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TLR signaling that induces weak inflammatory response and SHIP1 enhances osteogenic functions.

Muthukuru M, Darveau RP - Bone Res (2014)

Bottom Line: Our results suggest that osteoblasts stimulated by P3C, poorly induced IL-1β but strongly upregulated SHIP1 and enhanced osteogenic mediators.On the contrary, EcLPS significantly induced IL-1β and osteogenic mediators were not induced.In conclusion, these results suggest that induction of weak inflammatory response through TLR2 (with SHIP1 activity) and mutant TLR4 ligands could enhance osteogenesis.

View Article: PubMed Central - PubMed

Affiliation: West Virginia University, Department of Periodontics, School of Dentistry , Morgantown, WV, USA.

ABSTRACT
Toll-like receptor (TLR)-mediated inflammatory response could negatively affect bone metabolism. In this study, we determined how osteogenesis is regulated during inflammatory responses that are downstream of TLR signaling. Human primary osteoblasts were cultured in collagen gels. Pam3CSK4 (P3C) and Escherichia coli lipopolysaccharide (EcLPS) were used as TLR2 and TLR4 ligand respectively. Porphyromonas gingivalis LPS having TLR2 activity with either TLR4 agonism (Pg1690) or TLR4 antagonism (Pg1449) and mutant E. coli LPS (LPxE/LPxF/WSK) were used. IL-1β, SH2-containing inositol phosphatase-1 (SHIP1) that has regulatory roles in osteogenesis, alkaline phosphatase and mineralization were analyzed. 3α-Aminocholestane (3AC) was used to inhibit SHIP1. Our results suggest that osteoblasts stimulated by P3C, poorly induced IL-1β but strongly upregulated SHIP1 and enhanced osteogenic mediators. On the contrary, EcLPS significantly induced IL-1β and osteogenic mediators were not induced. While Pg1690 downmodulated osteogenic mediators, Pg1449 enhanced osteogenic responses, suggesting that TLR4 signaling annuls osteogenesis even with TLR2 activity. Interestingly, mutant E. coli LPS that induces weak inflammation upregulated osteogenesis, but SHIP1 was not induced. Moreover, inhibiting SHIP1 significantly upregulated TLR2-mediated inflammatory response and downmodulated osteogenesis. In conclusion, these results suggest that induction of weak inflammatory response through TLR2 (with SHIP1 activity) and mutant TLR4 ligands could enhance osteogenesis.

No MeSH data available.


Related in: MedlinePlus

Potency of the inflammatory response negatively regulates and overrules TLR2-induced osteogenic functions. Three-dimensional osteoblasts cultured in collagen gel cultures were stimulated for one week with either 1 μg⋅mL−1 of P3C along with 0–100 ng⋅mL−1 of TNF-α (a–d). Regulation of ALP was determined through histochemical (a) and biochemical (b) methods. In vitro mineralization and calcium deposition was analyzed by histochemical (c) and biochemical (d) methods. All analyses were performed in triplicate and images were captured at ×1 magnification. When stimulated with TNF-α, a dose-dependent downmodulation of ALP expression and Ca deposition that is induced by P3C is shown. *P≤0.001, t-test.
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fig5: Potency of the inflammatory response negatively regulates and overrules TLR2-induced osteogenic functions. Three-dimensional osteoblasts cultured in collagen gel cultures were stimulated for one week with either 1 μg⋅mL−1 of P3C along with 0–100 ng⋅mL−1 of TNF-α (a–d). Regulation of ALP was determined through histochemical (a) and biochemical (b) methods. In vitro mineralization and calcium deposition was analyzed by histochemical (c) and biochemical (d) methods. All analyses were performed in triplicate and images were captured at ×1 magnification. When stimulated with TNF-α, a dose-dependent downmodulation of ALP expression and Ca deposition that is induced by P3C is shown. *P≤0.001, t-test.

Mentions: In this study, we aimed to determine whether low doses of EcLPS have any positive influence on osteogenic functions. The data suggests that EcLPS employed at 1 ng⋅mL−1 moderately induced ALP (Figure 4a and 4b) and mineralization (Figure 4c and 4d) and this was downmodulated in a dose-dependent manner. We subsequently determined how mutant LPS structures that are weak TLR4 agonists (and therefore, weak inducers of inflammatory response) regulate osteogenic mediators. Mutant EcLPS structures namely, LPxF, LPxE and WSK were used along with P3C (positive control) and canonical EcLPS (negative control). When compared with EcLPS, osteoblasts stimulated with LPxF, LPxE or WSK significantly induced the expression of ALP (Figure 4e and 4f) and upregulated in vitro mineralization (Figure 5g and 5h). However, the induction of ALP and mineral deposition was more potent when osteoblasts were stimulated with P3C (Figure 5e and 5h). Moreover, SHIP1 was not significantly upregulated when osteoblasts were stimulated with any of the mutant LPS (i.e., LPxF, LPxE or WSK) relative to stimulating with P3C (Figure 5j). Despite lower induction of SHIP1, IL-1β was also not strongly induced by these mutant LPS structures relative to stimulation with the canonical EcLPS (Figure 5i). These results suggest that, irrespective of the potency of LPS structures that target TLR4, SHIP1 expression is not under the control of TLR4 signaling. Therefore, upregulation of osteogenic mediators in response to LPxF, LPxE or WSK, could be facilitated due to a weak inflammatory response mediated by these mutant LPS structures.


