This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
c-Src Controls Mouse Embryonic Osteogenic Differentiation Through Regulation of Stat1 Stability
by
Zahra Alvandi
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Department of Laboratory Medicine and Pathobiology University of Toronto
1.5.1 Regulation of Runx2 Transcriptional Activity via Post-Translational Modifications .........................................................................................................15
1.5.2 Regulation of Runx2 Transcriptional Activity via Runx2-Interacting Partners ....16
1.6 Regulation of Osteogenic Differentiation by c-Src ...........................................................18
1.6.1 c-Src Structure and Function .................................................................................19
v
1.7 Hypothesis and Objectives .................................................................................................21
3.1 c-Src Activity Inhibits Osteogenic Differentiation in mESCs ............................................28
3.1.1 Osteogenic Differentiation in Mouse Embryonic Stem Cells ...................................28
3.1.2 c-Src Expression Profile in Mouse Embryonic Stem Cells During Osteogenic Differentiation ........................................................................................................31
3.1.3 Inhibition of c-Src at Different Periods During ES Cells Osteogenic Differentiation ........................................................................................................32
3.1.4 Overexpression of p-Y416-c-Src in MC3T3-E1s and its Effect on Osteogenic Differentiation ........................................................................................................41
3.2.1 The Effect of c-Src Activity on Runx2 Target Genes’ Expression ..........................45
3.2.2 The Effect of c-Src Activity on Runx2 Expression ..................................................46
vi
3.2.3 c-Src Activity and its Effect on Runx2 Subcellular Localization .............................49
3.3.4 Inhibition of c-Src activity lowers Runx2 and Stat1 interaction ...............................51
3.3 c-Src Regulates Osteogenic Differentiation Through Stat1 ................................................53
3.3.1 c-Src effect on Runx2 localization and transcriptional activity during osteogenic differentiation is significantly reduced in the absence of Stat1 .............................53
3.3.2 c-Src Activity and its Effect on Stat1 Subcellular Localization ...............................55
3.3.3 The Effect of c-Src Activity on Stat1 Expression and its Half-life ..........................57
3.3.4 The Effect of c-Src Activity on Stat1 Degradation ...................................................61
We showed earlier that inhibition of c-Src between day 6 to 10 significantly increases osteogenic
differentiation in ESCs evidenced by a significant increase in OC mRNA expression and level of
mineralization of day 21 osteo-nodules. We also confirmed the specificity of c-Src inhibition
using specific c-Src siRNAs in MC3T3-E1 cells which also resulted in an increase of the OC
mRNA level and overall mineralization level assess by ARS. Furthermore, using lenitiviral
system we showed that overexpression of constitutively active c-Src inhibits osteogenic
differentiation in MC3T3-E1 cells. Next, we addressed the questions of how c-Src activity may
inhibits osteogenic differentiation. More importantly, we were interested to know the
significance of c-Src in osteogenic differentiation between day 6 to 10. As earlier
discussed, Runx2 initiates osteogenesis in a manner that is precisely controlled temporally and
spatially, and loss of Runx2 expression at this early stage impairs osteogenic differentiation in
bone development. For this reason, Runx2 became an interesting target of regulatory role of c-
Src in osteogenic differentiation for further investigation.
3.2.1 The Effect of c-Src Activity on Runx2 Target Genes’ Expression
It has been shown by others that Runx2 is involved in the transcriptional activation of many
promoters including those of COL1a1, BSPII, and OC (Ducy et al. 1997; Kern et al. 2001).
Considering Runx2 significance in transcription regulation of osteogenic markers, I tested Runx2
target genes expression including BSPII, COL1A1, and OC in response to inhibition of c-Src
activity. To do so, differentiating mESCs were either treated with PP2, the specific inhibitor of c-
Src, or PP3 (the inactive analog of PP2), or DMSO (control solvent) as negative controls for 24
hours. Lysates were then collected and subjected to qPCR analysis using specific primers listed in
table A-1. Results showed that inhibition of c-Src activity by PP2 significantly increased mRNA
expression of BSPII and COL1A by almost 1.5-fold (p<0.01, and p<0.001, respectively) and of
OC by more than 2 folds (p<0.01) when compared to the DMSO condition (Figure 3-9A). Results
of PP2 treated cells were significantly different from PP3 condition, however there were no
significant difference between DMSO and PP3 conditions. Next, I investigated to see whether or
not this significant change of the mRNA expressions is due to an increased transcriptional activity
of Runx2 at the promoters of the tested Runx2 target genes. Therefore, treated cells with PP2, PP3,
or DMSO were subjected to ChIP analysis. Anti- Runx2 antibody was used to immunoprecipitate
46
Runx2 protein binding to the extracted chromatin, and anti- POL II and anti-IgG served as a
positive and a negative control in the ChIP assay, respectively. The efficiency of the antibodies
was examined in a separate IP assay following WB analysis (data not shown). PCR primers
targeting COL1A, OC, and BSPII promoters are listed in table A-2. COL2 primer served as a
negative control as its promoter activity is not directly regulated by Runx2. Results of the
conducted ChIP assay revealed that when c-Src activity is inhibited under PP2 treatment condition,
the promoter occupancy of the COL1A, BSPII, and OC by Runx2 are significantly increased by
3.9, 9.3, and 3.4, respectively when compared to the DMSO control condition (Figure 3-9B). No
significant changes were observed in the IgG level among the conditions for the tested genes.
Furthermore, Runx2 occupancy of COL2 promoter was below detection limit which confirmed the
specificity of the observed phenomenon for Runx2 target genes.
3.2.2 The Effect of c-Src Activity on Runx2 Expression
Next, I asked whether or not the increased level of Runx2 transcriptional activity is due to an
elevated level of Runx2 expression. Therefore, first to test whether c-Src would regulate Runx2
expression between day 6 to 10 of osteogenic differentiation in ES cells, I treated the
differentiating cells either with PP2 (c-Src specific inhibitor) or PP3 (the inactive analog of PP2)
and DMSO (control solvent) as controls between days 6 to 10. Next, I collected cells at day 5, 7,
9, and 11 for all the mentioned conditions and subjected the lysates to qPCR and WB analysis.
Inhibition of c-Src activity in mESCs did not affect Runx2 mRNA and protein level (Figures 3-
10A and 3-10B). To further confirm that the effects of c-Src inhibition on Runx2 were specific c-
Src, I used two different c-Src specific siRNAs to reduce c-Src production in MC3T3-E1 cells
following the protocol as described in the material and method section. I performed twice
transfections on MC3T3-E1 cells with c-Src specific siRNAs every second day during
osteogenic differentiation for 5 days. SilencerTM select negative control siRNA served as control.
