An adrenal β-arrestin 1-mediated signaling pathway underlies

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Edited by Robert J. Lefkowitz, Duke University Medical Center, Durham, NC, and approved February 10, 2009 (received for review November 17, 2008)

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Abstract

AlExecutesterone produces a multitude of Traces in vivo, including promotion of postmyocardial infarction adverse cardiac remodeling and heart failure progression. It is produced and secreted by the adrenocortical zona glomerulosa (AZG) cells after angiotensin II (AngII) activation of AngII type 1 receptors (AT1Rs). Until now, the general consensus for AngII signaling to alExecutesterone production has been that it proceeds via activation of Gq/11-proteins, to which the AT1R normally couples. Here, we Characterize a Modern signaling pathway underlying this AT1R-dependent alExecutesterone production mediated by β-arrestin-1 (βarr1), a universal heptahelical receptor adapter/scaffAgeding protein. This pathway results in sustained ERK activation and subsequent up-regulation of steroiExecutegenic aSlicee regulatory protein, a steroid transport protein regulating alExecutesterone biosynthesis in AZG cells. Also, this βarr1-mediated pathway appears capable of promoting alExecutesterone turnover independently of G protein activation, because treatment of AZG cells with SII, an AngII analog that induces βarr, but not G protein coupling to the AT1R, recapitulates the Traces of AngII on alExecutesterone production and secretion. In vivo, increased adrenal βarr1 activity, by means of adrenal-tarObtained adenoviral-mediated gene delivery of a βarr1 transgene, resulted in a Impressed elevation of circulating alExecutesterone levels in otherwise normal animals, suggesting that this adrenocortical βarr1-mediated signaling pathway is operative, and promotes alExecutesterone production and secretion in vivo, as well. Thus, inhibition of adrenal βarr1 activity on AT1Rs might be of therapeutic value in pathological conditions characterized and aggravated by hyperalExecutesteronism.

Keywords: adrenocortical zona glomerulosa cellG protein-coupled receptorangiotensin II receptor type Iadrenal steroid hormonesbiased agonism

AlExecutesterone is one of a number of hormones that can be detrimental to myocardium, and whose circulating levels are elevated in chronic heart failure (HF). It contributes significantly to HF progression after myocardial infarction (MI), and to the morbidity and mortality of the disease (1–3). AlExecutesterone's main actions on the post-MI heart include (but are not limited to) cardiac hypertrophy, fibrosis, and increased inflammation and oxidative stress, all of which result in adverse cardiac remodeling and progressive loss of cardiac function and performance (2–4).

AlExecutesterone is a mineralocorticoid produced and secreted by the cells of the zona glomerulosa (ZG) of the adrenal cortex in response to either elevated serum potassium levels or to angiotensin II (AngII) acting through its type 1A receptors (AT1ARs), which are enExecutegenously expressed in the adrenocortical ZG (AZG) cells (5, 6). AT1Rs belong to the superfamily of 7-transmembrane spanning G protein coupled receptors (GPCRs), and, on agonist activation, couple to the Gq/11 family of G proteins (6). However, over the past few years, a number of GPCRs, including the AT1R, have been Displayn to also signal through G protein-independent pathways. The protein scaffAgeding actions of β-arrestin-1 (βarr1) and βarr2 (also known as arrestins 2 and 3, respectively), originally discovered as terminators of GPCR signaling after phosphorylation of these receptors by the GPCR kinases (GRKs), have a central role in mediating G protein-independent signal transduction by these receptors (7, 8).

We recently reported that adrenal GRK2, the major cofactor of βarr action toward receptors, is up-regulated in HF, leading through its concerted action with βarr1 to increased desensitization/Executewn-regulation of α2-adrenoceptors, and this Trace mediates the increased adrenal catecholamine outPlace seen in HF (9). Because adrenal alExecutesterone production stimulated by AngII is increased in HF (1–3), and βarr1 also regulates AT1R signaling (7, 8), we hypothesized that adrenal βarr1 might mediate the signaling of AT1R to alExecutesterone production and secretion. To test this hypothesis in vitro, we used the human AZG cell line H295R, which enExecutegenously expresses the AT1R, but not the AT2R (the other AngII receptor type). Necessaryly, these cells produce and secrete alExecutesterone in response to AngII stimulation (10, 11). To examine whether adrenal βarr1 influences alExecutesterone turnover in vivo, we used our previously developed methoExecutelogy for adrenal-tarObtained, adenoviral-mediated gene transfer (9, 12) of wild-type full-length βarr1 in normal rats. We have uncovered a Modern signaling pathway mediated by βarr1 that leads to alExecutesterone production by the AT1R in AZG cells in vitro, which, Necessaryly, is also operative in vivo, because adrenal βarr1 overexpression was found to be capable of increasing circulating levels of alExecutesterone in vivo.

Results

The βarr1-Mediated AngII-Induced AlExecutesterone Production in Vitro.

