Microenvironmental stiffness directs microtubule perturbation in chondrocyte mitosis via ILK-refilinB/Smad3 axis

Abstract

Cells actively sense and transduce microenvironmental mechanical inputs into chemical signals via cytoskeletal rearrangements. During these mechanosensation and mechanotransduction processes, the role of the actin cytoskeleton is well-understood, whereas the role of the tubulin cytoskeleton remains largely elusive. Here, we report the dynamic changes in microtubules in response to microenvironmental stiffness during chondrocyte mitosis. Mechanical stiffness was found to be coupled with microtubule generation, directing microtubule dynamics in mitotic chondrocytes. Refilin B was found to be a key regulator of microtubule assembly in chondrocytes in response to mechanical stiffness. It was found to play its role in microtubule formation via the p-Smad3 signaling pathway. Additionally, integrin-linked kinase (ILK), triggered by mechanical stiffness, was found to play an indispensable role in the process of microtubule dynamics mediated by refilin B. Our data emphasizes stiffness-mediated dynamic changes in the microtubules of chondrocytes in a quiescent state (G0) and at anaphase, which improves our understanding of the mechanical regulation of microtubule assembly during the chondrocyte cell cycle and provides insights into microenvironment mechanics during tissue maintenance, wound healing, and disease occurrence.

References

[1] De Belly H, Paluch EK, Chalut KJ. Interplay between mechanics and signalling in regulating cell fate. Nat. Rev. Mol. Cell Biol., 2022, 23: 465-480

[2] Nava MMet al.. Heterochromatin-driven nuclear softening protects the genome against mechanical stress-induced damage. Cell, 2020, 181: 800-817.e22

[3] Song Y, Soto J, Li S. Mechanical regulation of histone modifications and cell plasticity. Curr. Opin. Solid State Mater. Sci., 2020, 24: 100872

[4] Guillot C, Lecuit T. Mechanics of epithelial tissue homeostasis and morphogenesis. Science, 2013, 340: 1185-1189

[5] Huang S, Ingber DE. Cell tension, matrix mechanics, and cancer development. Cancer Cell, 2005, 8: 175-176

[6] Geiger Bet al.. Transmembrane crosstalk between the extracellular matrix-cytoskeleton crosstalk. Nat. Rev. Mol. Cell Biol., 2001, 2: 793-805

[7] DiStefano TJ, Illien-Jünger S, Iatridis JC. Homeostasis disrupted by strain mechanosensing. Nat. Biomed. Eng., 2019, 3: 951-952

[8] Hegde RS, Keenan RJ. The mechanisms of integral membrane protein biogenesis. Nat. Rev. Mol. Cell Biol., 2022, 23: 107-124

[9] Chen B, Ji B, Gao H. Modeling active mechanosensing in cell-matrix interactions. Annu. Rev. Biophys., 2015, 44: 1-32

[10] Bergert Met al.. Force transmission during adhesion-independent migration. Nat. Cell Biol., 2015, 17: 524-529

[11] Kavallaris M. Microtubules and resistance to tubulin-binding agents. Nat. Rev. Cancer, 2010, 10: 194-204

[12] Akhmanova A, Kapitein LC. Mechanisms of microtubule organization in differentiated animal cells. Nat. Rev. Mol. Cell Biol., 2022, 23: 541-558

[13] Janke C, Magiera MM. The tubulin code and its role in controlling microtubule properties and functions. Nat. Rev. Mol. Cell Biol., 2020, 21: 307-326

[14] Walton T, Wu H, Brown A. Structure of a microtubule-bound axonemal dynein. Nat. Commun., 2021, 12 477

[15] Nachury MV, Mick DU. Establishing and regulating the composition of cilia for signal transduction. Nat. Rev. Mol. Cell Biol., 2019, 20: 389-405

[16] Benoit MPMHet al.. Structural basis of mechano-chemical coupling by the mitotic kinesin KIF14. Nat. Commun., 2021, 12 3637

[17] Rafiq NBMet al.. A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions. Nat. Mater., 2019, 18: 638-649

[18] Kreitzer G, Myat MM. MicrotubuLe motors in establishment of epithelial cell polarity. Cold Spring Harb. Perspect. Biol., 2018, 10: a027896

[19] Singh Aet al.. Polarized microtubule dynamics directs cell mechanics and coordinates forces during epithelial morphogenesis. Nat. Cell Biol., 2018, 20: 1126-1133

[20] Conde C, Cáceres A. Microtubule assembly, organization and dynamics in axons and dendrites. Nat. Rev. Neurosci., 2009, 10: 319-332

