jkeithc82

CAMP- ANDROGEN RECEPTOR CROSSTALK

an article by Karl 'Nandi' Hoffman RIP


Androgens, acting via their receptors, regulate the development and maintenance of the differentiated function of the male reproductive system. The androgen receptor (AR) is a member of a large family of transcription factors known as the steroid/thyroid receptor superfamily. This superfamily includes receptors for steroid and thyroid hormones and the retinoids, as well as orphan receptors for which the ligands are not known. The receptors are ligand-dependent transcription factors and have distinct functional domains involved in transcriptional activation and repression, DNA binding, and steroid binding. In the presence of ligand (steroid hormone), the receptor is activated, resulting in stimulation or repression of transcription of genes that are under steroid hormone control. Hormone antagonists (either natural or synthetic molecules) compete with the agonist for binding to the receptor and block transcriptional activation by the receptor.

Before addressing the issue at hand, how cAMP (cyclic adenosine monophosphate) induced phosphorylation (addition of a phosphate or, PO4 group) of transcription factors enhances the gene transcription process, I’d like to briefly review how cells create proteins such a the contractile proteins of muscle tissue, enzymes, receptors, etc. essentially anything composed of amino acids, from the genes encoded along the strands of DNA found in the cellular nucleus.

Genes, we know, are stretches of DNA that code for proteins. Many genes are targeted by androgens; classic examples are the genes that code for the proteins that make up the androgen receptor (AR) itself. Androgens are capable of inducing the transcription of these genes, making more androgen receptors. This is the process of androgen receptor upregulation!

Part of the gene (depicted below) consists of a promoter region that contains a subsection called the androgen response element (ARE). When the ligand (androgen) enters the cell and binds to the AR, it induces a conformation change in the ligand-receptor complex that allows the complex to recognize and bind to the specific nucleotide sequence comprising the ARE. There it proceeds to recruit transcription factors called coactivators, which act as "power boosters" that amplify transcription, as well as other transcription factors, which are proteins that are required to initiate transcription of the target gene via RNA polymerase II. The receptor/ligand along with the various transcription factors ultimately form a large complex that serves as a sort of platform for RNA polymerase to dock with, allowing the polymerase to begin transcribing the gene. The messenger RNA (mRNA) created from the DNA template of the gene then leaves the nucleus and enters the cytoplasm, where in the process known as translation, the mRNA in turn serves as a template for the construction of a specific protein.

The exons in the gene depicted below contain the segments of DNA that actually code for the protein that will ultimately be transcribed.




Fig 1. Generic gene structure showing exon (protein coding region),
RNA polymerase II bound to gene; TATA box; and promoter with
bound transcription factors. The androgen/AR complex would bind
to a specific region within the promoter, the Androgen Response
Element (ARE). From (4)


Upstream from the exon is the region of the promoter called the TATA box. It contains a sequence of seven bases TATAAAA and is a common feature of promoters found in all genes. The base sequences in the remaining upstream portion of the promoter vary from gene to gene.

We mentioned the importance of transcription factors to the whole process. Although transcription is performed by RNA polymerase, the enzyme needs other proteins to produce the transcript. These factors are either associated directly with RNA polymerase or add in building the actual transcription apparatus. So a simple definition of a transcription factor is any protein other than RNA polymerase that is required for transcription. As mentioned in the introduction, the androgen receptor itself is by this definition also a member of the collection of transcription factors.

There is accumulating evidence that the activity of numerous transcription factors is regulated by reversible phosphorylation (1). Phosphorylation is the addition of a phosphate (PO4) group to a protein or a small molecule. In higher organisms, protein phosphorylation is one of the most important regulatory events controlling metabolic processes. Many enzymes and receptors are switched "on" or "off" by phosphorylation and dephosphorylation. Phosphorylation is catalyzed by various specific so-called protein "kinases", whereas "phosphatases" dephosphorylate (remove a phosphate from) specific target proteins.

In addition to general transcription factors being regulated by phosphorylation, the hormone receptor itself (remember it’s a transcription factor too, by definition) may be phosphorylated upon ligand (hormone) binding, altering its activity (2).

Compounds that stimulate phosphorylation or inhibit dephosphorylation were found to enhance steroid dependent transcription induced by progesterone, estrogen, glucocorticoids, and finally, androgens (3). In (3), the synthetic phosphorylating agent 8-Br-cAMP allowed for a threefold increase in the transcriptional efficiency of testosterone. In other words

Observant readers have likely noticed that 8-Br-cAMP is a derivative of the ubiquitous, naturally occurring signal transducer cAMP (cyclic adenosine monophosphate) that is found throughout the body and is involved in numerous hormonal and chemical signaling cascades. One with which we are intimately familiar is the activation of the lipolytic enzyme Hormone Sensitive Lipase (HSL) by beta receptor agonists, resulting in fat mobilization. Here cAMP activates Protein Kinase A (remember a kinase adds a phosphate group to a target protein) which phosphorylates and hence activates the enzyme HSL.



