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Steroid 21-hydroxylase expression and activity in human lymphocytes
Molecular and Cellular Endocrinology 127 (1997) 11 – 18
Steroid 21-hydroxylase expression and activity in human lymphocytes Zhifeng Zhou a, Veena R. Agarwal b, Naznin Dixit a, Perrin White c, Phyllis W. Speiser a,* a
Department of Pediatrics, North Shore Uni6ersity Hospital, New York Uni6ersity School of Medicine, 300 Community Dr., Manhasset, NY 11030, USA b Cecil H. and Ida Green Center for Reproducti6e Biology Sciences, The Uni6ersity of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235 -9051, USA c Department of Pediatrics, The Uni6ersity of Texas South-Western Medical Center at Dallas, Dallas, TX 75235 -9051, USA Received 24 July 1996; accepted 25 September 1996
Abstract Steroid 21-hydroxylase encoded by CYP21 is expressed in adrenal cortex. Mutations in CYP21 cause potentially lethal congenital adrenal hyperplasia (CAH). Earlier observations suggested alternative sources of 21-hydroxylase activity, although its genetic source remains unclear. We found a novel source of CYP21 expression in normal human cultured B lymphocytes. The quantity of 21-hydroxylase transcript was reduced in B cell lines of CAH subjects compared with that in normal B-lymphoblastoid cells. No CYP21 transcript was detected in lymphocytes from a CAH patient with homozygous CYP21 deletion. Cultured lymphoid cells, including those carrying homozygous CYP21 deletion, and peripheral blood leukocytes converted both 17-hydroxyprogesterone to 11-deoxycortisol and progesterone to deoxycorticosterone. We conclude that lymphocytes express CYP21, but also possess a 21-hydroxylase distinct from CYP21. Activity of this isozyme may partially compensate for severe adrenal 21-hydroxylase deficiency. © 1997 Elsevier Science Ireland Ltd. Keywords: Steroid 21-monooxygenase; Adrenal hyperplasia, Congenital; Isozymes; Lymphocytes; Phenotype
1. Introduction Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia (CAH). This enzyme deficiency disrupts cortisol and aldosterone production and indirectly promotes excess adrenal secretion of androgen precursors. Clinical severity of this form of CAH ranges from potentially lethal salt-wasting adrenal insufficiency and severe female genital ambiguity, to subclinical androgen excess [1]. Most phenotypic variation among 21-hydroxylase
Abbre6iations: CAH, congenital adrenal hyperplasia; CYP21, gene encoding steroid 21-hydroxylase; kb, kilobases; nt, nucleotide; RT, reverse transcription; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TLC, thin layer chromatography. * Corresponding author. Tel.: + 1 516 5624635; fax: + 1 516 5624029; e-mail: [email protected]
deficient patients results from allelism in CYP21 [2–4] although there are notable exceptions [2,5–7]. The most common genetic lesions in CYP21 encoding adrenal steroid 21-hydroxylase are deletions of 30 kb (encompassing the 3% end of the CYP21P pseudogene, all of C4B, and the first 5–7 exons of CYP21 producing a non-functional chimeric pseudogene) [8,9], or a single base change, nucleotide (nt) 656 A G. The latter mutation at position − 13 in the second intron causes activation of a novel splice acceptor at position −19, leading to retention of a portion of the intron and premature termination of translation [10]. These mutations cause severe or classic CAH with inadequate aldosterone synthesis and consequent salt-wasting. A point mutation in the fourth exon changes isoleucine172 to asparagine (N172) and is associated with classic simple virilizing (i.e., non-salt-wasting) CAH. Mild non-classic CAH is most often due to substitution of leucine for valine at codon 281 (V281L) [11].
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Since the early 1970s, it has been suspected that more than one 21-hydroxylase enzyme exists in humans [12]. In vivo experiments using radiolabeled 21-hydroxylase precursors showed that extra-adrenal 21-hydroxylation occurs with considerable inter-individual variability in healthy adults [13]. Subsequent ribonuclease protection studies with RNA probes ruled out CYP21 as the source of such extraadrenal 21-hydroxylase activity [14]. Further clinical studies in patients affected with salt-wasting CAH due to deletions and other CYP21 null mutations showed clear evidence of adrenal 21-hydroxylase activity, i.e., increased aldosterone synthesis with advancing age, which could not be attributed to CYP21 [5]. To date the isozyme(s) and gene(s) for this putative alternative human 21-hydroxylase have not been characterized. Although 21-hydroxylase activity has been identified [15] in a number of non-adrenal tissues, including lymphoid tissues such as thymic epithelium and spleen [16], hemodynamic considerations make it implausible that 21-hydroxylase in a tissue such as the kidney or gonad could compensate for a severe intra-adrenal 21hydroxylase deficiency [13]. Leukocytes, numerous both in the circulation and in tissues, could conceivably produce clinically detectable levels of 21- hydroxylated steroids in the presence of an active enzyme. We therefore examined CYP21 expression and 21-hydroxylase activity in lymphoid cells.
2. Materials and methods
2.1. Subjects and tissue sources B-lymphoblastoid cell lines were established from six patients diagnosed with 21-hydroxylase deficiency by clinical and hormonal criteria [17]. Genotyping was performed by Southern blotting in the case of the homozygous deletion [18], and otherwise by multiplex ligase detection reaction [19]. B-lymphoblastoid cell lines from normal individuals were selected at random from among reference samples in a tissue typing laboratory; HLA types of control specimens were not characteristically associated with classic or non-classic 21-hydroxylase deficiency (obtained courtesy of Drs B. Dupont and S.Y. Yang, Memorial-Sloan-Kettering Cancer Center). In view of the low prevalence of severe CYP21 mutations such as deletions ( 0.003) and the nt 656 G allele (0.006) [20] in the general population, control samples were not genotyped. Fresh peripheral blood leukocytes were obtained from four normal individuals. All human samples were obtained with informed consent and approval of the institutional review board.
