Calpain-10 Activity Underlies Angiotensin II-Induced Aldosterone Production in an Adrenal Glomerulosa Cell Model
Mutsa Seremwe, Rick G. Schnellmann, and Wendy B. Bollag
Charlie Norwood Veterans Administration Medical Center (W.B.B.), Augusta, Georgia 30904; Department of Physiology (M.S., W.B.B.) and Section of Dermatology (W.B.B.), Department of Medicine, Georgia Regents University, Augusta, Georgia 30912; and Department of Drug Discovery and Biomedical Sciences (R.G.S.), Medical University of South Carolina, and Ralph H. Johnson VA Medical Center (R.G.S.), Charleston, South Carolina 29425
Aldosterone is a steroid hormone important in the regulation of blood pressure. Aberrant pro- duction of aldosterone results in the development and progression of diseases including hyper- tension and congestive heart failure; therefore, a complete understanding of aldosterone pro- duction is important for developing more effective treatments. Angiotensin II (AngII) regulates steroidogenesis, in part through its ability to increase intracellular calcium levels. Calcium can activate calpains, proteases classified as typical or atypical based on the presence or absence of penta-EF-hands, which are involved in various cellular responses. We hypothesized that calpain, in particular calpain-10, is activated by AngII in adrenal glomerulosa cells and underlies aldosterone production. Our studies showed that pan-calpain inhibitors reduced AngII-induced aldosterone production in 2 adrenal glomerulosa cell models, primary bovine zona glomerulosa and human adrenocortical carcinoma (HAC15) cells, as well as CYP11B2 expression in the HAC15 cells. Although AngII induced calpain activation in these cells, typical calpain inhibitors had no effect on AngII- elicited aldosterone production, suggesting a lack of involvement of classical calpains in this pro- cess. However, an inhibitor of the atypical calpain, calpain-10, decreased AngII-induced aldoste- rone production. Consistent with this result, small interfering RNA (siRNA)-mediated knockdown of calpain-10 inhibited aldosterone production and CYP11B2 expression, whereas adenovirus- mediated overexpression of calpain-10 resulted in increased AngII-induced aldosterone produc- tion. Our results indicate that AngII-induced activation of calpain-10 in glomerulosa cells underlies aldosterone production and identify calpain-10 or its downstream pathways as potential targets for the development of drug therapies for the treatment of hypertension. (Endocrinology 156: 2138-2149, 2015)
A Idosterone, a mineralocorticoid hormone responsible for regulating fluid and electrolyte balance, is in- volved in blood pressure control. Excessive production of aldosterone results in the development and progression of hypertension, and increases the risk of cardiac fibrosis, congestive heart failure, and renal failure and stroke, all of
which can lead to premature death and disability. The addition of mineralocorticoid receptor antagonists to standard therapies has been shown to reduce morbidity and mortality rates in chronic heart failure and acute myo- cardial infarction patients, suggesting the involvement of aldosterone in cardiovascular disease (1).
Abbreviations: AnglI, angiotensin II; (DABCYL)-TPLK~SPPPSPR-(EDANS), [4-((4-(dimethyl- amino) phenyl) azo) benzoic acid, succinimidyl ester]-threonine-proline-leucine-lysine- ~serine-proline-proline-serine-proline-arginine-[5-((2-aminoethyl) amino) naphthalene- 1-sulfonic acid]; DMSO, dimethyl sulfoxide; FRET, fluorescence resonance energy transfer; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; KRB+, bicarbonate-buffered Kreb’s Ringer containing 2.5 mM sodium acetate; MDL, MDL-28170; MOI, multiplicity of infection; 22ROH, 22R-hydroxycholesterol; qRT-PCR, quantitative real-time PCR; siRNA, small interfering RNA; StAR, steroidogenic acute reg- ulatory protein; T2D, type 2 diabetes; ZG, zona glomerulosa.
Aldosterone biosynthesis occurs in the zona glomeru- losa (ZG) of the adrenal cortex upon stimulation of the ZG cells with angiotensin II (AngII), increased extracellular potassium (K+) levels or ACTH. The main secretagogues, AngII and elevated extracellular K+ levels, activate signal transduction pathways that increase cytosolic Ca2+ levels and underlie aldosterone production (2). The initial rate- limiting step in steroidogenesis requires steroidogenic acute regulatory protein (StAR) protein, which mediates translocation of cholesterol from the outer to the inner mitochondrial membrane, at which site the side-chain cleavage enzyme complex that initiates steroidogenesis is located (3). The final stages of aldosterone biosynthesis occur by the action of the aldosterone synthase enzyme (encoded by CYP11B2), which is the late rate-limiting step in aldosterone production (4). AngII increases the expres- sion of both StAR and CYP11B2 (4), as do increased K+ levels (5).
