Antisense Inhibition of Epidermal Growth Factor Receptor Decreases Expression of Human Surfactant Protein A

Abstract

Epidermal growth factor (EGF) stimulates surfactant protein A (SP-A) synthesis in fetal lung tissue through ligand binding to the EGF receptor. We hypothesized that inhibition of EGF receptor messenger RNA (mRNA) would block SP-A expression in human fetal lung tissue during alveolar type II cell differentiation in vitro. Midtrimester human fetal lung explants were maintained in serum-free Waymouth's medium for 3 to 5 d in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to the initiation codon region of EGF receptor mRNA. Sense and scrambled ONs similarly modified were used as additional controls. The concentration of EGF receptor mRNA was semiquantitatively determined by reverse transcriptase/polymerase chain reaction (RT-PCR). We found a significant 3-fold decrease in EGF receptor mRNA levels in the antisense-treated groups compared with the control group with no effect in the sense condition. Immunohistochemical staining revealed a decrease in the amount of staining for EGF receptor protein in distal pulmonary epithelial cells in the antisense-treated groups compared with either control or sense conditions. Treatment with antisense EGF receptor ON decreased both SP-A mRNA and protein compared with controls with no effect in the sense condition. The ONs did not affect tissue viability as measured by the release of lactate dehydrogenase. We conclude that selective degradation of EGF receptor mRNA with antisense ON treatment results in a decrease in SP-A expression in human fetal lung. These findings support the critical importance of the EGF receptor for the regulation of SP-A gene expression during human alveolar type II cell differentiation.

Antisense inhibition of surfactant protein A decreases tubular myelin formation in human fetal lung in vitro

“…The decreased connectivetissue cellularity observed was most likely a nonspecific (sequence independent) effect of the phosphorothioate backbone. This same finding was previously observed in human fetal lung explants exposed to different ON sequences that had the same phosphorothioate backbone (12). Phosphorothioate compounds have been found to interact nonspecifically with a number of cellular proteins (24), in part, because of their polyanionic nature, which mimics the charge density of heparin (10,32).…”

Role of epidermal growth factor and epidermal growth factor receptor on hemidesmosome complex formation and integrin subunit β4

“…Antisense oligo strategies to inhibit growth factors or their receptors have been performed successfully in embryonic tissue culture (Potts et al 1991;Seth et al 1993;Klein et al 2000). Both in vitro and organ culture models were used and transfected at concentrations shown to cause no detectable loss of viability.…”

Effect of Exogenous Surfactant on the Development of Surfactant Synthesis in Premature Rabbit Lung

“…We examined how exposure to exogenous surfactant affects the development of surfactant phospholipid synthesis and surfactant protein mRNA levels. The fine mincing of tissue in this model (Ͻ1-mm cubes) appears to create a large potential surface area for type II cell exposure to media components, a feature successfully used by others to specifically target treatments to the developing type II cell (40). It is possible that in vivo a larger population of type II cells would be exposed to the surfactant treatments; this would possibly enhance the effects observed here.…”