Linda Aumo, Marte Rusten, Gunnar Mellgren, Marit Bakke, Aurélia E. Lewis, Practical Roles of Protein Kinase A (PKA) and Change Protein Straight Activated by 3′,5′-Cyclic Adenosine 5′-Monophosphate (cAMP) 2 (EPAC2) in cAMP-Mediated Actions in Adrenocortical Cells, Endocrinology, Quantity 151, Problem 5, 1 Could 2010, Pages 2151–2161, https://doi.org/10.1210/en.2009-1139
It has lengthy been acknowledged that cAMP is a ubiquitous second messenger regulating many key mobile processes and that its actions are mediated by the cAMP effector protein kinase A (PKA). Nevertheless, a few decade in the past, two further cAMP sensors, trade proteins instantly activated by cAMP (EPAC)-1 and -2 (additionally termed cAMP-binding guanine trade elements I and II) have been found (1, 2). Since then, quite a few research have demonstrated that EPAC1/2 mediate a mess of mobile capabilities induced by cAMP, i.e. proliferation, differentiation, adhesion, apoptosis, and transcription (3, 4). Within the endocrine system, the invention of EPAC1/2 was vital to elucidate a few of the results mediated by cAMP in hormone signaling that weren’t mediated by PKA (reviewed in Ref. 4), for instance in granulosa cells (5) and thyrocytes (6–8). Furthermore, EPAC2 has been implicated in insulin secretion in pancreatic β-cells (9, 10), within the launch of α-MSH within the pituitary gland (11), and within the regulation of proglucagon expression in intestinal L cells (12). Each EPAC1 and -2 include a cAMP-binding area (CBD) just like the one discovered within the PKA regulatory subunit, however the two isoforms differ by the presence of 1 additional CBD within the N terminus of EPAC2. Binding of cAMP prompts EPAC1/2, which then act as guanine trade elements for the small G proteins Rap1 and Rap2 by catalyzing the trade of sure GDP for GTP (reviewed in Ref. 13). The additional N-terminal CBD in EPAC2 has low affinity for cAMP (14), and as a substitute, this area could also be concerned in plasma membrane localization (15). EPAC1 and -2 are differentially expressed (1). EPAC1 is discovered ubiquitously, whereas the expression sample of EPAC2 is restricted to sure areas of the mind, neuroendocrine tissues, and endocrine glands together with the adrenal and testis (1, 2, 9). Just lately, EPAC2 was described to exist as three completely different splicing variants, which differ solely at their N termini: the full-length protein EPAC2A; EPAC2B, which lacks the N terminus CBD; and EPAC2C, which lacks the N terminus CBD and the Dishevelled-Egl-10-Plekstrin (DEP) (15, 16).
Within the adrenal cortex, the biosynthesis of steroid hormones is regulated primarily by the pituitary-derived hormone ACTH. ACTH induces the transcription of steroidogenic enzymes concerned within the stepwise conversion of ldl cholesterol to corticosteroids (17). Binding of ACTH to its receptor results in the activation of a number of signaling pathways (18, 19). The pathway by far the very best characterised includes the activation of adenylyl cyclases adopted by an elevation of cAMP manufacturing. Though cAMP has been acknowledged as the primary second messenger recapitulating the results of ACTH on steroid hormone output, the downstream signaling occasions are solely partially understood (17–19). cAMP induces the activation of PKA and subsequent phosphorylation of goal substrates, such because the ldl cholesterol ester hydrolase and the steroidogenic acute regulatory protein (StAR) (20). These posttranslational modifications activate ldl cholesterol ester hydrolase and StAR and result in an elevated availability of free ldl cholesterol and its supply to the internal mitochondrial membrane. This permits for the primary enzymatic response by the cytochrome P450 (CYP) ldl cholesterol facet chain cleavage enzyme (CYP11A1), which converts ldl cholesterol to pregnenolone. Pregnenolone is additional transformed to aldosterone, cortisol, or androstenedione within the completely different zones of the adrenal gland, relying upon the expression of the completely different CYP steroidogenic enzymes (CYP17, CYP21, CYP11B1, and CYP11B2) and of the 3β-hydroxysteroid dehydrogenase (21).
