Genomic elements involved in transcriptional regulation of the rabbit surfactant protein-A gene.

Abstract

Expression of the surfactant protein-A (SP-A) gene is lung specific and is developmentally and hormonally regulated in fetal lung tissue. Cyclic AMP analogs and glucocorticoids stimulate transcriptional activity of the SP-A gene in fetal rabbit lung tissue in culture; an additive effect is observed when the agents are added in combination. To analyze the genomic regions that regulate SP-A promoter activity, fusion genes comprised of -1766, -991, -378, and -47 basepairs (bp) of DNA flanking the 5'-end of the SP-A gene, the transcription initiation site, and 20 bp of exon I linked to the human GH (hGH) structural gene were subcloned into a replication-defective human adenovirus vector and transfected into differentiated rat type II cells in primary culture. SP-A promoter activity was analyzed by RIA of hGH protein in the culture medium. In type II cells transfected with SP-A-1766:hGH and SP-A-991:hGH fusion genes, hGH production was induced 30- to 40-fold by (Bu)2AMP (Bt2cAMP; 1 mM). When type II cells were transfected with the SP-A-378:hGH fusion gene, basal levels of expression were reduced by more than 50%; however, Bt2cAMP caused an 11-fold increase in hGH production. In type II cells transfected with the SP-A-47:hGH fusion gene, basal levels of hGH production were essentially undetectable, and no stimulatory effect of Bt2cAMP was apparent. Cyclic AMP stimulation of expression of the SP-A-1766:hGH, SP-A-991:hGH, and SP-A-378:hGH fusion genes was limited to type II pneumonocytes in primary culture and was absent in two lung adenocarcinoma cell lines (NCl-H358 and A549), which do not express SP-A, and in cAMP-responsive adrenal Y1 cells. Mutations of a putative cAMP-responsive element (TGACCTCA) at -261 bp revealed its functional importance in mediating cAMP regulation of SP-A gene expression. Unexpectedly, dexamethasone (Dex; 10(-7) M) antagonized the stimulatory effect of Bt2cAMP on expression of SP-A:hGH fusion genes containing from -378 to -1766 bp of 5'-flanking DNA as well as that of a fusion gene construct containing -991 bp of 5'-flanking DNA, the first exon, the first intron, and 20 bp of the second exon (SP-A-991+670:hGH). The inhibitory effect of Dex was dose dependent, with half-maximal inhibition occurring at a Dex concentration of 8 x 10(-10) M. The inhibitory effect of Dex was prevented by the glucocorticoid receptor antagonist RU486.(ABSTRACT TRUNCATED AT 400 WORDS)

Cyclic AMP-responsive Expression of the Surfactant Protein-A Gene Is Mediated by Increased DNA Binding and Transcriptional Activity of Thyroid Transcription Factor-1

“…We previously observed that type II cells spontaneously differentiate when the human fetal lung is placed in organ culture in serum-free medium and that the rate of type II cell differentiation is enhanced by treatment with cyclic AMP analogues (9). After culture, the tissues were digested with collagenase for preparation of lung type II cells as described (20,37). The human lung adenocarcinoma cell line A549 (ATCC CCL 185) was maintained in Waymouth's MB752/1 medium containing fetal bovine serum (10%, v/v).…”

“…Fusion genes were constructed composed of 255 bp of bSP-A2 5Ј-flanking DNA with or without TBE mutations plus 40 bp of the first exon, linked to the human growth hormone (hGH) structural gene, as reporter. These fusion genes were then incorporated into the genome of a replication-defective human adenovirus 5 for highly efficient and reproducible transfer by infection (20,37). Equal amounts of recombinant adenoviruses were introduced into primary cultures of rat fetal lung type II cells incubated in the absence or presence of Bt 2 cAMP.…”

Increased Low-Density Lipoprotein Oxidation and Impaired High-Density Lipoprotein Antioxidant Defense Are Associated With Increased Macrophage Homing and Atherosclerosis in Dyslipidemic Obese Mice

“…25 The control recombinant adenovirus AdRR5 has been described elsewhere. 26 Plaque-forming units 5ϫ10 8 of AdLCAT or AdRR5 virus were injected into the tail vein of LDLR Ϫ/Ϫ ;ob/ob mice.…”

Role of Plasminogen System Components in Focal Cerebral Ischemic Infarction

“…18 -20 After transfection, recombinant viral plaques were harvested and amplified as described. [21][22][23] Large-scale production of recombinant adenovirus was performed as described. 21 The kinetics and organ distribution of tPA and PAI-1 expression after adenoviral transfer by intravenous bolus injection have been described in detail elsewhere.…”