Integrins in epithelial cell polarity: using antibodies to analyze adhesive function and morphogenesis

Abstract

Epithelial cells polarize in response to cell–substratum and cell–cell adhesive interactions. Contacts between cells and proteins of the extracellular matrix are mediated by integrin receptors. Of the 24 recognized integrin heterodimers, epithelial cells typically express four or more distinct integrins, with the exact complement dependent on the tissue of origin. Investigation of the roles of integrins in epithelial cell polarization has depended on the use of function-blocking antibodies both to determine ligand specificity of individual integrins and to disrupt and redirect normal morphogenesis. In this article we describe techniques for employing function-blocking anti-integrin antibodies in adhesion assays of the polarized Madin–Darby canine kidney (MDCK) cell line and to demonstrate the involvement of β1 integrins in collagen-induced tubulocyst formation. These techniques can be easily expanded to other antibodies and epithelial cell lines to characterize specific functions of individual integrins in epithelial morphogenesis.

Introduction

Epithelial cells polarize in response to adhesive contacts with the substratum and neighboring cells [1]. Early studies using the epithelial Madin–Darby canine kidney (MDCK) cell line indicated that contact with the culture substratum was sufficient to induce a rudimentary degree of apical polarity, as determined by immunolocalization of antigens normally present on the apical plasma membrane domain [2]. Full polarization required, though, cooperation between cell–cell contacts mediated by E-cadherin and cell–substratum adhesion [2]. This was demonstrated dramatically with suspended aggregates of MDCK cells [3], [4], [5]. Single MDCK cells lack apical–basal polarity. On formation of cell clusters linked by E-cadherin in the absence of any obvious cell–substratum contacts, apical proteins became segregated to the free surface of the clusters while basolateral proteins were restricted to areas of cell–cell contact [4]. Full polarization, including placement of tight junctions at the intersection of the apical and lateral plasma membranes, required deposition of collagen at the basal surfaces of cells, that is, in the center of the clusters [4].

In all cell types, the primary group of receptors for extracellular matrix proteins are integrins [6]. Integrins are a family of heterodimeric transmembrane glycoproteins consisting of at least 24 different combinations of α and β subunits. While eight different β subunits have been identified in mammals, the most abundant groups of integrins are those with the β1 (12 examples) and β2 (3 examples) chains. Integrins of the β2 family are expressed only in leukocytes. In epithelial cells, most integrins are β1-containing heterodimers, including α1β1, α2β1, α6β1, and α3β1, all receptors for collagen and laminin isoforms. Many other epithelial cells express α6β4, which is epithelial specific, as well as α5β1 and αVβ3. The latter are receptors for interstitial matrix or serum proteins and interact with their ligands via the RGD peptide motif [6].

The bulk of the integrin molecule is exposed outside the cell where it interacts with ligands via both the α and β subunits [6]. This was recently demonstrated directly by solution of the X-ray crystal structure of the extracellular domain of integrin αVβ3 in association with a peptide ligand [7], [8]. Most integrin α and β subunits have short cytoplasmic segments that serve as a locus for the assembly of multimeric signaling complexes and actin-binding proteins, leading to indirect interaction with basal actin filaments [6]. Several of the signaling molecules are tyrosine kinases such as focal adhesion kinase (FAK) and src, indicative of integrins’ proximal position in a variety of signaling cascades. Altogether, more than 50 different proteins have been reported to be present in focal adhesions [9]. Other proteins associate with integrins in the plane of the membrane, most notably those of the tetraspannin superfamily (TM4SF) [10].

The one exception to the general pattern of integrin engagement with the actin cytoskeleton is the integrin β4 subunit. Integrin β4, which has been reported to interact only with the α6 subunit, has a particularly long cytoplasmic tail [11], [12]. In certain epithelial cells such as keratinocytes, β4 is found in hemidesmosomes where it associates with intermediate filaments instead of actin [11], [12]. In these circumstances it plays an important mechanical role in anchoring the cells to the underlying tissue. However, there is evidence that β4, like other integrins, is also a significant signaling molecule that is able to modulate its cytoplasmic partners and, in some cases, associate with the actin cytoskeleton [11], [12].

