Oxidized Lipids Attenuate Osteogenic Potential Of Bone Marrow Stromal Cells By Binding To LRP6

BackgroundConsiderable epidemiological evidence has demonstrated the close correlation of osteoporosis to hyperlipidemia.Specifically,63%of osteoporotic patients have hyperlipidemia.Considerable epidemiological evidence has demonstrated the close correlation of osteoporosis to hyperlipidemia.Serum lipid levels negatively correlate with whole-body bone mineral content(BMC),bone mineral density(BMD)and mass.People with high low-density lipoprotein(LDL)concentrations are also more likely to suffer a non-vertebral fracture.In animal models,diet-induced hyperlipidemia is associated with a reduction in BMD and BMC in both mice and canines.Thus,delineating the mechanisms involved in bone loss in hyperlipidemia will help to develop new strategies to simultaneously treat both disorders.ObjectiiveAim 1:Determine the.major cellular target of oxidized-lipids in bone or bone marrow.Aim 2:Determine the molecular targert that mediates the adverse effect of oxidized-lipids in bone marrow.Methods1.Construct mice animal model of hyperlipidemia and examine its effect on bone phenotypeWe took advantage of the male wild type C57BL/6 and LDLR knock out mice,which were started on a high fat diet(HFD;Western high fat diets)for 3 months at 3 months of age.Also,half of each group was given chow food diet to compare effects of the high fat diet versus chow food diet.At the end of the experiment,animals were euthanized;sera,tissues,bone marrow supernatant and bone marrow BMSCs were collected.We examined whether HFD would lead to hyperlipidemia by serum level of total cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL),high density lipoprotein(HDL)and malondialdehyde(MDA).Then,we assessed the bone phenotype by micro-CT and histomorhometric analysis.At last,we isolated the BMSCs from bone marrow and cultured with osteogenic differentiation medium in vitro to assess the function of BMSCs by CFU-F,CFU-ob,Alizarin red staining and qRT-PCR.2.Determine the major cellular target of oxidized-lipids in bone or bone marrowMice were i.v.injected with Dil-oxLDL or Dil-nativeLDL 40ug per mouse,4 hours before euthanasia.Femurs were immediately collected,and then operated frozen slice and immunohistochemical statning;cells were isolated from the bone marrow for FACS and osteogenic function analysis.Moreover,BMSCs treated with oxidized lpids in vitro to examine whether it would decreased osteoblast differentiation capacity or blunted responses to BMP,Wnt,and PTH.3.Deternine the molecular targert that mediates the adverse effect of oxidized-lipids in bone marrowWe examined whether LRP6 on the plasma membrane of BMSCs can directly bind oxlipids.MQuse bone marrow MSCs were incubated with DiI-nLDL or DiI-oxLDL for different time periods and then analyzed visually and by flow ceytometry.To further confirn the binding of ox-lipids to LRP6 and identify the interaction domain(s),we employed the established protein lipid overlay assay(PLO),in which the in vitro binding of the same amount of nLDL and oxLDL to full length LRP6 or the intracellular domain of LRP6(LRP6C)were detected.To examine whether oxLDL-induced LRP6 endoctyosis is through caveolin-or clathrin-mediated pathways,we studied the effects of pharmacological inhibitors of clathrin-mediated and caveolae-mediated endocytosis on LRP6 expression on the BMSCs surface after the cells were stimulated with nLDL or oxLDL.At last we increased LRP6 on Cell surface with MESD trasfection or MDC to block endocytosis pathway and examined whether it would restore the declined osteogenic capacity of BMSCs by Oxidized Lipids.Results1.Osteogenic capacity of bone marrow BMSCs is impairetd in hyperlipidemic miceWe took advantage of the wild type C57L/B6 and Idlr-/-mice,which develop different degrees of hyperlipidemia,bone loss,and increased lipid oxidation after fed Western high fat diets(HFD)for 3 months.The mice are named "WT-H" and "ldlr-/-H",respectively.The serum level of malonaldehyde(MDA),a stable end product of lipid peroxidation,increased in WT-H mice and the elevation was much more dramatic in ldlr-/-H mice relative to their littermates fed standard chow diets(the mice are named "WT-C" and "ldlr-/-C",respectively).Micro-CT analysis showed that both WT-H and Idlr-/-H mice had low bone mass with reduced trabecular bone volume,thickness,and number relative to their littermates fed chow diet.The trabecular spacing was increased in WT-H mice but unchanged in ldlr-/-H mice compared with that in WT-C and ldlr-/-C mice,respectively.The numbers of bone surface osteocalcin+osteoblasts were also reduced in WT-H mice and ldlr-/-H mice,and the reduction was much more profound in ldlr-/-H mice.The numbers of TRAP+osteoclasts were not significantly changed after HFD treatment in both wild type C57L/B6 and ldlr-/-mice relative to the chow diet-treated