Retinoic Acid-induced Epidermal Transdifferentiation in Skin

Retinoids function as important regulatory signaling molecules during development, acting in cellular growth and differentiation both during embryogenesis and in the adult animal. In 1953, Fell and Mellanby first found that excess vitamin A can induce transdifferentiation of chick embryonic epidermis to a mucous epithelium (Fell, H.B.; Mellanby, E. Metaplasia produced in cultures of chick ectoderm by high vitamin A. However, the molecular mechanism of this transdifferentiation process was unknown for a long time. Recent studies demonstrated that Gbx1, a divergent homeobox gene, is one of the target genes of all-trans retinoic acid (ATRA) for this transdifferentiation. Furthermore, it was found that ATRA can induce the epidermal transdifferentiation into a mucosal OPEN ACCESS 159 epithelium in mammalian embryonic skin, as well as in chick embryonic skin. In the mammalian embryonic skin, the co-expression of Tgm2 and Gbx1 in the epidermis and an increase in TGF-β2 expression elicited by ATRA in the dermis are required for the mucosal transdifferentiation, which occurs through epithelial-mesenchymal interaction. Not only does retinoic acid (RA) play an important role in mucosal transdifferentiation, periderm desquamation, and barrier formation in the developing mammalian skin, but it is also involved in hair follicle downgrowth and bending by its effect on the Wnt/β-catenin pathway and on members of the Runx, Fox, and Sox transcription factor families.


Introduction
Skin is composed of an epidermis, which is an epithelium derived from ectodermal tissue, and an underlying dermis, which is a connective tissue derived from mesenchyme of mesodermal origin.During the formation of skin and its appendages, e.g., feathers, scales, and hair, the epithelium, and mesenchyme are inducers and targets of each other [1].Retinoic acids (RAs) including all-trans, 9-cis, and other derivatives, an active metabolite of retinol, regulate cell proliferation, differentiation, and morphogenesis during normal development of the skin [2][3][4][5][6][7].When human keratinocytes are grown on their dermal equivalent (fabricated collagen lattices), physiologic concentrations (1-10 nm) of RA result in an epithelium very similar to that in normally keratinized epidermis; but a higher concentration (>0.1 μm) of RA reduces epidermal maturation and produces parakeratosis, and a deficiency of RA leads to hyperkeratosis [8].Similar to the results obtained from keratinocyte cultures, retinol deficiency in the rat can cause squamous metaplasia and keratinization in a wide variety of nonkeratinized and secretory epithelia [9,10]; and excess retinol can induce epidermal mucous metaplasia in skin cultured from chick embryos [11].An amount of 16.7 μm of ATRA induces glandular metaplasia in mouse embryonic upper-lip skin instead of hair vibrissa follicle [12,13].Many of the abnormalities in pattern formation and organ formation that result from the addition of exogenous RA during embryogenesis are related in part to the ability of retinoids to change the pattern of expression of the clusters of homeobox genes in the embryo [14][15][16][17].Many homeobox genes have been shown to change their expression in the skin as a response to RA [18][19][20].It was demonstrated over a decade ago that Gbx1 (gastrulation brain homeobox 1), a divergent homeobox gene, is one of the target genes of RA for the mucous transdifferentiation [21].Our recent study found that 1 μm ATRA can induce mucous transdifferentiation in the mammalian embryonic skin as well as in chick embryonic skin.The molecular mechanism of this transdifferentiation was examined in the mammalian embryonic skin.Interestingly, ATRA-induced expression of Tgm2 (transglutaminase 2) and Gbx1 in the epidermis and that of TGF-β2 expression in the dermis are required for the mucosal transdifferentiation [22].Other than the transdifferentiation, RA also plays an important role in barrier formation and hair follicle formation in skin [6,7,[23][24][25][26][27].This review highlights our current understanding of the role of retinoic acid in the epidermal transdifferentiation of skin and its appendages.

