Y1 Adrenal Cells Research Articles

Regulation of adrenal and ovarian steroidogenesis by miR-132.

miR-132 is hormonally regulated in steroidogenic cells of the adrenal gland, ovary and testis. Here, we examined the potential role of miR-132 in the control of steroidogenesis. Transfection of Y1 adrenal cells with miR-132 increased mRNAs of 3β-HSD and 20α-HSD enzymes, which catalyze the sequential conversion of pregnenolone to progesterone to biologically inactive 20α-hydroxyprogesterone (20α-OHP). Overexpression of miR-132 reduced MeCP2 and StAR protein expression, basal progestin (progesterone and 20α-OHP) production, but enhanced their production in response to cAMP stimulation. Use of [3H] pregnenolone and free-diffusible 22(R)-hydroxycholesterol further confirmed that miR-132 promotes the production of 20α-OHP by upregulating 3β-HSD and 20α-HSD. Evidence is also presented that StAR is a direct target of miR-132. Transient transfection of Y1 cells with miR-132 demonstrated that miR-132 induction of 3β-HSD and 20α-HSD was accompanied by significant suppression of one of its target gene products, MeCP2. In contrast, co-expression of miR-132 plus MeCP2 protein partially blocked the ability of miR-132 to upregulate the expression and function of 3β-HSD and 20α-HSD. Moreover, suppression of MeCP2 protein with siRNA resulted in increased expression of 3β-HSD and 20α-HSD, further demonstrating that miR-132 induces the expression of these two enzymes via inhibition of MeCP2. Likewise, overexpression of miR-132 increased 20α-OHP production with and without HDL loading, while knockdown of miR-132 resulted in a significant decrease of 20α-OHP production by granulosa cells. In conclusion, our data suggest that miR-132 attenuates steroidogenesis by repressing StAR expression and inducing 20α-HSD via inhibition of MeCP2 to generate a biologically inactive 20α-OHP.

Cell-Specific Polymorphism and Hormonal Regulation of DNA Methylation in Scavenger Receptor Class B, Type I

The scavenger receptor class B, type I (SR-BI), is a cell-surface glycoprotein that mediates selective uptake of high density lipoprotein (HDL)-derived cholesteryl ester. SR-BI plays an important role in cellular delivery of cholesterol. Both human and rodent SR-BI are expressed most abundantly in the liver parenchymal cells and steroidogenic cells of the adrenal gland and gonads, where the selective pathway exhibits its highest activity. In steroidogenic cells, the expression of SR-BI is regulated by trophic hormones (adrenocorticotropic hormone or gonadotropins luteinizing hormone or follicle-stimulating hormone) in concert with the regulation of steroid hormone production. DNA methylation has been implicated in a large number of biological processes mainly by regulating gene expression. The SR-BI promoter contains one CpG island (CGI) in its promoter and seven CGIs in its intronic regions. Here, we studied the DNA methylation status of SR-BI gene and provide evidence that the DNA methylation is cell specific in this gene promoter as well as in intronic regions. The DNA methylation in the SR-BI promoter is subject to N(6), 2'-O-dibutyryladenosine3':5'-cyclic monophosphate regulation in mouse adrenal Y1 cells and mouse Leydig tumor cells (MLTCs). The seven intron CGIs are methylated differentially in Y1 cells, MLTCs, ovarian granulosa cells, and mouse liver hepa 1-6 cells. Our experiments raised the possibility that DNA methylation participates in hormonal regulation of SR-BI expression in a tissue-specific manner. We further suggest that the cell-specific DNA methylation in SR-BI intronic regions may be associated with specific biological function(s) of these regions, including regulation of gene expression.

A Novel Role of Salt-Inducible Kinase 1 (SIK1) in the Post-Translational Regulation of Scavenger Receptor Class B Type 1 Activity.

