E-cadherin roles in animal biology: A perspective on thyroid hormone-influence

E-cadherin function control via EC

The mature classical E-cadherin is a single-pass transmembrane glycoprotein with five ectodomains (EC1–5), each made up of ~110 amino acid residues. Their NH2-terminal ectodomain mediates adhesive binding to cadherins present on the surfaces of neighboring cells. Their four interdomain junctions are characterized by three calcium binding sites ─DXD, DRE, and DXNDNAPXF sequence motifs─ [207]. Cadherin function requires calcium to rigidify the EC domain [27, 112, 146] and to stabilize the protein [156, 159] avoiding proteolysis [79, 190], and allowing its proper localization [57, 156, 159].

E-cadherin mediates principally in cell-cell homophilic interactions through self-recognition of the conserved histidine-alanine-valine (HAV) sequence within EC1 [14] (Fig. 1), and a strand-swapping interface. In this surface, a Tryptophan in position 2 (W2/Trp2) inserts into a hydrophobic pocket from another cis (from the same cell surface) or trans (from an adjacent cell) cadherin [100, 111, 182]. Although another cis dimerization has been postulated [15], several models disagree on the roles and numbers of the inner EC domains involved in cadherin homophilic interaction [31, 66, 112, 160].

Cleavage of the ~130 amino acids prodomain of immature cadherin represents the switch from a non adhesive to the functional form [67]. Even though the specific E-cadherin endoprotease has not been identified yet [145], the E-cadherin proprotein contains a furin-cleavable motif [SSPGLRRQKR] (Fig. 1) [73, 136], and sequence specificity for other mammalian convertases [161]. In contrast, mature E-cadherins are inactivated by cleavage in the EC domain mediated by matrix metalloproteinases [37] and others proteases [89, 153], in a process known as ectodomain shedding, thus promoting the invasion [44, 122, 187] (Fig. 1).

In addition, it has also been reported that E-cadherins can adhere heterophilically to integrins αEβ7 and α2β1 [30, 176, 205], killer-cell lectin-like receptors G1 [173], and numerous infectious agent proteins that target E-cadherins as an entry receptor [5, 36, 132].

E-cadherin function control via CD

In active functionally adhesion complexes E-cadherin associates with intracellular components forming AJs. E-cadherin CD interacts with several cytoplasmic proteins, being the catenins the best understood: β-catenin or γ-catenin, α-catenin, and p120-catenin (p120-ctn) [41, 128, 133, 134, 165]. A homolog of β-catenin, γ-catenin/plakoglobin [152], can substitute it under some circumstances [38]. When they are fully incorporated in complexes with cadherins, these three catenins associate with a stoichiometry of one of each catenin per cadherin molecule [90, 147]. β-catenin binds directly to the distal ~72 amino acids of the E-cadherin CD through a 30-amino acid “core”, anchoring indirectly to α-catenin (Fig. 1) [76]. P120-ctn binds independently to the ~29 amino acid membrane-proximal region of the cadherin CD [165, 199] (Fig. 1). α-Catenin associates with the actin cytoskeleton [167, 214] and numerous adaptor proteins to strengthen cell-cell adhesion [1, 199].

Several cleavage fragments of E-cadherin CD and disassembling of their adhesive complexes have been reported. During apoptosis or calcium influx, presenilin 1 (PS1)/γ-secretase cleaves between human E-cadherin residues Leu731 and Arg732 [127] and caspase-3 cleaves on site 747-DTRD-750 and releases a 25-kDa fragment [93, 183]. In addition, during tumoral progression calpain mediates E-cadherin proteolysis [168] (Fig. 1).

