R-spondin 2 (Rspo2) is a novel osteogenic factor that acts cooperatively with BMP and Wnt/β-catenin signaling to promote osteoblast differentiation and bone formation. The study found that Rspo2 increases osteoblast gene expression, alkaline phosphatase activity, and mineralization independently of Wnt11. Rspo2 expression is necessary for the osteogenic effects of Wnt11, as knockdown of Rspo2 inhibited Wnt11-induced osteoblast differentiation and gene expression. Rspo2 acts through both β-catenin dependent and independent pathways to enhance bone formation.

1. R-spondin 2 is a Novel Osteogenic Factor Required for
Wnt11 Mediated Osteoblast Differentiation
Michael S. Friedman1, Sivan M. Oyserman1, Weijun Luo2,and Kurt D. Hankenson3
Thermogenesis1, University of Michigan Biomedical Engineering2, University of Pennsylvania School of Veterinary Medicine3
Abstract Results Results Results
The four members of the R-spondin family share structural features including two conserved
cysteine rich domains and a thrombospondin type I repeat. R-spondin family members can
activate canonical Wnt signaling as determined by stabilization of beta-catenin and increased
Rspo2 Activates β-catenin in Pre-osteoblasts Rspo2 Increases Mineralization Independent of Tcf Knockdown of Rspo2 Expression Inhibits Bsp and
TOPFLASH reporter activity. R-spondins are known to interact with Frizzled and LRP family Phex Expression
members and to enhance the signaling of Wnt ligands. During development, R-spondin 2 is
highly expressed in the apical ectodermal ridge, and knockout mice which die at birth, have Wnt11 MC3T3 Control MC3T3 dnTCF4 Rspo2 Expression: Rspo2 Knockdown Phex Expression: Rspo2 miR
limb patterning defects. We previously reported that Wnt11 increases osteoblast differentiation
GFP Rspo2 30 35
Fold Cha nge Gene Expression
Fold Change Gene Expression
25 30
and activates Rspo2 expression in MC3T3 E1.14 pre-osteoblasts. We hypothesized that Rspo2
may be involved in Wnt11-induced osteoblast differentiation.
Control 4 nM 2 nM Control 4 nM 2 nM 20
25
Rspo2 was retrovirally overexpressed in MC3T3 E1.14 pre-osteoblasts that were subsequently
β−catenin 15 Control
BMP
20
15
Control
BMP
10
cultured in osteogenic conditions. We observed that Rspo2 potently synergized with BMP to *
GFP 10
enhance alkaline phosphatase expression and matrix mineralization (Alizarin red S). We 5 5
examined osteoblast-specific gene expression in Rspo2 overexpressing cells relative to Wnt11 0 0
Wnt11/LacZmiR Wnt11/Rspo2 miR Wnt11/LacZmiR Wnt11/Rspo2 miR
and GFP control cells, with and without BMP treatment. BMP-treated Rspo2 cells show
statistically significant higher levels of osterix, Bsp, Dmp1, and Phex expression relative to BMP Lamin A
treated Wnt11 and GFP control cells. Wnt11 8
Dmp1 Expression: Rspo2 Knockdown
300
BSP Expression: Rspo2 Knockdown
Fold Change Gene Expression
Fold Change Gene Expression
7
To evaluate if Rspo2-mediated gene expression was dependent on Tcf/beta-catenin signaling, 6
250
we retrovirally overexpressed dominant/negative Tcf4 in Rspo2 overexpressing cells. dnTcf4 5
200
expression in Rspo2 cells significantly decreases osterix expression to levels comparable to Control (GFP), Wnt11, or Rspo2 overexpressing MC3T3 were cultured in 6 well plates for 2 4 Control 150 Control
*
GFP control cells. Phex expression is also decreased in Rspo2-dnTcf4 cells relative to Rspo2 days. The cells were subsequently cultured in osteoinductive medium for 3 days. The cells Rspo2 3
BMP
100
BMP
cells, but remains higher than Wnt11 overexpressing cells. were subsequently lysed for protein analysis and separated into nuclear, cytosolic, and 2
membrane fraction. Western blot for β-catenin was performed on the nuclear fraction.
50
1
To determine if Rspo2 expression was necessary for Wnt11-enhanced osteoblast 0 0
differentiation, we used retroviral-based micro RNAi to knock-down expression of Rspo2. GFP Wnt11/LacZmiR Wnt11/Rspo2 miR Wnt11/LacZmiR Wnt11/Rspo2 miR
cells were infected with a control LacZ miRNAi retrovirus, while Wnt11 cells were infected with
either a LacZ control miRNAi retrovirus or the Rspo2 miRNAi retrovirus. Alkaline phosphatase MC3T3 E1 control (Wnt11/LacZmiR), and Rspo2 miRNAi knockdown cells were plated in 6-well
induction and mineralization were evaluated as markers of osteoblast differentiation. Knock-
Q-PCR Validation of Wnt11 Induced Gene Expression MC3T3 GFP, MC3T3 Wnt11, MC3T3 GFP dnTCF, and Wnt11 dnTCF cells were cultured in dishes, cultured for 3 days, and subsequently placed in osteo-inductive media with or without
down of Rspo2 expression in Wnt11 cells decreases Wnt11-enhanced alkaline phosphatase osteoinductive media with or without the indicated concentration of rhBMP6 for 3 days. The rhBMP6. All cells were harvested for RNA 9 days after the initiation of BMP treatment. Values
induction to near control levels. Importantly, knock-down of Rspo2 expression resulted in a Rspo2 Expression Phex Expression media was changed ever 3 days thereafter and harvested for alizarin red S staining represent fold-change in gene expression of BMP treated control or Rspo2 knockdown cells
relative to untreated controls and normalized to β-actin, as determined by qRT-PCR (delta-delta
140 250
nearly complete blockade of mineralization. approximately 16 days after post induction.