Salt-inducible kinase 1 (SIK1) is a serine/threonine kinase that belongs to the stress- and energy-sensing AMPK family of kinases. SIK1 expression is rapidly induced in Y1 adrenal cells in response to ACTH via the cAMP-PKA signaling cascade, and it has been suggested that an increased level of SIK1 expression inhibits adrenal steroidogenesis by repressing the cAMP-dependent transcription of steroidogenic proteins, CYP11A1 and StAR, by attenuating CREB transcriptional activity. Here we show that SIK1 stimulates adrenal steroidogenesis by modulating the selective HDL-CE transport activity of SR-B1. Overexpression of SIK1 increases cAMP-stimulated and SR-B1-mediated selective HDL-BODIPY-CE uptake in cell lines without impacting SR-B1 protein levels, whereas knockdown of SIK1 attenuated cAMP-stimulated selective HDL-BODIPY-CE uptake. SIK1 forms a complex with SR-B1 by interacting with its cytoplasmic C-terminal domain, and in vitro kinase activity measurements indicate that SIK1 can phosphorylate the C-terminal domain of SR-B1. Among potential phosphorylation sites, SIK1-catalyzed phosphorylation of Ser496 is critical for SIK1 stimulation of the selective CE transport activity of SR-B1. Mutational studies further demonstrated that both the intact catalytic activity of SIK1 and its PKA-catalyzed phosphorylation are essential for SIK1 stimulation of SR-B1 activity. Finally, overexpression of SIK1 caused time-dependent increases in SR-B1-mediated and HDL-supported steroid production in Y1 cells; however, these effects were lost with knockdown of SR-B1. Taken together, these studies establish a role for SIK1 in the positive regulation of selective HDL-CE transport function of SR-B1 and steroidogenesis and suggest a potential mechanism for SIK1 signaling in modulating SR-B1-mediated selective CE uptake and associated steroidogenesis.

Low-dose Bisphenol A Activates Cyp11a1 Gene Expression and Corticosterone Secretion in Adrenal Gland via the JNK Signaling Pathway.

Certain commonly used compounds that interfere with the functions of the endocrine system are classified as endocrine-disrupting chemicals (EDCs). Bisphenol A (BPA) is an EDC that is widely used in food containers. BPA levels in human sera are commonly observed to be approximately 1-100 nM. Compared with the effects of BPA on the gonads, its effects on the adrenal gland are poorly understood. To investigate the influence of BPA on steroidogenesis, we examined the activity of the steroidogenic gene Cyp11a1 and its regulatory pathways in mouse Y1 adrenal cortex cells. Treatment with BPA at < 100 µM did not cause cell death. However, increased promoter activity and protein expression of Cyp11a1 were induced by low doses of BPA (10-1000 nM). Moreover, BPA induced c-Jun phosphorylation, and a specific inhibitor of c-Jun N-terminal kinase (JNK) significantly suppressed BPA-induced steroidogenesis. Thus, treatment of adrenal cells with low doses of BPA activated Cyp11a1 and increased corticosterone production through the JNK/c-Jun signaling pathway. Identical results were observed in rats after BPA injection. The abnormal induction of hormone synthesis by BPA in the adrenal gland might be linked to human metabolic defects and neuropsychiatric disorders.

Ectopically Expressed Pro-group X Secretory Phospholipase A2 Is Proteolytically Activated in Mouse Adrenal Cells by Furin-like Proprotein Convertases: IMPLICATIONS FOR THE REGULATION OF ADRENAL STEROIDOGENESIS

Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA2 is produced as a pro-enzyme (pro-GX sPLA2) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA2 activation have not been identified. We previously reported that GX sPLA2 negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA2 expression construct (FLAG-pro-GX sPLA2), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA2 processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA2 processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA2 processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA2 to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA2 processing and sPLA2 activity in Y1 cells, and it significantly attenuated GX sPLA2-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA2 is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.

Stimulation of StAR expression by cAMP is controlled by inhibition of highly inducible SIK1 via CRTC2, a co-activator of CREB

In mouse steroidogenic cells the activation of cholesterol metabolism is mediated by steroidogenic acute regulatory protein (StAR). Here, we visualized a coordinated regulation of StAR transcription, splicing and post-transcriptional processing, which are synchronized by salt inducible kinase (SIK1) and CREB-regulated transcription coactivator (CRTC2). To detect primary RNA (pRNA), spliced primary RNA (Sp-RNA) and mRNA in single cells, we generated probe sets by using fluorescence in situ hybridization (FISH). These methods allowed us to address the nature of StAR gene expression and to visualize protein–nucleic acid interactions through direct detection. We show that SIK1 represses StAR expression in Y1 adrenal and MA10 testis cells through inhibition of processing mediated by CRTC2. Digital image analysis matches qPCR analyses of the total cell culture. Evidence is presented for spatially separate accumulation of StAR pRNA and Sp-RNA at the gene loci in the nucleus. These findings establish that cAMP, SIK and CRTC mediate StAR expression through activation of individual StAR gene loci.

