Arleen P. Loewy

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.

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)-&agr; preferentially upregulate pro–matrix metalloproteinase 9 (pro-MMP9) activity. TNF-&agr; 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-&agr; induction of pro-MMP9 was inhibited by anti-OPN antibody and apocynin. Superoxide and the oxylipid product 8-isoprostaglandin F2 &agr;-isoprostane (8-IsoP) were increased by OPN treatment, and anti-OPN antibody suppressed 8-IsoP production. Like OPN and TNF-&agr;, 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-&agr; 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.

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.

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.

Ets Domain Transcription Factor PE1 Suppresses Human Interstitial Collagenase Promoter Activity by Antagonizing Protein−DNA Interactions at a Critical AP1 Element†

In MC3T3E1 calvarial osteoblasts, fibroblast growth factor receptor (FGFR) signaling elicits multiple transcriptional responses, including upregulation of the interstitial collagenase/matrix metalloproteinase 1 (MMP1) promoter. FGF responsiveness maps to a bipartite Ets/AP1 element at base pairs -123 to -61 in the human MMP1 promoter. Under basal conditions, the MMP1 promoter is repressed in part via protein-DNA interactions at the Ets cognate, and minimally two mechanisms convey MMP1 promoter upregulation by FGF2: (a) transcriptional activation via Fra1/c-Jun containing DNA-protein interactions at the AP1 cognate and (b) derepression of promoter activity regulated by the Ets cognate. To identify osteoblast Ets repressors that potentially participate in gene expression in the osteoblast, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis of mRNA isolated from MC3T3E1 cells, using degenerative amplimers to the conserved Ets DNA binding domain to survey the Ets genes expressed by these cells. Six distinct Ets mRNAs were identified: Ets2, Fli1, GABPalpha, SAP1, Elk1, and PE1. Of these, only PE1 has extensive homology to the known Ras-regulated Ets transcriptional repressor, ERF. Therefore, we cloned and characterized PE1 cDNA from a mouse brain library and performed functional analysis of this particular Ets family member. A 2 kb transcript was isolated from brain that encodes a approximately 57 kDa protein; the predicted protein contains the known N-terminal Ets domain of PE1 and a novel C-terminal domain with signficant homology to murine ERF. The murine PE1 open reading frame (ORF) is much larger than the previously reported human PE1 ORF. Consistent with this, affinity-purified rabbit anti-mouse PE1 antibody specifically recognizes an approximately 66 kDa protein present only in the nuclear fraction of MC3T3E1 osteoblasts. Recombinant PE1 binds authentic AGGAWG Ets DNA cognates, and transient transfection studies demonstrate that PE1 represses MMP1 promoter activity. Surprisingly, although deletion of the MMP1 Ets cognate at nucleotides -88 to -83 abrogates FGF2 induction, it does not prevent suppression of the AP1-dependent MMP1 promoter by PE1. PE1 regulation maps to the MMP1 promoter region -75 to -61, suggesting that PE1 suppresses transcription via protein-protein interactions with AP1. Consistent with this, recombinant GST-PE1 specifically inhibits the formation of protein-DNA interactions on the MMP1 AP1 site (-72 to -66) when present in an admixture with MC3T3E1 crude nuclear extract. In toto, these data indicate that PE1 participates in the transcriptional regulation of the MMP1 promoter in osteoblasts. As observed with other transcriptional repressors of MMP1 gene expression, transcriptional suppression by PE1 occurs via inhibition of AP1-dependent promoter activity.