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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

SHIP1 inhibition enhances inflammatory response in osteoblasts. Human primary osteoblasts between passages of 3–5 were cultured and incubated with 3AC at doses of 0–100 µmol⋅mL−1. 3AC dose-dependent cytotoxicity in osteoblasts was determined through tryphan blue exclusion method (a). Subsequently, osteoblasts were stimulated with 1 µg⋅mL−1 of P3C and were incubated with varying doses of 3AC (1–10 µmol⋅mL−1) and the levels of IL-1β gene expression and SHIP1 protein expression were determined and 3AC induced dose-dependent downmodulation of SHIP1 and upregulation of IL-1β (data not shown). Unstimulated osteoblasts or osteoblasts stimulated with P3C/Pg1449 alone or stimulated P3C/Pg1449 along with 10 µmol⋅mL−1 of 3AC were employed to determine the gene expression of IL-1β (b) and protein expression of SHIP1 (c). P3C- and Pg1449-induced SHIP1 expression was downregulated when osteoblasts were stimulated with AC3 (c). Osteoblasts stimulated with P3C/Pg1449 in the presence of AC3 to inhibit SHIP1 further upregulated the levels of IL-1β expression (b). All experiments were performed in triplicate and shown are the means along with standard error.
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fig6: SHIP1 inhibition enhances inflammatory response in osteoblasts. Human primary osteoblasts between passages of 3–5 were cultured and incubated with 3AC at doses of 0–100 µmol⋅mL−1. 3AC dose-dependent cytotoxicity in osteoblasts was determined through tryphan blue exclusion method (a). Subsequently, osteoblasts were stimulated with 1 µg⋅mL−1 of P3C and were incubated with varying doses of 3AC (1–10 µmol⋅mL−1) and the levels of IL-1β gene expression and SHIP1 protein expression were determined and 3AC induced dose-dependent downmodulation of SHIP1 and upregulation of IL-1β (data not shown). Unstimulated osteoblasts or osteoblasts stimulated with P3C/Pg1449 alone or stimulated P3C/Pg1449 along with 10 µmol⋅mL−1 of 3AC were employed to determine the gene expression of IL-1β (b) and protein expression of SHIP1 (c). P3C- and Pg1449-induced SHIP1 expression was downregulated when osteoblasts were stimulated with AC3 (c). Osteoblasts stimulated with P3C/Pg1449 in the presence of AC3 to inhibit SHIP1 further upregulated the levels of IL-1β expression (b). All experiments were performed in triplicate and shown are the means along with standard error.

Mentions: Data in Figures 2 and 3 suggest that TLR2 enhanced osteogenic mediators and levels of SHIP1. We therefore aimed to determine the role of SHIP1 in regulating osteogenesis. 3AC, an inhibitor of SHIP1,19 was employed to determine how SHIP1 regulated IL-1β (Figure 6) and osteogenic mediators (discussed in Figure 7). On the bases of dose-dependent cytotoxicity in osteoblasts (Figure 6a), 1–10 µmol⋅mL−1 3AC was employed and expression of IL-1β that was weakly induced by TLR2 was significantly upregulated when SHIP1 was inhibited by 3AC in a dose-dependent manner (data not shown). P3C and Pg1449 (TLR2 agonist but TLR4 antagonist) were employed as these ligands significantly induced SHIP1 and EcLPS was used as a negative control to determine how inhibiting SHIP1 regulates the inflammatory response. Since P3C and Pg1449 were potent inducers of SHIP1 (by activating TLR2), a significant upregulation of IL-1β was noted when 3AC was employed to inhibit SHIP1 (Figure 6b). Since SHIP1 was not potently upregulated through TLR4 when stimulated with EcLPS, IL-1β was only slightly upregulated when SHIP1 was inhibited (Figure 6b). Malachite Green assay, a fluorescent polarization assay that detects the 5′-inositol phosphatase activity of SHIP1, was employed to measure SHIP1 levels. Inhibition of SHIP1 activity with 3AC was profound when osteoblasts were stimulated with P3C or Pg1449 (Figure 6c).


