Kelly E. Beazley

Transglutaminase 2–Mediated Activation of β-Catenin Signaling Has a Critical Role in Warfarin-Induced Vascular Calcification

Objective— Accumulating experimental evidence implicates &bgr;-catenin signaling and enzyme transglutaminase 2 (TG2) in the progression of vascular calcification, and our previous studies have shown that TG2 can activate &bgr;-catenin signaling in vascular smooth muscle cells (VSMCs). Here we investigated the role of the TG2/&bgr;-catenin signaling axis in vascular calcification induced by warfarin. Methods and Results— Warfarin-induced calcification in rat A10 VSMCs is associated with the activation of &bgr;-catenin signaling and is independent of oxidative stress. The canonical &bgr;-catenin inhibitor Dkk1, but not the Wnt antagonist Wif-1, prevents warfarin-induced activation of &bgr;-catenin, calcification, and osteogenic transdifferentiation in VSMCs. TG2 expression and activity are increased in warfarin-treated cells, in contrast to canonical Wnt ligands. Vascular cells with genetically or pharmacologically reduced TG2 activity fail to activate &bgr;-catenin in response to warfarin. Moreover, warfarin-induced calcification is significantly reduced on the background of attenuated TG2 both in vitro and in vivo. Conclusion— TG2 is a critical mediator of warfarin-induced vascular calcification that acts through the activation of &bgr;-catenin signaling in VSMCs. Inhibition of canonical &bgr;-catenin pathway or TG2 activity prevents warfarin-regulated calcification, identifying the TG2/&bgr;-catenin axis as a novel therapeutic target in vascular calcification.

Ferritoid, a Tissue-specific Nuclear Transport Protein for Ferritin in Corneal Epithelial Cells

Previously we reported that ferritin in corneal epithelial (CE) cells is a nuclear protein that protects DNA from UV damage. Since ferritin is normally cytoplasmic, in CE cells, a mechanism must exist that effects its nuclear localization. We have now determined that this involves a nuclear transport molecule we have termed ferritoid. Ferritoid is specific for CE cells and is developmentally regulated. Structurally, ferritoid contains multiple domains, including a functional SV40-type nuclear localization signal and a ferritin-like region of ∼50% similarity to ferritin itself. This latter domain is likely responsible for the interaction between ferritoid and ferritin detected by co-immunoprecipitation analysis. To test functionally whether ferritoid is capable of transporting ferritin into the nucleus, we performed cotransfections of COS-1 cells with constructs for ferritoid and ferritin. Consistent with the proposed nuclear transport function for ferritoid, co-transfections with full-length constructs for ferritoid and ferritin resulted in a preferential nuclear localization of both molecules; this was not observed when the nuclear localization signal of ferritoid was deleted. Moreover, since ferritoid is structurally similar to ferritin, it may be an example of a nuclear transporter that evolved from the molecule it transports (ferritin).

Transglutaminase Inhibitors Attenuate Vascular Calcification in a Preclinical Model

Objective—In vitro, transglutaminase-2 (TG2)–mediated activation of the &bgr;-catenin signaling pathway is central in warfarin-induced calcification, warranting inquiry into the importance of this signaling axis as a target for preventive therapy of vascular calcification in vivo. Methods and Results—The adverse effects of warfarin-induced elastocalcinosis in a rat model include calcification of the aortic media, loss of the cellular component in the vessel wall, and isolated systolic hypertension, associated with accumulation and activation of TG2 and activation of &bgr;-catenin signaling. These effects of warfarin can be completely reversed by intraperitoneal administration of the TG2-specific inhibitor KCC-009 or dietary supplementation with the bioflavonoid quercetin, known to inhibit &bgr;-catenin signaling. Our study also uncovers a previously uncharacterized ability of quercetin to inhibit TG2. Quercetin reversed the warfarin-induced increase in systolic pressure, underlying the functional consequence of this treatment. Molecular analysis shows that quercetin diet stabilizes the phenotype of smooth muscle and prevents its transformation into osteoblastic cells. Conclusion—Inhibition of the TG2/&bgr;-catenin signaling axis seems to prevent warfarin-induced elastocalcinosis and to control isolated systolic hypertension.

