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The Effects of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol on Human Osteoblast Proliferation and Differentiation.

Smith M, Wilson R, O'Brien S, Tufarelli C, Anderson SI, O'Sullivan SE - PLoS ONE (2015)

Bottom Line: Inhibition of endocannabinoid degradation enzymes to increase endocannabinoid tone resulted in similar increases in ALP production. 2-arachidonoylglycerol also decreased osteocalcin secretion.Anandamide did not affect collagen or osteocalcin, but reduced calcium deposition.Our results have demonstrated a clear involvement of anandamide and 2-arachidonoylglycerol in modulating the activity of human osteoblasts, with anandamide increasing early cell differentiation and 2-AG increasing early, but decreasing late osteoblast-specific markers of differentiation.

View Article: PubMed Central - PubMed

Affiliation: Division of Medical Sciences & Graduate Entry Medicine, School of Medicine, Royal Derby Hospital, University of Nottingham, Derby DE22 3DT, United Kingdom.

ABSTRACT
The endocannabinoid system is expressed in bone, although its role in the regulation of bone growth is controversial. Many studies have examined the effect of endocannabinoids directly on osteoclast function, but few have examined their role in human osteoblast function, which was the aim of the present study. Human osteoblasts were treated from seeding with increasing concentrations of anandamide or 2-arachidonoylglycerol for between 1 and 21 days. Cell proliferation (DNA content) and differentiation (alkaline phosphatase (ALP), collagen and osteocalcin secretion and calcium deposition) were measured. Anandamide and 2-arachidonoylglycerol significantly decreased osteoblast proliferation after 4 days, associated with a concentration-dependent increase in ALP. Inhibition of endocannabinoid degradation enzymes to increase endocannabinoid tone resulted in similar increases in ALP production. 2-arachidonoylglycerol also decreased osteocalcin secretion. After prolonged (21 day) treatment with 2-arachidonoylglycerol, there was a decrease in collagen content, but no change in calcium deposition. Anandamide did not affect collagen or osteocalcin, but reduced calcium deposition. Anandamide increased levels of phosphorylated CREB, ERK 1/2 and JNK, while 2-arachidonoylglycerol increased phosphorylated CREB and Akt. RT-PCR demonstrated the expression of CB2 and TRPV1, but not CB1 in HOBs. Anandamide-induced changes in HOB differentiation were CB1 and CB2-independent and partially reduced by TRPV1 antagonism, and reduced by inhibition of ERK 1/2 and JNK. Our results have demonstrated a clear involvement of anandamide and 2-arachidonoylglycerol in modulating the activity of human osteoblasts, with anandamide increasing early cell differentiation and 2-AG increasing early, but decreasing late osteoblast-specific markers of differentiation.

No MeSH data available.


Related in: MedlinePlus

Effect of anandamide or 2-AG treatment on intracellular signalling in HOBs.Cells were grown for 24 hours in vitro, then treated with vehicle, 10μM anandamide or 10μM 2-AG for 20 minutes and multiplex analysis used to measure the level of phosphorylated (A) CREB, (B) Akt, (C) STAT3, (D) p70s6k, (E) NFκB, (F) ERK 1/2, (G) JNK, (H) STAT5 and (I) p38 expressed as mean fluorescence intensity (MFI) normalised to the (J) protein content of each sample. Data given as mean ± S.E.M., n = 6, from 1 experiment. *P<0.05, ** P<0.01, ***P<0.001, **** P<0.0001, compared to vehicle; # # P<0.01, compared to anandamide; one—way ANOVA with Tukey’s multiple comparisons test.
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pone.0136546.g005: Effect of anandamide or 2-AG treatment on intracellular signalling in HOBs.Cells were grown for 24 hours in vitro, then treated with vehicle, 10μM anandamide or 10μM 2-AG for 20 minutes and multiplex analysis used to measure the level of phosphorylated (A) CREB, (B) Akt, (C) STAT3, (D) p70s6k, (E) NFκB, (F) ERK 1/2, (G) JNK, (H) STAT5 and (I) p38 expressed as mean fluorescence intensity (MFI) normalised to the (J) protein content of each sample. Data given as mean ± S.E.M., n = 6, from 1 experiment. *P<0.05, ** P<0.01, ***P<0.001, **** P<0.0001, compared to vehicle; # # P<0.01, compared to anandamide; one—way ANOVA with Tukey’s multiple comparisons test.

Mentions: To establish the intracellular signalling pathways that might be activated by endocannabinoids in HOBs, confluent cells were treated with 10μM anandamide or 10μM 2-AG for 20 minutes, and the cells were analysed for changes in levels of phosphorylated signalling molecules. The signalling proteins was normalised to total protein content for each sample, which was consistent for each treatment group (Fig 5J). Anandamide significantly increased the levels of phosphorylated (p)-CREB (P<0.0001, Fig 5A), p-ERK 1/2 (P<0.0001, Fig 5F) and p-JNK (P<0.05, Fig 5G). 2-AG increased the levels of p-Akt (P<0.001, Fig 5B) and p-CREB (P<0.01, Fig 5A) compared to the cultures treated with vehicle. Although both endocannabinoids significantly increased the levels of p-CREB, osteoblasts treated with anandamide had significantly higher levels than those treated with 2-AG (P<0.01, Fig 5A). Neither endocannabinoid had an effect on the levels of p-NFκB, p-p38, p-p70s6k, p-STAT3 or p-STAT5 (Fig 5).


