Regulation of surfactant protein and defensin mRNA expression in cultured ovine type II pneumocytes by all‐trans retinoic acid and VEGF

Beta‐defensins and surfactant proteins are components of the pulmonary innate immune system. Their gene expression is regulated by development, hormones, growth and immunoregulatory factors. It was our hypothesis that growth and differentiation factors such as all‐trans retinoic acid (RA) and vascular endothelial growth factor (VEGF) may affect expression of selected innate immune genes by respiratory epithelial cells. Ovine JS7 cells (alveolar type II pneumocytes) were incubated in serum‐free Dulbecco's modified Eagle's medium (DMEM) complete media that contained: no treatment (negative control), RA (500 nM), or VEGF (100 ng/ml) for 6, 12 or 24 h incubation. Total RNA was isolated, cDNA synthesized, and relative mRNA levels of surfactant protein A (SP‐A) and SP‐D, and sheep beta‐defensin‐1 (SBD‐1) were determined by real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR). Cells had significantly increased expression of SP‐D mRNA at 6 h and 24 h, decreased expression of SP‐A mRNA at 12 h, and unchanged levels of SBD‐1 mRNA after the treatment with RA compared with their respective negative controls. VEGF did not alter the expression of the three innate immune genes. These findings suggest that SP‐A and SP‐D have different transcription regulation pathways, and that expression of SBD‐1 is not inducible by RA similar to its human homolog HBD‐1. The lack of changes induced by VEGF treatment suggests that VEGF does not have a direct effect on epithelial cells, but may affect gene expression indirectly.

[1]  P. McCray,et al.  Human airway epithelia express a beta-defensin. , 1997, American journal of respiratory cell and molecular biology.

[2]  J. Demartini,et al.  Alveolar Type II Cells Expressing Jaagsiekte Sheep Retrovirus Capsid Protein and Surfactant Proteins Are the Predominant Neoplastic Cell Type in Ovine Pulmonary Adenocarcinoma , 2002, Veterinary pathology.

[3]  G. Conner,et al.  Bronchial tissue kallikrein activity is regulated by hyaluronic acid binding. , 1999, American journal of respiratory cell and molecular biology.

[4]  J. Davoust,et al.  CD208/dendritic cell-lysosomal associated membrane protein is a marker of normal and transformed type II pneumocytes. , 2004, The American journal of pathology.

[5]  J. Rosenbloom,et al.  TGF-β1 inhibits surfactant component expression and epithelial cell maturation in cultured human fetal lung. , 1998, American journal of physiology. Lung cellular and molecular physiology.

[6]  David K. Meyerholz,et al.  Enhanced Surfactant Protein and Defensin mRNA Levels and Reduced Viral Replication during Parainfluenza Virus Type 3 Pneumonia in Neonatal Lambs , 2004, Clinical Diagnostic Laboratory Immunology.

[7]  Michaela R. Hoffmeyer,et al.  PR-39, a potent neutrophil inhibitor, attenuates myocardial ischemia-reperfusion injury in mice. , 2000, American journal of physiology. Heart and circulatory physiology.

[8]  M. Frye,et al.  Expression of human beta-defensin-1 promotes differentiation of keratinocytes. , 2001, Journal of molecular medicine.

[9]  M. Holtzman,et al.  A cryptic, microsomal-type arachidonate 12-lipoxygenase is tonically inactivated by oxidation-reduction conditions in cultured epithelial cells. , 1993, The Journal of biological chemistry.

[10]  L. D. Nielsen,et al.  KGF regulates pulmonary epithelial proliferation and surfactant protein gene expression in adult rat lung. , 2000, American journal of physiology. Lung cellular and molecular physiology.

[11]  E. Nabeyrat,et al.  Protective role of retinoic acid from antiproliferative action of TNF-α on lung epithelial cells , 2002 .

[12]  A. Persson,et al.  Surfactant protein D: subcellular localization in nonciliated bronchiolar epithelial cells. , 1992, The American journal of physiology.

[13]  M. Ackermann,et al.  Growth of differentiated ovine tracheal epithelial cells in vitro. , 2004, Journal of veterinary medicine. A, Physiology, pathology, clinical medicine.

[14]  E. Crouch.  Surfactant protein-D and pulmonary host defense , 2000, Respiratory research.

[15]  J. Sharp,et al.  Three-step procedure for isolation of epithelial cells from the lungs of sheep with jaagsiekte. , 1987, Research in veterinary science.

[16]  C. Daniels,et al.  The pattern of surfactant cholesterol during vertebrate evolution and development: does ontogeny recapitulate phylogeny? , 2003, Reproduction, fertility, and development.

[17]  K. Brogden,et al.  Repeated intravenous doses of all-trans-retinoyl beta-D-glucuronide is not effective in the treatment of bacterial bronchopneumonia in lambs but is devoid of gross and acute toxicity. , 2002, Medical science monitor : international medical journal of experimental and clinical research.

