A non-canonical role for p27Kip1 in restricting proliferation of corneal endothelial cells during development

Autoři: Dennis M. Defoe aff001;  Huiying Rao aff002;  David J. Harris, III aff001;  Preston D. Moore aff001;  Jan Brocher aff004;  Theresa A. Harrison aff001
Působiště autorů: Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States of America aff001;  Department of Ophthalmology, Fujian Provincial Hospital, Fujian, Fuzhou, Peoples Republic of China aff002;  Graduate Biomedical Research Program, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States of America aff003;  BioVoxxel, Ludwigshafen, Germany aff004
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226725


The cell cycle regulator p27Kip1 is a critical factor controlling cell number in many lineages. While its anti-proliferative effects are well-established, the extent to which this is a result of its function as a cyclin-dependent kinase (CDK) inhibitor or through other known molecular interactions is not clear. To genetically dissect its role in the developing corneal endothelium, we examined mice harboring two loss-of-function alleles, a null allele (p27) that abrogates all protein function and a knockin allele (p27CK) that targets only its interaction with cyclins and CDKs. Whole-animal mutants, in which all cells are either homozygous knockout or knockin, exhibit identical proliferative increases (~0.6-fold) compared with wild-type tissues. On the other hand, use of mosaic analysis with double markers (MADM) to produce infrequently-occurring clones of wild-type and mutant cells within the same tissue environment uncovers a roughly three- and six-fold expansion of individual p27CK−/CK and p27−/− cells, respectively. Mosaicism also reveals distinct migration phenotypes, with p27−/− cells being highly restricted to their site of production and p27CK−/CK cells more widely scattered within the endothelium. Using a density-based clustering algorithm to quantify dispersal of MADM-generated clones, a four-fold difference in aggregation is seen between the two types of mutant cells. Overall, our analysis reveals that, in developing mouse corneal endothelium, p27 regulates cell number by acting cell autonomously, both through its interactions with cyclins and CDKs and through a cyclin-CDK-independent mechanism(s). Combined with its parallel influence on cell motility, it constitutes a potent multi-functional effector mechanism with major impact on tissue organization.

Klíčová slova:

Alleles – Cell cycle and cell division – Cell cycle inhibitors – Cornea – Cyclins – Endothelial cells – Endothelium – Mutant genotypes


1. Kreutziger GO. Lateral membrane morphology and gap junction structure in rabbit corneal endothelium. Exp Eye Res. 1976; 23:285–93. doi: 10.1016/0014-4835(76)90129-9 976372

2. Hirsch M, Renard G, Faure J-P, Pouliquen Y. Study of the ultrastructure of the rabbit corneal endothelium by the freeze-fracture technique: apical and lateral junctions. Exp Eye Res. 1977 Sep; 25(3):277–88. doi: 10.1016/0014-4835(77)90094-x 590370

3. Ringvold A, Davanger M, Olsen EG. On the spatial organization of the cornea endothelium. Acta Ophthalmol. 1984 Dec; 62(6):911–8. doi: 10.1111/j.1755-3768.1984.tb08442.x 6524316.

4. Forest F, Thuret G, DuMollard JM, Peoc’h M, Perrache C, He Z. Optimization of immunostaining on flat mounted human corneas. Mol Vis. 2015 Dec 30; 21:1345–1356. 26788027

5. Harrison TA, He Z, Boggs K, Thuret G, Liu HX, Defoe DM. Corneal endothelial cells possess an elaborate multipolar shape to maximize the basolateral to apical membrane area. Mol Vis. 2016 Jan 16;22:31–9. 27081293.

6. He Z, Forest F, Gain P, Rageade D, Bernard A, Acquart S, et al. 3D map of the human corneal endothelial cell. Sci Rep. 2016; Jul 6;6:29047. doi: 10.1038/srep29047 27381832

7. Bonanno JA. Molecular mechanisms underlying the corneal endothelial pump. Exp Eye Res. 2012;95:2–7. doi: 10.1016/j.exer.2011.06.004 21693119.

8. Wilson RS, Roper-Hall MJ. Effect of age on the endothelial cell count in the normal eye. Br J Ophthalmol. 1982 Aug;66(8):513–5. doi: 10.1136/bjo.66.8.513 7104267.

9. Murphy C, Alvarado J, Juster R, Maglio M. Prenatal and postnatal cellularity of the human corneal endothelium. A quantitative histologic study. Invest Ophthalmol Vis Sci 1984 Mar;25(3):312–22. 6698749.

10. Joyce NC. Proliferative capacity of the corneal endothelium. Prog Retin Eye Res. 2003 May;22(3):359–89. doi: 10.1016/s1350-9462(02)00065-4 12852491.

