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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

Osteoblasts resist upregulation of TLR2 and TLR4 in response to TLR ligands. Human primary osteoblasts between passages of 3–5 and blood monocytes were cultured as described in the section on ‘Materials and methods’. Osteoblasts or monocytes were either unstimulated or stimulated with either P3C (1 µg⋅mL−1) or EcLPS (1 µg⋅mL−1) for 1 week. Total RNA was extracted and quantitated and its purity confirmed as described in the section on ‘Materials and methods’. cDNA was synthesized from identical quantities of toral RNA from all the samples. The purity of cDNA was determined by analysis of the OD260/OD280 ratio. mRNA quantitation was performed by real-time RT-PCR analysis. Levels of β-actin mRNA served as an internal control to normalize samples. All analyses were performed in triplicate. RT-PCR was performed as described in the section on ‘Materials and methods’ to determine the expression of TLR2 (a, i) and TLR4 (b, j). Surface expressions of TLR2 (c–e; k–m) and TLR4 (f–h; n–p) were analyzed through flow cytometry as described in materials and methods. Isotype-matched antibody controls (Iso) are shown for TLR2 (c, k) and TLR4 (f, n) in osteoblasts and monocytes. Shown is representative data from two individual experiments. *P=0.017 and **P=0.004; t-test.
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fig1: Osteoblasts resist upregulation of TLR2 and TLR4 in response to TLR ligands. Human primary osteoblasts between passages of 3–5 and blood monocytes were cultured as described in the section on ‘Materials and methods’. Osteoblasts or monocytes were either unstimulated or stimulated with either P3C (1 µg⋅mL−1) or EcLPS (1 µg⋅mL−1) for 1 week. Total RNA was extracted and quantitated and its purity confirmed as described in the section on ‘Materials and methods’. cDNA was synthesized from identical quantities of toral RNA from all the samples. The purity of cDNA was determined by analysis of the OD260/OD280 ratio. mRNA quantitation was performed by real-time RT-PCR analysis. Levels of β-actin mRNA served as an internal control to normalize samples. All analyses were performed in triplicate. RT-PCR was performed as described in the section on ‘Materials and methods’ to determine the expression of TLR2 (a, i) and TLR4 (b, j). Surface expressions of TLR2 (c–e; k–m) and TLR4 (f–h; n–p) were analyzed through flow cytometry as described in materials and methods. Isotype-matched antibody controls (Iso) are shown for TLR2 (c, k) and TLR4 (f, n) in osteoblasts and monocytes. Shown is representative data from two individual experiments. *P=0.017 and **P=0.004; t-test.

Mentions: Osteoblasts express TLRs and play a role in inflammatory response by secreting cytokines. However, little is known about the constitutive expression of TLRs and how TLR expression is regulated in osteoblasts. In this study, the expression of TLR2 and TLR4 in osteoblasts was compared with that of peripheral blood monocytes. Synthetic P3C and EcLPS were employed as TLR2 and TLR4 ligands respectively. Unstimulated cells or osteoblasts/monocytes stimulated with either P3C or EcLPS were analyzed for the expression of TLR2 and TLR4 through real-time RT-PCR and flow cytometry. Relative to blood monocytes (Figure 1i–1k and 1n), osteoblasts (Figure 1a–1c and 1f) have low constitutive mRNA and protein levels for TLR2 and TLR4. P3C was a poor inducer of either TLR2 or TLR4 in osteoblasts (Figure 1a–1b, 1d and 1g). P3C upregulated TLR2 relative to TLR4 in monocytes (Figure 1i, 1j, 1l and 1o). Monocytes upregulated TLR2 and TLR4 in response to EcLPS (Figure 1i, 1j, 1m and 1p). However, relative to monocytes, osteoblasts did not upregulate TLR2 and TLR4 when stimulated with EcLPS (Figure 1a, 1b, 1e and 1h).