Quercetin attenuates warfarin-induced vascular calcification in vitro independently from matrix Gla protein.

Background: Molecular mechanism(s) of warfarin-induced vascular calcification are not well known. Results: Inhibition of β-catenin signaling with shRNA or quercetin prevents osteoblastic transformation and calcification in VSMCs and calcification in aortic rings treated with warfarin, independent from MGP and protein carboxylation. Conclusion: Quercetin inhibits vascular calcification via β-catenin signaling. Significance: Quercetin may be instrumental in treatment of warfarin-induced vascular calcification. Warfarin can stimulate vascular calcification in vitro via activation of β-catenin signaling and/or inhibition of matrix Gla protein (MGP) carboxylation. Calcification was induced in vascular smooth muscle cells (VSMCs) with therapeutic levels of warfarin in normal calcium and clinically acceptable phosphate levels. Although TGF/BMP and PKA pathways are activated in calcifying VSMCs, pharmacologic analysis reveals that their activation is not contributory. However, β-catenin activity is important because inhibition of β-catenin with shRNA or bioflavonoid quercetin prevents calcification in primary human VSMCs, rodent aortic rings, and rat A10 VSMC line. In the presence of quercetin, reactivation of β-catenin using the glycogen synthase kinase-3β (GSK-3β) inhibitor LiCl restores calcium accumulation, confirming that quercetin mechanism of action hinges on inhibition of the β-catenin pathway. Calcification in VSMCs induced by 10 μm warfarin does not associate with reduced levels of carboxylated MGP, and inhibitory effects of quercetin do not involve induction of MGP carboxylation. Further, down-regulation of MGP by shRNA does not alter the effect of quercetin. These results suggest a new β-catenin-targeting strategy to prevent vascular calcification induced by warfarin and identify quercetin as a potential therapeutic in this pathology.

Corneal epithelial nuclear ferritin: Developmental regulation of ferritin and its nuclear transporter ferritoid

The corneal epithelium is exposed to reactive oxygen species that are potentially deleterious to nuclear DNA. However, our previous studies show that corneal epithelial cells have a novel, developmentally regulated mechanism for protection from such damage that involves having the iron‐sequestering molecule, ferritin, in the nucleus. Nuclear localization of ferritin is achieved through the action of a tissue‐specific nuclear transporter, ferritoid, which is itself a ferritin family member. Here, we show that during development ferritoid appears before ferritin. At this time, ferritoid is cytoplasmic, suggesting that its nuclear transport function requires an interaction with ferritin. To examine the developmental regulation of these two interacting components, cultured corneas were treated with the iron chelator deferoxamine. The results show that, while iron‐mediated translational regulation is involved in the synthesis of both molecules, ferritoid is also transcriptionally regulated, demonstrating that these family members—whose functions depend upon one another—are regulated differently. Developmental Dynamics 237:2529–2541, 2008.

Wnt16 Attenuates TGFβ-Induced Chondrogenic Transformation in Vascular Smooth Muscle

