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Role of MAPKs in TGF-β1-induced maturation and mineralization in human osteoblast-like cells

Objectives: Our study aimed to clarify the role of mitogen-activated protein kinases (MAPKs) in transforming growth factor (TGF)-β1-stimulated mineralization in the human osteoblast-like MG63 cells.

Methods: The viability of MG63 cells under TGF-β1 stimulation was assessed by MTS assay. Western blotting determined TGF-β1-mediated activation of extracellular signal-related protein kinase (ERK), p38, and c-Jun amino-terminal kinase (JNK). Mineralization-related gene expression was examined by quantitative real-time PCR, and mineral deposition levels were evaluated by alizarin red S staining.

Results: TGF-β1 had no effect on MG63 cell proliferation. Activation of p38 was observed at 3 h post TGF-β1 stimulation. Moreover, JNK phosphorylation was upregulated by TGF-β1 from 1 to 6 h post stimulation, but had no activation on ERK phosphorylation throughout the experimental period. Treatment with JNK inhibitor diminished the alizarin red S-stained area in a dose-dependent manner. Mineral deposition was unaffected by MEK inhibitor, whereas p38 inhibitor increased the red-stained area. Gene expression levels of ALP and BSP were significantly decreased under treatment with JNK inhibitor and p38 inhibitor. The MEK inhibitor had no effect on the TGF-β1-mediated upregulation of ALP and BSP. Althoughall three inhibitors suppressed expression of COL I, none were found to stimulate expression of OCN.

Conclusions: Human osteoblast-like MG63 cells maturation and mineralization are induced through JNK activation of MAPK signaling in response to TGF-β1.

 

Comments:

It seems like your study has found that in human osteoblast-like MG63 cells, the stimulation of transforming growth factor (TGF)-β1 leads to the activation of specific mitogen-activated protein kinases (MAPKs) and subsequently influences mineralization.

Here's a breakdown of your findings:

1. **Cell Viability**: TGF-β1 didn't affect the proliferation of MG63 cells.

2. **MAPK Activation**:
   - **p38**: Showed activation at 3 hours post TGF-β1 stimulation.
   - **JNK**: Upregulated by TGF-β1 from 1 to 6 hours post stimulation.
   - **ERK**: No activation observed throughout the experimental period.

3. **Inhibitor Effects**:
   - **JNK Inhibitor**: Reduced alizarin red S-stained area (dose-dependent effect) and decreased gene expression levels of ALP and BSP.
   - **p38 Inhibitor**: Increased the red-stained area and decreased ALP and BSP gene expression levels.
   - **MEK Inhibitor**: Had no effect on mineralization but suppressed COL I expression.

4. **Gene Expression**:
   - **ALP and BSP**: Significantly decreased under treatment with JNK and p38 inhibitors, not affected by MEK inhibitor.
   - **COL I**: Suppressed by all three inhibitors.
   - **OCN**: Not stimulated by any of the inhibitors.

5. **Conclusions**:
   - MG63 cells' maturation and mineralization are induced through JNK activation of MAPK signaling in response to TGF-β1.
  
Your study suggests that TGF-β1 stimulates mineralization in these cells by primarily activating JNK within the MAPK pathway. This finding could have implications for understanding the mechanisms behind bone development and potentially for developing therapies targeting these pathways.

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