Vascular smooth muscle LRP6 limits arteriosclerotic calcification in diabetic LDLR-/- mice by restraining noncanonical Wnt signals.

RATIONALE: 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. METHODS AND 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.

Targeted reduction of vascular Msx1 and Msx2 mitigates arteriosclerotic calcification and aortic stiffness in LDLR-deficient mice fed diabetogenic diets.

When fed high-fat diets, male LDLR(-/-) mice develop obesity, hyperlipidemia, hyperglycemia, and arteriosclerotic calcification. An osteogenic Msx-Wnt regulatory program is concomitantly upregulated in the vasculature. To better understand the mechanisms of diabetic arteriosclerosis, we generated SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice, assessing the impact of Msx1+Msx2 gene deletion in vascular myofibroblast and smooth muscle cells. Aortic Msx2 and Msx1 were decreased by 95% and 34% in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) animals versus Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) controls, respectively. Aortic calcium was reduced by 31%, and pulse wave velocity, an index of stiffness, was decreased in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice vs. controls. Fasting blood glucose and lipids did not differ, yet SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) siblings became more obese. Aortic adventitial myofibroblasts from SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice exhibited reduced osteogenic gene expression and mineralizing potential with concomitant reduction in multiple Wnt genes. Sonic hedgehog (Shh) and Sca1, markers of aortic osteogenic progenitors, were also reduced, paralleling a 78% reduction in alkaline phosphatase (TNAP)-positive adventitial myofibroblasts. RNA interference revealed that although Msx1+Msx2 supports TNAP and Wnt7b expression, Msx1 selectively maintains Shh and Msx2 sustains Wnt2, Wnt5a, and Sca1 expression in aortic adventitial myofibroblast cultures. Thus, Msx1 and Msx2 support vascular mineralization by directing the osteogenic programming of aortic progenitors in diabetic arteriosclerosis.

Dkk1 and MSX2-Wnt7b signaling reciprocally regulate the endothelial-mesenchymal transition in aortic endothelial cells.

OBJECTIVE: Endothelial cells (ECs) can undergo an endothelial-mesenchymal transition with tissue fibrosis. Wnt- and Msx2-regulated signals participate in arteriosclerotic fibrosis and calcification. We studied the impact of Wnt7, Msx2, and Dkk1, a Wnt7 antagonist, on endothelial-mesenchymal transition in primary aortic ECs. APPROACH AND RESULTS: Transduction of aortic ECs with vectors expressing Dkk1 suppressed EC differentiation and induced a mineralizing myofibroblast phenotype. Dkk1 suppressed claudin 5, PECAM, cadherin 5 (Cdh5), Tie1, and Tie2. Dkk1 converted the cuboidal cell monolayer into a spindle-shaped multilayer and inhibited EC cord formation. Myofibroblast and osteogenic markers, SM22, type I collagen, Osx, Runx2, and alkaline phosphatase, were upregulated by Dkk1 via activin-like kinase/Smad pathways. Dkk1 increased fibrotic mineralization of aortic ECs cultured under osteogenic conditions--the opposite of mesenchymal cell responses. Msx2 and Wnt7b maintained morphology and upregulated markers of differentiated ECs. Deleting EC Wnt7b with the Cdh5-Cre transgene in Wnt7b(fl/fl);LDLR(-/-) mice upregulated aortic osteogenic genes (Osx, Sox9, Runx2, and Msx2) and nuclear phospho-Smad1/5, and increased collagen and calcium accumulation. CONCLUSIONS: Dkk1 enhances endothelial-mesenchymal transition in aortic ECs, whereas Wnt7b and Msx2 signals preserve EC phenotype. EC responses to Dkk1, Wnt7b, and Msx2 are the opposite of mesenchymal responses, coupling EC phenotypic stability with osteofibrogenic predilection during arteriosclerosis.

TNFR1-activated reactive oxidative species signals up-regulate osteogenic Msx2 programs in aortic myofibroblasts.