Calpains are intracellular Ca2+-dependent cysteine proteases that are active at neutral pH (6). Calpains can be classified based on penta-EF-hand structures, which allow for the distinction between classical/typical and nonclas- sical/atypical calpains: nonclassical calpains have a loosely defined T-domain instead of the penta-EF-hands found in the classical calpains (7). These classes can be further subdivided into ubiquitous and tissue-specific cal- pains (reviewed in Refs. 7, 8), and to date, 16 calpain genes have been identified in mammals. Several mechanisms are thought to regulate cellular calpain activity including au- tolysis, phosphorylation, interactions with phospholipids, activator proteins or the small calpain subunit and inhi- bition by calpastatin, an endogenous calpain inhibitor (9). The physiological roles of calpains include effects on cy- toskeletal remodeling, signal transduction, gene expres- sion, cell cycle, apoptosis and long-term potentiation. Aberrant increases in intracellular Ca2+ lead to hyper- activation of calpains, which is associated with various pathologies that can be categorized as either genetic dis- eases or Ca2+ homeostasis-linked diseases. Calpain pa- thologies with a genetic background include limb girdle muscular dystrophy type 2A, gastric cancer and type 2 dia- betes (T2D), whereas calpain pathologies that are linked to aberrant Ca2+ homeostasis include neurodegenerative dis- orders, cataract formation, atrial fibrillation, myocardial in- farction, and hypertension. Due to the involvement of cal- pains in multiple pathologies, calpains are now targeted for the development of therapeutic treatments.
Calpain-10 is the most extensively studied atypical cal- pain and has been identified as a T2D susceptibility gene as well as an important mediator of insulin secretion (10). Calpain-10 is ubiquitously expressed in human and ani- mal tissues and has been detected in the cytosol, nucleus
and mitochondria of cultured cells (11). Human cal- pain-10 has up to 8 different variants as a result of alter- native splicing, with calpain-10a being the most abun- dant. Calpain-10 has been associated with renal cell death, ryanodine-induced apoptosis, pancreatic ß-cell exocyto- sis, glucose transporter type 4 vesicle translocation, cata- ractogenesis, and T2D. Moreover, mitochondrial cal- pain-10 has been shown to play a role in the regulation of the mitochondrial electron transport chain, and cal- pain-10 overexpression leads to mitochondrial dysfunc- tion (12). Calpain-10 is also required for cell viability, and a decrease in calpain-10 levels is observed in aging kidneys of rats, mice, and humans, associated with a decrease in renal function (13).
Calpains have been shown to contribute to the devel- opment of AngII-induced cardiovascular remodeling (14), and inhibition of calpain activity prevents endothelial dys- function, myocardial and vascular hypertrophy and tissue fibrosis in AngII-induced hypertension (15, 16). However, to date no study has addressed the role of calpain in ag- onist-induced aldosterone production in adrenal glomeru- losa cells. Because key regulators of aldosterone produc- tion increase cytosolic Ca2+ levels (2, 17, 18), and there is a Ca2+ requirement for activation of calpains (6), it seems possible that AngII could increase calpain activity in these cells. We hypothesized that calpain, and in particular cal- pain-10, is activated by AngII in adrenal glomerulosa cells and underlies increased aldosterone production. In this study, we are the first to identify the involvement of cal- pains in agonist-induced aldosterone production.
Materials and Methods
Materials
Calpain inhibitors and the fluorescent substrate were pur- chased from EMD Millipore. Primary antibodies were pur- chased from the next suppliers, anticalpain-10 antibody was purchased from Abcam, anti-CYP11B2 antibody was a generous gift from Dr Celso Gomez-Sanchez (University of Mississippi, Jackson, MS), and the anti-glyceraldehyde 3-phosphate dehy- drogenase (GAPDH) antibody was purchased from Cell Signal- ing Technology, Inc. All secondary antibodies were purchased from LI-COR Biosciences, and antibody information is provided in Supplemental Table 1. A detailed description of all reagents and methods used in this study is included in Supplemental Materials.
Methods
Culture of human adrenocortical carcinoma cells
Human adrenocortical carcinoma cells (HAC15 cells) were cultured as previously described (19, 20). Before experiments the HAC15 cells were incubated overnight in low-serum medium (containing 0.1% Cosmic Calf serum). Cells were then treated
with pan-calpain inhibitors or typical calpain inhibitors in the presence and absence of the indicated agonist; all treatments contained 0.1% dimethyl sulfoxide (DMSO). Supernatants were collected and frozen for subsequent measurement of aldosterone production. Cells were processed for the techniques described below, with greater detail provided in Supplemental Methods.
Culture of primary bovine adrenal ZG cells
Bovine adrenal ZG (bovine ZG) cells were isolated as previ- ously described (21, 22). For experimental treatments cells were pretreated with pan-calpain inhibitors, typical calpain inhibitors or vehicle (0.1% DMSO) in equilibrated bicarbonate-buffered Kreb’s Ringer containing 2.5mM sodium acetate (KRB+) fol- lowed by treatment with equilibrated KRB+ in the presence or absence of 10nM AngII or with or without 15mM K+ (for which KCl was isoosmotically substituted for NaCl). Supernatants were collected and frozen for subsequent measurement of aldo- sterone production.
