Branching morphogenesis: Rac signaling “PIX” tubulogenesis. Focus on “Pak1 regulates branching morphogenesis in 3D MDCK cell culture by a PIX and β1-integrin-dependent mechanism”
2010; American Physical Society; Volume: 299; Issue: 1 Linguagem: Inglês
10.1152/ajpcell.00145.2010
ISSN1522-1563
Autores Tópico(s)Developmental Biology and Gene Regulation
ResumoEDITORIAL FOCIBranching morphogenesis: Rac signaling "PIX" tubulogenesis. Focus on "Pak1 regulates branching morphogenesis in 3D MDCK cell culture by a PIX and β1-integrin-dependent mechanism"James A. MarrsJames A. MarrsDepartment of Biology, Center for Regenerative Biology and Medicine, Indiana University-Purdue University Indianapolis, Indianapolis, IndianaPublished Online:01 Jul 2010https://doi.org/10.1152/ajpcell.00145.2010This is the final version - click for previous versionMoreSectionsPDF (377 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat branching morphogenesis mechanisms are regulated during organogenesis, tissue maintenance, and repair (17). We are also beginning to understand the broad influence of these morphogenesis signaling mechanisms during physiological processes and disease progression. Branching morphogenesis mechanisms are conserved between vertebrates and invertebrates. Common features include regulation of cell-to-cell adhesion, cell-to-extracellular matrix adhesion, growth factor and paracrine signaling, cell migration, differentiation, cell growth, and cell survival. Many developmental model systems, from Drosophila tracheal branching to metanephric kidney development, are actively being exploited to understand fundamental mechanisms of branching morphogenesis (17).In addition to in vivo development models, cell culture models were developed that show incredible utility and versatility for examining cellular events of branching morphogenesis (22). Monolayer and cyst culture models using Madin-Darby canine kidney (MDCK) cells can recapitulate several aspects of branching morphogenesis, including important adhesion and polarity regulatory processes (4). An article by Hunter and Zegers (10) illustrates the power of the three-dimensional MDCK cell cyst model in the analysis of branching morphogenesis mechanisms.Branching MorphogenesisMany epithelial organs develop complex three-dimensional architecture by an amazing and complex process called branching morphogenesis. A variety of developmental model systems of branching morphogenesis are being actively studied, including kidney, lung, and pancreas development (2, 6). Tracheal branching morphogenesis in Drosophila uses the powerful molecular genetic experimental toolbox developed in this model (1, 2). Fundamental cellular mechanisms of branching morphogenesis are shared between different epithelial organs and between vertebrates and invertebrates. Vascular endothelial branching morphogenesis also share many of these conserved features (1, 2). Therefore, understanding cellular mechanisms of branching morphogenesis will be widely applicable to cellular events during development and in normal tissue maintenance.Kidney developmental biology has a rich tradition of studying inductive signals for epithelial differentiation and branching morphogenesis (5, 24). In early mouse embryos (E11), the ureteric bud emerges from the Wolffian duct and grows into the metanephric mesenchyme (Fig. 1A). Through an extensive series of experiments, Grobstein, Saxén, Ekblom, and other investigators (25) showed that there are reciprocal inductive interactions between the ureteric bud epithelium and the metanephric mesenchyme. The ureteric bud induces the metanephric mesenchyme to form distinct nephron cell types found from the glomerulus to distal convoluted tubule. The ureteric bud epithelium produces the collecting system. Metanephric mesenchyme cells induce the ureteric bud to branch. The newly branched ureteric bud induces more metanephric mesenchyme epithelial induction (Fig. 1A). Waves of these differentiation cycles produce the millions of nephrons needed for vertebrates to exist on dry land.Fig. 1.Comparison between branching morphogenesis during mammalian kidney development and during tubulogenesis in vitro. A: coordinate growth factor and integrin signaling regulates branching morphogenesis during metanephric kidney development. Interfering with either growth factor or integrin/extracellular matrix signaling produces similar phenotypes in conditional knockout mice, failure in ureteric bud branching morphogenesis (see text). B: branching morphogenesis is modeled using HGF induced tubulogenesis with the kidney epithelial cell line Madin-Darby canine kidney (MDCK) cells grown in collagen suspension as cysts. MDCK cell epithelial cells form polarized monolayers when grown on solid supports, but when single cell suspensions of MDCK cells are polymerized in type I collagen, then cells divide and differentiate to form a spherical, multicellular cyst that has a monolayer epithelium than secretes and binds a basement membrane facing the collagen matrix, and a free apical cell surface facing a fluid-filled lumen. HGF induces cells within these MDCK cysts to send out extensions that ultimately leads to tubulogenesis (see text). Hunter and Zegars (10) demonstrate that HGF and integrin-dependent tubule initiation and growth pathways are initiated by Pak1-mediated PIX signals.Download figureDownload PowerPointThe importance of cell adhesion molecules in metanephric kidney development were recognized in the earliest studies by Grobstein, Saxén, Ekblom, and others (8). Mesenchyme-to-epithelial transformation induced by signals from the ureteric bud