F-spondin regulates neuronal survival through activation of disabled-1 in the chicken ciliary ganglion.

The extracellular membrane-associated protein F-spondin has been implicated in cell-matrix and cell-cell adhesion and plays an important role in axonal pathfinding. We report here that F-spondin is expressed in non-neuronal cells in the embryonic chicken ciliary ganglion (CG) and robustly promotes survival of cultured CG neurons. Using deletion constructs of F-spondin we found that the amino-terminal Reelin/Spondin domain cooperates with thrombospondin type 1 repeat (TSR) 6, a functional TGFß-activation domain. In ovo treatment with blocking antibodies raised against the Reelin/Spondin domain or the TSR-domains caused increased apoptosis of CG neurons during the phase of programmed cell death and loss of about 30% of the neurons compared to controls. The Reelin/Spondin domain receptor - APP and its downstream signalling molecule disabled-1 are expressed in CG neurons. F-spondin induced rapid phosphorylation of disabled-1. Moreover, both blocking the central APP domain and interference with disabled-1 signalling disrupted the survival promoting effect of F-spondin. Taken together, our data suggest that F-spondin can promote neuron survival by a mechanism involving the Reelin/Spondin and the TSR domains.

TGFbeta induces GDNF responsiveness in neurons by recruitment of GFRalpha1 to the plasma membrane.

We have previously shown that the neurotrophic effect of glial cell line-derived neurotrophic factor (GDNF) in vitro and in vivo requires the presence of transforming growth factor (TGF)beta. Using primary neurons (chick E8 ciliary) we show that the combination of GDNF plus TGFbeta promotes survival, whereas the single factors do not. This cooperative effect is inhibited by blocking the extracellular signal-regulated kinase (ERK)/MAPK pathway, but not by interfering with the PI3 kinase signaling cascade. Although there is no functional GDNF signaling in the absence of TGFbeta, pretreatment with TGFbeta confers GDNF responsiveness to the cells. This is not due to upregulation of GDNF receptors mRNA and protein, but to TGFbeta-induced recruitment of the glycosyl-phosphatidylinositol-anchored GDNF receptor (GFR)alpha1 to the plasma membrane. This is supported by the fact that GDNF in the presence of a soluble GFRalpha1 can promote survival in the absence of TGFbeta. Our data suggest that TGFbeta is involved in GFRalpha1 membrane translocation, thereby permitting GDNF signaling and neurotrophic effects.

Rapid signalling by androgen receptor in prostate cancer cells.

Androgens are important growth regulators in prostate cancer. Their known mode of action in target cells requires binding to a cytoplasmic androgen receptor followed by a nuclear translocation event and modulation of the expression of specific genes. Here, we report another mode of action of this receptor. Treatment of androgen responsive prostate cancer cells with dihydrotestosterone leads to a rapid and reversible activation of mitogen-activated protein kinases MAPKs (also called extracellular signal-regulated kinases or Erks). Transient transfection assays demonstrated that the androgen receptor-mediated activation of MAP kinase results in enhanced activity of the transcription factor Elk-1. This action of the androgen receptor differs from its known transcriptional activity since it is rapid and insensitive to androgen antagonists such as hydroxyflutamide or casodex. Biochemical studies as well as analyses with dominant negative mutants showed the involvement of kinases such as MAPK/Erk kinase, phosphatidyl-inositol 3-kinase and protein kinase C in the androgen receptor-mediated activation of MAP kinase. These results demonstrate a novel regulatory action of the androgen receptor and prove that in addition to its known transcriptional effects, it also uses non-conventional means to modulate several cellular signalling processes.

Mechanism of androgen receptor activation and possible implications for chemoprevention trials.