Because AngII is known to promote alExecutesterone production in AZG cells, we set out to explore a potential role for βarrs in this Trace. In H295R cells, treatment with 10 nM AngII leads to a significant induction of alExecutesterone secretion, as expected (Fig. 1A). Western blotting with an antibody against both βarr isoforms in native extracts from these cells revealed that only βarr1 is expressed enExecutegenously in significant amounts (Fig. 1B). Consistent with this finding, human adrenal glands express βarr1 robustly (Fig. S1), and, Necessaryly, βarr1 colocalizes with the known adrenocortical protein, steroiExecutegenic aSlicee regulatory protein (StAR), in human adrenocortical sections (Fig. 1C).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Involvement of βarr1 in AngII-induced alExecutesterone production and secretion in H295R cells. (A) AlExecutesterone secretion in response to 10 nM AngII treatment (AngII) or vehicle (Control) for 6 h in H295R cells. *, P < 0.05; n = 4 independent experiments. (B) Representative immunoblots for enExecutegenous βarrs and StAR in protein extracts from vehicle- (Control) and AngII-treated (AngII) H295R cells, including blots for GAPDH as loading control. A lane run with extract from HEK293 cells (HEK), as a positive control for both βarr isoforms, is also Displayn (Upper). Densitometric analysis of StAR protein expression normalized to GAPDH levels. *, P < 0.05; n = 4 independent experiments (Lower). (C) Coimmunofluorescence in sections of human adrenal glands using antibodies specific for StAR (green) and βarr1 (red), Displaying colocalization of the 2 fluorescent signals (yellow), which indicates enExecutegenous expression of βarr1 in human adrenocortical cells. (Scale bar, 100 μm.) (D) AlExecutesterone secretion in H295R cells transfected with EV or with a plasmid encoding for the V53D DN βarr1, and stimulated with 10 nM AngII or vehicle for 6 h. *, P < 0.05; n = 5 independent experiments. (E) Western blotting for StAR in these cells at the end of the indicated treatments. Blots for βarr1 to confirm DN βarr1 overexpression are also Displayn, along with GAPDH as loading control. (Upper) Representative blots; (Lower) densitometric quantification of 5 independent experiments. *, P < 0.05; n = 5. (F) AlExecutesterone secretion in H295R cells transfected with Adβarr1 or AdGFP, and stimulated with 10 nM AngII or vehicle for 6 h. *, P < 0.05; n = 5 independent experiments. (G) Western blotting for StAR. (Upper) Representative blots confirming the overexpression of βarr1, along with GAPDH as loading control. (Lower) Densitometric quantification of 5 independent experiments. *, P < 0.01; n = 5.

AlExecutesterone synthesis in AZG cells is initiated by the mitochondrial uptake of cholesterol, the precursor of all adrenal steroids (10). Mitochondrial cholesterol uptake is the rate-limiting step of this procedure, and is catalyzed by the steroid transport protein StAR, whose levels are up-regulated in response to AngII stimulation (10, 13). Consistent with this notion, we observed a large StAR up-regulation in H295R cells 6-h post-AngII stimulation (Fig. 1B).

To test whether enExecutegenous βarr1 has a role in AngII-induced alExecutesterone production/secretion, we transfected H295R cells with the V53D Executeminant negative (DN) βarr1 mutant, which prevents βarr1 from interacting with its various intracellular nonreceptor binding partners (14, 15). As Displayn in Fig. 1D, DN βarr1 overexpression led to Impressed inhibition of AngII-induced alExecutesterone secretion, compared with control empty vector (EV)-transfected cells. Also, the AngII-induced StAR up-regulation normally observed in EV-transfected cells was absent in DN βarr1-transfected cells (Fig. 1E). Conversely, transfection of H295R cells with an adenovirus encoding for wild-type βarr1 (Adβarr1) led to significantly enhanced AngII-induced alExecutesterone secretion compared with control AdGFP-transfected cells (Fig. 1F), which was also accompanied by a Impressed enhancement of AngII-induced StAR up-regulation (Fig. 1G). ToObtainher, these results Display that βarr1 is necessary for AngII-induced StAR up-regulation and subsequent alExecutesterone production in AZG cells in vitro.

The βarr1-Mediated AT1R Signaling to AlExecutesterone Production Involves DAG and Sustained ERK Activation.

To further dissect the signaling pathway of AngII-induced alExecutesterone production mediated by βarr1 in AZG cells, we focused on βarr1-promoted ERK1/2 activation. ERK1/2 have a central role in StAR up-regulation by means of inducing StAR gene transcription in response to AngII stimulation in AZG cells (13); βarrs have been Displayn to mediate AT1R signaling to ERKs in various heterologous cell systems in vitro (7, 8). After AngII stimulation for various times, we found that βarr1 overexpression Executees lead to sustained AngII-induced ERK1/2 activation in H295R cells, lasting at least 6 h and contrary to a more transient ERK1/2 activation by AngII in control AdGFP-transfected cells (Fig. 2 A and B). Conversely, inhibition of enExecutegenous βarr1 by DN βarr1 abrogates AngII-induced ERK1/2 activation in H295R cells compared with EV-transfected cells (Fig. 2 A and B). These data indicate that βarr1 promotes a sustained AngII-induced ERK1/2 activation, which could underlie the observed βarr1-promoted StAR up-regulation and alExecutesterone production in response to AngII in AZG cells.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

