HMGB1 Induces Secretion Of Matrix Vesicles By Macrophages To Enhance Ectopic Mineralization

Objective:Ectopic mineralization(EM)is characterized by inappropriate deposition of calcium phosphate complexes in soft tissues.EM is commonly seen in physiological aging process and several common disorders,including infection,atherosclerosis,diabetes,and chronic renal disease.Based on the mechanisms leading to mineralization,EM is conventionally classified into:(a)Metabolic EM which arises from sustained hyperphosphatemia and/or hypercalcemia causing widespread mineral deposition;(b)Dystrophic EM which represents a response following tissue/organ insults,such as microbial or viral infection,autoimmune diseases and cancer.Extracellular matrix vesicles(MVs)act to initiate the mineralization process leading to physiological and pathological mineralization as following: Firstly,initial formation of hydroxyapatite(HA)in MVs;Secondly,HA crystals grow within MVs until membrane rupture.Once exposed to the extracellular matrix,they act as loci for the formation of new crystals via homologous nucleation.High mobility group box 1(HMGB1)is a nuclear constituent bound to chromatin in almost all eukaryotic cells.HMGB1 acts as a damage associated molecular pattern when released by dying cells or secreted by activated cells.Extracellular HMGB1 has drawn increasing attention with recognition of its role in the pathogenesis of autoimmune and inflammatory diseases.Recent studies have also shown that HMGB1 is a critical mediator of thrombosis.In addition,HMGB1 is known as a bone-active cytokine involving in both bone remodeling and EM pathogenesis.There is evidence that HMGB1 accumulates extracellularly in the area associated with macrophages infiltration and calcification in calcific aortic valve stenosis.Moreover,HMGB1 directly mediates the osteoblastic differentiation of human dental pulp cells and calcification process of vascular smooth muscle cells(VSMC)in patients with diabetes.However,it remains unknown whether HMGB1 is involved in MVs secretion that initiates mineral deposition of EM.Thus,we have studied the role of HMGB1 in formation and secretion of MVs leading to EM and explored the signaling mechanism.Methods:1.Both murine macrophage-like cells RAW264.7 and mouse peritoneal macrophages were cultured for experiments.CCK-8 assay was applied to evaluate the cell viability of HMGB1 on RAW264.7 cells.To test the effects of HMGB1 on mineralization of RAW264.7 cells,cells were treated with HMGB1 under high Ca/Pi conditions(Ca:2mM,Pi:2.7mM)and calcium deposition was determined by Alizarin Red staining and von Kossa staining.Effects of HMGB1 on the mRNA expression of the mineralization-related markers were examined by real-time quantitative PCR.Effects of HMGB1 on the protein expression of the Runx2 and OPN were detected by immunofluorescence staining.2.Transmission electron microscopic images show MVs with no crystalline hydroxyapatite,with early mineralized electron dense materials near the inner leaflet and with matured crystal-like structures in the vesicle interior.Size distribution of MVs was determined by dynamic light scattering method.Effect of HMGB1 on secretion of MVs from RAW264.7 or mouse peritoneal macrophages was determined by flow cytometry.TNAP activity,considered as a marker of MVs maturation was also determined.Control-MVs or HMGB1-MVs from RAW264.7 cells initiated mineralization in type I collagen-coated dishes was determined by both Alizarin Red staining and von Kossa staining.Control-MVs and HMGB1-MVs were subcutaneously injected in either side of the mice dorsal area respectively for 7 days.MVs induced mineral deposits in the subcutaneous tissues were determined by histological analysis and von Kossa staining.3.RAW264.7 cells were treated with 800 ng/ml HMGB1 for 24 h.The activity of neutral sphingomyelinase(nSMase)and acid sphingomyelinase(aSMase)of cell homogenate were examined using a sphingomyelinase assay kit.Effects of inhibition of nSMase2 with GW4869 on HMGB1-induced MVs secretion and mineralization were also determined.RAW264.7 cells were treated with 800 ng/ml HMGB1 