It has been proposed that while β-catenin and plakoglobin facilitate indirect interactions between classic cadherins and the actin cytoskeleton at AJs in vivo, p120-ctn subfamily members induce the lateral (cis) clustering of cadherins [209], and the tethering of signaling or regulatory entities, such as kinases and phosphatases [118]. P120-ctn also stabilizes cadherins at the cell membrane by modulating cadherin membrane trafficking (endocytosis-recycling) and degradation [21, 139, 211]. Catenins are modified by kinases and/or phosphatases that are enriched at cell-cell contacts [6, 42, 52, 118, 203]. E-cadherins, in turn, are normally maintained in a tyrosine-dephosphorylated state through the action of phosphatases that are crucial for stabilizing AJs [51, 135]. In some cell contexts, phosphatase inhibition promotes release of E-cadherins and β-catenins from cell-cell contacts, enabling cytoplasmic catenins to relocate and function in the nucleus and promoting E-cadherin proteolysis [86]. Like phosphatase inhibition, receptor tyrosine kinase stimulation disassembles cell contacts mediated by cadherin─β-catenin─α-catenin [18, 203]. In addition, p120-ctn is an important modulator of RhoGTPase activities, such as RhoA, Rac1 and Cdc42 [3, 4, 142] and gene transcription [43, 95, 149, 162, 180]. On the other hand, p120-ctn interacts with tubulin influencing microtubule stability and dynamics, and thereby affects cell motility and directional migration independently of the cadherin adhesion system [81, 170] (Fig. 2).

As for α-catenin, it modulates the actin-cytoskeleton organization because it can occur either in a monomeric or a homodimeric form [13, 50, 65]. Upon the application of force on epithelia, the conformational change of monomeric αE-catenin uncovers the vinculin binding site, allowing vinculin to bind, and recruit additional F-actin to the cadherin-catenin complexes [126, 214]. Additionally, ZO-1, spectrin, vinculin [199] and Eplin [1] assemble F-actin to E-cadherin-catenin complexes. In parallel, the non-junctional cytosolic homodimeric αE-catenin pool inhibits the Arp2/3 complex, reducing membrane dynamics by preventing F-actin branch-formation [13].

Establishment of adherens junctions

Epithelial AJs are built on a foundation of homophilic contacts between (E or P)-cadherin clusters on the surface of adjacent epithelial cells [62, 117]. Cadherins alone are not sufficient for AJ formation; rather, cooperation between nectins and cadherins is required [166, 188] (Fig. 3). Appearently, the trans-interacting nectin inhibits non-trans-interacting E-cadherin endocytosis through afadin, Rap1, and p120-ctn, thereby further non-trans-interacting E-cadherin accumulates in the nectin-based cell-cell adhesion sites for AJ formation [74]. Once AJs have been established through intracellular partner binding to the cytoskeleton, E-cadherin contacts modulate actin filament organization at the underlying cortex [10, 50, 155] and microtubules network [130, 131].

It is known that catenins, including β-catenins, α-catenins, and p120-catenins, bind to immature E-cadherins while traveling through the endoplasmic reticulum and Golgi apparatus [40, 71]. Following synthesis in the rough endoplasmic reticulum and phosphorylation of the cytoplasmic domain, the E-cadherin pro-region is cleaved by furin proteases in the trans-Golgi network, an event that is mandatory for the mature cadherin to function in adhesion [59, 75, 116] (Fig. 2). Via golgin-97 dependent tubulovesicular carriers, E-cadherins leave the Golgi complex [124], and subsequently fuse with an intermediate recycling endosome, in route to the basolateral plasma membrane. This pathway for E-cadherins trafficking is tightly integrated with other proteins involved in epithelial polarity, such as Sec5, 6, and 15 [107, 193], Rab 11 [123], PALS (Protein Associated with Lin Seven 1), and aquaporin 3 (AQP3) [137], and it determines lumen formation [49] (Fig. 2).

The function and regulation of E-cadherins post-translational modifications in vivo have remained poorly defined. While the phosphorylations to eight serine residue cluster within a region that binds β-catenin modulate the β-catenin affinity and strengthen cell–cell adhesion [181], the E-cadherin cytoplasmic O-glycosylation (O-GlcNAc) blocks its cell surface transport, reducing intercellular adhesion [218]. In addition, E-cadherins have four consensus sites Asn-X-Ser/Thr for N-glycosylation in EC4 and EC5 domains [102], most of them conserved among species (Fig. 1). It has been established that, while the N-glycans at Asn 633/483 are essential for E-cadherin folding, trafficking, and proper expression [217], their modification with complex N-glycans weakens AJs [121, 157] (Fig. 2).

