Y1 Cells Research Articles

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.

Differential Regulation of Glucocorticoid Synthesis in Murine Intestinal EpithelialVersusAdrenocortical Cell Lines

Glucocorticoids are steroid hormones with important functions in development, immune regulation, and glucose metabolism. The adrenal glands are the predominant source of glucocorticoids; however, there is increasing evidence for extraadrenal glucocorticoid synthesis in thymus, brain, skin, and vascular endothelium. We recently identified intestinal epithelial cells as an important source of glucocorticoids, which regulate the activation of local intestinal immune cells. The molecular regulation of intestinal glucocorticoid synthesis is currently unexplored. In this study we investigated the transcriptional regulation of the steroidogenic enzymes P450 side-chain cleavage enzyme and 11beta-hydroxylase, and the production of corticosterone in the murine intestinal epithelial cell line mICcl2 and compared it with that in the adrenocortical cell line Y1. Surprisingly, we observed a reciprocal stimulation pattern in these two cell lines. Elevation of intracellular cAMP induced the expression of steroidogenic enzymes in Y1 cells, whereas it inhibited steroidogenesis in mICcl2 cells. In contrast, phorbol ester induced steroidogenic enzymes in intestinal epithelial cells, which was synergistically enhanced upon transfection of cells with the nuclear receptors steroidogenic factor-1 (NR5A1) and liver receptor homolog-1 (NR5A2). Finally, we observed that basal and liver receptor homolog-1/phorbol ester-induced expression of steroidogenic enzymes in mICcl2 cells was inhibited by the antagonistic nuclear receptor small heterodimer partner. We conclude that the molecular basis of glucocorticoid synthesis in intestinal epithelial cells is distinct from that in adrenal cells, most likely representing an adaptation to the local environment and different requirements.

A Second Generation of Double Mutant Cholera Toxin Adjuvants: Enhanced Immunity without Intracellular Trafficking

Nasal application of native cholera toxin (nCT) as a mucosal adjuvant has potential toxicity for the CNS through binding to GM1 gangliosides in the olfactory nerves. Although mutants of cholera toxin (mCTs) have been developed that show mucosal adjuvant activity without toxicity, it still remains unclear whether these mCTs will induce CNS damage. To help overcome these concerns, in this study we created new double mutant CTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). Confocal microscopic analysis showed that intracellular localization of dmCTs differed from that of mCTs and nCTs in intestinal epithelial T84 cells. Furthermore, both dmCTs exhibited very low toxicity in the Y1 cell assay and mouse ileal loop tests. When mucosal adjuvanticity was examined, both dmCTs induced enhanced OVA-specific immune responses in both mucosal and systemic lymphoid tissues. Interestingly, although both dmCT E112K/KDEV and dmCT E112K/KDGL showed high Th2-type and significant Th1-type cytokine responses by OVA-specific CD4+ T cells, dmCT E112K/KDEV exhibited significantly lower Th1-type cytokine responses than did nCT and dmCT E112K/KDGL. These results show that newly developed dmCTs retain strong biological adjuvant activity without CNS toxicity.