The Effects of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol on Human Osteoblast Proliferation and Differentiation.

Smith M, Wilson R, O'Brien S, Tufarelli C, Anderson SI, O'Sullivan SE - PLoS ONE (2015)

Effect of anandamide or 2-AG treatment on intracellular signalling in HOBs.Cells were grown for 24 hours in vitro, then treated with vehicle, 10μM anandamide or 10μM 2-AG for 20 minutes and multiplex analysis used to measure the level of phosphorylated (A) CREB, (B) Akt, (C) STAT3, (D) p70s6k, (E) NFκB, (F) ERK 1/2, (G) JNK, (H) STAT5 and (I) p38 expressed as mean fluorescence intensity (MFI) normalised to the (J) protein content of each sample. Data given as mean ± S.E.M., n = 6, from 1 experiment. *P<0.05, ** P<0.01, ***P<0.001, **** P<0.0001, compared to vehicle; # # P<0.01, compared to anandamide; one—way ANOVA with Tukey’s multiple comparisons test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587563&req=5

pone.0136546.g005: Effect of anandamide or 2-AG treatment on intracellular signalling in HOBs.Cells were grown for 24 hours in vitro, then treated with vehicle, 10μM anandamide or 10μM 2-AG for 20 minutes and multiplex analysis used to measure the level of phosphorylated (A) CREB, (B) Akt, (C) STAT3, (D) p70s6k, (E) NFκB, (F) ERK 1/2, (G) JNK, (H) STAT5 and (I) p38 expressed as mean fluorescence intensity (MFI) normalised to the (J) protein content of each sample. Data given as mean ± S.E.M., n = 6, from 1 experiment. *P<0.05, ** P<0.01, ***P<0.001, **** P<0.0001, compared to vehicle; # # P<0.01, compared to anandamide; one—way ANOVA with Tukey’s multiple comparisons test.
Mentions: To establish the intracellular signalling pathways that might be activated by endocannabinoids in HOBs, confluent cells were treated with 10μM anandamide or 10μM 2-AG for 20 minutes, and the cells were analysed for changes in levels of phosphorylated signalling molecules. The signalling proteins was normalised to total protein content for each sample, which was consistent for each treatment group (Fig 5J). Anandamide significantly increased the levels of phosphorylated (p)-CREB (P<0.0001, Fig 5A), p-ERK 1/2 (P<0.0001, Fig 5F) and p-JNK (P<0.05, Fig 5G). 2-AG increased the levels of p-Akt (P<0.001, Fig 5B) and p-CREB (P<0.01, Fig 5A) compared to the cultures treated with vehicle. Although both endocannabinoids significantly increased the levels of p-CREB, osteoblasts treated with anandamide had significantly higher levels than those treated with 2-AG (P<0.01, Fig 5A). Neither endocannabinoid had an effect on the levels of p-NFκB, p-p38, p-p70s6k, p-STAT3 or p-STAT5 (Fig 5).

Bottom Line: Inhibition of endocannabinoid degradation enzymes to increase endocannabinoid tone resulted in similar increases in ALP production. 2-arachidonoylglycerol also decreased osteocalcin secretion.Anandamide did not affect collagen or osteocalcin, but reduced calcium deposition.Our results have demonstrated a clear involvement of anandamide and 2-arachidonoylglycerol in modulating the activity of human osteoblasts, with anandamide increasing early cell differentiation and 2-AG increasing early, but decreasing late osteoblast-specific markers of differentiation.

View Article: PubMed Central - PubMed

Affiliation: Division of Medical Sciences & Graduate Entry Medicine, School of Medicine, Royal Derby Hospital, University of Nottingham, Derby DE22 3DT, United Kingdom.

ABSTRACT
The endocannabinoid system is expressed in bone, although its role in the regulation of bone growth is controversial. Many studies have examined the effect of endocannabinoids directly on osteoclast function, but few have examined their role in human osteoblast function, which was the aim of the present study. Human osteoblasts were treated from seeding with increasing concentrations of anandamide or 2-arachidonoylglycerol for between 1 and 21 days. Cell proliferation (DNA content) and differentiation (alkaline phosphatase (ALP), collagen and osteocalcin secretion and calcium deposition) were measured. Anandamide and 2-arachidonoylglycerol significantly decreased osteoblast proliferation after 4 days, associated with a concentration-dependent increase in ALP. Inhibition of endocannabinoid degradation enzymes to increase endocannabinoid tone resulted in similar increases in ALP production. 2-arachidonoylglycerol also decreased osteocalcin secretion. After prolonged (21 day) treatment with 2-arachidonoylglycerol, there was a decrease in collagen content, but no change in calcium deposition. Anandamide did not affect collagen or osteocalcin, but reduced calcium deposition. Anandamide increased levels of phosphorylated CREB, ERK 1/2 and JNK, while 2-arachidonoylglycerol increased phosphorylated CREB and Akt. RT-PCR demonstrated the expression of CB2 and TRPV1, but not CB1 in HOBs. Anandamide-induced changes in HOB differentiation were CB1 and CB2-independent and partially reduced by TRPV1 antagonism, and reduced by inhibition of ERK 1/2 and JNK. Our results have demonstrated a clear involvement of anandamide and 2-arachidonoylglycerol in modulating the activity of human osteoblasts, with anandamide increasing early cell differentiation and 2-AG increasing early, but decreasing late osteoblast-specific markers of differentiation.

No MeSH data available.


Related in: MedlinePlus