Crossroads of integrins and cadherins in epithelia and stroma remodeling

Adhesion events mediated by cadherin and integrin adhesion receptors have fundamental roles in the maintenance of the physiological balance of epithelial tissues, and it is well established that perturbations in their normal functional activity and/or changes in their expression are associated with tumorigenesis. Over the last decades, increasing evidence of a dynamic collaborative interaction between these complexes through their shared interactions with cytoskeletal proteins and common signaling pathways has emerged not only as an important regulator of several aspects of epithelial cell behavior, but also as a coordinated adhesion module that senses and transmits signals from and to the epithelia surrounding microenvironment. The tight regulation of their crosstalk is particularly important during epithelial remodeling events that normally take place during morphogenesis and tissue repair, and when defective it leads to cell transformation and aggravated responses of the tumor microenvironment that contribute to tumorigenesis. In this review we highlight some of the interactions that regulate their crosstalk and how this could be implicated in regulating signals across epithelial tissues to sustain homeostasis.     

The Role of the Microenvironment in Mammary Gland Development and Cancer

The mammary gland is composed of a diverse array of cell types that form intricate interaction networks essential for its normal development and physiologic function. Abnormalities in these interactions play an important role throughout different stages of tumorigenesis. Branching ducts and alveoli are lined by an inner layer of secretory luminal epithelial cells that produce milk during lactation and are surrounded by contractile myoepithelial cells and basement membrane. The surrounding stroma comprised of extracellular matrix and various cell types including fibroblasts, endothelial cells, and infiltrating leukocytes not only provides a scaffold for the organ, but also regulates mammary epithelial cell function via paracrine, physical, and hormonal interactions. With rare exceptions breast tumors initiate in the epithelial compartment and in their initial phases are confined to the ducts but this barrier brakes down with invasive progression because of a combination of signals emitted by tumor epithelial and various stromal cells. In this article, we overview the importance of cellular interactions and microenvironmental signals in mammary gland development and cancer.The mammary gland is composed of a combination of multiple cell types that together form complex interaction networks required for the proper development and functioning of the organ. The branching milk ducts are formed by an outer myoepithelial cell layer producing the basement membrane (BM) and an inner luminal epithelial cell layer producing milk during lactation. The ducts are surrounded by the microenvironment composed of extracellular matrix (ECM) and various stromal cell types (e.g., endothelial cells, fibroblasts, myofibroblasts, and leukocytes). Large amount of data suggest that cell-cell and cell-microenvironment interactions modify the proliferation, survival, polarity, differentiation, and invasive capacity of mammary epithelial cells. However, the molecular mechanisms underlying these effects are poorly understood. The purification and comprehensive characterization of each cell type comprising normal and neoplastic human breast tissue combined with hypothesis testing in cell culture and animal models are likely to improve our understanding of the role these cells play in the normal functioning of the mammary gland and in breast tumorigenesis. In this article, we overview cellular and microenvironmental interactions that play important roles in the normal functioning of the mammary gland and their abnormalities in breast cancer.     

Development of functional thymic epithelial cells occurs independently of lymphostromal interactions

The thymus provides a specialised microenvironment for the development of T-cell precursors. This developmental programme depends upon interactions with stromal cells such as thymic epithelial cells, which provide signals for proliferation, survival and differentiation. In turn, it has been proposed that development of thymic epithelial cells themselves is regulated by signals produced by developing thymocytes. Evidence in support of this symbiotic relationship, termed thymic crosstalk, comes from studies analysing the thymus of adult mice harbouring blocks at specific stages of thymocyte development, where it is difficult to separate mechanisms regulating the initial development of thymic epithelial cells from those regulating their maintenance. To distinguish between these processes, we have analysed the initial developmental programme of thymic epithelial cells within the embryonic thymus, in either the presence or absence of normal T-cell development. We show that keratin 5+8+ precursor epithelial cells present in the early thymic rudiment differentiate into discrete cortical and medullary epithelial subsets displaying normal gene expression profiles, and acquire functional competence, independently of signals from T-cell precursors. Thus, our findings redefine current models of thymus development and argue against a role for thymocyte-epithelial cell crosstalk in the development of thymic epithelial progenitors.     

Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation

Tumors arise from cells that have sustained genetic mutations resulting in deregulation of several of their normal growth-controlling mechanisms. Much of the research concerning the origins of cancer has focused on the genetic mutations within tumor cells, treating tumorigenesis as a cell-autonomous process governed by the genes carried by the tumor cells themselves. However, it is increasingly apparent that the stromal microenvironment in which the tumor cells develop profoundly influences many steps of tumor progression. In various experimental tumor models, the microenvironment affects the efficiency of tumor formation, the rate of tumor growth, the extent of invasiveness, and the ability of tumor cells to metastasize. In carcinomas, the influences of the microenvironment are mediated, in large part, by paracrine signaling between epithelial tumor cells and neighboring stromal fibroblasts. In this review, we summarize recent advances in understanding the paracrine signaling interactions between epithelial cancer cells and associated fibroblasts and examine the effects of these bidirectional interactions on various aspects of carcinoma formation. We note, however, that paracrine signaling between other cell types within the carcinomas, such as endothelial cells and inflammatory cells, may play equally important roles in tumor formation and we will refer to these heterotypic interactions where relevant.     

R-cadherin influences cell motility via Rho family GTPases

Classical cadherins are the transmembrane proteins of the adherens junction and mediate cell-cell adhesion via homotypic interactions in the extracellular space. In addition, they mediate connections to the cytoskeleton by means of their association with catenins. Decreased cadherin-mediated adhesion has been implicated as an important component of tumorigenesis. Cadherin switching is central to the epithelial to mesenchymal transitions that drive normal developmental processes. Cadherin switching has also been implicated in tumorigenesis, particularly in metastasis. Recently, cadherins have been shown to be engaged in cellular activities other than adhesion, including motility, invasion, and signaling. In this study, we show that inappropriate expression of R-cadherin in tumor cells results in decreased expression of endogenous cadherins (cadherin switching) and sustained signaling through Rho GTPases. In addition, we show that R-cadherin induces cell motility when expressed in epithelial cells and that this increased motility is dependent upon Rho GTPase activity.     

Molecular and functional interdependence of the urokinase-type plasminogen activator system with integrins

The serine protease urokinase-type plasminogen activator (uPA), its inhibitor PAI-1, and its cellular receptor uPA-R (CD87) are of crucial importance during cellular invasion and migration, required for a variety of physio- and pathophysiological processes. It has become increasingly evident in recent years that the uPA/uPA-R-system has far more functional properties than plasminogen activation alone. This is reflected by its involvement in cellular events such as proliferation, adhesion, migration, and chemotaxis. Since uPA-R lacks a transmembrane domain and thus on its own is not capable of transmitting signals into cells, association and functional cooperation with other signaling molecules/receptors is needed. In this respect, one group of adhesion and signaling receptors, the integrins, have been identified which constitute, together with the uPA/uPA-R-system, an interdependent biological network by which the uPA/uPA-R-system broadly affects integrin functions and vice versa. Moreover, there is a growing body of evidence that cellular uPA, uPA-R, and PAI-1 expression is under control of specific ECM/integrin interactions and also that integrins are regulated by components of the uPA/uPA-R-system. By this multifaceted crosstalk, cells may modulate their proteolytic, adhesive, and migratory activities and monitor ECM integrity in their microenvironment.     

Phagocytic cells of the thymic reticulum interact with thymocytes via extracellular matrix ligands and receptors

We previously showed that, in the context of thymic epithelial cells, thymocyte migration is partially controlled by extracellular matrix (ECM)-mediated interactions. Herein we evaluated whether these interactions could be involved in cell migration related events in the context of non-epithelial cells of the thymic microenvironment, the phagocytic cells of the thymic reticulum (PTR). We first showed, by immunocytochemistry, cytofluorometry, and RT-PCR, that PTR produce ECM components, including fibronectin and laminin, and express the corresponding integrin-type receptors, VLA-4, VLA-5, and VLA-6. Thymocytes adhere onto PTR monolayers, with immature CD4(+)CD8(+) cells being predominant. Importantly, such an adhesion is partially mediated by ECM ligands and receptors, since it was impaired by anti-ECM or anti-ECM receptor antibodies. Conjointly, our data reveal that the ECM-dependence for thymocyte adhesion onto the thymic microenvironment is not restricted to the epithelial cells, being also seen when they encounter non-epithelial phagocytic cells.     

