Adenovirus-mediated Increase of Exogenous and Inhibition of Endogenous fosB Gene Expression in Cultured Pulmonary Arterial Smooth Muscle Cells

Abstract

Modification of gene expression in pulmonary arterial smooth muscle cells (PASMCs) could be a valuable tool for investigating the role of specific gene products in normal and pathological PASMC growth, and a novel potential therapy for pulmonary vascular diseases. To examine the direct role of fosB protein in PASMC growth, adenovirus (Ad) vectors were used to transfer sense or antisense full-length fosB cDNAs to cultured PASMCs to modify fosB expression, and investigate the effects of this modification on PASMC growth. The full-length fosB cDNA under the control of the cytomegalovirus (CMV) early gene promoter was constructed into an E1 region-deleted, replication-deficient human type 5 Ad vector in either sense or antisense orientation. Forty-eight hours after infection with the sense construct (Ad.S. fosB) at 3 plaque-forming units per cell (pfu/cell), PASMCs expressed abundant fosB mRNA and fosB protein. FosB protein was detected by immunohistochemistry in approximately 95% of the infected cells. PASMCs infected with Ad.S. fosB at ratios of 0.1, 0.2, 0.5, 1, 3, and 10 pfu/cell showed a dose-dependent increase in fosB mRNA expression, with half-maximal and maximal expression at 1 and 10 pfu/cell, respectively. The increase in fosB mRNA expression was detected as early as 8 h and persisted for 25 days after infection. Forty-eight hours after infection with the antisense construct (Ad.A. fosB) at 3 pfu/cell, very low levels of fosB mRNA were detected by Northern blotting, in which the double-stranded fosB cDNA was labeled and used as a hybridization probe. FosB protein was undetectable by Western blotting or immunocytochemical analyses in the Ad.A. fosB infected cells. Cytopathical effects were observed when PASMCs were infected with either Ad.S. fosB or Ad.A. fosB at ratios of 10 pfu/cell or higher. Infection of serum-deprived PASMCs with Ad.S. fosB or Ad.A. fosB alone at 3 pfu/cell did not affect cellular growth. These results show that adenoviral vectors containing sense or antisense fosB cDNA expression units can be used to effectively modify fosB gene expression. Although changes in fos-B gene expression did not affect cellular growth, this model system offers a very effective method for elucidating the biological roles of other gene products and studying the pathways of PASMC gene regulation and signal transduction.