title: Wnt inhibition induces persistent increases in intrinsic stiffness of human trabecular meshwork cells.
abstract: Wnt antagonism has been linked to glaucoma and intraocular pressure regulation, as has increased stiffness of human trabecular meshwork (HTM) tissue. We have shown culturing HTM cells on substrates that mimic the elevated stiffness of glaucomatous tissue leads to elevated expression of the Wnt antagonist secreted frizzled related protein 1 (SFRP1), suggesting a linkage between SFRP1 and HTM mechanobiology. In this study, we document biomechanical consequences of Wnt antagonism on HTM cells. Cells were treated with the Wnt antagonists (SFRP1, KY02111, and LGK-974) for 8 days and allowed to recover for 4 days. After recovery, intrinsic cell stiffness and activation of the Wnt pathway via β-catenin staining and blotting were assayed. Basal cell stiffness values were 3.71 ± 0.37, 4.33 ± 3.07, and 3.07 ± kPa (median ± S.D.) for cells derived from 3 donors. Cell stiffness increased after 0.25 μg/mL (4.32 ± 5.12, 8.86 ± 8.51, 4.84 ± 3.15 kPa) and 0.5 μg/mL (16.75 ± 5.59, 13.18 ± 7.99, and 8.54 ± 5.77 kPa) SFRP1 treatment. Stiffening was observed after 10 μM KY02111 (10.72 ± 5.63 and 6.57 ± 5.53 kPa) as well as LGK-974 (9.60 ± 7.41 and 11.40 ± 9.24 kPa) treatment compared with controls (3.79 ± 1.01 and 5.16 ± 2.14 kPa). Additionally, Wnt inhibition resulted in decreased β-catenin staining and increased phosphorylation at threonine 41 after recovery. In conclusion, this work demonstrates a causal relationship between Wnt inhibition and cell stiffening. Additionally, these findings suggest transient Wnt inhibition resulted in durable modulation of the mechanical phenotype of HTM cells. When placed in context with previous results, these findings provide a causal link between Wnt antagonism and cell stiffness and suggest a feedback loop contributing to glaucoma progression.
authors: Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, United States.
authors: Joshua T
authors: JT
authors: Morgan
authors: Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, United States.
authors: Vijay Krishna
authors: VK
authors: Raghunathan
authors: Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, United States.
authors: Yow-Ren
authors: YR
authors: Chang
authors: Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, United States; Department of Ophthalmology & Vision Science, School of Medicine, University of California, Davis, United States.
authors: Christopher J
authors: CJ
authors: Murphy
authors: Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, United States. Electronic address: prussell@ucdavis.edu.
authors: Paul
authors: P
authors: Russell
mesh_terms: D002478:Cells, Cultured; D055119:Elastic Modulus / Q000502:physiology*; D004548:Elasticity; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D008565:Membrane Proteins / Q000494:pharmacology; D018625:Microscopy, Atomic Force; D015398:Signal Transduction / Q000502:physiology; D014129:Trabecular Meshwork / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D051153:Wnt Proteins / Q000502:physiology*; D060449:Wnt Signaling Pathway / Q000502:physiology*; D051176:beta Catenin / Q000378:metabolism; D000094742:Secreted Frizzled-Related Proteins
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D051153:Wnt Proteins; D051176:beta Catenin; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins
keywords: Atomic force microscopy; Cell stiffness; Human trabecular meshwork; Secreted frizzled related protein; Wnt
references: J Ocul Pharmacol Ther. 2014 Mar-Apr;30(2-3):254-66
references: 24456002
references: Exp Eye Res. 2006 Apr;82(4):545-57
references: 16386733
references: J Neurosci Res. 2008 Mar;86(4):797-812
references: 17941050
references: Clin Cancer Res. 2003 Apr;9(4):1291-300
references: 12684397
references: Phys Rev Lett. 2000 Jul 24;85(4):880-3
references: 10991422
references: EMBO Rep. 2002 Jan;3(1):63-8
references: 11751573
references: Br J Ophthalmol. 2006 Mar;90(3):262-7
references: 16488940
references: FEBS Lett. 1998 Jun 23;430(1-2):12-6
references: 9678586
references: Mol Vis. 2010;16:122-9
references: 20111673
references: Cell Rep. 2012 Nov 29;2(5):1448-60
references: 23103164
references: Biosci Rep. 2014;34(5). pii: e00142. doi: 10.1042/BSR20140119
references: 25208913
references: Lab Invest. 2006 Feb;86(2):106-15
references: 16341020
references: Ophthalmology. 1984 Jun;91(6):564-79
references: 6462622
references: Biomaterials. 1996 Sep;17(17):1647-57
references: 8866026
references: Cell. 2002 Mar 22;108(6):837-47
references: 11955436
references: Exp Eye Res. 1993 Jun;56(6):683-92
references: 8595810
references: J Cell Biol. 2009 Jul 27;186(2):219-28
references: 19620634
references: J Glaucoma. 1997 Apr;6(2):123-32
references: 9098821
references: Genesis. 2008 Feb;46(2):92-103
references: 18257070
references: J Cell Sci. 2003 Jul 1;116(Pt 13):2627-34
references: 12775774
references: Invest Ophthalmol Vis Sci. 2009 Mar;50(3):1255-63
references: 18952927
references: N Engl J Med. 1993 Apr 15;328(15):1097-106
references: 8455668
references: Invest Ophthalmol Vis Sci. 2013 Jan;54(1):378-86
references: 23258147
references: Mol Vis. 2008;14:1513-6
references: 18728789
references: J Cell Biochem. 2005 Dec 15;96(6):1212-30
references: 16149051
references: Invest Ophthalmol Vis Sci. 2011 Apr;52(5):2147-52
references: 21220561
references: Exp Gerontol. 2005 Aug-Sep;40(8-9):745-8
references: 16051457
references: Exp Cell Res. 2014 Oct 15;328(1):164-71
references: 24992043
references: J Glaucoma. 1995 Jun;4(3):183-8
references: 19920666
references: Biophys J. 2002 May;82(5):2798-810
references: 11964265
references: Mol Cell Biol. 2012 Nov;32(21):4388-99
references: 22927647
references: PLoS One. 2010;5(4):e10184
references: 20419129
references: Neurobiol Dis. 2010 May;38(2):148-53
references: 19765659
references: Biomaterials. 2011 Mar;32(9):2417-23
references: 21220171
references: J Clin Invest. 2008 Mar;118(3):1056-64
references: 18274669
references: Cell Tissue Res. 2012 Jan;347(1):279-90
references: 22101332
references: Invest Ophthalmol Vis Sci. 2012 Oct;53(11):7043-51
references: 22956608
references: Cell. 2006 Nov 3;127(3):469-80
references: 17081971
references: Oncol Rep. 2013 Sep;30(3):1430-8
references: 23799613
references: J R Soc Interface. 2005 Dec 22;2(5):455-63
references: 16849205
references: Cold Spring Harb Perspect Biol. 2012 Dec;4(12). pii: a007880. doi: 10.1101/cshperspect.a007880
references: 23209147
references: Exp Eye Res. 2005 Jul;81(1):1-4
references: 15978248
references: Proc Natl Acad Sci U S A. 2013 Dec 10;110(50):20224-9
references: 24277854