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Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Texas Southwestern Medical Center (J.C.C., B.M.D., S.Y., W.E.R.), Dallas, Texas 75390; and Department of Pharmaco-Biology, University of Calabria (V.P.), 87036 Arcavacata di Rende, Italy
Address all correspondence and requests for reprints to: William E. Rainey, Ph.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9032. E-mail: william.rainey{at}utsouthwestern.edu.
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Abstract |
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Introduction |
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In vivo, the expression of CYP11B2 is regulated, as is aldosterone synthesis, by the level of circulating ANG II and K+ (4, 5, 6). In vitro studies have demonstrated that ANG II and K+ induction of CYP11B2 protein and mRNA result by a direct action on the adrenal cell (7, 8, 9, 10). ANG II and K+ share the ability to increase intracellular glomerulosa cell calcium concentrations, which represents a possible mechanism of controlling CYP11B2 expression. Indeed pharmacological agents that increase intracellular calcium induce the expression of CYP11B2 (7, 8, 10). Many aspects of calcium signaling occur through the activation of the calcium-binding protein calmodulin (CaM). By its binding to calcium, CaM is able to influence several intracellular signaling pathways, including the activation of the multifunctional CaM-dependent protein kinase I (CaMKI), CaMKII, and CaMKIV (11, 12, 13). CaM is expressed at high levels in the adrenal cortex (14, 15), and its role in the production of steroid hormones has been suggested by several experimental paradigms (16). Using aldosterone-producing cell models, there is considerable evidence that CaM and CaMK are important for the acute regulation of aldosterone production (17, 18, 19, 20, 21). In addition, antagonists of CaM and CaMK inhibit K+ induction of CYP11B2 mRNA levels (10). Herein, we have used reporter constructs prepared with the 5'-flanking region of the CYP11B2 to determine the roles of CaM and CaMK in transcriptional regulation. CaM and CaMKI were able to regulate the transcription of CYP11B2 through a cAMP response element found in the 5'-flanking DNA. CaMKI expression in the adrenal and in H295R adrenocortical cells was demonstrated using Northern blot, immunohistochemistry, and Western analysis. These findings support the hypothesis that CaMKI plays an important role in K+ and ANG II regulation of the adrenal capacity to produce aldosterone through the regulation of CYP11B2 transcription.
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Materials and Methods |
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Immunoblotting analysis
Total cell and tissue lysates from H295R cells, human adrenal cortex, and brain were prepared as previously described (22). H295R nuclear extracts were prepared as previously described (24). PAGE was carried out on the samples with 4–12% bis-Tris NuPage gels (Invitrogen, Carlsbad, CA). Proteins were electrophoretically transferred onto nylon membranes by wet transfer for 1 h at 25 V. After transfer, membranes were incubated for 1 h at room temperature with CaMKI antibody. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and immunoreactive bands were visualized. The CaMKI antibody (CC77) was provided by Dr. Angus Nairn (Rockefeller University, NY) and has previously been characterized with regard to detection of CaMKI using immunoblot analysis (25). The fusion protein tag hemagglutinin (HA) antibody (sc-7392) mapping to the internal region of the influenza HA protein specific for proteins containing the HA tag was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Reporter constructs, expression vectors, and cell transfection
The constructs containing human CYP11B2 5' flanking DNA were fused immediately upstream of the firefly luciferase gene in the reporter vector pGL3-Basic (Promega Corp.). A series of human CYP11B2 (pB2) 5'-flanking DNA containing progressive deletions, -1521, -870, -347, -149, and -64 bp, were subcloned by restriction digestion into the pGL3-Basic vector as previously described (26). Empty pGL3-Basic vector was used as a control vector to measure basal activity. The constructs containing the constitutively active and wild-type full-length CaMKI, CaMKII, and CaMKIV (27, 28) were provided by Richard Maurer, Oregon Health Sciences University (Portland, OR). Truncation of CaMKI at Lys295 (CaMKI-295), CaMKII at Leu290 (CaMKII-290), and CaMKIV at Leu313 (CaMKIV-313) removes an autoinhibitory regulatory region of the enzymes and results in a constitutively active protein kinase. The constitutively active isoforms of the CaMK were subcloned into the empty RSV-globin expression vector. Empty RSV-globin vector was used to ensure that DNA concentrations were constant in each transfection.
