non-classical actions of testosterone: an update

Non-classical actions of testosterone:an updateFaisal Rahman and Helen C. Christian

Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, UK OX1 3QX

Review TRENDS in Endocrinology and Metabolism Vol.18 No.10

Androgens are known to exert their effects via genomicsignalling, which involves intracellular androgen recep-tors that modulate gene expression on steroid binding.Whereas non-classical estrogen effects are well estab-lished, it is only recently that non-classical, rapid, mem-brane-initiated testosterone actions have receivedattention. Non-classical effects of testosterone havenow been demonstrated convincingly in several tissues,in particular in the reproductive, cardiovascular, immuneand musculoskeletal systems. There is evidence for theparticipation of the classical intracellular androgen re-ceptor and for involvement of novel, membrane-associ-ated androgen receptors in the non-classical actions oftestosterone. Here we discuss evidence for rapid testos-terone actions, which have clinical implications in ferti-lity, cardiovascular disease and the treatment of prostatecancer.

Introduction: non-classical effects of testosteroneAndrogens are essential for the propagation of the speciesand for establishing and maintaining the quality-of-life ofmales through support of sexual behaviour and function,muscle strength and sense of well-being [1]. Testosterone,the principal androgen, and its metabolite, 5a-dihydrotes-tosterone (DHT), are known to mediate their effectsthrough binding to the intracellular androgen receptor(AR). Initial [3H]-androgen binding assays in 1975 demon-strated 3H binding to AR in most tissues [2]. The humanAR was cloned in 1988 [3,4], and as for other members ofthe nuclear receptor superfamily, work in the 1990sshowed that the AR acts as a ligand-inducible transcrip-tion factor modulating transcription of target genes [1,5].This mechanism of steroid action became known as thegenomic pathway. However, rapid non-genomic effects ofandrogens that occur too quickly to be explained by theclassical AR genomic pathway also emerged and are nowgenerally accepted as contributing to the physiologicaleffects of the steroids [6]. Whereas genomic effects takehours or days to produce their actions (reflecting the timerequired to alter protein synthesis), rapid steroid effectsare activated within seconds or minutes. For example, oneof the first reports of rapid action of testosterone byYamada et al. in 1979 demonstrated increased hypothala-mic neuron firing within seconds of testosterone appli-cation in adult male rats [7], and our own studies inanterior pituitary, also in adult male rats, showed that

Corresponding author: Christian, H.C. ([email protected]).Available online 9 November 2007.

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testosterone elicits rapid release of prolactin within fiveminutes [8].

Unlike genomic actions, these non-genomic actions arenot removed by inhibition of transcription or translationand are often activated by membrane-impermeant steroidconjugates. However, rapid pathways of androgen actionmight ultimately act tomodulate transcriptional activity ofARs or other transcription factors, so the term non-classi-cal, rather than non-genomic, will be used in this review.Whereas estrogen has been shown to exert a vast array ofrapid functional effects on different cells and tissues [9,10],the rapid actions of testosterone have received less atten-tion until recently. Early suggestions were that rapidactions of testosterone occurred via aromatisation to estro-gen, but many reports have since verified that acute tes-tosterone actions are not affected by aromatase or estrogenreceptor inhibition. In this review we concentrate on rapidtestosterone actions occurring in physiologically relevantcontexts in the reproductive, cardiovascular, immune andmusculoskeletal systems, and their pathological and thera-peutic implications.