Though PKA clearly performs a key position in steroidogenesis, a number of research have pointed to cAMP indicators mediated independently of PKA within the adrenal cortex (22–24). Instruments that enable the particular activation of PKA and EPAC1/2 now exist [i.e. N6-benzoyl-cAMP (N6-Bnz) and 8-p-chlorophenylthio-2′-O-methyl-cAMP (8CPT-2M), respectively] (25) however haven’t beforehand been used to dissect cAMP-induced signaling cascades in adrenocortical cells. On this research, we set up that the EPAC pathway is practical in these cells and exploit the PKA- and EPAC1/2-specific analogs to discriminate between the involvement of those two pathways in corticosteroid manufacturing, adjustments in cell morphology, microfilament group, and migration. We discovered that a number of cAMP-dependent processes in steroidogenic cells, such because the expression of StAR and steroid hydroxylases, the induction of the transcription issue nerve development factor-inducible clone-B (NGFI-B), and the manufacturing of steroid hormones, are stimulated by cAMP in a PKA-dependent trend. In distinction, the activation of each pathways results in the lack of stress fibers, change of cell form, and a marked lower in migration.
Supplies and Strategies
Anti-Rap1 (sc-65), anti-EPAC2 (sc-9384 and sc-25633, epitope amino acids 1-220; sc-9383, inner epitope; all three antibodies can detect each EPAC2A and -2B), anti-NGFI-B (sc-5569), anti-StAR (sc-25806), anti-phospho-Tyr 204-ERK1/2 (sc-7383), anti-ERK1 (sc-94), and anti-ERK2 (sc-153) have been from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CYP17 was a beneficiant present from Dr. M. R. Waterman (Vanderbilt College, Nashville, TN). Anti-CYP11B1 was kindly offered by Dr. Hiroshi Takemori, (College of Osaka, Osaka, Japan). Anti-CYP11A1 (ab-67355) and anti-β-actin (ab-6276) have been from Abcam (Cambridge, UK). Antimouse CYP11A1 was from Millipore (Billerica, MA) (AB1294). The EPAC- and PKA-specific cAMP analogs, 8CPT-2M and N6-Bnz, respectively, have been from Biolog Life Science Institute (Bremen, Germany). Forskolin, ACTH (amino acids 1-39) and isobutyl 3-methylxanthine (IBMX) have been from Sigma-Aldrich Norway AS (Oslo, Norway). Flag-tagged pCMV2-EPAC2A and pCMV2-EPAC2B expression vectors have been form items from Dr. S. Seino (Kobe College Graduate College of Medication, Kobe, Japan).
Cell tradition
Murine adrenocortical tumor cells (Y1) (26), Sertoli cells (MSC-1) (27), prepubertal Sertoli cells (SMAT-1) (28), and monkey kidney COS-1 cells (American Kind Tradition Assortment, Manassas, VA) have been cultured in DMEM supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). Human adrenocortical carcinoma NCI-H295R (H295R) cells (29) have been obtained from American Kind Tradition Assortment and cultured as described beforehand (30). Mouse Leydig tumor cells (MA-10) have been cultured in a 1:1 combination of DMEM (excessive glucose) and HAM F12, 15% horse serum, 20 mm HEPES, and 40 μg/ml gentamicin (31). SMAT-1 cells have been kindly offered by N. di Clemente (Université Paris-Sud, Paris, France). COS-1 cells have been transfected with Flag-EPAC2A or Flag-EPAC2B with SuperFect (QIAGEN, Valencia, CA) at 1:4 (micrograms DNA to microliters SuperFect).
