Tissue-nonspecific alkaline phosphatase (TNALP) is a glycoprotein expressed by osteoblasts that promotes bone mineralization. TNALP catalyzes the hydrolysis of the mineralization inhibitor inorganic pyrophosphate and ATP to provide inorganic phosphate, thus controlling the inorganic pyrophosphate/inorganic phosphate ratio to enable the growth of hydroxyapatite crystals. N-linked glycosylation of TNALP is essential for protein stability and enzymatic activity and is responsible for the presence of different bone isoforms of TNALP associated with functional and clinical differences. The site-specific glycosylation profiles of TNALP are, however, elusive. TNALP has 5 potential N-glycosylation sites located at the asparagine (N) residues 140, 230, 271, 303, and 430. The objective of this study was to reveal the presence and structure of site-specific glycosylation in TNALP expressed in osteoblasts. Calvarial osteoblasts derived from Alpl^+/− expressing SV40 Large T antigen were transfected with soluble epitope-tagged human TNALP. Purified TNALP was analyzed with a lectin microarray, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and liquid chromatography with tandem mass spectrometry. The results showed that all sites (n = 5) were fully occupied predominantly with complex-type N-glycans. High abundance of galactosylated biantennary N-glycans with various degrees of sialylation was observed on all sites, as well as glycans with no terminal galactose and sialic acid. Furthermore, all sites had core fucosylation except site N271. Modelling of TNALP, with the protein structure prediction software ColabFold, showed possible steric hindrance by the adjacent side chain of W270, which could explain the absence of core fucosylation at N271. These novel findings provide evidence for N-linked glycosylation on all 5 sites of TNALP, as well as core fucosylation on 4 out of 5 sites. We anticipate that this new knowledge can aid in the development of functional and clinical assays specific for the TNALP bone isoforms.

BackgroundThe pathobiology of the non-destructive inflammatory bowel disease (IBD) lymphocytic colitis (LC) is poorly understood. We aimed to define an LC-specific mucosal transcriptome to gain insight into LC pathology, identify unique genomic signatures, and uncover potentially druggable disease pathways.MethodsWe performed bulk RNA-sequencing of LC and collagenous colitis (CC) colonic mucosa from patients with active disease, and healthy controls (n = 4-10 per cohort). Differential gene expression was analyzed by gene-set enrichment and deconvolution analyses to identify pathologically relevant pathways and cells, respectively, altered in LC. Key findings were validated using reverse transcription quantitative PCR and/or immunohistochemistry. Finally, we compared our data with a previous cohort of ulcerative colitis and Crohn's disease patients (n = 4 per group) to distinguish non-destructive from classic IBD.ResultsLC can be subdivided into channelopathic LC, which is governed by organic acid and ion transport dysregulation, and inflammatory LC, which is driven by microbial immune responses. Inflammatory LC displays an innate and adaptive immunity that is limited compared to CC and classic IBD. Conversely, we noted a distinct induction of regulatory non-coding RNA species in inflammatory LC samples. Moreover, compared with CC, water channel and cell adhesion molecule gene expression decreased in channelopathic LC, whereas it was accentuated in inflammatory LC and associated with reduced intestinal epithelial cell proliferation.ConclusionsWe conclude that LC can be subdivided into channelopathic LC and inflammatory LC that could be pathomechanistically distinct subtypes despite their shared clinical presentation. Inflammatory LC exhibits a dampened immune response compared to CC and classic IBDs. Our results point to regulatory micro-RNAs as a potential disease-specific feature that may be amenable to therapeutic intervention. image

The forkhead box family transcription factor FOXQ1 is highly induced in several types of carcinomas, where it promotes epithelial-to-mesenchymal transition and tumour metastasis. The molecular mechanisms that lead to FOXQ1 deregulation in cancer are incompletely understood. Here, we used CRISPR/Cas9-based genomic locus proteomics (GLoPro) and promoter reporter constructs to discover transcriptional regulators of FOXQ1, and identified the tumour suppressor p53 as a negative regulator of FOXQ1 expression. ChIP-qPCR as well as complementary gain and loss-of-function assays in model cell lines indicated that p53 binds close to the transcription start site of the FOXQ1 promoter, and that it suppresses FOXQ1 expression in various cell types. Consistently, pharmacological activation of p53 using nutlin-3 or doxorubicin reduced FOXQ1 mRNA and protein levels in cancer cell lines harboring wild-type p53. Finally, we observed that p53 mutations are associated with increased FOXQ1 expression in human cancers. Altogether, these results suggest that loss of p53 function - a hallmark feature of many types of cancer - de-represses FOXQ1, which in turn promotes tumour progression.

