The Mechanism Of Fam134b Deficiency Regulating Osteoclast Differentiation And Function By ER-stress

Objective:Herculoosteolysis is a rare bone-destroying disease involving the distal finger/phalanges.The pathologic features were semilunar defect at the margin of the tuberosity of the digit/toe,accompanied by osteosclerosis.With the increase of age,the patient’s condition deteriorates and ends with amputation,but this cannot fundamentally cure the disease and relieve the pain of the patient.Therefore,it is of great significance to discover the pathogenic mutation and further clarify its mechanism for the early diagnosis and rational treatment of inherited acromelysis.In this study,we studied the effect of FAM134B on osteoclast differentiation and its potential molecular mechanism by starting with a family gene FAM134B,which has never been reported before,in the hope of providing possible therapeutic strategies for the treatment of bone loss diseases.Methods:1.The expression of FAM134B was interfered with in RAW264.7 cells and human peripheral blood CD14⁺monocytes by adenovirus infection or si RNA transfection,respectively,to induce osteoclast differentiation.TRAP staining and bone absorption assay were used to evaluate the osteoclast differentiation and determine the effect of FAM134B deficiency on osteoclast differentiation.Rt-pcr and Western blot were used to detect the expression of osteoclast differentiation related molecules downstream of RANKL pathway.2.Expression of er stress-related molecules in osteoclasts was detected by RT-PCR and Western blot to determine the changes of ER stress pathway.3.Expression of TRPV4 was detected by RT-PCR and Western blot,and GSK2193874 was used to inhibit Ca²⁺influx mediated by TRPV4 to save excessive osteoclast formation and bone resorption.TRAP staining and bone resorption test were used to evaluate osteoclast differentiation.4.The expression of Fam134b was disturbed by adenovirus infection in mouse chondrocyte line ATDC5.Endoplasmic reticulum expansion was observed by transmission electron microscopy and cell apoptosis was detected by flow cytometry.Results:1.The expression of Fam134b was down-regulated in RAW264.7 cells and osteoclast generation was induced.TRAP staining showed that TRAP+multinuclear osteoclast generation was increased,and bone matrix absorption experiment showed that bone absorption capacity was correspondingly enhanced.Western Bolt showed that A/B expression of osteoclast formation related molecules p-JNK,NFATc1 and autophagy key molecule LC3 was increased.2.After FAM134B deficiency,the expression of er stress-related molecule ATF6increased,while IRE1a and PERK expressions did not change significantly.3.TRAP staining of OE-Fam134b group showed that the formation of TRAP+polynuclear osteoclasts was significantly reduced,and bone resorption capacity was also decreased by bone matrix absorption test.4.The expression of TRPV4 was significantly increased in osteoclast induced by FAM134B deletion,and inhibition of Ca²⁺influx mediated by TRPV4 by GSK2193874reduced osteoclast formation and bone resorption capacity in sh-Fam134b group.5.Interference of Fam134b expression in ATDC5 cells,and endoplasmic reticulum expansion was observed by transmission electron microscopy.Flow cytometry showed an increase in early apoptotic cells.Conclusion:1.Fam134b gene plays an important role in the regulation of osteoclast differentiation,and its deletion can lead to increased osteoclast formation and enhanced bone resorption capacity by activating JNK and autophagy.2.In osteoclasts,loss of FAM134B,as an endoplasmic reticulum autophagy receptor,causes ATF6-mediated ER stress,which plays an important role in the differentiation of fam134B-deficient osteoclasts3.Ca²⁺influx mediated by TRPV4 is an important pathway to promote the differentiation of Fam134B-deficient osteoclasts.4.Pharmacological inhibition of TRPV4 with GSK2193874 can rescue the increased osteoclast differentiation caused by FAM134B deficiency,and GSK2193874 is expected to be a therapeutic agent for clinical diseases related to decreased bone mineral density.5.Loss of FAM134B can cause endoplasmic reticulum stress and apoptosis of ATDC5 cells.