ACTH receptor: Ectopic expression, activity and signaling

Failure in obtaining expression of functional adrenocorticotropic hormone receptor (ACTHR, or melanocortin 2 receptor, MC2R) in non-adrenal cells has hindered molecular analysis of ACTH signaling pathways. Here, we ectopically expressed the mouse ACTHR in Balb/c mouse 3T3 fibroblasts to analyze ACTH signaling pathways involved in induction of fos and jun genes. Natural constitutive expression of the MC2R accessory protein (MRAP) in Balb3T3 and other mouse 3T3 fibroblasts (NIH, Swiss and 3T3-L1) renders these fibroblastic lines suitable for ectopic expression of ACTHR in its active form properly inserted into the plasma membrane at levels similar to those found in mouse Y1 adrenocortical tumor cells. The Y1 cell line is a cultured cell system well known for stably displaying normal adrenal specific metabolic pathways, ACTHR expression and ACTH functional responses. Thirty-nine sub-lines expressing ACTHR (3T3-AR transfectants) were selected for geneticin-resistance and clonally isolated after transfection of ACTHR-cDNA (in the pSVK3 mammalian plasmidial vector) into Balb3T3 fibroblasts. In addition, sixteen clonal sub-lines of Balb3T3 (3T3-0 transfectants) carrying the pSVK3 empty vector were likewise isolated. Fourteen 3T3-AR and four 3T3-0 clones were screened for response to ACTH(39) in comparison with Y1 adrenocortical cells. Eight 3T3-AR clones responded to ACTH(39) with activation of adenylate cyclase and induction of c-Fos protein, but the levels of, respectively, activation and induction were not strictly correlated. Other fos and jun genes were also induced by ACTH(39) in 3T3-AR transfectants, which express levels of ACTHR protein similar to parental Y1 cells. Signaling pathways relevant to c-Fos induction was extensively investigated in 3 clones: 3T3-AR01 and -07 and 3T3-04. In Y1 cells, specific inhibitors (H89/PKA; PD98059/MEK; Go6983/PKC and SP600125/JNK) show that signals initiated in the ACTH/ACTHR-system activate 4 pathways to induce the c-fos gene, namely: (a) cAMP/PKA/CREB; (b) MEK/ERK1/2; (c) PKC and d) JNK1/2. In 3T3-AR transfectants, both inhibitors PD98059 and Go6983 proved completely ineffective to inhibit c-Fos induction by ACTH(39), implying that MEK/ERK and PKC pathways are not involved in this process. On the other hand, SP600125 caused 85% inhibition of c-Fos induction by ACTH(39) and, in addition, ACTH(39) promotes JNK1/2 phosphorylation, suggesting that JNK is a major signaling pathway mediating c-Fos induction by ACTH(39) in these cells. In addiction, PKA inhibitor H89 also inhibits c-Fos induction in 3T3-AR7 cells by ACTH(39), implicating activation of the cAMP/PKA/CREB pathway in c-Fos induction by ACTH(39). However, the cAMP derivatives db-cAMP and 8Br-cAMP, do not promote CREB phosphorylation and c-Fos induction in parental Balb3T3 and 3T3-AR transfectants, confirming previous report by others. In conclusion, expression of active ACTHR in Balb3T3 fibroblasts renders these cells responsive to ACTH with activation of cAMP/PKA/CREB and JNK pathways and, also, induction of genes from the fos and jun families. These results show that Balb 3T3-AR sublines are useful cellular systems for genetic analysis of ACTH-signaling pathways. However, activation of cAMP/PKA/CREB and JNK pathways and induction of fos and jun genes are not yet sufficient to enable ACTH for interference in morphology, migration and proliferation of Balb3T3 fibroblasts as it does in Y1 adrenocortical cells.

Delineation of Type I Protein Kinase A-selective Signaling Events Using an RI Anchoring Disruptor

Control of specificity in cAMP signaling is achieved by A-kinase anchoring proteins (AKAPs), which assemble cAMP effectors such as protein kinase A (PKA) into multiprotein signaling complexes in the cell. AKAPs tether the PKA holoenzymes at subcellular locations to favor the phosphorylation of selected substrates. PKA anchoring is mediated by an amphipathic helix of 14-18 residues on each AKAP that binds to the R subunit dimer of the PKA holoenzymes. Using a combination of bioinformatics and peptide array screening, we have developed a high affinity-binding peptide called RIAD (RI anchoring disruptor) with >1000-fold selectivity for type I PKA over type II PKA. Cell-soluble RIAD selectively uncouples cAMP-mediated inhibition of T cell function and inhibits progesterone synthesis at the mitochondria in steroid-producing cells. This study suggests that these processes are controlled by the type I PKA holoenzyme and that RIAD can be used as a tool to define anchored type I PKA signaling events.