The Wnt/fl-catenin pathway is a critical regulator of devel-opment and stem cell maintenance. Mounting evidence suggests that the outcome of Wnt signaling is determined by the collab-orative action of multiple transcription factors, including members of the highly conserved forkhead box (FOX) protein family. However, the contribution of FOX transcription factors to Wnt signaling has not been investigated in a systematic manner. Here, we performed complementary screens of all 44 human FOX proteins to identify new Wnt pathway regulators. By combining fl-catenin reporter assays with Wnt pathway-focused qPCR arrays and proximity proteomics of selected candidates, we determine that most FOX proteins are involved in the regu-lation of Wnt pathway activity. As proof-of-principle, we addi-tionally characterize class D and I FOX transcription factors as physiologically relevant regulators of Wnt/fl-catenin signaling. We conclude that FOX proteins are common regulators of Wnt/ fl-catenin-dependent gene transcription that may control Wnt pathway activity in a tissue-specific manner.

The forkhead box transcription factor FOXQ1 contributes to the pathogenesis of carcinomas. In colorectal cancers, FOXQ1 promotes tumour metastasis by inducing epithelial-to-mesenchymal transition (EMT) of cancer cells. FOXQ1 may exacerbate cancer by activating the oncogenic Wnt/beta-catenin signalling pathway. However, the role of FOXQ1 in the Wnt pathway remains to be resolved. Here, we report that FOXQ1 is an activator of Wnt-induced transcription and regulator of beta-catenin target gene expression. Upon Wnt pathway activation, FOXQ1 synergises with the beta-catenin nuclear complex to boost the expression of major Wnt targets. In parallel, we find that FOXQ1 controls the differential expression of various Wnt target genes in a beta-catenin-independent manner. Using RNA sequencing of colorectal cancer cell lines, we show that Wnt signalling and FOXQ1 converge on a transcriptional programme linked to EMT and cell migration. Additionally, we demonstrate that FOXQ1 occupies Wnt-responsive elements in beta-catenin target gene promoters and recruits a similar set of co-factors to the beta-catenin-associated transcription factor Tcf711. Taken together, our results indicate a multifaceted role of FOXQ1 in Wnt/beta-catenin signalling, which may drive the metastasis of colorectal cancers.

Simple Summary Cancer is caused by a breakdown of cell-to-cell communication, which results in the unrestricted expansion of cells within a tissue. In many cases, tumor growth is maintained by the continuous activation of cell signaling programs that normally drive embryonic development and wound repair. In this review article, I discuss how one of the largest human protein families, namely FOX proteins, controls the activity of the Wnt pathway, a major regulatory signaling cascade in developing organisms and adult stem cells. Evidence suggests that there is considerable crosstalk between FOX proteins and the Wnt pathway, which contributes to cancer initiation and progression. A better understanding of FOX biology may therefore lead to the development of new targeted treatments for many types of cancer. Aberrant activation of the oncogenic Wnt signaling pathway is a hallmark of numerous types of cancer. However, in many cases, it is unclear how a chronically high Wnt signaling tone is maintained in the absence of activating pathway mutations. Forkhead box (FOX) family transcription factors are key regulators of embryonic development and tissue homeostasis, and there is mounting evidence that they act in part by fine-tuning the Wnt signaling output in a tissue-specific and context-dependent manner. Here, I review the diverse ways in which FOX transcription factors interact with the Wnt pathway, and how the ectopic reactivation of FOX proteins may affect Wnt signaling activity in various types of cancer. Many FOX transcription factors are partially functionally redundant and exhibit a highly restricted expression pattern, especially in adults. Thus, precision targeting of individual FOX proteins may lead to safe treatment options for Wnt-dependent cancers.

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

The CCNY gene, which encodes cyclin Y, has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Cyclin Y promotes Wnt/β-catenin signaling and autophagy, which are critical for intestinal epithelial cell (IEC) homeostasis, and may thereby contribute to wound repair in colitis. However, whether cyclin Y has an essential function in IECs is unknown. We, therefore, investigated the epithelial injury response and mucosal regeneration in mice with conditional knock-out of Ccny in the intestinal epithelium. We observed that Ccny-deficient mice did not exhibit any differences in cell proliferation and disease activity compared to wild-type littermates in the dextran sulfate sodium (DSS) colitis model. Complementary in vitro experiments showed that loss of CCNY in model IECs did not affect Wnt signaling, cell proliferation, or autophagy. Additionally, we observed that expression of the cyclin-Y-associated cyclin-dependent kinase (CDK) 14 is exceedingly low specifically in IEC. Collectively, these results suggest that cyclin Y does not contribute to intestinal epithelial homeostasis, possibly due to low levels of specific CDKs in these cells. Thus, it is unlikely that CCNY mutations are causatively involved in IBD pathogenesis.

