DEPARTMENT OF VETERANS AFFAIRS UNITED STATES OF AMERICA
Published in final edited form as: Mol Cell Endocrinol. 2016 September 15; 433: 138-146. doi:10.1016/j.mce.2016.05.018.
VLDL-activated cell signaling pathways that stimulate adrenal cell aldosterone production
Ying-Ying Tsai2, William E. Rainey2,3, Maribeth H. Johnson4, and Wendy B. Bollag1,2 1Charlie Norwood VA Medical Center, One Freedom Way, Augusta, GA 30904
2Department of Physiology, Medical College of Georgia at Augusta University, 1120 15th Street, Augusta, GA 30912
3Current address: Departments of Molecular & Integrative Physiology and Internal Medicine, University of Michigan, 7744 Med Sci II, Ann Arbor, MI 48109
4Department of Biostatistics and Epidemiology, Medical College of Georgia at Augusta University, 1120 15th Street, Augusta, GA 30912
Abstract
Aldosterone plays an important role in regulating ion and fluid homeostasis and thus blood pressure, and hyperaldosteronism results in hypertension. Hypertension is also observed with obesity, which is associated with additional health risks, including cardiovascular disease. Obese individuals have high serum levels of very low-density lipoprotein (VLDL), which has been shown to stimulate aldosterone production; however, the mechanisms underlying VLDL-induced aldosterone production are still unclear. Here we demonstrate in human adrenocortical carcinoma (HAC15) cells that submaximal concentrations of angiotensin II and VLDL stimulate aldosterone production in an additive fashion, suggesting the possibility of common mechanisms of action. We show using inhibitors that VLDL-induced aldosterone production is mediated by the PLC/IP3/PKC signaling pathway. Our results suggest that PKC is upstream of the extracellular signal-regulated kinase (ERK) activation previously observed with VLDL. An understanding of the mechanisms mediating VLDL-induced aldosterone production may provide insights into therapies to treat obesity-associated hypertension.
Keywords
adrenal cortex; aldosterone; obesity; phospholipase C; protein kinase C; VLDL; zona glomerulosa
1. Introduction
The mineralocorticoid hormone aldosterone is responsible for sodium retention that directly impacts body fluid levels and blood pressure. Excessive aldosterone production causes about
Publisher’s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
8% of the diagnosed cases of hypertension [1], a disorder that is associated with an increased risk of cardiovascular disease, stroke, visual loss and renal disease [2,3]. In addition, hyperaldosteronism contributes to congestive heart failure and cardiac fibrosis [4], which increases patient morbidity and mortality. Aldosterone is synthesized from cholesterol stores in adrenal zona glomerulosa cells and is mainly regulated by the traditional secretagogues angiotensin II (AngII) and serum potassium levels. (ACTH can also potently increase aldosterone production in vitro but its effect in vivo is transient.) The two key rate- limiting steps in aldosterone biosynthesis are steroidogenic acute regulatory (StAR) protein, which is involved in transporting cholesterol to the inner mitochondrial membrane to initiate steroid hormone synthesis, and aldosterone synthase (CYP11B2), which catalyzes the final reactions in aldosterone production.
Globally, overweight and obesity are reaching epidemic proportions. According to reports from the World Health Organization, currently 2 billion adults are overweight and 600,000 million of these people are obese [5]. Obesity is linked to serious health risks, for example, hypertension, type II diabetes, vascular disease, stroke and some forms of cancer [6]. In obese patients plasma levels of lipoproteins, such as triglycerides, low-density lipoprotein and very-low-density lipoprotein (VLDL) are typically elevated (dyslipidemia) in association with the increased risk of cardiovascular disease and hypertension. Previous studies have indicated a positive correlation between triglyceride levels and serum aldosterone in both normal and metabolic-syndrome subjects [7]. Indeed, several studies have indicated that aldosterone levels are a major link between obesity and hypertension [8- 11]. Thus, aldosterone levels are typically elevated in obese individuals and drop upon weight loss [11], with a correlation between decreases in serum aldosterone levels and in blood pressure with the reduction in weight [12]. Greater amounts of visceral fat are also suggested to result in increased aldosterone production [13]; adipose tissue can secrete a variety of adipokines, such as leptin, tumor necrosis factor-a, interleukin-6, complement- C1q, TNF-related protein 1 and fatty acids. In obese patients these adipokines have been suggested to be factors that stimulate aldosterone production via an alternative aldosterone regulatory system [14-16].
Very-low-density lipoprotein (VLDL) is synthesized in the liver as a light-density lipoprotein with a composition of approximately 50% triglyceride (TG) and 10% protein [17]. In addition to its well-known function of transporting fatty acids and triglyceride from the liver to peripheral tissues, VLDL has been suggested to activate several signaling cascades in different tissues, including adrenocortical cells. Indeed, we have previously shown that VLDL stimulates aldosterone secretion in a variety of zona glomerulosa cell models, including primary cultures of human adrenocortical and bovine glomerulosa cells [18] and in the human adrenocortical cell line H295R and its clone [19], HAC15 [20]. This effect of VLDL to increase aldosterone biosynthesis is related to its ability to increase the expression of CYP11B2 (the gene encoding aldosterone synthase) and steroidogenic acute regulatory (StAR) protein levels. In addition, VLDL stimulates CYP11B1 expression; however, it has no effect on cortisol production either in the H295R or in primary human adrenocortical cells although it increases DHEA production [18]. The expression of other steroidogenic genes, such as 17a-hydroxylase (CYP17), is not altered by VLDL [18]. Saha et al. [21,22] also demonstrated an ability of VLDL, both native and modified in vitro, to
Mol Cell Endocrinol. Author manuscript; available in PMC 2017 September 15.
stimulate CYP11B2 expression and aldosterone production. VLDL acted, at least in part, through scavenger receptor, class B, type 1 (SR-B1), cAMP-dependent protein kinase (protein kinase A or PKA), extracellular signal-regulated kinase-1 and -2 (ERK1/2) and Janus kinase-2 (JAK2) [21]. In other cases, VLDL appears to be altered in the circulation in vivo (e.g., in patients with diabetes) and this modified VLDL can also stimulate cell signaling events [23]. In addition to VLDL, high-density lipoprotein (HDL) has also been found to induce aldosterone production [24], although VLDL is more effective than HDL [18]. Others have reported that LDL can also stimulate aldosterone biosynthesis [21], although this effect has been somewhat controversial (reviewed in [25]).
