RESUMEN
Initial landmark studies in the design of synthetic hydrogels for intestinal organoid culture identified precise matrix requirements for differentiation, namely decompression of matrix-imposed forces and supplementation of laminin. But beyond stating the necessity of laminin, organoid-laminin interactions have gone largely unstudied, as this ubiquitous requirement of exogenous laminin hinders investigation. In this work, we exploit a fast stress relaxing, boronate ester based synthetic hydrogel for the culture of intestinal organoids, and fortuitously discover that unlike all other synthetic hydrogels to date, laminin does not need to be supplemented for crypt formation. This highly defined material provides a unique opportunity to investigate laminin-organoid interactions and how it influences crypt evolution and organoid function. Via fluorescent labeling of non-canonical amino acids, we further show that adaptable boronate ester bonds increase deposition of nascent proteins, including laminin. Collectively, these results advance the understanding of how mechanical and matricellular signaling influence intestinal organoid development.
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Pancreatic ductal adenocarcinoma (PDAC) presents at advanced stages and is refractory to most treatment modalities. Wnt signaling activation plays a critical role in proliferation and chemotherapeutic resistance. Minimal media conditions, growth factor dependency, and Wnt dependency were determined via Wnt inhibition for seven patient derived organoids (PDOs) derived from pancreatic tumor organoid libraries (PTOL). Organoids demonstrating response in vitro were assessed in vivo using patient-derived xenografts. Wnt (in)dependent gene signatures were identified for each organoid. Panc269 demonstrated a trend of reduced organoid growth when treated with ETC-159 in combination with paclitaxel or gemcitabine as compared with chemotherapy or ETC-159 alone. Panc320 demonstrated a more pronounced anti-proliferative effect in the combination of ETC-159 and paclitaxel but not with gemcitabine. Panc269 and Panc320 were implanted into nude mice and treated with ETC-159, paclitaxel, and gemcitabine as single agents and in combination. The combination of ETC-159 and paclitaxel demonstrated an anti-tumor effect greater than ETC-159 alone. Extent of combinatory treatment effect were observed to a lesser extent in the Panc320 xenograft. Wnt (in)dependent gene signatures of Panc269 and 320 were consistent with the phenotypes displayed. Gene expression of several key Wnt genes assessed via RT-PCR demonstrated notable fold change following treatment in vivo. Each pancreatic organoid demonstrated varied niche factor dependencies, providing an avenue for targeted therapy, supported through growth analysis following combinatory treatment of Wnt inhibitor and standard chemotherapy in vitro. The clinical utilization of this combinatory treatment modality in pancreatic cancer PDOs has thus far been supported in our patient-derived xenograft models treated with Wnt inhibitor plus paclitaxel or gemcitabine. Gene expression analysis suggests there are key Wnt genes that contribute to the Wnt (in)dependent phenotypes of pancreatic tumors, providing plausible mechanistic explanation for Wnt (in)dependency and susceptibility or resistance to treatment on the genotypic level.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Ratones , Humanos , Gemcitabina , Vía de Señalización Wnt , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Ratones Desnudos , Proliferación Celular , Línea Celular Tumoral , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Organoides/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Taste buds on the tongue contain taste receptor cells (TRCs) that detect sweet, sour, salty, umami and bitter stimuli. Like non-taste lingual epithelium, TRCs are renewed from basal keratinocytes, many of which express the transcription factor SOX2. Genetic lineage tracing has shown that SOX2+ lingual progenitors give rise to both taste and non-taste lingual epithelium in the posterior circumvallate taste papilla (CVP) of mice. However, SOX2 is variably expressed among CVP epithelial cells, suggesting that their progenitor potential may vary. Using transcriptome analysis and organoid technology, we show that cells expressing SOX2 at higher levels are taste-competent progenitors that give rise to organoids comprising both TRCs and lingual epithelium. Conversely, organoids derived from progenitors that express SOX2 at lower levels are composed entirely of non-taste cells. Hedgehog and WNT/ß-catenin are required for taste homeostasis in adult mice. However, manipulation of hedgehog signaling in organoids has no impact on TRC differentiation or progenitor proliferation. By contrast, WNT/ß-catenin promotes TRC differentiation in vitro in organoids derived from higher but not low SOX2+ expressing progenitors.