[18]  D. Voelker,et al.  KGF increases SP-A and SP-D mRNA levels and secretion in cultured rat alveolar type II cells. , 1998, American journal of respiratory cell and molecular biology.

[19]  Andreas Peschel,et al.  Chemokines Meet Defensins: the Merging Concepts of Chemoattractants and Antimicrobial Peptides in Host Defense , 2002, Infection and Immunity.

[20]  O. L. Miakotina,et al.  Mechanism of all trans-retinoic acid and glucocorticoid regulation of surfactant protein mRNA. , 1998, American journal of physiology. Lung cellular and molecular physiology.

[21]  C. Speer,et al.  The role of transforming growth factor beta in lung development and disease. , 2004, Chest.

[22]  J. M. Snyder,et al.  Insulin regulation of messenger ribonucleic acid for the surfactant-associated proteins in human fetal lung in vitro. , 1992, Endocrinology.

[23]  David K. Meyerholz,et al.  Adenovirus-Mediated Gene Therapy Enhances Parainfluenza Virus 3 Infection in Neonatal Lambs , 2004, Journal of Clinical Microbiology.

[24]  S. Boas,et al.  Serum vascular endothelial growth factor is elevated in cystic fibrosis and decreases with treatment of acute pulmonary exacerbation. , 2000, American journal of respiratory and critical care medicine.

[25]  M. Frye,et al.  Expression of human β-defensin-1 promotes differentiation of keratinocytes , 2001, Journal of Molecular Medicine.

[26]  M. Salathe,et al.  Coupling of [Ca2+]i and ciliary beating in cultured tracheal epithelial cells. , 1995, Journal of cell science.

[27]  P. Ballard,et al.  Ontogeny of tracheal fluid, pulmonary surfactant, and plasma corticoids in the fetal lamb. , 1975 .

[28]  Robert Bals,et al.  Antimicrobial Peptides , 2012, Drugs.

[29]  F. Speleman,et al.  Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes , 2002, Genome Biology.

[30]  R. Hancock,et al.  Cationic antimicrobial peptides and their multifunctional role in the immune system. , 2000, Critical reviews in immunology.

[31]  Christina H. Park,et al.  Human beta-defensin-2 production in keratinocytes is regulated by interleukin-1, bacteria, and the state of differentiation. , 2002, The Journal of investigative dermatology.

[32]  E. Nabeyrat,et al.  Retinoic acid-induced proliferation of lung alveolar epithelial cells: relation with the IGF system. , 1998, American journal of physiology. Lung cellular and molecular physiology.

[33]  P. Carmeliet,et al.  Loss of HIF-2α and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice , 2002, Nature Medicine.

[34]  H. Clark,et al.  Structural requirements for SP-D function in vitro and in vivo: therapeutic potential of recombinant SP-D. , 2002, Immunobiology.

[35]  A. Jobe,et al.  Developmental and glucocorticoid regulation of surfactant protein mRNAs in preterm lambs. , 1999, American journal of physiology. Lung cellular and molecular physiology.

[36]  J. Sharp,et al.  Pathology of ovine pulmonary adenocarcinoma. , 2003, Current topics in microbiology and immunology.

[37]  K. Goss,et al.  VEGF induces airway epithelial cell proliferation in human fetal lung in vitro. , 2001, American journal of physiology. Lung cellular and molecular physiology.

[38]  D. Voelker,et al.  Jaagsiekte Sheep Retrovirus Proviral Clone JSRVJS7, Derived from the JS7 Lung Tumor Cell Line, Induces Ovine Pulmonary Carcinoma and Is Integrated into the Surfactant Protein A Gene , 2001, Journal of Virology.

[39]  R. Creasy,et al.  Appearance of paoproteins of pulmonary surfactant in human amniotic fluid. , 1975, Journal of applied physiology.

[40]  Silvio Albertini,et al.  Gene expression in two hepatic cell lines, cultured primary hepatocytes, and liver slices compared to the in vivo liver gene expression in rats: possible implications for toxicogenomics use of in vitro systems. , 2003, Toxicological sciences : an official journal of the Society of Toxicology.

[41]  L. Brochard,et al.  Vascular endothelial growth factor synthesis in the acute phase of experimental and clinical lung injury. , 2001, The European respiratory journal.

[42]  C. Speer,et al.  The Role of Transforming Growth Factor β in Lung Development and Disease , 2004 .

[43]  C. Delacourt,et al.  Effects of vascular endothelial growth factor on isolated fetal alveolar type II cells. , 2004, American journal of physiology. Lung cellular and molecular physiology.

[44]  K. Rabe,et al.  Defensins: key players or bystanders in infection, injury, and repair in the lung? , 1999, The Journal of allergy and clinical immunology.

[45]  L. Schwichtenberg,et al.  Differential gene induction of human beta-defensins (hBD-1, -2, -3, and -4) in keratinocytes is inhibited by retinoic acid. , 2004, The Journal of investigative dermatology.