The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice

Recent work has identified LDL receptor-related family members, Lrp5 and Lrp6, as co-receptors for the transduction of Wnt signals. Our analysis of mice carrying mutations in both Lrp5 and Lrp6 demonstrates that the functions of these genes are redundant and are essential for gastrulation. Lrp5;Lrp6 double homozygous mutants fail to establish a primitive streak, although the anterior visceral endoderm and anterior epiblast fates are specified. Thus, Lrp5 and Lrp6 are required for posterior patterning of the epiblast, consistent with a role in transducing Wnt signals in the early embryo. Interestingly, Lrp5(+/-);Lrp6(-/-) embryos die shortly after gastrulation and exhibit an accumulation of cells at the primitive streak and a selective loss of paraxial mesoderm. A similar phenotype is observed in Fgf8 and Fgfr1 mutant embryos and provides genetic evidence in support of a molecular link between the Fgf and Wnt signaling pathways in patterning nascent mesoderm. Lrp5(+/-);Lrp6(-/-) embryos also display an expansion of anterior primitive streak derivatives and anterior neurectoderm that correlates with increased Nodal expression in these embryos. The effect of reducing, but not eliminating, Wnt signaling in Lrp5(+/-);Lrp6(-/-) mutant embryos provides important insight into the interplay between Wnt, Fgf and Nodal signals in patterning the early mouse embryo.     

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

The protein p21(Cip1, Waf1, Sdi1) is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase delta. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.     

Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling

Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/β-catenin pathway. Here, we show that RNF43 suppresses both Wnt/β-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/β-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/β-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/β-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/β-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.     

Hepatocyte Growth Factor (Hgf) Stimulates Low Density Lipoprotein Receptor-related Protein (Lrp) 5/6 Phosphorylation and Promotes Canonical Wnt Signaling

While Wnt and Hgf signaling pathways are known to regulate epithelial cell responses during injury and repair, whether they exhibit functional cross-talk is not well defined. Canonical Wnt signaling is initiated by the phosphorylation of the Lrp5/6 co-receptors. In the current study we demonstrate that Hgf stimulates Met and Gsk3-dependent and Wnt-independent phosphorylation of Lrp5/6 at three separate activation motifs in subconfluent, de-differentiated renal epithelial cells. Hgf treatment stimulates the selective association of active Gsk3 with Lrp5/6. In contrast, Akt-phosphorylated inactive Gsk3 is excluded from this association. Hgf stimulates β-catenin stabilization and nuclear accumulation and protects against epithelial cell apoptosis in an Lrp5/6-dependent fashion. In vivo, the increase in Lrp5/6 phosphorylation and β-catenin stabilization in the first 6–24 h after renal ischemic injury was significantly reduced in mice lacking Met receptor in the renal proximal tubule. Our results thus identify Hgf as an important transactivator of canonical Wnt signaling that is mediated by Met-stimulated, Gsk3-dependent Lrp5/6 phosphorylation.     

Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP''s and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway.     

Functional Consequences of Wnt-induced Dishevelled 2 Phosphorylation in Canonical and Noncanonical Wnt Signaling

Dishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594–609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2 P4m mutant showed a modest increase in canonical Wnt/β-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ϵ as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser⁵⁹⁴, Thr⁵⁹⁵, and Ser⁵⁹⁷ of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at the WIP sites reduces Dvl accumulation in puncta and attenuates β-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth.     

The role of Lrp5/6 in cardiac valve disease: experimental hypercholesterolemia in the ApoE-/- /Lrp5-/- mice

Lrp5/6 co-receptor is known to play a role in bone formation and lipid metabolism. This gene encodes a member of the low-density lipoprotein (LDL) receptor gene family. This study tests the hypothesis that Lrp5/6 is necessary for the development of valve calcification in experimental hypercholesterolemia. Experimental hypercholesterolemia mouse models were tested: Lrp5(-/-) /ApoE(-/-):Lrp5(-/-) /ApoE(-/-) mice (n = 180). Group I (n = 60) normal diet, Group II (n = 60) 0.25% chol diet (w/w), and Group III (n = 60) 0.25% (w/w) chol diet + atorv for the development of calcification by MicroCT and Synchrotron MicroCT Scan and by Masson trichrome stain. Finally gene expression for Lrp5, Lrp6, and Runx2 PCR was performed to evaluate the expression in the control and the cholesterol valves. The ApoE(-/-) cholesterol treated mice developed calcification and increase in Lrp5, Runx2 (P < 0.05) as compared to control. The Lrp5(-/-) mice developed no calcification by MicroCT and Synchrotron and positive gene expression for Lrp5/6 or Runx2. The double knockout ApoE(-/-):Lrp5(-/-) developed mild mineralization in the cholesterol treated valves with an increase in Lrp6 and Runx2 expression(P < 0.05). There was no mineralization in the right sided hearts valves. In conclusion Lrp5/6 is necessary for calcification in the aortic valve in the presence of experimental hypercholesterolemia. These data demonstrate the first mouse genetic evidence for the LDL-Density-Pressure theory in cardiac valves.     

