Peer reviewers: Atsushi Nakajima, MD, Professor, Gastroenterology Division, Yokohama City University Hospital, 3-9 Fukuura, Kanazawaku, Yokohama, Kanagawa 236, Japan; Qing Zhu, MD, PhD, NIH/NIAID, 10/11N104, 10 Center Dr., Bethesda, MD 20814, United States
Published online Apr 15, 2010. doi: 10.4291/wjgp.v1.i1.3
Revised: March 18, 2010
Accepted: March 25, 2010
Published online: April 15, 2010
The ability of cells to interact with extracellular matrix macromolecules is at the forefront of the regulation of cell phenotype and organization. Indeed most if not all cells bear specific cell surface receptors for these molecules, namely the integrins, which are specific for the ligation of various macromolecules such as the laminins, fibronectins and tenascins. It is now well established that integrins can regulate a variety of biological activities, most notably cell cycle and tissue-specific gene expression. In the intestine, several observations suggest functional roles for cell-matrix interactions in the regulation of epithelial cell functions. This article focuses on integrin α6β4 as a paradigm to illustrate the importance as well as the complexity of integrins in the mediation of cell-matrix interactions. Indeed, α6β4 has been well-characterized for its involvement as a link between the cytoskeleton and extracellular matrix molecules as well as in the activation of a variety of intracellular signalization processes in cooperation with growth factor receptors. Furthermore, recent studies show that distinct forms of α6 and β4 subunits are expressed in the human intestine and, more importantly, recent work provides experimental evidence that various forms of α6β4 can differentially regulate intestinal epithelial cell functions under both normal and pathological conditions. For instance, it has been discovered that colorectal cancer cells express a hybrid form of α6β4 that is never seen in normal cells. Although further work is needed, integrin α6β4 is emerging as a key regulator of intestinal functions in both intestinal health and disease.
The digestive epithelium is a highly organized tissue that requires only 3 to 5 d to be completely replaced. The regulation of its renewal and expression of digestive and absorptive functions depends on a number of factors[1-3]. Among these, the extracellular matrix macromolecules such as the fibronectins, tenascins and laminins play an important role[1-5]. As for other epithelia, the intestinal epithelium lies on a specialized thin extracellular matrix, the basement membrane (BM) (Figure 1). BM composition defines the microenvironment required for the expression of cell functions such as proliferation, migration, tissue-specific gene expression and apoptosis. Indeed, cell-matrix interactions are mediated by a variety of membrane receptors, many of which are members of the integrin superfamily[7-9].
The intestinal epithelium is a particularly attractive system for deciphering the role of cell-matrix interactions in the regulation of cell functions. In both the normal small and large intestine, the epithelial renewal unit consists of spatially well-separated progenitor and functional cell populations[2,5,10,11]: the crypt-villus axis in the small intestine and the glandular-surface epithelium axis in the colon (Figure 2). Analysis of the expression patterns of integrins and their ligands along the epithelial renewal units of the healthy and pathologic human small intestine and colon has provided crucial basic information on the potential implication of each of these molecules relative to cell state and has identified fundamental differences between the human and rodents[3-5,12-15]. Used in concert with the well-characterized experimental human cell models that replicate the intestinal cell life cycle throughout the epithelial renewal unit, these data have led to significant progress in our understanding of the implications of cell-matrix interactions on the regulation of intestinal cell functions.
The human intestinal BM contains all major macromolecules typical of basal lamina such as the type IV collagens and laminins as well as other non-exclusive components such as the fibronectins[4,13]. An important feature of the intestinal BM is that its composition varies considerably along the renewal unit[5,17-31]. For instance, two functionally important laminins, LM-211 and LM-511, are subject to a reciprocal pattern of expression along the small intestinal crypt-villus axis, being restricted to the proliferative and differentiated compartments, respectively. Other laminins are also subject to unique spatial and temporal patterns of expression[28,30,31]. Interestingly, patterns of laminin expression are altered in various intestinal pathologies including the chronic inflammatory bowel diseases and cancer[34,35] (see for a review). Taken together, these observations suggest functional roles for these interactions in the regulation of intestinal cell functions.
