Activation and expression of endogenous CREB‐regulated transcription coactivators (CRTC) 1, 2 and 3 in the rat adrenal gland

Abstract The activation and nuclear translocation of cAMP‐response element binding protein (CREB)‐regulated transcription coactivator (CRTC)2 occurs in the rat adrenal gland, in response to adrenocorticotrophic hormone (ACTH) and stressors, and has been implicated in the transcriptional regulation of steroidogenic acute regulatory protein (StAR). We have recently demonstrated the activation of CRTC isoforms, CRTC1 and CRTC3, in adrenocortical cell lines. In the present study, we aimed to determine the activation and expression of the three CRTC isoforms in vivo in relation to Star transcription, under basal conditions and following a robust endotoxic stress challenge. Rat adrenal glands and blood plasma were collected following i.v. administration of either an ultradian‐sized pulse of ACTH or administration of lipopolysaccharide, as well as under unstressed conditions across the 24‐hour period. Plasma ACTH and corticosterone (CORT) were measured and the adrenal glands were processed for measurement of protein by western immunoblotting, RNA by a quantitative reverse transcriptase‐polymerase chain reaction and association of CRTC2 and CRTC3 with the Star promoter by chromatin immunoprecipitation. An increase in nuclear localisation of CRTC2 and CRTC3 followed increases in both ultradian and endotoxic stress‐induced plasma ACTH, and this was associated with increased CREB phosphorylation and corresponding increases in Star transcription. Both CRTC2 and CRTC3 were shown to associate with the Star promoter, with the dynamics of CRTC3 binding corresponding to that of nuclear changes in protein levels. CRTC isoforms show little variation in ultradian expression or variation across 24 hours, although evidence of long‐term down‐regulation following endotoxic stress was found. We conclude that co‐transcription factors CRTC2 and, more clearly, CRTC3 appear to act alongside phosphorylated CREB in the generation of ultradian pulses of Star transcription, essential for the maintenance of basal StAR expression. Similarly, our findings suggest CRTC2 and CRTC3 mediate Star transcriptional initiation following an endotoxic stressor; however, other transcription factors are likely to be responsible for the long‐term up‐regulation of adrenal Star transcription.

