Frank Bulens

Involvement of Sp1 in basal and retinoic acid induced transcription of the human tissue-type plasminogen activator gene

Transcription of the human tissue‐type plasminogen activator (t‐PA) gene is regulated by a multi‐hormonal responsive enhancer at −7 kb. Transient co‐transfections of Drosophila SL2 and human HT1080 fibrosarcoma cells with t‐PA reporter constructs showed that Sp1 and Sp3 activate the t‐PA promoter. Moreover Sp1 (but not Sp3) binding to the promoter is involved in induction by retinoic acid (RA), a response mediated through the enhancer. The role of Sp1 is specific, since mutation of the CRE element in the promoter did not affect response to RA. In contrast, the glucocorticoid induction mediated by the enhancer is independent of these Sp1 and CRE elements.

1,25-Dihydroxyvitamin D3 induction of the tissue-type plasminogen activator gene is mediated through its multihormone-responsive enhancer

Tissue‐type plasminogen activator (t‐PA) is a positive modulator of the plasminogen‐plasmin system, which is involved in bone remodeling. In the present study, 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] was found to stimulate t‐PA gene expression in ROS17/2.8 osteosarcoma cells. Transient transfection analysis and in vitro DNA binding studies identified two vitamin D‐responsive elements (VDRE) in the human t‐PA enhancer. The first VDRE (bp −7175 to −7146) comprised an inverted palindrome separated by 9 bp (IP9) overlapping a palindrome separated by 3 bp. The second VDRE (bp −7315 to −7302) is an IP2 element overlapping the previously identified retinoic acid‐responsive element. 1,25(OH)2D3 treatment of primary osteoblasts derived from t‐PAlacZ transgenic mice containing 9 kb of 5′ sequence of the human t‐PA gene increased the number of lacZ‐positive cells, fitting with the probability model of enhancer function.

Induction of t-pa synthesis with intravenous bolus injection of vitamin-a palmitate in vitamin-a deficient rats

Abstract Retinoic acid and vitamin A palmitate induce tissue-type plasminogen activator (t-PA) synthesis in cultured human umbilical vein endothelial cells (HUVEC) in vitro without alteration of plasminogen activator inhibitor-] (PAI-1) synthesis. Therefore the effect of intravenous bolus injection of water-miscible vitamin A palmitate on the blood fibrinolytic system was investigated in normal and vitamin A deficient rats. In 5 normal rats, injection of 200000 IU/kg of vitamin A palmitate did not induce a significant increase in euglobulin fibrinolytic activity, t-PA antigen and PAI activity in plasma nor of t-PA and PAI-1 mRNA in the heart within 240 min after injection. In groups of 3–6 vitamin A deficient rats, injection of 200000IU/kg of vitamin A palmitate induced a significant increase in t-PA antigen in plasma (1.9-fold after 60 min and 2.9-fold after 120 min), but no significant increase in plasma euglobulin fibrinolytic activity. A 2-fold increase in PAI activity was observed 90 min after injection of both vitamin A palmitate as well as of its solvent, suggesting that this induction was non-specific. The increase in t-PA antigen coincided with a significant increase in t-PA mRNA in heart tissue: 2.3-fold after 60 min and 4.3-fold after 120 min. PAI-1 mRNA in heart tissue increased 3.6-fold 90 min after injection with vitamin A palmitate but, surprisingly, not after injection of solvent. It is concluded that intravenous bolus injection of vitamin A palmitate induces a specific increase of t-PA mRNA in the heart and of t-PA antigen secretion in plasma of vitamin A deficient rats.

Hormonal regulation of the expression of fibrinolytic components in HT1080 fibrosarcoma and endothelial cells

Summary The effects of the synthetic glucocorticoid dexamethasone (DEX) and of all-trans-retinoic acid (RA) on the expression of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and the plasminogen activator inhibitor (PAI)-1 were investigated in the HT1080 fibrosarcoma cell line and in human umbilical vein endothelial (HUVE) cells. DEX significantly reduced the u-PA steady state mRNA level in HT1080 cells to 0.41±0.08-fold control. It induced the t-PA transcript 2.2±0.36-fold and the 2.8 and 3.4 kb PAI-1 transcripts 2.5±0.13-fold and 2.8±0.48-fold, respectively. RA increased both PAI-1 transcripts to a minor but significant extent (1.3±0.03-fold and 1.2±0.03-fold), and the t-PA and u-PA transcripts more strongly (11±0.4-fold and 2.1±0.02-fold). When added together, DEX enhanced the induction of t-PA mRNA by RA to 34±1.5-fold, inhibited the RA-mediated induction of u-PA mRNA to 0.85±0.02-fold control, and further increased the levels of the 2.8 and 3.4 kb PAI-1 transcripts to 3.6±0.13-fold and 5.0±0.05-fold, respectively. The secretion of the corresponding antigens in the conditioned medium paralleled the changes in mRNA. The effects of DEX and RA were abolished by simultaneous treatment with cycloheximide, suggesting that they required protein synthesis. In HUVE cell culture only a minor effect on t-PA-related antigen secretion was observed when DEX was added alone (1.2±0.02-fold), but DEX increased the induction by RA from 6.2±0.1-fold to 9.6±0.15-fold. In contrast, DEX and RA, alone or in combination, had very little or no effect on the generation of PAI-1-related antigen (maximally 1.2±0.4-fold). It is concluded that the glucocorticoid and the RA signal transduction pathways can interact in a complex manner resulting in a differential regulation of the fibrinolytic parameters.

THYROID HORMONE INHIBITS TRANSCRIPTION OF THE HUMAN TISSUE-TYPE PLASMINOGEN ACTIVATOR GENE PROMOTER

Abstract Objective : to determine if triiodothyronine (T3) regulates the human tissue-type plasminogen activator (t-PA) gene through its upstream DNA sequences. Design : transient transfection analysis of t-PA reporter constructs and expression vectors for thyroid hormone receptor (TR) and retinoid X receptor (RXR) in HT 1080 fibrosarcoma and SK-N-SH neuroblastoma cells. Results : 10 −6 to 10 −9 M T3 was found to inhibit the expression of human t-PA promoter reporter constructs fivefold in transfected HT1080 fibroblast and SK-N-SH neuronal cells. Unliganded TR and RXR were shown to induce the basal level of the t-PA promoter twofold. Repression by T3 required the TR DNA binding domain. The region of the t-PA promoter mediating this repression was located within bp - 110-- 7 and comprised a sequence specifically interacting with TR homodimers. However, deletion of this sequence did not relieve T3 repression. Conclusion : T3 represses the human t-PA promoter activity in HT1080 and SK-N-SH cells. However, binding of TR homodimers to the minimal T3 responsive region was shown not to be involved in the T3 repression, suggesting the existence of an alternative T3 inhibitory mechanism.