cryosectioning-method-for-microdissection-of-murine-colonic-mucosa

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Emory University, Atlanta, Georgia

Cdc42 is a small GTPase that is required for cell polarity establishment in eukaryotes as diverse as budding yeast and mammals. Par6 is also implicated in metazoan cell polarity establishment and asymmetric cell divisions. Cdc42.GTP interacts with proteins that contain a conserved sequence called a CRIB motif. Uniquely, Par6 possesses a semi-CRIB motif that is not sufficient for binding to Cdc42. An adjacent PDZ domain is also necessary and is required for biological effects of Par6. Here we report the crystal structure of a complex between Cdc42 and the Par6 GTPase-binding domain. The semi-CRIB motif forms a beta-strand that inserts between the four strands of Cdc42 and the three strands of the PDZ domain to form a continuous eight-stranded sheet. Cdc42 induces a conformational change in Par6, detectable by fluorescence resonance energy transfer spectroscopy. Nuclear magnetic resonance studies indicate that the semi-CRIB motif of Par6 is at least partially structured by the PDZ domain. The structure highlights a novel role for a PDZ domain as a structural scaffold.

Structure of Cdc42 in a complex with the GTPase-binding domain of the cell polarity protein, Par6.

Zonula occludens (ZO)-1 was the first tight junction protein to be cloned and has been implicated as an important scaffold protein. It contains multiple domains that bind a diverse set of junction proteins. However, the molecular functions of ZO-1 and related proteins such as ZO-2 and ZO-3 have remained unclear. We now show that gene silencing of ZO-1 causes a delay of approximately 3 h in tight junction formation in Madin-Darby canine kidney (MDCK) epithelial cells, but mature junctions seem functionally normal even in the continuing absence of ZO-1. Depletion of ZO-2, cingulin, or occludin, proteins that can interact with ZO-1, had no discernible effects on tight junctions. Rescue of junction assembly using murine ZO-1 mutants demonstrated that the ZO-1 C terminus is neither necessary nor sufficient for normal assembly. Moreover, mutation of the PDZ1 domain did not block rescue. However, point mutations in the Src homology 3 (SH3) domain almost completely prevented rescue. Surprisingly, the isolated SH3 domain of ZO-1 could also rescue junction assembly. These data reveal an unexpected function for the SH3 domain of ZO-1 in regulating tight junction assembly in epithelial cells and show that cingulin, occludin, or ZO-2 are not limiting for junction assembly in MDCK monolayers.

Zonula occludens-1 function in the assembly of tight junctions in Madin-Darby canine kidney epithelial cells.

E-cadherin forms calcium-dependent homophilic intercellular adhesions between epithelial cells. These contacts regulate multiple aspects of cell behavior, including the organization of intercellular tight junctions (TJs). To distinguish between the roles of E-cadherin in formation versus maintenance of junctions, Madin-Darby canine kidney (MDCK) cells were depleted of E-cadherin by RNA interference. Surprisingly, reducing E-cadherin expression had little effect on the protein levels or localization of adherens junction (AJ) or TJ markers. The cells underwent morphological changes, as the normally flat apical surface swelled into a dome. However, apical-basal polarity was not compromised, transmembrane resistance was normal, and zonula occludin protein 1 dynamics at the TJs were unchanged. Additionally, an E-cadherin/Cadherin-6 double knockdown also failed to disrupt established TJs, although beta-catenin was lost from the cell cortex. Nevertheless, cells depleted of E-cadherin failed to properly reestablish cell polarity after junction disassembly. Recovery of cell-cell adhesion, transepithelial resistance, and the localization of TJ and AJ markers were all delayed. In contrast, depletion of alpha-catenin caused long-term disruption of junctions. These results indicate that E-cadherin and Cadherin-6 function as a scaffold for the construction of polarized structures, and they become largely dispensable in mature junctions, whereas alpha-catenin is essential for the maintenance of functional junctions.

Depletion of E-cadherin disrupts establishment but not maintenance of cell junctions in Madin-Darby canine kidney epithelial cells.