AngII regulates aldosterone production by activating, via the AngII receptor, type 1 (AT1R), a phosphoinositide-specific phospholipase C (PLC), which hydrolyzes the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate to generate two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) ([26-28][29]). IP3 then releases calcium from the endoplasmic reticulum (ER) and activates calcium/calmodulin-dependent protein kinase. On the other hand, DAG stimulates PKC activity, which has been suggested to sustain AngII-elicited aldosterone production in bovine adrenal glomerulosa cells [26,28,30,31]. Sustained calcium influx is required to maintain aldosterone production in response to AngII, such that inhibition of voltage-dependent calcium channels decreases hormone-induced steroidogenesis (reviewed in [29]).
In the present study we examined the role of the PLC/PKC signaling pathway in VLDL- regulated aldosterone production. Our findings demonstrate that VLDL increased DAG levels; in addition, a PLC inhibitor, as well as two PKC inhibitors and an IP3 receptor inhibitor, reduced aldosterone production and CYP11B2 expression stimulated by VLDL. Taken together, our results suggest that VLDL induces PLC-mediated phosphoinositide hydrolysis and the resultant production of IP3 and DAG to mediate aldosterone production. Finally, our data suggest that PKC downstream of PLC and phosphoinositide hydrolysis mediates VLDL-induced activation of ERK, a protein kinase that has also been reported to be involved in steroidogenesis [32].
2. Materials and Methods
2.1. Cell culture
A clone of the H295R cell line, human adrenocortical (HAC15) cells [33] were cultured in DMEM/F12 (Gibco) with 10% Cosmic calf serum (Thermo Scientific), 0.1% gentamicin (Invitrogen), 1% penicillin/streptomycin (Life Technologies), and 1% ITS (insulin- transferrin-selenous acid with linoleic acid and bovine serum albumin) (Becton Dickinson Labware). Cells were plated in 6- or 12-well plates and cultured at 37℃ for 2 days. Before experimentation, cells were incubated with low-serum experimental medium (0.1% Cosmic calf serum) for 24 hours. For inhibitor studies, cells were preincubated with either the PLC inhibitor U73122 (Enzo Life Sciences), the IP3 receptor inhibitor xestospongin C (Cayman Chemicals) or one of two PKC inhibitors, Ro31-8220 or Gö6983 (Calbiochem) for 30 minutes before treatment with or without VLDL (Kalen Biomedical, LLC) or AngII (Sigma- Aldrich) for the appropriate time periods. None of the inhibitors used in this study exhibited
toxicity towards HAC15 cells (Supplemental Figure 1), determined using MTT assays as described below.
2.2. Cell toxicity assay
Cell toxicity was determined using an MTT cell proliferation assay kit (Vybrant). Briefly, HAC15 cells were plated in 96-well microplates with growth medium for 48 hours; after 24 hours in low-serum medium the cells were incubated with fresh low-serum medium with or without inhibitors for 30 minutes prior to stimulation with or without AngII for 24 hours in low-serum medium. Cells were than incubated with the MTT solution provided in the kit at 37°℃ for 4 hours. After labeling, SDS-HCI solution was added to solubilize the cells, which were incubated at 37℃ for another 4-18 hours. The absorbance at 570 nm was measured in the SDS lysate using a PolarSTAR plate reader.
2.3. Aldosterone production
After a 24-hour treatment of the HAC15 cells with or without VLDL or AngII (after a 30- minute pretreatment with or without PLC, IP3 receptor or PKC inhibitors), the supernatants were collected and assayed with a solid-phase radioimmunoassay kit (Siemens Products, Los Angeles, CA) according to the supplier’s directions.
2.4. RNA Extraction, cDNA Synthesis, and Real-Time RT-PCR
RNA was extracted from cells using an RNeasy kit (5 PRIME, Gaithersburg, MD) following the protocols of the manufacturer. RNA concentration was determined using a Nanodrop instrument (NanoDrop Technologies, Wilmington, DE). Total RNA was reverse transcribed with a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA) following the manufacturer’s recommendations. Primer/probe sets for the amplification of target sequences were purchased from Applied Biosystems, and PCR amplifications were performed using the ABI Step One Fast Real-Time PCR System using the reaction parameters recommended by the manufacturer: 20 ul total volume consisting of primer/ probe mix and Fast Reagent Master Mix (Applied Biosystems), with 18S or cyclophilin (PPIA; Applied Biosystems) used as an endogenous normalization control gene. Water served as the negative control. The relative gene expression was calculated by the 44Ct method and the resultant values normalized to a calibrator. In every experiment the calibrator was the control (untreated or vehicle-treated) sample. Final results were expressed as n-fold difference in gene expression relative to the housekeeping gene and calibrator and were analyzed and plotted as the means ± SEM of these fold values.
2.5. Western analysis
After treatment with or without inhibitors for 30 minutes, followed by stimulation with VLDL for 30 minutes, 3 hours or 24 hours, human adrenocortical cells (HAC15) were harvested with warm lysis buffer [0.1875MTris-HCl (pH 8.5), 3% SDS, 1.5 mM EGTA]. Sample buffer containing ß-mercaptoethanol, glycerol and bromphenol blue was added to constitute Laemmli buffer, and equal volumes of boiled sample were loaded on to a 10% SDS gel. Resolved samples were then transferred to FL transfer membrane (Immobilon-FL). After blocking with LI-COR blocking buffer (1:1 diluted with PBS) for 1 hour, the
membranes were incubated with anti-CREM or -pCREM, (1:1000, Abcam), anti-StAR (1:10000, Abcam) or anti-total ERK or -pERK (1:1000, Cell Signaling) antibodies overnight at 4℃, followed by the appropriate secondary antibody (1:3000, LI-COR) for 45 minutes. Signal development and quantitation were performed using an Odyssey imaging system (LICOR, Biosciences, Lincoln, NE).
