1,25-Dihydroxyvitamin D3 has opposite effects on the expression of parathyroid secretory protein and parathyroid hormone genes

Previous studies have shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] decreases levels of mRNA for prepro-PTH as well as PTH secretion after chronic exposure (24-48 h) of parathyroid cells in tissue culture. We have now extended these studies to determine the effects of the vitamin D3 metabolite on parathyroid secretory protein (PSP) gene expression. Primary cultures of bovine parathyroid cells were incubated with 10(-8) M 1,25-(OH)2D3 for periods of time ranging from 24-72 h. As observed in earlier experiments, prepro-PTH mRNA decreased to less than 50% of the control value after 72 h. In marked contrast, PSP mRNA showed a 2.5-fold increase by 24 h and greater than 7-fold stimulation by 72 h. In the same studies, PTH secretion was suppressed (to 60% of control), while PSP secretion was increased by 40% over control values. Exposure to high (2.5 mM) or low (0.5 mM) calcium had no effect on PSP mRNA, even though low calcium stimulated the secretion of PSP while high calcium suppressed secretion. These studies showed that 1,25-(OH)2D3 has opposite effects on the gene expression of PSP and PTH in bovine parathyroid cells in tissue culture.     

Regulation of apoptosis by Caspases under oxidative stress conditions in mice testicular cells: in vitro molecular mechanism

Exposure to various toxicants is known to cause apoptosis in various cell types. The spermatogenic cells are particularly sensitive to various deleterious conditions including toxicant exposure. The affected cells might undergo apoptosis; however, the mechanisms may be different for different kinds of insults to the cells. In the present study, we looked into the mechanisms involved in apoptosis after exposure of testicular cells from mice to two different chemicals, diethyl maleate (DEM) and tert-butyl hydroperoxide (TBHP). For the study, cells were maintained for 4 h under various treatments: control (media only), 0.25 mM DEM, 0.5 mM DEM, 0.25 mM TBHP, and 0.5 mM TBHP. The treated cells were then harvested for various estimations, viz. viability, reduced and oxidized glutathione, redox ratio, free radical generation, and ethidium bromide/acridine orange co-staining. mRNA was extracted for RT-PCR analysis of Caspase 3, Caspase 8, Caspase 9, p53, p21, Bax, and Bcl-2. It was observed that both the treatments resulted in decreased levels of reduced glutathione and a concomitant increase in the oxidized form and ROS levels in a dose-dependent manner. The apoptotic cell death was evident from ethidium bromide/acridine orange staining. The mRNA expression pattern of various Caspases showed progressive increase in Caspase 3 and Caspase 9 mRNA in both the treatments in a dose-dependent manner, whereas there was no change in Caspase 8 mRNA expression. p53, p21, and Bax also showed increased expression, whereas Bcl-2 expression remained unchanged in DEM treatments and increased significantly in both TBHP treatments. Hence, the present study indicates the involvement and activation of various apoptotic factors, particularly Caspase 3 and 9 along with p53, in response to exposure of testicular cells to DEM and TBHP.     

Diethylmaleate and buthionine sulfoximine, glutathione-depleting agents, differentially inhibit expression of inducible nitric oxide synthase in endotoxemic mice.

Diethylmaleate (DEM) and buthionine sulfoximine (BSO), glutathione (GSH)-depleting agents, reduced the metabolic activity and the protein level of iNOS in both macrophages and hepatocytes activated by lipopolysaccharide (LPS). In this study, we examined the effects of DEM and BSO on iNOS expression in LPS-treated mice under the assumption that the level of GSH may alter the expression of nitric oxide synthase. Serum levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were also determined. DEM markedly decreased the levels of hepatic GSH in response to LPS. Treatment of mice with DEM significantly reduced serum nitrite/nitrate levels and hepatic iNOS protein and mRNA induction by LPS. Although BSO inhibited the level of hepatic GSH in LPS-treated mice, the agent did not alter serum nitrite/nitrate levels and hepatic iNOS expression. DEM completely inhibited an increase of serum IL-1beta level by LPS, whereas BSO failed to inhibit it. Neither DEM nor BSO significantly affected the induction of serum TNF-alpha level by LPS. These results showed that DEM and BSO differentially affect the expression of iNOS in endotoxemic mice, suggesting the possibility that suppression of iNOS expression by DEM may be associated with the inhibition of IL-1beta but not of TNF-alpha.     