The ability of integrins available on the cell surface to bind ligand is regulated. The classic example of this is the integrin αIIbβ3 found in large amounts on the surface of platelets [13]. In normal circumstances this integrin does not bind its ligand fibrinogen. However, when platelets are activated, cytoplasmic signals induce a conformational change in αIIbβ3 transmitted through the membrane that enables it to bind fibrinogen, von Willebrand factor, and fibronectin. While this example of integrin “affinity activation” is particularly dramatic, it is now believed that many if not all integrins undergo similar regulation of ligand binding, and a general mechanism has been proposed [13]. In the inactive state, it is believed that the α-subunit cytoplasmic tail sterically shields the β-subunit tail, and that this association renders the extracellular parts of the integrin molecule unable to bind ligand. Activation is then achieved when proteins such as talin, a component of focal adhesions, binds to the β subunit, releasing it from the α-subunit interaction [13]. These conformational changes are then communicated across the membrane to move the extracellular segments into a conformation able to bind ligand.

The potential involvement of integrins in epithelial cell polarization was first suggested by studies of the developing mouse kidney. The kidney tubular epithelium is formed when mesenteric mesenchymal cells are induced by the invading ureteric bud [14]. Following induction, the mesenchyme condenses into small aggregates of cells that initially form a hollow epithelial ball. Subsequently, this ball lengthens into a tubule to form the primordial nephron. Individual tubular epithelial cell types then differentiate [14]. Elements of this process from induction to formation of the initial epithelium can occur in organ culture [14]. Using this experimental system, Ekblom and colleagues blocked epithelialization of the kidney anlagen with function-blocking antibodies against either integrin α6 or laminin [15], [16]. Because only α6β1 and not α6β4 was expressed, these experiments demonstrated the importance of α6β1 in early epithelial differentiation. Subsequent studies implicating integrins in the development of epithelial cell polarity took advantage of the ability of collagen gels to invert the polar organization of the MDCK cell line [3]. When grown in suspension, MDCK cells form multicellular aggregates oriented with the apical side out [3]. When embedded in collagen gels, these aggregates reverse polarity. Using function-blocking antibodies against the integrin β1 subunit, Ojakian and Schwimmer showed that polarity reversal was dependent on β1 integrin function [17]. In an analogous process, confluent monolayers of MDCK cells form complex double-layered structures called tubulocysts when overlain with collagen gels [18], [19], [20]. This form of polarity inversion was also shown by Zuk and Matlin and the Ojakian group to depend on β1 integrins mislocalized to the apical domain of the epithelial cell, with the α2β1 collagen receptor of particular importance [18], [20].

More recently, studies in cultured mammary cells have provided compelling evidence for the involvement of the integrin α6β4 in epithelial polarization [21], [22]. Normal human mammary epithelial cells form polarized cysts when cultured in a reconstituted basement membrane gel rich in laminin 1. When incubated in either blocking antibodies against β4 or the α6 subunit, the cysts fail to polarize [22]. Because the cells do not express α6β1, these results clearly implicate the β4 integrin in the polarization process. Expression of either β4 lacking a cytoplasmic tail or a hemidesomosal localization signal also prevents polarization, suggesting that either the unique cytoskeletal or signaling environment of the hemidesmosome is critical to polarization mediated by α6β4 [22].

While these results are significant, the entire story of integrin involvement in epithelial polarization is just beginning to unfold. It is clear that there is a certain amount of redundancy in the function of integrins in polarization. As described previously, α6β1 is apparently important in the development of the immature renal epithelium. While this might suggest that this integrin, which shares both a subunit and laminin affinity with α6β4, might subserve the same function in the kidney as α6β4 in the mammary gland, mice lacking the α6 gene develop to birth with apparently normal kidneys, succumbing to skin blistering caused by the lack of hemidesmosomes [23]. Thus, the apparent requirement for α6 integrin in polarization or epithelial differentiation, whether in co mplex with β1 or β4, cannot be strict. Furthermore, none of the critical downstream events following adhesion have been elucidated.

As this brief discussion indicates, progress in understanding the role of integrins in epithelial polarization has been made through the use of a variety of function-blocking anti-integrin antibodies. In addition to these applications, anti-integrin antibodies have also been useful in determining integrin ligand binding specificity and in assessing the polarity of integrin expression on the surface of epithelial cells, both essential elements of the functional analysis of integrins. In this article, we describe methods that use utilize anti-integrin antibodies for ligand/adhesion analysis and inhibition of morphogenesis, all employing the MDCK cell system. In addition, we provide a critical analysis of the various types of anti-integrin antibodies available, with comments on their usefulness for different types of experiments.