George Whipple Laboratory for Cancer Research, Departments of Pathology (C.A.H., C.C.) and Urology (C.C.), University of Rochester, Rochester, New York 14642
Address all correspondence and requests for reprints to: Dr. Chawnshang Chang, George Whipple Laboratory for Cancer Research, University of Rochester, Department of Pathology, Box 626, 601 Elmwood Avenue, Rochester, New York 14642. E-mail: chang{at}urmc.rochester.edu.
ABSTRACT
The biological activity of testosterone and dihydrotestosterone is thought to occur predominantly through binding to the androgen receptor (AR), a member of the nuclear receptor superfamily that functions as a ligand-activated transcription factor. However, androgens have also been reported to induce the rapid activation of kinase-signaling cascades and modulate intracellular calcium levels. These effects are considered to be nongenomic because they occur in cell types that lack a functional AR, in the presence of inhibitors of transcription and translation, or are observed to occur too rapidly to involve changes in gene transcription. Such nongenomic effects of androgens may occur through AR functioning in the cytoplasm to induce the MAPK signal cascade. In addition, androgens may function through the sex hormone binding globulin receptor and possibly a distinct G protein-coupled receptor to activate second messenger signaling mechanisms. The physiological effect of nongenomic androgen action has yet to be determined. However, it may ultimately contribute to regulation of transcription factor activity, including mediation of the transcriptional activity of AR.
THE APPROPRIATE REGULATION of androgen activity is necessary for a range of developmental and physiological processes, particularly male sexual development and maturation, as well as the maintenance of male reproductive organs and of spermatogenesis (1, 2, 3, 4). The principle steroidal androgens, testosterone (T) and its metabolite 5-dihydrotestosterone (DHT), are thought to predominantly mediate their biological effects through binding to the androgen receptor (AR). AR, in common with other members of the nuclear receptor superfamily, functions as a ligand-inducible transcription factor. The binding of T or DHT to AR induces receptor dimerization, facilitating the ability of AR to bind to its cognate response element and recruit coregulators to promote the expression of target genes (1, 5, 6, 7). In addition to this transcriptional or genomic mode of action by steroids, an increasing body of evidence suggests that androgens, like progesterone and estrogen, can exert rapid, nongenomic effects (8, 9, 10). Nongenomic steroid activity typically involves the rapid induction of conventional second messenger signal transduction cascades, including increases in free intracellular calcium, and activation of protein kinase A (PKA), protein kinase C (PKC), and MAPK. Second messenger induction by nongenomic steroid action is insensitive to inhibitors of transcription and translation. Commonly, these effects are observed to occur within seconds to minutes, considered to be too rapid to involve changes in transcription and protein synthesis. For example, the production of full-length mRNA of immediate early genes, such as c-fos, is observed 510 min after stimulation, with c-fos protein detectable 3045 min after stimulation (11). The initiation of these second messenger cascades may ultimately serve to modulate the transcriptional activity of AR or other transcription factors. The nongenomic action of androgens has been implicated in a number of cellular effects, including gap junction communication, aortic relaxation, and neuronal plasticity (12, 13, 14, 15). However, some of these experiments were conducted at unphysiologically high levels of androgen (10 µM1 M T or DHT). The biological relevance of such observations is not clear, and these effects are not included in this review.
