Transcriptional regulation of the human prostatic acid phosphatase gene

Tissue-specific and androgen-dependent regulation of the promoter constructs in cell lines and transgenic mice

Jingdong Shan

Research Center for Molecular Endocrinology
WHO Collaborating Center for Research on Reproductive Health, Faculty of Medcine
Biocenter Oulu
University of Oulu

Abstract

Human prostatic acid phosphatase (hPAP) was the first laboratory parameter used for prostate cancer diagnosis, whereas the mechanisms behind the androgen regulation and tissue-specific expression of this prostate epithelium-specific differentiation antigen are not yet clear.

In this study, a transient transfection model and transgenic animal model have been set up for functional analysis of the promoter and first intron region of the hPAP gene. The promoter constructs covering the region-734/+467 of the gene were functional in both prostatic and nonprostatic cells. Although hPAP constructs included two putative AREs with in vitro AR-binding ability at -178 and +336, androgen treatment had little effect on the promoter activity of the gene in transiently transfected cells. The hPAP fragment -734/+467 could trigger the expression of the CAT reporter gene and restrict the expression mainly in the prostates of transgenic mice.

The DNA-binding site with the sequence GAAAATATGATA of a regulatory protein involved in prostate-specific and androgen receptor-dependent gene expression was identified from the rPB promoter. The exact same 12 bp sequence was found in the first intron +1144/+1155 of the hPAP gene. Five homologous sequence, A, B, C, D and E, were located in the -734/+467 region of the hPAP gene, where site C and E could bind the regulatory protein in EMSA. Deletion of site C decreased the transcriptional activities significantly compared to those of corresponding wild-type constructs in LNCaP cells when androgens were present. Deletion of site E or both sites D and E increased the promoter activity in LNCaP when androgens were absent.

In conclusion, androgens could not directly regulate hPAP expression via receptor-binding to the AREs in LNCaP cells. The promoter and first intron fragment -734/+467 of the hPAP gene could direct and restrict the gene expression mainly in prostate epithelium. A prostatic regulatory protein binds to multiple sites with the GAAAATATGATA or homologous sequences along the regulatory areas of the hPAP gene with different affinities, modulating the prostate-specific expression of the gene in a bidirectional manner, depending on the hormone status.

Table of Contents

Acknowledgements

Abbreviations

List of original publications

1. Introduction

2. Review of the literature

2.1. Phosphatases

2.2. Human prostatic acid phosphatase

2.2.1. Gene structure of hPAP
2.2.2. Protein structure of hPAP
2.2.3. Heterogeneity of hPAP
2.2.4. Physiological function of hPAP
2.2.5. Tissue-specific expression of hPAP
2.2.6. Androgen regulation of hPAP
2.2.7. Other regulators of the hPAP gene

2.3. Androgen receptor action

2.3.1. Androgen receptor
2.3.2. Selective DNA binding by androgen receptor

2.4. Prostate-specific gene expression

2.4.1. Prostate-specific promoters
2.4.2. Prostate-specific enhancers
2.4.3. Androgen receptor and prostate-specific gene expression
2.4.4. Prostate-specific regulatory proteins

3. Outlines of the present study

4. Materials and methods

4.1. Cell culture
4.2. Preparation of plasmid constructs for reporter gene analysis
4.3. Transient transfection assays
4.4. CAT, β -galactosidase, and protein assays
4.5. RNA extraction and slot blotting
4.6. Nuclear extracts
4.7. EMSAs and competition assay
4.8. UV-cross-linking analysis
4.9. DNase I footprinting
4.10. Generation and detection of transgenic mice
4.11. In situ hybridization
4.12. Western blotting and ECL detection

5.1. Transfecting well-differentiated prostatic cancer cell line LNCaP
5.2. Promoter activity of the hPAP gene and comparison with promoters of the PSA and hK2 genes
5.3. Identification of the DNA-binding site of a regulatory protein involved in prostate-specific and androgen receptor-dependent gene expression
5.4. The prostatic regulatory protein and tissue-specific regulation of hPAP promoter constructs

6. Discussion

6.1. Promoter activity and hormonal regulation of the hPAP gene
6.2. The putative prostatic regulatory protein, DNA binding sites and prostate-specific expression of the hPAP gene
6.3. LNCaP model and transgenic mice model
6.4. Conclusion

List of Tables
1. Classification of androgen response elements

List of Figures
1. Minimal reaction mechanism of rPAP. Modified from Lindqvist et al. 1994.
2. Proposed model: Interaction of hPAP and ErbB-2 is involved in androgen-mediated cell proliferation. (a) In hPAP (PAcP)-expressed human prostate epithelia, ErbB-2 has a low level of tyrosine phosphorylation, due to dephosphorylation by cellular hPAP. Tyrosine-dephosphorylated ErbB-2 is inactive and cannot transduce cell growth signals. (b) Androgen induces a cascade of transcriptional signals which include one pathway to suppress the expression of cellular hPAP, and another pathway to activate ErbB-2-specific tyrosine kinases. Tyrosine-phosphorylated ErbB-2 proteins form homodimers and subsequently activate the downstream MAP kinase signalling pathway, leading to a rapid growth rate. Modified from Meng et al. 2000.
3. Schematic structure of the androgen receptor. The androgen receptor gene was cloned in 1988 by Chang and co-workers (1988) and Lubahn et al. (1988 a, b) and soon after by several others (Tilley et al. 1989, Trapman et al. 1988, Brinkmann et al. 1989). There are eight exons encoding the receptor with a large exon 1 required for transactivation and exons 2-8 encoding a DNA-binding domain, hinge region, and hormone binding domain.
4. Proposed models for AR transactivation. (a) Androgens diffuse into target cells and bind to androgen receptors which exist in the form of complexes with heat shock proteins (hsps, marked with blocks) in the cytoplasm. (b) Androgen receptors were dissociated from hsps, dimerized, translocated into the nucleus. Upon binding to an androgen response element (ARE), the receptor dimer recruits coactivators such as p160 family and CBP/p300 that possess histone acetyltransferase activity or recruits histone acetyltransferase p/CAF to form an active preinitiation complex and interacts with basal transcription machinery to trigger the transcription of the target genes.
5. Location of the prostate-specific DNA-binding site GAAAATATGATA related sequences and potential androgen response elements in the schematic representation of the regulatory region of the hPAP gene. Homologs of GAAAATATGATA (A-F) were found using FINDPATTERNS (in https://seqweb.csc.fi) and are shown by black boxes with the nucleotide location underneath. Binding capacities of the elements for the prostatic protein in vitro are also marked (-, +, +++). AREs are shown by white boxes with the sequence position above. In vitro androgen receptor binding capacities are indicated by + or ++ above AREs.

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