3.1 BMP-2 peptide promotes the differentiation of DPSCs to osteoblasts more effectively than full-length BMP-2
In this study, DSPCs (Dental Pulp Stem Cells) were induced to differentiate into osteoblasts with active dimeric human BMP-2 or BMP-2 peptide, a short peptide derived from its carboxyterminal region, that has been shown to promote osteogenesis more effectively than the full-length protein (personal communication with Dr. S. Karoulias). Four groups were tested: a) a negative control group, consisting of untreated DPSCs cultured in DMEM, b) a positive control group, consisting of DPSCs that were induced to differentiate with StemPro® Osteogenesis Complete Medium, c) DPSCs that were induced to differentiate with Osteogenesis Medium and were also treated with 50 ng/ml active dimeric human BMP-2, and d) DPSCs that were induced to differentiate with Osteogenesis Medium and were also treated with 50 ng/ml BMP2 peptide.
During the formation of their extracellular matrix (approximately 7th to 14th day of osteogenesis), proliferating osteoblasts exhibit high expression of the enzyme Alkaline Phosphatase (ALP). The detection of its activity is used frequently as a marker of successful differentiation of hMSCs into osteoblasts. ALP activity in the extracellular matrix of proliferating osteoblasts can be detected by chromogenic reaction (NBT-BCIP staining). Fourteen days after the addition of growth factor BMP-2 or BMP-2 peptide in the culture medium, ALP activity was detectable in all groups (Fig. 1a-d). However, increased activity was observed in cells that were induced with either BMP2 or peptide 3 (Fig. 1c,d) compared to positive control cells (Fig. 1b), as indicated by multiple blue areas of reduced NBT. Negative control cells exhibited the highest level of alkaline phosphatase activity, which was comparable to the BMP2-treated and BMP-2 peptide-treated groups. The morphology of the positive control and the treated groups was indicative of proliferating osteoblasts (oval-shaped), while negative control cells maintained the elongated, fibroblast-like morphology that is typical of DPSCs.
At the final stage of osteogenic differentiation, osteoblasts form hydroxyapatite deposits in the extracellular matrix (mineralization). On the 21st day of osteogenesis, when mineralization is complete, successful bone tissue formation can be verified by staining the extracellular hydroxyapatite deposits with Alizarin Red S, a dye that binds to calcium cations. The Alizarin Red Assay showed that both the BMP-2-treated and BMP-2 peptide-treated cells exhibited higher concentration of calcium deposits compared to positive control cells. The highest concentration was observed in cells treated with BMP-2 peptide (Fig. 1e-h).
The successful induction of osteogenesis was also verified by monitoring with qPCR the marker gene BGLAP that codes for bone ECM glycoprotein, osteocalcin (Fig. 2). The mRNA levels of BGLAP in untreated DPSCs on the 7th Day of culture were set as the “reference levels”. In untreated cells, BGLAP expression was diminished towards Day 21, by 33-fold, after a small rise (7-fold). Treatment with factors included in the Osteogenesis medium increased the levels by 280-fold on Day 7, but a decline to reference levels followed in the course of the weeks. However, treatment with BMP-2 increased the expression of osteocalcin by 50-fold on Day 21 and BMP2 peptide by almost 500-fold, compared to reference levels.
3.2 Effect of BMP-2 and BMP-2 peptide on the expression of ZNF217, BMP4 and cell-cycle inhibitor p21 gene, CDKN1A, in DPSCs and osteoblasts
The mRNA levels of ZNF217, BMP4 and cell-cycle inhibitor gene CDKN1A (p21) were monitored by real-time qPCR on the 7th, 14th and 21st day after induction of osteogenesis with BMP2 or BMP-2 peptide. Relative quantification against reference gene RPLP0 was performed. In untreated DPSCs (negative control), ZNF217 expression declined 2.5-fold by Day 14 and 6-fold by Day 21, compared to Day 7 (Fig. 3a). Induction of differentiation with Osteogenesis Medium caused a temporary 2-fold decrease on Day 7, followed by a 4-fold increase on Day 14 and by a 4-fold decrease on the gene’s mRNA levels. However, treatment with BMP-2 diminished the expression of the gene in the proliferating osteoblasts during the second week of differentiation. Treatment with BMP-2 peptide had almost immediate effect, reducing the expression on Day 7 by almost 6-fold compared to reference levels. The expression returned to almost reference levels upon Day 21. These results suggest that initial treatment of DPSCs with 50 ng/ml exogenous BMP-2 or BMP-2 peptide can profoundly impact the oncogene’s expression during the first two weeks of osteogenesis.
In untreated DPSCs, BMP-4 expression remained unchanged during the weeks. (Fig. 3b). Induction of differentiation with osteogenesis medium was accompanied by an increase by 3- up to 6.5-fold in the gene’s expression. Treatment with BMP-2 or BMP-2 peptide had an almost immediate and profound effect on BMP-4 mRNA levels. BMP-2-treated proliferating osteoblasts exhibited 57-fold increase in the gene’s expression on Day 7, compared to Day 7 untreated cells. BMP-2 peptide-treated proliferating osteoblasts had an almost 1800-fold increase on the same day. In the BMP-2-treated group, the expression dropped to the positive control group’s levels after three weeks (Day 21). However, the BMP-2 peptide-treated group seemed to regain a high BMP-4 expression level by Day 21 (24-fold). These results suggest that initial treatment of DPSCs with exogenous BMP-2 may enhance the production of BMP-4 during the first week of osteogenic differentiation. However, similar treatment with 50 ng/ml BMP-2 peptide seems to impact the gene’s expression almost 17 times as much as BMP-2 and to cause a second increase at the last stage of differentiation.