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Papilas Gustativas , beta Catenina , Animales , Ratones , beta Catenina/metabolismo , Células Epiteliales/metabolismo , Proteínas Hedgehog/metabolismo , Lengua/metabolismoRESUMEN
Spatiotemporally coordinated transformations in epithelial curvature are necessary to generate crypt-villus structures during intestinal development. However, the temporal regulation of mechanotransduction pathways that drive crypt morphogenesis remains understudied. Intestinal organoids have proven useful to study crypt morphogenesis in vitro, yet the reliance on static culture scaffolds limits the ability to assess the temporal effects of changing curvature. Here, a photoinduced hydrogel cross-link exchange reaction is used to spatiotemporally alter epithelial curvature and study how dynamic changes in curvature influence mechanotransduction pathways to instruct crypt morphogenesis. Photopatterned curvature increased membrane tension and depolarization, which was required for subsequent nuclear localization of yes-associated protein 1 (YAP) observed 24 hours following curvature change. Curvature-directed crypt morphogenesis only occurred following a delay in the induction of differentiation that coincided with the delay in spatially restricted YAP localization, indicating that dynamic changes in curvature initiate epithelial curvature-dependent mechanotransduction pathways that temporally regulate crypt morphogenesis.
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Intestinos , Mecanotransducción Celular , Mucosa Intestinal/metabolismo , Organoides , MorfogénesisRESUMEN
Roundabout 4 (Robo4) is a transmembrane receptor that expresses specifically in endothelial cells. Soluble Robo4 was reported in the human plasma and mouse serum and is inhibitory towards FGF- and VEGF-induced angiogenesis. It remains unknown how soluble Robo4 is generated and if soluble Robo4 regulates additional angiogenic signaling. Here, we report soluble Robo4 is the product of constitutive ectodomain shedding of endothelial cell surface Robo4 by disintegrin metalloproteinases ADAM10 and ADAM17 and acts to inhibit angiogenic Slit3 signaling. Meanwhile, the ligand Slit3 induces cell surface receptor Robo4 endocytosis to shield Robo4 from shedding, showing Slit3 inhibits Robo4 shedding to enhance Robo4 signaling. Our study delineated ADAM10 and ADAM17 are Robo4 sheddases, and ectodomain shedding, including negative regulation by its ligand Slit3, represents a novel control mechanism of Robo4 signaling in angiogenesis.
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Células Endoteliales , Proteínas de la Membrana , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Animales , Células Endoteliales/metabolismo , Ligandos , Proteínas de la Membrana/metabolismo , Ratones , Neovascularización Patológica/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
3D organoid models have recently seen a boom in popularity, as they can better recapitulate the complexity of multicellular organs compared to other in vitro culture systems. However, organoids are difficult to image because of the limited penetration depth of high-resolution microscopes and depth-dependent light attenuation, which can limit the understanding of signal transduction pathways and characterization of intimate cell-extracellular matrix (ECM) interactions. To overcome these challenges, phototransfer by allyl sulfide exchange-expansion microscopy (PhASE-ExM) is developed, enabling optical clearance and super-resolution imaging of organoids and their ECM in 3D. PhASE-ExM uses hydrogels prepared via photoinitiated polymerization, which is advantageous as it decouples monomer diffusion into thick organoid cultures from the hydrogel fabrication. Apart from compatibility with organoids cultured in Matrigel, PhASE-ExM enables 3.25× expansion and super-resolution imaging of organoids cultured in synthetic poly(ethylene glycol) (PEG) hydrogels crosslinked via allyl-sulfide groups (PEG-AlS) through simultaneous photopolymerization and radical-mediated chain-transfer reactions that complete in <70 s. Further, PEG-AlS hydrogels can be in situ softened to promote organoid crypt formation, providing a super-resolution imaging platform both for pre- and post-differentiated organoids. Overall, PhASE-ExM is a useful tool to decipher organoid behavior by enabling sub-micrometer scale, 3D visualization of proteins and signal transduction pathways.
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Microscopía , Organoides , Compuestos Alílicos , Materiales Biocompatibles/metabolismo , Matriz Extracelular , Hidrogeles/metabolismo , Organoides/metabolismo , SulfurosRESUMEN
The epithelial lining of the intestine, particularly the stem cell compartment, is affected by harsh conditions in the luminal environment and also is susceptible to genotoxic agents such as radiation and chemotherapy. Therefore, the ability for intestinal epithelial cells to revert to a stem cell state is an important physiological damage response to regenerate the intestinal epithelium at sites of mucosal injury. Many signaling networks involved in maintaining the stem cell niche are activated as part of the damage response to promote cellular plasticity and regeneration. The relative contribution of each cell type and signaling pathway is a critical area of ongoing research, likely dependent on the nature of injury as well as the regional specification within the intestine. Here, we review the current understanding of the multicellular cooperation to restore the intestinal epithelium after damage.
