Article |
Address correspondence to Elaine Fuchs, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Ave., Box 300, New York, NY 10021. Tel.: (212) 327-7953. Fax: (212) 327-7954. E-mail: Fuchs{at}rockefeller.edu
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Abstract |
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Key Words: Wnt signaling; epidermis; hair follicle; ß-catenin; morphogenesis
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Introduction |
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Recent studies have implicated members of the Wnt signaling pathway in the epithelialmesenchymal exchanges that specify morphogenesis of the hair follicle (for review see Hardy, 1992; Fuchs et al., 2001). In mouse, TOP-ßgalactosidase (TOPGAL) activity was detected in embryonic skin at sites of hair follicle formation, and postnatally, TOPGAL was active in the stem cell compartment (bulge) at the beginning of each hair cycle and also in the precursor cells that differentiate into hairs (DasGupta and Fuchs, 1999). The studies of Kishimoto et al. (2000) suggest that Wnts maintain the inductive ability of mesenchyme to stimulate hair cell fate at the start of each cycle (Kishimoto et al., 2000).
Evidence that Wnt signaling is functionally important for hair follicle morphogenesis stems from transgenic mice engineered to express a constitutively stable and active form of ß-catenin under the control of an epidermal keratin promoter (K14) (Gat et al., 1998). In epidermis, its expression spontaneously induced aberrant hair follicles throughout the interfollicular epidermis between the preexisting follicles (Gat et al., 1998). Similarly, in chick embryos, what appeared to be sustained activation of ß-catenin led to excessive feather and scale morphogenesis (Noramly et al., 1999; Widelitz et al., 2000). Conversely, ablation of Lef1 gene expression in mouse skin or ß-catenin expression in K14-expressing cells of mouse skin impairs hair follicle morphogenesis (van Genderen et al., 1994; Huelsken et al., 2001). Based on these findings, Wnt signaling seems to coax skin stem cells along a hair (epidermal appendage) cell fate. Consistent with this notion is the finding that the hair-specific keratin promoters are bona fide target genes of Lef/ß-catenin signaling (Zhou et al., 1995; Merrill et al., 2001). Additionally, concomitant with the development of a hair placode is an upregulation in transcription of both Lef1 and ß-catenin genes (Zhou et al., 1995; Noramly et al., 1999; Widelitz et al., 2000; Huelsken et al., 2001).
Although Wnt signaling seems to promote hair cell fates, an absence of or interference with Wnt signaling seems to favor epidermal and sebocyte cell fates. Thus, a K14-conditional loss of function mutation in the ß-catenin gene locus resulted in a blockage of embryonic hair development if lost early and formation of epidermal-like cysts in place of hair follicles if lost later (Huelsken et al., 2001). In addition, transgenic mice expressing K14-NLef1, unable to bind ß-catenin, yielded epidermal and sebocyte differentiation at sites where postnatal hair cycling was expected (Merrill et al., 2001; Niemann et al., 2002).
The picture emerging from these studies suggests that setting the levels of ß-catenin plays a crucial role in deciding if stem cells or their progeny will contribute to structures below the bulge (hair cell types) or above the bulge (sebaceous gland, upper outer root sheath [ORS], and epidermis). These findings have prompted us to wonder whether epidermal and hair follicle cells are truly equivalent with respect to their internal ability to translate a Wnt signal, or whether there might be important internal differences that might be overridden and obscured by overexpressing or ablating ß-catenin in mice. If, as recent evidence implies, stem cell progeny migrate from the bulge and specify cell fates in different environments (Taylor et al., 2000; Oshima et al., 2001), do they maintain equivalency with respect to their ability to respond to a stabilized ß-catenin signal, or does this chemistry change? Is it simply differences in Wnt/Wnt receptors, or are there distinct differences in the way hair and epidermal cells respond to increased ß-catenin expression?
Placed in a broader context of cell lineage determination, the heart of this problem focuses on the extent to which the levels of ß-catenin's varied interacting partners might differ in cells and how this impacts on the manner in which the cell responds to a Wnt signal and/or stabilized ß-catenin. The skin becomes an interesting model system to tackle this problem, because not only is the hair versus epidermal fate manipulated by overexpression or underexpression of ß-catenin, but in addition, at least some of ß-catenin's associates, including Lef1/Tcf levels (Zhou et al., 1995; DasGupta and Fuchs, 1999; Merrill et al., 2001) and intercellular junction proteins (Nanba et al., 2000) are known to be differentially expressed in these two cell types.