Fig 2. Schematic showing cAMP mediated signaling leading
to activation of HSL and increased lipolysis. Beta receptor
agonists like norepinephrine, ephedrine, and clenbuterol
bind to the beta receptor, activating adenylate cyclase,
which in turn leads to a cascade of events ultimately
resulting in lipolysis, or the freeing-up of stored fat so that
it can be used as fuel.


Forskolin is a well-known activator of cAMP and PKA, and it too is capable of enhancing androgen initiated gene transcription (4). In (4) a synthetic system was created where yeast expressed the AR. Adding forskolin to the already potent androgen receptor agonist R1881 (methyltrienolone) enhanced the transcriptional capabilities of methyltrienolone. The mechanism is believed to be one where forskolin ultimately led to stronger binding of the ligand/receptor complex to the gene’s androgen response element on the DNA strand.

Many people report anecdotally that forskolin seems to preserve or even increase muscle mass while it is being used as part of a fat loss regimen. Forskolin and Green Tea based thermogenics have become popular since ephedrine was banned as a supplement. Increasing the effiency of testosterone could be one mechnanism for the observed mild anabolic effect. That and the fact that forskolin directly stimulates Leydig cell testosterone production (5) as well as hypothalamic GnRH (Gonadotropin Releasing Hormone) secretion (6).

Technically speaking, according to our definition of transcription factors, the androgen receptor itself is a transcription factor. It too is subject to phosphorylation, which seems to be critical for the proper functioning of the receptor. When natural or synthetic androgens bind to the AR, they promote its phosphorylation and allow the receptor/ligand complex to function as an androgen agonist. In contrast, agents such as bicalutamide and estramustine inhibit the receptor phosphorylation and act as antagonists (7).

Thus far we have seen how cAMP stimulated phosphorylation of the androgen receptor and other transcription factors enhances gene transcription. Somewhat surprisingly, the human AR can be activated in the absence of androgens by agents that directly modulate cAMP/ protein kinase A activity (8).

In (8) the authors examined the effects of adding forskolin and R1881 to monkey kidney cells or human prostate epithelial cells transfected with human AR along with a reporter gene. In the case of the kidney cells, addition of either 10 (-10) M R1881 or 2 x 10(-5) M forskolin activated the AR and resulted in transcription of the reporter gene. So here forskolin is acting as if it were a true androgen. The extent of AR activation with forskolin was 73% of that seen with R1881. Similar results were seen in the human prostate cells. The authors attributed the action of forskolin to increased binding of the AR to the reporter gene. This is consistent with the results reported in (4) above.

The authors of (8) summarized their results thusly:

<blockquote>"In this report, we demonstrate that the human AR can be activated in the absence of androgens through an alternate signaling pathway involving forskolin, a stimulator of adenylate cyclase. This ligand-independent activation of the AR was observed in both monkey kidney CV1 cells as well as in human prostate PC-3 cells transiently transfected with the AR expression vector and with two separate androgen-inducible reporter constructs (one an artificial promoter and the other, a natural context promoter). The cyclic AMP analog, 8-bromo-cyclic AMP, can also activate the AR in a ligand-independent manner (data not shown), albeit to a lower extent than forskolin (for 8-bromo-cyclic AMP, activation seen is between 20 and 25% of that with R1881).

"The implications of these findings for alternate activation of the AR may be important in understanding the progression of prostate cancer. We propose that activation of the AR may occur (by signaling pathways that modulate phosphorylation) even after androgen withdrawal therapy in individuals with prostate cancer, resulting in the androgen-independent growth of these cells. Our data suggest that these ligand-independent pathways may be blocked efficiently in vitro with the anti-androgens, casodex and flutamide." (8)</blockquote>

In a study similar to (8), Sadar examined the effects of adding forskolin at a concentration of 1 microM to a human prostate cancer cell line expressing the AR. Note that the concentration of forskolin is an order of magnitude smaller than what was employed in the previous study. Sadar found that this relatively low concentration of forskolin was able to promote transcription of the PSA (Prostate Specific Antigen) gene comparable with that of R1881 (9).

Despite the temptation to assume that forskolin may exert anabolic effects in muscle tissue by phosphorylating transcription regulatory factors, caution must be exercised. Transcription factors and cofactors can be cell specific. Androgens exert different actions in different tissues in part by recruiting the regulatory factors specific to those cells. In some cells lacking the appropriate factors, androgens may exert no action at all.