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Plasticidad de la Célula , Nicho de Células Madre , Homeostasis , Intestinos , RegeneraciónRESUMEN
Intestinal crypts have great capacity for repair and regeneration after intestinal stem cell (ISC) injury. Here, we define the cellular remodeling process resulting from ISC niche interruption by transient Notch pathway inhibition in adult mice. Although ISCs were retained, lineage tracing demonstrated a marked reduction in ISC function after Notch disruption. Surprisingly, Notch ligand-expressing Paneth cells were rapidly lost by apoptotic cell death. The ISC-Paneth cell changes were followed by a regenerative response, characterized by expansion of cells expressing Notch ligands Dll1 and Dll4, enhanced Notch signaling, and a proliferative surge. Lineage tracing and organoid studies showed that Dll1-expressing cells were activated to function as multipotential progenitors, generating both absorptive and secretory cells and replenishing the vacant Paneth cell pool. Our analysis uncovered a dynamic, multicellular remodeling response to acute Notch inhibition to repair the niche and restore homeostasis. Notably, this crypt regenerative response did not require ISC loss.
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Intestinos/citología , Intestinos/fisiología , Receptores Notch/metabolismo , Regeneración , Nicho de Células Madre , Animales , Apoptosis , Proteínas de Unión al Calcio/metabolismo , Proliferación Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones Transgénicos , Modelos Biológicos , Células Madre/citología , Células Madre/metabolismoRESUMEN
Intestinal organoids are useful in vitro models for basic and translational studies aimed at understanding and treating disease. However, their routine culture relies on animal-derived matrices that limit translation to clinical applications. In fact, there are few fully defined, synthetic hydrogel systems that allow for the expansion of intestinal organoids. Here, an allyl sulfide photodegradable hydrogel is presented, achieving rapid degradation through radical addition-fragmentation chain transfer (AFCT) reactions, to support routine passaging of intestinal organoids. Shear rheology to first characterize the effect of thiol and allyl sulfide crosslink structures on degradation kinetics is used. Irradiation with 365 nm light (5 mW cm-2 ) in the presence of a soluble thiol (glutathione at 15 × 10-3 m), and a photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate at 1 × 10-3 m), leads to complete hydrogel degradation in less than 15 s. Allyl sulfide hydrogels are used to support the formation of epithelial colonies from single intestinal stem cells, and rapid photodegradation is used to achieve repetitive passaging of stem cell colonies without loss in morphology or organoid formation potential. This platform could support long-term culture of intestinal organoids, potentially replacing the need for animal-derived matrices, while also allowing systematic variations to the hydrogel properties tailored for the organoid of interest.
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Compuestos Alílicos/química , Hidrogeles/química , Hidrogeles/metabolismo , Organoides/metabolismo , Fotólisis , Compuestos de Sulfhidrilo/química , Sulfuros/química , Animales , Mucosa Intestinal/citología , Luz , Ratones , Reología , Resistencia al Corte , SolubilidadRESUMEN
Intestinal organoid protocols rely on the use of extracellular scaffolds, typically Matrigel, and upon switching from growth to differentiation promoting media, a symmetry breaking event takes place. During this stage, the first bud like structures analogous to crypts protrude from the central body and differentiation ensues. While organoids provide unparalleled architectural and functional complexity, this sophistication is also responsible for the high variability and lack of reproducibility of uniform crypt-villus structures. If function follows form in organoids, such structural variability carries potential limitations for translational applications (e.g., drug screening). Consequently, there is interest in developing synthetic biomaterials to direct organoid growth and differentiation. It has been hypothesized that synthetic scaffold softening is necessary for crypt development, and these mechanical requirements raise the question, what compressive forces and subsequent relaxation are necessary for organoid maturation? To that end, allyl sulfide hydrogels are employed as a synthetic extracellular matrix mimic, but with photocleavable bonds that temporally regulate the material's bulk modulus. By varying the extent of matrix softening, it is demonstrated that crypt formation, size, and number per colony are functions of matrix softening. An understanding of the mechanical dependence of crypt architecture is necessary to instruct homogenous, reproducible organoids for clinical applications.
