title: Vascular smooth muscle LRP6 limits arteriosclerotic calcification in diabetic LDLR-/- mice by restraining noncanonical Wnt signals.
abstract: BACKGROUND
  Wnt signaling regulates key aspects of diabetic vascular disease.
  
  
  OBJECTIVE
  We generated SM22-Cre;LRP6(fl/fl);LDLR(-/-) mice to determine contributions of Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6) in the vascular smooth muscle lineage of male low-density lipoprotein receptor-null mice, a background susceptible to diet (high-fat diet)-induced diabetic arteriosclerosis.
  
  
  RESULTS
  As compared with LRP6(fl/fl);LDLR(-/-) controls, SM22-Cre;LRP6(fl/fl);LDLR(-/-) (LRP6-VKO) siblings exhibited increased aortic calcification on high-fat diet without changes in fasting glucose, lipids, or body composition. Pulse wave velocity (index of arterial stiffness) was also increased. Vascular calcification paralleled enhanced aortic osteochondrogenic programs and circulating osteopontin (OPN), a matricellular regulator of arteriosclerosis. Survey of ligands and Frizzled (Fzd) receptor profiles in LRP6-VKO revealed upregulation of canonical and noncanonical Wnts alongside Fzd10. Fzd10 stimulated noncanonical signaling and OPN promoter activity via an upstream stimulatory factor (USF)-activated cognate inhibited by LRP6. RNA interference revealed that USF1 but not USF2 supports OPN expression in LRP6-VKO vascular smooth muscle lineage, and immunoprecipitation confirmed increased USF1 association with OPN chromatin. ML141, an antagonist of cdc42/Rac1 noncanonical signaling, inhibited USF1 activation, osteochondrogenic programs, alkaline phosphatase, and vascular smooth muscle lineage calcification. Mass spectrometry identified LRP6 binding to protein arginine methyltransferase (PRMT)-1, and nuclear asymmetrical dimethylarginine modification was increased with LRP6-VKO. RNA interference demonstrated that PRMT1 inhibits OPN and TNAP, whereas PRMT4 supports expression. USF1 complexes containing the histone H3 asymmetrically dimethylated on Arg-17 signature of PRMT4 are increased with LRP6-VKO. Jmjd6, a demethylase downregulated with LRP6 deficiency, inhibits OPN and TNAP expression, USF1: histone H3 asymmetrically dimethylated on Arg-17 complex formation, and transactivation.
  
  
  CONCLUSIONS
  LRP6 restrains vascular smooth muscle lineage noncanonical signals that promote osteochondrogenic differentiation, mediated in part via USF1- and arginine methylation-dependent relays.
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Su-Li
authors.initials: SL
authors.lastname: Cheng
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Bindu
authors.initials: B
authors.lastname: Ramachandran
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Abraham
authors.initials: A
authors.lastname: Behrmann
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Jian-Su
authors.initials: JS
authors.lastname: Shao
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Megan
authors.initials: M
authors.lastname: Mead
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Carolyn
authors.initials: C
authors.lastname: Smith
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Karen
authors.initials: K
authors.lastname: Krchma
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Yoanna
authors.initials: Y
authors.lastname: Bello Arredondo
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Attila
authors.initials: A
authors.lastname: Kovacs
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Kapil
authors.initials: K
authors.lastname: Kapoor
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Laurence M
authors.initials: LM
authors.lastname: Brill
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Ranjan
authors.initials: R
authors.lastname: Perera
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.).
authors.forename: Bart O
authors.initials: BO
authors.lastname: Williams
authors.affiliation: From the Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Cardiovascular Pathobiology, Orlando, FL (S.-L.C., B.R., A.B., M.M., C.S., Y.B.A., K.K., L.M.B., R.P., D.A.T.); MD Anderson Cancer Center, Cancer Biology, Houston, TX (J.-S.S.); Washington University, Department of Medicine, St. Louis, MO (K.K., A.K.); and Van Andel Research Institute, Department of Cancer and Cell Biology, Grand Rapids, MI (B.O.W.). dwight.towler@utsouthwestern.edu.
authors.forename: Dwight A
authors.initials: DA
authors.lastname: Towler
mesh_terms: D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001161:Arteriosclerosis / Q000209:etiology / Q000378:metabolism / Q000517:prevention & control*; D002114:Calcinosis / Q000209:etiology / Q000378:metabolism / Q000517:prevention & control*; D003921:Diabetes Mellitus, Experimental / Q000150:complications* / Q000473:pathology; D004041:Dietary Fats / Q000009:adverse effects; D051157:Frizzled Receptors / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology; D006657:Histones / Q000378:metabolism; D060488:Low Density Lipoprotein Receptor-Related Protein-6 / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000503:physiopathology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism* / Q000473:pathology; D053495:Osteopontin / Q000096:biosynthesis / Q000235:genetics; D019899:Paracrine Communication; D025941:Protein Interaction Mapping; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism; D011956:Receptors, Cell Surface; D011973:Receptors, LDL / Q000172:deficiency* / Q000235:genetics; D051720:Upstream Stimulatory Factors / Q000502:physiology; D059289:Vascular Stiffness / Q000502:physiology; D060449:Wnt Signaling Pathway
chemical_list: D004041:Dietary Fats; D051157:Frizzled Receptors; C492388:Fzd10 protein, mouse; D006657:Histones; D060488:Low Density Lipoprotein Receptor-Related Protein-6; C555476:Lrp6 protein, mouse; C498708:Ptdsr protein, mouse; D011956:Receptors, Cell Surface; D011973:Receptors, LDL; C506896:Spp1 protein, mouse; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; D053495:Osteopontin; C018524:N,N-dimethylarginine; D001120:Arginine; C546338:Prmt1 protein, mouse; D011484:Protein-Arginine N-Methyltransferases
keywords: LRP6; USF1; Wnt; arteriosclerosis; signal transduction; type 2 diabetes mellitus; vascular calcification