In this paper we show that transcription factors Ets-1 and Ets-2 recruit transcription adapter proteins p300 and CBP (cAMP-responsive element-binding protein) during the transcriptional activation of the human stromelysin promoter, which contains palindromic Ets-binding sites. Ets-2 and p300/CBP exist as a complex in vivo. Two regions of p300/CBP between amino acids (a.a.) 328 and 596 and a. a. 1678 and 2370 independently can interact with Ets-1 and Ets-2 in vitro and in vivo. Both these regions of p300/CBP bind to the transactivation domain of Ets-2, whereas the C-terminal region binds only to the DNA binding domain of Ets-2. The N- and the C-terminal regions of CBP (a.a. 1-1097 and 1678-2442, respectively) which lack histone acetylation activity independently are capable of coactivating Ets-2. Other Ets family transcription factors failed to cooperate with p300/CBP in stimulating the stromelysin promoter. The LXXLL sequence, reported to be important in receptor-coactivator interactions, does not appear to play a role in the interaction of Ets-2 with p300/CBP. Previous studies have shown that the stimulation of transcriptional activation activity of Ets-2 requires phosphorylation of threonine 72 by the Ras/mitogen-activated protein kinase signaling pathway. We show that mutation of this site does not affect its capacity to bind to and to cooperate with p300/CBP.

The cAMP-response element-binding protein (CREB)-binding protein and p300 are two highly conserved transcriptional coactivators and histone acetyltransferases that integrate signals from diverse signal transduction pathways in the nucleus and also link chromatin remodeling with transcription. In this report, we have examined the role of p300 in the control of the G 1 phase of the cell cycle in nontransformed immortalized human breast epithelial cells (MCF10A) and fibroblasts (MSU) by using adenovirus vectors expressing p300-specific antisense sequences. Quiescent MCF10A and MSU cells expressing p300-specific antisense sequences synthesized p300 at much reduced levels and exited G1 phase without serum stimulation. These cells also showed an increase in cyclin A and cyclin A-and E-associated kinase activities characteristic of S phase induction. Further analysis of the p300-depleted quiescent MCF10A cells revealed a 5-fold induction of c-MYC and a 2-fold induction of c-JUN. A direct target of c-MYC, CAD, which is requiredfor DNA synthesis, was also found to be up-regulated, indicating that up-regulation of c-MYC functionally contributed to DNA synthesis. Furthermore, S phase induction in p300-depleted cells was reversed when antisense c-MYC was expressed in these cells, indicating that up-regulation of c-MYC may directly contribute to S phase induction. Adenovirus E1A also induced DNA synthesis and increased the levels of c-MYC and c-JUN in serum-starved MCF10A cells in a p300-dependent manner. Our results suggest an important role of p300 in cell cycle regulation at G1 and raise the possibility that p300 may negatively regulate early response genes, including c-MYC and c-JUN, thereby preventing DNA synthesis in quiescent cells.

Abstract-A number of studies suggest that moderate consumption of red wine may be more effective than other alcoholic beverages in decreasing the risk of coronary heart disease mortality. The phytochemical resveratrol found in wine, derived from grapes, has been thought to be responsible for cardiovascular benefits associated with wine consumption because it was shown to have antioxidant and antiplatelet activities. In the present investigation, we examined the effect of resveratrol on induction of tissue factor (TF) expression in vascular cells that were exposed to pathophysiological stimuli. The data presented herein show that resveratrol, in a dose-dependent manner, inhibited the expression of TF in endothelial cells stimulated with a variety of agonists, including interleukin-1␤ (IL-1␤), tumor necrosis factor-␣ (TNF␣) and lipopolysaccharide (LPS). A similar inhibition of TF induction was also seen in LPS stimulated monocytes that were pretreated with resveratrol before their stimulation with LPS. In addition, resveratrol was shown to inhibit the LPS-induced expression of TNF␣ mRNA in endothelial cells and of TNF␣ and IL-1␤ mRNA in monocytes. Nuclear run-on analysis in endothelial cells showed that resveratrol inhibited TF expression at the level of transcription.

Binding of plasma factor VII(a) to tissue factor (TF) initiates the coagulation cascade. In health, TF is not expressed in endothelial cells. However, endothelial cells express TF in response to lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF alpha), and other biological stimuli. TF expression by endothelial cells is implicated in thrombotic disorders in patients with a variety of clinical disorders. In the present study, we demonstrate that curcumin (diferulolylmethane), a known anticarcinogenic and anti-inflammatory agent, inhibited phorbol 12-myristate 13-acetate (PMA), LPS, TNF alpha, and thrombin-induced TF activity and TF gene transcription in human endothelial cells. The present data show that curcumin prevented the activation of c-Rel/p65, which is essential for TF gene activation in endothelial cells, by impairing the proteolytic degradation inhibitor protein, I kappa B alpha. The data also show that curcumin downregulated AP-1 binding activity. The present studies are the first to demonstrate that PMA, but not LPS, TNF alpha, and thrombin, induced Egr-1 binding to the second serum-responsive region (SRR-2) of TF promoter and that curcumin inhibited the PMA-induced Egr-1 binding to SRR-2. Overall, the data suggest that the anticarcinogenic and anti-inflammatory properties of curcumin may be related to its ability to inhibit cellular gene expression regulated by transcription factors NF-kappa B, AP-1, and Egr-1.

Tissue factor (TF) is a transmembrane receptor that serves as a cofactor for factor VIIa and initiates the extrinsic pathway of blood coagulation. Under normal physiological conditions, TF is expressed in extravascular and perivascular cells but not in vascular endothelial cells and monocytes. TF can be induced in these cells by inflammatory regulators and other stimulators, such as LPS, thrombin, oxidized lipoproteins, and certain growth factors. An earlier study showed that growing primary cultures of human umbilical vein endothelial cells (HUVECs) with endothelial cell growth supplement (ECGS) and heparin had impaired the ability of monolayers to express surface membrane TF activity after perturbation. The mechanism by which ECGS suppressed TF activity was not known. In the present study, we investigated the effect of recombinant acidic and basic fibroblast growth factors (aFGF and bFGF) on the induction of TF in a HUVEC cell line and a fibroblast cell line. Both aFGF and bFGF suppressed the phorbol myristate acetate-induced expression of TF in endothelial cells but not the serum-induced expression of TF in fibroblast cells. Diminished expression of the cell surface TF activity observed in endothelial cells grown with aFGF or bFGF was due to the accumulation of a lower number of TF mRNA transcripts. TF mRNA stability was not altered in HUVECs grown with aFGF or bFGF. Nuclear run-on experiments revealed that the transcription of TF and several other genes that play an important role in inflammation and angiogenesis was reduced in the endothelial cells that were cultured with aFGF or bFGF. The diminished expression of TF may be part of a generalized response of endothelial cells to FGF that facilitates migration of endothelial cells during angiogenesis.