Irisin Regulated Bone Metabolism in Gain-of- and Loss-of-function Mouse Models

Xiaofang Zhu1, 2, Qisheng Tu1, Jin Zhang1, Guofang Shen2, Jake Jinkun Chen1

Introduction

Irisin, a recently identified novel hormone-like myokine, is the cleaved and secreted portion of fibronectin-type III domain-containing 5 (FNDC5). It was initially reported that irisin plays an important role in converting the white adipose tissue (WAT) to brown adipose tissue (BAT) and regulating energy expenditure (Boström et al. 2012). Recent studies also reported the involvement of irisin in many other physiological and pathological conditions, such as type II diabetes mellitus (T2DM) (Zhang et al. 2014), renal disease (Ebert et al.2014), hippocampal neurogenesis (Curlik et al. 2013), and osteoporotic fractures (Anastasilakis et al. 2014). In our previous study, increased levels of irisin were found in different regions of femoral bones in exercising mice, and that leads us to hypothesize that irisin regulates bone metabolism. In this study, we explore the effects of myokine irisin in bone formation and further established an irisin knockout mouse line to determine the mechanisms of irisin regulating bone metabolism.

Materials and Methods

Cell culture experiments. MC3T3-E1 cells (ATCC, Manassas, VA) were cultured with osteogenic induction. MC3T3-E1 cells were serum-starved overnight and then treated with 50 µg/ml of ascorbic acid (AA) in the presence or absence of irisin for 7, 10 and 14 days. The formation of bone nodules was monitored by alizarin red staining followed by

1 Division of Oral Biology, School of Dental Medicine, Tufts University, Boston, Massachusetts, UNITED STATES 2 Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, CHINA melting bone nodules with 10% (v/v) cetylpyridinium chloride and determination of absorbance at 562 nm. RAW264.7 osteoclast precursor cells (ATCC, Manassas, VA) were cultured, and then serum-starved overnight and cultured in the presence or absence of irisin and/or 50 ng/ml RANKL (Pepro Tech) for 1, 3 or 6 days. After 6 days cells were fixed and stained for TRAP.

Irisin injection treatment. C57BL/6J mice (000664, Jackson Laboratory) were used.

Irisin protein (3.24 ng/mouse; n=6 mice; Phoenix Inc) or saline (n=6 mice) were injected

IP daily for two weeks, and sacrificed on the day after the last injection.

fl/fl Construction and verification of the Osx-Cre/Irisin mice. We designed the targeting strategy (Fig. 3A) and constructed the C57BL/6 mice carrying irisin gene bordered with two loxP sequences (irisinfl/fl). Osx-Cre/irisinfl/fl mice were generated by crossing irisinfl/fl mice with Sp7-Cre mice (006361, Jackson Laboratory), the transgenic mice expressing

Cre recombinase gene under SP7 promoter and specifically expressed in osteoblastic lineage. Both the ES cells for constructing irisinfl/fl mice and the irisinfl/fl mice were genotyped by Southern Blot and PCR, and the homozygous achieved on the third generation. (Fig. 3B) qRT-PCR and western blot analyses. Total RNA was isolated using the RNeasy Mini Kit

(Qiagen, CA). One µg of total RNA was used for reverse transcription. Whole protein lysates were prepared by using RIPA lysis buffer (Santa Cruz Biotechnology Inc).

Nuclear proteins were purified using a nuclear extraction kit (EMD Millipore, Germany).

Micro-computed tomography (µCT) analysis. The mice were anesthetized by isoflurane inhalation (1.5-2.0%) and assessed using a micro-computed tomography (µCT) system

(SkyScan1172; Bruker-microCT, Belgium). The morphometric indices are calculated. Results

Irisin induces osteoblast differentiation, mineralization and β-catenin protein expression in the MC3T3-E1 osteoblast cell cultures. When the MC3T3-E1 cells were treated with recombinant irisin, the mRNA expression of OSX, RUNX2, SATB2, BSP and collagen I was upregulated (Fig. 1A). In addition, irisin significantly increased the mineralization of

MC3T3-E1 cells at 6 weeks. (Fig. 1B) In agreement with irisin activating bone formation signaling mechanisms, higher β-catenin protein expression was detected in MC3T3-E1 cultures treated with irisin and AA for 3 or 6 hrs than in those cultured without irisin (Fig.

1C), and increased the nuclear β-catenin levels (Fig. 1D) at all times investigated.

Irisin inhibits RANKL-induced osteoclast differentiation. The expression of the osteoclast differentiation markers including TRAP and cathepsin K significantly decreased at 3

Fig 1. (A) Irisin increased AA induced osteoblast differentiation osteogenic genes expression at 10 days. (B) Mineralization assay of

MC3T3-E1cells cultured with or without irisin for 3 and 6 weeks. (C) Total β-catenin protein expression in MC3T3-E1 treated in the presence of AA for 1, 3 and 6 hours with or without irisin. (D) β-catenin protein expression in nuclear extracts of MC3T3-E1 cultures. (E) qRT-PCR analysis of osteoclast differentiation markers in RAW264.7 cells treated with RANKL and/or irisin for 3 days.