Then, I collected the lysates and subjected them to western blotting. Results showed both
siRNAs successfully downregulated the expression of c-Src and subsequently p-Y416-c-Src.
However, lower c-Src activity due to the decreased level of c-Src phosphorylation at Y416 did
not result in any significant change in Runx2 protein level when compared to the control
condition (Figure 3-10C).
47
A B
Figure 3-9 Inhibition of c-Src activity increases Runx2 transcriptional activity in mESCs.
(A) Day 10 differentiating cells from mESCs were treated with PP2 (10 µM), or PP3 (10 µM),
or DMSO for 2 hours. Lysates were collected and their total RNA were extracted. mRNA
expression of Runx2 target genes osteogenic markers including BSPII, COL1A1, and OC were
quantified by qPCR analysis. Values for PP3 and PP2 treated cells were normalized to their
corresponding DMSO condition. Mean values of triplicates were graphed for the indicated
conditions. One-way ANOVA was conducted for each primer pairs and p values were calculated.
(B) Lysates from described assay were also subjected to ChIP analysis using Runx2 antibody.
Precipitated immunocomplexes were analyzed by qPCR using BSPII, COL1A1, and OC primer
pairs. Values for PP3 and PP2 treated cells were normalized to their corresponding DMSO
condition. Mean values of triplicates were graphed and subjected to one-way ANOVA for
statistical analysis. Graphs represent pooled data from three independent assays.
*, **, ***, and **** indicate p<0.05, p<0.01, p<0.001, and p<0.0001, respectively.
48
A B
C
Figure 3-10 Inhibition of c-Src does not affect Runx2
expression in mESCs and MC3T3-E1s.
c-Src activity was inhibited using PP2 (10 µM) in
differentiating mESCs during day to 10 of osteogenesis. PP3
(10 µM) and DMSO served as negative controls. Lysates were
collected at the indicated times. (A) RNA was extracted for
qPCR analysis using Runx2 primer pairs. All values were
normalized to the corresponding L32 housekeeping gene and
fold changes were calculated by normalizing the quantified
values against their DMSO corresponding condition and mean
values of triplicates (±SD) were graphed.
One-way ANOVA was performed for each day data set where calculated p values showed no
statistical significant difference among tested conditions. The graph shows pooled data from
three independent experiments. (B) Lysates from day 5, 7, and 10 were subjected to WB analysis
using Runx2 antibody. P-Y416-c-Src antibody was applied to assess c-Src activity among
indicated conditions. (C) MC3T3-E1 cells were transfected with two distinct specific c-Src
siRNA along with Silencer ™ select negative control. Lysates were collected 48 hours post-
transfection and subjected to WB analysis using the indicated antibodies.
49
3.2.3 c-Src Activity and its Effect on Runx2 Subcellular Localization
Since a change in Runx2 mRNA and protein level was not observed following c-Src inhibition
neither in ESCs nor in MC3T3-E1s. However, reported from other studies levels of Runx2 are
often not well correlated with its transcriptional activity (Franceschi et al. 2003). Therefore, I
decided to examine whether or not c-Src inhibition would have any effect on Runx2 subcellular
localization. To do so I employed immunofluorescence (IF) staining using anti-Runx2 antibody
in mESCs treated with PP2 (10 µM), or PP3 (10 µM), or DMSO for two hours. IF staining
showed that distribution of Runx2 is more restricted to the nucleus of differentiating mESCs
when c-Src activity was inhibited with PP2 in comparison to PP3 or DMSO control conditions
(Figure 3-11A). To confirm these results, I used a biochemical fractionation to separate the
cytoplasmic and nuclear protein fractions from the treated mESCs with PP2, PP3 and DMSO to
examine the sub-cellular localization of Runx2. Nuclear fractions were then subjected to WB
analysis using anti-Runx2 antibody. Histone 3 (H3) served as the loading control for nuclear
fractions. Anti-GAPDH antibody was applied to assess the level of contamination with
cytoplasmic fractions. Consistent with the IF results, Runx2 remained mostly cytoplasmic under
both control conditions (DMSO and PP3), whereas it was re-localized to the nucleus upon PP2
treatment (Figure 3-11B). To further investigate the specificity of c-Src contribution into nuclear
localization of Runx2, I used c-Src specific siRNAs to down regulate the expression and activity
of c-Src in MC3T3-E1 cells. SilencerTM select negative control siRNA was used to transfect the
cells to further test the specificity of the assay. After 48 hours of transfection, nuclear fractions
were extracted and subjected to WB analysis using anti-Runx2 antibodies. Results showed that
upon downregulation of c-Src, nuclear localization of Runx2 was increased in comparison to the
control condition (figure 3-11C).
50
Figure 3-11 Runx2 nuclear localization is increased
when c-Src activity is inhibited.
(A) Day 10 ES differentiating cells were treated with PP2
(10 µM), or PP3 (10 µM), or DMSO for 2 hours, fixed and
stained with Runx2 antibody. DAPI used as a marker for
nuclei staining. Runx2 nuclear localization was increased
upon inhibition of c-Src activity by PP2. (B) Day 10 ES
differentiating cells were treated with PP2 (10 µM), or PP3
(10 µM), or DMSO for 2 hours. Lysates were collected and
fractions were isolated. Extracted nuclear fractions were by WB using Runx2 antibody. H3 and GAPDH served as loading controls of nuclear and cytoplasmic
fractions, respectively. (C) MC3T3-E1 cells were either treated with PP2 (10 µM) or DMSO for two
hours or subjected to transfection with c-Src specific siRNA or silencer™ negative select. Lysates of all
for conditions were fractionated and analyzed by immunoblotting using Runx2 antibody.
Results shown are representative experiments from three independent assays.
10µm
51
3.3.4 Inhibition of c-Src activity lowers Runx2 and Stat1 interaction
One major mechanism that affects protein localization is through interaction with other protein(s)
that stably reside in a cellular compartment. Given the earlier observation regarding the
inhibitory effect of c-Src on Runx2 nuclear localization, I hypothesized that c-Src has regulatory
role on Runx2-Stat1 interaction. To examine my hypothesis, first I tested the interaction of
Runx2 and Stat1 when c-Src activity was inhibited with a co-IP assay. In this assay, day 10
differentiating EBs were treated with PP2 for 2 hours. PP3 and DMSO were served as controls.