mice.Consistently,bone histomorphometry analysis showed reduced osteoblast number and surface,but unchanged osteoclast number and surface in the mice treated with HFD compared with those treated with chow diet.We then assessed the functional changes in BMSCs collected from the bone marrow of the mice.We first measured the level of ROS in bone marrow MSCs.BMSCs isolated from WT-H and ldir-/-H mice had relatively normal CFU-F but different degrees of reduction in CFU-Ob relative to CHD fed mice,indicating a decline in osteogenic potential of the cells but sustained colony-forming capacity.Reduced osteoblast differentiation of BMSCs from HFD fed mice was also observed after the cells were incubated with osteogenic medium and detected by Alizarin red staining.Therefore,bone marrow SSCs in WT-H and ldlr-/-H mice showed different degrees of increase in lipid oxidation and decrease in osteoblast differentiation capacity.2.BMSCs is a major cellular target of oxidized lipids in bone marrow in miceWe observed BMSCs in bone marrow could specificly uptake the oxLDL but not the nativeLDL;macrophage did not attend the oxLDL clearance in bone marrow.Furthermore,we examined the functional changes of the cultured BMSCs treated with oxLDL or POVPC,an individual oxlipid.50 μg/ml oxLDL or POVPC significantly reduced the colony numbers of MSCs in CFU-Ob assays relative to vehicle-or same amount of nLDL-treated cells.However,none of the treatment changed the colony numbers in CFU-F assays,indicating that oxlipids may have marginal effects on MSC growth/proliferation.The adverse effects of oxLDL and POVPC on osteoblast differentiation of MSCs were further confirmed by performing ALP and Alizarin Red staining and measuring osteoblast differentiation markers Runx2 and osteocalc’in mRNA levels after the cells incubated with osteogenic medium.BMPs,wnts,and PTH are known factors to stimulate osteoblastie bone formation.We examined the response of BMSCs to these factors with or without the treatment of oxLDL.OxLDL almost abolished Smadl phosphorylation,β-catenin accumulation,and cAMP production in MSCs stimulated by BMP2,wnt3a and PTH,respectively.Runx2 and Osterix expression stimulated by BMP4 was also abolished by oxLDL.3.Oxidized lipids bind to LRP6 on plasma membrane of BMSCs and induce LRP6 endocytosis,this progress attenuate the osteogenic function of BMSCs.Bone marrow CD45-lepR+ cells(non-hematopoietic stem cells containing BMSCs)isolated from ldlr-/-H mice showed dramatically reduced cell surface LRP6 levels relative to the same population from ldlr-/-C mice detected by flow cytometry analysis.While the total LRP6 protein expression were no significant different.The results suggest that LRP6 on plasma membrane of BMSCs may sense the oxidized lipids in bone environment and undergo endocytosis.Mouse bone marrow MSCs were incubated with DiI-nLDL or DiI-oxLDL for different time periods and then analyzed visually and by flow cytometry.While Dil-nLDL was almost washed off,Dil-oxLDL was retained on the membrane surface of MSCs and colocalized with LRP6.Consistently,FACS analysis showed much more DiI-oxLDL than DiI-nLDL uptake by the cells,indicating that LRP6 specifically binds oxLDL but not nLDL.OxLDL but not nLDL has strong interaction with full length LRP6.Neither nLDL nor oxLDL interacted with LRP6C,indicating that oxLDL specifically binds to the extracellular domain of LRP6.To further localize the domain(s)within LRP6 that mediate the oxLDL-LRP6 interaction,we purified GST fusion proteins containing the four individual YWTD propeller regions of LRP6 extracellular domain(E1,E2,E3,and E4)from E.coli,then detected the binding of LDL,oxLDL,and POVPC with each purified GST fusion protein by PLO assay.OxLDL had strong interaction,and POVPC had weak interaction with GST-E1.The interaction of nLDL with any of the four LRP6 regions was not detected.Therefore,the first YWTD-propeller region in LRP6(E1)is the oxlipids binding domain.Consistent with the flow cytometry result,BMSCs incubated with oxLDL but not nLDL had reduced LRP6 expression on plasma membrane.Imporrtantly,the reduction in LRP6 expression on plasma membrane induced by oxLDL was antagonized by clathrin-dependent endocytosis inhibitor monodansylcadaverine(MDC)but not by Filipin Ⅲ.The results suggest that Ox-lipid-induced endocytosis of LRP6 is dependent on clathrin-but not caveolin-mediated pathway.And increased LRP6 on cell surface restored the declined osteogenic capacity of bone marrow MSCs by oxidized lipids.Conclusion1.Mice fed with high fat diet(western diet)3 months could lead to the hyperlipidemia;it is much easier in LDLR KO mice;Hyperlipidemia could lead to osteoporosis in mice.2.BMSCs is a major cellular target of oxidized lipids in bone marrow in mice.3.Oxidized lipids bind to LRP6 on plasma membrane of BMSCs and induce LRP6 endocytosis,this progress attenuate the osteogenic function of BMSCs,.