Recent evidence has linked intestinal permeability to mucosal inflammation, but molecular studies are lacking. Candidate regulatory molecules localized within the tight junction (TJ) include Junctional Adhesion Molecule (JAM-A), which has been implicated in the regulation of barrier function and leukocyte migration. Thus, we analyzed the intestinal mucosa of JAM-A-deficient (JAM-A(-/-)) mice for evidence of enhanced permeability and inflammation. Colonic mucosa from JAM-A(-/-) mice had normal epithelial architecture but increased polymorphonuclear leukocyte infiltration and large lymphoid aggregates not seen in wild-type controls. Barrier function experiments revealed increased mucosal permeability, as indicated by enhanced dextran flux, and decreased transepithelial electrical resistance in JAM-A(-/-) mice. The in vivo observations were epithelial specific, because monolayers of JAM-A(-/-) epithelial cells also demonstrated increased permeability. Analyses of other TJ components revealed increased expression of claudin-10 and -15 in the colonic mucosa of JAM-A(-/-) mice and in JAM-A small interfering RNA-treated epithelial cells. Given the observed increase in colonic inflammation and permeability, we assessed the susceptibility of JAM-A(-/-) mice to the induction of colitis with dextran sulfate sodium (DSS). Although DSS-treated JAM-A(-/-) animals had increased clinical disease compared with controls, colonic mucosa showed less injury and increased epithelial proliferation. These findings demonstrate a complex role of JAM-A in intestinal homeostasis by regulating epithelial permeability, inflammation, and proliferation.

JAM-A regulates permeability and inflammation in the intestine in vivo.

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (-/-) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (-/-) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.

Annexin A1 regulates intestinal mucosal injury, inflammation, and repair.

Epithelial and endothelial tight junctions act as a rate-limiting barrier between an organism and its environment. Continuing studies have highlighted the regulation of the tight junction barrier by cytokines. Elucidation of this interplay is vital for both the understanding of physiological tight junction regulation and the etiology of pathological conditions. This review will focus on recent advances in our understanding of the molecular mechanisms of tight junctions modulation by cytokines.

Cytokine regulation of tight junctions.

Junctional adhesion molecule-A (JAM-A) is a transmembrane tight junction protein that has been shown to regulate barrier function and cell migration through incompletely understood mechanisms. We have previously demonstrated that JAM-A regulates cell migration by dimerization of the membrane-distal immunoglobulin-like loop and a C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding motif. Disruption of dimerization resulted in decreased epithelial cell migration secondary to diminished levels of beta1 integrin and active Rap1. Here, we report that JAM-A is physically and functionally associated with the PDZ domain-containing molecules Afadin and PDZ-guanine nucleotide exchange factor (GEF) 2, but not zonula occludens (ZO)-1, in epithelial cells, and these interactions mediate outside-in signaling events. Both Afadin and PDZ-GEF2 colocalized and coimmunoprecipitated with JAM-A. Furthermore, association of PDZ-GEF2 with Afadin was dependent on the expression of JAM-A. Loss of JAM-A, Afadin, or PDZ-GEF2, but not ZO-1 or PDZ-GEF1, similarly decreased cellular levels of activated Rap1, beta1 integrin protein, and epithelial cell migration. The functional effects observed were secondary to decreased levels of Rap1A because knockdown of Rap1A, but not Rap1B, resulted in decreased beta1 integrin levels and reduced cell migration. These findings suggest that JAM-A dimerization facilitates formation of a complex with Afadin and PDZ-GEF2 that activates Rap1A, which regulates beta1 integrin levels and cell migration.

Junctional adhesion molecule A interacts with Afadin and PDZ-GEF2 to activate Rap1A, regulate beta1 integrin levels, and enhance cell migration.

Wnt signaling pathways regulate proliferation, motility, and survival in a variety of human cell types. Dickkopf-1 (Dkk-1) is a secreted Wnt antagonist that has been proposed to regulate tissue homeostasis in the intestine. In this report, we show that Dkk-1 is secreted by intestinal epithelial cells after wounding and that it inhibits cell migration by attenuating the directional orientation of migrating epithelial cells. Dkk-1 exposure induced mislocalized activation of Cdc42 in migrating cells, which coincided with a displacement of the polarity protein Par6 from the leading edge. Consequently, the relocation of the microtubule organizing center and the Golgi apparatus in the direction of migration was significantly and persistently inhibited in the presence of Dkk-1. Small interfering RNA-induced down-regulation of Dkk-1 confirmed that extracellular exposure to Dkk-1 was required for this effect. Together, these data demonstrate a novel role of Dkk-1 in the regulation of directional polarization of migrating intestinal epithelial cells, which contributes to the effect of Dkk-1 on wound closure in vivo.

Dkk-1 inhibits intestinal epithelial cell migration by attenuating directional polarization of leading edge cells.