Synthesis of cDNA probes and Northern analysis for CaMKI and glyceraldehyde-3-phosphate dehydrogenase (G3PDH)
A specific human CaMKI probe (326 bp encompassing nucleotides 406–730 of the published cDNA) was designed to specifically hybridize with CaMKI. Random prime labeling (29) of CaMKI cDNA was performed using a Redi-Prime II kit (Amersham Pharmacia Biotech, Piscataway, NJ). G3PDH cDNA prepared using PCR was labeled in the same manner. We used a Redi-Prime reaction mix using exonuclease-free Klenow and Redivue [32P]deoxy-CTP (Amersham Pharmacia Biotech) for 10 min at 37 C to produce cDNA probes ready for hybridization.
Polyadenylated [poly(A)+] RNA was isolated from H295R cells using a poly(A)+ RNA purification kit from Amersham Pharmacia Biotech. RNA was then applied to an oligo(deoxythymidine)-cellulose spin column for purification of poly(A)+ RNA. Total RNA was isolated from human fetal brain and adult adrenal cortex in a one step 4 M guanidinium isothiocyanate extraction and 5.7 M cesium chloride ultracentrifugation at 42,000 rpm (SW60 rotor, Beckman, Palo Alto, CA) for 16–24 h. RNA from brain (10 µg total RNA), human adrenals [2 µg poly(A)+ RNA] and H295R cells [2 µg poly(A)+ RNA] were electrophoresed on a denaturing formaldehyde/1.5% agarose gel, transferred to a Hybond N+ membrane, and fixed by UV cross-linking. Hybridization of CaMKI and G3PDH cDNA was performed overnight at 42 C and subjected to a series of stringent posthybridization washes.
Immunohistochemical analysis
Before immunohistochemical staining, the sections were deparaffinized with xylene and rehydrated with a graded series of diluted ethanol in deionized water. The sections were allowed to equilibrate in 10 mM PBS solution. Endogenous tissue peroxidases were neutralized with a 1:9 solution of 30% hydrogen peroxide in absolute methanol. Thereafter, the sections were washed by immersing into PBS containing 0.1% BSA for 5 min. Sections were incubated overnight with primary antibody (anti-CaMKI, diluted 1:100) in a humidified chamber at 4 C. The CaMKI antibody (CC77) was provided by Dr. Angus Nairn (Rockefeller University, New York, NY) and has previously been characterized with regard to detection of CaMKI using immunohistochemistry (25). After washing with PBS, sections were incubated with a biotinylated donkey antirabbit secondary antibody for 30 min and a horseradish peroxidase-streptavidin enzyme conjugate for 10 min, with washes of PBS in between. Color development was achieved by exposing the treated tissue sections to 0.6% hydrogen peroxide and chromagen, 3,3'-diaminobenzidine tetrahydrochloride, and sections were then counterstained using hematoxylin. In each immunohistochemical study controls included tissues known to express CaMKI (brain) and sections with the primary antiserum omitted. Three different human adrenal tissue samples were analyzed using the CaMKI antibody, and the pattern of immunoreactivity was similar in each.
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Results |
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). Calmidazolium also inhibited ANG II stimulation of CYP11B2 reporter gene activity, but the inhibition was not complete as was seen for K+ (Fig. 1B). Induction of reporter expression by dbcAMP (Fig. 1C) was unaffected by calmidazolium. These data support the hypothesis that CaM is needed for both K+ and ANG II induction of CYP11B2 expression.
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) and inhibited ANG II (Fig. 1B) induction of luciferase reporter activity by 90%. As was observed for calmidazolium, inhibition of CaMK did not affect dbcAMP-regulated expression (Fig. 1C). Thus, all of the K+-stimulated and the majority of ANG II-stimulated CYP11B2 transcription appears to be regulated by a calcium-CaM-CaMK pathway.