Dynamic interactions between cells and their extracellular matrix mediate embryonic development

Cells and their surrounding extracellular matrix microenvironment interact throughout all stages of life. Understanding the continuously changing scope of cell‐matrix interactions in vivo is crucial to garner insights into both congenital birth defects and disease progression. A current challenge in the field of developmental biology is to adapt in vitro tools and rapidly evolving imaging technology to study cell‐matrix interactions in a complex 4‐D environment. In this review, we highlight the dynamic modulation of cell‐matrix interactions during development. We propose that individual cell‐matrix adhesion proteins are best considered as complex proteins that can play multiple, often seemingly contradictory roles, depending upon the context of the microenvironment. In addition, cell‐matrix proteins can also exert different short versus long term effects. It is thus important to consider cell behavior in light of the microenvironment because of the constant and dynamic reciprocal interactions occurring between them. Finally, we suggest that analysis of cell‐matrix interactions at multiple levels (molecules, cells, tissues) in vivo is critical for an integrated understanding because different information can be acquired from all size scales. Mol. Reprod. Dev. 77: 475–488, 2010. © 2010 Wiley‐Liss, Inc.     

Cell-cell adhesion impacts epithelia response to substrate stiffness: Morphology and gene expression

Monolayer epithelial cells interact constantly with the substrate they reside on and their surrounding neighbors. As such, the properties of epithelial cells are profoundly governed by the mechanical and molecular cues that arise from both the substrate and contiguous cell neighbors. Although both cell-substrate and cell-cell interactions have been studied individually, these results are difficult to apply to native confluent epithelia, in which both jointly regulate the cell phenotype. Specifically, it remains poorly understood about the intertwined contributions from intercellular adhesion and substrate stiffness on cell morphology and gene expression, two essential microenvironment properties. Here, by adjusting the substrate modulus and altering the intercellular adhesion within confluent kidney epithelia, we found that cell-substrate and cell-cell interactions can mask each other's influence. For example, we found that epithelial cells exhibit an elongated morphological phenotype only when the substrate modulus and intercellular adhesions are both reduced, whereas their motility can be upregulated by either reduction. These results illustrate that combinatorial changes of the physical microenvironment are required to alter cell morphology and gene expression.     

ECM和YAP/TAZ在决定细胞命运的过程中的作用机制

近年来,有研究表表明从细胞微环境中转化而来的机械信号可以调控细胞形状和影响细胞的命运。然而,这些机械信号转化成调节细胞生物过程的信号的机制仍然不是十分清楚。最新研究已阐明细胞可通过来自细胞外基质(extracellular matrix,ECM)的机械信号和细胞行为调控之间的相互作用来募集Hippo信号通路中的核心组件YAP/TAZ的作用机制。此外,研究发现在Wnt和Hippo信号之间的串扰是调节细胞命运的核心。这些机制可以解释力学微环境的信号是如何调节细胞行为和决定细胞命运的。本文重点对ECM和YAP/TAZ在决定细胞命运的过程中的作用机制展开系统综述。     

Reconstruction of human mammary tissues in a mouse model

Establishing a model system that more accurately recapitulates both normal and neoplastic breast epithelial development in rodents is central to studying human breast carcinogenesis. However, the inability of human breast epithelial cells to colonize mouse mammary fat pads is problematic. Considering that the human breast is a more fibrous tissue than is the adipose-rich stroma of the murine mammary gland, our group sought to bypass the effects of the rodent microenvironment through incorporation of human stromal fibroblasts. We have been successful in reproducibly recreating functionally normal breast tissues from reduction mammoplasty tissues, in what we term the human-in-mouse (HIM) model. Here we describe our relatively simple and inexpensive techniques for generating this orthotopic xenograft model. Whether the model is to be applied for understanding normal human breast development or tumorigenesis, investigators with minimal animal surgery skills, basic cell culture techniques and access to human breast tissue will be able to generate humanized mouse glands within 3 months. Clearing the mouse of its endogenous epithelium with subsequent stromal humanization takes 1 month. The subsequent implantation of co-mixed human epithelial cells and stromal cells occurs 2 weeks after humanization, so investigators should expect to observe the desired outgrowths 2 months afterward. As a whole, this model system has the potential to improve the understanding of crosstalk between tissue stroma and the epithelium as well as factors involved in breast stem cell biology tumor initiation and progression.     

