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Reply to comments to “Letter to the Editor: comment on Azmahani et al. steroidogenic enzymes, their related transcription factors and nuclear receptors in human sebaceous glands under normal and pathological conditions”
Journal of Steroid Biochemistry & Molecular Biology 155 (2016) 178–180
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry & Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb
Discussion
Reply to comments to “Letter to the Editor: comment on Azmahani et al. steroidogenic enzymes, their related transcription factors and nuclear receptors in human sebaceous glands under normal and pathological conditions” Abdullah Azmahania,b , Yasuhiro Nakamuraa,* , Saulo J.A. Felizolaa , Yohei Ozawaa,c, Kazue Isea , Takayoshi Inoued , Keely M. McNamaraa , Masao Doie , Hitoshi Okamurae, Christos C. Zouboulisf , Setsuya Aibag , Hironobu Sasanoa a
Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan Faculty of Medicine and Health Sciences, University Sultan Zainal Abidin, Kuala Terengganu, Terengganu, Malaysia Division of Advanced Surgical Science and Technology, Tohoku University Graduate School of Medicine, Sendai, Japan d Biological Science Laboratories, Kao Corporation, Haga, Tochigi, Japan e Department of Systems Biology, School of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan f Departments of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Dessau, Germany g Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan b c
The authors regret to report that in this article there was a mistake in the primers used for steroidogenic factor 1 (SF-1). As Dr. Pivonello kindly indicated, the primers did correspond to splicing factor-1 not steroidogenic factor 1. This erratum seeks to make the relevant corrections to the quantitative RT-PCR (qPCR) method and respective results in Azmahani et al. [1]. With the updated primers we did find that SF-1 was, indeed, detected in H295R adrenocortical cells but not in SZ95 sebocytes with the full details of both method and results given below. We must apologize to all readers of our original manuscript for this careless error in the selection of primers. Table 3 provides the information of corrected primers for SF-1 or steroidogenic factor-1 encoded by the NR5A1 gene (nuclear receptor subfamily 5, group A, member 1) gene forward, 50 AGGAGAACGTTTGGTACAATCGACATCC-30 , reverse, 50 -ACACA0 CATTTCAAGGAGTGTCTAGAAGCTT-3 generating a 144-bp product. We verified the qPCR product produced by these primers by using sequencing of the SF-1. PCR products were purified with a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and were visualized on 1% agarose gels with ethidium bromide. Sequencing of PCR products with the same forward and reverse primers were performed according to manufacturer’s recommendations using a
DOI of original article: http://dx.doi.org/10.1016/j.jsbmb.2014.07.010 * Corresponding author at: Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. Fax: +81 22 717 8051. E-mail address: [email protected] (Y. Nakamura). http://dx.doi.org/10.1016/j.jsbmb.2015.09.028 0960-0760/ ã 2015 Elsevier Ltd. All rights reserved.
BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies Japan, Tokyo). The cycling conditions were 96 C for 1 min, followed by 96 C for 10 s, 50 C for 5 s, and then 60 C for 4 min using a BIOER thermal cycler (Bioer Technology, Beijing city, China). Sequenced products were purified by a BigDye XTerminator Purification Kit (Life Technologies Japan, Tokyo) and analyzed by an ABI 3500xL Genetic Analyzer (Life Technologies Japan, Tokyo). Forward and reverse sequences of samples were reviewed using Sequence Scanner Software 2 (Life Technologies Japan, Tokyo). Sequences were analyzed and compared with currently used qPCR. Sequences were also verified using a BLAST search to further confirm the qPCR results. Our sequence analysis did reveal match primers of NR5A1 in H295R adrenocortical cells NCBI Reference Sequence: NM_004959.4 (data not shown). Supplementary figure demonstrated the verification of PCR product on agarose gel. Our results demonstrated that SF-1 primers were detected in H295R adrenocortical cells but not in SZ95 sebocytes. Corrected results of the SF-1 primers set of Azmahani et al. are reported in Table 3 and Fig. 8. Fig. 8 reports the corrected results of SZ95 sebocytes and H295R adrenocortical cells that were described in Section Result 3.4 of Azmahani et al. Results of our present study did reveal that SF-1 expression was detected in H295R cells but not in SZ95 sebocytes. Section Result 3.4 and Discussion should be replaced by the following paragraph. 3.4. qPCR analysis The expression of 3b-HSD1, CYP11A1, StAR, 17b-HSD5, 5a-red1, NGFI-B and GATA6 mRNA was detected in both SZ95 sebocytes and H295R adrenocortical cells (Fig. 8A). However, 3b-HSD2, CYP17A1, GATA4, SF-1 and 5a-red2 were not detectable in SZ95 sebocytes in
A. Azmahani et al. / Journal of Steroid Biochemistry & Molecular Biology 155 (2016) 178–180 Table 3 Primer and probe sequences of the PCR products for the genes examined in the present study. mRNA
Sequence
3b-HSD1
FWD 50 -AGAAGAGCCTCTGGAAAACACATG-30 REV 50 -TAAGGCACAAGTGTACAGGGTGC-30 PROBE 50 -FAM-CCATACCCACACAGC-NFQ-MGB-30
3b-HSD2
FWD 50 -AGAAGAGCCTCTGGAAAACACATG-30 REV 50 -CGCACAAGTGTACAAGGTATCACCA-30 PROBE 50 -VIC-TCCATACCCGTACAGCA-NFQ-MGB-30
CYP11A1
FWD 50 -GAGATGGCACGCAACCTGAAG-30 REV 50 -CTTAGTGTCTCCTTGATGCTGGC-30
StAR
FWD 50 -GAGAAGTCTTGCTTTATGGGCTCAAGAATG-30 REV 50 -GGTGCCTATGAAAGCAATAGGGAAACATGT-30
17b-HSD5
FWD 5 -GTCATCCGTATTTCAACCGGAGTAAATTGC-3 REV 50 -CATCGTTTGTCTCGTTGAGATCCCAGA-30
5a-red1
FWD 50 -CCAATGGCGCTTCTCTATGGACTTTGTAAA-30 REV 50 -CCCAGAGCTTGAAATTCTGACCTGTTACA-30
0
179
Table 3 (Continued) mRNA
Sequence
5a-red2
FWD 50 -GGCGACAGTTGCTTCTTAGCTATTGCT-30 REV 50 -GTGGGTATGAAGCCACACATGTACTTGGAT-30
CYP17A1
FWD 50 -TGAGTTTGCTGTGGACAAGG-30 REV 50 -TCCGAAGGGCAAATAGCTTA-30
NGFI-B
FWD 50 -CCTCCACCCATCATTGACAAGATCTTCATG-30 REV 50 -GCTTATTTACAGCACGGCGCTATGTGTTA -30
GATA4
FWD 50 -TCGAAGCAAACAAACACAACACAACAGAAT-30 REV 50 -CAGCTCTGCTGAGAAAGAGGAAGGAG-30
GATA6
FWD 50 -TCATGTGCGTTCATGGAGAAGATCACTT-30 REV 50 -ACTGACTTCAGATCAGCCACACAATATGA-30
SF-1SF1
FWD 50 -AGGAGAACGTTTGGTACAATCGACATCC-30 REV 50 -ACACACATTTCAAGGAGTGTCTAGAAGCTT-30
RPL13A
FWD 50 -CCTGGAG-GAGAAGAGGAAAG-30 REV 50 -TTGAGGACCT-CTGTGTATTT-30
0
Fig. 8. The relative expression levels of steroidogenic enzyme mRNAs. (Panel A) The mRNA levels of 3b-HSD1, 3b-HSD2, CYP11A1, StAR, 17b-HSD5, 5a-red1, CYP17A1, NGFI-B, GATA6 and SF-1 in SZ95 sebocytes and H295R cells. A significant difference was defined by **P < 0.01 and ***P < 0.001. The standard error is defined in each group. ND: not detectable. (Panel B–G) mRNA expression of the steroidogenic enzymes 3b-HSD1, CYP11A1, StAR, 5a-red1, 17b-HSD5 and NGFI-B in SZ95 sebocytes for 3 h, 6 h, 12 h or 24 h in response to forskolin or vehicle treatment. Six samples were analysed from two independent groups. Each group n = 3. The significant difference was defined as follows: *P < 0.05 and **P < 0.01 compared with untreated cells. The standard error is defined in each group. B: basal; F: for skolin.