Androgens are pivotal regulators of prostate cell growth, differentiation and function, and their actions are believed to be involved in prostate cancer development. The androgen-signaling pathway in the prostate gland is therefore one of the possible sites of intervention in prostate cancer prevention efforts. The central element of androgen signaling in the cell is the androgen receptor (AR), a member of the superfamily of nuclear receptors. Binding of androgen to its ligand-binding domain transforms the receptor to an active transcription factor that regulates gene expression by interacting with specific regulatory elements in the promoters of genes. In addition to this genomic action, the AR also interacts with other signaling pathways through protein-protein interaction, for example with AP-1 or Ets transcription factors. It is not only the action of androgenic hormones, but also the interactions with growth factor and protein kinase A-signaling pathways that can induce activation of AR. Moreover, these ligand-independent activators act synergistically together with low concentrations of androgens. The effects of long-term androgen deprivation on androgen signaling have been investigated in the LNCaP cell culture system. Long-term culture in a steroid-free medium results in a subline showing a hyperreactive AR characterized by increased AR expression and enhanced AR transcriptional activity in an environment with low levels of androgen hormones. It is not yet clear if similar changes also occur in normal or premalignant prostate epithelial cells and are thus relevant for prevention trials which interfere with androgen hormone signaling.

Expression, structure, and function of androgen receptor in advanced prostatic carcinoma.

BACKGROUND: Endocrine therapy for prostate cancer aims to reduce the levels of circulating androgen or to inhibit androgen action by blocking the androgen receptor in the prostate, or both. Studies in various animal and human prostate cancer models suggested that there may be a downregulation of androgen receptor during prostate cancer progression. Recent work, however, showed androgen receptor expression in all stages of prostate cancer. The presence of mutant androgen receptors in a portion of prostate cancers and receptor activation in the absence of androgen or in the presence of low androgen concentrations is discussed within this context. METHODS: This review attempts to summarize the literature on androgen receptor expression in vitro and in vivo, as well as structural and functional alterations and communication between androgen signal transduction cascade and other signaling pathways. CONCLUSIONS: Prostate tumors adapt to an environment with low androgen supply by using a hyperactive androgen receptor. The mechanisms involved are mutations of the androgen receptor generating receptors with broadened activation spectrum, increased receptor expression, and activation by interaction with other signaling pathways.

The androgen receptor as mediator of gene expression and signal transduction pathways.

The current model of action of androgens involves activation of a cytoplasmic receptor that migrates into the nucleus to regulate the expression of specific genes, either positively or negatively. While positive regulation requires direct binding of the receptor to DNA, negative regulation occurs mainly through protein-protein interactions of the receptor and other transcription factors. More recent findings have shown that the receptor also mediates non-conventional responses attributed hitherto only to activated growth factor receptors. These actions proceed principally through activation of cytoplasmic kinases and they suggest that in addition to its genomic functions, the androgen receptor also regulates non-genomic processes.

Hyperactive androgen receptor in prostate cancer: what does it mean for new therapy concepts?

Investigations on androgen signaling alterations in the late stages of prostate cancer revealed new molecular mechanisms that may be in part responsible for failure of endocrine therapy. Both primary and metastatic lesions from prostate cancer express androgen receptor protein. Amplification of androgen receptor gene occurs in a subset of prostate cancer patients. Several point mutations of androgen receptor gene have been described; they generate receptors which are functionally activated by androgens, other steroids, and even by antihormones. The frequency of androgen receptor mutations may be high in tumor metastases. Functional activity of androgen receptor is influenced by nonsteroidal factors, such as peptide growth factors and second messengers. Thus, prostate cancer cells adapt to low androgen environment by various mechanisms utilizing androgen receptor. Therefore, new strategies for switching off the androgen receptor are needed.

Androgen receptor-Ets protein interaction is a novel mechanism for steroid hormone-mediated down-modulation of matrix metalloproteinase expression.

Matrix metalloproteinases belong to a family of structurally related enzymes that plays important role in tissue morphogenesis, differentiation, and wound healing. Their expression is negatively regulated by several members of the steroid hormone receptor family. This is thought to occur through interaction of the steroid receptors with the transcription factor AP-1 that is otherwise required for positive regulation. Here, we demonstrate that AP-1 is not always a target for down-regulation of expression of matrix metalloproteinases by steroid receptors. Androgen receptor negatively regulates matrix metalloproteinase-1 expression not through AP-1 but through a family of Ets-related transcription factors that are also required for positive regulation. This negative regulation is specific for the androgen receptor. It does not require the DNA binding activity but needs amino-terminal sequences of the receptor. These results identify a novel regulatory pathway for negative regulation utilized by a member of the steroid hormone receptor family for down-regulating the expression of matrix metalloproteinases.