Role of extracellular matrix and YAP/TAZ in cell fate determination

The mechanical signals transduced from cellular microenvironment can regulate cell shape and affect cell fate determination. However, how these mechanical signals are transduced to regulate biological processes of cells has remained elusive. Recent studies had elucidated a novel mechanism through which the interactions between mechanical signals from extracellular matrix and cell behavior regulation converged on the function of core components in Hippo signaling pathway, including YAP and TAZ in mammals. Moreover, several very recent studies have found a new crosstalk between Wnt and Hippo signaling in the regulation of cell fate determination. Such mechanism may explain how mechanical signals from microenvironment can regulate cell behavior and determine cell fate.     

Integrin-linked kinase tunes cell–cell and cell-matrix adhesions to regulate the switch between apoptosis and EMT downstream of TGFβ1

Epithelial-mesenchymal transition (EMT) is a morphogenetic process that endows epithelial cells with migratory and invasive potential. Mechanical and chemical signals from the tumor microenvironment can activate the EMT program, thereby permitting cancer cells to invade the surrounding stroma and disseminate to distant organs. Transforming growth factor β1 (TGFβ1) is a potent inducer of EMT that can also induce apoptosis depending on the microenvironmental context. In particular, stiff microenvironments promote EMT while softer ones promote apoptosis. Here, we investigated the molecular signaling downstream of matrix stiffness that regulates the phenotypic switch in response to TGFβ1 and uncovered a critical role for integrin-linked kinase (ILK). Specifically, depleting ILK from mammary epithelial cells precludes their ability to sense the stiffness of their microenvironment. In response to treatment with TGFβ1, ILK-depleted cells undergo apoptosis on both soft and stiff substrata. We found that knockdown of ILK decreases focal adhesions and increases cell–cell adhesions, thus shifting the balance from cell–matrix to cell–cell adhesion. High cell–matrix adhesion promotes EMT whereas high cell–cell adhesion promotes apoptosis downstream of TGFβ1. These results highlight an important role for ILK in controlling cell phenotype by regulating adhesive connections to the local microenvironment.     

The role of interleukin-1 in interactive senescence and age-related human endometrial cancer

The causes of the age-related increase in cancer rates are poorly understood. One cause could be age-related changes in the stromal/epithelial cell interactions that facilitate tumorigenesis. We tested the hypothesis that aging of human endometrial stromal fibroblasts (ESF) alters their influence over endometrial epithelial cells. ESF from adults were found to inhibit anchorage-independent proliferation, to restrain colony outgrowth, and to induce formation of normal tissue architecture by human endometrial cancer cells. As ESF age, these inhibitory influences on malignant-like behaviors by epithelial cells are altered, becoming stimulatory. Age-related change in interleukin-1alpha (IL-1alpha) expression is a molecular determinant of ESF/epithelial cell interactions. Levels of IL-1alpha and IL-1-induced mRNAs increase in ESF with age. Treatment with IL-1 accelerates age-related changes in mRNA abundance and loss of ESF restraint over malignancy-associated behaviors by epithelial cells. Transfection of ESF with the intracellular IL-1 receptor antagonist preserved the young phenotype with respect to interactions with epithelial cells and prevented age-associated increases in groalpha and IL-8 mRNA levels. Our results indicate that aging of ESF is accompanied by an interactive senescence that alters ESF signaling to cancer cells and could contribute to increased cancer rates by providing a microenvironment that is more conducive to tumorigenesis.     

Tissue architecture: the ultimate regulator of epithelial function?

