Activity of a phage-modified RNA polymerase at hybrid promoters. Effects of substituting thymine for hydroxymethyluracil in a phage SP01 middle promoter.

Transcription of bacteriophage SP01 middle promoters is specifically initiated by a complex of the Bacillus subtilis host's RNA polymerase core (E) with the SP01 gene 28 transcription-regulating protein, gp28. Normal SP01 DNA contains hydroxymethyluracil (hmUra) in place of thymine and E . gp28 preferentially transcribes hmUra-containing DNA. Hybrid DNA molecules containing an SP01 middle promoter, PM25 . 1, have been constructed in which one DNA strand contains T and the other hmUra. The major feature of these reciprocal hybrid promoters is that one has, predominantly, T substituted for hmUra in the central -35 recognition sequence in the transcribed strand, while the other has, predominantly, T substituted for hmUra in the -10 recognition sequence in the non-transcribed strand. Binding by the E . gp28 RNA polymerase and transcription of these hybrid promoters and of the normal, all-hmUra, promoter have been compared. Both hybrid promoters are weaker than the normal PM25 . 1 promoter, but the hybrid promoter with T substituted in the -10 sequence is the weakest of the set. The DNase I footprint of the normal PM25 . 1 promoter shows temperature-dependent protection of a relatively long stretch of DNA downstream from the transcriptional start site, correlating with a thermal transition of transcriptional activity of promoter complexes. The stronger of the hybrid promoters also undergoes this transition, but the weaker does not. We discuss these findings in terms of protein-DNA interactions determining specificity for a modified nucleotide at this promoter.

Extracellular glutathione and gamma-glutamyl transpeptidase prevent H2O2-induced injury by 2,3-dimethoxy-1,4-naphthoquinone.

Quinones are intracellular H2O2 generators that have been used extensively in models of oxidant injury; however, their toxicity is mediated partially through direct conjugation with glutathione (GSH). To focus upon the action of extracellular GSH in preventing H2O2-mediated toxicity, we used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which cannot conjugate with GSH but does continuously generate H2O2 through redox cycling. A eukaryotic cell line (3T3-GGT) stably overexpressing gamma-glutamyl transpeptidase (GGT) activity was used to study the role of GGT in utilizing extracellular GSH against DMNQ-induced oxidative stress. DMNQ (0 to 150 microM) caused a dose-dependent decrease of intracellular GSH and adenosine 5'-triphosphate (ATP) in both control and 3T3-GGT cells. The rate of H2O2 escape into the medium during DMNQ exposure was also the same in both cell lines. Administration of GSH helped to maintain intracellular GSH and supported resistance to ATP depletion caused by DMNQ in 3T3-GGT cells but not in control cells. The protective effect of extracellular GSH was completely prevented by acivicin, an inhibitor of GGT. Our results suggest that GGT-dependent breakdown of extracellular GSH for subsequent intracellular resynthesis helped to maintain cellular GSH levels and increased cellular resistance against DMNQ-induced oxidative injury.

Nuclear factors from expressing tissues interact in vitro with a rat alpha-2u globulin gene intron.

Members of the rat alpha 2u globulin gene family are expressed in a tissue-specific manner. Using a DNase I footprinting assay, we find that expressing tissues (liver, lachrymal, and salivary gland) contain nuclear proteins that interact specifically with two sites in the third intron of a cloned gene. These sequences are similar to a CCAAT-like sequence in the 5'-flanking region of the gene and the binding site for the transcription factor Sp1. Extracts of nonexpressing tissues (kidney, testis, brain, and spleen) do not show footprinting activity at these sites. Whereas hepatic expression of alpha 2u globulin is restricted to normal adult males, nuclear extracts of liver from all animals (adult and juvenile, male and female, intact and hypophysectomized) produce similar footprints. Footprints by extracts of lachrymal and salivary glands, tissues that express a subset of alpha 2u globulin genes distinct from the hepatic set, are also similar to the liver activity. The findings suggest that the third intron of an alpha 2u globulin gene contains sites that interact with tissue-specific factors to establish the potential for hormonal and developmental control of expression.

