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Endocrinology Vol. 140, No. 5 2382-2386
Copyright © 1999 by The Endocrine Society

ARTICLES

Adrenomedullin Gene Is Abundantly Expressed and Directly Regulated by Androgen in the Rat Ventral Prostate1

E. Bradley Pewitt2, Riffat Haleem and Zhou Wang3

Department of Urology (E.B.P., R.H., Z.W.), Department of Molecular Pharmacology and Biological Chemistry (Z.W.), and The Robert H. Lurie Cancer Center (Z.W.), Northwestern University Medical School, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: Zhou Wang, Department of Urology, Tarry 11–715, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611-3009. E-mail: wangz{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A gene-expression screen, looking for androgen response genes in the rat ventral prostate, has identified adrenomedullin (AM), a 52-amino acid pluripotent peptide hormone, first isolated from pheochromocytoma. Northern blot analysis demonstrates that the level of expression in the prostate is reduced at least 25-fold by castration, with the majority of the decrease occurring in the first day, and that androgen replacement in seven-day castrated rats stimulates expression to supernormal levels, with the majority of the increase occurring within 14 h. The level of expression in the prostate is at least 50-fold higher than in the adrenal gland and cardiac atria, tissues previously reported to have the highest level of expression in the rat. In prostate organ culture, androgen was able to induce AM expression; and this induction resists protein synthesis inhibition, indicating that AM is a direct androgen response gene in the prostate. In situ hybridization of normal rat prostate tissue showed that AM expression is localized in the epithelial cells. Our analysis demonstrates that AM, a multifunctional peptide hormone, is abundantly expressed and directly regulated by androgen in the prostate epithelial cells. Thus, AM has the potential to play a crucial function in androgen action in the prostate.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IDENTIFICATION OF A comprehensive collection of androgen response genes in the prostate promises to provide a solid foundation to further the understanding of androgen action. In addition, this collection would have the potential to provide novel targets for intervention in androgen action. To that end, a gene expression screen (1, 2) was executed using the rat ventral prostate as a model system. Messenger RNA (mRNA) was isolated from the ventral prostate of 7-day castrate young adult male rats and the prostates of 7-day castrate rats 14 and 48 h after androgen replacement. These three populations of message were used to perform the expression screen (2). One of the identified androgen-response genes is adrenomedullin (AM).

AM is a pluripotent 52-amino acid peptide hormone, first isolated from a pheochromocytoma using its ability to elevate cAMP in platelets as an assay (3). The AM gene, represented by a single copy on chromosome 11 in the human (4), is translated into a 185-amino acid preprohormone that undergoes posttranslational modification, creating AM and pro-ADM N-terminal 20 peptide (5, 6, 7). Both AM and pro-ADM N-terminal 20 peptide are {alpha}-amidated peptides. AM possesses a single intramolecular disulfide cysteine-to-cysteine bond, forming a six-membered ring. Calcitonin gene related peptide (CGRP) has an analogous ring structure, has C-terminal amidation, and shares an overall 27% similarity with AM (5). In fact, AM can bind to the CGRP receptor, and the activity of AM can be blocked by the specific CGRP receptor antagonist CGRP 8–37 in a subpopulation of tissues (8, 9). However, a specific receptor for AM, not antagonized by CGRP 8–37, has been identified and cloned from rat lung; the gene encodes for a peptide sequence indicative of a member of the G protein-coupled receptor super family (10). The involvement of a G protein in the AM-signaling pathway is supported by studies in vascular smooth muscle, demonstrating that the ability of AM to elevate cAMP is augmented by GTP, abrogated by cholera toxin, and not affected by pertusis toxin (11). Although AM was first isolated on the basis of its ability to raise cAMP, there is evidence suggesting that AM may also activate alternate second-messenger systems, e.g. intracellular calcium, nitric oxide, and PG synthesis (5).

Diverse biological functions for AM have been described (5). It can function as a hypotensive vasodilatory agent, a growth factor, even as an antimicrobial (12). It has been suggested that AM plays a role during embryogenesis in the control of growth, differentiation, and invasion (13). It has been shown to modulate the levels of other growth factors and hormones. It can modulate thirst when injected intracranially. Serum levels are elevated in sepsis, renal insufficiency, pregnancy (14), acute myocardial infarction, and hypertension. It is found at high levels in amniotic fluid (12). It is expressed in the tall columnar ciliated cells of the respiratory epithelium and is likely secreted (15). It has been shown to have bronchodilatory activity (16). It has recently been shown to act as an autocrine/paracrine survival factor preventing endothelial cells from undergoing apoptosis in response to serum withdrawal (17).

