Su-Li Cheng

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.

Erk is essential for growth, differentiation, integrin expression,and cell function in human osteoblastic cells

Extracellular signal-regulated kinases (Erks), members of the mitogen-activated protein kinase superfamily, play an important role in cell proliferation and differentiation. In this study we employed a dominant negative approach to determine the role of Erks in the regulation of human osteoblastic cell function. Human osteoblastic cells were transduced with a pseudotyped retrovirus encoding either a mutated Erk1 protein with a dominant negative action against both Erk1 and Erk2 (Erk1DN cells) or the LacZ protein (LacZ cells) as a control. Both basal and growth factor-stimulated MAPK activity and cell proliferation were inhibited in Erk1DN cells. Expression of Erk1DN protein suppressed both osteoblast differentiation and matrix mineralization by decreasing alkaline phosphatase activity and the deposition of bone matrix proteins. Cell adhesion to collagen, osteopontin, and vitronectin was decreased in Erk1DN cells as compared with LacZ cells. Cell spreading and migration on these matrices were also inhibited. In Erk1DN cells, expression of αβ1, αvβ3, and αvβ5 integrins on the surface was decreased. Metabolic labeling indicated that the synthesis of these integrins was inhibited in Erk1DN cells. These data suggest that Erks are not only essential for the growth and differentiation of osteoblasts but also are important for osteoblast adhesion, spreading, migration, and integrin expression.

Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein‐2

We have examined the effects of BMP‐2 on the expression of bone matrix proteins in both human bone marrow stromal cells (HBMSC) and human osteoblasts (HOB) and their proliferation and mineralization. Both HBMSC and HOB express BMP‐2/‐4 type I and type II receptors. Treatment of these two cell types with BMP‐2 for 4 weeks in the presence of β‐glycerophosphate and ascorbic acid results in mineralization of their matrix. BMP‐2 increases the mRNA level and activities of alkaline phosphatase and elevates the mRNA levels and protein synthesis of osteopontin, bone sialoprotein, osteocalcin, and α1(I) collagen in both cell types. Whereas the mRNA level of decorin is increased, the mRNA concentration of biglycan is not altered by BMP‐2. No effect on osteonectin is observed. The effect of BMP‐2 on bone matrix protein expression is dose dependent from 25 to 100 ng/ml and is evident after 1–7 days treatment. In the presence of BMP‐2, proliferation of HBMSC and HOB is decreased under either serum‐free condition or in the presence of serum. Thus, BMP‐2 has profound effects on the proliferation, expression of most of the bone matrix proteins and the mineralization of both relatively immature human bone marrow stromal preosteoblasts and mature human osteoblasts. J. Cell. Biochem. 67:386–398, 1997.

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.

Induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene in a rabbit spinal fusion model.

Abstract. Bone marrow-derived mesenchymal stem cells are pluripotential cells that have the capacity to differentiate into an osteoprogenitor line. It has been demonstrated that BMP-2 can enhance this differentiation process. In an attempt to prolong the transforming effect of BMP-2, we used an adenoviral vector carrying the human BMP-2 gene to transduce marrow-derived mesenchymal stem cells of New Zealand white rabbits. Assays on tissue culture demonstrated that these cells indeed produced the BMP-2 protein. These transduced stem cells were then autologously reimplanted into the donor rabbits. The cells were placed in the intertransverse process area of five rabbits. In one out of the five rabbits, this resulted in the production of new bone which was demonstrable on both radiographic and histologic examination. We conclude that it is possible to successfully transduce mesenchymal stem cells with the gene for BMP-2 such that these cells will produce the BMP-2 protein in vitro. Further, the transduction results in transformation of these cells into an osteoprogenitor line capable of producing bone in vivo. These data suggest the feasibility of employing gene therapy using recombinant adenoviral vectors as a tool for enhancing spine fusion. Further work to improve the fidelity and longevity of the gene transfer is warranted.

β-Catenin and BMP-2 Synergize to Promote Osteoblast Differentiation and New Bone Formation

Mutations of critical components of the Wnt pathway profoundly affect skeletal development and maintenance, probably via modulation of β‐catenin signaling. We tested the hypothesis that β‐catenin is involved in mesenchymal lineage allocation to osteogenic cells using a β‐catenin mutant with constitutive transcriptional activity (ΔN151). Although this stable β‐catenin had no effects by itself on osteogenic differentiation of multipotent embryonic cell lines, it synergized with bone morphogenetic protein‐2 (BMP‐2) resulting in dramatic stimulation of alkaline phosphatase activity, osteocalcin gene expression, and matrix mineralization. Likewise, ΔN151 and BMP‐2 synergistically stimulated new bone formation after subperiosteal injection in mouse calvaria in vivo. Conversely, ΔN151 prevented adipogenic differentiation from pre‐adipocytic or uncommitted mesenchymal cells in vitro. Intriguingly, the synergism with BMP‐2 on gene transcription occurred without altering expression of Cbfa1/Runx2, suggesting actions independent or downstream of this osteoblast‐specific transcription factor. Thus, β‐catenin directs osteogenic lineage allocation by enhancing mesenchymal cell responsiveness to osteogenic factors, such as BMP‐2, in part via Tcf/Lef dependent mechanisms. In vivo, this synergism leads to increased new bone formation.

