Study of the Androgen Receptor Isoforms in Human Prostate-the Mechanism of Formation, Molecular Structure and the Significance in Prostate
|Course||Department of Urology|
|Keywords||Prostate androgen receptor isoforms benign prostate hyperplasia transitional zone peripheral zone|
Study of the androgen receptor isoforms in human prostate-----the mechanism of formation , molecular structure and the significance inprostateIntroductionPrsotate is the only organ that keeps growing in aging male and it depends on the androgen throughout all the stage of its development. Androgen also plays a crucial role in the etiology of benign prostate hyperplasia and prostatic cancer. Androgen action is mediated by a high-affinity androgen receptor （AR） that is expressed in target tissues. Although it is known that AR is a single protein encoded by a single gene, but there are a good many physiological and pathological phenomenons that can not been explained by a single AR.A few of subtypes or isoforms of andogen receptor have been found in several vertebrates. Japanese scientists in separate laboratories found two subtypes of AR in the fish of Japanese eel and rainbow trout, which is encoded by two genes. Two androgen receptor mRNA isoforms, alpha and beta were identified in the larynx of male Xenopus laevis. Northern blot analyses reveal that the beta isoforra is transiently expressed during early juvenile stages, whereas the alpha transcript is expressed throughout postmetamorphic life. In the Atalantic Croker and Kell Bass, two different ARs were reported which dispaying markly different steroid binding specificities. This is the first time people make sure that there are different AR isoforms with different affinity for T and DHT in a single body.Only a few study of AR isoforms in human were performed. But heterogeneous of AR protein has been detected for a long time. The AR doublet of 110-112KD is found in the LNCaP cell in SDS-PAGE, and it is believed to reflect posttranslation phosphorylation. Transfer the AR cDNA to the COS-1 cell to translating AR protein, two AR protein were found with different Mt, 94KD and 76KD. Both of them can bind to the androgen with high affinity. In the PCa cell line CWR22 which derived from recurrent cancer, two AR proteins also were identified. Wilson CM found two isoforms of AR in human genital skin fibrolasts,named AR-A and AR-B. Which were translated from the same AR gene. In human colon tissues , two AR isoforms were identified, and the altered expression in the colon cancer tissues contribute to the carcinogenesis. A new member of AR family has been identified in several human tissues. It is located in the membrane of the cell, but the traditional AR is in the nucleus or cytoplasm. The function and molecular structure is entirely distinguished with the the traditional AR. All these things strongly suggested that the isoforms of androgen receptor are present in human body assuredly. And different isoforms play different functions. For prostate, which depends on androgen for all of the life and put up alternative response to T and DHT, the study of isoforms of androgen receptor is of much more importance.In the past ten years, we have demonstrated that AR isoforms are present in normal and pathologic prostate. We employed high resolution isoelectric focusing（IEF） with H3 labelled DHT integerating covalently to AR. The findings suggested that four types of AR isoforms were detected with PI values at 6. 5, 6. 0, 5. 8 and 5. 3 in normal human prostate. There was a different expression between prostate transitional zone （TZ） and peripheral zone （PZ）. The isoforms of PI5. 8 was lack in the pathological specimens, such as BPH and PCa. But the mechanism from which the isoform generated and the molecular character of the isoform is not clear.The AR, glucocorticoid receptor（GR）, mineralocorticoid receptor（MR）, progesterone receptor（PR）, estrogen receptor（ER） and other steroid receptors make up the family of nuclear steroid receptors. With this receptor family, diversity is generated in at least two ways: in some instance receptors that respond to the same ligand are encoded by different genes, such as ER-α and ER-β . In other cases, isoforms are derived from a single gene, such as PR-A and PR-B. In some instance, both mechanisms appear to operate. The different recptors and isoforms are believed to subserve different functions.In this study , we will investigate the possible mechanism and the molecular character of the AR isoforms expressed in prostate. And the clinical significance is also studied.Objective:First of all, we adopt Western Blot to confirm the expression of ARisoforms in prostate. According to the possible mechanism from which the isoforms of steroid receptors derive, modern molecular-biology techniques are employed to investigate the possible mechanism of formation and the molecular structure of AR isoforms. Second , to make sure if the AR isoforms found in the prostate express popularly in other genital organs which depend on androgen too. And the expression pattern is also investigated. Third , to investigate the expression of AR isoforms in normal prostate, BPH tissues, especially the difference between the normal prostate transitional zone （TZ） and peripheral zone （PZ）, prostate transitional zone and BPH. And the clinical significance of AR isoforms in the development of prostate and etiology of BPH is explored. Methods:19 normal prostate glands were obtained from adult donors （mean age 26. 