Measurement of aldosterone or cortisol production
Upon treatment with the appropriate agents, cells were col- lected and solubilized in 0.3M NaOH to determine protein con- tent using the Bio-Rad protein assay (Bio-Rad Laboratories) with BSA as standard. Aldosterone content of the supernatants was assayed using a solid-phase RIA kit (Siemens) or a cortisol en- zyme immunoassay kit (Oxford Biomedical Research).
RNA extraction and cDNA synthesis
Total RNA was extracted from cells using the PerfectPure RNA Cultured Cell kit according to the protocols of the manufacturer (5 Prime). Purity, quantification and integrity of the RNA were deter- mined using a Nanodrop instrument (Thermo Scientific). Total RNA was reverse transcribed using Iscript cDNA synthesis kits according to the manufacturer’s instructions.
Quantitative real-time PCR (qRT-PCR) analysis
qRT-PCR amplifications were performed using an ABI Step- One Plus Fast Real-Time PCR system (Applied Biosystems by Life Technologies) according to the reaction parameters recom- mended by the manufacturer. All primers for the amplification of target sequences were purchased from Applied Biosystems and are listed in Supplemental Table 2. Relative gene expression was calculated by the 8-8 cycle threshold (Ct) (44Ct) method, and the results are expressed as the fold difference in gene expression normalized to the endogenous housekeeping gene (cyclophilin A) and relative to untreated samples.
Semiquantitative RT-PCR analysis
For semiquantitative RT-PCR analysis, primers were designed using Oligoperfect Designer (Life Technologies) and purchased from Integrated DNA Technologies (Supplemental Table 3). RT- PCR of cDNA was performed using RED Extract-N-AMP PCR reaction mix according to the manufacturer’s protocol. The ampli- fied products were resolved on a 1% Tris-acetate-EDTA agarose gel and visualized using a Syngene UV transilluminator.
Western blot analysis
After treatment cells were harvested and lysed. The proteins were separated with 10% SDS-PAGE and probed with antical-
pain-10 (1:1000 dilution), anti-aldosterone synthase (encoded by CYP11B2) (1:100 dilution) and anti-GAPDH (1:5000 dilu- tion) primary antibodies at 4℃. Protein bands were visualized by IRDye-conjugated secondary antibodies with the Odyssey-SA imaging system (LI-COR Biosciences). Fluorescent bands were quantified using the Odyssey-SA software, and data were nor- malized to GAPDH levels.
In vitro calpain activity assay
Calpain activity was measured using the fluorogenic sub-
strate, [4-((4-(dimethylamino) phenyl) azo) benzoic acid, suc-
cinimidyl ester]-threonine-proline-leucine-lysineserine-
proline-proline-proline-serine-proline-arginine-[5-((2-aminoethyl)
amino) naphthalene-1-sulfonic acid], or (DABCYL)-TPLKSP-
PPSPR-(EDANS) (Calbiochem by EMD Millipore) (23). Fluo-
rescence was measured using a Tecan Spectrafluor Plus fluores-
cent reader (Tecan, Mannedorf, Switzerland) with 320-nm
excitation and 480-nm emission wavelengths at 30℃ for 30
minutes. The initial velocity of the rate of increase in fluorescence
was determined by calculating the slope after subtracting the
background fluorescence.
Small interfering RNA (siRNA) silencing in HAC15 cells
We generated pooled siRNA by combining 2 mission prede- signed siRNAs (Sigma-Aldrich) that target calpain-10, SASI_Hs01_ 00149015 (RefSeqID NM_023083) and SASI_Hs01_00088088 (RefSeqID NM_023085). Cultured HAC15 cells were transfected with pooled calpain-10 siRNA and control siRNA with green fluorescent protein (GFP) by nucleofection (Lonza Group Ltd) as described in detail in Supplemental Methods. The efficiency of transfection was monitored by microscopically monitoring the percentage of GFP-positive cells upon transfection with control siRNA.
Adenoviral amplification and infection of HAC15 cells
Calpain-10 recombinant adenovirus purchased from Signa- gen was infected into Ad293 cells (Agilent Technologies). After purification as described previously (see reference 35 below), HAC15 cells were infected with the calpain-10- or GFP-express- ing adenoviral constructs at multiplicities of infection (MOIs) of 25, 50, and 100. The efficiency of infection was monitored by microscopically determining the percentage of GFP-positive cells, after 6 hours of infection.
Statistical analysis
All experiments were performed independently and repeated a minimum of 3 times in duplicate or triplicate. The values were statistically analyzed by one way-ANOVA, with a Student-New- mann-Keuls post hoc test, using Prism software (Graph Pad Soft- ware, Inc), with statistical significance assigned at P < . 05.