Objective— Phenotypic plasticity of vascular smooth muscle cells (VSMCs) contributes to cardiovascular disease. Chondrocyte-like transformation of VSMCs associates with vascular calcification and underlies the formation of aortic cartilaginous metaplasia induced in mice by genetic loss of matrix Gla protein (MGP). Previous microarray analysis identified a dramatic downregulation of Wnt16 in calcified MGP-null aortae, suggesting an antagonistic role for Wnt16 in the chondrogenic transformation of VSMCs. Approach and Results— Wnt16 is significantly downregulated in MGP-null aortae, before the histological appearance of cartilaginous metaplasia, and in primary MGP-null VSMCs. In contrast, intrinsic TGF&bgr; is activated in MGP-null VSMCs and is necessary for spontaneous chondrogenesis of these cells in high-density micromass cultures. TGF&bgr;3-induced chondrogenic transformation in wild-type VSMCs associates with Smad2/3-dependent Wnt16 downregulation, but Wnt16 does not suppress TGF&bgr;3-induced Smad activation. In addition, TGF&bgr;3 inhibits Notch signaling in wild-type VSMCs, and this pathway is downregulated in MGP-null aortae. Exogenous Wnt16 stimulates Notch activity and attenuates TGF&bgr;3-induced downregulation of Notch in wild-type VSMCs, prevents chondrogenesis in MGP-null and TGF&bgr;3-treated wild-type VSMCs, and stabilizes expression of contractile markers of differentiated VSMCs. Conclusions— We describe a novel TGF&bgr;-Wnt16-Notch signaling conduit in the chondrocyte-like transformation of VSMCs and identify endogenous TGF&bgr; activity in MGP-null VSMCs as a critical mediator of chondrogenesis. Our proposed model suggests that the activated TGF&bgr; pathway inhibits expression of Wnt16, which is a positive regulator of Notch signaling and a stabilizer of VSMC phenotype. These data advance the comprehensive mechanistic understanding of VSMC transformation and may identify a novel potential therapeutic target in vascular calcification.

Two sides of MGP null arterial disease: chondrogenic lesions dependent on transglutaminase 2 and elastin fragmentation associated with induction of adipsin.

Background: MGP inhibits tissue calcification, but underlying mechanisms are understudied. Results: In MGP null mice, TG2 ablation prevents calcifying cartilaginous vascular lesions but does not affect elastocalcinosis and elastin fragmentation associated with increased elastase adipsin. Conclusion: MGP acts via two distinct mechanisms. Significance: Our study identifies TG2 and adipsin as potential therapeutic targets in vascular disease linked to MGP deficiency. Mutations in matrix Gla protein (MGP) have been correlated with vascular calcification. In the mouse model, MGP null vascular disease presents as calcifying cartilaginous lesions and mineral deposition along elastin lamellae (elastocalcinosis). Here we examined the mechanisms underlying both of these manifestations. Genetic ablation of enzyme transglutaminase 2 (TG2) in Mgp−/− mice dramatically reduced the size of cartilaginous lesions in the aortic media, attenuated calcium accrual more than 2-fold, and doubled longevity as compared with control Mgp−/− animals. Nonetheless, the Mgp−/−;Tgm2−/− mice still died prematurely as compared with wild-type and retained the elastocalcinosis phenotype. This pathology in Mgp−/− animals was developmentally preceded by extensive fragmentation of elastic lamellae and associated with elevated serine elastase activity in aortic tissue and vascular smooth muscle cells. Systematic gene expression analysis followed by an immunoprecipitation study identified adipsin as the major elastase that is induced in the Mgp−/− vascular smooth muscle even in the TG2 null background. These results reveal a central role for TG2 in chondrogenic transformation of vascular smooth muscle and implicate adipsin in elastin fragmentation and ensuing elastocalcinosis. The importance of elastin calcification in MGP null vascular disease is highlighted by significant residual vascular calcification and mortality in Mgp−/−;Tgm2−/− mice with reduced cartilaginous lesions. Our studies identify two potential therapeutic targets in vascular calcification associated with MGP dysfunction and emphasize the need for a comprehensive approach to this multifaceted disorder.

Transglutaminase 2 Promotes PDGF-Mediated Activation of PDGFR/Akt1 and β-catenin Signaling in Vascular Smooth Muscle Cells and Supports Neointima Formation