TLR signaling that induces weak inflammatory response and SHIP1 enhances osteogenic functions.

Muthukuru M, Darveau RP - Bone Res (2014)

Potency of the inflammatory response negatively regulates and overrules TLR2-induced osteogenic functions. Three-dimensional osteoblasts cultured in collagen gel cultures were stimulated for one week with either 1 μg⋅mL−1 of P3C along with 0–100 ng⋅mL−1 of TNF-α (a–d). Regulation of ALP was determined through histochemical (a) and biochemical (b) methods. In vitro mineralization and calcium deposition was analyzed by histochemical (c) and biochemical (d) methods. All analyses were performed in triplicate and images were captured at ×1 magnification. When stimulated with TNF-α, a dose-dependent downmodulation of ALP expression and Ca deposition that is induced by P3C is shown. *P≤0.001, t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472124&req=5

fig5: Potency of the inflammatory response negatively regulates and overrules TLR2-induced osteogenic functions. Three-dimensional osteoblasts cultured in collagen gel cultures were stimulated for one week with either 1 μg⋅mL−1 of P3C along with 0–100 ng⋅mL−1 of TNF-α (a–d). Regulation of ALP was determined through histochemical (a) and biochemical (b) methods. In vitro mineralization and calcium deposition was analyzed by histochemical (c) and biochemical (d) methods. All analyses were performed in triplicate and images were captured at ×1 magnification. When stimulated with TNF-α, a dose-dependent downmodulation of ALP expression and Ca deposition that is induced by P3C is shown. *P≤0.001, t-test.
Mentions: In this study, we aimed to determine whether low doses of EcLPS have any positive influence on osteogenic functions. The data suggests that EcLPS employed at 1 ng⋅mL−1 moderately induced ALP (Figure 4a and 4b) and mineralization (Figure 4c and 4d) and this was downmodulated in a dose-dependent manner. We subsequently determined how mutant LPS structures that are weak TLR4 agonists (and therefore, weak inducers of inflammatory response) regulate osteogenic mediators. Mutant EcLPS structures namely, LPxF, LPxE and WSK were used along with P3C (positive control) and canonical EcLPS (negative control). When compared with EcLPS, osteoblasts stimulated with LPxF, LPxE or WSK significantly induced the expression of ALP (Figure 4e and 4f) and upregulated in vitro mineralization (Figure 5g and 5h). However, the induction of ALP and mineral deposition was more potent when osteoblasts were stimulated with P3C (Figure 5e and 5h). Moreover, SHIP1 was not significantly upregulated when osteoblasts were stimulated with any of the mutant LPS (i.e., LPxF, LPxE or WSK) relative to stimulating with P3C (Figure 5j). Despite lower induction of SHIP1, IL-1β was also not strongly induced by these mutant LPS structures relative to stimulation with the canonical EcLPS (Figure 5i). These results suggest that, irrespective of the potency of LPS structures that target TLR4, SHIP1 expression is not under the control of TLR4 signaling. Therefore, upregulation of osteogenic mediators in response to LPxF, LPxE or WSK, could be facilitated due to a weak inflammatory response mediated by these mutant LPS structures.

Bottom Line: Our results suggest that osteoblasts stimulated by P3C, poorly induced IL-1β but strongly upregulated SHIP1 and enhanced osteogenic mediators.On the contrary, EcLPS significantly induced IL-1β and osteogenic mediators were not induced.In conclusion, these results suggest that induction of weak inflammatory response through TLR2 (with SHIP1 activity) and mutant TLR4 ligands could enhance osteogenesis.

View Article: PubMed Central - PubMed

Affiliation: West Virginia University, Department of Periodontics, School of Dentistry , Morgantown, WV, USA.

ABSTRACT
Toll-like receptor (TLR)-mediated inflammatory response could negatively affect bone metabolism. In this study, we determined how osteogenesis is regulated during inflammatory responses that are downstream of TLR signaling. Human primary osteoblasts were cultured in collagen gels. Pam3CSK4 (P3C) and Escherichia coli lipopolysaccharide (EcLPS) were used as TLR2 and TLR4 ligand respectively. Porphyromonas gingivalis LPS having TLR2 activity with either TLR4 agonism (Pg1690) or TLR4 antagonism (Pg1449) and mutant E. coli LPS (LPxE/LPxF/WSK) were used. IL-1β, SH2-containing inositol phosphatase-1 (SHIP1) that has regulatory roles in osteogenesis, alkaline phosphatase and mineralization were analyzed. 3α-Aminocholestane (3AC) was used to inhibit SHIP1. Our results suggest that osteoblasts stimulated by P3C, poorly induced IL-1β but strongly upregulated SHIP1 and enhanced osteogenic mediators. On the contrary, EcLPS significantly induced IL-1β and osteogenic mediators were not induced. While Pg1690 downmodulated osteogenic mediators, Pg1449 enhanced osteogenic responses, suggesting that TLR4 signaling annuls osteogenesis even with TLR2 activity. Interestingly, mutant E. coli LPS that induces weak inflammation upregulated osteogenesis, but SHIP1 was not induced. Moreover, inhibiting SHIP1 significantly upregulated TLR2-mediated inflammatory response and downmodulated osteogenesis. In conclusion, these results suggest that induction of weak inflammatory response through TLR2 (with SHIP1 activity) and mutant TLR4 ligands could enhance osteogenesis.

No MeSH data available.


Related in: MedlinePlus