Signaling and maintainance of adherens cell-cell junctions

Chemical [3, 85, 86, 149] and mechanical [110, 214] signals from cell-cell junctions can be transduced through E-cadherin-CD via β-, α- and p120-catenin to the cytoplasm or nucleus. Mechanical stimulation generated by tumor cell proliferation leads to β-catenin phosphorylation via Src kinase at the site of its interaction with E-cadherin, increasing β-catenin nuclear localization, and upregulating the oncogenes Myc and Twist1 [204]. In physiological state, the formation of junctional complexes triggers activation of the phosphatidylinositol-3-kinase (PI3K)–Akt–protein kinase B pathway [151] because the PI3K-p85 subunit associates with AJs through direct binding to β-catenin [206] (Fig. 3). After recruitment of PI3K, phosphatidylinositol-(3,4,5)-triphosphate (PIP3) is generated, and guanine nucleotide exchange factors, that contain PIP3-binding pleckstrin homology domains, are recruited to the membrane and activate Rac1 [105, 210] or Cdc42 [94]. This stimulates membrane actin dynamics adjacent to the initial contact site, increasing the probability of additional E-cadherin engagements. α-Catenins, in turn, homodimerizes and are released from the cadherin─catenin complexes to bind at actin and antagonize Arp2/3 function, inhibiting actin branching and facilitating belt formation of unbranched actin filaments. Simultaneously, other actin-binding proteins such as vinculin, afadin and α-actinin link with actin cytoskeleton, and β-catenin and p120-ctn link with tubulin cytoskeleton to route vesicles of E-cadherin─catenin to the cell-cell contact sites. Meanwhile, PtdIns(3,4,5)P3 accumulation in the membrane signals for the formation and expansion of the basolateral surface, whereas Rac1 promotes polarity orientation and lumen formation. In tissue and cell-type specific contexts, Rac1 also triggers the activation of downstream signaling effectors, promoting cell cycle arrest of confluent epithelial cells, and survival of polarized epithelial cells [169]. Therefore, cadherins function in tissue morphogenesis by controlling both cell-cell adhesion and cell signaling. In this way, cadherins are involved in determining cell shape, position, migration [33, 68, 92, 125, 186], polarity [16, 200] and proliferation [96, 172], as well as tissular folding [201, 202]. In adherens junctions, E-cadherins and catenins determine cell-cell contact survival and epithelia function [11].

AJs are very dynamic structures that undergo constant remodeling. This can be low-scale remodelling involving replacement of individual or groups of molecules within the adhesive clusters without disrupting steady-state intercellular adhesions. It can also be large-scale junctional rearrangements that accompany breakdown and reformation of cell-cell contacts [82].

Growth factors (GFs) as specific regulators of E-cadherin─catenin traffic

GFs are responsible for crosstalk between cell proliferation, migration, and adhesion. GFs bind to their specific receptor, cause cell-cell dissociation coupled to E-cadherin endocytosis and recycling back to the cell surface by several mechanisms [22, 23, 148] (Fig. 3). Co-regulation of cadherins and GF signaling is prominent in epithelial to mesenchymal transitions (EMT) and tumorigenesis [20, 88]. E-cadherins, in turn, modulate signal transduction by interacting with receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR) [29, 163]. Among them, thyroid hormones (THs) modulate energy metabolism, having a great influence on growth and development by independent mechanisms [54]. An increasing number of studies show the THs action in different parallel signaling pathways via membrane receptors, cytoplasmic partners and thyroid hormone receptors (TRs) [72, 106]. TRs heterodimerize with retinoid X receptors (RXRs) and bind to T3 response elements (TRE) located within the genomic regions of target genes [212]. In the absence of T3, TRs interact with co-repressor proteins to inhibit TH-regulated target gene transcription (Fig. 4). Following T3 binding, co-repressors are displaced and co-activator proteins are recruited to the ligand-bound TR complex, so as to facilitate T3-dependent activation of the target genes. Complexity increases because the diploid organisms have THRA and THRB genes that encode the TRα and TRβ isoforms respectively, which are ubiquitously expressed [109, 175, 212, 213]. Moreover, depending on species, tissue or experimental systems, there are predominant TR cell isoforms, and each gene can generate different proteins using different promoters and/or alternative splicing [109, 175, 213]. In addition to functions mediated by TRs, THs also exert rapid non-genomic actions that are initiated at the cell membrane. For example, the αvβ3-integrin binds to THs, activating the mitogen-activated protein kinase (MAPK) cascade [45, 119] for modulating the membrane ion channels, Na⁺/K⁺ exchanger and Ca²⁺ATPase, as well as the actin cytoskeletal components [99] (Fig. 4).

