help button home button Endocrine Society Molecular Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2005-0479
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
20/10/2265    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Heemers, H. V.
Right arrow Articles by Swinnen, J. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Heemers, H. V.
Right arrow Articles by Swinnen, J. V.
Molecular Endocrinology 20 (10): 2265-2277
Copyright © 2006 by The Endocrine Society

Minireview

Androgen Activation of the Sterol Regulatory Element-Binding Protein Pathway: Current Insights

Hannelore V. Heemers, Guido Verhoeven and Johannes V. Swinnen

Laboratory for Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium

Address all correspondence and requests for reprints to: Johannes V. Swinnen, Laboratory for Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail: Johan.Swinnen{at}med.kuleuven.be.


   ABSTRACT
TOP
 ABSTRACT
DIRECT AND INDIRECT EFFECTS...
 THE SREBP PATHWAY
 ANDROGEN ACTIVATION OF THE...
 ANDROGEN ACTIVATION OF THE...
 CONCLUSIONS
 REFERENCES
 
The cellular effects of androgens are mediated by a cognate receptor, the androgen receptor. Typically, the androgen receptor is viewed to exert its activity by binding to androgen response elements located in or near the promoter region of target genes, thereby directly affecting the expression of these genes. However, increasing evidence indicates that androgens may also indirectly influence the expression of genes that do not contain androgen response elements by modulating the activity of secondary transcription factors, mediating the expression of growth factors acting in a paracrine or autocrine fashion, or by inducing changes in the production of other hormones. These indirect effects of androgens can induce cascade-like actions and may play an important role in more complex processes involving coordinated responses of genes, cells, and organs. Previously, our laboratory has identified and characterized a novel indirect mechanism of androgen action involving proteolytical activation of the key lipogenic transcription factor sterol regulatory element-binding protein (SREBP), resulting in the coordinate up-regulation of entire cellular lipogenic pathways. Interestingly, activation of SREBPs by androgens occurs not only under normal physiological conditions but has also been observed in a growing number of pathologies, and more in particular in the setting of steroid-regulated cancers, where increased lipogenesis has been shown to have remarkable diagnostic and prognostic potential and is considered a prime target for novel therapeutic approaches. This review aims to analyze current insights into the molecular mechanism(s) underlying androgen activation of the SREBP pathway and to ascertain the extent to which this phenomenon can be generalized to androgen-responsive cell systems.


   DIRECT AND INDIRECT EFFECTS OF ANDROGENS
TOP
 ABSTRACT
 DIRECT AND INDIRECT EFFECTS...
THE SREBP PATHWAY
 ANDROGEN ACTIVATION OF THE...
 ANDROGEN ACTIVATION OF THE...
 CONCLUSIONS
 REFERENCES
 
ANDROGENS, THE MAIN male sex steroids, are synthesized, for the most part, by the testes and, to a lesser extent, by the adrenals. They are the critical factors responsible for the development of the male phenotype during embryogenesis and for the achievement of sexual maturation at puberty. In adulthood, androgens remain essential for the maintenance of male reproductive function and behavior. Apart from their effects on reproduction, androgens affect a wide variety of nonreproductive tissues such as skin, bone, muscle, blood, and adipose tissue (1, 3).

Androgens exert most of their effects by interacting with a specific receptor, the androgen receptor (AR), a member of the large superfamily of ligand-activated nuclear hormone receptors. Binding of androgens causes release of heat shock proteins and induces a conformational change in the AR, allowing nuclear localization, increased phosphorylation, homodimer formation, and interaction with DNA. The activated AR binds to specific recognition sequences known as "androgen response elements" (AREs) located in or near androgen-regulated genes, where it recruits the components necessary for the assembly of a productive transcriptional complex, an event ultimately resulting in altered expression of target genes (2, 4, 5, 6, 7, 8, 9, 10).

Although many of the effects of androgens are indeed mediated by direct interaction of the AR with cis-acting AREs in the affected target genes (direct effects), it is becoming increasingly clear that androgens may also indirectly influence the expression of a variety of genes that do not necessarily contain AREs. Such indirect effects may take different forms, involving modulations in the expression or activity of secondary transcription factors, the production of growth factors acting in an autocrine or paracrine fashion, or changes in the expression of other hormones. As such, indirect effects of androgens may induce cascade-like actions and may play an important role in more complex effects involving coordinated responses of genes, cells, and organs (11).

While searching for androgen-regulated genes and cellular processes in prostate cancer (PCa) cells, our laboratory previously identified a novel indirect mechanism of androgen action involving activation of the key lipogenic transcription factor sterol regulatory element (SRE)-binding protein (SREBP). Androgen treatment of the human prostate adenocarcinoma cell line LNCaP was shown to induce a coordinated up-regulation of the expression of multiple genes belonging to the pathways of both cholesterol and fatty acid synthesis, an event accompanied and triggered by proteolytical maturation of SREBPs (12). This review aims to discuss current insights into the molecular mechanisms underlying androgen activation of the SREBP pathway as well as to explore to what extent the effects observed in LNCaP cells can be generalized to other androgen-responsive cell systems.


   THE SREBP PATHWAY
TOP
 ABSTRACT
 DIRECT AND INDIRECT EFFECTS...
 THE SREBP PATHWAY
ANDROGEN ACTIVATION OF THE...
 ANDROGEN ACTIVATION OF THE...
 CONCLUSIONS
 REFERENCES
 
SREBP Family Members
SREBPs are a family of transcription factors that have independently been characterized as central mediators of cellular cholesterol homeostasis (13, 14) and as regulators of fatty acid biosynthesis and uptake (15). Originally, three members of the SREBP family were identified: SREBP-1a, SREBP-1c, and SREBP-2. SREBP-1a and -1c are derived from a single gene, located at 17p11.2, through the use of alternate transcription start sites encoding alternative first exons that splice into a common second exon (16, 17). SREBP-1c has been cloned independently and was designated "adipocyte determination and differentiation factor-1" (15). The third member of the SREBP family, designated "SREBP-2," is encoded by a separate gene on chromosome 22q13 (18, 19). SREBP-1a, -1c, and -2 isoforms share a similar tripartite structure consisting of an N-terminal basic helix-loop-helix leucine zipper transcription factor domain, a central transmembrane region, and a C-terminal regulatory domain (20). More recently, a fourth SREBP protein, an isoform of SREBP-2 referred to as SREBP2gc, has been described. Unlike other SREBP isoforms, expression of SREBP2gc remains restricted to male germ cells, where it regulates transcription of spermatogenic genes in a cell- and stage-specific manner. Moreover, SREBP2gc, described as a shortened version of the N-terminal portion of SREBP2, is constitutively active and not subject to feedback control by sterols (as discussed below) (21, 22).

Activation Mechanism of SREBPs
Although alternative mechanisms of SREBP activation have been described, the originally described mechanism of SREBP activation, regulated by cellular sterol levels, is by far the best characterized.

Classical Sterol-Regulated SREBP Activation
Unlike other members of the basic helix-loop-helix leucine zipper class of transcription factors, SREBPs are synthesized as precursor proteins that are inserted into intracellular membranes such as those of the endoplasmic reticulum and the nuclear envelope (20). By means of their C-terminal domain, the newly synthesized SREBP precursor proteins immediately form a complex with an escort protein designated "SREBP cleavage-activating protein (SCAP)," an integral membrane protein anchored into the membranes of the endoplasmic reticulum (23). According to current insights, SCAP in turn interacts with a retention protein complex (24), consisting of at least two endoplasmic reticulum proteins [insulin-induced gene (insig)-1 and -2 (25, 26)], that serve to retain the SREBP/SCAP complex in the membranes of the endoplasmic reticulum where the SREBP precursor is kept in its inactive state. The SREBP/SCAP/insig complex is stabilized in the presence of cholesterol. A decrease in the intracellular cholesterol/sterol levels changes the conformation of a sterol sensor domain in SCAP, weakening its interaction with the insig retention proteins and permitting SCAP to escort SREBP precursor to the Golgi compartment (24, 25, 26, 27, 28). Within this cell organelle SREBP encounters two proteases [site 1 protease (S1P) and site 2 protease (S2P) (29, 30)] that act successively to release an N-terminal SREBP fragment by a mechanism of regulated intramembrane proteolysis (31). This transcriptionally active SREBP segment translocates to the nucleus [nuclear SREBP (nSREBP)] where it binds to SREs in the promoter region of an ever expanding set of SREBP target genes involved in the synthesis, metabolism, and uptake of fatty acids and cholesterol (Fig. 1Go). Depending on the SREBP isoform in question, a distinct set of lipogenic target genes is switched on. Whereas SREBP-1a is a potent activator of all SREBP-responsive genes, the roles of SREBP-1c and SREBP-2 are more restricted. SREBP-1c isoforms preferentially enhance transcription of fatty acid biosynthetic genes, whereas SREBP-2 mainly controls the expression of genes involved in cholesterologenesis (32, 33, 34, 35). As mentioned, SREBP2gc is different from the other SREBP isoforms in terms of structure and activation. Because it consists of most of the SREBP2 N-terminal transcription factor domain, this particular isoform exists as a soluble and constitutively active transacting factor that is not subject to feedback control by sterols to drive transcription of cell- and stage-specific SREBP target genes during spermatogenesis (21, 22).


