Glucagon-like peptide 2 decreases osteoclasts by stimulating apoptosis dependent on nitric oxide synthase

Abstract

Objectives

Glucagon-like peptide 2 (GLP2) is involved in the regulation of energy absorption and metabolism. Despite the importance of the GLP2 signalling mechanisms on osteoclast, little has been studied on how GLP2 works during osteoclastogenesis.

Materials and Methods

RAW264.7 cells were infected with rLV-Green-GLP2. The induction of osteoclasts was performed by RANKL. TRAP were detected by RT-PCR, Western blotting and staining. Total nitric oxide and total NOS activity were measured. Cells apoptosis was detected by Hoest33258 and Annix V staining. Animal test, chromatin immunoprecipitation (CHIP), co-immunoprecipitation(IP) and luciferase reporter assay were also performed.

Results

We indicate that GLP2 is associated with osteoporosis-related factors in aged rats, including BALP, TRAP, IL6, TNFα, Nitric Oxide (NO), iNOS, calcitonin and occludin. Moreover, GLP2 is demonstrated to result in negative action during proliferation of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts. Furthermore, GLP2 decreases osteoclasts induced from monocyte/macrophage cells RAW264.7 as well as the serum TRAP activity in aged rats. Mechanistic investigations reveal GLP2 enhances the expression of iNOS through stimulating the activity of TGFβ-Smad2/3 signalling in osteoclasts. In particular, inhibition of TGFβ fully abrogates this function of GLP2 in osteoclasts. Strikingly, overexpression of GLP2 significantly increases the product of nitric oxide via iNOS which promotes apoptosis of osteoclasts by decreasing bcl2 or increasing caspase3. Thereby, the ability of GLP2 to regulate apoptosis depends on TGFβ-Smad2/3-iNOS-NO signalling pathway since total NOS inhibitor L-NMMA specifically inhibits the actions by GLP2.

Conclusions

GLP2 induces apoptosis via TGFβ-Smad2/3 signalling, which contributes to the inhibition of the proliferation of osteoclasts and which may provide potential therapeutic targets for the treatment of osteoporosis.

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