Androgen has been shown to stimulate proliferation of osteoblast-like MC3T3-E1 cells. However, the molecular mechanism responsible for this effect remains to be elucidated. In the present study we demonstrate herein the non-genomic effect of androgen on osteoblast-like MC3T3-E1 cells involving activation of a PI(3)K/Akt signaling pathway and stimulating proliferation. In studies of steroids signaling, 5a-dihydrotestosterone (DHT), testosterone and 17b-estradiol but not dexamethasone or progesterone induced a rapid and transient phosphorylation of Akt in MC3T3-E1 cells. The androgen-induced Akt activation reached to the climax after 15 min and gradually diminished to baseline after 60 min. This induction of androgen was unaffected by actinomycin D and was specifically blocked by androgen receptor (AR) antagonist hydroxyflutamide (HF) or transfection of siRNA-AR. Treatment of MC3T3-E1 cells with PI(3)K inhibitor LY294002 or transfection with kinase-deficient Akt blocked androgen-induced cells proliferation. Moreover, androgen-induced activation of Akt was abolished by inhibitors of Src kinase, Gi-protein and phospholipase C showing the involvement of these effectors in androgen signaling pathway. Further, androgen-induced activation of Akt was dependent on intracellular calcium as shown by the effect of EGTA and intracellular calcium chelator BAPTA/AM. Fluorescence microscopy showed translocation of phospho-Akt from cytosol into nucleus after androgen treatment but no change in the subcellular distribution of phospho-Akt when HF or LY294002 pretreatment was administered to the cells. These results strongly suggest that phosphorylation of Akt in osteoblast cells is mediated by androgen receptor and the androgen-induced translocation of Akt is an important step in the androgen/AR signaling pathway that mediates osteoblast cells proliferation.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 71-83).

Steroid hormones play an important role in modulating behavioral responses to various social stimuli. However, relatively little is known about how hormones respond to social stimuli and their modulation of subsequent behavior. Variation in the hormonal regulation of behavior across species has complicated the overall understanding of the hormone-behavior dynamic. In order to further elucidate the interplay of hormones and behavior in social situations, we exposed males of the monogamous convict cichlid Amatitliana nigrofasciata to three social stimuli: gravid female, intruder male and nonsocial neutral stimulus. We used a repeated exposure paradigm to create behavioral profiles and explore how sex steroid hormones respond to and regulate social behavior. Results show clear behavioral profiles in different social situations with 11-KT acting as the active androgen, increasing in response to social stimuli. Pharmacological manipulations using androgen and estrogen receptor agonists and antagonists exposed complex control over digging behavior based on social context, showed a unique decrease in aggressive behavior due to blocking the androgen receptors and a ubiquitous drug effect on vertical display. Results create well defined context-specific behavior profiles and extends our understanding of particular social behavior and how sex steroid hormones are involved in social situations and the behavioral response. / text

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity.

During the lifetime of an organism, key events are orchestrated by a confluence of environmental, social, and physiological factors to promote reproductive success. Steroid hormones are critical regulators of fundamental aspects of reproductive life history, including gametogenesis, secondary sexual characteristics, sexual behavior, territory establishment and defense, and parenting. The steroid hormones investigated herein (testosterone (T), 11-ketotestosterone (KT), 17b-estradiol (E2) and cortisol) are linked through steroidogenic conversion pathways. This dissertation utilized an integrative approach to investigate the neuroendocrine and social contexts that regulate transitions among phenotypes in a bi-directionally hermaphroditic haremic fish, Lythrypnus dalli. Conventional sex roles are reversed, such that only males provide nest care, females exhibit intra-sexual competition and male reproductive success is associated with female courtship solicitation. Females living in stable social groups maintain dramatic differences in status, morphology, and tissue T, KT, E2, and cortisol. Parasitic male morphs, mini males, do not defend territories and have morph-typical water-borne and tissue profiles of T, E2, and KT. Two life history transitions, socially induced sex change and male parenting, are associated with increase in rates of behavior and KT levels. The regulation of these life history transitions by KT was investigated via two types of endocrine manipulations. Coupling systemic KT implants with a social context permissive to sex change caused rapid, but transient effects on agonistic behavior in dominant females, and secondary effects on subordinates during a period of social instability. Despite elevated brain and systemic KT 5 d after implant, overall rates of aggressive behavior remained unaffected, demonstrating a key role for context in regulating steroid associated changes in behavior. Intracerebroventricular inhibition of the enzyme 11b-hydroxysteroid dehydrogenase, reduced KT, elevated cortisol, and reduced male parenting behavior. 11-Ketotestosterone rapidly rescued parenting when administered along with the inhibitor, while cortisol had no effects on parenting. During reduced male nest attendance caused by KT inhibition, dominant, but not subordinate females, exhibited transient parenting and elevated brain KT. Taken together, rapid and/or local modulation of steroids allows for context-specific regulation of dynamic changes in behavior in an environment that requires an organism to successfully coordinate multiple activities to enhance fitness.

