Differential regulation of redox responsive transcription factors by the nephrocarcinogen 2,3,5-Tris(glutathion-S-yl)hydroquinone.

2,3,5-Tris(glutathion-S-yl)hydroquinone [TGHQ] is a potent nephrotoxicant and nephrocarcinogen, and induces a spectrum of mutations in human and bacterial cells consistent with those attributed to reactive oxygen species (ROS). Studies were conducted to determine whether the oxidative stress induced by TGHQ in renal proximal tubule epithelial cells (LLC-PK(1)) modulates transcriptional activities widely implicated in transformation responses, namely 12-O-tetradecanoyl phorbol 13-acetate (TPA) responsive element (TRE)- and nuclear factor kappa B (NF-kappaB)-binding activity. TGHQ increased TRE- and NF-kappaB-binding activity in a concentration- and time-dependent manner. Catalase fully inhibited peak TGHQ-mediated TRE- and NF-kappaB-binding activity. In contrast, although deferoxamine fully inhibited TGHQ-mediated TRE-binding activity, it had only a marginal effect on NF-kappaB-binding activity. Collectively, these data indicate that TGHQ modulates TRE- and NF-kappaB-binding activity in an ROS-dependent fashion. Cycloheximide and actinomycin D fully inhibited TGHQ-mediated TRE-binding activity, but in the absence of TGHQ increased NF-kappaB-binding activity. Although protein kinase C (PKC) is widely implicated in stress response signaling, pretreatment of cells with PKC inhibitors (H-89, calphostin C) did not modulate TGHQ-mediated DNA-binding activities. In contrast, pretreatment of cells with (PD098059), a mitogen activated protein kinase kinase (MEK) inhibitor, markedly reduced TGHQ-mediated TRE-binding activity, but enhanced TGHQ-mediated NF-kappaB-binding activity. We conclude that TGHQ-mediated TRE- and NF-kappaB-binding activities are ROS-dependent. Although there is a common requirement for hydrogen peroxide (H(2)O(2)) in the regulation of these DNA-binding activities, there appears to be divergent regulation after H(2)O(2) generation in renal epithelial cells.

Stress- and growth-related gene expression are independent of chemical-induced prostaglandin E(2) synthesis in renal epithelial cells.

Cellular stress can initiate prostaglandin (PG) biosynthesis which, through changes in gene expression, can modulate cellular functions, including cell growth. PGA(2), a metabolite of PGE(2), induces the expression of stress response genes, including gadd153 and hsp70, in HeLa cells and human diploid fibroblasts. PGs, gadd153, and hsp70 expression are also influenced by the cellular redox status. Polyphenolic glutathione conjugates retain the ability to redox cycle, with the concomitant generation of reactive oxygen species. One such conjugate, 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), is a potent nephrotoxic and nephrocarcinogenic metabolite of the nephrocarcinogen, hydroquinone. We therefore investigated the effects of TGHQ on PGE(2) synthesis and gene expression in a renal proximal tubular epithelial cell line (LLC-PK(1)). TGHQ (200 microM, 2 h) increases PGE(2) synthesis (2-3-fold) in LLC-PK(1) cells with only minor (5%) reductions in cell viability. This response is toxicant-specific, since another proximal tubular toxicant, S-(1, 2-dichlorovinyl)-L-cysteine (DCVC), stimulates PGE(2) synthesis only after massive (68%) reductions in cell viability. Consistent with the ability of TGHQ to generate an oxidative stress, both deferoxamine mesylate and catalase protect LLC-PK(1) cells from TGHQ-mediated cytotoxicity. Only catalase, however, completely blocks TGHQ-mediated PGE(2) synthesis, implying a major role for hydrogen peroxide in this response. TGHQ induces the early (60 min) expression of gadd153 and hsp70. However, while inhibition of cyclooxygenase with aspirin prevents TGHQ-induced PGE(2) synthesis, it does not affect TGHQ-mediated induction of gadd153 or hsp70 expression. In contrast, a stable PGE(2) analogue, 11-deoxy-16, 16-dimethyl-PGE(2) (DDM-PGE(2)), which protects LLC-PK(1) cells against TGHQ-mediated cytotoxicity, modestly elevates the levels of gadd153 and hsp70 expression. In addition, catalase and, to a lesser extent, deferoxamine mesylate block TGHQ-induced gene expression. Therefore, although TGHQ-induced generation of reactive oxygen species is required for PGE(2) synthesis and stress gene expression, acute TGHQ-mediated increases in gadd153 and hsp70 mRNA levels are independent of PGE(2) synthesis.

