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Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan
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Introduction |
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-hydroxycholesterol, which bypasses the mitochondrial outer membranes, was required for progesterone production. Nevertheless, these results clearly indicate that SF-1 is a key factor in steroidogenic cell differentiation.
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Result |
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We prepared an adenovirus construct containing bovine SF-1 (bSF-1), Adx-bSF-1. It became apparent from the results of this experiment that long-term cultured BMCs infected with Adx-bSF-1 could produce a significant amount of multiple steroids. Long-term (123 days) cultured BMCs from male GFP mice were infected with Adx-bSF-1 or Adx-LacZ as a control, and incubated for 7 days. The steroid contents in the medium accumulated for the next 4 days were then measured. The BMCs infected with Adx-bSF-1 produced a significant amount of progesterone (P4), deoxycorticosterone (DOC), corticosterone (B), 17-hydroxyprogesterone (17-OH P4), 11-deoxycortisol (S), dehydroepiandrosterone (DHEA), 
4-androstenedione (4-A) and testosterone (T), while those infected with Adx-LacZ did not (Fig. 2A). All of the steroid contents in the medium from the control cells infected with Adx-LacZ were undetectable except S. The slight detection of S in the control medium is probably due to the cross reactivity (9.5%) of the antibodies against S with hydrocortisone in the cultured medium, considering no significant production of precursor steroids
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We also observed a quite similar steroid profile of BMCs from 129SVJ mice (data not shown), suggesting little strain difference in steroidogenic capacity of BMCs.
An immunocytochemical study of cultured BMCs from 129SVJ mice confirmed the actual expression of P450scc using a specific antibody against cytochrome P450scc (Fig. 2C). The cells did not react with preimmune serum as a negative control (data not shown).
Characterization of the cell lineage of steroidogenic BMCs
The flowcytometry experiment revealed surface markers of the above mentioned steroidogenic BMCs (Fig. 3); that is, it revealed the negative expressions of CD45, which is specific to haematopoietic cells. The monocyte/macrophage marker, CD11b was also negative. Although the character of mouse mesenchymal stem cell has not been fully clarified and controversial (Jiang et al. 2002; Sun et al. 2003), one of such potential markers, CD44 was negative in our BMCs. In addition, the experiment revealed the positive expression of c-kit and Sca-1, which are haematopoietic and mesenchymal stem/progenitor markers. These results suggest that steroid-producing cells originate from multipotent and immature stem cells.
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Since ACTH-R expression has been confirmed, we next tested the responsiveness of BMCs to ACTH by measuring P4 and DOC secretion. We infected long-term (100 days) cultured BMCs from GFP mice with Adx-bSF-1 or Adx-LacZ, and then at 3–4 day intervals after infection they were stimulated with 2.4 nM–2.4 µM ACTH. After each stimulation the medium was collected for measurement of steroid content. ACTH stimulated the production of these steroids in a dose-dependent manner from the BMCs infected with Adx-bSF-1 (Fig. 4A), but not in the cells infected with Adx-LacZ (data not shown). These findings indicate that the introduction of bSF-1 into long-term cultured BMCs leads to transformation into steroidogenic cells, which in a basal state as well as in response to ACTH are capable of producing multiple steroid hormones. The induction of the mRNAs of steroidogenic enzymes, namely, P450scc, 3ß-HSD, P450c21, P450c11 and 17ß-HSD, by treatment with 2.4 µM ACTH for 4 days was also confirmed by real-time PCR (Fig. 4B). The unique induction of 17ß-HSD type 3 mRNA by ACTH might support the direct stimulation of testosterone production by ACTH in foetal and neonatal mouse testis (O'shaughnessy et al. 2003).
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Finally, we tested how long steroid production lasts in transformed BMCs after Adx-bSF-1 infection. We infected long-term (180 days) cultured BMCs with Adx-bSF-1 or Adx-LacZ, and then measured P4 and DOC levels in the cultured medium every 3–4 days. We observed a significant production of P4 and DOC until at least 112 days (Fig. 5A). Considering the relatively short half-life of adenovirus (2-3 weeks), this long-term steroid production is unexpected. It is reasonable to speculate the possible induction of endogenous SF-1 during culture, which might contribute to continuous long-term steroid production. However, this is unlikely because we could not prove the induction of endogenous mouse SF-1 expression during the experimental period (until 49 days after the infection) by RT-PCR, but we could detect adenovirus-induced bSF-1 expression (Fig. 5B).
