Integrin Alpha11 (ITGA11)
Integrin α11 is the last member of the integrin family to be discovered. This integrin subunit was initially named αmt, since it was first identified in human fetal myotube cultures (Gullberg et al. 1995). In 1999, the β1-associated α11integrin was characterized as a collagen-binding integrin and human α11 cDNA was sequenced (Velling et al. 1999). Two years later, Tiger et al. described the α11β1 integrin as a collagen receptor involved in cell migration and collagen reorganization (Tiger et al. 2001). The generation of α11−/− integrin mice was a major advance in the efforts to elucidate α11 integrin function in health and disease (Popova et al. 2007a, b; Zeltz and Gullberg 2016).
Gene and Protein Structure of the α11 Integrin
The human α11 integrin gene (ITGA11) is localized on chromosome 15q23 and consists of 130 kb, whereas the mouse α11 integrin gene (Itga11; length of 106 kb) has been mapped to chromosome 9. Both the human and mouse genes contain 30 exons and 29 introns, similar to genes encoding the other αI domain integrins. The signal peptide is split by two exons, exon 1 and 2. Unlike some other integrin genes, exon 1 in ITGA11 does not solely contain an UTR sequence, but also encodes the major part of the signal peptide, similar to the organization observed in the α10 integrin gene (Bengtsson et al. 2001). The I domain is encoded by four exons (exons 6–9) and flanking introns are in phase 1. An inserted region of 22 amino acids distinguishes α11 from other integrin α chains and is present in calf-1 domain (Velling et al. 1999). The inserted sequence contains two cysteines, suggesting that the sequence forms a loop structure. Comparison with ITGA2 exon 20 (GenBank:AC016619.5) shows that the homologous sequence starts in a position immediately after the insert observed in exon 20 of ITGA11. Interestingly, comparison with other integrin α-chains has identified this region as being involved in α/β chain interactions with the ability to influence integrin activation (Xie et al. 2004). Future studies will reveal the possible importance of the 22 inserted amino acids.
The cytoplasmic tail in integrin α chains has a conserved GFFXX sequence, which in human α11 corresponds to the sequence GFFRS. It is interesting to note that for those integrin α chains that undergo alternative splicing in the cytoplasmic tail, the alternative exons also encode GFFKR, supporting the view that GFFKR together with the cytoplasmic tail denotes a functional unit. Biochemical analysis of the exact border of the transmembrane domain has shown that GFFXX residues can be buried in the transmembrane domain, dependent on integrin chain tilting and activation status. A look at the gene structure for α11 shows that if the homologous sequence GFFX is considered to be part of the transmembrane domain, the majority of this domain is encoded by exon 29, with the final four residues being encoded by the terminating exon 30, which also encodes the cytoplasmic tail. When the GFFXX sequence is regarded as being part of the integrin α chain cytoplasmic tail together with the preceding conserved KL residues, the human α11 cytoplasmic tail contains 24 amino acids (Velling et al. 1999).
The closely related ITGA2 has been shown to display polymorphisms in the promoter region, also identified as risk factors for thrombotic disease (Bray 2000). Based on the high expression of integrin α11 in the periodontal ligament (PDL), it was hypothesized that single nucleotide polymorphisms (SNPs) in ITGA11 might predispose to periodontitis. Analyses of a limited set of patients with juvenile periodontitis however failed to identify polymorphism in the ITGA11 basal promoter (Barczyk et al. 2009). Other studies of ITGA11 SNPS have suggested a link to tick infection in cattle (Porto Neto et al. 2010); the identified SNP is however in a large internal intron, so it is unclear how this SNP could affect α11 expression and function. More recent data suggest that polymorphism in human ITGA11 gene correlated with prepubertal hypertension (Parmar et al. 2016). At this stage, it is also unclear how the identified SNP would correlate with α11 expression and function in relation to blood pressure control. Further studies of the promoter will be instructive in determining the regions in the upstream region that direct the cell-specific expression of α11 observed in vivo, the underlying mechanism for its mechanosensitivity, and finally the regions mediating responsiveness to fibrogenic growth factors.
