Mechanosignaling activation of TGFβ maintains intervertebral disc homeostasis

Abstract

Intervertebral disc (IVD) degeneration is the leading cause of disability with no disease-modifying treatment. IVD degeneration is associated with instable mechanical loading in the spine, but little is known about how mechanical stress regulates nucleus notochordal (NC) cells to maintain IVD homeostasis. Here we report that mechanical stress can result in excessive integrin α_vβ₆-mediated activation of transforming growth factor beta (TGFβ), decreased NC cell vacuoles, and increased matrix proteoglycan production, and results in degenerative disc disease (DDD). Knockout of TGFβ type II receptor (TβRII) or integrin α_v in the NC cells inhibited functional activity of postnatal NC cells and also resulted in DDD under mechanical loading. Administration of RGD peptide, TGFβ, and α_vβ₆-neutralizing antibodies attenuated IVD degeneration. Thus, integrin-mediated activation of TGFβ plays a critical role in mechanical signaling transduction to regulate IVD cell function and homeostasis. Manipulation of this signaling pathway may be a potential therapeutic target to modify DDD.

Figure 1

Mechanical instability induces activation of TGFβ, and reduces NC cell vacuoles. (a) Safranin O-Fast green staining of IVD sections from wild-type mice from E-16.0 to 4 months after birth. (b) Immunostaining of IVD sections showing the expression of aggrecan. n=6 per time point. (c) The height of IVD indicated by the double-headed arrow was measured in a. (d) Lumbar spine instability mouse model (LSI). Mouse L₃–L₅ spinous processes were resected along with the supraspinous and interspinous ligaments to induce instability of lumbar spine. (This diagram was drawn by the author and has been published in Scientific Reports: http://www.nature.com/articles/srep27093/figure/1). (e) Representative Safranin O staining images of the IVD sections showing the changes of NC cells in LSI and sham-operated 2-month-old mice at 0, 1, 2 and 4 weeks (w) post surgery. (f) Representative immunostaining images of IVD sections with antibody against pSmad2/3 (brown). Hematoxylin stains nuclei purple. (g) Evaluation of IVD degeneration by IVD score. (h) Quantification of pSmad2/3⁺ cells in f. n=6 per group. Data are shown as mean±s.d. *P<0.05, **P<0.01 (two-sided Student’s t test).

Figure 2

Mechanical instability stimulates extracellular aggrecan. (a) Immunofluorescence staining for CCN2 expression (red). DAPI stains nuclei blue. (b) Enlarged images of CCN2 expression in NP, AF and EP at 2 weeks post surgery. (c) Quantification of CCN2 expression in a. (d) Representative images of immunofluorescence staining for aggrecan (Acan, red). DAPI stains nuclei blue. (e) Quantitative analysis of Acan⁺ area in NC cells. n=6 per group. Data are shown as mean±s.d. *P<0.05, **P<0.01 (two-sided Student’s t test).

Figure 3

Integrin α_Vβ₆ induces TGFβ activation in response to mechanical stress. (a–h) Representative images and quantification of immunostaining of IVD sections with antibodies against (a, b) α_Vβ₆, (c, d) β8, (e, f) α_Vβ₅, and (g, h) α_Vβ₃ (brown) in LSI and sham mice. Hematoxylin stains nuclei purple. n=6 per group. Data are shown as mean±s.d. *P<0.05, **P<0.01 (two-sided Student’s t test). (i) Immunostaining for pSmad2/3 in the NC cells (brown). Hematoxylin stains nuclei purple. (j) Safranin O staining of IVD sections from an IVD ex vivo compression model with application of either Veh (rows 1 and 3), recombinant mouse TGFβ1 (row 2), RGD peptide, TGFβ or α_Vβ₆ neutralizing antibodies (bottom 3 rows). (k) Quantification of pSmad2/3⁺ cells in i. (l) Western blot analysis of pSmad2 and total Smad2 levels in the IVD. n=6 per group in histological examination. Representative image from three independent experiments were conducted for (l). Data are shown as mean±s.d. *P<0.05, **P<0.01 (ANOVA).

Figure 4

Mechanical stress regulates NC cell function. (a, b) Immunofluorescence staining of IVD sections for (a) CCN2 and (b) Acan in the NP (red). DAPI stains nuclei blue. (c, d) Quantification of CCN2 and Acan expression in a and b. (e) Expression of Acan mRNA in NP tissues by quantitative RT-PCR (qRT-PCR). n=6 per group in histological examination. Three independent experiments performed in triplicate were conducted for e. Data are shown as mean±s.d. *P<0.05, **P<0.01 (ANOVA).