Figure 1
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 1. Sterol-Regulated Activation Mechanism of SREBPs

SREBPs are synthesized as inactive precursor proteins, anchored into the membranes of the endoplasmic reticulum (ER), where they from a complex with SCAP. SCAP interacts with a retention protein complex that includes insig 1 (1 ) and insig 2 (2 ). The retention complex holds the SREBP/SCAP inside the endoplasmic reticulum. When the cell is in need of lipids, interaction between SCAP and the retention protein complex is lost, allowing the SREBP/SCAP complex to migrate to the Golgi complex. In this cell organelle, the SREBP precursor protein encounters two proteases (site 1 and site 2 protease) that cleave the precursor protein leading to the release of a N-terminal SREBP segment (nSREBP). This nSREBP translocates to the nucleus where it binds as a dimer to SREs in the promoter region of key lipogenic genes involved in both the synthesis of fatty acids and cholesterol, thereby activating their transcription and restoring the cellular lipid content.

 
Other Mechanisms of Posttranslational SREBP Activation
As investigation into the regulation of the SREBP pathway goes on, a picture of ever increasing complexity emerges. Apart from the sterol-mediated mechanism of SREBP activation, SREBP precursor proteins can also be proteolytically activated in a sterol-independent manner by cysteine proteases belonging to the caspase family (36, 37). These proteases cleave SREBPs in a single step at a site located closer to the N terminus of the protein than the recognition site for S2P, generating a SREBP fragment that is smaller than mature SREBP resulting from cellular cholesterol depletion but that remains transcriptionally active. Although SREBP activation occurs early in the apoptotic program (38), its exact role during apoptosis remains elusive.

Other Factors Affecting SREBP Transcription
In addition to the posttranscriptional sterol- and caspase-mediated regulation of SREBPs, several different means of transcriptional regulation have been described, including feed-forward regulation of SREBP-1c and SREBP-2 by SREs present in the promoter regions of these genes (39, 40), selective up-regulation of SREBP-1c transcription by liver X receptors (41, 42), nuclear receptors that can be activated by a variety of sterols, and modulation of phosphatidylinositol 3-kinase-dependent SREBP-1c transcription by nutrients and hormones (43). Moreover, SREBP-mediated transcription may be modulated by a number of other mechanisms. Transcriptional stimulation by mature SREBP, in itself a fairly weak transacting factor, has been shown to depend on interaction with several cofactors, and cross-talk with steroid receptors, such as the thyroid receptor and the estrogen receptor, has been reported (44, 45). In addition, posttranslational phosphorylation of nSREBP by growth factors or insulin has been described, resulting in an increase in SREBP-mediated transcriptional activation (46, 47, 48). Finally, some evidence suggests independent regulation of signaling by SREBP-1 and -2 isoforms, a finding that might further complicate an already complex situation (49).


   ANDROGEN ACTIVATION OF THE SREBP PATHWAY
TOP
 ABSTRACT
 DIRECT AND INDIRECT EFFECTS...
 THE SREBP PATHWAY
 ANDROGEN ACTIVATION OF THE...
ANDROGEN ACTIVATION OF THE...
 CONCLUSIONS
 REFERENCES
 
Induction of Lipogenesis by Androgens
Prompted by the identification of the gene encoding diazepam-binding inhibitor/acetyl-coenzyme A (CoA)-binding protein (DBI/ACBP), a small (10 kDa) protein involved in several aspects of cellular lipid metabolism, as an androgen-regulated gene in the human prostatic adenocarcinoma cell line LNCaP (50, 51), our laboratory started to examine the impact of androgen stimulation on cellular lipid metabolism in PCa cells. Exposure of LNCaP cells to androgens induced a marked accumulation of neutral lipids such as cholesteryl esters and triacylglycerols (52). Interestingly, the accumulated lipids appeared to be largely derived from de novo synthesis, rather than, for instance, uptake from the extracellular medium, suggesting that androgens might stimulate the expression and/or activity of one or more enzymes involved in endogenous lipogenesis. This contention was supported by the demonstration that androgens indeed up-regulated both the expression and the activity of fatty acid synthase (FAS), an enzyme that plays a key role in the synthesis of fatty acids, as well as the expression of several other enzymes belonging to the metabolic pathway of fatty acid synthesis such as ATP citrate lyase, acetyl-CoA-carboxylase (ACC), and malic enzyme (12, 53). In addition, multiple genes encoding enzymes involved in the biosynthesis of cholesterol including the gene encoding 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)-synthase, HMG-CoA-reductase, and farnesyl diphosphate synthase (FPPS) turned out to be induced by androgens (12). In support of the involvement of the AR in lipid accumulation, expression as well as activity of lipogenic enzymes, the steroid specificity of these events reflected the aberrant ligand specificity of the mutated AR (T877A) expressed in LNCaP cells. Moreover, the AR antagonist Casodex (bicalutamide) abolished the stimulatory effects of androgens. Consistent with requirement of AR, no androgen-induced lipogenic response was observed in the AR-negative PCa cell lines PC-3 and DU145 (Refs. 50 and 53 and our unpublished results).

Androgen-Induced Lipogenesis Mediated by Activation of the SREBP Pathway
The observation that androgens coordinately stimulate the expression of entire clusters of lipogenic genes suggested that the expression of these genes might not be controlled by a classical direct mechanism of androgen action, i.e. mediated by direct interaction of the AR with AREs in the affected genes, but rather through an indirect mechanism involving a common intermediary factor. Based on their central role in the coordinate control of lipogenic gene expression, the involvement of SREBPs as potential mediators of the lipogenic effects of androgens in LNCaP cells was investigated. Interestingly, early experiments revealed that LNCaP cells responded to androgen stimulation by an induction of the expression of SREBP-1 and SREBP-2 transcripts (1.3- and 2.1-fold stimulation, respectively), SREBP precursor protein levels (1.5- and 4-fold), and an increase in the nuclear content of mature SREBP-1 (up to 6-fold, dependent on the experiment). Moreover, the androgen-induced expression of lipogenic genes, such as the gene encoding FAS, was mediated by and dependent on cis-acting elements harboring SREBP-binding sites (12). Interestingly, also androgen regulation of the DBI/ACBP gene required an intact SREBP-binding site in the promoter region of this gene (54). Taken together, these data suggested the existence of a novel indirect mechanism of androgen action, mediated by activation of the SREBP pathway. Follow-up studies using promoter reporter constructs derived from other lipogenic genes, as well as dominant-negative SREBP forms, corroborated and expanded on these initial findings (Ref. 55 and our unpublished results). The precise mechanisms by which androgens exerted their effects on the SREBP pathway, however, remained elusive at that time.