2.6. Measurement of DAG levels
HAC15 cells were labeled in serum-free medium containing 5 µCi/ml [3H]oleic acid for 20 hours. The prelabeled cells were then equilibrated for 30 minutes in serum-free medium before preincubation with or without the PLC inhibitor U73122. After a 30-minute preincubation with U73122, the cells were stimulated with 100 µg/mL VLDL with/without inhibitor for 30 minutes. Reactions were terminated by adding 0.2% SDS containing 5 mM EDTA, and then phospholipids were extracted into chloroform/methanol containing acetic acid (1:2:0.04 vol/vol/vol). After drying under nitrogen gas, the samples were resuspended in chloroform/methanol (2:1) and spotted onto heat-activated silica gel 60 thin-layer chromatography plates (0.25mm thickness aluminum backed with concentrating zone). Phospholipids were separated using a mobile phase consisting of benzene/ethyl acetate (7:3 vol/vol) and visualized with autoradiography using En3Hance (PerkinElmer, Waltham, MA). Spots corresponding to 1,2-DAG, identified by comigration with authentic standards visualized with iodine vapor, were excised, placed in liquid scintillation fluid and counted.
2.7. Statistical Analysis
Experimental fold values from a minimum of 3 experiments (and performed at least in duplicate) were graphed as the means ± SEM of the mean values from each experiment and analyzed by ANOVA using the program Prism (GraphPad Software, San Diego, CA) with a Newman-Keuls post-hoc test. For Figure 1, a three VLDL (0, 30,100 µg/mL) by four AngII (0, 0.1, 1, 10 nM) ANOVA was performed on the ACt values, i.e., the Ct value for CYP11B2 minus that for the housekeeping gene (18S or PPIA), or the log of the raw aldosterone values. An interaction was explored and experiment was included to account for variation. No interactions were determined for either parameter, but there were significant additive effects for both VLDL and AngII. Significant differences between pairs of means within a treatment were determined using a Tukey’s multiple comparison test. SAS@ 9.3 (SAS Institute, Inc., Cary, NC) was used for these analyses.
3. Results
3.1. VLDL and angiotensin II act in an additive manner to induce CYP11B2 expression and aldosterone production
Both VLDL and angiotensin II (AngII) can induce aldosterone production in HAC15 cells, and we wished to determine whether the two agonists can act together to induce steroidogenesis in an additive or synergistic manner. We treated HAC15 cells with different doses of AngII (0, 0.1, 1 and 10 nM) with various concentrations of VLDL (0, 30 and 100 ug/mL) for 24 hours in low-serum medium and isolated RNA for qRT-PCR analysis and collected the medium for aldosterone assay. Our data (Figure 1A and B) showed that VLDL and AngII act in an additive fashion to stimulate CYP11B2 expression (p<0.001 and
p=0.0495, respectively) and aldosterone production (p<0.001 for both). Thus, at the highest concentrations of VLDL and AngII, VLDL induced an approximate 70-fold increase, AngII elicited a 6-fold rise and the combination produced an about 100-fold increase in CYP11B2, whereas the values for aldosterone production were 4-fold, 9-fold and 13-fold, respectively. This result suggested that VLDL and AngII induce aldosterone production via similar pathways.
3.2. The PLC inhibitor U73122 reduced VLDL-induced CYP11B2 mRNA expression and aldosterone production in HAC15 cells
To determine whether PLC is involved in VLDL-induced aldosterone production, we used the PLC inhibitor U73122. After pretreatment and incubation with or without VLDL in the presence or absence of U73122 for 24 hours, we found that U73122 significantly inhibited VLDL-induced CYP11B2 mRNA expression (Figure 2A), reducing the VLDL-induced increase from more than 100-fold to approximately 70-fold over control, and aldosterone production (Figure 2B), to a value of approximately 4-fold over control from a VLDL- elicited increase of about 10-fold.
3.3. VLDL induced an increase in radiolabeled DAG levels, and the PLC inhibitor U73122 blocked this increase in HAC15 cells
It is known that AngII binds to AT1R and activates PLC in glomerulosa cells (reviewed in [29]). PLC then increases the levels of DAG, which promotes PKC association with the cell membrane and activation [34]. Here we showed that after a 30-minute stimulation of HAC15 cells with VLDL, DAG levels were elevated by approximately 30% whereas the PLC inhibitor U73122 blocked this VLDL-induced increase (Figure 3). This result suggests that VLDL stimulated aldosterone production, at least in part, by regulating PLC and DAG signaling.
3.4. The IP3 receptor inhibitor xestospongin C decreased VLDL-induced CYP11B2 mRNA expression and aldosterone production in HAC15 cells
In addition to DAG, PLC activity also generates IP3, which binds to IP3 receptors in the ER and lead to the release of calcium efflux from this store as well as increased cytosolic calcium levels. To determine whether VLDL-induced IP3 production and binding to its receptor could underlie aldosterone biosynthesis, HAC15 cells were pretreated with or without the IP3 receptor inhibitor xestospongin C [35] prior to incubation in the presence or absence of VLDL. Consistent with results showing increased DAG levels (Figure 3) and increased cytosolic calcium levels [18], xestospongin C significantly decreased VLDL- induced CYP11B2 mRNA expression (Figure 4A) and aldosterone production (Figure 4B), with VLDL producing an increase of almost 40-fold for CYP11B2 expression and about 4- fold for aldosterone and xestospongin C decreasing these values to approximately 12-fold and 2-fold.
3.5. The PKC inhibitors Ro31-8220 and Go6983 reduced VLDL-induced CYP11B2 mRNA expression and aldosterone production in HAC15 cells
It has previously been shown that inhibition of the effector enzyme for calcium, CaMK, decreases VLDL-stimulated CYP11B2 expression [18]. On the other hand, a downstream effector of DAG is the PKC family of serine/threonine protein kinases. Therefore, we used two PKC inhibitors: Ro31-8220 and Gö6983 to determine whether PKC also plays a role in VLDL-induced aldosterone production. As with the PLC inhibitor, we showed that the two PKC inhibitors significantly decreased VLDL-induced CYP11B2 mRNA expression (Figure 5A) and aldosterone production (Figure 5B). Thus, VLDL induces CYP11B2 expression to about 60-fold over control, and Ro31-8220 and Gö6983 reduce this increase to 20- and 40- fold, respectively. Similar, VLDL stimulated aldosterone production about 3-fold and Ro31-8220 and Gö6983 inhibited this increase by about 50 and 75%, respectively.