In LDLR(-/-) mice fed high-fat diabetogenic diets, osteogenic gene-regulatory programs are ectopically activated in vascular myofibroblasts and smooth muscle cells that promote arteriosclerotic calcium deposition. Msx2-Wnt signaling pathways previously identified as important for craniofacial skeletal development are induced in the vasculature by TNF, a prototypic cytokine mediator of the low-grade systemic inflammation of diabesity. To better understand this biology, we studied TNF actions on Msx2 in aortic myofibroblasts. TNF up-regulated Msx2 mRNA 4-fold within 3 h but did not regulate Msx1. Although IL-1ß could also induce Msx2 expression, TNF-related apoptosis inducing ligand, receptor activator of nuclear factor-κB ligand, and IL-6 were inactive. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (Nox) activity and genetically induced Nox deficiency (p47phox(-/-)) reduced Msx2 induction, indicating contributions of reactive oxygen species (ROS) and redox signaling. Consistent with this, rotenone, an antagonist of mitochondrial complex I, inhibited TNF induction of Msx2 and Nox2, whereas pyruvate, an anapleurotic mitochondrial metabolic substrate, enhanced induction. Moreover, the glutathione peroxidase-mimetic ebselen abrogated this TNF response. Treatment of aortic myofibroblasts with hydrogen peroxide up-regulated Msx2 mRNA, promoter activity, and DNA-protein interactions. In vivo, SM22-TNF transgenic mice exhibit increased aortic Msx2 with no change in Msx1. Dosing SM22-TNF mice with either 20 ng/g Nox1 + 20 ng/g Nox2 antisense oligonucleotides or low-dose rotenone reduced arterial Msx2 expression. Aortic myofibroblasts from TNFR1(-/-) mice expressed levels of Msx2 that were 5% that of wild-type and were not inducible by TNF. Wnt7b and active ß-catenin levels were also reduced. By contrast, TNF-inducible Msx2 expression was not reduced in TNFR2(-/-) cells. Finally, when cultured under mineralizing conditions, TNFR1(-/-) aortic myofibroblasts exhibited reduced calcification compared with wild-type and TNFR2(-/-) cells. Thus, ROS metabolism contributes to TNF induction of Msx2 and procalcific responses in myofibroblasts via TNFR1. Strategies that reduce vascular Nox- or mitochondrially activated ROS signals may prove useful in mitigating arteriosclerotic calcification.

Vascular calcification and aortic fibrosis: a bifunctional role for osteopontin in diabetic arteriosclerosis.

OBJECTIVE: Calcification and fibrosis reduce vascular compliance in arteriosclerosis. To better understand the role of osteopontin (OPN), a multifunctional protein upregulated in diabetic arteries, we evaluated contributions of OPN in male low-density lipoprotein receptor (LDLR)-/- mice fed a high-fat diet. METHODS AND RESULTS: OPN had no impact on high-fat diet-induced hyperglycemia, dyslipidemia, or body composition. However, OPN-/-;LDLR-/- mice exhibited an altered time-course of aortic calcium accrual-reduced during initiation but increased with progression-versus OPN+/+;LDLR-/- controls. Collagen accumulation, chondroid metaplasia, and mural thickness were increased in aortas of OPN-/-;LDLR-/- mice. Aortic compliance was concomitantly reduced. Vascular reexpression of OPN (SM22-OPN transgene) reduced aortic Col2A1 and medial chondroid metaplasia but did not affect atherosclerotic calcification, Col1A1 expression, collagen accumulation, or arterial stiffness. Dosing with the proinflammatory OPN fragment SVVYGLR upregulated aortic Wnt and osteogenic gene expression, increased aortic ß-catenin, and restored early-phase aortic calcification in OPN-/-;LDLR-/- mice. CONCLUSIONS: OPN exerts stage-specific roles in arteriosclerosis in LDLR-/- mice. Actions phenocopied by the OPN metabolite SVVYGLR promote osteogenic calcification processes with disease initiation. OPN limits vascular chondroid metaplasia, endochondral mineralization, and collagen accumulation with progression. Complete deficiency yields a net increase in arteriosclerotic disease, reducing aortic compliance and conduit vessel function in LDLR-/- mice.

