Nuclear ferritin mediated regulation of JNK signaling in corneal epithelial cells.

Corneal epithelial (CE) cells are exposed to environmental insults (e.g., UV-irradiation), yet they suffer little damage. Our previous studies suggest that chicken CE cells have a novel form of protection involving having ferritin in a nuclear location where it can bind to DNA and sequester free iron. Here we describe another potential nuclear ferritin-mediated protective mechanism: the down-regulation of the JNK signaling pathway. The JNK pathway has been shown by others to promote apoptosis in response to cell damage and also to be activated in CE cell lines following exposure to UV radiation. Here we show in COS7 reporter cell lines that the expression of ferritin in a nuclear localization significantly down-regulates the JNK pathway (p = 5.7 × 10(-6)), but has no effect on the NFkB or the Erk pathways. In organ cultures of embryonic chicken corneas, we observed that inhibiting the synthesis of nuclear ferritin in CE cells, using the iron-chelating molecule deferoxamine, led to an increase in JNK signaling, as measured by phospho-JNK levels compared to CE cells with nuclear ferritin. Furthermore, the chemical inhibition of the JNK pathway using the molecule AS601245 decreased the production of nuclear ferritin. Taken together, these observations suggest that in CE cells a feedback-loop exists in which JNK signaling increases the production of nuclear ferritin and, in turn, nuclear ferritin decreases the activity of the JNK signaling pathway.

Effects of dietary quercetin on female fertility in mice: implication of transglutaminase 2.

Use of the dietary supplement quercetin is on the rise. Because previous studies imply an inhibitory effect of quercetin on male fertility, we explored the effects of this flavonoid on fertility in female mice. Birth outcomes, and ovarian morphology in 4-week-old offspring, were assessed in mice receiving dietary quercetin (5mgkg-1day-1) for 9 months during two breeding periods: from 2 to 6 months (prime reproductive age) and 8 to11 months of age. Quercetin increased birth spacing, leading to a 60% reduction in the number of litters, but enhanced folliculogenesis in ovaries of female offspring. While in young females quercetin caused an almost 70% increase in litter size, in older animals this effect was reversed. Consistent with the inhibitory activity of quercetin on the enzyme transglutaminase 2 (TG2), genetic ablation of TG2 in mice mirrors the effects of quercetin on birth outcomes and follicular development. Further, TG2-null mice lack responsiveness to quercetin ingestion. Our study shows for the first time that dietary quercetin can cause reduced reproductive potential in female mice and implies that TG2 may regulate ovarian ageing.

Wnt16 attenuates TGFß-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 TGFß is activated in MGP-null VSMCs and is necessary for spontaneous chondrogenesis of these cells in high-density micromass cultures. TGFß3-induced chondrogenic transformation in wild-type VSMCs associates with Smad2/3-dependent Wnt16 downregulation, but Wnt16 does not suppress TGFß3-induced Smad activation. In addition, TGFß3 inhibits Notch signaling in wild-type VSMCs, and this pathway is downregulated in MGP-null aortae. Exogenous Wnt16 stimulates Notch activity and attenuates TGFß3-induced downregulation of Notch in wild-type VSMCs, prevents chondrogenesis in MGP-null and TGFß3-treated wild-type VSMCs, and stabilizes expression of contractile markers of differentiated VSMCs. CONCLUSIONS: We describe a novel TGFß-Wnt16-Notch signaling conduit in the chondrocyte-like transformation of VSMCs and identify endogenous TGFß activity in MGP-null VSMCs as a critical mediator of chondrogenesis. Our proposed model suggests that the activated TGFß 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.

BMP2 cross-linked by transglutaminase 2 to collagen-plla scaffold promotes osteogenic differentiation in mesenchymal stem cells.

Transglutaminase 2 (TG2) was used to attach biologically-active BMP2 to collagen type I-coated poly-L-lactic acid (PLLA) nanofibrous scaffolds. Irreversibly cross-linked BMP2 retained its activity and induced Smad-dependent gene expression in cells seeded on PLLA-BMP2 scaffolds. These modified scaffolds promote osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) cultured in low-serum and growth factor free medium and support deposition of the calcified matrix and induction of the molecular osteogenic markers Runx2, osteopontin, osteonectin and bone sialoprotein. Importantly, the PLLA-BMP2 scaffolds did not support chondrogenic differentiation in hBMSCs as there was no expression of chondrogenic markers aggrecan, Sox 9, and collagen type II, and no deposition of cartilaginous glycosaminoglycan-rich matrix. Thus, TG2-mediated cross-linking of BMP2 to a scaffold is a novel approach to induce osteoblast-specific programming of hBMSCs in a spatially controlled manner.