The nongenomic effect of steroids could potentially be induced directly by the steroid in the absence of a receptor, through a nontranscriptional effect of the classical steroid receptor, or by a distinct nonclassical receptor that is possibly associated with the plasma membrane. Hydrophobic steroids, including estrogen, progesterone, T, and DHT can interact with the polar heads of membrane phospholipids to influence membrane fluidity (16, 17, 18). However, in these studies membrane fluidity was only altered at unphysiologically high levels of steroids (10100 µM) (17, 18), raising doubts as to whether this contributes a significant effect in vivo. To date, the reported nongenomic effects of steroids at physiological concentrations appear to be receptor mediated. AR, the progesterone receptor (PR), and the estrogen receptor (ER) are able to activate the MAPK ERK through a mechanism independent of their transcriptional activity (19, 20, 21). In addition to the classical AR, androgens can also stimulate second messenger cascades through at least one membrane receptor. Membrane receptor-mediated events are typically not blocked by antagonists of the classical AR, can be observed in AR-negative cells, and can be stimulated by T coupled to high molecular weight compounds such as BSA that retard its diffusion across the cell membrane. Consistent with this mode of action, T and DHT can activate cAMP and PKA through the sex hormone binding globulin (SHBG) receptor. Androgens have also been observed to stimulate an elevation in intracellular Ca2+ through a G protein-coupled receptor, although it remains to be determined whether this receptor is distinct from the SHBG receptor. The nongenomic, rapid stimulation of second messenger cascades by androgen may ultimately exert a biological effect through modulation of the transcriptional activity of AR or other transcription factors. Such modulation may occur through direct phosphorylation of transcriptional activators or their coregulators.
NONGENOMIC ACTION BY AR
Recently, AR, PR, ER, and ERß have been found to interact with the intracellular tyrosine kinase c-Src, triggering c-Src activation (19, 20, 21). c-Src is normally targeted to the inner surface of the plasma membrane by myristylation and palmitoylation of its amino terminus. The tyrosine kinase activity of c-Src is autoinhibited by the interaction between the tryosine kinase domain and the Src homology 2 (SH2) and Src homology 3 (SH3) domains. Disruption of these intramolecular interactions by proteins binding to the SH2 or SH3 domains, or through dephosphorylation tyrosine 527 of the SH2 domain, results in activation of the c-Src kinase. One of the targets of c-Src is the adapter protein Shc, an upstream regulator of the MAPK pathway. The c-Src-mediated activation of MAPK is involved in multiple cellular processes, including migration, proliferation, and differentiation (22, 23).
In response to DHT or the synthetic androgen R1881, AR interacts with the SH3 domain of c-Src (19, 20). The association of AR with c-Src results in stimulation of c-Src kinase activity within 1 min in the AR-positive LNCaP prostate cancer cell line in response to 10 nM R1881 (19). R1881 treatment also resulted in stimulation of two members of the MAPK signaling cascade, Raf-1 and ERK-2 within 2 and 5 min, respectively. Similarly, treatment of the AR positive osteocytic cell line MLO-Y4 with 10 nM DHT results in phosphorylation and activation of ERK within 2 min (20). The rapidity of ERK-2 activation suggests that R1881 stimulates the MAPK pathway through a nongenomic mechanism. Androgen induction of c-Src/Raf/ERK signaling is abrogated by inhibition of c-Src kinase activity or treatment with antiandrogens (19, 20). In the AR-negative COS-1 cells, transfection of AR is necessary to induce the activity of c-Src/Raf/ERK in response to R1881 (19). However, AR can also function cooperatively with ER or ERß to induce c-Src kinase activity as part of a terniary complex composed of c-Src, ER, and AR (19, 20) (Fig. 1
).
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SHBG
In the serum, only 0.54% of steroid hormones are free and not complexed with serum proteins (28). The majority of serum T and DHT (approximately 60%) is complexed with SHBG, with the remainder bound to albumin (28, 29). SHBG is also capable of binding estradiol (E2) but with a lower affinity than androgens (28, 30). A cell surface receptor for SHBG has been functionally identified in a number of tissues including the prostate, testis, breast, and liver (31, 32, 33), although SHBG binding has not been detected in lymphocytes and muscle (31). To date, the SHBG receptor has not been cloned, but is thought to either be a G protein-coupled receptor or functionally linked to one (34, 35). In LNCaP cells, membrane binding of SHBG is reduced by treatment with the nonhydrolyzable GTP analog Gpp(NH)p (34, 35). The binding of Gpp(NH)p to the heterotrimeric G protein complex results in its dissociation from the associated receptor and causes a reduction in receptor ligand binding (36, 37). Therefore, the reduction in SHBG membrane binding in the presence of Gpp(NH)p suggests that the SHBG receptor is coupled to G proteins (34, 35). One of the best characterized signal transduction mechanisms of G protein-coupled receptors is through the modulation of adenylyl cyclase and cAMP production. Overexpression of dominant-negative mutants of the GS subunit of the G protein complex decreases E2-SHBG mediated stimulation of a cAMP-responsive promoter, again suggesting that the SHBG receptor is coupled to G proteins (34, 35).