Effects of RA on Morphogenesis of Chick Embryonic Skin
RA is known to regulate cell proliferation, differentiation, and morphogenesis during the normal development of many tissues [16,28].Two cutaneous structures of chick, i.e., scales and feathers, have been studied extensively to define the mechanism of retinoid-induced morphogenetic change [11,14,15,20,[29][30][31]. Dhouailly et al. (1980) showed that the single injection of 125 μg (417 μm) of ATRA into the amniotic cavity of chick embryos at embryonic Hamburger-Hamilton (HH) stage 36 (day 10), which correspond to the beginning of scale morphogenesis, leads to the formation of feathers on chick foot scales [29].An amount of 2.5 μm of RA also modulates axis orientation and phenotypes of skin appendages [14].Using chick embryonic skin cultured in vitro, we examined the mechanism of the mucous transdifferentiation induced by RA and showed that in chick embryonic tarsometatarsal skin, this transdifferentiation to a mucous epithelium is induced by the interaction of the epidermis with the dermis, when dermal fibroblasts are pretreated with retinol (Figure 1) [21,[32][33][34][35]. Furthermore, 1 μm ATRA inhibits feather bud formation and induces the transdifferentiation of the epidermis of chick embryonic dorsal skin to a mucous one [36].In the mucous transdifferentiated epidermis, PAS-positive materials, mucous granules, and a discontinuous basement membrane are observed (Figure 1).The discrepancy between the results of Dhouailly et al. (1980) and ours may be ascribed to the difference in the RA concentrations used, the processing time, chicken embryonic stages, and culture conditions (in ovo or in vitro).

Effects of RA on the Expression of Homeobox Genes in Chick Embryonic Skin
Many of the abnormalities in pattern formation and organ formation that result from the addition of exogenous RA during embryogenesis are related in part to the ability of retinoids to change the pattern of expression of the clusters of homeobox (Hox) genes in the embryo [14,17].Hox genes are master control genes that specify the body plan and regulate the development and morphogenesis of higher organisms [37].Apart from these classic homeobox genes, there are other groups of homeobox genes (divergent homeobox genes), which are located outside the Hox loci and also play an important role in regulating growth and differentiation during the development of many organs.Using a degenerate RT-PCR (reverse transcriptase-polymerase chain reaction)-based screening method, we previously isolated divergent homeobox genes Gbx1 [21], Hex (currently Hhex) [38][39][40], and HB9 (currently Mnx1) [41,42] from chick embryonic tarsometatarsal scale skin in addition to Hox genes.Furthermore, among the many homeobox genes isolated, Gbx1 shows a strong increase in expression in the epidermis of tarsometatarsal scale skin during the course of retinol-induced epidermal transdifferentiation to mucosal epithelium; and its expression is induced by the interaction of the epidermis with dermal fibroblasts pretreated with retinol [21].
In the feather bud, homeobox genes other than Gbx1 are reported to be expressed, such as Gbx2 [43]; Msx-1 and Msx-2 [19], HB9 [42]; Hox b-4, Hox a-7, and Hox c-8 [44]; Dlx 2, 3, 5 [45]; and Hex [39,40].Gbx2 is a homeobox gene closely related to Gbx1 and is required for proper segregation of early regional identities anterior and posterior to the mid-hindbrain boundary in the case of vertebrates [46].[11].Epidermal mucous metaplasia of 13-day-old chick embryonic tarsometatarsal skin (HH stage 39) can be induced by culturing the skin with excess retinol for only 8 h and then without retinol for six days [34], after which the mucous granules and a discontinuous basement membrane are observed.Retinol primarily affects the dermal cells [32,35], which then transform the epidermal cells into mucus-secreting cells [33,34], suggesting the importance of epithelial-mesenchymal interaction in retinol-induced epidermal mucous metaplasia.
Studies on Gbx1 have concentrated on the brain or neurons.In mice, during development Gbx1 is expressed in the central nervous system [47,48]; and combined expression of Lhx7 and Gbx1 plays a role in the development of the cholinergic system of the basal forebrain [46].In zebrafish, Gbx1 and Otx2 are expressed in the neuroectoderm [48].It was recently shown for the first time that the Gbx1 gene is also expressed in the feather bud of chick embryonic dorsal skin [36].Treatment of organ cultures of chick embryonic dorsal skin with 1 μm ATRA for 24 h induces transdifferentiation of the epidermis to mucosal epithelium with a concomitant increase in Gbx1 mRNA expression in the epidermis.
We showed that treatment of organ cultures of chick embryonic dorsal skin with 1 μm ATRA at HH stage 31 or 33 inhibits feather-bud formation and induces transdifferentiation of the epidermis to a non-keratinized stratified mucous epithelium concomitant with an increase in Gbx1 expression as microvilli develop on the upper surface of the epithelium [36].In addition, a well-developed Golgi apparatus and PAS-positive materials are observed in the treated epidermis.These results are consistent with our previous reports stating that epidermal mucous transdifferentiation is induced by RA in chick embryonic tarsometatarsal skin at HH stage 39 along with an increase in Gbx1 expression and that typical goblet cells are induced in it by retinol [21,32,34].
Modulation of the axis orientation of feather buds by treatment with 1 μm ATRA throughout the culture period results in a higher frequency of small feather buds and many buds showing random orientations [14].However, when 1 μm ATRA was used for treatment for only one day, along with 10 nm hydrocortisone which induces the differentiation (keratinization) of epidermis [57], and the skin was cultured for an additional four days without these chemicals, a different result was obtained.ATRA did not modulate the axis orientation, but rather caused the transdifferentiation of the epidermis to a mucous epithelium [36].Thus RA has important roles in transdifferentiation, as well as in modulation of pattern formation.