produce condensates of mesenchyme expressing new cell-to-cell and cell-to-extracellular matrix adhesion molecules.Branching morphogenesis events similar to those in the metanephric kidney ureteric bud are seen in other developing organs, including Caenorhabditis elegans and Drosophila (17). Combinations of molecular genetic techniques and advanced imaging methods were used to dissect cellular events in these models, which shows that tubulogenesis is highly conserved (1).In the submandibular gland, lung, and metanephric kidney, branching morphogenesis requires coordinated extracellular matrix/integrin adhesion signaling with tyrosine kinase growth factor receptor signaling (3, 13, 32, 36). Significantly, ureteric bud branching morphogenesis was blocked in conditional knockout mice lacking β1-integrin expression in the ureteric bud (32, 36), and these β1-integrin knockout ureteric bud epithelial cells do not respond to glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor (FGF) (36). Growth factor and integrin signaling pathways activate a Rac1 GTPase and Pak1 kinase-dependent pathway (27). Like mammalian branching morphogenesis, Drosophila also uses adhesion and growth factor-mediated signaling mechanisms for tracheal development branching decisions (1, 2), suggesting that these mechanisms are conserved among metazoans.Cell Culture Models of Branching MorphogenesisCell culture models are able to recapitulate numerous aspects of branching morphogenesis. These in vitro branching morphogenesis models are also induced by tyrosine kinase growth factor receptor (HGFR, EGFR, etc.) activation (4). Many epithelial cells (primary cultures and cell lines) will form an epithelial cyst when suspended in extracellular matrix materials like type I collagen. MDCK cysts are used extensively to examine cyst and tubule morphogenesis (22). This cell culture model has helped us gain a more detailed and complete picture of branching morphogenesis.Collagen suspension cysts from epithelial cell lines like MDCK cells arise from a single cell, growing into a spherical, fluid-filled cyst (Fig. 1B, top) with the basal cell surface facing the surrounding extracellular matrix outside the sphere, with the lateral cell-to-cell contacts forming junctional complexes, and with the apical cell surface facing the fluid-filled lumen. The ability of MDCK and other epithelial cells to self-organize into multicellular, polarized, and transporting epithelium is a part of their differentiated epithelial state. Stages of cyst development are highly programmed, which has allowed numerous laboratories to use this model to examine cell polarity and morphogenesis mechanisms (22).Expanding the utilization of these cyst models and applying new technologies to these experiments should continue to yield new, fundamental knowledge of cellular mechanisms of morphogenesis. MDCK cells can be modified for genetic loss-of-function and gain-of-function experiments (4). Green fluorescent protein (GFP) fusion proteins are expressed and used to analyze dynamic cellular processes when combined with time-lapse imaging and other advanced imaging applications (9). Advances in GFP imaging technology, for example, using fluorescence resonance energy transfer (FRET) or biosensor probes to measure intracellular signaling (28) will be increasingly applied to the MDCK cyst system to dissect cell polarity mechanisms in high spatial and temporal resolution.Additional MDCK cell culture methods are used to experimentally manipulate cell polarity, in particular, showing the importance of extracellular matrix adhesion. In addition to growing MDCK cell cysts in a collagen suspension, MDCK cells can be grown in a media suspension, and the cysts that develop have apical domains facing the growth medium and basal domains facing the interior lumen, where basement membrane is laid down (30). These cysts that develop in media suspension can be embedded in a collagen matrix, forcing cells to reverse their polarity by degrading extracellular matrix within the lumen and reorganizing the junctional complexes and membrane proteins to have apical domains facing into the lumen and basal domains facing the new collagen matrix (31). Another alternative culture method that produces similar polarity reversal events in synchrony is achieved by growing MDCK cells in monolayer culture, and then, a collagen matrix can be polymerized on top of the monolayer (26, 37). In response to the collagen overlay, apical proteins are internalized, and new apical domains assemble on the lateral cell surface. The apical domains forming at lateral domains expand and fuse with other apical domains forming in other cells. Eventually, these collagen overlay cultures generate two complete monolayers that face one another and share a common lumen. These MDCK cell models helped characterize adhesion cues required during cyst formation.Exposing MDCK cysts to hepatocyte growth factor (HGF) induces tubulogenesis. The steps in tubule formation were extensively characterized (35). HGF-treated MDCK cell cysts proceed through a series of events that strongly resemble events seen in other branching morphogenesis models like ureteric bud branching (Fig. 1, A and B). First, basal extensions grow and penetrate the collagen matrix. The role of matrix metaloproteases in mammary duct branching morphogenesis is a well-studied mechanism for this invasive behavior (20), and it would be interesting to understand the regulatory mechanisms used for MDCK cell collagen invasion during tubulogenesis. MDCK cells proliferate and continue to penetrate the collagen gel to form a chain