To determine the extent to which hair and epidermal cells might differ in their internal ability to process and respond to a given level of ß-catenin, we sought a strategy for manipulating ß-catenin signaling in a way in which we would expect to see the same phenotypic outcomes in mouse epidermal and hair cells if they responded equivalently, but different outcomes if they harbored distinctly different means of receiving the same signal. Rather than stabilizing ß-catenin and seeing hair follicle morphogenesis in epidermis as well as hair follicles (Gat et al., 1998), or ablating ß-catenin and seeing epidermal differentiation in hair follicles as well as in epidermis (Huelsken et al., 2001), we searched for conditions in which epidermal cells responded in one way and hair cells in another. We settled on engineering mice to express a COOH terminally truncated form of the constitutively stabilized version of ß-catenin that we had used earlier in generating our super-furry mice (Gat et al., 1998).
The COOH terminus of ß-catenin is required for transactivation, and it binds the transcriptional activator p300/CBP as well as the chromatin remodeling factor Brg-1 (Molenaar et al., 1996; Orsulic and Peifer, 1996; van de Wetering et al., 1997; Huber et al., 1997; Hsu et al., 1998; Hecht et al., 1999, 2000; Barker et al., 2001). Indeed, ß-catenin mutants lacking the COOH terminus can be deficient in signaling not only in vitro, but also in vivo (Orsulic and Peifer, 1996; Cox et al., 1999; Hecht et al., 1999; Vleminckx et al., 1999; Barker et al., 2001; Tutter et al., 2001). This said, prior studies on ß-catenin signaling in Xenopus and in Drosophila had demonstrated that overexpression of membrane-tethered or NH2/COOH terminally truncated forms of ß-catenin could potentiate signaling depending on its interacting partners within the cell. This led to the hypothesis that these forms of ß-catenin might act by displacing endogenous ß-catenin from intercellular junctions, freeing it to be utilized in transactivation (Miller and Moon, 1997; Cox et al., 1999).
By expressing a single protein with potentially dominant negative and dominant positive character, we find that different cells within the skin behave unidirectionally towards this protein. However, strikingly and ironically, whereas the follicle stem cells and their progeny respond in a dominant negative fashion to the transgene product, the epidermal cells respond in a dominant positive fashion. Moreover, we show that by changing transgene expression midstream in the process, the process becomes reversible, and cells can revert back to their original lineage. Our findings have important and broad implications for how cells respond to and translate Wnt/ß-catenin signals and how cell type specificity and cell fate commitment is achieved.
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Results |
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Similar to that seen previously for TOPFLASH (Gat et al., 1998), Nßcatenin and Lef1 on their own each had only a modest effect on TOPGAL reporter activity, but together, they strongly transactivated (Fig. 1 C). These effects were dependent on the presence of functional Lef1 binding sites, as evidenced by the lack of activation with FOPGAL mutant in the Lef1 binding sites (Fig. 1 C). In contrast, Lef1 and
N
Cßcatenin together exhibited only slightly higher levels than Lef1 alone, although the activity was consistently above background (Fig. 1 C). Thus, despite the fact that
N
Cßcatenin lacks the transactivating domain, some activation was still observed when it was coexpressed with Lef1 in keratinocytes. We will return to this point again later.
When equal amounts of Nßcatenin and
N
Cßcatenin were expressed together with Lef1,
N
Cßcatenin suppressed the activation of the TOPGAL reporter by twofold (Fig. 1 D). Similar inhibitory effects were observed with Tcf3, although because Tcf3 normally functions as a repressor, this was more difficult to demonstrate (unpublished data). The dominant negative effects of
N
Cßcatenin were expected, given that
N
Cßcatenin still contained the armadillo repeats, able to interact with the Tcf/Lef1 DNA binding proteins (Huber et al., 1997; Graham et al., 2000; Tutter et al., 2001). Thus, irrespective of which Lef1/Tcf family member
N
Cßcatenin was combined with,
N
Cßcatenin impaired the transactivation potential conferred to by stabilized ß-catenin.
Expression of N
Cßcatenin in the epidermis, follicle ORS, and stem cell compartment of transgenic mice
Given the ability of N
Cßcatenin to act in a dominant negative fashion in interfering with the activity of functional Lef1/ßcatenin complexes in keratinocytes in vitro, we then turned towards using this transgene to assess the consequences of diminished Wnt signaling on hair organogenesis and postnatal cycling. For purposes of comparison to the previously generated K14-
Nßcatenin and K14-Cre conditional ß-cateninnull mice, we used the K14 promoter to drive the expression of
N
Cßcatenin in the dividing cells of the epidermis, the ORS, and the stem cell compartment of hair follicles.
