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Endocrinology, doi:10.1210/en.2007-1808
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Endocrinology Vol. 149, No. 9 4717-4725
Copyright © 2008 by The Endocrine Society

Adipose Tissue-Derived and Bone Marrow-Derived Mesenchymal Cells Develop into Different Lineage of Steroidogenic Cells by Forced Expression of Steroidogenic Factor 1

Shigeki Gondo, Taijiro Okabe, Tomoko Tanaka, Hidetaka Morinaga, Masatoshi Nomura, Ryoichi Takayanagi, Hajime Nawata and Toshihiko Yanase

Department of Medicine and Bioregulatory Science (S.G., T.O., T.T., H.M., M.N., R.T., T.Y.), Graduate School of Medical Science (H.N.), Kyushu UniversityHigashi-ku, Fukuoka-city, Fukuoka-pref 812-8582, Japan; and Gondo Clinic (S.G.), Tosu-city, Saga-pref 841-0015, Japan

Address all correspondence and requests for reprints to: Toshihiko Yanase, M.D., Ph.D., Department of Medicine and Bioregulatory Science, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka-city, Fukuoka-pref 812-8582, Japan. E-mail: yanase{at}intmed3.med.kyushu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Steroidogenic factor 1 (SF-1)/adrenal 4 binding protein is an essential nuclear receptor for steroidogenesis, as well as for adrenal and gonadal gland development. We have previously clarified that adenovirus-mediated forced expression of SF-1 can transform long-term cultured mouse bone marrow mesenchymal cells (BMCs) into ACTH-responsive steroidogenic cells. In the present study, we extended this work to adipose tissue-derived mesenchymal cells (AMCs) and compared its steroidogenic capacity with those of BMCs prepared from the identical mouse. Several cell surface markers, including potential mesenchymal cell markers, were identical in both cell types, and, as expected, forced expression of SF-1 caused AMCs to be transformed into ACTH-responsive steroidogenic cells. However, more elaborate studies revealed that the steroidogenic property of AMCs was rather different from that of BMCs, especially in steroidogenic lineage. In response to increased SF-1 expression and/or treatment with retinoic acid, AMCs were much more prone to produce adrenal steroid, corticosterone rather than gonadal steroid, testosterone, whereas the contrary was evident in BMCs. Such marked differences in steroidogenic profiles between AMCs and BMCs were also evident by the changes of steroidogenic enzymes. These novel results suggest a promising utility of AMCs for autologous cell regeneration therapy for patients with steroid insufficiency and also a necessity for appropriate tissue selection in preparing mesenchymal stem cells according to the aim. The different steroidogenic potency of AMCs or BMCs might provide a good model for the clarification of the mechanism of tissue- or cell-specific adrenal and gonadal steroidogenic cell differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROIDOGENIC factor 1 (SF-1)/adrenal 4 binding protein (Ad4BP) is a nuclear receptor, which is essential for steroid synthesis as a ubiquitous transcription factor of various steroidogenic enzymes (1, 2, 3). The transcriptional activity of SF-1 can be dramatically up-regulated by the cAMP-protein kinase A signal pathway (4). Knockout (KO) mice for SF-1 show agenesis of both the adrenal glands and gonads, as well as decreased expression levels of LH and FSH in the pituitary gonadotroph, indicating that SF-1 is an essential factor for differentiation of the pituitary-adrenal or pituitary-gonadal axis (3, 5, 6, 7).

