Ya-Hua Zhuang

EVIDENCE FOR ENHANCED UBIQUITIN-MEDIATED PROTEOLYSIS OF THE CHICKEN PROGESTERONE RECEPTOR BY PROGESTERONE

Genomic actions of progesterone are mediated via A and B isoforms of the progesterone receptor (PR). One major factor controlling PR level is progesterone causing negative autoregulation (down-regulation) of the receptor protein. In this work we studied the mechanism whereby progesterone exerts its effects on PR level in the chicken oviduct. We found that progesterone does not markedly regulate PR mRNA expression. Furthermore, we demonstrate here for the first time that PR is a target for ubiquitylation and that the proportion of ubiquitylated PR is increased by progesterone treatment. Our data suggest that ligand-induced down-regulation of PR involves enhanced degradation of receptor protein by ubiquitin-proteasome system in vivo.

Subcellular location of androgen receptor in rat prostate, seminal vesicle and human osteosarcoma MG-63 cells

Location of the androgen receptor (AR) before and after dihydrotestosterone (DHT) administration was studied in 6 castrated and 2 normal male rats, as well as in MG-63 human osteosarcoma cell culture. Two days after castration, rats were injected with DHT and sacrificed 0, 6 and 24 h later. Cryosections of ventral prostate and seminal vesicle were stained with a polyclonal anti-AR antibody. Cultured MG-63 cells were also stained similarly. The intensity of immunoreaction was measured semiquantitatively by computer-assisted image analysis. In both normal and castrated rats, a positive reaction was seen mainly in the nuclei of epithelial cells and stromal cells of the prostate and seminal vesicle, as well as in those of smooth muscle cells of the seminal vesicle. AR immunoreactivity was up-regulated by DHT, it decreased clearly in both organs after castration. Nuclear AR and its up-regulation by androgen were also seen in MG-63 cells. At the immunoelectron microscopy, silver enhanced gold particles were predominantly found in the heterochromatin of cell nuclei. Treatment with DHT caused a decondensation of the heterochromatin and AR was more dispersed. Thus, AR appears to be nuclear independently of the ligand.

Immunolocalization of retinoic acid receptors in rat, mouse and human ovary and uterus

We raised an antibody against a synthetic peptide corresponding to amino acids 155-174 of human retinoic acid receptor alpha (RAR-alpha). The sequence is highly homologous in all RARs and their isoforms. When mouse and human RARs (alpha, beta and gamma) expressed in Cos cell were analysed with immunoblot, all receptors gave a specific 51 K signal. Mouse RAR-gamma gave an additional signal corresponding to 58 K. In human teratocarcinoma cells (F9) both 51 and 58K molecule sizes were detected. The RAR expression in F9 cells was slightly down-regulated in charcoal-stripped culture medium and returned to normal level after retinoic acid treatment. The 51 K protein was found in all ovarian and uterine samples, but the quantity of the 58 K protein varied in different species and organs, being highest in the mouse uterus and the rat and human ovary. Using immunohistochemistry the RARs were found in the nuclear compartment. In the rat uterus, positive immunoreaction was found mainly in the nuclei of epithelial, uterine glandular and stromal cells. In the rat ovary, positive reaction was found in the nuclei of germinal epithelial, follicular and stromal cells.

Mechanisms of action of sex steroid hormones: Basic concepts and clinical correlations

The review deals with the clinically important aspects of the basic mechanisms of sex steroid hormones. Steroids can act through two basic mechanisms: genomic and non-genomic. The classical genomic action is mediated by specific intracellular receptors, whereas the primary target for the non-genomic one is the cell membrane. Many clinical symptoms seem to be mediated through the non-genomic route. Furthermore, membrane effects of steroid and other factors can interfere with the intranuclear receptor system inducing or repressing steroid-and receptor-specific genomic effects. These signalling pathways may lead to unexpected hormonal or anti-hormonal effects in patients treated with certain drugs. Steroid receptors (SRs) are members of a large family of nuclear transcription factors that regulate gene expression by binding to their cognate steroid ligands, to the specific enhancer sequences of DNA (steroid response elements) and to the basic transcription machinery. SRs are phosphoproteins, which are further phosphorylated after ligand binding. The role of phosphorylation in receptor transaction is complex and may not be uniform to all SRs. However, phosphorylation/dephosphorylation is believed to be a key event regulating the transcriptional activity of steroid receptors. SR activities can be affected by the amount of SR in the cell nuclei, which is modified by the rate of transcription and translation of the SR gene as well as by proteolysis of the SR protein. There is an auto- and heteroregulation of receptor levels. Some of the SRs appear to bind specific protease inhibitors and exhibit protease activity. The physiological significance of this weak proteolytic activity is not clear. Some SRs are expressed as two or more isoforms, which may have different effects on transcription. Receptor isoforms are different translation or transcription products of a single gene. Isoform A of the progesterone receptor is a truncated form of PR isoform B originating from the same gene, but it is able to suppress not only the gene enhancing activity of PR-B but also that of other steroid receptors. From the clinical point of view, it is important to note that the final hormonal effect in a target tissue is dependent on the cross talk between different nuclear steroid receptors and on expression of receptor isoforms.