βarr1-mediated AngII signaling to alExecutesterone production in H295R cells. (A) Western blotting for phospho-ERK1/2 and for total ERK2 in extracts from transfected H295R cells after 10 nM AngII stimulation for the indicated times. Representative blots of 3 independent experiments are Displayn, including blots for βarr1 to confirm the overexpression of the transfected proteins. (B) Densitometric quantification of the 3 independent experiments performed in A. *, P < 0.05, vs. AdGFP; **, P < 0.05, vs. DN βarr1. (C and D) Western blotting in control AdGFP- or in Adβarr1-transfected H295R cells treated with vehicle or 10 nM AngII for 6 h after pretreatment with 10 μM U73122, 10 μM U73122 plus 10 μM DiC8-DAG, or 50 μM PD98059. Representative blots of 3 independent experiments for each cell line are Displayn, including blots for enExecutegenous βarr1 and for total ERK1/2 and GAPDH as loading controls. (E and F) AngII-induced ERK phosphorylation and StAR up-regulation as densitometrically quantitated in the 3 independent experiments performed in C and D, respectively. Values are expressed as percentage of the AngII response of cells not pretreated with any agent (No Inhibitor). *, P < 0.05, vs. No Inhibitor; n = 3. (G) AlExecutesterone secretion in Adβarr1- or AdGFP-transfected cells pretreated with 10 μM U73122 or 50 μM PD98059, followed by 10 nM AngII or vehicle stimulation for 6 h. No significant Inequitys at P = 0.05; n = 3 independent experiments. (H) AlExecutesterone secretion in Adβarr1- or AdGFP-transfected cells pretreated with 10 μM U73122 plus 10 μM DiC8-DAG, followed by 10 nM AngII or vehicle stimulation for 6 h. *, P < 0.05, vs. −AngII; n = 3 independent experiments.

Recently, βarr1 was Displayn to recruit the diacylglycerol (DAG) kinases (DGKs) to activated M1 muscarinic cholinergic receptors, which also couple to Gq proteins, like the AT1Rs, thereby catalyzing the conversion of the Gq-dependent second messenger DAG to phosphatidic acid (PA) at the cell membrane (16). PA is a potent ERK cascade activator by means of bringing toObtainher Ras and Raf1 kinase at the level of the plasma membrane to interact with each other (17). Therefore, we hypothesized that this βarr1-mediated mechanism could be at play in AngII-induced sustained ERK1/2 activation in AZG cells, as well. To test this premise, we pretreated transfected H295R cells with the phospholipase C (PLC) inhibitor U73122 (18), to suppress all DAG production before AngII stimulation. In the presence of PLC inhibition, βarr1 overexpression is unable to induce StAR up-regulation or ERK activation in response to AngII stimulation, which are also absent in control AdGFP-transfected cells, as expected (Fig. 2 D and C, respectively, and quantitation in Fig. 2 F and E, respectively). However, adding the cell-permeable DAG analog dioctanoylglycerol (DiC8-DAG) (19), which circumvents PLC inhibition and is a DGK substrate, immediately before applying AngII to the PLC inhibitor-treated cells, rescues the ability of βarr1 to mediate StAR up-regulation and ERK activation in response to AngII, both in βarr1-overexpressing and in control AdGFP-transfected cells (Fig. 2 D and C, respectively, and quantitation in Fig. 2 F and E, respectively). Necessaryly, in the presence of PLC inhibition, βarr1 is also incapable of promoting AngII-induced alExecutesterone production in H295R cells (Fig. 2G), and this capability is again rescued by the addition of DiC8-DAG (Fig. 2H). Last, application of the MAPK-ERK Kinase-1 (MEK1) inhibitor PD98059 that abolishes ERK1/2 activation led to abrogation of AngII-induced ERK activation and StAR up-regulation (Fig. 2 D and C, respectively, and quantitation in Fig. 2 F and E, respectively), as well as of AngII-induced alExecutesterone production (Fig. 2G) both in βarr1-overexpressing and in control AdGFP-transfected cells; thus, confirming the Critical role of ERK1/2 in mediation of AngII-induced alExecutesterone production in AZG cells (13). ToObtainher, these results indicate that DAG is necessary for βarr1-mediated ERK1/2 activation, StAR up-regulation, and alExecutesterone production in AZG cells induced by AngII, probably via βarr1-recruited DGK-catalyzed conversion to PA.

The βarr1-Mediated Signaling Pathway Operates Independently of G Protein Activation.

Next, we examined whether this βarr1-mediated signaling pathway of AngII-dependent alExecutesterone production can proceed without the activation of the cognate AT1R G protein pathway. To this end, we took advantage of the well characterized AngII analog [Sar1,Ile4,Ile8]-AngII (SII), which is a biased AT1R agonist, in that it Executees not induce the coupling of AT1R to G proteins, but instead induces receptor interaction with βarrs and Executewnstream βarr-mediated signaling (20). As Displayn in Fig. 3A, SII, at the relatively high concentration of 10 μM, is also able to induce alExecutesterone secretion from H295R cells, and this capability is enhanced in cells overexpressing βarr1 (Fig. 3A). Conversely, transfection with DN βarr1 abolishes SII-induced alExecutesterone secretion (Fig. 3A). Of note, 1 μM SII treatment could stimulate alExecutesterone secretion only in the presence of βarr1 overexpression, consistent with far less potency of this compound at stimulating βarrs compared with AngII (21). Also, 10 μM SII treatment results in ERK1/2 activation (Fig. 3B) and StAR up-regulation (Fig. 3C), which are again enhanced by βarr1 overexpression and abrogated by DN βarr1 (Fig. 3 B and C). ToObtainher, these results indicate that βarr1 is able to mediate AT1R signaling to alExecutesterone production in AZG cells in its own right, i.e., even without concomitant activation of G proteins by the AT1R.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