For steroids to induce cAMP through SHBG and the SHBG receptor, SHBG must first bind to the SHBG receptor and then bind to the steroid. SHBG molecules already bound to steroid are not able to interact with the SHBG receptor (35, 38). Androgens and E2 are able to rapidly induce cAMP. In MCF-7 cells, DHT and E2 can induce cAMP within 5 and 15 min, respectively (39, 40). Similarly, in LNCaP cells both E2 and DHT can increase cellular cAMP within 15 min (41). In serum-free media or in fetal calf serum containing media from which SHBG has been removed, the addition of SHBG and the presence of cellular SHBG receptors is necessary for steroid induction of cAMP (31).
The induction of cAMP by SHBG results in the activation of PKA in both prostate and breast cancer cells (42, 43). AR transcriptional activity is enhanced by PKA stimulation, even at very low levels of androgen (44, 45, 46). Proliferation of the AR positive, ER negative ALVA-41 human prostate cancer cell line can be induced in response to both DHT and E2 (47, 48). In this cell line, SHBG is required by both steroids to increase cell growth (47, 48). In organ cultures of human prostate explants, the DHT induction of the AR target gene prostate-specific antigen (PSA) can be mimicked by stimulation of the SHBG-PKA pathway. PSA expression in this system can be induced by treatment with both SHBG and E2, whereas SHBG or E2 alone had no effect (43). E2-SHBG stimulation of PSA secretion was not inhibited by antiestrogens but was blocked by the antiandrogens cyproterone acetate (CPA) and hydroxyflutamide (HF) (43). Because the affinity of SHBG for DHT is 60% higher than for CPA or HF (49), E2-SHBG induction of PSA appears to function through transcriptional activation of AR in the absence of exogenous androgen (43), possibly through stimulation of PKA. Similar results have been obtained in a canine prostate explant model (50). Together, these observations suggest that the proliferation of prostate cells and the transcriptional activation of AR may be enhanced by DHT-SHBG interactions through DHT-SHBG induction of signal transduction cascades. In addition, normally weak adrenal androgens or E2 may contribute to AR transcriptional activity through stimulation of SHBG signaling.
AR does not contain a consensus PKA phosphorylation site and is not directly phosphorylated by PKA (44). However, the enhancement of AR transcription by activation of PKA in the presence of an intact SHBG signaling system may be through increased interaction of AR coregulators with AR (Fig. 1). Phosphorylation of steroid receptor coregulators has been found to influence their ability to interact with the steroid receptor or recruit other coregulatory complexes (27, 51). Although to date no AR coregulators have been found to be direct targets of PKA phosphorylation, SRC-1 has been shown to be phosphorylated by MAPK in response to activation of PKA (27). SRC-1 phosphorylation enhances its ability to increase steroid receptor transcriptional activity by facilitating SRC-1-CBP interaction (27). Therefore, it is possible that androgen-SHBG stimulation of PKA may result in alteration of the phosphorylation status of AR and AR coregulators and thus modulate AR transcriptional activity.