The biological activities of BM macromolecules depend on the repertoire of specific receptors expressed by the cells involved. Numerous receptors for extracellular matrix molecules have been identified but the integrins are considered to be the main mediators of cell-matrix interactions[8,9]. Indeed, the integrins act as fully functional membrane receptors that can trigger cytoskeletal rearrangements and a variety of intracellular signaling events leading to changes in gene expression[36-38]. Integrins are a superfamily of transmembrane αβ heterodimer glycoproteins that represent a major and ubiquitous class of receptors. There are at least a dozen of them that can interact with extracellular matrix molecules, of which many are expressed by human intestinal epithelial cells[4,5,12-14]. As for their ligands, integrin expression is highly regulated along the intestinal epithelial renewal unit. For instance, the α2β1 and α7β1 integrins are predominantly expressed in the proliferative cells of the glands and newly differentiated cells, respectively. Further examples involve other β1 integrins such as α5β1, α8β1 and α9β1[18,40-43].
Surprisingly, α6β4, one of the best characterized integrins involved in the regulation of many cell functions such as proliferation, migration and survival in both health and disease[36,44-47], was initially found to be uniformly distributed at the base of epithelial cells from the bottom of the glands to the tip of the small intestinal villus and the surface epithelium of the colon[25,48,49]. The difficulty in interpreting the ubiquitous expression of α6β4 arose from the existence of splicing variants for both α6 (α6A-B) and β4 (β4A-E) and proteolytically processed forms[51,52]. As reviewed herein, recent studies have shown that distinct forms of the α6 and β4 subunits are expressed in the intestine and, more importantly, recent work provides experimental evidence that various forms of α6β4 can differentially regulate intestinal epithelial cell functions under both normal and pathological conditions.
The α6β4 integrin is expressed at the base of most epithelial cells where it serves as a laminin receptor[53-55]. This integrin is considered to be an exception among the integrins in both structural and functional aspects. One of the most peculiar features of α6β4 is the atypically long cytoplasmic domain of its β subunit, which is involved in the formation of hemidesmosomes as well as in complex signaling functions[57,58].
The cytoplasmic domain of β4 can interact with the keratin network via plectin to initiate the formation of hemidesmosomes, which are specialized structures that mediate the attachment of epithelial cells to laminins in the BM[47,59-61]. Although structurally complex, hemidesmosomes are dynamic structures that can rapidly disassemble under specific circumstances such as cell division or migration[62,63]. Cellular α6β4 redistribution[64,65] appears to be regulated by phosphorylation in response to growth factor stimulation[44,59] and involves interaction of α6β4 with the actin cytoskeletal network[45,66,67].
The signaling functions of α6β4 have received much attention. Indeed, its β subunit behaves as a binary tyrosine kinase receptor. Signal transduction is mediated by the activation of a member of the Src kinase family which combines with the juxtamembrane segment of the β4 cytoplasmic domain. Then, Src kinase phosphorylates 5 major tyrosine phosphorylation sites located in the signalling domain of β4[68,69]. Phosphorylation of tyrosine 1526 mediates the recruitment of the adaptor protein Shc and activation of the Ras-MEK-Erk pathway as well as the PI3-K pathway and its targets including Akt, Rac and mTOR[70-72] while phosphorylation of tyrosine 1440 induces the recruitment of Shp2 phosphatase which favours the activation of the Src kinase Shp2. Furthermore, phosphorylation of serines 1356, 1360 and 1364 by PKC is involved in the disassembly of hemidesmosomes, the recruitment of the 14-3-3 proteins and the association with Ron. Cooperation with growth factor receptors appears to play an important role in β4 signalization. Indeed, α6β4 can associate with several tyrosine kinase receptors such as EGF, ErbB2, Met and Ron[64,75-77], which once activated, lead to the phosphorylation of the cytoplasmic domain of β4. Conversely, α6β4 can promote the phosphorylation of associated tyrosine kinase receptors via Src activation[73,76]. Interestingly, both series of signals appear to be necessary to generate a sustained intracellular response suggesting that in normal cells, ligation of α6β4 to laminin is required for amplifying the signal generated by the tyrosine receptor kinases. In tumor cells, receptor tyrosine kinases are often mutated or amplified and α6β4 is frequently over-expressed. Cooperation between deregulated β4 and receptor tyrosine kinases could contribute to tumoral growth and invasion[64,77-79].
