Biomedical subjects

J V Swinnen

Publications and source records attributed to J V Swinnen.

At least 19 recordsLinked to original sources

PubMed2002

The estrogen receptor ligand ICI 182,780 does not impair the bone-sparing effects of testosterone in the young orchidectomized rat model.

Testosterone (T) can affect bone metabolism not only directly, but also via its metabolites, estrogen or dihydrotestosterone, produced by enzymes present in bone. Therefore, the aim of this study was to investigate whether the high-affinity estrogen receptor ligand ICI 182,780 (ICI) impaired the bone-protective action of T in 3-month-old orchidectomized (Orch) rats, studied during an experimental period of 3 months. As expected, Orch significantly decreased trabecular bone volume in the proximal tibial metaphysis (-52%), as measured by histomorphometry, and had a similar negative effect on volumetric bone mineral density (BMD) in the distal femoral metaphysis (-53%), as assessed by peripheral quantitative computed tomography (pQCT). The loss of bone induced by Orch was completely prevented by T administration. Moreover, the Orch-associated increases of biochemical markers of bone turnover (serum osteocalcin, urinary deoxypyridinoline, and calcium excretion) did not occur when Orch rats received T. Administration of ICI in combination with T did not impair this bone-sparing effect. Cortical bone parameters (as determined by pQCT), body weight gain, and body composition (as measured by dual-energy X-ray absorptiometry) were not affected by T or ICI in combination with T. Furthermore, no differences were observed in serum concentrations of insulin-like growth factor-I or glucose homeostasis. In conclusion, ICI does not impair the long-term bone-protective effects of T in orchidectomized male rats, suggesting that testosterone can mediate its effect on the male skeleton directly via the androgen receptor. The absence of effects on body growth via the growth hormone--insulin-like growth factor-I axis may be a possible explanation for the lack of skeletal effects of this selective estrogen receptor antagonist.

Animals↗

PubMed2001

Testosterone prevents orchidectomy-induced bone loss in estrogen receptor-alpha knockout mice.

To examine the role of the estrogen receptor-alpha (ERalpha) during male skeletal development, bone density and structure of aged ERalphaKO mice and wild-type (WT) littermates were analyzed and skeletal changes in response to sex steroid deficiency and replacement were also studied. In comparison to WT, ERalphaKO mice had smaller and thinner bones, arguing for a direct role of ERalpha to obtain full skeletal size in male mice. However, male ERalphaKO mice had significantly more trabecular bone as assessed both by pQCT and histomorphometry, indicating that ERalpha is not essential to maintain cancellous bone mass. Six weeks following orchidectomy (ORX), both WT and ERalphaKO mice showed high-turnover osteoporosis as revealed by increases in serum osteocalcin and decreases in trabecular (-38% and -58% in WT and ERalphaKO, respectively) and cortical bone density (-5% and -4% in WT and ERalphaKO, respectively). Administration of testosterone propionate (T, 5 mg/kg/day) completely prevented bone loss both in ERalphaKO and in WT mice. As expected, estradiol (E2, 60 microg/kg/day) replacement did not prevent cancellous bone loss in ORX ERalphaKO mice. However, E2 stimulated bone formation at the endocortical surface in ORX ERalphaKO, suggesting that osteoblasts may respond to nonERalpha-mediated estrogen action. In conclusion, although functional ERalpha may play a significant role during male skeletal development, this receptor does not seem essential for androgen-mediated skeletal maintenance in older male mice.

Animals↗

PubMed2001

Effects and characterization of paracrine factors produced by human prostate stromal cells in bioassays using rat Sertoli cells, LNCaP tumor cells, and cultured prostate epithelial cells.