It may very well turn out that forskolin/cAMP are incapable of enhancing androgen action in muscle tissue, or activating the AR in muscle, due to the specific nature of the transcription factors found in muscle cells. Clearly further research is needed to answer this question.

One well-characterized transcription factor found in skeletal muscle is CREB (cAMP response Element Binding Protein). As the figure below illustrates, CREB is activated by cAMP:





Skeletal muscle differentiation is the process through which muscle precursor cells differentiate, or turn on the appropriate genes, that allow them to function as fully developed muscle tissue. This process is regulated by the MyoD family of basic helix-loop-helix (bHLH) transcription factors, including MyoD, Myf5, myogenin, and MRF4. MyoD is responsible for activating a number of these genes, and it turns out that MyoD associates with CREB, and CREB activity is required for MyoD to stimulate transcription (10). So in this sense cAMP does contribute to muscle growth and development.

So in summary we see that in certain cells forskolin is capable of both acting in concert with the AR to promote gene expression, and more interestingly, is even capable activating androgen responsive genes in the absence of androgens.

Interestingly, as an aside, testosterone itself appears to act in certain cells to activate CREB. Testosterone is essential for the Sertoli cells in the testes to produce spermatozoa (spermatogenesis). In the classical view of androgen action, binding of androgen to the intracellular androgen receptor (AR) produces a conformational change in AR such that the receptor–steroid complex has high affinity for specific DNA regulatory elements and is able to stimulate gene transcription. Fix, et.al. were able to demonstrate that testosterone can act by means of an alternative, rapid, and sustainable mechanism in Sertoli cells that is independent of AR–DNA interactions. Specifically, the addition of physiological levels of testosterone to Sertoli cells stimulates the so called mitogen-activated protein kinase signaling pathway (MAPK) and causes phosphorylation of the cAMP response element binding protein transcription factor (CREB) on serine 133, a modification known to be required for Sertoli cells to support spermatogenesis. Androgen-mediated activation of mitogen-activated protein kinase and cAMP response element binding protein occurs within 1 min, extends for at least 12 h.

So here we have yet another example of how androgens can work independently of AR binding, so called "non-genomic" androgen action (11). The significance of testosterone regulation of the CREB transcription factor for the support of spermatogenesis is highlighted by the observation that spermatozoa are not produced if CREB phosphorylation is inhibited in Sertoli cells. Blocking of CREB phosphorylation in Sertoli cells may prove to be an attractive target for male contraception (11).




References

Hunter T, Karin M Regulation of transcription by phosphorylation. Cell 70:375-385 1992

Orti E, Bodwell JB, Munck A. Phosphorylation of steroid hormone receptors. Endocr Rev 13:105-128 1992

Ikonen T, Palvimo JJ, Kallio PJ, Reinikainen P, Janne OA. Stimulation of androgen-regulated transactivation by modulators of protein phosphorylation. Endocrinology. 1994 Oct;135(4):1359-66

Rana S, Bisht D, Chakraborti PK. Synergistic activation of yeast-expressed rat androgen receptor by modulators of protein kinase-A. J Mol Biol. 1999 Feb 26;286(3):669-81.

Luo L, Chen H, Zirkin BR 2001 Leydig cell aging: steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme. J Androl 22:149-156

Martinez de la Escalera G, Choi AL, Weiner RI 1995 Signaling pathways involved in GnRH secretion in GT1 cells. Neuroendocrinology 61:310-317

Wang LG, Liu XM, Kreis W, Budman DR Phosphorylation/dephosphorylation of androgen receptor as a determinant of androgen agonistic or antagonistic activity. Biochem Biophys Res Commun. 1999 May 27;259(1):21-8.

Nazareth LV, Weigel NL. Activation of the human androgen receptor through a protein kinase A signaling pathway. J Biol Chem. 1996 Aug 16;271(33):19900-7.

Sadar MD. Androgen-independent induction of prostate-specific antigen gene expression via cross-talk between the androgen receptor and protein kinase A signal transduction pathways. J Biol Chem. 1999 Mar 19;274(12):7777-83.

Magenta A, Cenciarelli C, De Santa F, Fuschi P, Martelli F, Caruso M, Felsani A. MyoD stimulates RB promoter activity via the CREB/p300 nuclear transduction pathway. Mol Cell Biol. 2003 Apr;23(8):2893-906.

Fix C, Jordan C, Cano P, Walker WH. Testosterone activates mitogen-activated protein kinase and the cAMP response element binding protein transcription factor in Sertoli cells. Proc Natl Acad Sci U S A. 2004 Jul 27;101(30):10919-24

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