Primary cilia are sensory antennae located at the cell surface which mediate a variety of extracellular signals involved in development, tissue homeostasis, stem cells and cancer. Primary cilia are found in an extensive array of vertebrae cells but can only be generated when cells become quiescent. The small intestinal epithelium is a rapidly self-renewing tissue organized into a functional unit called the crypt–villus axis, containing progenitor and differentiated cells, respectively. Terminally differentiated villus cells are notoriously devoid of primary cilia. We sought to determine if intestinal crypts contain a quiescent cell population that could be identified by the presence of primary cilia. Here we show that primary cilia are detected in a subset of cells located deep in the crypts slightly above a Paneth cell population. Using a normal epithelial proliferative crypt cell model, we show that primary cilia assembly and activity correlate with a quiescent state. These results provide further evidence for the existence of a quiescent cell population in the human small intestine and suggest the potential for new modes of regulation in stem cell dynamics.

Epilepsy or seizure disorder is among the least understood chronic medical conditions affecting over 65 million people worldwide. Here, we show that disruption of the polycystic kidney disease 2-like 1 (Pkd2l1 or Pkdl), encoding polycystin-L (PCL), a non-selective cation channel, increases neuronal excitability and the susceptibility to pentylenetetrazol-induced seizure in mice. PCL interacts with β2-adrenergic receptor (β2AR) and co-localizes with β2AR on the primary cilia of neurons in the brain. Pkdl deficiency leads to the loss of β2AR on neuronal cilia, which is accompanied with a remarkable reduction in cAMP levels in the central nervous system (CNS). The reduction of cAMP levels is associated with a reduction in the activation of cAMP response element-binding protein, but not the activation of Ca(2+)/calmodulin-dependent protein kinase II, Akt or mitogen-activated protein kinases. Our data, thus, indicate for the first time that a ciliary protein complex is required for the control of neuronal excitability in the CNS.

The small intestine consists of two histological compartments composed of the crypts and the villi. The function of the adult small intestinal epithelium is mediated by four different types of mature cells: enterocytes, goblet, enteroendocrine and Paneth. Undifferentiated cells reside in the crypts and produce these four types of mature cells. The niche-related Wnt and Bmp signaling pathways have been suggested to be involved in the regulation and maintenance of the stem cell microenvironment. In our laboratory, we isolated the first normal human intestinal epithelial crypt (HIEC) cell model from the human fetal intestine and in this study we investigated the expression of a panel of intestinal stem cell markers in HIEC cells under normal culture parameters as well as under conditions that mimic the stem cell microenvironment. The results showed that short term stimulation of HIEC cells with R-spondin 1 and Wnt-3a±SB-216763, a glycogen synthase kinase 3β (GSK3β) inhibitor, induced β-catenin/TCF activity and expression of the WNT target genes, cyclin D2 and LGR5. Treatment of HIEC cells with noggin, an antagonist of BMP signaling, abolished SMAD2/5/8 phosphorylation. Inducing a switch from inactive WNT/active BMP toward active WNT/inactive BMP pathways was sufficient to trigger a robust intestinal primordial stem-like cell signature with predominant LGR5, PHLDA1, PROM1, SMOC2 and OLFM4 expression. These findings demonstrate that even fully established cultures of intestinal cells can be prompted toward a CBC stem cell-like phenotype. This model should be useful for studying the regulation of human intestinal stem cell self-renewal and differentiation.

The integrin α6 subunit pre-messenger RNA undergoes alternative splicing to generate two different splice variants, named α6A and α6B, having distinct cytoplasmic domains. In the human colonic gland, these splice variants display different patterns of expression suggesting specific functions for each variant. We have previously found an up-regulation of the α6β4 integrin in colon adenocarcinomas as well as an increase in the α6A/α6B ratio, but little is known about the involvement of α6Aβ4 versus α6Bβ4 in this context. The aim of this study was to elucidate the function of the α6Aβ4 integrin in human colorectal cancer (CRC) cells. Expression studies on a panel of primary CRCs confirmed that the up-regulation of the α6 subunit in CRC is a direct consequence of the increase of the α6A variant. To investigate the functional significance of an α6A up-regulation in CRC, we specifically knocked down its expression in well-established CRC cell lines using a small-hairpin RNA approach. Results showed a growth rate reduction in all α6A knockdown CRC cell lines studied. The α6A silencing was also found to be associated with a significant repression of a number of Wnt/β-catenin pathway end points. Moreover, it was accompanied by a reduction in the capacity of these cells to develop tumours in xenografts. Taken together, these results demonstrate that the α6A variant is a pro-proliferative form of the α6 integrin subunit in CRC cells and appears to mediate its effects through the Wnt/β-catenin pathway.