and the phosphorylated and total forms of ERK1/2,p38 MAPK and JNK were assessed by western blot analysis.Effects of inhibitors for ERK1/2(PD98059),p38MAPK(SB203580)or JNK(SP600125)on the activity of nSMase,MVs secretion and mineralization were determined.Real-time PCR analysis of mRNA expression in RAW264.7 cells after knockdown of TLR2,TLR4 or RAGE,respectively.Western blot analysis was performed for the p38 MAPK phosphorylated forms and corresponding non-phosphorylated proteins.The activity of nSMase,MVs secretion and mineralization were also determined.Results:1.HMGB1 enhances mineralization and expression of the mineralization-related markers of macrophages in culture1)HMGB1(0–800 ng/ml)treatment did not affect the viability of RAW264.7 cells2)A significant increase in calcium deposition was observed following HMGB1 treatment at 800 ng/ml under high Ca/Pi conditions(Ca:2mM,Pi:2.7mM)as determined by Alizarin Red staining and von Kossa staining.3)HMGB1(800 ng/ml)also significantly increased mRNA expression of the mineralization-related markers TNAP,Runx2,osteopontin and BMP2.Comparable changes in Runx2 and OPN protein expression were also observed by immunofluorescence staining.2.HMGB1 enhances MVs secretion which initiate mineralization both in vitro and in vivo1)TEM analysis identified MVs derived from HMGB1-treated RAW264.7 cells as membrane-bound vesicles,showing hydroxyapatite nucleation on the inside membrane and within vesicles after incubation in elevated Ca/Pi.2)Particle size analysis revealed that RAW264.7 cells released a population of vesicles with diameter between 50 to 500 nm and there was no effect of HMGB1 on the size-distribution of MVs.3)Treatment with HMGB1 at 800 ng/ml significantly enhanced MVs secretion as quantified by flow cytometry analysis.4)TNAP activity was higher in HMGB1-induced MVs.The cellular TNAP activity was <10% of that in MVs and was unaffected by HMGB1 treatment.5)In mouse peritoneal macrophages HMGB1 also stimulated production of MVs,TNAP activity in the MVs,and increased mineral deposits in calcifying condition as revealed by Alizarin Red staining and von Kossa staining6)Histological analysis revealed that only HMGB1-MVs induced mineral deposits in the subcutaneous tissues of mice.These deposits were rough in morphology and associated with dermal collagen fibers.3.The mechamism by which HMGB1 provoke MVs secretion from macrophage and subsequent mineralization in calcifying conditions.1)Activity of nSMase,but not aSMase,was upregulated by HMGB1.HMGB1 also enhanced expression of nSMase2 at the mRNA level in RAW264.7 cells.GW4869(nSMase2 inhibitor)dramatically reduced both TNAP activity recovered in MVs pellets and MVs release induced by HMGB1.As expected,these effects were accompanied by an abrogation of mineralization induced by HMGB12)Immunoblotting revealed HMBG1 treatment induced strong phosphorylation of ERK1/2,p38 MAPK and JNK.Our results showed that only SB-239063,but not PD-98059 and SP-600125,effectively prevented nSMase2 activation induced by HMGB1.SB-239063 also suppressed HMGB1-induced TNAP loading into MVs,MVs secretion and subsequent mineralization in elevated Ca/Pi conditions.3)Knockdown of RAGE by siRNA largely abrogated HMGB1-induced phosphorylation of p38 MAPK and activation of nSMase.TLR2 or TLR4 knockdown,however,did not have such effects.In consistent with the effects on p38 MAPK or nSMase,only RAGE-si RNA treatment decreased TNAP activity loading into MVs,prevented MVs secretion and reduced mineralization of RAW264.7 cells in response to HMGB1.Conclusion:Our study revealed a novel mechanism by which HMGB1 induces biomineralization.Specifically,we demonstrate for the first time that HMGB1 induces MVs secretion by macrophages and leads to subsequent mineralization in vitro and in vivo.This is attributable,at least in part,to the activation of signaling pathway involving p38 MAPK and nSMase2 following interaction of HMGB1 with RAGE.Our findings promise further testing of therapeutic interventions to decrease pathological ectopic mineralization by blocking the macrophage derived matrix vesicles secretion induced by HMGB1.