p21 is a cell-cycle inhibitor belonging to the Cip/Kip family of Cyclin-dependent kinase inhibitors. The expression of its gene, CDKN1A, was quantified and correlated with the expression pattern of ZNF217 and BMP-4. The mRNA levels of CDKN1A in untreated DPSCs on the 7th day of culture were set as “reference levels” (Fig. 3c). In untreated cells, CDKN1A expression declined over the weeks, in a manner that resembled ZNF217, 15-fold by Day 14 and 24-fold by Day 21, compared to reference levels (Day 7). This suggests that the DPSCs continued to multiply until Day 21, retaining their undifferentiated state. During differentiation induced by the osteogenesis medium, CDKN1A mRNA levels decreased 3.3-fold compared to reference levels on Day 7, increased up to reference level by Day 14 followed by a second, 6-fold, decrease by Day 21. The evident increase in CDKN1A mRNA levels compared to untreated DPSCs in expected of the gradual entry of osteoblasts into a quiescent, differentiated state. Unexpectedly, in BMP-2-treated cells, CDK1NA expression dropped up to 86-fold by Day 21. In BMP-2 peptide-treated cells, CDKN1A expression dropped 14-fold on the 14th day but returned to the levels of the positive control in the late stage of differentiation.
3.3 Effect of BMP-2 and BMP-2 peptide on the expression of ZNF217, BMP4 and cell-cycle inhibitor p21 gene, CDKN1A, in MCF-7 breast cancer cells
The mRNA levels of ZNF217, BMP4 and cell-cycle inhibitor gene CDKN1A (p21) were monitored by real-time qPCR 6, 12, 24 and 48 hours after treatment with 100 ng/ml BMP2 or different concentrations of BMP-2 peptide (45.2 ng/ml, 22.6 ng/ml and 4.52 ng/ml). The mRNA levels of ZNF217 in untreated MCF-7 at 6 h of culture were set as the “reference levels” for ZNF217. Similarly, the mRNA levels of BMP4 and CDKN1A in untreated MCF-7 at 6 h of culture were set as the reference levels for each of these genes, respectively.
In untreated MCF-7 cells, ZNF217 mRNA levels quadrupled at 12 h compared to 6-h reference control (Fig. 4a), then decreased at 24 h and 48 h. Treatment with BMP-2 caused only a 2-fold increase at 12 h, but a 5-fold increase at 24 h, compared to reference levels. Therefore, BMP-2-treated MCF-7 seemed to display induction of the oncogene’s expression later than the untreated group. Treatment with 45.2 ng/ml BMP-2 peptide caused an early 2-fold increase at 6 h, followed by 4-fold increase at 12 h before returning to reference levels at 24 h. Finally, at 48 h, an increase was observed, compared to 48 h negative control. The lowest concentration of BMP-2 peptide also seemed to have an upregulatory effect on ZNF217 that lasted until 48 hours later. However, an intermediate concentration of 22.6 ng/ml BMP-2 peptide had a different effect. It slowed the upregulation that was observed in the untreated group at 12 h, causing only 3.5-fold increase at this time point. At 24 h, ZNF217 mRNA levels, decreased almost to reference level, while the untreated group had a 2-fold increase at the same time point. Finally, at 48 h, the group treated with 22.6 ng/ml BMP-2 peptide had approximately the same ZNF217 mRNA level as the untreated group.
In untreated MCF-7, BMP-4 was downregulated 3-fold at 24 h compared to the 6-h reference level, but then increased almost 5-fold between 24 h and 48 h. (Fig. 4b). Treatment with BMP-2 caused severe downregulation of the BMP-4 gene at 24 h, when its mRNA levels were undetectable. However, between 24 h and 48 h, the expression increased, reaching the same levels as 48-h untreated cells. MCF-7 cells that were treated with 45.2 ng/ml or 22.6 ng/ml BMP-2 peptide seemed to retain BMP-4 expression approximately at reference levels during the first 24 hours and showed a 2.5-fold increase at 48 h. On the contrary, MCF-7 treated with the lowest concentration of BMP-2 peptide, 4.52 ng/l (equimolar to 100 ng/ml BMP-2), showed 2-fold reduction in BMP-4 mRNA levels at 6 h, followed by gradual increase that reached 4-fold the reference level at 48 h.
CDKN1A expression was monitored at 6 h, 12 h, 24 h and 48 h and was correlated with the expression of ZNF217. In untreated MCF-7, CDKN1A mRNA levels dropped 334-fold at 24 h and returned to reference levels at 48 h (Fig. 4c). However, treatment with 100 ng/ml BMP-2 or the higher concentrations of BMP-2 peptide seemed to lessen this downregulatory phenomenon. BMP-2-treated MCF-7 showed only 36-fold reduction in CDKN1A expression at 24 h, MCF-7 treated with 45.2 ng/ml BMP-2 peptide showed 42-fold and, those treated with 22.6 ng/ml Peptide 3, 54-fold reduction in CDKN1A mRNA levels at 24 h. Cells treated with the lowest concentration of BMP-2 peptide (4.52 ng/ml) showed approximately 220-fold downregulation of CDKN1A expression at 24 h. The MCF-7 groups that had been treated with BMP-2 peptide also showed a small reduction at 6 h (2-fold up to 7-fold), which was not observed in the BMP-2 treated group. Ιt was also observed that the cells that had been treated with 4.52 ng/ml or 22.6 ng/ml had a 3-fold increase in CDKN1A expression at 48 h, compared to reference levels, which was not observed in the rest of the groups.