The glucocorticoid-glucocorticoid receptor signal transduction pathway, transforming growth factor-beta, and embryonic mouse lung development in vivo.

Lung morphogenesis has been shown to be regulated by glucocorticoids (CORT). Because CORT has been primarily thought to affect fetal lung development, previous studies have focused on the role of CORT receptor (GR)-mediated regulation of fetal lung development. Although endogenous CORT increases during embryonic and fetal stages and exogenous CORT treatment in vivo and in vitro clearly accelerates embryonic lung development, little is known about the morphoregulatory role of the embryonic CORT-GR signal transduction pathway during lung development. In this study, we characterize the embryonic mouse CORT-GR pathway and demonstrate: stage-specific in situ patterns of GR immunolocalization; similarity in GR relative mobility with progressive (E13 --> E17) development; that embryonic GR can be activated to bind a GR response element (GRE); significantly increasing levels of functional GR with increasing lung maturation; and the presence of heat shock protein (hsp) 70 and hsp90 from early (E13) to late (E17) developmental stages. These results support the purported importance of the embryonic CORT-GR signal transduction pathway in progressive lung differentiation. To demonstrate that the embryonic CORT-GR directed pathway plays a role in lung development, early embryonic (E12) lungs were exposed to CORT in utero and surfactant-associated protein A (SP-A) expression was analyzed; CORT treatment up-regulates SP-A mRNA expression and spatiotemporal protein distribution. Finally, to determine whether CORT-GR-directed pulmonary morphogenesis in vivo involves the modulation of growth factors, we studied the effect of CORT on TGF-beta gene expression. Northern analysis of TGF-beta 1, TGF-beta 2, and TGF-beta 3 transcript levels in vivo indicates that CORT regulates the rate of lung morpho- and histodifferentiation by down-regulating TGF-beta 3 gene expression.

The CORT-GR signal transduction pathway and CORT-induced cleft palate in H-2 congenic mice.

A significant association between genes at or near the H-2 complex and glucocorticoid (CORT)-induced cleft palate in mice has been conclusively demonstrated. In addition, the frequency of CORT-induced cleft palate in heterozygous offspring from reciprocal crosses has been shown to be dependent on maternal H-2 haplotype. Although CORT responses are known to be mediated through the glucocorticoid receptor (GR), the involvement of H-2 haplotype-specific variation in the embryonic CORT-GR signal transduction pathway in CORT responsiveness remains uncertain. In this study, we characterized the embryonic (E13 to E15) CORT-GR pathway in developing B10 (H-2b) and B10.A (H-2a) mouse palates. Northern analysis demonstrates similar GR transcripts and mRNA steady-state levels in embryonic B10 and B10.A mouse palates. Palatal GR M(r) and pI, as determined by Western analysis, are similar between strains and among gestational days. We also analyzed possible haplotype-specific qualitative or quantitative differences in the ability of the GR to bind a glucocorticoid response element (GRE); no significant differences in the GR-GRE complex or in the levels of activated GR are seen between strains or among gestational days. Based on these results, we conclude that H-2 associated differences in susceptibility to CORT-induced cleft palate are not associated with either variation in embryonic GR or increasing gestational age. To determine if endogenous differences in maternal circulating corticosterone levels are related to differential embryonic CORT responsiveness, the levels of serum corticosterone were determined by RIA in pregnant dams on day 13 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoids, tumor necrosis factor-alpha, and epidermal growth factor regulation of pulmonary morphogenesis: a multivariate in vitro analysis of their related actions.

