TATA-flanking sequences influence the rate and stability of TATA-binding protein and TFIIB binding.

The kinetics of TATA-binding protein (TBP) and TFIIB binding were measured on a series of promoter constructs that had varying sequences within and flanking the TATA box. The flanking sequences were found to influence TBP stability even though they do not contact the protein. This occurs by altering the decay rate rather than the association rate. TFIIB association is accompanied by protein-protein cooperativity as indicated by the simultaneous release of both proteins in challenge experiments. The sequence of the TATA box and the sequences that flank it can influence the kinetics of the TFIIB.TBP.DNA complex. TFIIB can contribute to tighter TATA binding in two ways. It always slows the decay rate of TBP, but it can also increase the rate of association at promoters with certain combinations of TATA and flanking sequences. The results imply that the interplay between the TATA box and flanking elements leads to variations in the kinetics of preinitiation complex formation that may account for the observed effects of all of these diverse sequences on transcription.

Roles for non-TATA core promoter sequences in transcription and factor binding.

Sequence blocks within the core region were swapped among RNA polymerase II promoters to explore effects on transcription in vitro. The pair of blocks flanking TATA strongly influenced general transcription, with an additional effect on promoter activation. These flanking elements induced a change in the ratio of activated to basal transcription, whereas swapping TATA and initiator sequences only altered general transcription levels. Swapping the flanking blocks influenced binding by general transcription factors TBP and TFIIB. The results suggest that the architecture of the extended core sequence is important in determining promoter-specific effects on both general transcription levels and the tightness of regulation.

Promoter activation via a cyclic AMP response element in vitro.

Transcription activation via activating transcription factor cyclic AMP response element binding (ATF/CREB) sites in vitro was explored using transcription and permanganate assay for open complex formation. These sites were used to drive transcription from an adenovirus major late core sequence. Under conditions where activation is strong, 20-50-fold, ATF/CREB is required for preinitiation complexes to reach the open complex stage. Complete opening requires activator, ATP, and initiating nucleotides. In exploration of postinitiation steps, no stimulation of promoter clearance was observed but a modest stimulation of the rate of continuous transcription occurred. High amounts of DNA template, commonly used in in vitro studies, allows some templates to open without activator, but leaves the nucleotide requirements intact. This leads to a drastic lowering of the dependence on ATF/CREB. Taken together, the data indicate that ATF/CREB activates this system primarily by stimulating the formation of functional preinitiation complexes.

Prostaglandin synthase 1 and prostaglandin synthase 2 both participate in activation-induced prostaglandin D2 production in mast cells.

Activation of MMC-34 cells, a murine mast cell line, or bone marrow-derived mast cells by aggregation of IgE cell surface receptors or addition of calcium ionophore stimulates prostaglandin (PG) D2 synthesis and secretion. An initial rapid burst of PGD2 synthesis, complete within 30 min, is followed by a slower subsequent production of PGD2 that reaches a maximum 4 to 8 h after activation in MMC-34 cells. PG synthase 1 (PGS-1) message and protein are expressed constitutively in MMC-34 cells and are not modulated by exposure to calcium ionophore or aggregation of IgE receptors. In contrast, activation of MMC-34 or bone marrow-derived mast cells induces expression of the PG synthase 2 (PGS-2) gene. PGS-2 induction following mast cell activation is blocked by dexamethasone. The initial PGD2 burst in activated MMC-34 cells is prevented by aspirin pretreatment, suggesting that constitutive PGS-1 present in mast cells before activation is responsible for the early PGD2 production in response to activation. In contrast, the later phase of PGD2 production is blocked by dexamethasone, cycloheximide, or NS-398, a PGS-2-specific nonsteroidal anti-inflammatory drug that inhibits PGS-2 enzyme activity but not PGS-1 activity. These data demonstrate that mast cell activation results 1) in the induction of PGS-2 gene expression, and 2) in both PGS-1-dependent PGD2 synthesis and PGD2 synthesis that is dependent on the activation-induced synthesis and activity of PGS-2.

Immediate-early gene expression in response to hypertrophic and proliferative stimuli in pulmonary arterial smooth muscle cells.