Interactions between the cell basal membrane domain and the basement membrane are involved in several cell functions including proliferation, migration and differentiation. Intestinal epithelial cells can interact with laminin, a major intestinal basement membrane glycoprotein, via several cell-surface laminin-binding proteins including integrin and non-integrin receptors. The 37/67kDa laminin receptor (37/67LR) is one of these but its role in normal epithelial cells is still unknown. The aim of this study was to characterise the expression pattern and determine the main function of 37/67LR in the normal human small intestinal epithelium. Immunolocalization studies revealed that 37/67LR was predominantly present in the undifferentiated/proliferative region of the human intestinal crypt in both the immature and adult intestine. Using a human intestinal epithelial crypt (HIEC) cell line as experimental model, we determined that 37/67LR was expressed in proliferative cells in both the cytoplasmic and membrane compartments. Small-interfering RNA-mediated reduction of 37/67LR expression led to HIEC cell-cycle reduction and loss of the ability to adhere to laminin-related peptides under conditions not altering ribosomal function. Taken together, these findings indicate that 37/67LR regulates proliferation and adhesion in normal intestinal epithelial cells independently of its known association with ribosomal function.

The proto-oncogene Src is an important protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and survival. Here, we investigated the involvement of Src family kinases (SFKs) in human intestinal cell differentiation. We first observed that Src activity peaked in early stages of Caco-2/15 cell differentiation. Inhibition of SFKs with PP2, a selective SFK inhibitor, accelerated the overall differentiation program. Interestingly, all polarization and terminal differentiation markers tested, including sucrase-isomaltase, lactase-phlorizin hydrolase and E and Li-cadherins were found to be significantly up-regulated after only 3days of treatment in the newly differentiating cells. Further investigation of the effects of PP2 revealed a significant up-regulation of the two main intestinal epithelial cell-specific transcription factors Cdx2 and HNF1α and a reduction of polycomb PRC2-related epigenetic repressing activity as measured by a decrease in H3K27me3, two events closely related to the control of cell terminal differentiation in the intestine. Taken together, these data suggest that SFKs play a key role in the control of intestinal epithelial cell terminal differentiation.

have previously shown that mouse embryonic fibroblasts (MEFs) null for Klf4 is genetically unstable as evidenced by the presence of high level of DNA damage, centrosome amplification, and aneuploidy.AIM: To investigate role and possible mechanisms of Klf4 in preserving genetic stability.METHODS: To determine the ability of Klf4 restoration in correcting the observed genetic instability in MEFS null for Klf4 (Klf4-/-), we overexpressed Klf4 in Klf4-/-MEFs and compared the results to wild type (Klf4+/+) and non-transfected Klf4-/-MEFs.Cell cycle profiles and proliferation curve was performed to determine growth characteristics.Quantification of chromosome number and immunohistochemical staining of centrosomes were performed to assess chromosome integrity.To determine the role of Klf4 in correcting DNA damage, we overexpressed or not Klf4 in Klf4-/-MEFs followed by γ-irradiation or not in parallel with Klf4+/+ and Klf4-/-MEFs.Using immunohistochemistry, γH2AX and p53BP1 foci were counted at 0, 1h, 4h and 24h after γ-irradiation.The levels of p53, p21, cyclin E, Cdk2 and γH2AX in Klf4+/+ and Klf4-/-MEFs, with and without Klf4 overexpression, were determined by Western blotting.RESULTS: Overexpression of Klf4 in Klf4-/-MEFs suppressed cell proliferation compared to untransfected Klf4+/+ and untransfected Klf4-/-MEFs.Centrosome staining showed a 10-fold decrease in the percent of cells with ≥3 centrosomes after overexpressing Klf4 in Klf4-/-MEFs compared to untransfected Klf4-/-MEFs.A higher percentage of Klf4-/-MEFs cells were detected with increased and sustained ≥5 γH2AX and p53BP1 foci following γ-irradiation compared to irradiated Klf4+/+ MEFs.Overexpression of Klf4 in Klf4-/-MEFs reduced the percentage of cells with ≥5 γH2AX foci to a level similar to that in Klf4+/+ MEFs, while the percent of cells with ≥5 p53BP1 foci were reduced to the same level as irradiated Klf4+/+ but at a faster rate.Karyotype analysis showed that Klf4-transfected Klf4-/-MEFs had a 25% decrease in number of cells exhibiting aneuploidy in contrast to untransfected Klf4-/-MEFs.Western blot analysis showed that p53, cyclin E, Cdk2 and γH2AX levels were reduced and p21 was elevated following Klf4 overexpression in Klf4-/-MEFs.CONCLUSION: Results of this study demonstrate that overexpression of Klf4 in MEFs null for the Klf4 alleles display evidence of improved genetic stability as illustrated by reduced DNA damage, aneuploidy, and centrosome amplification.These results indicate that KLF4 plays a crucial role in preserving genetic stability.

