Transgenic mouse model for conditional expression of influenza hemagglutinin-tagged human SLC20A1/PIT1


Autoři: Sampada Chande aff001;  Bryan Ho aff001;  Jonathan Fetene aff001;  Clemens Bergwitz aff001
Působiště autorů: Section of Endocrinology and Metabolism, Yale University School of Medicine, New Haven, CT, United States of America aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223052

Souhrn

To further investigate the role of the phosphate (Pi) transporter PIT1 in Pi homeostasis and tissue mineralization, we developed a transgenic mouse expressing the C-terminal influenza hemagglutinin (HA) epitope-tagged human PIT1 transporter under control of the cytomegalovirus/chicken beta actin/rabbit beta-globin gene (CAG) promotor and a loxP-stop-loxP (LSL) cassette permitting conditional activation of transgene expression (LSL-HA-hPITtg/+). For an initial characterization of this conditional mouse model, germline excision of the LSL cassette was performed to induce expression of the transgene in all mouse tissues (HA-hPIT1tg/+). Recombination was confirmed using genomic DNA obtained from blood samples of these mice. Furthermore, expression of HA-hPIT1 was found to be at least 10-fold above endogenous mouse Pit1 in total RNA isolated from multiple tissues and from cultured primary calvaria osteoblasts (PCOB) estimated by semi-quantitative RT-PCR. Robust expression of the HA-hPIT1 protein was also observed upon immunoblot analysis in most tissues and permits HA-mediated immunoprecipitation of the transporter. Characterization of the phenotype of HA-hPIT1tg/+ mice at 80 days of age when fed a standard chow (0.7% Pi and 1% calcium) showed elevated plasma Pi, but normal plasma iPTH, iFGF23, serum calcium, BUN, 1,25-dihydroxy vitamin D levels and urine Pi, calcium and protein excretion when compared to WT littermates. Likewise, no change in bone mineral density was observed upon uCT analysis of the distal femur obtained from these mice. In conclusion, heterozygous overexpression of HA-hPIT1 is compatible with life and causes hyperphosphatemia while bone and mineral metabolism of these mice are otherwise normal.

Klíčová slova:

Kidneys – Mouse models – Osteocytes – Phosphates – Polymerase chain reaction – Urine – Bone and mineral metabolism – Fibroblast growth factor


Zdroje

1. Chande S, Bergwitz C. Role of phosphate sensing in bone and mineral metabolism. Nat Rev Endocrinol. 2018;14:637–55. doi: 10.1038/s41574-018-0076-3 30218014

2. Chavkin NW, Chia JJ, Crouthamel MH, Giachelli CM. Phosphate uptake-independent signaling functions of the type III sodium-dependent phosphate transporter, PiT-1, in vascular smooth muscle cells. Exp Cell Res. 2015;333(1):39–48. doi: 10.1016/j.yexcr.2015.02.002 25684711; PubMed Central PMCID: PMC4387109.

3. Bon N, Frangi G, Sourice S, Guicheux J, Beck-Cormier S, Beck L. Phosphate-dependent FGF23 secretion is modulated by PiT2/Slc20a2. Mol Metab. 2018;11:197–204. Epub 2018/03/20. S2212-8778(17)31089-X [pii] doi: 10.1016/j.molmet.2018.02.007 29551636; PubMed Central PMCID: PMC6001877.

4. Bon N, Couasnay G, Bourgine A, Sourice S, Beck-Cormier S, Guicheux J, et al. Phosphate (Pi)-regulated heterodimerization of the high-affinity sodium-dependent Pi transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pi sensing independently of Pi uptake. Journal of Biological Chemistry. 2018;293(6):2102–14. doi: 10.1074/jbc.M117.807339 29233890

5. Yamada S, Giachelli CM. Vascular calcification in CKD-MBD: Roles for phosphate, FGF23, and Klotho. Bone. 2016. Epub 2016/11/17. S8756-3282(16)30345-3 [pii] doi: 10.1016/j.bone.2016.11.012 27847254.