Effects of Gbx1 on Chick Skin
As ATRA induces Gbx1 mRNA expression in the epidermis, we also examined whether Gbx1 alone (without ATRA) could induce epidermal transdifferentiation. Feather buds were elongated by transient transfection of the dorsal epidermis with Gbx1 cDNA, indicating stimulation of epidermal cell proliferation by Gbx1; however, no transdifferentiation was induced [36].These results suggest that Gbx1 alone does not induce mucous transdifferentiation and that other signals playing a key role cooperatively with Gbx1 are involved in the epidermal to mucous transdifferentiation.

ATRA Induces Transdifferentiation of Rat Embryonic Epidermis to Mucosal Epithelium with Up-Regulation of Esophageal Markers MUC4 and Keratin 4
To determine the molecular mechanism of the transdifferentiation induced by ATRA, we established a culture system using rat embryonic skin in which ATRA induces epidermal transdifferentiation that is accompanied by the expression of markers of esophageal epithelium.We examined what genes are up-regulated and down-regulated in the 16E rat embryonic skin by ATRA.Representative genes up-regulated after 24 h ATRA treatment are shown in Table 1.The remainder of the up-regulated genes is shown in Supplementary Table 1.The down-regulated genes are shown in Supplementary Table 2. Bioinformatic pathway analysis of the microarray data identifies the up-regulated and down-regulated genes involved in retinol signaling pathway.Stra6 (stimulated by retinoic acid gene 6), Cyp2b3, d1 and Cyp26a1, b1 (cytochrome P450, family 2 and 26), Lrat (lecithin-retinol acyltransferase), and Ugt2b (UDP glycosyltransferase 2 family, polypeptide B) are increased in expression at the mRNA level (Table 1 and Supplementary Table 1), while Adh7 (alcohol dehydrogenase 7), Cyp1a1, Cyp2c12, Cyp3a23/3a1, and Ugt1a1(UDP glucuronosyltransferase 1 family, polypeptide A1) are decreased (Supplementary Table 2).Other than these up-regulated genes, not only Tgm2 but also Mucin 4 (MUC4) and keratin 4 (K4), which are markers of esophageal cells, are increased in expression at the mRNA level [22].K4 and MUC4 are expressed in non-keratinized squamous stratified epithelium and in respiratory and digestive epithelium, respectively [58,59].Cytokeratin, an intermediate filament observed mainly in epithelial cells, is an essential cytoskeleton component involved in the maintenance of cell morphology.The cytoskeleton of epithelia is formed by 20 subtypes of cytokeratins whose expression depends primarily on the epithelial cell type and degree of differentiation [60].As keratin filaments, e.g., tonofibrils, K1 and K10 are not at all observed throughout the epidermis; but both K4 and MUC4 proteins are expressed in RA-induced transdifferenti ated epithelial cells, but not in control epidermal cells [22].According to the results by electron microscopic and immunofluorescent analyses on tonofibrils, we consider that all the cells in the epidermis are induced by ATRA to undergo transdifferentiation.This transdifferentiation of whole epidermal tissue to mucosal tissue occurs without any gene transfection and requires only a one-day treatment with ATRA.Thus, this is an exciting model for induction of tissue organization, regeneration, and transdifferentiation because the generation of these transdifferentiated cells is fast and efficient.