Formation of the epithelial barrier and apico-basal cell polarity represent two characteristics and mutually dependent features of differentiated epithelial monolayers. They are controlled by special adhesive structures, tight junctions (TJs), and polarity protein complexes that define the apical and the basolateral plasma membrane. The functional interplay between TJs and polarity complexes remains poorly understood. We investigated the role of Scribble, a basolateral polarity protein and known tumor suppressor, in regulating TJs in human intestinal epithelium. Scribble was enriched at TJs in T84 and SK-CO15 intestinal epithelial cell monolayers and sections of normal human colonic mucosa. siRNA-mediated knockdown of Scribble in SK-CO15 cells attenuated development of epithelial barrier and inhibited TJ reassembly independently of other basolateral polarity proteins Lgl-1 and Dlg-1. Scribble selectively co-imunoprecipitated with TJ protein ZO-1, and ZO-1 was important for Scribble recruitment to intercellular junctions and TJ reassembly. Lastly, Scribble was mislocalized from TJs and its expression down-regulated in interferon-gamma-treated T84 cell monolayers and inflamed human intestinal mucosa in vivo. We conclude that Scribble is an important regulator of TJ functions and plasticity in the intestinal epithelium. Down-regulation of Scribble may mediate mucosal barrier breakdown during intestinal inflammation.

Tumor suppressor scribble regulates assembly of tight junctions in the intestinal epithelium.

Inflammatory cytokines have been proposed to regulate epithelial homeostasis during intestinal inflammation. We report here that interferon-gamma (IFN-gamma) regulates the crucial homeostatic functions of cell proliferation and apoptosis through serine-threonine protein kinase AKT-beta-catenin and Wingless-Int (Wnt)-beta-catenin signaling pathways. Short-term exposure of intestinal epithelial cells to IFN-gamma resulted in activation of beta-catenin through AKT, followed by induction of the secreted Wnt inhibitor Dkk1. Consequently, we observed an increase in Dkk1-mediated apoptosis upon extended IFN-gamma treatment and reduced proliferation through depletion of the Wnt coreceptor LRP6. These effects were enhanced by tumor necrosis factor-alpha (TNF-alpha), suggesting synergism between the two cytokines. Consistent with these results, colitis in vivo was associated with decreased beta-catenin-T cell factor (TCF) signaling, loss of plasma membrane-associated LRP6, and reduced epithelial cell proliferation. Proliferation was partially restored in IFN-gamma-deficient mice. Thus, we propose that IFN-gamma regulates intestinal epithelial homeostasis by sequential regulation of converging beta-catenin signaling pathways.

Interferon-gamma regulates intestinal epithelial homeostasis through converging beta-catenin signaling pathways.

Expression of the tight junction protein junctional adhesion molecule-A (JAM-A) has been linked to proliferation and tumour progression. However, a direct role for JAM-A in regulating proliferative processes has not been shown. By using complementary in vivo and in vitro approaches, we demonstrate that JAM-A restricts intestinal epithelial cell (IEC) proliferation in a dimerization-dependent manner, by inhibiting Akt-dependent β-catenin activation. Furthermore, IECs from transgenic JAM-A(-/-)/β-catenin/T-cell factor reporter mice showed enhanced β-catenin-dependent transcription. Finally, inhibition of Akt reversed colonic crypt hyperproliferation in JAM-A-deficient mice. These data establish a new link between JAM-A and IEC homeostasis.

JAM-A regulates epithelial proliferation through Akt/β-catenin signalling.

Coordinated regulation of cell proliferation is vital for epithelial tissue homeostasis, and uncontrolled proliferation is a hallmark of carcinogenesis. A growing body of evidence indicates that epithelial tight junctions (TJs) play a role in these processes, although the mechanisms involved are poorly understood. In this study, we identify and characterize a novel plasma membrane pool of cyclin D1 with cell-cycle regulatory functions. We have determined that the zonula occludens (ZO) family of TJ plaque proteins sequesters cyclin D1 at TJs during mitosis, through an evolutionarily conserved class II PSD-95, Dlg, and ZO-1 (PDZ)-binding motif within cyclin D1. Disruption of the cyclin D1/ZO complex through mutagenesis or siRNA-mediated suppression of ZO-3 resulted in increased cyclin D1 proteolysis and G(0)/G(1) cell-cycle retention. This study highlights an important new role for ZO family TJ proteins in regulating epithelial cell proliferation through stabilization of cyclin D1 during mitosis.

Tight function zonula occludens-3 regulates cyclin D1-dependent cell proliferation.