Results
The pan-calpain inhibitors, calpeptin and MDL- 28170 (MDL), inhibit agonist-induced aldosterone production in primary bovine ZG (bovine ZG) cells
To determine whether calpain plays a role in agonist- induced aldosterone production in adrenal glomerulosa
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cells, we investigated the effect of pan-calpain inhibitors in primary cultures of bovine ZG cells. Bovine ZG cells were stimulated with medium containing 15mM K+ or 10nM AngII, secretagogues known to stimulate aldosterone pro- duction in these cells (21). The calpain inhibitor calpeptin reduced aldosterone production in AngII- and K+-treated cells (Figure 1A). Cells pretreated with calpeptin exhibited an approximately 50% inhibition (AngII) and 80% inhi- bition (K+) of agonist-induced aldosterone secretion. To ensure that the inhibitory response was not a result of nonspecific cytotoxicity, glomerulosa cells were treated with 22R-hydroxycholesterol (22ROH) for 1 hour. Wa- ter-soluble 22ROH is a cholesterol analog that bypasses normal signaling pathways regulating the StAR-mediated rate-limiting step of cholesterol shuttling from the outer to the inner mitochondrial membrane where steroidogenesis is initiated. Therefore, a comparison of steroid synthesis occurring upon exposure to 22ROH in the presence or absence of calpeptin provides an indication of possible nonspecific effects on adrenal glomerulosa cell viability/ health or steroidogenic enzyme activity. Calpeptin had no significant effect on 22ROH-mediated aldosterone pro- duction (Figure 1A). Thus, the ability of calpeptin to in- hibit agonist-induced aldosterone production in bovine ZG cells was not due to nonspecific cytotoxicity.
When bovine ZG cells were treated with MDL similar results were obtained, whereby the increase in the aldo- sterone secretory response induced by elevated K+ levels and AngII was inhibited by pretreatment with MDL. After a 1-hour treatment with agonist in the presence of MDL, there was an approximately 85% and 45% inhibition of
K+- and AngII-elicited aldosterone production, respectively (Figure 1B). Furthermore, the inhibition of agonist-induced steroidogenesis was not a result of cytotoxicity, as indi- cated by the inability of MDL to in- hibit 22ROH-mediated aldosterone production.
The pan-calpain inhibitors, calpeptin and MDL, inhibit agonist-induced aldosterone production in human adrenocortical carcinoma HAC15 cells
To confirm the involvement of calpain in agonist-elicited aldoste- rone production in another cell model of the ZG (19), we treated a human adrenocortical carcinoma cell line, HAC15 cells, with the pan- calpain inhibitors calpeptin and MDL in the presence and absence of elevated K+ levels and AngII, and monitored aldosterone production after 24 hours. HAC15 cells are a subclone of the H295R cell line (24) that exhibit a greater aldosterone biosynthetic re- sponse to AngII (20). Aldosterone production induced by K+ and AngII was inhibited by calpeptin 55% and 40%, respectively, in HAC15 cells (Figure 2A). Similar results were observed with MDL (Figure 2B), although MDL was a more effective inhibitor of agonist-induced aldosterone production (80% for K+; 70% for AngII) than calpeptin in HAC15 cells. Furthermore, the inhibition of aldosterone by the pan-calpain inhibitors was not due to cytotoxicity as there was no effect of the inhibitors on 22ROH-mediated aldosterone production (Supplemental Figure 1) and a trypan blue exclusion assay indicated that there was no sig- nificant difference in cell viability among the treatment groups (data not shown). We suggest a potential role for calpain in agonist-induced aldosterone production. Based on the more robust response to AngII, we elected to focus on this agonist in subsequent experiments.
AngII increases calpain activity in HAC15 cells
AngII has been reported to increase calpain activity in vitro and in vivo in cardiovascular tissue (15, 16). We employed an in vitro calpain activity assay that uses a fluorescence resonance energy transfer (FRET)-based fluorogenic substrate (DABCYL)-TPLK~SPPPSPR-(EDANS) (23) to measure calpain activity in HAC15 cells. This sub- strate is believed to be a more selective substrate for cal- pains vs other proteases than are other available peptide
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substrates. After treatment, HAC15 cells were harvested and lysed in a buffer solution as described in Methods, and the relative fluorescence per second due to calpain cleav- age of the substrate was monitored as a measure of calpain activity. AngII increased calpain activity to approximately 25 fluorescent units per second when compared with a control of about 19 fluorescent units per second, and this activity was attenuated by calpeptin alone and in the pres- ence of AngII to approximately 5 fluorescent units per second (Figure 3A). Cumulative values obtained from multiple experiments indicated that AngII stimulated cal- pain activity and this activity was blocked by calpeptin (Figure 3B).