K14-N
Cßcatenin mice were generated in the CD-1 strain, and PCR of tail DNAs confirmed the identity of mice that carried the transgene. Expression of
N
Cßcatenin protein was confirmed by immunoblot analysis of proteins isolated from whole newborn skin of transgenic and control littermate animals, as well as from primary keratinocytes derived K14-
N
Cßcateninexpressing mice. In transgenic keratinocytes in vivo and in vitro, the transgenic protein of
66.5 kD was detected by the anti-HA antibody (Fig. 2 A). Immunofluorescence analysis using the HA antibody on frozen skin sections confirmed the expression of the transgene specifically in the basal epidermal layer (Fig. 2 B) and the follicle ORS and the bulge (Bu) (Fig. 2, B' and C, schematic of follicle). Two independently derived mouse lines behaved similarly in this and all subsequent analyses, thereby attributing the observed effects to transgene expression and not chromosomal integration site.
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The outermost layer of cells surrounding the follicular cysts exhibited morphological features typical of the basal layer of the epidermis (Fig. 4, compare A, epidermis, with B, cyst). Progressively more inward, the cyst resembled terminally differentiating epidermis. Large suprabasal-like cells were seen within the range between the outer basal-like layer and the middle of the cyst, whereas cells packed with keratohyalin granules were seen further inward. Enucleated squamous-like cells were found at the cyst center. Immunofluorescence with antibodies against a variety of epidermal differentiation markers confirmed the epidermal character of the cysts (Fig. 4, C and D). The basal-like cells at the periphery of the cyst were positive for K5 and K14, the suprabasal cells stained with antibodies against the spinous layer marker keratin 1 (K1), and the granular cells labeled with antibodies against filaggrin and loricrin, typically seen in the epidermal granular layer.
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Overall, the marked morphological and biochemical resemblance of these cysts to masses of differentiating epidermal cells paralleled the description of the cysts seen when ß-catenin was conditionally ablated in K14-expressing cells of mice (Huelsken et al., 2001) and when transgenic mice were engineered to express a K14-NLef1 transgene, encoding a form of Lef1 unable to associate with ß-catenin (Merrill et al., 2001; Niemann et al., 2002). In contrast to the striking effects on the hair cycle, the skin epidermis of K14-
N
Cßcatenin mice was only modestly affected (Fig. 4 A). The patterns of K5, K1, and filaggrin expression in transgenic epidermis appeared largely normal (unpublished data), although the spinous layer and granular layers were somewhat thickened in some areas. The increased thickening was attributed to enhanced proliferation, as judged by staining with an antibody against Ki67, present in the nuclei of proliferating basal epidermal cells (unpublished data). These changes aside, the overall spatial and temporal pattern of epidermal differentiation was largely similar between WT and K14-
N
Cßcatenin transgenic skin, and in some areas, no major differences were noted (Fig. 3, D and E).
Expression of N
Cßcatenin generates a failure to initiate Lef1/Tcf/ßcatenin-mediated signaling in the bulge
Given the similarities between the epidermal cysts of N
Cßcatenin and ß-cateninnull skin, and the inhibitory effects of
N
Cßcatenin on Lef1/Tcf/ß-cateninregulated gene transcription in vitro, it seemed most likely that the cysts arose from a failure to activate this transcription pathway. To test this, we mated our previously generated TOPGAL transgenic mice on the background of the
N
Cßcatenin transgenic mice, and examined the double transgenic offspring for their ability to transactivate TOPGAL. In normal TOPGAL mice, a few cells within the bulge of many hair follicles expressed ß-galactosidase at the start of the first postnatal hair cycle, when the mesenchymal dermal papilla was abutted against the epithelial stem cell compartment (Fig. 5 A; DasGupta and Fuchs, 1999). When bred against the background of our previously generated K14-
Nßcatenin transgenic mice, very strong TOPGAL activity was observed in most bulge cells undergoing hair cycle initiation (Fig. 5 B). In striking contrast, the bulges at the equivalent stage of
N
Cßcatenin transgenic follicles were usually devoid of ß-galactosidase activity (Fig. 5 C). The finding that the cells in the bulge compartment of
N
Cßcatenin expressing follicles were silent for TOPGAL activity and resulted in epidermal cyst formation provided compelling evidence that a loss of Tcf/Lef1/ß-cateninmediated signaling activity in the bulge leads or contributes to the acquisition of epidermal cell fates.