Transglutaminase 2 promotes PDGF-mediated activation of PDGFR/Akt1 and ß-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 ß-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/ß-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 ß-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 ß-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 ß-catenin pathways in VSMCs. This study identifies TG2 as a potential therapeutic target for blocking neointima in blood vessels.

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-ß3 stimulation in vitro, and to characterize the associated alterations in cell signaling. METHODS AND RESULTS: Molecular analysis revealed activation of ß-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 ß-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.

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

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.

Induction of chondrogenic differentiation in mesenchymal stem cells by TGF-beta cross-linked to collagen-PLLA [poly(L-lactic acid)] scaffold by transglutaminase 2.

Transglutaminase-mediated cross-linking has been employed to optimize the mechanical properties and stability of tissue scaffolds. We have characterized tissue transglutaminase (TG2)-mediated cross-linking as a useful tool to deliver biologically-active TGF to mesenchymal stem cells (MSCs) and direct their differentiation towards a chondrogenic lineage. TGF-ß3 is irreversibly cross-linked by TG2 to collagen type II-coated poly(L-lactic acid) nanofibrous scaffolds and activates Smad phosphorylation and Smad-dependent expression of a luciferase reporter. Human bone marrow-derived MSCs cultured on these scaffolds deposit cartilaginous matrix after 14 days of culture at 50 % efficiency compared to chondrogenesis in the presence of soluble TGF-ß3. These findings are significant because they suggest a novel approach for the programming of MSCs in a spatially controlled manner by immobilizing biologically active TGF-ß3 via cross-linking to a collagen-coated polymeric scaffold.

Transglutaminase inhibitors attenuate vascular calcification in a preclinical model.

OBJECTIVE: In vitro, transglutaminase-2 (TG2)-mediated activation of the ß-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 ß-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 ß-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. CONCLUSIONS: Inhibition of the TG2/ß-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.

Warfarin can stimulate vascular calcification in vitro via activation of ß-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, ß-catenin activity is important because inhibition of ß-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 ß-catenin using the glycogen synthase kinase-3ß (GSK-3ß) inhibitor LiCl restores calcium accumulation, confirming that quercetin mechanism of action hinges on inhibition of the ß-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 ß-catenin-targeting strategy to prevent vascular calcification induced by warfarin and identify quercetin as a potential therapeutic in this pathology.

Implication for transglutaminase 2-mediated activation of ß-catenin signaling in neointimal vascular smooth muscle cells in chronic cardiac allograft rejection.

BACKGROUND: Cardiac allograft vasculopathy (CAV) remains the main cause of long-term transplant rejection. CAV is characterized by hyperproliferation of vascular smooth muscle cells (VSMCs). Canonical ß-catenin signaling is a critical regulator of VSMC proliferation in development; however, the role of this pathway and its regulation in CAV progression are obscure. We investigated the activity of ß-catenin signaling and the role for a putative activating ligand, transglutaminase 2 (TG2), in chronic cardiac rejection. METHODS: Hearts from Bm12 mice were transplanted into C57BL/6 mice (class II mismatch), and allografts were harvested 8 weeks after transplantation. Accumulation and sub-cellular distribution of ß-catenin protein and expression of several components of ß-catenin signaling were analyzed as hallmarks of pathway activation. In vitro, platelet-derived growth factor treatment was used to mimic the inflammatory milieu in VSMC and organotypic heart slice cultures. RESULTS: Activation of ß-catenin in allografts compared with isografts or naïve hearts was evidenced by the augmented expression of ß-catenin target genes, as well as the accumulation and nuclear localization of the ß-catenin protein in VSMCs of the occluded allograft vessels. Expression of TG2, an activator of ß-catenin signaling in VSMCs, was dramatically increased in allografts. Further, our ex vivo data demonstrate that TG2 is required for VSMC proliferation and for ß-catenin activation by platelet-derived growth factor in cardiac tissue. CONCLUSIONS: ß-Catenin signaling is activated in occluded vessels in murine cardiac allografts. TG2 is implicated as an endogenous activator of this signaling pathway and may therefore have a role in the pathogenesis of CAV during chronic allograft rejection.

Transglutaminase 2-mediated activation of ß-catenin signaling has a critical role in warfarin-induced vascular calcification.