RT-PCR
mRNA was remoted from Y1 and H295R cells utilizing the FastTrack 2.0 equipment (Invitrogen, Carlsbad, CA). mRNA (50 ng) was reverse transcribed (30 min at 42 C) utilizing a reverse transcription system (Promega, Madison, WI; A3500). The ensuing cDNA was amplified utilizing Taq DNA polymerase (New England Biolabs, Beverly, MA). The primers for EPAC1 have been as described (2). The next primers have been used for EPAC2: ahead 5′-ATTAATGGACGCCTGTTTGC-3′ (nucleotides 2393-2412 in EPAC2A) and reverse 5′-CCTCCTCAGGAACAAATCCA-3′ (nucleotides 2643-2625 in EPAC2A). PCR merchandise have been separated on 3% agarose gels stained with ethidium bromide.
Western immunoblotting
Entire-cell extracts have been ready by lysis in RIPA buffer [50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 1 mm phenylmethylsulfonyl fluoride, 2 mm sodium orthovanadate, and 2 μg/ml aprotinin and leupeptin]. Equal quantities of protein have been separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes have been blocked in 5% milk and incubated in a single day in main antibodies and subsequently with antimouse/rabbit antibodies conjugated to horseradish peroxidase (1:10,000) for 1 h. Antibody detection was carried out utilizing SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) in accordance with the producer’s directions and scanned with an LAS-3000 imaging system (Fuji movie, Tokyo, Japan). Densitometry measurements have been achieved for every protein examined utilizing ImageJ 1.42q (http://rsb.data.nih.gov/ij/). Quantifications of the ratios between every protein and β-actin have been carried out and proven as fold values in contrast with management. Means ± sd are introduced as histograms.
Rap1 activation assay
Y1 and H295R cells have been plated on 10-cm plates in serum-supplemented medium and grown for two–3 d. Cells have been rinsed in PBS and incubated in serum-free medium for twenty-four h and subsequently handled as indicated within the determine legend (see Fig. 3). Cells have been washed twice in PBS, lysed in Rap buffer [50 mm Tris-HCl (pH 7.2), 150 mm NaCl, 10 mm MgCl2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 2 mm sodium orthovanadate, and 2 μg/ml aprotinin and leupeptin] and incubated with glutathione S-transferase (GST) fused ral guanine nucleotide dissociation stimulator (RalGDS) Rap1-binding area (RBD) coupled to glutathione beads (300 μl) (Pharmacia, Piscataway, NJ) for 1 h at 4 C. GST-RalGDS-RBD was expressed and purified as described beforehand (32). After three washes with Rap lysis buffer, sure proteins have been eluted with SDS pattern buffer for five min at 95 C, separated by SDS-PAGE on a 12% gel and subjected to Western blotting utilizing a Rap1-specific antibody. Quantifications of the ratios between GTP-Rap1 and whole Rap1 have been carried out and proven as fold values in contrast with management. Means ± sd have been introduced as histograms.
Immunostaining
H295R cells, seeded on coverslips, have been fastened in 3.7% formaldehyde in PBS for 15 min, washed twice with PBS, permeabilized with 0.1% Triton X-100 in PBS for 15 min (EPAC2 staining) or 4 min (actin staining), and incubated with blocking buffer (1% BSA in PBS) for 1 h. Cells have been incubated with phalloidin conjugated to Alexa 488 (1:40) in PBS for 20 min at room temperature or with anti-EPAC2 (sc-9383) diluted in 0.1% BSA in PBS (1:25) in a single day at 4 C, adopted by incubation with Cy5-conjugated antigoat antibody (Molecular Probes, Eugene, OR) diluted in 0.1% BSA in PBS (1:200) and mounted with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) after in depth washing in PBS. Management staining with secondary antibody alone below the identical staining and publicity situations confirmed no unspecific staining. Confocal photos of EPAC2-stained cells have been acquired with a Zeiss LSM 510 Meta confocal laser scanning microscope geared up with a ×63, numerical aperture 1.4 oil immersion goal and utilizing 633- and 405-nm laser traces. Pictures of actin-stained cells have been obtained with a Nikon TE 2000 fluorescence microscope utilizing a ×63 oil immersion goal.
Migration
Migration was decided after scratch wounding of H295R cell monolayers. Cells have been handled with forskolin or the PKA- and EPAC-specific analogs within the presence and absence of IBMX, instantly after creating the wound. Pictures from three fields per experiment (n = 2) have been taken after 48 h, and the wound areas have been quantified with the ImageJ software program.
Measurements of cortisol and aldosterone
H295R cells plated on six-well plates have been grown for two d, incubated in DMEM/F12 serum-free and phenol red-free medium for twenty-four h, and subsequently handled as indicated in Desk 1, and conditioned media have been collected for cortisol and aldosterone measurements. Cortisol was analyzed by a solid-phase, aggressive chemiluminescence enzyme immunoassay on Immulite 2000 from Diagnostics Merchandise Corp. (Los Angeles, CA). Aldosterone was analyzed by the Coat-A-Depend process, a solid-phase RIA from Diagnostics Merchandise. Cross-reactivity with the antialdosterone antiserum was 0.0004% for 11-deoxycortisol, 0.0005% for DHEA, 0.006% for 11-deoxycorticosterone, and 0.002% for corticosterone, as outlined by the producer. Cross-reactivity was not detectable for cortisol and androstenedione.
Statistics
The information are introduced because the imply ± sd. Statistics have been carried out utilizing the GraphPad INSTAT 3 software program (GraphPad Software program, Inc., San Diego, CA) and one-way ANOVA and Bonferroni as submit take a look at, or Scholar’s t take a look at.
Outcomes
Adrenocortical cells categorical EPAC2B
Northern blot analyses have beforehand demonstrated the presence of EPAC2 however not of EPAC1 in adrenal glands (1, 9). To find out the putative presence of EPAC1/2 in adrenocortical-derived tumor cell traces (murine Y1 cells and human H295R cells), we carried out RT-PCR analyses. As proven in Fig. 1A, we discovered that these cell traces expressed solely EPAC2. RNA ready from NIH-3T3 cells was used as a optimistic management for EPAC1 (Fig. 1A) (2). In a current research, Seino et al. (15) demonstrated that the adrenal cortex expressed a shorter EPAC2 mRNA ranging from exon 5 (referred to as isoform EPAC2B). We subsequently validated the existence of this shorter variant on the protein degree in H295R and Y1 cells by Western immunoblotting, as proven in Fig. 1B. Cell extracts from COS-1 cells transfected with Flag-EPAC2A or Flag-EPAC2B have been used as controls. EPAC2B was additionally detected in testicular Leydig cells (MA-10) however not in nonsteroidogenic testicular Sertoli cells (SMAT-1 and MSC-1) or in fibroblasts (NIH-3T3) (Fig. 1C). The subcellular localization of EPAC2B was evaluated by immunofluorescence staining in H295R cells (Fig. 1D). A punctate staining sample was detected within the nucleus and to a lesser diploma within the cytoplasm. Management labeling with secondary antibodies alone confirmed no background staining (knowledge not proven). We noticed the identical sample of staining in Y1 cells (knowledge not proven).
The EPAC-specific cAMP analog prompts Rap1 in adrenocortical cells
To research whether or not the EPAC2B signaling pathway is practical in adrenocortical cells, we decided whether or not the EPAC-specific cAMP analog (8CPT-2M) led to the activation of Rap1. Y1 and H295R cells have been incubated with forskolin (an adenylyl cyclase activator that elevates cAMP ranges) or the EPAC-specific cAMP analog, and cell extracts have been analyzed for the presence of lively Rap1 (GTP-bound Rap1) by Western immunoblotting. As proven in Fig. 2, each brokers elevated the extent of lively Rap1 in each cell traces, whereas the degrees of whole Rap1 remained unchanged. This demonstrates that the EPAC2B signaling cascade is practical in these cells and that EPAC2B prompts Rap1 like EPAC2A regardless of the dearth of the N-terminal CBD.
Activation of each PKA and EPAC2B induces cell rounding, together with IBMX
In response to cAMP-elevating brokers, adrenocortical cells change from a flat and adherent form to a spherical form with occasional arborization (33). To judge the involvement of PKA and EPAC2B in cAMP-mediated results on cell morphology, Y1 (Fig. 3A) and H295R (Fig. 3B) cells have been incubated with forskolin or with the cAMP analogs particular for PKA or EPAC. As a result of artificial cAMP analogs have been reported to be hydrolyzed by phosphodiesterases (PDEs) (34), the PKA- and EPAC-specific analogs have been employed each within the presence and absence of the overall PDE inhibitor IBMX. In Y1 cells, the impact of forskolin on cell rounding was dramatic and fast (first noticed inside 1 h; proven after 6 h in Fig. 3A), as reported beforehand (35, 36). Cell rounding was reproduced by each the PKA- and EPAC-specific cAMP analogs when mixed with IBMX, albeit extra potently with the PKA-specific analog (as a result of results have been noticed at 100 μm with the PKA analog and required 500 μm for the EPAC analog). IBMX had no impact alone in Y1 cells. In H295R cells, the impact of forskolin was much less dramatic and slower than in Y1 cells (cell rounding was first detected after 16 h; proven after 24 h in Fig. 3B). IBMX had a reasonable impact by itself in H295R cells, however the PKA- and EPAC-specific analogs, when mixed with IBMX, had a dramatic impact and have been equally potent on this cell line. These outcomes point out that each PKA and EPAC2B are concerned within the attribute adjustments in cell form mediated by cAMP and that this impact relies upon the concomitant inhibition of PDEs with IBMX.
Activation of each PKA and EPAC2B induces lack of stress fibers and inhibits migration together with IBMX
Modifications in cell morphology are sometimes accompanied by a reorganization of the actin cytoskeleton, which may itself coordinate cell migration (reviewed in Ref. 37). We subsequently evaluated the involvement of PKA and EPAC2B in cAMP-induced actin reorganization (Fig. 4A) and cell migration (Fig. 4B). In Fig. 4A, H295R cells have been incubated with forskolin or with the cAMP analogs particular for PKA or EPAC within the presence or absence of IBMX and stained with phalloidin to detect filamentous actin (F-actin). Management cells introduced attribute actin stress fibers (as indicated with arrows), as beforehand reported in Y1 and rat glomerulosa cells (38, 39). Remedy with forskolin resulted in lack of stress fibers and the relocation of actin to restricted areas on the cell periphery (as indicated with arrowheads). This impact was mimicked when cells have been incubated with both of the PKA- and EPAC-specific analogs when mixed with IBMX. A modest impact was additionally noticed with IBMX alone. Related outcomes have been obtained in Y1 cells (knowledge not proven).
The consequences of the PKA- and EPAC-specific analogs on cell migration have been evaluated in H295R cells by scratch assays. Forskolin inhibited cell migration (by 30 ± 5%, Fig. 4B). Apparently, the EPAC-specific analog additionally decreased cell invasion into the wound (by 14 ± 0.3%), and within the presence of IBMX, the EPAC-specific analog decreased migration much more than forskolin (by 42 ± 9%). The PKA-specific analog had no impact by itself however inhibited migration severely when IBMX was current (36 ± 7%). IBMX additionally had an impact by itself (22 ± 0.9%). Taken collectively, these experiments present that activation of each PKA and EPAC2B induce adjustments in cell morphology and actin transforming and that these results are accompanied by a discount of cell motility. Brokers that elevate cAMP ranges are recognized to inhibit adrenocortical cell proliferation by stopping DNA synthesis (40, 41). We didn’t observe any impact by the EPAC-specific analog in both cell line when analyzed by fluorescence-activated cell sorting to look at their cell cycle distribution. As well as, we didn’t observe any impact on whole protein synthesis. This could spotlight the truth that our knowledge on migration should not resulting from variation both on cell division or on hypertrophy.
Activation of PKA, however not of EPAC2B, will increase the protein ranges of steroidogenic elements
To match the roles of PKA and EPAC2B within the induction of StAR and steroidogenic enzymes, H295R cells (Fig. 5) and Y1 cells (Fig. 6) have been incubated for twenty-four h with the cAMP analogs particular for PKA or EPAC within the presence or absence of IBMX in addition to with forskolin alone. In H295R cells, the protein ranges of StAR and the CYP enzymes CYP11A1, CYP17, and CYP11B1 have been analyzed by Western immunoblotting (Fig. 5B) and quantified (Fig. 5C). As anticipated, forskolin elevated the degrees of StAR, CYP11A1, and CYP17. This impact was reproduced by the PKA-specific cAMP analog (at 500 μm) and was not dependent upon the impact of IBMX. In distinction, the EPAC-specific cAMP analog had no impact alone. Of word, the StAR ranges have been elevated when the EPAC-specific cAMP analog and IBMX have been employed collectively, however this induction was not considerably completely different from the impact of IBMX alone. The degrees of CYP11B1 weren’t induced by any agent. We noticed, nonetheless, the presence of a doublet that was obvious solely in cells handled with forskolin or the PKA-specific analog. To the very best of our data this impact has not been reported earlier than however could warrant additional investigation. Mouse adrenocortical cells don’t categorical CYP17 (42), and subsequently solely the protein ranges of StAR and CYP11A1 have been decided in Y1 cells (Fig. 6). Once more, forskolin elevated the degrees of StAR and CYP11A1, and this impact was mimicked by the PKA-specific analog (at 500 μm) however not by the EPAC-specific analog. In distinction to H295R cells, the induction by the PKA-specific analog required the presence of IBMX, however there was no impact by IBMX alone (Fig. 6). The degrees of β-actin weren’t affected by any therapy in both cell line.
NGFI-B is an immediate-early gene that’s quickly induced by cAMP and ACTH in adrenocortical cells (43, 44). This nuclear receptor is implicated in cAMP-dependent transcription of a number of steroid hydroxylase genes (44–46) and is thus an vital consider cAMP-induced steroidogenesis. As demonstrated in Fig. 7, the degrees of NGFI-B have been robustly elevated by forskolin after 2 h of therapy in each cell traces, in concordance with earlier research (43, 44). The PKA-specific analog additionally elevated the degrees of NGFI-B. IBMX had no impact by itself however enhanced significantly the results of the PKA-specific analog, thereby recapitulating the results of forskolin. The EPAC-specific analog had no impact in both the presence or absence of IBMX. These outcomes point out subsequently that the cAMP-induced expression of steroidogenic enzymes in addition to that of NGFI-B is mediated by PKA.
Activation of PKA, however not of EPAC2B, induces the synthesis of cortisol and aldosterone
To additional characterize the roles of PKA and EPAC pathways in steroidogenesis, H295R cells have been handled with forskolin alone or with the cAMP analogs particular for PKA and EPAC within the absence or presence of IBMX. The degrees of cortisol and aldosterone have been subsequently measured within the media (Desk 1). The PKA-specific analog induced the manufacturing of each cortisol and aldosterone in a dose-dependent method, and this was additional enhanced by IBMX. As anticipated, forskolin additionally induced the manufacturing of each hormones. In distinction, as predicted from the dearth of impact on the expression of StAR and the steroid hydroxylases, the EPAC-specific analog had no impact on cortisol or aldosterone secretion.
Dialogue
Just lately, EPAC2 was described to exist as three completely different splicing variants, which differ solely at their N termini: EPAC2A (full size), which is expressed within the cerebral cortex and pancreas; EPAC2B, which lacks the N-terminal CBD and has, till now, been detected solely within the adrenal cortex; and EPAC2C, which lacks the N-terminal CBD and the Dishevelled-Egl-10-Plekstrin (DEP) area and is particularly expressed within the liver (15, 16). On this research, we have now proven that the EPAC2B protein is expressed in Y1 and H295R cells (derived from adrenocortical cells) and MA-10 cells (derived from testicular Leydig cells). Neither EPAC1 nor EPAC2A have been detected in adrenocortical cells. The discovering that therapy with the EPAC-specific analog prompts Rap1 in Y1 and H295R cells (Fig. 2) demonstrates that EPAC2B is functionally lively in adrenocortical cells regardless of the dearth of the N-terminal CBD. That is in settlement with a earlier report asserting that the low-affinity N-terminal CBD isn’t required for cAMP-induced activation of EPAC2 (14). It’s fascinating to notice that whereas EPAC2A (that comprises the N-terminal CBD) was proven to be localized to the cytoplasm, close to the plasma membrane (15), EPAC1 and EPAC2B are discovered within the nucleus (47, 48) (Fig. 1D), indicating that the N-terminal a part of EPAC2A could also be concerned in mobile distribution of the protein. Apparently, the localization of EPAC1 has just lately been linked to the nuclear/cytoplasmic trafficking of the DNA-dependent protein kinase (48).
In a number of organic techniques, EPAC1/2 potentiates the optimistic results of PKA on cell differentiation (e.g. for neurite extension in pheochromocytoma cells (49) and adipose conversion of preadipocytes (50) and cell division (in thyrocytes;51). Contemplating that the EPAC2B variant seems to be particularly expressed in cells with steroidogenic capability, this prompted us to systematically look at the contribution of the PKA and EPAC pathways in steroidogenesis. We discovered that the biosynthesis of each cortisol and aldosterone was mediated by PKA and never by EPAC2B in adrenocortical cells (Desk 1), as beforehand indicated by comparative research in Y1 and Kin-8 cells (cell line derived from Y1 cells that harbor an inactivating mutation within the regulatory subunit of PKA) (52). As well as, EPAC2B was not capable of potentiate the results of PKA on steroid biosynthesis when the cAMP analogs have been mixed at suboptimal concentrations (knowledge not proven). Though EPAC2B can be expressed in testicular cells, PKA was additionally the only inducer of steroid manufacturing in MA-10 cells (knowledge not proven). In sum, PKA clearly performs a dominant position within the synthesis of steroid hormones. We can’t exclude, nonetheless, that different cAMP-induced pathways can also be concerned, notably resulting in aldosterone synthesis as a result of the Ca2+/calmodulin kinases have been proven to play a significant position in cAMP-induced aldosterone synthesis in zona glomerulosa cells (24).
According to the results on steroid hormones synthesis, the PKA-specific cAMP analog, however not the EPAC-specific cAMP analog, stimulated the expression of various elements concerned within the metabolism of ldl cholesterol, (i.e. StAR, NGFI-B, CYP11A1, and CYP17). The outcomes on the regulation of CYP11A1 and CYP17 expression are in settlement with earlier reviews both evaluating Y1 and Kin-8 cells (CYPT11A1) (53, 54) or utilizing a pharmacological inhibitor of PKA in Leydig cells (CYP17) (55), which present that cAMP-induced transcription of the corresponding genes relies upon PKA activation. We have now additionally noticed that the CYP17 promoter is activated by the PKA- however not the EPAC-specific cAMP analog in H295R cells (knowledge not proven). ACTH and cAMP each induce transcription from the NGFI-B gene (43, 44) and regulate its phosphorylation state (56). Pharmacological inhibition of PKA confirmed that cAMP induced the transcription of NGFI-B in corticotropes (57) in a PKA-dependent method, and our knowledge now clarified the first involvement of PKA within the induction of NGFI-B on the protein degree. StAR ranges have been additionally elevated solely after PKA activation, which is according to earlier research (54). The regulation of StAR expression by cAMP is not like that of steroid hydroxylases. PKA isn’t concerned within the transcriptional expression of StAR however slightly capabilities by regulating its ranges posttranslationally, presumably by regulating its stability (54). In assist of those knowledge, we have now noticed that the activation of neither PKA nor EPAC may recapitulate the results of cAMP-elevating brokers on the StAR promoter exercise in luciferase reporter assays in Y1 cells (knowledge not proven). Different signaling molecules downstream of cAMP are subsequently prone to be required for StAR transcription, and ERK1/2 (58) and Ca2+/calmodulin kinases (24) have been proven to be doable candidates.
cAMP is well-known to induce attribute adjustments within the cell form and concomitant reorganization of F-actin microfilaments in adrenocortical cells (reviewed in Ref. 18). These practical responses have beforehand been attributed partly to PKA (52, 59). For example, Kin-8 cells exhibit solely a partial resistance to cell rounding after 8-Br-cAMP therapy (52). Though it isn’t excluded that Kin-8 cells have residual PKA exercise, this end result helps the information introduced in Figs. 3 and 4, particularly that the EPAC-specific analog, along with the PKA-specific analog, can reproduce the results of cAMP on cell rounding and the concomitant reorganization of F-actin microfilaments. Our outcomes are additionally according to the impact of the EPAC-specific analog on the breakdown of actin stress fibers in endothelial cells (60–63) and in prostate most cancers cells (64). Activation of EPAC2B additionally considerably inhibited cell migration in H295R cells (Fig. 4B), and that is in keeping with a current research by Grandoch et al. (64) exhibiting that the EPAC-specific cAMP analog inhibited the migration of prostate most cancers cells. As has been extensively reported beforehand, we discovered that PKA additionally inhibits migration (reviewed in Ref. 65). Each the PKA- and EPAC-activated pathways required simultaneous inhibition of PDEs to attain an analogous degree of response as with forskolin for the regulation of cell form and actin transforming (Figs. 3 and 4A). Artificial cAMP analogs are hydrolyzed by PDEs (34, 66), which could clarify the requirement of IBMX in these experiments. Within the scratch assay, IBMX had a major impact by itself on cell migration (Fig. 4B), and we can’t exclude a direct position for PDEs in actin transforming and migration. Accordingly, a current research demonstrates that the inhibition of PDE4 (utilizing the particular inhibitor rolipram) stimulated integrin-induced actin meeting and decreased cell migration, clearly pointing to a extra direct position for PDE4 (67). To the very best of our data, there aren’t any reviews on the expression and the precise operate of PDE4 within the adrenal cortex. PDE2, PDE8B, and PDE11A have been proven to be expressed within the adrenal cortex (68–70), and specifically, the exercise of PDE2 was demonstrated to be tightly related to the regulation of cAMP ranges in rat glomerulosa cells (71). The aforementioned PDE members could subsequently be believable candidates for the results noticed with IBMX.
In abstract, we have now proven that PKA and EPAC2B have distinct but in addition overlapping practical roles in adrenocortical cells. PKA is clearly the mediator of cAMP-induced results on steroidogenesis. Nevertheless, each PKA and EPAC2B have an effect on the actin cytoskeleton integrity and cell migration. This research is the primary report exhibiting the involvement of the splicing variant EPAC2B in these cAMP-mediated responses and additional establishes a job for EPAC1/2 in actin reorganization and migration. Moreover, the position of EPAC2B in cell motility could have wider implications, corresponding to for the group of the adrenal gland and for adrenal most cancers cell invasion.
Acknowledgments
The superb technical help of Torild Ellingsen and Anne Sellevold is extremely appreciated. Confocal imaging was carried out with the assistance of Hege Avsnes Dale on the Molecular Imaging Heart (Practical Genomics Program, Norwegian Analysis Council), College of Bergen.
Abbreviations – “protein kinase a function”
“protein kinase a function”