The epithelial tight junction (TJ) is the apical-most intercellular junction and serves as a gatekeeper for the paracellular pathway by permitting regulated passage of fluid and ions while restricting movement of large molecules. In addition to these vital barrier functions, TJ proteins are emerging as major signaling molecules that mediate crosstalk between the extracellular environment, the cell surface, and the nucleus. Biochemical studies have recently determined that epithelial TJs contain over a hundred proteins that encompass transmembrane proteins, scaffolding molecules, cytoskeletal components, regulatory elements, and signaling molecules. Indeed, many of these proteins have defined roles in regulating epithelial polarity, differentiation, and proliferation. This review will focus on recent findings that highlight a role for TJ proteins in controlling cell proliferation during epithelial homeostasis, wound healing, and carcinogenesis.

Regulation of epithelial proliferation by tight junction proteins.

Epithelial adhesive cell-to-cell contacts contain large, plasma membrane-spanning multiprotein aggregates that perform vital structural and signaling functions. Three prominent adhesive contacts are the tight junction, adherens junction, and the desmosome. Each junction type has unique cellular functions and a complex molecular composition. In this review, we comment on recent and exciting advances in our understanding of junction composition and function.

Epithelial adhesive junctions.

Tight junctions (TJs) are dynamic, multiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiological development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a biosensor of TJ remodeling, we observed increased claudin 4 fluorescence recovery after photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon γ and tumor necrosis factor α increased the proportion of mobile claudin 4 in the TJ. Up-regulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin-binding incompatibility.

Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins.

Tight Junctions (TJs) are multi-molecular complexes in epithelial tissues that regulate paracellular permeability. Within the TJ complex, claudins proteins span the paracellular space to form a seal between adjacent cells. This seal allows regulated passage of ions, fluids, and solutes, contingent upon the complement of claudins expressed. With as many as 27 claudins in the human genome, the TJ seal is complex indeed. This review focuses on changes in claudin expression within the epithelial cells of the gastrointestinal tract, where claudin differentiation results in several physiologically distinct TJs within the lifetime of the cell. We also review mechanistic studies revealing that TJs are highly dynamic, with the potential to undergo molecular remodeling while structurally intact. Therefore, physiologic Tight Junction plasticity involves both the adaptability of claudin expression and gene specific retention in the TJ; a process we term claudin switching.

Article	Year
Structure of Cdc42 in a complex with the GTPase-binding domain of the cell polarity protein, Par6. The EMBO journal\| PubMed ID: 12606577	2003
Zonula occludens-1 function in the assembly of tight junctions in Madin-Darby canine kidney epithelial cells. Molecular biology of the cell\| PubMed ID: 16436508	2006
Depletion of E-cadherin disrupts establishment but not maintenance of cell junctions in Madin-Darby canine kidney epithelial cells. Molecular biology of the cell\| PubMed ID: 17093058	2007
JAM-A regulates permeability and inflammation in the intestine in vivo. The Journal of experimental medicine\| PubMed ID: 18039951	2007
Annexin A1 regulates intestinal mucosal injury, inflammation, and repair. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 18802107	2008
Cytokine regulation of tight junctions. Biochimica et biophysica acta\| PubMed ID: 18952050	2009
Junctional adhesion molecule A interacts with Afadin and PDZ-GEF2 to activate Rap1A, regulate beta1 integrin levels, and enhance cell migration. Molecular biology of the cell\| PubMed ID: 19176753	2009
Dkk-1 inhibits intestinal epithelial cell migration by attenuating directional polarization of leading edge cells. Molecular biology of the cell\| PubMed ID: 19776352	2009
Tumor suppressor scribble regulates assembly of tight junctions in the intestinal epithelium. The American journal of pathology\| PubMed ID: 19959811	2010
Interferon-gamma regulates intestinal epithelial homeostasis through converging beta-catenin signaling pathways. Immunity\| PubMed ID: 20303298	2010
JAM-A regulates epithelial proliferation through Akt/β-catenin signalling. EMBO reports\| PubMed ID: 21372850	2011
Tight function zonula occludens-3 regulates cyclin D1-dependent cell proliferation. Molecular biology of the cell\| PubMed ID: 21411630	2011
Regulation of epithelial proliferation by tight junction proteins. Annals of the New York Academy of Sciences\| PubMed ID: 22731724	2012
Epithelial adhesive junctions. F1000prime reports\| PubMed ID: 24592313	2014
Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins. Molecular biology of the cell\| PubMed ID: 25031428	2014
Claudin switching: Physiological plasticity of the Tight Junction. Seminars in cell & developmental biology\| PubMed ID: 25957515	2015

Christopher T. Capaldo

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