Cloning and expression of Xenopus Lrp5 and Lrp6 genes

LRP5 and LRP6 comprise a subfamily of lipoprotein-receptor related proteins that function as co-receptors for Wnt proteins. Mutation of human LRP5 is responsible for osteoporosis-pseudoglioma syndrome and disruption of Lrp6 in mice causes similar effects to mutation of several different Wnt genes. We have cloned Xenopus homologues of Lrp5 and Lrp6 (Xlrp5, Xlrp6) and examined their expression during embryogenesis. Both genes are expressed maternally and ubiquitously through early development. At later stages, Xlrp5 is found in the eye, forebrain, hindbrain, branchial arches and the tip of the tail bud. Xlrp6 is expressed throughout the central nervous system, branchial arches, in the eye and otic vesicle. Both genes are also expressed at the intersomitic boundary. These results suggest roles for Wnt signaling via LRP proteins in these tissues.     

The Imprinted Gene PEG3 Inhibits Wnt Signaling and Regulates Glioma Growth

The imprinted gene PEG3 confers parenting and sexual behaviors, alters growth and development, and regulates apoptosis. However, a molecular mechanism that can account for the diverse functions of Peg3/Pw1 is not known. To elucidate Peg3-regulated pathways, we performed a functional screen in zebrafish. Enforced overexpression of PEG3 mRNA during zebrafish embryogenesis decreased β-catenin protein expression and inhibited Wnt-dependent tail development. Peg3/Pw1 also inhibited Wnt signaling in human cells by binding to β-catenin and promoting its degradation via a p53/Siah1-dependent, GSK3β-independent proteasomal pathway. The inhibition of the Wnt pathway by Peg3/Pw1 suggested a role in tumor suppression. Hypermethylation of the PEG3 promoter in primary human gliomas led to a loss of imprinting and decreased PEG3 mRNA expression that correlated with tumor grade. The decrease in Peg3/Pw1 protein expression increased β-catenin, promoted proliferation, and inhibited p53-dependent apoptosis in human CD133⁺ glioma stem cells. Thus, mammalian imprinting utilizes Peg3/Pw1 to co-opt the Wnt pathway, thereby regulating development and glioma growth.     

The Wnt Co-Receptor Lrp6 Is Required for Normal Mouse Mammary Gland Development

Canonical Wnt signals are transduced through a Frizzled receptor and either the LRP5 or LRP6 co-receptor; such signals play central roles during development and in disease. We have previously shown that Lrp5 is required for ductal stem cell activity and that loss of Lrp5 delays normal mammary development and Wnt1-induced tumorigenesis. Here we show that canonical Wnt signals through the Lrp6 co-receptor are also required for normal mouse mammary gland development. Loss of Lrp6 compromises Wnt/β-catenin signaling and interferes with mammary placode, fat pad, and branching development during embryogenesis. Heterozygosity for an inactivating mutation in Lrp6 is associated with a reduced number of terminal end buds and branches during postnatal development. While Lrp6 is expressed in both the basal and luminal mammary epithelium during embryogenesis, Lrp6 expression later becomes restricted to cells residing in the basal epithelial layer. Interestingly, these cells also express mammary stem cell markers. In humans, increased Lrp6 expression is associated with basal-like breast cancer. Taken together, our results suggest both overlapping and specific functions for Lrp5 and Lrp6 in the mammary gland.     

Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions

Canonical Wnt/beta-catenin signaling has central roles in development and diseases, and is initiated by the action of the frizzled (Fz) receptor, its coreceptor LDL receptor-related protein 6 (Lrp6), and the cytoplasmic dishevelled (Dvl) protein. The functional relationships among Fz, Lrp6 and Dvl have long been enigmatic. We demonstrated previously that Wnt-induced Lrp6 phosphorylation via glycogen synthase kinase 3 (Gsk3) initiates Wnt/beta-catenin signaling. Here we show that both Fz and Dvl functions are critical for Wnt-induced Lrp6 phosphorylation through Fz-Lrp6 interaction. We also show that axin, a key scaffolding protein in the Wnt pathway, is required for Lrp6 phosphorylation via its ability to recruit Gsk3, and inhibition of Gsk3 at the plasma membrane blocks Wnt/beta-catenin signaling. Our results suggest a model that upon Wnt-induced Fz-Lrp6 complex formation, Fz recruitment of Dvl in turn recruits the axin-Gsk3 complex, thereby promoting Lrp6 phosphorylation to initiate beta-catenin signaling. We discuss the dual roles of the axin-Gsk3 complex and signal amplification by Lrp6-axin interaction during Wnt/beta-catenin signaling.     

