[Frontiers In Bioscience, Landmark, 24, 1316-1329, June 1, 2019]

Kruppel-like factor 6 regulates Sertoli cell blood-testis barrier

Xiu-Xia Wang1, Yan Zhang1,2, Xiao-Yu Li1, Jian Li1, Ji-Xin Tang1, Yuan-Yuan Li1,3, Shou-Long Deng1, Chuen-Yan Cheng4, Yi-Xun Liu1,2

1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beichen west Road, Chaoyang District, Beijing 100101, China, 2Changsha Reproductive Medicine Hospital, Changsha, China, 3University of Chinese Academy of Sciences, Beijing 100049, China, 4The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, 1230 York Ave, New York 10065, USA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Animals
3.2. Primary mouse SC isolation
3.3. Knockdown of Klf6 by siRNA and shRNA
3.4. TUNEL assay
3.5. RNA extraction and RT-PCR
3.6. Western blot analysis
3.7. Immunofluorescent staining
3.8. Assessment of SC tight junction permeability barrier in vitro
3.9. Electron microscopy
3.10. Luciferase assay
3.11. Statistics
4. Results
4.1. Klf6 is expressed in Sertoli cells of mouse testis
4.2. Klf6 is required for the proliferative activity of cultured SCs
4.3. Klf6 regulates the integrity and permeability of BTB
4.4. Klf6 regulates the expression of BTB proteins in SCs
4.5. Zo-1 and Claudin-3 are direct targets of transcription factor Klf6
5. Discussion
6. Acknowledgement
7. References

1. ABSTRACT

Blood-testis barrier (BTB) that is constructed by testicular Sertoli cells (SCs) is essential for spermatogenesis. Krüppel-like factor 6 (Klf6), a nuclear transcription regulator, is reported to be associated with tight junction molecules of BTB between SCs during spermatogenesis; however, the specific regulatory role and mechanism of Klf6 in BTB regulation are still unknown. Here, we primarily confirmed the temporal and spatial expression patterns of Klf6 in mouse testes. Then, Klf6 was silenced in mouse cultured SCs using either Klf6-siRNA or Klf6-shRNA lentivirus. We mainly found that: (i) Klf6 was indispensable for the proliferative activity of mouse SCs; (ii) Klf6 regulated the integrity and permeability of BTB; (iii) Klf6 knockdown led to the significant upregulation of Zo-1, Claudin-11 and Vimentin, and downregulation of Claudin-3. Furthermore, Zo-1 and Claudin-3, participated in the tight junction remolding, were determined as targets of transcription factor Klf6 by luciferase assay. In summary, our findings suggest that Klf6 regulates the BTB assembly and disassembly via mainly targeting Zo-1 and Claudin-3 in mouse SCs.

7. REFERENCES

1. B. E. Smith and R. E. Braun: Germ cell migration across Sertoli cell tight junctions. Science, 338(6108), 798-802 (2012)
DOI: 10.1126/science.1219969

2. M. Chihara, S. Otsuka, O. Ichii, Y. Hashimoto and Y. Kon: Molecular dynamics of the blood–testis barrier components during murine spermatogenesis. Molecular reproduction and development, 77(7), 630-639 (2010)
DOI: 10.1002/mrd.21200

3. M. D. Griswold: The central role of Sertoli cells in spermatogenesis. In: Seminars in cell & developmental biology. Elsevier,(1998)
DOI: 10.1006/scdb.1998.0203

4. M. Dym and D. W. Fawcett: The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biology of reproduction, 3(3), 308-326 (1970)
DOI: 10.1093/biolreprod/3.3.3.08

5. N. Defamie, I. Berthaut, B. Mograbi, D. Chevallier, J.-P. Dadoune, P. Fénichel, D. Segretain and G. Pointis: Impaired gap junction connexin43 in Sertoli cells of patients with secretory azoospermia: a marker of undifferentiated Sertoli cells. Laboratory investigation, 83(3), 449 (2003)
DOI: 10.1097/01.lab.0000059928.82702.6d