DDM-PGE(2)-mediated cytoprotection in renal epithelial cells by a thromboxane A(2) receptor coupled to NF-kappaB.

The present studies were conducted to determine the pharmacological nature of a cytoprotective 11-deoxy-16,16-dimethyl-PGE(2) (DDM-PGE(2)) receptor in LLC-PK(1) cells. DDM-PGE(2)-mediated cytoprotection against 2,3,5-(trisglutathion-S-yl)hydroquinone (TGHQ)-mediated cytotoxicity can be reproduced using thromboxane A(2) (TXA(2)) receptor (TP) agonists (U46619 and IBOP), and the cytoprotective response to DDM-PGE(2) and TP agonists is inhibited by TP antagonists (SQ-29,548 and ISAP). Western blot analysis using an antipeptide antibody against the human platelet TP receptor (55 kDa) identified a particulate associated 54-kDa protein. DDM-PGE(2)-mediated 12-O-tetradecanoyl phorbol-13-acetate (TPA) responsive element (TRE) binding activity is not inhibited by cyclooxygenase inhibitors (aspirin and indomethacin) or a TXA(2) synthase inhibitor (sulfasalazine), suggesting that the biological response to DDM-PGE(2) is not dependent on de novo TXA(2) biosynthesis. Peak DDM-PGE(2)- and U46619-mediated TRE binding activity and nuclear factor-kappaB (NF-kappaB) binding activity are inhibited by SQ-29,548. The full cytoprotective response to DDM-PGE(2) requires an 8-h pulse with agonist. DDM-PGE(2)-mediated TRE and NF-kappaB binding activity remain elevated in the presence of agonist and rapidly decay following agonist washout, suggesting a direct correlation between DDM-PGE(2)-mediated cytoprotection and persistent DNA binding activities. TPA, a protein kinase C activator, induces cytoprotection and a persistent increase of NF-kappaB binding activity. DDM-PGE(2)-mediated cytoprotection and NF-kappaB binding activity but not TRE binding activity are inhibited by sulfasalazine. We conclude that the DDM-PGE(2) receptor is a TP receptor and that the cytoprotective response may be mediated in part by NF-kappaB.

Secondary osteoporosis. Diagnostic considerations.

This article discusses the important secondary causes of osteoporosis that contribute significantly to bone loss and that seem to increase fracture risk, including hypogonadism, endogenous and exogenous thyroxine excess, hyperparathyroidism, malignancies, gastrointestinal diseases, medications, vices, and connective tissue diseases.

c-Ha-rasEJ transfection in vascular smooth muscle cells circumvents PKC requirement during mitogenic signaling.

In view of the prominent role of protein kinase C (PKC) in the regulation of vascular smooth muscle cell (VSMC) growth and differentiation, the present studies were conducted to assess the impact of c-Ha-rasEJ transfection on PKC-dependent growth programming. PKC activity was elevated in the cytosolic and particulate compartments of c-Ha-rasEJ VSMC, relative to naive or pSV2neo vector controls. Constitutive and 12-O-tetradecanoyl phorbol 13-acetate (TPA)-inducible binding to a TPA-responsive element (TRE) was also enhanced in c-Ha-rasEJ VSMC. Fetal bovine serum (FBS) did not increase TRE-binding activity in serum-starved c-Ha-rasEJ VSMC but increased TRE-binding activity in pSV2neo VSMC. FBS-mediated TRE-binding activity was dramatically decreased in serum-starved pSV2neo VSMC pretreated with 100 ng/ml TPA for 24 h to downregulate PKC activity. c-Ha-rasEJ VSMC exhibited a marked proliferative advantage over controls under both restrictive and growth-permissive serum conditions. PKC downregulation did not influence the mitogenic response to serum in c-Ha-rasEJ VSMC but ablated [3H]thymidine incorporation into DNA in naive or pSV2neo vector counterparts. Western blot analysis demonstrated increased expression of extracellular signal-regulated kinase 2 (ERK2), but not ERK1, in c-Ha-rasEJ VSMC, relative to pSV2neo control. Immunoblots of serum-starved and PKC-depleted c-Ha-rasEJ VSMC demonstrated a dramatic increase in the phosphorylated form of ERK2, relative to pSV2neo controls. These data suggest that oncogenic c-Ha-rasEJ circumvents a requirement for a TPA-responsive PKC isoform(s) during mitogenic stimulation of VSMC.

