ISOELECTRIC FOCUSING STUDIES OF STEROID BINDING PROTEINS

Steroid binding proteins are an obvious choice in the search for genetic factors in plasma that might predispose to upper body obesity, a risk factor for non-insulin dependent diabetes and cardiovascular disease. The two steroid binding proteins studied by isoelectric focusing were sex hormone binding globulin (SHBG), the transport protein for sex hormones and corticosteroid binding globulin (CBG), the transport protein for corticosteroids. Auto-radiography and immunoblotting on polyacrylamide gels were used to detect polymorphism in SHBG. Immunoblotting on agarose gels was used to visualize corticosteroid binding globulin. SHBG showed similar structural variation in American Caucasians, American Blacks and Canadian Indians. Two alleles (1, 2) were hypothesized with highly polymorphic frequencies in all three ethnic groups. CBG was not found to be polymorphic, but two variants were found in Caucasian male twins and in a Black individual. The finding of a good assay and a polymorphic system for SHBG are the first steps for additional studies into disease associations. ^

RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2

Steroids, thyroid hormones, vitamin D3, and retinoids are lipophilic small molecules that regulate diverse biological effects such as cell differentiation, development, and homeostasis. The actions of these hormones are mediated by steroid/nuclear receptors which function as ligand-dependent transcriptional regulators. Transcriptional activation by ligand-bound receptors is a complex process requiring dissociation and recruitment of several additional cofactors. We report here the cloning and characterization of receptor-associated coactivator 3 (RAC3), a human transcriptional coactivator for steroid/nuclear receptors. RAC3 interacts with several liganded receptors through a mechanism which requires their respective ligand-dependent activation domains. RAC3 can activate transcription when tethered to a heterologous DNA-binding domain. Overexpression of RAC3 enhances the ligand-dependent transcriptional activation by the receptors in mammalian cells. Sequence analysis reveals that RAC3 is related to steroid receptor coactivator 1 (SRC-1) and transcriptional intermediate factor 2 (TIF2), two of the most potent coactivators for steroid/nuclear receptors. Thus, RAC3 is a member of a growing coactivator network that should be useful as a tool for understanding hormone action and as a target for developing new therapeutic agents that can block hormone-dependent neoplasia.

Potentially predictive and manipulable blood serum correlates of aging in the healthy human male: Progressive decreases in bioavailable testosterone, dehydroepiandrosterone sulfate, and the ratio of insulin-like growth factor 1 to growth hormone

A cross-sectional survey was made in 56 exceptionally healthy males, ranging in age from 20 to 84 years. Measurements were made of selected steroidal components and peptidic hormones in blood serum, and cognitive and physical tests were performed. Of those blood serum variables that gave highly significant negative correlations with age (r > −0.6), bioavailable testosterone (BT), dehydroepiandrosterone sulfate (DHEAS), and the ratio of insulin-like growth factor 1 (IGF-1) to growth hormone (GH) showed a stepwise pattern of age-related changes most closely resembling those of the age steps themselves. Of these, BT correlated best with significantly age-correlated cognitive and physical measures. Because DHEAS correlated well with BT and considerably less well than BT with the cognitive and physical measures, it seems likely that BT and/or substances to which BT gives rise in tissues play a more direct role in whatever processes are rate-limiting in the functions measured and that DHEAS relates more indirectly to these functions. The high correlation of IGF-1/GH with age, its relatively low correlation with BT, and the patterns of correlations of IGF-1/GH and BT with significantly age-correlated cognitive and physical measures suggest that the GH–IGF-1 axis and BT play independent roles in affecting these functions. Serial determinations made after oral ingestion of pregnenolone and data from the literature suggest there is interdependence of steroid metabolic systems with those operational in control of interrelations in the GH–IGF-1 axis. Longitudinal concurrent measurements of serum levels of BT, DHEAS, and IGF-1/GH together with detailed studies of their correlations with age-correlated functional measures may be useful in detecting early age-related dysregulations and may be helpful in devising ameliorative approaches.