Androgen Activation of the SREBP Pathway Mediated by the Structural Elements Required for Its Sterol Regulation
A first clue toward understanding the mechanism by which androgen treatment of LNCaP cells leads to activation of SREBP precursor proteins came from the observation that the terminal steps involved in androgen-induced activation of the SREBP pathway are identical to those involved in the activation of the pathway by sterol depletion. Indeed, comparison of the size of the endogenous nSREBP fragments generated after androgen exposure or sterol depletion demonstrated that androgen-induced maturation of SREBPs depends on the presence of an intact cleavage site 1, an intact cleavage site 2, and the presence of the SCAP-interacting regulatory C-terminal region of the SREBP precursor. This was confirmed by mutagenesis studies using ectopically expressed recombinant SREBP precursors in which the cleavage sites for S1P or S2P were altered or the entire C-terminal domain responsible for interaction with the transporter protein SCAP was deleted (Ref. 56 and our unpublished results). Despite these findings, androgen stimulation of SREBP maturation appeared independent of the sterol content of LNCaP cells. Because isoform-specific antibodies against amino-terminal SREBP fragments were not available, our studies on the maturation of endogenous SREBPs could not distinguish between nSREBP-1a and nSREBP-1c. Similarly, the lack of a suitable antibody directed against SREBP-2 limited these studies to SREBP-1. However, as androgen exposure of LNCaP cells does lead to increased nuclear levels of herpes simplex virus-tagged recombinant SREBP-2 and stimulates transcription of typical SREBP-2 target genes such as HMG-CoA-synthase (55, 56), one may reasonably assume that androgens also induce maturation of the SREBP-2 precursor and that this maturation proceeds along the same lines as that of SREBP-1.

Additional studies ruled out the possibility that androgen-mediated activating mechanisms of the SREBP pathway depend on structural elements other than those involved in its sterol regulation. Detailed examination of the size of the androgen-generated nSREBPs had already excluded the possibility of caspase-mediated SREBP precursor cleavage. Moreover, specific mutation of the cleavage site for caspases in SREBP precursor proteins did not affect androgen-mediated SREBP maturation (our unpublished results). Furthermore, consistent with literature reports, no activation of caspases was noted upon exposure of LNCaP cells to the doses of androgens used (our unpublished results). In addition, the banding pattern of immunoreactive nSREBP Western blots, typically apparent as a cluster of bands, did not change dramatically upon androgen exposure, making it unlikely that androgen treatment induces major posttranslational modifications in the nSREBP fragment (Refs. 12 and 55 and our unpublished results).

Androgen-Induced Increase in Cellular SCAP Levels Leads to SREBP Activation
Further investigation into the molecular mechanism(s) governing induction of SREBP maturation by androgens revealed that the action of androgens not only relies on the presence of the same structural elements but is, in fact, mediated by alterations in the expression of key elements within the SREBP pathway itself. Indeed, androgens were shown to notably increase the expression of SCAP, the escort protein that mediates the translocation of SREBP precursors from the endoplasmic reticulum to the Golgi apparatus, the place were they are cleaved and activated. This increase was observed both at the mRNA and the protein level. Underscoring the importance of up-regulation of SCAP in the androgen stimulation of lipogenic gene expression, overexpression of SCAP was shown to increase transcription of key genes involved in both fatty acid (FAS) and cholesterol synthesis (HMG-CoA-synthase). Forced overexpression of SCAP led to nuclear accumulation of mature SREBPs and SCAP-induced transcriptional activation of lipogenic genes depended entirely on the presence of intact SREs in the promoter regions of these genes and was counteracted by dominant-negative SREBP forms lacking either the ability to bind to SREs or the capacity to drive transcription (55).

In addition to SCAP, the mRNA levels of SREBP-1c and SREBP-2 were also enhanced after androgen treatment of LNCaP cells (55). However, whereas SCAP proved to be a primary target of androgen action, evidence indicates that the changes in expression of SREBP-1c and SREBP-2 are the result of previously described auto-regulatory effects of nSREBP. First, SREBP-1c and SREBP-2 mRNA levels were elevated after androgen-independent alterations in nSREBP levels whereas SCAP expression was not affected despite the presence of a putative SRE in the promoter region (57) of the SCAP gene. Second, mutation of SREBP-binding elements in the promoter regions of the SREBP-1c gene prevented androgen-induced expression of this gene, as did addition of dominant-negative SREBP forms. Third, time course studies revealed that induction of SCAP expression coincided with nuclear translocation of SREBPs and preceded the increase in expression of lipogenic genes as well as the genes encoding SREBP-1c and SREBP-2 (our unpublished results).

With regard to the impact of androgen exposure on other key components of the SREBP pathway in LNCaP cells, expression of SREBP-1a and S1P remained essentially unaltered whereas a slight increase in the expression of S2P was noted upon androgen stimulation (55). Because successful cleavage at site 2 has previously been shown to depend entirely on completed cleavage at site 1 (20, 31), it would seem highly unlikely that a moderate increase in S2P levels by themselves would account for a substantial part in androgen activation of the SREBP pathway. Some contribution to this event, however, cannot be excluded at this time.

SCAP as a Direct Target for the AR in the SREBP Pathway
In view of the central role of SCAP in the maturation of SREBP precursor proteins and induction of the lipogenic program by androgens in LNCaP cells, our laboratory set out to pinpoint the molecular mechanism by which androgens affect SCAP expression in these cells. As preliminary studies using the transcription inhibitor actinomycin D suggested that the regulation of SCAP expression by androgens depended, at least in part, on transcriptional control (55), a systematic search for regulatory sequences in the SCAP gene that might mediate the effects of androgens was initiated. Although no androgen-regulatory regions could be identified in a 6-kb fragment of the 5'-upstream region of the SCAP gene, a region within intron 8 was able to transfer androgen responsiveness (58). Interestingly, gel retardation experiments indicated a direct interaction between this region and the DNA-binding domain (DBD) of the AR. Further analysis revealed the presence of a sequence aagaGGAAGAaaaTGTACCtctt, bearing resemblance to the 5'-TGTTCT-3' consensus core-binding motif of a canonical ARE. This sequence proved to be necessary and sufficient for AR binding and conferring transcriptional activation to reporter genes. Moreover, genomic fragments of the SCAP gene containing this particular motif were able to drive androgen-induced transcription of reporter genes independently of the promoter context. Mutation in or deletion of either the right half-site or the left half-site of the ARE sequence abrogated binding of the AR and abolished androgen-regulated reporter gene activity (58). Characterization of this newly identified SCAP ARE revealed that it displays all the hallmarks of a classical ARE. Contrary to so-called "specific AREs," which selectively recognize the AR and are structured as partial direct repeats (59), the SCAP ARE is organized as an (imperfect) inverted repeat and binds the DBD of the glucocorticoid receptor (another class I steroid hormone receptor) with an affinity that is only slightly lower than that observed for the AR DBD [dissociation constant (Ks) = 300 nM vs. 150 nM for AR binding] (58). The identification of an ARE in intron 8 of SCAP supports the contention of SCAP as a direct target for the AR on the SREBP pathway. Although not the most common localization, intronic localization of androgen-responsive regions has also been reported for the genes encoding the C1 and C3 component of prostatic binding protein, the gene encoding ß-glucuronidase, as well as the human GH gene (60, 61, 62, 63). Because only about 70% of the sequence of the SCAP gene could be cloned and studied, the existence of additional AREs in some of the uninvestigated areas of the gene cannot be excluded. In addition, stable introduction of the AR in otherwise androgen-unresponsive PC-3 PCa cells failed to evoke a full androgen response of the SCAP gene (our unpublished results), suggesting that additional factors are needed to permit androgen-induced lipogenesis in LNCaP cells.