Among genomic mechanisms, it is known that transcriptional repression of E-cadherins is mediated either by promoter CpG hypermethylation [60] or activation of repressors binded at E-boxes or brain-specific silencers, such as Snail and Slug [194], Twist [208] and E12/E47 (E2A gene product) [154]. In contrast, the miR-200 family directly targets repressors ZEB1 and ZEB2, promoting E-cadherin expression upregulation [103, 104] (Fig. 1). In hormonal signaling routes, it is known that the activated androgen receptor binds to promoter and also represses E-cadherin gene expression, promoting metastasis by EMT [120].

Conversely, our results suggest that T3-TRs promote E-cadherin, β- and α-catenin gene expression in vivo and EMT inhibition [56, 85]. E-cadherin locus up-regulation can be mediated by an enhancer element at intron 1 [12, 25, 69, 70]. In addition, Stemler Stemmler et al. [185] reveal a complex mechanism of gene regulation at mouse E-cadherin locus intron 2. While in differentiated epithelia intron 2 sequences are required both to initiate transcriptional activation and additionally to maintain E-cadherin expression during embryogenesis, the level of intron 2-dependent E-cadherin expression is relative to the tissues and developmental timing. Thus, early embryogenesis requires intron 2 for the onset of expression, but at later stages, a second mechanism initiates E-cadherin expression independently of intron 2, although for high-level expression the support of the intron 2 enhancer elements is still required. The onset of the second wave of expression was detected in the surface ectoderm differentiate to form skin, and in the gut endoderm (around E12.5). A locus control region (LCR), in turn, might influence gene activity for proper activation and downregulation, sited upstream of cadherin clusters [184, 185] with a vital role for the large intron 2 (Fig. 1). Striking, cadherin superfamily genes display a higher average total intron number and significantly longer introns than other genes and across the entire vertebrate lineage [144]. Particularly, the human genome has an uncommon high frequency of MIR and MaLR regulatory-associated repetitive elements at 5’-located introns, concomitant with increased de novo intronic transcription. Therefore, these intronic-specific sites may constitute targets of cadherin superfamily expression regulation, both in homeostasis and illness.

Thus, searching for some of those physiological needs to sustain epithelial life, we have analyzed cell adhesion molecule response to 3,5,3’-triiodothyronine (T3), detecting morphometric evidences of gene upregulation exerted by T3 on E-cadherin, β- and α-catenin expression in different epithelial cell types of the metamorphosing anuran foregut [85]. Coincidentally, mouse β-catenin gene upregulation and transrepression by TH-TR [61, 158], as well as the impact of TH signaling in development, homeostasis and cancer susceptibility of mouse intestine [178] have been reported. T3-TRα1 binds directly to β-catenin gene-intron 1 specific TRE in the intestine, increasing its expression in an epithelial cell-autonomous way [158] (Fig. 5). This is parallel to positive regulation of proliferation-controlling genes, such as type D cyclins and c-myc, which are known targets of the Wnt/β-catenin pathway [158], synergizing Wnt pathway and inducing crypt cell proliferation and promoting tumorigenesis [179]. In contrast, CTNNB1 transrepression is mediated by binding of the TRβ-RXR complexes on TREs located in the human promoter between −807 and −772 (Fig. 5) [61]. Therefore, liganded TRβ acts as a tumor suppressor via inhibition of the expression of a potent tumor promoter, the CTNNB1 gene.

