Control of CYP11B2 Gene Expression through Differential Regulation of Its Promoter by Atypical and Conventional Protein Kinase C Isoforms*

Jean-Guy LeHouxDagger, Gilles Dupuis, and Andrée Lefebvre

From the Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada

Received for publication, October 18, 2000, and in revised form, December 12, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We reported previously that the protein kinase C (PKC) inhibitor GF109203X stimulated the hamster CYP11B2 promoter activity in transfected NCI-H295 cells. PKCalpha , -epsilon , and -zeta were detected in hamster adrenal zona glomerulosa and NCI-H295 cells, and PKCtheta in NCI-H295 cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) inhibited basal and stimulated cytochrome P450 aldosterone synthase mRNA expression by angiotensin (AII), dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP), or KCl in NCI-H295 cells. Basal CYP11B2 promoter activity was inhibited in cells cotransfected with constitutively active (CA) PKCalpha , -epsilon , and -theta mutants, whereas it was increased with CA-PKCzeta . Dominant negative (DN) PKCalpha , -theta , -epsilon , and -zeta mutants stimulated the promoter activity. AII-, KCl-, and Bt2cAMP-stimulatory effects were abolished in cells cotransfected with CA-PKCalpha , -epsilon , or -theta . The effect of Bt2cAMP was abolished by CA-PKCzeta but AII and KCl were still able to enhance the promoter activity. DN-PKCalpha , -epsilon , -theta , or -zeta did not inhibit these effects. Gö6976 enhanced promoter activity, providing further evidence that PKCalpha was involved. Various CYP11B2 promoter constructs were used to identify the area associated with TPA and PKC inhibition. TPA and CA-PKCalpha , -epsilon , or -theta abolished the effects of AII, KCl, and Bt2cAMP on the activity of -102 and longer constructs. In summary, our findings suggest that the hamster CYP11B2 gene is under differential control by conventional (alpha ) and atypical (zeta ) PKC.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Aldosterone synthase is the product of the CYP11B2 gene and is responsible for the final step of aldosterone synthesis in most mammalian adrenal glands (1). This key regulatory enzyme is under the control of many factors including angiotensin II (AII),1 adrenocorticotropin (ACTH), and changes in blood Na+ and K+ concentrations (1). The hamster adrenal CYP11B2 gene has been cloned and its promoter activity studied (2, 3). In transfected human adrenocortical carcinoma NCI-H295 cells, the first 161 base pairs of the 5'-flanking region of the hamster CYP11B2 were shown to be sufficient for maximal promoter activity and for the stimulatory effect of AII, KCl, and Bt2cAMP (4). In comparison, the first 129 base pairs of the 5'-flanking region of the human CYP11B2 gene were also shown to be responsive to the stimulatory action of AII, KCl and dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) (5).

The involvement of protein kinase C (PKC) in the expression of adrenal steroidogenic enzymes has been studied using activators and inhibitors of PKC. For example, 12-O-tetradecanoylphorbol-13-acetate (TPA) has been reported to increase the mRNA levels of 3-beta -hydroxysteroid dehydrogenase (6) and cytochrome P450 21-hydroxylase in the NCI-H295 cell line (7, 8). In addition, phorbol esters have been shown to decrease the mRNA levels of cytochrome P450 17alpha -hydroxylase (7) and cytochrome P450 side chain cleavage (7) and to abolish the enhancing effect of BAYK 8644 on cytochrome P450 11beta -hydroxylase (P450C11) and cytochrome P450 aldosterone synthase (P450aldo) (9). Steroidogenesis is also affected by inhibition of PKC. For instance, staurosporine has been reported to stimulate steroidogenesis in Y-1 cells (3, 10), to elevate the levels of mRNA of cytochrome P450 side chain cleavage and P450C11 (10), and to stimulate the activity of the hamster CYP11B2 promoter in transfected Y-1 cells (3). We have reported that GF109203X, a specific PKC inhibitor, has stimulated the output of aldosterone and the activity of the hamster CYP11B2 promoter in NCI-H295-transfected cells (4). In addition, staurosporine has been reported to reverse the inhibitory effect of TPA on aldosterone production in rat adrenal glomerulosa cells and to enhance the stimulatory effects of AII and K+, but not of ACTH, in these cells (11).

PKC comprises several isoforms of serine/threonine kinases that can be divided in three families (12-14). Members of the conventional family (PKCalpha , -beta 1, -beta 2, and -gamma ) are calcium-dependent and are activated by phosphatidylserine and diacylglycerol. Members of the novel family (PKCdelta , -epsilon , -theta , and -eta ) do not require calcium for their activity but are activated by diacylglycerol, whereas members of the atypical family (PKCzeta and -lambda /iota ) do not respond to calcium or diacylglycerol.

Current knowledge supports the interpretation that PKC is involved in the regulation of steroidogenesis at least at two separate enzymatic steps of aldosterone synthesis, namely the transformation of cholesterol to pregnenolone (10, 15) and the conversion of deoxycorticosterone to aldosterone (3, 4). However, the role of individual PKC isozymes in the regulation of aldosterone synthesis remains unknown. We have addressed this question by investigating the role of PKC isoforms on the regulation of the hamster CYP11B2 gene promoter activity. We show here that cotransfection of NCI-H295 cells with constitutively active (CA) or dominant negative (DN) mutants of PKC isoforms, along with the CYP11B2 gene promoter, resulted in a differential regulation of promoter activity of conventional (alpha ) and atypical (zeta ) PKC.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tissues-- Adult male Syrian Golden hamsters (120 ± 10 g) and 2-month-old Long Evans male rats were purchased from Charles River Inc. (St. Constant, Quebec, Canada) and kept on Purina rat chow and tap water ad libitum. Animals were sacrificed in accordance with the ethical standards of our local institutional review committee. The adrenal glands were removed and freed of fatty tissue. The zona glomerulosa was separated from the zona fasciculata-reticularis and the medulla by the method of Giroud et al. (16). Three adult adrenal glands were obtained from 50-, 52-, and 55-year-old renal transplant donors, in accordance with the Human Subject Review Committee of our institution.

Reagents-- The PKC inhibitors GF109203X and Gö6976 were purchased from Calbiochem, Cedarlane Laboratories Ltd. (Hornsby, Ontario, Canada). TPA, AII, and Bt2cAMP were obtained from Sigma-Aldrich (St. Louis, MO).