Non-classical testosterone signalling pathwaysSertoli cells

Testosterone has important roles in the reproductivesystem, particularly in male sexual development andmaturation, as well as in the maintenance of male repro-ductive organs and of spermatogenesis [11]. Locatedwithin the seminiferous tubules of the testes, Sertoli cellssupport and nourish the adjacent germ cells by providingnutrients and growth factors required for their maturationinto spermatozoa (via spermatogenesis). Testosterone isimportant for the regulation of spermatogenesis throughdirect actions on Sertoli cells, which involve both non-classical and classical signalling pathways [12]. Evidencefor two non-classical testosterone signalling pathways inSertoli cells has accumulated, namely the stimulation ofcalcium influx and activation of the mitogen-activatedprotein kinase (MAPK) pathway (Figure 1). Applicationof testosterone to rat Sertoli cells at concentrations withinthe physiological range measured in seminiferous tubulefluid (175–365 nM) [13,14] produces rapid and prolongedincreases in intracellular Ca2+ ([Ca2+]i) [15,16]. Treatmentwith testosterone conjugated to bovine serum albumin (T–BSA), a membrane-impermeable analogue, mimics theactions of testosterone and indicates plasma membraneaction of testosterone. Increases in [Ca2+]i are abolished byEGTA (ethylene glycol tetraacetic acid) and L-type Ca2+

channel current inhibitors (ICa-L), indicating extracellular

d. doi:10.1016/j.tem.2007.09.004

mailto:[email protected]

http://dx.doi.org/10.1016/j.tem.2007.09.004

Figure 1. Non-classical testosterone signalling pathways in Sertoli cells. 1. Testosterone (T) binds to membrane-associated androgen receptor (AR). 2. Activation of PLC to

produce Ins(1,4,5)P3 (IP3) and DAG. 3. Inhibition of K+ATP-channel by IP3 and/or DAG leading to membrane depolarisation. 4. Ca2+ influx leads to increase in [Ca2+]i. 5.[Ca2+]i

acts through an unknown pathway for ERK activation. Ca2+ influx might also have other effects (e.g. cytoskeletal). 6. A further pathway for ERK activation. Testosterone

binds to the membrane-associated receptor, which associates with and activates Src kinase. 7. Direct activation of EGFR by SRC (activated by testosterone-bound AR)

activates Ras, which in turn activates a RAF kinase. 8. RAF kinase activates MEK, which in turn activates ERK. 9. The ERK pathway phosphorylates CREB to modulate gene

expression leading to effects on spermatogenesis. Abbreviations: AR, membrane associated androgen receptor; CREB, cAMP response element binding protein; DAG,

diacylglycerol; EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; GPCR, G-protein-coupled receptor; IP3, inositol trisphosphate; PLC, phospholipase C;

SRC, Src kinase; RAS, Ras GTPase protein; RAF, Raf kinase; T, testosterone; ? indicates gaps in details of pathways that require further study.

372 Review TRENDS in Endocrinology and Metabolism Vol.18 No.10

Ca2+ influx through L-type Ca2+ channels [16,17]. Inaddition, testosterone (100–500nM) induces rapid mem-brane depolarisation of Sertoli cells [17,18]. This effect isabolished by co-treatment with diazoxide, which stimu-lates the ATP-sensitive K+ current (IK-ATP) and the effect ismimicked by the IK-ATP inhibitor glibenclamide, indicatingthat testosterone induces depolarisation by inhibition ofthe IK-ATP. Pharmacological studies suggest a mechanismin which rapid activation of Gq protein-mediated phospho-lipase C (PLC) signalling by testosterone inhibits the IK-

ATP, resulting in membrane depolarisation and, in turn,activation of ICa-L and Ca2+ influx [17,18]. A relativelysmall (1–2 mV) depolarisation over 30–60s activates suffi-cient ICa-L to produce a dramatic 50–100 nM increase in[Ca2+]i. It is possible that testosterone also decreasesthe ICa-L activation threshold and/or stimulates Ca2+

release from intracellular stores to achieve this. Althoughthe cellular targets have not been fully determined, the risein [Ca2+]i might influence the cytoskeleton and cell–celljunctions to regulate Sertoli cell mobility and metamor-phosis, gene transcription, and secretions [17]. Exper-iments using classical AR antagonists, RNA interferenceand testicular feminised (tfm) rats (lacking functional AR),showed that nuclear AR are required for testosterone-induced rises in [Ca2+]i in Sertoli cells [15,16].

