USSCs which were introduced by Kogler and colleagues (2004) have certainadvantages over the other stem cells which might candidate them for the celltherapy. To date not enough knowledge is however available on the signalingnetworks regulating USSCs proliferation and differentiation. We therefore soughtto partially unravel the role of TGF-? signaling pathway in the osteogenic differentiationof USSCs.
In the present study, we showed for the first time that TGF-? pathwayis active in USSCs and its inhibition increases their osteogenicdifferentiation, 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 othermembers of the family such as BMP in vivo and in vitro 12-15, others showed that TGF-?1 had inhibitoryeffects on osteogenic markers such as alkaline phosphatase in mesenchymal stem cells16-19. The variable effects of TGF-?1 on osteogenesisappear to be dose or stage dependent 8,20,21. For example TGF-?1 has been shown to inducemigration and proliferation during the early stage but inhibits mineralization anddifferentiation of the osteoblasts during the late stage of bone formation 6,9,21. Tang and Alliston (2013) alsopointed out to another mechanism of TGF-? signaling action which resulted inmore osteogenic differentiation during the early stages compared to the late.They showed that activation of TGF-? pathway led to the smad 3 entrance to thenucleus through binding to histone deacetylases 4 and 5, thus forming a complexthat prevents the action of transcription factor Runx2 as well as theexpression of osteo-specific genes during the late stage of osteogenicdifferentiation 22.
To evaluate if TGF-?1 differentially affects osteogenesis in USSCs, we treatedthe cells with TGF-?1 antagonist, SB431542, for 3.5 days at different timepoints within 21 days of culture. Our results showed that TGF-?1 inhibition increasedthe osteogenic differentiation throughout, but more prominently at the early stages. Evaluating Runx2 expressionas an important transcription factor for the osteoblast differentiation, we alsoshowed that SB significantly increased the level of Runx2 in USSCs. Consistently,Alliston and colleagues (2001) showed the increased expression of Runx2 followingthe TGF-?1 inhibition in primary osteoblasts and osteoblast like cell lines 23, suggesting a unanimous mechanism of TGF-?1 actionon osteogenesis in a variety of stem cells. The marked increase during theearly stages might be reflected from TGF-?1 interaction with BMP 24.
Considering that TGF-? and BMP pathways share smad4for their signal transduction, it is conceivable that blockade of one increasethe accessibility of smad4 for the other, hence to one active pathway over theother.We have further showed that matrix secretion was increased followingthe 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, theeffect of TGF-?1 inhibition on osteogenesis was however less pronounced duringthe late stages. Several studies have reported that the activity ofendogenousTGF-?1 and the receptors of this pathway is reduced during the late stageof differentiation 19,25, hence the increased osteogenic differentiationdue to the inhibition of this pathway by SB in the late stage, is not as greatas that of the early stage.
For further research in future, it is important to study the level ofgene expressions of alkaline phosphatase and other genes related to osteogenicdifferentiation following the treatments with SB and TGF-?1. Also, one coulduse siRNA to block the expression of TGF-?1 and examine if the same statusholds true for the osteogenic differentiation.