2.2. RNA isolation Peripheral blood leukocytes were isolated as described [21]. B-lymphoblastoid cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin plus 100 mg/ml streptomycin, and 2 mM glutamine (Gibco, Grand Island, NY), until a cell count of about 2×108 cells was attained. RNA extraction was performed as described [22], and about 20 mg of poly(A) + RNA was isolated using oligo (dT) [PolyATract mRNA Isolation Systems, Promega, Madison, WI). RNA was extracted by the same method from snap-frozen normal adult human adrenal gland procured at nephrectomy.
2.3. Re6erse Transcriptase Polymerase Chain Reacton RNA (1 mg poly(A) + RNA from cultured cells, or 5 mg total adrenal RNA) was treated with DNase I (Gibco) at room temperature for 15 min to remove genomic DNA. The DNase was then inactivated by heating to 90°C for 5 min. RNA was denatured at 65°C for 2 min and annealed to random hexamers (Gibco) by slow cooling to 37°C. RNA was reverse-transcribed (RT) using Superscript II (Gibco) at 42°C for 1 h in the presence of 166 mM dNTP, 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, and 5 U RNasin (Promega). cDNA was amplified using the polymerase chain reaction (PCR) in the presence of 5 pmol of each primer, 200 mM dNTPs, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 0.001% (w/v) gelatin, 2U Taq polymerase (Perkin Elmer-Roche, Branchburg, NJ), 0.3 mCi [a-P 32 ]dCTP (Dupont-New England Nuclear, Boston, MA) in a total volume of 10 ml. Primers were synthesized using an Applied Biosystems DNA synthesizer Model 381A with reagents from the manufacturer. The 5% sense primer was 5%-ACCTGTCCTTGGGAGACTAC-3% representing nt 2373– 2393 of the CYP21 sequence in GenBank (underline shows area of 8 bp deletion in exon 3 of CYP21P conferring specificity of primer for CYP21 ), and the 3% antisense primer was 5%-TAGGCAGGCATTAAGTTGTCG-3% representing nt 2798–2819. PCR conditions consisted of 30 cycles of denaturation at 94°C for 30 s, followed by annealing at 60°C for 1 min and extension at 72°C for 1 min 30 s. A final 5 min extension was performed. All reactions were performed on a Gene Amp PCR System 9600 (Perkin–Elmer). The expected size of CYP21 PCR-amplified cDNA was 246 bp. Onefourth of PCR products were analyzed on 5% non-denaturing polyacrylamide gels alongside end-labeled molecular weight markers (Msp I-digested pBR322, New England Biolabs, Beverly, MA). Gels were autoradiographed with Kodak X-Omat film (Eastman Kodak, Rochester, NY), and quantitative analysis was done with a Bio-Rad Imaging System GS 363 and Molecular
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Fig. 1. (A) Expression of CYP21 transcripts in lymphoid cells. Reverse-transcription (RT) followed by polymerase chain reaction (PCR) specific for CYP21 was performed as described in text. Selective gene amplification was accomplished with CYP21 -specific primers. PCR products were sequenced, confirming the authenticity of amplified material. Samples in lanes 1 and 2 were derived from normal human lymphoblastoid cell lines; samples in lanes 3 – 8 were derived from CYP21 mutant CAH lymphoblastoid cell lines, respectively homozygous for the nt 656 G splice mutation (lanes 3 and 4), the leucine to valine mutation at codon 281, L281 (lanes 5 and 6), the asparagine to isoleucine mutation at codon 172, N172 (lane 7), and the 30 kb mutation including CYP21 (lane 8). Lanes 9 and 10 were derived from normal human breast and fibroblast cell lines, respectively. Lane 11 is from normal human adrenal tissue. Upper panel shows an amplified band at 246 bp specific for CYP21. Lower panel depicts results of separate RT-PCR amplification of a 306 bp GAPDH gene segment. The latter is a ubiquitously expressed gene used to standardize expression levels. (B) Relative quantities of CYP21 mRNA in cell lines compared with adrenal mRNA on an arbitrary scale measuring intensity of radio-labeled PCR-amplified bands. Numbering and lane identification is as noted above. (C) Comparison of CYP21 mRNA expression in cell lines vs. adrenal. Relative quantity is indicated in arbitrary units. The percent of the apparent level of adrenal gene expression is shown. This experiment was representative of three separate experiments which produced similar results.
Image software (Bio-Rad, Hercules, CA). Results shown in Fig. 1 are representative of three experiments which produced similar proportions of CYP21 transcripts. To check the integrity and comparative quantity of templates, the housekeeping gene, glyceraldehyde-3phosphate dehydrogenase (GAPDH) was chosen as an endogenous marker. Primers were designed from the GAPDH sequence in GenBank: (1) 5% end sense primer
5%-CGGAGTCAACGGATTTGGTCGTAT-3% and (2) 3% end antisense primer 5%AGCCTTCTCCATGGTGGTGAAGAC-3%. Template for GAPDH primers was the same as used for CYP21 amplification. PCR conditions were as described above, but GAPDH amplifications were performed separately from the CYP21 PCR reactions for 22 cycles. Expected size of these PCR products was 306 bp.
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2.4. Sequencing PCR-amplified putative CYP21 cDNA was directly sequenced to verify its identity. Double stranded PCR amplified templates were subjected to electrophoresis in 1.5% high melting temperature agarose (USB, Cleveland, OH) gels, and purified by means of a DNA gel extraction kit (Qiagen, La Jolla, CA). PCR primers were used as sequencing primers. DNA sequencing was performed with the Taq DyeDeoxy fluorescent dye terminator cycle sequencing kit (ABI, Foster City, CA). Products were loaded onto an ABI 373A sequencer according to the manufacturer’s instructions.