Induction of cystine and glutamate transport activity in human fibroblasts by diethyl maleate and other electrophilic agents

The transport activity for cystine and glutamate in cultured human diploid fibroblasts is enhanced in response to diethyl maleate treatment. The enhancement is time- and dose-related, with a lag of about 3 h, and maximum enhancement (approximately 3-fold increase in the rate of uptake) is attained after 1 to 2 days of incubation of the cells with 0.1 mM diethyl maleate. The enhancement of the transport activity is accompanied by an increase in the Vmax and little change in the Km, and it requires RNA and protein synthesis. Other electrophilic agents, such as cyclohex-2-en-1-one, ethacrynic acid, 1,2-epoxy-3-(p-nitro-phenoxy)propane, and sulfobromophthalein, similarly enhance the transport activity. These electrophiles are known as agents that interact with glutathione. For example, diethyl maleate at high concentrations, i.e. 1 mM, depletes intracellular glutathione and injures the cells. However, at relatively low concentrations diethyl maleate and other electrophilic compounds do cause increases in the intracellular levels of glutathione which we attribute to the enhanced uptake of cystine. It is suggested that the transport system for cystine and glutamate is involved in a protective mechanism of cells against an electrophilic attack.     

Response of leptin mRNA to 24-h food deprivation and refeeding is influenced by age in rats

To obtain an insight into the influence of aging on leptin gene expression, the responses of leptin mRNA in retroperitoneal and epididymal adipose tissues and plasma leptin concentrations to 24-h food deprivation and refeeding were examined in 2-, 10- and 24-month-old normal rats. The basal level of leptin gene expression in retroperitoneal adipose tissue was significantly higher in 10- and 24-month-old rats than that in 2-month-old rats, while the level in epididymal adipose tissue was highest in 10-month-old rats for all three age groups. The basal concentrations of plasma leptin was significantly higher in 10- and 24-month-old rats than those in 2-month-old rats. The 24-h food deprivation was followed by a significant reduction in leptin mRNA expression in both retorperitoneal and epididymal adipose tissues for all three age groups. The leptin gene expression was restored to control levels 24 h following refeeding in the 2- and 10-month-old rats, but failed to be restored in the 24-month-old rats. In addition, the time course of recovery for leptin mRNA expression by refeeding to the control levels differed between the retroperitoneal and the epididymal adipose tissue in 2- and 10-month-old rats. The concentrations of plasma leptin 24 h following refeeding were compatible with the leptin mRNA levels in adipose tissues in three age groups. These results suggest that the expression of the leptin gene in response to food-deprivation and refeeding is influenced by an animal's age and that this expression is different for different regions of white adipose tissue.     

Dexamethasone up-regulates mannose receptor activity by increasing mRNA levels.

The macrophage mannose receptor is highly susceptible to modulation by a variety of inflammatory and anti-inflammatory agents. Previous studies have demonstrated that mannose receptor activity is dramatically enhanced in rat bone marrow macrophages following treatment with dexamethasone. In the present study we have investigated potential mechanisms that might be involved in this up-regulation. Uptake of ligands by the mannose receptor was increased 2.5-fold in a dose- and time-dependent manner. Maximal stimulation was seen following treatment of macrophages with 0.1-1.0 microgram/ml of dexamethasone for 24-48 h. Dexamethasone treatment increased both the number of cell surface binding sites and total cellular binding activity to 250% of control levels. In addition, total receptor protein as measured by immunoprecipitation was increased 2.5-fold. Neither the maturation rate nor the turnover rate of the protein was altered by dexamethasone treatment. Using an oligonucleotide probe derived from sequence data from the cloned human receptor cDNA, we investigated the effect of dexamethasone on the expression of mannose receptor mRNA. Following incubation with dexamethasone for 12-24 h, the level of mRNA was significantly increased. These results demonstrate that dexamethasone treatment of rat bone marrow macrophages induces synthesis of new receptor protein through an increase in the level of mannose receptor mRNA.     