3.6. PKC mediated VLDL-induced steroidogenic acute regulatory protein levels and transcription factor phosphorylation (activation)
A previous study [18] suggested that VLDL increases the levels of steroidogenic acute regulatory protein (StAR), which is involved in transporting cholesterol from the outer to the inner mitochondrial membrane, the early rate-limiting step during steroid hormone synthesis [29]. We further explored the role of PKC in this VLDL-induced increase in StAR levels as well as the activity of the transcription factor, cAMP responsive element modulator (CREM), which can regulate StAR expression [36]. After pretreatment with the PKC inhibitors Ro31-8220 and Gö6983 for 30 minutes, cells were treated with VLDL for either 30 minutes (Figure 6A) or 3 hours (Figure 6B); the phosphorylation (activation) of the transcription factor CREM and the levels of StAR protein were then monitored. VLDL induced a more than 2-fold increase in CREM phosphorylation and the PKC inhibitors returned this phosphorylation to control levels without affecting basal phosphorylation. Similarly, StAR protein levels were also significantly enhanced approximately 2-fold by VLDL, and this increase was essentially completely inhibited by the inhibitors. These results suggest that PKC activity may be involved in VLDL-induced CREM phosphorylation (activation) and StAR protein expression.
3.7. PKC inhibitors reduced VLDL-Induced ERK activation in HAC15 cells
Previous studies indicated that LDL and VLDL induce the phosphorylation (activation) of ERK1/2 as a downstream signaling molecule to transmit signals for regulating the expression of different genes involved in aldosterone production [21,37]. Although the mechanism of ERK-1/2 activation was not determined in this prior work, in the case of VLDL-induced activation of ERK-1/2 in HepG2 cells, a likely involvement of PKC was demonstrated [38]. Therefore, to determine the role of PKC in this VLDL-induced ERK activation, we pretreated cells with the PKC inhibitors Ro31-8220 and Gö6983 for 30 minutes and with VLDL for an additional 30 minutes. Our results showed that VLDL induced an approximate 50% increase in ERK-1/2 phosphorylation and that the inhibitors completely inhibited this VLDL-induced phosphorylation (activation) of ERK-1/2 (Figure 7).
4. Discussion
The plasma levels of VLDL are typically high in obese individuals; previous studies have shown that VLDL induces aldosterone biosynthesis in several glomerulosa cell models [18,20,21], suggesting a possible mechanism linking obesity, hypertension and aldosterone levels [5]. In these studies VLDL was found to stimulate steroidogenic acute regulatory (StAR) protein and CYP11B2 and transcription factor expression [18]. VLDL has also been reported to enhance aldosterone production and CYP11B2 expression in part by recruiting PKA, ERK1/2 and JAK2 in the H295R human adrenocortical carcinoma cell line [21]. We have also shown a role for phospholipase D signaling in VLDL-induced aldosterone production in HAC15 cells, a clonal derivative of the H295R cell line [20]. In addition, VLDL increases intracellular calcium levels in H295R cells [18] thereby inducing CYP11B2 expression. Nevertheless, it is not clear whether the calcium involved is released from intracellular stores or enters the cell from the extracellular fluid (or both), although data demonstrating that VLDL-induced CYP11B2 expression can be blocked by a voltage- dependent calcium channel inhibitor [18] suggest a key role for calcium influx. However, a calcium channel antagonist also inhibits chronic aldosterone production in response to AngII despite the importance of ER calcium release in steroidogenesis stimulated by this agonist (reviewed in [29]). Our current data suggest that VLDL also releases calcium from the ER, since interfering with stimulation of the IP3 receptor inhibits CYP11B2 and aldosterone production in response to VLDL (Figure 4). VLDL-induced CYP11B2 expression is also reduced by an inhibitor of calcium/calmodulin-dependent protein kinase (CaMK) [18], indicating that VLDL likely induces aldosterone production via similar pathways to those utilized by AngII. Our data using the PLC inhibitor U73122 also supports this idea since this inhibitor reduced VLDL-induced aldosterone production and CYP11B2 mRNA expression (Figure 2), indicating that VLDL activates PLC. Such an idea is also consistent with our results that VLDL and AngII induce aldosterone production in an additive manner (Figure 1). A previous study in our laboratory [20] showed that VLDL does not directly interact with the AT1R to increase aldosterone production. Saha et al. [22] have reported that VLDL exerts its effects, at least in part, through SR-B1, although there may be other receptors as well (e.g., VLDL receptor, the LDL receptor [39] and CD36 [40]). Thus, although they do not share the same receptor, our results suggest that VLDL and AngII, at some level, share similar signaling pathways leading to aldosterone production.
To confirm an ability of VLDL to activate PLC suggested by our results with U73122, we next focused on the second messengers generated by the PLC signaling pathway, DAG and IP3. Our results showed that VLDL significantly increased DAG levels, and this increase was inhibited by the PLC inhibitor (Figure 3). In addition, the ability of the IP3 receptor inhibitor to decrease aldosterone production and CYP11B2 expression (Figure 4) argues for an important signaling role for IP3 in mediating VLDL’s action. Next we determined whether PKC, which is a downstream effector activated by DAG, is involved in VLDL- induced aldosterone production. Our results demonstrated that upon treatment with either of the two PKC inhibitors, Ro31-8220 (a pan-PKC inhibitor) and Gö6983 (a classical PKC inhibitor), aldosterone production and CYP11B2 expression were inhibited (Figure 5), indicating that PKC is involved in VLDL-induced aldosterone production. In AngII
signaling PKC lies upstream of the serine-threonine protein kinase, protein kinase D (PKD), which has been shown to be activated by AngII and to mediate aldosterone production in AngII-treated H295R human adrenocortical carcinoma cells and primary cultures of bovine adrenal glomerulosa cells (e.g., [41,42]). PKD possesses two cysteine-rich domains that allow its direct activation by DAG and additionally the enzyme can be transphosphorylated and activated by novel PKC isoenzymes [43]. Therefore, given the parallels between the signaling pathways activated by VLDL and AngII, as well as the fact that PKD is a downstream mediator of PKC action [44-46], it seems possible that VLDL might also activate PKD. Experiments are in progress to investigate this possibility.