2.5. Lymphocytes’ 21 -hydroxylase acti6ity Approximately 106 cells were incubated for 24 h in the presence of 1 mCi tritiated 17-hydroxyprogesterone (Dupont-New England Nuclear, Boston, MA). For experiments involving progesterone, 2 × 106 cells were incubated with 5 mCi tritiated progesterone. All experiments were repeated three times; negative controls consisted of medium without cells incubated with radio-labeled steroid. After 24 h, cells were precipitated by centrifugation, and 1 nmol each of 21-hydroxylase substrate and product (Sigma, St. Louis, MO) were added to the cell-free medium in 2 ml ethanol. The medium was then extracted with 2 vol. of ethyl acetate. The dried organic phase was resuspended in 30 ml ethyl acetate, spotted on a silica gel plate (Sigma Polygram G/UV254), and resolved in chloroform: acetone (70:30). Radioactivity was measured by imaging autoradiograms of thin layer chromatography (TLC) plates (BioRad imaging system GS 363, Hercules, CA). Protein was assayed in parallel experiments as described [23]. Activity was calculated as picomoles of product/mg protein/h. To verify the authenticity of 21-hydroxylated steroid product detected on thin layer chromatography, specific radioimmunoassay for 11-deoxycortisol was performed on extracted cell medium from parallel incubations done with non-radioactive 17-hydroxyprogesterone (Endocrine Sciences Laboratory, Calabasas Hills, CA). Cross-reactivity with 17-hydroxyprogesterone is 2.5%, and less than 2% for other steroids.
3. Results
3.1. CYP21 expression Fig. 1 shows relatively prominent expression of CYP21 transcripts in human lymphoid cells studied by RT-PCR. The level of expression from normal Blymphoblastoid cells normalized by GAPDH expression was 10.4 and 16.9% (average about 13.5%) of the
level found in the normal human adrenal. In contrast, B-lymphoblastoid cells derived from CAH patients with non-deletional mutations showed lower levels of CYP21 expression (average9S.D., 2.391%, range 0.2–6%) compared with the level observed in the adrenal. There was no evidence of CYP21 expression in the cells derived from the individual with a homozygous deletion of CYP21 ; primers were designed to be specific for the deleted portion of CYP21. Normal human breast tissue also did not express CYP21 ; a low level of expression was detected in normal human fibroblasts (0.7% compared with adrenal). Performing PCR for 30 cycles maintained the reaction in the linear range and did not alter the observed CYP21 product ratios among the various samples. Performing 25 cycles proved suboptimal for product detection, and 40 cycles were supra-maximal. To exclude the possibility of exogenous CYP21 cDNA contamination in tissue-derived RNA, negative controls were designed omitting reverse transcriptase from RT reactions. By omitting RNA from the RT reactions, and by omitting cDNA from PCR reactions, the possibility of CYP21 cDNA contamination in RT or PCR reagents was excluded. All these negative controls showed no amplification of CYP21 (not shown), confirming that the CYP21 cDNA transcripts identified were derived from the tissues indicated. The identity of the amplified bands was confirmed to be CYP21 based on direct sequencing (not shown) of PCR products of appropriate size which co-migrated with a similar sized PCR product derived from adrenal cDNA.
3.2. Steroid 21 -hydroxylase acti6ity Conversion of 17-hydroxyprogesterone to 11-deoxycortisol was detected in cultured B-lymphoblastoid cells from normal individuals, from CAH patients homozygous for the CYP21 mutation at nt 656 G (splice mutation), patients homozygous for the leucine to valine mutation at codon 281 (L281), and a patient homozygous for deletion of CYP21 (Fig. 2). Similar activity was seen in peripheral blood leukocytes (not shown). Cultured human breast cells and fibroblasts showed 5–10-fold lower 21-hydroxylase activity. Negative controls in which no cells were added showed no enzyme activity. Conversion of progesterone to deoxycorticosterone could also be detected after incubation with a larger number of cells, and a higher concentration of radiolabeled progesterone (Fig. 3). The identities of the hormonal products of 21-hydroxylation were confirmed when these experiments were repeated using a different solvent system (chloroform:ethyl acetate, 80:20). Authenticity of 21-hydroxylated product was further confirmed by RIA detection of deoxycorticosterone in cell media extract, but not in control media lacking cells.
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Fig. 2. (A) Conversion of 17-hydroxyprogesterone to 11-deoxycortisol by lymphocytes. Thin layer chromatography was performed after incubating cells with tritiated 17-hydroxyprogesterone as described in text. Lanes 1 – 3 represent results in cultured normal human B-lymphoblastoid cells; lanes 4 – 6 represent cultured B-lymphoblastoid cells respectively derived from CAH patients homozygous for the L281 mutation, the nt 656 G splice mutation, and the 30 kb deletion including most of CYP21. Lanes 7 and 8 show cultured human breast and fibroblasts, respectively. Lane 9 represents a sample derived from an incubation to which no cells were added. Arrows at left show position of cold marker steroids: 17-hydroxyprogesterone and 11-deoxycortisol. The third radioactive spot represents an unidentified steroid metabolite. (B) Steroid 21-hydroxylation product 11-deoxycortisol is graphed for each cell line in fmol/mg protein/h. This experiment depicts the results of three separate experiments.
Human placenta, cultured rodent skeletal muscle, and monkey kidney epithelial COS cells also showed levels of 21-hydroxylase activity similar to that of cultured breast and fibroblasts (not shown).