Western Blot Analysis of PKC Isoforms-- NCI-H295 cells were harvested in Laemmli's sample buffer (17), and tissues were homogenized in the same buffer. Samples were passed through a 26-gauge needle, heated in boiling water for 10 min, and centrifuged (12,000 × g) for 10 min. Triton X-100-soluble and -insoluble fractions of NCI-H295 cells or hamster adrenal zona glomerulosa were prepared according to the technique described by Garcia-Paramio et al. (12). Thin human adrenal slices were obtained using a Stadie-Riggs tissue slicer (Thomas Scientific, St. Laurent, Quebec, Canada). The first slices consisted of the adrenal capsule and the zona glomerulosa, and the next slices consisted of the zona fasciculata (18). Contamination of the zona glomerulosa by the zona fasciculata was estimated to be ~20%, based on P450 17alpha -hydroxylase expression, as determined by Western blotting analysis, using the same quantity of proteins from each zone preparation (data not shown). Proteins were separated by electrophoresis on 7.5% polyacrylamide gel and analyzed by Western blotting as described previously (19, 20) using the following polyclonal antibodies: anti-rabbit PKCalpha , anti-rabbit PKCbeta 1 (Sigma-Aldrich), anti-rabbit PKCbeta 2, anti-mouse PKCgamma , anti-rabbit PKCepsilon , and anti-rabbit PKCzeta (Sigma-Aldrich), anti-mouse PKCdelta and anti-mouse PKCtheta (Transduction Laboratories, Lexington, KY), and anti-rabbit PKClambda (N-17) (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Immunoreactive proteins were detected using ECL+ light emitting reagents (Amersham Pharmacia Biotech UK Ltd, Little Chalfont, Buckinghamshire, United Kingdom). Autoradiograms were observed by exposing the blots to Kodak X-Omat RP films.

In Situ Detection of PKC Isoforms-- Adrenal glands were excised from three different animals. Human adult adrenal glands were also used. The glands were fixed in buffered neutral formalin solution for 24 h. The fixed adrenal glands were dehydrated by successive treatments with graded aqueous ethanol, cleared in toluene, and embedded in paraffin. Five to seven 5-µm-thick sections were prepared according to the usual histologic procedures. Sections were freed of paraffin, hydrated, and treated with NH4Cl in 50 mM PBS (20 min) to block aldehydic groups. After two washes, tissue sections were incubated for 2 h at room temperature with the respective anti-PKCalpha , -epsilon , or -zeta antibodies (diluted in PBS containing 1% bovine serum albumin), and then washed twice. Tissues sections were then incubated for 30 min with a fluorescein-conjugated goat anti-rabbit IgG (Roche Molecular Biochemicals, Laval, Quebec, Canada) diluted 1/50, washed in PBS for 5 min, and then mounted in glycerol-PBS (9:1) containing 0.1% phenylenediamine (21). Control sections were incubated with the second antibody only. Paraffin-embedded sections were examined with a Reichert Polyvar 2 microscope equipped for epifluorescence.

RNA Analysis-- Total RNA was isolated from NCI-H295 cells using RNeasy® (Qiagen Inc., Mississauga, Ontario, Canada) and treated with DNase I. Two hundred ng total RNA was reversed transcribed and amplified using a Titan One Tube RT-PCR Kit (Roche Diagnostics, Laval, Quebec, Canada) following the manufacturer's protocol. A 392-base pair (bp) fragment corresponding to exons 1 and 2 of P450C11 and P450aldo was amplified using the following primers: 5'-ATGGCACTCAGGGCAAAGGCA-3' (sense) and 5'-CAAGAACACGCCACATTTGTGC-3' (antisense) (7). The PCR products were then digested with BglI (P450aldo-specific) and separated on a 2% agarose gel containing ethidium bromide. GAPDH was used as an internal control; a 239-bp fragment containing a BglI restriction site was amplified using 5'-CATCCTGGGCTACACTGAGC-3' (sense) and 5'-TCTCTCTTCCTCTTGTGCTC-3' (antisense) primers. The BglI digestion distinguished P450C11 from P450aldo by cleaving the 392-bp P450aldo fragment to 307- and 85-bp fragments; the conversion of the GAPDH 239-bp fragment to 176- and 63-bp fragments demonstrates complete BglI digestion. The marker pBR322 DNA-MspI digest was purchased from New England Biolabs Inc. (Beverly, MA).

Generation of PKC Mutants-- CA- and DN-PKC mutants were engineered by point mutation using a PCR technique (22). In the case of the CA mutants, a specific alanine residue in the pseudo-substrate region was exchanged for a glutamic acid residue (PKCalpha A25E, PKCepsilon A159E, PKCtheta A148E, PKCzeta A119E). The pseudo-substrate region refers to the consensus sequence XRXX(S/T)XRX found in the phosphorylation sites of prominent PKC substrates. However, in the pseudo-substrate region of PKC, one alanine residue replaces a serine or threonine residue normally found in the substrate motif. It is generally thought that the pseudo-substrate region interacts with the catalytic (substrate binding) site of PKC and maintains the enzyme in an inactive state (23). In the case of the DN mutants, a critical lysine in the 5'-(pyro)-triphosphates of adenosine binding site was exchanged for an alanine residue (PKCalpha K368A, PKCepsilon 75 437A, PKCtheta K409A, PKCzeta K281A) (24, 25). The pcDNA3 vector (Invitrogen, Carlsbad, CA) was used as the expression vector.

Deletion Plasmids of the CYP11B2 Promoter-- The modified pCAT basic vector (Promega, Madison, WI) was used to construct CYP11B2 gene deletion plasmids as we have described previously (3, 4). The modifications consisted of the removal of an AP-1 consensus sequence and of an AP-1 like sequence. In this study -83-, -102-, -134-, -167-, -328-, -350-, -486-, and -2600-CYP11B2-CAT deletion plasmids were used (4). The sequence 5'-CAGGGACAGC-3' of the -102-CYP11B2 deletion plasmid was changed to 5'-tggtacccat-3' to produce the -102 mutant (mut-102).