Lysates were then prepared and subjected to an IP analysis using Runx2 antibody. Precipitated
complexes were then subjected to WB analysis with both Runx2 and Stat1 antibodies. As shown
in figure 3-12A, upon inhibition of c-Src activity with PP2, Runx2 interaction with Stat1 is
significantly reduced compared to the control conditions. To further examine the specificity of c-
Src inhibition and effect on Runx2-Stat1 interaction, I used specific c-Src siRNAs to inhibit c-
Src expression and activity this time in MC3T3-E1 cells. 48 hours post-transfection MC3T3-E1
cells were then collected and subjected to the co-IP assay in which immune complexes were
immunoprecipitated this time with Stat1 antibody to further confirm the specificity of the IP
assay. Precipitated immunocomplexes were subjected to WB analysis using both anti-Runx2 and
anti-Stat1 antibodies. Results are presented in the figure 3-12B which showed that although Stat1
antibody precipitated comparable level of Stat1 in both control and c-Src down-regulated
MC3T3-E1 cells, however Runx2 interacting with Stat1 was remarkably lower in cells with
(A) c-Src activity was inhibited in day 10 of osteogenic differentiation in mESCs using PP2 (10
µM) for 2 hours. PP3 (10 µM) and DMSO served as controls. Lysates were collected and
subjected to IP assay using Runx2 antibody. Precipitated immunocomplexes were then analyzed
by WB using both Runx2 and Stat1 antibodies. (B) c-Src expression was down regulated in
MC3T3-E1 cells. Silencer™ select negative siRNA served as negative control. 48 hours post-
transfection cells were lysed and IP assay was performed using Stat1 antibody.
Immunocomplexes were subjected to WB analysis using Stat1 and Runx2 antibodies.
Results shown are representative experiments from three independent assays.
A B
53
3.3 c-Src Regulates Osteogenic Differentiation Through Stat1
3.3.1 c-Src effect on Runx2 localization and transcriptional activity during osteogenic differentiation is significantly reduced in the absence of Stat1
To test whether or not c-Src exerts its effect on osteogenic differentiation at least in part through
regulation of Stat1-Runx2 interaction, I decided to examine whether or not inhibition of c-Src
would still be effective on enhancement of osteogenic differentiation when Stat1 is depleted in
MC3T3-E1 cells. Therefore MC3T3-E1 cells were transfected by Stat1 specific siRNA or to
transiently downregulate Stat1. SilencerTM select negative control siRNA served as control. 48
hours post-transfection, transfected cells were either treated with DMSO or PP2 for 2 hours.
Cells were then collected and subjected to fractionation assay to examine the effect of c-Src
inhibition by PP2 on subcellular localization of Runx2 in absence and presence of Stat1. Nuclear
fractions were further analyzed by WB using Runx2 antibody. GAPDH and H3 served as
cytoplasmic and nuclear markers, respectively. Results showed that downregulation of Stat1
resulted in an increase in the level of nuclear Runx2 almost comparable to the control condition
upon inhibition in c-Src activity by PP2. Also, the results indicated that in the absence of Stat1,
the inhibition of c-Src activity was not as effective in terms of increase in Runx2 nuclear
localization (Figure 3-13A). To further examine the effect of c-Src activity in the absence and
presence of Stat1 on Runx2 transcriptional activity and osteogenic differentiation, I decided to
evaluate the expression of OC as the specific markers of osteoblasts and Runx2 target by the end
of a 14-day differentiation assay. Therefore, MC3T3-E1 cells were transfected with Stat1 every
other day for 3 times with either Stat1 specific siRNA or SilencerTM select negative control
siRNA. Transfected cells were treated with PP2 (10 µM) or DMSO for 6 days starting from day
3 (48 hours post-transfection). On day 14, the final day of osteogenic differentiation, total RNA
from differentiated MC3T3-E1 cells for the experimented conditions were extracted and
subjected to qPCR analysis to test the expression level of OC. Analyzed data were normalized to
the average value of control condition in which cells were transfected with negative control
siRNA and were treated with DMSO. Graphed data are shown in figure 3-13B. Data showed
that inhibition of c-Src activity with PP2 resulted in a statistically significant increase of OC
mRNA (p<0.01). Also, downregulation of Stat1 resulted in a significant higher expression of OC
mRNA with (p<0.01) or without (p<0.05) inhibition of c-Src activity by PP2. However, the
54
difference between OC mRNA level of -/+ PP2 treatment of transfected MC3T3-E1 cells with
specific Stat1 siRNA was not significant (p=0.2). The results of this experiment collectively
suggest that the regulatory effect of c-Src on Runx2 subcellular localization is through Stat1
protein as an interacting partner of Runx2.
10.0
0.5
1.0
1.5
2.0
2.5
OC
Fol
d ch
ange
Stat siRNA - - + + PP2 - + - +
**
***
Figure 3-13 c-Src inhibitory role on osteogenic differentiation is Stat1-dependent.
(A) MC3T3-E1 cells were transfected with Stat1 specific siRNA along with Silencer™ select
negative control siRNA. 48 hours post transfection cells were treated either with PP2 (10 µM) or
DMSO for 2 hours. Cells were lysed and subjected to fractionation assay. Nuclear fractions were
analyzed by WB analysis using Stat1 and Runx2 antibodies. H3 and GAPDH served as loading
controls for nuclear and cytoplasmic fractions. (B) MC3T3-E1 cells were subjected to
transfection three times every 48 hours. Transfected cells were either treated with PP2 (10 µM)
or DMSO for 6 days. Lysates were collected at day 14 of osteogenic differentiation and total
RNAs were extracted. RNAs were analyzed by qPCR using OC primer pairs. OC mRNA
expression was quantified and triplicate values for each condition were normalized to average of
triplicate values from DMSO treated negative siRNA transfected cells and graphed. One-way
ANOVA was conducted and p values were calculated. *, and ** indicate p<0.5 and p<0.1.
Results shown are representative experiment from three independent assays.