of cells. This chain of cells then transforms into a tubule using cellular mechanisms that were first characterized in Drosophila tracheal development and zebrafish vascular development (1). Pairs of cells undergo a characteristic intercalation and tubule outgrowth events. There are four steps identified during the intercalation process. Initially, two cells pair to form a lumen, and second, the adherens junctions reach around the lumen on each end of the lumen. Third, the two cells displace one another, where one cell moves upstream and the other downstream in the tubule. Finally, the two cells are completely extended with adherens junctions reoriented to maximize tubule extension. Variations on these programmed events appear to be conserved in all branching morphogenesis in epithelial and endothelial cells.Rho family GTPase (Rho, Rac, and Cdc42) signaling is critical in the establishment and maintenance of epithelial cell polarity (29). Rho, Rac, and Cdc42 signaling control cytoskeletal and junctional complex assembly. Integrin and HGF signaling activate Rho family GTPase signaling (33). Integrin adhesion with extracellular matrix molecules provide cell polarity cues (16). MDCK cystogenesis and tubulogenesis models were instrumental in showing that Rac1 regulates cell-to-extracellular matrix adhesion and epithelial differentiation (7, 18, 19, 23, 26, 27, 33, 37). MDCK cells expressing dominant negative Rac1 mutant protein have severe defects in epithelial polarity mechanisms (11, 12, 15, 18), and these cysts do not form lumens nor tubules when treated with HGF (18). Experiments performed by Hunter and Zegers (10) show that the Rac signaling effector molecule Pak1 functions within a complex that includes the protein PIX to initiate tubulogenesis, which also depends on integrin signals.Rac, Pak, and PIX: Branching MorphogenesisBranching morphogenesis in Drosophila trachea and mouse kidney development require Rac signaling mechanisms, which are recapitulated in the MDCK branching morphogenesis model. An important effector for Rac signaling is Pak1, a serine-threonine p21 (Rho family GTPase)-activated kinase family member, and HGF and integrin signaling activate Rac-Pak pathways (27). Hunter and Zegers (10) report that expressing a full-length kinase dead dominant-negative Pak1 mutant (DN-Pak1) induces HGF-independent precocious tubulogenesis in MDCK cysts. DN-Pak1-induced tubulogenesis requires integrin-mediated extracellular matrix adhesion, and DN-Pak1 induces myosin contractility. Hunter and Zegers (10) also determined that the tubulogenesis phenotype induced by DN-Pak1 expression requires binding of the PIX protein (a Rac guanine nucleotide exchange factor; Rac GEF) and a component of the Pak1-PIX-GIT complex. This protein complex regulates focal contact turnover and cell migration behaviors in several cell types (34). To prevent Pak1 recruitment to the complex, Hunters and Zegers (10) expressed DN-Pak1 molecule with two-point mutations that prevented PIX binding, which they called DN-Pak1-ΔPIX. Cysts expressing DN-Pak1-ΔPIX do not induce precocious tubules (10), indicating that tubulogenesis requires appropriately regulated Pak1 signaling and Pak1/PIX/GIT complex assembly.These important findings from the Zegers laboratory (10) should also be considered within the context of their recent findings that cadherin-mediated cell-to-cell adhesion and Pak1 signals from integrin-mediated cell-to-extracellular matrix adhesion coordinately regulate contact-mediated inhibition of cell proliferation (15). Contact-mediated inhibition of cell proliferation activity also requires normal regulation of the Pak1-PIX-GIT complex. Cadherin- and integrin-mediated adhesion signaling mechanisms also converge on phosphoinositide 3 kinase (PI3 kinase) signaling mechanisms (15, 21). PI3 kinase signaling reciprocally can regulate cadherin-mediated cell-to-cell adhesion and integrin-mediated cell-to-extracellular matrix adhesion mechanisms, suggesting that these mechanisms represent a cross-talk signaling pathway to coordinate cell adhesion, proliferation, and migration.There are very few toeholds into the puzzle of cross-talk signaling mechanisms that permit coordinate regulation of cell-to-cell matrix and cell-to-extracellular adhesion mechanisms. Thus findings from the Zegers laboratory (10, 15) represent a very significant advance within this research avenue. These coordinate regulatory mechanisms are utilized during normal tissue development and tissue maintenance, but we are learning that tipping the balance of cell differentiation is a part of many disease processes. The epithelial-to-mesenchymal transitions that occur during tumor progression and metastasis represent a disease process that scrambles the normal balance between cell-to-cell adhesion, cell-to-extracellular matrix adhesion, cell proliferation, and cell survival (14). In vitro models like MDCK cystogenesis and tubulogenesis will continue to provide important insight into difficult and complex cellular processes. Similarly, application of new genetic, epigenetic, imaging and other technologies to these models should generate more detailed information about branching morphogenesis mechanisms in the near future.DISCLOSURESNo conflict of interest, financial or otherwise, are declared by the author(s).REFERENCES1. Affolter M , Caussinus E. Tracheal branching morphogenesis in Drosophila: new insights into cell behaviour and organ architecture. Development 135: 2055–2064, 2008.Crossref | PubMed | ISI | Google Scholar2. Affolter M , Zeller R , Caussinus E. 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