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Discussion |
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Mouse adrenal produces mineralocorticoids like DOC and B because of the adrenal-specific expression of P450c11, but does not produce C19 steroids like DHEA and sex steroids because of the lack of P450c17 expression in this tissue. On the other hand, mouse gonad (testis or ovary) possesses the reverse expression profile of the above two enzymes, namely gonad-specific P450c17 expression and the absent expression of P450c11, resulting in the predominant production of C19 steroids without any productions of mineralocorticoids. In the present study, we demonstrated, for the first time, that BMCs could differentiate into steroidogenic cells in response to ACTH under condition of adenovirus-mediated forced expression of SF-1. BMCs originally expressed ACTH receptor which was a type transcribed in adipose tissue but not in adenal gland. The steroid profile of the cultured BMCs showed a mixed pattern of mouse adrenal and gonadal steroidogenesis, namely the simultaneous productions of DOC, B, DHEA, 4-A and T. Interestingly, P450c17 is expressed in human adrenal but not in mouse adrenal, thus the significant expression of P450c17 in the BMCs and the significant production of 17-hydroxylated steroid, S may also suggest a mixed steroid profile beyond species. These findings might suggest the multipotency of BMCs during steroidogenic cell differentiation and the possibility of a common origin for steroidogenic tissues, namely, stem cells. Although little is known about the origin of these stem cells, a previous study on the expression profile of SF-1 indicated that the undifferentiated adrenal cortex and gonads of early stage fetuses originate from common adreno-genital primordium (Hatano et al. 1996).
In the long term culture, a significant production of P4 and DOC was surprisingly observed until at least 112 days. Interestingly, there was a relatively continuous production of DOC in contrast to a sharp decline in P4 production from days 18 to 25 (Fig. 5A). We need to clarify in the future whether this time-dependent change in the steroid profile might reflect a BMC differentiation process or just reflect a phenomenon due to a difference of the steroidogenic enzyme stability. Although the half-life of adenovirus is 2–3 weeks and we could not detect the induction of endogenous mouse SF-1, steroidogenesis lasted much longer than we expected. The continuous bSF-1 expression by adenovirus infection, even if at a low level, might be enough to maintain BMCs for long-term multiple steroid production. Another possibility is that SF-1 expression may be indispensable for the initiation of steroidogenesis by inducing steroidogenic enzymes, but may not be so critical for its maintenance.
The multipotency of BMCs to differentiate into either adrenal or gonadal steroidogenic cells might provide an important model for the future clarification of the mechanisms of tissue-, zone- or cell-specific adrenal and gonadal steroidogenic cell differentiation. An additional undifferentiated zone between the zona glomerulosa and zona fasciculate has been suggested as an adrenocortical stem cell zone which expresses SF-1 (Mitani et al. 2003). The presence of intra-adrenal stem cell has been also suggested from the success of xenotransplanted adrenocortical tissue formation from clonal or immortalized bovine adrenocortical cells in immunodeficient mice (Thomas et al. 1997, 2000). One plausible speculation might be that bone marrow-derived stem cells settle in the adrenocortical stem cell zone, where SF-1 expression might become possible.
Cell origin of steroidogenic BMCs
In the present study, we expanded a relatively purified BMC population by culturing the BMCs for 120–180 days (over 12–18 passages) and then the cells were subjected to the experiment to investigate steroidogenic property after infection with Adx-bSF-1. Although the BMCs in our experiment still constitute a heterogeneous population, the analysis of the cell surface markers highly suggested a possibility that the steroid-producing cells originate from multipotent and immature stem cells. Importantly, the long-term cultured BMCs differentiated into an osteoblastic phenotype by treatment with 0.05 mM ascorbic acid, 10 mMß-glycerophosphate and 0.1 µM dexamethasone (Pittenger et al. 1999) as shown by alkaline phosphatase staining (data not shown), suggesting that the character of the steroidogenic cells may be much closer to mesenchymal BMC lineages (Pittenger et al. 1999). However, the exact origin of these stroidogenic cells remains unclear and warrants further investigation.
Therapeutic potential for patients with steroid insufficiency
Steroid hormone replacement therapy has become well established and provides many benefits to patients with adrenal insufficiency or hypogonadism. However, such treatments must be continued throughout an individual's lifetime, especially in cases of adrenal insufficiency. Therefore, alternative therapies such as gene therapy or ex vivo steroidogenic cell transplantation might be beneficial. In addition to the above-mentioned successful reports of xenotransplantation of adrenocortical cells (Thomas et al. 1997, 2000), the feasibility of gene therapy for congenital adrenal hyperplasia has been demonstrated. Namely, a single intra-adrenal injection of an adenoviral vector encoding CYP21 has been shown to compensate for the biochemical and endocrine alterations in 21-hydroxylase deficient mice (Tajima et al. 1999). Although more extensive studies are required, autograft cell transplantation of the SF-1-induced steroidogenic BMCs presented in this study may provide another therapeutic possibility for adrenal insufficiency. To determine their applicability in therapeutic treatments, the limits and biological importance of these BM-derived steroidogenic cells need to be assessed.