BACKGROUND AND AIMS: The pathophysiology of the inflammatory bowel disease collagenous colitis (CC) is poorly described. Our aim was to use RNA sequencing of mucosal samples from patients with active CC, CC in remission, refractory CC, ulcerative colitis (UC), and controls to gain insight into CC pathophysiology, identify genetic signatures linked to CC, and uncover potentially druggable disease pathways.

METHODS: We performed whole transcriptome sequencing of CC samples from patients before and during treatment with the corticosteroid drug budesonide, CC steroid-refractory patients, UC patients, and healthy controls (n=9-13). Bulk mucosa and laser-captured microdissected intestinal epithelial cell (IEC) gene expression were analyzed by gene-set enrichment and gene-set variation analyses to identify significant pathways and cells, respectively, altered in CC. Leading genes and cells were validated using reverse transcription quantitative PCR and/or immunohistochemistry.

RESULTS: We identified an activation of the adaptive immune response to bacteria and viruses in active CC that could be mediated by dendritic cells. Moreover, IECs display hyperproliferation and increased antigen presentation in active CC. Further analysis revealed that genes related to the immune response (DUOX2, PLA2G2A, CXCL9), DNA transcription (CTR9), protein processing (JOSD1, URI1) and ion transport (SLC9A3) remained dysregulated even after budesonide-induced remission. Budesonide-refractory CC patients fail to restore normal gene expression, and displayed a transcriptomic profile close to UC.

CONCLUSIONS: Our study confirmed the implication of innate and adaptive immune responses in CC, governed by IECs and dendritic cells, respectively; and identified ongoing epithelial damage. Refractory CC could share pathomechanisms with UC.

Forkhead box (FOX) family transcription factors play essential roles in development, tissue homeostasis, and disease. Although the biology of several FOX proteins has been studied in depth, it is unclear to what extent these findings apply to even closely related family members, which frequently exert overlapping but non-redundant functions. To help address this question, we have generated a uniform, ready-to-use expression library of all 44 human FOX transcription factors with a convenient peptide tag for parallel screening assays. In addition, we have generated multiple universal forkhead box reporter plasmids, which can be used to monitor the transcriptional activity of most FOX proteins with high fidelity. As a proof-of-principle, we use our plasmid library to identify the DNA repair protein XRCC6/Ku70 as a selective FOX interaction partner and regulator of FOX transcriptional activity. We believe that these tools, which we make available via the Addgene plasmid repository, will considerably expedite the investigation of FOX protein biology.

BACKGROUND AND AIMS: Diarrhoea is a common, debilitating symptom of gastrointestinal disorders. Pathomechanisms probably involve defects in trans-epithelial water transport, but the role of aquaporin [AQP] family water channels in diarrhoea-predominant diseases is unknown. We investigated the involvement of AQPs in the pathobiology of collagenous colitis [CC], which features chronic, watery diarrhoea despite overtly normal intestinal epithelial cells [IECs].

METHODS: We assessed the expression of all AQP family members in mucosal samples of CC patients before and during treatment with the corticosteroid drug budesonide, steroid-refractory CC patients and healthy controls. Samples were analysed by genome-wide mRNA sequencing [RNA-seq] and quantitative real-time PCR [qPCR]. In some patients, we performed tissue microdissection followed by RNA-seq to explore the IEC-specific CC transcriptome. We determined changes in the protein levels of the lead candidates in IEC by confocal microscopy. Finally, we investigated the regulation of AQP expression by corticosteroids in model cell lines.

RESULTS: Using qPCR and RNA-seq, we identified loss of AQP8 expression as a hallmark of active CC, which was reverted by budesonide treatment in steroid-responsive but not refractory patients. Consistently, decreased AQP8 mRNA and protein levels were observed in IECs of patients with active CC, and steroid drugs increased AQP8 expression in model IECs. Moreover, low APQ8 expression was strongly associated with higher stool frequency in CC patients.

CONCLUSION: Down-regulation of epithelial AQP8 may impair water resorption in active CC, resulting in watery diarrhoea. Our results suggest that AQP8 is a potential drug target for the treatment of diarrhoeal disorders.

