The effects of mechanical forces on intestinal physiology and pathology

The epithelial and non-epithelial cells of the intestinal wall experience a myriad of physical forces including strain, shear, and villous motility during normal gut function. Pathologic conditions alter these forces, leading to changes in the biology of these cells. The responses of intestinal epithelial cells to forces vary with both the applied force and the extracellular matrix proteins with which the cells interact, with differing effects on proliferation, differentiation, and motility, and the regulation of these effects involves similar but distinctly different signal transduction mechanisms. Although normal epithelial cells respond to mechanical forces, malignant gastrointestinal epithelial cells also respond to forces, most notably by increased cell adhesion, a critical step in tumor metastasis. This review will focus on the phenomenon of mechanical forces influencing cell biology and the mechanisms by which the gut responds these forces in both the normal as well as pathophysiologic states when forces are altered. Although more is known about epithelial responses to force, information regarding mechanosensitivity of vascular, neural, and endocrine cells within the gut wall will also be discussed, as will, the mechanism by which forces can regulate epithelial tumor cell adhesion.     

Comparison of two cell-adhesion molecules, uvomorulin and cell-CAM 105

Two cell adhesion molecules, cell-CAM 105 and uvomorulin (UM), were compared by analysing their antigenic structures, their activity in cell aggregation assays and their expression in various tissues. Cell-CAM 105 is a membrane glycoprotein which mediates the intercellular adhesion of reaggregating rat hepatocytes, and UM was first described to be involved in the compaction of preimplantation mouse embryos and embryonal carcinoma cells. UM is not only expressed during embryonic development but also in various adult tissues including liver, epithelia of lung, gut, kidney and uterus. A similar distribution for UM was found in rat tissues on cell types where cell-CAM 105 is known to be present. Our studies show that (i) cell-CAM 105 and UM are distinct and different proteins; (ii) uvomorulin is involved in the compaction of rat preimplantation embryos but Fab anti-UM has no effect on reaggregating rat hepatocytes, where Fab anti-cell CAM is effective; (iii) distribution studies show that UM is expressed on a broader range of epithelial cells while cell-CAM 105 is more restricted to hepatocytes and simple epithelia. In cases where both cell adhesion molecules are expressed on the same cell types they can be localized to different parts of the cell surface.     

Changes in adhesive properties of epithelial cells during early morphogenesis of the mammary gland

Previous studies have demonstrated that cell adhesion systems are downregulated in epithelial buds at the earliest stages of submandibular gland and hair follicle development, but are restored at subsequent stages. Here it is shown that epithelial cell adhesion systems are also remodeled during early mammary gland development. Immunofluorescence and electron microscopy of the mouse mammary bud demonstrated that cell-cell adhesion systems were hardly detectable, with significant downregulation of expression of desmosomal molecules, but not of E-cadherin and beta-catenin. Hemidesmosomal structures were also rarely found, although their component molecules were expressed. Differences in cell adhesivity between cells of the mammary bud and those of the overlying epidermis were shown by the finding that the mammary cells formed smaller aggregates than the epidermal cells and were not randomly mixed with the epidermal cells. At subsequent stages, the mammary epithelium restored cell-cell adhesion systems along with de novo expression of tight junction molecules. These data, together with previous findings, indicate that remodeling of epithelial cell adhesion systems is a general feature underlying the early development of several ectoderm-derived organs and support the idea that segregation and rearrangements of cells are involved in early epithelial morphogenesis.     

Adhesion and Cytosolic Dye Transfer between Macrophages and Intestinal Epithelial Cells

Activated macrophages (MQ) found in the intestinal lesions of patients with inflammatory bowel disease (IBD) secrete many inflammatory mediators which can regulate intestinal epithelial cell (IEC) function. However, little is known about direct MQ-IEC interactions. Two potential mechanisms by which cells may interact are through specific receptor-ligand binding of adhesion molecules, such as integrins or cadherins, and by exchange of cytoplasmic substances through transmembraneous channels called gap junctions. We investigated whether Mø could adhere to epithelial cells in culture and form transmembrane communication channels as defined by dye transfer. Primary cultures of murine Mø and a Mø cell line, P388D1, adhered to Mode-K and IEC6, but not CMT-93 IEC. Antibody blocking studies determined that P388D1-Mode-K binding was partially dependent on β2 integrin (CD18) function, Mode-K constitutively expressed CD106 (VCAM-1) and cell associated fibronectin, while P388D1 expressed low levels of CD49d/CD29 (VLA4) but blocking antibodies to these surface molecules did not inhibit P388D1-Mode-K adherence. Transfer of calcein dye from MQ to IEC was quantitated by flow cytometry and was dependent on Mø-IEC adhesion. Dye transfer was concentration dependent in that the fluorescence intensity of Mode-K was proportional to the number of adherent P388D1 cells as well as the dye load of the Mø. These results indicate that Mø interact with IEC by adhesion and possibly through gap junctions and may thus regulate IEC function by direct cell-cell communication.     