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Experimental procedures |
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We prepared a recombinant adenovirus vector derived from the human type 5-adenovirus using a commercially available Adenovirus Expression Vector Kit (Takara, Osaka, Japan). Bovine SF-1/Ad4BP cDNA (Honda et al. 1993) was provided by Prof K Morohashi (National Institute for Basic Biology, Okazaki, Japan), restricted by BamHI and EcoRI, blunt-ended, and inserted into the SwaI site of the recombinant cosmid vector, pAxCAwt (Takara), which contains a CAG promoter. The recombinant SF-1 adenovirus (Adx-bSF-1) was obtained according to the manufacturer's protocol based on an original report (Miyake et al. 1996).
Long-term bone marrow culture and adenovirus treatment
We obtained bone marrow cells (BMCs) from a 3-month old male B6-GFP (green fluorescence protein) mouse, C57BL/6Tg14 (act-EGFP) osbY01, donated by Dr Yamada (Kyoto University, Kyoto, Japan). In some experiments, we prepared BMCs from a 4 month-old male 129SVJ mouse. We cultured the BMCs according to a previous method (Dexter et al. 1977), with some modifications. Briefly, fresh whole bone marrow was harvested by flushing the bones from mice in medium A. Medium A includes -MEM containing 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.0125 µg/mL, Amphotericin B (Sigma-aldrich, Irvine, UK), 10–7 M hydrocortisone (Nikkenkayaku, Japan), and 20% donor horse serum (Lot 6603F or 7307F, ICN Biochemicals, Aurora, OH, USA). We seeded the harvested cells in 75 cm2 tissue culture flasks (Nalge Nunc, Rochester, NY, USA), incubated them at 37 °C in 5% CO2 in air with medium A. Only the adherent cells were maintained for several weeks, trypsinized and stored in a cell banker at –80 °C until use. When needed, we cultured the stored BMCs for 120–180 days (over 12–18 passages) with medium A, aiming at the expansion of a relatively purified cell population. From this cell population, 5 x 105 BMCs were seeded again in 60 mm dish (Nunc) with medium A and when the BMCs became subconfluent, the cells were infected with adenovirus at approximately 10 plaque-forming units/cell. As a control for all experiments, we infected the BMCs with recombinant adenovirus expressing ß-galactosidase (Adx-LacZ). After the infection, the cells were also maintained with medium A.
Measurements of the steroid content in the medium secreted from the BMCs
We measured the progesterone (P4), deoxycorticosterone (DOC), corticosterone (B), 17-hydroxyprogesterone (17-OHP4), 11-deoxycortisol (S), dehydroepiandrosterone (DHEA), 4-androstenedione (4-A) and testosterone (T) contents secreted into the culture medium with the collaboration of SRL Co. Ltd. (Tokyo, Japan) using commercial RIA kits (Diagnostic Products Corp., LA, USA) and respective specific RIA systems developed by SRL (Den et al. 1978). The secretions of P4 and DOC in the medium were also confirmed in the presence or absence of synthetic 1–24 ACTH (Shionogi Co., Osaka, Japan). The detection limits of P4, DOC, B, 17-OHP4, S, DHEA, 4-A and T were less than 0.1 ng/mL, 0.02 ng/mL, 20.0 ng/mL, 0.1 ng/mL, 0.04 ng/mL, 0.2 ng/mL, 0.1 ng/mL and 0.05 ng/mL, respectively.