4. Discussion We have demonstrated the presence of CYP21 expression and 21-hydroxylase activity in human lymphocytes. The finding of steroid 21-hydroxylase activity in lymphocytes is not entirely surprising, per se. Previously, other steroidogenic activities such as 5a-reductase, 17b-hydroxysteroid dehydrogenase [24], 3aand 3b-hydroxysteroid dehydrogenase [25], have all been detected in human lymphocytes. Steroid metabo-
lizing enzymes other than 21-hydroxylase are also apparently active in all the cells we examined, although we have not identified their specific hormonal products. Interestingly, we found 21-hydroxylase activity in a B-lymphoblastoid cell line from a CAH patient with 21-hydroxylase deficiency due to homozygous deletion of CYP21. This enzymatic activity is apparently comparable with that found in lymphocytes with intact CYP21. Moreover, breast cells do not express CYP21, yet a low level of 21-hydroxylase activity is measurable. Thus, these data provide compelling confirmation of physiologic activity of a 21- hydroxylase not encoded by CYP21. We are pursuing genetic characterization of this isozyme. Expression of CYP21 was more prominent in Blymphoblastoid cell lines of normal subjects compared
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Fig. 3. (A) Conversion of progesterone to deoxycorticosterone by lymphocytes. Thin layer chromatography was performed after incubating cells with tritiated progesterone as described in text. Note that twice as many cells and a 5-fold greater concentration of radio-labeled steroid precursor were added in these experiments. Lanes 1–3 represent results in cultured normal human B-lymphoblastoid cells; lanes 4 – 6 represent cultured B-lymphoblastoid cells respectively derived from CAH patients homozygous for the L281 mutation, the nt 656 G splice mutation, and the 30 kb deletion including most of CYP21. Lane 7 represents a sample derived from an incubation to which no cells were added. Arrows at left show position of cold marker steroids: progesterone and deoxycorticosterone. The other radioactive spots represent unidentified steroid metabolites. (B) Steroid 21-hydroxylation product deoxycorticosterone is graphed for each cell line in fmol/mg protein/h. This experiment depicts the results of three separate experiments.
with those from CAH patients who had point mutations, yet 21-hydroxylase activity was no different in lymphoid cells of CAH patients without CYP21 deletions compared with controls. Patients with severely deleterious mutations (deletions and splice mutations at nt 656) who showed lymphocytic enzyme activity had demonstrated severe salt-wasting disease and continuing need for steroid replacement, indicating that 21hydroxylase activity was profoundly deficient in their adrenal glands. Therefore, it would seem that lymphocyte 21-hydroxylase activity is not sufficient to completely correct cortisol and aldosterone deficiency. The existence of more than one human 21-hydroxylase enzyme has long been suspected based on physio-
logic studies in healthy and adrenalectomized adults, and in pregnant women. Among healthy adults fractional conversion of progesterone to DOC, an index of extra-adrenal 21-hydroxylation not mediated by CYP21, may vary by a factor of 15 [16]. It is interesting to note that hormonal assays of blood and urine from CAH patients of varying ages have identified 21-hydroxylated steroids [16,26]. Indeed, several adolescent CAH patients with homozygous deletion or deletion/ null mutation in CYP21 were found to produce moderate quantities of aldosterone, a 21-hydroxylated steroid [5]. Thus, it is plausible that ectopic 21-hydroxylase activity in lymphocytes is a source of inter-individual variation in steroid synthesis and, consequently, in phe-
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notype among CAH patients with similar CYP21 genotypes. The reason for apparently lower levels of CYP21 transcription in the CAH cell lines is not entirely clear. Prior experiments showed that the N172 mutant gene produced mRNA levels comparable with the wild-type gene [27]. These experiments were done using a transient expression system in cultured murine adrenocortical tumor cells, followed by RNase protection. Thus, the experimental conditions were different in several respects from those reported here. It is plausible that these mutant cell lines could have associated mutations in the promoter region which decrease transcription. The region − 167 bp upstream of the transcriptional start site has been identified as crucial for the 5-fold lower activity of the CYP21 pseudogene promoter [28,29]. We were unable to detect sequence polymorphisms in this region of the CYP21 promoter in any of the four CAH cell lines we examined (unpublished observations). With respect to reduced expression of CYP21 bearing the splice mutation (nt 656 G), there are other examples of 5% splice and termination mutations resulting in low abundance of transcript due to aberrant pre-mRNA processing [30]. In conclusion, we have observed CYP21 expression in lymphocytes, as well as a genetically distinct form of 21-hydroxylase activity in these cells. These data suggest an alternative pathway for steroid 21-hydroxylation, confirming earlier in vivo studies. Furthermore, it would appear that there are endogenously produced ‘immunosteroids’ in lymphoid tissue similar to neurosteroids observed in brain [31]. Steroids play an important role in immune modulation [32]. Physiologic concentrations of glucocorticoids decrease all lymphocyte subsets — particularly B cells, modify lymphocyte distribution [33], and induce apoptosis in thymocytes [34]. CYP21 is located in the midst of the Class III major histocompatibility complex (MHC) on the short arm of chromosome 6 in close proximity to the HLA region [35]. It is therefore interesting to speculate that there may be some functional importance to this genetic arrangement, conserved throughout evolution since some time before mammalian speciation. Indeed, in mice an androgen dependent gene has been identified in the MHC region [36]. The presence of steroidogenic enzymes in lymphocytes suggests that local steroid production may contribute to endocrine modulation of the immune system. It will be interesting to examine the other steroidogenic enzymes expressed in human lymphocytes and to determine their role.
Acknowledgements This work was supported in part by grants HD 00072 (PWS) and DK 37867 (PCW) from the National Insti-
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tutes of Health, the Hausman Fund of the Department of Pediatrics, North Shore University Hospital, and the Genentech Foundation. We also acknowledge the support of Eli Lilly and Company and Mr and Mrs John Payson. We thank Drs Thomas Degnan and Robert Pergolizzi and the staff of the Core Laboratory of the Department of Research, North Shore University Hospital for oligonucleotide synthesis, Drs Bo Dupont and Soo Young Yang for the lymphoblastoid cell lines, and Drs Nicholas Chiorazzi, Peter Gregersen, Savita Pahwa and Soe Than for lymphocyte samples. Helen Hsu’s technical assistance was greatly appreciated.