Regulation of AM expression has been investigated in vascular epithelial and smooth-muscle cells (18, 19, 20). The level of AM expression is stimulated, most strikingly, by TNF{alpha}, TNFß, IL-1{alpha}, IL-1ß, and lipopolysaccharide, whereas forskolin and cAMP were shown to down-regulate expression, suggesting a negative feedback mechanism. The steroid hormones aldosterone, hydrocortisone, and thyroid hormone also stimulate AM expression. However, estradiol and testosterone demonstrated no effect on AM expression in these cells.

In the present study, we characterize androgen regulation of AM expression in the rat ventral prostate as a first step in understanding its biological role in the prostate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RNA isolation and Northern blot
Total RNA was isolated by the guanidinium/CsCl gradient method (21). RNA was fractionated by electrophoresis through formaldehyde-agarose gels (1.4%) and transferred to nylon membrane (MAGNA, Micron Separations Inc., Westborough, MA) by capillary flow transfer overnight. The RNA was cross-linked to the membrane by UV irradiation. Labeled probes were prepared using hexamer oligonucleotide random priming in the presence of {alpha}-32P-deoxycytidine triphosphate (22). Two different templates were used in the probe labeling reaction. The first one, a short complementary DNA (cDNA) fragment, isolated during the gene expression screen, is 356 bp in length, spanning 322–678 bp of the published sequence for the rat AM full-length cDNA (23). This short cDNA fragment was cloned into the EcoRI site of pBluescript (Stratagene, La Jolla, CA). The second was the full-length cDNA isolated from a {lambda}ZAP cDNA phage library constructed from normal rat ventral prostate mRNA. The full-length AM cDNA is approximately 1.4 kb in length, and the 500 bases at the 5' end of the cDNA insert were confirmed by sequence analysis. Northern blot hybridization was performed overnight at 42 C in a buffer containing 5 x SSPE, 2 x Denhardt’s solution, 0.1% SDS, 100 µg/ml denatured salmon sperm DNA, and 50% formamide. Blots were washed at 65 C, three times, for 30 min, with an excess of wash buffer consisting of 0.2 x saline-sodium citrate (SSC) and 0.1% SDS.

Organ culture
The ventral prostate was harvested from 7-day castrate young adult male Sprague Dawley rats (250–350 g, from Harlan Sprague Dawley, Inc., Indianapolis, IN). The prostates were minced with a scalpel to create uniform fragments, 1 mm in all dimensions. The minces were then scattered on lens paper supported at the media-air interface with a stainless steel screen in a 100-mm culture plate and incubated in 5% CO2 at 37 C. Care was taken to ensure that the minces were in good contact with the media but not submerged. The media consists of M-199 (with Earle’s salts, L-glutamine, and 2.2 g/liter sodium bicarbonate, without phenol red, from Gibco BRL, Gaithersburg, MD), with the addition of 10% charcoal-stripped FBS, penicillin G sodium at 200 U/ml, and streptomycin sulfate at 0.2 mg/ml, in the presence or absence of cycloheximide (50 µg/ml) and anisomycin (80 µg/ml). After treatment, the minces were harvested and frozen immediately in liquid N2, and the RNA was isolated by the guanidinium/CsCl gradient method.

In situ hybridization
In situ hybridization was carried out essentially as previously described (24). Tissue was harvested and fixed overnight in 4% paraformaldehyde in SPB (3% sucrose, 0.15 mM CaCl2, in 0.06 M phosphate buffer, pH 7.4) at 4 C. The tissue was then rinsed in 10% sucrose in SPB and incubated overnight at 4 C in 30% sucrose in SPB. The tissue was cryosectioned and placed on ProbeOn plus microscope slides (Fisher Biotech, Pittsburgh, PA). Before hybridization, the slides were washed with PBS, fixed in 4% paraformaldehyde, digested with proteinase K at 20 µg/ml in PBS, refixed in 4% paraformaldehyde, rewashed in PBS, and then acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0.

The full-length AM cDNA, inserted into EcoRI and XhoI site between T3 and T7 promoters in pBluescript II SK plasmid vector, was used for in situ probe synthesis. The plasmid, purified by CsCl double banding, was linearized with EcoRI or XhoI and proteinase K treated. The purified linear DNA templates were used in the synthesis of both sense and antisense digoxygenin-labeled riboprobes using either T3 or T7 RNA polymerase (Promega Corp., Madison, WI) as previous described (25). The size of the riboprobes was reduced to approximately 250 bp using limited alkaline hydrolysis (26).