Cell Cultures-- NCI-H295 cells were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium (1:1) supplemented with 1% ITS Premix (Becton Dickinson Labware, Bedford, MA), 2% UtroSer SF (Bio Sepra S.A., Villeneuve la Garenne, France), 200 µg/ml streptomycin, and 200 units/ml penicillin G. The cells were grown at 37 °C under 95% air, 5% CO2.

Transient Transfections and CAT Assays-- Transient transfections were performed in NCI-H295 cells as described previously (4). NCI-H295 cells (5 × 105) were seeded into six-well tissue culture plates and grown until 80-90% confluence. The cells were transiently transfected for 5 h with plasmid constructions using the SuperFect transfection reagent (Qiagen), according to the manufacturer's protocol. After transfection, the cells were incubated with medium containing 2% UtroSer SF for 16 h to allow recovery and expression of foreign DNA. Media were changed to low serum media (Dulbecco's modified Eagle's medium/Ham's F-12 containing 0.1% UtroSer SF), and the cells were incubated for 16 h in the presence/absence of AII (10-7 M), KCl (16 mM), Bt2cAMP (1 mM), or TPA (0.16 µM). Cell lysates were prepared by freeze-thawing, and CAT assays were performed using [14C]chloramphenicol as described previously (4). In experiments where CAT activity was too high, the assays were performed with smaller amounts of cell proteins to keep the rate of acetylation linear. Quantification was done using a Storm 860 laser scanner instrument (Molecular Dynamics, Sunnyvale, CA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Immunodetection of PKC-- Western blotting analysis was used to determine which PKC isoforms were expressed in hamster adrenal glands and in NCI-H295 cells. Adrenal glands of humans and rats were also included in our analysis to determine the similarity of PKC expression among species. Rat brain preparations were used as control (Fig. 1, left lane). Results showed that the PKCalpha , -epsilon , and -zeta isoforms were present in hamster adrenal inner zones and zona glomerulosa, whereas PKCbeta 1, -beta 2, -gamma , -theta , -lambda , and delta  were absent. PKCalpha , -epsilon , -theta , and -zeta and low levels of PKClambda were detected in NCI-H295 cells, whereas PKCbeta 1, -beta 2, -gamma , and -delta were absent. PKCalpha , -epsilon , and -zeta were revealed in rat zona glomerulosa and inner zones, in human zona glomerulosa and zona fasciculata-reticularis. Very low levels of PKClambda and -theta were also detected in adult human adrenal preparations. PKCdelta was present in the human adrenal zona fasciculata and in very low levels in rat brain. The ubiquitous presence of PKCalpha , -epsilon , and -zeta in the adrenal cortex of species studied is indicative of their physiological importance in this tissue. Indirect immunofluorescence was also used to investigate the expression of PKC isozymes in paraffin sections of the hamster and the human adrenal cortex. Results showed (Fig. 2) that PKCalpha , -epsilon , and -zeta were present in hamster and human adrenal zona glomerulosa and, to a lesser extent, in the zona fasciculata.



View larger version (84K):
[in this window]
[in a new window]
 
Fig. 1.   Immunoblot of adrenal gland preparations probed with anti-PKC antibodies. The zona glomerulosa (ZG) of rat and hamster (Ham) adrenal glands were separated from inner zones (IZ) containing the medulla. Adult human adrenal zona glomerulosa and zona fasciculata (ZF), NCI-H295 cells (C), and rat brain (Rb) were also used. Tissues were homogenized, and 50 µg of proteins of each preparation were used for immunodetection with antibodies directed against PKCalpha , -beta 1, -beta 2, -gamma , -delta , -epsilon , -theta , -lambda , and -zeta , respectively.



View larger version (177K):
[in this window]
[in a new window]
 
Fig. 2.   Indirect immunofluorescence detection of PKC in micrographs of paraffin sections of hamster and human adrenal glands. A, the fluorescein-conjugated antibody revealed PKCalpha in the zona glomerulosa and in some cells of the zona fasciculata. B, PKCepsilon was revealed in zona glomerulosa and in some cells of the zona fasciculata. C, PKCzeta was revealed in the zona glomerulosa and in some cells of the zona fasciculata. Hum, human; Ham, hamster.

We have analyzed the activation state of PKC in NCI-H295 cells and in hamster adrenal zona glomerulosa. Activated PKCs associate with cellular components and their distribution can be revealed in detergent Triton X-100-insoluble fraction (26). Furthermore, activation of PKC isoforms makes them susceptible to dephosphorylation and degradation (27). In NCI-H295 cells, PKCtheta and -epsilon isoforms were found in the Triton X-100-soluble (nonactivated) fraction whereas the alpha  and zeta  isoforms were distributed in the Triton X-100-soluble and -insoluble fractions (Fig. 3). Similarly, in hamster adrenal zona glomerulosa, PKCalpha was also found in both the Triton X-100-soluble and -insoluble fraction. Furthermore, PKCepsilon and -zeta were detected in the detergent-insoluble fraction. These data suggest that, under basal conditions, PKCalpha , -epsilon , and -zeta are activated in hamster adrenal zona glomerulosa and that PKCalpha and -zeta are also activated in NCI-H295 cells. In contrast, PKCepsilon and -theta were found to be mostly associated with the Triton X-100-soluble fraction in NCI-H295 cells and therefore presumably nonactivated.



View larger version (64K):
[in this window]
[in a new window]
 
Fig. 3.   Determination of the activation state of PKCs in NCI-H295 cells and in hamster adrenal zona glomerulosa. NCI-H295 (H295) cells and hamster adrenal zona glomerulosa were fractionated into Triton X-100-soluble (S) and Triton X-100-insoluble (I) fractions. Equivalent samples of each fraction were separated by electrophoresis and then subjected to immunoblot assays with antibodies specific to each PKC. Rat brain (Rb) was used as control.

TPA Inhibition of P450aldo mRNA Expression in NCI-H295 Cells-- The presence of phorbol ester/diacylglycerol (DAG)-sensitive PKC in NCI-H295 cells led us to investigate the effect of TPA on the P450aldo mRNA expression. This was done by reverse transcriptase-PCR performed as described under "Experimental Procedures." Fig. 4A shows the PCR-amplified products. BglI digestion distinguished P450aldo from P450C11 bands, and the complete BglI digestion was demonstrated by GAPDH (Fig. 4, B and C). TPA inhibited basal and stimulated P450aldo mRNA expression by AII, Bt2cAMP, or KCl. Although TPA inhibited AII- and KCl-stimulated P450C11 mRNA expression, it did not inhibit its basal or Bt2cAMP-stimulated expression.