11. Laing RA, Sanstrom MM, Berrospi AR, Leibowitz HM. Changes in the corneal endothelium as a function of age. Exp Eye Res. 1976 Jun;22(6):587–94. doi: 10.1016/0014-4835(76)90003-8 776638.

12. Laule A, Cable MK, Hoffman CE, Hanna C. Endothelial cell population changes of human cornea during life. Arch Ophthalmol. 1978 Nov;96(11):2031–5. doi: 10.1001/archopht.1978.03910060419003 718491.

13. Sherrard ES. The corneal endothelium in vivo: its response to mild trauma. Exp Eye Res. 1976 Apr;22(4):347–57. doi: 10.1016/0014-4835(76)90227-x 954871.

14. Yee RW, Matsuda M, Schultz RO, Edelhauser HF. Changes in the normal corneal endothelial cellular pattern as a function of age. Curr Eye Res. 1985 Jun;4(6):671–8. doi: 10.3109/02713688509017661 4028790.

15. Ikebe H, Takamatsu T, Itoi M, Fujita S. Age-dependent changes in nuclear DNA content and cell size of presumably normal human corneal endothelium. Exp Eye Res. 1986 Aug;43(2):251–8. doi: 10.1016/s0014-4835(86)80093-8 3758224.

16. Losick VP, Jun AS, Spradling AC. Wound-induced polyploidization: regulation by Hippo and JNK signaling and conservation in mammals. PLoS One 2016 Mar 9;11(3):e0151251. doi: 10.1371/journal.pone.0151251 26958853.

17. Mishima S. Clinical investigations on the corneal endothelium-XXXVIII Edward Jackson Memorial Lecture. Am J Ophthalmol. 1982 Jan;93(1):1–29. doi: 10.1016/0002-9394(82)90693-6 6801985.

18. Schmedt T, Silva MM, Ziaei A, Jurkunas U. Molecular bases of corneal endothelial dystrophies. Exp Eye Res. 2012 Feb;95(1):24–34. doi: 10.1016/j.exer.2011.08.002 21855542.

19. Tan DT, Dart JK, Holland EJ, Kinoshita S. Corneal transplantation. Lancet. 2012 May 5;379(9827):1749–61. doi: 10.1016/S0140-6736(12)60437-1 22559901.

20. Tuft SJ, Coster DJ. The corneal endothelium. Eye. 1990;4(Pt 3):389–424. doi: 10.1038/eye.1990.53 2209904.

21. Joyce NC, Harris DL, Zieske JD. Mitotic inhibition of corneal endothelium in neonatal rats. Invest Ophthalmol Vis Sci. 1998 Dec;39(13):2572–83. 9856767.

22. Gordon SR. Changes in extracellular matrix proteins and actin during corneal endothelial growth. Invest Ophthalmol Vis Sci 1990 Jan;31(1):94–101. 2404898.

23. Joyce NC, Harris DL, Mello DM. Mechanisms of mitotic inhibition in corneal endothelium: contact inhibition and TGF-beta2. Invest Ophthalmol Vis Sci. 2002 Jul;43(7):2152–9. 12091410.

24. Yoshida K, Kase S, Nakayama K, Nagahama H, Harada T, Ikeda H, et al. Involvement of p27KIP1 in the proliferation of the developing corneal endothelium. Invest Ophthalmol Vis Sci. 2004 Jul;45(7):2163–7. doi: 10.1167/iovs.03-1238 15223790.

25. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 1999 Jun 15;13(12):1501–12. doi: 10.1101/gad.13.12.1501 10385618.

26. Russo AA, Jeffrey PD, Patten AK, Massagué J, Pavletich NP. Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature 1996 Jul 25;382(6589):325–31. doi: 10.1038/382325a0 8684460.

27. Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, et al. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27Kip1-deficient mice. Cell. 1996 May 31;85(5):733–44. doi: 10.1016/s0092-8674(00)81239-8 8646781.

28. Kiyokawa H, Kineman RD, Manova-Todorova KO, Soares VC, Hoffman ES, Ono M, et al. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27Kip1. Cell. 1996 May 31;85(5):721–32. doi: 10.1016/s0092-8674(00)81238-6 8646780.

29. Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, et al. Mice lacking p27Kip1 display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell. 1996 May 31;85(5):707–20. doi: 10.1016/s0092-8674(00)81237-4 8646779.

30. Besson A, Gurian-West M, Chen X, Kelly-Spratt KS, Kemp CJ, Roberts JM. A pathway in quiescent cells that controls p27Kip1 stability, subcellular localization, and tumor suppression. Genes Dev 2006 Jan 1;20(1):47–64. doi: 10.1101/gad.1384406 16391232.