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

Muthukuru M, Darveau RP - Bone Res (2014)

Osteoblasts resist upregulation of TLR2 and TLR4 in response to TLR ligands. Human primary osteoblasts between passages of 3–5 and blood monocytes were cultured as described in the section on ‘Materials and methods’. Osteoblasts or monocytes were either unstimulated or stimulated with either P3C (1 µg⋅mL−1) or EcLPS (1 µg⋅mL−1) for 1 week. Total RNA was extracted and quantitated and its purity confirmed as described in the section on ‘Materials and methods’. cDNA was synthesized from identical quantities of toral RNA from all the samples. The purity of cDNA was determined by analysis of the OD260/OD280 ratio. mRNA quantitation was performed by real-time RT-PCR analysis. Levels of β-actin mRNA served as an internal control to normalize samples. All analyses were performed in triplicate. RT-PCR was performed as described in the section on ‘Materials and methods’ to determine the expression of TLR2 (a, i) and TLR4 (b, j). Surface expressions of TLR2 (c–e; k–m) and TLR4 (f–h; n–p) were analyzed through flow cytometry as described in materials and methods. Isotype-matched antibody controls (Iso) are shown for TLR2 (c, k) and TLR4 (f, n) in osteoblasts and monocytes. Shown is representative data from two individual experiments. *P=0.017 and **P=0.004; t-test.
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fig1: Osteoblasts resist upregulation of TLR2 and TLR4 in response to TLR ligands. Human primary osteoblasts between passages of 3–5 and blood monocytes were cultured as described in the section on ‘Materials and methods’. Osteoblasts or monocytes were either unstimulated or stimulated with either P3C (1 µg⋅mL−1) or EcLPS (1 µg⋅mL−1) for 1 week. Total RNA was extracted and quantitated and its purity confirmed as described in the section on ‘Materials and methods’. cDNA was synthesized from identical quantities of toral RNA from all the samples. The purity of cDNA was determined by analysis of the OD260/OD280 ratio. mRNA quantitation was performed by real-time RT-PCR analysis. Levels of β-actin mRNA served as an internal control to normalize samples. All analyses were performed in triplicate. RT-PCR was performed as described in the section on ‘Materials and methods’ to determine the expression of TLR2 (a, i) and TLR4 (b, j). Surface expressions of TLR2 (c–e; k–m) and TLR4 (f–h; n–p) were analyzed through flow cytometry as described in materials and methods. Isotype-matched antibody controls (Iso) are shown for TLR2 (c, k) and TLR4 (f, n) in osteoblasts and monocytes. Shown is representative data from two individual experiments. *P=0.017 and **P=0.004; t-test.
Mentions: Osteoblasts express TLRs and play a role in inflammatory response by secreting cytokines. However, little is known about the constitutive expression of TLRs and how TLR expression is regulated in osteoblasts. In this study, the expression of TLR2 and TLR4 in osteoblasts was compared with that of peripheral blood monocytes. Synthetic P3C and EcLPS were employed as TLR2 and TLR4 ligands respectively. Unstimulated cells or osteoblasts/monocytes stimulated with either P3C or EcLPS were analyzed for the expression of TLR2 and TLR4 through real-time RT-PCR and flow cytometry. Relative to blood monocytes (Figure 1i–1k and 1n), osteoblasts (Figure 1a–1c and 1f) have low constitutive mRNA and protein levels for TLR2 and TLR4. P3C was a poor inducer of either TLR2 or TLR4 in osteoblasts (Figure 1a–1b, 1d and 1g). P3C upregulated TLR2 relative to TLR4 in monocytes (Figure 1i, 1j, 1l and 1o). Monocytes upregulated TLR2 and TLR4 in response to EcLPS (Figure 1i, 1j, 1m and 1p). However, relative to monocytes, osteoblasts did not upregulate TLR2 and TLR4 when stimulated with EcLPS (Figure 1a, 1b, 1e and 1h).

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