View larger version (110K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of TPA on the mRNA levels of P450 11beta -hydroxylase (C11) and P450 aldosterone synthase (AS) in NCI-H295 cells. NCI-H295 cells were incubated for 16 h without (control, Ctr) or with AII (10-7 M), Bt2cAMP (1 mM) or KCl (16 mM), in the absence (-) or presence (+) of TPA (0.16 µM). Two hundred ng of total RNA were reverse transcribed using specific primers to both P450C11, P450aldo, and to GAPDH, and PCR-amplified for 30 cycles. A, the PCR products were separated through a 2% agarose gel containing ethidium bromide. B and C, the PCR products from two independent experiments were digested with BglI. M corresponds to DNA markers of indicated molecular sizes.

Effect of TPA on the Hamster CYP11B2 Promoter Activity in Transfected NCI-H295 Cells-- NCI-H295 cells transiently transfected with the hamster -2600-CYP11B2-CAT deletion plasmid were exposed to TPA and CAT activity determined as before (4). Results showed (Fig. 5) that TPA decreased CAT activity. Importantly, TPA abolished the stimulatory effects of AII, Bt2cAMP, or KCl, suggesting that activation of phorbol ester/DAG-sensitive PKC has a negative effect on the regulation of the CYP11B2 promoter activity. The KCl concentration used for stimulation in the above experiments was 16 mM. Although less efficient, lower concentrations of KCl (4-7 mM) were still able to stimulate the promoter activity (data not shown).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of TPA on the hamster CYP11B2 promoter activity. Transfected NCI-H295 cells with the hamster -2600-CYP11B2-CAT deletion plasmid were incubated with AII (10-7 M), TPA (0.16 µM), Bt2cAMP (cAMP) (1 mM), or KCl (16 mM). Incubations in the presence of either AII and TPA, cAMP and TPA, or KCl and TPA were also done. Results shown are measurements of three different experiments (mean ± S.E.) performed in triplicate.

Effect of Gö6976 on the Hamster CYP11B2 Promoter Activity in Transfected NCI-H295 Cells-- The inhibitor Gö6976 specifically blocks the action of conventional PKCs (28). This characteristic was used to discriminate between the action of the unique conventional PKCalpha and that of other DAG-dependent PKCs expressed in hamster adrenal and NCI-H295 cells. At 10-6 M Gö6976 enhanced CAT activity (percentage of acetylation: mean ± S.E., control 8.43 ± 1.37; Gö6976: 16.51 ± 1.43, n = 3, performed in triplicate). These observations provided further evidence that PKCalpha was involved in suppression of the hamster CYP11B2 promoter activity.

Effect of CA-PKCalpha and DN-PKCalpha Mutants on the CYP11B2 Promoter Activity in Transfected NCI-H295 Cells-- To confirm the suppressive effect of PKCalpha on the expression of CYP11B2, cotransfection experiments were performed using CA-PKCalpha and DN-PKCalpha mutants. Results indicated that CYP11B2 promoter activity was inhibited when cotransfected with CA-PKCalpha (Fig. 6A). The inhibition was also observed when the cells had been transfected with 0.5 or 1 µg of PKCalpha -containing plasmid (Fig. 7). Of interest, expression of CA-PKCalpha also inhibited, in a plasmid concentration-dependent manner, the stimulatory effect of AII (Fig. 7). In contrast, expression of DN-PKCalpha enhanced promoter activity (Fig. 6A).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of CA- and DN-PKC mutants on the basal CYP11B2 promoter activity. NCI-H295 cells were cotransfected with the hamster -2600-CYP11B2-CAT deletion plasmid and pcDNA3 or cotransfected with pcDNA3 harboring different mutated PKC DNAs. Results shown are mean ± S.E. of three to five experiments performed in triplicate (n = 5 for CA-alpha , DN-alpha , DN-zeta ; n = 4 for CA-epsilon , DN-epsilon , DN-theta ; n = 3 for CA-theta and CA-zeta ). A: PKCalpha and PKCepsilon mutants; B: PKCtheta and PKCzeta mutants.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 7.   CA-PKCalpha decreases the hamster -2600-CYP11B2-CAT expression in transfected NCI-H295 cells. NCI-H295 cells were cotransfected with 2 µg of the hamster -2600-CYP11B2-CAT deletion plasmid and 1 µg of pcDNA3 per well (0). In other wells, increasing quantities (0.05-1 µg) of pcDNA3 harboring the mutated CA-PKCalpha DNA was substituted to equivalent quantities of pcDNA3. Results shown are measurements of three different samples (mean ± S.E.). Filled columns, treated angiotensin II (10-7 M).

CA-PKCalpha also abolished the stimulatory effects of KCl and Bt2cAMP on CAT activity (Fig. 8A), whereas DN-PKCalpha increased the basal promoter activity (Fig. 6A) and did not prevent the stimulatory effects of AII, KCl and Bt2cAMP (Fig. 8A). The fact that the DN mutant enhanced rather than decreased these effects adds weight to the hypothesis that PKCalpha suppresses the hamster CYP11B2 promoter activity.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 8.   Effects of CA- and DN-PKC mutants on the CYP11B2 promoter activity in transfected NCI-H295 cells stimulated by AII, Bt2cAMP, or KCl. NCI-H295 cells were cotransfected with the hamster -2600-CYP11B2-CAT deletion plasmid and pcDNA3 or pcDNA3 harboring CA-PKCalpha or DN-PKCalpha DNAs (A) or pcDNA3 or pcDNA3 harboring CA-PKCepsilon or DN-PKCepsilon DNAs (B). C, pcDNA3 or pcDNA3 harboring CA-PKCzeta or DN-PKC zeta  DNAs. Cells were incubated for 16 h with AII (10-7 M), Bt2cAMP (cAMP) (1 mM), or KCl (16 mM). Results are mean ± S.E. of six independent experiments performed in triplicate for cells cotransfected with pcDNA3 and three experiments for cells cotransfected with PKCs.

