pendent GR phosphorylation site, serine . Serine  is hyperphosphorylated under an array of stressful conditions, including glucose starvation, oxidative stress, UV irradiation, and osmotic shock. Interestingly, cells expressing a mutant GR incapable of phosphorylation at Ser (SerGR) had a redirection of the transcriptional response to hormone and an altered activation of distinct biological pathways. These alterations in gene expression profiles Silybin result from changes in the ability of phosphorylated SerGR (phosphoSerGR) to interact with the  zeta signaling proteins on different gene promoters within chromatin. Our results suggest that the level of cellular stress prior to hormone stimulation, as measured by changes in the phosphorylation of Ser, dictates which genes will respond to glucocorticoids.

MATERIALS AND METHODS Reagents, custom antibodies, and plasmids.  cetirizine  Dexamethasone (Dex) (pregnadien 9fluoromethyl,triol,0dione) was purchased from Steraloids (Newport, RI). Doxycycline, hydrogen peroxide, and lipopolysaccharide (LPS) were purchased from Sigma (St. Louis, MO). Tumor necrosis factor alpha (TNF) and transforming growth factor  (TGF) were purchased from R&D Systems (Minneapolis, MN). The kinase inhibitors SB080, LY900, KT70, SP00, and compound C were purchased from Calbiochem (San Diego, CA). Rabbit antiphosphoSerGR antibodies were produced by using peptides made by AnaSpec (San Jose, CA), and the antisera were produced by Covance (Denver, PA). pTREhGR SA and pTREhGR SD were generated by the sitedirected mutagenesis of pTREhGR  using a QuikChange kit (Stratagene, La Jolla, CA). Nterminally Flagtagged GR  (pcDNAhGR Flag) was described previously .

The knockdown of p8  was accomplished with Mission short hairpin RNA (shRNA) lentiviral transduction particles (Sigma), and zeta knockdowns were accomplished with small  Chlorogenic acid inhibitor  interfering RNA (siRNA) SMARTpools purchased from Dharmacon (Lafayette, CO). An expression vector containing pCMV zeta was purchased from Open Biosystems (Huntsville, AL), and pCMV DN zeta was created by PCR as previously described (). All cloning and mutagenesis products were verified by DNA sequencing at the DNA Sequencing Core at the National Institute of Environmental Health Sciences (NIEHS). Cell culture and stable cell line production. UOS, A9, H9C, hepatocytoma (HTC), and HeLa cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM)–F medium Chlorogenic acid 327-97-9 supplemented with 0% fetal calf serum. Geneticin (00 g/ml; Invitrogen) hygromycin (0 g/ml; Invitrogen) was used to establish UOS cell lines stably expressing a polyclonal mixed population of wildtype hGR (WThGR ), SAhGR, and SDhGR similar to that described previously.

Receptor levels were compared by Western blot analysis, and cell populations expressing comparable receptor levels were used. Results were validated with the use of three spectrum approach independent mixed populations for each mutant of the GR. Some cell treatments were done in glucosefree DMEM (Invitrogen) supplemented with  g/liter glucose and/or 0% fetal bovine serum (FBS) or 0 mM mannitol.