Prunetin signals via G-protein-coupled receptor, GPR30(GPER1): Stimulation of adenylyl cyclase and cAMP-mediated activation of MAPK signaling induces Runx2 expression in osteoblasts to promote bone regeneration.
Prunetin is found in red clover and fruit of Prunus avium (red cherry). The effect of prunetin on osteoblast function, its mode of action and bone regeneration in vivo were investigated. Cultures of primary osteoblasts, osteoblastic cell line and HEK293T cells were used for various in vitro studies. Adult female rats received drill-hole injury at the femur diaphysis to assess the bone regenerative effect of prunetin. Prunetin at 10nM significantly (a) increased proliferation and differentiation of primary cultures of osteoblasts harvested from rats and (b) promoted formation of mineralized nodules by bone marrow stromal/osteoprogenitor cells. At this concentration, prunetin did not activate any of the two nuclear estrogen receptors (α and β). However, prunetin triggered signaling via a G-protein-coupled receptor, GPR30/GPER1, and enhanced cAMP levels in osteoblasts. G15, a selective GPR30 antagonist, abolished prunetin-induced increases in osteoblast proliferation, differentiation and intracellular cAMP. In osteoblasts, prunetin up-regulated runt-related transcription factor 2 (Runx2) protein through cAMP-dependent Erk/MAP kinase activation that ultimately resulted in the up-regulation of GPR30. Administration of prunetin at 0.25mg/kg given to rats stimulated bone regeneration at the site of drill hole and up-regulated Runx2 expression in the fractured callus and the effect was comparable to human parathyroid hormone, the only clinically used osteogenic therapy. We conclude that prunetin promotes osteoinduction in vivo and the mechanism is defined by signaling through GPR30 resulting in the up-regulation of the key osteogenic gene Runx2 that in turn up-regulates GPR30.
Division of Endocrinology and Center for Research in Anabolic Skeletal Targets in Health and Illness, Central Drug Research Institute, Council of Scientific and Industrial Research, Lucknow 226021, India.
Kainat
K
Khan
Division of Endocrinology and Center for Research in Anabolic Skeletal Targets in Health and Illness, Central Drug Research Institute, Council of Scientific and Industrial Research, Lucknow 226021, India.
Subhashis
S
Pal
Division of Biochemistry, Central Drug Research Institute, Council of Scientific and Industrial Research, Lucknow 226021, India.
Manisha
M
Yadav
Division of Medicinal and Process Chemistry, Central Drug Research Institute, Council of Scientific and Industrial Research, Lucknow 226021, India.
Rakesh
R
Maurya
Division of Biochemistry, Central Drug Research Institute, Council of Scientific and Industrial Research, Lucknow 226021, India.
Arun Kumar
AK
Trivedi
Division of Biochemistry, Central Drug Research Institute, Council of Scientific and Industrial Research, Lucknow 226021, India.
Sabyasachi
S
Sanyal
Division of Endocrinology and Center for Research in Anabolic Skeletal Targets in Health and Illness, Central Drug Research Institute, Council of Scientific and Industrial Research, Lucknow 226021, India. Electronic address: n_chattopadhyay@cdri.res.in.
Naibedya
N
Chattopadhyay
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001861:Bone Regeneration; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D005260:Female; D005269:Femur / Q000473:pathology; D017102:Fracture Healing; D057809:HEK293 Cells; D006801:Humans; D007529:Isoflavones / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010012:Osteogenesis; D010281:Parathyroid Hormone / Q000378:metabolism; D027861:Prunus; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D015854:Up-Regulation; D055114:X-Ray Microtomography
D050659:Core Binding Factor Alpha 1 Subunit; C109584:GPER1 protein, human; C520305:Gper1 protein, rat; D007529:Isoflavones; D010281:Parathyroid Hormone; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C493717:Runx2 protein, rat; C083295:prunetin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases
Bone formation; Estrogen signaling; Fracture healing; G protein coupled receptor 30; Isoflavonoid