Identification of a Multihormone Responsive Enhancer Far Upstream from the Human Tissue-type Plasminogen Activator Gene

Abstract

A 2.4-kilobase (kb) DNA fragment, located 7.1 kb upstream from the human tissue-type plasminogen activator (t-PA) gene (t-PA2.4), acts as an enhancer which is activated by glucocorticoids, progesterone, androgens, and mineralocorticoids. Transient expression of t-PAchloramphenicol acetyltransferase reporter constructs in HT1080 human fibrosarcoma cells identified a glucocorticoid responsive unit with four functional binding sites for the glucocorticoid receptor, located between bp ؊7,501 and ؊7,974. The region from bp ؊7,145 to ؊9,578 (t-PA2.4) was found to confer a cooperative induction by dexamethasone and all-trans-retinoic acid (RA) to its homologous and a heterologous promoter, irrespective of its orientation. The minimal enhancer, defined by progressive deletion analysis, comprised the region from ؊7.1 to ؊8.0 kb (t-PA0.9) and encompassed the glucocorticoid responsive unit and the previously identified RA-responsive element located at ؊7. Vascular patency is the result of a dynamic equilibrium between blood coagulation and fibrinolysis. Injury of the vessel wall can initiate the blood clotting cascade resulting in the formation of a hemostatic clot. To counterbalance fibrin deposition, blood contains the fibrinolytic system, one main function of which is to dissolve blood clots in the circulation. It is composed of the inactive precursor plasminogen, which can be converted into the proteolytic enzyme plasmin by the plasminogen activators (PAs) 1 tissue-type and urokinase-type PA (t-PA and u-PA, respectively) leading to the degradation of fibrin. Fibrinolytic activity can be inhibited both at the level of the PAs and plasmin by PA inhibitors (PAI-1 and -2) and ␣ 2 -antiplasmin, respectively (for a review, see ref. 1). Phenotypic analysis of mice deficient for either t-PA, u-PA, or both suggested that t-PA and u-PA were complementary, not only in the prevention of uncontrolled fibrin deposition in vivo but also in distinct processes which require local extracellular proteolytic activity, such as wound healing and gonadotropin-induced ovulation, as reviewed elsewhere (2).Dexamethasone, a synthetic glucocorticoid, and androgens increase t-PA synthesis in vitro (3-5) and in vivo (6, 7). The level of t-PA expression in breast carcinoma cells correlates with the endogenous level of progesterone receptor (8), and progesterone induces t-PA-related antigen secretion in primary human endometrial cells (9). Vitamin A, retinoic acid (RA), and some of its (synthetic) analogues induce t-PA-related antigen secretion by human umbilical vein endothelial cells in vitro (10 -12) and in the plasma as well as in specific tissues of vitamin A-deficient rats in vivo (11,13,14), suggesting that circulating RA might regulate t-PA expression in the vessel wall. The induction of t-PA expression by glucocorticoids and RA can be reproduced in HT1080 human fibrosarcoma cells, and interestingly, both agents induce t-PA mRNA steady state levels and t-PA-related antigen secretion in a cooperative manner.2 Both steroid hormones and RA are t...

The t-PA -7351C>T enhancer polymorphism decreases Sp1 and Sp3 protein binding affinity and transcriptional responsiveness to retinoic acid

“…23 Mutation of the 2 Sp1 sites in the t-PA promoter has been reported to completely abolish RA induction through the enhancer, 11 while the RA response is independent of the intervening sequence between the enhancer and the initiation element. 7 These observations suggest a DNA looping mechanism bringing the enhancer and the proximal promoter in physical contact. By self-association, Sp1 can join distant DNA segments, thereby causing the intervening DNA to loop.…”

“…4 The t-PA enhancer mediates induction of t-PA gene transcription in response to vitamin A (retinoic acid, RA) as well as to all steroid hormones except estrogens. 7 The RA response is mediated by an RA-responsive element (RARE) comprising a DR5 element located in the vicinity of the polymorphic site (ie, at Ϫ7319). The steroid hormone response is mediated through a hormone responsive unit consisting of 4 glucocorticoid responsive elements (GREs) located upstream of the polymorphic site (ie, at Ϫ7960, Ϫ7942, Ϫ7703, and Ϫ7501).…”

Androgen Control of Cell Proliferation and Cytoskeletal Reorganization in Human Fibrosarcoma Cells

“…The HT-1080 cell line was established in 1974 from a tumor biopsy taken from the acetabulum of a 35-year-old male who had not received chemotherapy and died of metastatic disease without an autopsy 3 months after diagnosis (12). It has been shown that HT-1080 cells express functional glucocorticoid receptor, but lack AR, progesterone receptor, and mineralocorticoid receptor (17). Because androgen and glucocorticoid responses are partially overlapping in a variety of cell types (18 -20), we reasoned that ectopic expression of human AR in HT-1080 cells might recapitulate some or all of the steroid-induced cytoskeletal changes seen with glucocorticoid receptor, and moreover, provide a null genetic background to investigate molecular determinants of AR signaling.…”

Retinoic Acid Induces Expression of the Thyroid Hormone Transporter, Monocarboxylate Transporter 8 (Mct8)

“…6). RAREs have been reported both within the proximal promoter (5,33,34) and outside of the proximal promoter (35,36).…”