Stable expression of SF-1 has been shown to direct embryonic stem cells toward the steroidogenic lineage. However, this steroidogenic capacity was very limited because progesterone (P4) was the only steroid produced in the presence of an exogenous substrate, 20{alpha}-hydroxycholesterol (8). Bone marrow mesenchymal cells (BMCs) contain pluripotent progenitor cells, which differentiate into multiple lineages (9). In a previous study, we reported that adenovirus-mediated forced expression of SF-1 (Adx-bSF-1) could transform mouse primary long-term cultured BMCs into steroidogenic cells possessing the capability for mixed character of adrenal and gonadal steroid production, which was supported by the expression of the responsible enzyme genes (10). More recently, we have reported that primary cultured human BMCs infected with Adx-bSF-1 could also produce adrenal- and gonadal-type steroids (11). The steroidogenic profiles of both mouse and human BMCs were broadly similar except for a greater induction of both the ACTH receptor (ACTH-R) and LH receptor (LH-R) in human BMCs infected with Adx-bSF-1 than in mouse BMCs. Although the mechanism is not fully characterized, the ultimate regulation for the final differentiation to adrenal and/or gonadal cell is thought to require a series of transcription factors, including SF-1 in a cascade of sexual differentiation (12, 13). Importantly, among several factors (including: SF-1; Wilms tumor 1; dosage sensitive sex reversal, adrenal hypoplasia congenital, critical region on the X chromosome gene 1; pre-B-cell leukemia homeobox 1; cAMP response element-binding protein/p300-interacting transactivator with glutamic acid/aspartic acid-rich C-terminal domain 2; and wingless-type mouse mammary tumor virus integration site family member 4), only the introduction of SF-1 enabled the human BMCs to express P450scc, and to produce cortisol (F) and testosterone (T), suggesting that SF-1 is a master regulator for the production of steroidogenic cells from human BMCs (11). Similar to our data, a recent report demonstrated that cAMP stimulation of a cloned mouse BMC line, KUM9 and human BMC line, hMSC-hTERT-E6/E7, stably expressing SF-1 induced cell differentiation into cells with a testis type and adrenal type steroidogenic capacity, respectively (14). These results suggest a promising utility of BMCs as a regenerative source of steroidogenic cells.

Adipose tissue is also a mesodermally derived organ that contains a stromal cell population containing microvascular endothelial cells, smooth muscle cells, and mesenchymal stem cells (15, 16). Adipose tissue-derived mesenchymal cells (AMCs) share many characteristics with bone marrow cells, including extensive proliferative potential and the ability to undergo multilineage differentiation (17). AMCs, like BMCs, have differentiated into bone, cartilage, fat, muscle, heart, blood vessels, and nerve (16). However, no reports have described the differentiation capability into steroidogenic cells. Adipose tissue is much easier to access than bone marrow and may be a more promising source for the future clinical application of autologous cell transplantation therapy for patients with steroid hormone deficiency. In this regard, we applied the previous technique to AMCs and compared their steroidogenic profiles with those of mouse BMCs. In addition, we have focused on the effect of retinoic acid (RA) on differentiation of AMCs or BMCs to steroidogenic cells because RA has been suggested to play important roles in stem cell differentiation (18) and sexual differentiation, including steroidogenesis (19, 20, 21, 22, 23).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cell line
C57BL/6J (B6) mice were purchased from Charles River (Yokohama, Japan). At all times the animals were treated according to National Institutes of Health guidelines. The Y1 mouse adrenocortical tumor cell line was purchased from Japanese Cell Research Bank (Tokyo, Japan). Y1 cells were maintained in DMEM (Life Technologies, Tokyo, Japan) supplemented with 10% fetal bovine serum at 37 C in 5% CO2.

Long-term mesenchymal cell culture and adenovirus treatment
To prepare recombinant SF-1 adenovirus (Adx-bSF-1) and recombinant adenovirus expressing β-galactosidase (Adx-LacZ) as a control, we followed the same protocol as previously described (10). We prepared BMCs and AMCs from an identical 4-month-old male B6 mouse. The method to isolate BMCs was exactly the same as previously described (10). On the other hand, to isolate AMCs, visceral fat was harvested, treated with 2% collagenase in DMEM/F12, and washed three times with medium A by centrifuge at 600 rpm. Medium A includes {alpha}-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, Tokyo, Japan), and 20% donor horse serum (lot 6603F or 7307F; ICN Biochemicals, Aurora, OH) (10). We seeded the harvested cells in 75-cm2 tissue culture flasks (Nalgene Nunc, Rochester, NY) and incubated them at 33 C in 5% CO2 in air with medium A. Only adherent cells were maintained for several weeks, trypsinized and stored in cell banker at –80 C until use. When needed, we cultured the stored BMCs or AMCs for more than 200 d (almost 20 passages) with medium A, aiming at the expansion of a relatively purified cell population. From this cell population, 1 x 105 BMCs or AMCs were seeded again in collagen one-coated six-well plates (Iwaki, Tokyo, Japan) with medium A. When the cells became subconfluent, they were infected with an appropriate dose of adenovirus expressing bovine SF-1 (Adx-bSF-1). As a control for all experiments, we infected the BMCs or AMCs with recombinant Adx-LacZ. After the infection the cells were also maintained, and the steroid contents in the culture medium incubated from d 14–18 were measured. A summary of the experimental protocol is shown in Fig. 1Go.