6. Beck-Cormier S, Lelliott CJ, Logan JG, Lafont DT, Merametdjian L, Leitch VD, et al. Slc20a2, Encoding the Phosphate Transporter PiT2, Is an Important Genetic Determinant of Bone Quality and Strength. J Bone Miner Res. 2019:e3691. Epub 2019/02/06. doi: 10.1002/jbmr.3691 30721528.

7. Yamada S, Wallingford MC, Borgeia S, Cox TC, Giachelli CM. Loss of PiT-2 results in abnormal bone development and decreased bone mineral density and length in mice. Biochem Biophys Res Commun. 2018;495(1):553–9. Epub 2017/11/15. S0006-291X(17)32249-0 [pii] doi: 10.1016/j.bbrc.2017.11.071 29133259; PubMed Central PMCID: PMC5739526.

8. Couasnay G, Bon N, Devignes CS, Sourice S, Bianchi A, Veziers J, et al. PiT1/Slc20a1 Is Required for Endoplasmic Reticulum Homeostasis, Chondrocyte Survival, and Skeletal Development. J Bone Miner Res. 2018. Epub 2018/10/23. doi: 10.1002/jbmr.3609 30347511.

9. Forand A, Koumakis E, Rousseau A, Sassier Y, Journe C, Merlin JF, et al. Disruption of the Phosphate Transporter Pit1 in Hepatocytes Improves Glucose Metabolism and Insulin Signaling by Modulating the USP7/IRS1 Interaction. Cell Rep. 2016;16(10):2736–48. Epub 2016/08/30. S2211-1247(16)31058-0 [pii] doi: 10.1016/j.celrep.2016.08.012 27568561.

10. Beck L, Leroy C, Beck-Cormier S, Forand A, Salaun C, Paris N, et al. The phosphate transporter PiT1 (Slc20a1) revealed as a new essential gene for mouse liver development. PLoS One. 2010;5(2):e9148. doi: 10.1371/journal.pone.0009148 20161774.

11. Festing MH, Speer MY, Yang HY, Giachelli CM. Generation of mouse conditional and null alleles of the type III sodium-dependent phosphate cotransporter PiT-1. Genesis. 2009;47(12):858–63. Epub 2009/11/03. doi: 10.1002/dvg.20577 19882669; PubMed Central PMCID: PMC2794919.

12. Bourgine A, Pilet P, Diouani S, Sourice S, Lesoeur J, Beck-Cormier S, et al. Mice with hypomorphic expression of the sodium-phosphate cotransporter PiT1/Slc20a1 have an unexpected normal bone mineralization. PLoS One. 2013;8(6):e65979. Epub 2013/06/21. doi: 10.1371/journal.pone.0065979 [pii]. 23785462; PubMed Central PMCID: PMC3681848.

13. Suzuki A, Ammann P, Nishiwaki-Yasuda K, Sekiguchi S, Asano S, Nagao S, et al. Effects of transgenic Pit-1 overexpression on calcium phosphate and bone metabolism. J Bone Miner Metab. 2010;28(2):139–48. doi: 10.1007/s00774-009-0121-3 19795094.

14. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A. 1980;77(12):7380–4. Epub 1980/12/01. doi: 10.1073/pnas.77.12.7380 6261253; PubMed Central PMCID: PMC350507.

15. Pruett-Miller SM. It's CRISPR Clear: Off-Target Study Misses the Mark. CRISPR J. 2018;1:130–1. Epub 2019/04/26. doi: 10.1089/crispr.2018.29013.mil 31021203.

16. Tasic B, Hippenmeyer S, Wang C, Gamboa M, Zong H, Chen-Tsai Y, et al. Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc Natl Acad Sci U S A. 2011;108(19):7902–7. Epub 2011/04/06. doi: 10.1073/pnas.1019507108 [pii]. 21464299; PubMed Central PMCID: PMC3093482.