6. X.-Y. Li, Y. Zhang, X.-X. Wang, C. Jin, Y.-Q. Wang, T.-C. Sun, J. Li, J.-X. Tang, A. Batool and S.-L. Deng: Regulation of blood–testis barrier assembly in vivo by germ cells. The FASEB Journal, 32(3), 1653-1664 (2017)
DOI: 10.1096/fj.201700681R

7. E. A. Fischer, M.-C. Verpont, L. A. Garrett-Sinha, P. M. Ronco and J. A. Rossert: Klf6 is a zinc finger protein expressed in a cell-specific manner during kidney development. Journal of the American Society of Nephrology, 12(4), 726-735 (2001)
DOI: 10.1053/jren.2001.23138

8. D. Slavin, V. Sapin, F. Lopez-Diaz, P. Jacquemin, N. Koritschoner, B. Dastugue, I. Davidson, B. Chatton and J. Bocco: The Krüppel-like core promoter binding protein gene is primarily expressed in placenta during mouse development. Biology of reproduction, 61(6), 1586-1591 (1999)
DOI: 10.1095/biolreprod61.6.1.586

9. A. B. Bialkowska, V. W. Yang and S. K. Mallipattu: Krüppel-like factors in mammalian stem cells and development. Development, 144(5), 737-754 (2017)
DOI: 10.1242/dev.145441

10. G. Narla, K. E. Heath, H. L. Reeves, D. Li, L. E. Giono, A. C. Kimmelman, M. J. Glucksman, J. Narla, F. J. Eng and A. M. Chan: KLF6, a candidate tumor suppressor gene mutated in prostate cancer. Science, 294(5551), 2563-2566 (2001)
DOI: 10.1126/science.1066326

11. J. Ma, Y. Yao, P. Wang, Y. Liu, L. Zhao, Z. Li, h. Li and Y. Xue: MiR-181a regulates blood-tumor barrier permeability by targeting Krüppel-like factor 6. Journal of Cerebral Blood Flow & Metabolism, 34(11), 1826-1836 (2014)
DOI: 10.1038/jcbfm.2014.152

12. H. Cai, Y. Ren, X. X. Li, J. L. Yang, C. P. Zhang, M. Chen, C. H. Fan, X. Q. Hu, Z. Y. Hu and F. Gao: Scrotal heat stress causes a transient alteration in tight junctions and induction of TGF‐β expression. International journal of andrology, 34(4pt1), 352-362 (2011)
DOI: 10.1096/fj.201700681R

13. X.-X. Li, S.-R. Chen, B. Shen, J.-L. Yang, S.-Y. Ji, Q. Wen, Q.-S. Zheng, L. Li, J. Zhang and Z.-Y. Hu: The heat-induced reversible change in the blood-testis barrier (BTB) is regulated by the androgen receptor (AR) via the partitioning-defective protein (Par) polarity complex in the mouse. Biology of reproduction, 89(1) (2013)
DOI: 10.1095/biolreprod.113.109405

14. E. O’Keefe: siRNAs and shRNAs: Tools for protein knockdown by gene silencing. World Lab (2013)
DOI: 10.13070/mm.cn.3.197

15. M. A. Tremblay, R. E. Mendoza-Villarroel, N. M. Robert, F. Bergeron and J. J. Tremblay: KLF6 cooperates with NUR77 and SF1 to activate the human INSL3 promoter in mouse MA-10 leydig cells. Journal of molecular endocrinology, JME-15-0139 (2016)
DOI: 10.1530/JME-15-0139

16. A. Sandelin, W. Alkema, P. Engström, W. W. Wasserman and B. Lenhard: JASPAR: an open‐access database for eukaryotic transcription factor binding profiles. Nucleic acids research, 32(suppl_1), D91-D94 (2004)
DOI: 10.1093/nar/gkh012

17. A. Khan, O. Fornes, A. Stigliani, M. Gheorghe, J. A. Castromondragon, R. D. L. Van, A. Bessy, J. Chèneby, S. R. Kulkarni and G. Tan: JASPAR 2018: update of the open-access database of transcription factor binding profiles and its web framework. Nucleic Acids Research, 77(21), e43 (2017)
DOI: 10.1158/0008-5472.CAN-17-0331