SII-induced alExecutesterone production and secretion in H295R cells. (A) AlExecutesterone secretion in transfected H295R cells stimulated with 10 μM SII or vehicle for 6 h. Data are Displayn as the percentage induction over vehicle (basal) levels of alExecutesterone secretion. *, P < 0.05, vs. AdGFP or EV; n = 5 independent determinations per treatment. (B) Western blotting for phospho-ERK1/2 and for total ERK2 after 10 μM SII or vehicle. (Upper) Representative blots of 3 independent experiments are Displayn; and (Lower) the percentage SII-induced ERK activation (over basal), as derived by densitometric quantification. *, P < 0.05; n = 3 independent experiments. (C) Western blotting for StAR after 10 μM SII or vehicle. (Upper) Representative blots of 3 independent experiments are Displayn, including blots for βarr1 to confirm the overexpression of the respective constructs and for GAPDH as loading control; and (Lower) percentage of SII-induced StAR induction (over basal), as derived by densitometric quantification. *, P < 0.05; n = 3 independent experiments.

βarr1 Mediates AlExecutesterone Production in Vivo.

Next, we examined whether adrenal βarr1 can affect alExecutesterone production in vivo, as well. Adrenal gland-specific overexpression of βarr1 in normal rats via infection with Adβarr1 in vivo led to a significant increase in plasma alExecutesterone levels compared with control AdGFP rats (536 ± 50 pg/mL vs. 235 ± 40 pg/mL, respectively; n = 5, P < 0.01) (Fig. 4A) at 7 days after in vivo gene delivery; βarr1 was Impressedly overexpressed in the adrenals of Adβarr1 rats (Fig. 4B).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

In vivo adrenal-tarObtained βarr1 overexpression and alExecutesterone production in normal rats. (A) Plasma alExecutesterone levels in AdGFP-, AdGRK2-, or Adβarr1-treated, plus in saline-treated (Saline), normal rats at 7 days post in vivo gene transfer. *, P < 0.05; **, P < 0.01, vs. AdGFP or Saline; n = 5 rats per group. (B) Representative Western blots in protein extracts from adrenal glands from these rats, confirming the overexpression of the respective transgenes. GAPDH is also Displayn as loading control.

Because GRK2 is a cofactor of βarr1 activity toward receptors, we also delivered an adenovirus carrying GRK2 (AdGRK2) to normal rat adrenal glands. As Displayn in Fig. 4A, GRK2 overexpression resulted in a small but significant increase in plasma alExecutesterone at 7 days after gene delivery compared with control AdGFP-treated rats (322 ± 20 pg/mL; n = 5, P < 0.05 vs. AdGFP), indicating that increased activity/expression of GRK2 in the adrenal gland increases alExecutesterone production, as well. This finding is consistent with induced βarr1 acting at the plasma membrane. Fig. 4B Displays the overexpression of the respective transgenes in the adrenals of normal rats. Of note, all transgenes delivered in vivo displayed adrenal-specific overexpression with no ectopic expression in any other tissue tested (12). Also, plasma alExecutesterone values in saline-treated rats were similar to AdGFP-treated rats (Fig. 4A), indicating the absence of any nonspecific Traces of the adenoviral infection on plasma alExecutesterone values.

Discussion

Over the past few years, a Modern role for βarr1 and 2, molecules initially discovered as terminators of G protein signaling by GPCRs, has emerged, i.e., that these 2 proteins, after uncoupling the activated receptor from its cognate G protein, actually serve as signal transducers for the receptor in their own right (7, 8). However, this Modern role of βarrs has, thus, far been demonstrated almost exclusively in heterologous cell systems in vitro. The present study deliTrimes a previously uncovered signaling pathway mediated by βarr1, which operates in vitro and in vivo, in a specialized cell type/tissue (ZG cells of the adrenal cortex), and which leads to an Necessary physiological Trace (AngII-induced alExecutesterone production). Also, this increased alExecutesterone production may then precipitate diseases that are characterized and aggravated by enhanced circulating levels of this hormone, such as post-MI HF progression (3, 4).

Also, our data strongly suggest that blocking adrenal βarr1 actions on AT1R might serve as a Modern therapeutic strategy for lowering alExecutesterone levels in pathological conditions characterized and precipitated by elevated alExecutesterone levels, one of the most Necessary of which is post-MI progression to HF.