Withinsteroidogeniczonafasciculatacellsoftheadrenalcortex, CORTissynthesiseddenovouponACTHactivationofsteroidogenic enzymes.Theactivityandexpressionoftheseenzymes,particularly that of rate-limiting steroidogenic acute regulatory (StAR) protein, is tightly regulated by ACTH, predominantly through phosphorylation by the cAMP/PKA pathway. [2][3][4][5] Turnover of StAR protein israpid,withtheactiveformofStARhavingahalf-lifeofapproxi-mately5-15minutes. 3,6 InadditiontorapidactivationofStARand other steroidogenic enzymes through phosphorylation, ACTH also stimulates the transcription of Starandthesteroidogenicenzymes to replenish and maintain expression. [7][8][9] AkeytranscriptionfactorinvolvedintheregulationofStar tran-scriptioniscAMPresponseelementbindingprotein(CREB),directly regulatedbyACTHthroughphosphorylation. 10,11 Additionally,ACTH can indirectly activate CREB through post-transcriptional modification of co-transcription factors, including the well-characterised CREB binding protein/p300 (CBP/p300) and CREB-regulated transcriptioncoactivator(CRTC,previouslyknownasTORC).CRTCisa co-transcription factor that enhances binding of CREB to the gene promoterthroughitsbindingtotheCREBbZIPdomain,workingindependently of CBP. 12 CRTCissequesteredinthecytoplasmuntilactivatedbydephosphorylation,followingincreasedintracellularcAMP levels. 13,14 This allows CRTC to translocate into the nucleus and bind CREB at the transcription site. 15 Three isoforms (CRTC1, 2 and 3) have been identified, with CRTC2 and CRTC3 shown to be the most highly expressed in the adrenal gland. 12,16 CRTC2 has long been implicated in the regulation of Star transcription in vitro 13,14 and in vivo, where nuclear levels of phosphorylated CREB (pCREB) and CRTC2 in the rat adrenal gland have been shown to increase in response to bothhighandlowdoseACTHandrestraintstress. 17,18 More recently, using murine adrenocortical cell lines, we have demonstrated that, in additiontoCRTC2,ACTHalsostimulatesrapidnucleartranslocation of CRTC1 and CRTC3, and that both CRTC2 and CRTC3 bind at the StarpromoterinresponsetoACTH,suggestingarolefortheseisoforms in mediating the initiation of Star transcription. 16 Underbasal(unstressed)conditions,bothACTHandCORTlevels fluctuate in both an ultradian and a circadian manner 19 and we have shown that circadian and ultradian dynamics are evident within the steroidogenic pathway (both at the level of protein activation and gene transcription) and reflect ACTH and CORT profiles. 20,21 Furthermore, we have shown that the adrenal gland steroidogenic pathway is also activated in response to endotoxic stress. 21 The present study aimed to examine whether the activity and expression of the endogenous CRTC isoforms in vivo in the rat adrenal gland exhibit a similar pattern of activity and expression as observed for other key regulators of CORT synthesis. Specifically, given the differential activation across CRTC isoforms observed in vitro, we aimed to determine the extent to which endogenously expressed CRTC1, CRTC2 and CRTC3 may be activated by translocation and associated with the Star promoter during the initiation and sustained transcription of Star in response to an ACTH ultradian pulse and followingacuteendotoxicstress.Furthermore,becauseseveralkey steroidogenic regulators exhibit an ultradian and circadian pattern of expression in basal condition, as well as in response to stress, we investigatedwhetherCRTC1-3alsoexhibitstrongendogenousregulation of transcription and expression. Gloucester, UK), as described previously. 22 Following 5-7 days recovery after surgery, experiments were commenced at 9.00 am. Rats wereadministeredwitheitheranultradian-sizedACTHpulse(10ng ina100-µLvolumeof0.9%salinesolution;AlliancePharmaceuticals, Ltd, Chippenham, UK; n = 5-7 independent rats per group) or LPS Forinvestigationofam-pm variation, rats were maintained under a normal light/dark schedule as described above. Rats were killed usingisofluraneevery4hoursat1.00am,5.00am, 9.00 am, 1.00 pm, 5.00pm and 9.00 pm (n =5or6pertimepoint).

| Animals
Immediately after death, as indicated above, trunk blood was
RNA was tested using a NanoDrop spectrophotometer (Thermo  Absence of detection when omitting the reverse transcription enzyme Superscript III (Invitrogen) indicated a lack of genomic DNA contamination.

| Protein extraction and western immunoblotting
Whole cell lysate extraction was performed as described previously. 20

| Statistical analysis
For these experiments, no animals were excluded from the study.
Rats were allocated to treatment/timepoint groups using randomi-    The acrophase was then taken as the clock time corresponding to the peak value in the fitted curve. 20  24 Ultradian activity of CRTC isoforms was investigated by isolating and quantifying the adrenal gland nuclear cell fraction to measure activation by nuclear translocation ( Figure 1E). ACTH had no significant effect on cytosolic levels of CRTC1 or CRTC2, although it did affect CRTC3 levels, which were maximal by 60 minutes following ACTH administra-  30minutesbyTukey'spost-hoctest;CRTC3P =0.0615minutesvs

| CRTC activation in response to an endotoxic stressor
To activate the adrenal gland response to endotoxic stress, rats were injected i.v. with LPS ( Figure 2). As previously reported, there was asignificantincreaseinplasmaACTH(F 7,11.79 =50.25;P < 0.0001

| Interaction of CRTC2 and CRTC3 at the Star promoter
To determine whether significant increases in adrenal nuclear CRTC2 and CRTC3 in response to ACTH were associated with increased binding of these isoforms at the Starpromoter,ChIPassayswereper-