[21] Sakakibara Aet al.. Microtubule dynamics in neuronal morphogenesis. Open Biol., 2013, 3: 130061

[22] Subramanian Aet al.. Mechanical force regulates tendon extracellular matrix organization and tenocyte morphogenesis through TGFbeta signaling. Elife, 2018, 7: e38069

[23] Chinipardaz Zet al.. Role of primary cilia in bone and cartilage. J. Dent. Res., 2022, 101: 253-260

[24] Quadri N, Upadhyai P. Primary cilia in skeletal development and disease. Exp. Cell Res., 2023, 431: 113751

[25] Zhou Cet al.. Runx1 protects against the pathological progression of osteoarthritis. Bone Res., 2021, 9: 50

[26] Du Xet al.. The role of TGF-beta3 in cartilage development and osteoarthritis. Bone Res., 2023, 11: 2

[27] Kumar Set al.. Viscoelastic retraction of single living stress fibers and its impact on cell shape, cytoskeletal organization, and extracellular matrix mechanics. Biophys. J., 2006, 90: 3762-3773

[28] Yang Yet al.. Lipid metabolism in cartilage and its diseases: a concise review of the research progress. Acta Biochim. Biophys. Sin., 2021, 53: 517-527

[29] Lee HPet al.. Mechanical confinement regulates cartilage matrix formation by chondrocytes. Nat. Mater., 2017, 16: 1243-1251

[30] Xie Jet al.. Anterior cruciate ligament transection-induced cellular and extracellular events in menisci: implications for osteoarthritis. Am. J. Sports Med., 2018, 46: 1185-1198

[31] Chang SHet al.. Excessive mechanical loading promotes osteoarthritis through the gremlin-1-NF-κB pathway. Nat. Commun., 2019, 10 1442

[32] Wu Let al.. Lysophosphatidic acid mediates fibrosis in injured joints by regulating collagen type I biosynthesis. Osteoarthr. Cartil., 2015, 23: 308-318

[33] Zhang Xet al.. Targeting downstream subcellular YAP activity as a function of matrix stiffness with Verteporfin-encapsulated chitosan microsphere attenuates osteoarthritis. Biomaterials, 2020, 232: 119724

[34] Park JSet al.. Mechanical regulation of glycolysis via cytoskeleton architecture. Nature, 2020, 578: 621-626

[35] Saraswathibhatla A, Indana D, Chaudhuri O. Cell-extracellular matrix mechanotransduction in 3D. Nat. Rev. Mol. Cell Biol., 2023, 24: 495-516

[36] Zhou Cet al.. Microenvironmental stiffness mediates cytoskeleton re-organization in chondrocytes through laminin-FAK mechanotransduction. Int. J. Oral. Sci., 2022, 14: 15

[37] Xin Yet al.. Biophysical properties of corneal cells reflect high myopia progression. Biophys. J., 2021, 120: 3498-3507

[38] Prosser SL, Pelletier L. Mitotic spindle assembly in animal cells: a fine balancing act. Nat. Rev. Mol. Cell Biol., 2017, 18: 187-201

[39] Nam KTet al.. Gastric tumor development in Smad3-deficient mice initiates from forestomach/glandular transition zone along the lesser curvature. Lab, 2012, 92: 883-895

[40] Yan Cet al.. Microtubule acetylation is required for mechanosensation in drosophila. Cell Rep., 2018, 25: 1051-1065.e6

[41] Shan Bet al.. Requirement of HDAC6 for transforming growth factor-beta1-induced epithelial-mesenchymal transition. J. Biol. Chem., 2008, 283: 21065-21073

[42] Nishida Net al.. Structural basis for two-way communication between dynein and microtubules. Nat. Commun., 2020, 11 1038

[43] Raja MKet al.. Elevated synaptic vesicle release probability in synaptophysin/gyrin family quadruple knockouts. Elife, 2019, 8: e40744

[44] Baudier J, Jenkins ZA, Robertson SP. The filamin-B-refilin axis—spatiotemporal regulators of the actin-cytoskeleton in development and disease. J. Cell Sci., 2018, 131: jcs213959

[45] Gay Oet al.. RefilinB (FAM101B) targets FilaminA to organize perinuclear actin networks and regulates nuclear shape. Proc. Natl. Acad. Sci. USA, 2011, 108: 11464-11469

[46] Mizuhashi Ket al.. Filamin-interacting proteins, Cfm1 and Cfm2, are essential for the formation of cartilaginous skeletal elements. Hum. Mol. Genet., 2014, 23: 2953-2967

[47] Gay Oet al.. Refilins: a link between perinuclear actin bundle dynamics and mechanosensing signaling. Bioarchitecture, 2011, 1: 245-249