CaMK regulation of CYP11B2 transcription
To better define which of the CaMK are responsible for the regulation of CYP11B2 expression, H295R cells were cotransfected with the pB2-1521 reporter construct and expression vectors containing the full-length coding sequences of CaMKI, CaMKII, and CaMKIV. Three concentrations of CaMK-containing vectors (0.3, 1, and 1.5 µg/ml) were tested, and 1 µg/ml vector was chosen for the study (data not shown). Of the three CaMK expression vectors, only CaMKI significantly increases basal reporter activity (Fig. 2). However, it has been shown that wild-type CaMK-expressed kinases can be further activated by agents that increase intracellular calcium to maximally influence target gene transcription (28). Therefore, we treated cells that were cotransfected with the CaMK vectors and CYP11B2 reporter construct with ionomycin (1 µM), which increases intracellular calcium. As shown in Fig. 2, ionomycin increased reporter construct activity driven by the CYP11B2 promoter. Ionomycin stimulation of reporter activity was highest in cells cotransfected with the CaMKI expression vector, where reporter levels increased by 5-fold above basal. CaMKII- and CaMKIV-transfected cells were not significantly affected by ionomycin. One disadvantage with the use of the full-length CaMK expression system is that it requires increased intracellular calcium to have maximal kinase activity. Increasing intracellular calcium in the H295R cell activates not only the transfected CaMKI, but also any CaMKI that is already expressed in H295R cells. To try and avoid this issue we designed experiments to examine more directly the role of each CaMK using constitutively active forms of CaMKI, -II, and -IV.
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). CaMKI-295 increased CYP11B2 reporter construct expression 6-fold over basal, whereas CaMKIV-313 caused a 1.6-fold increase in reporter activity. CaMKII-290 caused an inhibition of reporter expression. CaMKI-295 stimulated reporter expression in a concentration-dependent manner, with the highest effect seen using 2 µg/ml CaMKI-295 expression vector (range tested, 0.5–3.0 µg/ml; data not shown). To ensure that the effects of the CaMK vectors were not the result of variable expression, we monitored cellular levels of transfected CaMK using Western analysis with HA-tagged CaMK constructs. All three isoforms were abundantly expressed, with CaMKI expression slightly less than CaMKII or -IV (data not shown).
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). These constructs were transiently transfected into H295R cells with CaMKI-295 or empty RSV vector. Figure 4 shows the luciferase activity of these constructs as a percentage of basal activity. Luciferase activities of each construct containing 5'-flanking sequences from -1521 to -347 bp were similar. The 6-fold increase in pB2-1521 reporter expression with CaMKI-295 decreased to a 1.2-fold stimulation over basal when the CYP11B2 5'-flanking fragment was deleted from -347 to -64 bp. This result indicates that DNA sequences located between -64 and -347 bp are essential for CaMKI-stimulated gene expression.
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).
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). Relative levels of CaMKI in adrenal lysate, H295R cell lysate, and nuclear extracts were compared with that in brain lysate as a positive control. Two protein bands (
40 and 45 kDa) were detected in both H295R and adrenal cell lysates, which probably represent different isoforms of CaMKI that have previously been described (31). CaMKI (45 kDa) was the predominant form present in H295R cell lysates and was expressed in adrenal lysate at levels almost as high as those observed in the positive control (brain lysate). Levels of CaMKI were also examined using nuclear extract from the H295R cells (Fig. 6A). The level of immunoreactive 45-kDa CaMKI was less in the nuclear extract than in the whole cell lysate. However, the level of 40-kDa CaMKI was increased in the nuclear extract from H295R cells.
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). RNA from multiple cultures of H295R cells and three adrenals were pooled to isolate poly(A+) RNA, which was used for Northern analysis. Brain RNA, due to its high expression of CaMKI, was used as a positive control. Transcripts for CaMKI (1.4 kb) were detected in RNA isolated from adrenal, H295R, and brain, demonstrating that the adrenal and H295R models express this kinase.