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The Wnt signaling pathway is of paramount importance for development and disease. However, the tissue-specific regulation of Wnt pathway activity remains incompletely understood. Here we identify FOXB2, an uncharacterized forkhead box family transcription factor, as a potent activator of Wnt signaling in normal and cancer cells. Mechanistically, FOXB2 induces multiple Wnt ligands, including WNT7B, which increases TCF/LEF-dependent transcription without activating Wnt coreceptor LRP6 or beta-catenin. Proximity ligation and functional complementation assays identified several transcription regulators, including YY1, JUN, and DDX5, as cofactors required for FOXB2-dependent pathway activation. Although FOXB2 expression is limited in adults, it is induced in select cancers, particularly advanced prostate cancer. RNA-seq data analysis suggests that FOXB2/WNT7B expression in prostate cancer is associated with a transcriptional program that favors neuronal differentiation and decreases recurrence-free survival. Consistently, FOXB2 controls Wnt signaling and neuroendocrine differentiation of prostate cancer cell lines. Our results suggest that FOXB2 is a tissue-specific Wnt activator that promotes the malignant transformation of prostate cancer.

Chronic inflammatory bowel diseases, including Crohns disease and ulcerative colitis, are a major health burden worldwide. Numerous conserved signaling pathways control tissue injury and repair during colitis, but owing to the complexity of the inflammatory process, their individual contribution remains poorly understood. A key regulatory pathway in the intestinal mucosa is Wnt/beta-catenin signaling, which acts as the central organizer of epithelial stem cell identity and maintenance. Apart from this core function, there is mounting evidence that the Wnt pathway is highly interconnected with numerous other signaling cascades, and that combinatorial signaling events shape epithelial homeostasis and tissue regeneration. Here we provide an updated view of how Wnt signaling intersects with major inflammatory pathways, with a particular focus on intestinal inflammation. Elucidating the reciprocal actions of Wnt ligands and cytokines has the potential to reveal new treatment options for chronic colitis and other inflammatory disorders.

Spermiogenesis is the final phase during sperm cell development in which round spermatids undergo dramatic morphological changes to generate spermatozoa. Here we report that the serine/threonine kinase Stk33 is essential for the differentiation of round spermatids into functional sperm cells and male fertility. Constitutive Stk33 deletion in mice results in severely malformed and immotile spermatozoa that are particularly characterized by disordered structural tail elements. Stk33 expression first appears in primary spermatocytes, and targeted deletion of Stk33 in these cells recapitulates the defects observed in constitutive knockout mice, confirming a germ cell-intrinsic function. Stk33 protein resides in the cytoplasm and partially co-localizes with the caudal end of the manchette, a transient structure that guides tail elongation, in elongating spermatids, and loss of Stk33 leads to the appearance of a tight, straight and elongated manchette. Together, these results identify Stk33 as an essential regulator of spermatid differentiation and male fertility.

Angiopoietin-like 4 (ANGPTL4) is a secreted signaling protein that is implicated in cardiovascular disease, metabolic disorder, and cancer. Outside of its role in lipid metabolism, ANGPTL4 signaling remains poorly understood. Here, we identify ANGPTL4 as a Wnt signaling antagonist that binds to syndecans and forms a ternary complex with the Wnt co-receptor Lipoprotein receptor-related protein 6 (LRP6). This protein complex is internalized via clathrin-mediated endocytosis and degraded in lysosomes, leading to attenuation ofWnt/b-catenin signaling. Angptl4 is expressed in the Spemann organizer of Xenopus embryos and acts as a Wnt antagonist to promote notochord formation and prevent muscle differentiation. This unexpected function ofANGPTL4 invites reinterpretation of its diverse physiological effects in light of Wnt signaling and may open therapeutic avenues for human disease.

The canonical Wnt/beta-catenin signaling pathway is a central regulator of development and tissue homeostasis. In the intestine, Wnt signaling is primarily known as the principal organizer of epithelial stem cell identity and proliferation. Within the last decade, numerous scientific breakthroughs have shed light on epithelial self-organization in the gut, and organoids are now routinely used to study stem cell biology and intestinal pathophysiology. The contribution of non-epithelial cells to Wnt signaling in the gut has received less attention. However, there is mounting evidence that stromal cells are a rich source of Wnt pathway activators and inhibitors, which can dynamically shape Wnt signaling to control epithelial proliferation and restitution. Elucidating the extent and mechanisms of paracrine Wnt signaling in the intestine has the potential to broaden our understanding of epithelial homeostasis, and may be of particular relevance for disorders such as inflammatory bowel diseases and colitis-associated cancers.