The mouse lung commences development on embryonic day 11 as an epithelial evagination from the posterior pharyngeal wall into undifferentiated mesenchyme, this epithelium bifurcating to form the lung primordium. Branching morphogenesis, as well as terminal differentiation, requires epithelial-mesenchymal interactions utilizing precise regulatory controls. Not surprisingly, specific hormones and growth factors appear to play a key role in this regulation. We report here a series of experiments designed to investigate morphodifferentiation (epithelial branching number, generation number, and fractal dimension) and histodifferentiation (cell morphology and SP-A immunolocalization), as they relate to glucocorticoid (CORT)-regulation of growth factor function and expression (Northern analysis). These experiments were conducted in embryonic lung primordia (E11.5-E12) cultured under defined conditions in the presence of single or combined CORT, TNF-alpha, and EGF supplementation. EGF supplementation enhances branching morphogenesis, but not immunodetectable SP-A expression, in embryonic lung primordia cultured for 4 or 7 days. TNF-alpha supplementation also enhances branching morphogenesis on days 4 or 7 in vitro; on day 7, SP-A expression is also enhanced. By contrast, the introduction of exogenous CORT to embryonic explants cultured 4 or 7 days markedly alters morphodifferentiation and histodifferentiation. Early on it would appear to enhance morphodifferentiation by changing the process of branching, while contemporaneously initiating precocious SP-A expression; later on, it alters morphogenesis by continued terminal differentiation of normal lung epithelium and a singular transdifferentiation of lung mesenchyme into an epithelioid morphotype expressing SP-A. This is correlated with a CORT-induced, highly significant, down-regulation of TGF-beta 2 and TGF-beta 3 transcripts. Explants supplemented with CORT + TNF-alpha or CORT + EGF.demonstrate a microanatomy and SP-A expression pattern identical to that seen with CORT supplementation alone. EGF inhibits the accelerated lung maturation normally seen in the presence of exogenous TNF-alpha alone, suggesting a relationship between these two seemingly disparate regulatory pathways.

Developmental expression and CORT-regulation of TGF-beta and EGF receptor mRNA during mouse palatal morphogenesis: correlation between CORT-induced cleft palate and TGF-beta 2 mRNA expression.

Glucocorticoids (CORT) have been shown to induce cleft palate in mice. Although the pathogenetic pathway of CORT-induced cleft palate has been investigated for several decades, the molecular details remain to be elucidated. Since growth factors have been shown to regulate palate morphogenesis, and the expression of several growth factors or their receptors, e.g. TGF-beta, EGF receptor (EGF-R), are known to be modulated by CORT, we postulate that CORT modulation of growth factor (or receptor) gene expression is a key mechanism involved in CORT-induced cleft palate. To test this hypothesis, we analyzed the steady-state levels (Northern and RNase protection) and developmental expression (in situ hybridization) of four CORT-responsive genes--TGF-Beta 1, TGF- beta 2, TGF-beta 3, and EGF receptor (EGF-R)--in developing mouse palates in the presence or absence of exogenous CORT. Pregnant B10.A dams were injected on day 12 of gestation with CORT or sham-injected and embryonic palates were collected at 1, 2, and 3 days postinjection (E13-E15). During mouse palate development, significant increases in TGF-beta 1 and TGF-beta 3 mRNA levels, as well as significant decrease in TGF-beta 2 mRNA levels, are detected; no significant difference in EGF-R transcript level is observed with progressive development. In CORT-exposed palates, we demonstrate no significant differences in the direction or magnitude of change with time in TGF-beta 1, TGF-beta 3, and EGF-R mRNA levels compared to controls. However, CORT delays by 1 day the down-regulation of palatal TGF-beta 2 transcript normally seen on day 14 of gestation. TGF-beta 2 is known to inhibit cell proliferation. The level of TGF-beta 2 mRNA, the only isoform primarily expressed in the palatal mesenchyme, significantly decreases with progressive palatal development; this down-regulation of TGF-beta 2 expression is associated with increased mesenchymal cell proliferation and palatal shelf growth. CORT, at a critical stage of palatogenesis, induces a delay in the normal down-regulation of TGF-beta 2 gene expression. Given that CORT is known to inhibit mesenchymal cell proliferation and palatal shelf growth, we conclude that the CORT-induced delay in the normal down-regulation of TGF-beta 2 gene expression is probably key event in the pathogenesis of CORT-induced cleft palate.

