No abstract

Cadmium triggers apoptosis of LLC-PK1 cells through induction of endoplasmic reticulum (ER caused activation of the activating transcription factor 6-CCAAT/enhancer-binding protein-homologous protein (CHOP) and the inositol-requiring ER-to-nucleus

Cadmium is a widely distributed nephrotoxic metal that causes renal tubular injury. In this report, we investigated involvement of endoplasmic reticulum (ER) stress and individual unfolded protein responses in cadmium-initiated apoptosis of tubular epithelial cells. Cadmium chloride (CdCl 2 ) induced expression of endogenous ER stress markers, GRP78, GRP94 and CHOP in vitro and in vivo, and subsequently caused cytological changes typical of apoptosis. Attenuation of ER stress by transfection with ER chaperone GRP78 or ORP150 suppressed CdCl 2 -triggered apoptosis. In response to CdCl 2 , phosphorylation of RNAdependent protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2a (eIF2a) was observed. Enhanced phosphorylation of eIF2a attenuated, whereas inhibition of eIF2a exacerbated CdCl 2 -induced apoptosis. Activating transcription factor 6 (ATF6) was also activated by CdCl 2 and blockade of this process suppressed induction of CHOP and thereby improved cell survival. CdCl 2 also triggered activation of the inositol-requiring ER-to-nucleus signal kinase 1 (IRE1)-X-box-binding protein 1 (XBP1) pathway and inhibition of XBP1 attenuated apoptosis independent of GRP78 and CHOP. c-Jun N-terminal kinase (JNK), another molecule downstream of IRE1, was also phosphorylated by CdCl 2 and its inhibition attenuated apoptosis. These results evidenced bidirectional regulation of apoptosis in cadmium-exposed cells. The ATF6 and IRE1 pathways cooperatively caused apoptosis via induction of CHOP, activation of XBP1 and phosphorylation of JNK, and the PERK-eIF2a pathway counteracted the proapoptotic processes.

Activity of secreted alkaline phosphatase (SEAP) produced by transfected cells is rapidly down-regulated by endoplasmic reticulum (ER) stress independent of transcriptional regulation. This phenomenon was observed in a wide range of cell types triggered by various ER stress inducers. The magnitude of the decrease in SEAP was proportional to the extent of ER stress and inversely correlated with the induction of endogenous ER stress markers grp78 and grp94. In contrast to SEAP, activity of secreted luciferase was less susceptible to ER stress. The decrease in SEAP activity by ER stress was caused by abnormal post-translational modification, accelerated degradation and reduced secretion of SEAP protein. In transgenic mice constitutively producing SEAP, systemic induction of ER stress led to reduction in serum SEAP. In these mice, administration with lipopolysaccharide caused rapid, transient decrease in serum SEAP activity, and it was correlated with up-regulation of grp78 in several organs including the spleen, lung, kidney, liver and heart. These results elucidated for the first time a possible involvement of transient, systemic ER stress in endotoxemia and provided evidence for usefulness of ER stress responsive alkaline phosphatase for real-time monitoring of ER stress in vitro and in vivo.

Cigarette smoke contains low levels of agonists for the aryl hydrocarbon receptor (AhR; also called the dioxin receptor). However, little is understood about the whole potential of cigarette smoke for activating AhR. In this report, we evaluated the total ''dioxin-like'' activity of cigarette smoke using in vitro and in vivo reporter systems. Cigarette smoke extract (CSE) was prepared from seven cigarette brands (1-20 mg tar content) and subjected to in vitro bioassay based on the xenobiotic-responsive element (XRE) as the sensor and secreted alkaline phosphatase (SEAP) as the reporter. Exposure of reporter cells to CSE triggered activation of XRE in a dose-dependent manner, which was suppressed by functional inhibition of AhR. Direct, brief exposure of the cells to cigarette smoke similarly induced activation of XRE. Using 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) as the standard, the XRE-activating potential (XAP) of individual smoke was evaluated quantitatively. Positive correlation was observed between the tar content and XAP values. The XAP values estimated were extremely high with a range from 18.5 to 51.2 ng 2,3,7,8-TCDD equivalent per cigarette. To further estimate XAP of cigarette smoke in vivo, we generated transgenic reporter mice that secrete SEAP under the control of XRE. After exposure of the mice to smoke, serum levels of SEAP were significantly elevated within 12 hours, peaked at 24 hours, and declined thereafter. These results evidenced for the first time that cigarette smoke has unexpectedly high dioxin-like potential that triggers the AhR-XRE pathway in vitro and in vivo. (Cancer Res 2006; 66(14): 7143-50)

No abstract

Expression of nephrin, a crucial component of the glomerular slit diaphragm, is downregulated in patients with proteinuric glomerular diseases. Using conditionally immortalized reporter podocytes, we found that bystander macrophages as well as macrophage-derived cytokines IL-1b and TNF-a markedly suppressed activity of the nephrin gene promoter in podocytes. The cytokine-initiated repression was reversible, observed on both basal and inducible expression, independent of Wilms' tumor suppressor WT1, and caused in part via activation of the phosphatidylinositol-3-kinase/Akt pathway. These results indicated a novel mechanism by which activated macrophages participate in the induction of proteinuria in glomerular diseases.

Downregulation of nephrin in podocytes leads to development of proteinuria in human and experimental kidney diseases. However, little is understood about pathophysiologic substances that regulate nephrin expression. In this report, we established conditionally immortalized reporter podocytes REPON for sensitive, continuous monitoring of nephrin gene expression. A murine podocyte cell line harboring a temperature-sensitive simian virus 40 large T antigen was stably transfected with a gene encoding secreted alkaline phosphatase (SEAP) under the control of the 5.4 or 8.3 kb nephrin gene promoter. The established reporter cells REPON5.4 and REPON8.3 were exposed to various pathophysiologic substances, and culture media were subjected to SEAP assay to identify regulators of nephrin gene expression. Among the bioactive substances tested, three physiological ligands of nuclear receptors including all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, and dexamethasone significantly activated the nephrin gene promoter in a dose-dependent manner. These effects were observed in both REPON5.4 and REPON8.3 and were associated with upregulation of nephrin mRNA. The effects of these substances were synergistic, and the maximum effect was observed by combination of three agents. In contrast, inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha as well as phorbol ester significantly downregulated the activity of the nephrin promoter as well as nephrin gene expression. These results elucidated the bidirectional regulation of nephrin by distinct pathophysiologic substances and may provide molecular bases for explaining how proteinuria is induced under pathologic situations and why some ligands for nuclear receptors have the anti-proteinuric potential.