USSCs which were introduced by Kogler and colleagues (2004) have certain
advantages over the other stem cells which might candidate them for the cell
therapy. To date not enough knowledge is however available on the signaling
networks regulating USSCs proliferation and differentiation. We therefore sought
to partially unravel the role of TGF-? signaling pathway in the osteogenic differentiation
of USSCs. In the present study, we showed for the first time that TGF-? pathway
is active in USSCs and its inhibition increases their osteogenic
differentiation, especially during the early stages.

TGF-?1 differentially affects the osteogenesis;
while some researchers have shown that TGF-?1 induces osteogenesis, either alone or together with other
members of the family such as BMP in vivo and in vitro 12-15, others showed that TGF-?1 had inhibitory
effects on osteogenic markers such as alkaline phosphatase in mesenchymal stem cells
16-19. The variable effects of TGF-?1 on osteogenesis
appear to be dose or stage dependent 8,20,21. For example TGF-?1 has been shown to induce
migration and proliferation during the early stage but inhibits mineralization and
differentiation of the osteoblasts during the late stage of bone formation 6,9,21.

 Tang and Alliston (2013) also
pointed out to another mechanism of TGF-? signaling action which resulted in
more osteogenic differentiation during the early stages compared to the late.
They showed that activation of TGF-? pathway led to the smad 3 entrance to the
nucleus through binding to histone deacetylases 4 and 5, thus forming a complex
that prevents the action of transcription factor Runx2 as well as the
expression of osteo-specific genes during the late stage of osteogenic
differentiation 22.

To evaluate if TGF-?1 differentially affects osteogenesis in USSCs, we treated
the cells with TGF-?1 antagonist, SB431542, for 3.5 days at different time
points within 21 days of culture. Our results showed that TGF-?1 inhibition increased
the osteogenic differentiation throughout, but more prominently at the early stages.

 Evaluating Runx2 expression
as an important transcription factor for the osteoblast differentiation, we also
showed that SB significantly increased the level of Runx2 in USSCs. Consistently,
Alliston and colleagues (2001) showed the increased expression of Runx2 following
the TGF-?1 inhibition in primary osteoblasts and osteoblast like cell lines 23, suggesting a unanimous mechanism of TGF-?1 action
on osteogenesis in a variety of stem cells. The marked increase during the
early stages might be reflected from TGF-?1 interaction with BMP 24. Considering that TGF-? and BMP pathways share smad4
for their signal transduction, it is conceivable that blockade of one increase
the accessibility of smad4 for the other, hence to one active pathway over the

We have further showed that matrix secretion was increased following
the TGF-?1 inhibition throughout, more pronouncedly however during the early stages
(days 0-3.5 and 3.5-7). According to our result and in line with others, the
effect of TGF-?1 inhibition on osteogenesis was however less pronounced during
the late stages. Several studies have reported that the activity of
endogenousTGF-?1 and the receptors of this pathway is reduced during the late stage
of differentiation 19,25, hence the increased osteogenic differentiation
due to the inhibition of this pathway by SB in the late stage, is not as great
as that of the early stage.

For further research in future, it is important to study the level of
gene expressions of alkaline phosphatase and other genes related to osteogenic
differentiation following the treatments with SB and TGF-?1. Also, one could
use siRNA to block the expression of TGF-?1 and examine if the same status
holds true for the osteogenic differentiation.


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