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Intestinos , Organoides , Matriz Extracelular , Mucosa Intestinal , Reproducibilidad de los ResultadosRESUMEN
Acute intestinal inflammation includes the early accumulation of neutrophils (PMN). Based on recent evidence that PMN infiltration "imprints" changes in the local tissue environment through local oxygen depletion and the release of adenine nucleotides, we hypothesized that the interaction between transmigrating PMN and intestinal epithelial cells (IECs) results in inflammatory acidification of the tissue. Using newly developed tools, we revealed that active PMN transepithelial migration (TEM) significantly acidifies the local microenvironment, a decrease of nearly 2 pH units. Using unbiased approaches, we sought to define acid-adaptive pathways elicited by PMN TEM. Given the significant amount of adenosine (Ado) generated during PMN TEM, we profiled the influence of Ado on IECs gene expression by microarray and identified the induction of SLC26A3, the major apical Cl-/HCO3- exchanger in IECs. Utilizing loss- and gain-of-function approaches, as well as murine and human colonoids, we demonstrate that Ado-induced SLC26A3 promotes an adaptive IECs phenotype that buffers local pH during active inflammation. Extending these studies, chronic murine colitis models were used to demonstrate that SLC26A3 expression rebounds during chronic DSS-induced inflammation. In conclusion, Ado signaling during PMN TEM induces an adaptive tissue response to inflammatory acidification through the induction of SLC26A3 expression, thereby promoting pH homeostasis.
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Acidosis/inmunología , Antiportadores/metabolismo , Colitis/inmunología , Inflamación/inmunología , Mucosa Intestinal/fisiología , Intestinos/inmunología , Neutrófilos/inmunología , Transportadores de Sulfato/metabolismo , Acidosis/inducido químicamente , Adaptación Fisiológica , Adenosina/metabolismo , Animales , Antiportadores/genética , Células Cultivadas , Colitis/inducido químicamente , Modelos Animales de Enfermedad , Humanos , Enfermedades del Sistema Inmune , Inflamación/inducido químicamente , Trastornos Leucocíticos , Ratones , Activación Neutrófila , Dodecil Sulfato de Sodio , Transportadores de Sulfato/genética , Migración Transendotelial y Transepitelial , Regulación hacia ArribaRESUMEN
BACKGROUND & AIMS: Loss of leucine-rich repeat-containing G-protein-coupled receptor 5-positive crypt base columnar cells provides permissive conditions for different facultative stem cell populations to dedifferentiate and repopulate the stem cell compartment. In this study, we used a defensin α4-Cre recombinase (Defa4Cre) line to define the potential of Paneth cells to dedifferentiate and contribute to intestinal stem cell (ISC) maintenance during normal homeostasis and after intestinal injury. METHODS: Small intestine and enteroids from Defa4Cre;Rosa26 tandem dimer Tomato (tdTomato), a red fluoresent protein, (or Rosa26 Enhanced Yellow Fluorescent Protein (EYFP)) reporter, Notch gain-of-function (Defa4Cre;Rosa26 Notch Intracellular Domain (NICD)-ires-nuclear Green Fluorescent Protein (nGFP) and Defa4Cre;Rosa26reverse tetracycline transactivator-ires Enhanced Green Fluorescent Protein (EGFP);TetONICD), A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) loss-of-function (Defa4Cre;ADAM10flox/flox), and Adenomatous polyposis coli (APC) inactivation (Defa4Cre;APCflox/flox) mice were analyzed. Doxorubicin treatment was used as an acute intestinal injury model. Lineage tracing, proliferation, and differentiation were assessed in vitro and in vivo. RESULTS: Defa4Cre-expressing cells are fated to become mature Paneth cells and do not contribute to ISC maintenance during normal homeostasis in vivo. However, spontaneous lineage tracing was observed in enteroids, and fluorescent-activated cell sorter-sorted Defa4Cre-marked cells showed clonogenic enteroid growth. Notch activation in Defa4Cre-expressing cells caused dedifferentiation to multipotent ISCs in vivo and was required for adenoma formation. ADAM10 deletion had no significant effect on crypt homeostasis. However, after acute doxorubicin-induced injury, Defa4Cre-expressing cells contributed to regeneration in an ADAM10-Notch-dependent manner. CONCLUSIONS: Our studies have shown that Defa4Cre-expressing Paneth cells possess cellular plasticity, can dedifferentiate into multipotent stem cells upon Notch activation, and can contribute to intestinal regeneration in an acute injury model.