Direct Inhibition of GSK3β by the Phosphorylated Cytoplasmic Domain of LRP6 in Wnt/β-Catenin Signaling

Wnt/β-catenin signaling plays a central role in development and is also involved in a diverse array of diseases. Binding of Wnts to the coreceptors Frizzled and LRP6/5 leads to phosphorylation of PPPSPxS motifs in the LRP6/5 intracellular region and the inhibition of GSK3β bound to the scaffold protein Axin. However, it remains unknown how GSK3β is specifically inhibited upon Wnt stimulation. Here, we show that overexpression of the intracellular region of LRP6 containing a Ser/Thr rich cluster and a PPPSPxS motif impairs the activity of GSK3β in cells. Synthetic peptides containing the PPPSPxS motif strongly inhibit GSK3β in vitro only when they are phosphorylated. Microinjection of these peptides into Xenopus embryos confirms that the phosphorylated PPPSPxS motif potentiates Wnt-induced second body axis formation. In addition, we show that the Ser/Thr rich cluster of LRP6 plays an important role in LRP6 binding to GSK3β. These observations demonstrate that phosphorylated LRP6/5 both recruits and directly inhibits GSK3β using two distinct portions of its cytoplasmic sequence, and suggest a novel mechanism of activation in this signaling pathway.     

The role of Lrp5/6 in cardiac valve disease: LDL-density-pressure theory

Atherosclerosis and osteoporosis are the leading causes of mortality and morbidity in the World. Recent epidemiologic studies have demonstrated that these disease processes develop in parallel. Evidence indicates that hyperlipidemia plays a paradoxical role in both disease processes. However, the mechanism is not understood. This prospectus hypothesizes the role of lipids activate atherosclerosis within the bone and the heart to initiate the development of diseases in both of these tissues. The Prospectus on the Lrp 5/6 receptors provides a foundation for the mechanisms involved in the Lrp5/6 mediated disease biology. The LDL-Density-Pressure theory: the Role of Lrp5/6 provides a biological and a hemodynamic approach towards understanding the development of valvular heart disease and the implications in the field of bone molecular biology. This prospectus will review the current literature, provide a basis for the development of valve disease and indicate future therapeutic pathways for this disease process in the future.     

MK-801 Increases Extracellular 5-Hydroxytryptamine in Rat Hippocampus and Striatum In Vivo

The effect of MK-801 (0.25 or 0.5 mg/kg) on the extracellular concentration of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in rat hippocampus and striatum was studied using intracerebral dialysis. The dialysate 5-HT concentration was dose-dependently increased by MK-801 in both regions. In the hippocampus, at the higher drug dose a slow increase in the 5-HIAA level was observed, and this became significant 3 h after treatment. In contrast to this, the extracellular 5-HIAA content in the striatum was significantly decreased 150 min after administration of both doses of MK-801. The data are discussed in the light of the known behavioural effects of MK-801 and possible N-methyl-D-aspartic acid receptor regulation of 5-HT release.     

The Wnt signaling receptor Lrp5 is required for mammary ductal stem cell activity and Wnt1-induced tumorigenesis

Canonical Wnt signaling has emerged as a critical regulatory pathway for stem cells. The association between ectopic activation of Wnt signaling and many different types of human cancer suggests that Wnt ligands can initiate tumor formation through altered regulation of stem cell populations. Here we have shown that mice deficient for the Wnt co-receptor Lrp5 are resistant to Wnt1-induced mammary tumors, which have been shown to be derived from the mammary stem/progenitor cell population. These mice exhibit a profound delay in tumorigenesis that is associated with reduced Wnt1-induced accumulation of mammary progenitor cells. In addition to the tumor resistance phenotype, loss of Lrp5 delays normal mammary development. The ductal trees of 5-week-old Lrp5-/- females have fewer terminal end buds, which are structures critical for juvenile ductal extension presumed to be rich in stem/progenitor cells. Consequently, the mature ductal tree is hypomorphic and does not completely fill the fat pad. Furthermore, Lrp5-/- ductal cells from mature females exhibit little to no stem cell activity in limiting dilution transplants. Finally, we have shown that Lrp5-/- embryos exhibit substantially impaired canonical Wnt signaling in the primitive stem cell compartment of the mammary placodes. These findings suggest that Lrp5-mediated canonical signaling is required for mammary ductal stem cell activity and for tumor development in response to oncogenic Wnt effectors.     