CAG-repeat expansion in androgen receptor in Kennedy's disease is not a loss of function mutation.

Expansion of CAG trinucleotide repeats in androgen receptor gene is present in patients with a rare X-linked inherited form of motor neuron disorder termed Kennedy's disease or spinal and bulbar muscular atrophy (SBMA). This is a late onset progressive disease often associated with mild signs of androgen insensitivity. Defects in androgen receptor (AR) action have been linked to the expansion of the CAG trinucleotide repeats and postulated to be the cause of the disease. We have identified a trinucleotide repeat of 45 in the N-terminus of the AR in two brothers with SBMA and several members in their family (range in the general population is 11-35). Treatment of the patients with androgens failed to improve their clinical symptoms and provided no hint of an anomalous function of the AR. Consistently, functional analysis of the mutant receptor showed hormone binding, transactivation and transrepression potentials identical to that of the wild-type receptor. These results together argue against SBMA being a loss of function mutation of the AR.

Activation of two mutant androgen receptors from human prostatic carcinoma by adrenal androgens and metabolic derivatives of testosterone.

The androgen receptor (AR) plays a central regulatory role in prostatic carcinoma and is a target of androgen ablation therapy. Recent detection of mutant receptors in tumor specimens suggest a contribution of AR alterations to progression towards androgen independence. In a specimen derived from metastatic prostate cancer we have reported a point mutation in the AR gene that leads to a single amino acid exchange in the ligand binding domain of the receptor. Another amino acid exchange resulting from a point mutation was also identified 15 amino acids away from our mutation. This mutation was detected in the AR gene isolated from an organ-confined prostatic tumor. Here we report the functional characterization of the two mutant receptors in the presence of adrenal androgens and testosterone metabolites. These studies were performed by cotransfecting androgen-responsive reporter genes and either the wild-type or mutant AR expression vectors into receptor negative DU-145 and CV-1 cells. The indicator genes used consisted of the promoter of the androgen-inducible prostate-specific antigen gene or the C' Delta9 enhancer fragment from the promoter of the mouse sex-limited protein driving the expression of the bacterial chloramphenicol acetyl transferase gene. Cotransfection-transactivation assays revealed that the adrenal androgen androstenedione and two products of testosterone metabolism, androsterone and androstandiol, induced reporter gene activity more efficiently in the presence of the mutant receptors than in the presence of the wild-type receptor. No difference between wild-type and mutant receptors was observed in the presence of the metabolite androstandione. The interaction of receptor-hormone complexes with target DNA was studied in vitro by electrophoretic mobility shift assays (EMSA). Dihydrotestosterone and the synthetic androgen mibolerone induced a faster migrating complex with all receptors, whereas the androgen metabolite androstandione induced this complex only with the two mutant receptors. Androsterone and androstandiol were inactive in the EMSA. These aberrant properties of the mutant receptors in the presence of adrenal androgens and products of androgen metabolism may be of importance in the course of the prostate cancer, especially during androgen ablation therapy.

Mutant androgen receptors in prostatic tumors distinguish between amino-acid-sequence requirements for transactivation and ligand binding.

Mutant androgen receptors are thought to contribute to hormone resistance in prostate carcinoma. The part they play in this process, however, is ill-defined. Here we report on transactivation by 2 mutant androgen receptors from prostatic tumors with single amino-acid exchanges in their hormone-binding domains. These exchanges enhance the transactivation property of the receptors, particularly to androsterone and androstanediol, 2 metabolized derivatives of testosterone present in the prostate. Additionally, they enhance the transactivation potential of the mutant receptors to hydroxyflutamide, an anti-androgen frequently used in hormone ablation therapy. The increased transactivation by the mutant receptors did not result from altered affinity of the receptors to the inducing ligands nor from measurable changes in conformation of the liganded receptors. Thus the single amino-acid exchanges identify differences in amino-acid-sequence requirements for transactivation and ligand binding in the hormone-binding domain of the androgen receptor. These results provide new insights into ligand-dependent transactivation, and form a framework for the search for effective antagonists to be used in prostate-cancer therapy.