| Abstract |
Background
NF-Y is a CCAAT-binding transcription factor consisting of three subunits, NF-YA, NF-YB and NF-YC, which are all necessary for DNA binding. NF-YB and NF-YC form a stable dimer which acts as a binding surface for NF-YA. NF-YA is characterized as the regulatory subunit of the complex because of its differential expression during the cell cycle. NF-YA appears to have a complex role in hematopoietic stem cell (HSC) biology. So far, there are no available data on NF-Y role in mesenchymal stem cells (MSCs).
Aims
The evaluation of NF-YA expression levels during the in vitro expansion of human bone marrow mesenchymal stem cells (BM-MSCs) and the assessment of its possible role during the in vitro differentiation of BM-MSCs into the adipogenic and osteogenic lineages.
Methods
BM-MSCs were isolated from posterior iliac crest aspirates of hematologically healthy individuals undergoing orthopedic surgeries after written informed consent. BM-MSCs were in vitro expanded until passage-10 (P10). Total RNA was isolated from BM-MSCs at different passages (P2, P6, and P10) for the evaluation of NF-YA mRNA levels.
BM-MSCs in vitro differentiations into adipocytes and osteoblasts were induced using the appropriate culture media. Total RNA and proteins were isolated from differentiated BM-MSCs, and NF-YA levels, as well as lineage-specific marker genes levels were evaluated. The relative gene expression was evaluated by real-time PCR. All PCR results are expressed as 2-ΔCt. Protein levels of NF-YA were immunodetected by Western blot. Moreover, differentiations were verified using appropriate cytochemical stains.
In order to specifically knockdown NF-YA expression, BM-MSCs were transduced with shRNA for NF-YA (shNF-YA) lentiviral particles and adipocytic as well as osteoblastic differentiations were induced. Total RNA was isolated from transduced BM-MSCs at several time-points during differentiations.
Results
A statistically significant (p=0.0296) reduction in the mRNA levels of NF-YA was found in P10, as compared to P3 BM-MSC cultures (n=9).
During adipogenic differentiation, NF-YA mRNA and protein levels were found to significantly increase (p=0.0478) in day 16, as compared to the onset of differentiation induction (day 0), (n=14). Furthermore, NF-YA expression levels were strongly correlated
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with PPARG (r=0.74, p΄&λτ0.0001), C/EBPA (r=0.76, p΄&λτ0.0001) and LPL (r=0.74, p΄&λτ0.0001) transcription levels.
Regarding osteogenesis, in contrast with adipogenesis, no statistically significant change was detected in NF-YA mRNA levels (n=9).
In order to evaluate if NF-YA is functionally related to in vitro adipogenesis and osteogenesis of BM-MSCs, NF-YA knockdown was performed and adipocytic as well as osteoblastic differentiations were induced.
Throughout adipocytic differentiation process, the relative mRNA levels of NF-YA in shNF-YA-transduced BM-MSCs were reduced to 51.03% compared with control (non-transduced) cells. At day 21 of adipogenesis, a reduction of 31.22%, 70.06% and 74.3% was also observed in PPARG, C/EBPA and LPL mRNA levels, respectively. At last, Oil Red O staining indicated a lack of lipid droplets accumulation in shNF-YA-transduced BM-MSCs compared with control cells.
During osteogenesis, NF-YA mRNA levels in BM-MSCs transduced with shNF-YA were reduced to 52.37% compared to control cells. Although a reduction of 52.03% and 83.95% was observed in OSC and BSP relative mRNA levels respectively, the transcription levels of DLX5, RUNX2, OSX and ALP remained unaffected after NF-YA knockdown, in shNF-YA-transduced cells compared to control cells. Furthermore, Alizarin Red S and Von Kossa stainings did not reveal any significant difference between shNF-YA-transduced BM-MSCs compared with control cells.
Summary/Conclusion
We have found for the first time that NF-YA mRNA levels decrease after prolonged in vitro expansion of BM-MSCs.
We have shown for the first time that NF-YA, the regulatory subunit of the heterotrimeric transcription factor NF-Y, is implicated in the in vitro differentiation of BM-MSCs into the adipogenic cell-lineage. More specifically, during adipogenesis, NF-YA mRNA and protein levels were found to be elevated. Moreover NF-YA mRNA expression was positively correlated with PPARG, C/EBPA and LPL transcription levels. Furthermore, after NF-YA silencing, BM-MSCs were characterized by aberrant potential of in vitro adipocytic differentiation. Our results imply an emerging role of NF-Y in BM-MSCs adipogenesis.
In contrast, during induction of osteogenesis we did not observe a significant change regarding the expression levels of NF-YA. Additionally, transcription levels of most osteogenesis marker genes checked, remained unaffected, after NF-YA-silencing. Cytochemical stains, Alizarin Red S and Von Kossa, did not reveal any significant difference between shNF-YA transduced BM-MSCs and control cells.
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