During the lifetime of an organism, key events are orchestrated by a confluence of environmental, social, and physiological factors to promote reproductive success. Steroid hormones are critical regulators of fundamental aspects of reproductive life history, including gametogenesis, secondary sexual characteristics, sexual behavior, territory establishment and defense, and parenting. The steroid hormones investigated herein (testosterone (T), 11-ketotestosterone (KT), 17b-estradiol (E2) and cortisol) are linked through steroidogenic conversion pathways. This dissertation utilized an integrative approach to investigate the neuroendocrine and social contexts that regulate transitions among phenotypes in a bi-directionally hermaphroditic haremic fish, Lythrypnus dalli. Conventional sex roles are reversed, such that only males provide nest care, females exhibit intra-sexual competition and male reproductive success is associated with female courtship solicitation. Females living in stable social groups maintain dramatic differences in status, morphology, and tissue T, KT, E2, and cortisol. Parasitic male morphs, mini males, do not defend territories and have morph-typical water-borne and tissue profiles of T, E2, and KT. Two life history transitions, socially induced sex change and male parenting, are associated with increase in rates of behavior and KT levels. The regulation of these life history transitions by KT was investigated via two types of endocrine manipulations. Coupling systemic KT implants with a social context permissive to sex change caused rapid, but transient effects on agonistic behavior in dominant females, and secondary effects on subordinates during a period of social instability. Despite elevated brain and systemic KT 5 d after implant, overall rates of aggressive behavior remained unaffected, demonstrating a key role for context in regulating steroid associated changes in behavior. Intracerebroventricular inhibition of the enzyme 11b-hydroxysteroid dehydrogenase, reduced KT, elevated cortisol, and reduced male parenting behavior. 11-Ketotestosterone rapidly rescued parenting when administered along with the inhibitor, while cortisol had no effects on parenting. During reduced male nest attendance caused by KT inhibition, dominant, but not subordinate females, exhibited transient parenting and elevated brain KT. Taken together, rapid and/or local modulation of steroids allows for context-specific regulation of dynamic changes in behavior in an environment that requires an organism to successfully coordinate multiple activities to enhance fitness.

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity.

Prostate cancer is the second leading cause of death from cancer in Australian men. As prostate cancer cells are reliant on androgens for growth and survival, the standard therapy for metastatic disease is androgen ablation therapy (AAT). AAT inhibits androgen signalling by altering androgen synthesis or prevent binding of androgens to their intracellular mediator, the androgen receptor (AR). Although initially effective, virtually all patients relapse, beyond which there are limited treatment options. The failure of AAT is not necessarily due to a decreased requirement for androgen signalling, but rather the AR is able to maintain signalling and tumour growth in an androgen-depleted environment. Therefore novel strategies that directly target the AR may provide a more effective therapeutic approach. We have endeavoured to suppress AR activity in prostate cancer cells by utilising a dominant negative AR. The most effective dominant negative construct developed, ARi41O, lacks amino acids 39-410 in the AR amino terminal transactivation domain. In studies of transcriptional activity, ARi410 has no intrinsic activity but inhibits the activity of wild type AR (wtAR) and also clinically relevant AR variants, by up to 95%. The objective of this thesis was to characterise the mechanisms of action of ARi410 and assess the functional effects of introducing this dominant negative receptor into prostate cancer cells. To investigate the mechanism by which ARi410 suppresses AR activity, a robust and sensitive AR inhibition assay was developed. This assay revealed that ARi410 is a potent inhibitor of AR activity on three independent AR-regulated promoters, regardless of the level of AR expression. Furthermore, while ARi410 can inhibit AR activity, it does not alter AR protein levels. By using ARi410 variants with mutations and/or deletions in regions of functional importance, the AR inhibition assay was also used to identify the critical regions of ARi410 required for its dominant negative activity. These studies demonstrate that the dominant negative activity of ARi41 0 is ligand-dependent, requires dimerisation through the ligand binding domain (LBD) and an intact DNA-binding domain (DBD). Further investigation into the mechanism of dominant negative activity revealed that ARi410 does not alter the subcellular localisation of AR, as both receptors are predominantly cytoplasmic in the absence of ligand and rapidly co-localise to the nucleus in response to androgens. Furthermore, an interaction between AR and ARi410 was observed in the presence and absence of ligand, and electrophoretic mobility shift assays demonstrated that AR and ARi410 form heterodimers on DNA. These studies led to the conclusion that the mechanism of dominant negative activity by ARi4I0 involves the formation of inactive receptor heterodimers that assemble on DNA and suppress AR activity. To determine the functional consequence of expressing the dominant negative androgen receptor in prostate cancer cells, an adenoviral method of gene delivery was developed. Adenoviral expression of ARi410 in LNCaP prostate cancer cells did not allow assessment of cell viability due to cell-specific toxicity of the viral vectors when expressed long-term. However, short-term expression of ARi410 in LNCaP cells resulted in inhibition of AR signalling, as determined by reduced expression of the androgen regulated genes apolipoprotein D and kallikrein 2. Importantly, this finding is consistent with the inhibitory activity of ARi410 observed using synthetic AR-regulated reporter genes in the AR inhibition assay, and demonstrates that ARi410 can effectively suppress endogenous AR signalling. The results of this thesis indicate that heterodimerisation between AR and ARi410 is the most likely mechanism of dominant negative inhibition of AR function by ARi410, and that the DBD and dimerisation through the LBD are required for optimal dominant negative activity. Furthermore, this thesis has demonstrated that ARi410 is an effective inhibitor of AR signalling and provides a basis for further functional studies and evaluation of the dominant negative androgen receptor in vitro and in vivo. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1338478 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2009. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 May 17.