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The pan-calpain inhibitors, calpeptin and MDL, inhibit AngII-induced CYP11B2 expression in HAC15 cells
The 2 key proteins regulating al- fff dosterone production in adrenal glo- ** merulosa cells are StAR, which me- fff diates the initial rate-limiting step of translocation of cholesterol into the mitochondria, and aldosterone syn- 10nM AnglI AnglI + MDL thase (encoded by CYP11B2), which catalyzes the final reactions in al- dosterone biosynthesis (4). We de- termined the effect of pan-calpain inhibitors on the AngII-induced ex- pression of StAR and CYP11B2. Al- though the expression of the StAR transcript was increased in response to AngII as expected, calpain inhib- itors had no effect on this increase (data not shown). In contrast, CYP11B2 mRNA expression was increased upon stimulation with AngII at 24 hours, and calpeptin and MDL inhibited CYP11B2 expression by approxi- mately 60% and 30%, respectively (Figure 4A). The levels of aldosterone synthase in HAC15 cells were also deter- mined using Western blot analysis. AngII increased aldo- sterone synthase levels, and the calpain inhibitors returned the AngII-stimulated levels to a value not statistically dif- ferent from the control (Figure 4B), with MDL inhibiting the aldosterone synthase increase by about 30%. Al- though a 24-hour exposure was selected for these exper- iments, it is possible that this time point was not optimal, as the inhib- itory effect of the calpain inhibitors * on CYP11B2 mRNA expression was maximal at 12 hours (Supplemental Figure 2). Collectively, our results suggest that calpain regulates aldo- fff
sterone production, in part by regu- lating CYP11B2 expression.
The typical calpain inhibitors PD150606 and cell-permeant calpastatin peptide do not inhibit AngII-induced aldosterone production in HAC15 cells
Calpains are classified as either typical or atypical based on the pres- ence or absence of the penta-EF domain in the C terminus. To deter- mine the subclass of the calpain in-
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volved in AngII-mediated aldosterone production, we per- formed additional studies using inhibitors that are more specific to typical calpains. Wang et al (25) identified a class of calpain inhibitors that include PD150606 [3-(4- iodophenyl)-2-mercapto-(Z)-2-propenoic acid], which binds to the penta-EF-hand domain of calpains instead of to the active site of the protease, allowing for selectivity against calpains that possess this penta-EF-domain, ie, typical calpain isozymes. Based on previously reported inhibitor concentrations (26), we treated HAC15 cells with 1μM, 3μ.Μ, 10μM, and 30μM PD150606 and found that this agent had no effect on basal aldosterone production except at the 30uM treatment dose, at which concentration it actually increased aldosterone produc- tion (Figure 5A). PD150606 had no significant effect on AngII-induced aldosterone production at any dose, whereas the positive control MDL significantly inhibited the response (Figure 5B). In bovine ZG cells, PD150606 had no effect on aldosterone production either in the ab- sence or the presence of AngII (data not shown), providing evidence that the calpain involved in inducing aldosterone production was not a typical calpain.
To verify the results with PD150606, we used a syn- thetic peptide derived from the endogenous calpain inhib- itor calpastatin, calpastatin peptide, which is fused to an 11 poly-arginine peptide to allow for improved cell per- meability (27). HAC15 cells were treated with calpastatin peptide at 2.5uM and 5uM doses in the presence and
absence of AngII. A dose of 2.5 uM alone increased aldosterone produc- tion 6-fold when compared with control; 5 uM calpastatin peptide en- hanced basal aldosterone produc- tion by approximately 11-fold (Fig- ure 5C). However, in the presence of AngII, there was no effect of cal- pastatin peptide on aldosterone pro- duction at either dose, confirming the importance of an atypical rather than a typical calpain isoform in AngII-induced aldosterone production.
The atypical calpain-5, calpain-7, and calpain-10 are expressed in HAC15 cells
We examined the expression of atypical calpains in HAC15 cells by semiquantitative RT-PCR and found calpain-5, calpain-7, and calpain-10 were expressed (Supplemental Figure 3). Because the mitochondrion is a key organelle in steroidogenesis and cal- pain-10 exhibits cytosolic, nuclear, and mitochondrial localization (12), we hypothesized that mitochondrial calpain-10 mediates AngII-mediated aldoste- rone production.
The calpain-10 inhibitor, CYGAK, inhibits AngII- induced aldosterone production in HAC15 cells
The 5 amino acid peptide, cysteine-tyrosine-glycine-al- anine-lysine (CYGAK) has been shown to be a potent and efficacious calpain-10 inhibitor in renal proximal tubular cells, inhibiting both cytosolic and mitochondrial cal- pain-10 (28). Treatment of HAC15 cells with and without 10uM CYGAK in the presence and absence of 10nM An- gII for 24 hours significantly inhibited AngII-induced al- dosterone production (by approximately 20%) (Figure 6A). Inhibition of calpain-10 by CYGAK also tended to inhibit CYP11B2 mRNA expression in AngII-stimulated cells when compared with AngII alone. A two-way ANOVA analysis of 8Ct values revealed a small main ef- fect of CYGAK to inhibit CYP11B2 expression, although in individual pairwise comparisons, statistical significance was not achieved due to the sample size (Figure 6B).
Calpain-10 overexpression increases AngII-induced aldosterone production but has no effect on AngII-induced CYP11B2 mRNA expression in HAC15 cells
To verify the calpain-10 inhibitor studies, we infected HAC15 cells with calpain-10-expressing adenovirus vs a
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GFP-expressing control vector for 6 hours, after which we replaced the media with low-serum medium for an ad- ditional 18 hours and then stimulated with or without
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10nM AngII for 24 hours. There was an increase in aldosterone production in response to AngII treatment (Figure 7A). Uninfected AngII-treated HAC15 cells and An- gII-treated GFP vector-infected cells produced comparable amounts of al- dosterone, indicating that there was no effect of adenoviral infection. In the presence of AngII cells overex- pressing calpain-10 at different MOIs exhibited a concentration-de- pendent increase in aldosterone pro- duction (Figure 7A).
