6.2. The putative prostatic regulatory protein, DNA binding sites and prostate-specific expression of the hPAP gene

A 12-bp binding site located at -251/-240 in the rat Pb promoter was identified from the 5’-flanking area (-426/+52) of the gene, which has been shown to be sufficient for targeting gene expression to the prostatic epithelium (Greenberg et al. 1994, 1995). Deletion of this element from the PB promoter construct significantly decreased the androgen induction of the promoter in prostatic cells but not in nonprostatic cells. Although the -251/-240 was important for androgen induction in prostatic cells, at least 20 5’-flanking nucleotides were needed for maximal effect. However, these flanking nucleotides did not bind any sequence-specific proteins of their own. These nucleotides may help the putative prostatic regulatory protein to bind to its response element (III). Similar observation has been seen in the NF-κ B response element of the human intercellular adhesion molecule-1 (ICAM-1) gene (Paxton et al. 1997). ICAM-1 is induced when NF-κ B binds to a modified NF-κ B site after the stimulation of tumor necrosis factor-α (TNFα). In addition to this binding site, both specific 5’- and 3’- flanking sequences are necessary for TNFα induction. When either of the AREs at the rPB -286/+52-CAT construct are mutated, androgen induction is almost completely lost (Kasper et al. 1994), although the constructs contain the DNA-binding site of the putative prostatic regulatory protein. These results suggest that the regulatory protein is not obligatory for the function of AR in the rPB promoter. However, the presence of this factor can duplicate or even triplicate the effect of androgens, indicating that there is a synergistic action between the prostatic transcription factor and AR (III).

The exact same 12-bp sequence has also been found in the first intron of the hPAP gene. EMSA shows that the prostatic regulatory protein could also bind to this element (F: +1144/+1155), suggesting that the transcription factor could regulate hPAP expression (II). Five homolog sequences, sites A (-580/-569), B (-257/-246), C (-151/-140), D (+218/+229), and E (+244/+255), have been found in the hPAP -734/+467 region, which has been shown to trigger the reporter gene expression and restrict the expression mainly in prostate epithelium. EMSA showed that the putative prostatic regulatory protein could bind to sites C and E, but to not A, B, or D (Fig.5). The behavior of site C is similar to that of the prostatic binding site in the rPB promoter, since the transcription efficiency was decreased in the presence of androgen after the deletion of site C from the hPAP constructs. The location of site C at -151/-140 of the hPAP proximal promoter, 13 nucleotides downstream from an ARE, is similar to the position of the prostate-specific binding site at -251/-239 of the PB promoter, which is only three nucleotides away from one downstream ARE. However, it is currently uncertain whether androgen action and site C-mediated function are connected directly or indirectly. Sites A and B at the proximal promoter of hPAP are unable to bind transcription factors in vitro, which does not, however, completely disqualify the possibility of a regulatory role for the sites in vivo. Sites D and E are located in the first intron of the hPAP gene. Surprisingly, the deletion of site E from the hPAP -734/+467 construct caused an increase in transcriptional activity in the absence of androgens compared to the wild-type construct. Further deletion of the site D concomitant to site E was able to intensify the effect in hormone-depleted medium. This could mean that site E is a low-affinity binding site for the prostatic regulatory protein and possibly the close proximity of sites D and E improves the binding capacities of both sites in vivo (IV). Relatively few prostate-specific transcription factors have been characterized so far: one is Nkx3.1, which is an androgen-regulated, prostate-specific homeobox protein (He et al. 1997, Prescott et al. 1998). Nkx3.1 preferentially binds to the TAAGTA sequence (Steadman et al. 2000); another is prostate-derived Ets-factor (PDEF, Oettgen et al. 2000). Among the Ets family, PDEF prefers binding to a GGAT rather than a GGAA core. PDEF could be a potential partner of Nkx3.1 (Chen et al. 2002). Coimmunoprecipitation analyses demonstrated that Nkx3.1 and PDEF are physically associated in prostate epithelial cells. Cotransfection analyses revealed that Nkx3.1 can abolish the transcriptional activation function of PDEF on the PSA promoter. The binding sites of Nkx3.1 and PDFE are clearly different from GAAAATATGATA, the binding site of the putative prostatic regulatory protein, suggesting that the prostatic regulatory protein could be a new prostate-specific transcription factor.

Site C is located in the promoter region, while sites D and E are located in the first intron of the hPAP gene. The orientation of site E is opposite to other homolog sites present in the hPAP gene. Furthermore, the distance between site E and the nearest ARE in the hPAP gene is about six times longer than in the case of site C. These could open the possibility of differential regulatory responses for the elements. Our recent studies have indicated that the closer the prostate-specific binding site of rPB is to an ARE, the more powerful the activating effect is in the presence of androgens (unpublished data). AR is a potential connecting molecule in the detected bidirectional gene regulation via C and D/E elements binding to the prostatic transcription factor. Anti-androgen flutamide could block the transcriptional activation of the hPAP -734/+467 construct after the deletion of E and D elements in hormone-depleted conditions, suggesting a mechanism of the ligand-independent action for AR (IV). Interestingly, a bidirectional regulation process has also been seen in the case of the glycoprotein hormone α-subunit gene. Pituitary cell type-dependent activation and repression is mediated though an upstream regulator of the gene. Differences in the regulatory mechanisms were observed, but elements mediating the activation or repression seemed to be closely juxtaposed or even overlapped (Wood et al. 1999).

A sequence sharing approximately 80% identity with the GAAAATATGATA sequence was located in the hPSA (-4196/-4185) enhancer area. The DNA fragment containing this GAAGATATTATC sequence could not form a specific DNA-protein complex when LNCaP or PC-3 nuclear extracts were used in EMSA (II). The prostatic regulatory protein binds to element F of the hPAP gene with high affinity, while it binds with moderate affinity to the binding site in rPB promoter and with low affinity to elements C and E of the hPAP gene (IV). Preliminary methylation interference experiments using the hPAP fragment containing element F as a probe suggested that the thirtieth nucleotide C is also involved in DNA-protein interaction (unpublished data). This could explain why element F has the highest affinity with the prostatic transcription factor. The existance of multiple binding sites for a regulatory protein with different affinities within one gene is quite common. At least eight high and low affinity AR binding sites have been identified in the promoter and enhancer regions and have been implicated in the androgen-mediated regulation of PSA. Inspection of the PSA promoter and enhancer sequence revealed the existence of at least 11 putative Ets-binding sites (Oettgen et al. 2000). Some of these sites are in close proximity to AR-binding sites, and EMSA demonstrated that PDEF could bind to some of these sites with different affinities.

The importance of the putative bidirectional regulation of the whole hPAP gene through the GAAAATATGATA and homologous sequence during different biological conditions is not yet clear. The first indication of the androgen-dependency of hPAP expression is the appearance of the protein following sexual maturation (Yam 1974). This could mean that in normal prostatic cells, the hPAP gene is activated when the androgen concentration in the circulation reaches the levels of adulthood. Therefore, a mechanism controlling the expression of the hPAP gene could exist where the multiple prostate-specific DNA-binding sites mediate either activation or repression, depending on the relevant physiological level of androgens in the serum.