Stat1
55
3.3.2 c-Src Activity and its Effect on Stat1 Subcellular Localization
Our findings so far suggested that c-Src could have a regulatory role on Stat1 such that it
facilitates Stat1 interaction with Runx2 or prevents it. One possible way would be that c-Src
affects the phosphorylation of Stat1 which could in turn affect Stat1 interaction with other
molecules including Runx2. It is well established knowledge now that Stat1 undergoes
dimerization upon phosphorylation of Tyr 701 and translocate to the nucleus. Therefore, if c-Src
affects Stat1 phosphorylation at Y701, it could alter Stat1 sub cellular localization which
potentially affects Stat1 interaction with Runx2 in cytoplasm. Therefore, I asked whether or not
Stat1 phosphorylation status of Y701 is affected by c-Src activity. To address this question, I
conducted an experiment in which day 10 differentiating mESCs were treated with PP2 (10µM),
or PP3 (10 µM), or DMSO for 2 hours. Treated cells were then collected and subjected to WB
analysis using Y701 phospho-specific Stat1, Stat1, and GAPDH antibodies. Strikingly, inhibition
of c-Src by PP2 resulted in Stat1 phosphorylation at Y701 suggesting that c-Src activity
chelation using BAPTA-AM enhances it via phosphorylation of p-Y416-c-Src. These findings
strongly suggest a regulatory role for CRT upstream of c-Src during osteogenic differentiation
and should be further investigated.
4.3.1 Regulation of Stat1 Degradation by ERK Downstream of c-Src
Phosphorylation of Stat1 on serine 727 is required for Stat1 transcriptional activity (O'Shea et al.
2002) and degradation (Soond et al. 2008). Active p42/p44 MAPK-ERK has shown to
phosphorylate STAT1 on serine 727 and targets it for proteasomal degradation (Soond et al.
2008). Using WB analysis, I showed that inhibition of c-Src activity resulted in an increase in
ERK activity (Figure A6). Therefore, enhanced activity of ERK in response to c-Src inhibition
potentially increases p-S727-Stat1 level and enhances Stat1 degradation. This observation may
suggest a role for ERK downstream of c-Src to regulate Stat1 stability and should be further
examined.
4.3.1 Stat1 Proteasomal Degradation by c-Src/SIAH2
E3 ubiquitin ligase seven-in-absentia-2 (SIAH2) has been shown to indirectly abrogate the
tyrosine phosphorylation of Stat1through tyrosine-kinase 2 (TYK2) degradation (Muller et al.
2014). Phosphorylation and activation of SIAH2 by c-Src has also been shown in an independent
study (Sarkar et al. 2012). c-Src regulatory effect on Stat1 proteasomal degradation through
SIAH2 should be further investigated.
74
References
Aguila, H.L., and Rowe, D.W. 2005. Skeletal development, bone remodeling, and hematopoiesis. Immunol Rev. 208: 7-18.
Akiyama, H. Kim, J.E., Nakashima, K., Balmes, G., Iwai, N., Deng J.M., Zhang, Z., Martin, J.F., Behringer, R.R., Nakamura, T., Crombrugghe, B. 2005. Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors. Proc. Natl. Acad. Sci. U S A 102: 14665-70.
Akiyama, H. and de Crombrugghe, B. 2009. Transcriptional control of chondrocyte differentiation. 147-170.
Arumugam, B., Vairamani, M., Partridge, N.C., and Selvamurugan, N. 2018. Characterization of Runx2 phosphorylation sites required for TGF-beta1-mediated stimulation of matrix metalloproteinase-13 expression in osteoblastic cells. J Cell Physiol. 233: 1082-1094.
Bain, J., McLauchlan, H., Elliott, M., and Cohen, P. 2003. The specificities of protein kinase inhibitors: an update. Biochem. J. 371: 199-204.
Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C.J., McLauchlan, H., Klevernic, I., Arthur, J.S., Alessi, D.R., and Cohen, P. 2007. The selectivity of protein kinase inhibitors: a further update. Biochem. J. 408: 297-315.
Baksh D., Song, L., Tuan, R.S. 2004. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med. 8: 301-16.
Berendsen A.D., Oslen, BR. 2015. Bone development. Bone 80: 14-18.
Bhattacharyya, S., Yu, H., Mim, C., and Matouschek, A. 2014. Regulated protein turnover: snapshots of the proteasome in action. Nat Rev Mol Cell Biol. 15: 122-133.
Bianco, P. 2014. "Mesenchymal" stem cells. Annu Rev Cell Dev Biol. 30: 677-704.
Bonewald, L.F. 2011. The amazing osteocyte. J Bone Miner Res. 26: 229-38.
Bouet, G., Bouleftour, W., Juignet, L., Linossier, M.T., Thomas, M., Vanden Bossche, A., Aubin, J.E., Vico, L., Marchat, D., and Malaval, L. 2015. The impairment of osteogenesis in bone sialoprotein (BSP) knockout calvaria cell cultures is cell density dependent. PLoS One 10: e0117402.
Buttery, L.D., Bourne, S, Xynos J.D., Wood, H, Hughes F.J., Hughes S.P., Episkopou, V, and Polak, J.M. 2001. Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells. Tissue Eng. 7: 89-99.
Choi, Y.H., Han, Y., Lee, S.H., Cheong, H., Chun, K.H., Yeo, C.Y., and Lee, K.Y. 2015. Src enhances osteogenic differentiation through phosphorylation of Osterix. Mol Cell Endocrinol. 15: 85-97.
75
Colnot, C. 2009. Skeletal cell fate decisions within periosteum and bone marrow during bone regeneration. J Bone Miner Res. 24: 274-282.
Compton, J.T. and Lee, F.Y. 2014. A review of osteocyte function and the emerging importance of sclerostin. J Bone Joint Surg Am. 96: 1659-68.
Csobonyeiova, M., Polak, S., Zamborsky, R., and Danisovic, L. 2017. iPS cell technologies and their prospect for bone regeneration and disease modeling: A mini review. J Adv Res. 8: 321-327.
Das, A.T., Tenenbaum, L., and Berkhout, B. 2016. Tet-On Systems For Doxycycline-inducible Gene Expression. Curr Gene Ther. 16: 156-167.
Dingwall, M., Marchildon, F., Gunanayagam, A., Louis, C.S., and Wiper-Bergeron, N. 2011. Retinoic acid-induced Smad3 expression is required for the induction of osteoblastogenesis of mesenchymal stem cells. Differentiation 82: 57-65.
Dragoo J.L., Samimi, B., Zhu, M., Hame, SL. Thomas, B.J., Lieberman, J.R., Hedrick, M.K. Benhaim, P. 2003. Tissue-engineered cartilage and bone using stem cells from human infrapatellar fat pads. J Bone Joint Surg Br. 85: 740-7.