Overexpression of calpain-10 was confirmed by monitoring cal- pain-10 protein and mRNA levels. Calpain-10 was successfully overex- pressed at all 3 MOIS. The cal- pain-10 protein bands at 50 and 54 kDa increased with increasing MOI from 25 to 100 MOI (Figure 7B). Furthermore, infection with cal- pain-10 adenovirus increased ex- pression of the 75-kDa calpain iso- form, which has been reported to be the calpain-10a isoform (29, 30); however, we failed to visualize this 75-kDa band in uninfected or GFP- infected HAC15 cells. At MOIs 25 and 50, there was a 2.5-fold increase in calpain-10 protein expression when compared with GFP vector-infected cells in the absence and presence of AngII. Further- more, overexpression of calpain-10 at MOI 50 led to a 110-fold increase in calpain-10 mRNA when com- pared with vector-infected cells (Figure 7C). We also examined the effect of overexpression of cal- pain-10 on CYP11B2 mRNA levels and found that whereas AngII in- creased CYP11B2 expression in both vector- and calpain-10-infected cells when compared with untreated cells, there was no significant differ- AnglI + 10mM CYGAK ence in the AngII-induced increase in CYP11B2 expression with cal- pain-10 overexpression (Figure 7D). However, calpain-10 overexpres- sion alone resulted in an approxi- mate 5-fold increase in CYP11B2 mRNA levels under basal (un-
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treated) conditions (Figure 7D), with no effect on basal aldosterone production. This disparate outcome likely re- sults from the lack of an effect of calpain-10 overexpres- sion on StAR (data not shown), the early rate-limiting step in steroidogenesis.
Knockdown of calpain-10 inhibits AngII-induced CYP11B2 mRNA expression levels and aldosterone production in transfected HAC15 cells
We also determined the effect of down-regulating cal- pain-10 using siRNA. HAC15 cells were transfected with pooled-calpain-10 siRNA using an AMAXA Nucleofec- tor, and the cells were allowed to recover for 48 hours before treatment with or without AngII for 24 hours. HAC15 cells transfected with GFP-expressing scrambled siRNA served as control. AngII-mediated aldosterone
production was inhibited with calpain-10 knockdown when compared with HAC15 cells transfected with scram- bled siRNA followed by AngII treatment (Figure 8A). Ex- pression results indicated successful knockdown of cal- pain-10 mRNA by more than 50% when compared with HAC15 cells that had been transfected with scrambled siRNA (Figure 8B). Knockdown of calpain-10 siRNA also inhibited AngII-induced CYP11B2 expression when com- pared with AngII-treated scrambled siRNA (Figure 8C). As the identity between the coding sequence of the other atypical calpains that we found to be expressed in HAC15 cells (calpain-5 and calpain-7) and capain-10 (variant 3) is only approximately 41%-50%, the ability of calpain-10 siRNA to down-regulate calpain-5 or calpain-7 is un- likely, although the possibility of such an effect was not eliminated.
A
B
Aldosterone production (Fold over sc-siRNA)
CAPN10 mRNA expression (Fold over sc-sirna)
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20-
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ff
0.75-
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SC-SIRNA 40nM
SİRNA CAPN10 40nM
AnglI +sc-siRNA 40nM
AnglI +siRNA CAPN 10 40nM
SC-SiRNA 40nM
CAPN10 siRNA 40nM
C
CYP11B2 mRNA expression (Normalised to sc-siRNA)
35-
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SC-SIRNA 40nM
CAPN10 siRNA 40nM
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AnglI +CAPN10 siRNA 40nM
Relevance of calpain-10 to adrenal steroidogenesis in vivo
Our results provide evidence of the importance of cal- pain-10 to regulating aldosterone production in in vitro models of ZG cells. To determine whether these data are potentially relevant to the situation in vivo, we examined calpain-10 protein expression in the human adrenal cortex in situ. Normal adrenal tissue obtained from indi- viduals undergoing adrenalectomy was subjected to im- munohistochemical procedures and calpain-10 immuno- reactivity determined. Calpain-10 staining was observed throughout the adrenal cortex, with perhaps a slight pre- dominance in the region immediately subjacent to the cap- sule, which showed no immunoreactivity (Supplemental Figure 4).
Calpain-10 and cortisol production
Immunohistochemistry indicated that calpain-10 pro- tein was distributed throughout the adrenal cortex, sug-
gesting the possibility that it might play a role in the production of other adrenal steroids. HAC15 cells are dedifferentiated and can be induced to produce all of the adrenal steroids; in fact, they produce greater amounts of cortisol than aldosterone (19). To investigate whether cal- pain-10 regulates adrenal steroiodo- genesis in general, cortisol levels were measured in the supernatants from cells treated with and without AngII in the presence or absence of pan-calpain inhibitors. Supplemen- tal Figure 5 illustrates that the pan- calpain inhibitors, calpeptin and MDL, had no effect on cortisol pro- duction in response to AngII, sug- gesting that the involvement of cal- pain-10 in steroidogenesis may be restricted to the adrenal ZG.