[48] Wu Het al.. Cell-dependent pathogenic roles of filamin B in different skeletal malformations. Oxid. Med. Cell Longev., 2022, 2022: 8956636

[49] Zheng Let al.. Filamin B represses chondrocyte hypertrophy in a Runx2/Smad3-dependent manner. J. Cell Biol., 2007, 178: 121-128

[50] Tang PMet al.. Smad3 promotes cancer progression by inhibiting E4BP4-mediated NK cell development. Nat. Commun., 2017, 8 14677

[51] Kanchanawong P, Calderwood DA. Organization, dynamics and mechanoregulation of integrin-mediated cell-ECM adhesions. Nat. Rev. Mol. Cell Biol., 2023, 24: 142-161

[52] Cabodi Set al.. Integrin signalling adaptors: not only figurants in the cancer story. Nat. Rev. Cancer, 2010, 10: 858-870

[53] Legate KRet al.. ILK, PINCH and parvin: the tIPP of integrin signalling. Nat. Rev. Mol. Cell Biol., 2006, 7: 20-31

[54] Löer Bet al.. The NHL-domain protein Wech is crucial for the integrin-cytoskeleton link. Nat. Cell Biol., 2008, 10: 422-428

[55] Park Het al.. Integrin-linked kinase controls retinal angiogenesis and is linked to Wnt signaling and exudative vitreoretinopathy. Nat. Commun., 2019, 10 5243

[56] Chen Het al.. The role of fibroblast growth factor 8 in cartilage development and disease. J. Cell Mol. Med., 2022, 26: 990-999

[57] Duan Met al.. TGF-β2 enhances nanoscale cortex stiffness via condensation of cytoskeleton-focal adhesion plaque. Biophys. J., 2025, 124: 336-350

[58] Huang Het al.. Fibroblast growth factor 8 (FGF8) induces mitochondrial remodeling in chondrocytes via ERK/AMPK signaling pathway. FASEB J., 2025, 39: e70501

[59] Ji Qet al.. Single-cell RNA-seq analysis reveals the progression of human osteoarthritis. Ann. Rheum. Dis., 2019, 78: 100-110

[60] Jeon OHet al.. Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nat. Med., 2017, 23: 775-781

[61] Varela-Eirin Met al.. Cartilage regeneration and ageing: Targeting cellular plasticity in osteoarthritis. Ageing Res. Rev., 2018, 42: 56-71

[62] Muthu Set al.. Failure of cartilage regeneration: emerging hypotheses and related therapeutic strategies. Nat. Rev. Rheumatol., 2023, 19: 403-416

[63] Zhou Cet al.. Hydrogel platform with tunable stiffness based on magnetic nanoparticles cross-linked GelMA for cartilage regeneration and its intrinsic biomechanism. Bioact. Mater., 2022, 25: 615-628

[64] Huang, M. et al., FGF8 promotes lipid droplet accumulation via the FGFR1/p-p38 axis in chondrocytes. Acta Biochim. Biophys. Sin. https://doi.org/10.3724/abbs.2025075 (2025).

[65] Bernabei Iet al.. Cartilage calcification in osteoarthritis: mechanisms and clinical relevance. Nat. Rev. Rheumatol., 2023, 19: 10-27

[66] Zhou Cet al.. Biomimetic fibers based on equidistant micropillar arrays determines chondrocyte fate via mechanoadaptability. Adv. Health. Mater., 2023, 12: e2301685

[67] Lee Wet al.. Inflammatory signaling sensitizes Piezo1 mechanotransduction in articular chondrocytes as a pathogenic feed-forward mechanism in osteoarthritis. Proc. Natl. Acad. Sci. USA, 2021, 118: e2001611118

[68] Duan Met al.. Stiffened fibre-like microenvironment based on patterned equidistant micropillars directs chondrocyte hypertrophy. Mater. Today Biol., 2023, 20: 100682

[69] Liu Yet al.. The role of mechano growth factor in chondrocytes and cartilage defects: a concise review. Acta Biochim. Biophys. Sin., 2023, 55: 701-712

[70] Chen Cet al.. Substrate stiffness together with soluble factors affects chondrocyte mechanoresponses. ACS Appl Mater. Interfaces, 2014, 6: 16106-16116

[71] Zhou Cet al.. Compliant substratum modulates vinculin expression in focal adhesion plaques in skeletal cells. Int. J. Oral. Sci., 2019, 11: 18

[72] Zhang Tet al.. Softening substrates promote chondrocytes phenotype via RhoA/ROCK pathway. ACS Appl. Mater. Interfaces, 2016, 8: 22884-22891

