Ifnβ Regulates Human Bone Marrow Derived Mesenchymal Stem Cell Differentiation: Interaction with Canonical Wnt Signaling

Research Article

J Stem Cells Res, Rev & Rep. 2014;1(1): 1005.

Ifnβ Regulates Human Bone Marrow Derived Mesenchymal Stem Cell Differentiation: Interaction with Canonical Wnt Signaling

Weimin Qiu1*, Li Chen1 and Moustapha Kassem1-3

1Department of Endocrinology, Odense University Hospital, Denmark

2Department of Anatomy, King Saud University, Saudi Arabia

3Department of Health Sciences, University of Copenhagen, Denmark

*Corresponding author: Weimin Qiu, Department of Endocrinology, Molecular Endocrinology Laboratory (KMEB), Odense University Hospital, Winsløws parken 25.1, DK-5000 Odense C, Denmark

Received: Aug 07, 2014; Accepted: Aug 20, 2014; Published: Aug 21, 2014


Interferons (IFNs) are multifunctional secreted cytokines involved in antiviral defense, cell growth regulation and immune activation. In addition, IFNs have been reported to play a regulatory role in bone homeostasis and bone resorption. However, the effects of IFNs on osteoblastic cells and bone formation are not clear. In the current study, we demonstrated that IFNβ decreased Human Bone Marrow derived mesenchymal Stem Cell (hBMSC) proliferation and their osteoblast differentiation but enhanced adipocyte differentiation by inhibiting Wnt signaling. Wnt signaling inhibition was mediated by two mechanisms: upregulation of Wnt antagonist DKK via canonical IFN signaling and reduction of stabilized nuclear β-catenin via reduced AKT phosphorylation. Targeting IFNβ signaling in hBMSC is a potential approach to enhance bone formation.


Keywords: Human bone marrow derived mesenchymal stem cell; IFNβ; Wnt; Bone homeostasis


Osteoimmunology is an interdisciplinary research area focusing on interaction of the immune system with bone and initially transpired due to the close proximity of bone and bone marrow organ. An increasing number of immune related cytokines have been reported to play a regulatory role in bone biology [1,2]. Among these molecules are Interferons (IFNs) which are multifunctional secreted cytokines involved in antiviral defense, cell growth regulation and immune activation. IFNs are categorized into two classes: type I IFN are produced in direct response to virus infection and are comprised of IFNα, IFNβ, and type II IFN consists of IFNγ which is produced in response to the recognition of infected cells by activated T lymphocytes and natural killer cells [3]. All type I IFNs share a common type I IFN receptor whereas IFNγ binds to type II IFN receptor [4]. Several studies have demonstrated that IFNs exert direct effects on bone cells. IFNs regulate osteoclast formation and osteoclastic bone resorption [2,5]. IFNγ promotes bone formation and rescues osteoporosis-related bone loss in ovariectomized mice [6,7]. IFNγ has also been reported to inhibit adipogenesis of Human Bone Marrow derived mesenchymal Stem Cells (hBMSC) in vitro and to prevent bone marrow fat infiltration in ovariectomized mice [8]. Mice deficient in IFNα receptor 1 (IFNAR1) (IFNAR1-/-) develop osteoporosis due to enhanced osteoclastic bone resorption [9]. A similar low bone mass phenotype was observed in IFNβ-/- mice (9). IFNβ has been reported to inhibit osteoblast-mediated mineralized matrix formation in vitro - effects that were abolished by 1α-25- dihydroxyvitamin D3 treatment [10, 11].

During the recent years, Wnt signaling has been recognized as the principal signaling pathway regulating osteoblastic cell differentiation and functions as well as bone formation [12]. Wnt signaling determines MSC differentiation fate to osteoblast or adipocyte lineages [13,14]. Patients with inactivating or activating mutations in Wnt coreceptor lipoprotein related receptor protein 5 (LRP5) causing inactivation or activation of Wnt signaling [15] exhibit either osteoporosis (a disease known as osteoporosis-pseudoglioma syndrome) or high bone mass, respectively [16-19]. Finally, in a number of genetic mouse models, inducible increase or decrease in skeletal Wnt signaling resulted in corresponding changes in bone mass [20-23]. The interaction between IFNs and Wnt signaling has been reported in a number of non-skeletal systems. Nava et al. have shown that, in intestinal epithelium cells, IFNγ induced Wnt signaling by activating PI3K/ AKT/β-catenin pathway and increased expression of DKK1 [24]. Also, IFNα inhibited Wnt signaling in pre-neoplastic rat liver by promoting β-catenin binding to FOXO instead of TCF protein [25] and by upregulating β-catenin nuclear export factor RanBP3 [26]. The interaction of IFN and Wnt signaling in bone has not previously been studied.