Vitamin C inhibits diethylmaleate-induced L-cystine transport in human vascular smooth muscle cells

Adaptive increases in intracellular glutathione (GSH) in response to oxidative stress are mediated by induction of L-cystine uptake via the anionic amino acid transport system x(c)(-). The recently cloned transporter xCT forms a heteromultimeric complex with the heavy chain of 4F2 cell surface antigen (4F2hc/CD98). Depletion of GSH by the electrophile diethylmaleate (DEM) induces the activity and expression of xCT in peritoneal macrophages. We here examine the effects of vitamin C on induction of xCT by DEM in human umbilical artery smooth muscle cells. DEM caused time- (3-24 h) and concentration- (25-100 microM) dependent increases in L-cystine transport, with GSH depleted by 50% after 6 h and restored to basal values after 24 h. xCT mRNA levels increased after 4 h DEM treatment with negligible changes detected for 4F2hc mRNA. DEM caused a rapid (5-30 min) phosphorylation of p38(MAPK). Inhibition of p38(MAPK) by SB203580 (10 microM) enhanced DEM-induced increases in L-cystine transport and GSH, whereas inhibition of p42/p44(MAPK) (PD98059, 10 microM) had no effect. Pretreatment of cells with vitamin C (100 microM, 24 h) attenuated DEM-induced adaptive increases in L-cystine transport and GSH levels. Inhibition of p38(MAPK), but not p42/p44(MAPK), reduced the cytoprotective action of vitamin C. Our findings suggest that DEM induces activation of xCT via intracellular signaling pathways involving p38(MAPK), and that vitamin C, in addition to its antioxidant properties, may modulate this signaling pathway to protect smooth muscle cells from injury.     

Acrolein causes inhibitor kappaB-independent decreases in nuclear factor kappaB activation in human lung adenocarcinoma (A549) cells

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde to which humans are exposed in various situations. In the present study, the effects of sublethal doses of acrolein on nuclear factor kappaB (NF-kappaB) activation in A549 human lung adenocarcinoma cells were investigated. Immediately following a 30-min exposure to 45 fmol of acrolein/cell, glutathione (GSH) and DNA synthesis and NF-kappaB binding were reduced by more than 80%. All parameters returned to normal or supranormal levels by 8 h post-treatment. Pretreatment with acrolein completely blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of NF-kappaB. Cells treated for 1 h with 1 mM diethyl maleate (DEM) showed a 34 and 53% decrease in GSH and DNA synthesis, respectively. DEM also reduced NF-kappaB activation by 64% at 2 h post-treatment, with recovery to within 22% of control at 8 h. Both acrolein and DEM decreased NF-kappaB function approximately 50% at 2 h after treatment with TPA, as shown by a secreted alkaline phosphatase reporter assay. GSH returned to control levels by 8 h after DEM treatment, but proliferation remained significantly depressed for 24 h. Interestingly, DEM caused a profound decrease in NF-kappaB binding, even at doses as low as 0.125 mM that had little effect on GSH. Neither acrolein nor DEM had any effect on the levels of phosphorylated or nonphosphorylated inhibitor kappaB-alpha (IkappaB-alpha). Furthermore, acrolein decreased NF-kappaB activation in cells depleted of IkappaB-alpha by TPA stimulation in the presence of cycloheximide, demonstrating that the decrease in NF-kappaB activation was not the result of increased binding by the inhibitory protein. This conclusion was further supported by the finding that acrolein modified NF-kappaB in the cytosol prior to chemical dissociation from IkappaB with detergent. Together, these data support the conclusion that the inhibition of NF-kappaB activation by acrolein and DEM is IkappaB-independent. The mechanism appears to be related to direct modification of thiol groups in the NF-kappaB subunits.     