C-Ki-ras oncogene amplification and FGF2 signaling pathways in the mouse Y1 adrenocortical cell line

The mouse Y1 adrenocortical tumor cell line is highly responsive to FGF2-(Fibroblast Growth Factor 2) and possesses amplified and over-expressed c-Ki-ras proto-oncogene. We previously reported that this genetic lesion leads to high constitutive levels of activation of the c-Ki-Ras-GTP-->PI3K-->Akt signaling pathway (Forti et al. 2002). On the other hand, activation levels of another important pathway downstream of c-Ki-Ras-GTP, namely, Raf-->MEK-->ERK, remain strictly dependent on FGF2 stimulation (Rocha et al. 2003). Here we show that, first, FGF2 transiently up-regulates the c-Ki-Ras-GTP-->PI3K-->Akt pathway, in spite of its high basal levels. Second, c-Ki-Ras-GTP transient up-regulation likely underlies activation of the ERK1/2 pathway by FGF2. Third, c-Ki-Ras-GTP high basal levels suppress activation of the c-H-Ras onco-protein. But, Y1 cells, expressing dominant negative mutant RasN17, display a rapid and transient up-regulation of c-H-Ras-GTP upon FGF2 treatment. Elucidation of FGF2-signaling pathways in Y1 tumor cells can uncover new targets for drug development of interest in cancer therapy.

Allosteric Activation of the Follicle-stimulating Hormone (FSH) Receptor by Selective, Nonpeptide Agonists

The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC50's = 20 microm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 2 nm) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 10.5 nm). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy.

Bioluminescence Color Modulation of Vibrio fischeri Strain Y1 Coupled with Alterable Levels of Endogenous Yellow Fluorescent Protein and Its Fluorescence Imaging

Bioluminescence (BL) (lambda(max) approximately 535 nm) of Vibrio fischeri strain Y1 has been previously characterized in terms of the fluctuation in intracellular levels of yellow fluorescent protein (YFP). In this study fluorescence microscopic analysis has revealed that yellow fluorescence, as well as blue fluorescence attributable to a luciferase intermediate, is localized to the periphery of V. fischeri Y1 cells. This finding indicates that both YFP and the luciferase are present in the vicinity of the cell membrane. By using cyanide to enhance yellow BL, it has been shown that BL modulation is coupled with the fluctuations in the intracellular levels of YFP and the primary emitter. On the basis of the BL characterization, combined with results of a sedimentation experiment, it has been shown that larger cells produce a relatively stronger yellow BL. Two-dimensional gel electrophoresis of cell-protein extracts has shown that the YFP level is more alterable than the luciferase level. It is postulated that the yellow BL modulation takes place in connection with cell growth.

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.

SF-1 (Nuclear Receptor 5A1) Activity Is Activated by Cyclic AMP via p300-Mediated Recruitment to Active Foci, Acetylation, and Increased DNA Binding

Steroidogenic factor 1 (SF-1) is a nuclear receptor essential for steroidogenic gene expression, but how its activity is regulated is unclear. Here we demonstrate that p300 plays an important role in regulating SF-1 function. SF-1 was acetylated in vitro and in vivo by p300 at the KQQKK motif in the Ftz-F1 (Fushi-tarazu factor 1) box adjacent to its DNA-binding domain. Mutation of the KQQKK motif reduced the DNA-binding activity and p300-dependent activation of SF-1. When stimulated with cyclic AMP (cAMP), adrenocortical Y1 cells expressed more p300, leading to additional SF-1 association with p300 and increased SF-1 acetylation and DNA binding. It also increased SF-1 colocalization with p300 in nuclear foci. Collectively, these results indicate that SF-1 transcriptional activity is regulated by p300 in response to the cAMP signaling pathway by way of increased acetylation, DNA binding, and recruitment to nuclear foci.