Figure 5

Conditional knockout of TGFβ type II receptor prohibits functional transition of NC cells. (a, b) Immunostaining of IVD sections from TgfbrII^−/− and their TgfbrII^+/+ littermates with antibodies against TβRII and pSmad2/3 (brown). Hematoxylin stains nuclei purple. (c, d) Quantification of TβRII- and pSmad2/3-positive cells in a and b. (g, h) Immunofluorescence staining of IVD sections for CCN2 (g) and Acan (h) (red). DAPI stains nuclei blue. (e, f) Quantification of CCN2 and Acan in the NC cells in g and h. (i) Safranin O-fast green staining of L_3–4 IVDs in TgfbrII^−/− mice and their TgfbrII^+/+ littermates (4-week-old). (j) Quantitative analysis of IVD volumes from μCT scan. n=6 per group. Data are shown as mean±s.d. *P<0.05, **P<0.01 (two-sided Student’s t test).

Figure 6

Conditional knockout of integrin α_v impedes functional transition of NC cells. (a, b) Immunostaining of IVD sections from α_v^−/− mice and their wild-type α_v^+/+ littermates (4-week-old) with antibodies against α_v (a) and β₆ (b) (brown). Hematoxylin stains nuclei purple. (c, d) Immunostaining for pSmad2/3 (c) and α_Vβ₆ (d) in NC cells. (e, f) Immunostaining for CCN2 (e) and Acan (f) in NC cells. (g, h) Quantification of pSmad2/3 (c) and α_Vβ₆ (d) expression in NC cells. (i, j) Quantification of CCN2 (i) and α_Vβ₆ (j) expression in NC cells. (k) Western blot analysis of α_v level in NC cells. (l) Western blot analysis of pSmad2 and Smad2 levels in NC cells. (m) Safranin O-fast green staining of L_3–4 IVDs showing enlarged vacuoles with less proteoglycans (red orange) in α_v^−/− mice relative to their α_v^+/+ littermates (4-week-old). (n) Safranin O staining images of the IVDs from sham-operated or LSI TgfbrII^−/− mice and α_v^−/− mice (8-week-old) at 2 weeks post surgery. n=3 per group. Data are shown as mean±s.d. *P<0.05, **P<0.01 (two-sided Student’s t test).

Figure 7

Inhibition of excess active TGFβ attenuates IVD degeneration in LSI mice. (a) IVD score in sham, LSI mice with injection of either Veh or TβRI inhibitor (SB, 1.0 mg·kg⁻¹). (b, e) Immunostaining and quantification of pSmad2/3 in the NP (brown). Hematoxylin stains nuclei purple. (c) Quantification of CCN2 areas in g. (d) Safranin O staining of L_4–5IVD section. (f, g) Immunofluorescence staining of IVD sections for Acan (f) and CCN2 (g) (red). DAPI stains nuclei blue. n=6 per group. Data are shown as mean±s.d. *P<0.05, **P<0.01, ^#P<0.05, ^##P<0.01 (ANOVA).

Figure 8

Inhibition of excess active TGFβ attenuates IVD degeneration in CIC rats and CSI mice. (a, c, d, h) Local administration of TGFβ neutralizing antibody (1D11, 0.7 μg) into the NP of rat caudal IVD compression model (CIC, 12-week-old) prevented IVD degeneration. (a) Rat CIC model. Rats were attached with the loading device. The axial stress loaded from the distal side produced a compressive pressure of 1.3 MPa on C_8–9 and C_9–10 IVDs. The C_8–9 of loaded IVDs were injected with an alginate bead containing 1D11 while the C_9–10 IVDs with that containing vehicle. (c) Safranin O staining of Caudal 8th-9th (C_8–9) IVD with 1D11 or vehicle treatment. Immunostaining and quantification of pSmad2/3⁺ cells (d, h) in the NP of CIC rat (brown). Hematoxylin stains nuclei purple. n=6 per group. (b, e g, i k) Caudal SI(CSI) mouse model was induced by full-depth annular stab and NP removal of the C_7–8 IVD. (b) CSI mouse model. The instability of caudal spine was induced by full-depth annular stab and NP removal of the C_7–8 IVD. The adjacent C_8–9 IVDs were chosen for observation. The adjacent C_8–9 IVDs were chosen for observation. (e) Safranin O staining of IVD section. Immunostaining of C_8–9IVD sections with antibodies against (f) pSmad2/3 and (g) CCN2; (i) IVD degeneration was evaluated by IVD score at 4 weeks post surgery. Quantification of (j) pSmad2/3⁺ cells and (k) CCN2⁺ expression in f and g. n=6 per group. Data are shown as mean±s.d. *P<0.05, **P<0.01, ^#P<0.05, ^##P<0.01 (ANOVA). (l) Model showing mechanosignaling activation of TGFβ controls IVD homeostasis.