Thyroid hormone receptor-associated proteins and general positive cofactors mediate thyroid hormone receptor function in the absence of the TATA box-binding protein-associated factors of TFIID

Coactivators previously implicated in ligand-dependent activation functions by thyroid hormone receptor (TR) include p300 and CREB-binding protein (CBP), the steroid receptor coactivator-1 (SRC-1)-related family of proteins, and the multicomponent TR-associated protein (TRAP) complex. Here we show that two positive cofactors (PC2 and PC4) derived from the upstream stimulatory activity (USA) cofactor fraction act synergistically to mediate thyroid hormone (T3)-dependent activation either by TR or by a TR-TRAP complex in an in vitro system reconstituted with purified factors and DNA templates. Significantly, the TRAP-mediated enhancement of activation by TR does not require the TATA box-binding protein-associated factors of TFIID. Furthermore, neither the pleiotropic coactivators CBP and p300 nor members of the SRC-1 family were detected in either the TR-TRAP complex or the other components of the in vitro assay system. These results show that activation by TR at the level of naked DNA templates is enhanced by cooperative functions of the TRAP coactivators and the general coactivators PC2 and PC4, and they further indicate a potential functional redundancy between TRAPs and TATA box-binding protein-associated factors in TFIID. In conjunction with earlier studies on other nuclear receptor-interacting cofactors, the present study also suggests a multistep pathway, involving distinct sets of cofactors, for activation of hormone responsive genes.

Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions

The evolution of novelty in tightly integrated biological systems, such as hormones and their receptors, seems to challenge the theory of natural selection: it has not been clear how a new function for any one part (such as a ligand) can be selected for unless the other members of the system (e.g., a receptor) are already present. Here I show—based on identification and phylogenetic analysis of steroid receptors in basal vertebrates and reconstruction of the sequences and functional attributes of ancestral proteins—that the first steroid receptor was an estrogen receptor, followed by a progesterone receptor. Genome mapping and phylogenetic analyses indicate that the full complement of mammalian steroid receptors evolved from these ancient receptors by two large-scale genome expansions, one before the advent of jawed vertebrates and one after. Specific regulation of physiological processes by androgens and corticoids are relatively recent innovations that emerged after these duplications. These findings support a model of ligand exploitation in which the terminal ligand in a biosynthetic pathway is the first for which a receptor evolves; selection for this hormone also selects for the synthesis of intermediates despite the absence of receptors, and duplicated receptors then evolve affinity for these substances. In this way, novel hormone-receptor pairs are created, and an integrated system of increasing complexity elaborated. This model suggests that ligands for some “orphan” receptors may be found among intermediates in the synthesis of ligands for phylogenetically related receptors.

Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator.

The estrogen receptor (ER), a member of a large superfamily of nuclear hormone receptors, is a ligand-inducible transcription factor that regulates the expression of estrogen-responsive genes. The ER, in common with other members of this superfamily, contains two transcription activation functions (AFs)--one located in the amino-terminal region (AF-1) and the second located in the carboxyl-terminal region (AF-2). In most cell contexts, the synergistic activity of AF-1 and AF-2 is required for full estradiol (E2)-stimulated activity. We have previously shown that a ligand-dependent interaction between the two AF-containing regions of ER was promoted by E2 and the antiestrogen trans-hydroxytamoxifen (TOT). This interaction, however, was transcriptionally productive only in the presence of E2. To explore a possible role of steroid receptor coactivators in transcriptional synergism between AF-1 and AF-2, we expressed the amino terminal (AF-1-containing) and carboxyl-terminal (AF-2-containing) regions of ER as separate polypeptides in mammalian cells, along with the steroid receptor coactivator-1 protein (SRC-1). We demonstrate that SRC-1, which has been shown to significantly increase ER transcriptional activity, enhanced the interaction, mediated by either E2 or TOT, between the AF-1-containing and AF-2-containing regions of the ER. However, this enhanced interaction resulted in increased transcriptional effectiveness only with E2 and not with TOT, consistent with the effects of SRC-1 on the full-length receptor. Our results suggest that after ligand binding, SRC-1 may act, in part, as an adapter protein that promotes the integration of amino- and carboxyl-terminal receptor functions, allowing for full receptor activation. Potentially, SRC-1 may be capable of enhancing the transcriptional activity of related nuclear receptor superfamily members by facilitating the productive association of the two AF-containing regions in these receptors.

The insect neuropeptide prothoracicotropic hormone is released with a daily rhythm: re-evaluation of its role in development.

Prothoracicotropic hormone (PTTH) is the central cerebral neurohormone in insect development. Its release has been believed for decades to be confined to one (or two) critical moments early in each developmental stage at which time it triggers prolonged activation of the prothoracic glands to synthesize and release the steroid molting hormones (ecdysteroids), which elicit developmental responses in target tissues. We used an in vitro assay for PTTH released from excised brains of the bug Rhodnius prolixus and report that release of PTTH does occur at the expected time on day 6, but that this release is merely the first in a daily rhythm of release that continues throughout most of the 21 days of larval-adult development. This finding, together with reports of circadian control of ecdysteroid synthesis and titer throughout this time, raises significant challenges to several features of the current understanding of the hormonal control of insect development. New questions are raised concerning the function(s) of PTTH, its relationship with the prothoracic glands, and the significance of circadian rhythmicity throughout this endocrine axis. The significance of the reported observations derives from the set of entirely new questions they raise concerning the regulation of insect development.

