ADENOVIRUS-MEDIATED INCREASE OF EXOGENOUS AND INHIBITION OF ENDOGENOUS FOSB GENE-EXPRESSION IN CULTURED PULMONARY ARTERIAL SMOOTH-MUSCLE CELLS

Modification of gene expression in pulmonary arterial smooth muscle ce lls (PASMCs) could be a valuable tool for investigating the role of sp ecific gene products in normal and pathological PASMC growth, and a no vel potential therapy for pulmonary vascular diseases. To examine the direct role of fosB protein in PASMC growth, adenovirus (Ad) vectors w ere used to transfer sense or antisense full-length fosB cDNAs to cult ured 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 construc ted into an E1 region-deleted, replication-deficient human type 5 Ad v ector in either sense or antisense orientation. Forty-eight hours afte r infection with the sense construct (Ad.S.fosB) at 3 plaque forming u nits per cell (pfu/cell), PASMCs expressed abundant fosB mRNA and fosB protein. FosB protein was detected by immunohistochemistry in approxi mately 95% of the infected cells. PASMCs infected with Ad.S. fosB at r atios 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 expres sion at 1 and 10 pfu/cell, respectively. The increase in fosB mRNA exp ression was detected as early as 8 h and persisted for 25 days after i nfection. Forty-eight hours after infection with the antisense constru ct (Ad.A.fosB) at 3 pfu/cell, very low levels of fosB mRNA were detect ed by Northern blotting, in which the double-stranded fosB cDNA was la beled and used as a hybridization probe. FosB protein was undetectable by Western blotting or immunocytochemical analyses in the Ad.A.fosB i nfected cells. Cytopathical effects were observed when PASMCs were inf ected with either Ad.S.fosB or Ad.A.fosB at ratios of 10 pfu/cell or h igher. 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 expre ssion units can be used to effectively modify fosB gene expression. Al though 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. (C) 1996 Academic Press Limited