Alterations in the Expression Pattern of insig Retention Proteins: A Novel Way for Androgens to Stimulate SREBP Activation?
Although SCAP is a pivotal element in the control of SREBP signaling, many elements in this signaling pathway remain incompletely understood and may turn out to be additional targets for androgen action. In this respect, exciting new data in our laboratory suggest that androgens may further modulate SREBP signaling by inducing changes in the expression pattern of key components of the retention protein complex. Indeed, the expression of insig-1 and insig-2, two recently identified components of the postulated retention protein complex, was shown to be subject to androgen regulation (Fig. 2AGo). Interaction of these insig proteins and SCAP has been shown to determine activation of the SREBP pathway. Ribonuclease protection assays on RNA derived from LNCaP cells revealed opposite effects of androgens on the expression of insig-1 and -2 mRNA levels: whereas androgens increased insig-1 mRNA, insig-2 mRNA levels were markedly repressed. Infection of LNCaP cells with an adenoviral construct encoding a dominant-active SREBP form led to a marked increase in insig-1 levels, indicating that insig-1 expression is up-regulated secondary to SREBP maturation. Insig-2 levels, on the other hand, were unaffected by changes in nSREBP levels (Fig. 2BGo). Similar results were obtained using LNCaP cell lines that inducibly express nSREBP-1c (data not shown). These data are reminiscent of the situation in mouse liver where activation of the SREBP pathway by insulin stimulation is accompanied by down-regulation of a liver-specific insig-2 transcript and up-regulation of insig-1 along with other SREBP target genes (64). The impact of androgen-regulated expression of insig proteins and particularly the down-regulation of insig-2 on the activity of the SREBP pathway in LNCaP cells, as well as the mechanism by which androgens affect insig-2 expression, are currently under investigation. Preliminary experiments using actinomycin D suggest that the effects of androgens on insig-2 expression occur, at least in part, at the transcriptional level (our unpublished results).


Figure 2
View larger version (40K):
[in this window]
[in a new window]
  		Fig. 2. Effect of Androgens on the Expression of Essential Components of the SREBP Pathway

A, LNCaP cells were cultured in the presence (+) or absence (–) of 10–8 M R1881 for 2 d. Total RNA was isolated and Northern blot analysis was performed with probes for SREBP-1a, SREBP-1c, SREBP-2, S1P, S2P, and SCAP. Hybridization with a probe for 18S rRNA was performed to exclude potential loading differences (data not shown). mRNA expression levels of insig-1 and insig-2 were evaluated by ribonuclease protection assay. Results shown are representative of two independent experiments. B, LNCaP cells were cultured either without adenovirus (no virus), with adenovirus expressing a dominant-active form of SREBP-1(Ad-DA, 30 pfu/cell), with adenovirus without insert (Ad-Null), or with adenovirus expressing green fluorescent protein (GFP) as a control. The next day, cells were harvested followed by RNA isolation and ribonuclease protection analysis of insig-1 and insig-2 expression. Ad, Adenovirus.

 
Androgen Activation of the SREBP Pathway: Current Working Hypothesis
Taken together, the data discussed above led us to formulate a working hypothesis in which androgens, although using the structural experiments needed for sterol regulation of SREBP activation, induce maturation of SREBP precursors by an alternative mechanism. In the classical sterol-regulated pathway of SREBP activation, a decrease in intracellular concentrations of sterols changes the conformation of the sterol-sensing part of SCAP, resulting in weakening of the interaction with retention complex proteins and translocation of the SREBP/SCAP complex to the Golgi apparatus, where SREBP precursors are cleaved and activated. In the alternative pathway proposed here, increased expression of SCAP, induced by direct transcriptional stimulation by the AR, causes a shift in the normal cellular equilibrium between SCAP and components of its retention protein complex, favoring translocation of SREBP precursors to the Golgi apparatus, followed by cleavage and activation. Selective changes in the expression of insig retention proteins might further stimulate this process (Fig. 3Go). In support of this model, ectopical overexpression of either SCAP (fragments) or insig proteins has recently been shown to be able to induce or repress, respectively, transcriptional activation of SREBPs (24, 55, 65).


Figure 3
View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3. Androgen Activation of the SREBP Pathway: Current Working Hypothesis

Androgen-induced changes in the expression of the escort protein SCAP and its retention proteins result in a shift in the normal cellular equilibrium between SCAP and insig proteins, facilitating translocation of SREBP precursors to the Golgi apparatus where they are cleaved and activated.

 

   ANDROGEN ACTIVATION OF THE SREBP PATHWAY CAN BE GENERALIZED TO OTHER ANDROGEN-RESPONSIVE CELL SYSTEMS
TOP
 ABSTRACT
 DIRECT AND INDIRECT EFFECTS...
 THE SREBP PATHWAY
 ANDROGEN ACTIVATION OF THE...
 ANDROGEN ACTIVATION OF THE...
CONCLUSIONS
 REFERENCES
 
Androgen Regulation of Lipogenic Gene Expression by Activation of the SREBP Pathway Is a Common Mechanism in PCa Cells
Since our initial observations, androgen regulation of lipogenic gene expression in LNCaP cells has been confirmed by several independent DNA microarray studies, as has the role of the AR in this process (66, 67, 68). To ascertain whether this phenomenon can be generalized to other androgen-responsive PCa cells, we extended our investigations to two recently established and independent PCa cell lines, MDA-PCa-2a and PC346c. PC346c cells, derived from a primary untreated tumor, express a wild-type AR (69). The AR found in MDA-PCa-2a cells, on the other hand, displays two mutations in its ligand-binding domain: T887A (found also in the AR of LNCAP cells) and L701H (70). In both these PCa cell lines, androgens provoke a coordinate increase in the expression of genes involved in fatty acid synthesis and cholesterol synthesis (Refs. 55 and 56 and our unpublished results). Similar observations were done in the MDA-PCa-2b cell line, a PCa cell line derived from the same bone metastasis as MDA-PCa-2a cells, but displaying different genetic features and a different phenotype (Ref. 71 and our unpublished results). The absolute expression levels and the relative fold induction of these genes, however, were different in the different cell lines studied. Moreover, when investigated in MDA-PCa-2a and PC346c cells, the up-regulation of the expression of both transiently and endogenously expressed lipogenic genes was accompanied by and depended on the maturation of SREBP precursors and the presence of intact SREBP-binding sites in the promoter region of these genes (Ref. 55 and our unpublished results). Interestingly, in all three PCa cell lines studied, up-regulation of SREBP signaling appeared to be the result of an androgen-induced imbalance between SCAP and its retention proteins. An increase of SCAP expression comparable to the one observed in LNCaP cells was observed in MDA-PCa-2a cells, whereas the androgen-induced decrease in insig-2 expression was seen in both MDA-PCa-2a and PC346c cells. Moreover, as in LNCaP cells, androgen treatment of MDA-PCa-2a and PC346c increased the expression of SREBP-1c, SREBP-2, insig-1, and S2P but did not notably affect SREBP-1a and S1P mRNA levels (see Table 1Go) (Refs. 55 and 56 and our unpublished results).


View this table:
[in this window]
[in a new window]
  		Table 1. Overview of the Effects of Androgens on Key Components of the SREBP Pathway

 
Taken together, these data suggest that androgen stimulation of lipogenic gene expression by a mechanism involving SREBP activation is a common feature in androgen-responsive PCa cell lines. Interestingly, several lines of evidence suggest a role for SREBP-mediated lipogenesis also in the clinical setting of PCa. First, overexpression of lipogenic genes, usually characterized by expression of the key lipogenic gene FAS, is a very early and common event in the development of PCa (72). Expression of FAS and corresponding lipogenic activity are already increased in prostatic intraepithelial neoplasia lesions, generally considered to be the premalignant precursor of prostate cancer, and is further elevated as the tumor progresses. Overexpression is seen in almost all invasive prostate cancers where it correlates with a high proliferative index and correlates with a poor prognosis. Second, along with FAS, other genes encoding lipogenic enzymes (e.g. acetyl-CoA-carboxylase) have been shown to be overexpressed in PCa, indicating the possibility of coordinated and central control governing the expression of gene clusters belonging to the same metabolic pathways (73). Third, initial observations in our laboratory suggested that coordinated up-regulation of FAS and ACC mRNA levels in a panel of prostate cancer specimens is accompanied by a slight, but distinct, increase in the mRNA expression of the SREBP-1 gene, a potential indication of activation of the SREBP pathway (Ref. 73 and our unpublished results). More recently, the finding of up-regulation of SREBP transcripts and precursor proteins in PCa cells has been confirmed by immunohistochemical studies by other research groups (74). Because antibodies specifically recognizing mature SREBP are not yet available, immunohistochemical SREBP detection does not allow discrimination between mature SREBPs and SREBP precursors, which are also anchored into the membranes of the nuclear envelope. Therefore, evaluation of the expression status of active SREBPs in clinical PCa specimens is, at present, impossible.

