An Hsp27-related, Dominant-negative-acting Intracellular Estradiol-binding Protein

Abstract

New World primates (NWPs) exhibit a compensated form of resistance to gonadal steroid hormones. We demonstrated recently that estrogen resistance in NWP cells was associated with the overexpression of two proteins, a nonreceptor-related, dominant-negative-acting estrogen response element (ERE)-binding protein (ERE-BP) and an intracellular estradiol-binding protein (IEBP). Based on the N-terminal sequences of tryptic fragments of IEBP isolated from a 17␤-estradiol (E 2 ) affinity column we cloned a full-length cDNA for IEBP from the estrogen-resistant NWP cell line, B95-8. Subsequent sequence analysis revealed 87% sequence identity between the deduced peptide for IEBP and human Hsp27. When hormone-responsive, wild-type Old World primate (OWP) cells were transiently transfected with IEBP cDNA, E 2 -directed ERE reporter luciferase activity was reduced by 50% compared with vector only-transfected OWP cells (p < 0.0018). When IEBP and ERE-BP were cotransfected, ERE promoter-reporter activity was reduced by a further 60% (p < 0.0001). Electrophoresis mobility shift analyses showed that IEBP neither bound to ERE nor competed with the estrogen receptor (ER) for binding to ERE. However, there was evidence of protein-protein interaction of IEBP and ER␣; IEBP was coimmunoprecipitated with anti-ER␣ antibody in wildtype cells stably transfected with IEBP. A specific interaction between ER␣ and IEBP was confirmed in glutathione S-transferase pull-down and yeast two-hybrid assays. Data indicate that the Hsp27-related IEBP interacts with the ligand binding domain of the ER␣. In summary, by inhibiting the ER␣-E 2 interaction, IEBP acts to squelch ER␣-directed ERE-regulated transactivation and promote estrogen resistance in NWP cells.Compared with Old World primates (OWPs), 1 including man, New World primates (NWPs) represent an evolutionarily distinct infraorder of primates confined to the South and Central American subcontinents. A central point of distinction between NWPs and OWPs resides in the fact that NWPs display relative resistance to adrenal, gonadal, and vitamin D sterol/steroid hormones (1-11). The precise mechanism for this remains unclear but does not involve aberrant expression of nuclear receptors for specific steroid hormones (12, 13), the principal cause of hormone resistance in humans (14). Instead, hormone resistance in NWP cells appears to be the result of epigenetic factors, which result either in low affinity receptorsteroid binding kinetics or attenuation of receptor-DNA interaction. For example, studies of glucocorticoid resistance in NWP cells have shown increased expression of the heat shock protein (Hsp)90-associated FK506-binding immunophilin FKBP51, which inhibits ligand binding to glucocorticoid receptors (GR) (15). By contrast, vitamin D resistance in NWPs appears to be caused by aberrant expression of a dominantnegative-acting vitamin D response element-binding protein (VDRE-BP), the protein being homologous to human heterogeneous nuclear ribonucleoprotein A (hnRNPA) (16). Estrogen resistance in ...

Potential Contributions of Heat Shock Proteins to Temperature-Dependent Sex Determination in the American Alligator

“…This process can be modified by various intracellular proteins as they can either complex with the ER machinery to augment or inhibit ERE binding or interact directly with the ERE thus altering estrogenic signaling. HSP27 is capable of suppressing ERE-mediated transcription at the ERE by acting as an intracellular estradiol-binding protein in mammals [Chen et al, 2004[Chen et al, , 2008. The HSP70 family can be divided into 2 subfamilies, a cognate or constitutive type (HSC70) and an inducible form (HSP70).…”

“…one-month-old gonadal tissue from alligators HSP27 mRNA was significantly elevated in testicular tissue when compared to ovarian tissue of the same age. HSP27 can function as a suppressor of estrogen response element (ERE)-mediated transcription by competing with the estrogen receptor (ER) [Chen et al, 2004[Chen et al, , 2008; i.e., estrogenic signaling requires that the ligand (under normal conditions an endogenous estrogen) binds the ER which then dimerizes and translocates to the DNA where it binds to an ERE in the promoter of an estrogen-inducible gene. This process can be modified by various intracellular proteins as they can either complex with the ER machinery to augment or inhibit ERE binding or interact directly with the ERE thus altering estrogenic signaling.…”

Bag‐1M inhibits the transactivation of the glucocorticoid receptor via recruitment of corepressors

“…GCUNC45, an Hsp90 cochaperone, effectively blocks the progression of progesterone receptor chaperoning in the presence of Hsp90beta [31]. Hsp27 was known to dysregulate the interaction of estrogen receptor with chromatin and acts as a suppressor of estrogen-induced transcription [32,33]. Hsp27 was also shown to be phosphorylated in an androgen-dependent manner and enhanced the AR stability, shuttling and transactivation, thereby promoted the survival of prostate cancer cells [34].…”

Ligand Regulation and Nuclear Receptor Action

“…Based on characterization of the IDBP and IEBP in NWP cells, it was assumed that hsc70 and hsp27 act as intracellular 'decoys' for the relatively high levels of 1,25(OH) 2 D and E 2 present in NWPs, thereby attenuating over-exuberant VDR or ERα signaling. This is certainly true of hsp27 which suppresses E 2 -ERα-mediated gene transcription when over-expressed in human cells [92]. By contrast, studies using various in vitro models showed that over-expression of hsc70-like IDBP cloned from NWP cells enhanced 1,25(OH) 2 D-VDR-mediated gene transcription [93].…”