ATRA Increases Expression of Tgm2 and Gbx1 mRNA and Protein in Rat Embryonic Epidermis
We focused on three genes, i.e., Tgm2, Gbx1, and TGF-β, because we [21] and others [61,62] showed these genes are induced by RA.At first we examined the expression of two of them, Tgm2 and Gbx1, in rat embryonic skin.The expression of both Tgm2 and Gbx1 mRNAs is increased in the epidermis of the skin cultured for one day with ATRA and then for four days without RA [22].The expression of both Tgm2 and Gbx1 proteins is also increased throughout the epidermis cultured for one day in the presence of ATRA and then for an additional four days in its absence.Tgm2 protein is detected in the cytoplasm, nuclei, and the area of cell adhesion in the intermediate and upper layers of the epidermis, reflecting the multifunctional nature of this protein [63][64][65][66]; whereas the expression of Gbx1 protein is seen mainly in the nuclei of the epidermal skin cells.Interestingly, in situ transamidase activity of Tgm2 is not detected in RA-treated Tgm2 protein [22], suggesting that the enzymatic activity might be dispensable and that other potential functions of Tgm2 [63] (discussed below) might be involved in transdifferentiation.In fact, RA-induced transdifferentiation is not inhibited by a transamidase inhibitor, ZM449829 [67], in the range of 100-300 nm (unpublished data).
To examine the function of Tgm2 and Gbx1 in the epidermis, we transfected the epidermis with these genes by electroporation.Epidermal keratinization and expression of K10, which is specifically expressed in the epidermis, are inhibited.In addition, rounded cells in the upper layer of the epidermis are observed in the skin by overexpression of both Tgm2 and Gbx1 [22].However, epidermal keratinization is observed in the skin by overexpression of either Tgm2 or Gbx1 [22].However, neither K4 nor MUC4 expression is observed in the skin overexpressing both Tgm2 and Gbx1 genes [22].Thus these findings indicated that co-expression of Tgm2 and Gbx1 in the epidermis is required for ATRA-induced mucous transdifferentiation but that Tgm2 or Gbx1 alone cannot induce esophagus-like mucosal transdifferentiation. RA is a consistent inducer of Tgm2 expression in various cells and tissues [68][69][70].In mammalian epidermal cells, 3 μM RA induces tissue transglutaminase (Tgm2) [71].Tgm2 knock-out mice do not have any defects in their keratinocyte differentiation program [72].Tgm2 is the most diverse and ubiquitous enzyme of the transglutaminase family [73] and has been implicated in diverse processes such as inflammation, wound healing, apoptosis, neurodegenerative disorders, and cancer [63,74,75].The most characteristic enzyme function of the transglutaminase family is calcium-dependent transamidation activity, resulting in the formation of ε-(g-glutamyl) lysine cross-links between proteins and, thus, polymerization.Tgm1, Tgm3, and Tgm5 of the transglutaminase family are found in mammalian keratinocytes and play an important role in the formation of the stratum corneum in the skin by the introduction of cross-links between proteins [76][77][78].In addition to its transamidation activity, Tgm2 functions as a signal-transducing GTP-binding protein [64] and as a protein-disulfide isomerase that regulates adenylate cyclase [66].
Hence, Gbx1 may be a key regulator of epithelial differentiation in addition to its known role in brain development.We showed immunohistochemically that the expression of Gbx1 protein starts to increase in the nuclei of all cells of the epidermis after one day in culture.The expression of Gbx1 mRNA in the epidermis is up-regulated not only after 8 h of treatment with RA, but also by the physical interaction of untreated skin with the RA-pretreated dermal fibroblasts in which these fibroblasts actively migrate into the dermis, suggesting that Gbx1 expression would appear to be regulated by some unidentified factor in the dermis (Figure 2) [21,22].

Involvement of TGF-β Signaling Pathway in ATRA-Induced Epidermal Transdifferentiation to Mucous Epithelium in Rat Embryonic Skin
RA added to cultures of human scalp hair follicles increases the expression of TGF-β in the dermal sheath, dermal papilla, and hair follicle [62].In addition to Tgm2 and Gbx1, ATRA also increases the expression of both TGF-β mRNA and protein in the dermis of rat embryonic skin, but does so only slightly in the epidermis [22].As ATRA increases the expression of TGF-β in the dermis, this finding suggests that the interaction between epidermis and dermis might have an important role in epidermal transdifferentiation to a mucous epithelium.Furthermore, SB431542, a specific inhibitor of ser/thr kinase of the TGF-β type II receptor [79], and pan-TGF-β1, -β2, -β3 mAb, a neutralizing antibody, partially suppress the expression of K4 and MUC4, indicating that the TGF-β signaling pathway is involved in ATRA-induced epidermal transdifferentiation to a mucous epithelium (Figure 2).RA up-regulates the expression of Tgm2 and TGF-β in the epidermis and dermis, respectively, after 8-24 h of treatment, which is followed by the appearance of a discontinuous basement membrane.On the other hand, Gbx1 expression is up-regulated in the epidermis after 8 h by an unknown factor, which is induced in the dermis by RA [21].The TGF-β signaling pathway activated by TGF-β and acting together with Gbx1 induces epidermal transdifferentiation to an esophagus-like mucosal epithelium [22] (reproduced with permission from Int.J. Dev.Biol.2011, 55, 933-943).

