Artikel
TGF-β1 impairs mechanosensation of human osteoblast via HDAC6-mediated shortening and distortion of primary cilia
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Veröffentlicht: | 23. Oktober 2017 |
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Gliederung
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Objectives: Transforming growth factor β (TGF-β) is a critical regulator of bone density owing to its multiple effects on cell growth and differentiation. However, its mode of action is not completely understood, as both reduced and chronically elevated TGF-β1 levels are associated with retarded bone formation. Previously, we have shown that TGF-β1 effectively blocks bone morphogenetic protein induced osteogenic differentiation into osteoblasts by up-regulating histone deacetylase (HDAC) activity. The current study aimed at investigating the underlying mechanisms by identifying the involved HDACs and their cellular effects.
Methods: Primary human osteoblasts were isolated from spongy bone by collagenase digestion. During osteogenic differentiation cells were mechanically stimulated by fluid flow. HDAC expression was determined by (q)RT-PCR and Western blot. HDAC6 activity was chemically inhibited by tubacin or CAY10603. Primary cilia were visualized by immunofluorescent staining of acetylated α-tubulin. Osteoblast function was characterized by AP activity and matrix mineralization. Data sets were compared by Kruskall Wallis test followed by Dunn's multiple comparison test.
Results and Conclusion: Exposure to TGF-β1 significantly induced expression of HDAC6 (21.5-fold, p<0.001) both on gene and protein level. Being most abundant in the cytoplasm HDAC6 effectively deacetylates microtubule structures. Thus, TGF-β1-induced expression of HDAC6 led to deformation and shortening (-23%, p<0.001) of primary cilia as well as to reduced numbers of ciliated cells (4-fold). Primary cilia sense mechanical stimuli, e.g. fluid flow, which stimulates osteogenic differentiation. Thus, compromised primary cilia in TGF-β1-treated cells were associated with reduced osteogenic differentiation, i.g. reduced AP activity (-67%, p<0.001) and matrix mineralization (-48%, p<0.001), despite of exposure to fluid flow conditions. Chemical inhibition of HDAC6 with Tubacin significantly improved tubulin acetylation and restored primary cilia length (Figure 1 [Fig. 1]). These cells showed significantly improved osteogenic function under fluid flow conditions. Summarizing our results, TGF-β1 impairs human osteoblast differentiation partially via HDAC6-mediated distortion and/or shortening of primary cilia. This knowledge opens up new treatment options for patients with chronically elevated TGF-β1-levels (e.g. diabetics), which frequently suffer from decreased bone mineral density and delayed fracture healing despite adequate mechanical stimulation.