The expression of estrogen receptors in rat genioglossus muscle-derived satellite cells and its relationship to intracellular Ca(2+) mobilization.

Genioglossus (GG) is the most important pharyngeal dilator muscle in maintaining upper airway (UA) patency in human; therefore, its dysfunction plays an important role in pathogenesis of sleep-related breathing disorder. Recently, the expression of estrogen receptors (ERs) on mRNA and protein level has been evidenced in GG muscle; however, the cellular localization of two subtypes of ER in GG myoblasts remains unclear. The present study was designed to clarify the expression and cellular distribution of ERs in rat GG muscle-derived satellite cells (MDSCs) and further probe the effect of ERs expression on regulation of intracellular Ca(2+). The immunocytochemistry revealed positive staining for both ERalpha and ERbeta in nuclei and cytoplasm of GG MDSCs. Noticeably, positive signals for ERalpha and ERbeta were comparable in cytoplasm, whereas the positive staining of ERalpha in nuclear was obviously strong than that of ERbeta. More intriguingly, by using Fluo 4-AM as a fluorescent Ca(2+) indicator and 17beta-estradiol (E2) as a stimulant, we observed that the level of intracellular Ca(2+) was not affected by E2 application, which implied that Ca(2+) signaling may not be involved in ER-mediated estrogenic effects on GG MDSCs. Taken together, the present study clearly indicates the differential cellular localization of ERs in rat GG MDSCs; moreover, ER-mediated estrogenic effect in rat GG MDSCs bears no relationship to intracellular Ca(2+) mobilization. In addition, the GG MDSCs express both ERalpha and ERbeta and therefore, provide a suitable and convenient in vitro cell model for investigating the molecular mechanisms of estrogenic effects on rat GG muscle.

Androgen and estrogen (alpha) receptor localization on periaqueductal gray neurons projecting to the rostral ventromedial medulla in the male and female rat.

The periaqueductal gray (PAG) is involved in many gonadal steroid-sensitive behaviors, including responsiveness to pain. The PAG projects to the rostral ventromedial medulla (RVM), comprising the primary circuit driving pain inhibition. Morphine administered systemically or directly into the PAG produces greater analgesia in male compared to female rats, while manipulation of gonadal hormones alters morphine potency in both sexes. It is unknown if these alterations are due to steroidal actions on PAG neurons projecting to the RVM. The expression of androgen (AR) and estrogen (ERalpha) receptors in the PAG of female rats and within this descending inhibitory pathway in both sexes is unknown. The present study used immunohistochemical techniques (1) to map the distribution of AR and ERalpha across the rostrocaudal axis of the PAG; and (2) to determine whether AR and/or ERalpha were colocalized on PAG neurons projecting to the RVM in male and female rats. AR and ERalpha immunoreactive neurons (AR-IR, ERalpha-IR) were densely distributed within the caudal PAG of male rats, with the majority localized in the lateral/ventrolateral PAG. Females had significantly fewer AR-IR neurons, while the quantity of ERalpha was comparable between the sexes. In both sexes, approximately 25-50% of AR-IR neurons and 20-50% of ERalpha-IR neurons were retrogradely labeled. This study provides direct evidence of the expression of steroid receptors in the PAG and the descending pathway driving pain inhibition in both male and female rats and may provide a mechanism whereby gonadal steroids modulate pain and morphine potency.

Translational machinery and protein folding: evidence of conformational variants of the estrogen receptor alpha.

As an approach to understand how translation may affect protein folding, we analyzed structural and functional properties of the human estrogen receptor alpha synthesized by different eukaryotic translation systems. A minimum of three conformations of the receptor were detected using limited proteolysis and a sterol ligand-binding assay. The receptor in vitro translated in rabbit reticulocyte lysate was rapidly degraded by protease, produced major bands of about 34kDa and showed a high affinity for estradiol. In a wheat germ translation system, the receptor was more slowly digested. Two soluble co-existing conformations were evident by different degradation patterns and estradiol binding. Our data show that differences in the translation machinery may result in alternative conformations of the receptor with distinct sterol binding properties. These studies suggest that components of the cellular translation machinery itself might influence the protein folding pathways and the relative abundance of different receptor conformers.

Understanding ligand binding effects on the conformation of estrogen receptor alpha-DNA complexes: a combinational quartz crystal microbalance with dissipation and surface plasmon resonance study.

Estrogen receptors are ligand-activated transcription factors that regulate gene expression by binding to specific DNA sequences. To date, the effect of ligands on the conformation of estrogen receptor alpha (ERalpha)-DNA complex remains a poorly understood issue. In our study, we are introducing the quartz crystal microbalance with dissipation monitoring (QCM-D) as a new alternative to study the conformational differences in protein-DNA complexes. Specifically, we have used QCM-D, in combination with surface plasmon resonance (SPR) spectroscopy, to monitor the binding of ERalpha to a specific DNA (estrogen response element, ERE) and a nonspecific DNA in the presence of either the agonist ligand, 17b-estradiol, the partial antagonist ligand, 4-hydroxytamoxifen, or vehicle alone. Both with presence and absence of ligand, the specific ERalpha-ERE complexes are observed to adopt a more compact conformation compared to nonspecific complexes. This observation is well correlated to the biophysical changes occurring during protein-DNA interaction shown by past structural and mechanism studies. Notably, pretreatment of ERalpha with E2 and 4OHT affects not only the viscoelasticity and conformation of the protein-DNA complex but also ERalpha binding capacity to immobilized ERE. These results affirm that ligands have remarkable effects on ERalpha-DNA complexes. Understanding these effects will provide insight into how ligand binding promotes subsequent events required for gene transcription.

Radiation damage to DNA-protein specific complexes: estrogen response element-estrogen receptor complex.

The exposure of a DNA-protein regulatory complex to ionising radiation induces damage to both partner biomolecules and thus can affect its functioning. Our study focuses on a complex formed by the estrogen response element (ERE) DNA and the recombinant human estrogen receptor alpha (ER), which mediates the signalling of female sex hormones, estrogens. The method of native polyacrylamide retardation gel electrophoresis is used to study the stability of the complex under irradiation by low LET radiation ((60)Co gamma rays) and the ability of the separately irradiated partners to form complexes. The relative probabilities of ERE DNA strand breakage and base damages as well as the probabilities of damages to the ER binding domain are calculated using the Monte Carlo method-based model RADACK.

On-chip biosensing of estrogen receptor-alpha at single molecular level.

A novel method for detecting interaction between DNA and DNA-binding protein at single molecular level has been proposed. In this study, estrogen receptor-alpha (ER-alpha) was used for biosensing as the proof-example. A 518 bp-long (ca. 176 nm) DNA probe labeled with streptavidin at its 5'-terminus was prepared by inserting a consensus oligonucleotide sequence that binds to ER-alpha. A solution containing ER-alpha was dropped onto the Ni-treated mica substrate on which the DNA prove was previously immobilized, and it was observed by AFM. Specific binding of ER-alpha could be observed by measuring the distance between the site where binding occur, to the streptavidin label.