Myokine Irisin promotes osteogenesis by activating BMP/SMAD signaling via αV integrin and regulates bone mass in mice

Abstract

Irisin is well-known to contribute to bone homeostasis due to its bidirectional regulation on osteogenesis and osteoclastogenesis. However, the mechanisms of irisin involved in mesenchymal stem/stromal cells (MSCs)-derived osteogenesis are still under investigated. Fibronectin type III domain-containing protein 5 (FNDC5) is the precursor protein of irisin, compare with wild type (WT) littermates, FNDC5^-/- mice lost bone mass significantly, collectively evidenced by the decrease of bone mineral density (BMD), impaired bone formation and reduced N-terminal propertied of type I procollagen (P1NP) in sera. Meanwhile, the bone resorbing of FNDC5^-/- mice has enhanced accompanied by increased tartrate phosphatase (TRAP) staining cells morphologically and cross-Linked C-telopeptide of type 1 collagen (CTX) level in sera. In vitro study showed that lack of irisin impeded the MSC-derived osteogenesis of FNDC5^-/- mice. The addition of irisin promote the osteogenesis of WT and irisin-deficient MSCs, by activating αV integrin-induced ERK/STAT pathway, subsequently enhancing bone morphogenetic protein 2 (BMP2) expression and BMP/SMAD signaling activation. Taken together, these findings further indicate that irisin regulates bone homeostasis. Moreover, irisin promotes MSC-derived osteogenesis by binding to αV integrin and activating BMP/SMAD signaling consequently. Thus, irisin may be a promising therapeutic target for osteoporosis and bone defects.

Keywords: BMP/SMAD signaling; Irisin; Osteogenesis; mesenchymal stem/stromal cell; αV integrin..

Figure 1

FNDC5 deficiency in mice reduced bone mass. (A) 3D and (B) 2D reconstruction of micro-CT images of femurs. (C) Bone parameters of femurs at indicative time point. BV/TV (%), Tb.Th (mm), Tb.N (1/mm), Tb.Sp, BMD (mg/cm³), (n=6, *P < 0.05, **P <0.01, ***P <0.001).

Figure 2

FNDC5 deficiency in mice reduced bone formation and increased bone resorption. (A) H&E-staining of femur section. (B) Masson trichrome staining showing new bone collagen fibers in blue and red for mature bone. (C) Immunohistochemical staining indicating Osterix expression level in femur. (D) TRAP staining of femur sections. (E) P1NP, CTX in serum determined by ELISA. (n=6, *P < 0.05, **P <0.01, ***P <0.001).

Figure 3

R-irisin promoted osteogenic differentiation in vitro. (A) ALP staining of BMSCs after osteogenic induction. (B) AKP activity. (C) Alizarin Red staining. (D) Semi-quantitative analysis of mineralized nodules. (E) qRT-PCR analysis of OPN, RUNX2, ALP, and Osterix after osteogenic induction for 7 days. (n=5, **P <0.01, ***P <0.001, ****P <0.0001).

Figure 4

The clonal formation efficiency and osteogenic differentiation of bone marrow cells in WT and irisin deficiency mice. (A) Crystal violet staining and the number of cell colonies analysis after 7 days culture. (B) ALP staining and ALP positive area analysis after 7 days osteogenesis. (C) ARS staining and semi-quantitative analysis after 14 days osteogenesis (n=5, ****P <0.0001).

Figure 5

R-irisin rescued the reduced osteogenic differentiation of BMSCs caused by the deficiency of FNDC5. (A) ALP staining after osteogenic induction for 7 days. (B) AKP activity. (C) ARS staining after osteogenic induction for 14 days. (D) Quantitative analysis of ARS staining. (E) qRT-PCR analysis of the relative messenger RNA (mRNA) expression of the osteogenesis-related genes, including RUNX2, ALP, Osterix and OPN after osteogenic induction for 7 days (n=5, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001).

Figure 6

Endogenous and exogenous irisin restrain osteoclast activation. (A) qRT-PCR analysis of DC-STAMP, NFATc1, TRAP and OSCAR of WT mice after induction for 3 days. (B) TRAP staining of BMMs from WT mice after induction for 6 days. (C) qRT-PCR analysis of OSCAR, TRAP, DC-STAMP, and NFATc1 of BMMs from WT and FNDC5^-/- mice after osteoclastogenesis induction under the intervention of r-irisin for 3 days. (D) TRAP staining (**P <0.01, ***P <0.001, ****P <0.0001).

Figure 7

Irisin promoted the phosphorylation of ERK/STAT through binding to integrin receptor αV and up-regulates the expression of BMP2 to enhance osteogenic differentiation. (A) Cell lysates were subjected to immunoblotting analysis. Protein expression levels of integrin αV, p-Erk1/2, p-STAT3, BMPR2, p-Smad1/5/9, and Smad4 of BMSCs induced into osteoblasts for 3 days in the presence of r-irisin and SB273005. (B) ALP and ARS staining. (C) Protein expression levels of BMP2, Smad1/5/9, p-Smad1/5/9 and Smad4 after 3 days of osteogenic induction of BMSCs treated with r-irisin, and the ratio of p-Smad1/5/9 to Smad1/5/9 of the protein grayscale values. (D) Immunohistochemical staining of p-Smad1/5/9 in the femur of WT and FNDC5^-/- mice (n=6, *P <0.05, ***P <0.001, ****P <0.0001).

Scheme 1

Schematic illustration indicating FNDC5-deficient mice experienced bone loss by inhibiting osteogenic differentiation and promoting osteoclast activation. (A) Schematic diagram of the mechanism of irisin regulating bone formation. Irisin combined with its receptor integrin αV on the surface of BMSCs, then in turn activated the phosphorylation of ERK and STAT3, promotes an increase of BMP2. It banded to the receptor BMPR2 on the membrane surface to activate the phosphorylation of Smad1/5/9 and promote osteogenic differentiation.