Ducy, P., Zhang, R., Geoffroy, V., Ridall, A.L., and Karsenty, G. 1997. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. 89: 747-54.
Canalis, E. 2008. Notch Signaling in Osteoblasts. Sci. Signal. 1: pe17.
Espada, J., and Martin-Perez, J. 2017. An Update on Src Family of Nonreceptor Tyrosine Kinases Biology. Int Rev Cell Mol Biol. 331: 83-122.
Feng, X., and Teitelbaum, S.L. 2013. Osteoclasts: New Insights. Bone Res. 1: 11-26.
Fihria A., Lenb, C., Varmac, RS., Solhy, A.L.2017. Hydroxyapatite: A review of syntheses, structure and applications in heterogeneous catalysis. Coordination Chemistry Reviews 347: 48-76.
Franceschi, R.T., Xiao, G., Jiang, D., Gopalakrishnan, R., Yang, S., and Reith, E. 2003. Multiple signaling pathways converge on the Cbfa1/Runx2 transcription factor to regulate osteoblast differentiation. Connect Tissue Res. 44: 109-16.
Gao, C., Guo, H., Mi, Z., Grusby, M.J., and Kuo, P.C. 2007. Osteopontin induces ubiquitin-dependent degradation of STAT1 in RAW264.7 murine macrophages. J Immunol. 178: 1870-81.
Gessin, J.C., Brown, L.J., Gordon, J.S., and Berg, R.A. 1993. Regulation of collagen synthesis in human dermal fibroblasts in contracted collagen gels by ascorbic acid, growth factors, and inhibitors of lipid peroxidation. Exp Cell Res. 206: 283-90.
76
Hamidouche, Z, Hay, E., Vaudin, P., Charbord, P., Schule, R., Marie, P.J., and Fromigue, O. 2008. FHL2 mediates dexamethasone-induced mesenchymal cell differentiation into osteoblasts by activating Wnt/beta-catenin signaling-dependent Runx2 expression. FASEB J. 22: 3813-22.
Heng, B.C., Cao, T., Stanton, L.W., Robson, P., and Olsen, B. 2004. Strategies for directing the differentiation of stem cells into the osteogenic lineage in vitro. J Bone Miner Res. 19: 1379-94.
Huang, W., Yang, S., Shao, J., and Li, Y.P. 2007. Signaling and transcriptional regulation in osteoblast commitment and differentiation. Front Biosci. 12: 3068-92.
Id, B.H., Lagneaux, L.F., Najar, M.F., Piccart, M.F., Ghanem, G.F., Body J.J., Journe, F. 2010. The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts. BMC Cancer 17: 298.
Jaenisch, R., and Young, R. 2008. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132: 567-582.
Kague, E., Roy, P., Asselin, G., Hu, G., Stanley, A., Albertson, C., Simonet, J., and Fisher, S. 2016. Osterix/Sp7 limits cranial bone initiation sites and is required for formation of sutures. Dev Biol. 413: 160-72.
Kanke, K., Masaki, H., Saito, T., Komiyama, Y., Hojo, H., Nakauchi, H., Lichtler, A.C., Takato, T., Chung, U.I., and Ohba, S. 2014. Stepwise differentiation of pluripotent stem cells into osteoblasts using four small molecules under serum-free and feeder-free conditions. Stem Cell Reports 2: 751-760.
Keller, G. 2005. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev. 19: 1129-1155.
Kern, B., Shen, J., Starbuck, M., and Karsenty, G. 2001. Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes. J Biol Chem. 276: 7101-7.
Kim, H.J. and Park, J.S. 2017. Usage of Human Mesenchymal Stem Cells in Cell-based Therapy: Advantages and Disadvantages. Dev Repord. 21: 1-10.
Kim, J.H., Liu, X., Wang, J., Chen, X., Zhang, H., Kim, S.H., Cui, J., Li, R., Zhang, W., Kong, Y., Zhang, J., Shui, W., Lamplot, J., Rogers, M.R., Zhao, C., Wang, N., Rajan, P., Tomal, J., Statz, J., Wu, N., Luu, H.H., Haydon, R.C., and He, TC. 2013. Wnt signaling in bone formation and its therapeutic potential for bone diseases. Ther. Adv. Musculoskelet. Dis. 5: 13-31.
77
Kim, S., Koga, T., Isobe, M., Kern, B.E., Yokochi, T., Chin, Y.E., Karsenty, G., Taniguchi, T., and Takayanagi, H. 2003. Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation. Genes Dev. 17: 1979-1991.
Kim, T.K., and Maniatis, T. 1996. Regulation of interferon-gamma-activated STAT1 by the ubiquitin-proteasome pathway. Science 273: 1717-1719.
Kobayashi, H., Gao, Y.H., Ueta, C., Yamaguchi, A., and Komori, T. 2000. Multilineage differentiation of Cbfa1-deficient calvarial cells in vitro. Biochem Biophys Res Commun. 273: 630-6.
Komori, T. 2005. Regulation of skeletal development by the Runx family of transcription factors. J Cell Biochem. 95: 445-53.
Komori, T. 2006. Regulation of osteoblast differentiation by transcription factors. J Cell Biochem. 99: 1233-9.
Komori, T. 2010. Regulation of osteoblast differentiation by Runx2. Adv Exp Med Biol 658: 43-49.
Komori, T. 2011. Signaling networks in RUNX2-dependent bone development. J Cell Biochem. 112: 750-5.
Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., Bronson, R.T., Gao, Y.H., Inada, M., Sato, M., Okamoto, R., Kitamura, Y., Yoshiki, S., and Kishimoto, T. 1997. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 89: 755-64.
Kumar, Y., Kapoor, I., Khan, K., Thacker, G., Khan, M.P., Shukla, N., Kanaujiya, J.K., Sanyal, S., Chattopadhyay, N., and Trivedi, A.K. 2015. E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation. J Biol Chem. 290: 30975-87.
Langenbach, F., and Handschel, J. 2013. Effects of dexamethasone, ascorbic acid and beta-glycerophosphate on the osteogenic differentiation of stem cells in vitro. Stem Cell Res Ther. 4: 117.
Lee J.A., Parrett, B.M., Conejero, J.A., Laser, J., Chen, J., Kogon , A.J., Nanda, D., Grant, R.T., Breitbart, A.S. 2003. Biological alchemy: engineering bone and fat from fat-derived stem cells. Ann Plast Surg. 50: 610-7.