Wnt/beta-catenin signaling plays a key role in embryonic development, stem cell biology, and neurogenesis. However, the mechanisms of Wnt signal transmission, notably how the receptors are regulated, remain incompletely understood. Here we describe that the Parkinson's disease-associated receptor GPR37 functions in the maturation of the N-terminal bulky beta-propellers of the Wnt co-receptor LRP6. GPR37 is required for Wnt/beta-catenin signaling and protects LRP6 from ER-associated degradation via CHIP (carboxyl terminus of Hsc70-interacting protein) and the ATPase VCP GPR37 is highly expressed in neural progenitor cells (NPCs) where it is required for Wnt-dependent neurogenesis. We conclude that GPR37 is crucial for cellular protein quality control during Wnt signaling.

The canonical Wnt signaling pathway is of paramount importance in development and disease. An emergent question is whether the upstream cascade of the canonical Wnt pathway has physiologically relevant roles beyond beta-catenin-mediated transcription, which is difficult to study due to the pervasive role of this protein. Here, we show that transcriptionally silent spermatozoa respond to Wnt signals released from the epididymis and that mice mutant for the Wnt regulator Cyclin Y-like 1 are male sterile due to immotile and malformed spermatozoa. Post-transcriptional Wnt signaling impacts spermatozoa through GSK3 by (1) reducing global protein poly-ubiquitination to maintain protein homeostasis; (2) inhibiting septin 4 phosphorylation to establish a membrane diffusion barrier in the sperm tail; and (3) inhibiting protein phosphatase 1 to initiate sperm motility. The results indicate that Wnt signaling orchestrates a rich post-transcriptional sperm maturation program and invite revisiting transcription-independent Wnt signaling in somatic cells as well.

Regional expression of Wingless/Int (Wnt) genes plays a central role in regulating intestinal development and homeostasis. However, our knowledge of such regional Wnt proteins in the colon remains limited. To understand further the effect of Wnt signaling components in controlling intestinal epithelial homeostasis, we investigated whether the physiological heterogeneity of the proximal and distal colon can be explained by differential Wnt signaling. With the use of a Wnt signaling-specific PCR array, expression of 84 Wnt-mediated signal transduction genes was analyzed, and a differential signature of Wnt-related genes in the proximal versus distal murine colon was identified. Several Wnt agonists (Wnt5a, Wnt8b, and Wnt11), the Wnt receptor frizzled family receptor 3, and the Wnt inhibitory factor 1 were differentially expressed along the colon length. These Wnt signatures were associated with differential epithelial cell proliferation and migration in the proximal versus distal colon. Furthermore, reduced Wnt/beta-catenin activity and decreased Wnt5a and Wnt11 expression were observed in mice lacking commensal bacteria, an effect that was reversed by conventionalization of germ-free mice. Interestingly, myeloid differentiation primary response gene 88 knockout mice showed decreased Wnt5a levels, indicating a role for Toll-like receptor signaling in regulating Wnt5a expression. Our results suggest that the morphological and physiological heterogeneity within the colon is in part facilitated by the differential expression of Wnt signaling components and influenced by colonization with bacteria.

The proinflammatory cytokine interferon gamma (IFNgamma ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. beta-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNgamma inhibits IEC proliferation despite sustained activation of Akt/beta-catenin signaling. Here we show that inhibition of Akt/beta-catenin-mediated cell proliferation by IFNgamma is associated with the formation of a protein complex containing phosphorylated beta-catenin 552 (pbeta-cat552) and 14.3.3zeta. Akt1 served as a bimodal switch that promotes or inhibits beta-catenin transactivation in response to IFNgamma stimulation. IFNgamma initially promotes beta-catenin transactivation through Akt-dependent C-terminal phosphorylation of beta-catenin to promote its association with 14.3.3zeta. Augmented beta-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3zeta to translocate 14.3.3zeta/beta-catenin from the nucleus, thereby inhibiting beta-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.

