Peptide binding identifies an ERalpha conformation that generates selective activity in multiple in vitro assays.

Drugs such as tamoxifen, which act at the estrogen receptor (ER), have very different in vitro and in vivo effects from those of the native hormone. Previous research has established that different ligands induce distinct conformational changes in the ER, thus affecting the interactions of the receptor with cell-specific co-activating or co-repressing proteins (cofactors) and estrogen response elements (EREs), thus potentially driving differing biological effects. Affinity-selected peptides have been used to probe the conformational changes that occur within the ER upon binding various ligands. In this study, the authors characterize the ability of several peptides to be recruited to liganded ER under cellular conditions. Approximating ER conformation via recruitment of this peptide to the ER is concluded to be a better predictor of the agonist nature of an ER ligand under these different cellular contexts than is a canonical cotransfection transactivation assay.

Rosiglitazone induction of Insig-1 in white adipose tissue reveals a novel interplay of peroxisome proliferator-activated receptor gamma and sterol regulatory element-binding protein in the regulation of adipogenesis.

Insulin-induced gene 1 (INSIG-1) is a key regulator in the processing of the sterol regulatory element-binding proteins (SREBPs). We demonstrated that Insig-1 is regulated by peroxisome proliferator-activated receptor gamma (PPARgamma) providing a link between insulin sensitization/glucose homeostasis and lipid homeostasis. Insig-1 was identified as a PPARgamma target gene using microarray analysis of mRNA from the white adipose tissue of diabetic (db/db) animals treated with PPARgamma agonists. Insig-1 was induced in subcutaneous (9-fold) and epididymal (4-fold) fat pads from db/db mice treated for 8 days with the PPARgamma agonist rosiglitazone (30 mg/kg/day). This in vivo response was confirmed in differentiated C3H10T1/2 adipocytes treated with rosiglitazone. To elucidate the molecular mechanisms regulating INSIG-1 expression, we cloned and characterized the human INSIG-1 promoter. Co-expression of PPARgamma and RXRalpha transactivated the INSIG-1 promoter in the presence of PPARgamma agonists. This induction was attenuated when a dominant negative PPARgamma construct was transfected into cells. Furthermore, a PPARgamma antagonist repressed the transactivation of the INSIG-1 promoter-reporter construct. Truncations of the promoter resulted in the identification of a PPAR response element that mediated the regulation of the promoter. We demonstrated with recombinant proteins that the PPARgamma/RXRalpha heterodimer binds directly to this PPAR response element. In addition to regulation by PPARgamma/RXRalpha, we demonstrated that the INSIG-1 promoter is regulated by transcriptionally active SREBP. The sterol response element was identified 380 base pairs upstream of the transcriptional start site. These findings suggest that the regulation of Insig-1 by PPARgamma agonists could in turn regulate SREBP processing and thus couple insulin sensitizers with the regulation of lipid homeostasis.

Retinoic acid receptor ligands based on the 6-cyclopropyl-2,4-hexadienoic acid.

A series of novel cyclopropanyl methyl hexadienoic acid retinoids was designed and prepared. These compounds exhibited either selective activity as RXR agonists or pan-agonists on one or more of each of the RAR and RXR isoforms. The most potent pan-agonist 5a (RAR's EC(50)=17-59 nM; RXR's EC(50)=6-14 nM) showed good antiproliferative properties in the in vitro cancer cell lines, ME 180 and RPMI 8226.