Taken together, these data suggest that the α6β4 integrin plays an important role in normal cells where it is involved in the formation of hemidesmosomes as well in the regulation of a variety of intracellular signalization processes. In tumor cells, cooperation of over-expressed α6β4 with various growth factor receptors enhances signals leading to the promotion of cellular events linked to tumor progression. However, fundamental questions such as the precise mechanisms involved remain open[44,45,47].
A better understanding of how α6β4 functions under both normal and pathological conditions would have significant impact on the diagnosis and/or treatment of epithelium-related diseases. Among these, cancers in general and more particularly colorectal cancers, which is one of the major causes of death by cancer[80,81], appear the most prominent pathologies. However, until recently, α6β4 was considered to be expressed ubiquitously in the human intestinal epithelium and its up- or down-regulation in colorectal cancer was controversial[34,35,82,83]. The data obtained in our laboratory over the last few years have led to a different concept. Indeed, the discovery of distinct forms of the α6β4 integrin, which are functionally distinct and differentially expressed in relation to the cell state, suggests an additional level of complexity for this integrin.
Although important, the intrinsic signalling potential of β4 appears to be less than the complete α6β4 heterodimer, suggesting that the α6 subunit has a more important role than thought initially[84-89]. The α6 subunit is involved in signalling by different ways including via the association with proteins such as CD151[87,90], other proteins that interact with its GFFKR motif or with its PDZ domain at the C-terminal end[84,88].
The α6 integrin mRNA undergoes alternative splicing to yield two distinct isoforms termed α6A and α6B that exhibit distinct cytoplasmic domains and dissimilar spatial and temporal patterns of tissue expression[92-96]. For instance, α6A is found in the mammary gland and in basal keratinocytes while α6B is the predominant variant in the kidney. These distinct patterns of expression for α6A and α6B have been conserved in many species. Their importance is also suggested from work showing the distinct capacity of the two variants to initiate intracellular signalling events[97,98] and the ability to migrate onto laminin when associated with the β1 integrin subunit; the α6Aβ1 integrin being considered to be the "active" variant relative to α6Bβ1[97-101].
Until recently, nothing was known of the importance of α6Aβ4 and α6Bβ4. Based on the fact that α6 only dimerizes with β4 in intestinal cells[102,103] and that both α6 variants are expressed in this tissue, the intestinal epithelium was used to characterize any functional differences between the α6Aβ4 and α6Bβ4 integrins. First, distinct patterns of expression of the α6A and α6B variants were found in the normal intestine. In both the small intestine and colon, proliferative cells of the crypt were found to predominantly express α6A while the differentiated cells of the villus, the Paneth cells, as well as the upper gland and surface epithelial cells of the colon were found to express α6B[103,104]. A similar relationship was observed in intestinal experimental cell models. Second, in addition to an upregulation of the total amounts of α6 subunit, a predominant expression of α6A in relation to α6B was found in both primary colon tumors and adenocarcinoma cell lines suggesting that the enhanced α6A/α6B ratios may be linked to the proliferative status of colon cancer cells. Further studies have shown that manipulating the cellular balance of the two α6 variants can alter cell proliferation and influence transcriptional activities related to cell proliferation but not differentiation[103,104]. More specifically, the data suggest that a predominant expression of α6A could favour cancer cell proliferation by a) directly activating the TCF4/β-catenin pathway, a well-documented pathway for colorectal cancer progression[105-108] and b) competing with the inhibitory effect of α6B on cell proliferation and c-Myc activity.