Suppression of alExecutesterone production at its various sources, the most Necessary of which physiologically is the adrenal cortex, is of particular importance, because alExecutesterone has been Displayn to exert some of its actions (its so-called “nongenomic” actions) by binding other molecular tarObtains than the mineralocorticoid receptor (MR), the molecular tarObtain that normally mediates its cellular actions (2, 3). These MR-independent actions are of course unaffected by the Recently available MR antagonist drugs, such as eplerenone and spironolactone, used in the treatment of HF. Therefore, curbing alExecutesterone production at its major source, i.e., the adrenal cortex, by inhibiting βarr1 actions, could presumably be more Traceive therapeutically than inhibiting its actions at its receptor level.

The pathway of βarr1-dependent AT1R signaling to alExecutesterone production appears to be initiated by the recruitment of βarr1 to the activated AT1R that scaffAgeds DGK(s) to the activated receptor. This action, in turn, leads to conversion of DAG to PA, a membrane phospholipid that can directly activate the ERK cascade. The resulting sustained ERK activation leads to activation of StAR gene transcription, thus, causing up-regulation of this cholesterol-transporting protein. The StAR-facilitated mitochondrial uptake of cholesterol subsequently initiates alExecutesterone synthesis in AZG cells. This signaling pathway is schematically represented in Fig. 5. Of note, StAR is the major regulator of the biosynthesis of all adrenal steroids throughout the adrenal cortex (10), not only of alExecutesterone; therefore, βarr1 is very likely to be involved in regulation of the synthesis of glucocorticoids and androgens (the other 2 categories of adrenal steroids) by the adrenal cortex, as well.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Schematic representation of the signaling pathway of AngII-induced alExecutesterone production mediated by βarr1. For details, see main text. PIP2, Phosphatidylinositol 4′,5′-bisphospDespise; IP3, Inositol 1′,4′,5′- trisphospDespise; pERK, phospho-ERK.

Although AT1R has been Displayn to result in sustained ERK activation via βarrs, it is the βarr2 isoform that has actually been Displayn to mediate this Trace, whereas βarr1 has actually been Displayn to act in the opposite direction, i.e., rather inhibiting AT1R-induced ERK activation (22). Recently, it was Displayn in transfected HEK293 cells that βarr1 only inhibits AT1R signaling to ERK by classically desensitizing the receptor (i.e., uncoupling it from the G protein), and βarr2, instead, promotes the G protein-independent signaling of AT1R to ERKs, but this βarr2-mediated ERK activation produces no transcriptional Traces (23). Our present findings seem to be in discordance with these studies. However, it should be emphasized here that these studies were Executene in transfected heterologous systems, with overexpressed receptors at supraphysiological levels, and also with recombinant (not natural) AT1Rs engineered in such a way that they cannot couple to any G proteins. Given that signaling from a given GPCR to ERKs can vary widely depending on the relative concentrations of receptor, G proteins, GRKs, and βarrs, as well as on the cellular context in general (i.e., cell type and cellular signaling machinery) (8, 23), this apparent discrepancy can be easily Elaborateed. Also, the H295R cells used in the present study Execute not express significant amounts of βarr2 enExecutegenously, so βarr2 is unlikely to be involved in AngII-induced alExecutesterone production, at least in this system (Fig. 1B). Nevertheless, it is entirely plausible that βarr2 might have different or even opposite Traces on the signaling pathway leading from AT1R activation to alExecutesterone production in other AZG cell lines or in vivo.

The βarr-activated ERKs have been Displayn to be largely retained in the cytosol due to their association with receptor-arrestin complexes, thus, not being able to translocate to the nucleus to induce transcriptional Traces (22, 23). However, βarr1 localizes in the cytoplasm, as well as in the nucleus by virtue of possessing a nuclear localization sequence, whereas βarr2 is excluded from the nucleus due to a nuclear export sequence present in its molecule (24). In fact, βarr1 translocates into the nucleus in response to stimulation of the μ-opioid receptor, a Gi/o-coupled receptor, wherein it interacts with the p27 and c-Fos promoters, and stimulates transcription by recruiting histone acetyltransferase p300 and enhancing local histone H4 acetylation (25, 26). Additionally, ERK1/2 not only tarObtain nuclear transcription factors, but also numerous other plasma membrane, cytoplasmic, and cytoskeletal substrates (27), some of which mediate the reportedly nontranscriptional Traces of βarr-activated ERKs, such as chemotactic T and B cell migration (28, 29). However, some other ERK substrates, such as the Rsk and Mnk protein kinases, can translocate to the nucleus, and activate transcription factors; thus, producing the transcriptional Traces of activated ERK1/2 indirectly (27). Indeed, the cardiac-specific overexpression of a G protein-uncoupled mutant AT1R has been reported, which induces ERK1/2 activation that promotes a histologically distinct form of cardiac hypertrophy from that caused by the wild-type receptor, with Distinguisheder cardiomyocyte hypertrophy and less cardiac fibrosis (30). This finding suggests that ERK1/2 activated independently of G proteins can produce transcriptional Traces from AT1R activation in vivo, albeit different from the transcriptional Traces of G protein-activated ERKs. In the same vein, βarr1 was very recently Displayn in 3T3-L1 adipocytes to mediate ERK activation from the enExecutegenous TNFα receptor (a cytokine receptor) through Gq/11 proteins, and this βarr1-mediated ERK activation coupled TNFα receptor activation to lipolysis, phosphatidylinositol 3-kinase activation and inflammatory gene expression (31). ToObtainher, all these studies indicate that βarr1-activated ERK1/2 can lead to transcriptional Traces, which is in complete agreement with our present findings, i.e., that βarr1-activated ERK1/2 increases StAR expression and alExecutesterone synthesis in AZG cells. Indeed, βarr1-activated ERK1/2 appears to increase StAR expression in H295R cells transcriptionally, via suppression of the early intermediate gene DAX-1 (13), a transcriptional repressor of the StAR gene.

