Conversion of mechanical force into TGF-β-mediated biochemical signals

Abstract

Mechanical forces influence homeostasis in virtually every tissue [1, 2]. Tendon, constantly exposed to variable mechanical force, is an excellent model in which to study the conversion of mechanical stimuli into a biochemical response [3-5]. Here we show in a mouse model of acute tendon injury and in vitro that physical forces regulate the release of active transforming growth factor (TGF)-β from the extracellular matrix (ECM). The quantity of active TGF-β detected in tissue exposed to various levels of tensile loading correlates directly with the extent of physical forces. At physiological levels, mechanical forces maintain, through TGF-β/Smad2/3-mediated signaling, the expression of Scleraxis (Scx), a transcription factor specific for tenocytes and their progenitors. The gradual and temporary loss of tensile loading causes reversible loss of Scx expression, whereas sudden interruption, such as in transection tendon injury, destabilizes the structural organization of the ECM and leads to excessive release of active TGF-β and massive tenocyte death, which can be prevented by the TGF-β type I receptor inhibitor SD208. Our findings demonstrate a critical role for mechanical force in adult tendon homeostasis. Furthermore, this mechanism could translate physical force into biochemical signals in a much broader variety of tissues or systems in the body.

Figure 1. Loss of tensile loading causes tenocyte cell death in adult Achilles tendons

(A) – (D) Characterization of adult ScxGFP transgenic mice. (A) Achilles tendons (Ac, arrows) in 10-wk-old ScxGFP transgenic mice express a robust ScxGFP signal (green) under fluorescence stereomicroscopy. (B) Histological analysis of Achilles tendons in 10-wk-old ScxGFP transgenic mice. HE-stained sections (left panels) and the same areas with GFP/UV filters (right panels: green, ScxGFP; blue, DAPI [cell nuclei]). Upper panels: adult Achilles tendon. Note that aligned tenocytes express ScxGFP. Middle panels: Myotendinous junction at proximal Achilles tendon. ScxGFP is expressed only in tenocytes (arrow heads); myocytes (M) are completely negative for ScxGFP. Lower panels: Distal insertion of Achilles tendon. ScxGFP is expressed only in tenocytes. Chondrocytes (arrow heads) at the calcaneus are completely negative for ScxGFP. Bars = 50 μm. (C) Only primary tenocytes from ScxGFP transgenic mice express ScxGFP in vitro. Left panels: Phase contrast micrographs of primary tenocytes, chondrocytes and osteoblasts. Right panels: Fluorescence micrographs of the same area with a GFP filter. Bar = 100 μm. (D) RT-PCR analysis of gene expression profiles related to tendon and cartilage in primary adult wild-type mouse tenocytes and dermal skin fibroblasts. Tenocyte markers Scx and tenomodulin and chondocyte marker Sox9 are not expressed in skin fibroblasts. α1, α1 integrin; α11, α11 integrin; Col I, collagen type I; GAPDH, glyceraldehyde 3-phosphate dehydrogenase (as a control gene). (E) – (G) Acute tensile loading-loss model by complete transection of adult Achilles tendons. (E) Left panels: Illustrations of the complete transection model. Achilles tendon (Ac) was completely transected at 2 mm proximal to the calcaneus (Ca) (white line in picture and red line in drawing, respectively). Middle and right panels: Histological analysis of intact (untreated) and transected tendon tissues at 3 d after operation. HE-stained sections and the same areas with GFP/UV filters (green, ScxGFP; blue, DAPI). Sections were prepared approximately 3 mm from calcaneus in both transected and intact tendons as shown in left panels. Note that very few cells retain ScxGFP expression in comparison with normal intact adult tendon. Bar = 50 μm. (F) Analysis of cell death. Expression of ScxGFP (green; left panels), TUNEL staining (red; middle panels) and merged images (right panels) with DAPI (blue) at 1, 2, and 24 h after complete transection. Upper panels: 1 h after transection. ScxGFP expression is diminished, and TUNEL-positive cells appear at the edge of the tendon (arrows). Middle panels: 2 h after transection. ScxGFP expression is significantly diminished, and TUNEL-positive cells expand to the proximal side of the Achilles tendon. Lower panels: 24 h after transection. Extensive cell death results in the formation of an acellular region (arrowheads). Bar = 100 μm. (G) Analysis of TUNEL-positive cells in non-transected (Control) and transected tendons. Error bars represent standard deviation (n = 4; field = 0.07 mm²). *, P < 0.05; **, P < 0.001: significantly different compared to the number of positive cells in control non-transected tendons.