Recent evidence showed that non-classical effects oftestosterone can regulate numerous transcription factors,including cAMP response element binding protein (CREB),viaMAPK signalling pathways that are independent of ARbinding to DNA [19]. Phosphorylated CREB is an essential

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factor for Sertoli cells to support spermatogenesis [20], andtherefore for germ cell survival. Fix et al. [19] demon-strated that activation of ERK1/2 (extracellular receptorkinase 1/2) leads to phosphorylation of the transcriptionfactor CREB within one min of testosterone (100–250 nM)application, which subsequently induced the expression ofthree CREB-regulated genes in Sertoli cells, namely thoseencoding CREB itself, lactate dehydorogenase-A (LDH-A)and early growth response 1 (Egr1). Experiments usingclassical AR antagonists, RNA interference and tfm ratshave also shown that nuclear AR are required for testos-terone-induced ERK and CREB phosphorylation [19] pro-viding further evidence that non-classical actions oftestosterone in Sertoli cells require AR.

How do classical ARs activate the MAPK pathway?Immunocytochemistry has now demonstrated that a popu-lation of classical ‘nuclear’ ARs in Sertoli cells localise to theplasma membrane, and that AR associates with, and acti-vates, the tyrosine kinase Src kinase after testosteronestimulation, which subsequently activates theMAPK path-way through the EGF receptor [21]. Although testosteronecan elevate cAMP by binding to sex hormone binding glo-bulin (SHBG) and its receptor [22,23], activation of ERK1/2is independent of a cAMP-mediated pathway. Physiologi-cally it is likely that the non-classical actions of testosteronein Sertoli cells have the potential to regulate the expressionof many more genes than what is possible by direct AR-promoter interactions, and that they function in tandemwith the classical actions to maintain full spermatogenesis.Furthermore, the non-classical pathways might provide

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novel targets for male contraceptives and for treatment ofmale infertility.

Prostate cells

The prostate gland relies on androgen action for its de-velopment and its maintenance in adulthood. Prostatecancer is initially an androgen-dependent condition, andandrogens act genomically to increase transcription ofprostate-specific proteins such as prostate-specific antigen(PSA). Androgen ablation is one therapeutic approach thatinhibits prostate cancer growth [24]. However, prostatecancers can become androgen-independent, as seen inpatients who do not respond to hormone ablative treat-ments. Non-classical androgen signalling has been exten-sively studied in the LNCaP human prostate cancer cellline as a model for prostate cancer. Treating LNCaP cellswith T or T–BSA stimulates a rise in [Ca2+]i within oneminute [16,25] and actin cytoskeleton reorganisation andPSA secretion within 10 min [26]. Flow cytometric analysisof T–BSA–FITC (fluoroscein isothiocyanate) binding stu-dies have shown specific, saturable binding to LNCaPcells. Although the identity of the binding protein is notknown, it is not thought to be the classical AR becauseplasma membrane labelling was not detectable byimmunocytochemistry for AR. Other studies have shownthat treatment of LNCaP cells with androgen producesanti-apoptotic effects by inducing the rapid formation of acytosolic signalling complex containing the classical AR,estrogen receptor (ER) and non-receptor tyrosine kinaseSrc. This complex, in turn, activates the ERK1/2 andCREBpathway [27]. A scaffolding protein termed ‘modulator ofnon-genomic activity of estrogen receptor’ (MNAR) hasbeen shown to be associated with this signalling complexand is upregulated constitutively in LNCaP cells that havebecome androgen-independent [28]. Recent studies inLNCaP cells have also shown that membrane-associatedclassical AR activates the phosphoinositide-3 (PI3)-kinase/serine threonine kinase Akt signalling cascade [29], amajor pathway of growth signalling. Additional parallelsignalling pathways activated by androgens at the mem-brane via SHBG binding to prostate cells and subsequentactivation of cAMP and protein kinase A have also beendemonstrated [22,30]. Furthermore, in normal canineprostate, estrogen can amplify or substitute for androgensin activating the AR via the SHBG receptor, resulting inelevation of cAMP and activation of the canine equivalentof PSA, arginine esterase [30]. This pathway might have arole in benign prostatic hyperplasia because a similarsignalling pathway resulting in secretion of PSA has beenobserved in human benign hyperplastic prostate tissue[31]. Further clarification of the non-classical signallingcascades activated by androgens and their relationship toclassical genomic signalling should improve understand-ing of prostate cancer progression and development ofnovel treatment strategies.