MEMBRANE ANDROGEN RECEPTORS
The existence of a novel membrane-bound androgen receptor has been postulated by a number of authors based on the detection of specific androgen binding to plasma membranes in different cell types (52, 53, 54). The ability of androgens to rapidly modulate the activity of ion channels and intracellular calcium levels has been observed in several cell types. However, it has not yet been determined whether these nongenomic effects are mediated through a membrane androgen receptor or are acting through SHBG or a c-Src kinase-AR complex. Unfortunately, this putative membrane receptor has not yet been further purified or cloned, preventing a definitive characterization. A human membrane receptor for progesterone has been cloned (55, 56) and a heteromeric membrane receptor for anabolic androgens has recently been isolated (57). The identification of distinct membrane receptors for other steroid hormones suggests a novel membrane receptor for androgens may also exist.
The ability of T to induce an increase in intracellular Ca2+ occurs rapidly and is linked to a G protein-coupled receptor. In T-cells and IC-21 macrophage cells, membrane binding of T induced an increase in intracellular of Ca2+ within seconds (53, 54). In IC-21 cells, the Ca2+ mobilization is due to release from intracellular Ca2+ stores and can be induced by T conjugated to BSA, which retards the free diffusion across the plasma membrane. IC-21 cells do not express the classical AR and Ca2+ mobilization in these cells is insensitive to CPA or HF. However, the T-induced Ca2+ increase is sensitive to the G protein receptor antagonist pertussis toxin, suggesting that the membrane androgen-binding protein in these cells is either a G protein receptor or its function is closely linked to one (53). Intracellular Ca2+ is also increased in murine splenic T-cells within seconds of treatment with T or a T-BSA conjugate (54, 58). T-cells possess a very low level of AR, but the AR that is present apparently is unable to translocate to the nucleus, suggesting they may not be transcriptionally active (54). In contrast to IC-21 cells, the increase in intracellular Ca2+ in T-cells results from an influx through nonvoltage-gated Ca2+ channels. However, T induction of Ca2+ influx is also sensitive to pertussis toxin and insensitive to CPA, suggesting that this effect is not mediated by AR in T-cells, but rather by a G protein-coupled receptor (54, 58).
The ability of physiological levels of T to induce an influx of Ca2+ has also been reported in primary cultures of rat Sertoli cells and osteoblasts (59, 60). In contrast to T-cells, Ca2+ influx in these cell types is sensitive to verapamil indicating the involvement of voltage gated Ca2+ channels. The ability of T or T-BSA to increase cellular Ca2+ in osteoblasts is sexually dimorphic. T is only able to induce Ca2+ influx in male primary osteoblasts but not female-derived cells (60). Ca2+ influx in female osteoblasts can be mediated nongenomically by E2, whereas male osteoblasts are E2 insensitive (60). The basis of this sexual dimorphism is not known, but it is possible that separate membrane receptors for E2 and T exist (10).
Androgen-mediated stimulation of voltage-linked Ca2+ channels has also been reported in Sertoli cells and LNCaP cells (61, 62). In LNCaP cells, intracellular Ca2+ increases less than a minute after the addition of DHT (61). Intracellular Ca2+ increases in Sertoli cells within 4 min of T treatment (62). Although T induction of Sertoli cell Ca2+ influx could be inhibited by HF, it was not influenced by CPA (62). It remains to be determined whether these effects are mediated through a novel androgen-binding protein or whether not all AR transcriptional antagonists function as antagonists of nongenomic AR function.
Ca2+ functions as an ubiquitous second messenger and modulation of intracellular Ca2+ levels regulates a wide range of cellular processes, including proliferation, apoptosis, motility, and gene expression (63). The elevation of intracellular Ca2+ is detected by specific Ca2+ sensor molecules, including PKC and calmodulin (CaM), to induce signal transduction cascades and modulation of transcription factor activity (64) (Fig. 1). The specific cellular response to elevation of Ca2+ levels is highly dependent of the strength and duration of the Ca2+ spike (63, 65). In the case of androgen induction of Ca2+ mobilization, relatively little is known about the ultimate cellular effect. The sustained elevation of intracellular Ca2+ with Ca2+ ionophores or inhibitors of Ca2+-ATPase have been found to reduce AR expression (66) and promote apoptosis in prostate cancer cells (67). The effect of physiological exposure to androgen on Ca2+-mediated functions remains to be investigated.