Human adrenal expression of CaMKI was further studied using immunohistochemistry (Fig. 7). CaMKI immunoreactivity was localized to the zona glomerulosa of the human adrenal, which is the same zone that expresses CYP11B2. A small amount of immunoreactivity was observed in the outer part of the zona fasciculata, and no staining was observed in the capsule, reticularis, or medulla of the adrenal. There was also no reactivity in the adrenal when sections were analyzed in the absence of primary antibody.
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Discussion |
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Primary cultures of rat adrenal cells and the H295R adrenal cell line have been useful to dissect the signaling pathways used by ANG II and K+ to regulate aldosterone production. In these cell culture models, treatment with ANG II and K+ increases CYP11B2 protein and mRNA levels as well as transcription of the CYP11B2 gene (8, 9, 10, 37, 38, 39). The effects of K+ and ANG II could be mimicked by calcium channel activation using BAYK8644, suggesting that cellular calcium levels are important in this process (7). Supporting the role of calcium, the calcium channel blocker nifedipine completely blocked K+ and partially blocked ANG II stimulation of CYP11B2 mRNA levels (8). Together, these experiments provide strong evidence that intracellular calcium levels are involved in agonist induction of CYP11B2 gene transcription, mRNA levels, and protein expression.
Intracellular calcium induces several signaling pathways through the activation of CaM. CaM appears important for activation of CYP11B2 transcription, as calmidazolium also inhibited the K+ and ANG II induction of reporter activity driven by 5'-flanking DNA from the CYP11B2 gene. CaM can activate a number of kinases that exhibit a broad range of protein substrates, including CaMKI, -II, and -IV. To determine whether the CaMKs were important in the regulation of CYP11B2 expression, we used KN93, an inhibitor of this family of multifunctional CaMK (40). This CaMK inhibitor did not exhibit nonspecific effects on steroidogenesis (17) that have previously been observed for its predecessor, KN62 (23). KN93 treatment completely inhibited K+ induction of CYP11B2 mRNA (10) and CYP11B2 reporter transcription, as shown in Fig. 1. KN93 also inhibited ANG II stimulation of reporter gene expression and partially inhibited the effects of ANG II on CYP11B2 mRNA levels (10). In contrast, treatment with calmidazolium or KN93 was without effect on cAMP induction of CYP11B2 mRNA (10) or transcription. These experiments suggest that one of the key pathways used by ANG II and K+ to regulate CYP11B2 expression is the calcium-CaM-CaMK cascade.
By its binding to calcium, CaM is able to influence several intracellular signaling pathways, including the activation of the multifunctional CaMKI, -II, and -IV (11, 12, 13). To examine the effects of CaMKI, -II, and -IV on CYP11B2 transcription, we used expression vectors encoding the complete coding sequence of each kinase. Of the three CaMK tested, only CaMKI increased basal CYP11B2 reporter expression. Increasing intracellular calcium with ionomycin was most effective at increasing CaMKI induction of CYP11B2-driven reporter activity. The data obtained using coexpression of wild-type CaMK in the H295R cell could be influenced by endogenously expressed kinase that would also be activated by ionomycin. Therefore, we designed experiments using truncated versions of CaMK that did not require ionomycin treatment for activation. An interesting feature of CaMKI, -II, and -IV is the ability to produce a constitutively active form of each enzyme by truncating the protein in a manner that removes an autoinhibitory domain. Truncation of CaMKI at Lys295, CaMKII at Leu290, and CaMKIV at Leu313 removes the autoinhibitory regulatory region and results in a constitutively active protein kinase (28). We cotransfected expression vectors containing constitutively active CaMKI-295, -II-290, or -IV-313 with the pB2-1521bp reporter construct. Cotransfection with CaMKI-295 caused a 6-fold increase in reporter gene expression, CaMKIV-313 also showed a modest stimulation, whereas CaMKII-290 inhibited CYP11B2 reporter gene expression. These data further support a role for CaMKI in the regulation of CYP11B2 transcription.