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Steroid 21-hydroxylase expression and activity in human lymphocytes Zhifeng Zhou a, Veena R. Agarwal b, Naznin Dixit a, Perrin White c, Phyllis W. Speiser a,* a
Department of Pediatrics, North Shore Uni6ersity Hospital, New York Uni6ersity School of Medicine, 300 Community Dr., Manhasset, NY 11030, USA b Cecil H. and Ida Green Center for Reproducti6e Biology Sciences, The Uni6ersity of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235 -9051, USA c Department of Pediatrics, The Uni6ersity of Texas South-Western Medical Center at Dallas, Dallas, TX 75235 -9051, USA Received 24 July 1996; accepted 25 September 1996
Abstract Steroid 21-hydroxylase encoded by CYP21 is expressed in adrenal cortex. Mutations in CYP21 cause potentially lethal congenital adrenal hyperplasia (CAH). Earlier observations suggested alternative sources of 21-hydroxylase activity, although its genetic source remains unclear. We found a novel source of CYP21 expression in normal human cultured B lymphocytes. The quantity of 21-hydroxylase transcript was reduced in B cell lines of CAH subjects compared with that in normal B-lymphoblastoid cells. No CYP21 transcript was detected in lymphocytes from a CAH patient with homozygous CYP21 deletion. Cultured lymphoid cells, including those carrying homozygous CYP21 deletion, and peripheral blood leukocytes converted both 17-hydroxyprogesterone to 11-deoxycortisol and progesterone to deoxycorticosterone. We conclude that lymphocytes express CYP21, but also possess a 21-hydroxylase distinct from CYP21. Activity of this isozyme may partially compensate for severe adrenal 21-hydroxylase deficiency. © 1997 Elsevier Science Ireland Ltd. Keywords: Steroid 21-monooxygenase; Adrenal hyperplasia, Congenital; Isozymes; Lymphocytes; Phenotype
1. Introduction Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia (CAH). This enzyme deficiency disrupts cortisol and aldosterone production and indirectly promotes excess adrenal secretion of androgen precursors. Clinical severity of this form of CAH ranges from potentially lethal salt-wasting adrenal insufficiency and severe female genital ambiguity, to subclinical androgen excess [1]. Most phenotypic variation among 21-hydroxylase
Abbre6iations: CAH, congenital adrenal hyperplasia; CYP21, gene encoding steroid 21-hydroxylase; kb, kilobases; nt, nucleotide; RT, reverse transcription; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TLC, thin layer chromatography. * Corresponding author. Tel.: + 1 516 5624635; fax: + 1 516 5624029; e-mail: [email protected]
deficient patients results from allelism in CYP21 [2–4] although there are notable exceptions [2,5–7]. The most common genetic lesions in CYP21 encoding adrenal steroid 21-hydroxylase are deletions of 30 kb (encompassing the 3% end of the CYP21P pseudogene, all of C4B, and the first 5–7 exons of CYP21 producing a non-functional chimeric pseudogene) [8,9], or a single base change, nucleotide (nt) 656 A G. The latter mutation at position − 13 in the second intron causes activation of a novel splice acceptor at position −19, leading to retention of a portion of the intron and premature termination of translation [10]. These mutations cause severe or classic CAH with inadequate aldosterone synthesis and consequent salt-wasting. A point mutation in the fourth exon changes isoleucine172 to asparagine (N172) and is associated with classic simple virilizing (i.e., non-salt-wasting) CAH. Mild non-classic CAH is most often due to substitution of leucine for valine at codon 281 (V281L) [11].
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Since the early 1970s, it has been suspected that more than one 21-hydroxylase enzyme exists in humans [12]. In vivo experiments using radiolabeled 21-hydroxylase precursors showed that extra-adrenal 21-hydroxylation occurs with considerable inter-individual variability in healthy adults [13]. Subsequent ribonuclease protection studies with RNA probes ruled out CYP21 as the source of such extraadrenal 21-hydroxylase activity [14]. Further clinical studies in patients affected with salt-wasting CAH due to deletions and other CYP21 null mutations showed clear evidence of adrenal 21-hydroxylase activity, i.e., increased aldosterone synthesis with advancing age, which could not be attributed to CYP21 [5]. To date the isozyme(s) and gene(s) for this putative alternative human 21-hydroxylase have not been characterized. Although 21-hydroxylase activity has been identified [15] in a number of non-adrenal tissues, including lymphoid tissues such as thymic epithelium and spleen [16], hemodynamic considerations make it implausible that 21-hydroxylase in a tissue such as the kidney or gonad could compensate for a severe intra-adrenal 21hydroxylase deficiency [13]. Leukocytes, numerous both in the circulation and in tissues, could conceivably produce clinically detectable levels of 21- hydroxylated steroids in the presence of an active enzyme. We therefore examined CYP21 expression and 21-hydroxylase activity in lymphoid cells.
2. Materials and methods
2.1. Subjects and tissue sources B-lymphoblastoid cell lines were established from six patients diagnosed with 21-hydroxylase deficiency by clinical and hormonal criteria [17]. Genotyping was performed by Southern blotting in the case of the homozygous deletion [18], and otherwise by multiplex ligase detection reaction [19]. B-lymphoblastoid cell lines from normal individuals were selected at random from among reference samples in a tissue typing laboratory; HLA types of control specimens were not characteristically associated with classic or non-classic 21-hydroxylase deficiency (obtained courtesy of Drs B. Dupont and S.Y. Yang, Memorial-Sloan-Kettering Cancer Center). In view of the low prevalence of severe CYP21 mutations such as deletions ( 0.003) and the nt 656 G allele (0.006) [20] in the general population, control samples were not genotyped. Fresh peripheral blood leukocytes were obtained from four normal individuals. All human samples were obtained with informed consent and approval of the institutional review board.