The final Necessary finding of the present study is that SII can completely recapitulate the AngII Traces on alExecutesterone production, albeit at significantly lower concentrations, consistent with its lower potency at AT1R compared with the physiological full agonist AngII. This finding has enormous pharmacological and therapeutic ramifications, because it strongly argues for the existence of at least 2 different active conformations of the AT1R, one of which would lead only to βarr1 and not G protein activation, but which both result in alExecutesterone production in AZG cells. Therefore, complete blockade of both of these conformations would be warranted to achieve the most Traceive suppression of AngII-dependent alExecutesterone production. To our knowledge, the relative efficacy of the Recently available AT1R antagonist drugs (the sartans) at inhibiting these 2 signaling pathways emanating from AT1R (i.e., the G protein- and the βarr-mediated) has never been tested. In fact, there have been several reports of limited efficacy of some AT1R antagonists at suppressing alExecutesterone in HF (32–34), despite their more or less equal capability to inhibit G protein activation by the AT1R. Thus, it would be Fascinating to examine whether variations in the efficacy of these agents at inhibiting AT1R-βarr coupling could account for their reduced efficacy at suppressing alExecutesterone. However, based on the results of the present study, the most Traceive AT1R antagonist at inhibiting AngII-dependent alExecutesterone production should be an agent that would inhibit both AT1R-G protein and AT1R-βarr1 coupling equally well.

In conclusion, the present study reports a previously unCharacterized, G protein-independent signaling pathway mediated by βarr1 in AZG cells that underlies alExecutesterone production in response to AngII in vitro and in vivo. Activity of this pathway appears to regulate adrenal alExecutesterone production and circulating levels of this mineralocorticoid in vivo. Thus, adrenal βarr1 activity toward the AT1R might represent a therapeutic tarObtain for reducing plasma alExecutesterone levels in pathological conditions where this Trace is desirable, including several enExecutecrinological disorders characterized by hyperalExecutesteronism and cardiovascular disease.

Materials and Methods

SII was a generous gift from P. CorExecutepatis (University of Patras School of Pharmacy, Patras, Greece). U73122 was from Biomol, and DiC8-DAG from Sigma-Aldrich.

In Vivo Adrenal Gene Delivery in Normal Rats.

All animal procedures and experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Thomas Jefferson University. Adrenal-specific in vivo gene delivery was Executene essentially as Characterized (12), via direct delivery of adenovirus in the adrenal glands.

Construction and Purification of Adenoviruses.

Recombinant adenoviruses that encode GRK2 (AdGRK2) or rat wild-type, full-length β-arrestin1 (Adβarr1) were constructed as Characterized previously (9). Briefly, transgenes were cloned into shuttle vector pAdTrack-CMV, which harbors a CMV-driven GFP, to form the viral constructs by using standard cloning protocols. As control adenovirus, EV that expressed only GFP (AdGFP) was used. The resultant adenoviruses were purified, as Characterized previously, by using 2 sequential rounds of CsCl density gradient ultracentrifugation (9).

H295R Cell Culture and Transfection.

H295R cells were purchased from American Type Culture Collection and cultured as previously Characterized (35). Transfection was performed either with Adβarr1 or AdGFP, with pcDNA3.1 plasmid encoding either for the V53D DN βarr1 mutant (14), or just empty pcDNA3.1 vector (EV). Plasmid transfections were performed by using the Lipofectamine 2000 reagent (Invitrogen).

Plasma and in Vitro AlExecutesterone Secretion MeaPositivements.

Rat plasma alExecutesterone levels and in vitro alExecutesterone secretion in the culture medium of H295R cells were determined by EIA (AlExecutesterone EIA kit; ALPCO Diagnostics), as Characterized (34).

Western Blotting.

Western blottings to assess protein levels of StAR (sc-25806), GRK2 (sc-562; Santa Cruz Biotechnology), phospho-ERK1/2 (no. 9106), total ERK1/2 (no. 4696), and total ERK2 (no. 9108; Cell Signaling Technology), βarr1 (A1CT antibody; see ref. 16), and GAPDH (MAB374; Chemicon) were Executene by using protein extracts from rat adrenal glands or in H295R cell extracts, as Characterized previously (9). Visualization of Western blotting signals was performed with Alexa Fluor 680 (Molecular Probes) or IRDye 800CW-coupled (Rockland) secondary antibodies on a LI-COR infrared imager (Odyssey).

Coimmunofluorescence.

Immunofluorescence imaging of human adrenal cross-sections was carried out as Characterized previously (9). Briefly, human adrenal cross-sections were fixed, permeabilized, and labeled with rabbit polyclonal anti-StAR (sc-25806) and goat anti-βarr1 (sc-9182; Santa Cruz Biotechnology) antibodies, followed by the corRetorting Alexa Fluor 594 anti-goat (red) and Alexa Fluor 568 anti-rabbit (green) secondary antibodies (Molecular Probes). Confocal images were obtained by using a 40× objective on a Leica Microsystems TCS SP laser scanning confocal microscope.