Glucocorticoids, TGF-beta, and embryonic mouse salivary gland morphogenesis.

Branching morphogenesis is a fundamental embryologic process in many developing organs: salivary gland, lung, pancreas, mammary gland, and kidney. Glucocorticoids (CORT) are known to regulate morphogenesis in several branching organs (e.g., lung); we hypothesize that CORT is also important to embryonic mouse salivary gland morphogenesis. We studied the CORT-glucocorticoid receptor (GR) signal transduction pathway during embryonic mouse submandibular gland development. Western analysis demonstrates that the 96-kDa GR is present in E14 to E18 submandibular glands. The embryonic GR is functional, as defined by its ability to bind a DNA CORT response element, for all gestational ages evaluated (E14 to E18); increasing GR levels are observed with progressive development. The level of endogenous corticosterone in embryonic submandibular glands was also determined. Using radioimmunoassays corticosterone is first detected on day 15 of gestation; there is a progressive increase in steroid levels from day 15 to 18 of gestation. In addition, we investigated the effect of exogenous CORT on submandibular gland morphogenesis in vivo and in vitro. In vivo experiments were performed using maternal injection of CORT or sham injection on day 12 of gestation with sacrifice 72 hr post-injection (E15). CORT treatment significantly increases embryonic submandibular gland growth in vivo. To further analyze the effect of exogenous CORT on embryonic submandibular gland morphogenesis, E13 mouse submandibular glands were cultured under serum-free, chemically-defined conditions. A significant (P < 0.05) enhancement of mean branching ratios (72 hr/0 hr) is detected in CORT-supplemented explants (10(-5) M to 10(-8) M) compared to control explants; doses more dilute than 10(-8) M CORT do not significantly (P > 0.10) increase the induction of branching. In addition, CORT administration to embryonic mice in utero enhances the expression of acinar-cell-specific mucin protein. These data indicate that the CORT-GR signal transduction pathway plays an important role in salivary gland morphogenesis. Northern analysis of TGF-beta 1, TGF-beta 2, and TGF-beta 3 transcript levels in vivo confirms the presence of TGF-beta 1 and TGF-beta 2 and identifies TGF-beta 3 in embryonic submandibular glands. To begin to identify a molecular mechanism of CORT-GR mediated submandibular gland morphodifferentiation, we studied the effect of CORT on growth factor gene expression, specifically TGF-beta. Northern analysis suggests that the CORT-GR signal transduction pathway modulates the rate of morphogenesis by regulating TGF-beta 2 and TGF-beta 3 mRNA expression.

Hormone-sensitive lipase overexpression increases cholesteryl ester hydrolysis in macrophage foam cells.

Atherosclerosis is a complex physiopathologic process initiated by the formation of cholesterol-rich lesions in the arterial wall. Macrophages play a crucial role in this process because they accumulate large amounts of cholesterol esters (CEs) to form the foam cells that initiate the formation of the lesion and participate actively in the development of the lesion. Therefore, prevention or reversal of CE accumulation in macrophage foam cells could result in protection from multiple pathological effects. In this report, we show that the CE hydrolysis catalyzed by neutral cholesterol ester hydrolase (nCEH) can be modulated by overexpression of hormone-sensitive lipase (HSL) in macrophage foam cells. For these studies, RAW 264.7 cells, a murine macrophage cell line, were found to be a suitable model of foam cell formation. HSL expression and nCEH activity in these cells and in peritoneal macrophages were comparable. In addition, antibody titration showed that essentially all nCEH activity in murine macrophages was accounted for by HSL. To examine the effect of HSL overexpression on foam cell formation, RAW 264.7 cells were stably transfected with a rat HSL cDNA. The resulting HSL overexpression increased hydrolysis of cellular CEs 2- to 3-fold in lipid-laden cells in the presence of an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. Furthermore, addition of cAMP produced a 5-fold higher rate of CE hydrolysis in cholesterol-laden, HSL-overexpressing cells than in control cells and resulted in nearly complete hydrolysis of cellular CEs in only 9 hours, compared with <50% hydrolysis in control cells. Thus, HSL overexpression stimulated the net hydrolysis of CEs, leading to faster hydrolysis of lipid deposits in model foam cells. These data suggest that HSL overexpression in macrophages, alone or in combination with ACAT inhibitors, may constitute a useful therapeutic approach for impeding CE accumulation in macrophages in vivo.

gamma-Glutamyl transpeptidase is increased by oxidative stress in rat alveolar L2 epithelial cells.