Down-regulation of pituitary receptors for luteinizing hormone-releasing hormone (LH-RH) in rats by LH-RH antagonist Cetrorelix.

Antagonists of luteinizing hormone-releasing hormone (LH-RH), unlike the LH-RH agonists, suppress gonadotropins and sex steroid secretion immediately after administration, without initial stimulatory effects. [Ac-D-Nal(2)1,D-Ph(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-R H (SB-75; Cetrorelix) is a modern, potent antagonistic analog of LH-RH. In this study, the binding characteristics of receptors for LH-RH in membrane fractions from rat anterior pituitaries were investigated after a single injection of Cetrorelix at a dose of 100 microg per rat. To determine whether the treatment with Cetrorelix can affect the concentration of measurable LH-RH binding sites, we applied an in vitro method to desaturate LH-RH receptors by chaotropic agents such as manganous chloride (MnCl2) and ammonium thiocyanate (NH4SCN). Our results show that the percentages of occupied LH-RH receptors at 1, 3, and 6 h after administration of Cetrorelix were approximately 28%, 14%, and 10%, respectively, of total receptors. At later time intervals, we could not detect occupied LH-RH binding sites. Ligand competition assays, following in vitro desaturation, demonstrated that rat pituitary LH-RH receptors were significantly (P < 0.01) down-regulated for at least 72 h after administration of Cetrorelix. The lowest receptor concentration was found 3-6 h after Cetrorelix treatment and a recovery in receptor number began within approximately 24 h. The down-regulation of LH-RH binding sites induced by Cetrorelix was accompanied by serum LH and testosterone suppression. Higher LH-RH receptor concentrations coincided with elevated serum hormone levels at later time intervals. Our results indicate that administration of LH-RH antagonist Cetrorelix produces a marked down-regulation of pituitary receptors for LH-RH and not merely an occupancy of binding sites.

Steroid receptor heterodimerization demonstrated in vitro and in vivo.

The mineralocorticoid and glucocorticoid receptors (MR and GR, respectively) are members of the intracellular receptor superfamily that bind as homodimers to the same hormone response elements (HREs). Physiological evidence suggests that MR and GR interact with each other in cells that express both receptors, implying that they might directly interact in the regulation of transcription initiation. Indeed, we have found that coexpressed MR and GR interact functionally at the transcriptional level and furthermore that they interact physically through heterodimer formation at a shared HRE in vitro and in vivo. We suggest from these findings that heterodimerization may play an important role in steroid receptor transcriptional regulation.

Interactions between the retinoid X receptor and a conserved region of the TATA-binding protein mediate hormone-dependent transactivation.

The retinoid X receptor (RXR) participates in a wide array of hormonal signaling pathways, either as a homodimer or as a heterodimer, with other members of the steroid and thyroid hormone receptor superfamily. In this report the ligand-dependent transactivation function of RXR has been characterized, and the ability of RXR to interact with components of the basal transcription machinery has been examined. In vivo and in vitro experiments indicate the RXR ligand-binding domain makes a direct, specific, and ligand-dependent contact with a highly conserved region of the TATA-binding protein. The ability of mutations that reduce ligand-dependent transcription by RXR to disrupt the RXR-TATA-binding protein interaction in vivo and in vitro suggests that RXR makes direct contact with the basal transcription machinery to achieve activation.

Estradiol and the inhibition of hypothalamic gonadotropin-releasing hormone pulse generator activity in the rhesus monkey.

In mammals, gonadal function is controlled by a hypothalamic signal generator that directs the pulsatile release of gonadotropin-releasing hormone (GnRH) and the consequent pulsatile secretion of luteinizing hormone. In female rhesus monkeys, the electrophysiological correlates of GnRH pulse generator activity are abrupt, rhythmic increases in hypothalamic multiunit activity (MUA volleys), which represent the simultaneous increase in firing rate of individual neurons. MUA volleys are arrested by estradiol, either spontaneously at midcycle or after the administration of the steroid. Multiunit recordings, however, provide only a measure of total neuronal activity, leaving the behavior of the individual cells obscure. This study was conducted to determine the mode of action of estradiol at the level of single neurons associated with the GnRH pulse generator. Twenty-three such single units were identified by cluster analysis of multiunit recordings obtained from a total of six electrodes implanted in the mediobasal hypothalamus of three ovariectomized rhesus monkeys, and their activity was monitored before and after estradiol administration. The bursting of all 23 units was arrested within 4 h of estradiol administration although their baseline activity was maintained. The bursts of most units reappeared at the same time as the MUA volleys, the recovery of some was delayed, and one remained inhibited for the duration of the study (43 days). The results indicate that estradiol does not desynchronize the bursting of single units associated with the GnRH pulse generator but that it inhibits this phenomenon. The site and mechanism of action of estradiol in this regard remain to be determined.