Oocytes

One of the best understood systems in which non-classicalactions of androgens are physiologically relevant is thematuration of Xenopus oocytes. The maturation process isstriking because the actions of androgens occur in the


complete absence of genomic transcription, and thereforethe signalling pathway is driven by a non-genomic mech-anism. Inhibition of androgen synthesis in vivo reducesoocyte maturation and delays ovulation [32]. The actionsare mediated in part by classical Xenopus ARs, localised inthe plasma membrane, cytoplasm and nucleus; the ARantagonist flutamide and RNA interference eliminationof AR specifically reduced androgen-mediated maturation[33]. G protein Gbg signalling, which activates potassiuminflux through G protein-regulated inward rectifying chan-nels (GIRKs), maintains Xenopus oocytes in meioticarrest. Testosterone (via AR) overcomes this inhibitionby attenuating Gbg signalling [34]. Non-genomic androgensignalling is important for Xenopus oocyte maturation, butis it important in mammalian oocytes? Recent studiesindicate that these signalling pathways are conserved invertebrates. Addition of testosterone to mouse oocytes heldinmeiotic arrest in vitro triggeredmeiosis and activation ofMAPK cell cycle activators independent of transcription.Furthermore, ARs were detected in mouse oocytes, and thenon-classical testosterone effects were blocked by finaster-ide [35,36].Whether these non-classical signals are import-ant for mammalian oocyte maturation and ovulation invivo is not known, but human females with excess exogen-ous or endogenous androgens have unregulated folliclegrowth and infertility [36,37], and non-classical actionsmight be involved.

Immune system

Testosterone has a suppressive effect on immuneresponses and increases susceptibility to many infectiousdiseases [38]. For example, in experimental models ofmalaria with Plasmodium chabaudi or protozoan infectionwith Leishmania donovani, prior testosterone treatmentresults in increased infection rates [38–40]. Testosteroneprofoundly influences host defence, and non-classicalactions of testosterone in T cells and macrophages arenow understood to mediate these effects in part. In murinesplenic T cells, testosterone (1–10 nM) stimulates Ca2+

influx within seconds [41,42] through Ni2+-sensitive vol-tage-independent Ca2+ channels. By contrast, murinemacrophages of the cell line IC-21 respond to testosteronewith predominantly intracellular Ca2+ mobilisationmediated through a membrane receptor G protein-coupledreceptor (GPCR) PLC signalling pathway [43]. Howdo non-classical testosterone calcium signalling pathways med-iate immunosuppressive actions? Although testosteroneactivation of Ca2+ signalling does not independently acti-vate MAPK signalling pathways, c-fos transcription, ornitric oxide (NO) production, testosterone has beenshown to reduce lipopolysaccharide and Leishmania dono-vani-induced elevations in MAPK phosphorylation, c-Fospromoter activity and nitric oxide (NO) production inmacrophages, inhibition of which increased pathogen in-fection rates [44]. The suppressive effect of testosteronewas prevented by the cell permeable Ca2+ chelator BAPTA-AM, emphasizing the crucial role of [Ca2+]i in these actions[40,44]. These actions of testosterone might contribute tothe sexual dimorphism of the immune response in thatmales have a greater susceptibility to infectious diseasescompared with females [38,39]. However, the details of the


molecular pathways activated by testosterone mediatingimmunosuppression inmacrophages require further inves-tigation.