Although a definite role for the AR in the activation of SREBPs and in the up-regulation of lipogenic gene expression in neoplastic prostate cells in vivo remains to be determined, several observations, both in PCa xenografts as well as in patient-derived material, do suggest involvement of this transcription factor in activation of the lipogenic program in PCa in vivo. Castration of male mice carrying CWR22 or LNCaP PCa xenografts results in an immediate decrease in FAS expression in the xenograft tissue that can be restored by androgen treatment (75, 76). In xenografts corresponding to more advanced stages of PCa, so-called androgen depletion-independent (ADI) PCa (77), however, the androgen dependency of FAS expression is lost, and FAS remains highly expressed despite castration. In this regard FAS expression mirrors the expression pattern of other typical AR-regulated genes such as the gene encoding prostate-specific antigen. Growth factor-mediated and androgen-independent activation of the AR may be one of the mechanisms responsible for the latter phenotype. In this regard it should be mentioned that data from our laboratory confirm that, at least in PCa cells in culture, lipogenic gene expression can be up-regulated by growth factors such as epidermal growth factor and that this up-regulation also involves signaling through the SREBP pathway and is accompanied by an increase in SCAP expression (Ref. 78 and our unpublished results). In addition to elucidating the role of the AR in neoplastic lipogenesis, studies using LNCaP xenografts have confirmed the activation of SREBP precursors and a role for SCAP in this process during the natural progression of PCa. After an initial decline in the expression levels of most of these genes, levels of not only FAS, but also ACBP/DBI, FPPS, SREBP-1a, SREBP-1c, and SREBP-2 increased significantly during progression to the ADI state. Importantly, levels of SCAP decreased after castration and increased markedly at androgen depletion independence (76). In this respect, the LNCaP xenograft model mimics the expression profiles of LNCaP and LNCaP-Rf cells, a subline of LNCaP established after long-term androgen deprivation and considered to be a valuable model system for the study of ADI PCa (Ref. 79 and Schmidt, L.J., and D.J. Tindall, personal communication). To our knowledge, expression levels of insig proteins in these tumors have not yet been examined. High levels of SREBP-1 were noted in specimens from patients with varying grades of disease, emphasizing the importance of the SREBP pathway in the clinical setting of PCa. After hormone withdrawal, tumor levels of SREBP-1 decreased significantly. At the ADI stage, tumors expressed significantly higher levels of SREBP-1 (76).

As to why AR-mediated stimulation of this lipogenic pathway could be associated with PCa, recent observations in our laboratory indicate that the majority of the newly synthesized lipids are phospholipids enriched in saturated and monounsaturated fatty acyl chains that tend to partition into detergent-resistant membrane microdomains. Because these raft aggregates are implicated in key cellular processes, such as intracellular trafficking, signal transduction, and cell migration, it is expected that increased lipogenesis in PCa cells affects several crucial aspects of PCa cell biology and actively contributes to the progression of PCa (Ref. 80 and our unpublished results).

Androgen Regulation of Lipogenic Gene Expression by Activation of the SREBP Pathway in Normal, Noncancerous Androgen-Responsive Cells
Because lipogenic enzymes are expressed at aberrantly high basal levels in PCa cells, we wondered whether androgen stimulation of lipogenic gene expression, mediated by the SREBP pathway, could represent a more general physiological regulation in noncancerous androgen-responsive cells as well. Using adult male Wistar rats as an experimental paradigm, we tested whether modulation of androgen levels by castration and/or readministation of androgens affects the expression of genes belonging to the pathways of both fatty acid synthesis (FAS and ACC) and cholesterol synthesis (HMG-CoA-reductase and FPPS) in androgen-responsive tissues. In the ventral prostate, a prime target tissue for androgen action, a marked decrease was noted in both mRNA and protein levels of all four genes tested, as well as expression of DBI/ACBP, after castration for 4 d (81, 82). Administration of testosterone or the nonaromatizable androgen dihydrotestosterone completely restored lipogenic gene expression, implicating the AR in the observed effects. Immunohistochemical staining for FAS demonstrated that the effects of androgens on lipogenic gene expression were not merely the result of androgen-dependent changes in prostate tissue composition (82). Similar effects were noted on the expression of these lipogenic genes in the dorsolateral prostate (our unpublished results) and in the lacrimal gland (82), an androgen-responsive organ that, contrary to prostatic tissue, does not depend strictly on the presence of androgens for the maintenance of its structural integrity. Recent reports of androgen-stimulated expression of genes involved in fatty acid biosynthesis as well as cholesterologenesis in these tissues confirm our initial findings (83, 84, 85). Moreover, similar effects have also been reported in organs and tissues as diverse as sebaceous glands, meibomian glands, and adipose tissue, both in rodents and in humans (86, 87, 88, 89, 90).

Given the coordinated manner in which the androgen-related changes in lipogenic gene expression take place in these androgen-responsive tissues, we investigated whether they might be the result of activation of the SREBP pathway. Due to the apparently very low expression levels, effects of modulating serum androgen levels on mature SREBP levels in the lacrimal gland could not be detected. In the ventral prostate, however, castration gave rise to a marked decrease in nSREBP content, which could be restored completely by administration of testosterone (82). A more recent publication reports similar androgen-induced accumulation of nSREBP in sebaceous glands, confirming our initial findings (88). Although these data do not provide definite proof for a functional relationship between the observed increases in both nSREBP levels and the expression of lipogenic enzymes in vivo, they certainly point toward such a relationship. Further confirmation of this relationship might require selective interference with the SREBP pathway (using as-yet-unavailable inhibitors, RNA interference...) or the development of transgenic animals in which the expression of essential components of the SREBP pathway is impaired in selected tissues. In the absence of such tools, the finding, that, in both the ventral prostate and the lacrimal gland, androgens regulate the mRNA levels of SREBP-1c and SREBP-2, corroborates the hypothesis of involvement of activated SREBPs. Consistent with observations in PCa cell lines, SREBP-1a mRNA expression remained unaffected (our unpublished results). Interestingly, several literature reports describe androgen-stimulated lipogenic gene expression in other tissues and organs to be accompanied by comparable changes in SREBP isoform expression levels (87, 88). Moreover, in some, but not all, tissues these changes occurred along with an androgen-dependent increase in SCAP expression. In this respect, examination of the impact of modulating androgen levels on the expression of insig retention proteins should prove to be informative.

With regard to the potential functional significance of the described in vivo androgen-mediated changes in lipogenic genes expression, it should be stressed that in acinar cells of sebaceous glands, meibomian gland, prostate, and seminal vesicles, androgens have major stimulatory effects on the production, accumulation, and/or secretion of lipids. In most of these tissues the composition of these lipids is complex, consisting of cholesteryl esters, diacylglycerides, triglycerides, and wax esters. Depending on the tissue type, these lipids have different physiological functions, and dysregulation of their production may be related to pathological conditions as diverse as acne and dry eye syndrome (86, 87, 91, 92, 93, 94, 95).


   CONCLUSIONS
TOP
 ABSTRACT
 DIRECT AND INDIRECT EFFECTS...
 THE SREBP PATHWAY
 ANDROGEN ACTIVATION OF THE...
 ANDROGEN ACTIVATION OF THE...
 CONCLUSIONS
REFERENCES
 
Taken together, the data discussed above clearly demonstrate that activation of the metabolically critical SREBP pathway represents a general mechanism for androgens to exert control over the entire cellular lipogenic program, both under normal physiological and neoplastic conditions. By stimulating maturation of SREBP precursor proteins, androgens are able to indirectly drive transcription of genes constituting complete lipid-synthesizing pathways, allowing a profound impact on cellular lipid homeostasis and a substantial amplification of the initial androgenic signal. Moreover, current literature reports support the contention that the molecular mechanism responsible for the androgen-induced maturation of SREBP precursors that we initially unraveled in LNCaP PCa cells, consisting of an androgen-mediated disturbance in the normal cellular equilibrium between the escort protein SCAP and its retention proteins, is also conserved in other androgen-sensitive cell systems. These findings put the SCAP-retention protein axis forward as an interesting therapeutic target for the treatment of diseases characterized by altered lipid synthesis and governed by androgens such as acne, prostate cancer, and dry eye disease.