Corticotropin (ACTH) regulates alternative RNA splicing in y1 mouse adrenocortical tumor cells☆

The stimulatory effect of ACTH on gene expression is well documented and is thought to be a major mechanism by which ACTH maintains the functional and structural integrity of the gland. Previously, we showed that ACTH regulates the accumulation of over 1200 transcripts in Y1 adrenal cells, including a cluster with functions in alternative splicing of RNA. On this basis, we postulated that some of the effects of ACTH on the transcription landscape of Y1 cells are mediated by alternative splicing. In this study, we demonstrate that ACTH regulates the alternative splicing of four transcripts – Gnas, Cd151, Dab2 and Tia1. Inasmuch as alternative splicing potentially affects transcripts from more than two-thirds of the mouse genome, we suggest that these findings are representative of a genome-wide effect of ACTH that impacts on the mRNA and protein composition of the adrenal cortex.

Progress toward a novel pro‐drug approach for temporal manipulation of protein kinase A with a commercially available ruthenium catalyst (968.1)

Transition metal catalysts have been demonstrated in principle for temporal and spatial control of reactions, but this technology has yet to be broadly applied as a tool for biological study and cellular manipulation. Here we demonstrate the use of a ruthenium complex catalyst as a novel approach for spatial and temporal release of a pro‐drug inside cells, namely the transition of caged H89 to active H89, a Protein Kinase A (PKA) inhibitor. We synthesized caged H89 by protecting H89 as an allyl carbamate. We designed the system to intracellularly generate an active H89 inhibitor from caged H89 following addition of the ruthenium metal catalyst with the goal of inhibiting the catalytic activity of PKA and therefore reducing the phosphorylation of perilipin 1. The ruthenium catalyst activity was monitored by assessing the phosphorylation of perilipin 1, which is phosphorylated by PKA on 6 serine residues and not phosphorylated by other kinases. Experiments were performed on cell lines expressing endogenous perlipin 1, differentiated 3T3L1 cells and Y1 adrenal cells. Our results show that in its active state, H89 inhibited phosphorylation activity of PKA on substrates such as perilipin whereas the caged form of H89 lacked inhibitory activity. Experiments are planned for use of the ruthenium catalyst after the H89 and caged H89 incubation steps to determine the ability of the catalyst to activate caged H89 intracellularly.Grant Funding Source: Supported by The Royal Society of Chemistry

Abstract 432: Post-translational Regulation of Group X Secretory Phospholipase A2

Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes phospholipids within membrane bilayers, liberating free fatty acids and lysophospholipids. We reported previously that C57BL/6 mice deficient in GX sPLA2 (GX KO) have increased plasma corticosterone levels under both basal and adrenocorticotropic hormone (ACTH) stimulated conditions (Shridas et al. 2010, J. Biol. Chem. 285:20031-9). Conversely, overexpression of GX sPLA2 but not a catalytically inactive mutant, suppresses steroid production in mouse Y1 adrenal cells. GX sPLA2 is produced as a proenzyme that contains an N-terminal 11 amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase. While propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for GX sPLA2 activation have not been identified. In this study we report that ACTH treatment increases the phospholipase activity secreted by Y1 adrenal cells stably expressing GX sPLA2 (Y1-GX cells). Western blot analysis showed an increase in the ratio of mature/total GX sPLA2 in the media of ACTH-treated Y1-GX cells. Use of broad specificity proprotein convertase inhibitors confirmed the involvement of furin-like proteases in the processing and subsequent activation of GX sPLA2. Results from qRT-PCR analysis indicated that furin and PACE4, but not other members of the convertase family, are upregulated by ACTH in Y1 cells. siRNA-mediated gene targeting indicated that both furin and PACE4 mediate GX sPLA2 activation under both basal and ACTH-induced conditions. These results clearly demonstrate a role for furin and PACE4 in the proteolytic activation of GX sPLA2 in Y1 adrenal cells.