17. Li Y, Caballero D, Ponsetto J, Chen A, Zhu C, Guo J, et al. Response of Npt2a knockout mice to dietary calcium and phosphorus. PLOS ONE. 2017;12(4):e0176232. doi: 10.1371/journal.pone.0176232 28448530

18. Farrell KB, Tusnady GE, Eiden MV. New structural arrangement of the extracellular regions of the phosphate transporter SLC20A1, the receptor for gibbon ape leukemia virus. J Biol Chem. 2009;284(43):29979–87. Epub 2009/09/01. doi: 10.1074/jbc.M109.022566 [pii]. 19717569; PubMed Central PMCID: PMC2785626.

19. Lu Y, Xie Y, Zhang S, Dusevich V, Bonewald LF, Feng JQ. DMP1-targeted Cre expression in odontoblasts and osteocytes. J Dent Res. 2007;86(4):320–5. Epub 2007/03/27. 86/4/320 [pii]. doi: 10.1177/154405910708600404 17384025.

20. Bakker AD, Klein-Nulend J. Osteoblast Isolation from Murine Calvaria and Long Bones. In: Helfrich MH, Ralston SH, editors. Bone Research Protocols. Totowa, NJ: Humana Press; 2012. p. 19–29.

21. Sekiguchi S, Suzuki A, Asano S, Nishiwaki-Yasuda K, Shibata M, Nagao S, et al. Phosphate overload induces podocyte injury via type III Na-dependent phosphate transporter. Am J Physiol Renal Physiol. 2011;300(4):F848–56. Epub 2011/02/11. doi: 10.1152/ajprenal.00334.2010 [pii]. 21307129.

22. Couasnay G, Bon N, Devignes CS, Sourice S, Bianchi A, Veziers J, et al. PiT1/Slc20a1 Is Required for Endoplasmic Reticulum Homeostasis, Chondrocyte Survival, and Skeletal Development. J Bone Miner Res. 2019;34(2):387–98. Epub 2018/10/23. doi: 10.1002/jbmr.3609 30347511.

23. Beck L, Leroy C, Salaun C, Margall-Ducos G, Desdouets C, Friedlander G. Identification of a Novel Function of PiT1 Critical for Cell Proliferation and Independent of Its Phosphate Transport Activity. Journal of Biological Chemistry. 2009;284(45):e99959.

24. Fedde KN, Blair L, Silverstein J, Coburn SP, Ryan LM, Weinstein RS, et al. Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia. J Bone Miner Res. 1999;14(12):2015–26. Epub 2000/01/05. jbm594 [pii] doi: 10.1359/jbmr.1999.14.12.2015 10620060; PubMed Central PMCID: PMC3049802.

25. Yadav MC, Bottini M, Cory E, Bhattacharya K, Kuss P, Narisawa S, et al. Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte-Derived Matrix Vesicles in Phospho1(-/-) and Phospho1/Pi t1 Double-Knockout Mice. J Bone Miner Res. 2016;31(6):1275–86. doi: 10.1002/jbmr.2790 26773408; PubMed Central PMCID: PMC4891278.

26. Khoshniat S, Bourgine A, Julien M, Weiss P, Guicheux J, Beck L. The emergence of phosphate as a specific signaling molecule in bone and other cell types in mammals. Cell Mol Life Sci. 2011;68(2):205–18. doi: 10.1007/s00018-010-0527-z 20848155.

27. Takashi Y, Fukumoto S. FGF23 beyond Phosphotropic Hormone. Trends Endocrinol Metab. 2018;29(11):755–67. Epub 2018/09/16. S1043-2760(18)30150-4 [pii] doi: 10.1016/j.tem.2018.08.006 30217676.

28. Michigami T, Kawai M, Yamazaki M, Ozono K. Phosphate as a Signaling Molecule and Its Sensing Mechanism. Physiol Rev. 2018;98(4):2317–48. Epub 2018/08/16. doi: 10.1152/physrev.00022.2017 30109818.


Článek vyšel v časopise

PLOS One


2019 Číslo 10