Interestingly, the mechanism governing activation of the SREBP pathway could also be relevant for therapeutic intervention in conditions that are regulated by steroids other than androgens. For example, progestagens induce lipogenic gene expression in several breast cancer cell lines as well as in preadipocytes in culture, and activation of SREBPs has been also implicated in this phenomenon (96). Moreover, increased expression of lipogenic genes has been observed in several steroid-sensitive tumors including breast cancer and ovarian cancer (97, 98). Evidence for the presence of an activated SREBP pathway in these tumors is emerging (99, 100). To our knowledge, a role for SCAP and/or its retention protein complex has not yet been investigated in these settings. In view of our observation that the SCAP ARE we identified is able to bind class I receptors other than the AR (58), it is tempting to speculate that several steroid hormones may be able to activate the SREBP pathway by changing the equilibrium between SCAP and its retention proteins.


   FOOTNOTES
 
This work was supported by Cancer Research Grants from Fortis Bank Insurance and VIVA, by a grant "Geconcerteerde Onderzoeksactie van de Vlaamse Gemeenschap," by research grants from the Fund for Scientific Research, Flanders (Belgium) (F.W.O.), by a grant Interuniversity Poles of Attraction Programme, Belgian State, Prime Minister’s Office, Federal Office for Scientific, Technical and Cultural Affairs, and by a specialization grant from the Institute for the Encouragement of Innovation by Science and Technology in Flanders (to H.V.H.).

Current address for H.V.H.: Department of Urology Research, Mayo Clinic and Foundation, Rochester, Minnesota 55905.

Author Disclosure Summary: H.V.H., G.V., and J.V.S. have nothing to declare.

First Published Online February 2, 2006

Abbreviations: ACC, Acetyl-CoA-carboxylase; ADI, androgen depletion-independent; AR, androgen receptor; ARE, androgen response element; CoA, coenzyme A; DBD, DNA-binding domain; DBI/ACBP, diazepam-binding inhibitor/acetyl-CoA-binding protein; FAS, fatty acid synthase; FPPS, farnesyl diphosphate synthase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; insig, insulin-induced gene; nSREBP, nuclear SREBP; PCa, prostate cancer; S1P, site 1 protease; SCAP, SREBP cleavage-activating protein; SRE, sterol regulatory element; SREBP, SRE-binding protein.

Received for publication November 28, 2005. Accepted for publication January 23, 2006.


   REFERENCES
TOP
 ABSTRACT
 DIRECT AND INDIRECT EFFECTS...
 THE SREBP PATHWAY
 ANDROGEN ACTIVATION OF THE...
 ANDROGEN ACTIVATION OF THE...
 CONCLUSIONS
 REFERENCES
 

  1. George FW, Wilson JD 1986 Hormonal control of sexual development. Vitam Horm 43:145–196[Medline]
  2. Quigley CA, De Bellis A, Marschke KB, el-Awady MK, Wilson EM, French FS 1995 Androgen receptor defects: historical, clinical, and molecular perspectives. Endocr Rev 16:271–321[CrossRef][Medline]
  3. De Moor P, Verhoeven G, Heyns W 1973 Permanent effects of foetal and neonatal testosterone secretion on steroid metabolism and binding. Differentiation 1:241–253
  4. Tsai MJ, 0'Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  5. McEwan IJ, Gustafsson J 1997 Interaction of the human androgen receptor transactivation function with the general transcription factor TFIIF. Proc Natl Acad Sci USA 94:8485–8490[Abstract/Free Full Text]
  6. Lee DK, Duan HO, Chang C 2000 From androgen receptor to the general transcription factor TFIIH. Identification of cdk activating kinase (CAK) as an androgen receptor NH(2)-terminal associated coactivator. J Biol Chem 275:9308–9313[Abstract/Free Full Text]
  7. Lee DK, Chang C 2003 Molecular communication between androgen receptor and general transcription machinery. J Steroid Biochem Mol Biol 84:41–49[CrossRef][Medline]
  8. Heinlein CA, Chang C 2002 Androgen receptor (AR) coregulators: an overview. Endocr Rev 23:175–200[Abstract/Free Full Text]
  9. Heinlein CA, Chang C 2004 Androgen receptor in prostate cancer. Endocr Rev 25:276–308[Abstract/Free Full Text]
  10. Wang L, Hsu CL, Chang C 2005 Androgen receptor corepressors: an overview. Prostate 63:117–130[CrossRef][Medline]
  11. Verhoeven G, Swinnen JV 1999 Indirect mechanisms and cascades of androgen action. Mol Cell Endocrinol 151:205–212[CrossRef][Medline]
  12. Swinnen JV, Ulrix W, Heyns W, Verhoeven G 1997 Coordinate regulation of lipogenic gene expression by androgens: evidence for a cascade mechanism involving sterol regulatory element binding proteins. Proc Natl Acad Sci USA 94:12975–12980[Abstract/Free Full Text]
  13. Briggs MR, Yokoyama C, Wang X, Brown MS, Goldstein JL 1993 Nuclear protein that binds to sterol regulatory element of low density lipoprotein receptor promoter. I. Identification of the protein and delineation of its target nucleotide sequence. J Biol Chem 268:14490–14496[Abstract/Free Full Text]
  14. Wang X, Briggs MR, Hua X, Yokoyama C, Goldstein JL, Brown MS 1993 Nuclear protein that binds sterol regulatory element of the low density lipoprotein receptor promoter. II. Purification and characterisation. J Biol Chem 268:14497–14504[Abstract/Free Full Text]
  15. Tontonoz P, Kim JB, Graves RA, Spiegelman BM 1993 ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation. Mol Cell Biol 13:4753–4759[Abstract/Free Full Text]
  16. Yokoyama C, Wang X, Briggs MR, Admon A, Wu J, Hua X, Goldstein JL, Brown MS 1993 SREBP-1, a basic helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene. Cell 75:187–197[CrossRef][Medline]
  17. Hua X, Wu J, Goldstein JL, Brown MS, Hobbs HH 1995 Structure of the human gene encoding sterol regulatory element binding protein-1 (SREBF-1) and localisation of SREBF-1 and SREBF-2 to chromosomes 17p11.2 and 22q13. Genomics 25:667–673[CrossRef][Medline]
  18. Hua X, Yokoyama C, Wu J, Briggs MR, Brown MS, Goldstein JL, Wang X 1993 SREBP-2, a second basic helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element. Proc Natl Acad Sci USA 90:11603–11607[Abstract/Free Full Text]
  19. Miserez AR, Cao G, Probst LC, Hobbs HH 1997 Structure of the human gene encoding sterol regulatory element binding protein 2 (SREBF-2). Genomics 40:31–40[CrossRef][Medline]
  20. Brown MS, Goldstein JL 1997 The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331–340[CrossRef][Medline]
  21. Wang H, Liu F, Millette CF, Kilpatrick DL 2002 Expression of a novel, sterol-insensitive form of sterol regulatory element binding protein 2 (SREBP2) in male germ cells suggests important cell- and stage-specific functions for SREBP targets during spermatogenesis. Mol Cell Biol 24:8478–8490
  22. Wang H, San Agustin JT, Witman GB, Kilpatrick DL 2004 Novel role for a sterol response element binding protein in directing spermatogenic cell-specific gene expression. Mol Cell Biol 24:10681–10688[Abstract/Free Full Text]
  23. Sakai J, Nohturfft A, Cheng D, Ho YK, Brown MS, Goldstein JL 1997 Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein. J Biol Chem 272:20213–20221[Abstract/Free Full Text]
  24. Yang T, Goldstein JL, Brown MS 2000 Overexpression of membrane domain of SCAP prevents from inhibiting SCAP-SREBP exit from endoplasmic reticulum. J Biol Chem 275:29881–29886[Abstract/Free Full Text]
  25. Yang T, Espenshade PJ, Wright ME, Yabe D, Gong Y, Aebersold R, Goldstein JL, Brown MS 2002 Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell 110:489–500[CrossRef][Medline]
  26. Yabe D, Brown MS, Goldstein JL 2002 Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins. Proc Natl Acad Sci USA 99:12753–12758[Abstract/Free Full Text]
  27. Nohturfft A, DeBose-Boyd RA, Scheek S, Goldstein JL, Brown MS 1999 Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi. Proc Natl Acad Sci USA 96:11235–11240[Abstract/Free Full Text]
  28. Sever N, Lee PCW, Song B-L, Rawson RB, DeBose-Boyd RA 2004 Isolation of mutant cells lacking Insig-1 through selection with SR-12813, an agent that stimulates degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. J Biol Chem 279:43136–43147[Abstract/Free Full Text]
  29. Sakai J, Rawson RB, Espenshade PJ, Cheng D, Seegmiller AC, Goldstein JL, Brown MS 1999 Molecular identification of the sterol-regulated luminal protease that cleaves SREBPs and controls lipid composition of animal cells. Mol Cell 2:505–514
  30. Rawson RB, Zelenski NG, Nijhawan D, Ye J, Sakai J, Hasen MT, Chang TY, Brown MS, Goldstein JL 1997 Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs. Mol Cell 1:47–57[CrossRef][Medline]
  31. Brown MS, Ye J, Rawson RB, Goldstein JL 2000 Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell 100:391–398[CrossRef][Medline]
  32. Shimano H, Horton JD, Hammer RE, Shimomura I, Brown MS, Goldstein JL 1996 Overproduction of cholesterol and fatty acids cause massive liver enlargement in transgenic mice expressing truncated SREBP-1a. J Clin Invest 98:1575–1584[Medline]
  33. Shimano H, Horton JD, Shimomura I, Hammer RE, Brown MS, Goldstein JL 1997 Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells. J Clin Invest 99:846–854[Medline]
  34. Horton JD, Shimomura I, Brown Ms, Hammer RE, Goldstein JL, Shimano H 1998 Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element binding protein-2. J Clin Invest 101:2331–2339[Medline]
  35. Amemiya-Kudo M, Shimano H, Hasty AH, Yahagi N, Yoshikawa T, Matsuzaka T, Okazaki H, Tamura Y, Iizuka Y, Ohashi K, Osuga J, Harada K, Gotoda T, Sato R, Kimura S, Ishibashi S, Yamada N 2002 Transcriptional activities of nuclear SREBP-1a, -1c, and -2 to different target promoters of lipogenic and cholesterogenic genes. J Lipid Res 43:1220–1235[Abstract/Free Full Text]
  36. Wang X, Pai J-T, Wiedenfeld EA, Medina JC, Slaughter CA, Goldstein JL, Brown MS 1995 Purification of an interleukin-1ß converting enzyme related cysteine protease that cleaves sterol regulatory element-binding proteins between the leucine zipper and transmembrane domains. J Biol Chem 270:18044–18050[Abstract/Free Full Text]
  37. Pai J-T, Brown MS, Goldstein JL 1996 Purification and cDNA cloning of a second apoptosis related cysteine protease that cleaves and activates sterol regulatory element binding proteins. Proc Natl Acad Sci USA 93:5437–5442[Abstract/Free Full Text]
  38. Higgins ME, Ioannou YA 2001 Apoptosis-induced release of mature sterol regulatory element-binding proteins activates sterol-responsive genes. J Lipid Res 42:1939–1946[Abstract/Free Full Text]
  39. Sato R, Inoue J, Kawabe Y, Komada T, Tokano T, Maeda M 1996 Sterol-dependent transcriptional regulation of sterol-regulatory element-binding protein-2. J Biol Chem 271:26461–26464[Abstract/Free Full Text]
  40. Amemiya-Kudo M, Shimano H, Yoshikawa T, Yahagi N, Hasty AH, Okazaki H, Tamura Y, Shionairi F, Iizuka Y, Ohashi K, Osuga J, Harada K, Gotoda T, Sato R, Kimura S, Ishibashi S, Yamada N 2000 Promoter analysis of the mouse sterol regulatory element-binding protein-1c gene. J Biol Chem 275:31078–31085[Abstract/Free Full Text]
  41. Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccano JM, Shimomura I, Shan B, Brown MS, Goldstein JL, Mangelsdorf DJ 2000 Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXR{alpha} and LXRß. Genes Dev 14:2819–2830[Abstract/Free Full Text]
  42. Yoshikawa T, Shimano H, Amemiyo-Kudo M, Yahagi N, Hasty AH, Matsuzaka T, Okazaki H, Tamura Y, Iizuka Y, Ohashi K, Osuga JI, Harada K, Gotoda T, Kimura S, Ishibashi S, Yamada N 2001 Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatory element-binding protein-1c gene promoter. Mol Cell Biol 21:2991–3000[Abstract/Free Full Text]
  43. Fleischmann M, Iynedjsan PB 2000 Regulation of sterol regulatory-element binding protein 1 gene expression in liver: role of insulin and protein kinase B/cAkt. Biochem J 349:13–17[CrossRef][Medline]