PGE2-mediated cytoprotection in renal epithelial cells: evidence for a pharmacologically distinct receptor.

Although the exact mechanism of prostaglandin E2 (PGE2)-mediated cytoprotection has not been elucidated, its ability to induce cytoprotection in cell culture suggests this action occurs at the cellular level. The present studies were conducted to determine whether PGE2 induces protection against 2,3,5-(trisglutathion-S-yl)-hydroquinone [2,3,5-(trisglutathion-S-yl)-HQ]-mediated cytotoxicity in a renal proximal tubule epithelial cell line (LLC-PK1) and to delineate the cellular and molecular mechanisms associated with this response. Pretreatment of LLC-PK1 cells with 0.01-40 microM PGE2 for 24 h fully protects against a moderately toxic concentration of 2,3,5-(trisglutathion-S-yl)-HQ. PGE2-mediated cytoprotection is observed in cells pretreated at pH 7.4 but not at pH 7.8. However, cytoprotection is observed in LLC-PK1 cells pretreated with the PGE2 analog, 11-deoxy-16,16-dimethyl PGE2 (DDM-PGE2) but not with the PGE2 receptor [E-prostanoid (EP)] agonists 17-phenyltrinor PGE2 (EP1), 11-deoxy PGE1 (EP2/EP4), sulprostone (EP1/EP3), PGE1, or PGA2. 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C (PKC), also induces cytoprotection, supporting a role for this pathway in the cytoprotective response. PGE2, DDM-PGE2, and TPA all induce the binding of nuclear proteins to a TPA responsive element (TRE), whereas analogs that did not induce cytoprotection (PGE1, 17-phenyltrinor PGE2, sulprostone) were without effect. DDM-PGE2- and TPA-mediated cytoprotection and TRE binding activity are inhibited by N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89), a PKC inhibitor. These data suggest that cytoprotection by PGE2 and DDM-PGE2 in LLC-PK1 cells is mediated by a PKC-coupled receptor, which is pharmacologically distinct from the presently classified EP receptor subtypes.

Induction of highly proliferative phenotypes in cultured glomerular mesangial cells by benzo[a]pyrene alone or in combination with methoxamine.

Recent studies have suggested that aromatic hydrocarbons can initiate glomerular mesangial cell (GMC) injury and contribute to the onset of renal disease. The present studies were conducted to assess the impact of benzo[a]pyrene (BaP), a ubiquitous polycyclic aromatic hydrocarbon, on the proliferation of GMCs. Challenge of cultured GMCs with BaP (0.3-30 microM) for 24 h was associated with concentration-dependent decreases in DNA synthesis, a response mediated by selective interference with early G1 cell cycle progression. One cycle of sequential treatment with 3 microM BaP for 24 h followed by challenge with 10 microM methoxamine (MeoA), a growth-promoting alpha 1-adrenergic agonist, for an additional 24 h attenuated the inhibitory response elicited by BaP alone. Following three rounds of sequential treatment with BaP and MeoA, GMCs exposed to BaP alone or BaP/MeoA exhibited enhanced proliferation rates relative to controls. BaP/MeoA cells acquired the greatest proliferative enhancement and exhibited unregulated c-jun and c-fos gene expression under growth-arrested and serum-stimulated conditions. Marked increases in specific AP-1 binding to a synthetic oligonucleotide were observed upon serum stimulation of quiescent cultures of BaP/MeoA cells relative to controls or any of the other treatment groups. These data demonstrate that sequential treatment with BaP in combination with MeoA is associated with induction of highly proliferative phenotypes in GMCs characterized by differential expression of growth-related protooncogenes.