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Overall, the effects of N
Cßcatenin on epidermal cells was as a positive stimulator of ß-catenin signaling, thereby activating the hair cell fate and eventually leading to hair cell tumors, or pilomatricomas in all transgenic mice. This was in marked contrast to the transgene's effects as an inhibitor of ß-catenin signaling in the follicle precursor cells, where the outcome was epidermal cyst formation. The opposing effects were at least in part due to differences in transcriptional activation of Lef/Tcf/ß-cateninregulated genes, as revealed by the fact that the epidermal cysts at sites anticipated for hair follicles were TOPGAL negative, whereas the follicle-like structures at sites anticipated for epidermis were TOPGAL positive. Most remarkably, the opposing effects of the transgene were not random, but rather were displayed in a highly cell typespecific fashion.
N
Cßcatenin is able to interact with E-cadherin and with members of the ß-catenin degradation machinery
Previous studies have shown that N
Cßcatenin can associate with Lef/Tcf family members, and our in vitro studies show that it can influence transactivation potential of Lef-regulated genes in keratinocytes. To assess how
N
Cßcatenin is capable of having positive effects on epidermal cells at the same time as it has negative effects on hair cells, it was necessary to confirm that this protein is also able to associate with candidate cytoplasmic partners, including the adherens junction protein E-cadherin and APC, the protein that targets ß-catenin for the proteosome degradation pathway.
Cell lysates from skin and calcium-treated primary keratinocytes and from skin of WT and transgenic mice were first adjusted to have equal levels of E-cadherin in the starting lysate extracts for WT and transgenic (Fig. 7). AntiE-cadherin antibody was then used in a pulldown assay to bind E-cadherin complexes to protein Gconjugated Sepharose beads. These complexes contained both HA-tagged N
Cßcatenin and endogenous ß-catenin (Fig. 7). In skin and in primary keratinocytes, the overall E-cadherin pulled down in the assays was consistently higher in the transgenic versus the WT samples, despite the roughly comparable levels in the aliquots used for pulldown.
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The ratio of nuclear N
Cßcatenin to endogenous ß-catenin is lower in epidermal than in hair cells, and yet K14 promoter activity is higher in epidermal than in hair cells
Without a COOH-terminal activation domain, ß-catenin is unable to function with Lef1/Tcf family members to transactivate genes. To understand how N
Cßcatenin might be eliciting positive effects on interfollicular and upper ORS epithelium, we first examined the location of
N
Cßcatenin when transiently expressed in keratinocytes. To distinguish transgenic from endogenous ß-catenin, we used an anti-HA antibody to recognize the epitope tag of the transgene product and an antiß-catenin antibody directed against the COOH terminus of ß-catenin to specifically recognize the endogenous ß-catenin protein by immunofluorescence. The results are summarized in Fig. 8. Whereas only
25% of primary keratinocytes were transfected,
40% of them exhibited nuclear as well as cytoplasmic localization of the transgene product (Fig. 8 A,
N
Cßcat in green). Interestingly, 100% of transfected cells exhibited nuclear localization of endogenous ß-catenin (Fig. 8, A and B, endogenous ß-catenin in red). Under the low-calcium conditions used here, endogenous ß-catenin in untransfected and transfected cells was always cytoplasmic. However, in stark contrast to the nuclei of transfected cells, the nuclei of untransfected cells were negative for endogenous ß-catenin. These findings suggest that by expressing
N
Cßcatenin protein in keratinocytes, endogenous ß-catenin is translocated to the nucleus, a point which we confirmed by immunoblot analyses (unpublished data). Moreover, the data demonstrate at a molecular level that the transgene product can displace endogeous ß-catenin from the cytoplasmic pool, and stimulate a process that results in its accumulation in the nucleus. The generation of nuclear ß-catenin in transfected cells is likely to explain why our transfections with
N
Cßcatenin always gave a slight enhancement of TOPGAL activity when coexpressed with Lef1 (Fig. 1 C).
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In WT epidermis in vivo, ß-catenin is normally restricted to cellcell borders, and is not found in the cytoplasm or nucleus (Fig. 8 D). However, in the hair follicle, cytoplasmic and nuclear ß-catenin are readily detected in the hair progenitor cells, referred to as precortex (Fig. 8 E). The precortex (Fig. 8, arrowheads) is where activation of Lef1/ß-catenin regulated genes, including the hair-specific keratins and TOPGAL, typically occurs (DasGupta and Fuchs, 1999; Merrill et al., 2001).
The pattern of endogenous ß-catenin was dramatically different in N
Cßcatenin epidermis (Fig. 8 F). In addition to cellcell border staining, which was seen with antibodies against both the HA-tagged
N
Cßcatenin gene product and endogenous ß-catenin, cytoplasmic staining was also prominent, particularly for endogenous ß-catenin (Fig. 8 F). In the TOPGAL-positive, flower-like downgrowths of
N
Cßcatenin skin, nuclear ß-catenin was also detected (Fig. 8 G, arrowheads). In these downgrowths, the ratio of FITC (transgene product) to Texas red (endogenous ß-catenin) was always higher at cell borders than in the nuclei (Fig. 8 G', inset, higher magnification). The preferential detection of endogenous nuclear ß-catenin was consistent with the presence of Lef1 and TOPGAL activity in these downgrowths.