differentiation, thus showing that in analogy to the intestine, esophageal stemness is lost.Using RNA micro-arrays, we identified a gene signature of 47 genes that were lost in both individual cell lines upon induction of ER stress.Of these genes, we found 29 genes to be restricted to the basal layer of the mouse esophagus.Out of these 29, nine genes show expression in only a small proportion of the basal cells, potentially marking stem cells.Conclusion: ER stress depletes esophageal precursor cells.Our in vitro screen combined with in situ hybridization identified nine genes that are specifically expressed in a subset of proliferating genes, thereby potentially marking esophageal stem cells.

The crypt-villus axis constitutes the functional unit of the small intestine, where mature absorptive cells are confined to the villi, and stem cells and transit amplifying and differentiating cells are restricted to the crypts. The polycomb group (PcG) proteins repress differentiation and promote self-renewal in embryonic stem cells. PcGs prevent transcriptional activity by catalysing epigenetic modifications, such as the covalent addition of methyl groups on histone tails, through the action of the polycomb repressive complex 2 (PRC2). Although a role for PcGs in the preservation of stemness characteristics is now well established, recent evidence suggests that they may also be involved in the regulation of differentiation. Using intestinal epithelial cell models that recapitulate the enterocytic differentiation programme, we generated a RNAi-mediated stable knockdown of SUZ12, which constitutes a cornerstone for PRC2 assembly and functionality, in order to analyse intestinal cell proliferation and differentiation. Expression of SUZ12 was also investigated in human intestinal tissues, revealing the presence of SUZ12 in most proliferative epithelial cells of the crypt and an increase in its expression in colorectal cancers. Moreover, PRC2 disruption led to a significant precocious expression of a number of terminal differentiation markers in intestinal cell models. Taken together, our data identified a mechanism whereby PcG proteins participate in the repression of the enterocytic differentiation program, and suggest that a similar mechanism exists in situ to slow down terminal differentiation in the transit amplifying cell population.

