Expression of steroidogenic enzymes and metabolism of steroids in COS-7 cells known as non-steroidogenic cells
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OPEN
Received: 6 October 2017
Accepted: 15 January 2018
Published: xx xx xxxx
Expression of steroidogenic
enzymes and metabolism of
steroids in COS-7 cells known as
non-steroidogenic cells
Mitsuki Nozaki1, Shogo Haraguchi1,2, Takuro Miyazaki2, Daichi Shigeta1, Noriko Kano1, XiaoFeng Lei2, Joo-ri Kim-Kaneyama2, Hiroyuki Minakata3, Akira Miyazaki2 & Kazuyoshi Tsutsui1
The COS-7 (CV-1 in Origin with SV40 genes) cells are known as non-steroidogenic cells because they
are derived from kidney cells and the kidney is defined as a non-steroidogenic organ. Therefore, COS-7
cells are used for transfection experiments to analyze the actions of functional molecules including
steroids. However, a preliminary study suggested that COS-7 cells metabolize [3H]testosterone to [3H]
androstenedione. These results suggest that COS-7 cells are able to metabolize steroids. Therefore, the
present study investigated the expression of steroidogenic enzymes and the metabolism of steroids
in COS-7 cells. RT-PCR analyses demonstrated the expressions of several kinds of steroidogenic
enzymes, such as cytochrome P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase/
Δ5-Δ4 isomerase, cytochrome P450 7α-hydroxylase, cytochrome P450 17α-hydroxylase/17,20-lyase,
17β-hydroxysteroid dehydrogenase, 5α-reductase, cytochrome P450 21-hydroxylase, cytochrome
P450 11β-hydroxylase, and cytochrome P450 aromatase in COS-7 cells. In addition, steroidogenic
enzymes 3β-HSD, P4507α, 5α-reductase, P450c17, P450c21, P450c11β, and 17β-HSD actively
metabolized various steroids in cultured COS-7 cells. Finally, we demonstrated that 17β-HSD activity
toward androstenedione formation was greater than other steroidogenic enzyme activities. Our results
provide new evidence that COS-7 cells express a series of steroidogenic enzyme mRNAs and actively
metabolize a variety of steroids.
The COS-7 (CV-1 in Origin with SV40 genes) cell line was developed by Prof. Yakov Gluzman in the early 1980s.
It is derived from the CV-1 African green monkey kidney fibroblast cell line transformed by a mutant strain
of Simian Virus 40 (SV40) that codes for the wild-type T-antigen1,2. This cell line has unique characteristics of
fibroblast-like growth and virus susceptibility1,2. These characteristics make COS-7 cells a popular research tool
and an excellent choice for DNA plasmid transfection experiments1–5.
Many previous studies have reported that COS-7 cells are non-steroidogenic cells6–8. The COS-7 cell line is
derived from kidney cells and the kidney is defined as a non-steroidogenic organ9,10. Therefore, COS-7 cells have
been used for transfection experiments to analyze the functions of steroidogenic genes11–13, steroid receptors14–16,
and the effects of steroids on functional molecules17,18.
A preliminary study in our laboratory suggested that COS-7 cells actively metabolize [3H]testosterone to [3H]
androstenedione (S. Haraguchi et al., unpublished observations). In addition, the expression of steroidogenic
enzymes in the kidney of humans19–21 and rodents22–24 has been reported. These results suggest that COS-7 cells
may metabolize steroids.