Interestingly, in the early stages of epidermal cyst formation, we occasionally captured follicle-like structures where intense anti-HA staining, reflective of N
Cßcatenin, was detected in the nuclei of the precortex (Fig. 9 E). At these early stages, HA nuclear staining was sometimes also seen in the bulge compartment (Fig. 9 F). These findings are consistent with the dominant negative action of
N
Cßcatenin in transcriptional regulation and with our failure to detect TOPGAL activity in the bulge or the precortex of the
N
Cßcatenin follicles. In epidermal cysts, peripheral basal-like cells showed intense border staining for the K14 promoter-driven transgene product, paralleling the intensity seen in the K14-positive epidermal basal layer and the ORS (Fig. 9 D, arrows). In these regions, some nuclei stained with HA antibodies (Fig. 9 D'', inset, arrows; higher magnification of such HA-positive nuclei). The more central, spinous-like cells did not display nuclear HA staining, and in fact expressed considerably less
N
Cßcatenin, consistent with the switch from K5/K14 to K1/K10 and the downregulation of K14 promoter activity in these layers. These results provide additional evidence that expression of
N
Cßcatenin in the basal-like cells of developing cysts is high and able to block the transactivating functions of ß-catenin.
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Interestingly, despite considerable ß-catenin in the cytoplasm of spinous epidermal cells (e.g., Fig. 8 F), AE13 and TOPGAL staining was not seen in differentiating epidermis. Thus, although the epidermal cysts appeared morphologically and biochemically similar to epidermis, they retained a certain level of atypical plasticity, and were able to revert midstream from an epidermal to a hair program of terminal differentiation.
To summarize the data presented in Figs. 8 and 9, there were marked differences in the relative levels of N
Cßcatenin and endogenous ß-catenin in the nuclei of different epithelial cells within the skin of the transgenic mice. The ratio of nuclear
N
Cßcatenin to endogenous ß-catenin seemed to be highest in hair follicle cells and lowest in epidermal basal cells and upper ORS cells. These differences correlated well with whether the transgene acted in a negative or positive fashion to influence TOPGAL activity. These differences also correlated well with the type of epithelial cell within the skin and its relative stage of differentiation along a particular lineage. The differences did not correlate with K14 promoter activity.
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Discussion |
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In ß-cateninnull skin, plakoglobin is able to associate with E-cadherin to assemble adherens junctions (Aberle et al., 1996; Huber et al., 1997). In N
Cßcatenin skin,
N
Cßcatenin and endogenous ß-catenin both localized to adherens junctions. Despite these differences in armadillo proteins at cellcell junctions, the ß-cateninnull and
N
Cßcatenin mice displayed similar epidermal cysts, suggesting that the cysts arose from a failure to activate ß-catenin/Lef1/Tcfregulated downstream target genes, rather than changes in intercellular junctions per se. Consistent with this notion is our finding that TOPGAL activation was largely silent during the postnatal hair cycles that led to epidermal cyst formation at the expense of hair differentiation. These findings were in good agreement with, and extended those of, Huelsken et al. (2001) and Niemann et al. (2002).
Our in vitro and in vivo assays tell us that elevated levels of Nßcatenin, but not
N
Cßcatenin, can activate TOPGAL in the stem cell compartment of the hair follicle. The failure to transactivate ß-catenin/Lef/Tcfregulated genes in the bulge of K14-
N
Cßcatenin mice did not appear to affect the downgrowth of the secondary hair germs. However, the process of their subsequent development into hair follicle was severely perturbed.
The nuclear HA staining and reduced endogenous ß-catenin staining of the precortical cells of the K14-N
Cßcatenin secondary hair germs provided an explanation for the dominant negative effects of the transgene on hair cells. However, the strong HA staining could not be explained by K14 promoter activity, which is actually very low in matrix and precortical cells (Byrne et al., 1994; Wang et al., 1997). Moreover, the strong nuclear HA staining could not be explained by a simple canonical Wnt signaling mechanism, which should have increased nuclear levels of endogenous ß-catenin without affecting levels of
N
Cßcatenin. Rather, the preferential concentration of nuclear
N
Cßcatenin over endogenous ß-catenin seemed to be a reflection of a novel mechanism that either specifically stabilizes ß-catenin through its armadillo repeats or preferentially translocates the truncated ß-catenin to the nucleus. Although the molecular nature of the underlying mechanism remains to be explored, its existence is one that seemed to occur preferentially in the bulge, matrix and precortex, i.e., cells known to express members of the Tcf/Lef1 family of HMG proteins.
