👤 Tetsuya Tagami

Cigarette smoke extract (CSE) contains many toxicants and may derange the physiological processes, such as cholesterol metabolism. We examined the impact of CSE on transcriptional regulation mediated peroxisome proliferator-activated receptors (PPARs) and its interaction with cofactors to elucidate differences in the molecular mechanism between CSE and other agonists of PPARs. We constructed several mutant PPARs (mPPARs) with amino acid substitution in the ligand-binding domain, which according to the molecular modeling, may affect the binding of agonists. In transient expression assays, each wild-type peroxisome proliferator-activated receptor (PPAR) mediated transcription stimulated by CSE was faintly yet significantly elevated compared to the control. The CSE-induced transcriptional activation was abolished in the H323A, H323Y, S342A, and H449A mPPARγs, although the activation elevated by pioglitazone was reserved. In the mPPARγ with Y473A and mPPARβ/δs with H286Y and Y436A, the pioglitazone-induced or L165041-activated transcriptional elevations were decreased and were lower than that of CSE-induced stimulation. These results suggested that CSE activated both mutant PPARs to be selectively different from those ligands. Mammalian two-hybrid assay illustrated that CSE could mildly recruit SRC1 or GRIP1 to the wild-type PPARγ. Representative ingredients, such as acrolein and crotonaldehyde present in CSE, could stimulate PPAR isoforms even at the toxicological concentrations and might possibly contribute to stimulatory effects. CSE mildly regulates the cholesterol metabolism-related genes, such as low density lipoprotein (LDL) receptor and Liver X receptor (LXR)β. In conclusion, these CSE effects the nuclear hormone receptors and their cofactors thereby disturbing metabolic phenomena. Therefore, CSE might be involved in cholesterol metabolism. Show less

The iodothyronine deiodinases are selenoenzymes that regulate the activity of thyroid hormone via specific inner- or outer-ring deiodination. In humans, type 1 deiodinase (D1) is highly expressed in the liver, but the mechanism by which its gene expression is regulated remains to be elucidated. Liver X receptor α (LXRα), a transcription factor of the nuclear receptor superfamily, is highly expressed in the liver, where it functions as a sensor for excess intracellular oxysterols. LXRα interacts with other nuclear receptors on promoters of genes that contain a binding core sequence for nuclear receptors. In addition, it is reported that the promoter of the gene encoding human D1 (hDIO1) contains the core sequence for one of nuclear receptors, thyroid hormone receptor (TR). We investigated the involvement of LXRα in the regulation of hDIO1, in the liver. We performed hDIO1 promoter-reporter assays using a synthetic LXR agonist, T0901317, and compared promoter activity between a human liver carcinoma cell line, HepG2, and a clone of human embryonic kidney cells, TSA201. We defined the region between nucleotides -131 and -114, especially nucleotides -126 and -125, of the hDIO1 promoter as critical for basal and LXRα-mediated specific transcriptional activation in HepG2 cells. An increase in hDIO1 expression was observed in LXRα-stimulated cells, but absent in cycloheximide-treated cells, indicating that new protein synthesis is required for LXRα-mediated regulation of hDIO1. On the other hand, electrophoretic mobility shift assays revealed that LXRα and RXRα bound to the hDIO1 promoter. We also demonstrated that LXRα and TRβ compete with each other on this specific region of the promoter. In conclusion, our results indicated that LXRα plays a specific and important role in activation of TH by regulating D1, and that LXRα binds to and regulates the hDIO1 promoter, competing with TRβ on specific sequences within the promoter. Show less