HDLG5/KIAA0583, encoding a MAGUK‐family protein, is a primary progesterone target gene in breast cancer cells

The steroid hormone progesterone is known to have profound effects on growth and differentiation of normal and malignant breast epithelial cells. The biologic actions of progesterone are exerted through the nuclear progesterone receptor‐mediated control of target gene transcription. We utilized differential display polymerase chain reaction (DD‐RT‐PCR) to identify genes whose expression is altered in response to progestins in cultured breast cancer cells. Here we report identification of a gene encoding a member of the MAGUK protein family, hDlg5 (also known as KIAA0583 and P‐dlg), as being the primary progestin target gene in MCF‐7 breast cancer cells. Quantitative real‐time RT‐PCR analysis showed a rapid and strong upregulation of hDlg5 mRNA in cells treated with synthetic progestin medroxyprogesterone acetate (MPA) in the presence of estrogen in MCF‐7, T47D and ZR‐75‐1 cells. The induction was abrogated by antiprogestin RU486. hDlg5 mRNA was also upregulated by progesterone, R5020 and dexamethasone. Protein synthesis inhibitor cycloheximide failed to block progestin‐mediated induction of the hDlg5 gene. hDlg5 is a member of the growing family of MAGUKs (membrane‐associated guanylate kinase homologs) and is to our knowledge the first member of the family reported to be hormonally regulated. hDlg5 is one of the human homologs of the Drosophila gene dlg [lethal(1)discs‐large], which was initially identified as a tumor suppressor gene. The Dlg has a well‐established role in cell growth control and maintenance of cell adhesion and cell polarity. Domain profile analysis revealed that hDlg5 has 2 additional PDZ domains than previously reported.

Spermatogenesis in the vitamin A-deficient rat : Possible interplay between retinoic acid receptors, androgen receptor and inhibin α-subunit

In order to understand the mechanisms of retinol action on the testis, testicular retinoic acid receptor alpha, beta(RAR alpha and beta), androgen receptor (AR) and inhibin alpha-subunit were studied in normal, vitamin A-deficient (VAD) and vitamin A-supplemented rats by immunohistochemistry and immunoblotting. Compared to the normal testis, expression of 110 K AR was up-regulated by vitamin A withdrawal, whereas 51 K RAR alpha remained unchanged. An additional 55 K RAR alpha signal was observed. Readministration of retinol caused a marked decrease of AR in the VAD testis. By 24 h, AR declined to below the normal level. Although the 51 K RAR alpha signal remained unchanged, the 55 K band was slightly up-regulated at 6 h after retinol administration. A 51 K RAR beta protein was seen in the VAD but in not the normal testis. The intensity of the 51 K RAR beta band remained constant before and after the administration of retinol, but it had a slight up-shift at 6 h after retinol injection, suggesting post-translational modification of the receptor. The inhibin alpha-subunit of 18 K protein was undetectable in the VAD testis and increased to above normal level at 24 h after retinol administration. Immunohistochemically, nuclear AR immunostaining was more intense in the VAD testis than in the normal testis. The intensity of immunostaining declined in all AR-positive cells after the injection of retinol, but the decrease was more evident in Sertoli than in other cells. At 24 h after retinol the immunostaining was undetectable in most Sertoli cells. The regulation of the inhibin alpha-subunit by retinol in the cytoplasm of Sertoli cells detected by immunohistochemistry was correlated to the results in immunoblotting. These results suggest a possible interplay between retinoids, androgen and inhibin signalling systems in Sertoli cells in the regulation of spermatogenesis during retinol action.

Immunochemical and in situ hybridization analyses of retinoic acid receptor α, β, and γ in murine Harderian and submandibular glands

Retinoic acid (RA), through its cognate receptors (retinoic acid receptors,RARs), plays an important role in the ontogenesis and maintenance of the normal function of murine Harderian and submandibular glands. In the present study, autoradiography was used to study RA binding to these glands. Both glands showed high radioactive labelling after [14C]-RA administration in normal and partially vitamin A-deficient (VAD) mice. The peak uptake was at 6 h after [14C]-RA administration in normal mice and at 0.5 h in VAD mice. At 24 h, RA binding remained high in normal mice, while it decreased significantly in VAD mice. In western blots with an antibody recognizing all forms of RARs, a band of molecular weight 51 kDa was seen in homogenates of both glands. Immunohistochemically, RAR staining was found in the nuclei of the glandular cells. The Harderian gland exhibited more intense staining than the submandibular gland. In the latter, the most intense staining was seen in the acinar cells, followed by the intercalated duct cells. The granular convoluted tubule showed weak immunostaining and the striated duct was negative. In the Harderian gland, RAR immunostaining was observed in both type I and II cells, but only part of them stained with RAR antibody. The expression of RARα, β, and γ transcripts was studied by in situ hybridization using specific oligonucleotide probes. The cell-specific expression of RARα mRNA in the submandibular gland corresponded to the RAR proteins detected by immunohisto-chemistry, while the RARβ transcript was mainly seen in the striated duct. The transcripts of RARα and β were evenly distributed in type I and II glandular cells of the Harderian gland. RARγ labelling was below detectable levels in both glands. This result suggests that RA and RARs regulate the functions of Harderian and submandibular glands in a cell-specific manner.