Abstract
Simple SummaryPro-androgenic substances such as testosterone are often used to treat muscle- or bone-related disorders. Their interactions with the classical androgen receptor, however, can trigger a number of undesirable effects. It would therefore be of great benefit if the positive androgenic effects could be obtained by circumventing the classical androgen receptor. ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance. Using in silico methods, we identified and verified the extracellular localization of its androgen binding site and designed small peptides that fit in it that do not interact with the AR. All peptides were found to be pro-androgenic; they stimulate mineralization in osteoblastic cells and myogenesis in myoblasts. Thus, these peptides might serve as testosterone surrogates in the treatment of osteogenic or myogenic disorders.ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance that may mediate certain physiological responses to androgens. Using in silico methods, six tetrapeptides with the best docking properties at the testosterone binding site of ZIP9 were synthesized and further investigated. All tetrapeptides displaced T-BSA-FITC, a membrane-impermeable testosterone analog, from the surface of mouse myogenic L6 cells that express ZIP9 but not the classical androgen receptor (AR). Silencing the expression of ZIP9 with siRNA prevented this labeling. All tetrapeptides were found to be pro-androgenic; in L6 cells they stimulated the expression of myogenin, triggered activation of focal adhesion kinase, and prompted the fusion of L6 myocytes to syncytial myotubes. In human osteoblastic SAOS-2 cells that express AR and ZIP9, they reduced the expression of alkaline phosphatase and stimulated mineralization. These latter effects were prevented by silencing ZIP9 expression, indicating that the osteoblast/osteocyte conversion is exclusively mediated through ZIP9. Our results demonstrate that the synthetic tetrapeptides, by acting as ZIP9-specific androgens, have the potential to replace testosterone or testosterone analogs in the treatment of bone- or muscle-related disorders by circumventing the undesirable effects mediated through the classical AR.
Highlights
The physiological effects of testosterone are mediated through the classical nuclear androgen receptors (AR), and through membrane-bound receptors that trigger various cytosolic signaling cascades upon binding of the steroid hormone
Three models were generated for ZIP9 protein using the SWISS-MODEL server and a structural comparison was made (Table 1)
In the last two decades, great efforts have been made to develop and characterize selective androgen receptor modulators (SARMs) that can activate their receptor in a tissuespecific manner and promote the beneficial effects of androgens while avoiding unwanted side effects as much as possible [59]
Summary
The physiological effects of testosterone are mediated through the classical nuclear androgen receptors (AR), and through membrane-bound receptors that trigger various cytosolic signaling cascades upon binding of the steroid hormone. One of these receptors is ZIP9, a zinc transporter from the family of ZRT/IRT-like proteins ZIP9 mediates testosterone signaling events of physiological relevance through interactions with G-proteins [1,2,3]. It is known that in spermatogenic cells the non-classical signaling of testosterone, which involves the activation of a signaling cascade that contains the elements Erk1/2, CREB, and ATF-1 [4], is mediated through ZIP9. Recent investigations demonstrate a direct involvement of ZIP9 in the proliferation and migratory activity of the human metastatic prostate cancer cell line LNCaP [8]
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