Quantitative real-time PCR
We performed quantitative analysis of the mRNA expressions of StAR, ACTH receptor (ACTH-R) and various steroidogenic enzymes including P450scc, P450c17, P450C11, P450C21, P450ald, 3ß-HSD, and 17ß-HSD type 3 by real-time PCR using a LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) as previously described (Mukasa et al. 2003). We isolated the total RNA from the cultured BMCs and Y-1 cells using a RNeasy mini kit (Qiagen), and from mouse testis and adrenals using Isogen (Wako Pure Chemical Industries, Osaka Japan). We synthesized first-strand complimentary DNA using 5 µg of total RNA as a template and carried out PCR in a LightCycler according to the manufacturer's instructions. The sense/anti-sense primers used were previously reported (Ilgar et al. 1997; Jennifer & Holly 2002; Mukai et al. 2002; Kubo et al. 2004): StAR 5'-TAG CTG AAG ATG GAC AGA CTT GC-3'/5'-GAC CTT GAA AGG CTC AGG AAG AAC-3'; P450scc, 5'-AAC TTG AAG GTA CAG GAG ATG CTG C-3'/5'-CAT CAG GAT GAG GCT GAA CTT GGT C; 3ß-HSD, 5'-CAG ACC ATC CTA GAT GT-3'/5'-AGG AAG CTC ACA GTT TCC A-3'; P450c21, 5'-CTT CAC GAC TGT GTC CAG GAC TTG-3'/5'-CAG CAG AGT GAA GGC CTG CAG CAG-3'; P450c11, 5'-AAG AAA ACT TAG AGT CCT GGG ATT-3'/5'-GTG TCA GTG CTT CCA GCA ATG AGT-3'; P450ald, 5'-AAG AAC ATT TCG ATG CCT GGG ATG-3'/5'-GTG TCA ACG CTC CCA GCG GTG AGC-3'; 17ß-HSD, 5'-CAT TTG AGT TGG CCA GAC ATG G-3'/5'-GGA GCA TTC CAA CGT TGT TGA C-3'; ACTH-receptor, 5'-CCA AGG AGA GGA GCA TTA TTG G-3'/5'- CAG GAC AAT CGG AGT TAT TTC TTG CGG-3'; ACTH-R 1a, 5'-CAG TCA TCT TGC CGA GAA AG-3'/5'-CAG ACT GCC CAA CAT GTC-3'; ACTH-R 1f, 5'-CAA GGG AGG GCA GAA ACT G-3'/5'-CAG ACT GCC CAA CAT GTC-3'; ß-actin, 5'-GCA ATG CCT GGG TAC ATG GTG G-3'/5'-GTC GTA CCA CAG GCA TTG TGA TGG-3'. PCR conditions are available on request. Threshold values were obtained where fluorescent intensity was in the geometric phase of amplification, as determined with LightCycler Software Ver.3.5. Products were verified on a 2% agarose gel. We verified the nucleotide sequences of each PCR product by direct sequencing using the appropriate primers. Relative expression levels of the mRNA were calibrated to those of ß-actin and its ratio to the control mouse adrenocortical Y-1 cells or mouse adrenal or testis.
Flowcytometry
The protocol essentially followed a previously described method (Hirase et al. 2000). Briefly, 3 x 105 BMCs were incubated with either PE (phycoerythin)-conjugated anti-mouse c-kit, CD11b, CD34, CD44, CD45, and Sca-1 monoclonal antibodies (BD Biosciences, Japan) or an isotype-matched PE-conjugated rat IgG (BD Biosciences) for 30 min at 4 °C. The cells were finally analysed on a FACScan flow cytometer (Becton Dickinson).
Immunocytochemistry
We conducted an immunocytochemical study of the BMCs from 129SVJ mice with an antibody against rabbit anti-cytochrome P450scc (RDI, NJ, USA) using Zenon Rabbit IgG labelling kits (Molecular Probes, Inc., OR, USA) or preimmune serum. The cells were plated on to collagen I-type membranes (Asahi technoglass, Tokyo, Japan) in 35 mm dishes and fixed with 4% paraformaldehyde at 4 °C for 1 h. After this, we completely followed the manufacturer's protocol. The fluorescence was observed using fluorescence microscopy (BX-51; Olympus, Tokyo, Japan).
Statistics
One-factor ANOVA was used for statistical evaluation. P < 0.05 was considered statistically significant.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: nawata{at}intmed3.med.kyushu-u.ac.jp
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Received: 27 July 2004
Accepted: 12 September 2004
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) or absence (
) of 2.4 µM ACTH. After infection with Adx-bSF-1 (day 0), BMCs were treated with 2.4 µM ACTH on day 0, 4 and 7 and cultured for 4 days. On day 11, total RNAs of the cells were extracted and real-time PCRs were performed. Values represent the mean ± SD (n = 3). Relative mRNA expression levels were calibrated to ß-actin. A relative ratio to the expression of the control Y-1 cells is expressed in the cases of P450scc and 3ß-HSD; to that of the mouse adrenal is expressed in the cases of P450c21, P450c11 and ACTH-R; and to that of the mouse testis is expressed in the cases of 17ß-HSD type3. *P < 0.05, **P < 0.01 vs. control (absence of ACTH).