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Plasticidad de la Célula , Integrasas/metabolismo , Intestinos/lesiones , Intestinos/patología , Células de Paneth/metabolismo , Receptores Notch/metabolismo , alfa-Defensinas/metabolismo , Proteína ADAM10/metabolismo , Adenoma/patología , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Alelos , Animales , Desdiferenciación Celular , Linaje de la Célula , Células Clonales , Doxorrubicina , Eliminación de Gen , Homeostasis , Hiperplasia , Ratones , Mitosis , Células Madre Multipotentes/metabolismo , Organoides/crecimiento & desarrollo , Organoides/patología , RegeneraciónRESUMEN
Colorectal cancer is treated with antibodies blocking epidermal growth factor receptor (EGF-R), but therapeutic success is limited. EGF-R is stimulated by soluble ligands, which are derived from transmembrane precursors by ADAM17-mediated proteolytic cleavage. In mouse intestinal cancer models in the absence of ADAM17, tumorigenesis was almost completely inhibited, and the few remaining tumors were of low-grade dysplasia. RNA sequencing analysis demonstrated down-regulation of STAT3 and Wnt pathway components. Because EGF-R on myeloid cells, but not on intestinal epithelial cells, is required for intestinal cancer and because IL-6 is induced via EGF-R stimulation, we analyzed the role of IL-6 signaling. Tumor formation was equally impaired in IL-6-/- mice and sgp130Fc transgenic mice, in which only trans-signaling via soluble IL-6R is abrogated. ADAM17 is needed for EGF-R-mediated induction of IL-6 synthesis, which via IL-6 trans-signaling induces ß-catenin-dependent tumorigenesis. Our data reveal the possibility of a novel strategy for treatment of colorectal cancer that could circumvent intrinsic and acquired resistance to EGF-R blockade.
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Proteína ADAM17/metabolismo , Receptores ErbB/metabolismo , Interleucina-6/metabolismo , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Transducción de Señal , Proteína ADAM17/deficiencia , Poliposis Adenomatosa del Colon/metabolismo , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Microbioma Gastrointestinal , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Intestinales/genética , Intestino Delgado/patología , Antígeno Ki-67/metabolismo , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Estadificación de Neoplasias , Organoides/patología , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción STAT3/metabolismo , Carga Tumoral , beta Catenina/metabolismoRESUMEN
The host metalloprotease meprin ß is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin ß must be proteolytically shed from epithelial cells. Hence, regulation of meprin ß shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin ß activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin ß and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin ß activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin ß into its active form, impairing meprin ß shedding and its function as a mucus-detaching protease.
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Adhesinas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Metaloendopeptidasas/metabolismo , Metaloproteasas/metabolismo , Secuencia de Aminoácidos/genética , Animales , Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Femenino , Cisteína-Endopeptidasas Gingipaínas , Células HEK293 , Humanos , Masculino , Metaloendopeptidasas/genética , Ratones Transgénicos , Mucina 2/genética , Mucina 2/metabolismoRESUMEN
A disintegrin and metalloproteinases (ADAMs) are a family of mSultidomain, membrane-anchored proteases that regulate diverse cellular functions, including cell adhesion, migration, proteolysis and other cell signaling events. Catalytically-active ADAMs act as ectodomain sheddases that proteolytically cleave type I and type II transmembrane proteins and some GPI-anchored proteins from the cellular surface. ADAMs can also modulate other cellular signaling events through a process known as regulated intramembrane proteolysis (RIP). Through their proteolytic activity, ADAMs can rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g. inflammation) and play a central role in coordinating intercellular communication. Dysregulation of these processes through aberrant expression, or sustained ADAM activity, is linked to chronic inflammation, inflammation-associated cancer and tumorigenesis. ADAM10 was the first disintegrin-metalloproteinase demonstrated to have proteolytic activity and is the prototypic ADAM associated with RIP activity (e.g. sequential Notch receptor processing). ADAM10 is abundantly expressed throughout the gastrointestinal tract and during normal intestinal homeostasis ADAM10 regulates many cellular processes associated with intestinal development, cell fate specification and maintenance of intestinal stem cell/progenitor populations. In addition, several signaling pathways that undergo ectodomain shedding by ADAM10 (e.g. Notch, EGFR/ErbB, IL-6/sIL-6R) help control intestinal injury/regenerative responses and may drive intestinal inflammation and colon cancer initiation and progression. Here, I review some of the proposed functions of ADAM10 associated with intestinal crypt homeostasis and tumorigenesis within the gastrointestinal tract in vivo. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.