BACKGROUND: Prostatic stroma affects both proliferation and differentiation of epithelial cells but the factors involved remain poorly understood. In order to identify and characterize potential paracrine mediators, we studied the effects of human prostate fibroblast-conditioned media (PFCM) in three bioassay systems. METHODS: The first bioassay uses transferrin secretion by cultured rat Sertoli cells as an endpoint for differentiating activity. Factors active in this (heterologous) assay were compared to PModS, a mediator of mesenchymal-epithelial interactions in the testis, also produced by rat prostate stromal cells. The two other (homologous) bioassays use LNCaP tumor cells or subcultured human prostate epithelial cells (PEC) as targets. Differentiation is evaluated by prostate-specific antigen (PSA) secretion and reverse transcriptase-polymerase chain reaction (RT-PCR) for a number of markers of epithelial function. Proliferation is assayed by measurements of DNA and thymidine incorporation. RESULTS: PFCM markedly stimulates transferrin production by Sertoli cells. The main factor(s) involved are acid stable and bind to heparin. However, both their size (approximately 37 kDa) and their behavior on reversed-phase chromatography differ from that of PModS. Although PFCM increases total RNA content of LNCaP, it does not increase or restore differentiated function of LNCaP or PEC. Proliferative effects are observed in LNCaP and these effects cannot be neutralized by an antiserum directed against basic fibroblast growth factor (bFGF). Antiproliferative effects are observed in PEC and these effects are largely due to transforming growth factor-beta (TGF-beta). CONCLUSIONS: PFCM provokes differentiating effects in a Sertoli cell bioassay, but unlike with rat stromal cells, the factor(s) involved differ from PModS. In the two homologous systems studied, differentiating effects could not be demonstrated and discordant effects were noted on proliferation. Various bioassay systems will be required to identify the spectrum of mediators present in PFCM.

Animals↗

PubMed2001

E2F activity is biphasically regulated by androgens in LNCaP cells.

Androgens exert a peculiar biphasic dose-dependent influence on the proliferation of LNCaP cells, a widely used model to study androgen effects on prostate cancer cells. Low concentrations of androgen stimulate proliferation, but high concentrations inhibit proliferation and induce strong expression of differentiation markers. In order to gain more insight into the molecular mechanisms that underlie these changes we studied the influence of a wide concentration range of the synthetic androgen R1881 on several cell cycle- and differentiation-related parameters. Low concentrations (0.1 nM), known to promote LNCaP cell proliferation, induce an increase of Retinoblastoma protein phosphorylation, accompanied by an increase of E2F-1 protein levels and E2F activity and by increased expression of the E2F-target gene products E2F-1 and cyclin A. High concentrations of R1881 (10 nM) induce strong expression of the differentiation marker prostate-specific antigen. Retinoblastoma protein is largely hypophosphorylated, resulting in low E2F activity and low concentrations of E2F-1 and cyclin A mRNA. Finally, there is a strong increase of p27(KIP1) protein, but not of p27(KIP1) mRNA. These results indicate that the biphasic dose response of LNCaP proliferation to androgen is closely reflected in Rb phosphorylation, E2F activity and p27(KIP1) protein expression.

Androgens↗

PubMed2001

Androgens stimulate lipogenic gene expression in prostate cancer cells by activation of the sterol regulatory element-binding protein cleavage activating protein/sterol regulatory element-binding protein pathway.

Using two independent prostate cancer cell lines (LNCaP and MDA-PCa-2a), we demonstrate that coordinated stimulation of lipogenic gene expression by androgens is a common phenomenon in androgen-responsive prostate tumor lines and involves activation of the sterol regulatory element-binding protein (SREBP) pathway. We show 1) that in both cell lines, androgens stimulate the expression of fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase, two key lipogenic genes representative for the fatty acid and the cholesterol synthesis pathway, respectively; 2) that treatment with androgens results in increased nuclear levels of active SREBP; 3) that the effects of androgens on promoter-reporter constructs derived from both lipogenic genes (fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase) depend on the presence of intact SREBP-binding sites; and 4) that cotransfection with dominant-negative forms of SREBPs abolishes the effects of androgens. Related to the mechanism underlying androgen activation of the SREBP pathway, we show that in addition to minor effects on SREBP precursor levels, androgens induce a major increase in the expression of sterol regulatory element-binding protein cleavage-activating protein (SCAP), an escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of proteolytical activation in the Golgi. Both time course studies and overexpression experiments showing that increasing levels of SCAP enhance the production of mature SREBP and stimulate lipogenic gene expression support the contention that SCAP plays a pivotal role in the lipogenic effects of androgens in tumor cells.

Androgens↗

PubMed2001

Increased lipogenesis in steroid-responsive cancer cells: mechanisms of regulation, role in cancer cell biology and perspectives on clinical applications.

Steroid hormones have a strong influence on the biology of several common human cancers, including cancer of the prostate, breast, endometrium and ovarium. To gain more insight into this process, a screening for androgen-regulated genes was set up in prostate cancer cells. In addition to their well known effects on cell survival, proliferation and differentiated function, androgens were found to markedly stimulate the expression of several lipogenic enzymes. In clinical cancer samples these enzymes are markedly overexpressed in comparison to normal tissues, allowing them to be used as cancer markers and as potential targets for antineoplastic therapy. Investigation of the underlying mechanisms of gene regulation revealed that androgens stimulate lipogenic gene expression through a novel indirect mechanism involving Sterol Regulatory Element-binding Proteins (SREBPs), lipogenic transcription factors that play a key role in the fundamental feedback mechanism of cellular lipid homeostasis. Interestingly, also growth factors, whose signaling pathways are frequently dysregulated and constitutively activated as prostate cancer progresses towards a more advanced disease, stimulate lipogenesis through the same SREBP-mediated mechanism. While studies on the role of enhanced intermediary metabolism in cancer cell biology are progressing, these findings provide important new insights into the long-known dysregulation of intermediary metabolism in cancer cells and open new perspectives for clinical diagnosis and therapeutic intervention.

Androgens↗

PubMed2000

Stimulation of tumor-associated fatty acid synthase expression by growth factor activation of the sterol regulatory element-binding protein pathway.

Increased expression of fatty acid synthase (FAS) is observed in a clinically aggressive subset of various common cancers and interference with FAS offers promising opportunities for selective chemotherapeutic intervention. The mechanisms by which FAS expression is (up)-regulated in these tumors remain, however, largely unknown. Recently we demonstrated that in LNCaP prostate cancer cells FAS expression is markedly elevated by androgens via an indirect pathway involving sterol regulatory element-binding proteins (SREBPs). Here, we also show that growth factors such as EGF are able to stimulate FAS mRNA, protein and activity. Several observations also indicate that the effects of EGF on FAS expression are ultimately mediated by SREBPs. EGF stimulates SREBP-1c mRNA expression and induces an increase in mature nuclear SREBP-1. Moreover, in transient transfection studies EGF stimulates the transcriptional activity of a 178 bp FAS promoter fragment harboring a complex SREBP-binding site. Deletion or mutation of this binding site abolishes these effects and ectopic expression of dominant negative SREBP-1 inhibits FAS expression and induction in intact LNCaP cells. Given the frequent dysregulation of growth factor signaling in cancer and the key role of SREBP-1 in lipid homeostasis, growth factor-induced activation of the SREBP pathway is proposed as one of the mechanisms responsible for up-regulation of lipogenic gene expression in a subset of cancer cells.

Binding Sites↗

PubMed2000

Apparent coactivation due to interference of expression constructs with nuclear receptor expression.

Transient cotransfection in COS-7 cells, a standard approach to demonstrate coactivation, was used to study the coactivation properties of NuRIP183, a new nuclear receptor interacting protein of 183 kDa, isolated by a yeast two-hybrid screening. Transfection with a NuRIP183 expression construct strongly increased the ligand-dependent response of reporter constructs for several nuclear receptors when compared to transfection with the empty expression vector. A more detailed study, however, revealed major changes in the expression level of the nuclear receptors in cotransfection experiments, indicating that the observed changes in receptor activity were not due to coactivation but to differences in receptor concentration caused by interference from the cotransfected expression constructs with the expression of the receptor. Such interference, which is inversely related to the length of the insert, was observed, not only in COS-7 cells but also in CV-1 and MCF-7 cells, using different transfection techniques (FuGENE-6 and calcium phosphate) and different expression vectors (pSG5, pcDNA1.1 and pIRESneo). These data cast some doubt on coactivation of nuclear receptors based on similar cotransfection experiments without measurement of receptor concentration. Moreover, it is recommended to limit the amounts of (co)transfected expression plasmid and to avoid the use of empty expression plasmid as a control. Finally, one should be aware of similar misleading results in other experimental set-ups based on cotransfection.

Animals↗

PubMed2000

Selective activation of the fatty acid synthesis pathway in human prostate cancer.

A substantial subset of breast, colorectal, ovarian, endometrial and prostatic cancers displays markedly elevated expression of immunohistochemically detectable fatty acid synthase, a feature that has been associated with poor prognosis and that may be exploited in anti-neoplastic therapy. Here, using an RNA array hybridisation technique complemented by in situ hybridisation, we report that in prostate cancer fatty acid synthase expression is up-regulated at the mRNA level together with other enzymes of the same metabolic pathway. Contrary to the observations that in many cell systems (including androgen-stimulated LNCaP prostate cancer cells) fatty acid and cholesterol metabolism are co-ordinately regulated so as to supply balanced amounts of lipids for membrane biosynthesis, storage or secretion, no changes in the expression of genes involved in cholesterol synthesis were found. These findings point to selective activation of the fatty acid synthesis pathway and suggest a shift in the balance of lipogenic gene expression in a subgroup of prostate cancers.

Acetyl-CoA Carboxylase↗

PubMed2000

Progestins and androgens increase expression of Spot 14 in T47-D breast tumor cells.

Enhanced expression of fatty acid synthase and other lipogenic enzymes has been observed in a subset of breast cancers with poor prognosis. This phenomenon has been related to amplification of a gene on chromosome region 11q13 encoding Spot 14, a putative regulator of lipogenic enzyme expression. In this paper we demonstrate that the induction of lipogenesis by progestins and androgens in the breast cancer cell line T47-D is accompanied by a marked increase in the expression of Spot 14. These data corroborate the correlation between Spot 14 expression and increased lipogenesis. Moreover they show that apart from gene amplification there is another steroid-regulated pathway that may enhance Spot 14 expression and lipogenesis in tumor cells.

Breast Neoplasms↗

PubMed2000

An aged rat model of partial androgen deficiency: prevention of both loss of bone and lean body mass by low-dose androgen replacement.

The aim of this study was to evaluate the effects of different doses of androgen replacement, both on body composition and bone, in an aged male orchidectomized rat model. Testosterone was administered by 0.5, 1, and 2.5-cm sc SILASTIC implants (release of, respectively, 11.5, 23, and 55 microg/day) to aged (12 months old, +/- 550 g) male orchidectomized Wistar rats during a 15-week experimental period. T 0.5 only partially prevented decrease of ventral prostate and seminal vesicle weight, compared with an intact group that received an empty implant (Intact). The 1-cm implant (T 1) completely prevented decrease of both seminal vesicles and ventral prostate weight. The 2.5-cm implant (T 2.5) was clearly supraphysiological, as demonstrated by significant hypertrophy of both androgen-sensitive organs. Serum testosterone was lower in T 0.5 and T 1 (0.38 +/- 0.06 ng/ml and 0.92 +/- 0.06 ng/ml, respectively) and higher in T 2.5 (2.4 +/- 0.28. ng/ml), compared with both Intact (1.6 +/- 0.23 ng/ml) and the baseline group(1.6 +/- 0.11 ng/ml). As expected, orchidectomized rats that received an empty SILASTIC implant had significantly lower bone mineral content (-7.9%), apparent density (-5.7%), and lean body mass (-10.8%), as measured by dual-energy x-ray absorptiometry, without significant changes in body weight and fat mass, compared with Intact. Also, cancellous (-50.3%) and cortical (-1.8%) volumetric density, as measured by peripheral quantitative computed tomography, were decreased in the tibia. Bone turnover, as measured by serum osteocalcin and urinary deoxypyridinoline excretion, was increased in orchidectomized rats that received an empty SILASTIC implant. T 0.5 prevented all changes, not only in bone mineral content, density, and turnover but also in lean body mass. Moreover, there were no significant differences, for all these parameters, between the different doses of testosterone replacement. In conclusion, low-dose androgen replacement does not lead to lower bone mineral density, higher bone turnover, and lower lean body mass in aged male rats, whereas complete androgen deficiency does. Therefore, the threshold concentration of testosterone necessary for prevention of both bone and lean body mass loss in aged male rats is clearly lower than for prostate and seminal vesicles.

Aging↗

PubMed1999

Androgens down-regulate the expression of the human homologue of paternally expressed gene-3 in the prostatic adenocarcinoma cell line LNCaP.

mRNA differential display polymerase chain reaction analysis was used to screen systematically for novel androgen-regulated genes in the human prostatic adenocarcinoma cell line LNCaP. A 232 bp PCR fragment was found to be consistently down-regulated by the synthetic androgen R1881. Sequencing revealed complete identity with the human homologue of mouse Paternally expressed gene 3 (Peg3), an imprinted gene that plays an important role as a downstream mediator of the effects of tumor necrosis factor (TNF). The down-regulation of Peg3 mRNA by androgens was confirmed by Northern blot hybridization. The effect proved time and dose dependent with maximal repression (3.5-fold) after 24 h of treatment with 10(-8) M R1881. The steroid specificity of Peg3 mRNA regulation reflected the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells, supporting the involvement of the androgen receptor in the repression process. Basal expression of Peg3 mRNA was almost completely abolished by the protein synthesis inhibitor cycloheximide. Experiments with Actinomycin D suggested that androgens act at a transcriptional level rather than by changing the stability of Peg3 mRNA. Comparison of the expression of Peg3 mRNA in 50 different human tissues revealed ubiquitous expression, but low levels in the prostate. The highest levels were observed in endocrine tissues such as ovary, placenta, adrenal and pituitary. High levels were also noted in various parts of the brain. No detectable levels of Peg3 mRNA were observed in two other androgen receptor-positive prostate tumor cell lines (MDA PCa-2a and -2b), and in the poorly differentiated and androgen receptor-negative prostate tumor lines PC-3 and DU-145. It is concluded that both androgens and loss of differentiation may affect the expression of Peg3, a mediator of the effects of TNF. Further experiments will be required to explore whether these changes affect the responsiveness of prostate tumor cells to TNF.

Adenocarcinoma↗

PubMed1999

Androgens decrease and retinoids increase the expression of insulin-like growth factor-binding protein-3 in LNcaP prostatic adenocarcinoma cells.

Changes in circulating levels of insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been related to prostate cancer, but the nature and the significance of this relationship remains elusive. Recent reports suggest that modulation of the production of IGFBP-3 by retinoids may affect growth of breast and prostate tumor cells. In the present study we explored whether androgens (R1881), retinoids (all-trans- and 9-cis-retinoic acid: atRA and 9cRA), deltanoids (1alpha,25-dihydroxycholecalciferol: VD3) and thyroid hormone (triiodothyronine: T3) influence the production of IGFBPs by LNCaP prostatic adenocarcinoma cells and whether the observed changes affect tumor cell growth. Northern blot experiments demonstrated that LNCaP cells express IGFBP-2, -3, -4 and (to a small extent) -5. IGFBP-4 and -5 were not measurably affected by the mentioned agonists. At a growth promoting concentration (10(-10) M), R1881 increased IGFBP-2 transcript levels two- to three-fold and this effect was neutralized by atRA and VD3. Similar effects could not be demonstrated, however, at the protein level using Western ligand blotting. R1881 decreased and atRA increased the mRNA levels of IGFBP-3 and these effects were confirmed by Western ligand blotting and by radioimmunoassay. The effects of atRA were mimicked by 9cRA and by a specific RAR agonist but not by a RXR agonist. VD3 and T3 had no significant effect on IGFBP-3 secretion but respectively enhanced or decreased the effect of 9cRA. The effects of retinoids required high concentrations (10(-6)-10(-5) M) that also induced growth inhibition. R1881, however, decreased IGFBP-3 at growth promoting (10(-10) M) as well as at growth inhibitory (10(-8) M) concentrations. Moreover, under serum-free conditions, we were unable to demonstrate any growth modulating effect of IGFBP-3. It is concluded that several agonists acting by nuclear receptors affect IGFBP-3 secretion by LNCaP cells but that the functional significance of these changes warrants further investigation.

Adenocarcinoma↗

PubMed1999

The differentiation-related gene 1, Drg1, is markedly upregulated by androgens in LNCaP prostatic adenocarcinoma cells.

A differential display technique was used to identify androgen-regulated genes in LNCaP prostatic adenocarcinoma cells. One of the genes markedly upregulated by androgens proved to be identical to differentiation-related gene 1 (Drg1; also described as RTP, Cap43 and rit42), a gene whose expression has recently been shown to be diminished in colon, breast and prostate tumors. We show that Drg1 is abundantly expressed in the (androgen-exposed) human prostate and that its expression is stimulated some 14-fold in androgen-treated LNCaP cells. The ligand specificity of the induction reflects the altered specificity of the mutated androgen receptor in LNCaP. In androgen receptor negative tumor lines basal expression is slightly higher than in LNCaP but inducibility is absent. These data suggest that Drg1 is a novel marker of androgen-induced differentiation in the human prostate.

Adenocarcinoma↗

PubMed1999

Indirect mechanisms and cascades of androgen action.

Androgens are the main hormones responsible for the induction of the male phenotype. This process involves a complex combination of reversible and irreversible effects. As for other steroid hormones, many of the effects of androgens are mediated by a specific intracellular receptor that interacts with cis-acting regulatory regions (AREs) in the affected genes. Apart from these direct effects, however, androgens may indirectly affect the expression of a variety of genes that do not necessarily contain AREs. Indirect effects may be related to androgen-induced changes in the concentration or activity of secondary transcription factors, autocrine and paracrine mediators, and circulating hormones. Such indirect effects may induce cascade-like actions and may play an important role in more complex effects of androgens involving coordinated responses of genes, cells and organs.

Androgens↗

PubMed1999

Heregulins or neu differentiation factors and the interactions between peritubular myoid cells and Sertoli cells.

Interactions between (mesenchymal) peritubular myoid cells and (epithelial) Sertoli cells play an important role in the control of Sertoli cell function and spermatogenesis. The factors involved, however, have only partially been identified. Heregulins or neu differentiation factors (NDFs) mediate mesenchymal-epithelial interactions in a variety of tissues, but their role in the testis has not been investigated. Here we demonstrate that recombinant human heregulin-alpha (Her-alpha) and Her-beta stimulate transferrin and androgen-binding protein production by cultured rat Sertoli cells up to 2.5-fold. These effects are more pronounced than those of previously identified growth factors active in this assay, such as insulin-like growth factor I and basic fibroblast growth factor. Combination with these factors results in additive effects and in marked morphological changes in the Sertoli cell cultures, including formation of tubule-like structures. Stimulation of androgen-binding protein secretion is paralleled by increased levels of the corresponding messenger RNA. This parallelism was less consistent for transferrin. Semiquantitative RT-PCR indicates that the expression of NDF-alpha and NDF-beta is more pronounced in peritubular cells than in Leydig or Sertoli cells. Conversely, the main receptors for heregulins/NDFs, HER-3 and HER-4, are predominantly expressed in Sertoli cells. A displacement assay confirms the presence of high-affinity binding sites for [125I]Her-beta on intact Sertoli cells and reveals parallel displacement curves for Her-beta, Her-alpha, and concentrated peritubular cell-conditioned medium (PTCM; estimated ED50 values: 1 ng/ml, 50 ng/ml, and 130 microg protein/ml, respectively), indicating that peritubular cells secrete one or more factors able to compete for heregulin receptors. It is concluded that heregulins/NDFs may play a role in mesenchymal-epithelial interactions in the testis. Estimates of the concentrations of heregulins in PTCM, however, make it unlikely that they contribute significantly to the effects observed with low concentrations of PTCM and ascribed to the putative mediator PModS (peritubular factor that modulates Sertoli cell function). Further investigations will be required to define the exact role of heregulins in the testis.

Androgen-Binding Protein↗

PubMed1999

Interaction of the putative androgen receptor-specific coactivator ARA70/ELE1alpha with multiple steroid receptors and identification of an internally deleted ELE1beta isoform.

Steroid-regulated gene transcription requires the coordinate physical and functional interaction of hormone receptors, basal transcription factors, and transcriptional coactivators. In this context ARA70, previously called RFG and ELE1, has been described as a putative coactivator that specifically enhances the activity of the androgen receptor (AR) but not that of the glucocorticoid receptor (GR), the progesterone receptor, or the estrogen receptor (ER). Here we describe the cloning of the cDNA for ELE1/ARA70 by RT-PCR from RNA derived from different cell lines (HeLa, DU-145, and LNCaP). In accordance with the previously described sequence, we obtained a 1845-bp PCR product for the HeLa and the LNCaP RNA. Starting from T-47D RNA, however, an 860-bp PCR product was obtained. This shorter variant results from an internal 985-bp deletion and is called ELE1beta; accordingly, the longer isoform is referred to as ELE1alpha. The deduced amino acid sequence of ELE1alpha, but not that of ELE1beta, differs at specific positions from the one previously published by others, suggesting that these two proteins are encoded by different nonallelic genes. ELE1alpha is expressed in the three prostate-derived cell lines examined (PC-3, DU-145, and LNCaP), and this expression is not altered by androgen treatment. Of all rat tissues examined, ELE1alpha expression is highest in the testis. This is also the only tissue in which we could demonstrate ELE1beta expression. Both ELE1alpha and ELE1beta interact in vitro with the AR, but also with the GR and the ER, in a ligand-independent way. Overexpression of either ELE1 isoform in DU-145, HeLa, or COS cells had only minor effects on the transcriptional activity of the human AR. ELE1alpha has no intrinsic transcription activation domain or histone acetyltransferase activity, but it does interact with another histone acetyltransferase, p/CAF, and the basal transcription factor TFIIB. The interaction with the AR occurs through the ligand-binding domain and involves the region corresponding to the predicted helix 3. Mutation in this domain of leucine 712 to arginine greatly reduces the affinity of the AR for ELE1alpha but has only moderate effects on its transcriptional activity. Taken together, we have identified two isoforms of the putative coactivator ARA70/ELE1 that may act as a bridging factor between steroid receptors and components of the transcription initiation complex but which lack some fundamental properties of a classic nuclear receptor coactivator. Further experiments will be required to highlight the in vivo role of ELE1 in nuclear receptor functioning.

Acetyltransferases↗

PubMed1998

Identification of diazepam-binding Inhibitor/Acyl-CoA-binding protein as a sterol regulatory element-binding protein-responsive gene.

Diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP), a highly conserved 10-kDa polypeptide, has been implicated in various physiological processes including gamma-aminobutyric acid type A receptor binding, acyl-CoA binding and transport, steroidogenesis, and peptide hormone release. Both in LNCaP prostate cancer cells and 3T3-L1 preadipocytes, the expression of DBI/ACBP is stimulated under conditions that promote lipogenesis (treatment with androgens and insulin, respectively) and that involve the activation of sterol regulatory element-binding proteins (SREBPs). Accordingly, we investigated whether DBI/ACBP expression is under the direct control of SREBPs. Analysis of the human and rat DBI/ACBP promoter revealed the presence of a conserved sterol regulatory element (SRE)-like sequence. Gel shift analysis confirmed that this sequence is able to bind SREBPs. In support of the functionality of SREBP binding, coexpression of SREBP-1a with a DBI/ACBP promoter-reporter gene resulted in a 50-fold increase in transcriptional activity in LNCaP cells. Disruption of the SRE decreased basal expression and abolished SREBP-1a-induced transcriptional activation. In agreement with the requirement of a co-regulator for SREBP function, transcriptional activation by SREBP-1a overexpression was severely diminished when a neighboring NF-Y site was mutated. Cholesterol depletion or androgen treatment, conditions that activate SREBP function in LNCaP cells, led to an increase in DBI/ACBP mRNA expression and SRE-dependent transcriptional activation. These findings indicate that the promoter for DBI/ACBP contains a functional SRE that allows DBI/ACBP to be coregulated with other genes involved in lipid metabolism.

Animals↗