GPCR/EGFR Cross Talk Is Conserved in Gonadal and Adrenal Steroidogenesis but Is Uniquely Regulated by Matrix Metalloproteinases 2 and 9 in the Ovary

Previous work has demonstrated that cross talk between G protein-coupled LH receptors and epidermal growth factor receptors (EGFR) is essential for LH-induced steroid production in ovarian follicles and testicular Leydig cells. Here we demonstrate that G protein-coupled receptor (GPCR)/EGFR cross talk is also required for ACTH-induced steroidogenesis in Y1 adrenal cells. Moreover, we confirm that the signaling pathway from GPCR to Erk activation is conserved in all three steroidogenic tissues. ACTH or LH induces Gα(s), resulting in elevated cAMP and protein kinase A activation. cAMP/protein kinase A then triggers EGFR trans-activation, which promotes Erk signaling and subsequent steroidogenesis. Interestingly, although EGFR trans-activation is conserved in all three tissues, the specific mechanisms regulating this receptor cross talk differ. ACTH and LH trigger matrix metalloproteinase (MMP)-mediated release of EGFR ligands in adrenal and gonadal cells, respectively. However, this extracellular, ligand-dependent EGFR transactivation is required only for LH-induced steroidogenesis in ovarian follicles, reflecting the unique requirement of cell-cell cross talk for ovarian steroid production. Furthermore, MMP2 and MMP9 appear to regulate LH-induced steroidogenesis in mouse ovarian follicles, because a specific MMP2/9 inhibitor as well as the MMP2/9 inhibitor doxycycline suppress LH-induced follicular steroid production in vitro. Notably, although EGFR or MMP inhibition minimally affects estrous cycling in female mice, they attenuate ovarian steroidogenesis in response to LHR overstimulation in vivo. These results may have implications with regard to EGFR inhibitor use in various cancers as well as in polycystic ovarian syndrome, where excess LH-driven ovarian androgen production might be controlled by MMP2/9 inhibition.

Dax-1 and Steroid Receptor RNA Activator (SRA) Function as Transcriptional Coactivators for Steroidogenic Factor 1 in Steroidogenesis

The nuclear receptor steroidogenic factor 1 (SF-1) is essential for adrenal development and steroidogenesis. The atypical orphan nuclear receptor Dax-1 binds to SF-1 and represses SF-1 target genes. Paradoxically, however, loss-of-function mutations of Dax-1 also cause adrenal hypoplasia, suggesting that Dax-1 may function as an SF-1 coactivator under some circumstances. Indeed, we found that Dax-1 can function as a dosage-dependent SF-1 coactivator. Both SF-1 and Dax-1 bind to steroid receptor RNA activator (SRA), a coactivator that functions as an RNA. The coactivator TIF2 also associates with Dax-1 and synergistically coactivates SF-1 target gene transcription. A naturally occurring Dax-1 mutation inhibits this transactivation, and the mutant Dax-1-TIF2 complex mislocalizes in living cells. Coactivation by Dax-1 is abolished by SRA knockdown. The expression of the steroidogenic gene products steroidogenic acute regulatory protein (StAR) and melanocortin 2 receptor is reduced in adrenal Y1 cells following the knockdown of endogenous SRA. Similarly, the knockdown of endogenous Dax-1 downregulates the expression of the steroidogenic gene products CYP11A1 and StAR in both H295R adrenal and MA-10 Leydig cells. These findings reveal novel functions of SRA and Dax-1 in steroidogenesis and adrenal biology.

Fibroblast Growth Factor 2 Restrains Ras-Driven Proliferation of Malignant Cells by Triggering RhoA-Mediated Senescence

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant Y1 adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated beta-galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Y1 adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either Y1 or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Ras-dependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RhoA-GTP. Surprisingly, attempts to select FGF2-resistant cells from the Y1 and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Ras-dependent malignant cells could rarely overcome.