Molecular insights into N
Cßcatenin's gain of function phenotypes in the epidermis and upper ORS of the skin
An important and revealing feature of our N
Cßcatenin transgenic mice was the surprising existence of a gain-of-function phenotype within the interfollicular and upper ORS epithelium of the skin. By all criteria examined, these
N
Cßcatenin-expressing cells behaved analogously to the equivalent cells of the
Nßcatenin mice. Thus, whereas
N
Cßcatenin elicited a dominant negative effect on keratinocytes in vitro and on skin epithelial stem cells and follicle cells in vivo, it elicited a positive effect on interfollicular and upper ORS epithelium. The interfollicular and upper ORS epithelium are thought to be derived from the stem cells in the bulge. How can we account for the remarkable difference between stem cells and their progeny in the way they respond to this single transgene protein?
Several key observations help us to understand this paradox. One important clue is that despite the inability of N
Cßcatenin/Lef/Tcf complexes to transactivate Lef1/Tcf regulated genes on their own, TOPGAL was ectopically activated in the epidermis and the ORS at sites of hair germ like invaginations. Conversely, as detected through TOPGAL activity, normal Lef/Tcf/ß-catenin regulated gene activity was impaired in the bulge and precortical cells of postnatal transgenic follicles. Because the transgene protein lacks a transactivation domain, its positive actions on TOPGAL activity must then be indirect. In this regard, our in vitro studies revealed endogenous ß-catenin in the nuclei of
N
Cßcatenintransfected cells, and in vivo, nuclear endogenous ß-catenin was seen in areas where TOPGAL was activated.
How then does N
Cßcatenin lead to generation of critical threshold levels of stabilized ß-catenin in interfollicular epidermal cells and in the upper ORS, but not in the bulge or in the precortex? Our analyses left us to conclude that it is a property inherent to the character of each epithelial skin cell type that determines how the cell will respond to
N
Cßcatenin. Thus, ironically, hair folliclelike invaginations were restricted to transgenic upper ORS and epidermis, whereas epidermal transdifferentiation was restricted to transgenic cells that normally would adopt a hair cell fate.
At sites of epithelial downgrowth in the interfollicular epidermis or upper ORS, the pools of cytoplasmic and nuclear endogenous ß-catenin seemed to increase. One way in which this might be achieved is that in epidermis, N
Cßcatenin localizes strongly to cellcell junctions, where it may displace endogenous ß-catenin and raise its cytoplasmic pool. Localization of the transgene protein to cellcell junctions sequesters it, physically restraining it from interfering in the nucleus. Intriguingly, cellcell junctions are fewer in hair follicle precursor cells (Nanba et al., 2000), perhaps increasing the likelihood that
N
Cßcatenin will enter the nuclei of these cells and act to impair Lef1/Tcf gene activity.
Another contributing factor is likely to be N
Cßcatenin's association with APC (for review see Bienz, 1999; Mimori-Kiyosue and Tsukita, 2001). Through
N
Cßcatenin-mediated sequestering of APC or other members of the ß-catenin degradation machinery, endogenous ß-catenin could be stabilized. The preferential concentration of endogenous ß-catenin in the nuclei of cells at epithelial downgrowths suggests that
N
Cßcatenin may preferentially associate with either or both of these interacting partners.
Our transient transfection experiments with cultured keratinocytes demonstrate that even under conditions where cellcell junctions do not form, endogenous ßcatenin from the cytoplasm can translocate to the nucleus when cells express the transgene. This finding is compatible with a displacement mechanism that involves N
Cßcatenin-mediated sequestration of either a component of the proteosome degradation machinery or a component that keeps endogenous ß-catenin from entering the nucleus. APC has been implicated in both processes, and its ability to associate with transgenic and endogenous ß-catenin makes it a good candidate for explaining these observations. Overall, our in vitro and in vivo observations corroborated similar studies in Xenopus (Miller and Moon, 1997) and in Drosophila (Cox et al., 1999), where positive actions of a membrane-tethered form of ß-catenin also prompted investigators to posit that competition for E-cadherin and APC may displace endogenous ß-catenin into the nucleus. A related parallel stems from altering E-cadherin levels, which also results in phenotypes consistent with sequestering signaling-competent ß-catenin away from the nucleus (Orsulic et al., 1999; Ciruna and Rossant, 2001).