18. S. Kojima, S. Hayashi, K. Shimokado, Y. Suzuki, J. Shimada, M. P. Crippa and S. L. Friedman: Transcriptional activation of urokinase by the Krüppel-like factor Zf9/COPEB activates latent TGF-β1 in vascular endothelial cells. Blood, 95(4), 1309-1316 (2000)
DOI: 10.1016/S0016-5085(13)62748-1

19. V. G. Warke, M. P. Nambiar, S. Krishnan, K. Tenbrock, D. A. Geller, N. P. Koritschoner, J. L. Atkins, D. L. Farber and G. C. Tsokos: Transcriptional activation of the human inducible nitric-oxide synthase promoter by Krüppel-like factor 6. Journal of Biological Chemistry, 278(17), 14812-14819 (2003)
DOI: 10.1074/jbc.M300787200

20. W. Su, D. D. Mruk and C. Y. Cheng: Regulation of actin dynamics and protein trafficking during spermatogenesis–insights into a complex process. Critical reviews in biochemistry and molecular biology, 48(2), 153-172 (2013)
DOI: 10.3109/10409238.2012.758084

21. D. Günzel and A. S. Yu: Claudins and the modulation of tight junction permeability. Physiological Reviews, 93(2), 525-69 (2013)
DOI: 10.1152/physrev.00019.2012

22. J. Meng, R. W. Holdcraft, J. E. Shima, M. D. Griswold and R. E. Braun: Androgens regulate the permeability of the blood–testis barrier. Proceedings of the National Academy of Sciences, 102(46), 16696-16700 (2005)
DOI: 10.1073/pnas.0506084102

23. P. Chakraborty, F. William Buaas, M. Sharma, B. E. Smith, A. R. Greenlee, S. M. Eacker and R. E. Braun: Androgen-dependent Sertoli cell tight junction remodeling is mediated by multiple tight junction components. Molecular Endocrinology, 28(7), 1055-1072 (2014)
DOI: 10.1210/me.2013-1134

24. N. Matsumoto, A. Kubo, H. Liu, K. Akita, F. Laub, F. Ramirez, G. Keller and S. L. Friedman: Developmental regulation of yolk sac hematopoiesis by Krüppel-like factor 6. Blood, 107(4), 1357-1365 (2006)
DOI: 10.1182/blood-2005-05-1916

25. M. Long, S. Yang, S. Dong, X. Chen, Y. Zhang and J. He: Characterization of semen quality, testicular marker enzyme activities and gene expression changes in the blood testis barrier of Kunming mice following acute exposure to zearalenone. Environmental Science and Pollution Research, 24(35), 27235-27243 (2017)
DOI: 10.1007/s11356-017-0299-1

26. I. Junichi, U. Kazuaki, T. Sachiko, F. Mikio and T. Shoichiro: Requirement of ZO-1 for the formation of belt-like adherens junctions during epithelial cell polarization. Journal of Cell Biology, 176(6), 779-786 (2007)
DOI: 10.1083/jcb.200612080

27. N. P. Chung and C. Y. Cheng: Is cadmium chloride-induced inter-Sertoli tight junction permeability barrier disruption a suitable in vitro model to study the events of junction disassembly during spermatogenesis in the rat testis? Endocrinology, 142(5), 1878 (2001)
DOI: 10.1210/endo.142.5.8.145

28. L. Su, D. D. Mruk, W. M. Lee and C. Y. Chenga: Differential effects of testosterone and TGF-β3 on endocytic vesicle-mediated protein trafficking events at the blood–testis barrier. Experimental Cell Research, 316(17), 2945-2960 (2010)
DOI: 10.1016/j.yexcr.2010.07.018

Key Words: Kruppel-like factor 6, Klf6, Blood-testis barrier, Sertoli cells, Zo-1; Claudin-3

Send correspondence to: Xiu-Xia Wang, 1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China, Tel: 86-010-64807038; Fax: 86-010-64807038, E-mail: wangxiuxia@ioz.ac.cn