Abstract Colon cancer is a major cause of death by cancer in North America. Recently, we found that both subunits of the integrin α6β4 are up-regulated in colon primary tumours. The α6 integrin subunit exists as two distinct splice isoforms, α6A and α6B. It is the α6A isoform that was shown to be associated with the proliferative cell population of the normal human colon and up-regulated in colorectal cancer. The aim of this study was to analyze the role of integrin α6Aβ4 in human colon cancer, more precisely its effect on cell proliferation and its mechanism of regulation. To investigate the function of integrin α6Aβ4 in colon cancer, expression of splice variant α6A was specifically abolished, using shRNA technology, in colon adenocarcinoma cell lines (Caco-2/15, T84, SW480, SW620, DLD-1 and HT29). The function of splice variant α6A of the α6β4 integrin was investigated in vitro and in tumour xenografts. In all colon cancer cell lines tested, knockdown of the integrin α6A subunit resulted in a decrease of up to 90% of the α6A variant without affecting the levels of the α6B variant. Furthermore, no compensatory expression of α6Bβ4 integrin was observed following the abolition of the α6A subunit. In the majority of colon cancer cell lines, the knockdown of α6A resulted in a significant decrease in proliferation as show by BrdU incorporation. Moreover, it was accompanied by reduced activity of the Wnt/β-catenin pathway as measured by a decrease of responsive β-catenin/TCF promoter activity and increased β-catenin phosphorylation at Ser37/Thr41 by GSK3β. Preliminary data confirmed that in these cells, the Wnt/β-catenin pathway can be stimulated by pharmacological inhibition of GSK3β (SB216763), as displayed by a decrease of β-catenin phosphorylation at Ser37/Thr41 and an increase in responsive β-catenin/TCF promoter activity. Analysis of splice variants of the α6 integrin subunit in human colorectal cancer revealed that α6A was increased in 80% of tumours as compared to their matched resection margins and a significant correlation was observed between expression of α6A and c-myc in the tumours. Furthermore, to determine the role of the integrin α6A subunit on colon cancer tumorigenesis in vivo, control and α6A knockdown T84, SW620, HT29 and DLD-1 cells were injected into nude mice. Results showed that abolition of the integrin α6A subunit led to a significant reduction in tumour growth for T84, SW620 and HT29 colon cell lines. Moreover, analysis of tumours showed an increase in β-catenin phosphorylation at Ser37/Thr41 in T84 and HT29. Our results show for the first time that splice variant A of the integrin α6 subunit regulates the Wnt/β-catenin pathway to promote cancer cell growth. Since 80% of colon cancers display an up-regulation of integrin α6A expression, targeting this integrin in colon cancers may offer new therapeutic value. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 30. doi:1538-7445.AM2012-30

Recently, autophagy has been found to be strongly activated in colon cancer cells, but few studies have addressed the normal colon mucosa. The aim of this study was to characterize autophagy in normal human intestinal cells. We used the expression of LC3-II and BECN1 as well as SQSTM1 as markers of autophagy activity. Using the normal human intestinal epithelial crypt (HIEC) cell experimental model, we found that autophagy was much more active in undifferentiated cells than in differentiated cells. In the normal adult colonic mucosa, BECN1 was found in the proliferative epithelial cells of the lower part of the gland while SQSTM1 was predominantly found in the differentiated cells of the upper part of the gland and surface epithelium. Interestingly, the weak punctate pattern of SQSTM1 expression in the lower gland colocalized with BECN1-labeled autophagosomes. The usefulness of SQSTM1 as an active autophagy marker was confirmed in colon cancer specimens at the protein and transcript levels. In conclusion, our results show that autophagy is active in the colonic gland and is associated with the intestinal proliferative/undifferentiated and progenitor cell populations.

Collagen VI is a heterotrimer composed of three α chains (α1, α2, α3) widely expressed throughout various interstitial matrices. Collagen VI is also found near the basement membranes of many tissues where it serves as an anchoring meshwork. The aim of this study was to investigate the distribution and role of collagen VI at the epithelial–stromal interface in the intestine. Results showed that collagen VI is a bona fide epithelial basal lamina component and constitutes the major collagen type of epithelial origin in this organ. In vitro, collagen VI co-distributes with fibronectin. Targeted knockdown of collagen VI expression in intestinal epithelial cells was used to investigate its function. Depletion of collagen VI from the matrix led to a significant increase in cell spreading and fibrillar adhesion formation coinciding with an upregulation of fibronectin expression, deposition and organization as well as activation of myosin light chain phosphorylation by the myosin light chain kinase and Rho kinase dependent mechanisms. Plating cells deficient for collagen VI on collagen VI rescued the phenotype. Taken together, these data demonstrate that collagen VI is an important basal lamina component involved in the regulation of epithelial cell behavior most notably as a regulator of epithelial cell–fibronectin interactions.

Integrin‐linked kinase (ILK) plays a role in integrin signaling‐mediated extracellular matrix (ECM)–cell interactions and also acts as a scaffold protein in functional focal adhesion points. In the present study, we investigated the expression and roles of ILK in human intestinal epithelial cells (IECs) in vivo and in vitro. Herein, we report that ILK and its scaffold‐function interacting partners, PINCH‐1, α‐parvin, and β‐parvin, are expressed according to a decreasing gradient from the bottom of the crypt (proliferative/undifferentiated) compartment to the tip of the villus (non‐proliferative/differentiated) compartment, closely following the expression pattern of the ECM/basement membrane component fibronectin. The siRNA knockdown of ILK in human IECs caused a loss of PINCH‐1, α‐parvin, and β‐parvin expression, along with a significant decrease in cell proliferation via a loss of cyclin D1 and an increase in p27 and hypophosphorylated pRb expression levels. ILK knockdown severely affected cell spreading, migration, and restitution abilities, which were shown to be directly related to a decrease in fibronectin deposition. All ILK knockdown‐induced defects were rescued with exogenously deposited fibronectin. Altogether, our results indicate that ILK performs crucial roles in the control of human intestinal cell and crypt–villus axis homeostasis—especially with regard to basement membrane fibronectin deposition—as well as cell proliferation, spreading, and migration. J. Cell. Physiol. 222: 387–400, 2010. © 2009 Wiley‐Liss, Inc.