In addition to the late rate-limiting enzyme CYP11B2, we also showed that VLDL increased the levels of the early rate-limiting protein StAR [18]. Based on previous studies indicating the involvement of the activating transcription factor/cAMP response element binding protein (CREB) family of transcription factors in AngII-induced StAR mRNA and protein expression [28], we postulated the involvement of one of these factors. Our findings demonstrated that the transcription factor CREM was phosphorylated (activated) via PKC signaling, as the two PKC inhibitors inhibited this event (Figure 6). These results, together with data in bovine adrenal glomerulosa cells indicating that activated CREB can associate with the StAR promoter [47], suggest that VLDL increases StAR expression and thus protein levels by activating CREM.
ERK1/2 is reported to activate cholesterol ester hydrolase, also known as hormone-sensitive lipase [32]. Previous studies have indicated that ERK1/2 regulates aldosterone production and can be activated by both VLDL [18] and LDL [37]. Here we showed that PKC inhibitors reduced VLDL-induced ERK 1/2 phosphorylation (activation), suggesting that PKC may control ERK 1/2 activity thereby regulating aldosterone biosynthesis (Figure 7). This result is consistent with findings in macrophages that PKC is upstream of ERK1/2 in a VLDL- induced signaling cascade [48]. Nevertheless, controversy remains as to whether ERK1/2 mediates aldosterone production, as some investigators have found no effect of ERK pathway inhibitors on agonist-induced steroidogenesis [49-52]. It is known that ERK1/2 can phosphorylate StAR on serine 232 and stimulate its cholesterol-transporting activity ([53] and reviewed in [54,55]), suggesting that this signaling pathway likely does promote aldosterone production, although additional studies are necessary to resolve this controversy. In addition, membrane polarization maintained by various potassium channels and calcium influx through voltage-dependent and store-operated calcium channels is also very important in regulating aldosterone production in response to a variety of agonists (reviewed in [29]).
In conclusion, the present study expands our understanding of the signaling pathways through which VLDL induces aldosterone production. Figure 8 summarizes the findings of the present study: the pathways activated by VLDL include calcium, PLC, DAG, PKC and ERK signaling to increase StAR and CYP11B2 expression through phosphorylation (activation) of members of the CREB family of transcription factors. Supplemental Figure 2 collates information from the current study as well as previous work examining the regulation of aldosterone secretion by VLDL, inferring information also from the signaling pathways utilized by angiotensin II. In summary, our results indicate that VLDL stimulates aldosterone production by similar mechanisms to those used by AngII. Although it is
difficult to directly extrapolate in vitro results to the organismal level, our data demonstrating a correlation between triglyceride levels (a surrogate for VLDL levels) and adrenal CYP11B2 expression in rats fed normal diets and high-fat, high-sucrose diets [18] suggests that this effect may also occur in vivo. Indeed, it should be noted that serum aldosterone levels exhibit a positive correlation with the triglyceride levels, which in fasted individuals are a surrogate measure of VLDL levels, in obese subjects [56]. Finally, treatment of hypertensive patients with or without metabolic syndrome with the mineralocorticoid receptor antagonist, epleronone, results in reductions in serum triglyceride levels concomitantly with decreases in blood pressure upon treatment [57]. Thus, future studies into the role of VLDL in regulating aldosterone production are clearly warranted.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This project was supported in part by VA Merit Award I01BX001344. Dr. Bollag is supported by a VA Research Career Scientist Award. The contents of this article do not represent the views of the Department of Veterans Affairs or the United States Government.
Abbreviations
| AngII | angiotensin II |
| CREB | cAMP response element binding protein |
| CREM | cAMP responsive element modulator |
| DAG | diacylglycerol |
| ERK | extracellular signal-regulated kinase |
| IP3 | inositol 1,4,5-trisphosphate |
| PLC | phospholipase C |
| PKC | protein kinase C |
| StAR | steroidogenic acute regulatory protein |
| VLDL | very low-density lipoprotein |
References
1. Brown NJ. This is not Dr. Conn’s aldosterone anymore. Trans Am Clin Climatol Assoc. 2011; 122:229-243. [PubMed: 21686229]
2. Hostetter TH, Rosenberg ME, Ibrahim HN, Juknevicius I. Aldosterone in renal disease. Curr Opin Nephrol Hypertens. 2001; 10:105-110. [PubMed: 11195042]
3. Funder JW, Reincke M. Aldosterone: a cardiovascular risk factor? Biochim Biophys Acta. 2010; 1802:1188-1192. [PubMed: 20713154]
4. Brilla CG, Maisch B, Zhou G, Weber KT. Hormonal regulation of cardiac fibroblast function. Eur Heart J. 1995; 16(Suppl C):45-50. [PubMed: 7556272]
Mol Cell Endocrinol. Author manuscript; available in PMC 2017 September 15.