For hybridization, the probe was diluted in hybridization solution (5 x SSC, 1 x Denhardt’s, 100 µg/ml salmon testis DNA, 50% formamide, and 250 µg/ml yeast transfer RNA), and the slides were hybridized overnight at 67 C in a sealed chamber humidified with 5 x SSC/50% formamide. The coverslips were removed, and the slides were washed in 0.2 x SSC at 72 C for 1 h. After washing in buffer B1 (0.1 M Tris (pH 7.6), 0.15 M NaCl), the slides were blocked in 10% horse serum in B1 at room temperature for 1 h. The slides were then incubated overnight at 4 C with a 2,000-fold dilution of antidigoxigenin-AP Fab fragments (Boehringer Mannheim, Mannheim, Germany) in B1 1% horse serum. After washing, the slides were developed with nitro blue tetrazolium (2.25 µl/ml) and 5-bromo-4-chloro-3-indolyl-phosphate, 4-toluidine salt (0.6 µg/ml) in alkaline phosphatase buffer (0.1 M Tris (pH 9.5), 0.05 M MgCl2, 0.1 M NaCl).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Androgen regulation of AM expression
Androgen regulation of AM expression was investigated in whole-animal studies. Northern blot analysis of total RNA from the ventral prostate of a series of rats after castration (Fig. 1AGo) demonstrates down-regulation of AM expression by 25-fold within 1 day. Androgen replacement in the 7-day castrate rat results in up-regulation of expression within 14 h (Fig. 1BGo). The results show that AM expression is rapidly and dramatically regulated by androgen in the prostate.



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  		Figure 1. Regulation of AM expression in the rat ventral prostate by androgen. A, Down-regulation by castration. The ventral prostate was harvested, and RNA was isolated for AM Northern blot analysis (probed with the short probe) at various times after castration. N represents RNA isolated from testis intact rats, and the subsequent lanes represent 1 day, 2 days, 3 days, 5 days, and 7 days after castration. Each lane contains 20 µg total RNA from a pool of at least three animals. B, Up-regulation by androgen replacement. Seven-day castrate rats were given testosterone (2 mg) by sc injection at time zero and every 24 h thereafter. The ventral prostate was harvested, and RNA was isolated for AM Northern blot analysis (short probe) at various times after the initial androgen replacement. Lane C, Total RNA from 7-day castrate rats. The subsequent lanes represent 14 h, 1 day, 2 days, 3 days, and 5 days of androgen replacement. Each lane contains 10 µg total RNA from a pool of at least three animals. The methylene blue staining (30 ) is depicted in the lower panels.

 
Tissue survey of androgen regulation of AM expression
Androgen regulation of expression was evaluated in a variety of tissues by Northern blot analysis of total RNA isolated from normal, 7-day castrate rats and from 7-day castrate rats after 2 days of androgen replacement (Fig. 2Go). AM expression in the normal prostate is at least 50-fold higher than in the normal adrenal gland and cardiac atria. In addition, AM is up-regulated by androgen only in the prostate. The increase in expression, 7 days after castration, in the seminal vesicles may reflect an enrichment of the cells that express AM.



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  		Figure 2. Tissue survey of androgen regulation. Total RNA was isolated from the indicated organs, harvested from testis intact animals (N), 7-day castrate animals (-), and 7-day castrate animals after 2 days of androgen replacement (+). In the upper panel, the Northern blot was probed with the full-length probe, demonstrating a band at 1.6 kb (AM), whereas the lower panel was probed with the short probe, also revealing a major band at 1.6 kb. Ten micrograms of total RNA was loaded in each lane, out of a pool of RNA from at least three animals. The methylene blue staining of the total RNA is indicated in the panels labeled: Total RNA.

 
Cycloheximide treatment in organ culture
The ability of androgen to stimulate AM expression in the absence of protein synthesis was examined in organ culture. Under the described conditions, cycloheximide and anisomycin were able to reduce (35S) methionine incorporation in trichloroacetic acid precipitable counts by 98%. Dihydrotestosterone (DHT) was able to stimulate AM expression in the absence of cycloheximide and anisomycin (Fig. 3Go). The baseline level of expression in the presence of cycloheximide is elevated relative to control, and the elevation is superinduced by DHT. This evidence indicates that AM is a direct androgen-response gene in the prostate.



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  		Figure 3. Androgen stimulation in organ culture. The ventral prostates from 7-day castrate rats were placed in organ culture, as described. After overnight incubation, either cycloheximide (CHX) or an equal volume of ethanol vector was added to each dish. Two hours later, DHT (final concentration, 1 µM) or ethanol vector was added. The tissue was harvested, and RNA was isolated after 24 h of androgen stimulation. The Northern blot (full-length probe) contains 10 µg total RNA in each lane.

 
In situ hybridization
In situ hybridization for AM was performed on normal rat ventral prostate (Fig. 4Go), revealing almost exclusive staining in the epithelial cell compartment. Staining of the 7-day castrate ventral prostate gave no specific staining, whereas the androgen-replaced prostate demonstrated very intense epithelial cell staining.