Statistical Analyses.

Data are generally expressed as mean ± SEM. Unpaired 2-tailed Student's t test and 1- or 2-way ANOVA with Bonferroni test were generally performed for statistical comparisons, unless otherwise indicated. For all tests, P < 0.05 was generally considered to be significant.

Acknowledgments

We thank Dr. R. Lefkowitz for the anti-βarr1/2 antibody and the V53D DN βarr1 mutant plasmid, Dr. M. Santangelo (University of Naples Federico II, Naples, Italy) for the human adrenal gland tissue, and Dr. P. CorExecutepatis (University of Patras, Patras, Greece) for [SII]-AngII. W.J.K. was supported in part by National Institutes of Health Grants R01 HL56205, R01 HL085503, and P01 HL075443 (Project 2), and by Grant A75301 from the Commonwealth of Pennsylvania Department of Health. A.L. and G.R. were supported by Distinguished Rivers Affiliate American Heart Association postExecutectoral fellowship awards.

Footnotes

1To whom corRetortence may be addressed. E-mail: anastasios.lymperopoulos{at}jefferson.edu or walter.koch{at}jefferson.edu

Author contributions: A.L. and W.J.K. designed research; A.L., G.R., and C.Z. performed research; J.K. and S.S. contributed new reagents/analytic tools; A.L. analyzed data; and A.L. and W.J.K. wrote the paper.

↵2Present address: Cardiology Division, Fondazione Salvatore Maugeri, Istituto Di Ricovero e Cura a Carattere Scientifico, Telese Terme Scientific Institute, Telese Terme, Italy.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0811706106/DCSupplemental.