Effect of CA-PKCepsilon and DN-PKCepsilon Mutants on the CYP11B2 Promoter Activity in Transfected NCI-H295 Cells-- The fact that PKCepsilon was present in NCI-H295 cells and, to a lesser extent in hamster adrenal zona glomerulosa (Figs. 1 and 2), led us to address whether this PKC plays a role on the hamster CYP11B2 promoter activity in cotransfected NCI-H295 cells. We found that CA-PKCepsilon had an inhibitory effect on CAT activity (Figs. 6A and 8B) similar to that of the CA-PKCalpha mutant. In contrast, DN-PKCepsilon had a stimulatory effect (Figs. 6A and 8B). Moreover, the stimulatory effects of AII, KCl, and Bt2cAMP were blocked in cells transfected with CA-PKCepsilon but not with DN-PKCepsilon (Fig. 8B). These results indicated that PKCepsilon could also be involved in the regulation of the CYP11B2 promoter activity in NCI-H295 cells.

Effect of CA-PKCzeta and DN-PKCzeta Mutants on the CYP11B2 Promoter Activity in Transfected NCI-H295 Cells Stimulated by AII, KCl, and Bt2cAMP-- The role of atypical PKCzeta , which is expressed in the hamster adrenal zona glomerulosa and NCI-H295 cells (Fig. 1), on the hamster CYP11B2 promoter activity was investigated in the next series of experiments. In contrast to results obtained with CA-PKCalpha , CA-PKCzeta stimulated the CYP11B2 promoter basal activity (Figs. 6B and 8C). In addition, cell stimulation with AII or KCl were not abolished in cotransfection experiments with CA-PKCzeta ; the effect of Bt2cAMP, however, was abolished. DN-PKCzeta increased the basal activity of the promoter and the stimulatory effects of AII, KCl, and Bt2cAMP were observed (Figs. 6B and 8C).

Cellular Distribution of CA-PKCalpha and DN-PKCalpha -- The aim of the next series of experiments was to obtain evidence of the state of activation of PKCalpha , CA-PKCalpha , and DN-PKCalpha by comparing their distribution in Triton X-100-soluble and -insoluble fractions in transfected NCI-H295 cells stimulated by AII or TPA. Fig. 9 showed that wild type PKCalpha was mainly present in the soluble fraction whereas faint protein bands were found in the insoluble fraction. The pattern of PKCalpha distribution was not altered when cells were exposed to AII. In contrast, TPA induced an increase of PKCalpha in the insoluble fraction. Although speculative, the slow and fast moving bands observed in the soluble and insoluble fractions are presumably phosphorylated and dephosphorylated protein, respectively, which is in agreement with results reported by Garcia-Paramio et al. (12). Marked differences were observed with preparations of cells transfected with CA-PKCalpha . In a situation where CAT activity was inhibited, the insoluble fraction of these cells contained large amounts of immunoreactive protein migrating faster than the single band detected in the soluble fraction, suggesting a translocation of PKCalpha within the cell and presumably dephosphorylation of the protein. The presence of AII did not change this profile. A similar profile was observed with TPA except for the appearance of a small fast migrating band in the soluble fraction. Fig. 9 also shows results obtained in the case of the DN form of PKCalpha in a situation where CAT activity was not inhibited. Without treatment, the main immunoreactive band found was a slow migrating protein in the soluble fraction. When the cells were treated with AII or TPA, a second fast migrating band was revealed in the soluble and in the insoluble fractions in addition to the main upper band. These data suggested that, upon stimulation, this mutant PKC was able to translocate and was presumably dephosphorylated.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 9.   Detergent analysis of the distribution of wild type (Wt) and PKCalpha mutants in stimulated transfected NCI-H295 cells. NCI-H295 cells transfected with wild type PKCalpha , CA-PKCalpha , or DN-PKCalpha were fractionated into Triton X-100-insoluble (I) and -soluble (S) fractions. Cells were incubated without (Ctr) or with TPA (0.16 µM) or AII (10-7 M). Equivalent amounts of proteins from each fraction were separated by electrophoresis. PKCalpha was analyzed by Western blotting.

Effect of TPA on the Activity of Different Deletion CYP11B2 Plasmids-- Eight different deletion plasmids were used (listed under "Experimental Procedures") to locate the promoter region responsible for TPA inhibition of the hamster promoter CYP11B2 gene. The basal, AII-, KCl-, and Bt2cAMP-stimulated activity profiles of the different deletion plasmids were similar to those reported previously by us (4). TPA slightly inhibited the enhancing effects of AII, KCl, and Bt2cAMP on the activity of the -83 deletion plasmid. However, the enhancing effects of AII, KCl, and Bt2cAMP on the activity of the -102 and longer CYP11B2 deletion plasmids was abolished by TPA (Fig. 10).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 10.   TPA inhibition of the hamster CYP11B2 promoter activity stimulated by AII, Bt2cAMP, and KCl. NCI-H295 cells were transfected with eight different (from -83- to-2600)-CYP11B2-CAT deletion plasmids. Transfected cells were incubated with AII (10-7 M), KCl (16 mM), or Bt2cAMP (cAMP) (1 mM) alone or combined with TPA (0.16 µM). Data are measurements of three different samples (mean ± S.E.).

Effect of CA-PKCalpha , -epsilon , -theta , and -zeta on the Activity of Different Deletion CYP11B2 Plasmids-- CA-PKCalpha , -epsilon , and -theta were able to prevent the enhancing effects of AII, KCl, and Bt2cAMP on the activity of the -102-CYP11B2 deletion plasmid (Fig. 11A) in cotransfection experiments, as observed in the case of TPA-treated cells. In contrast, the enhancing effect of CA-PKCzeta on the basal promoter activity was observed in cotransfection experiments using -102-CYP11B2 (Fig. 11B). In addition, promoter stimulation with AII or KCl was not abolished in cotransfection experiments with CA-PKCzeta , whereas the effect of Bt2cAMP was prevented.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 11.   Inhibition of the -102-CYP11B2 promoter activity by the constitutively active PKC mutants. NCI-H295 cells were cotransfected with the hamster -102-CYP11B2-CAT deletion plasmid and pcDNA3 or pcDNA3 harboring CA-PKCalpha , CA-PKCepsilon , CA-PKCtheta , or CA-PKCzeta DNAs. Cells were incubated for 16 h with or without AII (10-7 M), KCl (16 mM), or Bt2cAMP (cAMP) (1 mM). Results shown are (mean ± S.E.) from one experiment (A) and two experiments (B) performed in triplicate. A: DAG-dependent PKC-alpha , epsilon , and theta ; B: atypical PKCzeta .