In this study, we have analyzed the expression and localization of polycystin-1 in intestinal epithelial cells, a system lacking primary cilia. Polycystin-1 was found to be expressed in the epithelium of the small intestine during development and levels remained elevated in the adult. Dual-labelling indirect immunofluorescence revealed polycystin-1 at sites of cell–cell contact co-localizing with the desmosomes both in situ as well as in polarized Caco-2/15 cells. In unpolarized cultures of Caco-2/15 cells, polycystin-1 was recruited to the cell surface early during initiation of cell junction assembly. In isolated Caco-2/15 cells and HIEC-6 cell cultures, where junctional complexes are absent, polycystin-1 was found predominantly associated with the cytoskeletal elements of the intermediate filaments and microtubule networks. More precisely, polycystin-1 was seen as brightly labelled puncta decorating the keratin-18 positive filaments as well as the β-tubulin positive microtubules, which was particularly obvious in the lamellipodia. Treatment with the microtubule-disrupting agent, nocodazole, eliminated the microtubule association of polycystin-1 but did not seem to affect its association with keratin or the desmosomes. Taken together these data suggest that polycystin-1 is involved with the establishment of cell–cell junctions in absorptive intestinal epithelial cells and exploits the microtubule-based machinery in order to be transported to the plasma membrane.

Rapid and efficient healing of epithelial damage is critical to the functional integrity of the small intestine. Epithelial repair is a complex process that has largely been studied in cultured epithelium but to a much lesser extent in mucosa. We describe a novel method for the study of wound healing using a co-culture system that combined an intestinal epithelial Caco-2/15 cell monolayer cultured on top of human intestinal myofibroblasts, which together formed a basement membrane-like structure that contained many of the major components found at the epithelial-mesenchymal interface in the human intestine. To investigate the mechanism of restitution, small lesions were generated in epithelial cell monolayers on plastic or in co-cultures without disturbing the underlying mesenchymal layer. Monitoring of wound healing showed that repair was more efficient in Caco-2/15-myofibroblast co-cultures than in Caco-2/15 monolayers and involved the deposition of basement membrane components. Functional experiments showed that the addition of type I collagen or human fibronectin to the culture medium significantly accelerated wound closure on epithelial cell co-cultures. This system may provide a new tool to investigate the mechanisms that regulate wound healing in the intestinal epithelium.

The expression of apoptotic factors Bcl-2 and Bax were studied in the conjunctiva of diabetic patients with and without retinopathy. All patients underwent a complete ophthalmic examination including ocular fundus and retinal fluorescein angiography. The indirect immunoperoxidase method was performed on 15 normal conjunctiva taken during cataract surgery (group 1), on 40 eyes of 40 patients with type 2 diabetes without diabetic retinopathy (group 2) and 13 eyes of 13 patients with diabetic retinopathy (group 3). In normal human conjunctiva, Bax showed positive expression in epithelial, vascular and stromal cells whereas Bcl-2 staining was negative. In the conjunctiva of diabetic patients without diabetic retinopathy, Bax was widely, and strongly, expressed in epithelial cells, vascular endothelial cells, fibroblasts and infiltrating cells such as macrophages. For patients with diabetic retinopathy, Bax was consistently strong to very strong. Bcl-2 protein expression became weak to negative for diabetic patients both with and without diabetic retinopathy. Immunoreactivity was not correlated between Bcl-2 and Bax in the conjunctiva of diabetic patients. Bax was always localized in tissues characterized by a high rate of apoptosis, whereas, Bcl-2 was absent. Our results suggest that diabetic human conjunctiva, with its inflammatory phenomena, is considered as a privileged target for programmed cell death.