Collagen receptor control of epithelial morphogenesis and cell cycle progression.

To define the unique contributions of the alpha subunit cytoplasmic tails of the alpha(1)beta(1) and alpha(2)beta(1) integrin to epithelial differentiation and branching morphogenesis, a variant NMuMG cell line lacking alpha(1)beta(1) and alpha(2)beta(1) integrin expression was stably transfected with the full-length alpha(2) integrin subunit cDNA (X2C2), chimeric cDNA consisting of the extracellular and transmembrane domains of the alpha(2) subunit and the cytoplasmic domain of the alpha(1) subunit (X2C1), or alpha(2) cDNA truncated after the GFFKR sequence (X2C0). The X2C2 and X2C1 transfectants effectively adhered, spread, and formed focal adhesion complexes on type I collagen matrices. The X2C0 transfectants were less adherent to low concentrations of type I collagen, spread less well, and formed poorly defined focal adhesion complexes in comparison to the X2C2 and X2C1 transfectants. The X2C2 and X2C1 transfectants but not the X2C0 transfectants proliferated on collagen substrates. Only the X2C2 transfectants developed elongate branches and tubules in three-dimensional collagen gels and migrated on type I collagen. These findings suggest a unique role for the alpha(2) integrin cytoplasmic domain in postligand binding events and cooperative interactions with growth factors that mediate epithelial differentiation and branching morphogenesis. Either intact alpha(1) or alpha(2) integrin subunit cytoplasmic domain can promote cell cycle progression.

Differential expression of integrin alpha subunits supports distinct roles during lung branching morphogenesis.

Epithelial branching morphogenesis is a process by which a continuous epithelium, embedded in mesenchyme, forms tubules that extend and branch into the surrounding mesenchyme. The morphogenetic process is responsible for the architecture of many organs including the lung. Proper expression and function of extracellular matrix (ECM) molecules, such as collagens and laminins, are necessary for branching to occur normally. However, little is known about the role of epithelial cell surface molecules that mediate epithelial-matrix interactions during this process. We have studied the expression patterns of cell surface collagen and laminin integrin receptor alpha subunits, alpha 1, alpha 2, alpha 3, and alpha 6, in relation to that of collagen and laminin during lung branching morphogenesis. The alpha 1 integrin subunit was present on endothelia and smooth muscles around airways and large blood vessels. The mesenchyme expressed high levels of alpha 2 and alpha 6 but not alpha 3, whereas the epithelium expressed all three integrin subunits. In contrast to the widespread epithelial expression of alpha 3 and alpha 6, the epithelial expression of alpha 2 was restricted to branch tips. By performing in situ hybridization and immunofluorescence on serial sections, we found that alpha 2 protein expression on the epithelium correlated spatially and temporally with high level expression of collagen IV and laminin-1 mRNAs, suggesting that the alpha 2-expressing epithelial cells were in the process of producing and assembling their collagen and laminin matrices. While the expression of alpha 3 and alpha 6 on all lung epithelia suggests that these integrins may be important to lung epithelial development, the unique expression pattern of the alpha 2 subunit suggests that the alpha 2 beta 1 integrin may be important at branch tips either in the process of collagen/laminin synthesis and assembly or extension of the epithelial tubules into the mesenchyme.

The spatial and temporal expression of the alpha 2 beta 1 integrin and its ligands, collagen I, collagen IV, and laminin, suggest important roles in mouse mammary morphogenesis.

To begin to determine the role of the alpha 2 beta 1 integrin and its ligands, collagen I, collagen IV, and laminin, in mammary epithelial differentiation in vivo, we determined the expression of these molecules by in situ hybridization and immunofluorescence in the developing mouse mammary gland. Expression of collagen I, collagen IV, and laminin mRNAs in the mammary gland during puberty corresponded to the period of greatest growth of the gland, 4-7 weeks postnatally. Collagen I expression preceded collagen IV expression, both of which preceded laminin expression, suggesting an important temporal sequence of extracellular matrix (ECM) production. When growth of the epithelium ceased in the adult virgin gland, expression of all three mRNAs became undetectable. Following the onset of pregnancy these molecules were re-expressed with the same chronology observed during puberty. Collagen I, collagen IV, and laminin were expressed by stromal cells immediately surrounding the developing ductal epithelium. Surprisingly, we found no expression of ECM components in the epithelial cells, suggesting the mammary epithelium does not synthesize its own basement membrane. The distribution of collagen I was consistent with a role in duct formation, since collagen I was strikingly abundant around larger mammary ducts, but was sparse around growing endbuds or alveoli. Conversely, there was abundant laminin near growing endbuds and around alveoli, and less around large ducts, suggesting its role is different than collagen I. The alpha 2 beta 1 integrin was present on the basal, lateral, and apical surfaces of the mammary epithelium throughout postnatal development and pregnancy. The alpha 2 beta 1 integrin expression was strongest at midpregnancy, suggesting a role for alpha 2 beta 1 integrin in the alveolar formation that occurs at this time. The alpha 2 beta 1 integrin expression decreased dramatically in the lactating gland. Our results suggest that alpha 2 beta 1 integrin interactions with its temporally and spatially regulated ligands, collagen I, collagen IV, and laminin, could play an important role in mammary morphogenesis in vivo.

Complex patterns of expression suggest extensive roles for the alpha 2 beta 1 integrin in murine development.

The extracellular matrix plays important roles in embryogenesis. The integrin family of adhesion receptors may mediate critical cellular interactions with the extracellular matrix during development. In this study, we elucidated the developmental spatial and temporal expression pattern of the alpha 2 beta 1 integrin heterodimer, a cell surface receptor for collagens and laminin. We generated reagents for studying the alpha 2 beta 1 integrin and examined the developmental expression of the integrin in postimplantation mice. A partial length murine alpha 2 cDNA was isolated and the protein encoding region was found to be 82% homologous to that of the human alpha 2 cDNA. A synthetic peptide corresponding to the carboxy-terminus of murine alpha 2 was used to generate alpha 2-specific antiserum. The antiserum and riboprobes derived from both the alpha 2 cDNA and the previously characterized murine beta 1 subunit cDNA were used to determine the spatiotemporal expression of the alpha 2 subunit by immunocytochemistry and of the alpha 2 and beta 1 mRNAs by in situ hybridization. Both approaches gave concordant results. Expression of the alpha 2 integrin subunit was observed in both the maternal and embryonic components of the placenta, namely the perivascular and basal zone decidual cells and decidual cells and spongiotrophoblasts at the maternal/embryonic junction. Expression was also observed in cells actively producing and remodeling the extracellular matrix in the maternal uterus and in the developing gut, lens, cartilage, bone, and tooth of the embryo. Generally, expression of the alpha 2 integrin subunit was found in cells entering their later stages of differentiation such as in chondrocytes as they became hypertrophic, ameloblasts and odontoblasts as they became columnar and began to secrete the matrix of the tooth, endothelial cells after they formed tubules, in the lens just prior to and during lens fiber production, and in the collecting ducts of the kidney only after full gestation. Throughout embryogenesis, beta 1 mRNA was widely distributed and present in cell types expressing alpha 2 mRNA and protein. The developmental expression pattern of the alpha 2 beta 1 integrin suggests roles for the integrin in placental development and matrix assembly and remodeling.