Fold Change Gene Expression
Fold Change Gene Expression
120
200 Ct method).
We also evaluated levels of osteoblast associated gene expression in Wnt11/Rspo2 100
knockdown cells. By quantitative PCR, Rspo2 expression in knock-down cells was reduced by 80
150 *
Control Control
99% relative to the Wnt11-LacZ miRNA control cells. Knockdown of Rspo2 in untreated Wnt11
cells significantly decreased expression of Otoraplin/Cdrap, Bsp, and Phex. BMP treated
60
40
BMP 100 BMP
Rspo2 is Required for Increased AP Activity and Discussion
50
Wnt11-Rspo2 knockdown cells also demonstrate significantly reduced expression of BSP and
Phex. We conclude that Rspo2 expression is both necessary and sufficient to mediate many of
20
0 0
Mineralization by Wnt11
Control Wnt11 Rspo2 Rspo2 Control Wnt11 Rspo2 Rspo2
the effects of Wnt11-enhanced osteoblast differentiation. dnTCF dnTCF
We previously determined that Wnt11 increases BMP induced osteoblast differentiation and
800
activates expression of Rspo2. In these studies, we evaluated the role of Rspo2 in Wnt11
6 nM 2 nM mediated osteoblast differentiation. We found that Rspo2 increases alkaline phosphatase
Dmp1 Expression
Osterix Expression
activity and mineralization, independently of Wnt11. Similar to Wnt11, Rspo2 increases β-
Results
1000
7 700 BMP BMP
Control
Fold Change Gene Expression
Fold Change Gene Expression
6 catenin activitation, resulting in nuclear accumulation.
100
5 600
* Rspo2 overexpression, in combination with BMP, is also sufficient for increased Phex, Osx,
nmoles PNP/Hr
4
GFP/LacZ
Rspo2 Increases AP Acitivity and Mineralization 3
* Control
BMP
10 Control
BMP 500 Dmp1, and Bsp expression—all genes we previously reported as Wnt11 responsive genes. We
2
had previously shown that Tcf activity was required for increased Dmp1, Phex, and Bsp
1
Control
1 400 expression in Wnt11 overexpressing pre-osteoblasts. Interestingly, Tcf activity is only required
900 BMP
0 0.1 for increased Osx expression in Rspo2 overexpressing cells. Thus Rspo2 has effects that are
Control 5nM BMP 2.5nM BMP 1nM BMP Control Wnt11 Rspo2 Rspo2 Control Wnt11 Rspo2 Rspo2 300 Wnt11/LacZ independent of Wnt11 and Tcf signaling.
800 dnTCF dnTCF
*
700 ‡
200 Rspo2 is also necessary for increased osteoblast function and gene expression downstream of
GFP Rs Bsp Expression Wnt11. Retroviral based miRNAi knockdown of Rspo2 leads to decreased alkaline
10000
100
nmoles PNP/Hr
phosphatase activity and mineralization relative to Wnt11 control cells. Rspo2 is also required
Fold Change Gene Expression
600
po
2 1000
Wnt11/Rspo2 miR for increased expression of Phex and Bsp downstream of Wnt11.
500 0
Control
We conclude that Rspo2 is a novel mediator of osteoblast differentiation, acting cooperatively
400
BMP 100 Control GFP WNT11 WNT11-
Wnt-11 BMP with BMP, Wnt/Tcf/β-catenin signaling to regulate osteoblast gene expression and function.
RSPO2
300 10
miR
200 1
Control Wnt11 Rspo2 Rspo2
Rspo2
Acknowledgements
dnTCF
100
MC3T3 GFP/LacZ miR (control), MC3T3 Wnt11, and MC3T3 Wnt11-Rspo2 knockdown
0
MC3T3 E1 control, Wnt11, Rspo2, and Rspo2 dnTCF overexpressing cells were plated in 6- (Wnt11-Rspo2 miR) cells were plated in 6-well dishes and cultured for 3 days. The cells were
GFP WNT11 RSPO2 well dishes and cultured for 3 days. The cells were subsequently placed in osteo-inductive subsequently placed in osteo-inductive media with rhBMP6. The media was changed every 3 1. These studies were funded by NIH grant R01DE017471 to KDH
media with rhBMP6. The media was changed every 3 days thereafter and replaced with osteo- days thereafter and replaced with osteo-inductive media without rhBMP6. The cells were
2. All sample preparations, hybridizations, and microarray scans were performed by the
inductive media without rhBMP6. All cells were harvested for RNA 9 days after the initiation of harvested and alkaline phosphatase activity assessed 6 days after the initiation of BMP
University of Michigan Microarray Core facility.
MC3T3 control (GFP), Wnt11, or Rspo2-overexpressing E1 pre-osteoblasts were placed in BMP treatment. Values represent fold-change in gene expression of BMP treated GFP or treatment. Alizarin red S staining for mineral was performed 12-16 after the initiation of BMP
osteinductive conditions with or without BMP and harvested at day 6 for an alkaline Wnt11 cells relative to untreated controls and normalized to β-actin, as determined by qRT- treatment. 3. Support was also provided by the University of Michigan Microarray Gene Expression
phosphatase assay or at days 12-16 for alizarin red S staining. PCR (delta-delta Ct method). Analysis Musculoskeletal Research Core facility.