Regulation of IkappaB kinase epsilon expression by the androgen receptor and the nuclear factor-kappaB transcription factor in prostate cancer

Although several genes have been associated with prostate cancer progression, it is clear that we are far from understanding all the molecular events implicated in the initiation and progression of the disease to a hormone-refractory state. The androgen receptor is a central player in the initiation and proliferation of prostate cancer and its response to hormone therapy. Nuclear factor-kappaB has important proliferative and antiapoptotic activities that could contribute to the development and progression of cancer cells as well as resistance to therapy. In this study, we report that IkappaB kinase epsilon (IKKepsilon), which is controlled by nuclear factor-kappaB in human chondrocytes, is expressed in human prostate cancer cells. We show that IKKepsilon gene expression is stimulated by tumor necrosis factor-alpha treatment in LNCaP cells and is inhibited by transfection of a dominant-negative form of IkappaBalpha, which prevents the nuclear translocation of p65. Furthermore, we found that tumor necrosis factor-alpha-induced IKKepsilon expression is inhibited by an androgen analogue (R1881) in androgen-sensitive prostate cancer cells and that this inhibition correlates with the modulation of IkappaBalpha expression by R1881. We also noted constitutive IKKepsilon expression in androgen-independent PC-3 and DU145 cells. To our knowledge, this is the first report of an IkappaB kinase family member whose expression is modulated by androgen and deregulated in androgen receptor-negative cells.     

Identification of direct genomic targets downstream of the nuclear factor-kappaB transcription factor mediating tumor necrosis factor signaling

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-kappaB (NF-kappaB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kappaB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kappaB inhibitor to systematically identify NF-kappaB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kappaB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F)<0.001) and demonstrated a change in signal intensity of+/-3-fold relative to control. Of these, 28 were NF-kappaB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-kappaB-dependent genes and five genes not previously known to be NF-kappaB-dependent (Gro-beta and-gamma, IkappaBepsilon, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-kappaB regulated. Expression of constitutively active enhanced green fluorescent.NF-kappaB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-kappaB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IkappaBalpha/epsilon, Gro-beta/gamma, and Naf-1 promoters directly bound NF-kappaB/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-kappaB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.     

Gene expression of isoformic enzymes in arachidonate cyclooxygenase pathway and the regulation by tumor necrosis factor alpha during life cycle of adipocytes

Adipocytes serve not only as a storage depot of fats but also as endocrine cells secreting adipocytokines including tumor necrosis factor alpha (TNFalpha). Using preadipogenic 3T3-L1 cells, we attempt to determine the response of adipocytes at different stages of the life cycle to TNFalpha with respect to the gene expression of the arachidonate cyclooxygenase (COX) pathway and the role of endogenous prostaglandins (PGs). The gene expression analysis of the COX pathway revealed the marked increase in mRNA and protein levels of COX-2 in response to TNFalpha in preadipocytes, whereas COX-1 was expressed constitutively. Moreover, the cells at different cycle stages exhibited the specific gene expression of isoformic enzymes of prostaglandin (PG) synthases for PGs of the D(2), E(2), and F(2alpha) series upon exposure to TNFalpha. The treatment of preadipocytes with TNFalpha along with calcium ionophore A23187 resulted in the stimulated formation of PGE(2) and PGF(2alpha), attenuating the apoptotic cell death induced by TNFalpha alone. The response of adipocytes to synthesize these PGs declined during the differentiation and maturation phases. The cells during the differentiation phase were the most sensitive to TNFalpha in terms of the decrease in adipogenesis without the mediation of endogenous PGs. TNFalpha was also effective in suppressing adipogenesis during the maturation process. Taken together, TNFalpha can control cell number of preadipocytes as well as the size of fat storage in mature adipocytes. The action of TNFalpha on preadipocytes can be modulated by the production of endogenous PGs through the induction of COX-2.     

Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha

The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-kappaB ligand (RANKL). Tumor necrosis factor-alpha (TNF-alpha) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of alpha-lipoic acid (alpha-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, alpha-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-alpha (RANKL/TNF-alpha). alpha-LA abolished ROS elevation by RANKL or RANKL/TNF-alpha and inhibited NF-kappaB activation in osteoclast precursor cells. Specifically, alpha-LA reduced DNA binding of NF-kappaB but did not inhibit IKK activation. Furthermore, alpha-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-alpha in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-kappaB regulation and the antioxidant alpha-lipoic acid has a therapeutic potential for bone erosive diseases.     

Aggression and arginine vasopressin immunoreactivity regulation by androgen receptor and estrogen receptor alpha

In the following study, we asked which steroid receptors regulate aggression and arginine vasopressin (AVP) immunoreactivity (– ir) in several limbic regions. Using spontaneous mutant and knockout mice, we generated a novel cross of mice whose offspring lacked estrogen receptor α (ERα), androgen receptor (AR) or both ERα and AR. The wild-type (WT) males and females were compared with ERα knockout (ERαKO) male, mutated AR (Tfm) male and ERαKO/Tfm (double knockout; DKO) male littermates. Animals were gonadectomized and treated with 17β-estradiol (E2) prior to resident-intruder aggression tests. WT and Tfm males showed aggression whereas WT females, ERαKO and DKO males did not. In the lateral septum, WT and Tfm male brains had significantly denser AVP-ir as compared with WT females and DKO males. ERαKO male brains were intermediate in the amount of AVP-ir present. In the medial amygdala, brains from all genotypes had equivalent AVP-ir, except DKO males, which had significantly less AVP-ir. Overall, the expression of aggressive behavior coincided with AVP-ir in WT, Tfm and DKO males. However, in ERαKO males and WT females, the amount of AVP-ir was not associated with resident-intruder aggression. In sum we have shown that E2 acts via ERα to regulate aggression in male mice. In contrast both ERα and AR contribute to AVP-ir in limbic brain regions.     