The human α11 protein as a mature protein is composed of 1166 amino acids. The extracellular domain contains 7 FG-GAP repeats and a 195-amino-acid-long I domain inserted between the repeats 2 and 3. The I domain presents a metal ion-dependent adhesion site (MIDAS) motif and three potential divalent cation binding motifs. As already mentioned the short cytoplasmic tail of 24 amino acids contains the motif GFFRS instead of the conserved GFFKR sequence present in a majority of integrin α subunits. A 23-amino-acid-long transmembrane domain links the extracellular domain with the cytoplasmic domain (Velling et al. 1999). The mouse α11 integrin shows 89% overall identity with human α11 at the protein level, whereas in the I domain the sequence identity is 97% (Popova et al. 2004).
Expression and Regulation of the α11 Integrin Subunit
Distribution in developing normal tissues. The expression of integrin α11 was first described in cultured human fetal muscle cells in vitro (Gullberg et al. 1995). In human adult tissues, α11 mRNA is expressed in high levels in uterus and heart and in intermediate levels in skeletal muscle (Velling et al. 1999). However, in human and mouse embryos, no expression of α11 was detected in muscle cells (Tiger et al. 2001; Popova et al. 2004); instead the current data suggest that the α11-signal in Western- and Northern blotting is derived from fibroblasts. In the human embryo, α11 is present in fibroblasts around ribs, vertebrae, in intervertebral discs, and in keratocytes of the cornea of 8-week-old embryo. In the mouse embryo, α11 has been localized to the ectomesenchyme in the head including the periodontal ligament, in tendons and intestinal villi fibroblasts (Popova et al. 2004). Transient expression of α11 in odontoblasts has also been observed (Popova et al. 2007a). α11β1 expression is thus primarily restricted in vivo to subsets of fibroblasts but also to mesenchymal stem cells (MSCs). In bone marrow-derived nonhematopoetic MSCs, α11 was reported to be expressed on a small subfraction of cells able to undergo osteogenic differentiation (Kaltz et al. 2010). More notably, however, the subfraction of stem cells undergoing adipogenic differentiation did not express α11. In a separate study, it has been reported that MSCs depended on both α2 and α11 for osteogenic differentiation (Popov et al. 2011) and that knockdown of α2 and α11 in human mesenchymal stem cells will reduce ERK and Akt signaling and result in decreased cell proliferation and increased apoptosis (Popov et al. 2011). In summary in developing normal tissues, the α11 chain expression appears to be specific to mesenchymal nonmuscle cells in vitro and in vivo, but a complete characterization in adult tissues has not yet been performed.
α11 expression has also been reported in tumor tissue from melanoma and lung carcinoma. The high levels of α11 integrin expression in lung carcinoma in situ are derived from the cancer-associated fibroblasts and is thus in the lung not contributed by the cancer cells. Due to lack of good reagents, an overview of distribution and cell type expression in different tumor types is currently lacking.
Regulation of α11 expression. The α11 integrin proximal promoter, which is important for basal transcription, contains some regulatory elements which have been characterized in some detail. The presence of two Sp1 sites and an Ets-1 site is necessary to the control of the α11 integrin gene expression (Lu et al. 2006). TGF-β1 has been shown to upregulate the α11 expression in multiple cell lines including HT1080 fibrosarcoma cells and various fibroblast types. The responsiveness to TGF-β1 is dependent on Smad2/3 and Sp1 regulating transcription. The Smad-binding element SBE2 and the Sp1-binding site SBS1 are located in close region on the proximal promoter (nt −182/−176 and −140/−134, respectively). This proximity could promote a possible interaction between the Smad and Sp1 proteins. Activin A, which belongs to the TGF-β family, is involved in the upregulation of α11 in mouse embryonic fibroblasts (MEFs), in a mechanosensitive manner (Carracedo et al. 2010). This induction of α11 expression requires the Smad3 protein. Downregulation of α11 has been reported to occur in mesenchymal stem cells, human dermal fibroblasts, and mouse embryonic fibroblasts treated with FGF-2 (Varas et al. 2007; Carracedo et al. 2010; Grella et al. 2016). However, the responsive elements involved in this downregulation have not yet been determined in the α11 promoter. Type I interferons, including IFN-α and IFN-β, have also been described to regulate the α11 expression. IFNs are able to stimulate α11 mRNA and protein expression in the glioblastoma-derived cell line T98G (Leomil Coelho et al. 2006).
