Jian-Su Shao

Conditional inactivation of FGF receptor 2 reveals an essential role for FGF signaling in the regulation of osteoblast function and bone growth

Human craniosynostosis syndromes, resulting from activating or neomorphic mutations in fibroblast growth factor receptor 2 (FGFR2), underscore an essential role for FGFR2 signaling in skeletal development. Embryos harboring homozygous null mutations in FGFR2 die prior to skeletogenesis. To address the role of FGFR2 in normal bone development, a conditional gene deletion approach was adopted. Homologous introduction of cre recombinase into the Dermo1 (Twist2) gene locus resulted in robust expression of CRE in mesenchymal condensations giving rise to both osteoblast and chondrocyte lineages. Inactivation of a floxed Fgfr2 allele with Dermo1-cre resulted in mice with skeletal dwarfism and decreased bone density. Although differentiation of the osteoblast lineage was not disturbed, the proliferation of osteoprogenitors and the anabolic function of mature osteoblasts were severely affected.

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.

Aortic Msx2-Wnt Calcification Cascade Is Regulated by TNF-α–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-&agr; 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-&agr; in Ldlr−/− mice. Serum haptoglobin (inflammatory marker) was increased along with aortic expression of BMP2, Msx2, Wnt3a, and Wnt7a. Dosing with the TNF-&agr; 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-&agr; augmented by a transgene (SM22-TNF&agr;Tg) driven from the SM22 promoter upregulated aortic Msx2, Wnt3a, and Wnt7a. Furthermore, SM22-TNF&agr;Tg;TOPGAL mice exhibited greater aortic &bgr;-galactosidase reporter staining versus TOPGAL sibs, indicating enhanced mural Wnt signaling. In aortic myofibroblast cultures, TNF-&agr; upregulated Msx2, Wnt3a, Wnt7a, and ALP. ALP induction was inhibited by Dkk1, an antagonist of paracrine Wnt actions. Conclusions—TNF-&agr; promote aortic Msx2-Wnt programs that contribute to aortic calcium accumulation in T2DM.

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.

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

Abstract:  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.

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

We previously described anosteocalcin (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 NH2-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.

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 β-galactosidase expression (p ≤ 0.01), along with Wnt7a and Wnt7b, and reduced circulating Dkk1. To better understand molecular mechanisms, we studied C3H10T1/2 osteoprogenitor cells. As in bone, Msx2 increased Wnt7 genes and down-regulated Dkk1, while inducing the osteoblast gene alkaline phosphatase. Msx2-directed RNA interference increased Dkk1 expression and promoter activity, while reducing Wnt7a, Wnt7b, and alkaline phosphatase. Moreover, Msx2 inhibited Dkk1 promoter activity and reduced RNA polymerase association with Dkk1 chromatin. RNA interference-mediated knockdown of Wnt7a, Wnt7b, and LRP6 significantly reduced Msx2-induced alkaline phosphatase. Msx2 exerts bone anabolism in part by reducing Dkk1 expression and enhancing Wnt signaling, thus promoting osteogenic differentiation of skeletal progenitors.

Activation of Vascular Smooth Muscle Parathyroid Hormone Receptor Inhibits Wnt/β-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 &bgr;-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/&bgr;-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/&bgr;-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 &bgr;-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/&bgr;-catenin signaling and reduces vascular oxidative stress, thus limiting aortic type I collagen and calcium accrual in diabetic LDLR-deficient mice.

Osteogenic Regulation of Vascular Calcification

Abstract:  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.

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.