5. Kawarazaki W, Fujita T. The role of aldosterone in obesity-related hypertension. Am J Hypertens. 2016; 29:415-423. [PubMed: 26927805]
6. Kaidar-Person O, Bar-Sela G, Person B. The two major epidemics of the twenty-first century: obesity and cancer. Obes Surg. 2011; 21:1792-1797. [PubMed: 21842287]
7. Fujita T. Aldosterone in salt-sensitive hypertension and metabolic syndrome. J Mol Med (Berl). 2008; 86:729-734. [PubMed: 18437332]
8. Egan BM, Stepniakowski K, Goodfriend TL. Renin and aldosterone are higher and the hyperinsulinemic effect of salt restriction greater in subjects with risk factors clustering. Am J Hypertens. 1994; 7:886-893. [PubMed: 7826551]
9. Kidambi S, Kotchen JM, Krishnaswami S, Grim CE, Kotchen TA. Aldosterone contributes to blood pressure variance and to likelihood of hypertension in normal-weight and overweight African Americans. Am J Hypertens. 2009; 22:1303-1308. [PubMed: 19763119]
10. Nagase M, Fujita T. Mineralocorticoid receptor activation in obesity hypertension. Hypertens Res. 2009; 32:649-657. [PubMed: 19521418]
11. Briet M, Schiffrin EL. The role of aldosterone in the metabolic syndrome. Curr Hypertens Rep. 2011; 13:163-172. [PubMed: 21279740]
12. Rocchini AP, Katch VL, Grekin R, Moorehead C, Anderson J. Role for aldosterone in blood pressure regulation of obese adolescents. Am J Cardiol. 1986; 57:613-618. [PubMed: 3513521]
13. Krug AW, Vleugels K, Schinner S, Lamounier-Zepter V, Ziegler CG, et al. Human adipocytes induce an ERK1/2 MAP kinases-mediated upregulation of steroidogenic acute regulatory protein (StAR) and an angiotensin II-sensitization in human adrenocortical cells. Int J Obes (Lond). 2007; 31:1605-1616. [PubMed: 17452987]
14. Judd AM, Call GB, Barney M, McIlmoil CJ, Balls AG, et al. Possible function of IL-6 and TNF as intraadrenal factors in the regulation of adrenal steroid secretion. Ann N Y Acad Sci. 2000; 917:628-637. [PubMed: 11268391]
15. Jeon JH, Kim KY, Kim JH, Baek A, Cho H, et al. A novel adipokine CTRP1 stimulates aldosterone production. FASEB J. 2008; 22:1502-1511. [PubMed: 18171693]
16. Ehrhart-Bornstein M, Lamounier-Zepter V, Schraven A, Langenbach J, Willenberg HS, et al. Human adipocytes secrete mineralocorticoid-releasing factors. Proc Natl Acad Sci U S A. 2003; 100:14211-14216. [PubMed: 14614137]
17. Cushley RJ, Okon M. NMR studies of lipoprotein structure. Annu Rev Biophys Biomol Struct. 2002; 31:177-206. [PubMed: 11988467]
18. Xing Y, Rainey WE, Apolzan JW, Francone OL, Harris RB, et al. Adrenal cell aldosterone production is stimulated by very-low-density lipoprotein (VLDL). Endocrinology. 2012; 153:721- 731. [PubMed: 22186415]
19. Wang T, Rainey WE. Human adrenocortical carcinoma cell lines. Mol Cell Endocrinol. 2012; 351:58-65. [PubMed: 21924324]
20. Tsai YY, Rainey WE, Pan ZQ, Frohman MA, Choudhary V, et al. Phospholipase D activity underlies very-low-density lipoprotein (VLDL)-induced aldosterone production in adrenal glomerulosa cells. Endocrinology. 2014; 155:3550-3560. [PubMed: 24956203]
21. Saha S, Bornstein SR, Graessler J, Kopprasch S. Very-low-density lipoprotein mediates transcriptional regulation of aldosterone synthase in human adrenocortical cells through multiple signaling pathways. Cell Tissue Res. 2012; 348:71-80. [PubMed: 22331364]
22. Saha S, Willenberg HS, Bornstein SR, Graessler J, Kopprasch S. Diabetic lipoproteins and adrenal aldosterone synthesis — a possible pathophysiological link? Horm Metab Res. 2012; 44:239-244. [PubMed: 22147656]
23. Saha S, Schwarz PE, Bergmann S, Bornstein SR, Graessler J, et al. Circulating very-low-density lipoprotein from subjects with impaired glucose tolerance accelerates adrenocortical cortisol and aldosterone synthesis. Horm Metab Res. 2013; 45:169-172. [PubMed: 23047828]
24. Xing Y, Cohen A, Rothblat G, Sankaranarayanan S, Weibel G, et al. Aldosterone production in human adrenocortical cells is stimulated by high-density lipoprotein 2 (HDL2) through increased expression of aldosterone synthase (CYP11B2). Endocrinology. 2011; 152:751-763. [PubMed: 21239432]
25. Capponi AM. Regulation of cholesterol supply for mineralocorticoid biosynthesis. Trends Endocrinol Metab. 2002; 13:118-121. [PubMed: 11893525]
26. Barrett PQ, Bollag WB, Isales CM, McCarthy RT, Rasmussen H. Role of calcium in angiotensin II-mediated aldosterone secretion. Endocr Rev. 1989; 10:496-518. [PubMed: 2558878]
27. Ganguly A, Davis JS. Role of calcium and other mediators in aldosterone secretion from the adrenal glomerulosa cells. Pharmacol Rev. 1994; 46:417-447. [PubMed: 7899472]
28. Hattangady NG, Olala LO, Bollag WB, Rainey WE. Acute and chronic regulation of aldosterone production. Mol Cell Endocrinol. 2012; 350:151-162. [PubMed: 21839803]
29. Bollag WB. Regulation of aldosterone synthesis and secretion. Compr Physiol. 2014; 4:1017- 1055. [PubMed: 24944029]
30. Bollag WB, Barrett PQ, Isales CM, Liscovitch M, Rasmussen H. A potential role for phospholipase-D in the angiotensin-II-induced stimulation of aldosterone secretion from bovine adrenal glomerulosa cells. Endocrinology. 1990; 127:1436-1443. [PubMed: 1696885]
31. Kapas S, Purbrick A, Hinson JP. Role of tyrosine kinase and protein kinase C in the steroidogenic actions of angiotensin II, alpha-melanocyte-stimulating hormone and corticotropin in the rat adrenal cortex. Biochem J. 1995; 305(Pt 2):433-438. [PubMed: 7832756]
32. Cherradi N, Pardo B, Greenberg AS, Kraemer FB, Capponi AM. Angiotensin II activates cholesterol ester hydrolase in bovine adrenal glomerulosa cells through phosphorylation mediated by p42/p44 mitogen-activated protein kinase. Endocrinology. 2003; 144:4905-4915. [PubMed: 12960096]
33. Parmar J, Key RE, Rainey WE. Development of an adrenocorticotropin-responsive human adrenocortical carcinoma cell line. J Clin Endocrinol Metab. 2008; 93:4542-4546. [PubMed: 18713819]
34. Qin H, Frohman MA, Bollag WB. Phospholipase D2 mediates acute aldosterone secretion in response to angiotensin II in adrenal glomerulosa cells. Endocrinology. 2010; 151:2162-2170. [PubMed: 20219982]
35. Gafni J, Munsch JA, Lam TH, Catlin MC, Costa LG, et al. Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor. Neuron. 1997; 19:723-733. [PubMed: 9331361]
36. Sekine Y, Koike H, Nakano T, Nakajima K, Suzuki K. Remnant lipoproteins stimulate proliferation and activate MAPK and Akt signaling pathways via G protein-coupled receptor in PC-3 prostate cancer cells. Clin Chim Acta. 2007; 383:78-84. [PubMed: 17512923]
37. Ansurudeen I, Pietzsch J, Graessler J, Ehrhart-Bornstein M, Saha S, et al. Modulation of adrenal aldosterone release by oxidative modification of low-density lipoprotein. Am J Hypertens. 2010; 23:1061-1068. [PubMed: 20559286]