Differences between the growth regulatory pathways in primary rat adrenal cells and mouse tumor cell line

In this study, DNA synthesis, phosphorylation of ERK1/2 and CREB proteins, as well as induction of c-Fos protein, were examined in rat adrenocortical, glomerulosa and fasciculata/reticularis cells, as well as in the Y1 cell line. We found that FGF2 was mitogenic only in glomerulosa cells and although ACTH did not activate ERK1/2, it did activate CREB protein, indicating efficient transduction of signals initiated in the ACTH receptors of rat adrenocortical cells. The FGF2 activated ERK1/2 in rat adrenal cells by a mechanism that might be modulated by upstream PKA pathway phosphorylation of MEK and despite the nonmitogenic effect of ACTH on rat adrenal cells it effectively induces c-Fos protein. The results presented herein describe distinct differences between the ACTH and FGF2 signal transduction mechanisms seen in adrenocortical cells and those observed in the Y1 cell line, indicating that, in vitro, ACTH blockage of the mitogenic effect occurs in normal adrenal cells after induction of c-Fos protein.

Anthrax edema factor potency depends on mode of cell entry

Anthrax edema factor (EF) is a highly active calmodulin-dependent adenylyl cyclase toxin that can potently raise intracellular cAMP levels causing a broad range of tissue damage. EF needs anthrax protective antigen (PA) to enter into the host cell and together they form edema toxin. Here, we examine factors that are critical for edema toxin cell entry and potency. In Y1, 293T and mouse embryonic fibroblast cells, EF causes cell rounding, aggregation, and sometimes detachment via protein kinase A but not Epac. The rate-limiting step for these EF-mediated effects is cellular entry via the anthrax toxin receptor. Finally, EF potency is also enhanced if the EF adenylyl cyclase domain is transfected into host cells, even in the absence of the anthrax PA-binding domain. These results indicate that the effects of EF in cells can differ dependent upon the mode of cellular entry of the adenylyl cyclase.

An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein in the signal transduction pathway of steroidogenic hormones

We have described that, in adrenal and Leydig cells, the hormonal regulation of free arachidonic acid (AA) concentration is mediated by the concerted action of two enzymes: an acyl-CoA thioesterase (MTE-I or ARTISt) and an acyl-CoA synthetase (ACS4). In this study we analyzed the potential regulation of these proteins by hormonal action in steroidogenic cells. We demonstrated that ACS4 is rapidly induced by adrenocorticotropin (ACTH) and cAMP in Y1 adrenocortical cells. The hormone and its second messenger increased ACS4 protein levels in a time and concentration dependent way. Maximal concentration of ACTH (10 mIU/ml) produced a significant effect after 15 min of treatment and exerted the highest increase (3-fold) after 30 min. Moreover, (35)S-methionine incorporation showed that the increase in ACS4 protein levels is due to an increase in the de novo synthesis of the protein. On the contrary MTE-I protein levels in Y1 and MA-10 cells did not change after steroidogenic stimuli. In contrast with the effect observed on protein levels, stimulation of both cell lines did not change ACS4 RNA levels during the first hour of treatment, indicating that the effect of both stimuli is exerted at the level of ACS4 protein synthesis.StAR protein induction has a key role on the activation of steroidogenesis since this protein increases the rate of the limiting step of the whole process. In agreement with the fact that the inhibition of ACS4 activity by triacsin C blocks cAMP-stimulated progesterone production by MA-10 Leydig cells, here we demonstrated that ACS4 inhibition also reduces StAR protein levels. Moreover, exogenous AA was able to overcome the effect of triacsin C on both events, StAR induction and steroidogenesis. These results were confirmed by experiments using ACS4-targeted siRNA which result in a reduction in both ACS4 and StAR protein levels. The concomitant decrease in steroid production was overcome by the addition of AA to the knocked-out cells. In summary, this study suggests that in adrenal and Leydig cells the hormonal action prompts the synthesis of a labile protein, ACS4, which activity is involved in the regulation of AA release and is essential for steroidogenesis and StAR protein induction.