Mutation of the Ad2 cis-Element-- Bases -95 to -86 of the Ad2 cis-element (3) in the hamster -102-CYP11B2-CAT deletion plasmid, were all mutated (mut-102). As shown in Fig. 12, this mutation resulted in the decrease of basal and stimulated promoter activities. The effect of Bt2cAMP and AII on mut-102 were partially inhibited by TPA while that of KCl was completely inhibited. TPA completely abolished the stimulatory effect of the three regulators on wild type -102 deletion plasmid (Fig. 12).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 12.   Effect of mutating the Ad2-cis-element on the CYP11B2 promoter activity. NCI-H295 cells were transfected with the hamster -102-CYP11B2-CAT (-102) or the -102-mutated CYP11B2-CAT deletion plasmid (mut-102); bases -95 to -86 comprised in the Ad2 cis-element were all mutated. Cells were incubated for 16 h without (control, Ctr) or with AII (10-7 M), Bt2cAMP (cAMP) (1 mM), or KCl (16 mM) in the absence or presence of TPA (0.16 µM). Data shown are measurements of three different samples (mean ± S.E.).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies have shown that PKC are involved in the control of several adrenal steroidogenic genes (4-10). Evidence was derived from experiments using PKC activators, phorbol esters, and various PKC inhibitors. The present work was designed to determine which PKCs were involved in the control of the hamster CYP11B2 gene expression. We report that DAG-dependent PKC can suppress whereas the atypical PKCzeta can enhance the basal expression of this gene in transfected NCI-H295 cells.

High levels of expression of PKCalpha , low levels of PKCzeta , and very low levels of PKCepsilon were detected (Western blotting) in the hamster adrenal zona glomerulosa. Indirect immunofluorescence analysis confirmed the presence of PKCalpha , -zeta , and -epsilon in the hamster and human adrenal zona glomerulosa and in lower levels in the zona fasciculata. In comparison, these isoforms were found in the zona glomerulosa of rat and in NCI-H295 cells. Our results are in agreement with previous reports indicating that the rat adrenal zona glomerulosa expressed PKCalpha and -epsilon (28). PKC isoforms alpha , epsilon , and zeta  were also commonly expressed in the human adrenocortical NCI-H295 cell line (Fig. 1) and in the mouse adrenal cell line Y-1 (15). The expression of PKCalpha , -zeta , and -epsilon in the zona glomerulosa of all adrenal tissues studied is indicative of their specificity and functional importance in this zone. Moreover, the fact that under basal conditions PKCalpha , -epsilon , and -zeta were partially activated (as determined by their presence in the Triton X-100-insoluble fraction) in hamster adrenal zona glomerulosa was also indicative of their functional importance in these adrenocortical cells.

We have shown for the first time that the PKC activator TPA inhibited basal, AII-, Bt2cAMP-, and KCl-stimulated P450aldo mRNA level in NCI-H295 cells, thus validating this model for studying hamster CYP11B2 promoter activity. We also observed that TPA did not inhibit basal and cAMP-stimulated P450C11 mRNA levels, suggesting a differential control of mRNA expression of the CYP11B1 and CYP11B2 genes by phorbol ester/DAG-sensitive PKC in NCI-H295 cells.

In the present study, we have concentrated our efforts to elucidate the role of the three PKC isoforms alpha , epsilon , and zeta  present in the hamster adrenal zona glomerulosa, on the expression of the hamster CYP11B2 promoter activity. We have found that the PKC activator TPA inhibited the enhanced expression of the hamster CYP11B2 by AII, Bt2cAMP, or KCl, suggesting that phorbol ester/DAG-dependent PKC were involved in inhibition of the promoter activity.

We have used a combined approach to discriminate between the role of PKCalpha and -epsilon , present in hamster zona glomerulosa and in NCI-H295 cells, and of PKCtheta also present in NCI-H295 cells. The specific inhibitory action of Gö6976 on the classical PKC isoforms (28) resulted in stimulation of promoter activity. These observations led us to conclude that PKCalpha was responsible for inhibition of promoter activity since this PKC isoform is the only member of conventional PKC found in the zona glomerulosa and in NCI-H295 cells.

Additional evidence for the inhibitory action of phorbol ester/DAG-dependent PKC on the expression of the hamster CYP11B2 promoter activity was obtained in cotransfection experiments with CA-PKCalpha and DN-PKCalpha mutants. The data clearly showed not only an inhibition of promoter activity by the constitutively active mutants but also a blockade of the stimulatory effects of AII, KCl, or Bt2cAMP. As expected, inactive DN-PKCalpha isoform, showed an absence of inhibitory effect. These results were consistent with the interpretation that the inhibitory action of endogenous PKCalpha could be reversed by overexpression of the DN mutant form of PKCalpha . A dose dependence of the effects of the CA-PKCalpha expression vector on the promoter activity showed that inhibition was maximal at a concentration of 1 µg of transfected DNA. Dose-response studies were not done in the case of other PKCs since, at this concentration of transfected DNA, CA-PKCepsilon and CA-PKCtheta efficiently blocked the CYP11B2 promoter activity. It remains to be seen whether, in the case of these two mutant PKCs, a lower concentration of transfected DNA would be as effective.

The fact that PKCtheta was not detected in hamster adrenal glands rules out any interference between the PKCalpha and -theta isoforms in regulating CYP11B2 promoter activity in hamster adrenal zona glomerulosa. It cannot be excluded however, that PKCtheta could play a role in NCI-H295cells. To test this possibility we have overexpressed the CA-PKCtheta and DN-PKCtheta mutants in NCI-H295 cells. We found that the CA but not the DN mutant had an inhibitory effect on CAT activity similar to that of CA-PKCalpha . Moreover, the stimulatory effects of AII, KCl, and Bt2cAMP were all blocked when CA-PKCtheta was overexpressed (data not shown). It cannot be excluded that endogenous PKCtheta could have interfere and/or participate in the control of the hamster CYP11B2 expression in transfected NCI-H295 cells.

