Abstract
Keratin intermediate filaments convey mechanical stability and protection against stress to epithelial cells. Keratins are essential for colon health, as seen in keratin 8 knockout (K8−/−) mice exhibiting a colitis phenotype. We hypothesized that keratins support the nuclear envelope and lamina in colonocytes. K8−/− colonocytes in vivo exhibit significantly decreased levels of lamins A/C, B1, and B2 in a colon-specific and cell-intrinsic manner. CRISPR/Cas9- or siRNA-mediated K8 knockdown in Caco-2 cells similarly decreased lamin levels, which recovered after reexpression of K8 following siRNA treatment. Nuclear area was not decreased, and roundness was only marginally increased in cells without K8. Down-regulation of K8 in adult K8flox/flox;Villin-CreERt2 mice following tamoxifen administration significantly decreased lamin levels at day 4 when K8 levels had reduced to 40%. K8 loss also led to reduced levels of plectin, LINC complex, and lamin-associated proteins. While keratins were not seen in the nucleoplasm without or with leptomycin B treatment, keratins were found intimately located at the nuclear envelope and complexed with SUN2 and lamin A. Furthermore, K8 loss in Caco-2 cells compromised nuclear membrane integrity basally and after shear stress. In conclusion, colonocyte K8 helps maintain nuclear envelope and lamina composition and contributes to nuclear integrity.
| Original language | English (US) |
|---|---|
| Journal | Molecular biology of the cell |
| Volume | 33 |
| Issue number | 13 |
| DOIs | |
| State | Published - Nov 1 2022 |
Funding
We thank Gerhard Wiche (Max Perutz Labs, University of Vienna) for the kind donation of the plectin antibody, Jan Lammerding (Cornell University) for the plasmids used in nuclear integrity analysis, Cecilia Sahlgren and Freddy Suarez-Rodriguez (Åbo Akademi University [ÅAU]) for assistance with the orbital shaker model, Pekka Taimen (University of Turku) for fruitful discussions, Keijo Viiri (University of Tampere) for the Cre-villin mouse strains, Petra Fichtinger (Max Perutz Labs, Medical University of Vienna) for help with the LAP2α mice, and John Eriksson (Turku Bioscience Centre, University of Turku and ÅAU, and Biosciences/Cell Biology, ÅAU) and members of the Eriksson laboratory, especially Elin Torvaldson and Josef Gullmets, for vimentin−/− mice, reagents, and discussions. We are grateful to all members of the Toivola laboratory, especially Frank Weckström and Molly Feiring (Biosciences/Cell Biology, Faculty of Science and Engineering, Åbo Akademi University; ÅAU). Imaging was performed at the Cell Imaging and Cytometry Core at Turku Bioscience Centre (University of Turku and ÅAU) and Biocenter Finland. This work was financed by the Academy of Finland 140759/126161/332582/315139 (D.M.T.), the InFLAMES Flagship Programme of the Academy of Finland, Sigrid Juselius Foundation (D.M.T.), Turku Doctoral Programme in Molecular Biosciences at ÅAU (C-G.A.S., J.H.N.), Medicinska Understödsföreningen Liv och Hälsa Foundation (D.M.T., J.H.N., C-G.A.S.), EU FP7 IRG (D.M.T.), ÅAU Centers of Excellence of Cell Stress and Molecular Aging and Cellular Mechanostasis (D.M.T.), The Swedish Cultural Foundation in Finland (J.H.N., C-G.A.S.), Makarna Agneta och Carl-Erik Olins Foundation (J.H.N., C-G.A.S.), Victoria Foundation (C-G.A.S., J.H.N.), K. Albin Johansson Foundation (C-G.A.S., J.H.N.), Magnus Ehrnrooths stiftelse (J.H.N.) Kommersrå-det Otto A. Malms Donationsfond (J.H.N.), Waldemar von Frenckells Foundation (J.H.N.), the EuroCellNet COST Action (CA15214; D.M.T., R.F.), National Institutes of Health (NIH) PO1 GM-096971 (R.D.G.), and NIH RO-106023 (R.D.G.). We thank Gerhard Wiche (Max Perutz Labs, University of Vienna) for the kind donation of the plectin antibody, Jan Lammerding (Cornell University) for the plasmids used in nuclear integrity analysis, Cecilia Sahlgren and Freddy Suarez-Rodriguez (Åbo Akademi University [ÅAU]) for assistance with the orbital shaker model, Pekka Taimen (University of Turku) for fruitful discussions, Keijo Viiri (University of Tampere) for the Cre-villin mouse strains, Petra Fichtinger (Max Perutz Labs, Medical University of Vienna) for help with the LAP2α mice, and John Eriksson (Turku Bioscience Centre, University of Turku and ÅAU, and Biosciences/Cell Biology, ÅAU) and members of the Eriksson laboratory, especially Elin Torvaldson and Josef Gullmets, for vimentin−/− mice, reagents, and discussions. We are grateful to all members of the Toivola laboratory, especially Frank Weckström and Molly Feiring (Biosciences/Cell Biology, Faculty of Science and Engineering, Åbo Akademi University; ÅAU). Imaging was performed at the Cell Imaging and Cytometry Core at Turku Bioscience Centre (University of Turku and ÅAU) and Biocenter Finland. This work was financed by the Academy of Finland 140759/126161/332582/315139 (D.M.T.), the InFLAMES Flagship Programme of the Academy of Finland, Sigrid Juselius Foundation (D.M.T.), Turku Doctoral Programme in Molecular Biosciences at ÅAU (C-G.A.S., J.H.N.), Medicinska Understödsföreningen Liv och Hälsa Foundation (D.M.T., J.H.N., C-G.A.S.), EU FP7 IRG (D.M.T.), ÅAU Centers of Excellence of Cell Stress and Molecular Aging and Cellular Mechanostasis (D.M.T.), The Swedish Cultural Foundation in Finland (J.H.N., C-G.A.S.), Makarna Agneta och Carl-Erik Olins Foundation (J.H.N., C-G.A.S.), Victoria Foundation (C-G.A.S., J.H.N.), K. Albin Johansson Foundation (C-G.A.S., J.H.N.), Magnus Ehrnrooths stiftelse (J.H.N.) Kommersrådet Otto A. Malms Donationsfond (J.H.N.), Waldemar von Frenckells Foundation (J.H.N.), the EuroCellNet COST Action (CA15214; D.M.T., R.F.), National Institutes of Health (NIH) PO1 GM-096971 (R.D.G.), and NIH RO-106023 (R.D.G.).
ASJC Scopus subject areas
- General Medicine