Background: Phenotypic switch of vascular smooth muscle cells (VSMCs) accompanies neointima formation and associates with vascular diseases. Platelet-derived growth factor (PDGF)-induced activation of PDGFR/Akt1 and β-catenin signaling pathways in VSMCs has been implicated in vessel occlusion. Transglutaminase 2 (TG2) regulates these pathways and its levels are increased in the neointima. Objective: The aim of this study was to evaluate the role of TG2 in PDGF/β-catenin signaling cross-talk and assess its contribution to neointima. Methods: Aortic VSMCs from wild-type and TG2 knockout mice were tested in vitro for levels of VSMC markers, proliferation, migration and PDGF-induced activation of PDGFR/Akt1 and β-catenin pathways. Neointima in these mice was studied ex vivo in coronary vessels using a heart slice model and in vivo using a carotid artery ligation model. Results: Genetic deletion of TG2 attenuated the PDGF-induced phenotypic switch of aortic VSMCs, reduced their proliferation and migration rates, and inhibited PDGF-induced activation of PDGFR/Akt1 and β-catenin pathways in both ex vivo and in vivo neointima models. Importantly, genetic deletion of TG2 also markedly attenuated vessel occlusion. Conclusions: TG2 promotes neointima formation by mediating the PDGF-induced activation of the PDGFR/Akt1 and β-catenin pathways in VSMCs. This study identifies TG2 as a potential therapeutic target for blocking neointima in blood vessels.

Phosphorylation Regulates the Ferritoid―Ferritin Interaction and Nuclear Transport

Ferritin is an iron‐sequestering protein that is generally cytoplasmic; however, our previous studies have shown that in avian corneal epithelial (CE) cells ferritin is nuclear. We have also observed that this nuclear localization involves a tissue‐specific nuclear transporter that we have termed ferritoid, and that nuclear ferritin protects DNA from oxidative damage. Recently we have determined that ferritoid functions not only as a nuclear transporter, but also, within the nucleus, it remains associated with ferritin as a heteropolymeric complex. This ferritoid–ferritin complex has unique properties such as being half the size of a typical ferritin molecule and showing preferential binding to DNA. It is likely that the association between ferritoid and ferritin is involved both in the nuclear transport of ferritin and in determining certain of the properties of the complex; therefore, we have been examining the mechanisms involved in regulating the association of these two components. As the ferritoid sequence contains six putative phosphorylation sites, we have examined here whether phosphorylation is one such mechanism. We have determined that ferritoid in the nuclear ferritoid–ferritin complexes is phosphorylated, and that inhibition of this phosphorylation, using inhibitors of PKC, prevents its interaction with ferritin. Furthermore, in an experimental model system in which the nuclear transport of ferritin normally occurs (i.e., the co‐transfection of COS‐1 cells with full length constructs for ferritin and ferritoid), when phosphorylation sites in ferritoid are mutated, the interaction between ferritoid and ferritin is inhibited, as is the nuclear transport of ferritin. J. Cell. Biochem. 107: 528–536, 2009.

Attenuation of Chondrogenic Transformation in Vascular Smooth Muscle by Dietary Quercetin in the MGP-Deficient Mouse Model

Rationale Cartilaginous metaplasia of vascular smooth muscle (VSM) is characteristic for arterial calcification in diabetes and uremia and in the background of genetic alterations in matrix Gla protein (MGP). A better understanding of the molecular details of this process is critical for the development of novel therapeutic approaches to VSM transformation and arterial calcification. Objective This study aimed to identify the effects of bioflavonoid quercetin on chondrogenic transformation and calcification of VSM in the MGP-null mouse model and upon TGF-β3 stimulation in vitro, and to characterize the associated alterations in cell signaling. Methods and Results Molecular analysis revealed activation of β-catenin signaling in cartilaginous metaplasia in Mgp-/- aortae in vivo and during chondrogenic transformation of VSMCs in vitro. Quercetin intercepted chondrogenic transformation of VSM and blocked activation of β-catenin both in vivo and in vitro. Although dietary quercetin drastically attenuated calcifying cartilaginous metaplasia in Mgp-/- animals, approximately one-half of total vascular calcium mineral remained as depositions along elastic lamellae. Conclusion Quercetin is potent in preventing VSM chondrogenic transformation caused by diverse stimuli. Combined with the demonstrated efficiency of dietary quercetin in preventing ectopic chondrogenesis in the MGP-null vasculature, these findings indicate a potentially broad therapeutic applicability of this safe for human consumption bioflavonoid in the therapy of cardiovascular conditions linked to cartilaginous metaplasia of VSM. Elastocalcinosis is a major component of MGP-null vascular disease and is controlled by a mechanism different from chondrogenic transformation of VSM and not sensitive to quercetin.