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cAMP stimulation of rodent steroidogenic cells produces two StAR transcripts, a major 3.5 kb and a minor 1.6 kb mRNA, differing only in their 3′ untranslated regions (3′ UTR). They exhibit very different responses to stimulation and removal of 8-Br-cAMP, with the 3.5 kb form increasing and declining much more rapidly than the 1.6 kb form. The 3′ end of the 3.5 kb StAR mRNA contains three conserved AU-rich element (AURE) motifs that mediate fast mRNA turnover in over 900 genes in the human genome. In this paper, we explore post-transcriptional regulation in steroidogenic and non-steroidogenic cells using expression vectors containing StAR or luciferase with different StAR 3′ UTRs. We show that the basal steady-state levels of StAR or luciferase protein and mRNA are five to eight times lower with the 3′ UTR of 3.5 kb StAR compared with that of the 1.6 kb 3′ UTR. Examination of transcript stability by direct mRNA transfection showed only a 1.5-fold increase in the rate of cytoplasmic decay of the 3.5 kb mRNA relative to the 1.6 kb mRNA. However, the long 3′ UTR caused a fivefold decrease in the rate of appearance of mature cytoplasmic mRNA despite transcription from the same promoter. This is attributed to less efficient nuclear processing of immature transcripts prior to export to cytoplasm. Selective 3′ UTR sequence substitutions, deletions, and mutations showed that this loss of expression is produced additively by specific sequences in a 700-base basal instability region and by non-specific length effects. These mechanisms are selectively enhanced in steroidogenic cells. The AURE contribute a smaller basal destabilization effect selective for steroidogenic cells that is removed by their mutations. Inclusion of introns in the 3.5 kb StAR vector enhances StAR expression, suggesting the effects of introns complexes on nuclear processing. Br-cAMP provides an additional means to rapidly modulate StAR expression independent of transcription by attenuating the nuclear and cytoplasmic instability mechanisms within the extended 3′ UTR.

Adenosine deaminase (ADA) regulates cellular levels of adenosine and deoxyadenosine, and 17beta-estradiol (E(2)) induces ADA mRNA in MCF-7 human breast cancer cells. IGF-I also induces ADA gene expression in these cells, and induction of this response through IGF activation of estrogen receptor alpha (ERalpha) was further investigated. IGF and other polypeptide growth factors induce reporter gene expression in MCF-7 cells cotransfected with ERalpha expression plasmid and pADA211, a construct containing the -211 to +11 region of the ADA gene promoter which is required for high basal and E(2)-inducible activity. Deletion analysis of this promoter demonstrates that IGF activates ERalpha/Sp1 interactions with multiple GC-rich sites in the promoter and this response is abrogated in cells transfected with ERalpha containing mutations at Ser(118) or Ser(163). IGF induces both MAPK (mitogen-activated protein kinase) and PI3-K (phosphatidylinositol-3-kinase) phosphorylation cascades in MCF-7 cells; however, using a series of inhibitors and dominant negative constructs, our results show that induction of ADA by IGF activation of ERalpha/Sp1 is dependent on the MAPK signaling pathway.

Insulin-like growth factor-I (IGF-I), transforming growth factor alpha (TGFalpha) and epidermal growth factor (EGF) induced cathepsin D gene expression and reporter gene activity in MCF-7 human breast cancer cells transiently transfected with a construct (pCD1) containing a -2576 to -124 cathepsin D gene promoter insert. In contrast, IGF-I, but not TGFalpha or EGF, induced reporter gene activity in cells cotransfected with wild-type estrogen receptor (ER) expression plasmid and a construct (pCD2) containing estrogen-responsive downstream elements from -208 to -101. Promoter deletion and mutational analysis experiments identified four GC-rich sites and an imperfect palindromic estrogen responsive element required for IGF-I activation of the ER (ligand-independent). Subsequent studies with the mitogen-activated protein kinase (MAPK) inhibitor, PD98059, and a serine(118(-ER mutant confirmed the role of the MAPK pathway for IGF-I activation of the ER in MCF-7 cells. Thus, growth factor activation of ER can mediate transactivation vs ER/Sp1 binding to GC-rich sites and represents a novel pathway for ligand-independent ER action. The divergent pathways for IGF-I and TGFalpha/EGF activation of the ER observed in MCF-7 cells contrast with previous data indicating that pathways for growth factor activation of the ER are dependent on the gene and/or gene promoter and on cell context.

Heat shock protein 27 (Hsp 27) is expressed in mammary tumors and may play a role in tumor growth and response to anti-neoplastic drug therapy. 17beta-Estradiol (E2) induces Hsp 27 mRNA levels in MCF-7 human breast cancer cells, and we have investigated the comparative inhibitory mechanisms using the aryl hydrocarbon receptor (AhR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the direct-acting antiestrogen ICI 164,384. TCDD inhibited E2-induced Hsp 27 gene expression and analysis of the Hsp 27 gene promoter showed that the inhibitory response was associated with AhR interactions with a pentanucleotide motif at -3 to +2 in the promoter that corresponded to the core sequence of a dioxin responsive element. In contrast, ICI 164,384 induced Hsp 27 gene expression and reporter gene activity in MCF-7 cells and this represents one of the few examples of the estrogen receptor-alpha (ERalpha) agonist activity of the 'pure' antiestrogen ICI 164,384.