ABSTRACT Cell–cell communication is essential for tissue homeostasis, but its contribution to disease prevention remains to be understood. We demonstrate the involvement of connexin 43 (Cx43, also known as GJA1) and related gap junction in epithelial homeostasis, illustrated by polarity-mediated cell cycle entry and mitotic spindle orientation (MSO). Cx43 localization is restricted to the apicolateral membrane of phenotypically normal breast luminal epithelial cells in 3D culture and in vivo . Chemically induced blockade of gap junction intercellular communication (GJIC), as well as the absence of Cx43, disrupt the apicolateral distribution of polarity determinant tight junction marker ZO-1 (also known as TJP1) and lead to random MSO and cell multilayering. Induced expression of Cx43 in cells that normally lack this protein reestablishes polarity and proper MSO in 3D culture. Cx43-directed MSO implicates PI3K–aPKC signaling, and Cx43 co-precipitates with signaling node proteins β-catenin (CTNNB1) and ZO-2 (also known as TJP2) in the polarized epithelium. The distribution of Cx43 is altered by pro-inflammatory breast cancer risk factors such as leptin and high-fat diet, as shown in cell culture and on tissue biopsy sections. The control of polarity-mediated quiescence and MSO may contribute to the tumor-suppressive role of Cx43.

Summary Inositol-(1,4,5)-triphosphate receptors (InsP 3 Rs) are ligand-gated Ca 2+ channels that control Ca 2+ release from intracellular stores and play a central role in a wide range of cellular responses. In most epithelial cells, InsP 3 Rs are not uniformly distributed within the endoplasmic reticulum (ER) membrane, with the consequence that agonist stimulation results in compartmentalized Ca 2+ signals. Despite these observations, little is known about the mechanisms that regulate the intracellular localization of InsP 3 Rs. Here, we report that exogenously expressed InsP 3 R1–GFP and endogenous InsP 3 R3 interact with the K-Ras-induced actin-binding protein (KRAP) in both differentiated and undifferentiated Madin–Darby canine kidney (MDCK) cells. KRAP mediates InsP 3 R clustering in confluent MDCK cells and functions as an adapter, linking InsP 3 Rs to vimentin intermediate filaments. Upon epithelial differentiation, KRAP and vimentin are both required for InsP 3 R accumulation at the periphery of MDCK cells. Finally, KRAP associates with vimentin in chicken B lymphocytes and with keratins in a breast cancer cell line devoid of vimentin. Collectively, our data suggest that intermediate filaments in conjunction with KRAP may govern the localization of InsP 3 Rs in a large number of cell types (including epithelial cells) and in various physiological or pathological contexts.

Basement membranes contain two major molecular networks consisting of laminin and collagen IV. Previous antibody perturbation experiments suggest that the interaction between laminin and nidogen-1 is necessary for proper basement membrane formation and epithelial development, whereas results from gene ablation experiments in mice show that both basement membranes and general development are grossly normal in the absence of nidogen-1. To refine the perturbation approach, we produced F9-teratocarcinoma-cell-derived embryoid bodies in the presence of recombinantly expressed nidogen-binding sites localized within the γ1III3-5 laminin fragment. We found basement membranes were disrupted in γ1III3-5-expressing embryoid bodies. As a measurement of basement membrane function, we tested permeability and detected drastically increased diffusion rates in correlation with basement membrane disruption. Furthermore, TROMA-1 localization in embryoid bodies expressing the nidogen-binding site was altered, suggesting separation of epithelium-specific gene expression from the formation of the actual epithelium when occurring in the absence of an organized basement membrane.

Summary Mesenchyme tissue: cells, matrix and soluble factors, influence the morphogenesis, proliferation and differentiation of a variety of embryonic epithelia, e.g. in the tooth, skin, mammary and salivary glands. Mesenchyme derivatives also ‘maintain’ adult epithelia, e.g. the local proliferation rate and cytokeratin composition of oral mucosa. Abnormalities in such epithelial–mesenchymal interactions lead to a variety of pathologies such as premalignant lesions, e.g. leukoplakia, tumours and psoriasis, whilst therapeutic manipulation of such interactions can prevent the exfoliation of dental implants. In all of these systems it is critical to understand, at the cellular and molecular levels, how the mesenchyme signals to the epithelium and how the latter processes and responds to such signals. We have investigated such questions using the developing embryonic palate both as a model system and as an important organ: failure of mesenchymal signalling leads to the common and distressing birth defect of cleft palate. Bilateral palatal shelves arise from the maxillary processes of embryonic day 11 (E11) mice, grow initially vertically down the sides of the tongue, elevate on E13.8 to a horizontal position above the dorsum of the tongue and fuse with each other in the midline on E14. The medial edge epithelia of each shelf fuse with each other to form a midline epithelial seam, suprabasal cells die, and the basal (stem) cells synthesize extracellular matrix molecules and turn into mesenchymal cells. Simultaneously the oral epithelia differentiate into stratified squamous cells and the nasal epithelia into pseudostratified ciliated columnar cells. Oral, medial and nasal epithelial differentiation is specified by the underlying mesenchyme in vivo and in vitro . Signalling involves a bifurcating action of a combination of soluble growth factors e.g. TGF- α , TGF- β , PDGF and FGF on palatal epithelia and mesenchyme. These factors stimulate the synthesis of specific extracellular matrix molecules by palate mesenchyme cells, and the appearance of receptors for such molecules on epithelial cells. In this way, a combination of mesenchymal soluble factors and extracellular matrix molecules direct palatal epithelial differentiation. These signals act on epithelial basal (stem) cells, causing them to synthesize unique proteins, which may direct subsequent differentiation of daughter cells. In the most extreme example, namely the medial edge epithelia, these signals result in the basal epithelial cells transforming into mesenchymal cells, thus demonstrating that they are indeed multipotential stem cells.