Figure 1
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  		FIG. 1. Summary of the experimental protocol. Visceral fat tissue and bone marrow were obtained from an identical 4-month-old male B6 mouse. Visceral fat tissue was treated with collagenase and centrifuged. The cell pellet was cultured, and only the adherent cells were long-term cultured at 33 C for more than 200 d (~20 passages), aiming at the expansion of a relatively purified cell population. Whole bone marrow was harvested by flushing the bones; cultured and adherent cells were long-term cultured the same way as fat cells. From each cell population, 1 x 105 BMCs were seeded again in collagen one-coated six-well plates with medium A (see Materials and Methods), and, when the BMCs or AMCs became subconfluent, the cells were infected with an approximate dose of adenovirus. As a control for all experiments, we infected the BMCs with recombinant Adx-LacZ. After the infection, the cells were also maintained with medium A.

 
Measurement of steroid content in the medium secreted from AMCs
Concentrations of pregnenolone (P5), P4, corticosterone (B), aldosterone, 17{alpha}-hydroxypregnenolone (17-OH P5), F, aldosterone, and T secreted into the culture medium were measured with the collaboration of SRL Co. Ltd. (Tokyo, Japan) using commercial RIA kits (Diagnostic Products Corp., Los Angeles, CA) and respective specific RIA systems developed by SRL Co. (24). Dehydroepiandrosterone (DHEA) was measured using another enzyme immunoassay kit (Assay Designs, Ann Arbor, MI). The secretion of B and T in the medium was also confirmed in the presence or absence of synthetic 1–24 ACTH (Shionogi Co., Osaka, Japan). The detection limits of P5, P4, B, aldosterone, 17-OH P5, F, DHEA, and T were 100 pg/ml, 100 pg/ml, 15 pg/ml, 10 pg/ml, 20 pg/ml, 20 ng/ml, 15 pg/ml, and 50 pg/ml, respectively (10, 11). Aldosterone production was also measured by liquid chromatography electrospray ionization tandem mass spectrometry (25) in collaboration with Teikoku Hormone Medical Co. Ltd. (Tokyo, Japan). The limit of quantification of aldosterone by this method was 1 pg/ml. The steroid contents were corrected by the final cell number determined by a hemocytometer, namely per 104 cells.

Quantitative real- time PCR
We isolated the total RNA from the cultured BMCs, AMCs, and Y1 cells using an RNeasy mini kit (QIAGEN, GmbH, Hiden, Germany), and from testis and adrenals of 4-month-old male B6 mouse using Isogen (Wako Pure Chemical Industries, Osaka, Japan). In cultured system, after removal of the medium, the remaining cells were harvested with 0.01% trypsin-0.02% EDTA in PBS. After determination of the cell number by a hemocytometer, total RNA was extracted from the precipitated cells. We performed quantitative analysis of the mRNA expression of steroidogenic acute regulatory protein (StAR), ACTH-R, and various steroidogenic enzymes, including P450scc, P450c17, P450c11, P450c21, P450ald, 3β-hydroxysteroid dehydrogenase (HSD), and 17β-HSD type 3, by real-time PCR using a LightCycler (Roche Diagnostics GmbH, Mannheim, Germany), as described previously (26). We synthesized first-strand complimentary DNA using 5 µg total RNA as a template and performed PCR in a LightCycler according to the manufacturer’s instructions. The detailed sense/antisense PCR primers, except for LH-R, were previously reported (10). The sense/antisense PCR primers for mouse LH-R were obtained from Takara Biotechnology (Tokyo, Japan). 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 version 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 mRNA expression levels were calibrated to those of β-actin and its ratio to the control mouse adrenal or testis.

Flow cytometry
The protocol essentially followed a previously described method (27). Briefly, 3 x 105 BMCs and AMCs were incubated with either phycoerythrin (PE)-conjugated antimouse CD11b, CD34, CD44, CD45, c-kit and Sca-1 monoclonal antibodies (BD Biosciences, San Jose, CA), or an isotype-matched PE-conjugated rat IgG (BD Biosciences) for 30 min at 4 C. The cells were finally analyzed on a FACScan flow cytometer (BD Biosciences).

Immunofluorescence cell staining
We conducted immunofluorescence cell staining of AMCs using rabbit antibody against bovine SF-1 (kindly provided by Professor Morohashi, Kyushu University, Fukuoka, Japan), as previously described (11). Briefly, inoculated cells were plated onto CC2 treated chamber slides (Nalgene Nunc International Co., Naperville, IL), cultured for 18 d, and fixed with 4% paraformaldehyde at 4 C for 1 h. Immunofluorescence cell staining was then performed according to the manufacturer’s protocol. The fluorescence was observed using fluorescence microscopy (BX-51; Olympus, Tokyo, Japan).