Activation of vascular smooth muscle parathyroid hormone receptor inhibits Wnt/beta-catenin signaling and aortic fibrosis in diabetic arteriosclerosis.

RATIONALE: Vascular fibrosis and calcification contribute to diabetic arteriosclerosis, impairing Windkessel physiology necessary for distal tissue perfusion. Wnt family members, upregulated in arteries by the low-grade inflammation of "diabesity," stimulate type I collagen expression and osteogenic mineralization of mesenchymal progenitors via beta-catenin. Conversely, parathyroid hormone (PTH) inhibits aortic calcification in low-density lipoprotein receptor (LDLR)-deficient mice fed high fat diabetogenic diets (HFD). OBJECTIVE: We sought to determine the impact of vascular PTH receptor (PTH1R) activity on arteriosclerotic Wnt/beta-catenin signaling in vitro and in vivo. We generated SM-caPTH1R transgenic mice, a model in which the constitutively active PTH1R variant H223R (caPTH1R) is expressed only in the vasculature. METHODS AND RESULTS: The caPTH1R inhibited Wnt/beta-catenin signaling, collagen production, and vascular smooth muscle cell proliferation and calcification in vitro. Transgenic SM-caPTH1R;LDLR(+/-) mice fed HFD develop diabesity, with no improvements in fasting serum glucose, cholesterol, weight, body composition, or bone mass versus LDLR(+/-) siblings. SM-caPTH1R downregulated aortic Col1A1, Runx2, and Nox1 expression without altering TNF, Msx2, Wnt7a/b, or Nox4. The SM-caPTH1R transgene decreased aortic beta-catenin protein accumulation and signaling in diabetic LDLR(+/-) mice. Levels of aortic superoxide (a precursor of peroxide that activates pro-matrix metalloproteinase 9 and osteogenic signaling in vascular smooth muscle cells) were suppressed by the SM-caPTH1R transgene. Aortic calcification, collagen accumulation, and wall thickness were concomitantly reduced, enhancing vessel distensibility. CONCLUSIONS: Cell-autonomous vascular smooth muscle cell PTH1R activity inhibits arteriosclerotic Wnt/beta-catenin signaling and reduces vascular oxidative stress, thus limiting aortic type I collagen and calcium accrual in diabetic LDLR-deficient mice.

Msx2 exerts bone anabolism via canonical Wnt signaling.

Msx2 is a homeodomain transcription factor first identified in craniofacial bone and human femoral osteoblasts. We hypothesized that Msx2 might activate skeletal Wnt signaling. Therefore, we analyzed the effects of CMV-Msx2 transgene (Msx2Tg) expression on skeletal physiology and composition. Skeletal Msx2 expression was increased 2-3-fold by Msx2Tg, with expanded protein accumulation in marrow, secondary ossification centers, and periosteum. Microcomputed tomography established increased bone volume in Msx2Tg mice, with increased numbers of plate-like trabeculae. Histomorphometry revealed increased bone formation in Msx2Tg mice versus non-Tg siblings, arising from increased osteoblast numbers. While decreasing adipogenesis, Msx2Tg increased osteogenic differentiation via mechanisms inhibited by Dkk1, an antagonist of Wnt receptors LRP5 and LRP6. Bone from Msx2Tg mice elaborated higher levels of Wnt7 canonical agonists, with diminished Dkk1, changes that augment canonical signaling. Analysis of non-Tg and Msx2Tg siblings possessing the TOPGAL reporter confirmed this; Msx2Tg up-regulated skeletal beta-galactosidase expression (p

Vascular Bmp Msx2 Wnt signaling and oxidative stress in arterial calcification.