(F) Irisin effects on formation of TRAP+ cells with RANKL in different concentrations of irisin for 6 days. TRAP positive cells counted.

(G) Irisin effects in NFATc1 mRNA expression and NFATc1 protein expression. (H) Western blot image and quantification of calcineurin and p-AKT1 in RANKL–induced RAW264.7 cells treated with or without irisin for 0, 10, 30, and 60 minutes. days by irisin treatment (Fig. 1E). Additionally, irisin treatment significantly decreased the TRAP+ multinucleated cells in a dose dependent manner (Fig. 1F), and NFATc1 mRNA and the protein expression levels in the RANKL-treated RAW264.7 cells on day

6 (Fig. 1G). In addition, irisin inhibited the expression of calcineurin and phosphorylation of JNK signals at 60 min, the phosphorylation of Akt1 at 10 and 30 min in the RANKL- treated RAW264.7 cells (Fig. 1H).

Irisin regulates bone metabolism in vivo. Intraperitoneal (IP) injections of recombinant irisin induced the appearance of irisin-positive osteoblasts at the edge of the growth plate (Fig. 2A) and led to 3-fold higher irisin levels in circulation (Fig. 2B). µCT analyses of femoral bones revealed significant increases of bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and cortical thickness (Co.Th) in the irisin-treated group as compared with the saline-treated group (Figs. 2C, D). The irisin-treated group also demonstrated higher osteoblast numbers (Fig. 2E) than the control group.

Fig 2. (A) Representative immunohistochemistry with anti-irisin antibody shows irisin positive osteoblasts (arrows) were found on the edge of growth plate in irisin-treated mice. (B) Circulatory levels of irisin in control and irisin treated mice. (C) µCT images of the distal metaphyseal regions of femurs of control and irisin-treated mice (n=5). (D) Trabecular BV/TV, Tb.Th and Co.Th were measured by µCT in femurs of control and irisin-treated mice. Values are means ± SD of five mice/group (*p<0.05, vs control).

Bone development and mineralization were significantly delayed in Osx-Cre/ Irisinfl/fl mice. We successfully generated the Osx-Cre/irisinfl/fl conditional knockout mice, in which FNDC5/irisin gene was specifically deleted in osteoblastic lineage. Through the reconstruction of the µCT images, the mineralization of skull, hyoid, ribs, xiphoid and coccyx in cKO group were found to be significantly slower than in WT group at 6 and 10 weeks. The cortical bone mineral density (Co.BMD) and Tb.BV/TV in femurs in cKO group were remarkably lower than that in WT groups, while the Co.BS/BV were increased (p < 0.05)

Fig 3. (A) Irisin gene conditional knockout targeting strategy. Exon 2 was conditionally removed. (B) (Left) Southern blot confirmed that the loxP fragments were successfully inserted into the ES cells. (Right) PCR was used to genotype the irisinfl/fl mice. (C) The mineralization of skull, hyoid, ribs, xiphoid and coccyx of the irisin cKO mice (arrows) was delayed comparing with that in wild-type in

6 and 10 weeks. (D) Cortical BMD, BS/BV and Trabecular BV/TV were measured by µCT in femurs of WT group and cKO group on

6 and 10 weeks. Values are means ± SD of four mice/group (*p<0.05, vs control). Discussion

For the first time, our studies demonstrate that irisin increases osteoblastogenesis through the Wnt/β-catenin pathway and inhibites osteoclastogenesis by suppressing the

RANKL/NFATc1 pathway. Systemic administration of irisin increases trabecular bone volume, cortical bone thickness, and osteoblast numbers. Our newly established irisin conditional knockout mice showed delayed bone formation and mineralization. Irisin, as a new myokine, may play an important role in bone metabolism, representing a potential molecule for the prevention and treatment of bone diseases. On the other hand, the effect of the lacking of irisin in other systems in vivo, such as adipose metabolism, nervous system, are still unknown. We recently established the Ella-Cre/iriisnfl/fl mice by crossing irisinfl/fl mice with EIIa-Cre mice (003724, Jackson Laboratory), the transgenic mice for germ line deletion of loxP-flanked genes. The broader effects of irisin will be further investigated in our future work.

References

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Authorship Contribution

Xiaofang Zhu designed and performed experiments, analysed data and wrote this extended abstract; Jake Jinkun Chen, Qisheng Tu and Guofang Shen designed experiments; Jin Zhang designed experiments, and performed expriments.