Activation of the SCPx promoter in mouse adrenocortical Y1 cells

Sterol carrier protein X (SCPx) is a peroxisomal protein with both lipid transfer and thiolase activity. Treatment of mouse adrenal Y1 cells with cAMP for 24 h caused a significant induction of SCPx mRNA levels. Reporter gene studies demonstrated that treatment with cAMP and SF-1 was able to activate the SCPx promoter. Sequence analysis revealed the presence of three putative steroidogenic factor-1 (SF-1) binding motifs (designated SFB1, SFB2, and SFB3) and one CRE. Only SFB1 and SFB3 were able to bind recombinant SF-1 protein in electrophoretic mobility shift assays. The CRE was able to form a DNA/protein complex in the presence of Y1 nuclear extracts. Mutational analysis studies demonstrated that SFB3 is required for full activation of the SCPx promoter by cAMP treatment. Regulation of the SCPx gene by SF-1 and cAMP is similar to the regulatory mechanisms observed for other steroidogenic genes.

Dynasore, a Cell-Permeable Inhibitor of Dynamin

Dynamin is essential for clathrin-dependent coated vesicle formation. It is required for membrane budding at a late stage during the transition from a fully formed pit to a pinched-off vesicle. Dynamin may also fulfill other roles during earlier stages of vesicle formation. We have screened about 16,000 small molecules and have identified 1, named here dynasore, that interferes in vitro with the GTPase activity of dynamin1, dynamin2, and Drp1, the mitochondrial dynamin, but not of other small GTPases. Dynasore acts as a potent inhibitor of endocytic pathways known to depend on dynamin by rapidly blocking coated vesicle formation within seconds of dynasore addition. Two types of coated pit intermediates accumulate during dynasore treatment, U-shaped, half formed pits and O-shaped, fully formed pits, captured while pinching off. Thus, dynamin acts at two steps during clathrin coat formation; GTP hydrolysis is probably needed at both steps.

Global Profiles of Gene Expression Induced by Adrenocorticotropin in Y1 Mouse Adrenal Cells

ACTH regulates the steroidogenic capacity, size, and structural integrity of the adrenal cortex through a series of actions involving changes in gene expression; however, only a limited number of ACTH-regulated genes have been identified, and these only partly account for the global effects of ACTH on the adrenal cortex. In this study, a National Institute on Aging 15K mouse cDNA microarray was used to identify genome-wide changes in gene expression after treatment of Y1 mouse adrenocortical cells with ACTH. ACTH affected the levels of 1275 annotated transcripts, of which 46% were up-regulated. The up-regulated transcripts were enriched for functions associated with steroid biosynthesis and metabolism; the down- regulated transcripts were enriched for functions associated with cell proliferation, nuclear transport and RNA processing, including alternative splicing. A total of 133 different transcripts, i.e. only 10% of the ACTH-affected transcripts, were represented in the categories above; most of these had not been described as ACTH-regulated previously. The contributions of protein kinase A and protein kinase C to these genome-wide effects of ACTH were evaluated in microarray experiments after treatment of Y1 cells and derivative protein kinase A-defective mutants with pharmacological probes of each pathway. Protein kinase A-dependent signaling accounted for 56% of the ACTH effect; protein kinase C-dependent signaling accounted for an additional 6%. These results indicate that ACTH affects the expression profile of Y1 adrenal cells principally through cAMP- and protein kinase A- dependent signaling. The large number of transcripts affected by ACTH anticipates a broader range of actions than previously appreciated.

Characterization ofl-arginine transport in adrenal cells: effect of ACTH

Nitric oxide synthesis depends on the availability of its precursor L-arginine, which could be regulated by the presence of a specific uptake system. In the present report, the characterization of the L-arginine transport system in mouse adrenal Y1 cells was performed. L-arginine transport was mediated by the cationic/neutral amino acid transport system y+L and the cationic amino acid transporter (CAT) y+ in Y1 cells. These Na+-independent transporters were identified by their selectivity for neutral amino acids in both the presence and absence of Na+ and by the effect of N-ethylmaleimide. Transport data correlated to expression of genes encoding for CAT-1, CAT-2, CD-98, and y+LAT-2. A similar expression profile was detected in rat adrenal zona fasciculata. In addition, cationic amino acid uptake in Y1 cells was upregulated by ACTH and/or cAMP with a concomitant increase in nitric oxide (NO) production.