Statistics
One-way ANOVA was used for statistical evaluation. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Similar cell surface marker profiles were observed between BMCs and AMCs
Long-term (>200 d) cultured BMCs and AMCs were subjected to the analysis of cell surface markers by flow cytometry. Surface markers on both AMCs and BMCs were the same as far as several typical markers were examined. Namely, both were negative for CD11b, CD34, and CD45, low expressions of CD44 and c-kit, and high expression of Sca1, suggesting that both AMCs and BMCs share a common character as a mesenchymal cell. The actual flow cytometry data for AMCs are shown in Fig. 2Go.


Figure 2
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  		FIG. 2. Flow cytometric analysis of surface marker expression in cultured AMCs. The flow cytometry experiment was done before the infection with adenovirus using the same AMCs used in the experiment in Fig. 3AGo. There were 3 x 105 AMCs incubated with either PE-conjugated antimouse CD11b, CD34, CD44, CD45, c-kit, and sca-1 monoclonal antibodies, or an isotype-matched PE-conjugated rat IgG for 30 min at 4 C and finally analyzed on a FACScan flow cytometer as previously described (27 ). Fluorescence-2 (FL2) is a channel that collects the emitted light between 560 and 585 nm. FL2-H represents fluorescence level of PE. The cells were negative for CD11b, CD34, and CD45 but showed low expressions of CD44 and c-kit, and high expression of sca-1.

 
AMCs infected with Adx-bSF-1 were steroidogenic
Morphologically, the most cultured AMCs and BMCs initially showed spindle-type fibroblastic cell appearance and then developed a more round-shaped appearance by forced expression of SF-1 with Adx-bSF-1. Immunofluorescence staining using the SF-1 antibody revealed that all AMCs and BMCs infected with 100 multiplicity of infection (MOI) of Adx-bSF-1 predominantly express SF-1 in the nucleus, whereas no such staining was observed in AMCs infected with 100 MOI of Adx-LacZ. The actual staining data for AMCs only are shown in supplemental Fig. 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.

Next, the steroidogenic ability of either AMCs or BMCs transformed by the introduction of SF-1 was examined and compared with each other. The long-term cultured AMCs or BMCs obtained from an identical 4-month-old male B6 mouse were infected with 100 MOI of Adx-bSF-1 or Adx-LacZ as a control, and incubated for 14 d. The steroid content in the medium accumulated for the next 4 d (d 14–18) was then measured (Fig. 1Go).

As expected, a forced expression of SF-1 enabled both AMCs and BMCs to transform into steroidogenic cells. Because the steroidogenic properties of the BMCs were broadly similar, as we previously reported (10), only that of AMCs is shown in Fig. 3AGo. The AMCs infected with Adx-bSF-1 secreted a significant amount of P5, 17-OH P5, P4, B, aldosterone, F, DHEA, and T, whereas those infected with Adx-LacZ did not. All of the steroid contents in the medium from the control cells infected with Adx-LacZ were undetectable. Similar to our previous results observed in mouse BMCs (10), real-time RT-PCR of the AMCs used in the experiment described previously revealed very clear and significant expression of StAR, P450scc, 3β-HSD, P450c21, P450c11, P450c17, and 17β-HSD type 3 mRNA in the AMCs on d 18 after infection with Adx-bSF-1 (Fig. 3BGo). Despite the presence of a significant amount of aldosterone production in AMCs (Fig. 3AGo), we never succeeded to detect the expression of P450ald mRNA by real-time PCR (data not shown). This puzzling situation was also observed in mouse BMCs (10). The significant production of aldosterone from AMCs infected with Adx-bSF-1 but not from AMCs infected with Adx-LacZ was further demonstrated by a nonimmunoassay-based technique, namely, liquid chromatography ionization tandem mass spectrometer in collaboration with Teikoku Hormone Medical (21.8 ± 2.1 pg/104 cells vs. undetectable level, mean ± SD, n = 4). Thus, further studies are needed to optimize the PCR conditions to detect P450ald mRNA. ACTH-R was expressed even in cells infected with Adx-LacZ, though the expression level was very low (2% of the expression in the Y-1 cells), whereas it was decreased with Adx-bSF-1 infection (Fig. 3BGo). On the other hand, LH-R mRNA was not detected in both AMCs and BMCs infected with either Adx-LacZ or Adx-bSF-1, whereas it was surely detected in the mouse testis (data not shown). This is in striking contrast to human BMCs because human BMCs transformed by SF-1 showed a very clear induction of both ACTH-R and LH-R (11).