JAM4, a junctional cell adhesion molecule interacting with a tight junction protein,MAGI-1

MAGI-1 is a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. It interacts with various molecules and functions as a scaffold protein at cell junctions. We report here a novel MAGI-1-binding protein that we named junctional adhesion molecule 4 (JAM4). JAM4 belongs to an immunoglobulin protein family. JAM4 was colocalized with ZO-1 in kidney glomeruli and in intestinal epithelial cells. Biochemical in vitro studies revealed that JAM4 bound to MAGI-1 but not to ZO-1, whereas JAM1 did not bind to MAGI-1. JAM4 and MAGI-1 interacted with each other and formed clusters in COS-7 cells when coexpressed. JAM4 mediated calcium-independent homophilic adhesion and was accumulated at cell-cell contacts when expressed in L cells. MAGI-1, ZO-1, and occludin were recruited to JAM4-based cell contacts. JAM4 also reduced the permeability of CHO cell monolayers. MAGI-1 strengthened JAM4-mediated cell adhesion in L cells and sealing effects in CHO cells. These findings suggest that JAM4 together with MAGI-1 provides an adhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small intestinal epithelial cells.     

Stage-specific expression of the intermediate filament protein cytokeratin 13 in luminal epithelial cells of secretory phase human endometrium and peri-implantation stage rabbit endometrium

In preparation for blastocyst implantation, uterine luminal epithelial cells express new cell adhesion molecules on their apical plasma membrane. Since one mechanism epithelial cells employ to regulate membrane polarity is the establishment of specific membrane-cytoskeletal interactions, this study was undertaken to determine if new cytokeratin (CK) intermediate filament assemblies are expressed in endometrial epithelial cells during developmental stages related to blastocyst implantation. Type-specific CK antibodies were used for immunocytochemical and immunoblot analyses of 1) intermediate filament networks of the endometrial epithelium during embryo implantation in rabbits and 2) proliferative and secretory phases of the human menstrual cycle. CK18, a type I CK found in most simple epithelia, was expressed in all luminal and glandular epithelial cells of both the human and rabbit endometrium at all developmental stages analyzed; it was also strongly expressed in trophectoderm of the implanting rabbit blastocyst. In contrast, CK13, another type I cytokeratin, exhibited a regulated expression pattern in luminal, but not glandular, epithelial cells of secretory phase human and peri-implantation stage rabbit endometrium. Furthermore, in the rabbit implantation chambers, CK13 was predominantly localized at the cell apex of luminal epithelial cells, where it assembled into a dense filamentous network. These data suggest that the stage-specific expression of CK13 and a reorganization of the apical intermediate filament cytoskeleton of uterine luminal epithelial cells may play important functions in preparation for the implantation process.     

The homeodomain protein Barx2 promotes myogenic differentiation and is regulated by myogenic regulatory factors

The homeobox protein Barx2 is expressed in both smooth and skeletal muscle and is up-regulated during differentiation of skeletal myotubes. Here we use antisense-oligonucleotide inhibition of Barx2 expression in limb bud cell culture to show that Barx2 is required for myotube formation. Moreover, overexpression of Barx2 accelerates the fusion of MyoD-positive limb bud cells and C2C12 myoblasts. However, overexpression of Barx2 does not induce ectopic MyoD expression in either limb bud cultures or in multipotent C3H10T1/2 mesenchymal cells, and does not induce fusion of C3H10T1/2 cells. These results suggest that Barx2 acts downstream of MyoD. To test this hypothesis, we isolated the Barx2 gene promoter and identified DNA regulatory elements that might control Barx2 expression during myogenesis. The proximal promoter of the Barx2 gene contained binding sites for several factors involved in myoblast differentiation including MyoD, myogenin, serum response factor, and myocyte enhancer factor 2. Co-transfection experiments showed that binding sites for both MyoD and serum response factor are necessary for activation of the promoter by MyoD and myogenin. Taken together, these studies indicate that Barx2 is a key regulator of myogenic differentiation that acts downstream of muscle regulatory factors.     

Signaling from E-cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell-cell contact formation

E-cadherins are well characterized cell surface molecules expressed in epithelial cells, which play a major role in cell adhesion through the establishment of calcium-dependent homophilic interactions at sites of cell-cell contacts. They are also integral components of morphogenetic programs controlling the maintenance of the structural and functional integrity of epithelia. Accumulated evidence indicates that the E-cadherin-mediated cell adhesion system is highly regulated from inside the cells by a number of intracellular signaling pathways. Recently available information suggests that E-cadherins may also play a role in the transduction of signals from the outside of the cell to the cytoplasm. However, the nature of the biochemical routes regulated by E-cadherins is still largely unknown. In this study, we set out to explore the possibility that E-cadherins may regulate the activity of MAPK, a key signaling pathway involved in cell fate decisions, upon the formation of cell-cell contacts among neighboring cells. By using an immortalized non-tumorigenic keratinocyte cell line, HaCat, as a model system, we provide evidence that the assembly of calcium-dependent adherens junctions leads to a rapid and remarkable increase in the state of activation of MAPK and that this event is mediated by E-cadherins. Furthermore, we found that E-cadherins stimulate the MAPK pathway through the ligand-independent activation of epidermal growth factor receptors and the consequent activation of a biochemical route leading to the stimulation of MAPKs. These findings suggest that E-cadherins can initiate outside-in signal transducing pathways through the engagement of tyrosine kinase receptors for epidermal growth factor, thus providing a novel molecular mechanism whereby these cell adhesion molecules may ultimately control the fate of normal and transformed epithelial cells.     