Even though TH signaling controls the proliferation of the intestinal epithelial progenitors in both amphibians and mammals, it has been suggested that TH control on the Wnt/β-catenin pathway does not appear to play a central role in amphibians [24, 178]. However, our in vivo experiments contradict this hypothesis, since we have detected early upregulation (24 hs) of E-cadherin, β- and α-catenin genes in the Xenopus laevis gut [56]. During metamorphic climax, larval cell apoptosis coexists with pre-adult (juvenile) cell proliferation and differentiation, and with cell-cell junction assembly─disassembly, that require a complex signal network to control tissular homeostasis. We found that T3 modulates epithelial adhesive potential during gut remodeling in X. laevis development, early and directly activating E-cadherin, β-catenin and α-catenin genes, and downstream, modulating small GTPases and other proteins involved in adhesive epithelial properties. Using INSECT2.0 web server to predict the occurrence of Cis-Regulatory Modules (CRMs) [150], we found putative TERs in X. laevis E-cadherin, β-catenin and α-catenin genes, but not in the p120-ctn gene [56]. Among evaluated small GTPases, gastrointestinal Rac1-mRNA levels significantly increased at 24 hs T3-treatment correlated with heightened “lamellipodia” or membrane protrusions. In contrast, at 5 days post T3-treatment RhoA-mRNA levels decrease while the important Rap1-mRNA increasing suggests that membrane Rap1-dependent E-cadherin recycling occurs at metamorphic climax end. Cdc42 and Arp2 actin nucleation protein became constant both during T3-induced and natural metamorphosis [56].

THs exert profound effects on tissues. Among them, cell-type dependent proliferation and differentiation both in mammalian skin [101, 113, 164, 195] and gut [158, 178], as in anuran kidney [84], skin [86] and gut [55, 56, 85]. These effects are regulated by phosphorilation levels of several partner proteins [86] and modulation of gen expression [56, 85]. Among the non-genomic effects of thyroid hormones, it appears that T3 activates PKA to in turn induce β-catenin nuclear translocation by phosphorylation at Ser675 site, thereby β-catenin modulates cyclin-D1 gene transcription and induces cell proliferation (Fig. 5) [53]. In contrast, TH causes astrocyte differentiation through both initial PKA activity and later by phospho-MAP kinase (p-MAPK or p-ERK) [58]. Postbirth, Schwann cell E-cadherin expression is highly regulated through cAMP-PKA activation for maintaining structural integrity [39]. Thus, E-cadherin can negatively regulate, in an adhesion dependent manner, the ligand-dependent activation of divergent classes of RTKs, by inhibiting their ligand-dependent activation in association with a decrease in receptor mobility and in ligand-binding affinity [163] (Fig. 5).

Therefore, the function of THs and their TRs in cell proliferation, differentiation and apoptosis is not homogenous, because it depends strongly on the physio-pathological context; that is, the cell-type, ontogeny (progenitor or differentiated cell) and health (normal or tumoral cell). Although TH-dependent processes are highly coordinated, in turn, the local requirements cannot be governed by global mechanisms, such as an alteration of the thyroid gland function and variations in plasma TH concentrations. Instead, they require tissue-specific regulation mediated by deiodinases. Thus, in mammals, T3 induces type 2 deiodinase (D2) and E-cadherin expression, which sequesters β-catenin and reduces both β-catenin/TCF complex and type 3 deiodinase (D3) activation levels. Consequently, the local active T3-level increases and promotes cell differentiation and reduces its oncogenic effects in intestinal cells (Fig. 5) [48]. Recently, this hypothesis has been again supported by Catalano and coworkers [28], who have found that increased intracellular TH concentration through D3 depletion induces cell differentiation and sharply mitigates tumor formation.

In this context, it is possible to postulate that during organ remodeling T3 induces epithelial stem cell proliferation via EGF-EGFR and cAMP-PKA signaling, and in parallel, T3 leads to repression of this via in a subgroup of these stem cells, and simultaneously increases E-cadherin, β- and α-catenin transcription via TH-RT to differentiate them to pre-adult (juvenile) gut epithelia cells (Fig. 5). In addition, the erichment of junctional E-cadherins sequestrates β-catenins, reducing their β-catenin nuclear translocalization and D3 disponibility, thereby strengthening cell differentiation.