  44. Lopez D, Sanchez MD, Shea-Eaton W, McLean MP 2002 Estrogen activates the high-density lipoprotein receptor gene via binding to estrogen response elements and interaction with sterol regulatory element binding protein-1A. Endocrinology 143:2155–2168[Abstract/Free Full Text]
  45. Yin L, Zhang Y, Hillgartner FB 2002 Sterol regulatory element-binding protein-1 interacts with the nuclear thyroid hormone receptor to enhance acetyl-CoA carboxylase-{alpha} transcription in hepatocytes. J Biol Chem 277:19554–19565[Abstract/Free Full Text]
  46. Kotzka J, Muller-Wieland D, Koponen A, Njamen D, Kremer L, Roth G, Munck M, Knebel B, Krone W 1998 ADD1/SREBP-1c mediates insulin-induced gene expression linked to the MAP kinase pathway. Biochem Biophys Res Commun 249:375–379[CrossRef][Medline]
  47. Kotzka J, Muller-Wieland D, Roth G, Kremer L, Munck M, Schurmann S, Knebel B, Krone W 2000 Sterol regulatory element binding proteins (SREBP)-1a and SREBP-2 are linked to the MAP kinase cascade. J Lipid Res 41:99–108[Abstract/Free Full Text]
  48. Roth G, Kotzka J, Kremer L, Lehr S, Lohaus C, Meyer HE, Krone W, Muller-Wieland D 2000 MAP kinase Erk 1/2 phosphorylate sterol regulatory element-binding protein (SREBP)-1a at serine 117 in vitro. J Biol Chem 275:33302–33307[Abstract/Free Full Text]
  49. Hannah VC, Ou J, Luong A, Goldstein JL, Brown MS 2001 Unsaturated fatty acids down-regulate SREBP isoforms 1a and 1c by two mechanisms in HEK-293 cells. J Biol Chem 276:4365–4372[Abstract/Free Full Text]
  50. Swinnen JV, Esquenet M, Heyns W, Rombauts W, Verhoeven G 1994 Androgen regulation of the messenger RNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in the human prostatic adenomacarcinoma cell line LNCaP. Mol Cell Endocrinol 104:153–162[CrossRef][Medline]
  51. Swinnen JV, Esquenet M, Rosseels J, Claessens F, Rombauts W, Heyns W, Verhoeven G 1996 A human gene encoding diazepam-binding inhibitor/acyl-CoA-binding protein: transcription and hormonal regulation in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP. DNA Cell Biol 15:197–208[Medline]
  52. Swinnen JV, Van Veldhoven PP, Esquenet M, Heyns W, Verhoeven G 1996 Androgens markedly stimulate the accumulation of neutral lipids in the human prostatic adenocarcinoma cell line LNCaP. Endocrinology 137:4468–4474[Abstract]
  53. Swinnen JV, Esquenet M, Goossens K, Heyns W, Verhoeven G 1997 Androgens stimulate fatty acid synthase in the human prostate cancer cell line LNCaP. Cancer Res 57:1086–1090[Abstract/Free Full Text]
  54. Swinnen JV, Alen P, Heyns W, Verhoeven G 1998 Identification of diazepam-binding inhibitor/acyl-CoA-binding protein as a sterol regulatory element-binding protein responsive gene. J Biol Chem 273:19938–19944[Abstract/Free Full Text]
  55. Heemers H, Maes B, Foufelle F, Heyns W, Verhoeven G, Swinnen JV 2001 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. Mol Endocrinol 15:1817–1828[Abstract/Free Full Text]
  56. Heemers H, Heyns W, Verhoeven G, Swinnen JV 2005 Androgens stimulate the SREBP pathway in prostate cancer cells by inducing a shift in the SCAP-retention protein(s) balance. In: Li JJ, Li SA, Llombart-Bosch A, eds. Hormonal carcinogenesis. 1st ed. New York: Springer; 357–363
  57. Nakajima T, Hamakubo T, Kodama T, Inazama J, Emi M 1999 Genomic structure and chromosomal mapping of the human sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) gene. J Hum Genet 44:402–407[CrossRef][Medline]
  58. Heemers H, Verrijdt G, Organe S, Claessens F, Heyns W, Verhoeven G, Swinnen JV 2004 Identification of an androgen response element in intron 8 of the sterol regulatory element-binding protein cleavage-activating protein gene allowing direct regulation by the androgen receptor. J Biol Chem 279:30880–30887[Abstract/Free Full Text]
  59. Verrijdt G, Haelens A, Claessens F 2003 Selective DNA recognition by the androgen receptor as a mechanism for hormone-specific regulation of gene expression. Mol Genet Metab 78:175–185[CrossRef][Medline]
  60. Claessens F, Celis L, Peeters B, Heyns W, Verhoeven G, Rombauts W 1989 Functional characterization of an androgen response element in the first intron of the C3(1) gene of prostatic binding protein. Biochem Biophys Res Commun 164:833–840[CrossRef][Medline]
  61. Claessens F, Celis L, De Vos P, Peeters B, Heyns W, Verhoeven G, Rombauts W 1993 Intronic androgen response elements of prostatic binding protein genes. Biochem Biophys Res Commun. 191:688–694
  62. Lund SD, Gallagher PM, Wang B, Porter SC, Ganschow RE 1991 Androgen responsiveness of the murine ß-glucuronidase gene is associated with nuclease hypersensitivity, protein binding, and haplotype-specific sequence diversity within intron 9. Mol Cell Biol 11:5426–5434[Abstract/Free Full Text]
  63. Marschke KB, Tan J-A, Kupfer SR, Wilson EM, French FS 1995 Specificity of simple hormone response elements in androgen regulated genes. Endrocrine 3:819–825
  64. Yabe D, Komuro R, Liang G, Goldstein JL, Brown MS 2003 Liver-specific mRNA for Insig-2 down-regulated by insulin: implications for fatty acid synthesis. Proc Natl Acad Sci USA 100:3155–3160[Abstract/Free Full Text]
  65. Engelking LJ, Kuriyama H, Hammer RE, Horton JD, Brown MS, Goldstein JL, Liang G 2004 Overexpression of Insig-1 in the livers of transgenic mice inhibits SREBP processing and reduces insulin-stimulated lipogenesis. J Clin Invest 113:1168–1175[CrossRef][Medline]
  66. Nelson PS, Clegg N, Arnold H, Ferguson C, Bonham M, White J, Hood L, Lin B 2002 The program of androgen-responsive genes in neoplastic prostate epithelium. Proc Natl Acad Sci USA 99:11890–11895[Abstract/Free Full Text]
  67. Segawa T, Nau ME, Xu LL, Chilukuri RN, Makarem M, Zhang W, Petrovics G, Sesterhenn IA, McLeod DG, Moul JW, Vahey M, Srivastava S 2002 Androgen-induced expression of endoplasmic reticulum (ER) stress response genes in prostate cancer cells. Oncogene 21:8749–8758[CrossRef][Medline]
  68. Eder IE, Haag P, Basik M, Mousses S, Bektic J, Bartsch G, Klocker H 2003 Gene expression changes following androgen receptor elimination in LNCaP prostate cancer cells. Mol Carcinog 37:181–191[CrossRef][Medline]
  69. van Weerden WM, de Ridder CM, Verdaasdonk CL, Romijn JC, van der Kwast TH, Schroder FH, van Steenbrugge GJ 1996 Development of seven new human prostate tumor xenograft models and their histopathological characterization. Am J Pathol 149:1055–1062[Abstract]
  70. Zhao XY, Boyle B, Krishnan AV, Navone NM, Peehl DM, Feldman D 1999 Two mutations identified in the androgen receptor of the new human prostate cancer cell line MDA PCa 2a. J Urol 162:2192–2199[CrossRef][Medline]
  71. Navone NM, Olive M, Ozen M, Davis R, Troncoso P, Tu S, Johnston D, Pollack A, Pathak S, von Eschenbach AC, Logothetis CJ 1997 Establishment of two human prostate cancer cell lines derived from a single bone metastasis. Clin Cancer Res 3:2493–2500[Abstract]
  72. Swinnen JV, Roskams T, Joniau S, VanPoppel H, Oyen R, Baert L, Heyns W, Verhoeven G 2002 Overexpression of fatty acid synthase is an early and common event in the development of prostate cancer. Int J Cancer 98:19–22[CrossRef][Medline]
  73. Swinnen JV, Vanderhoydonc F, Elgamal AA, Eelen M, Vercaeren I, Joniau S, VanPoppel H, Baert L, Goossens K, Heyns W, Verhoeven G 2000 Selective activation of the fatty acid synthesis pathway in human prostate cancer. Int J Cancer 88:176–179[CrossRef][Medline]
  74. Rossi S, Graner E, Febbo P, Weinstein L, Bhattacharya N, Onody T, Bubley G, Balk S, Loda M 2003 Fatty acid synthase expression defines distinct molecular signatures in prostate cancer. Mol Cancer Res 1:707–715[Abstract/Free Full Text]
  75. Myers RB, Oelschlager DK, Weiss HL, Frost AR, Grizzle WE 2001 Fatty acid synthase: an early molecular marker of progression of prostatic adenocarcinoma to androgen independence. J Urol 165:1027–1032[CrossRef][Medline]
  76. Ettinger SL, Sobel R, Whitmore TG, Akbari M, Bradley DR, Gleave ME, Nelson CC 2004 Dysregulation of sterol response element-binding proteins and downstream effectors in prostate cancer during progression to androgen independence. Cancer Res 64:2212–2221[Abstract/Free Full Text]
  77. Roy-Burman P, Tindall DJ, Robins DM, Greenberg NM, Hendrix MJ, Mohla S, Getzenberg RH, Isaacs JT, Pienta KJ 2005 Androgens and prostate cancer: are the descriptors valid? Cancer Biol Ther 4:4–5[Medline]
  78. Swinnen JV, Heemers H, Deboel L, Foufelle F, Heyns W, Verhoeven G 2000 Stimulation of tumor-associated fatty acid synthase expression by growth factor activation of the sterol regulatory element-binding protein pathway. Oncogene 19:5173–5181[CrossRef][Medline]
  79. Murillo H, Huang H, Schmidt LJ, Smith DI, Tindall DJ 2001 Role of PI3K signaling in survival and progression of LNCaP prostate cancer cells to the androgen refractory state. Endocrinology 142:4795–4805[Abstract/Free Full Text]
  80. Swinnen JV, Van Veldhoven PP, Timmermans L, De Schrijver E, Brusselmans K, Vanderhoydonc F, Van de Sande T, Heemers H, Heyns W, Verhoeven G 2003 Fatty acid synthase drives the synthesis of phospholipids partitioning into detergent-resistant membrane microdomains. Biochem Biophys Res Commun 302:898–903[CrossRef][Medline]
  81. Swinnen JV, Vercaeren I, Esquenet M, Heyns W, Verhoeven G 1996 Androgen regulation of the messenger RNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in the rat. Mol Cell Endocrinol 118:65–70[CrossRef]