OBJECTIVE: Accumulating experimental evidence implicates ß-catenin signaling and enzyme transglutaminase 2 (TG2) in the progression of vascular calcification, and our previous studies have shown that TG2 can activate ß-catenin signaling in vascular smooth muscle cells (VSMCs). Here we investigated the role of the TG2/ß-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 ß-catenin signaling and is independent of oxidative stress. The canonical ß-catenin inhibitor Dkk1, but not the Wnt antagonist Wif-1, prevents warfarin-induced activation of ß-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 ß-catenin in response to warfarin. Moreover, warfarin-induced calcification is significantly reduced on the background of attenuated TG2 both in vitro and in vivo. CONCLUSIONS: TG2 is a critical mediator of warfarin-induced vascular calcification that acts through the activation of ß-catenin signaling in VSMCs. Inhibition of canonical ß-catenin pathway or TG2 activity prevents warfarin-regulated calcification, identifying the TG2/ß-catenin axis as a novel therapeutic target in vascular calcification.

Developmental regulation of the nuclear ferritoid-ferritin complex of avian corneal epithelial cells: roles of systemic factors and thyroxine.

Previously we observed that avian corneal epithelial cells protect their DNA from oxidative damage by having the iron-sequestering molecule ferritin - normally cytoplasmic - in a nuclear location. This localization involves a developmentally-regulated ferritin-like protein - ferritoid - that initially serves as the nuclear transporter, and then as a component of a ferritoid-ferritin complex that is half the size of a typical ferritin and binds to DNA. We also observed that developmentally, the synthesis of ferritin and ferritoid are regulated coordinately - with ferritin being predominantly translational and ferritoid transcriptional. In the present study we examined whether the mechanism(s) involved in this regulation reside within the cornea itself, or alternatively involve a systemic factor(s). For this, we explanted embryonic corneas of one age to the chorioallantoic membrane (CAM) of host embryos of a different age - all prior to the initiation of ferritin synthesis. Consistent with systemic regulation, the explants initiated the synthesis of both ferritin and ferritoid in concert with that of the host. We then examined whether this systemic regulation might involve thyroxine - a hormone with broad developmental effects. Employing corneal organ cultures, we observed that thyroxine initiated the synthesis of both components in a manner similar to that which occurs in vivo (i.e. ferritin was translational and ferritoid transcriptional).

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.

Nuclear ferritin: a ferritoid-ferritin complex in corneal epithelial cells.

PURPOSE: Ferritin is an iron storage protein that is generally cytoplasmic. However, in embryonic avian corneal epithelial (CE) cells, the authors previously observed that the ferritin was predominantly nuclear. They also obtained evidence that this ferritin protects DNA from oxidative damage by UV light and hydrogen peroxide and that the nuclear localization involves a tissue-specific nuclear transporter, termed ferritoid. In the present investigation, the authors have determined additional properties of the nuclear ferritoid-ferritin complexes. METHODS: For biochemical characterization, a combination of molecular sieve chromatography, immunoblotting, and nuclear-cytoplasmic fractionation was used; DNA binding was analyzed by electrophoretic mobility shift assay. RESULTS: The CE nuclear ferritin complex has characteristics that differentiate it from a "typical" cytoplasmic ferritin, including the presence of ferritin and ferritoid subunits; a molecular weight of approximately 260 kDa, which is approximately half that of cytoplasmic ferritin; its iron content, which is below our limits of detection; and its ability to bind to DNA. CONCLUSIONS: Within CE cell nuclei, ferritin and ferritoid are coassembled into stable complex(es) present in embryonic and adult corneas. Thus, ferritoid not only serves transiently as a nuclear transporter for ferritin, it remains as a component of a unique ferritoid-ferritin nuclear complex.

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.

Nuclear ferritin in corneal epithelial cells: tissue-specific nuclear transport and protection from UV-damage.

We have identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form that is indistinguishable from the cytoplasmic form of ferritin found in other cell types. Thus it most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and other tissues were UV irradiated, and damage to DNA was detected by an in situ 3'-end labeling assay. Consistent with the hypothesis, corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than the other cells types examined. However, when the expression of nuclear ferritin was inhibited the cells now became much more susceptible to UV-induced DNA damage. Since ferritin is normally cytoplasmic, corneal epithelial cells must have a mechanism that effects its nuclear localization. We have determined that this involves a nuclear transport molecule which binds to ferritin and carries it into the nucleus. This transporter, which we have termed ferritoid for its similarity to ferritin, has at least two domains. One domain is ferritin-like and is responsible for binding the ferritin; the other domain contains a nuclear localization signal that is responsible for effecting the nuclear transport. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage. In addition, since ferritoid is structurally similar to ferritin, it may represent an example of a nuclear transporter that evolved from the molecule it transports (i.e., ferritin).

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 approximately 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).