Results

Androgen Activation of the SREBP Pathway Relies on the Presence of the Structural Elements Required for its Sterol-Regulation. To unravel the mechanism(s) by which A treatment of PCA cells gives rise to an increase in the content of nSREBPs, we investigated whether the action of As involves the same segments ofthe SREBP precursor protein that are required for its sterol regulation (such as the cleavage sites for S1P and S2P and the SCAP-interacting carboxyterminal domain). By means of site-specific mutagenesis of an expression construct for SREBP-2 (pTK-HSV-BP2) expression constructs for SREBP-2 were generated that had undergone mutations (site 1 and site 2) or deletions (COOH terminus) at these crucial sites (Figure 2A). Recombinant SREBP was provided with an HSV epitope tag. Immunoblot analysis using an antibody directed against the HSV epitope was performed on total cell extracts (SREBP precursor protein expression), as well as on nuclear extracts (nSREBP expression) of LNCaP cells grown either in the presence or absence of As or in the presence or absence of sterols. As shown in Figure 2B, activation of SREBP was observed only in cells expressing wild type SREBP precursor proteins, indicating the importance ofintact recognition sites for S1P and S2P and the need for interaction with SCAP for As to cause maturation of SREBP precursors (Figure 2B).

Androgen Treatment of LNCaP and MDA-PCA-2a Cells Markedly Upregulates the Expression of the Transporter Protein SCAP. The data shown in Figure 2) suggest that the structural elements involved in the A-activation of SREBP precursor proteins are identical to those required for sterol-regulated SREBP activation. Thus, we investigated whether As affect the expression of components essential for the sterol-regulation ofthe SREBP pathway. In the three cell lines studied, slight increases in the mRNA expression for SREBP-1c and SREBP-2, known nSREBP target genes (11,12), and S2P were observed following A exposure. SREBP-1a and S1P were not affected (data not shown). In both LNCaP and MDA-PCa-2a cells, A treatment caused a more pronounced increase in the mRNA expression of SCAP. In PC346c cells such a stimulatory effect on the expression of SCAP was not observed (Figure 3).

Figure 3. As markedly stimulate the expression of SCAP in LNCaP and MDA-PCa-2a cells. Cells were cultured in the presence (+) or absence (-) of R1881 for 2

(LNCaP and MDA-PCa-2a cells) or 3 (PC346c cells) days. Total RNA was isolated and northern blot analysis was performed with a SCAP probe. Data shown are representative of two independent experiments.

Figure 2. Mutation of sites required for sterol regulation of SREBPs prevents A-induced SREBP activation.

Schematic representation of the locations of the mutations, as well as the HSV epitope tag (A). Total cell and nuclear extracts derived from LNCaP cells expressing either wild type SREBP-2 (wt) or SREBP-2 in which site 1 (Slmut) or 2 (S2mut) was mutated or in which the C-terminus was deleted (Ctermdel), incubated in the absence (-) or presence (+) of R1881, or in the +/- of sterols, were subjected to western blotting with an antibody directed against the HSV epitope (B). Results shown are representative of three independent experiments.

Figure 3. As markedly stimulate the expression of SCAP in LNCaP and MDA-PCa-2a cells. Cells were cultured in the presence (+) or absence (-) of R1881 for 2

(LNCaP and MDA-PCa-2a cells) or 3 (PC346c cells) days. Total RNA was isolated and northern blot analysis was performed with a SCAP probe. Data shown are representative of two independent experiments.

^ LNCaP

MDA-PCJ-2S

PC34fic

Overexpression of SCAP Leads to Activation of the SREBP Pathway. To explore whether increased SCAP expression is actively involved in the A-induced activation of the SREBP pathway, an expression vector encoding SCAP (pcDNAl.l-SCAP) was generated. COS-7 cells were transiently transfected with pcDNAl.l-SCAP and pTK-HSV-BP2. Two days later, nuclear extracts were prepared and immunoblot analysis was performed using an antibody directed against the HSV-epitope. Figure 4A shows that enhancing cellular SCAP levels does indeed result in a pronounced increase in mature nuclear SREBP. To investigate whether such a SCAP-induced elevation of nuclear SREBP levels is responsible for the observed increase in lipogenic gene expression, pcDNAl.l-SCAP was co-transfected into MDA-PCa-2a cells with a promoter-reporter construct derived from SYN. Co-transfection of pcDNAl.l-SCAP gave rise to a marked increase in SYN reporter activities. Mutation of SREs in this promoter fragment prevented SCAP-induced transcriptional activation of the reporter gene (Figure 4B). Co-transfection of MDA-PCa-2a cells with increasing amounts of an expression construct encoding a dominant-negative form ofSREBP-1 (DN-SREBP) gradually prevented the stimulatory effect of enhanced SCAP expression on both FAS (data not shown) and SYN (Figure 4C) promoter-reporter constructs. Similar SCAP-induced stimulatory effects on the transcriptional activation of these genes were observed in LNCaP cells (data not shown) (8).

Figure 4. Effect of increased cellular SCAP levels on nSREBP-dependent lipogenic gene transcription. (A) COS-7 cells were transiently transfected with pTK-HSV-BP2 and 20 ng of an expression construct encoding human SCAP (pcDNA1.1-SCAP).

Representative data from two independent experiments. (B) MDA-PCa-2a cells were transiently transfected with either a plasmid containing a luciferase reporter gene driven by a HMG-CoA-synthase (SYN) promoter fragment harboring two SREs (SRE-1 and SRE-2) (wt), or a similar construct in which either SRE-1 or SRE-2 was mutated (mut-1 and mut-2). Fourty ng of an expression construct encoding human SCAP (pcDNA1.1-SCAP) was added. (C) Increasing amounts of a plasmid encoding a dominant-negative form of SREBP (DN-SREBP) were added. Luciferase activity was expressed in relative luciferase units (RLU). The data represent the mean ± SEMs of incubations performed in triplicate.

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