7） and sampled according to McNeal s zonal anatomy. 20 BPH samples were obtained from patients undergoing retropubic prostatectomy, nodules near the urethral area were dissected for experimental analysis.1. Western Blot was employed to confirm the expression of AR isoforms, and specific antibody for the two terminals of AR protein, the antibody N20 for N extreme and C19 for C-extreme were employed to evaluate alternative translation start sites. We use RT-PCR to evaluate the exons splicing variants, the primers were designed spanning from the first to the eighth exons of AR gene. So for any leakage or duplicate of exons, the bands of PCR will vary from the expected length. Furthermore , Northern Blot was employed to study the transcription variants on mRNA level.2. Western Blot was employed to investigate the expression pattern in the the other genital organs which depend on androgen but appear different response to T and DHT.3. Total RNA was isolated by two-step protocol. Reversal transcript-polymerase chain reaction （ RT-PCR ） was used to investigate semi-quantitatively the expression of AR mRNA and TGF-β1 mRNA in different regions of normal prostate and BPH. Western Blot was employed to investigated the AR isoforms expression pattern in normal prostate and BPH. Inmmune-histochemistry was also used to make sure the difference of AR expression between epithelium and stroma in different prostate tissues.Results:1. Three AR isoforms separated by SDS-PAGE were found in prostate tissue and LNCaP cell whose molecular weight were 110KD, 95KD and 87KD. The AR of 95KD were found only in LNCaP cell , while the other two isoforms were expressed in both of prostate and LNCaP cell. The isoform of 87KD was identified by the antibody of C19 only, which means that it is truncated in N terminal. N20 can identify the 95KD isoform which can not be found by C19, which means it lose segment of the C terminal. The AR of 110KD can be identified by both N20 and C19. RT-PCR showed single bands , none splicing variants were found . Nothern Blot shows also the traditional band, no aberrant mRNA was identified.2. In the genital systems, both the isoforms of 110KD and 87KD are found generally, but the 95KD isoform is not identified. The expression pattern is similary. In the organs depending on DHT in the process of development such as penis and those depending on T such as epididymis and ejaculotary duct, the isoform of 110KD is all dominantly expressed. The isoforms of AR are found in bladder tumor also, and the 87KD/110KD is higher than the normal epithelium. But the difference is not significant statisticly.3. In normal prostate, the level of AR mRNA is higher in PZ than in TZ, but the relative level of the isoforms is of no differnence. And in both regions, AR is expressed dominantly in epithelium. The AR expression in the stroma of TZ and PZ has no difference. TGF-β1 mRNA is slightly higher in the TZ. But in BPH, AR and TGF-β1 increase significantly. 87KD/110KD is also higher than the TZ of normal prostate, which means that the isoform of 87KD is expressed more than TZ. So the response to the androgen in BPH may be changed. Conclusions:1. AR isoforms are confirmed to be expressed in prostate by SDS-PAGE. Three isoforms of AR are identified in prostate with variant molecular weight. From the findings of Western Blot, RT-PCR and Northern Blot, we can conclud that all of three isoforms are derived from the single mRNA. All of the isoforms have the same amino acid sequences in the middle part. The difference among them is the truncation in the N terminal and C terminal. 110KD is the full length translation. The 87KD is translated from the internal start site andthere is a lack of amino acid sequences in the N terminal. While the 95KD lacked the part of the sequences of C terminal.2. Both the isoforms of 110KD and 87KD are made sure to be expressed in the other genital organs. But the different response to the T and DHT can not be explained by the alternative expression of the isoforms, for their relative expression is similar. But this findings is got from the adult tissues, which can not represent the embryo development.3. During the development of prostate, PZ is the dominant share of the prostate in the stage of first accelerated growth, but TZ predominates with the aging gradually. Especially when BPH occurred, PZ has changed into the surgical capsule. Higher level of AR and lower TGF-β1 in PZ may contribute to this growth character in the first accelerated growth. In BPH, both of AR and TGF-β1 levels are much more higher than TZ of normal prostate. Furthermore, the relative level of the isoforms changes too. 87KD/110KD is higher than in TZ. All of which may changes the response to the androgen of prostate . The change of AR and the isoforms cooperate with TGF-β1 promote the proliferation of epithelium and stroma. So the balance between the apoptosis and proliferation may be destroyed, which facilitates the occurrence of BPH.