Discussion
We have shown for the first time that the pan-calpain inhibitors calpeptin and MDL reduce aldosterone pro- duction in response to elevated ex- tracellular K+ levels and AngII in pri- mary bovine ZG cells and HAC15 cells. Further exploration deter- mined that calpain-10 underlies in part the ability of AngII to stimulate steroidogenesis. Primary cultures of bovine ZG cells re- spond to agonist treatments with aldosterone secretion within minutes of exposure, whereas the HAC15 cells take hours as basal aldosterone synthase (encoded by CYP11B2) levels are low and expression of this key late rate-limiting enzyme must be induced. Therefore, use of these cells can provide insight into the mechanisms regu- lating the chronic aldosterone response.
The pan-calpain inhibitors did not completely return AngII-elicited aldosterone production to control levels, suggesting that calpain contributes to the induced aldo- sterone production but is not the sole molecular signal mediating the response. It is known that AngII stimulates an increase in intracellular Ca2+ levels (reviewed by Ref. 31), which presumably triggers calpain activation to in- crease aldosterone production. The pan-calpain inhibitors partially inhibited AngII-induced CYP11B2 mRNA ex- pression at all experimental time points (Supplemental
Figure 2). Maximal expression of CYP11B2, and maxi- mum inhibition by the calpain inhibitors, was observed at the 12-hour treatment time point but we chose the 24-hour time point for subsequent studies as this period of incu- bation yielded more robust aldosterone production. In ad- dition, we have also shown that calpain inhibition de- creased the CYP11B2 expression induced in response to AngII; calpain inhibitors also reduced the AngII-stimu- lated protein levels to a value not statistically different from the control. Nevertheless, it is not clear whether an ability to increase CYP11B2 expression is the sole, or even the primary, mechanism by which calpain increases AngII- induced aldosterone production. It seems possible that cal- pain could activate the aldosterone synthase enzyme en- coded by CYP11B2, leading to the observed inhibition of aldosterone production with calpain inhibitors. However, the evidence to date suggests that the activity of aldoste- rone synthase is regulated predominantly by changes in its expression/levels. It is also possible that there may be ac- tions of calpain at earlier events in steroiodogenesis; this idea is the subject of ongoing studies in the laboratory.
We were also able to show that AngII increased calpain activity in HAC15 cells using a FRET-based calpain-se- lective substrate, and this activity was inhibited by cal- peptin, indicating that AngII activated calpain. However, the calpain substrate is not specific for a particular calpain isoform; therefore, it is difficult to determine which spe- cific calpain isoform(s) is (are) activated. However, in sub- sequent experiments we showed that the calpain most likely involved in AngII-stimulated aldosterone produc- tion was an atypical calpain because the typical calpain inhibitors, PD150606 and calpastatin, had no effect on AngII-elicited aldosterone production in HAC15 cells. In- terestingly, PD150606 alone at 30/M induced a slight but significant increase in aldosterone production. Similarly, the synthetic calpastatin peptide, which mimics endoge- nous calpastatin to inhibit typical calpains, also induced an increase in the aldosterone secretory response in the absence of AngII. We suggest that typical calpains, if in- volved in glomerulosa cell function, may in fact have an inhibitory role in controlling the basal synthesis of aldo- sterone rather than AngII-elicited aldosterone production.
Our results indicated that the specific calpain-10 inhib- itor, CYGAK, inhibited AngII-induced aldosterone pro- duction. The percentage inhibition was approximately 20% of the AngII-elicited maximal response, a slightly lower degree of inhibition than was observed with the pan-calpain inhibitors, although we were somewhat lim- ited in the dosage of CYGAK that we could test as this newly synthesized inhibitor is not commercially available (28, 32). Nevertheless, this result provided evidence for a role of calpain-10 in AngII-elicited aldosterone produc-
tion, although potential roles for other atypical calpains are not excluded. We also showed that CYGAK tended to inhibit CYP11B2 mRNA expression, suggesting that cal- pain-10 may be able to regulate AngII-induced CYP11B2 expression.