Although PKCepsilon is present in very low levels in hamster adrenal zona glomerulosa and in NCI-H295 cells, its role on CYP112B2 expression remains to be elucidated. It is clear, however, that, when highly expressed, CA-PKCepsilon but not DN-PKCepsilon was able to inhibit CYP11B2 promoter activity and block the stimulatory effects of AII, KCl, and Bt2cAMP (Fig. 8B). It is therefore possible that, in hamster adrenal zona glomerulosa and NCI-H295 cells, endogenous PKCepsilon is involved in CYP11B2 gene expression. Garcia-Paramio et al. (12) have reported a lack of specificity of DN mutants of PKCalpha and -epsilon . Both mutants were able to attenuate the effect of CA-PKCalpha on the TPA responsive element-luciferase activity and to inhibit the normal accumulation of a fully phosphorylated PKCalpha in cotransfected COS-1 cells. Although low levels of PKCepsilon were found in NCI-H295 cells and in hamster adrenal glands, the degree of cross-talk between PKCepsilon and -alpha could not be evaluated until the turnover of PKCepsilon was assessed.

In contrast to the results on the action of DAG-dependent PKC, data obtained in the case of cotransfection experiments using PKCzeta mutants indicated that this PKC isoform did not play any negative regulatory role on the basal expression of hamster adrenal CYP11B2. CA-PKC zeta , however, did not prevent the enhancing effects of AII and KCl on promoter activity but inhibited the effect of Bt2cAMP, suggesting additional differential regulation between DAG-dependent PKC and atypical PKCzeta .

In agreement with our results, Reyland et al. (15) have reported that steroidogenesis in Y-1 cells inversely correlates with total PKC activity and with the expression of PKCalpha but not with PKCepsilon or -zeta . Induction of PKCalpha expression in these cells resulted in decreased cytochrome P450 side chain cleavage activity and reduced transcription of a cytochrome P450 side-chain cleavage promoter-luciferase construct. However, the same authors found no effects on cAMP induction of steroidogenesis, indicating that these pathways function independently to regulate steroidogenesis. In contrast, our study showed that the overexpression of the constitutively active PKCalpha inhibited the enhancing effect of Bt2cAMP on the CYP11B2 promoter activity. These observations suggest that genes involved in the regulation of the first step of steroidogenesis, the transformation of cholesterol to pregnenolone, and in the last step of aldosterone synthesis, the transformation of deoxycorticosterone to aldosterone, may be differently regulated by cAMP.

The fact that TPA and CA-PKCalpha , -epsilon , and -theta blocked the enhancing effect of AII on the CYP11B2 promoter activity indicated that AII did not act through the activation of DAG-dependent PKC. This finding is in agreement with the conclusion of Nakano et al. (30), who reported a full aldosterone response to AII despite marked cellular PKC depletion in rat adrenal zona glomerulosa cells after prolonged preincubation with TPA. Our results are also in agreement with those of Hajnóczky et al. (11), who showed that, regardless of whether PKC is activated by phorbol esters or physiological stimuli such as AII or K+, it exerts an inhibitory action on steroid production in rat adrenal zona glomerulosa cells. Therefore, the mode of action of stimuli enhancing CYP11B2 expression in adrenal zona glomerulosa remains to be fully understood. However, in bovine adrenal zona glomerulosa cells, it has been recently reported that AII stimulated Raf-1 kinase activity through Ras, independently of PKC activation and elevation of intracellular calcium (31), thus opening a new avenue of investigation for the action of this hormone.

AII binding to AII receptor type 1 stimulates inositol phosphate and DAG formation, activates PKC, and enhances aldosterone secretion in adrenal zona glomerulosa cells. At first sight, it seems paradoxical that AII stimulates aldosterone biosynthesis while activating DAG-dependent PKC that inhibit CYP11B2 expression and therefore P450aldo synthesis. As an explanation to this apparent discrepancy, we suggest that AII exerts a positive action on CYP11B2 expression by other signaling pathway(s) yet to be discovered, and a negative action through the PKC signaling pathway. One has to speculate that the negative regulatory action would be delayed in comparison to the positive action so producing a feedback control to restore normal levels of CYP11B2 expression and to end the enhancing AII action.

Results of Western blotting experiments showed that activated PKCalpha translocated to the Triton X-100-insoluble cell fraction, where it was presumably dephosphorylated. This observation was in agreement with previous reports indicating that PKCalpha is constitutively phosphorylated, the protein being fully active albeit in a latent effector-dependent state (26). PKCalpha is dephosphorylated and inactivated in a desensitization process following activation and is then degraded (27). Our results showed that mutants CA-PKCalpha and DN-PKCalpha could be dephosphorylated following stimulation with TPA, suggesting that phosphorylation and dephosphorylation could occur for both mutants.

Different deletion plasmids of the CYP11B2 promoter were used in an attempt to identify the region for TPA-dependent PKC inhibition. TPA only partially prevented the enhancing effects of AII, KCl, and Bt2cAMP on the activity of the -83-CYP11B2-CAT deletion plasmid (containing a CRE cis-element (Ref. 4)) and completely blocked their actions on the activity of the -102-CYP11B2-CAT and on that of longer deletion plasmids. The inhibition by TPA on the -102 deletion plasmid was mimicked by cotransfection with CA-PKCalpha , -epsilon , or -theta mutants. In contrast, the CA-PKCzeta mutant had a stimulatory effect on the basal -102-CYP11B2-CAT deletion plasmid activity. The hamster 102-CYP11B2-CAT deletion plasmid contains a cis-element (Ad2 (Ref. 4)) conserved among CYP11B2 genes of rat, mouse, and human species (3). Mutation of bases -86 to -95 of the Ad2 cis-element in the -102-CYP11B2-CAT deletion plasmid dramatically decreased basal and AII-, Bt2cAMP-, and KCl-stimulated promoter activity (Fig. 12). Similar results were obtained when the same mutation was done in the -486-CYP11B2-CAT deletion plasmid (data not shown), thus emphasizing the importance of this cis-element in the control of the gene expression. Although TPA attenuated the stimulatory effect of AII and Bt2cAMP to a lesser extent than in wild type, we cannot draw a firm conclusion on the role of this cis-element in TPA action due to the very low promoter activity of the mut-102.

In conclusion, our findings suggest that the hamster CYP11B2 gene is under differential control by conventional (alpha ) and atypical (zeta ) PKC.