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  		Figure 4. In situ hybridization. The ventral prostate of a testis-intact animal was harvested, and in situ hybridization was performed as described. The upper panel represents the antisense probe, whereas the lower panel represents the sense control on a serial section.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results presented in this study strongly suggest that AM expression is directly and markedly regulated in the rat ventral prostate epithelial cells by androgen. In addition, the level of expression in the prostate is high, relative to adrenal gland and cardiac atria, tissues previously reported to have the highest level of expression in the rat (27).

Steroid hormone regulation of AM expression has been investigated in two other studies, and androgen was found not to effect AM expression (18, 19). The cell types studied were vascular epithelial and endothelial cells isolated from the rat thoracic aorta. Our tissue survey also showed no androgen regulation outside the prostate. In the seminal vesicles, a secondary sex organ, there is a relative increase in AM expression 7 days after castration. This is likely caused by the dramatic involution that the tissue undergoes in response to castration and the enrichment of AM-expressing cells in the tissue. However, the seminal vesicles from the 7-day castrate rat did not show an increase in expression after 2 days of androgen replacement, conditions that resulted in an increase greater than 25-fold in the ventral prostate. During the first 2 days of androgen replacement, there is no significant change in cell number in either tissue, to contribute to the difference in expression. This suggests prostate-specific androgen regulation of AM expression.

The organ culture experiment is consistent with the previous studies (18, 19) that demonstrated superinduction of AM at the message level in the presence of cycloheximide. It was suggested that cycloheximide stabilizes AM mRNA by inhibiting the synthesis of a labile AU-binding protein responsible for AM mRNA degradation (18). In addition, androgen was able to further stimulate AM expression in prostate organ culture when the tissue was pretreated with cycloheximide, suggesting that androgen is capable of stimulating AM expression directly. Taken together, the lack of androgen regulation outside the prostate and the ability of androgen to stimulate expression in the absence of protein synthesis in the prostate, make likely the existence of a prostate-specific transcription factor that works in concert with the androgen receptor. As more direct androgen response genes are identified from the prostate and their promoter regions are sequenced, the nature of prostate specific androgen-dependent gene activation will be further illuminated.

These studies focus at the expression level. What happens at the protein and functional level in the prostate is yet to be investigated. However, some corollaries can be drawn with the function of AM described in other tissues. AM is expressed at high levels in the epithelial cells in the prostate, and it is likely that it is secreted. AM is similar to the defensin family of proteins with antimicrobial activity (12). AM has also been shown to be produced by the bladder epithelium and is measurable in the urine. Prostate secretion of AM may work in concert with bladder epithelial secretion to combat urinary tract infection.

In many tissues, AM function seems to be mediated through cAMP. The proliferative response of the tissue is likely to be dictated by the relative abundance of type I and type II regulatory subunits of cAMP-dependent protein kinase. In the rat ventral prostate, type I enzymatic activity is inhibited by castration, whereas type II is unchanged (28). The proliferative response to AM in the prostate may depend on the level of androgen in the tissue.

Tissue homeostasis in the prostate is a balance between cell proliferation and programmed cell death. Recently, AM has been shown to abrogate serum-deprivation-induced apoptosis in vascular smooth-muscle cell culture, through an autocrine/paracrine mechanism, independent of cAMP elevation (17). This suggests that a tonic level of AM production, maintained by androgen in the prostatic epithelial cells, has the potential to act, through an autocrine/paracrine mechanism, to mediate androgen-dependent tissue homeostasis.

AM may play a role in the adenocarcinoma of the prostate. It has the potential to act as an autocrine growth factor, as was shown in several other tumor cell lines (29). Expression of AM and its receptor has been identified in the androgen-independent prostatic tumor cell line DU-145 (29). In addition, AM has the potential to act as an angiogenesis factor, a vasodilatory factor, and a stimulator of endogenous metalloproteinases; all these properties could enhance the tumorigenicity of the cell line that produces it.

AM is a direct androgen-response gene, expressed in the rat ventral prostate epithelium, and the level of expression in the prostate is much higher than in the adrenal gland and cardiac atria. Although its functional role in the prostate is unclear at this time, it is likely that it functions as an antimicrobial and has the potential to play a crucial part in mediating the effects of androgen on growth and/or differentiation.


   Acknowledgments
 
We would like to thank Sharon Lang for her assistance in in situ hybridization; and our colleagues, for critical reading.


   Footnotes
 
1 This work was supported by NIH Grant R01-DK-51193. Back

2 A Pfizer, Inc. Research Fellow of the American Foundation for Urologic Disease. Back

3 Recipient of a Junior Faculty Research Award from The American Cancer Society. Back

Received August 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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