Another intriguing phenomenon that is likely relevant to our understanding of Lef1/Tcf/ß-catenin action in skin epithelium is the elevated Lef1 mRNA and protein expression which occurs in epithelial invaginations transgenic for either Nßcatenin (Gat et al., 1998) or
N
Cßcatenin (present study). This feature suggests the existence of a positive feedback mechanism that could lead to Lef1 transcription when ß-catenin levels are stabilized in epidermis. Because the Lef1 promoter contains functional Lef1/Tcf binding sites (Hovanes et al., 2001 and references therein), it is possible that displacement of endogenous ß-catenin frees it to associate with small amounts of Lef1 which could then initiate this cycle of upregulated Lef1 gene expression. Further experiments will be necessary to fully understand this mechanism. In addition, recent studies on E-cadherin have also provided new insights into how altering the levels of adherens junction proteins may modulate Wnt-mediated signaling in morphogenetic processes (Orsulic et al., 1999; Ciruna and Rossant, 2001).
A number of other ß-catenin interacting proteins have been identified in recent years, and a priori, some of these proteins may also be involved in accounting for the unexpected transactivating-like phenotype associated with N
Cßcatenin in skin. Consistent with this notion are transcriptional repressor proteins such as XSox17, XSox3, and the Smad proteins, which have been shown to physically interact with ß-catenin (Zorn et al., 1999; Schohl and Fagotto, 2002), and in this regard, the transgene protein might act to titrate out some of these repressors. In contrast, the repressor proteins reptin52 and pontin52 have recently been identified as COOH terminally interacting ß-cateninassociated repressor proteins, which would render them insensitive to the transgene protein (Bauer et al., 2000; Etard et al., 2000; Takemaru and Moon, 2000). However, for these proteins to be involved,
N
Cßcatenin would have to function by relief of ß-catenin/Tcf-mediated transcriptional repression, a mechanism previously invoked to explain certain positive phenotypes obtained with the
N
Cßcatenin mutant (Funayama et al., 1995; Miller and Moon, 1997). This seems unlikely for epidermis because: (a) the nuclear ß-catenin seen in the activated cells was mostly endogenous rather than transgene product; (b) the epidermis and upper ORS do not normally express appreciable levels of Lef1/Tcf family members; and (c) the Lef/Tcf member activated at these sites was Lef1, acting primarily as a coactivator and not a repressor. However, repression might still be relieved if endogenous nuclear ß-catenin competes for the binding of Tcf/Lef repressor proteins, e.g., Grg/Tle (Merrill et al., 2001).
Reconciling two opposing actions from the same transgene
Ironically, by expressing N
Cßcatenin, the epidermal-like cells ended up generating hair folliclelike phenotypes, whereas the hair folliclelike cells ended up generating epidermal-like phenotypes. These findings imply that the behavior of
N
Cßcatenin in epithelial cells of the skin is highly tailored to the particular stem cell lineage and stage of differentiation of the skin cell. The final outcome of epithelial skin cell fate then appears to be dictated not only by the levels and location of ß-catenin in a cell, but also the relative levels of other ß-catenin interacting factors in the cell. An interesting point that supports this conclusion is that by downregulation of transgene expression in differentiating epidermal cysts, the former hair cells turned epidermal cells reverted back to their hair cell fate, activating hair keratin gene expression. Such reversibility of cell fates was not observed in the bona fide epidermis, indicating that suppression of nuclear ß-catenin in a hair precursor cell required sustained transgene expression to be maintained, whereas epidermal precursor cells have some intrinsic mechanism to prevent this from happening.
Given the complexities of ß-catenin's interacting partners, a detailed picture of how epidermal and hair follicle precursor cells regulate intracellular levels of ß-catenin is beyond the scope of the present study. However, based on these new findings, we can already develop models that are consistent with our present knowledge of cellular differences in hair and epidermal precursor cells and that enable us to begin to envisage how such differences might influence the way in which these might respond to the transgene protein. In summary, the ability of N
Cßcatenin to act in both positive and negative fashions in a highly specialized and cell differentiationspecific fashion has uncovered a fascinating new frontier underlying the complex ways in which different cells receive and respond to changes in ß-catenin.
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Materials and methods |
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For analyses of cells where the transgene had integrated and was expressed at levels not lethal to the animal, we isolated primary keratinocytes from transgenic and control littermates and cultured them as previously described (Vasioukhin et al., 2000).
