This study presents the first direct evidence for the critical role of TG2-mediated activation of the β-catenin signaling pathway in warfarin-induced medial calcification. While under normal physiological condition vascular β-catenin signaling is generally inactive 46
, despite the expression of several canonical Wnt ligands in VSMCs 40
, its activation associates with vascular injury and remodeling 47, 48
. Previous studies have implicated the β -catenin signaling pathway in vascular calcification 49
based on the reported accumulation of the pathway proteins in calcific valve disease 27
and in diabetic calcifying lesions 26, 28, 50
. In addition, circumstantial evidence for β-catenin signaling in medial calcification is provided by the demonstrated abilities of MMPs to activate β-catenin 51
and promote calcification associated with elastin degradation 52, 53
. However, the mechanisms regulating the β-catenin pathway in valvular and vascular calcification have yet to be characterized 49, 54
, and the contribution of this pathway to calcification has not been demonstrated.
In this study, warfarin-induced calcification and osteogenic gene expression were studied in cultured aortic smooth muscle (A10) VSMCs, independent of the inflammatory response that commonly accompanies calcification in vivo
. Earlier studies investigated inhibition of the γ-carboxylation pathway by warfarin in cultured VSMCs and in arterial tissue ex vivo
. Here, we examined whether other mechanisms relevant to vascular calcification, such as oxidative stress and β-catenin signaling, are affected by warfarin and thus may mediate its effect. We did not observe induction of oxidative stress in VSMCs by warfarin, indicating that this mechanism is likely not involved in warfarin-mediated calcification in vitro
. However, we did find that warfarin potently activates β-catenin signaling. Further, we observed that the LRP5/6 antagonist Dkk1, which has been shown to inhibit osteogenic differentiation in mesenchymal cells and myofibroblasts 26, 55
, attenuates warfarin-induced β-catenin activity, expression of osteogenic genes, and calcium deposition in VSMCs. These findings provide novel, direct evidence for the key role of the LRP5/6-mediated canonical β-catenin pathway in osteoblastic transformation and calcification in VSMCs, and thus expand previous observations implicating β-catenin signaling in diverse types of vascular calcification 49
There is only a limited understanding of endogenous activators of β-catenin signaling in vascular calcification in general, and in warfarin-induced calcification in particular. Wnts 2, 3a, 7b and 8b are expressed by VSMCs 40
and Wnt7b and Wnt3a are increased in the calcifying arteries of the diabetic atherosclerotic LDLR-/- mice and in calcified heart valves 26, 27, 49
, indicative of the potential role of these canonical ligands in β-catenin activation. However, the levels of these Wnt ligands are not changed in calcification of the MGP-/- arteries 39
or during attenuation of β-catenin activity and calcification in the LDLR-/- mice by PTH 28
, and we found that they are also not affected by the calcification-inducing warfarin treatment of VSMCs. Further, warfarin-induced activation of β-catenin is not prevented by Wif-1, an antagonist of Wnt ligands. Therefore, other factors responsible for regulation of the canonical β-catenin pathway in vascular calcification should be considered.
Here we show that warfarin induces up-regulation and activation of TG2 – an enzyme that can bind canonical LRP5 receptor, activate β-catenin signaling, and enhance phosphate-induced calcification in VSMCs 23
. Using pharmacological and genetic approaches we show that catalytic activity of TG2 is critical for warfarin-induced activation of β-catenin signaling, osteoblast-like trans-differentiation of VSMCs, and calcium mineral deposition in vitro
and in vivo
, demonstrating the key role for the TG2/β-catenin axis in warfarin-induced calcification. Moreover, in addition to increased TG2-dependent protein cross-linking in the calcifying aortas of warfarin-treated rats 21
, previous studies showed the requirement for catalytic activity of TG2 in phosphate-induced aortic calcification ex vivo
and accumulation of TG2 in arterial walls of the MGP-/- mice 22
, indicating that such a mechanism may be involved in diverse variants of medial vascular calcification, although involvement of the β-catenin pathway in these cases still needs to be defined. Interestingly, in contrast to medial calcification, in atherosclerotic plaques elevated TG2 is associated with inflammatory cells and acts to regulate plaque stability, showing little correlation with vascular calcification 56-59
. Thus, biological activities of TG2 may be determined by the local milieu of various regulators that differ between the vascular wall layers.