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this analysis (Data not shown). The mRNA levels of StAR and CYP11A1 were significantly higher in H295R cells than in SZ95 sebocytes (P < 0.001). Moreover, the levels of 3b-HSD1, 5a-red1 and GATA6 were significantly higher in SZ95 sebocytes than H295R cells (P < 0.01).
that it is expressed, it may not be an important regulator of steroid metabolism or production in the sebaceous gland [7,12,33]. Once again we must appreciate the commentary from Dr. Pivonello and apologize to all readers of our original manuscript for the error.
4. Discussion
Appendix A. Supplementary data
Compared with the H295R adrenocortical cell line, SZ95 sebocytes expressed significantly lower levels of multiple steroidogenic enzymes (3b-HSD2, 17b-HSD5, CYP11A1 and StAR) as well as a lower level of the transcription factor NGFI-B. Interestingly, the levels of a subset of steroidogenic pathways (3b-HSD1 and 5a-red1) and transcription factor were significantly higher in SZ95 sebocytes than in H295R adrenocortical cells. Our results failed to demonstrate SF-1 in human sebocytes in both IHC and qPCR analysis, and although previous studies have suggested
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jsbmb.2015. 09.028. References [1] A. Azmahani, Y. Nakamura, S.J.A. Felizola, Y. Ozawa, K. Ise, T. Inoue, et al., Steroidogenic enzymes, their related transcription factors and nuclear receptors in human sebaceous glands under normal and pathological conditions, J. Steroid Biochem. Mol. Biol. 144 (2014) 268.
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry & Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb
Discussion
Reply to comments to “Letter to the Editor: comment on Azmahani et al. steroidogenic enzymes, their related transcription factors and nuclear receptors in human sebaceous glands under normal and pathological conditions” Abdullah Azmahania,b , Yasuhiro Nakamuraa,* , Saulo J.A. Felizolaa , Yohei Ozawaa,c, Kazue Isea , Takayoshi Inoued , Keely M. McNamaraa , Masao Doie , Hitoshi Okamurae, Christos C. Zouboulisf , Setsuya Aibag , Hironobu Sasanoa a
Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan Faculty of Medicine and Health Sciences, University Sultan Zainal Abidin, Kuala Terengganu, Terengganu, Malaysia Division of Advanced Surgical Science and Technology, Tohoku University Graduate School of Medicine, Sendai, Japan d Biological Science Laboratories, Kao Corporation, Haga, Tochigi, Japan e Department of Systems Biology, School of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan f Departments of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Dessau, Germany g Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan b c
The authors regret to report that in this article there was a mistake in the primers used for steroidogenic factor 1 (SF-1). As Dr. Pivonello kindly indicated, the primers did correspond to splicing factor-1 not steroidogenic factor 1. This erratum seeks to make the relevant corrections to the quantitative RT-PCR (qPCR) method and respective results in Azmahani et al. [1]. With the updated primers we did find that SF-1 was, indeed, detected in H295R adrenocortical cells but not in SZ95 sebocytes with the full details of both method and results given below. We must apologize to all readers of our original manuscript for this careless error in the selection of primers. Table 3 provides the information of corrected primers for SF-1 or steroidogenic factor-1 encoded by the NR5A1 gene (nuclear receptor subfamily 5, group A, member 1) gene forward, 50 AGGAGAACGTTTGGTACAATCGACATCC-30 , reverse, 50 -ACACA0 CATTTCAAGGAGTGTCTAGAAGCTT-3 generating a 144-bp product. We verified the qPCR product produced by these primers by using sequencing of the SF-1. PCR products were purified with a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and were visualized on 1% agarose gels with ethidium bromide. Sequencing of PCR products with the same forward and reverse primers were performed according to manufacturer’s recommendations using a
DOI of original article: http://dx.doi.org/10.1016/j.jsbmb.2014.07.010 * Corresponding author at: Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. Fax: +81 22 717 8051. E-mail address: [email protected] (Y. Nakamura). http://dx.doi.org/10.1016/j.jsbmb.2015.09.028 0960-0760/ ã 2015 Elsevier Ltd. All rights reserved.
BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies Japan, Tokyo). The cycling conditions were 96 C for 1 min, followed by 96 C for 10 s, 50 C for 5 s, and then 60 C for 4 min using a BIOER thermal cycler (Bioer Technology, Beijing city, China). Sequenced products were purified by a BigDye XTerminator Purification Kit (Life Technologies Japan, Tokyo) and analyzed by an ABI 3500xL Genetic Analyzer (Life Technologies Japan, Tokyo). Forward and reverse sequences of samples were reviewed using Sequence Scanner Software 2 (Life Technologies Japan, Tokyo). Sequences were analyzed and compared with currently used qPCR. Sequences were also verified using a BLAST search to further confirm the qPCR results. Our sequence analysis did reveal match primers of NR5A1 in H295R adrenocortical cells NCBI Reference Sequence: NM_004959.4 (data not shown). Supplementary figure demonstrated the verification of PCR product on agarose gel. Our results demonstrated that SF-1 primers were detected in H295R adrenocortical cells but not in SZ95 sebocytes. Corrected results of the SF-1 primers set of Azmahani et al. are reported in Table 3 and Fig. 8. Fig. 8 reports the corrected results of SZ95 sebocytes and H295R adrenocortical cells that were described in Section Result 3.4 of Azmahani et al. Results of our present study did reveal that SF-1 expression was detected in H295R cells but not in SZ95 sebocytes. Section Result 3.4 and Discussion should be replaced by the following paragraph. 3.4. qPCR analysis The expression of 3b-HSD1, CYP11A1, StAR, 17b-HSD5, 5a-red1, NGFI-B and GATA6 mRNA was detected in both SZ95 sebocytes and H295R adrenocortical cells (Fig. 8A). However, 3b-HSD2, CYP17A1, GATA4, SF-1 and 5a-red2 were not detectable in SZ95 sebocytes in
A. Azmahani et al. / Journal of Steroid Biochemistry & Molecular Biology 155 (2016) 178–180 Table 3 Primer and probe sequences of the PCR products for the genes examined in the present study. mRNA
Sequence
3b-HSD1
FWD 50 -AGAAGAGCCTCTGGAAAACACATG-30 REV 50 -TAAGGCACAAGTGTACAGGGTGC-30 PROBE 50 -FAM-CCATACCCACACAGC-NFQ-MGB-30
3b-HSD2
FWD 50 -AGAAGAGCCTCTGGAAAACACATG-30 REV 50 -CGCACAAGTGTACAAGGTATCACCA-30 PROBE 50 -VIC-TCCATACCCGTACAGCA-NFQ-MGB-30
CYP11A1
FWD 50 -GAGATGGCACGCAACCTGAAG-30 REV 50 -CTTAGTGTCTCCTTGATGCTGGC-30
StAR
FWD 50 -GAGAAGTCTTGCTTTATGGGCTCAAGAATG-30 REV 50 -GGTGCCTATGAAAGCAATAGGGAAACATGT-30
17b-HSD5
FWD 5 -GTCATCCGTATTTCAACCGGAGTAAATTGC-3 REV 50 -CATCGTTTGTCTCGTTGAGATCCCAGA-30
5a-red1
FWD 50 -CCAATGGCGCTTCTCTATGGACTTTGTAAA-30 REV 50 -CCCAGAGCTTGAAATTCTGACCTGTTACA-30
0
179
Table 3 (Continued) mRNA
Sequence
5a-red2
FWD 50 -GGCGACAGTTGCTTCTTAGCTATTGCT-30 REV 50 -GTGGGTATGAAGCCACACATGTACTTGGAT-30
CYP17A1
FWD 50 -TGAGTTTGCTGTGGACAAGG-30 REV 50 -TCCGAAGGGCAAATAGCTTA-30
NGFI-B
