論文の公開元へFamily with sequence similarity 20 member B regulates osteogenic differentiation of bone marrow mesenchymal stem cells on titanium surfaces
概要
主論文の要旨
Family with sequence similarity 20 member B regulates
osteogenic differentiation of bone marrow mesenchymal
stem cells on titanium surfaces
Family with sequence similarity 20 member B は
チタン表⾯における⾻髄由来間葉系幹細胞の
⾻分化能を制御する
名古屋大学大学院医学系研究科
頭頸部・感覚器外科学講座
（指導：日比 英晴
宋 昕蔓
総合医学専攻
顎顔面外科学分野
教授）
【Introduction】
Successful bone regeneration on titanium (Ti) surfaces is a key process in dental implant
treatment. Bone marrow mesenchymal stem cells (BMSCs) are fundamental cellular
components of this process, and their early recruitment, proliferation, and differentiation into
bone-forming osteoblasts are crucial. A proteoglycan (PG)-rich layer has been reported to exist
between Ti surfaces and bones; however, the molecules that could potentially affect the
formation of this layer remain unknown.
Family with sequence similarity 20 member B (FAM20B) is a newly identified kinase that
regulates the synthesis of glycosaminoglycans, an important component of the PG-rich layer.
And FAM20B is also closely associated with bone development.
The extracellular-regulated kinase (ERK1/2) pathway is important for maintaining bone
homeostasis, and has been reported to control osteogenic differentiation in response to
fibroblast growth factor 2 (FGF2) in BMSCs and affect the expression of osteoblast markers.
However, the effect of FAM20B on ERK1/2 signaling in BMSCs has not yet been reported.
Runt-related transcription factor 2 (RUNX2) is the main transcription factor for osteogenesis.
It contains a DNA-binding runt domain and plays a major role in the commitment of MSCs to
the osteoblastic lineage, regulating osteogenic differentiation and activating the expression of
genes related to osteogenesis.
We believe that FAM20B affects the osteogenic differentiation of BMSCs via ERK1/2 and
RUNX2. To test this hypothesis, we analyzed the function of FAM20B in BMSCs on Ti
surfaces.
【Materials and Methods】
The FAM20B knock down BMSCs cell lines (shBMSCs) were employed and all of cells
were cultured on Ti surfaces. The cells were then subjected to cell initial adhesion and
osteogenic differentiation. qRT-PCR, western blot, immunofluorescence, ALP activate assay,
reporter assay were used to explore the osteogenic-related target molecule expressions and
signaling pathway. Alizarin red staining was used to evaluate calcified nodules. The
transmission electron microscopy was also performed to analyze interface between Ti and
BMSCs.
【Results】
Topology of Ti surface
The morphology of the treated Ti surfaces (machined surfaces with ridges and valleys along
the treating direction) were clean and devoid of any contamination.
Establishing FAM20B knockdown cell lines
To investigate the role of FAM20B in osteogenesis, we obtained stable UE7T-13 cell lines
wherein FAM20B was knocked down. Two shRNA sequences and one control were separately
inserted into the UE7T-13 cell line using a lentivirus system. Furthermore, the cell proliferation
tests revealed that the depletion of FAM20B increased cell proliferation after the cells were
cultured on Ti surfaces for 48 h.
Effect of FAM20B on focal adhesion and PG-rich layer of BMSCs cultured on Ti surfaces
Quantification of the vinculin was done 1, 2, and 24 h after culturing the cells on Ti surfaces,
it can be observed that sh groups showed no significant difference in the area of expression of
vinculin from the control group at any time. After 14 days of culture, the area of the PG-rich
layer on the interface was observed to be lower in the sh groups than in the control group.
Effect of FAM20B on osteogenic differentiation of BMSCs cultured on Ti surfaces
The cells were cultured on Ti surfaces for seven days. Expression of osteogenic biomarkers
ALP and OCN were downregulated in sh groups. However, the expression of RUNX2 did not
show a difference in all groups. ALP staining was lighter in the sh groups. Similarly, ALP
activity also decreased in the sh groups after culturing on Ti surfaces for seven days. And lower
calcified nodules were observed in sh groups.
Effect of FAM20B on activation of ERK1/2 signaling of BMSCs cultured on Ti surfaces
pERK1/2 expression levels in the sh groups were lower than that in the control group while
maintaining the values for total ERK1/2.
In comparison to no FGF2 administration, FGF2
treatment promoted ALP expression in all groups, but the level of enhancement was higher in
the control group than in the sh group. Treatment with FGF2 increased the OCN expression in
the control group but not in the sh groups.
Effect of FAM20B on RUNX2 nuclear translocation
IF tests showed that while majority of the RUNX2 was in the nucleus for the control group,
it was in the cytosol for the sh groups when the cells were cultured on Ti surfaces for 24 h and
72 h. Likewise, the results of western blotting tests showed that the sh groups exhibited
decreased RUNX2 in the nucleus and increased RUNX2 in the cytosol when the cells were
cultured on Ti surfaces for 24 h and 72 h, but their total RUNX2 amount was similar to that of
the control group.
Effect of FAM20B on RUNX2 transcriptional activity
Transcriptional activity decreased in FAM20B siRNA groups compared to the control group.
And we found that the RUNX2-promoted activation of the OCN promoter was significantly
decreased by depletion of FAM20B.
【Discussion】
In this study, we established BMSC cell lines devoid of FAM20B and analyzed their function
in the osteogenic differentiation of BMSCs on Ti surfaces. After culturing for 14 days on Ti
surfaces, a reduced PG-rich layer was observed between the cells and the surfaces, which was
probably because of the incomplete formation of GAGs as a result of FAM20B depletion.
We also found that FAM20B depletion (a) resulted in decreased expression of osteogenesisrelated factors, (b) decreased ALP formation capacity and activity, and (c) reduced mineralization
ability in BMSCs cultured on Ti surfaces. These results suggest that FAM20B is an essential
component for osteogenic differentiation of BMSCs cultured on Ti surfaces.
In addition, depletion of FAM20B resulted in a reduced molecular level of pERK after FGF2
treatment. This further suggests that depletion of FAM20B inhibits the activation of the
ERK1/2 signaling pathway. In the present study, depletion of FAM20B was observed to inhibit
the osteogenic differentiation of BMSCs cultured on Ti surfaces, even though the expression
of RUNX2 remained unchanged. As a mechanism, the results of IF and Western blotting
showed that depletion of FAM20B in BMSCs inhibited nuclear translocation of RUNX2 and
reduced its transcriptional activity. Regarding the relationship between ERK activation and
RUNX2, ERK has been reported to not affect gene expression but stimulate transcriptional
activity. FAM20B was observed to reduce pERK1/2 signaling; this may have directly led to a
decrease in transcriptional activity.
To summarize, in this study, a part of the mechanism of bone formation on Ti surfaces has
been elucidated, which is the primary focus of dental implant treatment. Further research on
FAM20B is required for discovering a drug that will ensure successful dental implant treatment.
【Conclusion】
This study showed that FAM20B affected the osteogenic differentiation of BMSCs on Ti
surfaces through ERK1/2 and RUNX2 transcriptional activity.
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