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Proteína ADAM10/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Carcinogénesis/genética , Proteínas de la Membrana/genética , Neoplasias/genética , Proteolisis , Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Membrana Celular/genética , Humanos , Inflamación/genética , Inflamación/patología , Proteínas de la Membrana/metabolismo , Neoplasias/patologíaRESUMEN
BACKGROUND & AIMS: Total parenteral nutrition (TPN), a crucial treatment for patients who cannot receive enteral nutrition, is associated with mucosal atrophy, barrier dysfunction, and infectious complications. Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) improve intestinal epithelial cell (IEC) responses and attenuate mucosal atrophy in several TPN models. However, it remains unclear whether these 2 factors use distinct or overlapping signaling pathways to improve IEC responses. We investigated the interaction of GLP-2 and EGF signaling in a mouse TPN model and in patients deprived of enteral nutrition. METHODS: Adult C57BL/6J, IEC-Egfrknock out (KO) and IEC-pik3r1KO mice receiving TPN or enteral nutrition were treated with EGF or GLP-2 alone or in combination with reciprocal receptor inhibitors, GLP-2(3-33) or gefitinib. Jejunum was collected and mucosal atrophy and IEC responses were assessed by histologic, gene, and protein expression analyses. In patients undergoing planned looped ileostomies, fed and unfed ileum was analyzed. RESULTS: Enteral nutrient deprivation reduced endogenous EGF and GLP-2 signaling in mice and human beings. In the mouse TPN model, exogenous EGF or GLP-2 attenuated mucosal atrophy and restored IEC proliferation. The beneficial effects of EGF and GLP-2 were decreased upon Gefitinib treatment and in TPN-treated IEC-EgfrKO mice, showing epidermal growth factor-receptor dependency for these IEC responses. By contrast, in TPN-treated IEC-pi3kr1KO mice, the beneficial actions of EGF were lost, although GLP-2 still attenuated mucosal atrophy. CONCLUSIONS: Upon enteral nutrient deprivation, exogenous GLP-2 and EGF show strong interdependency for improving IEC responses. Understanding the differential requirements for phosphatidylinositol 3-kinase/phosphoAKT (Ser473) signaling may help improve future therapies to prevent mucosal atrophy.
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Efficient clearance of pro-inflammatory macrophages from tissues after resolution of a challenge is critical to prevent prolonged inflammation. Defects in clearance can contribute to conditions such as inflammatory bowel disease, and thus may be therapeutically targetable. However, the signaling pathways that induce termination of pro-inflammatory macrophages are incompletely defined. We tested whether the ErbB4 receptor tyrosine kinase, previously not known to have role in macrophage biology, is involved in this process. In vitro, pro-inflammatory activation of cultured murine and human macrophages induced ErbB4 expression; in contrast, other ErbB family members were not induced in pro-inflammatory cells, and other innate immune lineages (dendritic cells, neutrophils) did not express detectable ErbB4 levels. Treatment of activated pro-inflammatory macrophages with the ErbB4 ligand neuregulin-4 (NRG4) induced apoptosis. ErbB4 localized to the mitochondria in these cells. Apoptosis was accompanied by loss of mitochondrial membrane potential, and was dependent upon the proteases that generate the cleaved ErbB4 intracellular domain fragment, suggesting a requirement for this fragment and mitochondrial pathway apoptosis. In vivo, ErbB4 was highly expressed on pro-inflammatory macrophages but not neutrophils during experimental DSS colitis in C57Bl/6 mice. Active inflammation in this model suppressed NRG4 expression, which may allow for macrophage persistence and ongoing inflammation. Consistent with this notion, NRG4 levels rebounded during the recovery phase, and administration of exogenous NRG4 during colitis reduced colonic macrophage numbers and ameliorated inflammation. These data define a novel role for ErbB4 in macrophage apoptosis, and outline a mechanism of feedback inhibition that may promote resolution of colitis.
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Apoptosis/fisiología , Colon/metabolismo , Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Macrófagos/metabolismo , Receptor ErbB-4/metabolismo , Animales , Células Cultivadas , Colitis/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Ratones Endogámicos C57BL , Neurregulinas/metabolismo , Neutrófilos/metabolismo , Transducción de Señal/fisiologíaRESUMEN
A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis.