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

Abstract Objectives Glucagon‐like peptide 2 ( GLP 2) is involved in the regulation of energy absorption and metabolism. Despite the importance of the GLP 2 signalling mechanisms on osteoclast, little has been studied on how GLP 2 works during osteoclastogenesis. Materials and Methods RAW 264.7 cells were infected with rLV ‐Green‐ GLP 2. 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 GLP 2 is associated with osteoporosis‐related factors in aged rats, including BALP , TRAP , IL 6, TNF α, Nitric Oxide ( NO ), iNOS , calcitonin and occludin. Moreover, GLP 2 is demonstrated to result in negative action during proliferation of tartrate‐resistant acid phosphatase‐positive ( TRAP +) osteoclasts. Furthermore, GLP 2 decreases osteoclasts induced from monocyte/macrophage cells RAW 264.7 as well as the serum TRAP activity in aged rats. Mechanistic investigations reveal GLP 2 enhances the expression of iNOS through stimulating the activity of TGF β‐Smad2/3 signalling in osteoclasts. In particular, inhibition of TGF β fully abrogates this function of GLP 2 in osteoclasts. Strikingly, overexpression of GLP 2 significantly increases the product of nitric oxide via iNOS which promotes apoptosis of osteoclasts by decreasing bcl2 or increasing caspase3. Thereby, the ability of GLP 2 to regulate apoptosis depends on TGF β‐Smad2/3‐ iNOS ‐ NO signalling pathway since total NOS inhibitor L‐ NMMA specifically inhibits the actions by GLP2. Conclusions GLP 2 induces apoptosis via TGF β‐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.