The architecture of a tissue is defined by the nature and the integrity of its cellular and extracellular compartments, and is based on proper adhesive cell-cell and cell-extracellular matrix interactions. Cadherins and integrins are major adhesion-mediators that assemble epithelial cells together laterally and attach them basally to a subepithelial basement membrane, respectively. Because cell adhesion complexes are linked to the cytoskeleton and to the cellular signalling pathways, they represent checkpoints for regulation of cell shape and gene expression and thus are instructive for cell behaviour and function. This organization allows a reciprocal flow of mechanical and biochemical information between the cell and its microenvironment, and necessitates that cells actively maintain a state of homeostasis within a given tissue context. The loss of the ability of tumour cells to establish correct adhesive interactions with their microenvironment results in disruption of tissue architecture with often fatal consequences for the host organism. This review discusses the role of cell adhesion in the maintenance of tissue structure and analyses how tissue structure regulates epithelial function.     

Primary human epithelial cell culture system for studying interactions between female upper genital tract and sexually transmitted viruses, HSV-2 and HIV-1

Evidence from clinical and epidemiological studies indicates that women are disproportionately susceptible to sexually transmitted viral infections. To understand the underlying biological basis for this increased susceptibility, more studies are needed to examine the acute events in the female reproductive tract following exposure to viruses during sexual transmission. The epithelial lining of the female reproductive tract is the primary barrier that sexually transmitted viruses, such as HIV-1 and HSV-2 need to infect or traverse, in order to initiate and establish productive infection. We have established an ex-vivo primary culture system to grow genital epithelial cells from upper reproductive tract tissues of women. Using these cultures, we have extensively examined the interactions between epithelial cells of the female genital tract and HSV-2 and HIV-1. In this review, we describe in detail the experimental protocol to grow these cultures, monitor their differentiation and inoculate with HSV-2 and HIV-1. Prospective use of these cultures to re-create the microenvironment in the reproductive tract is discussed.     

LAP proteins: new gatekeepers of epithelial homeostasis

Cell proliferation and cell differentiation are balanced processes required for the correct development and maintenance of tissues, including epithelial tissues. Disruption of this balance by downregulation or loss of function of gatekeepers of epithelial homeostasis may unleash tumor suppressing activities leading ultimately to tumorigenesis. Among the newcoming actors involved in epithelial cell polarity, recent data shed light on the crucial role played by the LAP (LRR And PDZ) protein family. LAP proteins assemble receptors, cytoplasmic adaptors and enzymes in multimolecular networks important for the different steps of epithelial differentiation : adhesion, building of tight junctions and trafficking of proteins along the secretory pathway. Furthermore, genetic studies in invertebrates and vertebrates have installed LAP proteins not only as crucial determinants for epithelial integrity but also as key regulators of cell proliferation and embryonic development.     

The advantages of co-culture over mono cell culture in simulating in vivo environment

Breast cancer tissue consists of both carcinoma cells and stromal cells, and intratumoral stroma is composed of various cell types such as fibroblasts, adipocytes, inflammatory including lymphocytes and macrophage and lymphatic and blood capillaries including pericytes and endothelial cells. Recently, cell-cell communications or interactions among these cells have been considered to play an important role to cancer initiation, promotion, and progression. In particular, intratumoral fibroblasts are well known as cancer-associated fibroblast (CAF). CAF is considered to be different from normal fibroblasts in terms of promoting cancer progression through the cytokine signals. Carcinoma cell lines have contributed to the advancement of our understanding of cancer cell biology. Numerous researches have employed these carcinoma cell lines as a single- or mono-culture. However, it is also true that this mono-culture system cannot evaluate interactions between carcinoma and intratumoral stromal cells. Co-culture compositions of two different cell type of cancer tissues i.e., carcinoma cell lines and fibroblasts, were established in order to evaluate cell-cell interactions in these cancer microenvironment. This co-culture condition has the advantage of evaluating cell-cell interactions of cancer microenvironment. Therefore, in this review, we focused upon co-culture system and its application to understanding of various biological phenomenon as an ex vivo evaluation method of cancer microenvironment in breast cancer.