Tissue-specific calibration of extracellular matrix material properties by transforming growth factor-β and Runx2 in bone is required for hearing

Abstract

Physical cues, such as extracellular matrix stiffness, direct cell differentiation and support tissue-specific function. Perturbation of these cues underlies diverse pathologies, including osteoarthritis, cardiovascular disease and cancer. However, the molecular mechanisms that establish tissue-specific material properties and link them to healthy tissue function are unknown. We show that Runx2, a key lineage-specific transcription factor, regulates the material properties of bone matrix through the same transforming growth factor-β (TGFβ)-responsive pathway that controls osteoblast differentiation. Deregulated TGFβ or Runx2 function compromises the distinctly hard cochlear bone matrix and causes hearing loss, as seen in human cleidocranial dysplasia. In Runx2+/⁻ mice, inhibition of TGFβ signalling rescues both the material properties of the defective matrix, and hearing. This study elucidates the unknown cause of hearing loss in cleidocranial dysplasia, and demonstrates that a molecular pathway controlling cell differentiation also defines material properties of extracellular matrix. Furthermore, our results suggest that the careful regulation of these properties is essential for healthy tissue function.

Figure 1

Transforming growth factor-β overexpression disrupts cochlear bone matrix material properties and hearing. (A) The elastic modulus (GPa) of each bone is distinct. In the cochlear capsule, the (B) elastic modulus and (C) hardness of D4 cochlear bone matrix are reduced relative to WT (^*P<0.01). (D) D4 mice (open squares) have elevated ABR thresholds for click and pure tone stimuli relative to WT (filled circles) (^*P<0.001; ^#P<0.005). All error bars represent s.d. values. ABR, auditory brainstem response; TGFβ, transforming growth factor-β; WT, wild type.

Figure 2

Runx2 insufficiency in mouse models of cleidocranial dysplasia impairs hearing. (A) X-ray confirmed absent clavicles (arrows) in Runx2^+/− and D4 mice. (B) Runx2^+/− mice (open triangles) exhibit higher ABR thresholds than WTs (filled circles) (^*P<0.001). (C) CAP thresholds (click stimuli) were elevated in Runx2^+/− mice. (D) Expression of Runx2, OC and nAchR-α9 mRNA in cochlear bone or soft tissue. ABR, auditory brainstem response; CAP, compound action potential; mRNA, messenger RNA; OC, osteocalcin; WT, wild type.

Figure 3

Mouse models of cleidocranial dysplasia lack discernible defects in cochlear structures. (A) No differences between WT, Runx2^+/− and D4 cochleae were observed in the organ of Corti (orange arrow), spiral ligament, stria vascularis and spiral ganglia. (B) Phalloidin-stained organ of Corti showed normal organization with one IHC row and 3–4 OHC rows. Stereocilia (blue arrow) are normal in each strain. (C) Dissected ossicles (malleus (M), incus (I), stapes (S)) stained for bone and cartilage showed normal development and mineralization at 6 days of age. (D) Micro-CT shows that each ossicle and the joints (green arrow) between them are intact. Apparent differences are artefacts of imaging at scanner resolution limits. CT, computed tomography; IHC, inner hair cell; OHC, outer hair cell; WT, wild type.

Figure 4

Cochlear bone matrix material properties are regulated by Runx2 and are essential for hearing. The (A) elastic modulus and (B) hardness of cochlear bone matrix from Runx2^+/− mice is reduced relative to WT (^*P<0.01). (C) Reduced mineralization of Runx2^+/− tibial bone as assessed by XTM. (D) Multiple linear regression analysis comparing elastic modulus with ABR thresholds for each ear shows that elastic modulus is a critical determinant of auditory function (P<0.01, R²=0.77). ABR, auditory brainstem response; WT, wild type; XTM, X-ray tomography.

Figure 5

Defective Runx2^+/− bone matrix material properties, mineralization and hearing are rescued by inhibition of transforming growth factor-β. Quantitative reverse transcriptase–PCR shows the effect of (A) elevated TGFβ in D4 mice and (B) TGFβ inhibition in DNTβRII mice on Runx2-target gene expression in calvarial bone. X-rays, nanoindentation and TEM show that (C) clavicle dysplasia is not, but (D) elastic modulus (^*P<0.001) and (E) mineralization are rescued in DNTβRII;Runx2^+/− mice. (E) Arrows show sites lacking mineralization as detected through TEM. (F) ABR thresholds for Runx2^+/− (triangles), DNTβRII (diamonds) and DNTβRII;Runx2^+/− (crosses), or WT mice (filled circles) are shown (^*P<0.05). ABR, auditory brainstem response; TEM, transmission electron microscopy; TGFβ, transforming growth factor-β; WT, wild-type.