A plausible model for the warfarin-induced calcification mediated by the TG2/β-catenin axis is illustrated in . The observed up-regulation of TG2 transcript awaits further elucidation. One of the possible mechanisms of this transcriptional activation may be owing to the activation of the nuclear receptor PXR (NR1I2) by direct binding of the warfarin molecule 10
. Activated PXR may bind to the four putative PXR-binding sites 60
present in the 4-kb upstream promoter region of the mouse TG2
gene (our unpublished data, 2011). Direct activation of the TG2 catalytic activity by warfarin presents another level of regulation. These effects are specific for TG2, because other transglutaminases expressed in the TG2-/- arteries do not respond to warfarin treatment. The unique feature of the TG2 protein is allosteric inhibition of its catalytic activity 43
, which may be released by the conformational changes upon warfarin binding to tyrosine residues (similar to the described interaction of warfarin with the vitamin K epoxide reductase complex subunit 1) 15
. Yet another potential pathway, not shown in the figure, may involve inhibition of MGP function by warfarin leading to accumulation of TG2 in the vessel wall similar to the effect of MGP knockout 22
, but the molecular mechanisms that may underlie this effect are not yet clear. At the next step, up-regulated and activated TG2 interacts with LRP5/6 receptors and stimulates canonical β-catenin signaling in VSMCs, leading to expression of osteoblastic genes and enhanced calcification.
Figure 7 Proposed mechanism for warfarin-induced calcification of VSMCs. Warfarin enhances TG2 expression and activity, leading to accumulation and nuclear localization of β-catenin protein and increased β-catenin-dependent transcription of osteogenesis-related (more ...)
It is possible that in vivo
warfarin may also activate β-catenin in VSMCs through enhanced vascular BMP signaling, which is normally suppressed by γ-carboxylated MGP protein 61
but may be activated when warfarin inhibits vitamin K-dependent γ-carboxylation. While redox cues from immunity and inflammatory cells in the adventitia can also activate the BMP/Msx2-Wnt signaling in diabetic arteriosclerosis model 50, 61
, in clonal VSMC culture these cells are absent, and warfarin-induced calcification and activation of theTG2/β-catenin axis is not coupled to oxidative stress or changes in Msx2 expression. It is tempting to speculate that the MGP-BMP pathway which mediates cross-talk between endothelium and VSMCs 61
, the BMP/Msx2-Wnt cascade originating in the adventitia 49
, and the TG2/β-catenin signaling axis which is central for osteogenic trans-differentiation in VSMCs, complement each other to mediate the activation of β-catenin and vascular calcification in diverse manifestations of this manifold disorder.
, warfarin acts as an amplifier rather than inducer of the osteochondrogenic trans-differentiation in VSMCs because it requires elevated levels of inorganic phosphate (Pi). The critical role of increased serum phosphate in vascular calcification is thoroughly supported by previous reports 31, 33, 35, 36
. A new observation of our study is that warfarin augments destabilization of the VSMC phenotype induced by a modest increase in Pi from 1.2 to 1.6 mM, while earlier studies analyzed the effects of warfarin on arterial tissue in 2-3.8 mM Pi 5, 32
. Taking into account that each 0.33 mM increase in serum phosphate within the “normal” clinical range increases the likelihood of arterial calcification by 34% 62
, our findings indicate that warfarin-induced vascular calcification may substantially vary in the general population and can pose a significant risk in chronic kidney disease patients even in early stages of the disease.
In conclusion, this study provides the first direct evidence for the requirement of the canonical β-catenin pathway in vascular calcification, and newly identifies the important role of the TG2/β-catenin signaling axis in warfarin-induced vascular calcification in VSMCs, adding to the emerging list of pharmacotherapeutic targets in cardiovascular disease.