[73] Jaabar ILet al.. Deciphering pathological remodelling of the human cartilage extracellular matrix in osteoarthritis at the supramolecular level. Nanoscale, 2022, 14: 8691-8708

[74] Humphrey JD, Dufresne ER, Schwartz MA. Mechanotransduction and extracellular matrix homeostasis. Nat. Rev. Mol. Cell Biol., 2014, 15: 802-812

[75] Calvo Fet al.. Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. Nat. Cell Biol., 2013, 15: 637-646

[76] Campbell JJet al.. Loading alters actin dynamics and up-regulates cofilin gene expression in chondrocytes. Biochem. Biophys. Res. Commun., 2007, 361: 329-334

[77] Xu Tet al.. Regulation of PTHrP expression by cyclic mechanical strain in postnatal growth plate chondrocytes. Bone, 2013, 56: 304-311

[78] Knight MMet al.. Mechanical compression and hydrostatic pressure induce reversible changes in actin cytoskeletal organisation in chondrocytes in agarose. J. Biomech., 2006, 39: 1547-1551

[79] Cai Let al.. Biomaterial stiffness guides cross-talk between chondrocytes: implications for a novel cellular response in cartilage tissue engineering. ACS Biomater. Sci. Eng., 2020, 6: 4476-4489

[80] Sarasa-Renedo A, Tunç-Civelek V, Chiquet M. Role of RhoA/ROCK-dependent actin contractility in the induction of tenascin-C by cyclic tensile strain. Exp. Cell Res., 2006, 312: 1361-1370

[81] Pascarelli NAet al.. Changes in ultrastructure and cytoskeletal aspects of human normal and osteoarthritic chondrocytes exposed to interleukin-1beta and cyclical hydrostatic pressure. Int. J. Mol. Sci., 2015, 16: 26019-26034

[82] Walsh SK, Skala MC, Henak CR. Real-time optical redox imaging of cartilage metabolic response to mechanical loading. Osteoarthr. Cartil., 2019, 27: 1841-1850

[83] Gay Oet al.. Refilins are short-lived Actin-bundling proteins that regulate lamellipodium protrusion dynamics. Biol. Open, 2016, 5: 1351-1361

[84] Gay O, Nakamura F, Baudier J. Refilin holds the cap. Commun. Integr. Biol., 2011, 4: 791-795

[85] Nishiyama Aet al.. Lineage, fate, and fate potential of NG2-glia. Brain Res., 2016, 1638: 116-128

[86] Shi Yet al.. Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling. Cell, 1998, 94: 585-594

[87] Randall RAet al.. Different Smad2 partners bind a common hydrophobic pocket in Smad2 via a defined proline-rich motif. EMBO J., 2002, 21: 145-156

[88] Massagué J. TGFβ signalling in context. Nat. Rev. Mol. Cell Biol., 2012, 13: 616-630

[89] Duan Met al.. The role of TGF-beta2 in cartilage development and diseases. Bone Jt. Res, 2021, 10: 474-487

[90] Ehrlicher AJet al.. Mechanical strain in actin networks regulates FilGAP and integrin binding to filamin A. Nature, 2011, 478: 260-263

[91] Gordon CAet al.. NUSAP1 promotes invasion and metastasis of prostate cancer. Oncotarget, 2017, 8: 29935-29950

[92] Jones E, Pu H, Kyprianou N. Targeting TGF-beta in prostate cancer: therapeutic possibilities during tumor progression. Expert Opin. Ther. Targets, 2009, 13: 227-234

[93] Buffin E, Emre D, Karess RE. Flies without a spindle checkpoint. Nat. Cell Biol., 2007, 9: 565-572

[94] Chen Het al.. FGF19 induces the cell cycle arrest at G2-phase in chondrocytes. Cell Death Discov., 2023, 9: 250

[95] Martínez-Alonso D, Malumbres M. Mammalian cell cycle cyclins. Semin. Cell Dev. Biol., 2020, 107: 28-35

[96] Han, S., Pang, M. F. & Nelson, C. M. Substratum stiffness tunes proliferation downstream of Wnt3a in part by regulating integrin-linked kinase and frizzled-1. J. Cell Sci. 131, jcs210476 (2018).