31. Besson A, Hwang HC, Cicero S, Donovan SL, Gurian-West M, Johnson D, et al. Discovery of an oncogenic activity in p27Kip1 that causes stem cell expansion and a multiple tumor phenotype. Genes Dev 2007 Jan 1;21(1):1731–1746. doi: 10.1101/gad.1556607 17626791.

32. Defoe DM, Adams LB, Sun J, Wisecarver SN, Levine EM. Defects in retinal pigment epithelium cell proliferation and retinal attachment in mutant mice with p27Kip1 ablation. Mol Vis. 2007 Feb 27;13:273–86. 17356514.

33. Berton S, Pellizzari I, Fabris L, D’Andrea S, Segatto I, Canzonieri V, et al. Genetic characterization of p27kip1 and stathmin in controlling cell proliferation in vivo. Cell Cycle. 2014;13(19):3100–11. doi: 10.4161/15384101.2014.949512 25486569.

34. Fabris L, Berton S, Pellizzari I, Segatto I, D’Andrea S, Armenia J, et al. p27kip1 controls H-Ras/MAPK activation and cell cycle entry via modulation of MT stability. Proc Natl Acad Sci USA. 2015 Nov 10;112(45):13916–21. doi: 10.1073/pnas.1508514112 26512117.

35. Vlach J, Hennecke S, Amati B. Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27. EMBO J. 1997 Sep 1;16(17):5334–44. doi: 10.1093/emboj/16.17.5334 9311993.

36. Zong H, Espinosa JS, Su HH, Muzumdar MD, Luo L. Mosaic analysis with double markers in mice. Cell. 2005 May 6;121(3):479–92. doi: 10.1016/j.cell.2005.02.012 15882628.

37. Tang SH, Silva FJ, Tsark WM, Mann JR. A Cre/loxP-deleter transgenic line in mouse strain 129S1/SvimJ. Genesis. 2002 Mar;32(3):199–202. doi: 10.1002/gene.10030 11892008.

38. Espinosa JS, Tea JS, Luo L. Mosaic analysis with double markers (MADM) in mice. Cold Spring Harb Protoc. 2014 Feb 1;2014(2):182–9. doi: 10.1101/pdb.prot080366 24492775.

39. Zong H. Generation and applications of MADM-based mouse genetic mosaic system. Methods Mol Biol. 2014; 1194:187–201. doi: 10.1007/978-1-4939-1215-5_10 25064104.

40. Muzumdar MD, Luo L, Zong H. Modeling sporadic loss of heterozygosity in mice by using mosaic analysis with double markers (MADM). Proc Natl Acad Sci U S A. 2007 Mar 13;104(11)al:4495–500. doi: 10.1073/pnas.0606491104 17360552.

41. Lorincz A, Nusser Z. Molecular identity of dendritic voltage-gated sodium channels. Science. 2010 May 14;328:906–9. doi: 10.1126/science.1187958 20466935.

42. Espinosa JS, Luo L. Timing neurogenesis and differentiation: Insights from quantitative clonal analyses of cerebellar granule cells. J Neurosci. 2008 Mar 5;28(10):2301–12. doi: 10.1523/JNEUROSCI.5157-07.2008 18322077.

43. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012 June 28;9(7):676–682. doi: 10.1038/nmeth.2019 22743772.

44. Ester M, Kriegel H-P, Sander J, Xu X. A density-based algorithm for discovering clusters in large spatial databases with noise. In: Simoudis E, Han J, Fayyad UM, editors. Proceedings of the Second International Conference on Knowledge Discovery and Data Mining (KDD-96); AAAI Press; 1996. p. 226–231. CiteSeerX [ISBN 1-57735-004-9].

45. Germani F, Bergantinos C, Johnson LA. Mosaic analysis in Drosophila. Genetics. 2018 Feb;208(2):473–490. doi: 10.1534/genetics.117.300256 Review. 29378809.

46. Baldassarre G, Belletti B, Nicoloso MS, Schiappacassi M, Vecchione A, Spessotto P, et al. p27Kip1-stathmin interaction influences sarcoma cell migration and invasion. Cancer Cell. 2005 Jan;7(1):51–63. doi: 10.1016/j.ccr.2004.11.025 15652749.

47. Besson A, Gurian-West M, Schnidt A, Hall A, Roberts JM. p27Kip1 modulates cell migration through the regulation of RhoA activation. Genes Dev 2004 Apr 15;18(8):862–76. doi: 10.1101/gad.1185504 15078817.