2.2. RNA isolation Peripheral blood leukocytes were isolated as described [21]. B-lymphoblastoid cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin plus 100 mg/ml streptomycin, and 2 mM glutamine (Gibco, Grand Island, NY), until a cell count of about 2×108 cells was attained. RNA extraction was performed as described [22], and about 20 mg of poly(A) + RNA was isolated using oligo (dT) [PolyATract mRNA Isolation Systems, Promega, Madison, WI). RNA was extracted by the same method from snap-frozen normal adult human adrenal gland procured at nephrectomy.
2.3. Re6erse Transcriptase Polymerase Chain Reacton RNA (1 mg poly(A) + RNA from cultured cells, or 5 mg total adrenal RNA) was treated with DNase I (Gibco) at room temperature for 15 min to remove genomic DNA. The DNase was then inactivated by heating to 90°C for 5 min. RNA was denatured at 65°C for 2 min and annealed to random hexamers (Gibco) by slow cooling to 37°C. RNA was reverse-transcribed (RT) using Superscript II (Gibco) at 42°C for 1 h in the presence of 166 mM dNTP, 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, and 5 U RNasin (Promega). cDNA was amplified using the polymerase chain reaction (PCR) in the presence of 5 pmol of each primer, 200 mM dNTPs, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 0.001% (w/v) gelatin, 2U Taq polymerase (Perkin Elmer-Roche, Branchburg, NJ), 0.3 mCi [a-P 32 ]dCTP (Dupont-New England Nuclear, Boston, MA) in a total volume of 10 ml. Primers were synthesized using an Applied Biosystems DNA synthesizer Model 381A with reagents from the manufacturer. The 5% sense primer was 5%-ACCTGTCCTTGGGAGACTAC-3% representing nt 2373– 2393 of the CYP21 sequence in GenBank (underline shows area of 8 bp deletion in exon 3 of CYP21P conferring specificity of primer for CYP21 ), and the 3% antisense primer was 5%-TAGGCAGGCATTAAGTTGTCG-3% representing nt 2798–2819. PCR conditions consisted of 30 cycles of denaturation at 94°C for 30 s, followed by annealing at 60°C for 1 min and extension at 72°C for 1 min 30 s. A final 5 min extension was performed. All reactions were performed on a Gene Amp PCR System 9600 (Perkin–Elmer). The expected size of CYP21 PCR-amplified cDNA was 246 bp. Onefourth of PCR products were analyzed on 5% non-denaturing polyacrylamide gels alongside end-labeled molecular weight markers (Msp I-digested pBR322, New England Biolabs, Beverly, MA). Gels were autoradiographed with Kodak X-Omat film (Eastman Kodak, Rochester, NY), and quantitative analysis was done with a Bio-Rad Imaging System GS 363 and Molecular
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Fig. 1. (A) Expression of CYP21 transcripts in lymphoid cells. Reverse-transcription (RT) followed by polymerase chain reaction (PCR) specific for CYP21 was performed as described in text. Selective gene amplification was accomplished with CYP21 -specific primers. PCR products were sequenced, confirming the authenticity of amplified material. Samples in lanes 1 and 2 were derived from normal human lymphoblastoid cell lines; samples in lanes 3 – 8 were derived from CYP21 mutant CAH lymphoblastoid cell lines, respectively homozygous for the nt 656 G splice mutation (lanes 3 and 4), the leucine to valine mutation at codon 281, L281 (lanes 5 and 6), the asparagine to isoleucine mutation at codon 172, N172 (lane 7), and the 30 kb mutation including CYP21 (lane 8). Lanes 9 and 10 were derived from normal human breast and fibroblast cell lines, respectively. Lane 11 is from normal human adrenal tissue. Upper panel shows an amplified band at 246 bp specific for CYP21. Lower panel depicts results of separate RT-PCR amplification of a 306 bp GAPDH gene segment. The latter is a ubiquitously expressed gene used to standardize expression levels. (B) Relative quantities of CYP21 mRNA in cell lines compared with adrenal mRNA on an arbitrary scale measuring intensity of radio-labeled PCR-amplified bands. Numbering and lane identification is as noted above. (C) Comparison of CYP21 mRNA expression in cell lines vs. adrenal. Relative quantity is indicated in arbitrary units. The percent of the apparent level of adrenal gene expression is shown. This experiment was representative of three separate experiments which produced similar results.
Image software (Bio-Rad, Hercules, CA). Results shown in Fig. 1 are representative of three experiments which produced similar proportions of CYP21 transcripts. To check the integrity and comparative quantity of templates, the housekeeping gene, glyceraldehyde-3phosphate dehydrogenase (GAPDH) was chosen as an endogenous marker. Primers were designed from the GAPDH sequence in GenBank: (1) 5% end sense primer
5%-CGGAGTCAACGGATTTGGTCGTAT-3% and (2) 3% end antisense primer 5%AGCCTTCTCCATGGTGGTGAAGAC-3%. Template for GAPDH primers was the same as used for CYP21 amplification. PCR conditions were as described above, but GAPDH amplifications were performed separately from the CYP21 PCR reactions for 22 cycles. Expected size of these PCR products was 306 bp.
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2.4. Sequencing PCR-amplified putative CYP21 cDNA was directly sequenced to verify its identity. Double stranded PCR amplified templates were subjected to electrophoresis in 1.5% high melting temperature agarose (USB, Cleveland, OH) gels, and purified by means of a DNA gel extraction kit (Qiagen, La Jolla, CA). PCR primers were used as sequencing primers. DNA sequencing was performed with the Taq DyeDeoxy fluorescent dye terminator cycle sequencing kit (ABI, Foster City, CA). Products were loaded onto an ABI 373A sequencer according to the manufacturer’s instructions.