Expression of tissue-type plasminogen activator and its inhibitor couples with development of capillary network by human microvascular endothelial cells on matrigel

Human omental microvascular endothelial (HOME) cells seeded on Matrigel begin to migrate within 1 h, forming honeycomb-like structures and capillary-like networks within 18 h. Cross-sections of the capillary networks show them to be tube-like structures. Northern blot analysis showed that tissue-type plasminogen activator (t-PA) mRNA synthesis increased from the initial state at 0 h after seeding on Matrigel, reaching a steady state after 4 h. This elevated cellular t-PA mRNA level decreased markedly at 24 h. In contrast, the cellular plasminogen activator inhibitor-1 (PAI-1) mRNA level demonstrated biphasic curves during the 24 h after seeding on Matrigel: the PAI-1 mRNA level was increased eightfold initially at 4 h over that at O h, then declined, and again secondarily increased to greater than tenfold at 18 h. Cellular levels of both 72 kD type IV collagenase and tissue inhibitor of metalloproteinase (TIMP-2) mRNA were increased only a slightly within 2–4 h. These elevated mRNA levels were maintained for 18 h, while the TIMP-1 mRNA level increased up to 18 h, reaching around three times the level at O h. However, on collagen-coated dishes, cellular levels of t-PA, PAI-1, 72 kD type IV collagenase, TIMP-1, and TIMP-2 mRNA were not greatly changed during incubation for 24 h. On Matrigel, the cellular t-PA mRNA level at 18 h after seeding was greatly increased when treated with specific anti-transforming growth factor-β (TGF-β) antibody. In contrast, both PAI-1 and TIMP-1 mRNA levels at 18 h were reduced in the presence of anti-TGF-β antibody. Development of the capillary network on Matrigel was inhibited in the presence of anti-t-PA antibody. Epidermal growth factor (EGF) enhanced t-PA gene expression and TGF-β inhibited its expression in HOME cells cultured on collagen-coated dishes. On the other hand, TGF-β enhanced cellular expression of the PAI-1 gene. The formation of a capillary network by HOME cells on Matrigel appears to be balanced by angiogenic EGF and anti-angiogenic TGF-β through modulation of PA activity. © 1995 Wiley-Liss, Inc.     

Cellular glutathione depletion by diethyl maleate or buthionine sulfoximine: no effect of glutathione depletion on the oxygen enhancement ratio

The hypoxic and euoxic radiation response for Chinese hamster lung and A549 human lung carcinoma cells was obtained under conditions where their nonprotein thiols, consisting primarily of glutathione (GSH), were depleted by different mechanisms. The GSH conjugating reagent diethylmaleate (DEM) was compared to DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathionine biosynthesis. Each reagent depleted cellular GSH to less than 5% of control values. A 2-hr exposure to 0.5 mM DEM or a 4- or 24-hr exposure to BSO at 10 or 1 mM, respectively, depleted cellular GSH to less than 5% of control values. Both agents sensitized cells irradiated under air or hypoxic conditions. When GSH levels are lowered to less than 5% by both agents, hypoxic DEM-treated cells exhibited slightly greater X-ray sensitization than hypoxic BSO-treated cells. The D0's for hypoxic survival curves were as follows: control, 4.87 Gy; DEM, 3.22 Gy; and BSO, 4.30 Gy for the V79 cells and 5.00 Gy versus 4.02 Gy for BSO-treated A549 cells. The D0's for aerobic V79 cells were 1.70 Gy versus 1.13 Gy, DEM, and 1.43 Gy for BSO-treated cells. The D0's for the aerobic A549 were 1.70 and 1.20 for BSO-treated cells. The aerobic and anoxic sensitization of the cells results in the OER's of 2.8 and 3.0 for the DEM- and BSO-treated cells compared to 2.9 for the V79 control A549. BSO-treated cells showed an OER of 3.3 versus 3 for the control. Our results suggest that GSH depletion by either BSO or DEM sensitizes aerobic cells to radiation but does not appreciably alter the OER.