The molecular mechanisms that restore intestinal epithelial homeostasis during colitis are incompletely understood. Here, we report that during intestinal inflammation, multiple inflammatory cytokines promote the activity of a master regulator of cell proliferation and apoptosis, serine/threonine kinase CK2. Enhanced mucosal CK2 protein expression and activity were observed in animal models of chronic colitis, particularly within intestinal epithelial cells (IECs). The in vitro treatment of intestinal epithelial cell lines with cytokines resulted in increased CK2 expression and nuclear translocation of its catalytic alpha subunit. Similarly, nuclear translocation of CK2alpha was a prominent feature observed in colonic crypts from individuals with ulcerative colitis and Crohn's disease. Further in vitro studies revealed that CK2 activity promotes epithelial restitution, and protects normal IECs from cytokine-induced apoptosis. These observations identify CK2 as a key regulator of homeostatic properties of the intestinal epithelium that serves to promote wound healing, in part through inhibition of apoptosis under conditions of inflammation.

Mice lacking junctional adhesion molecule A (JAM-A, encoded by F11r) exhibit enhanced intestinal epithelial permeability, bacterial translocation, and elevated colonic lymphocyte numbers, yet do not develop colitis. To investigate the contribution of adaptive immune compensation in response to increased intestinal epithelial permeability, we examined the susceptibility of F11r(-/-)Rag1(-/-) mice to acute colitis. Although negligible contributions of adaptive immunity in F11r(+/+)Rag1(-/-) mice were observed, F11r(-/-)Rag1(-/-) mice exhibited increased microflora-dependent colitis. Elimination of T cell subsets and cytokine analyses revealed a protective role for TGF-beta-producing CD4(+) T cells in F11r(-/-) mice. Additionally, loss of JAM-A resulted in elevated mucosal and serum IgA that was dependent upon CD4(+) T cells and TGF-beta. Absence of IgA in F11r(+/+)Igha(-/-) mice did not affect disease, whereas F11r(-/-)Igha(-/-) mice displayed markedly increased susceptibility to acute injury-induced colitis. These data establish a role for adaptive immune-mediated protection from acute colitis under conditions of intestinal epithelial barrier compromise.

Epithelial cells form protective barriers that physically separate an organism from the outside world. Rather than being merely static, impregnable shields, epithelia are highly dynamic structures that can adjust their proliferation, differentiation, and death in response to intrinsic and extrinsic signals. The advantages as well as pitfalls of this flexibility are highlighted in inflammatory disorders such as inflammatory bowel diseases and psoriasis, which are characterized by a chronically dysregulated homeostasis of the epithelium. In recent years, it has become increasingly apparent that epithelial cells communicate with their surroundings through converging, integrated signaling cascades and that even minor alterations in these pathways can have dramatic pathologic consequences. In this review, we discuss how inflammatory cytokines and other signaling molecules, directly or through cross talk, regulate epithelial homeostasis in the intestine, and we highlight parallels and differences in a few other organs.

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 beta-catenin activation. Furthermore, IECs from transgenic JAM-A(-/-)/beta-catenin/T-cell factor reporter mice showed enhanced beta-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.

Background & Aims

Dkk1 is a secreted antagonist of the Wnt/β-catenin signaling pathway. It is induced by inflammatory cytokines during colitis and exacerbates tissue damage by promoting apoptosis of epithelial cells. However, little is known about the physiologic role of Dkk1 in normal intestinal homeostasis and during wound repair following mucosal injury. We investigated whether inhibition of Dkk1 affects the morphology and function of the adult intestine.

Methods

We used doubleridge mice (Dkk1d/d), which have reduced expression of Dkk1, and an inhibitory Dkk1 antibody to modulate Wnt/β-catenin signaling in the intestine. Intestinal inflammation was induced with dextran sulfate sodium (DSS), followed by a recovery period in which mice were given regular drinking water. Animals were killed before, during, or after DSS administration; epithelial homeostasis and the activity of major signaling pathways were investigated by morphometric analysis, bromo-2′-deoxyuridine incorporation, and immunostaining.

Results

Reduced expression of Dkk1 increased proliferation of epithelial cells and lengthened crypts in the large intestine, which was associated with increased transcriptional activity of β-catenin. Crypt extension was particularly striking when Dkk1 was inhibited during acute colitis. Dkk1d/dmice recovered significantly faster from intestinal inflammation but exhibited crypt architectural irregularities and epithelial hyperproliferation compared with wild-type mice. Survival signaling pathways were concurrently up-regulated in Dkk1d/d mice, including the AKT/β-catenin, ERK/Elk-1, and c-Jun pathways.

Conclusions

Dkk1, an antagonist of Wnt/β-catenin signaling, regulates intestinal epithelial homeostasis under physiologic conditions and during inflammation. Depletion of Dkk1 induces a strong proliferative response that promotes wound repair after colitis.

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.