Lee, Y.C., Huang, C.F., Murshed, M., Chu, K., Araujo, J.C., Ye, X., deCrombrugghe, B., Yu-Lee, L.Y., Gallick, G.E., and Lin, S.H. 2010. Src family kinase/abl inhibitor dasatinib suppresses proliferation and enhances differentiation of osteoblasts. Oncogene 29: 3196-3207.
Li, J., Zhang, H., Yang, C., Li, Y., and Dai, Z. 2016. An overview of osteocalcin progress. J Bone Miner Metab. 34: 367-79.
78
Long, F. 2012. Building strong bones: molecular regulation of the osteoblast lineage. Nature Reviews Molecular Cell Biology 13: 27-38.
Mackenzie, T.C. and Flake, A.W. 2001. Human mesenchymal stem cells persist, demonstrate site-specific multipotential differentiation, and are present in sites of wound healing and tissue regeneration after transplantation into fetal sheep. Blood Cells Mol Dis. 27: 601-4.
Malaval, L., Wade-Gueye, N.M., Boudiffa, M., Fei, J., Zirngibl, R., Chen, F., Laroche, N., Roux, J.P., Burt-Pichat, B., Duboeuf, F., Boivin, G., Jurdic, P., Lafage-Proust, M.H., Amedee, J, Vico, L., Rossant, J., and Aubin, J.E. 2008. Bone sialoprotein plays a functional role in bone formation and osteoclastogenesis. J Exp Med. 205: 1145-1153.
Maruyama, Z., Yoshida, C.A., Furuichi, T., Amizuka, N., Ito, M., Fukuyama, R., Miyazaki, T., Kitaura, H., Nakamura, K., Fujita, T., Kanatani, N., Moriishi, T., Yamana, K., Liu, W.F., Kawaguchi, H., Nakamura, K., and Komori, T. 2007. Runx2 determines bone maturity and turnover rate in postnatal bone development and is involved in bone loss in estrogen deficiency. Dev Dyn. 236: 1876-90.
Marzia, M., Sims, N.A., Voit, S., Migliaccio, S., Taranta, A., Bernardini, S., Faraggiana, T., Yoneda, T., Mundy, G.R., Boyce, B.F., Baron, R., and Teti, A. 2000. Decreased c-Src expression enhances osteoblast differentiation and bone formation. J Cell Biol. 151: 311-320.
Masson, A.O., Hess, R, O'Brien, K, Bertram, K.L., Tailor, P., Irvine, E., Ren, G., and Krawetz, R.J. 2015. Increased levels of p21((CIP1/WAF1)) correlate with decreased chondrogenic differentiation potential in synovial membrane progenitor cells. Mech Ageing Dev. 149: 31-40.
Meyn, M.A., Schreiner, S.J., Dumitrescu, T.P., Nau, G.J., and Smithgall, T.E. 2005. SRC family kinase activity is required for murine embryonic stem cell growth and differentiation. Mol Pharmacol. 68: 1320-30.
Michalak, M., Groenendyk, J., Szabo, E., Gold, L.I., and Opas, M. 2009. Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochem J. 417: 651-66.
Michalak, M., Robert Parker, J.M., and Opas, M. 2002. Ca2+ signaling and calcium binding chaperones of the endoplasmic reticulum. Cell Calcium. 32: 269-78.
Miller, SC, Saint-Georges, L, Bowman, BM, and Jee, WS. 1989. Bone lining cells: structure and function. Scanning Microsc. 3: 953-60.
Miraoui, H., Oudina, K., Petite, H., Tanimoto, Y., Moriyama, K., and Marie, P.J. 2008. Fibroblast growth factor receptor 2 promotes osteogenic differentiation in mesenchymal cells via ERK1/2 and protein kinase C signaling. J. Biol. Chem. 284: 4897-4904.
79
Muller, S., Chen, Y., Ginter, T., Schafer, C., Buchwald, M., Schmitz, L.M., Klitzsch, J., Schutz, A., Haitel, A., Schmid, K., Moriggl, R., Kenner, L., Friedrich, K., Haan, C., Petersen, I., Heinzel, T., and Kramer, O.H. 2014. SIAH2 antagonizes TYK2-STAT3 signaling in lung carcinoma cells. Oncotarget. 5: 3184-3196.
Murry, C.E. and Keller, G. 2008. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 132: 661-80.
Nakano, T., Kodama, H., and Honjo, T. 1994. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265: 1098-101.
Nakashima, K., Zhou, X., Kunkel, G., Zhang, Z., Deng, J.M., Behringer, R.R., and de Crombrugghe, B. 2002. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell. 108: 17-29.
O'Shea, J.J., Gadina, M., and Schreiber, R.D. 2002. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell 109: S121-31.
Olmsted-Davis, E.A., Gugala, Z., Camargo, F., Gannon, F.H., Jackson, K., Kienstra, K.A., Shine, H.D., Lindsey, R.W., Hirschi, K.K., Goodell, M.A., Brenner, M.K., Davis, A.R. 2003. Primitive adult hematopoietic stem cells can function as osteoblast precursors. Proc Natl Acad Sci U S A 100: 15877-82.
Ornitz, D.M. and Marie, P.J. 2015. Fibroblast growth factor signaling in skeletal development and disease. Genes Dev. 29: 1463-86.
Otto, F., Thornell, A.P., Crompton, T., Denzel, A., Gilmour, K.C., Rosewell, I.R., Stamp, G.W., Beddington, R., Mundlos, S., Olsen, B.R., Selby, P., and Owen, M.J. 1997. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell. 89: 765-71.
Owen,T.A., Aronow, M., Shalhoub,V, Baone, L.M., Wilming, L., Tassinari, M.S., Kennedy, M.B., Pockwinse, S., Lian, J.B., Stein, G.S. 1999. Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol. 143: 420-30.
Park, O.J., Kim, H.J., Woo, K.M., Baek, J.H., and Ryoo, H.M. 2010. FGF2-activated ERK mitogen-activated protein kinase enhances Runx2 acetylation and stabilization. J Biol Chem. 285: 3568-74.
Peruzzi, B., Cappariello, A., Del Fattore, A., Rucci, N., De Benedetti, F., and Teti, A. 2012. c-Src and IL-6 inhibit osteoblast differentiation and integrate IGFBP5 signalling. Nat Commun. 3: 630.
