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126 CEACAM6: A HORMONALLY REGULATED PROTEIN IN DIFFERENTIATING HUMAN FETAL LUNG TYPE II CELLS.
  1. P. L. Ballard1,
  2. L. W. Gonzales1,
  3. V. Kolla1,
  4. N. Bailey1
  1. 1Neonatology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA.

Abstract

Ceacam6 (carcinoembryonic antigen cell adhesion molecule) is a GPI-anchored membrane glycoprotein recently identified in human fetal lung undergoing in vitro hormone-induced differentiation of type II cells. The protein is a tumor biomarker in adult lung and selected other tissues, where it has antimicrobial activity; however, expression and function in lung have not been characterized. Undifferentiated epithelial cells were isolated by collagenase/trypsin digestion from human fetal lung (16-21 wk gestation) and cultured 1-5 d in the absence (control) or presence of DCI (dexamethasone, 10 nM; 8-Br-cAMP, 0.1 mM; isobutylmethylxanthine, 0.1 mM). Some cells in control media were treated with adenovirus Ad12A2 (0.5-6 pfu/cell) to overexpress thyroid transcription factor-1 (TTF-1) or were transfected by electroporation (AMAXA nucleofection technology) with siRNAs (200 nM) for TTF-1 or Ceacam6 to knockdown expression in the presence of DCI. CEACAM6 mRNA and protein were up-regulated ≈10-fold by exposure of fetal lung epithelial cells to either DCI or to a dose of Ad12A2 that produced a level of recombinant TTF-1 comparable with endogenous TTF-1 in DCI-treated cells. By confocal immunofluorescence, CEACAM6 localized to both intracellular vesicles, costaining with SP-B (ie, lamellar bodies), and to the plasma membrane of type II cells. Knockdown of TTF-1 (46 ± 4% of control) in DCI-treated cells reduced CEACAM6 mRNA and protein by 80% (n = 4). Knockdown of CEACAM6 expression by siRNA (88 ± 3%, n = 3) did not alter expression or staining patterns of TTF-1, SP-B or DC-LAMP (a lamellar body membrane protein). Secretion rates for CEACAM6 were similar for both DCI- and Ad12A2-treated cells, with no response to treatment with secretagogues that stimulate surfactant secretion. Treatment of cells with mannosamine, an inhibitor of GPI anchor synthesis, blocked ≈50% of CEACAM6 secretion, consistent with GPI-anchor involvement in the secretory mechanism. We conclude that CEACAM6 expression in primary fetal lung epithelial cells is regulated by glucocorticoid and cAMP and is dependent on TTF-1. CEACAM6 is associated with intracellular surfactant but is secreted primarily by a constitutive pathway independent of lamellar bodies. CEACAM6 may contribute to the innate defense mechanisms of the mature type II cell.

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