[97] Zhou Cet al.. Substrate mechanics dictate cell-cell communication by gap junctions in stem cells from human apical papilla. Acta Biomater., 2020, 107: 178-193

[98] Kawakami Aet al.. Apolipoprotein CIII induces expression of vascular cell adhesion molecule-1 in vascular endothelial cells and increases adhesion of monocytic cells. Circulation, 2006, 114: 681-687

[99] Pines Met al.. Mechanical force regulates integrin turnover in Drosophila in vivo. Nat. Cell Biol., 2012, 14: 935-943

[100] Peng Yet al.. Regulation of the integrin alphaVbeta3- actin filaments axis in early osteogenic differentiation of human mesenchymal stem cells under cyclic tensile stress. Cell Commun. Signal, 2023, 21 308

[101] Brancaccio Met al.. Integrin signalling: the tug-of-war in heart hypertrophy. Cardiovasc. Res., 2006, 70: 422-433

[102] Zou Ret al.. Role of integrin-linked kinase in static compressive stress-induced autophagy via phosphatidylinositol 3 kinase in human periodontal ligament cells. Int. J. Mol. Med., 2021, 48: 167

[103] Bangasser BLet al.. Shifting the optimal stiffness for cell migration. Nat. Commun., 2017, 8 15313

[104] Di Russo Jet al.. Integrin alpha5beta1 nano-presentation regulates collective keratinocyte migration independent of substrate rigidity. Elife, 2021, 10: e69861

[105] Goult BT, Yan J, Schwartz MA. Talin as a mechanosensitive signaling hub. J. Cell Biol., 2018, 217: 3776-3784

[106] Xie Jet al.. Compliant substratum changes osteocyte functions: the role of ITGB3/FAK/beta-catenin signaling matters. ACS Appl. Biol. Mater., 2018, 1: 792-801

[107] Chen Het al.. Mechanosensing by the alpha6-integrin confers an invasive fibroblast phenotype and mediates lung fibrosis. Nat. Commun., 2016, 7 12564

[108] Sanz-Ramos Pet al.. Identification of signalling pathways triggered by changes in the mechanical environment in rat chondrocytes. Osteoarthr. Cartil., 2012, 20: 931-939

[109] Yamamura Yet al.. Myocardin-related transcription factor contributes to renal fibrosis through the regulation of extracellular microenvironment surrounding fibroblasts. FASEB J., 2023, 37: e23005

[110] Liu S, Calderwood DA, Ginsberg MH. Integrin cytoplasmic domain-binding proteins. J. Cell Sci., 2000, 113: 3563-3571

[111] Olmos G, López-Ongil S, Ruiz Torres MP. Integrin-linked kinase: a new actor in the ageing process?. Exp. Gerontol., 2017, 100: 87-90

[112] Liu F. Smad3 phosphorylation by cyclin-dependent kinases. Cytokine Growth Factor Rev., 2006, 17: 9-17

[113] Sun Let al.. Low-dose paclitaxel ameliorates fibrosis in the remnant kidney model by down-regulating miR-192. J. Pathol., 2011, 225: 364-377

[114] Zheng Let al.. The role of metabolism in chondrocyte dysfunction and the progression of osteoarthritis. Ageing Res. Rev., 2021, 66: 101249

[115] Kan Set al.. FGF19 increases mitochondrial biogenesis and fusion in chondrocytes via the AMPKα-p38/MAPK pathway. Cell Commun. Signal, 2023, 21 55

[116] Hall JL, Cowan NJ. Structural features and restricted expression of a human alpha-tubulin gene. Nucleic Acids Res., 1985, 13: 207-223

[117] Gadadhar Set al.. The tubulin code at a glance. J. Cell Sci., 2017, 130: 1347-1353

[118] Arnold TD, Zang K, Vallejo-Illarramendi A. Deletion of integrin-linked kinase from neural crest cells in mice results in aortic aneurysms and embryonic lethality. Dis. Model Mech., 2013, 6: 1205-1212

[119] Akhtar N, Streuli CH. An integrin-ILK-microtubule network orients cell polarity and lumen formation in glandular epithelium. Nat. Cell Biol., 2013, 15: 17-27

[120] Zhang Let al.. Outer membrane vesicles derived from fusobacterium nucleatum trigger periodontitis through host overimmunity. Adv. Sci., 2024, 11: e2400882

[121] Cherif Het al.. Single-cell RNA-seq analysis of cells from degenerating and non-degenerating intervertebral discs from the same individual reveals new biomarkers for intervertebral disc degeneration. Int. J. Mol. Sci., 2022, 23: 3993

[122] Fu Wet al.. Cellular features of localized microenvironments in human meniscal degeneration: a single-cell transcriptomic study. Elife, 2022, 11: e79585

[123] Wang Xet al.. Comparison of the major cell populations among osteoarthritis, Kashin-Beck disease and healthy chondrocytes by single-cell RNA-seq analysis. Cell Death Dis., 2021, 12 551

Funding

National Natural Science Foundation of China (National Science Foundation of China)(81771047)

RIGHTS & PERMISSIONS

The Author(s)