The C-Terminal Domain of Cernunnos/XLF Is Dispensable for DNA Repair In Vivo

The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair.Nonhomologous end joining (NHEJ) represents the main pathway for solving DNA double-strand breaks (DSB) in mammals. The core of the NHEJ pathway is composed of seven proteins: Ku70, Ku80, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos) (reviewed in reference 18). Briefly, the Ku70-Ku80 heterodimer bound to broken DNA recruits the serine/threonine kinase DNA-PKcs. DNA-PK phosphorylates downstream effectors such as the nuclease Artemis. The X4-L4 complex carries out the final joining of synapsed DNA ends in association with Cernunnos (2, 6). Cernunnos was identified through cDNA functional complementation of a fibroblast cell line obtained from a human patient with immune deficiency and microcephaly (5). The same factor, called XLF, was identified through a yeast two-hybrid screen with X4 as a bait (2).Cernunnos is structurally related to X4 and consists of a globular head domain followed by a coiled-coil region and an unstructured C-terminal domain (2, 6, 12). One major difference between the structures of X4 and Cernunnos appears in the coiled-coil region. While this region is linear in X4, a hinge in the middle of the coiled-coil of Cernunnos folds back the end of the domain toward the head (3, 14).Cernunnos interacts with the X4-L4 complex in vivo and in vitro (2, 6). Cernunnos and X4 both appear to interact directly with L4, but the Cernunnos-L4 interaction seems to be very weak (7). In addition, purified Cernunnos associates with DNA in a sequence-independent manner (20) but in a DNA length-dependent manner, like X4 (15). Although the X4-L4 complex can ligate DNA in vitro (10), Cernunnos further improves this activity (11, 15, 16, 20). Cernunnos seems important, in particular, for the ligation of mismatched or noncohesive DNA ends, but not for that of compatible DNA ends, in vitro (10, 20).Cernunnos is therefore a “core” NHEJ component, but limited information is available about its precise function during DNA repair in vivo. We show here that although X4 and Cernunnos share sequence and structural homologies, their functions are distinct. We also demonstrate that Cernunnos requires L4 for its association with X4. Lastly, the Cernunnos C terminus is dispensable for DNA repair following ionizing radiation (IR) and V(D)J recombination.     

The Wnt Co-Receptor Lrp5 Is Required for Cranial Neural Crest Cell Migration in Zebrafish

During vertebrate neurulation, cranial neural crest cells (CNCCs) undergo epithelial to mesenchymal transition (EMT), delaminate from the neural plate border, and migrate as separate streams into different cranial regions. There, they differentiate into distinct parts of the craniofacial skeleton. Canonical Wnt signaling has been shown to be essential for this process at different levels but the involved receptors remained unclear. Here we show that the frizzled co-receptor low-density-lipoprotein (LDL) receptor-related protein 5 (Lrp5) plays a crucial role in CNCC migration and morphogenesis of the cranial skeleton. Early during induction and migration of CNCCs, lrp5 is expressed ubiquitously but later gets restricted to CNCC derivatives in the ventral head region besides different regions in the CNS. A knock-down of lrp5 does not interfere with induction of CNCCs but leads to reduced proliferation of premigratory CNCCs. In addition, cell migration is disrupted as CNCCs are found in clusters at ectopic positions in the dorsomedial neuroepithelium after lrp5 knock-down and transient CRISPR/Cas9 gene editing. These migratory defects consequently result in malformations of the craniofacial skeleton. To date, Lrp5 has mainly been associated with bone homeostasis in mammals. Here we show that in zebrafish, lrp5 also controls cell migration during early morphogenetic processes and contributes to shaping the craniofacial skeleton.     

TIEG1 is upregulated in Lrp5/6-mediated valve osteogenesis

We have previously demonstrated that Lrp5/6/β-catenin plays an important role in valve calcification with a specific osteogenic phenotype defined by increased bone mineral content and overall valve thickening. Recent studies indicate that TIEG1 may be involved in mediating the Wnt signaling pathway in bone, which is known to play critical roles in osteoblast differentiation and bone mineralization. Therefore, we sought to test the role of TIEG1 in mediating Wnt signaling, in an established model of hypercholesterolemic valve disease. Our previous model treated null mice with cholesterol diets: Lrp5 ^−/−/ApoE ^−/− mice versus wild-type control (n = 180). Group I (n = 60) normal diet, Group II (n = 60) 0.25% chol diet (w/w), and Group III (n = 60) 0.25% (w/w) chol diet + atorv was tested for gene expression for TIEG1, Lrp6, and Runx2. Real-time polymerase chain reaction confirmed that there is upregulation of the gene expression for TIEG1 and Runx2 in the hypercholesterolemic double knockout and single knockout valves as compared with controls with a mild increase in Lrp6. To confirm the mechanism, coexpression of β-catenin, TIEG1, and LEF1 in valve cells in vitro, led to the coactivation of the TOPFLASH reporter, which was further confirmed by the observation that TIEG1 and β-catenin colocalize with one another in the nucleus of valvular interstitial cells (VICs) following stimulation with transforming growth factor-β treatment, an established activator of TIEG1. Taken together, these data implicate an important role for TIEG1 in mediating valve osteogenesis.