MULTIPLE SPLICING EVENTS INVOLVED IN REGULATION OF HUMAN AROMATASE EXPRESSION BY A NOVEL PROMOTER, I.6

The expression of aromatase is regulated in a tissue-specific fashion through alternative use of multiple promoter-specific first exons. To date, eight different first exons have been reported in human aromatas e, namely I.1., I.2, I.3. I.4, I.5, PII, 2a, and If. Recently, we have found a new putative exon I in a RACE-generated Library of THP-1 cell s and have conducted studies to characterize this new exon I. We confi rmed that the constructs containing -1552/+17 or less flanking sequenc e of this exon function as a promoter in THP-1 cells, JEG-3 cells and osteoblast-like cells obtained from a human fetus. Results of transfec tion assays using a series of deletion constructs and mutation constru cts indicate that a l-bp mismatch of the consensus TATA-like box (TTTA AT) and the consensus sequence of the initiator site, which is located 45 bp downstream of the putative TATA box, were functioning cooperati vely as a core promoter. The putative transcription site was confirmed by the results of RT-PCR southern blot analysis. We examined the regu lation and the expression of this exon, 1.6, in several human cells an d tissues by RT-PCR Southern blot analysis. THP-1 cells (mononuclear l eukemic origin) and JEG-3 cells (choriocarcinoma origin) expressed exo n 1.6 in serum-free media. The level of expression was increased by se rum and phorbol myristyl acetate (PMA) in both cell lines. Adipose str omal cells also expressed exon I.6 in the presence of PMA. In fetal os teoblasts, the expression of exon 1.6 was increased most effectively b y serum and less so by dexamethasone (DEX) + IL-1 beta and DEX + IL-11 , whereas induction by serum was suppressed by the addition of DEX. Th e level of expression was low in granulosa cells in culture and did no t change with forskolin. On the other hand, dibutyryl cAMP suppressed PMA-stimulated expression of exon 1.6 in THP-1 cells and adipose strom al cells. This result supports the hypothesis that the expression of e xon 1.6 is regulated mainly via an AP-1 binding site that is found ups tream of the initiator site of the promoter region. Expression of exon I.6-specific transcripts was examined in several human tissues. Testi s and bone obtained from normal adults expressed exon I.6. Testicular tumor and hepatic carcinoma expressed high levels of exon 1.6, whereas granulosa cell tumor did not. Fetal liver and bone also showed a sign ificant level of exon 1.6 expression, but not so much as testicular tu mor and hepatic tumor. Several splicing variants of exon I.6 were dete cted especially in THP-1 and JEG-3 cells, and to a lesser extent in pr imary cultures and tissue samples. These variants were identified as a n unspliced form, a form spliced at the end of exon I.4, a form splice d at the end of exon I.3 (truncated) and a form spliced 220 bp downstr eam of the 3' end of exon I.6. The last variant revealed a new splicin g site. Because most of the splicing variants contain the sequence spe cific for exon I.3, RT-PCR specific for exon I.3 can coamplify these s plicing variants of exon I.6 transcripts. These results suggests that it is necessary to examine the expression of I.6 in tissues that are k nown to express exon I.3 such as breast adipose tissue, in which promo ter usage of exon I of the aromatase gene switches from exon I.4 to I. 3 in the course of malignant transformation.