In the present study, we investigated the IFN effect on hBMSC proliferation and differentiation and its interaction with Wnt signaling. We demonstrated that among different IFNs, IFNβ exhibited the most significant inhibitory effect on hBMSC proliferation, osteoblast differentiation but enhanced adipocyte differentiation. These effects were mediated through inhibition of Wnt signaling by upregulating the expression of Wnt antagonist DKK and reducing nuclear β-catenin.

Material and Methods

Recombinant proteins, antibodies and conditioned medium

Recombinant human IFNα-2a (Genscript), IFNβ and IFNγ (Peprotech) were reconstituted in 0.1% BSA in 100 μg/ml and stored at -80°C for use. Antibodies for total and phospho-AKT (Ser473), total and phospho-β-catenin (Ser552) were purchased from Cell Signaling. α-tubulin antibody and peroxidase-conjugated secondary antibody were purchased from Santa Cruz. Control Conditioned Medium (CCM) and Wnt3a conditioned medium (Wnt3a) were prepared as previously reported [14].

Cell culture and differentiation

The previous, well characterized hBMSC, which was isolated from a healthy male donor and immortalized by human telomerase approved by local ethics committee [27] was used in this study. The culture and differentiation of hBMSC had been described previously (14). Briefly, cells were cultured in Minimum Essential Medium (MEM) plus 10% FBS and 1% Penicillin/Streptomycin (Invitrogen). Osteogenic induction medium contains 10mM β-glycerophosphate, 50μg/ml 2-phoshate ascorbate, 10nM dexamethasone and 10nM vit D3. Adipogenic induction medium contains 10%FBS, 10%horse serum, 100nM dexamethasone, 450μM 1-methyl-3-isobutylxanthine (IBMX) (Sigma-Aldrich), 1 μM Rosiglitazone (BRL49653) (kindly provided by Novo Nordisk, Bagsvaerd Denmark), 3μg/ml human recombinant insulin (Sigma-Aldrich). In this study, 1ng/ml IFNα, β or γ were also used in corresponding hBMSC differentiation experiments.

Cell proliferation assay

Cell proliferation was determined by CellTiter-Blue reagent. Briefly, hBMSC cells were seeded at 3000 cells/cm2 in 96-well plate with normal culture medium. The next day, attached cell numbers were quantitated by CellTiter-Blue reagent (Promega) according to the instruction as day 0. Medium was changed in the remaining wells to normal culture medium with 0.1%BSA as control or with 1ng/ml or 100ng/ml IFNα, β or γ. Cell number was quantitated at day 1, 3, 5, 7 and 9 and the medium was changed every three days.

ALP activity assay and real-time qRT-PCR analysis

Analysis of osteoblastic and adipocytic marker genes have been described previously [14,28]. Briefly, total RNA was isolated by GenElute™ mammalian total RNA miniprep kit (Sigma) and quantitated by Nanodrop as instructed. Up to 1µg total RNA was reverse transcribed by using iScript cDNA synthesis kit (Bio-Rad) and gene expression was analyzed by using fast SYBR® green master mix (Applied Biosystem) on Step One PlusTM system (Applied Biosystem) according to the manufacturer’s protocol. The primers used in this study were listed in supplemental Table 1.

siRNA knock down

siRNA targeting human type I IFN receptors (IFNAR1 and IFNAR2) and AKT1 (siRNA ID# s7183, s7184, s659 respectively) as well as non-targeting siRNA were purchased from Ambion. 33nM siRNA was reverse transfected by LipofectaminTM 2000 (Invitrogen) according to the instruction.