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.

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.

C-Myc protein is stabilized by fibroblast growth factor 2 and destabilized by ACTH to control cell cycle in mouse Y1 adrenocortical cells

ACTH is the hormone known to control adrenal cortex function and maintenance in the intact animal but, in culture, it inhibits proliferation of adrenocortical cells from different mammalian species, a puzzle that has remained unsolved for nearly 30 years. In this paper we compare ACTH and fibroblast growth factor 2 (FGF2) antagonistic effects on the cell cycle in the Y1 cell line, a functional lineage of mouse adreno-cortical tumor cells. This cell line displays chronic high levels of c-Ki-Ras-GTP, high active constitutive levels of phosphatidylinositol 3-OH kinase/Protein Kinase B (PI3K/AKT) and low constitutive basal expression of c-Myc, which accounts for a minor deregulation of the cell cycle. In G0/G1-arrested Y1 cells, over-expression of the dominant negative mutant HaRasN17 drastically reduces c-Ki-Ras-GTP levels, eliminating basal c-Myc expression and basal S phase entry. PI3K/Akt seems to be the downstream pathway from c-Ki-ras for deregulation of c-Myc basal expression, since wortmannin abolishes c-Myc expression in serum-starved, G0/G1-arrested Y1 cells. FGF2 is a strong mitogen for Y1 cells, promoting -- in a manner dependent on the MEK/ERK pathway -- c-myc transcription induction, c-Myc protein stabilization and S phase entry in G0/G1-arrested Y1 cells. On the other hand, ACTH causes c-Myc protein destabilization, partially blocking S phase entry induced by FGF2, by a process dependent on the cAMP/protein kinase A (PKA) pathway. The whole pathway activated by ACTH to destabilize c-Myc protein in Y1 cells might comprise the following steps: ACTH receptor -->cAMP/PKA --> Akt deactivation -->GSK3 activity liberation --> c-Myc Thr58 phosphorylation. We demonstrate that c-Myc regulation is a central key in the cell cycle control by these factors, since enforced expression of c-Myc through the MycER chimera abrogates the ACTH inhibitory effect over FGF2-induced S phase entry.

Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells

Enterotoxigenic Escherichia coli (ETEC) is a prevalent cause of traveler's diarrhea and infant mortality in third-world countries. Heat-labile enterotoxin (LT) is secreted from ETEC via vesicles composed of outer membrane and periplasm. We investigated the role of ETEC vesicles in pathogenesis by analyzing vesicle association and entry into eukaryotic cells. Fluorescently labeled vesicles from LT-producing and LT-nonproducing strains were compared in their ability to bind adrenal and intestinal epithelial cells. ETEC-derived vesicles, but not control nonpathogen-derived vesicles, associated with cells in a time-, temperature-, and receptor-dependent manner. Vesicles were visualized on the cell surface at 4 degrees C and detected intracellularly at 37 degrees C. ETEC vesicle endocytosis depended on cholesterol-rich lipid rafts. Entering vesicles partially colocalized with caveolin, and the internalized vesicles accumulated in a nonacidified compartment. We conclude that ETEC vesicles serve as specifically targeted transport vehicles that mediate entry of active enterotoxin and other bacterial envelope components into host cells. These data demonstrate a role in virulence for ETEC vesicles.