38. Banfi C, Mussoni L, Rise P, Cattaneo MG, Vicentini L, et al. Very low density lipoprotein- mediated signal transduction and plasminogen activator inhibitor type 1 in cultured HepG2 cells. Circ Res. 1999; 85:208-217. [PubMed: 10417403]
39. Chappell DA, Fry GL, Waknitz MA, Muhonen LE, Pladet MW. Low density lipoprotein receptors bind and mediate cellular catabolismof normal very low density lipoproteins in vitro. J Biol Chem. 1993; 268:25487-25493. [PubMed: 8244984]
40. Calvo D, Gomez-Coronado D, Suarez Y, Lasuncion MA, Vega MA. Human CD36 is a high affinity receptor for the native lipoproteins HDL, LDL, and VLDL. J Lipid Res. 1998; 39:777-788. [PubMed: 9555943]
41. Olala LO, Seremwe M, Tsai YY, Bollag WB. A role for phospholipase D in angiotensin II-induced protein kinase D activation in adrenal glomerulosa cell models. Mol Cell Endocrinol. 2013; 366:31-37. [PubMed: 23178798]
42. Shapiro BA, Olala L, Arun SN, Parker PM, George MV, et al. Angiotensin II-activated protein kinase D mediates acute aldosterone secretion. Mol Cell Endocrinol. 2010; 317:99-105. [PubMed: 19961896]
43. Wang QJ. PKD at the crossroads of DAG and PKC signaling. Trends Pharmacol Sci. 2006; 27:317-323. [PubMed: 16678913]
44. Romero DG, Welsh BL, Gomez-Sanchez EP, Yanes LL, Rilli S, et al. Angiotensin II-mediated protein kinase D activation stimulates aldosterone and cortisol secretion in H295R human adrenocortical cells. Endocrinology. 2006; 147:6046-6055. [PubMed: 16973724]
45. Matthews SA, Rozengurt E, Cantrell D. Protein kinase D. A selective target for antigen receptors and a downstream target for protein kinase C in lymphocytes. J Exp Med. 2000; 191:2075-2082. [PubMed: 10859332]
46. Yuan J, Bae D, Cantrell D, Nel AE, Rozengurt E. Protein kinase D is a downstream target of protein kinase Ctheta. Biochem Biophys Res Commun. 2002; 291:444-452. [PubMed: 11855809]
47. Olala LO, Choudhary V, Johnson MH, Bollag WB. Angiotensin II-induced protein kinase D activates the ATF/CREB family of transcription factors and promotes StAR mRNA expression. Endocrinology. 2014; 155:2524-2533. [PubMed: 24708239]
48. Liu Z, Li H, Li Y, Wang Y, Zong Y, et al. Up-regulation of VLDL receptor expression and its signaling pathway induced by VLDL and beta-VLDL. J Huazhong Univ Sci Technolog Med Sci. 2009; 29:1-7. [PubMed: 19224153]
49. Natarajan R, Yang DC, Lanting L, Nadler JL. Key role of P38 mitogen-activated protein kinase and the lipoxygenase pathway in angiotensin II actions in H295R adrenocortical cells. Endocrine. 2002; 18:295-301. [PubMed: 12450322]
50. Haisenleder DJ, Ferris HA, Shupnik MA. The calcium component of gonadotropin-releasing hormone-stimulated luteinizing hormone subunit gene transcription is mediated by calcium/ calmodulin-dependent protein kinase type II. Endocrinology. 2003; 144:2409-2416. [PubMed: 12746302]
51. Munir I, Yen HW, Geller DH, Torbati D, Bierden RM, et al. Insulin augmentation of 17alpha- hydroxylase activity is mediated by phosphatidyl inositol 3-kinase but not extracellular signal- regulated kinase-1/2 in human ovarian theca cells. Endocrinology. 2004; 145:175-183. [PubMed: 14512432]
52. McNeill H, Whitworth E, Vinson GP, Hinson JP. Distribution of extracellular signal-regulated protein kinases 1 and 2 in the rat adrenal and their activation by angiotensin II. J Endocrinol. 2005; 187:149-157. [PubMed: 16214950]
53. Poderoso C, Converso DP, Maloberti P, Duarte A, Neuman I, et al. A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis. PLoS One. 2008; 3:e1443. [PubMed: 18197253]
54. Manna PR, Dyson MT, Stocco DM. Regulation of the steroidogenic acute regulatory protein gene expression: present and future perspectives. Mol Hum Reprod. 2009; 15:321-333. [PubMed: 19321517]
55. Poderoso C, Maloberti P, Duarte A, Neuman I, Paz C, et al. Hormonal activation of a kinase cascade localized at the mitochondria is required for StAR protein activity. Mol Cell Endocrinol. 2009; 300:37-42. [PubMed: 19007846]
56. Goodfriend TL, Egan B, Stepniakowski K, Ball DL. Relationships among plasma aldosterone, high-density lipoprotein cholesterol, and insulin in humans. Hypertension. 1995; 25:30-36. [PubMed: 7843750]
57. Sato A, Fukuda S. Clinical effects of eplerenone, a selective aldosterone blocker, in Japanese patients with essential hypertension. J Hum Hypertens. 2010; 24:387-394. [PubMed: 19865106]
| Highlights | |
|---|---|
| In glomerulosa cells VLDL increases aldosterone secretion and • diacylglycerol levels | |
| VLDL induces similar signal transduction mechanisms to those used by • angiotensin II | |
| • | Inhibitor studies show that VLDL induces aldosterone synthesis via phospholipase C |
| Protein kinase C inhibitors also reduce VLDL-induced steroidogenesis • VLDL-stimulated protein kinase C induces transcription factor activity • | |
VA Author Manuscript
A
+ Oug/mL VLDL
CYP11B2 Expression (-fold over control)
30µg/mL VLDL
B
150
100µg/mL VLDL
Aldosterone Production (-fold over control)
20-
a
a,b
a,b
b
a
a,b
b,c
C
100
t
15-
+
I
10
T
I
#
50
#
5-
*
0
*
0
0
0.1
1
10
0
0.1
1
10
Angll (nM)
Angll (nM)
HAC15 cells were treated with VLDL (0, 30 or 100 µg/mL) and/or AngII (0, 0.1, 1 or 10 nM) for 24 hours. (A) CYP11B2 expression was measured using qRT-PCR. (B) Aldosterone levels in the media were measured using a radioimmunoassay. Results represent the means ± SEM of 3 separate experiments. Statistical analyses were performed as described in the Methods section. There was no significant interaction observed between VLDL and AngII; however, there were significant additive effects for both VLDL and AngII across the dose range for aldosterone levels (p<0.001 for both) and CYP11B2 expression (p<0.001 and p=0.0495, respectively). Means for each concentration of VLDL were significantly different from each other across the dose range of AngII for both aldosterone and CYP11B2 values; for AngII 1 and 10 nM values for aldosterone production were significantly greater than 0, 0.1 nM was significantly less than 10, and 1 and 10 nM were not significantly different across all concentrations of VLDL. For CYP11B2 expression 10 nM AngII was statistically greater than 0 and 0.1 and 1 nM were intermediate and were not significantly different across all concentrations of VLDL. The symbols (*, # and }) and the letters (a, b and c), as indicated on the graphs, show pairs of means, with different letters representing significantly different (p<0.05) values (and similar letters indicating no difference) within a treatment by a Tukey’s multiple comparison test.