    ACKNOWLEDGEMENTS

We are indebted to Dr. W. E. Rainey for providing the NCI-H295 cells and to Dr. N. Gallo-Payet for supplying human adrenal glands. We also acknowledge the help of D. Martel.


    FOOTNOTES

* This work was supported in part by Medical Research Council of Canada Grant MT-10983 (to J.-G. L.) and a grant from the Heart and Stroke Foundation of Canada (to J.-G. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Chercheur Boursier de Carrière from the Fonds de la Recherche en Santé du Québec. To whom correspondence should be addressed.

Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M009495200


    ABBREVIATIONS

The abbreviations used are: AII, angiotensin II; PKC, protein kinase C; ACTH, adrenocorticotropin; Bt2cAMP, dibutyryl cyclic AMP; TPA, 12-O-tetradecanoylphorbol-13-acetate; DAG, diacylglycerol; P450aldo, cytochrome P450 aldosterone synthase; P450C11, cytochrome P450 11beta -hydroxylase; CA, constitutively active; DN, dominant negative; CAT, chloramphenicol acetyltransferase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. LeHoux, J. G., Bernard, H., Ducharme, L., Lefebvre, A., Shapcott, D., Tremblay, A., and Véronneau, S. (1996) Adv. Mol. Cell Biol. 14, 149-201
2. Véronneau, S., Bernard, H., Cloutier, M., Courtemanche, J., Ducharme, L., Lefebvre, A., Mason, J. I., and LeHoux, J. G. (1996) J. Steroid Biochem. Mol. Biol. 57, 125-139[CrossRef][Medline] [Order article via Infotrieve]
3. Coulombe, N., Lefebvre, A., and LeHoux, J. G. (1996) Endocr. Res. 22, 653-661[Medline] [Order article via Infotrieve]
4. LeHoux, J. G., and Lefebvre, A. (1998) J. Mol. Endocrinol. 20, 183-191[Abstract/Free Full Text]
5. Clyne, C. D., Zhang, Y., Slutsker, L., Mathis, J. M., White, P. C., and Rainey, W. E. (1997) Mol. Endocrinol. 11, 638-649[Abstract/Free Full Text]
6. Bird, I. M., Mathis, J. M., Mason, J. I., and Rainey, W. E. (1995) Endocrinology 136, 5677-5684[Abstract]
7. Staels, B., Hum, D. W., and Miller, W. L. (1993) Mol. Endocrinol. 7, 423-433[Abstract]
8. Bird, I. M., Mason, J. I., and Rainey, W. E. (1998) J. Clin. Endocrinol. Metab. 83, 1592-1597[Abstract/Free Full Text]
9. Bird, I. M., Mason, J. I., and Rainey, W. E. (1998) Endocrine Res. 24, 345-354[Medline] [Order article via Infotrieve]
10. Reyland, M. E. (1993) Mol. Endocrinol. 7, 1021-1030[Abstract]
11. Hanjnóczky, H., Várnai, P., Buday, L., Faragó, A., and Spät, A. (1992) Endocrinology 130, 2230-2236[Abstract]
12. Garcia-Paramio, P., Cabrerizo, Y., Bornancin, F., and Parker, P. J. (1998) Biochem. J. 333, 631-636[Medline] [Order article via Infotrieve]
13. Mellor, H., and Parker, P. J. (1998) Biochem. J. 332, 281-292[Medline] [Order article via Infotrieve]
14. Parekh, D. B., Ziegler, W., and Parker, J. P. (2000) EMBO J. 19, 496-503[Free Full Text]
15. Reyland, M. E., Williams, D. L., and White, E. K. (1998) Am. J. Physiol. 275, C780-C789[Abstract]
16. Giroud, C. J. P., Stachenko, J., and Venning, E. H. (1956) Proc. Soc. Exp. Biol. Med. 92, 154-158
17. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
18. Guillon, G., Trueba, M., Joubert, D., Grazzini, E., Chouinard, L., Côté, M., Payet, M. D., Manzoni, C. B., Robert, M., and Gallo-Payet, N. G. (1995) Endocrinology 136, 1285-1295[Abstract]
19. LeHoux, J. G., Lefebvre, A., Ducharme, L., LeHoux, J., Martel, D., and Brière, N. (1996) J. Endocrinol. 149, 341-349[Abstract]
20. LeHoux, J. G., Mason, J. I., and Ducharme, L. (1992) Endocrinology 131, 1874-1882[Abstract]
21. Calvert, R., Millane, G., and Beaulieu, J. F. (1994) Anat. Rec. 240, 358-366[Medline] [Order article via Infotrieve]
22. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve]
23. Pears, C. J., Kour, G., House, C., Kemp, B. E., and Parker, P. J. (1990) Eur. J. Biochem. 194, 89-94[Abstract]
24. Hug, H., and Sarre, T. F. (1993) Biochem. J. 291, 329-343[Medline] [Order article via Infotrieve]
25. Selbie, L. A., Schmitz-Peiffer, C., Sheng, Y., and Biden, T. J. (1993) J. Biol. Chem. 268, 24296-24302[Abstract/Free Full Text]
26. Whelan, R. D. H., and Parker, P. J. (1998) Oncogene 16, 1939-1944[CrossRef][Medline] [Order article via Infotrieve]
27. Hansra, G., Garcia-Paramio, P., Prevostel, C., Whelan, R. D. H., Bornancin, F., and Parker, P. J. (1999) Biochem. J. 342, 337-344[CrossRef][Medline] [Order article via Infotrieve]
28. Martiny-Baron, G., Kazanietz, M. G., Mischak, H., Blumberg, P. M., Kochs, G., Hug, H., Marmé, D., and Schächtele, C. (1993) J. Biol. Chem. 268, 9194-9197[Abstract/Free Full Text]
29. Natarajan, R., Lanting, L., Xu, L., and Nadler, J. (1994) Mol. Cell. Endocrinol. 101, 59-66[CrossRef][Medline] [Order article via Infotrieve]
30. Nakano, S., Carvallo, P., Rocco, S., and Aguilera, G. (1990) Endocrinology 126, 125-133[Abstract]
31. Smith, R. D, Baukal, A. J., Dent, P., and Catt, K. J. (1999) Endocrinology 140, 1385-1391[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.