2.5. Lymphocytes’ 21 -hydroxylase acti6ity Approximately 106 cells were incubated for 24 h in the presence of 1 mCi tritiated 17-hydroxyprogesterone (Dupont-New England Nuclear, Boston, MA). For experiments involving progesterone, 2 × 106 cells were incubated with 5 mCi tritiated progesterone. All experiments were repeated three times; negative controls consisted of medium without cells incubated with radio-labeled steroid. After 24 h, cells were precipitated by centrifugation, and 1 nmol each of 21-hydroxylase substrate and product (Sigma, St. Louis, MO) were added to the cell-free medium in 2 ml ethanol. The medium was then extracted with 2 vol. of ethyl acetate. The dried organic phase was resuspended in 30 ml ethyl acetate, spotted on a silica gel plate (Sigma Polygram G/UV254), and resolved in chloroform: acetone (70:30). Radioactivity was measured by imaging autoradiograms of thin layer chromatography (TLC) plates (BioRad imaging system GS 363, Hercules, CA). Protein was assayed in parallel experiments as described [23]. Activity was calculated as picomoles of product/mg protein/h. To verify the authenticity of 21-hydroxylated steroid product detected on thin layer chromatography, specific radioimmunoassay for 11-deoxycortisol was performed on extracted cell medium from parallel incubations done with non-radioactive 17-hydroxyprogesterone (Endocrine Sciences Laboratory, Calabasas Hills, CA). Cross-reactivity with 17-hydroxyprogesterone is 2.5%, and less than 2% for other steroids.
3. Results
3.1. CYP21 expression Fig. 1 shows relatively prominent expression of CYP21 transcripts in human lymphoid cells studied by RT-PCR. The level of expression from normal Blymphoblastoid cells normalized by GAPDH expression was 10.4 and 16.9% (average about 13.5%) of the
level found in the normal human adrenal. In contrast, B-lymphoblastoid cells derived from CAH patients with non-deletional mutations showed lower levels of CYP21 expression (average9S.D., 2.391%, range 0.2–6%) compared with the level observed in the adrenal. There was no evidence of CYP21 expression in the cells derived from the individual with a homozygous deletion of CYP21 ; primers were designed to be specific for the deleted portion of CYP21. Normal human breast tissue also did not express CYP21 ; a low level of expression was detected in normal human fibroblasts (0.7% compared with adrenal). Performing PCR for 30 cycles maintained the reaction in the linear range and did not alter the observed CYP21 product ratios among the various samples. Performing 25 cycles proved suboptimal for product detection, and 40 cycles were supra-maximal. To exclude the possibility of exogenous CYP21 cDNA contamination in tissue-derived RNA, negative controls were designed omitting reverse transcriptase from RT reactions. By omitting RNA from the RT reactions, and by omitting cDNA from PCR reactions, the possibility of CYP21 cDNA contamination in RT or PCR reagents was excluded. All these negative controls showed no amplification of CYP21 (not shown), confirming that the CYP21 cDNA transcripts identified were derived from the tissues indicated. The identity of the amplified bands was confirmed to be CYP21 based on direct sequencing (not shown) of PCR products of appropriate size which co-migrated with a similar sized PCR product derived from adrenal cDNA.
3.2. Steroid 21 -hydroxylase acti6ity Conversion of 17-hydroxyprogesterone to 11-deoxycortisol was detected in cultured B-lymphoblastoid cells from normal individuals, from CAH patients homozygous for the CYP21 mutation at nt 656 G (splice mutation), patients homozygous for the leucine to valine mutation at codon 281 (L281), and a patient homozygous for deletion of CYP21 (Fig. 2). Similar activity was seen in peripheral blood leukocytes (not shown). Cultured human breast cells and fibroblasts showed 5–10-fold lower 21-hydroxylase activity. Negative controls in which no cells were added showed no enzyme activity. Conversion of progesterone to deoxycorticosterone could also be detected after incubation with a larger number of cells, and a higher concentration of radiolabeled progesterone (Fig. 3). The identities of the hormonal products of 21-hydroxylation were confirmed when these experiments were repeated using a different solvent system (chloroform:ethyl acetate, 80:20). Authenticity of 21-hydroxylated product was further confirmed by RIA detection of deoxycorticosterone in cell media extract, but not in control media lacking cells.
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Fig. 2. (A) Conversion of 17-hydroxyprogesterone to 11-deoxycortisol by lymphocytes. Thin layer chromatography was performed after incubating cells with tritiated 17-hydroxyprogesterone as described in text. Lanes 1 – 3 represent results in cultured normal human B-lymphoblastoid cells; lanes 4 – 6 represent cultured B-lymphoblastoid cells respectively derived from CAH patients homozygous for the L281 mutation, the nt 656 G splice mutation, and the 30 kb deletion including most of CYP21. Lanes 7 and 8 show cultured human breast and fibroblasts, respectively. Lane 9 represents a sample derived from an incubation to which no cells were added. Arrows at left show position of cold marker steroids: 17-hydroxyprogesterone and 11-deoxycortisol. The third radioactive spot represents an unidentified steroid metabolite. (B) Steroid 21-hydroxylation product 11-deoxycortisol is graphed for each cell line in fmol/mg protein/h. This experiment depicts the results of three separate experiments.
Human placenta, cultured rodent skeletal muscle, and monkey kidney epithelial COS cells also showed levels of 21-hydroxylase activity similar to that of cultured breast and fibroblasts (not shown).