The genomic pathway via AR does not explainthe effects of testosterone in macrophages because ARhave not been detected in either tissue macrophages orthe mouse IC-21 macrophage cell line [39,43]. Although Tcells express AR, testosterone application does not result innuclear translocation, indicating that the classical genomicpathway of AR signalling is not present [41], although thecytoplasmic AR could be involved in mediating rapidresponses. In support of a membrane-initiated effect inT cells and macrophages, confocal imaging and flow cyto-metric analysis of fluoroscein-tagged T–BSA bindingshowed co-localisation of T–BSA with the plasma mem-brane marker ConA-Rhodamine [39,42,44]. Fifteen min-utes after T–BSA–FITC application, punctate fluorescencewas observed within macrophages, indicating receptor–ligand internalisation, which might be important for sig-nalling [43,44]. It is exciting to speculate that the bindingof androgen to novel surface receptors independent of theintracellular AR, followed by rapid Ca2+ influx, is theinitial event in the pathway mediating testosterone-induced susceptibility to Plasmodium chabaudi malariain mice. If so, selective inhibition of testosterone cell-sur-face receptors could be a potential target for future thera-pies for prevention of malaria infection.

Cardiovascular systemBecause males have a higher incidence of coronary arterydisease, and anabolic steroids have been associated withmyocardial ischaemia and hypertension in athletes,endogenous and exogenous androgens have been proposedas underlying causes of increased cardiovascular risk[45,46]. However, great interest has now been generatedby recent findings that testosterone is beneficial to thecardiovascular system [45,46]. Clinical studies consist-ently report that acute testosterone therapy reduces myo-cardial ischaemia within 30 min in men with coronaryartery disease (CAD), and evidence suggests that a directnon-classical vasodilator action of testosterone is one under-lying effector mechanism [47–49]. However, although invivo studies have shown acute vasodilator effects of testos-terone at nanomolar concentrations, pharmacological (i.e.high micromolar) concentrations are required for themajority of in vitro effects reported in animal studies [50].Organ bath studies have shown that testosterone causesvasorelaxation in precontracted pulmonary, aortic, mesen-teric and coronary vasculature in a variety of species withinminutes atmicromolar concentrations [50–52]. By contrast,testosterone rapidly increases coronary vascular flow andvesseldiameteratphysiological (nanomolar) concentrationsin CAD patients [48]. It has been suggested that becauseSHBG is involved in the presentation of testosterone toeffector proteins within the target membrane, the absenceof SHBG and blood-borne delivery of testosterone in in vitroexperiments might explain the marked loss of potencycomparedwith in vivo studies [50,53,54]. Receptor antagon-ist studies indicate that neither intracellular nor mem-brane-associated ARs are required for the rapidvasodilator effect [52] although lack of functional AR in


the tfm mouse is associated with reduced endothelial-dependent vasodilatation, indicating that genomic path-ways also influence vascular tone [55]. Clinical findings thattestosterone acts rapidly to reduce exercise-induced myo-cardial ischaemia in CAD patients indicate that testoster-one has potential therapeutic benefits in CAD [45–48].