Secreted autotransporter toxin produced by a diffusely adhering Escherichia coli strain causes intestinal damage in animal model assays

Bacterial diarrheal diseases remain a major cause of morbidity and mortality in developing countries. Diffusely adhering Escherichia coli (DAEC) is a newly proposed category of diarrheagenic E. coli based on epidemiological studies. Sat, a new virulence factor of some uropathogeic Escherichia coli, was described with a vacuolating cytotoxic action in bladder and kidney tissues. In the present study, we analyzed the Sat effects, produced by a DAEC strain in rabbit ileal intestinal tissue and cultured epithelial cells. We observed enterotoxic activity in rabbit ileum tissues by Ussing chamber assays, a pronounced fluid accumulation in rabbit ileum loops with villous necrosis observed in the histopathologic examination, and morphological changes in monolayer cultures of Y1 adrenal cells. Our results suggest that DAEC strains may be involved in diarrhea.

Peroxisome proliferator activated receptor gamma (PPARγ) agonists suppress adrenocortical tumor cell proliferation and induce differentiation

Thiazolidinediones (TZDs) as specific ligands for the PPARγ have been implemented into clinical practice for the treatment of type 2 diabetes mellitus. More recently, in vitro and pre-clinical in vivo findings have suggested that TZDs might also have favorable effects in the treatment of a variety of tumors as differentiation inducing agents. Adrenocortical carcinoma (ACC) is a rare tumor with poor prognosis due to a highly malignant phenotype and lack of effective treatment options. Since most effects of TZDs are dependent upon the presence of PPARγ in the target tissue, we studied a series of 32 adrenocortical tumors for the expression of PPARγ. Interestingly, PPARγ mRNA was detectable in all tumors including ACCs at similar levels, while ACTH receptor (MC2-R) mRNA as a marker of differentiation varied widely between the tumor entities. To further explore the functional significance of these findings, the adrenocortical tumor cell line NCI h295 was treated with the PPARγ agonists rosiglitazone and cell viability, proliferation, apoptosis and steroidogenesis was monitored. Incubation with rosiglitazone led to a decrease in cell viability as measured by the MTT assay in a time-and dose-dependent manner. This decrease was paralleled by a decrease of cellular proliferation (BrdU assay) and increase in apoptosis as measured by caspase 3/7 activity. In contrast, rosiglitazone did not affect cellular viability of murine adrenal Y1 cells that do not express PPARγ. On the molecular level, NCI h295 cells expressed higher levels of MC2-R mRNA upon treatment, while cyclin E mRNA was reduced, thus reflecting a shift towards a expression pattern found in less aggressive adrenocortical tumors in vivo. Taken together, these data indicate that TZDs have the potential to become an additional treatment option as differentiation inducing agents in patients with ACC.

Mucosal Adjuvant Properties of Mutant LT-IIa and LT-IIb Enterotoxins That Exhibit Altered Ganglioside-Binding Activities

LT-IIa and LT-IIb, the type II heat-labile enterotoxins of Escherichia coli, are closely related in structure and function to cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and E. coli, respectively. Recent studies from our group demonstrated that LT-IIa and LT-IIb are potent systemic and mucosal adjuvants. To determine whether binding of LT-IIa and LT-IIb to their specific ganglioside receptors is essential for adjuvant activity, LT-IIa and LT-IIb enterotoxins were compared with their respective single-point substitution mutants which have no detectable binding activity for their major ganglioside receptors [e.g., LT-IIa(T34I) and LT-IIb(T13I)]. Both mutant enterotoxins exhibited an extremely low capacity for intoxicating mouse Y1 adrenal cells and for inducing production of cyclic AMP in a macrophage cell line. BALB/c female mice were immunized by the intranasal route with the surface adhesin protein AgI/II of Streptococcus mutans alone or in combination with LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I). Both LT-IIa and LT-IIb potentiated strong mucosal and systemic immune responses against AgI/II. Of the two mutant enterotoxins, only LT-IIb(T13I) had the capacity to strongly potentiate mucosal anti-AgI/II and systemic anti-AgI/II antibody responses. Upon boosting with AgI/II, however, both LT-IIa(T34I) and LT-IIb(T13I) enhanced humoral memory responses to AgI/II. Flow cytometry demonstrated that LT-IIa(T34I) had no affinity for cervical lymph node lymphocytes. In contrast, LT-IIb(T13I) retained binding activity for T cells, B cells, and macrophages, indicating that this immunostimulatory mutant enterotoxin interacts with one or more unknown lymphoid cell receptors.

ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells

Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.