4. Discussion We have demonstrated the presence of CYP21 expression and 21-hydroxylase activity in human lymphocytes. The finding of steroid 21-hydroxylase activity in lymphocytes is not entirely surprising, per se. Previously, other steroidogenic activities such as 5a-reductase, 17b-hydroxysteroid dehydrogenase [24], 3aand 3b-hydroxysteroid dehydrogenase [25], have all been detected in human lymphocytes. Steroid metabo-
lizing enzymes other than 21-hydroxylase are also apparently active in all the cells we examined, although we have not identified their specific hormonal products. Interestingly, we found 21-hydroxylase activity in a B-lymphoblastoid cell line from a CAH patient with 21-hydroxylase deficiency due to homozygous deletion of CYP21. This enzymatic activity is apparently comparable with that found in lymphocytes with intact CYP21. Moreover, breast cells do not express CYP21, yet a low level of 21-hydroxylase activity is measurable. Thus, these data provide compelling confirmation of physiologic activity of a 21- hydroxylase not encoded by CYP21. We are pursuing genetic characterization of this isozyme. Expression of CYP21 was more prominent in Blymphoblastoid cell lines of normal subjects compared
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Fig. 3. (A) Conversion of progesterone to deoxycorticosterone by lymphocytes. Thin layer chromatography was performed after incubating cells with tritiated progesterone as described in text. Note that twice as many cells and a 5-fold greater concentration of radio-labeled steroid precursor were added in these experiments. Lanes 1–3 represent results in cultured normal human B-lymphoblastoid cells; lanes 4 – 6 represent cultured B-lymphoblastoid cells respectively derived from CAH patients homozygous for the L281 mutation, the nt 656 G splice mutation, and the 30 kb deletion including most of CYP21. Lane 7 represents a sample derived from an incubation to which no cells were added. Arrows at left show position of cold marker steroids: progesterone and deoxycorticosterone. The other radioactive spots represent unidentified steroid metabolites. (B) Steroid 21-hydroxylation product deoxycorticosterone is graphed for each cell line in fmol/mg protein/h. This experiment depicts the results of three separate experiments.
with those from CAH patients who had point mutations, yet 21-hydroxylase activity was no different in lymphoid cells of CAH patients without CYP21 deletions compared with controls. Patients with severely deleterious mutations (deletions and splice mutations at nt 656) who showed lymphocytic enzyme activity had demonstrated severe salt-wasting disease and continuing need for steroid replacement, indicating that 21hydroxylase activity was profoundly deficient in their adrenal glands. Therefore, it would seem that lymphocyte 21-hydroxylase activity is not sufficient to completely correct cortisol and aldosterone deficiency. The existence of more than one human 21-hydroxylase enzyme has long been suspected based on physio-
logic studies in healthy and adrenalectomized adults, and in pregnant women. Among healthy adults fractional conversion of progesterone to DOC, an index of extra-adrenal 21-hydroxylation not mediated by CYP21, may vary by a factor of 15 [16]. It is interesting to note that hormonal assays of blood and urine from CAH patients of varying ages have identified 21-hydroxylated steroids [16,26]. Indeed, several adolescent CAH patients with homozygous deletion or deletion/ null mutation in CYP21 were found to produce moderate quantities of aldosterone, a 21-hydroxylated steroid [5]. Thus, it is plausible that ectopic 21-hydroxylase activity in lymphocytes is a source of inter-individual variation in steroid synthesis and, consequently, in phe-
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notype among CAH patients with similar CYP21 genotypes. The reason for apparently lower levels of CYP21 transcription in the CAH cell lines is not entirely clear. Prior experiments showed that the N172 mutant gene produced mRNA levels comparable with the wild-type gene [27]. These experiments were done using a transient expression system in cultured murine adrenocortical tumor cells, followed by RNase protection. Thus, the experimental conditions were different in several respects from those reported here. It is plausible that these mutant cell lines could have associated mutations in the promoter region which decrease transcription. The region − 167 bp upstream of the transcriptional start site has been identified as crucial for the 5-fold lower activity of the CYP21 pseudogene promoter [28,29]. We were unable to detect sequence polymorphisms in this region of the CYP21 promoter in any of the four CAH cell lines we examined (unpublished observations). With respect to reduced expression of CYP21 bearing the splice mutation (nt 656 G), there are other examples of 5% splice and termination mutations resulting in low abundance of transcript due to aberrant pre-mRNA processing [30]. In conclusion, we have observed CYP21 expression in lymphocytes, as well as a genetically distinct form of 21-hydroxylase activity in these cells. These data suggest an alternative pathway for steroid 21-hydroxylation, confirming earlier in vivo studies. Furthermore, it would appear that there are endogenously produced ‘immunosteroids’ in lymphoid tissue similar to neurosteroids observed in brain [31]. Steroids play an important role in immune modulation [32]. Physiologic concentrations of glucocorticoids decrease all lymphocyte subsets — particularly B cells, modify lymphocyte distribution [33], and induce apoptosis in thymocytes [34]. CYP21 is located in the midst of the Class III major histocompatibility complex (MHC) on the short arm of chromosome 6 in close proximity to the HLA region [35]. It is therefore interesting to speculate that there may be some functional importance to this genetic arrangement, conserved throughout evolution since some time before mammalian speciation. Indeed, in mice an androgen dependent gene has been identified in the MHC region [36]. The presence of steroidogenic enzymes in lymphocytes suggests that local steroid production may contribute to endocrine modulation of the immune system. It will be interesting to examine the other steroidogenic enzymes expressed in human lymphocytes and to determine their role.
Acknowledgements This work was supported in part by grants HD 00072 (PWS) and DK 37867 (PCW) from the National Insti-
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tutes of Health, the Hausman Fund of the Department of Pediatrics, North Shore University Hospital, and the Genentech Foundation. We also acknowledge the support of Eli Lilly and Company and Mr and Mrs John Payson. We thank Drs Thomas Degnan and Robert Pergolizzi and the staff of the Core Laboratory of the Department of Research, North Shore University Hospital for oligonucleotide synthesis, Drs Bo Dupont and Soo Young Yang for the lymphoblastoid cell lines, and Drs Nicholas Chiorazzi, Peter Gregersen, Savita Pahwa and Soe Than for lymphocyte samples. Helen Hsu’s technical assistance was greatly appreciated.
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