Active debate remains regarding the mechanism(s) oftestosterone-induced vasorelaxation (Figure 2). Pharma-cological evidence indicates that testosterone-inducedvasorelaxation involves opening of K+ channels [47,52],including high conductance Ca2+-activated (BKCa) [55],voltage-sensitive [56] and ATP-sensitive K+ channels[57]. A Ca2+ channel antagonistic action of testosteronemight also be important for vasorelaxation. In rat aorticsmooth muscle cells, application of testosterone rapidlyinhibits activation of the ICa-L [54,58] but does not inhibitinositol trisphosphate (Ins(1,4,5)P3)-mediated Ca2+ mobil-isation [54]. It is not known whether testosterone targetsboth K+ and Ca2+ channels, or if action on one channelsubsequently modulates the function of the other. Thiscould be particularly important for modulating contractionby a wide variety of agonists in vivo. Alternatively, themechanism involved might depend on the vessel type (e.g.conductance versus resistance vessels) or the experimentalagonist used to induce contraction [59]. The role ofendothelium-derived NO in testosterone-induced vasodi-latation is not clear. Some studies using endothelium-denuded tissues and inhibitors of NO synthesis suggesta partial contribution [56], whereas others report no rolefor NO in testosterone-induced vasodilatation [52,55].Although vasodilatory effects have been shown with T–BSA in a variety of models, the effects of T–BSA on ionchannels have not been investigated and it is not known ifeffects on channels aremembrane-initiated or secondary tointracellular signalling [59].

In addition, testosterone and T–BSA have recently beenshown to act by rapid AR-independent pathways in cardi-omyocytes. Cardiomyocytes contain intracellular ARs thatregulate the expression of several genes, including L-typeCa2+ channel subunits [60,61]. Evidence indicates thattestosterone (10–100nM) rapidly stimulates an increasein intracellular Ca2+ (1–7 min) that persists in the absenceof extracellular Ca2+ in neonatal and adult isolated cardi-omyocytes [62]. The rise in [Ca2+]i is inhibited by pertussistoxin, U73122 (a PLC inhibitor) and xestospongin-C (anIns(1,4,5)P3 receptor inhibitor), suggesting the involve-ment of the GPCR–PLC–Ins(1,4,5)P3 pathway. In femaleadult rat myocytes whole-cell and single-channel analysisof ICa-L has shown that although chronic testosteronetreatment increases basal cardiac ICa-L by the genomicAR pathway, acute testosterone treatment decreasesL-type and T-type single channel activity and shortensaction potential duration [61–63]. Experiments using theAR antagonist flutamide have shown that although ARsare required for chronic actions of testosterone in cardio-myocytes, they are not required for the acute effects. Owingto the rapidity of the acute action, it has been speculatedthat testosterone might bind to a Ca2+ channel subunitdirectly to mediate the inhibition of currents, but as yetthere is no evidence to support this [61,63,64]. Together,these studies indicate that acute and chronic testosterone

Figure 2. Non-classical testosterone signalling in vascular smooth muscle cells inducing vasorelaxation. 1. Activation of one or more Kv, KATP, BKCa channels leading to

hyperpolarisation and inhibition of L-type calcium channels. 2. Direct inhibition of L-type calcium channel. The figure illustrates the involvement of a membrane-associated

androgen receptor, but it is also possible that testosterone acts directly on target proteins such as L-type calcium channels. 3. Endothelial-derived NO might activate the NO

pathway of smooth muscle relaxation. At most, NO is a partial contributor to the vasorelaxation response to testosterone. Abbreviations: AR, membrane associated

classical androgen receptor; NO, nitric oxide; R, novel plasma membrane receptor for testosterone; T, testosterone.


actions regulate cardiomyocyte Ca2+ handling, and anyshift in this control will influence cardiac contractility.Testosterone-induced increases in [Ca2+]i might have phys-iological genotropic effects and could modulate geneexpression pathophysiologically in cardiac hypertrophy,for example in steroid abuse by athletes, an area whichrequires further investigation. Testosterone can alsodirectly and acutely protect cardiomyocytes against ischae-mic injury by rapid opening of ATP-sensitive K+ channelsin the mitochondrial inner membrane [65,66], suggestingthat testosterone might also have an important role inrecovery after myocardial infarction.