48. Olson MF, Ashworth A, Hall A. An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science. 1995 Sep 1;269(5228):1270–2. doi: 10.1126/science.7652575 7652575

49. Yamamoto M, Marui N, Sakai T, Morii N, Kozaki S, Ikai K, et al. ADP-ribosylation of the rhoA gene product by botulinum C3 exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of the cell cycle. Oncogene. 1993 Jun;8(6):1449–55. 8502473.

50. Coleman ML, Marshall CJ, Olson MF. RAS and RHO GTPases in G1-phase cell-cycle regulation. Nat Rev Mol Cell Biol. 2004 May;5(5):355–66. doi: 10.1038/nrm1365 15122349.

51. Croft DR, Olson MF. The Rho GTPase effector ROCK regulates cyclin A, cyclin D1, and p27Kip1 levels by distinct mechanisms. Mol Cell Biol. 2006 Jun;26(12):4612–27. doi: 10.1128/MCB.02061-05 16738326.

52. Okumura N, Ueno M, Koizumi N, Sakamoto Y, Hirata K, Hamuro J, et al. Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor. Invest Ophthalmol Vis Sci. 2009 Aug;50(8):3680–7. doi: 10.1167/iovs.08-2634 19387080.

53. Okumura N, Nakano S, Kay EP, Numata R, Ota A, Sowa Y, et al. Involvement of cyclin D and p27 in cell proliferation mediated by ROCK inhibitors Y-27632 and Y-39983 during corneal endothelium wound healing. Invest Ophthalmol Vis Sci. 2014 Jan 15;55(1):318–29. doi: 10.1167/iovs.13-12225 24106120.

54. Okumura N, Koizumi N, Kay EP, Ueno M, Sakamoto Y, Nakamura S, et al. The ROCK inhibitor eye drop accelerates corneal endothelium wound healing. Invest Ophthalmol Vis Sci. 2013 Apr 3;54(4):2493–502. doi: 10.1167/iovs.12-11320 23462749.

55. Okumura N, Inoue R, Okazaki Y, Nakano S, Nakagawa H, Kinoshita S, et al. Effect of the Rho kinase inhibitor Y-27632 on corneal endothelial wound healing. Invest Ophthalmol Vis Sci. 2015 Sep;56(10):6067–74. doi: 10.1167/iovs.15-17595 26393474.

56. Okumura N, Okazaki Y, Inoue R, Kakutani K, Nakano S, Kinoshita S, et al. Effect of the Rho-associated kinase inhibitor eye drop (Ripasudil) on corneal endothelial wound healing. Invest Ophthalmol Vis Sci. 2016 Mar;57(3):1284–92. doi: 10.1167/iovs.15-18586 26998714.

57. Meekins LC, Rosado-Adames N, Maddala R, Zhao JJ, Rao PV, Afshari NA. Corneal endothelial cell migration and proliferation enhanced by Rho kinase (ROCK) inhibitors in in vitro and in vivo models. Invest Ophthalmol Vis Sci. 2016 Dec 1;57(15):6731–38. doi: 10.1167/iovs.16-20414 27951595.

58. Zhu YT, Chen HC, Chen SY, Tseng SC. Nuclear p120 catenin unlocks mitotic block of contact-inhibited human corneal endothelial monolayers without disrupting adherent junctions. J Cell Sci. 2012 Aug 1;125(Pt 15):3636–48. doi: 10.1242/jcs.103267 22505615.

59. McAllister SS, Becker-Hapak M, Pintucci G, Pagano M, Dowdy SF. Novel p27kip1 C-terminal scatter domain mediates Rac-dependent cell migration independent of cell cycle arrest functions. Mol Cell Biol. 2003 Jan;23(1):216–28. doi: 10.1128/MCB.23.1.216-228.2003 12482975.

60. Nagahara H, Vocero-Akbani AM, Snyder EL, Ho A, Latham DG, Lissy NA, et al. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat Med. 1998 Dec;4(12):1449–52. doi: 10.1038/4042 9846587.

61. Kawauchi T, Chihama K, Nabeshima Y, Hoshino M. Cdk5 phosphorylates and stabilizes p27kip1 contributing to actin organization and cortical neuronal migration. Nat Cell Biol. 2006 Jan;8(1):17–26. doi: 10.1038/ncb1338 16341208.

62. Nguyen L, Besson A, Heng JI, Schuurmans C, Teboul L, Parras C, et al. p27kip1 independently promotes neuronal differentiation and migration in the cerebral cortex. Genes Dev. 2006 Jun 1;20(11):1511–24. doi: 10.1101/gad.377106 16705040.

63. Sheaff RJ, Groudine M, Gordon M, Roberts JM, Clurman BE. CyclinE-CDK2 is a regulator of p27Kip1. Genes Dev. 1997 Jun 1;11(11):1464–78. doi: 10.1101/gad.11.11.1464 9192873.

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