Based on this background, in the present study, a series of experiments was conducted to demonstrate
the expression of steroidogenic enzymes and metabolism of steroids in COS-7 cells, which are known as
non-steroidogenic cells. Because pregnenolone formation is the first step in steroid synthesis25–27, we first investigated the formation of [3H]pregnenolone from [3H]cholesterol in cultured COS-7 cells. Steroidogenic acute
1
Laboratory of Integrative Brain Sciences, Department of Biology and Center for Advanced Biomedical Sciences of
Waseda University, Tokyo, 162-8480, Japan. 2Department of Biochemistry, Showa University School of Medicine,
Tokyo, 142-8555, Japan. 3Suntory Foundation for Life Sciences, Kyoto, 619-0284, Japan. Correspondence and
requests for materials should be addressed to S.H. (email: ) or K.T. (email: k-tsutsui@
waseda.jp)
SCIentIfIC REpOrTS | (2018) 8:2167 | DOI:10.1038/s41598-018-20226-2
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Primer
Forward primer 5′->3′
Reverse primer 5′->3′
StAR
GAGCTCTCTGCTCGGTTCTC
CGCCCTGATGACACCTTTCT
P450scc
CCCGATTTACAGGAAGTCAGC
TCTGTAGAGGATGCCACGGT
3β-HSD type I
GAGGGAGGAATTTTCCAAGCTCCA
GTCTTTCAGAGTCCACCCATCA
3β-HSD type VII
CCTTCTACAGGGGCAACGAA
GAACACCAGCAGCCAGTAGG
P4507α
GCCTGATCTGCCTAGAAAGCA
TGAATACCAAACAACAAGCGGT
P450c17
CGGCCTCAAGTGACAACTCT
GGTGATAGAGTCACTGCGGAA
17β-HSD type I
TCAGACCCATCCCAGAGCTT
CTCGATCAGGCTCAAGTGGAC
17β-HSD type II
CAAATGGACGTCACGAAGCC
CGTGCCTGCGATACTTGTTC
17β-HSD type III
CCAAAGCCTTTCTTGCGGTC
CACACAAACGCCTTGGAAGC
17β-HSD type IV
TGGCCAGCTATGATTCAGTGG
CAAAGCCAAAGGACAAGCGG
5α-Reductase type I
CGGGCATCGGTGCTTAATTT
GAGTGCATGACAGCAGGAGA
5α-Reductase type II
GTGCATTACTTCCACAGGACA
CAGCCCAAGGAAACAAACCG
5α-Reductase type III
TCAGTGCTGTGGAATGGCTT
CAGTGAATGACCACTCCTGCT
P450arom
TCCCTTTGGACGAAAGTGCT
CTGGTACCGCATGCTCTCAT
P450c11β
GATAGCCTGCATCCCCACAG
AGTTGTCGCCGTACTGGAAG
P450c21
CGACCTCCCCATCTATCTGC
GGGAAGAACTTGATCTTGTCTCCA
Table 1. Primers for PCR analyses.
regulatory protein (StAR; gene name Star) delivers cholesterol to the mitochondrial cytochrome P450 side-chain
cleavage enzyme (P450scc; gene name Cyp11a), which produces pregnenolone. RT-PCR analyses demonstrated
P450scc mRNA expression in COS-7 cells. We further demonstrated that the mRNAs of several kinds of steroidogenic enzymes 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD; gene name Hsd3b), cytochrome
P450 7α-hydroxylase (P4507α; gene name Cyp7b), cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17;
gene name Cyp17), 17β-hydroxysteroid dehydrogenase (17β-HSD; gene name Hsd17b), 5α-reductase (gene
name Srd5a), cytochrome P450 21-hydroxylase (P450c21; gene name Cyp21), cytochrome P450 11β-hydroxylase
(P450c11β; gene name Cyp11b1), and cytochrome P450 aromatase (P450arom; gene name Cyp19) are expressed
in COS-7 cells. In addition, steroidogenic enzymes 3β-HSD, P4507α, 5α-reductase, P450c17, P450c21, P450c11β,
and 17β-HSD were active in cultured COS-7 cells. Especially, androstenedione formation from testosterone catalyzed by 17β-HSD was greater than other steroidogenic enzyme activities. Our results provide new evidence that
COS-7 cells express a series of steroidogenic enzyme mRNAs and actively metabolize a variety of steroids.
Materials and Methods
COS-7 cells. The COS-7 cell line (JCRB9127) was purchased from the Japanese Collection of Research
Bioresources (JCRB) cell bank (Osaka, Japan). COS-7 cells were maintained in DMEM (043-30085; Wako, Osaka,
Japan) supplemented with 10% FBS (S1820-500; BioWest, Nuaill, France) and a 1% penicillin-streptomycin solution (Wako, Osaka, Japan) at 37 °C in a humidified 5% CO2-containing atmosphere. COS-7 cells were used in the
experiments between passages 3 and 15, or passages 30 and 40.
RT-PCR analyses of steroidogenic enzyme mRNAs. Total R (...truncated)