CAMP-dependent Protein Kinase Regulates Ubiquitin-Proteasome-mediated Degradation and Subcellular Localization of the Nuclear Receptor Coactivator GRIP1

Nuclear receptors and their coactivators are key regulators of numerous physiological functions. GRIP1 (glucocorticoid receptor-interacting protein) is a member of the steroid receptor coactivator family. Here, we show that GRIP1 is regulated by cAMP-dependent protein kinase (PKA) that induces its degradation through the ubiquitin-proteasome pathway. GRIP1 was down-regulated in transiently transfected COS-1 cells after treatment with 8-para-chlorophenylthio-cAMP or forskolin and 3-isobutyl-1-methylxanthine and in adrenocortical Y1 cells after incubation with adrenocorticotropic hormone. Pulse-chase experiments with transiently transfected COS-1 cells demonstrated that the half-life of GRIP1 was markedly reduced in cells overexpressing the PKA catalytic subunit, suggesting that activation of PKA increases the turnover of GRIP1 protein. The proteasome inhibitors MG132 and lactacystin abolished the PKA-mediated degradation of GRIP1. Using ts20 cells, a temperature-sensitive cell line that contains a thermolabile ubiquitin-activating E1 enzyme, it was confirmed that PKA-mediated degradation of GRIP1 is dependent upon the ubiquitin-proteasome pathway. Coimmunoprecipitation studies of COS-1 cells transfected with expression vectors encoding GRIP1 and ubiquitin using anti-GRIP1 and anti-ubiquitin antibodies showed that the ubiquitination of GRIP1 was increased by overexpression of PKA. Finally, we show that PKA regulates the intracellular distribution pattern of green fluorescent protein-GRIP1 and stimulates recruitment of GRIP1 to subnuclear foci that are colocalized with the proteasome. Taken together, these data demonstrate that GRIP1 is ubiquitinated and degraded through activation of the PKA pathway. This may represent a novel regulatory mechanism whereby hormones down-regulate a nuclear receptor coactivator.

Salt‐inducible kinase‐1 represses cAMP response element‐binding protein activity both in the nucleus and in the cytoplasm

Salt-inducible kinase-1 (SIK1) is phosphorylated at Ser577 by protein kinase A in adrenocorticotropic hormone-stimulated Y1 cells, and the phospho-SIK1 translocates from the nucleus to the cytoplasm. The phospho-SIK1 is dephosphorylated in the cytoplasm and re-enters the nucleus several hours later. By using green-fluorescent protein-tagged SIK1 fragments, we found that a peptide region (586-612) was responsible for the nuclear localization of SIK1. The region was named the 'RK-rich region' because of its Arg- and Lys-rich nature. SIK1s mutated in the RK-rich region were localized mainly in the cytoplasm. Because SIK1 represses cAMP-response element (CRE)-mediated transcription of steroidogenic genes, the mutants were examined for their effect on transcription. To our surprise, the cytoplasmic mutants strongly repressed the CRE-binding protein (CREB) activity, the extent of repression being similar to that of SIK1(S577A), a mutant localized exclusively in the nucleus. Several chimeras were constructed from SIK1 and from its isoform SIK2, which was localized mainly in the cytoplasm, and they were examined for intracellular localization as well as CREB-repression activity. A SIK1-derived chimera, where the RK-rich region had been replaced with the corresponding region of SIK2, was found in the cytoplasm, its CREB-modulating activity being similar to that of wild-type SIK1. On the other hand, a SIK2-derived chimera with the RK-rich region of SIK1 was localized in both the nucleus and the cytoplasm, and had a CREB-repressing activity similar to that of the wild-type SIK2. Green fluorescent protein-fused transducer of regulated CREB activity 2 (TORC2), a CREB-specific co-activator, was localized in the cytoplasm and nucleus of Y1 cells, and, after treatment with adrenocorticotropic hormone, cytoplasmic TORC2 entered the nucleus, activating CREB. The SIK1 mutants, having a strong CRE-repressing activity, completely inhibited the adrenocorticotropic hormone-induced nuclear entry of green fluorescent protein-fused TORC2. This suggests that SIK1 may regulate the intracellular movement of TORC2, and as a result modulates the CREB-dependent transcription activity. Together, these results indicate that the RK-rich region of SIK1 is important for determining the nuclear localization and attenuating CREB-repressing activity, but the degree of the nuclear localization of SIK1 itself does not necessarily reflect the degree of SIK1-mediated CREB repression.