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.

Human Osteoblasts Express a Repertoire of Cadherins, Which Are Critical for BMP‐2–Induced Osteogenic Differentiation

Direct cell–cell interactions are fundamental for tissue development and differentiation. We have studied the expression and function of cadherins in human osteoblasts during in vitro differentiation. Using reverse transcription‐polymerase chain reaction and mRNA hybridization, we found that human trabecular bone osteoblasts (HOBs), osteoprogenitor marrow stromal cells (BMCs), and the osteogenic sarcoma lines, SaOS‐2 and MG‐63, expressed mRNA for cadherin‐11 (C11) and N‐cadherin (N‐cad). HOBs and BMCs also expressed low levels of cadherin‐4 (C4) mRNA. C11 was the most abundant cadherin protein present in human osteoblasts, and its expression was unaffected by bone morphogenetic protein‐2 (BMP‐2) treatment of either BMCs or HOBs. Likewise, N‐cad mRNA did not change during BMP‐2 incubation. Conversely, C4 protein, undetectable in transformed cell lines, was down‐regulated by BMP‐2 treatment of normal cells. Both C11 and C4 were localized to sites of cell–cell contact in both HOBs and BMCs, colocalized with β‐catenin, and bands corresponding to cadherins were coimmunoprecipitated by a β‐catenin antibody, findings indicative of functional cadherins. A decapeptide containing the HAV motif of human N‐cad partially inhibited Ca2+‐dependent cell–cell adhesion and completely prevented BMP‐2–induced stimulation of alkaline phosphatase activity by BMCs. Thus, human osteoblasts and their progenitor cells express a repertoire of multiple cadherins. Cadherin‐mediated cell‐to‐cell adhesion is critical for normal human osteoblast differentiation.

In Vitro andIn Vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene

It has been well established that bone morphogenetic protein-2 (BMP-2) can induce bone formation bothin vivo andin vitro, although high concentrations (up to milligrams) of BMP-2 have been required to achieve this effectin vivo. Further, clinical applications are usually limited to a single dose at the time of implantation. In an attempt to prolong the transforming effect of BMP-2 we used a recombinant adenoviral vector carrying the human BMP-2 gene (Adv-BMP2) to transduce marrow-derived mesenchymal stem cells (MSC) of skeletally mature male New Zealand white rabbits. The pluripotential MSC were incubated with Adv-BMP2 overnight followed by culture in growth medium for 1 week. Assays on tissue cultures demonstrated that these Adv-BMP2 transduced MSC produced BMP-2 protein, differentiated into an osteoprogenitor line, and induced bone formationin vitro. These MSC had increased alkaline phosphatase activity, increased expression of type I collagen, osteopontin, and osteocalcin mRNA, and induced matrix mineralization compared with both nontransduced cells and cells transduced with a control adenoviral construct. To analyze the osteogenic potentialin vivo, Adv-BMP2-transduced MSC were autologously implanted into the intertransverse process space between L5 and L6 of the donor rabbits. The production of new bone was demonstrated by radiographic examination 4 weeks later in areas implanted with cells transduced with Adv-BMP2, whereas no bone was evident at sites implanted with cells transduced with the control adenoviral construct. Histological examination further confirmed the presence of new bone formation. These accumulated data indicate that it is possible to successfully transduce mesenchymal stem cells with a recombinant adenoviral vector carrying the gene for BMP-2 such that these cells will produce BMP-2, differentiate into an osteoprogenitor line, and induce bone formation bothin vitro andin vivo. Moreover, incubation of the Adv-BMP2-transduced cells for an additional 7 days in culture before transplantation enhances the success rate in bone formation (three out of three) as compared with our previous report (one out of five, Calcif Tissue Int 63:357–360, 1998).

αvβ Integrins Play an Essential Role in BMP‐2 Induction of Osteoblast Differentiation

Both integrins and BMP‐2 exert similar effects on osteoblasts. We examined the relationship between the αv‐containing integrins (αvβ) and BMP‐2 in osteoblast function. BMP‐2 stimulates αvβ expression. BMP‐2 receptors co‐localize/overlap with αvβ integrins, and the intact function of αvβ is essential in BMP‐2 activity.