Skeletal muscle

Testosterone is well known to increase muscle mass andstrength by inducing hypertrophy of type 1 and type 2muscle fibres and increasing myonuclear and satellite cellnumber [1,11]. For example, development of the perinealstriated muscles bulbocavernosus and levator ani is sexu-ally dimorphic and developmentally dependent on testos-terone, and has been widely used as a model for elucidatingAR-dependent classicalmechanisms [67]. As inSertoli cells,vascular smooth muscle, macrophages and T cells, testos-terone rapidlyacts to increase [Ca2+]i in skeletalmuscle cellsby a non-classical pathway. In cultured rat myotubules,testosterone (10nM) and T–BSA both produce a rapid(within 1 min) increase in [Ca2+]i, often accompanied byCa2+ oscillations, as visualised with confocal microscopy[68]. Similar to cardiomyocytes, pharmacological evidencehas demonstrated that aGPCR–PLC–Ins(1,4,5)P3 pathwaystimulates a rise in [Ca2+]i in skeletalmuscle cells. Further-more, T–BSA rapidly increasedMAPKERK1/2 phosphoryl-ation detectable after 1 min exposure,whichwas dependentonGPCR–PLC–Ins(1,4,5)P3pathwayactivationand [Ca2+]i,


as shown by inhibitor studies and use of the Ca2+ chelatorBAPTA-AM.Additionally, testosteronewas shown toact viaMEK (mitogen-induced extracellular kinase) and Ras tostimulate phosphorylation of ERK1/2 independently ofthe intracellular AR because the responses were not inhib-ited by the intracellular AR antagonist cyproterone. Bycontrast, testosterone-induced Ca2+ oscillations were de-pendent on extracellular Ca2+ influx via voltage-indepen-dent capacitative Ca2+ entry [68,69]. It is likely that therapid testosterone-induced Ca2+ signalling pattern andMAPKactivationof transcriptionpathwaysworkwithgeno-mic AR testosterone actions in the development of skeletalmuscle hypertrophy.

Receptors for non-classical testosterone actionNon-classical actions might occur through multiple recep-tors, and to date three possible receptor targets for andro-gens have been proposed [53,70]. First, testosterone boundto SHBG has been shown to act through a SHBG receptorin the LNCaP human prostatic cancer cell line [22,23]. Todate the SHBG receptor has not been cloned but it isthought to be a GPCR or functionally linked to this typeof receptor. Alternatively, testosterone might bind a mem-brane-associated classical AR or a membrane androgenreceptor distinct from the classical AR. Although severalgroups have reported specific binding of testosterone to theplasma membrane in different cell types [8,26,43,54], aspecific membrane protein through which testosteronemediates non-classical actions has not yet been purifiedor cloned. However, using cellular fractionation and immu-nohistochemistry techniques, membrane association ofclassical ARs has been illustrated in Xenopus oocytes[33], Sertoli cells [21] and T cells [42]. ARs lack a standardtransmembrane structure and hydrophobicity, and thus it


is likely that ARs interact with membrane proteins,enabling anchorage to the membrane or in the vicinityof the membrane. For example, estrogen receptors havepreviously been localised to caveolae and lipid raftsenriched with several signalling and scaffolding proteins[71]. A lipid raft association has recently been demon-strated for ARs in LNCap prostate cancer cells in whichAR preferentially interacts with Akt1, an importantmediator of growth signalling [29,72]. In the future it willbe interesting to determine other cell types in which a lipidraft association for AR is found. However, what is knownabout the structural elements and modifications of the ARthat enable translocation to the plasma membrane andlipid rafts? A conservedmechanism for translocation to theplasma membrane of AR, progesterone receptors A and Band estrogen receptors ERa and ERb involving a 9 aminoacid localisation motif in the ligand-binding domain hasrecently been identified in mutagenesis studies in ChineseHamster Ovary (CHO) cells [73]. The localisation sequencemediated palmitoylation of the AR, which enabled associ-ation with caveolin-1 (a protein marker for caveolae),subsequent membrane association and steroid signalling.Finally, the plasma concentration of testosterone in malesis 10–35 nM [74] and in females is 0.7–2 nM [75]. It isunlikely that dynamic minute-to-minute changes insystemic serum testosterone concentrations occur quicklyenough to explain rapid physiological responses via mem-brane-associated receptors in target cells. One explanationmight be that tissues produce testosterone locally, causingacute changes in local steroid concentrations, which hasbeen reported in the brain [76–78]. There is also an emer-ging literature regarding the brain that demonstrates thatandrogens influence aggression, anxiety and/or reward, inpart through non-classical actions [79,80]. In addition,endogenous and exogenous androgens are known to exertclassical and non-classical actions via modulation ofgamma amino butyric acid (GABA) receptors centrally tomediate male and female reproductive behaviors inrodents [81–83]. Further work is required to elucidatewhether the non-classical androgen signalling pathwaysdetailed in the peripheral systems discussed in detail hereoccur in the brain.

