Identification of GRP78 as a molecular target of medicarpin in osteoblast cells by proteomics.
Osteogenic activity was identified in medicarpin (Med), a natural pterocarpan. Further, it was decided to study the differentially regulated protein expression during osteoblast differentiation in the presence of Med. Using 2D proteomic approach, we found that Med treatment to osteoblasts significantly downregulated GRP78, an ER chaperone with anti-apoptotic properties which also controls the activation of unfolded protein response signaling, a pro-survival strategy for normal ER functioning. However, severe stress leads to triggering of apoptotic responses and signaling switches to pro-apoptotic. In order to elucidate the effect of Med downregulation of GRP78, osteoblasts were transfected with SiGRP78 or SiGRP78+ Med or Med alone. It was seen that mRNA and protein levels of ER stress markers like GRP78, ATF-4, and CHOP were decreased in all the three groups with maximum reduction in SiGRP78+ Med group. Med targets GRP78 by inhibiting mitochondrial-mediated apoptosis which is evident by reduced levels of cytochrome c, caspase-3, Bax/BCL2 ratio, and enhanced expression of survivin. Finally, Annexin-PI staining of apoptotic cells revealed that MED inhibition of GRP78 leads to reduced osteoblast apoptosis and increased osteoblast survival. Altogether, our data show that Med inhibits ER stress-induced apoptosis and promotes osteoblast cell survival by targeting GRP78.
Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, B.S. 10/1, Sector-10, Jankipuram Extension, Lucknow, India.
Jyoti
J
Kureel
Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, B.S. 10/1, Sector-10, Jankipuram Extension, Lucknow, India.
Aijaz A
AA
John
Division of Medicinal & Process Chemistry, CSIR-Central Drug Research Institute, B.S. 10/1, Sector-10, Jankipuram Extension, Lucknow, India.
Ashutosh
A
Raghuvanshi
Division of Medicinal & Process Chemistry, CSIR-Central Drug Research Institute, B.S. 10/1, Sector-10, Jankipuram Extension, Lucknow, India.
Pallavi
P
Awasthi
Division of Medicinal & Process Chemistry, CSIR-Central Drug Research Institute, B.S. 10/1, Sector-10, Jankipuram Extension, Lucknow, India.
Atul
A
Goel
Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, B.S. 10/1, Sector-10, Jankipuram Extension, Lucknow, India. divya_singh@cdri.res.in.
Divya
D
Singh
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects* / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D040901:Proteomics; D036343:Pterocarpans / Q000494:pharmacology*; D000094162:Phytoalexins
D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D036343:Pterocarpans; C000717248:Hspa5 protein, mouse; C047353:medicarpin; D000094162:Phytoalexins
Apoptosis; GRP78; Medicarpin; Osteoblast; Survival