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

Muthukuru M, Darveau RP - Bone Res (2014)

SHIP1 inhibition enhances inflammatory response in osteoblasts. Human primary osteoblasts between passages of 3–5 were cultured and incubated with 3AC at doses of 0–100 µmol⋅mL−1. 3AC dose-dependent cytotoxicity in osteoblasts was determined through tryphan blue exclusion method (a). Subsequently, osteoblasts were stimulated with 1 µg⋅mL−1 of P3C and were incubated with varying doses of 3AC (1–10 µmol⋅mL−1) and the levels of IL-1β gene expression and SHIP1 protein expression were determined and 3AC induced dose-dependent downmodulation of SHIP1 and upregulation of IL-1β (data not shown). Unstimulated osteoblasts or osteoblasts stimulated with P3C/Pg1449 alone or stimulated P3C/Pg1449 along with 10 µmol⋅mL−1 of 3AC were employed to determine the gene expression of IL-1β (b) and protein expression of SHIP1 (c). P3C- and Pg1449-induced SHIP1 expression was downregulated when osteoblasts were stimulated with AC3 (c). Osteoblasts stimulated with P3C/Pg1449 in the presence of AC3 to inhibit SHIP1 further upregulated the levels of IL-1β expression (b). All experiments were performed in triplicate and shown are the means along with standard error.
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fig6: SHIP1 inhibition enhances inflammatory response in osteoblasts. Human primary osteoblasts between passages of 3–5 were cultured and incubated with 3AC at doses of 0–100 µmol⋅mL−1. 3AC dose-dependent cytotoxicity in osteoblasts was determined through tryphan blue exclusion method (a). Subsequently, osteoblasts were stimulated with 1 µg⋅mL−1 of P3C and were incubated with varying doses of 3AC (1–10 µmol⋅mL−1) and the levels of IL-1β gene expression and SHIP1 protein expression were determined and 3AC induced dose-dependent downmodulation of SHIP1 and upregulation of IL-1β (data not shown). Unstimulated osteoblasts or osteoblasts stimulated with P3C/Pg1449 alone or stimulated P3C/Pg1449 along with 10 µmol⋅mL−1 of 3AC were employed to determine the gene expression of IL-1β (b) and protein expression of SHIP1 (c). P3C- and Pg1449-induced SHIP1 expression was downregulated when osteoblasts were stimulated with AC3 (c). Osteoblasts stimulated with P3C/Pg1449 in the presence of AC3 to inhibit SHIP1 further upregulated the levels of IL-1β expression (b). All experiments were performed in triplicate and shown are the means along with standard error.
Mentions: Data in Figures 2 and 3 suggest that TLR2 enhanced osteogenic mediators and levels of SHIP1. We therefore aimed to determine the role of SHIP1 in regulating osteogenesis. 3AC, an inhibitor of SHIP1,19 was employed to determine how SHIP1 regulated IL-1β (Figure 6) and osteogenic mediators (discussed in Figure 7). On the bases of dose-dependent cytotoxicity in osteoblasts (Figure 6a), 1–10 µmol⋅mL−1 3AC was employed and expression of IL-1β that was weakly induced by TLR2 was significantly upregulated when SHIP1 was inhibited by 3AC in a dose-dependent manner (data not shown). P3C and Pg1449 (TLR2 agonist but TLR4 antagonist) were employed as these ligands significantly induced SHIP1 and EcLPS was used as a negative control to determine how inhibiting SHIP1 regulates the inflammatory response. Since P3C and Pg1449 were potent inducers of SHIP1 (by activating TLR2), a significant upregulation of IL-1β was noted when 3AC was employed to inhibit SHIP1 (Figure 6b). Since SHIP1 was not potently upregulated through TLR4 when stimulated with EcLPS, IL-1β was only slightly upregulated when SHIP1 was inhibited (Figure 6b). Malachite Green assay, a fluorescent polarization assay that detects the 5′-inositol phosphatase activity of SHIP1, was employed to measure SHIP1 levels. Inhibition of SHIP1 activity with 3AC was profound when osteoblasts were stimulated with P3C or Pg1449 (Figure 6c).

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