Studies of fracture repair have revealed that paracrine endothelial-mesenchymal interactions direct bone formation that restores osseous integrity. Angiogenic growth factors and specific members of the bone morphogenetic protein (BMP) family mediate these interactions. Recently, these same signals have been shown to be critical in the vascular pathobiology of hypertension, diabetes, and atherosclerosis. In the arterial vasculature, mechanical and inflammatory redox signals, characteristic of hypertension and diabetes have emerged as a secretagogues for BMP production-with downstream activation of endothelial NADPH oxidases (Nox). Preliminary data now indicate that the paracrine signals provided by BMP and reactive oxygen species augment aortic myofibroblast Msx2-Wnt signaling and matrix turnover. The net mural response to these stimuli promotes osteogenic differentiation of calcifying vascular cells, moreover, oxidation of vascular LDL cholesterol generates oxysterols that trigger Runx2 activity via hedgehog pathways. Thus, BMP, Wnt, and hedgehog gene expression programs-osteogenic pathways highly familiar to the bone biologist-are elaborated in the arterial vasculature via redox-regulated mechanisms. In the brief review, we recount mounting evidence that points to oxidative stress as a major contributor to the pathobiology of diabetic arterial calcification.

Aortic Msx2-Wnt calcification cascade is regulated by TNF-alpha-dependent signals in diabetic Ldlr-/- mice.

OBJECTIVE: Aortic calcification is prevalent in type II diabetes (T2DM), enhancing morbidity and tracking metabolic syndrome parameters. Ldlr(-/-) mice fed high-fat "Westernized" diets (HFD) accumulate aortic calcium primarily in the tunica media, mediated via osteogenic morphogens and transcriptional programs that induce aortic alkaline phosphatase (ALP). Because elevated TNF-alpha is characteristic of obesity with T2DM, we examined contributions of this inflammatory cytokine. METHODS AND RESULTS: HFD promoted obesity, hyperglycemia, and hyperlipidemia, and upregulated serum TNF-alpha in Ldlr(-/-) mice. Serum haptoglobin (inflammatory marker) was increased along with aortic expression of BMP2, Msx2, Wnt3a, and Wnt7a. Dosing with the TNF-alpha neutralizing antibody infliximab did not reduce obesity, hypercholesterolemia, or hyperglycemia; however, haptoglobin, aortic BMP2, Msx2, Wnt3a, and Wnt7a and aortic calcium accumulation were downregulated by infliximab. Mice with vascular TNF-alpha augmented by a transgene (SM22-TNFalphaTg) driven from the SM22 promoter upregulated aortic Msx2, Wnt3a, and Wnt7a. Furthermore, SM22-TNFalphaTg;TOPGAL mice exhibited greater aortic beta-galactosidase reporter staining versus TOPGAL sibs, indicating enhanced mural Wnt signaling. In aortic myofibroblast cultures, TNF-alpha upregulated Msx2, Wnt3a, Wnt7a, and ALP. ALP induction was inhibited by Dkk1, an antagonist of paracrine Wnt actions. CONCLUSIONS: TNF-alpha promote aortic Msx2-Wnt programs that contribute to aortic calcium accumulation in T2DM.

Osteogenic regulation of vascular calcification.

Vascular calcification increasingly afflicts our aging and dysmetabolic population, predisposing patients to cardiovascular mortality and lower extremity amputation. Active osteogenic processes are evident in most histoanatomic variants, including elaboration of BMP2-Msx2 signals required for craniofacial bone formation. We developed an animal model of diet-induced diabetes, dyslipidemia, and vascular calcification. High-fat diets promote vascular calcification in male low-density lipoprotein receptor (LDLR)-deficient mice, with concomitant upregulation of aortic BMP2 and Msx2 gene expression. We wished to test if Msx2 exerts pro-calcific actions during vascular calcification, as it does in craniofacial bone. We studied CMV-Msx2Tg+;LDLR+ transgenic mice (C57Bl/6), a model previously demonstrated to recapitulate features of Msx2 signaling during craniosynostosis. After 16 weeks of fatty diets, vascular calcification was studied in CMV-Msx2Tg+ versus nontransgenic sibs. Only CMV-Msx2Tg+ mice fed high-fat diets exhibited vascular calcium accumulation by alizarin red staining, noted in the tunica media of coronary arteries and the aorta. Gene expression studies revealed that while Msx2 was expressed primarily in adventitial cells, alkaline phosphatase (ALP) expression and calcification occurred primarily in the tunica media. Msx2 promotes the elaboration of a pro-osteogenic milieu by upregulating expression of Wingless type (Wnt) ligands while downregulating the canonical antagonist, Dickkopf (Dkk1). Msx2 upregulates aortic Wnt signaling in vivo, revealed by the analysis of TOPGAL+ (Wnt reporter) versus CMV-Msx2Tg+; TOPGAL+ mice. Aortic Msx2 exerts pro-osteogenic signaling in vivo and in vitro, mediated in part via the enhancement of paracrine Wnt signaling. Strategies that selectively inhibit aortic Msx2-Wnt cascades may help diminish the initiation and progression of diabetic vascular disease.