Integrin α11 Functions
In Vitro Functions
The α11 integrin chain is exclusively associated with the β1 subunit at the cell surface, to form the α11β1 integrin. Tiger et al. first demonstrated that α11β1 promoted cell attachment and cell migration to collagen I (using mouse cells lacking collagen receptors transfected with human α11 cDNA) (Tiger et al. 2001). Integrin α11β1 displays certain collagen specificity, since it binds preferentially type I collagen, whereas it interacts with collagen IV with a low affinity. The α11I domain recognizes the triple-helical GFOGER sequence present in collagen I as well as the GLOGER motif (Zhang et al. 2003; Siljander et al. 2004). Thus, α11β1-mediated cell responses could differ depending on the GxxGER collagen motif that interacts with this integrin and the activation status of the integrin. Another study has identified the GLPGER motif of the recombinant Scl protein, a prokaryotic collagen, as an α11β1 -binding sequence (Caswell et al. 2008). The interaction between the cell surface streptococcal Scl1 and the human α11β1 integrin might increase host colonization by pathogenic bacteria, but the potential in vivo significance of this remains to be determined.
The role of α11β1 in PDGF-stimulated cell migration on collagen I coating seems to be cell type dependent. The C2C12 mouse satellite cells, stably transfected with human α11 integrin cDNA, showed a stronger chemotactic response to PDGF-BB, compared to C2C12 wild-type cells, which lack endogenous collagen receptors (Tiger et al. 2001). In contrast, MEFs depleted in α11β1 migrated more on collagen I in comparison to wild-type embryonic fibroblasts (Popova et al. 2004). However, in this last case, a compensatory mechanism, involving other collagen receptor, cannot be excluded.
Not surprisingly, since α11β1 is a collagen receptor with affinity for fibrillar collagens, α11β1 mediates contraction of collagen lattices (Tiger et al. 2001).
Under certain conditions, fibroblasts can be activated and differentiate into so-called myofibroblasts. Myofibroblasts are characterized by α-smooth muscle actin (αSMA) incorporated into stress fibers. Corneal fibroblasts, under action of TGF-β, overexpress αSMA. Since siRNA directed against the α11 integrin completely abrogated αSMA upregulation, it was shown that α11β1 also plays a role in myofibroblast differentiation (Carracedo et al. 2010). The regulation of myofibroblast differentiation by α11β1 has later been shown to be important both during wound healing (Schulz et al. 2015) and fibrosis (Civitarese et al. 2016), events where myofibroblasts play a central role (Zeltz and Gullberg 2016).
Integrin turnover is an essential process involved in cell adhesion and migration. Generally, integrins present on the cell surface are either released and used in new adhesion sites or internalized by endocytosis. Rab proteins, including Rab21, regulate the traffic of endocytotic vesicles via interaction with the cytoplasmic tail of α integrin subunit, as shown for integrin α2β1 (Pellinen et al. 2006). The C-terminal part of Rab21 has also been shown to bind to the cytoplasmic domain of α11 integrin, thus suggesting that α11β1 endocytosis depends on Rab21 activity.
In Vivo Functions in “Unchallenged Conditions” (Normal Development, Tissue Homeostasis)
Intriguingly, later data have shown that α11-deficent mice display reduced serum levels of IGF-1 (Blumbach et al. 2012). The mechanism for how a fibroblast-specific protein would affect the pituitary axis responsible for IGF-1 secretion is unclear at this stage. The study is however important since it stresses that α11-deficent mice are smaller already at birth, before the incisor eruption effect on body weight has come into play and further stresses that more detailed studies are needed to sort out the underlying molecular mechanism for reduced body weight observed in α11−/− mice at different ages.
In Vivo Functions in “Challenged Conditions” of Mouse Models (Wound Healing, Fibrosis, Tumor-Stroma Interactions)
The first indication that α11β1 might be involved in dermal wound healing was the observation that α11 integrin was strongly induced in mice 7 days after inflicting excisional wounds (Zweers et al. 2007). The contribution of α11β1 to wound healing has recently been determined using α11-deficient mice (Schulz et al. 2015), in which dermal wounds display reduced granulation tissue 7 days after excision due to a defect in myofibroblast differentiation. This finding is in agreement with the in vitro studies in which α11β1 was shown to contribute to TGF-β-induced myofibroblast differentiation (Carracedo et al. 2010). Integrin α11β1 is also important for the quality of the scar tissue, since α11−/− wounds displayed poorer tensile strength. This is indicative of an important role for α11β1 in collagen remodeling in granulation tissue. Despite the already described role of α2β1 in collagen reorganization in vitro (Zhang et al. 2006) and its expression on mouse dermal fibroblasts, α11β1 appears to be the major collagen receptor on dermal fibroblasts and contributes to early collagen remodeling in a TGF-β-dependent manner (Schulz et al. 2015). It is interesting in this context to note that although TGF-β is known to regulate α11 integrin through Smad signaling (Lu et al. 2010), the involvement of noncanonical c-Jun N-terminal kinase (JNK)-dependent TGF-β signaling was shown to be crucial for α11β1-dependent collagen remodeling. Further studies are needed to determine the link between α11β1 and JNK activation.