Interference with protein kinase C-related signal transduction in vascular smooth muscle cells by benzo[a]pyrene.

Elucidation of the mechanisms involved in the deregulation of vascular smooth muscle cell (SMC) growth and differentiation during the course of atherogenesis and the putative role of toxic injury in this process have been a subject of considerable interest in recent years. In this regard, we have recently shown that in vitro exposure of vascular (aortic) SMCs to benzo[a]pyrene (BaP), an atherogenic polycyclic aromatic hydrocarbon, initially delays cell cycle progression and inhibits cell proliferation and then causes permanent modulation to a highly proliferative state. To define the molecular basis of this response, we have examined critical components of the protein kinase C (PKC) signal transduction system upon exposure to BaP. Marked inhibition of serum-stimulated inositol phospholipid turnover was observed in growth-arrested SMC cultures challenged with 30 microM BaP for 24 h and then stimulated with 10% fetal bovine serum for 120 or 1800 s. Benzo[a]pyrene inhibited PKC-mediated phosphorylation of exogenous and endogenous proteins in the cytosolic and particulate fraction of cycling, as well as quiescent cultures. The PKC inhibitory response was observed as early as 0.5 h following BaP treatment and maintained for at least 5 days. Exposure of quiescent SMCs to 30 microM BaP inhibited the ability of serum to induce c-fos mRNA expression and decreased AP-1 binding to a 12-O-tetradecanoyl phorbol-13-acetate responsive element. Inhibition of PKC-related signal transduction was not due to generalized interference with cell cycle events since peak expression of the c-myc and c-Ha-ras protooncogenes following serum stimulation of quiescent cultures was unchanged, or slightly enhanced, by 30 microM BaP. Collectively, these data suggest that the ability of BaP to modulate growth and differentiation programs in vascular SMCs involves early interference with PKC-related mitogenic signal transduction.

Altered protein secretion and extracellular matrix deposition is associated with the proliferative phenotype induced by allylamine in aortic smooth muscle cells.

Repeated cycles of allylamine-induced aortic injury in vivo modulate the proliferative potential of smooth muscle cells (SMCs) during serial propagation in vitro. This modulation may be partly mediated by disturbances in polyphosphoinositide metabolism which afford allylamine-treated cells a growth advantage over control cells [Cox, Murphy and Ramos (1990) Exp. Mol. Pathol. 53, 52-63]. The present studies were conducted to further evaluate the mechanisms which mediate the enhanced proliferative potential of allylamine cells. Cellular growth and/or [3H]thymidine incorporation into DNA were evaluated in control and allylamine cells seeded on plastic culture dishes or glass coverslips in the presence of 0.1, 1 or 10% fetal bovine serum (FBS). On either substrate, incubation in 0.1% FBS for 48 h inhibited DNA synthesis in cultures of both cell types, but the inhibitory response was more pronounced in allylamine cells. Subsequent challenge with 10% FBS increased thymidine incorporation to a greater extent in allylamine cells. Interestingly, enhanced DNA synthesis of allylamine cells was associated with increased cell numbers only when seeded on a glass surface. The enhanced growth rate on glass was not due to increased plating efficiency since comparable attachment rates were observed for both cell types. Reseeding of control cells on glass substrates pre-coated by allylamine cells afforded control cells a growth advantage comparable with that observed for allylamine cultures. Conditioned media from growth-arrested, as well as cycling cultures, of allylamine cells stimulated DNA synthesis in cultures of either cell type to a greater extent than conditioned media from control counterparts. In addition, the responsiveness of allylamine cells to secreted products was enhanced relative to that of control cells. Metabolic labelling studies revealed that the synthesis and/or secretion of 52, 46, 33 and 28 kDa proteins was enhanced in allylamine cultures relative to controls, and that the expression of two proteins of 30 and 31 kDa only occurred in allylamine cultures. We conclude that the enhanced growth response of allylamine cells is associated with both altered protein secretion and differential extracellular matrix deposition.