LOX Regulates BMP9 Induced Osteogenic Differentiation Of Pluripotent Stem Cells Through HIF-1α/Wnt/β-Catenin Signaling

Osteoporosis is a kind of common orthopedic disease in clinic,mostly in the elderly and postmenopausal women group morbidity,and fracture is one of the most common complications of osteoporosis.One of the important causes of these diseases is bone loss,and increasing bone mass or inhibiting bone resorption is an effective measure to prevent these diseases.Pluripotent stem cells are a kind of cells with multi-directional differentiation potential and easy to culture,so they are often used as seed cells in the field of regenerative medicine.Bone morphogenetic protein 9(BMP9)is an important member of the BMPs family,which can induce osteogenic differentiation of pluripotent stem cells and the effect is stronger than other members of the BMPs family.Lysyloxidase(LOX)is a kind of copper-dependent amine oxidase,which may play a regulatory role in bone development,but the mechanism is not clear.The aim of this study was to investigate the effect of LOX on osteogenic differentiation of pluripotent stem cells induced by BMP9 and its mechanism.The effects of LOX on osteogenic differentiation of BMP9-induced pluripotent stem cells were determined by histochemical staining and Western blot.Micro-CT scanning and hematoxylin-eosin(he)staining were used to evaluate the heterotopic osteogenesis in nude mice;The effects of LOX and BMP9 on adipose differentiation were detected by oil red O staining and Western blot.Histochemical staining,PCR,Western blot and immunofluorescence were used to explore the mechanism.LOX is expressed in a variety of stem cells,but the expression level in C3H10T1/2cells and 3T3L-1 cells is relatively high.BMP9 could up-regulate the expression of LOX in 3T3L-1 cells.Overexpression of LOX significantly inhibited the effect of BMP9 on ALP activity,Runx2 expression and cell mineralization in 3T3L-1 cells.Inhibition or silencing of LOX promoted BMP9 induction of related osteogenic markers or ectopic osteogenesis,but inhibited BMP9 induction of C/EBP-α and PPAR-γexpression in 3T3L-1 cells.In addition,inhibition of LOX promoted BMP9up-regulation of HIF-1α expression;Overexpression of HIF-1α promoted BMP9-induced osteogenic differentiation of 3T3L-1 cells and promoted BMP9-induced ectopic osteogenesis of 3T3L-1 cells.Silencing HIF-1α could reduce the effect of LOX inhibition on BMP9-induced osteogenic differentiation of 3T3L-1 cells,but overexpression of HIF-1α could partially reverse the inhibitory effect of LOX on osteogenic differentiation.Inhibition of LOX and overexpression of HIF-1α could promote BMP9 to enhance Wnt/β-catenin signaling,and silencing of HIF-1α attenuated the effect of LOX inhibition on BMP9 to enhance Wnt/β-catenin signaling.LOX can inhibit BMP9-induced osteogenic differentiation of pluripotent stem cells,and inhibition of LOX can promote BMP9-induced osteogenic differentiation of pluripotent stem cells.The mechanism may be related to the inhibition of LOX and BMP9 induced adipose differentiation of pluripotent stem cells;At the same time,inhibition of LOX can also upregulate the expression of HIF-1α,thereby enhancing the Wnt/β-catenin signal.