The tripeptide glutathione (GSH) is used by cells to detoxify hydroperoxides, produced during oxidative stress, and is consumed in the process. Previous studies have indicated that cells can be protected against oxidative stress by extracellular GSH through its degradation catalyzed by the exoenzyme gamma-glutamyl transpeptidase (gamma GT) and its de novo synthesis within the cytosol. We hypothesized that gamma GT would be increased as part of the adaptation of cells to oxidative stress. We examined whether oxidative stress could increase gamma GT activity, protein, and mRNA in a lung epithelial cell line (L2). Cultures were subjected to H2O2-mediated toxicity by 15 min of exposure to the redox cycling quinone, menadione. Menadione (50 microM) caused an initial decrease (27 +/- 9% of baseline after 15 min) in intracellular GSH, followed by resynthesis to levels significantly higher than baseline (335 +/- 40% after 24 h, P < 0.001). This elevation was prevented by acivicin, a gamma GT inhibitor. Menadione also caused a dose-dependent increase in gamma GT enzymatic activity (715 +/- 125% of control at 24 h after 15 min of exposure to 100 microM menadione, P < 0.001) that was prevented by actinomycin D. Western blot analysis indicated increased levels of gamma GT protein with increasing menadione. A concentration-dependent increase in gamma GT-mRNA was also observed. Previous investigation has demonstrated that an increase in gamma GT activity enhances the capacity of cells to utilize extracellular GSH. The findings presented here are consistent with a role for gamma GT in cellular adaptation to oxidative stress.

Paradoxical effect on atherosclerosis of hormone-sensitive lipase overexpression in macrophages.

Foam cells formed from receptor-mediated uptake of lipoprotein cholesterol by macrophages in the arterial intima are critical in the initiation, progression, and stability of atherosclerotic lesions. Macrophages accumulate cholesterol when conditions favor esterification by acyl-CoA:cholesterol acyltransferase (ACAT) over cholesteryl-ester hydrolysis by a neutral cholesteryl-ester hydrolase, such as hormone-sensitive lipase (HSL), and subsequent cholesterol efflux mediated by extracellular acceptors. We recently made stable transfectants of a murine macrophage cell line, RAW 264.7, that overexpressed a rat HSL cDNA and had a 5-fold higher rate of cholesteryl-ester hydrolysis than control cells. The current study examined the effect of macrophage-specific HSL overexpression on susceptibility to diet-induced atherosclerosis in mice. A transgenic line overexpressing the rat HSL cDNA regulated with a macrophage-specific scavenger receptor promoter-enhancer was established by breeding with C57BL/6J mice. Transgenic peritoneal macrophages exhibited macrophage-specific 7-fold overexpression of HSL cholesterol esterase activity. Total plasma cholesterol levels in transgenic mice fed a chow diet were modestly elevated 16% compared to control littermates. After 14 weeks on a high-fat, high-cholesterol diet, total cholesterol increased 3-fold, with no difference between transgenics and controls. However, HSL overexpression resulted in thicker aortic fatty lesions that were 2.5-times larger in transgenic mice. HSL expression in the aortic lesions was shown by immunocytochemistry. Atherosclerosis was more advanced in transgenic mice exhibiting raised lesions involving the aortic wall, along with lipid accumulation in coronary arteries occurring only in transgenics. Thus, increasing cholesteryl-ester hydrolysis, without concomitantly decreasing ACAT activity or increasing cholesterol efflux, is not sufficient to protect against atherosclerosis. hormone-sensitive lipase overexpression in macrophages.