Cdx2 initiates histodifferentiation of the midgut endoderm

Null mutation or haploinsufficiency of Cdx2 results in the development of heterotopic lesions with a gastric phenotype in the midgut endoderm. Conversely transgenic expression of Cdx2 in the stomach causes the endoderm to differentiate into intestinal-type mucosa. We demonstrate that the mesoderm adjacent to intestinal heterotopic areas expresses stomach specific Barx1 while the surrounding mesoderm is Barx1 negative. We conclude that the initiation of gut histodifferentiation lies in the endodermal expression of Cdx2 and that endodermal/mesodermal cross-talk involving Barx1 with appropriate feedback loops results in the development of the postnatal gut phenotype.     

barx1 is necessary for ectomesenchyme proliferation and osteochondroprogenitor condensation in the zebrafish pharyngeal arches

Barx1 modulates cellular adhesion molecule expression and participates in specification of tooth-types, but little is understood of its role in patterning the pharyngeal arches. We examined barx1 expression during zebrafish craniofacial development and performed a functional analysis using antisense morpholino oligonucleotides. Barx1 is expressed in the rhombencephalic neural crest, the pharyngeal arches, the pectoral fin buds and the gut in contrast to its paralogue barx2, which is most prominently expressed in the arch epithelium. Additionally, barx1 transient expression was observed in the posterior lateral line ganglia and developing trunk/tail. We show that Barx1 is necessary for proliferation of the arch osteochondrogenic progenitors, and that morphants exhibit diminished and dysmorphic arch cartilage elements due to reductions in chondrocyte differentiation and condensation. Attenuation of Barx1 results in lost arch expression of osteochondrogenic markers col2a1, runx2a and chondromodulin, as well as odontogenic marker dlx2b. Further, loss of barx1 positively influenced gdf5 and chordin, markers of jaw joint patterning. FGF signaling is required for maintaining barx1 expression, and that ectopic BMP4 induces expression of barx1 in the intermediate region of the second pharyngeal arch. Together, these results indicate an essential role for barx1 at early stages of chondrogenesis within the developing zebrafish viscerocranium.     

Modulation of epithelial morphogenesis and cell fate by cell-to-cell signals and regulated cell adhesion

Cell-cell and cell-extracellular matrix (ECM) interactions control many developmental decisions of epithelial cell fate and morphogenesis. Protein tyrosine kinases are one class of regulatory molecules that have been implicated in the modulation of these processes. Several protein tyrosine kinases co-localize with cell-cell (cadherin) and cell-ECM (integrin) adhesion molecules at specific adhesion domains of epithelial cells. Protein tyrosine kinases may regulate epithelial development by modulating cell-cell and cell-ECM interactions and by relaying signals initiated by these interactions to other cellular components that determine cell structure and function.     

Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration

Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, we show that Netrin-1 is expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. We demonstrate that alpha6beta4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of alpha6beta4 and alpha3beta1 regulate the migration of CK19+/PDX1+ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells.     

Ep-CAM: a human epithelial antigen is a homophilic cell-cell adhesion molecule

The epithelial glycoprotein 40 (EGP40, also known as GA733-2, ESA, KSA, and the 17-1A antigen), encoded by the GA-733-2 gene, is expressed on the baso-lateral cell surface in most human simple epithelia. The protein is also expressed in the vast majority of carcinomas and has attracted attention as a tumor marker. The function of the protein is unknown. We demonstrate here that EGP40 is an epithelium-specific intercellular adhesion molecule. The molecule mediates, in a Ca(2+)- independent manner, a homophilic cell-cell adhesion of murine cells transfected with the complete EGP40 cDNA. Two murine cell lines were tested for the effects of EGP40 expression: fibroblastic L cells and dedifferentiated mammary carcinoma L153S cells. The expression of the EGP40 protein causes morphological changes in cultures of transfected cells--increasing intercellular adhesion of the transfectants--and has a clear effect on cell aggregating behavior in suspension aggregation assays. EGP40 directs sorting in mixed cell populations, in particular, causes segregation of the transfectants from the corresponding parental cells. EGP40 expression suppresses invasive colony growth of L cells in EHS-matrigel providing tight adhesions between cells in growing colonies. EGP40 can thus be considered a new member of the intercellular adhesion molecules. In its biological behavior EGP40 resembles to some extent the molecules of the immunoglobulin superfamily of cell adhesion molecules (CAMs), although no immunoglobulin-like repeats are present in the EGP40 molecule. Certain structural similarities in general organization of the molecule exist between EGP40 and the lin-12/Notch proteins. A possible role of this adhesion molecule in formation of architecture of epithelial tissues is discussed. To reflect the function of the molecule the name Ep-CAM for EGP40 seems appropriate.