It is unclear whether the G protein-coupled receptor mediating intracellular calcium levels in osteoblasts (60), IC-21 and T-cells (53, 54) is distinct from the SHBG receptor, which has also been suggested to be coupled to G proteins (34, 35). The SHBG receptor is associated with GS containing G protein complexes (34, 35). However, the intracellular Ca2+ increase in T- cells and IC-21 cells is sensitive to pertussis toxin, which does not inhibit the G
S subfamily (68). A number of receptors are known to be coupled to more than one G
subfamily (69). Therefore, it remains to be determined whether androgen action via G protein-coupled receptors occurs through a single receptor coupled to different G
subfamilies, possibly in a tissue- specific manner, or through separate receptors each linked to specific G protein complexes.
SUMMARY
The nongenomic action of androgens can be mediated by at least two androgen-binding proteins, the classical nuclear receptor for androgens AR, and SHBG. In both of these cases, the biological effect of the nongenomic stimulation of second messenger cascades may be the enhancement of AR transcriptional activation. Androgens also interact with a plasma membrane binding protein that may constitute a novel AR. Plasma membrane androgen-binding proteins have been associated with the modulation of intracellular calcium levels. The observations of nongenomic androgen-mediated calcium increase appears to occur through different mechanisms in different cell types. It has yet to be determined if this represents the function of different androgen-binding proteins with a distinct cell type distribution or if there is one membrane androgen-binding protein that conveys its signal in a manner subject to other cell type-specific differences. Future studies will need to address the extent to which the three nongenomic mechanisms of androgen action interact to result in a specific rapid androgen effect in a particular cell type. The cloning of the SHBG receptor and the androgen membrane-binding protein will greatly help to clarify these issues. The isolation of these membrane receptors will facilitate the development of antagonists of nongenomic androgen action. Such antagonists would be useful not only in the clarification of the biological significance and nongenomic steroid action but may also be useful therapeutically to modulate androgen action.
The delay in the isolation and cloning of the SHBG receptor and the membrane androgen receptor may be related to receptor solubility and/or lability, as has been reported for the aldosterone membrane receptor (8) and the membrane form of the ER (70). Photoaffinity labeling using T or DHT may assist in the purification of these membrane receptors and has been successfully used to isolate a heteromeric membrane complex that binds 17-alkylated androgens (57). It is possible that members of this complex may contribute to membrane binding of T and DHT. The functional linkage between some nongenomic androgen effects and G protein-coupled receptors suggest that membrane androgen receptors may be G protein-coupled receptors. Over 20 G protein-coupled receptors have been cloned using mammalian expression strategies with radiolabeled ligands (71), and this strategy may be useful in the isolation of membrane androgen receptors. Finally, the human membrane PR has been cloned and is possibly a member of a gene family (55). The ligand binding domains of the classical nuclear PR and AR are the most homologous among steroid nuclear receptors in terms of amino acid sequence identity and crystallographic structure (72, 73). It is possible that the membrane androgen receptor may also show homology to the membrane PR.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Yi-Fen Lee of the University of Rochester Department of Urology and Loretta L. Collins of the University of Rochester Department of Pathology for thoughtful and helpful discussions about the manuscript.
FOOTNOTES
Abbreviations: AR, Androgen receptor; CPA, cyproterone acetate; c-Src, p60c-src tyrosine kinase; DHT, 5-dihydrotestosterone; E2, estradiol; ER, estrogen receptor; HF, hydroxyflutamide; PKA, protein kinase A; PKC, protein kinase C; PR, progesterone receptor; PSA, prostate-specific antigen; SH2, Src homology 2; SH3, Src homology 3; SHBG, sex hormone binding globulin; SRC, steroid receptor coactivator; T, testosterone.
Received for publication February 14, 2002. Accepted for publication July 5, 2002.
REFERENCES