Figure 2. A gradual tensile loading-loss model by local injection of botulinum toxin A into adult Achilles tendons

(A) Left panel: Illustration of the botulinum toxin treatment. The toxin (6 U kg⁻¹ weight) was injected intramuscularly into medial and lateral sides of the gastrocnemius muscle (asterisks). Ac, Achilles tendon. Right panels: Time course of fluorescence micrographs in toxin-treated Achilles tendons of ScxGFP mice (green, ScxGFP; blue, DAPI; red, collagen type I and COMP). Bar = 50 μm. (B) Analysis of ScxGFP, collagen type I and COMP intensities shown in Figure 2A. Relative fluorescence intensities are shown relative to each control value of 100 (control tendon) (n = 5 for each group). Error bars represent standard deviation. *, P < 0.05; **, P < 0.01: significantly different compared to controls. (C) Biomechanical analysis of toxin-treated tendon tissues. Left panel: Resected Achilles tendon (Ac, arrowheads) for biomechanical experiments. Right panel: Typical force-distance curves in toxin-treated (botulinum toxin A, red line) and saline-treated (sham, blue line) tendon tissue at 1 wk post injection. Arrows indicate peak force. (D) Stiffness, peak force, and ultimate stress in untreated tendons (Control) and at 1 and 2 wks after toxin treatment. Error bars represent standard deviation (n = 5). Note that the toxin-treated Achilles tendons exhibit significantly reduced stiffness and peak force compared to untreated controls at 1 wk post treatment although ultimate stress is not significantly different. *, P < 0.05.

Figure 3. Mechanical force and Smad2-mediated signaling is required for maintenance of Scx expression in adult tenocytes in vitro

(A) Diagram of microfluidic chamber and regions. The composition is a bifurcating network of several generations with areas (I–IV) of different shear stresses (dyne cm⁻²), as indicated by the color legend. A steady flow of cell culture medium from the input tube (left white circle) to the output tube (right black circle) was supplied by a syringe pump. For a flow rate of 1 ml h⁻¹, the wall mechanical strain in each area was about 0.1 (area I), 0.14 (area II), 0.30 (area III), and 0.60 (area IV) dyne cm⁻², respectively. (B) ScxGFP expression in primary tenocytes at 7 d under different mechanical forces (flow rate, 1 ml hr⁻¹). Note that tenocytes in areas II (0.14 dyne cm⁻²), III (0.30 dyne cm⁻²) and IV (0.60 dyne cm⁻²) retain ScxGFP expression, whereas tenocytes in untreated controls and area I (0.1 dyne cm⁻²) show a marked decrease in ScxGFP levels. Bar = 50 μm. (C) Analysis of ScxGFP intensities in primary tenocytes at 7 d under the different mechanical forces shown in Figure 3B. GFP intensity of untreated tenocytes at 7 d after culture (No shear) was set to 1.0. Relative GFP intensities of tenocytes determined in each area are shown relative to untreated control. Results are the mean of measurements for 100 cells in each area. Error bars represent standard deviation. ScxGFP levels in areas II, III and IV are significantly higher than those in untreated controls. *, P < 0.05. (D) Left panels: ScxGFP in primary tenocytes at 7 d under 0.14 dyne cm⁻² mechanical force with or without 1.0 μM TGF-β type-I receptor inhibitor SD208. Note that SD208 markedly inhibits mechanical force-mediated ScxGFP induction. Bar = 100 μm. Right panel: Analysis of ScxGFP intensities. GFP intensity of tenocytes is shown relative to the control value of 100 (percent of control). Error bars represent standard deviation. Note that SD208 significantly (75.6%) inhibits mechanical force-mediated ScxGFP induction. *, P < 0.001. (E) Real-time PCR analysis of endogenous Scx mRNA expression in primary tenocytes at 7 d under 0.14 dyne cm⁻² mechanical force with or without (Control) 1.0 μM SD208. The intensity of Scx signals were normalized to that of 18S rRNA signals. Error bars represent standard deviation obtained by error propagation (n = 3). Note that SD208 significantly (72.0%) downregulates mechanical force-mediated endogenous Scx mRNA expression, and this downregulation correlates with ScxGFP levels shown in Figure 3D. *, P < 0.001. (F) Effect of SD208 on pSmad2 levels in primary tenocytes from wild-type mice at 7 d under mechanical force with or without (Control) 1.0 μM SD208 or without shear stress. Upper panels: Expression of pSmad2 (green; left panels), nuclear staining with DAPI (blue; middle panels) and merged images (right panels). Bar = 25 μm. Lower panel: Analysis of pSmad2 intensities. The intensity is shown relative to the control value of 100 (Control tenocytes under shear stress). Error bars represent standard deviation. *, P < 0.001.