We were then able to manipulate calpain-10 genetically and showed using adenovirus infection that we could suc- cessfully overexpress calpain-10. By Western blot analy- sis, we observed multiple calpain-10 protein bands. Pre- viously, multiple calpain-10 bands have been suggested to be splice variants (29). However, because the adenovirus expresses a mature calpain-10 cDNA, our result suggests that the multiple bands may instead be proteolytic prod- ucts of calpain-10. Adenoviral-mediated overexpression enhanced the levels of the 75-kDa protein as well as bands of other molecular weights. The 54-kDa band of cal- pain-10 has been reported to be the membrane-associated “isoform” that is linked to the cleavage of SNAP25 during insulin secretion from pancreatic ß-cells (33). This immu- noreactive band was of greatest intensity in the uninfected HAC15 cells; however, the literature does not specify which of the calpain-10 “splice variants” this band rep- resents. Importantly, overexpression of calpain-10 en- hanced AngII-induced aldosterone production in a dose- dependent manner. Despite enhancing AngII-induced aldosterone production, calpain-10 overexpression did not increase AngII-induced CYP11B2 expression, which was somewhat unexpected. It is possible that the time point that we selected for monitoring CYP11B2 expres- sion in this experiment was not ideal or that endogenous calpain-10 levels are already optimal for CYP11B2 ex- pression. Nevertheless, CYP11B2 mRNA levels were in- creased by overexpression of calpain-10 under basal con- ditions, without a corresponding enhancement in basal aldosterone production. In fact, CYP11B2 expression is the late rate-limiting step in aldosterone production; the early rate-limiting step is the movement of cholesterol from the outer to the inner mitochondrial membrane, re- quiring the activity of StAR. As we found no effect of calpain-10 inhibition on StAR expression or levels (data not shown), the absence of an enhancement of basal al- dosterone production by calpain-10 overexpression likely results from the lack of effect of calpain-10 on this early rate-limiting event in steroidogenesis.
Taking the opposite approach, we showed that siRNA- mediated calpain-10 knockdown inhibited AngII-elicited CYP11B2 mRNA expression and aldosterone produc- tion, corroborating our data using the calpain inhibitors. This result indicates that calpain-10 is important for An- gII-induced aldosterone production. Therefore, collec- tively our data suggest that calpain-10 is important in the regulation of chronic aldosterone biosynthesis. Although
our results suggest an involvement of calpain-10 in AngII- induced aldosterone production, our data do not directly identify the mechanism by which this enzyme modulates steroidogenesis. Calpains are often referred to as intracel- lular “modulator proteases” due to their ability to hydro- lyze their substrates in a limited fashion to transform or modulate their substrates’ structure and/or activity (34). The substrate (or substrates) of calpain-10 that is partially proteolyzed to induce steroidogenesis is unknown, al- though steroidogenesis activator peptide appears to be de- rived from glucose-regulated protein 78 by proteolysis (35). It is interesting to speculate that calpain-10 might be the protease that cleaves glucose-regulated protein 78 to generate the approximately 30-kDa steroidogenesis acti- vator peptide, which acts synergistically with GTP to en- hance steroidogenesis (36), although the inability of cal- pain inhibition to affect AngII-stimulated cortisol production argues against this possibility. Calpain’s ac- tions on cytoskeletal arrangement or vesicle trafficking could also be involved (14, 37). Thus, calpains have been shown to localize to focal adhesion complexes and cleave substrates, such as myristoylated alanine-rich C kinase substrate, resulting in disruption of its cross-linking ability (38). Future studies will investigate the means by which calpain-10 regulates aldosterone production, although difficulties in delineating these mechanisms will be sub- stantial, due to limitations in the available experimental techniques and reagents that can accurately identify cal- pain isoforms and/or activities.
In summary, we have shown for the first time that cal- pains are involved in the regulation of agonist-induced aldosterone production in adrenal glomerulosa cells. In addition, AngII increased calpain activity in these cells. We were able to identify the atypical calpain-10 as an isoform involved in steroidogenesis in adrenal glomerulosa cells. Overexpression of calpain-10 increased AngII-induced al- dosterone production and knockdown of calpain-10 by RNA interference inhibited aldosterone production and CYP11B2 expression, validating our results with calpain inhibitors. Therefore, AngII stimulates atypical cal- pain-10 activity to increase aldosterone production, likely in part by regulating the expression of CYP11B2. Al- though little is known about the role and mechanism of action of calpain in steroidogenesis, our novel data have shed light on a previously unknown signal regulating aldosterone production. Our results identify calpain-10 and its downstream pathways as potential targets for the development of drugs to inhibit aldosterone pro- duction to ultimately lead to new therapeutic treat- ments for hypertension.
Acknowledgments
We thank Ms Maribeth Johnson for her assistance with the two- way ANOVA statistical analysis and Ms Kimya Jones of GRU’s Georgia Esoteric Molecular (GEM) Lab’s Histology Services for immunohistochemical assistance. We also thank the GRU Tu- mor and Tissue Biorepository and the GRU Adrenal Center for providing normal human tissue samples. Finally, we thank Xun- sheng (Sara) Chen for her assistance with semiquantitative RT-PCR.
Address all correspondence and requests for reprints to: Dr Wendy B. Bollag, PhD, Department of Physiology, Medical Col- lege of Georgia at Georgia Regents University, 1120 15th Street, Augusta, GA 30912. E-mail: WB@gru.edu.
This work was supported in part by the National Institutes of Health Grant HL070046 and Veterans Affairs Merit Review Award BX001344 (to W.B.B.) and by National Institutes of Health Grants GM084147 and ES012239 and Veterans Affairs Merit Review Award BX000851 (to R.G.S.). W.B.B. is also sup- ported by a Veterans Affairs Research Career Scientist award.
Disclosure Summary: The authors have nothing to disclose.
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