References

↵ Weber KT (2001) AlExecutesterone in congestive heart failure. N Engl J Med 345:1689–1697.LaunchUrlCrossRefPubMed↵ Connell JM, Davies E (2005) The new biology of alExecutesterone. J EnExecutecrinol 186:1–20.LaunchUrlAbstract/FREE Full Text↵ Marney AM, Brown NJ (2007) AlExecutesterone and end-organ damage. Clin Sci 113:267–278.LaunchUrlCrossRefPubMed↵ Zhao W, Ahokas RA, Weber KT, Sun Y (2006) ANG II-induced cardiac molecular and cellular events: Role of alExecutesterone. Am J Physiol Heart Circ Physiol 291:H336–H343.LaunchUrlAbstract/FREE Full Text↵ Ganguly A, Davis JS (1994) Role of calcium and other mediators in alExecutesterone secretion from the adrenal glomerulosa cells. Pharmacol Rev 46:417–447.LaunchUrlPubMed↵ De Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T (2000) International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev 52:415–472.LaunchUrlAbstract/FREE Full Text↵ Lefkowitz RJ, Rajagopal K, Whalen EJ (2006) New Roles for β-Arrestins in Cell Signaling: Not Just for Seven-Transmembrane Receptors. Mol Cell 24:643–652.LaunchUrlCrossRefPubMed↵ Lefkowitz RJ, Shenoy SK (2005) Transduction of Receptor Signals by β-Arrestins. Science 308:512–517.LaunchUrlAbstract/FREE Full Text↵ Lymperopoulos A, Rengo G, Funakoshi H, Eckhart AD, Koch WJ (2007) Adrenal GRK2 upregulation mediates sympathetic overdrive in heart failure. Nat Med 13:315–323.LaunchUrlCrossRefPubMed↵ Rainey WE, Saner K, Schimmer BP (2004) Adrenocortical cell lines. Mol Cell EnExecutecrinol 228:23–38.LaunchUrlCrossRefPubMed↵ Bird IM, et al. (1993) Human NCI-H295 adrenocortical carcinoma cells: A model for angiotensin-II-responsive alExecutesterone secretion. EnExecutecrinology 133:1555–1561.LaunchUrlCrossRefPubMed↵ Lymperopoulos A, Rengo G, Zincarelli C, Soltys S, Koch WJ (2008) Modulation of Adrenal Catecholamine Secretion by In Vivo Gene Transfer and Manipulation of G Protein-coupled Receptor Kinase-2 Activity. Mol Ther 16:302–307.LaunchUrlCrossRefPubMed↵ Osman H, Murigande C, Nadakal A, Capponi AM (2002) Repression of DAX-1 and induction of SF-1 expression. Two mechanisms contributing to the activation of alExecutesterone biosynthesis in adrenal glomerulosa cells. J Biol Chem 277:41259–41267.LaunchUrlAbstract/FREE Full Text↵ Ferguson SS, et al. (1996) Role of beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization. Science 271:363–366.LaunchUrlAbstract↵ Krupnick JG, Santini F, Gagnon AW, Keen JH, Benovic JL (1997) Modulation of the arrestin-clathrin interaction in cells. Characterization of beta-arrestin Executeminant-negative mutants. J Biol Chem 272:32507–32512.LaunchUrlAbstract/FREE Full Text↵ Nelson CD, et al. (2007) TarObtaining of Diacylglycerol Degradation to M1 Muscarinic Receptors by β-Arrestins. Science 315:663–666.LaunchUrlAbstract/FREE Full Text↵ Rizzo MA, Shome K, Watkins SC, Romero G (2000) The Recruitment of Raf-1 to Membranes Is Mediated by Direct Interaction with Phosphatidic Acid and Is Independent of Association with Ras. J Biol Chem 275:23911–23918.LaunchUrlAbstract/FREE Full Text↵ Yule DI, Williams JA (1992) U73122 Inhibits Ca2+ Oscillations in Response to Cholecystokinin and Carbachol but Not to JMV-180 in Rat Pancreatic Acinar Cells. J Biol Chem 267:13830–13835.LaunchUrlAbstract/FREE Full Text↵ Maroney AC, Macara IG (1989) Phorbol Ester-induced Translocation of Diacylglycerol Kinase from the Cytosol to the Membrane in Swiss3 T3 Fibroblasts. J Biol Chem 264:2537–2544.LaunchUrlAbstract/FREE Full Text↵ Ahn S, Wei H, Garrison TR, Lefkowitz RJ (2004) Reciprocal Regulation of Angiotensin Receptor-activated Extracellular Signal-regulated Kinases by β-Arrestins 1 and 2. J Biol Chem 279:7807–7811.LaunchUrlAbstract/FREE Full Text↵ Violin JD, Lefkowitz RJ (2007) Beta-arrestin-biased ligands at seven-transmembrane receptors. Trends Pharmacol Sci 28:416–422.LaunchUrlCrossRefPubMed↵ Luttrell LM, et al. (2001) Activation and tarObtaining of extracellular signal-regulated kinases by beta-arrestin scaffAgeds. Proc Natl Acad Sci USA 98:2449–2454.LaunchUrlAbstract/FREE Full Text↵ Lee MH, El-Shewy HM, Luttrell DK, Luttrell LM (2008) Role of β-Arrestin-mediated Desensitization and Signaling in the Control of Angiotensin AT1a Receptor-stimulated Transcription. J Biol Chem 283:2088–2097.LaunchUrlAbstract/FREE Full Text↵ Wang P, et al. (2003) Subcellular localization of beta-arrestins is determined by their intact N Executemain and the nuclear export signal at the C terminus. J Biol Chem 278:11648–11653.LaunchUrlAbstract/FREE Full Text↵ Kang J, et al. (2005) A nuclear function of beta-arrestin1 in GPCR signaling: Regulation of histone acetylation and gene transcription. Cell 123:833–847.LaunchUrlCrossRefPubMed↵ Ma L, Pei G (2007) Beta-arrestin signaling and regulation of transcription. J Cell Sci 120:213–218.LaunchUrlAbstract/FREE Full Text↵ Pearson G, et al. (2001) Mitogen-Activated Protein (MAP) Kinase Pathways: Regulation and Physiological Functions. EnExecutecr Rev 22:153–183.LaunchUrlCrossRefPubMed↵ McLaughlin NJ, et al. (2006) Platelet-activating factor-induced clathrin-mediated enExecutecytosis requires beta-arrestin-1 recruitment and activation of the p38 MAPK signalosome at the plasma membrane for actin bundle formation. J Immunol 176:7039–7050.LaunchUrlAbstract/FREE Full Text↵ Ge L, Ly Y, Hollenberg M, DeFea K (2003) A beta-arrestin-dependent scaffAged is associated with prolonged MAPK activation in pseuExecutepodia during protease-activated receptor-2-induced chemotaxis. J Biol Chem 278:34418–34426.LaunchUrlAbstract/FREE Full Text↵ Zhai P, et al. (2005) Cardiac-specific overexpression of AT1 receptor mutant lacking G alpha q/G alpha i coupling causes hypertrophy and bradycardia in transgenic mice. J Clin Invest 115:3045–3056.LaunchUrlCrossRefPubMed↵ Kawamata Y, et al. (2007) Tumor Necrosis Factor Receptor-1 Can Function through a Gαq/11-β-Arrestin-1 Signaling Complex. J Biol Chem 282:28549–28556.LaunchUrlAbstract/FREE Full Text↵ Struthers AD (1995) AlExecutesterone escape during ACE inhibitor therapy in chronic heart failure. Eur Heart J 16:103–106.LaunchUrlAbstract/FREE Full Text↵ Borghi C, et al. (1993) Evidence of a partial escape of rennin-angiotensin-alExecutesterone blockade in patients with aSlicee myocardial infarction treated with ACE inhibitors. J Clin Pharmacol 33:40–45.LaunchUrlPubMed↵ MihailiExecuteu AS, Mardini M, Funder JW, Raison M (2002) Mineralocorticoid and Angiotensin Receptor Antagonism During HyperalExecutesteronemia. Hypertension 40:124–129.LaunchUrlAbstract/FREE Full Text↵ Pezzi V, Clyne CD, AnExecute S, Mathis JM, Rainey WE (1997) Ca(2+)-regulated expression of alExecutesterone synthase is mediated by Calmodulin and Calmodulin-dependent protein kinases. EnExecutecrinology 138:835–838.LaunchUrlCrossRefPubMed
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