Figure 4. Sudden interruption of continuous tensile loading destabilizes the ECM’s structural organization and allows consequent release of a significant amount of active TGF-β

(A) Active TGF-β bioassay in culture media from adult tenocyte cell lines cultured in a single network chamber for 5 d under different mechanical forces. Flow rate conditions for 60, 100 and 200 μl h⁻¹ corresponded to 0.01, 0.015 and 0.03 dyne cm⁻² mechanical force, respectively. Culture medium (100 μl) from each condition was used for the assay. The data show the amounts released in conditioned media for 1 h under different flow rates. Luciferase activity is presented as relative light units (RLU). Error bars represent standard deviation (n = 3). The values for active TGF-β released in the conditioned media over 1 h were 0.30 ± 0.018 pM under 60 μl h⁻¹ shear stress; 0.52 ± 0.18 pM under 100 μl h⁻¹ shear stress; and 1.01 ± 0.19 pM under 200 μl h⁻¹ shear stress. *, significantly different (P < 0.01) compared to the amount under strain at 60 μl h⁻¹ flow rate. (B) Effects of the cytokine TGF-β1 on primary tenocytes. Left and middle panels: Phase contrast and fluorescence micrographs of the same area with a GFP filter for each condition. Right panels: The assessment of cell death by TUNEL staining (red). Primary tenocytes were cultured for 48 h, then cytokines were added for a further 7 d. Note that the addition of TGF-β1 at a low concentration (2 ng ml⁻¹; 80 pM) retains the expression of ScxGFP. In contrast to other culture conditions, only a higher concentration (160 pM) leads to tenocyte cell death (13.0 ± 2.7 cells/field [n = 4; field = 0.15 mm²]). Retention of ScxGFP expression was seen at TGF-β1 concentrations as low as 20 pM (data not shown). Neither no addition (DMEM containing 1% FBS) nor 10% FBS maintained ScxGFP expression. Bars = 100 μm. (C) Ultrastructural analysis of collagen fibrils. Transmission electron micrographs of transverse sections (left panels) and morphometric analysis of fibrils (right panels) in intact (Control), completely transected, and toxin-injected adult Achilles tendon tissues at 2 and 24 h post treatment. Note that collagen fibril ultrastructure in tension-collapsing tendons by complete transection reveals irregular patterns by as early as 2 h post injury. Bar in left panel = 200 nm. (D) Active TGF-β bioassay in intact (untreated) and transected (at 1.5 h after complete transection) adult Achilles tendons. Luciferase activity is presented as relative light units (RLU). Error bars represent standard deviation (n = 3). Note that the transacted tendon tissues release significant amounts of active TGF-β. *, P < 0.05. (E) Effects of SD208 on cell death in vivo after complete transection of adult Achilles tendon. TUNEL staining (red; left panels), expression of ScxGFP (green; middle panels) and merged images (right panels) with DAPI (blue) in intact tendons and at 1.5 h after complete transection without (untreated) or with 1.0 μM SD208 treatment. Bar = 50 μm. (F) Analysis of TUNEL-positive cells at 1.5 h after complete transection without or with SD208 treatment. Error bars represent standard deviation (n = 4; field = 0.07 mm²). Note that SD208 significantly attenuates tenocyte cell death. *, P < 0.001.