Development of Skin in Retinoid Signaling Deficient Mice
RA is commonly used in the treatment of skin diseases such as acne and psoriasis, as well as in chemotherapy for leukemia.A frequent adverse effect of these therapies is RA-induced hair loss [62,80].The mechanisms to explain how excess RA arrests hair follicle growth have been explored [27,62].ATRA induces a catagen-like stage in human hair follicles, presumably via up-regulation of TGF-β2 in the dermal papilla, resulting in hair loss [62].In a mouse model which knocked out cytochrome P450, family 26, subfamily b, polypeptide 1 (Cyp26b1), which encodes an RA-degrading enzyme, the in vivo administration of RA to pregnant mice or its addition to skin cultures demonstrated that appropriate RA levels are important for periderm desquamation, embryonic skin differentiation, and barrier formation in the developing mammalian skin [26].Furthermore knock-out mouse lacking Cyp26b1 in their skin exhibit major hair follicle development defects [27].By acting on the Wnt/β-catenin pathway and on members of the Runx, Fox, and Sox transcription factor families, RA modulates pathways and factors implicated in hair follicle downgrowth and bending [27].In the human and mouse cicatricial alopecia and alopecia areata, retinoid metabolism is altered and genes involved in RA synthesis increases, while RA degradation genes are decreased [81,82].Appropriate RA metabolism is needed for the normal formation of the hair follicle [6,27,81,82].
Neutral lipid synthesis enzyme acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) functions as the major acyl-CoA:retinol acyltransferase (ARAT) in murine skin.When dietary retinol is abundant, DGAT1 deficiency results in elevated levels of RA in skin and cyclical hair loss [88].Deletion of DGAT1 specifically in the epidermis causes alopecia.DGAT1 functions as an ARAT in the skin, where it acts to maintain retinoid homeostasis and modulate RA signaling [88].

Concluding Remarks
RA plays important roles in skin development by affecting signaling pathways and the expression of many genes including homeobox genes.RA induces epidermal transdifferentiation in chick and rodent embryonic skin.We identified Gbx-1, Tgm2 and TGF-β as the key target genes regulated by ATRA during transdifferentiation. Thus far we don't have any information on which isoforms of RARs and RXRs are directly involved in the ATRA-induced transdifferentiation, and to what extent are 9-cis retinoic acid and non-genomic signals, i.e., posttranslational modification, important to the transdifferentiation.These issues remain to be explored.An embryonic skin culture system where RA induces epidermal transdifferentiation provides a useful model in which RA can direct cell reprogramming to another cell.As the generation of these transdifferentiated cells is fast, efficient, and devoid of tumorigenic pluripotent stem cells, these cells can provide a novel system for regenerative medicine.
Further study is required to elucidate the role of RA in the skin and skin appendage development by identifying additional developmental signaling pathways and transcriptional regulatory proteins regulated by RA.

Figure 1 .
Figure 1.Schematic diagram summarizing the effect of RA on epidermal differentiation of tarsometatarsal chick embryonic skin.Excess retinol can induce epidermal mucous metaplasia (transdifferentiation) in cultured skin obtained from chick embryos[11].Epidermal mucous metaplasia of 13-day-old chick embryonic tarsometatarsal skin (HH stage 39) can be induced by culturing the skin with excess retinol for only 8 h and then without retinol for six days[34], after which the mucous granules and a discontinuous basement membrane are observed.Retinol primarily affects the dermal cells[32,35], which then transform the epidermal cells into mucus-secreting cells[33,34], suggesting the importance of epithelial-mesenchymal interaction in retinol-induced epidermal mucous metaplasia.

Figure 2 .
Figure 2. Schematic diagram summarizing the effect of RA on epidermal mucosal transdifferentiation.RA up-regulates the expression of Tgm2 and TGF-β in the epidermis and dermis, respectively, after 8-24 h of treatment, which is followed by the appearance of a discontinuous basement membrane.On the other hand, Gbx1 expression is up-regulated in the epidermis after 8 h by an unknown factor, which is induced in the dermis by RA[21].The TGF-β signaling pathway activated by TGF-β and acting together with Gbx1 induces epidermal transdifferentiation to an esophagus-like mucosal epithelium[22] (reproduced with permission from Int.J. Dev.Biol.2011, 55, 933-943).

Table 1 .
List of representative genes up-regulated in 16E rat embryonic dorsal skin after 24 h incubation with 1 μm retinoic acid (RA) (Fold change).