Phillips, J.E., Gersbach, C.A., Wojtowicz, A.M., and Garcia, A.J. 2006. Glucocorticoid-induced osteogenesis is negatively regulated by Runx2/Cbfa1 serine phosphorylation. J Cell Sci. 119: 581-91.
80
Pineda, E.T., Nerem, R.M., and Ahsan, T. 2013. Differentiation patterns of embryonic stem cells in two- versus three-dimensional culture. Cells Tissues Organs 197: 399-410.
Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284: 143-147.
Rahman, M.S., Akhtar, N., Jamil, H.M., Banik, R.S., and Asaduzzaman, S.M. 2015. TGF-beta/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation. Bone Res. 14: 15005.
Rauch C., Brunet, A.C., Deleule, J., Farge, E. 2002. C2C12 myoblast/osteoblast transdifferentiation steps enhanced by epigenetic inhibition of BMP2 endocytosis. Am J Physiol Cell Physiol. 283: C235-45.
Regan, J., and Long, F. 2013. Notch signaling and bone remodeling. Curr Osteoporos Rep. 11: 126-129.
Reich, N.C. 2013. STATs get their move on. JAKSTAT. 2: e27080.
Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A., and Bongso, A. 2000. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 18: 399-404.
Roeder, E., Matthews, B.G., and Kalajzic, I. 2016. Visual reporters for study of the osteoblast lineage. Bone 92: 189-195.
Roskoski, R. 2005. Src kinase regulation by phosphorylation and dephosphorylation. Biochem Biophys Res Commun. 331: 1-14.
Rous, P. 1911. A Sarcoma of the fowl transmissible by an agent separable from the tumor cells. J Exp Med. 13: 397-411.
Rutkovskiy, A., Stenslokken, K., Vaage, I.J. 2016. Osteoblast Differentiation at a Glance. Med Sci Monit Basic Res. 22: 95-106.
Sarkar, T.R., Sharan, S., Wang, J., Pawar, S.A., Cantwell, C.A., Johnson, P.F., Morrison, D.K., Wang, J.M., and Sterneck, E. 2012. Identification of a Src tyrosine kinase/SIAH2 E3 ubiquitin ligase pathway that regulates C/EBP delta expression and contributes to transformation of breast tumor cells. Mol Cell Biol. 32: 320-332.
Schroeder, T.M., Kahler, R.A., Li, X., and Westendorf, J.J. 2004. Histone deacetylase 3 interacts with runx2 to repress the osteocalcin promoter and regulate osteoblast differentiation. J Biol Chem. 279: 41998-2007.
Sinha, K.M., and Zhou, X. 2013. Genetic and molecular control of osterix in skeletal formation. J Cell Biochem. 114: 975-84.
81
Soond, S.M., Townsend, P.A., Barry, S.P., Knight, R.A., Latchman, D.S., and Stephanou, A. 2008. ERK and the F-box protein betaTRCP target STAT1 for degradation. J Biol Chem. 283: 16077-83.
Soriano, P., Montgomery, C., Geske, R., and Bradley, A. 1991. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 64: 693-702.
Stehelin, D., Varmus, H.E., Bishop, J.M., and Vogt, P.K. 1976. DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature. 260: 170-3.
Stephens, A.S. and Morrison, N.A. 2014. Novel target genes of RUNX2 transcription factor and 1,25-dihydroxyvitamin D3. J Cell Biochem. 115: 1594-608.
Sterner, D.E. and Berger, S.L. 2000. Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev. 64: 435-59.
Stock, M., Schafer, H., Fliegauf, M., and Otto, F. 2004. Identification of novel genes of the bone-specific transcription factor Runx2. J Bone Miner Res. 19: 959-72.
Kim, S., Koga, T., Isobe, M., Kern, B.E., Yokochi, T, Chin, Y.E. Karsenty, G., Taniguchi, T. Takayanagi, H. 2003. Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation. 17: 1979-1991.
Surinder M.S., Townsend, P.A., Barry, S.P., Knight, R.A., Latchman, D.S., Stephanou, A. 2008. ERK and the F-box Protein ßTRCP Target STAT1 for Degradation. J Biol Chem. 283: 16077-16083.
Swaney, D.L., Beltrao, P., Starita, L., Guo, A., Rush, J., Fields, S., Krogan, N.J., and Villen, J. 2013. Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation. Nat Methods. 10: 676-682.
Tajima, K., Takaishi, H., Takito, J., Tohmonda, T., Yoda, M., Ota, N., Kosaki, N., Matsumoto, M., Ikegami, H., Nakamura, T., Kimura, T., Okada, Y., Horiuchi, K., Chiba, K., and Toyama, Y. 2010. Inhibition of STAT1 accelerates bone fracture healing. J Orthop Res. 28: 937-41.
Thirunavukkarasu, K., Mahajan, M., McLarren, K.W., Stifani, S., and Karsenty, G. 1998. Two domains unique to osteoblast-specific transcription factor Osf2/Cbfa1 contribute to its transactivation function and its inability to heterodimerize with Cbfbeta. Mol Cell Biol. 18: 4197-208.
Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282: 1145-7.
Trounson, A. 2002. Human embryonic stem cells: mother of all cell and tissue types. Reprod Biomed Online 4: 58-63.
82
Viguet-Carrin, S., Garnero, P., and Delmas, P.D. 2006. The role of collagen in bone strength. Osteoporos Int. 17: 319-36.
Vimalraj, S., Arumugam, B., Miranda, P.J., and Selvamurugan, N. 2015. Runx2: Structure, function, and phosphorylation in osteoblast differentiation. Int J Biol Macromol. 78: 202-8.
Wang, D., Christensen, K., Chawla, K., Xiao, G., Krebsbach, P.H., and Franceschi, R.T. 1999. Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential. J Bone Miner Res. 14: 893-903.
Wang, H., Pang, B., Li, Y.F., Zhu, D., Pang, T., and Liu, Y. 2012. Dexamethasone has variable effects on mesenchymal stromal cells. Cytotherapy 14: 423-30.
Wee, H.J., Huang, G., Shigesada, K., and Ito, Y. 2002. Serine phosphorylation of RUNX2 with novel potential functions as negative regulatory mechanisms. EMBO Rep. 3: 967-974.
WeinEmail M.N. 2017. Bone Lining Cells: Normal Physiology and Role in Response to Anabolic Osteoporosis Treatments. Current Molecular Biology Reports 3: 79-84.