Coronins, WD-repeat actin-binding proteins, are known to regulate cell motility by coordinating actin filament turnover in lamellipodia of migrating cell. Here we report a novel mechanism of Coronin 1C-mediated cell motility that involves regulation of cell-matrix adhesion. RNAi silencing of Coronin 1C in intestinal epithelial cells enhanced cell migration and modulated lamellipodia dynamics by increasing the persistence of lamellipodial protrusion. Coronin 1C-depleted cells showed increased cell-matrix adhesions and enhanced cell spreading compared to control cells, while over-expression of Coronin 1C antagonized cell adhesion and spreading. Enhanced cell-matrix adhesion of coronin-deficient cells correlated with hyperphosphorylation of focal adhesion kinase (FAK) and paxillin, and an increase in number of focal adhesions and their redistribution at the cell periphery. siRNA depletion of FAK in coronin-deficient cells rescued the effects of Coronin 1C depletion on motility, cell-matrix adhesion, and spreading. Thus, our findings provide the first evidence that Coronin 1C negatively regulates epithelial cell migration via FAK-mediated inhibition of cell-matrix adhesion.

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.

Infiltrating monocytes and macrophages contribute to the initiation and perpetuation of mucosal inflammation characteristic for human inflammatory bowel disease (IBD). Peripheral blood monocytes expressing the low-affinity Fcgamma receptor CD16 have been identified previously as a major proinflammatory cell population, based on their unique cytokine secretion profile. However, the contribution of these cells to the pathogenesis of inflammatory bowel disease remains to be elucidated. Thus, in this study we investigated whether the peripheral CD16(+) monocyte count correlates with common IBD disease parameters, and whether these cells infiltrate the intestinal mucosa under inflammatory conditions. We observed that CD16(+) peripheral blood monocytes are increased significantly in active Crohn's disease, particularly in patients with high Crohn's disease activity index and colonic involvement. Furthermore, we found that CD16(+) cells are a major contributor to the inflammatory infiltrate in Crohn's disease mucosa, although their spontaneous migration through primary human intestinal endothelial cells is limited. Our data suggest that lamina propria, but not peripheral blood, CD16(+) monocytes are a crucial proinflammatory cell population in IBD, and a potential target for anti-inflammatory therapy.

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.

In the intestine, a single layer of epithelial cells effectively separates potentially harmful luminal content from the underlying tissue. The importance of an intact mucosal layer is highlighted by pathological disorders of the gut such as inflammatory bowel disease, in which disruption of the epithelial barrier leads to severe inflammation of the submucosal tissue compartments. Epithelial barrier function is provided by tightly regulated intercellular junctions, which consist of a plethora of membrane-associated and transmembrane proteins organized in discreet, spatially restricted complexes. Classically, these complexes are known to be dynamic seals for fluids and small molecules, as well as to provide mechanical strength by anchoring cell-cell contacts to the cytoskeleton. Rather than just acting as simple gates and adapters, however, junctional complexes themselves can relay extracellular stimuli to the epithelium and initiate cellular responses such as differentiation and apoptosis. In this review, we will highlight recent studies by our group and others which discuss how junctional proteins can promote outside-to-inside signaling and modulate epithelial cell fate. Unraveling the complex crosstalk between epithelial cells and their intercellular junctions is essential to understanding how epithelial barrier function is maintained in vivo and might provide new strategies for the treatment of inflammatory disorders of the intestine.

Epithelial cell motility is critical for self-rejuvenation of normal intestinal mucosa, wound repair, and cancer metastasis. This process is regulated by the reorganization of the F-actin cytoskeleton, which is driven by a myosin II motor. However, the role of myosin II in regulating epithelial cell migration remains poorly understood. This study addressed the role of non-muscle myosin (NM) IIA in two different modes of epithelial cell migration: two-dimensional (2-D) migration that occurs during wound closure and three-dimensional (3-D) migration through a Matrigel matrix that occurs during cancer metastasis. Pharmacological inhibition or siRNA-mediated knockdown of NM IIA in SK-CO15 human colonic epithelial cells resulted in decreased 2-D migration and increased 3-D invasion. The attenuated 2-D migration was associated with increased cell adhesiveness to collagen and laminin and enhanced expression of beta1-integrin and paxillin. On the 2-D surface, NM IIA-deficient SK-CO15 cells failed to assemble focal adhesions and F-actin stress fibers. In contrast, the enhanced invasion of NM IIA-depleted cells was dependent on Raf-ERK1/2 signaling pathway activation, enhanced calpain activity, and increased calpain-2 expression. Our findings suggest that NM IIA promotes 2-D epithelial cell migration but antagonizes 3-D invasion. These observations indicate multiple functions for NM IIA, which, along with the regulation of the F-actin cytoskeleton and cell-matrix adhesions, involve previously unrecognized control of intracellular signaling and protein expression.