A
B
CYP11B2 Expression (fold over Control)
Aldosterone Production (fold over Control)
150
20
…
100
15.
…
* tt
…
10
50
*+
5
tt
0
Control
U73122
Angll
Angll + U73122
VLDL
VLDL + U73122
0
Control
U73122
Angll
Angll + U73122
VLDL
VLDL + U73122
Figure 2. A PLC inhibitor decreased VLDL-induced aldosterone production and CYP11B2 expression HAC15 cells were pretreated for 30 minutes with or without the PLC inhibitor U73122 (10 uM) or the DMSO vehicle, prior to incubation in the presence or absence of 100 µg/mL VLDL, or 10 nM AngII as a positive control, for 24 hours. (A) CYP11B2 expression was measured with qRT-PCR. (B) Aldosterone levels in the supernatant were measured using a 20 radioimmunoassay. Results represent the means ± SEM of 3 separate experiments; *p<0.05, *** p<0.001 versus the control, *p<0.05 versus AngII alone, ** p<0.01 versus VLDL alone.
[3H]Diacylglycerol Levels (fold over Control)
1.5
*
1.0
+
0.5
0
Control
VLDL
U73122
VLDL + U73122
A
B
45
5
CYP11B2 Expression (fold over Control)
Aldosterone Production (fold over Control)
40
35
4.
30
+
8
25
20
ttt
2
15
10
1
5
0
0
Control
VLDL
XC
VLDL + XC
Control
VLDL
XC
VLDL + XC
Figure 4. The IP3 receptor inhibitor xestospongin C decreased VLDL-induced aldosterone production and CYP11B2 expression HAC15 cells were pretreated for 30 minutes with or without the IP3 receptor inhibitor xestospongin C (XC) (10 µM) or the DMSO vehicle, prior to incubation in the presence or absence of 100 µg/mL VLDL for 24 hours. (A) CYP11B2 expression was measured with qRT-PCR. (B) Aldosterone levels in the supernatant were measured using a radioimmunoassay. Results represent the means ± SEM of 3 separate experiments;
*** p<0.001 versus the control, fp<0.05 and *** p<0.001 versus VLDL alone.
A
B
CYP11B2 Expression (fold over Control)
Aldosterone Production (fold over Control)
80
4
3 7 - N WA
…
…
60
T
*tt
*tt
40
*tt
tt
20
0
Control
VLDL
Ro31-8220
VLDL + Ro
Gö6983
VLDL + Gö
0
Control
VLDL
Ro31-8220
VLDL + Ro
Gö6983
VLDL + Gö
A
B
Control
VLDL
Ro31-8220
VLDL + Ro
Gö6983
VLDL + Gö
Control
VLDL
Ro31-8220
VLDL + Ro
Gö6983
VLDL + Gö
pCREM
StAR
CREM
ß-actin
Normalized pCREM Levels (fold over Control)
4
Normalized StAR Levels (fold over Control)
2.5;
3
2.0
ttt
ttt
1.5
2
+
tt
1.0
1
0.5
0
0.0
Control
VLDL
Ro31-8220
VLDL + Ro
Gö6983
VLDL + Gö
Control
VLDL
Ro31-8220
VLDL + Ro
Gö6983
VLDL + Gö
Control
VLDL
Ro31-8220
VLDL + Ro
Gö6983
VLDL + Gö
pERK
ERK
Normalized pERK Levels (fold over Control)
2.0
1.5
tt
1.0
tt
0.5
0.0
Control
VLDL
Ro31-8220
VLDL + Ro
Gö6983
VLDL + Gö
Figure 7. PKC inhibitors decreased VLDL-induced ERK phosphorylation (activation) in HAC15 cells HAC15 cells were pre-treated with the PKC inhibitors Ro31-8220, Gö6983 (5 uM) or DMSO vehicle for 30 minutes prior to treatment with or without VLDL (100 µg/mL) for 1 hour. Cells were then harvested for analysis of ERK phosphorylation. Shown is a representative blot as well as cumulative results of 3 independent experiments presented as means ± SEM of levels normalized to ERK; ** p<0.05 versus the control, ** p<0.001 versus VLDL alone.
VLDL
DAG
PIP 2 - U73122
PLC
PKC
Ro Gö
^ Ca2+
ERK
Ca2+
- XC
p-CREM
Ca2+
ER
Nucleus
Chol
CYP11B2 StAR
StAR
Mitochondria
CYP11B2
Aldosterone