Luciferase reporter cell lines and luciferase assay

To establish type I IFN and canonical Wnt luciferase reporter cell lines, hBMSC were first infected with lentivirus expressing Renilla Luciferase (GenTarget Inc.) at multiplicities of infection (MOI) of 25 in the presence of 6µg/ml polybrene, and then selected by 300ug/ml G418 (Invitrogen) for one week. Then cells were infected with Cignal Lenti ISRE Luc Reporter or Cignal Lenti TCF/LEF Luc Reporter (CLS-008L or CLS-018L, SABiosciences) respectively and selected by 0.8ug/ml puromycin for one week and then named as hBMSC-ISRE or hBMSC-TCF respectively.

For luciferase assay, reporter cells were seeded in 96-well plate at the density of 2x104 cells/cm2. The next day hBMSC-TCF cells were treated with 5% to 50% Wnt3a conditioned medium, or 20% Wnt3a conditioned medium with 1 or 100ng/ml IFNα, β or γ for 24 hours. To determine IFNAR1 and 2 knocking down efficiency, hBMSC-ISRE reporter cells were transfected by siRNA and treated with 1ng/ml IFNβ for 24 hours. To examine the effect of IFNAR1 and 2 knocking down on Wnt signaling, hBMSC-TCF cells were transfected by siRNA and treated with 20% Wnt3a conditioned medium plus 1ng/ml IFNβ for 24 hours. Luciferase activity was determined by dual luciferase assay (Promega) as described.

Expression analysis of Wnt components

For expression of Wnt components, hBMSC was cultured in 20% Wnt3a conditioned as control or 20% Wnt3a conditioned medium with 1ng/ml IFNβ for three days. Gene expression was analyzed by real-time qRT-PCR [14,28].

Western blot and immunofluorescence staining

hBMSC was treated with either 0.1% BSA as control or 1ng/ ml IFNα, β or γ for 24 hours. Western blot analysis was performed as described [14] and band intensity was quantitated by Image J software and normalized against tubulin. For immunofluorescence staining of nuclear β-catenin, hBMSC was treated with 20% Wnt3a conditioned medium without or with 1ng/ml IFNβ for 2 hours and fixed in PBS buffered formaldehyde (pH 7.0) for 10 minutes followed by incubation with TBS buffered Triton X-100 (pH 7.4) for 10 minutes. After blocking with PBS containing 10% donkey serum for 30 minutes, cells were incubated with anti-β-catenin antibody (1:100 dilution) for 1 hour and then incubated with Alex 488 conjugated donkey anti-Rb IgG (1:500 dilution) for 1 hour and images were taken by Leica fluorescent microscope.

Statistical analysis

Statistical testing was determined by Student’s t-test and P<0.05 was considered as significant.


IFNβ regulates hBMSC proliferation and differentiation

To determine the effect of type I and type II IFN on hBMSC proliferation, hBMSC were treated with IFNα, β or γ (1ng/ml or 100ng/ml) for 9 days. We observed that IFNβ inhibited hBMSC proliferation at day 3 (p<0.001) and there was no difference between the effects of 1 ng/ml or 100 ng/ml IFNβ. The inhibitory effects of IFNα and γ on hBMSC proliferation were first detected at high concentration (100 ng/ml) at day 3 (p<0.001) and day 5 respectively (p<0.05) (Figure 1A). During osteoblast differentiation, IFNα, β or γ (all 1ng/ml) inhibited early osteoblastic differentiation marker ALP’s activity, and among these, IFNβ exhibited the most pronounced effects (Figure 1B). Similarly, IFNβ significantly reduced the steady state gene expression levels of ALP and COL1A1 at day 7 (data not shown) and day 10 (Figure 1C) but not the expression of other osteogenic markers e.g. RUNX2, BSP and osteocalcin (data not shown). In vitro mineralization analysis by using alizarin red staining was not possible in this study due to significant inhibition of cell proliferation by IFNβ. Moreover, no significant inhibitory effects of IFNα and γ on the expression of ALP and COL1A1 were observed (Figure 1C). In contrast, IFNβ increased gene expression of adipocytic markers including CEBPA, PPARG2, AP2, APM1 and LPL (Figure 1D) with more pronounced effects as compared to IFNα and γ. These results demonstrate that IFNβ exerts the most significant effects on hBMSC proliferation and in vitro differentiation.