An osteopontin-NADPH oxidase signaling cascade promotes pro-matrix metalloproteinase 9 activation in aortic mesenchymal cells.

Osteopontin (OPN) is a cytokine upregulated in diabetic vascular disease. To better understand its role in vascular remodeling, we assessed how OPN controls metalloproteinase (MMP) activation in aortic adventitial myofibroblasts (AMFs) and A7r5 vascular smooth muscle cells (VSMCs). By zymography, OPN and tumor necrosis factor (TNF)-alpha preferentially upregulate pro-matrix metalloproteinase 9 (pro-MMP9) activity. TNF-alpha upregulated pro-MMP9 in AMFs isolated from wild-type (OPN(+/+)) mice, but pro-MMP9 induction was abrogated in AMFs from OPN(-/-) mice. OPN treatment of VSMCs enhanced pro-MMP9 activity, and TNF-alpha induction of pro-MMP9 was inhibited by anti-OPN antibody and apocynin. Superoxide and the oxylipid product 8-isoprostaglandin F(2) alpha-isoprostane (8-IsoP) were increased by OPN treatment, and anti-OPN antibody suppressed 8-IsoP production. Like OPN and TNF-alpha, 8-IsoP preferentially activated pro-MMP9. Superoxide, 8-IsoP, and NADPH oxidase 2 (Nox2) subunits were reduced in OPN(-/-) AMFs. Treatment of A7r5 VSMCs with OPN upregulated NADPH oxidase subunit accumulation. OPN structure/function studies mapped these activities to the SVVYGLR heptapeptide motif in the thrombin-liberated human OPN N-terminal domain (SLAYGLR in mouse OPN). Treatment of aortic VSMCs with SVVYGLR upregulated pro-MMP9 activity and restored TNF-alpha activation of pro-MMP9 in OPN(-/-) AMFs. Injection of OPN-deficient OPN(+/-) mice with SVVYGLR peptide upregulated pro-MMP9 activity, 8-IsoP levels, and Nox2 protein levels in aorta and increased panmural superoxide production (dihydroethidium staining). At equivalent hyperglycemia and dyslipidemia, 8-IsoP levels and aortic pro-MMP9 were reduced with complete OPN deficiency in a model of diet-induced diabetes, achieved by comparing OPN(-/-)/LDLR(-/-) versus OPN(+/-)/LDLR(-/-) siblings. Thus, OPN provides a paracrine signal that augments vascular pro-MMP9 activity, mediated in part via superoxide generation and oxylipid formation.

Molecular mechanisms of vascular calcification: lessons learned from the aorta.