LEM1, an ATP-binding-cassette transporter, selectively modulates the biological potency of steroid hormones.

The rat glucocorticoid receptor confers hormone-dependent transcriptional enhancement when expressed in yeast, thereby enabling the genetic identification of nonreceptor proteins that function in the hormone signal-transduction pathway. We isolated a yeast mutant, lem1, with increased sensitivity to dexamethasone and triamcinolone acetonide; responsiveness to a third agonist, deoxycorticosterone, is unaffected. Cloning of wild-type LEM1 revealed a putative transport protein of the ATP-binding cassette family. Dexamethasone accumulation is increased in lem1 cells, suggesting that wild-type LEM1 decreases dexamethasone potency by exporting this ligand. LEM1 appears to affect certain steroids and not others. We propose that transporters like LEM1 can selectively modulate the intracellular levels of steroid hormones. Differential activities of such transporters in mammalian cells might regulate hormone availability and thereby hormone signaling in a cell-type specific manner.

Sex steroid feedback regulation of pituitary gonadotropins during early secondary oocyte growth in coho salmon (Oncorhynchus kisutch) – a potential target of endocrine disruption.

Thesis (Ph.D.)--University of Washington, 2016-06

Chronic exposure to sub-lethal concentration of a glyphosate-based herbicide alters hormone profiles and affects reproduction of female Jundia (Rhamdia quelen)

This work was carried out to verify the effect of a glyphosate-based herbicide on Jundia hormones (cortisol, 17 beta-estradiol and testosterone), oocyte and swim-up fry production. Earthen ponds containing Jundia females were contaminated with glyphosate (3.6 mg/L); blood samples were collected from eight females from each treatment immediately before, or at 1, 10, 20 30 and 40 days following contamination. A typical post-stress rise in cortisol levels was observed at the 20th and 40th days following exposure to glyphosate. At the 40th day, 17 beta-estradiol was decreased in the exposed females. A similar number of oocytes were stripped out from females from both groups, however, a lower number of viable swim-up fry were obtained from the herbicide exposed females, which also had a higher liver-somatic index (LSI). The results indicate that the presence of glyphosate in water was deleterious to Rhamdia quelen reproduction, altering steroid profiles and egg viability. (c) 2006 Elsevier B.V. All rights reserved.