ConclusionThe study of non-classical testosterone actions is a swiftlyevolving field. A multitude of rapid, membrane-initiatedtestosterone effects have been demonstrated, but theextent to which rapid non-classical and genomic actionsof androgens interact is not clear. Future investigationsmight uncover the extent to which non-classical androgeneffects act in parallel or in series with genomic actions tomodify transcriptionally relevant molecules, such as coac-tivators [10,84]. In each of the systems discussed here,testosterone acts to increase [Ca2+]i and an area yet to beexplored is the possibility of testosterone-induced Ca2+

regulation of gene expression through Ca2+-sensitive pro-moter elements and transcription factors [e.g. NF-AT(nuclear factor of activated T cells), CARE (calciumresponse element)]. Furthermore, selective ARmodulators(SARMs) that specifically promote genomic versus non-genomic androgen responses have been characterised.


These include androstenediol and estren, which preferen-tially promote non-genomic signals, and R1881 and19-nortestosterone, which preferentially promote genomicsignalling [36,85,86]. SARMs that are preferentially ana-bolic and lack non-classical prostate effects hold promise asanabolic therapies to improve muscle mass and strength inchronic illness and aging [87]. The further understandingof non-classical actions and development of SARMs se-lective for genomic or non-classical signalling could holdtherapeutic potential for use in many conditions that posea high clinical burden, such as cardiovascular disease,infectious diseases and infertility.

AcknowledgementsThe authors acknowledge grant support from the Wellcome Trust.

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81 Frye, C.A. (2001) The role of neurosteroids and non-genomic effects ofprogestins and androgens in mediating sexual receptivity of rodents.Brain Res. Rev. 37, 201–222

82 Clark, A.S. et al. (2006) Sex and age specific effects of analbolicandrogenic steroidson reproductivebehaviors.BrainRes.1126,122–138

83 Henderson, L.P. (2007) Steroid modulation of GABA receptor-mediated transmission in the hypothalamus:effects on reproductivefunction. Neuropharmacology 52, 1439–1453

84 Vasudevan, N. et al. (2001) Earlymembrane estrogenic effects requiredfor full expression of slower genomic actions in a nerve cell line. Proc.Natl. Acad. Sci. U. S. A. 98, 12267–12271

85 Kousteni, S. et al. (2001) Nongenotropic, sex-non-specific signallingthrough the estrogen or androgen receptors: dissociation fromtranscriptional activity. Cell 104, 719–730

86 Haas, D. et al. (2005) The modulator of non-genomic actions of theestrogen receptor (MNAR) regulates transcription-independentandrogen receptor-mediated signaling: evidence that MNARparticipates in G protein-regulated meiosis in Xenopus laevisoocytes. Mol. Endocrinol. 19, 2035–2046

87 Bhasin, S. et al. (2006) Drug insight: testosterone and selectiveandrogen receptor modulators as anabolic therapies for chronicillness and aging. Nat. Clin. Pract. Endocrinol. Metab. 2, 146–159

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non-classical actions of testosterone: an update

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