Vascular calcification increasingly afflicts our aging and dysmetabolic population. Once considered a passive process, it has emerged as an actively regulated form of calcified tissue metabolism, resembling the mineralization of endochondral and membranous bone. Executive cell types familiar to bone biologists, osteoblasts, chondrocytes, and osteoclasts, are seen in calcifying macrovascular specimens. Lipidaceous matrix vesicles, with biochemical and ultrastructural "signatures" of skeletal matrix vesicles, nucleate vascular mineralization in diabetes, dyslipidemia, and uremia. Skeletal morphogens (bone morphogenetic protein-2 (BMP) and BMP4 and Wnts) divert aortic mesoangioblasts, mural pericytes (calcifying vascular cells), or valve myofibroblasts to osteogenic fates. Paracrine signals provided by these molecules mimic the epithelial-mesenchymal interactions that induce skeletal development. Vascular expression of pro-osteogenic morphogens is entrained to physiological stimuli that promote calcification. Inflammation, shear, oxidative stress, hyperphosphatemia, and elastinolysis provide stimuli that: (1) promote vascular BMP2/4 signaling and matrix remodeling; and (2) compromise vascular defenses that limit calcium deposition, inhibit osteo/chondrogenic trans-differentiation, and enhance matrix vesicle clearance. In this review, we discuss the biology of vascular calcification. We highlight how aortic fibrofatty tissue expansion (adventitia, valve interstitium), the adventitial-medial vasa, vascular matrix, and matrix vesicle metabolism contribute to the regulation of aortic calcium deposition, with greatest emphasis placed on diabetic vascular disease.

Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals.

In diabetic LDLR-/- mice, an ectopic BMP2-Msx2 gene regulatory program is upregulated in association with vascular calcification. We verified the procalcific actions of aortic Msx2 expression in vivo. CMV-Msx2 transgenic (CMV-Msx2Tg(+)) mice expressed 3-fold higher levels of aortic Msx2 than nontransgenic littermates. On high-fat diets, CMV-Msx2Tg(+) mice exhibited marked cardiovascular calcification involving aortic and coronary tunica media. This corresponded to regions of Msx2 immunoreactivity in adjacent adventitial myofibroblasts, suggesting a potential paracrine osteogenic signal. To better understand Msx2-regulated calcification, we studied actions in 10T1/2 cells. We found that conditioned media from Msx2-transduced 10T1/2 cells (Msx2-CM) is both pro-osteogenic and adipostatic; these features are characteristic of Wnt signaling. Msx2-CM stimulated Wnt-dependent TCF/LEF transcription, and Msx2-transduced cells exhibited increased nuclear beta-catenin localization with concomitant alkaline phosphatase induction. Msx2 upregulated Wnt3a and Wnt7a but downregulated expression of the canonical inhibitor Dkk1. Dkk1 treatment reversed osteogenic and adipostatic actions of Msx2. Teriparatide, a PTH1R agonist that inhibits murine vascular calcification, suppressed vascular BMP2-Msx2-Wnt signaling. Analyses of CMV-Msx2Tg(+) mice confirmed that Msx2 suppresses aortic Dkk1 and upregulates vascular Wnts; moreover, TOPGAL(+) (Wnt reporter); CMV-Msx2Tg(+) mice exhibited augmented aortic LacZ expression. Thus, Msx2-expressing cells elaborated an osteogenic milieu that promotes vascular calcification in part via paracrine Wnt signals.

Teriparatide (human parathyroid hormone (1-34)) inhibits osteogenic vascular calcification in diabetic low density lipoprotein receptor-deficient mice.

Cardiovascular calcification is a common consequence of diabetes. High fat diets induce diabetes and arterial calcification in male low density lipoprotein receptor (LDLR) -/- mice; calcification occurs via Msx2 signaling that promotes the osteogenic differentiation of arterial myofibroblasts. We studied regulation of arterial osteogenesis by human parathyroid hormone (PTH) (1-34) (also called teriparatide) in LDLR -/- mice fed diabetogenic diets for 4 weeks. LDLR -/- mice were treated with vehicle or 0.4 mg/kg of PTH(1-34) subcutaneously five times/week. Gene expression was determined from single aortas and hind limb RNA by fluorescence reverse transcription-PCR. Valve calcification was determined by histological staining of cardiac sections using image analysis to quantify valve leaflet mineralization. PTH(1-34) increased bone mineral content (by dual energy x-ray absorptiometry) in LDLR -/- mice, with induction of osseous osteopontin (OPN) expression and serum OPN levels (>150 nM); PTH(1-34) did not significantly change serum glucose, lipids, body weight, or fat mass. PTH(1-34) suppressed aortic OPN and Msx2 expression >50% and decreased cardiac valve calcification 80% (8.3 +/- 1.5% versus 1.4 +/- 0.5%; p < 0.001). Of the known circulating regulators of vascular calcification (OPN, osteoprotegerin, and leptin), PTH(1-34) regulated only serum OPN. We therefore studied actions of PTH(1-34) and OPN in vitro on cells induced to mineralize with Msx2. OPN (5-50 nM) reversed Msx2-induced mineralization. PTH(1-34) inhibited mineralization by 40% and down-regulated Msx2 in aortic myofibroblasts. PTH(1-34) inhibits vascular calcification and aortic osteogenic differentiation via direct actions and potentially via circulating OPN. PTH(1-34) exerts beneficial actions at early stages of macrovascular disease responses to diabetes and dyslipidemia.

MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors.

In the aorta, diabetes activates an osteogenic program that includes expression of bone morphogenetic protein-2 (BMP2) and the osteoblast homeoprotein Msx2. To evaluate BMP2-Msx2 signaling in vascular calcification, we studied primary aortic myofibroblasts. These cells express vascular smooth muscle cell (VSMC) markers, respond to BMP2 by up-regulating Msx2, and undergo osteogenic differentiation with BMP2 treatment or transduction with a virus encoding Msx2. The osteoblast factor osterix (Osx) is up-regulated 10-fold by Msx2, but Runx2 mRNA is unchanged; the early osteoblast marker alkaline phosphatase increases 50-fold with mineralized nodule formation enhanced 30-fold. Adipocyte markers are concomitantly suppressed. To better understand Msx2 actions on osteogenesis versus adipogenesis, mechanistic studies were extended to C3H10T1/2 mesenchymal cells. Msx2 enhances osteogenic differentiation in synergy with BMP2. Osteogenic actions depend upon intrinsic Msx2 DNA binding; the gain-of-function variant Msx2(P148H) directs enhanced mineralization, whereas the binding-deficient variant Msx2(T147A) is inactive. Adipogenesis (lipid accumulation, Pparg expression) is inhibited by Msx2. By contrast, suppression of adipogenesis does not require Msx2 DNA binding; inhibition occurs in part via protein-protein interactions with C/EBPalpha that control Pparg transcription. Thus, Msx2 regulates osteogenic versus adipogenic differentiation of aortic myofibroblasts. Myofibroblasts capable of both fates can be diverted to the osteogenic lineage by BMP2-Msx2 signaling and contribute to vascular calcification.

Rat enterocytes secrete SLPs containing alkaline phosphatase and cubilin in response to corn oil feeding.

Surfactant-like particles (SLP) are unilamellar secreted membranes associated with the process of lipid absorption and isolated previously only from the apical surface of enterocytes. In this paper, the intracellular membrane has been isolated from corn oil-fed animals, identified by its content of the marker protein intestinal alkaline phosphatase (IAP). Another brush-border protein, cubilin, and its anchoring protein megalin have been identified as components of extracellular SLP, but only cubilin is present to any extent in intracellular SLP. During fat absorption, IAP is modestly enriched in intracellular SLP, but full-length cubilin (migrating at 210 kDa in fat-fed mucosal fractions) falls by one-half, although fragments of cubilin are abundant in the intracellular SLP. Both IAP and cubilin colocalize to the same cells during corn oil absorption and colocalize around lipid droplets. This localization is more intense during feeding of corn oil with Pluronic L-81, a detergent that allows uptake of fatty acids and monoglycerides from the lumen, but blocks chylomicron secretion. Confocal microscopy confirms the colocalization of IAP and the ligand for cubilin, intrinsic factor. Possible roles for cubilin in intracellular SLP include facilitating movement of the lipid droplet through the cell and binding to the basolateral membrane before reverse endocytosis.

Osteopontin transcription in aortic vascular smooth muscle cells is controlled by glucose-regulated upstream stimulatory factor and activator protein-1 activities.