Modulation of the SHBG signalling axis

Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein that is the major sex steroid carrier-protein in the bloodstream and functions also as a key regulator of steroid bioavailability within target tissues, such as the prostate. Additionally, SHBG binds to prostatic cell membranes via the putative and unidentified SHBG receptor (RSHBG), activating a signal transduction pathway implicated in stimulating both proliferation and expression of prostate specific antigen (PSA) in prostate cell lines in vitro. A yeast-two hybrid assay suggested an interaction between SHBG and kallikrein-related protease (KLK) 4, which is a serine protease implicated in the progression of prostate cancer. The potential interaction between these two proteins was investigated in this PhD thesis to determine whether SHBG is a proteolytic substrate of KLK4 and other members of the KLK family including KLK3/PSA, KLK7 and KLK14. Furthermore, the effects from SHBG proteolytic degradation on SHBG-regulated steroid bioavailability and the activation of the putative RSHBG signal transduction pathway were examined in the LNCaP prostate cancer cell line. SHBG was found to be a proteolytic substrate of the trypsin-like KLK4 and KLK14 in vitro, yielding several proteolysis fragments. Both chymotrypsin-like PSA and KLK7 displayed insignificant proteolytic activity against SHBG. The kinetic parameters of SHBG proteolysis by KLK4 and KLK14 demonstrate a strong enzyme-substrate binding capacity, possessing a Km of 1.2 ± 0.7 µM and 2.1 ± 0.6 µM respectively. The catalytic efficiencies (kcat/Km) of KLK4 and KLK14 proteolysis of SHBG were 1.6 x 104 M-1s-1 and 3.8 x 104 M-1s-1 respectively, which were comparable to parameters previously reported for peptide substrates. N-terminal sequencing of the fragments revealed cleavage near the junction of the N- and C-terminal laminin globulin-like (G-like) domains of SHBG, resulting in the division of the two globulins and ultimately the full degradation of these fragments by KLK4 and KLK14 over time. Proteolytic fragments that may retain steroid binding were rapidly degraded by both proteases, while fragments containing residues beyond the steroid binding pocket were less degraded over the same period of time. Degradation of SHBG was inhibited by the divalent metal cations calcium and zinc for KLK4, and calcium, zinc and magnesium for KLK14. The human secreted serine protease inhibitors (serpins), α1-antitrypsin and α2-antiplasmin, inhibited KLK4 and KLK14 proteolysis of SHBG; α1-antichymotrypsin inhibited KLK4 but not KLK14 activity. The inhibition by these serpins was comparable and in some cases more effective than general trypsin protease inhibitors such as aprotinin and phenylmethanesulfonyl fluoride (PMSF). The binding of 5α-dihydrotestosterone (DHT) to SHBG modulated interactions with KLK4 and KLK14. Steroid-free SHBG was more readily digested by both enzymes than DHT-bound SHBG. Moreover, a binding interaction exists between SHBG and pro-KLK4 and pro-KLK14, with DHT strengthening the binding to pro-KLK4 only. The inhibition of androgen uptake by cultured prostate cancer cells, mediated by SHBG steroid-binding, was examined to assess whether SHBG proteolysis by KLK4 and KLK14 modulated this process. Proteolytic digestion eliminated the ability of SHBG to inhibit the uptake of DHT from conditioned media into LNCaP cells. Therefore, the proteolysis of SHBG by KLK4 and KLK14 increased steroid bioavailability in vitro, leading to an increased uptake of androgens by prostate cancer cells. Interestingly, different transcriptional responses of PSA and KLK2, which are androgen-regulated genes, to DHT-bounsd SHBG treatment were observed between low and high passage number LNCaP cells (lpLNCaP and hpLNCaP respectively). HpLNCaP cells treated with DHT-bound SHBG demonstrated a significant synergistic upregulation of PSA and KLK2 above DHT or SHBG treatment alone, which is similar to previously reported downstream responses from RSHBG-mediated signaling activation. As this result was not seen in lpLNCaP cells, only hpLNCaP cells were further investigated to examine the modulation of potential RSHBG activity by KLK4 and KLK14 proteolysis of SHBG. Contrary to reported results, no increase in intracellular cAMP was observed in hpLNCaP cells when treated with SHBG in the presence and absence of either DHT or estradiol. As a result, the modulation of RSHBG-mediated signaling activation could not be determined. Finally, the identification of the RSHBG from both breast (MCF-7) and prostate cancer (LNCaP) cell lines was attempted. Fluorescently labeled peptides corresponding to the putative receptor binding domain (RBD) of SHBG were shown to be internalized by MCF-7 cells. Crosslinking of the RBD peptide to the cell surfaces of both MCF-7 and LNCaP cells, demonstrated the interaction of the peptide with several targets. These targets were then captured using RBD peptides synthesized onto a hydrophilic scaffold and analysed by mass spectrometry. The samples captured by the RBD peptide returned statistically significantly matches for cytokeratin 8, 18 and 19 as well as microtubule-actin crosslinking factor 1, which may indicate a novel interaction between SHBG and these proteins, but ultimately failed to detect a membrane receptor potentially responsible for the putative RSHBG-mediated signaling. This PhD project has reported the proteolytic processing of SHBG by two members of the kallikrein family, KLK4 and KLK14. The effect of SHBG proteolysis by KLK4 and KLK14 on RSHBG-mediated signaling activation was unable to be determined as the reported signal transduction pathway was not activated after treatment with SHBG, in combination with either DHT or estradiol. However, the digestion of SHBG by these two proteases positively regulated androgen bioavailability to prostate cancer cells in vitro. The increased uptake of androgens is deleterious in prostate cancer due to the promotion of proliferation, metastasis, invasion and the inhibition of apoptosis. The increased bioavailability of androgens, from SHBG proteolysis by KLK4 and KLK14, may therefore promote both carcinogenesis and progression of prostate cancer. Finally, this information may contribute to the development of therapeutic treatment strategies for prostate cancer by inhibiting the proteolysis of SHBG, by KLK4 and KLK14, to prevent the increased uptake of androgens by hormone-dependent cancerous tissues.