The role of α11β1 in skin fibrosis is currently unknown, but α11 has been shown to have a pro-fibrotic role in diabetic cardiomyopathy, a condition in which high levels of glucose lead to the glycation of collagen, resulting in heart fibrosis (Civitarese et al. 2016; Zeltz and Gullberg 2016). In an experimental rat disease model, the interaction of cardiac fibroblasts with glycated collagen through α11β1 increased TGF-β expression, which in turn induced αSMA expression (Talior-Volodarsky et al. 2012). Interestingly, glycation appears to interfere with α11β1-mediated adhesion to collagen, while still stimulating myofibroblast differentiation (Talior-Volodarsky et al. 2015). The increased level of α11 under these conditions has been interpreted as an attempt by the cells to compensate for reduced adhesion. In the rat diabetes models, TGF-β is responsible for the α11β1-increased expression that is induced by Smad3 binding to an α11 promoter site, which is distinct from the site previously described to mediate TGF-β-stimulated α11 expression (Lu et al. 2010; Talior-Volodarsky et al. 2015).
One important role for α11β1 in tumorigenesis was first suggested in 2002, when Wang et al. identified this integrin as a novel candidate biomarker gene for non-small-cell lung cancer (NSCLC) (Wang et al. 2002). Using representational difference analysis, they showed that α11 was overexpressed in lung adenocarcinoma as compared with healthy lung tissue. Later it became clear that α11β1 was essentially overexpressed in the stroma of NSCLC, especially in CAFs (Zhu et al. 2007). A role for α11β1 in tumorigenesis was indicated in xenograft experiments where the mixing of tumor cells with α11-expressing fibroblasts was shown to stimulate tumor growth. In a different model, involving 3D heterospheroids composed of MEFs and lung carcinoma cells, a downregulation of the chemokine CXCL5 in the tumor cells in the absence of α11 was observed, suggesting that α11-expressing fibroblasts stimulated the autocrine secretion of CXCL5 in NSCLC cells (Lu et al. 2014). However, this is not the only role of α11β1 in tumorigenesis as indicated by our recent study, which emphasized its ability to remodel collagen matrices during wound healing (Schulz et al. 2015). In the heterospheroid model, fibroblasts synthesize collagen I independently of α11, but α11-expressing fibroblasts were shown to induce contraction of the collagen matrix. This reorganization of the matrix resulted in an increase in interstitial fluid pressure (Lu et al. 2014), suggested to contribute to a barrier for drug delivery in vivo. In another recent study, a NSCLC xenograft model has been described, in which α11β1-promoted collagen remodeling and a correlation between tumor tissue stiffness and α11 expression was observed (Navab et al. 2015). Here, a reduced activation of FAK and ERK in tumors grown in α11-deficient mice was also noted. An analysis of possible genes involved in regulating tissue stiffness directly or indirectly revealed a correlation between stromal α11 expression and LOXL1, an elastin and collagen cross-linking enzyme (Navab et al. 2015). As α11 is selectively expressed on fibroblasts, it is not surprising that its mRNA is upregulated by EMT-like events in various tumor models. So far, this induction of α11 mRNA has not been confirmed to translate to the protein level, and it is thus not yet known whether α11β1 affects the EMT process. Recently, however, ITGA11 was identified as an invasion promoting gene in a seven-gene signature that has been established for the leading invasive “trailblazer” cells in a spheroid-based model for collective breast cancer cell invasion (Westcott et al. 2015). These data indicate that the role of α11 in tumors might not be restricted to CAFs but could also extend to tumor cells that assume a mesenchymal invasive phenotype.
As described above, α11β1 has been reported to be upregulated in some tumor forms. However, the exact role of α11 in the tumor stroma during TGF-β1-dependent myofibroblast differentiation, tumor growth, and tumor metastasis remains to be determined.
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