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Decreasing NF-κB expression enhances odontoblastic differentiation and collagen expression in dental pulp stem cells exposed to inflammatory cytokines.

Hozhabri NS, Benson MD, Vu MD, Patel RH, Martinez RM, Nakhaie FN, Kim HK, Varanasi VG - PLoS ONE (2015)

Bottom Line: Nuclear Factor-κB activation in the presence of these cytokines decreased significantly in a dose-dependent manner by a Nuclear Factor-κB inhibitor (MG132) and p65 siRNA.Finally, dental pulp stem cells exposed to reduced Nuclear Factor-κB activity resulted in a significant increase in collagen (I)-α1 expression in the presence of these cytokines.In conclusion, a decrease in Nuclear Factor-κB in dental pulp stem cells in the presence of inflammatory cytokines enhanced odontoblastic differentiation and collagen matrix formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Center for Craniofacial Research and Diagnosis, Texas A&M University Baylor College of Dentistry, Dallas, Texas 75246, United States of America.

ABSTRACT
Inflammatory response in the dental pulp can alter the collagen matrix formation by dental pulp stem cells and lead to a delay or poor healing of the pulp. This inflammatory response is mediated by cytokines, including interleukin-1β and tumor necrosis factor-α. In this study, it is hypothesized that suppressing the actions of these inflammatory cytokines by knocking down the activity of transcription factor Nuclear Factor-κB will lead to dental pulp stem cell differentiation into odontoblasts and the production of collagen. Here, the role of Nuclear Factor-κB signaling and its reduction was examined during odontogenic behavior in the presence of these cytokines. The results showed a significant increase in Nuclear Factor-κB gene expression and p65 protein expression by interleukin-1β and tumor necrosis factor-α. Nuclear Factor-κB activation in the presence of these cytokines decreased significantly in a dose-dependent manner by a Nuclear Factor-κB inhibitor (MG132) and p65 siRNA. Down-regulation of Nuclear Factor-κB activity also enhanced the gene expression of the odontoblastic markers (dentin sialophosphoprotein, Nestin, and alkaline phosphatase) and displayed an odontoblastic cell morphology indicating the promotion of odontogenic differentiation of dental pulp stem cells. Finally, dental pulp stem cells exposed to reduced Nuclear Factor-κB activity resulted in a significant increase in collagen (I)-α1 expression in the presence of these cytokines. In conclusion, a decrease in Nuclear Factor-κB in dental pulp stem cells in the presence of inflammatory cytokines enhanced odontoblastic differentiation and collagen matrix formation.

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The effect of MG132 protease inhibitor and p65 siRNA on Collagen (I)-α1 (Col(I)α1) gene expression in DPSCs with low- and high-cytokine doses.DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Col(I)-α1 mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p < 0.05; **, ++, or ΔΔ for p < 0.01; ***, +++, or ΔΔΔ for p < 0.001.
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pone.0113334.g006: The effect of MG132 protease inhibitor and p65 siRNA on Collagen (I)-α1 (Col(I)α1) gene expression in DPSCs with low- and high-cytokine doses.DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Col(I)-α1 mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p < 0.05; **, ++, or ΔΔ for p < 0.01; ***, +++, or ΔΔΔ for p < 0.001.

Mentions: The results for type I collagen gene expression showed that the addition of MG132 to the DPSCs induced a ~8-fold and 2-fold increase in collagen expression with 0.1μM and 0.5μM MG132, respectively, compared to the control (Fig. 6A). P65 siRNA also elevated the collagen gene expression, producing a 1.5-fold and 3.5-fold increase in collagen (I) gene expression with 20 pM and 50 pM, respectively (Fig. 6B).


Decreasing NF-κB expression enhances odontoblastic differentiation and collagen expression in dental pulp stem cells exposed to inflammatory cytokines.

Hozhabri NS, Benson MD, Vu MD, Patel RH, Martinez RM, Nakhaie FN, Kim HK, Varanasi VG - PLoS ONE (2015)

The effect of MG132 protease inhibitor and p65 siRNA on Collagen (I)-α1 (Col(I)α1) gene expression in DPSCs with low- and high-cytokine doses.DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Col(I)-α1 mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p < 0.05; **, ++, or ΔΔ for p < 0.01; ***, +++, or ΔΔΔ for p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4309458&req=5

pone.0113334.g006: The effect of MG132 protease inhibitor and p65 siRNA on Collagen (I)-α1 (Col(I)α1) gene expression in DPSCs with low- and high-cytokine doses.DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Col(I)-α1 mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p < 0.05; **, ++, or ΔΔ for p < 0.01; ***, +++, or ΔΔΔ for p < 0.001.
Mentions: The results for type I collagen gene expression showed that the addition of MG132 to the DPSCs induced a ~8-fold and 2-fold increase in collagen expression with 0.1μM and 0.5μM MG132, respectively, compared to the control (Fig. 6A). P65 siRNA also elevated the collagen gene expression, producing a 1.5-fold and 3.5-fold increase in collagen (I) gene expression with 20 pM and 50 pM, respectively (Fig. 6B).

Bottom Line: Nuclear Factor-κB activation in the presence of these cytokines decreased significantly in a dose-dependent manner by a Nuclear Factor-κB inhibitor (MG132) and p65 siRNA.Finally, dental pulp stem cells exposed to reduced Nuclear Factor-κB activity resulted in a significant increase in collagen (I)-α1 expression in the presence of these cytokines.In conclusion, a decrease in Nuclear Factor-κB in dental pulp stem cells in the presence of inflammatory cytokines enhanced odontoblastic differentiation and collagen matrix formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Center for Craniofacial Research and Diagnosis, Texas A&M University Baylor College of Dentistry, Dallas, Texas 75246, United States of America.

ABSTRACT
Inflammatory response in the dental pulp can alter the collagen matrix formation by dental pulp stem cells and lead to a delay or poor healing of the pulp. This inflammatory response is mediated by cytokines, including interleukin-1β and tumor necrosis factor-α. In this study, it is hypothesized that suppressing the actions of these inflammatory cytokines by knocking down the activity of transcription factor Nuclear Factor-κB will lead to dental pulp stem cell differentiation into odontoblasts and the production of collagen. Here, the role of Nuclear Factor-κB signaling and its reduction was examined during odontogenic behavior in the presence of these cytokines. The results showed a significant increase in Nuclear Factor-κB gene expression and p65 protein expression by interleukin-1β and tumor necrosis factor-α. Nuclear Factor-κB activation in the presence of these cytokines decreased significantly in a dose-dependent manner by a Nuclear Factor-κB inhibitor (MG132) and p65 siRNA. Down-regulation of Nuclear Factor-κB activity also enhanced the gene expression of the odontoblastic markers (dentin sialophosphoprotein, Nestin, and alkaline phosphatase) and displayed an odontoblastic cell morphology indicating the promotion of odontogenic differentiation of dental pulp stem cells. Finally, dental pulp stem cells exposed to reduced Nuclear Factor-κB activity resulted in a significant increase in collagen (I)-α1 expression in the presence of these cytokines. In conclusion, a decrease in Nuclear Factor-κB in dental pulp stem cells in the presence of inflammatory cytokines enhanced odontoblastic differentiation and collagen matrix formation.

Show MeSH
Related in: MedlinePlus