Tumor necrosis factor receptor signaling. A dominant negative mutation suppresses the activation of the 55-kDa tumor necrosis factor receptor.

To investigate the signaling mechanism of the 55-kDa tumor necrosis factor (TNF) receptor a functional transfection based assay was developed. The human 55-kDa TNF receptor, stably expressed in mouse L929 cells, was demonstrated to be activated specifically by agonist antibodies and to initiate a signal for cellular cytotoxicity. A deletion mutant of the human TNF receptor lacking most of the cytoplasmic domain was found to be completely defective in generating the signal for cytotoxicity. Additionally, expression of the truncated receptor substantially suppressed signaling by endogenous mouse TNF receptors in response to TNF, but not in response to specific anti-murine TNF receptor antibodies. These results suggest that aggregation of 55-kDa TNF receptor intracellular domains, which are not associated in the absence of ligand, is an important component of the signal for cellular toxicity. This work also provides an example of a dominant negative mutation in a transmembrane receptor that lacks a tyrosine kinase domain, and suggests a more general utility of dominant negative mutations in the investigation of cytokine receptor function.     

SIMPL is a tumor necrosis factor-specific regulator of nuclear factor-kappaB activity

The IL-1 receptor-associated kinase (IRAK/mPLK) is linked to the regulation of nuclear factor-kappaB (NF-kappaB)-dependent gene expression. Here we describe a novel binding partner of IRAK/mPLK that we term SIMPL (signaling molecule that associates with the mouse pelle-like kinase). Overexpression of SIMPL leads to the activation of NF-kappaB-dependent promoters, and inactivation of SIMPL inhibits IRAK/mPLK as well as tumor necrosis factor receptor type I-induced NF-kappaB activity. Dominant inhibitory alleles of IkappaB kinase (IKKalpha or IKKbeta) block the activation of NF-kappaB by IRAK/mPLK and SIMPL. Furthermore, SIMPL binds IRAK/mPLK and the IKKs in vitro and in vivo. In the presence of antisense mRNA to SIMPL, the physical association between IRAK/mPLK and IKKbeta but not IRAK/mPLK and IKKalpha is greatly diminished. Moreover, dominant-negative SIMPL blocks IKKalpha- or IKKbeta-induced NF-kappaB activity. These results lead us to propose a model in which SIMPL functions to regulate NF-kappaB activity by linking IRAK/mPLK to IKKbeta/alpha-containing complexes.     

Cytokine-mediated regulation of ovarian function. Tumor necrosis factor alpha inhibits gonadotropin-supported ovarian androgen biosynthesis

Resident ovarian macrophages have been implicated in the regulation of ovarian function, presumably through local paracrine secretion of regulatory molecules (i.e. cytokines). One such macrophage product, tumor necrosis factor (TNF) alpha, has been shown to attenuate the gonadotropin-dependent differentiation of the somatic ovarian (estrogen-producing) granulosa cell. This study examines the possibility that TNF alpha may also regulate the adjacent androgen-producing theca interstitial cell. The basal accumulation of androsterone (the major androgenic steroid), synthesized by whole ovarian dispersates from immature rats, remained unchanged following treatment with TNF alpha (30 ng/ml) alone. In contrast, concurrent treatment with increasing concentrations of TNF alpha (0.03-30 ng/ml), yielded dose-dependent inhibition of the human chorionic gonadotropin (1 ng/ml)-stimulated accumulation of androsterone. This reversible and immunoneutralizable effect of TNF alpha was characterized by a minimal effective dose of 0.1 ng/ml, a median inhibitory dose of 0.9 ng/ml, a maximal inhibitory effect of 90%, and a minimal time requirement of less than or equal to 48 h. Comparable results were obtained when using highly purified theca interstitial cells, thereby indicating that TNF alpha is capable of exerting a direct inhibitory effect at the level of the ovarian androgen-producing cell. TNF alpha action was not accounted for by alterations in the plated viable cell mass. Instead, treatment with TNF alpha resulted in significant inhibition of the human chorionic gonadotropin-supported accumulation of cAMP, the putative second messenger of gonadotropin hormonal action. TNF alpha action at sites distal to cAMP generation was associated with profound inhibition of the conversion of the [3H]pregnanolone (3 alpha-hydroxy,5 alpha-pregnane-20-one) and [3H]17 alpha-hydroxypregnanolone (3 alpha, 17 alpha-dihydroxy,5 alpha-pregnane-20-one) substrates to androsterone, suggesting stimulation of 20 alpha-hydroxysteroid dehydrogenase activity, inhibition of 17 alpha-hydroxylase/17:20 lyase activity, or both. Taken together, these findings indicate that TNF alpha, acting at relatively low concentrations, is capable of inhibiting gonadotropin-supported ovarian androgen biosynthesis by selectively modulating the activity of relevant key steroidogenic enzymes. As such, these observations suggest that the theca interstitial cell is a site of TNF alpha reception and action and that TNF alpha, possibly of resident ovarian macrophage origin, may partake in the regulation of ovarian androgen production, an effect due in part to inhibition of the activity of the key steroidogenic enzymes 17 alpha-hydroxylase/17:20 lyase.