Figure 3
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  		FIG. 3. Panel A, Steroidogenic profiles of AMCs infected with Adx-bSF-1. Basal secretion of P5, P4, B, aldosterone, 17-OH P5, F, DHEA, and T in the culture medium of the AMCs. The cells were infected with 100 MOI of Adx-bSF-1 or Adx-LacZ as a control and cultured for 14 d. The steroid contents in the medium accumulated for the next 4 d were then measured. The figure represents averages of three independent experiments, and each experiment was done using three to four culture dishes. Panel B, Real-time PCR of StAR, ACTH-R, and steroidogenic enzymes. Relative mRNA expression levels were calibrated to β-actin. A relative ratio to the expression of the: control Y-1 cells (expressed as Y1 = 1) is expressed in the cases of StAR, ACTH-R, P450scc, and 3β-HSD; mouse adrenal (expressed as adrenal = 1) is expressed in the cases of P450c21 and P450c11; and mouse testis (expressed as testis = 1) is expressed in the cases of P450c17 and 17β-HSD type 3. No significant PCR products of P450scc, 3β-HSD, P450c11, P450c17, P450c21, and 17β-HSD type 3 were obtained from control cells infected with Adx-LacZ. Values are means ± SD (n = 3). ***, P < 0.001. S and L indicate BMCs transfected with Adx-bSF-1, and BMCs transfected with Adx-LacZ, respectively. N.D., Not detectable.

 
We next tested the responsiveness of AMCs to ACTH by measuring B and T secretion, and expression levels of several steroidogenic enzymes and proteins using real-time PCR. We infected long-term cultured AMCs with 20 MOI of Adx-bSF-1 or Adx-LacZ, and then stimulated them with 2.4 µM ACTH twice a week. After stimulation on d 14, the medium and cells were collected after incubation for another 4 d. In AMCs infected with Adx-bSF-1, the mRNA expressions of P450c11 and P450c17 were significantly induced by treatment with 2.4 µM ACTH for 4 d (Fig. 4AGo). The expression level of P450c21 mRNA tended to increase with ACTH, but it was not statistically significant. The mRNA expression levels of StAR and ACTH-R were unchanged by the same treatment. The varied responsiveness of steroidogenic enzymes and StAR to ACTH may reflect the different time course of respective mRNA expressions under this experimental condition. No inducible effect of ACTH on the expressions of mRNA for steroidogenic enzymes, StAR and ACTH-R was observed in AMCs infected with Adx-LacZ (data not shown). In almost accord with the aforementioned data, ACTH stimulated the production of B and T from the AMCs infected with Adx-bSF-1 (Fig. 4BGo), but not in the cells infected with Adx-LacZ (data not shown). The direct stimulation of T production by ACTH was also reported in fetal and neonatal mouse testis (28), and in human BMCs infected with Adx-bSF-1 (11). Interestingly, ACTH increased the B to T ratio in the medium. According to these findings, the basic steroidogenic character of AMCs infected with Adx-bSF-1 is very similar to that of BMCs in that both cell types can produce both adrenal- and gonadal-type steroids. However, the steroidogenic properties of AMCs and BMCs are not exactly the same under some conditions, as is described below.


Figure 4
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  		FIG. 4. Panel A, Effect of ACTH on mRNA expressions for StAR, ACTH-R, and steroidogenic enzymes. After infection with Adx-bSF-1 (MOI = 20) or Adx-LacZ (d 0), cells were treated with ACTH twice a week. After stimulation on d 14, the cells were collected after incubation for another 4 d, and total RNA was extracted for real-time PCR. Relative mRNA expression levels were calibrated to β-actin. A relative ratio to the expression of the: control Y-1 cells (expressed as Y1 = 1) is expressed in the cases of StAR and ACTH-R; mouse adrenal (expressed as adrenal = 1) is expressed in the cases of P450c21 and P450c11; and mouse testis (expressed as testis = 1) is expressed in the cases of P450c17. Panel B, Effect of ACTH on the secretion of B and T from cultured AMCs. After infection with Adx-bSF-1 (MOI = 20) or Adx-LacZ (d 0), cells were treated with ACTH twice a week. After stimulation on d 14, the medium was collected after incubation for another 4 d, and the steroid concentration was measured. Values are means ± SD (n = 3). *, P < 0.05; **, P < 0.01. (–) and (+) indicate control and 2.4 µM ACTH, respectively.

 
Steroidogenic profiles of BMCs and AMCs are different
We examined the difference in steroidogenic profiles between AMCs and BMCs by comparing the production of B as a marker of typical adrenal-type steroids and T as a marker of typical gonadal-type steroids. With the increase of MOI of infected Adx-bSF-1, the productions of B and T in both AMCs and BMCs were increased (Fig. 5AGo). However, the increased MOI of infected Adx-bSF-1 caused AMCs to produce relatively more B to T, resulting in an increased B to T ratio in AMCs. On the other hand, the same treatment caused BMCs to produce relatively more T to B, resulting in a decreased B to T ratio in BMCs (Fig. 5AGo). To know the reason for the difference in steroidogenic profiles between AMCs and BMCs, we examined the expression levels of steroidogenic enzymes P450c21, P450c11, P450c17, and 17β-HSD type 3 in both cells on d 18 after infection with Adx-bSF-1(MOI = 50) by real-time PCR (Fig. 5BGo). Expression levels of P450c21 and P450c11 in such cells were determined as the relative ratio to that in mouse adrenal tissue, whereas those of P450c17 and 17β-HSD were determined as the relative ratio to that in mouse testis tissue. AMCs showed about 25-, 10-, and 2.5-fold higher expression of P450c21, P450c11, and 17β-HSD, respectively, than BMCs. However, relative expression level of P450c17 was not statistically different between AMCs and BMCs (Fig. 5BGo). These results suggest that the relatively higher B to T ratio in AMCs with the increase of SF-1 expression is mainly due to higher expression of P450c21 and P450c11. Importantly, such a remarkable difference in steroidogenic profiles of BMCs and AMCs was reproducible in the same experiments using both cells prepared in different chances.


Figure 5
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  		FIG. 5. Panel A, Comparison of the dose effect of infected Adx-bSF-1 on steroid production between AMCs and BMCs. The cells were infected with Adx-bSF-1 (MOI =10, 50, or 100) and cultured for 14 d. The steroid contents of B and T in the medium were allowed to accumulate for the next 4 d and were then measured. Panel B, Comparison of mRNA expression levels of steroidogenic enzymes between AMCs and BMCs infected with Adx-bSF-1 (MOI = 50). Real-time PCR analysis of the expression levels of P450c11, P450c21, P450c17, and 17β-HSD type 3 mRNAs in AMCs and BMCs infected with Adx-bSF-1. The mRNA expression levels relative to the levels of β-actin are expressed. Values are means ± SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
RA potentiates the differentiation of AMCs into adrenal-type lineage and that of BMC into gonadal-type lineage of steroidogenic cells
We first examined the optimal dose of all-trans retinoic acid (ATRA) to see the effect on steroidogenesis in cultured AMCs. Across the concentration range (10–7 to 10–4 M) of ATRA tested, we obtained the maximum stimulation of B production at 10–5 M ATRA in cultured AMCs infected with 50 MOI of Adx-bSF-1. Treatment of the cells with 10–4 M ATRA caused cell death, and no production of B was obtained (supplemental Fig. 2). No production of B was observed from AMCs infected with 50 MOI of Adx-LacZ (data not shown). The same dose effect of ATRA on B production was observed in cultured BMCs infected with 50 MOI of Adx-bSF-1 (data not shown). When 10–5 M ATRA was added to BMC cells infected with 50 MOI of Adx-bSF-1, the treatment caused an increased production of both B and T (Fig. 6AGo). However, this treatment caused AMCs to produce relatively more B than T, thus resulting in an increased B to T ratio. On the other hand, the same treatment caused BMCs to produce relatively more T than B, resulting in a decreased B to T ratio (Fig. 6AGo). The expression levels of steroidogenic enzymes of both cells infected with 50 MOI of Adx-bSF-1 were determined by real-time PCR in the presence or absence of 10–5 M ATRA. Induction of P450c17 by ATRA in AMCs was observed to the same extent as observed in BMCs. The induction of P450c21 by ATRA was largely specific to AMCs. Although the induction of P450c11 by ATRA was observed in both AMCs and BMCs, the absolute induction value was much higher in AMCs. The induction of 17β-HSD type 3 by ATRA was specifically observed in BMCs, and the expression level was rather suppressed in AMCs (Fig. 6BGo). These results suggest that the relatively higher B to T ratio by ATRA in AMCs was due to the increased induction of P450c21 and P450c11, and down-regulation of 17β-HSD. In both BMCs and AMCs infected with Adx-bSF-1 or Adx-LacZ, no induction of endogenous SF-1 was observed in any of the experiments, including the experiments using ATRA (data not shown). The aforementioned different steroidogenic profile of BMCs and AMCs was reproducible even when we used both cells prepared in different chances.


Figure 6
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  		FIG. 6. Panel A, Comparison of the effect of ATRA (10–5 M) on the Adx-bSF-1 (MOI = 50)-induced steroid production between AMCs and BMCs. The cells were infected with Adx-bSF-1 and cultured for 14 d. The steroid contents of B and T in the medium were allowed to accumulate for the next 4 d and were then measured. Panel B, Comparison of mRNA expression levels of steroidogenic enzymes between AMCs and BMCs infected with Adx-bSF-1 in the presence or absence of 10–5 M ATRA. Real-time PCR analysis of the expression levels of P450c11, P450c21, P450c17, and 17β-HSD type 3 mRNAs in AMCs and BMCs infected with Adx-bSF-1. After the infection, cells were treated with either 10–5 M ATRA or vehicle for 18 d. The mRNA expression levels relative to the levels of β-actin are expressed. Values are means ± SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (–) and (+) indicate control and ATRA (10–5 M), respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In general, whatever the sources are, mesenchymal stem cells have differentiated commonly into multiple lineages, including bone, cartilage, fat, muscle, heart, blood vessels, and nerve (9, 16). In the present study, we showed that primary cultured mouse AMCs could also be transformed into steroidogenic cells by forced expression of SF-1, just like primary cultured BMCs (10, 11) and cloned BMCs (14). Although the superficial steroidogenic capacity of both cell types appeared to be very similar with respect to the type of steroids produced and steroidogenic enzymes expressed, more elaborate studies interestingly revealed that the steroidogenic property of AMCs was rather different from that of BMCs, especially in steroidogenic lineage. In particular, in response to SF-1 and/or RA, AMCs were more prone to produce adrenal steroid, B, rather than the gonadal steroid, T, whereas BMCs were prone to show the opposite. These findings uniquely tell us that the character of mesenchymal cells is not always the same but rather depends on the tissue source. Although little is known about the origin of steroidogenic stem cell, 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 adrenogenital primordium (29). Similarly, a common origin for steroidogenic mesenchymal stem cell may occur from some origins before the differentiation of AMCs and BMCs.

With regard to the cell surface markers analyzed in our study, both AMCs and BMCs were negative for monocyte/macrophage marker, CD11b, and hematopoietic stem cell marker, CD45, but low positive for c-kit and high positive for Sca-1, which are hematopoietic and mesenchymal stem/progenitor markers. Both AMCs and BMCs were positive for CD44, a potential mesenchymal stem cell marker, and negative for CD34. Although markers of mouse mesenchymal stem cells were not completely defined (9, 30), these results suggest that both AMCs and BMCs originate from multipotent immature mesenchymal stem cells. We previously reported that mouse BMCs were negative for the CD44 marker. This is probably due to the fact that the C57BL/6Tg14(act-EGFP)osbY01 mouse strain used in the study by Gondo et al. (10) was not the same as the mice in the present study. In an earlier report, CD34 and CD44 were suggested to be useful to classify AMCs and BMCs in the B6 mouse (31). However, in that study, BMCs and AMCs were collected from different mice and cultured in different conditions, making it somewhat difficult to conclude the utility of these markers. From our results showing the common expression pattern of CD34 and CD44 in both cell types, these markers may be less critical in the differentiation of AMCs and BMCs.

Adx-bSF-1-infected AMCs produced relatively more adrenal steroid, B, whereas Adx-bSF-1-infected BMCs produced more gonadal steroids, T, and such differences in the steroid profile are supported by differences in the expression levels of P450c21, P450c11, and 17β-HSD. These trends were dependent on the expression levels of SF-1, suggesting a dosage-sensitive effect of SF-1 on steroidogenic cell lineage. The phenotype of homozygous SF-1 KO mice shows agenesis of both adrenal glands and gonads (3, 5, 6, 7). However, a more predominant dosage-sensitive effect of SF-1 on adrenal rather than gonadal formation has been suggested from a finding of the predominant reduction of adrenal size in heterozygous SF-1 KO mice (32). A similar finding was recently reported in an experiment in which SF-1 transgenic mice harboring varied expression levels of SF-1 among tissues were applied to rescue homozygous SF-1 KO mice; the transgenic mice failed to rescue the adrenal gland but successfully rescued the gonad and spleen (33). Therefore, the steroidogenic cell lineage might be partly directed by SF-1 expression levels or SF-1 transcriptional activity in mesenchymal stem cells. In this sense, AMCs may need a relatively higher SF-1 expression level or greater SF-1 activity to be transformed into adrenal-type steroidogenic cells, but the situation may be the opposite for BMCs. The increased B to T ratio in the AMCs after treatment with ACTH may also reflect this concept because ACTH is well known to increase transcriptional activity of SF-1 through protein kinase A activation (4). However, this speculation may not be true in humans because 46XY human patients who have one allele SF-1 mutation present gonadal dysgenesis or impaired sexual differentiation but have apparently normal adrenocortical function (34, 35, 36). This phenotype of human SF-1 mutant does not match the phenotype of heterozygous SF-1 KO mice (32). Thus, in humans, gonadal formation seems to be more sensitive to SF-1 dosage rather than adrenal gland formation. Therefore, careful studies are needed in the future to determine better the steroidogenic lineage of human AMCs and BMCs.

