. Altered phosphatidylcholine metabolism in C3H10T1/2 cells transfected with the Harvey-ras oncogene. J Biol Chem. 1990 Apr 15;265(11):6042–6047. [PubMed] [Google Scholar]
  33. Tokmakjian S., Possmayer F. Pool sizes of the precursors for phosphatidylcholine synthesis in developing rat lung. Biochim Biophys Acta. 1981 Oct 23;666(1):176–180. doi: 10.1016/0005-2760(81)90103-x. [DOI] [PubMed] [Google Scholar]
  34. Utal A. K., Jamil H., Vance D. E. Diacylglycerol signals the translocation of CTP:choline-phosphate cytidylyltransferase in HeLa cells treated with 12-O-tetradecanoylphorbol-13-acetate. J Biol Chem. 1991 Dec 15;266(35):24084–24091. [PubMed] [Google Scholar]
  35. Wang Y., MacDonald J. I., Kent C. Regulation of CTP:phosphocholine cytidylyltransferase in HeLa cells. Effect of oleate on phosphorylation and intracellular localization. J Biol Chem. 1993 Mar 15;268(8):5512–5518. [PubMed] [Google Scholar]
  36. Warden C. H., Friedkin M. Regulation of choline kinase activity and phosphatidylcholine biosynthesis by mitogenic growth factors in 3T3 fibroblasts. J Biol Chem. 1985 May 25;260(10):6006–6011. [PubMed] [Google Scholar]
  37. Watkins J. D., Kent C. Phosphorylation of CTP:phosphocholine cytidylyltransferase in vivo. Lack of effect of phorbol ester treatment in HeLa cells. J Biol Chem. 1990 Feb 5;265(4):2190–2197. [PubMed] [Google Scholar]
  38. Watkins J. D., Kent C. Regulation of CTP:phosphocholine cytidylyltransferase activity and subcellular location by phosphorylation in Chinese hamster ovary cells. The effect of phospholipase C treatment. J Biol Chem. 1991 Nov 5;266(31):21113–21117. [PubMed] [Google Scholar]
  39. Weinhold P. A., Charles L., Rounsifer M. E., Feldman D. A. Control of phosphatidylcholine synthesis in Hep G2 cells. Effect of fatty acids on the activity and immunoreactive content of choline phosphate cytidylyltransferase. J Biol Chem. 1991 Apr 5;266(10):6093–6100. [PubMed] [Google Scholar]
  40. Weinhold P. A., Rounsifer M. E., Charles L., Feldman D. A. Characterization of cytosolic forms of CTP: choline-phosphate cytidylyltransferase in lung, isolated alveolar type II cells, A549 cell and Hep G2 cells. Biochim Biophys Acta. 1989 Dec 18;1006(3):299–310. doi: 10.1016/0005-2760(89)90017-9. [DOI] [PubMed] [Google Scholar]
  41. Wikenheiser K. A., Vorbroker D. K., Rice W. R., Clark J. C., Bachurski C. J., Oie H. K., Whitsett J. A. Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11029–11033. doi: 10.1073/pnas.90.23.11029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Xu Z. X., Smart D. A., Rooney S. A. Glucocorticoid induction of fatty-acid synthase mediates the stimulatory effect of the hormone on choline-phosphate cytidylyltransferase activity in fetal rat lung. Biochim Biophys Acta. 1990 May 1;1044(1):70–76. doi: 10.1016/0005-2760(90)90220-r. [DOI] [PubMed] [Google Scholar]
  43. Zimmermann L. J., Hogan M., Carlson K. S., Smith B. T., Post M. Regulation of phosphatidylcholine synthesis in fetal type II cells by CTP:phosphocholine cytidylyltransferase. Am J Physiol. 1993 Jun;264(6 Pt 1):L575–L580. doi: 10.1152/ajplung.1993.264.6.L575. [DOI] [PubMed] [Google Scholar]
  44. Zimmermann L. J., Lee W. S., Smith B. T., Post M. Cyclic AMP-dependent protein kinase does not regulate CTP:phosphocholine cytidylyltransferase activity in maturing type II cells. Biochim Biophys Acta. 1994 Feb 10;1211(1):44–50. doi: 10.1016/0005-2760(94)90137-6. [DOI] [PubMed] [Google Scholar]

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