FWD 50 -CCTCCACCCATCATTGACAAGATCTTCATG-30 REV 50 -GCTTATTTACAGCACGGCGCTATGTGTTA -30
GATA4
FWD 50 -TCGAAGCAAACAAACACAACACAACAGAAT-30 REV 50 -CAGCTCTGCTGAGAAAGAGGAAGGAG-30
GATA6
FWD 50 -TCATGTGCGTTCATGGAGAAGATCACTT-30 REV 50 -ACTGACTTCAGATCAGCCACACAATATGA-30
SF-1SF1
FWD 50 -AGGAGAACGTTTGGTACAATCGACATCC-30 REV 50 -ACACACATTTCAAGGAGTGTCTAGAAGCTT-30
RPL13A
FWD 50 -CCTGGAG-GAGAAGAGGAAAG-30 REV 50 -TTGAGGACCT-CTGTGTATTT-30
0
Fig. 8. The relative expression levels of steroidogenic enzyme mRNAs. (Panel A) The mRNA levels of 3b-HSD1, 3b-HSD2, CYP11A1, StAR, 17b-HSD5, 5a-red1, CYP17A1, NGFI-B, GATA6 and SF-1 in SZ95 sebocytes and H295R cells. A significant difference was defined by **P < 0.01 and ***P < 0.001. The standard error is defined in each group. ND: not detectable. (Panel B–G) mRNA expression of the steroidogenic enzymes 3b-HSD1, CYP11A1, StAR, 5a-red1, 17b-HSD5 and NGFI-B in SZ95 sebocytes for 3 h, 6 h, 12 h or 24 h in response to forskolin or vehicle treatment. Six samples were analysed from two independent groups. Each group n = 3. The significant difference was defined as follows: *P < 0.05 and **P < 0.01 compared with untreated cells. The standard error is defined in each group. B: basal; F: for skolin.
180
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this analysis (Data not shown). The mRNA levels of StAR and CYP11A1 were significantly higher in H295R cells than in SZ95 sebocytes (P < 0.001). Moreover, the levels of 3b-HSD1, 5a-red1 and GATA6 were significantly higher in SZ95 sebocytes than H295R cells (P < 0.01).
that it is expressed, it may not be an important regulator of steroid metabolism or production in the sebaceous gland [7,12,33]. Once again we must appreciate the commentary from Dr. Pivonello and apologize to all readers of our original manuscript for the error.
4. Discussion
Appendix A. Supplementary data
Compared with the H295R adrenocortical cell line, SZ95 sebocytes expressed significantly lower levels of multiple steroidogenic enzymes (3b-HSD2, 17b-HSD5, CYP11A1 and StAR) as well as a lower level of the transcription factor NGFI-B. Interestingly, the levels of a subset of steroidogenic pathways (3b-HSD1 and 5a-red1) and transcription factor were significantly higher in SZ95 sebocytes than in H295R adrenocortical cells. Our results failed to demonstrate SF-1 in human sebocytes in both IHC and qPCR analysis, and although previous studies have suggested
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jsbmb.2015. 09.028. References [1] A. Azmahani, Y. Nakamura, S.J.A. Felizola, Y. Ozawa, K. Ise, T. Inoue, et al., Steroidogenic enzymes, their related transcription factors and nuclear receptors in human sebaceous glands under normal and pathological conditions, J. Steroid Biochem. Mol. Biol. 144 (2014) 268.