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.

Members of the interleukin (IL)-12 family constitute subunits of IL-12, -23, and -27. These ILs represent pivotal mediators in the regulation of cell-mediated immune responses and in animal models of human inflammatory bowel disease. Recent work has suggested that intestinal endothelial cells might serve as a second line of defense in bacterial sensing of invading pathogens. The purpose of this study was to examine the production of IL-12 family members in intestinal endothelial cells (HIMEC). HIMEC were stimulated with proinflammatory agents (TNF-alpha, IFN-gamma, IL-1beta) and microbial antigens [LPS, lipoteichoic acid, peptidoglycan, CpG-DNA, flagellin, poly(I:C)]. Expression of IL-12 family members and of Toll-like receptor (TLR)3 in HIMEC was assessed by real-time RT-PCR, immunostaining, flow cytometry, and immunoblot analysis. HIMEC display an induction of Epstein-Barr virus-induced gene 3 (EBI3), IL-12p35, and IL-23p19, whereas no expression of IL-12p40 and IL-27p28 was detectable. The strongest induction was induced by proinflammatory factors known to utilize the NF-kappaB pathway, and expression of EBI3 and IL-23p19 was diminished by an NF-kappaB inhibitor. HIMEC display regulated expression of TLR3. Adhesion and transmigration assays showed proinflammatory responses after HIMEC stimulation. HIMEC are capable of producing IL-12 family members as a response to microbial stimuli. The TLR3 agonist, poly(I:C), was shown to enhance leukocyte adhesion in vitro in HIMEC. Our data suggest that the intestinal microvasculature is responsive to ligands of TLR3 expressed on intestinal endothelial cells, thereby adding to the regulation of adaptive immunity and leukocyte recruitment.

Defective apoptosis of mucosal cell populations seems to be a relevant pathogenetic mechanism in inflammatory bowel disease (IBD). It has been suggested that the induction of apoptosis in various effector cells may be a relevant therapeutic mechanism in IBD. Indeed, it was shown that different drugs used for treatment of IBD have the capacity to induce apoptosis in T cells or monocytes in vitro and in vivo. However, it remains unclear whether these observations are related to clinical efficacy of these agents. TNF-alpha is one of the most relevant proinflammatory mediators in IBD and anti-TNF treatment has been shown to be of particular benefit for patients with IBD. It could subsequently be shown that various anti-TNF-alpha agents, such as infliximab and adalimumab, can induce apoptosis in activated monocytes and lymphocytes in vitro and in vivo. This mechanism requires reverse signaling via transmembranous TNF, thereby eliciting a signal transduction cascade that results in programmed cell death. Although other mechanisms might also contribute to the clinical effect of anti-TNF-alpha, current data suggest that apoptosis is a relevant mechanism that is associated with clinical efficacy of anti-TNF agents. Induction of apoptosis in activated monocytes or T cells may be regarded as therapeutic tool not only for anti-TNF agents, but also for other drugs used in IBD. Future strategies should focus on identification of mechanisms that prevent apoptosis in the mucosa of patients with IBD and in targeting apoptotic pathways as a therapeutic strategy in IBD.

Vascular endothelial cadherin (VE-cadherin) mediates contact inhibition of cell growth in quiescent endothelial cell layers. Searching for proteins that could be involved in VE-cadherin signaling, we found the cytosolic C-terminal Src kinase (Csk), a negative regulator of Src family kinases. We show that Csk binds via its SH2 domain to the phosphorylated tyrosine 685 of VE-cadherin. VE-cadherin recruits Csk to cell contacts and both proteins can be co-precipitated from cell lysates of transfected cells and endothelial cells. Association of VE-cadherin and Csk in endothelial cells increased with increasing cell density. CHO cells expressing the tyrosine replacement mutant VE-cadherin-Y685F grow to higher cell densities than cells expressing wild-type VE-cadherin. Overexpression of Csk in these cells under an inducible promoter inhibits cell proliferation in the presence and absence of VE-cadherin, but not in the presence of VE-cadherin-Y685F. Reduction of Csk expression by RNA interference enhances endothelial cell proliferation. Our results suggest that the phosphorylated tyrosine residue 685 of VE-cadherin and probably the binding of Csk to this site are involved in inhibition of cell growth triggered by cell density.