| Expression and transcriptional activation of CRTC1-3
In addition to activating steroidogenic transcriptional regulators, ACTH also regulates the transcription of many transcription factorsinvolvedinsteroidogenesis,includingSF-1,Nur77andDAX-1. We then investigated whether CRTC transcription will be affected by endotoxic stress ( Figure 5B). hnRNA levels for all three isoforms were significantly altered following LPS injection (Crtc1

| D ISCUSS I ON
Inthepresentstudy,wedemonstratethatnucleartranslocationof endogenous CRTC2 and CRTC3 in the rat adrenal gland occurs in response to both basal, ultradian HPA axis activity, and following endotoxic stress, thus complimenting the findings from previous in vitro and in vivo studies in which activation of CRTC2 and CRCT3 wasshowninresponsetoACTHandrestraintstress. 13,14,[16][17][18] Here, we show that an increase in CRTC2 and CRTC3 nuclear localisation parallels, or precedes, the increase in CREB phosphorylation and has previously been reported 17 and, in the present study, we show that activation of CRTC2 and CRTC3 also occurs following endotoxic stress induced by LPS injection, with peak nuclear levels seen at30-60minutesafterexposure,returningtobasalby180minutes.
LowdoseACTHandendotoxicstressorinducednucleartranslocation of endogenous CRTC1, although with no statistical significance, suggesting that CRTC1 may still play a role in mediating ACTH-induced Star transcription. This potentially indicates that

CRTC1islessresponsivetoACTHsignallingthanCRTC2orCRTC3.
Elsewhere, CRTC1 has demonstrated a limited increase in nuclear translocationfollowingforskolintreatmentinhypothalamic4Bcells, HeLa cells and Hek293 cells. 15,26 Alternatively,constitutivenuclear localisation of CRTC1 may be responsible for the smaller increases in nuclearlocalisationdetectedfollowingACTHtreatment.However, adrenal gland expression of CRTC1 is lower than CRTC2 or CRTC3 in the adrenal gland, 12,16 suggesting that the isoform is of limited importance in this tissue. Further functional studies are therefore requiredtoestablishtheroleCRTC1playsinthesecells. Levels of the dominant-negative SF-1 inhibitor, DAX-1, however, were shown to be decreased between 3 and 4 hours, potentially leading to increased SF-1 activity at this time. 21 Furthermore, LPS has also been shown to rapidly increase rat adrenal hnRNA and mRNA levels of orphan nuclear receptors Nr4a3 (aka Nor1) and Nr4a1 (aka Nur77). 21 Nur77 has been shown to bind the Star pro- Conversely, other transcription factors, including SF-1, Nur77 and Nor1, exhibit circadian variation in expression. 20,21 Furthermore, hnRNA levels of Nr5a1, Nr4a1 and Nr4a3 are all increased in response to 10 ng of ACTH, behaving similarly to Star. 21 It appears that,asshownforCREB,CRTCactivityisregulatedbyACTHmainly at the level of post-translational modifications. Interestingly, while no circadian variation in CRTC2 expression has been shown in the mouseSCN,apeakinCrtc1mRNAhasbeenshownduringtheday. 46 Followinganendotoxicstressor,hnRNAlevelsforCrtc1, Crtc2 and Crtc3, as well as levels of both cytosolic and nuclear CRTC2 and CRTC3 protein, decreased significantly below baseline, suggesting negative-feedback on CRTC isoform expression in the adrenal glandasaresultofthelong-termup-regulationofStar transcription,

Furtherstudiesareneededtoexplorethishypothesis.
In conclusion, our data show that CRTC2 and, more distinctly, CRTC3 appear to be key co-transcription factors in the regulation of Startranscription,inresponsetobothbasalultradianACTHactivityandfollowingstress.OurresultssupportaroleforCRTC2and

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y
The data that support the findings of this study are available from thecorrespondingauthoruponreasonablerequest.