Extraction of proteins
For cultured cells: 100-mm plate of confluent cells was washed 2x in PBS. The cells were lysed with 500 µL either mild lysis buffer (1% Triton X-100 in PBS with 10 mM EDTA, 0.3 mg/mL freshly made PMSF, and protease inhibitor cocktail) or RIPA buffer (1% Triton X-100 in PBS with 10 mM EDTA, 150 mN NaCl, 1% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail; Roche]). Cells were scraped off the plate and transferred into a 1.5-mL eppendorf tube. The cell suspension was sonicated 3 x 15 s and centrifuged at 14,000 rpm at 4°C. The supernatant-soluble fraction was transferred into a fresh tube and equal volume of 2x sample buffer was added to it. The pellet was dissolved directly in 2x sample loading buffer and treated as the insoluble fraction.
For skin tissue: Frozen tissue was pulverized in a liquid Nitrogen-cooled Gevebesmascher and the powder scraped into a chilled microfuge tube. RIPA buffer (described above) was added to the tube and samples were processed as described above. Instead of adding the sample buffer, the protein extracts were stored at -80°C for future use in co-IPs and Western blotting.
Coimmunoprecipitation studies
Primary keratinocytes grown on tissue culture dishes were washed in ice-cold PBS. RIPA buffer was added to each plate and the cells scraped off and collected in a tube. Soluble fractions were isolated as described in the previous section. The soluble extracts were incubated with 50 µL protein-G Sepharose beads for 1 h at 4°C, to preclear the lysate. The samples were centrifuged for 1 min and transferred into a fresh microfuge tube. The protein samples were incubated with primary antibody (-E-cadherin 5 µL and
-APC 10 µL) O/N at 4°C. The antibodyE-cadherin/antibody-APC complex, was retrieved by incubating with 50 µL protein-G Sepharose for 1 h, at 4°C. The immunoprecipitated E-cadherin or APC was washed three times with cold lysis buffer and immediately used for immunoblotting. The same protocol was followed for coimmunoprecipitation studies of skin protein extracts as described above.
For immunoblotting, the following primary antibodies were used: -Ecad (1:1,000, Eccd2; Zymed);
-APC (1:1,000, C-20, Santa Cruz Biotechnology);
-HA (1:1,000; Roche Biochemicals). HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) and ECL (chemiluminescent solution) were used for detecting the bound antibody.
Generation of K14-N
Cßcatenin construct
A PCR strategy was used to create the COOH-terminal deletion on the K14-N87ß-catenin construct. The PCR product was flanked by SpeI site (contained within the ß-catenin cDNA) at the 5'-end and XbaI site at the 3'-end. It was first cloned into pCRII-Blunt, TOPO vector (Invitrogen) for sequence confirmation. The fragment was then excised by SpeI-XbaI double digestion and subcloned into the K14-
N87ß-catenin construct cut with SpeI-XbaI (which eliminates the COOH terminus of ß-catenin). Sequences of the primers used for the PCR reaction in mentioned in the list of primers. The primers used were SpeI-
CF.1 and
CR.1-HA-XbaI. cDNA sequence encoding the HA-tag was included in the reverse primer.
Immunohistochemistry
For immunofluorescence, 810-µm sections of frozen tissue were fixed in 4% PFA, treated with blocking solution (5% heat-inactivated normal goat serum, 1% BSA in 1x PBS), and incubated with the primary antibody (diluted in the blocking solution + 0.1% Triton X-100), as described previously (DasGupta and Fuchs, 1999). Fluorescein- or rhodamine-conjugated secondary antibodies (obtained from Jackson ImmunoResearch Laboratories) were used to detect primary bound antibody. Results were visualized under the Zeiss 410 confocal microscope or the Zeiss Axiophot (Zeiss) for fluorescent microscopy.
For immunofluorescence, the primary antibodies used were -Ecadherin (1:1,000, Eccd2; Zymed);
-HA (1:100; Roche);
-ß-catenin (1:1,000; 15
8; Sigma-Aldrich);
-K1 (1:250; Babco);
-filaggrin (1:1,000; Babco);
-AE13 (1:5; a gift from Dr. T.T. Sun [New York University, New York, NY]);
-AE15 (1:5; a gift from Dr. T.T. Sun);
-Lef1 (1:250; lab generated); and
-Tcf3 (1:100; lab generated). Rhodamine- or fluorescein-conjugated secondary antibodies (1:100; Jackson ImmunoResearch Laboratories) were used to detect the primary antibodies bound to their respective targets.
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Footnotes |
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* Abbreviations used in this paper: APC, adenomatous polyposis coli; ORS, outer root sheath; TOP, Tcf-optimal-promoter; TOPGAL, TOP-ßgalactosidase; WT, wild-type.
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Acknowledgments |
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This work was supported by a grant from the National Institutes of Health.
Submitted: 24 April 2002
Revised: 20 May 2002
Accepted: 24 May 2002
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References |
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