Remodeling of the pulmonary vascular tree in pulmonary hypertension is associated with hypertrophy and proliferation of smooth muscle cells. Since the stimuli and signaling pathways for these processes are not well understood, we used a rat pulmonary arterial smooth muscle cell line (PAC1) to examine the effects of thrombin and platelet-derived growth factor (PDGF) on cellular growth and immediate-early gene expression. Over 72 h, thrombin (1 unit/ml) caused hypertrophy as reflected by a 102 +/- 12% increase in protein synthesis and a 49 +/- 11% increase in protein content per cell, but no change in cell number. PDGF (2.5 ng/ml) stimulated proliferation as evidenced by an increase in cell number (doubling in 5 days), but no significant change in protein content per cell. Immediate-early gene expression was examined by Northern blotting: both thrombin and PDGF induced egr-1, c-fos, c-jun, junB, and fra-1 mRNAs within 15 min; the response was maximal at 30-60 min (increases ranging from 2.9- to 9.3-fold over control serum-deprived cells) and returned to base-line levels within 2-4 h. Neither agent affected junD mRNA levels. However, thrombin but not PDGF, caused an increase in fosB mRNA levels (7.7 +/- 4.0-fold higher than control, n = 12, p < 0.0005). The immediate-early gene response to both agonists was generally dependent on extracellular Ca2+, Na2+/H+ exchange, and protein kinase C activation, but not on cAMP. The exception was c-jun mRNA, the levels of which were not affected by inhibition of protein kinase C, but decreased significantly by prevention of cAMP formation. Thapsigargin-sensitive intracellular Ca2+ stores were necessary for the response to thrombin, but not to PDGF. These results demonstrate that thrombin is a hypertrophic agent and that PDGF is a proliferative agent in PAC1 cells. These two agonists stimulate increases in a variety of immediate-early gene mRNAs, but only thrombin induces fosB mRNA.

Agonist-selective regulation of polyphosphoinositide metabolism in pulmonary artery smooth muscle cells.

Using a rat pulmonary artery smooth muscle cell line (PAC1), detailed analysis of polyphosphoinositide (PPI) metabolism reveals receptor type-selective patterns in the formation of inositol phosphates and 3-hydroxyphosphorylated PPIs. Responses to several agonists that stimulate hypertrophy or proliferation were examined, and distinct categories of response profile were observed. Thrombin and angiotensin II stimulated the hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate and the formation of several cytosolic species of inositol phosphates without the activation of PI 3-hydroxykinase. The response to thrombin was distinctive because a very large production of inositol 1,4-bisphosphate was accompanied by hydrolysis of PI 4-phosphate. The response to platelet-derived growth factor (PDGF) was distinguished by the production of the PI 3-hydroxykinase product, PI 3,4,5-trisphosphate, and the appearance of PI 3-hydroxykinase activity in immunoprecipitates. PDGF treatment of PAC1 cultures did not produce accumulation of detectable amounts of inositol 1,4,5-trisphosphate, although a small sustained elevation in the level of inositol monophosphate and a gradual accumulation of inositol 1,3,4-trisphosphate were observed. Characterization of these distinctive responses permitted us to correlate agonist-regulated PPI metabolism with induction of immediate-early genes and stimulation of hypertrophy or proliferation of PAC1 cultures (Rothman, A., Wolner, B., Button, D., and Taylor, P. (1994) J. Biol. Chem. 269, 6399-6404). Thrombin-stimulated PPI turnover and the production of a high level of inositol bisphosphate may be early signals linked to the induction of fosB and PAC1 cell hypertrophy, whereas the activation of PI 3-hydroxykinase and the accumulation of PI 3,4,5-trisphosphate in response to PDGF appear to be associated with mitogenesis.

Initial 5-fluorouracil trabeculectomy in uncomplicated glaucoma.

The effectiveness of trabeculectomy with adjunctive low-dose 5-fluorouracil (5-FU) as the initial surgical procedure in uncomplicated glaucoma was evaluated retrospectively in a consecutive series of 52 patients (mean follow-up, 18.6 +/- 11.7 mos) and 74 control subjects. The cumulative 2-year success (intraocular pressure [IOP] less than 21 mmHg) was 100% in the 5-FU group and 78.9% in the control group (P = 0.01, Wilcoxon test). The 5-FU group had a mean postoperative IOP of 12.5 +/- 4.6 mmHg versus 17.4 +/- 5.7 mmHg in the control group at 2-year follow-up (P = 0.015, t test). Antiglaucoma medications were required in 5.8% of patients in the 5-FU group and in 41.9% of controls within 2 years (P less than 0.0001, Fisher's exact test). These results suggest that low-dose 5-FU at the time of initial trabeculectomy leads to a higher success rate, lower IOP, and less need for antiglaucoma medications postoperatively.

Late bleb-related endophthalmitis after trabeculectomy with adjunctive 5-fluorouracil.

The incidence of late-onset bleb-related endophthalmitis was evaluated retrospectively in 229 consecutive trabeculectomies performed with adjunctive 5-fluorouracil (5-FU) therapy. Mean follow-up was 23.7 +/- 16.3 months (range, 3 to 60 months). Thirteen eyes (5.7%) of 11 patients developed bleb-related endophthalmitis an average of 25.9 +/- 17.4 months (range, 5 to 58 months) after surgery. Infection occurred in 9 of 96 (9.4%) procedures performed from below and in 4 of 133 (3.0%) procedures performed superiorly (P = 0.05, Fisher's exact test). The relative risk of bleb-related endophthalmitis in trabeculectomy from below versus above is 4.0 after adjustment for age and sex (95% confidence interval = 1.1, 14.8). Trabeculectomy with adjunctive 5-FU performed from below carries an increased risk of late bleb-related infection. The incidence of late bleb-related endophthalmitis after 5-FU trabeculectomy appears to be higher than that for trabeculectomy without adjunctive 5-FU injections.