TGF-β1 promotes osteogenesis of mesenchymal stem cells via integrin mediated mechanical positive autoregulation

Abstract

Positive autoregulation (PAR), one type of network motifs, provides a high phenotypic heterogeneity for cells to better adapt to their microenvironments. Typical mechanosensitive proteins can also form PAR, e.g., integrin mediated PAR, but the role of such mechanical PAR in physiological development and pathological process remains elusive. In this study, we found that transforming growth factor β1 (TGF-β1) and integrin levels decrease with tissue softening after the development of paradentium in vivo in rat model of periodontitis (an inflammatory disease with bone defect). Interestingly, TGF-β1 could induce the formation of mechanical PAR involving the integrin-FAK-YAP axis in mesenchymal stem cells (MSCs) by both in vitro experiments and in silico computational model. The computational model predicted a mechanical PAR involving the bimodal distribution of focus adhesions, which enables cells to accurately perceive extracellular mechanical cues. Thus, our analysis of TGF-β1 mediated mechanosensing mechanism on MSCs may help to better understand the molecular process underlying bone regeneration.

Keywords: Biological sciences; Cell biology; Mathematical biosciences; Mechanobiology.

Graphical abstract

Figure 1

Decreased expression of TGF-β1 and integrin α_vβ₃ in the periodontal ligament (PDL) upon periodontitis (A) Schematic of periodontitis induction between the first and second molar of rat maxillary. Both the MicroCT and HE staining indicated obvious absorption of alveolar bone caused by periodontitis (decreased R/C value and bone mass). Scale bar, 200 μm. (B) The stiffness of healthy PDL and periodontitis PDL in rats. (C) Immunostaining for TGF-β1 in healthy and periodontitis PDL of rats (left) and the corresponding quantification of TGF-β1 expression (right). Scale bar, 20 μm. (D) Immunostaining for integrin α_vβ₃ and stro-1 expression in healthy and periodontitis PDL of rats (left) and corresponding quantification of integrin α_vβ₃ expression (right). Red, integrin α_vβ₃; green, stro-1, one of the markers for mesenchymal stem cells; blue, nucleus. Scale bar, 30 μm. (E) Meta-analysis of published gene expression omnibus (GEO) data analysis showing TGF-β1, TGF-β1 receptor, and integrin expression in healthy PDL and periodontitis PDL. R, root; C, crown; B, bone; D, dentin. ∗∗∗p < 0.001, ∗∗p < 0.01.

Figure 2

Integrin mediated adhesion formation and MSCs osteogenic differentiation correlated with substrate stiffness and TGF-β1 (A) Schematic of the design of cell experiment in vitro for MSCs osteogenesis regulated by TGF-β1 and integrin mediated signaling. (B) Schematic of PEG hydrogel cross-linking process. (C) Immunostaining of Runx2 expression in MSCs w/wo TGF-β1 on hydrogels with different stiffness and the quantification of Runx2 expression (right). Scale bar, 30 μm. (D) Immunostaining of integrin α_vβ₃ expression in MSCs w/wo TGF-β1 on hydrogels with different stiffness and the quantification of integrin α_vβ₃ (right). Scale bar, 30 μm. (E) Immunostaining of focal adhesion (FA; paxillin) in MSCs w/wo TGF-β1 on different stiffness hydrogels and the quantification of FA number (upper right) and FA length (lower right). Green, paxillin; red, F-actin; blue, nucleus. Scale bar, 10 μm. (F) Immunostaining of FAK Y397 phosphorylation expression in MSCs w/wo TGF-β1 on different stiffness hydrogels and the quantification of FAK Y397 phosphorylation level (lower). Scale bar, 30 μm ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

Figure 3

TGF-β1 promoted the integrin-YAP mediated mechanosensing pathway (A) Representative immunofluorescent images of YAP, F-actin, and nuclear lamin A/C-in MSCs w/wo TGF-β1 treatment or on different stiffness hydrogels. Green, YAP; red, F-actin; yellow, lamin A/C; blue, nucleus. Scale bar, 10 μm. (B) Quantification of YAP nuclear/cytoplasm ratio, cytoskeleton anisotropy, and nuclear thickness. (C) Immunostaining of YAP nuclear localization in MSCs after the inhibition of FAK Y397 w/wo TGF-β1 on soft hydrogel and quantification of YAP nuclear/cytoplasm ratio (right). Green, YAP; red, F-actin; blue, nucleus. Scale bar, 30 μm ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

Figure 4

TGF-β1 mediated integrin-YAP mechanical positive autoregulation (PAR) (A) Schematic of the proposed pathway of TGF-β1 mediated integrin-YAP mechanical PAR. (B) Immunostaining of FA (paxillin) expression in YAP silenced-MSCs on different stiffness hydrogels. Green, paxillin; blue, nucleus. Scale bar, 10 μm. (C) Quantification of the FA number (left) and FA length (right). (D) Immunofluorescent staining of FA (paxillin) expression in YAP silenced-MSCs with TGF-β1 treatment on the stiff hydrogel. Green, paxillin; blue, nucleus. siNC, control siRNA group. Scale bar, 10 μm. (E) Real-time PCR analysis of FA related genes expression in YAP silenced-MSCs with TGF-β1 treatment on stiff hydrogel, including vinculin, paxillin and talin1. (F) Western blot analysis of FA related protein expression in YAP silenced-MSCs with TGF-β1 treatment on stiff hydrogel, including vinculin, paxillin, and talin1. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

Figure 5

TGF-β1 and matrix stiffness mediated mechanical positive autoregulation (PAR) could induce the bimodal distribution of FA (A) Model assumption of PAR for mathematical model. (B) Schematic of the mathematical model. (C and D) The distribution evolution of FA length (clustered integrins) in our experiments and simulation. (E) The sensitivity analysis of the mathematical model. + represents a forward process in which the distribution changes as the parameter value increases; - represents a reverse process in which the distribution changes as the parameter value increases. (F) A schematic illustration of TGF-β1 and matrix stiffness induced the formation of integrin mediated mechanical PAR by YAP. TGF-β1 and stiff matrix activate focal adhesion proteins-talin, vinculin, and paxillin, they bind to and activate integrin, resulting in downstream mechanical signaling, such as FAK. This mechanical signaling creates tension on the actin fiber cytoskeleton which causes stretching of the nuclear pore and ensures nuclear localization of YAP. In the nucleus, YAP promotes the transcription of genes encoding for FA proteins. The successive feedback induces integrin mediated mechanical PAR. On the contrary MSCs on the soft substrate without TGF-β1 treatment experience less tension, forming an unstable focal adhesion complex. Tension-dependent stretching of actin fiber cytoskeleton is disrupted, causing cytoplasmic retention of YAP and less mechanical PAR.