Wu, T.R., Hong, Y.K., Wang, X.D., Ling, M.Y., Dragoi, A.M., Chung, A.S., Campbell, A.G., Han, Z.Y., Feng, G.S., and Chin, Y.E. 2002. SHP-2 is a dual-specificity phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues in nuclei. J Biol Chem. 277: 47572-47580.
Yu, Y., Al Mansoori, L., and Opas, M. 2015. Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro. Differentiation 89: 1-10.
Zaidi, S.K., Sullivan, A.J., Medina, R., Ito, Y., van Wijnen, A.J., Stein, J.L., Lian, J.B., and Stein, G.S. 2004. Tyrosine phosphorylation controls Runx2-mediated subnuclear targeting of YAP to repress transcription. EMBO J. 23: 790-799.
Zayzafoon, M. 2006. Calcium/calmodulin signaling controls osteoblast growth and differentiation. J Cell Biochem. 97: 56-70.
Zhang, S.Q., Yang, W., Kontaridis, M.I., Bivona, T.G., Wen, G., Araki, T., Luo, J., Thompson, J.A., Schraven, B.L., Philips, M.R., and Neel, B.G. 2004. Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment. Mol Cell. 13: 341-355.
Zhang, X., Simerly, C., Hartnett, C., Schatten, G., and Smithgall, T.E. 2014b. Src-family tyrosine kinase activities are essential for differentiation of human embryonic stem cells. Stem Cell Res. 13: 379-389.
Zhang, X., Simerly, C., Hartnett, C., Schatten, G., and Smithgall, T.E. 2014a. Src-family tyrosine kinase activities are essential for differentiation of human embryonic stem cells. Stem Cell Res. 13: 379-389.
Zheng, X.M., Resnick, R.J., and Shalloway, D. 2000. A phosphotyrosine displacement mechanism for activation of Src by PTPalpha. EMBO J. 19: 964-78.
83
Zhou, X., von der Mark, K., Henry, S., Norton, W., Adams, H., and de Crombrugghe, B. 2014. Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice. PLoS Genet. 10: e1004820.
Zhu, W., He, X., Hua, Y., Li, Q., Wang, J., and Gan, X. 2017. The E3 ubiquitin ligase WWP2 facilitates RUNX2 protein transactivation in a mono-ubiquitination manner during osteogenic differentiation. J Biol Chem. 292: 11178-11188.
Zou, L., Kidwai, F.K., Kopher, R.A., Motl, J., Kellum, C.A., Westendorf, J.J., and Kaufman, D.S. 2015. Use of RUNX2 expression to identify osteogenic progenitor cells derived from human embryonic stem cells. Stem Cell Reports 4: 190-198.
87
Appendices
88
Figure A-1
Figure A-1 Expression of pluripotency markers in early mES osteogenic differentiation
RNA expression of pluripotency markers including Oct3/4, Nanog, and Sox2 were measured by
qPCR in mESCs going through osteogenic differentiation for the indicated days. The primer
pairs are listed in table A-1. Data shown represent the mean (±SD) of triplicates. Data shown are
representative experiments of three independent assays.
Days of osteogenic differentiation
Rel
ativ
e m
RN
A E
xpre
ssio
n
89
Figure A-2
Figure A-2 Inhibition of c-Src activity by pharmacological inhibitors
(A and B) Three different inhibitors of c-Src were applied in the concentrations of 1µM and 10 µM in day
5 differentiating mES EBs for 2 and 24 hours. Lysates were collected and subjected to immunoblot
analysis using p-Y416-c-Src and c-Src antibodies. GAPDH served as loading control. (C) Day 5
differentiating EBs were treated with different concentrations of PP2, and PP3 (inactive analog PP2) of
including 100 nM, 1, 5, 10, and 20 µM for 2 hours. Adjusted volume of DMSO for each corresponding
concentration of PP2 served as the solvent control. Lysates were prepared and subjected to immunoblot
analysis using p-Y416-c-Src and c-Src antibodies. GAPDH served as loading control. (D) Band density
for each condition in the WB was quantified using ImageJ software and those for p-Y416-c-Src were
normalized to their corresponding GAPDH. Calculated values were graphed. Data shown are
representative experiments of three independent assays.
A B
C
D
D
90
Figure A-3
A
B
C
91
Figure A-3 Failed attempts for transfecting mESCs with c-Src siRNAs
Different c-Src siRNA from Ambion (A) and Thermo Fisher Scientific (B) along with GAPDH
specific siRNA as a positive control were applied using transfectamin to downregulate c-Src
activity in mESCs for the indicated concentrations. Lysates were collected and subjected to WB
analysis using c-Src antibody. GAPDH served as loading control. (C) mESCs were subjected to
transfection using electroporation with the indicated concentrations of c-Src and GAPDH
specific siRNAs. Lysates were analyzed by WB using c-Src and GAPDH antibodies. Data shown
are representative experiments of three independent assays.
92
Figure A-4
Figure A-4 Stat1 phosphorylation status on Y701 in early days of osteogenic differentiation
(A) Expression and phosphorylation status of proteins including p-Y701-Stat1, Stat1, p-Y527-c-
Src (inactive c-Src), p-Y416-c-Src (active c-Src), and c-Src were analyzed by immunoblotting.
GAPDH served as the loading control and is shown below its corresponding blots. (B) band
densities of p-Y701-Stat1 and p-Y416-c-Src were normalized to their corresponding total
protein, Stat1 and c-Src, and calculated values are shown in the graph. Data shown are
representative experiments of three independent assays.
Day (s) 3 5 6 7 8 9 10
p-Y416-c-
GAPDH
c-Src
Stat1
p-Y701-Stat1
GAPDH
p-Y527-c-
A B
93
Figure A-5
p-Y701-Stat1
Stat1
GAPDH
c-Src Y416
Whole cell lysates
Whole cell lysates
C
B A
Figure A-5 Shp2 inhibition increases Stat1 Y701
phosphorylation and Runx2 localization.
(A) Shp2 activity was inhibited using specific PHPS1
inhibitor (5 µM) for 2 hours. Expression and
phosphorylation status of proteins including p-Y701-
Stat1, Stat1, and p-Y416-c-Src were analyzed by
immunoblotting. GAPDH served as the loading
control. (B) Runx2 nuclear fraction in mESCs was
enhanced in response to Shp2 inhibition by PHPS1.
(C) Inhibition of c-Src activity by PP2 resulted in