The expression of the matrix cytokine osteopontin (OPN) is up-regulated in aortic vascular smooth muscle cells (VSMCs) by diabetes. OPN expression in cultured VSMCs is reciprocally regulated by glucose and 2-deoxyglucose (2-DG; inhibitor of cellular glucose metabolism). Systematic analyses of OPN promoter-luciferase reporter constructs identify a CCTCATGAC motif at nucleotides -80 to -72 relative to the initiation site that supports OPN transcription in VSMCs. The region -83 to -45 encompassing this motif confers basal and glucose- and 2-DG-dependent transcription on an unresponsive promoter. Competition and gel mobility supershift assays identify upstream stimulatory factor (USF; USF1:USF2) and activator protein-1 (AP1; c-Fos:c-Jun) in complexes binding the composite CCTCATGAC element. Glucose up-regulates both AP1 and USF binding activities 2-fold in A7r5 cells and selectively up-regulates USF1 protein levels. By contrast, USF (but not AP1) binding activity is suppressed by 2-DG and restored by glucose treatment. Expression of either USF or AP1 activates the proximal OPN promoter in A7r5 VSMCs in part via the CCTCATGAC element. Moreover, glucose stimulates the transactivation functions of c-Fos and USF1, but not c-Jun, in one-hybrid assays. Mannitol does not regulate binding, transactivation functions, USF1 protein accumulation, or OPN transcription. Thus, OPN gene transcription is regulated by USF and AP1 in aortic VSMCs, entrained to changes in cellular glucose metabolism.

Regulation of osteocalcin gene expression by a novel Ku antigen transcription factor complex.

We previously described an osteocalcin (OC) fibroblast growth factor (FGF) response element (FRE) DNA binding activity as a target of Msx2 transcriptional regulation. We now identify Ku70, Ku80, and Tbdn100, a variant of Tubedown-1, as constituents of the purified OCFRE-binding complex. Northern and Western blot analyses demonstrate expression of Ku and Tbdn100 in MC3T3E1 osteoblasts. FGF2 treatment regulates Ku, but not Tbdn100, protein accumulation. Gel supershift studies confirm sequence-specific DNA binding of Ku in the OCFRE complex; chromatin immunoprecipitation assays confirm association of Ku and Tbdn100 with the endogenous OC promoter. In the promoter region -154 to -113, the OCFRE is juxtaposed to OSE2, an osteoblast-specific element that binds Runx2 (Osf2, Cbfa1). Expression of the Ku.Tbdn100 complex up-regulates both the basal and Runx2-dependent transcription driven by this 42-bp OC promoter element, reconstituted in CV-1 cells. Synergistic transactivation occurs in the presence of activated FGF receptor 2 signaling. Msx2 suppresses Ku- and Runx2-dependent transcription; suppression is dependent upon the Msx2 homeodomain NH(2)-terminal arm and extension. Pull-down assays confirm physical interactions between Ku and these co-regulatory transcription factors, consistent with the functional interactions identified. Finally, cultured Ku70 -/- calvarial cells exhibit a profound, selective deficiency in OC expression as compared with wild-type calvarial cells, confirming the biochemical data showing a role for Ku in OC transcription. In toto, these data indicate that a novel Ku antigen complex assembles on the OC promoter, functioning in concert with Msx2 and Runx2 to regulate OC gene expression.

Analysis of human cellular retinol-binding protein II promoter during enterocyte differentiation.

Cellular retinol binding protein II (CRBP II) is a vitamin A-binding protein that is expressed specifically in small intestinal villus absorptive cells. Previous studies have shown that retinoic acid upregulates endogenous human CRBP II gene expression in differentiated Caco-2 cells. To better characterize the regulation of human CRBP II expression, we analyzed the ability of receptor-selective agonists to enhance transcription from the 5'-upstream flanking region of the human CRBP II gene. Stable transfection experiments showed that the proximal 2.8-kb region of the human CRBP II gene is sufficient for retinoic acid inducibility in differentiated Caco-2 cells. However, direct sequence analysis and transient transfection experiments indicate that, unlike the rat CRBP II promoter, the human CRBP II promoter is not a direct retinoid X receptor target. The results indicate that the retinoic acid responsiveness of the human CRBP II promoter is mediated by an indirect mechanism and that this mechanism is associated with enterocyte differentiation.