The specific abilities of α6A to activate pathways linked to cell growth and of α6B to inhibit proliferation are in accordance with their predominant expression in the proliferative and quiescent compartments, respectively, of both the normal small intestine and colon. Up-regulation of α6A expression in primary colon cancers and adenocarcinoma cell lines strongly suggests that the expression and ratio of the α6A and α6B splice variants are inherent to normal intestinal homeostasis and exploited by colon cancer cells.
As mentioned above, the β4 subunit, which is ubiquitously expressed by epithelial cells[12,46,55,109], possesses an unusual β integrin cytoplasmic domain. As for its partner α6, cytoplasmic splice variants have been described but these minor forms of β4 remain poorly characterized compared to the ubiquitous β4A form. On the other hand, a cytoplasmic variant of the β4A subunit that results from the proteolytic cleavage of the C-terminal domain (ctd) has been identified in the human intestinal epithelium. Interestingly, this β4ctd- variant was found to be associated with the proliferative/undifferentiated cells of the crypts in both the small intestine and colon while the non-cleaved β4ctd+ form was only detected in the differentiated cells of the villus and upper gland/surface epithelium in the small intestine and colon, respectively[102,110]. Furthermore, the α6β4ctd- form was not functional for adhesion on purified laminin-332, one of the preferred ligands for α6β4.
In colorectal cancer, an overall up-regulation of the expression of the β4 subunit has been found in relation to c-Myc in the primary tumors but the β4ctd- form was lost in both tumors and adenocarcinoma cell lines. Based on the fact that a mutation in β4 generating a deletion of this ctd domain is lethal in man, its function appears to be crucial. However, at present, the only potential role for the ctd domain is to self-attach to the connecting segment, a region located between the two pairs of type III fibronectin-like domains of the cytoplasmic β4 forming a loop[112,113] and/or to serve as a binding site for plectin[111,114]. The β4ctd- form, which generates an inactive α6β4 integrin for adhesion, appears to be an exclusive feature of normal proliferative intestinal cells. Its expression as a β4ctd+ form in differentiated normal intestinal cells as well as in adenocarcinoma cells thus raises fundamental questions. For instance, can the fact that the α6β4ctd- integrin is inactive for adhesion be linked to the possibility that the ctd domain is required to maintain a functional conformation of the integrin? While this hypothesis appears to be compatible with the various proposed models of α6β4[47,115-117], further work is needed to verify it. Another interesting challenge would be the identification of the precise mechanism involved in the intracellular cleavage of ctd. Indeed, the characterization of a hypothetical "β4ctd-ase" that could impair tumor growth and promotion may provide an interesting clue in the development of an anti-colorectal cancer therapy.
The complexity of the integrin α6β4 has only begun to be unravelled. The α6 and β4 subunits are both up-regulated in various tumor types including colorectal cancer and play key roles in the major intracellular signaling networks. Furthermore, the characterization of cytoplasmic variants for both subunits has revealed new elements to be considered in the equation. Indeed, as illustrated in Figure 3, these findings show on one hand that the α6β4 integrin is present under the α6Aβ4ctd- form (pro-proliferative but not functional for adhesion) in normal proliferative intestinal cells and under the α6Bβ4ctd+ form (anti-proliferative but functional for adhesion) in quiescent and differentiated intestinal cells. In the other hand, in human colorectal adenocarcinoma cells, the predominant form is α6Aβ4ctd+ (pro-proliferative and functional for adhesion), a hybrid form that is never seen in normal cells.
A better understanding of how α6β4 and its variants function under normal and pathological conditions, such as during the tumor progression process, would have significant impact on the diagnosis and treatment of many epithelial cancers including colorectal cancers.
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