A simple method to screen ligands of peroxisome proliferator-activated receptor delta.

Peroxisome proliferator-activated receptors (PPARs) are transcription factors that belong to the nuclear receptor superfamily directly modulating gene expression by binding to specific ligands. Recently, it has been reported that PPARdelta ligands play an essential role in improvement of metabolic disorders and skin disorders. We introduce an enzyme-linked immunosorbent assay (ELISA) to screen new PPARdelta ligands. This method is based on the activation mechanism of PPARdelta where the ligand binding to PPARdelta induces the interaction of the receptor with transcriptional co-activators. We optimized a simple ELISA method for screening PPARdelta ligands. Among co-activators such as SRC-1, TIF-2, and p300, PPARdelta had more strong binding with SRC-1 in an ELISA system. GW501516 and linoleic acid, the well-known ligands of PPARdelta, increased the binding between PPARdelta and co-activators in a ligand dose-dependent manner. The recruitment of co-activator SRC-1 was also more effective than those of TIF-2 and p300. We optimized and developed a novel and useful ELISA system for the mass screening of PPARdelta ligands. This screening system may be useful in the development of drugs for metabolic disorders and skin disorders.

LPS-induced CD53 expression: a protection mechanism against oxidative and radiation stress.

The CD53 antigen is a member of the tetraspanin membrane protein family that is expressed in the lymphoid-myeloid lineage. Its biological role remains unknown. Using microarrays, we identified CD53 as one of the principal genes up-regulated by exposure of macrophages to LPS. Northern blot analysis confirmed the induction of CD53 in RAW264.7 macrophages treated with LPS or SNAP (a nitric oxide donor). Cells stably transfected with sense CD53 cDNA had increased levels of intracellular GSH and lower levels of peroxide, and were more resistant to H2O2 and to UVB irradiation. Cells harboring antisense CD53 had the opposite properties. We propose that the induction of CD53 is a major mechanism by which macrophages protect themselves against LPS-induced oxidative stress and UVB irradiation.

Sauchinone, a lignan from Saururus chinensis, suppresses iNOS expression through the inhibition of transactivation activity of RelA of NF-kappaB.

Sauchinone, a known lignan, was isolated from the root of Saururus chinensis as an active principle responsible for inhibiting the production of NO in LPS-stimulated RAW264.7 cells by activity-guided fractionation. Sauchinone dose-dependently inhibited not only the production of NO, but also the expression of iNOS mRNA and protein in LPS-stimulated RAW 264.7 cells. Furthermore, sauchinone prevented LPS-induced NF-kappaB activation, which is known to play a critical role in iNOS expression, assessed by a reporter assay under the control of NF-kappaB. However, an electrophoretic mobility shift assay (EMSA) demonstrated that sauchinone did not suppress the DNA-binding activity of NF-kappaB or the degradation of IkappaB-alpha induced by LPS. Further analysis revealed that transactivation activity of RelA subunit of NF-kappaB was dose-dependently suppressed in the presence of sauchinone. Taken together, our results suggested that sauchinone could inhibit production of NO in LPS-stimulated RAW264.7 cells through the suppression of NF-kappaB by inhibiting transactivation activity of RelA subunit.

Differentiation-inducing effects of verticinone, an isosteroidal alkaloid isolated from the bulbus of Fritillaria ussuriensis, on human promyelocytic leukemia HL-60 cells.

The inducer of differentiation of human promyelocytic leukemia HL-60 cells is commonly accepted to have potential therapeutic importance. Verticinone, one of the major isosteroidal alkaloids from the bulbus of Fritillaria ussuriensis, was found to inhibit the growth of HL-60 cells by inducing these cells to differentiate toward granulocytes. Importantly, the combination of verticinone with all-trans retinoic acid (ATRA), a well-known inducer of HL-60 cells into granulocytic lineages, was more effective than either alone, suggesting its therapeutic use in minimizing the effective dose of ATRA.