The cis-elements that regulate agonist enhancement of CYP11B2 transcription have been previously studied (26, 30, 37). Deletion analysis demonstrated that DNA between -347 and -64 of the CYP11B2-flanking DNA caused a loss of CaMKI stimulation. We have previously shown that a critical cis-element in ANG II, K+, and cAMP stimulation of human CYP11B2 transcription is located at -72/-63 and is a near consensus CRE (26, 30). The mutation of this CRE abolished the CaMKI induction of reporter gene activity. This CYP11B2-proximal CRE is well conserved, with 100% sequence identity in mouse, rat, hamster, and human CYP11B2 genes (37). In humans, the CRE is also shared with the CYP11B1 gene, where it is critical for maximal agonist stimulation of transcription (30). Using electrophoretic mobility shift assay, the CYP11B2 CRE has been shown to bind members of the activating transcription factors (ATF-1 and ATF-2) and CRE-binding protein (CREB) (30). The ability of these transcription factors, particularly ATF-1 and CREB, to enhance transcription is partially regulated by their state of phosphorylation. Thus, one possibility is that activated CaMKI phosphorylates members of CREB or ATF-1, leading to increased CYP11B2 transcription. Phosphorylation of CREB and ATF-1 by CaMKI and -IV increases their ability to enhance transcription (41, 42). In contrast, CaMKII is unable to activate either ATF-1 or CREB. With this in mind, it is not surprising that CaMKII was not a positive regulator of CYP11B2 transcription. However, it should be noted that CaMKII might play a role in the activation of T-type calcium channels in bovine glomerulosa cells (43). In this case, CaMKII may influence cellular calcium levels and thereby the activation of both CaM and CaMKI. CaMKI could also influence CYP11B2 in an indirect mechanism by phosphorylation of cytoplasmic factors that then influence CYP11B2 transcription. This option must be considered, as most cell types have more CaMKI expressed in the cytoplasm than the nucleus.
Although CaMKI expression is considered to occur in most, if not all, tissues, no studies have been performed to examine adrenal tissue or adrenal cell expression. Therefore, immunohistochemistry, Northern, and Western analyses were used to determine whether the H295R cell or adrenal tissue express CaMKI. Using Northern analysis we demonstrated that RNA isolated from adrenal and H295R cells contained CaMKI mRNA. Western analysis demonstrated two immunoreactive proteins corresponding in size to 40 and 45 kDa. Previous reports have shown that there are multiple isoforms of CaMKI corresponding to these molecular masses (31). Importantly, CaMKI was also detected in H295R nuclear extracts, and recent studies have shown that nuclear expression of CaMKI varies in a tissue-specific manner (42). In addition, the amount of nuclear CaMKI, at least in neurons, can be controlled by agonist treatment (44). The expression of CaMKI in the nucleus would support a role for this kinase in the direct regulation of CRE-binding transcription factors that regulate CYP11B2. Our observation that CaMKI expression is localized to the adrenal zona glomerulosa where CYP11B2 is expressed and aldosterone is produced supports the hypothesis that CaMKI plays a role in vivo.
In conclusion, there is considerable evidence supporting a role for calcium signaling in ANG II- and K+-regulated aldosterone production. Herein, we demonstrate that calcium signaling through CaM and CaMKI is an important regulator of CYP11B2 transcription. Together, these findings provide evidence of a common signaling pathway through which ANG II and K+ may regulate the capacity of the adrenal glomerulosa to produce aldosterone.
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Acknowledgments |
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Footnotes |
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Abbreviations: ANG II, Angiotensin II; ATF, activating transcription factor; CaM, calmodulin; CaMK, calmodulin-dependent protein kinase; CRE, cAMP response element; CREB, CRE-binding protein; dbcAMP, dibutyryl cAMP; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; hemagglutinin; poly(A)+, polyadenylated; RSV, Rous sarcoma virus.
Received December 3, 2001.
Accepted for publication May 15, 2002.
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) or were treated for 6 h with ANG II (100 nM; 
). Mutation of the proximal CRE blocked both ANG II and CaMKI stimulation of reporter activity. Luciferase activity for each of the promoter constructs is presented as a percentage of the untreated reporter value. Results represent the mean ± SE of determinations from three to six independent experiments, each of which was performed in triplicate.