RA is well known to influence stem cell differentiation (8, 18). Recently, RA was important for the development of Sertoli, germ, and Leydig cells of fetal and neonatal rat (22, 23). RA also stimulates T secretion from human fetal testis organ culture over a short period of time by increasing the expression of StAR, P450scc, and P450c17 (21), although it decreased the total number of germ cells. In mature steroidogenic cells, RA also affects steroidogenesis by modifying several steroidogenic enzymes (19, 20). In this study we also demonstrated that RA may be an important factor to control the steroidogenic lineage of AMCs and BMCs because the treatment with ATRA caused a dramatic change in the steroidogenic profiles of both AMCs and BMCs. Namely, RA introduced an adrenal-type steroid profile by increasing B/T in AMCs, whereas it introduced a gonadal-type steroid profile by decreasing the ratio in BMCs, which is also supported by the expression profiles of steroidogenic enzymes. The major actions of retinoids are transduced through nuclear receptors known as RA receptors (RARs), which bind ATRA and 9-cis-RA, and retinoid X receptor (RXR), which binds 9-cis-RA. Liganded RAR and RXR are thought to act on the promoter of retinoid-responsive genes (37). The different steroidogenic profiles between AMCs and BMCs might reflect the cell-specific difference in the responsiveness to ATRA in some differentiation mechanisms of mesenchymal stem cells. Alternatively, such differences might be simply explained by the cell-specific difference in the transcriptional effect of ATRA on steroidogenic enzymes; different patterns of cofactor recruitment to RAR:RXR heterodimers on the steroidogenic genes between AMCs and BMCs might result in different steroidogenic lineage in both cells. To date, no factor has been found to determine the tissue-specific steroidogenic cell type; such factors would be useful to modify the cell type of mesenchymal stem cells, adrenal- or gonadal-type cells. RA may be one such candidate, and the clarification of the underlying mechanism may provide a clue molecule to determine the steroidogenic lineages.

Taken all together, at least in mice, AMCs may be more appropriate for adrenal-type steroidogenic cell regeneration, whereas BMC may be more appropriate for gonadal-type steroidogenic cell regeneration. This fact may be important when we consider the clinical application of mesenchymal stem cells for autologous cell regeneration therapy in the near future. In this case we should be careful about a concern of adrenocortical tumor formation associated with a relative overexpression or amplification of human tissue SF-1 (38), as also shown by adrenocortical tumor formation in SF-1 transgenic mice (39). Considering such concerns, a continuous effort should be made to develop this promising outcome into a therapeutic application, especially for patients with steroid insufficiency.


   Footnotes
 
This work was supported by a Health and Labor Sciences Research grant, and a grant from the Ministry of Education, Culture, Sports, Science and Culture (No. 16086207: Molecular Mechanisms of Sex Differentiation).

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 19, 2008

Abbreviations: ACTH-R, ACTH receptor; Adx-bSF-1, adenovirus-mediated forced expression of steroidogenic factor 1; Adx-LacZ, adenovirus expressing β-galactosidase; AMC, adipose tissue-derived mesenchymal cell; Ad4BP, adrenal 4 binding protein; ATRA, all-trans retinoic acid; B, corticosterone; BMC, bone marrow mesenchymal cell; B6, C57BL/6J; DHEA, dehydroepiandrosterone; F, cortisol; 17-OH P5, 17{alpha}-hydroxypregnenolone; HSD, hydroxysteroid dehydrogenase; KO, knockout; LH-R, LH receptor; MOI, multiplicity of infection; PE, phycoerythrin; P5, pregnenolone; P4, progesterone; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; SF-1, steroidogenic factor 1; StAR, steroidogenic acute regulatory protein; T, testosterone.

Received December 31, 2007.

Accepted for publication June 9, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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