The atypical RhoU/Wrch1 Rho GTPase controls cell proliferation and apoptosis in the gut epithelium.

BACKGROUND: The mammalian gut epithelium displays among the highest rates of self-renewal, with a turnover time of less than 5 days. Renewal involves concerted proliferation at the bottom of the crypt, migration and differentiation along the crypt-villus axis and anoïkis/shedding in the luminal epithelium. Renewal is controlled by interplay between signalling pathways, among which canonical and non-canonical Wnt signals play prominent roles. Overall 92% of colon tumours show increased canonical Wnt signalling resulting from mutations, established as major driver steps towards carcinogenesis. RESULTS: Here, we examined the physiological role of RhoU/Wrch1 in gut homeostasis. RhoU is an atypical Rho GTPase related to Cdc42/Rac1 and identified as a transcriptional target of non-canonical Wnt signalling. We found that RHOU expression is reduced in human colorectal tumour samples. We show that RhoU is mainly expressed in the differentiated compartment of the gut epithelium. Rhou specific invalidation in the mouse gut elicits cell hyperplasia and is associated in the colon with a highly disorganized luminal epithelium. Hyperplasia affects all cell types in the small intestine and colon and has a higher impact on goblet cells. Hyperplasia is associated with a reduction of apoptosis and an increased proliferation. RhoU knockdown in human DLD-1 colon cancer cells also elicits a higher growth index and reduces cell apoptosis. Last, loss of RhoU function in the mouse gut epithelium or in DLD-1 cells increases RhoA activity and the level of phosphorylated Myosin Light Chain-2, which may functionally link RhoU activity to apoptosis. CONCLUSION: RhoU is mostly expressed in the differentiated compartment of the gut. It plays a role in homeostasis as its specific invalidation elicits hyperplasia of all cell types. This mainly results from a reduction of apoptosis, through actomyosin-dependent mechanisms. SIGNIFICANCE: RhoU negatively controls cell growth in the intestinal epithelium. Since its expression is sensitive to non-canonical Wnt signals and is reduced in colorectal tumours, downregulating RhoU may thus have an instrumental role in tumour progression.

SOX9 has distinct regulatory roles in alternative splicing and transcription.

SOX9 is known as a crucial transcription factor for various developmental processes and for tissue homeostasis. We examined here its potential role in alternative splicing by analyzing global splicing changes, using RNA-seq of colon tumor cells. We show that SOX9 knockdown alters the splicing of hundreds of genes without affecting their expression levels, revealing that SOX9 controls distinct splicing and transcriptional programs. SOX9 does not affect splicing patterns through the control of splicing factors expression. We identify mutants that uncouple SOX9 splicing function from its transcriptional activity. We demonstrate that SOX9 binds to RNA and associates with several RNA-binding proteins, including the core exon junction complex component Y14. Half of SOX9 splicing targets are also modulated by Y14 and are no longer regulated by SOX9 upon Y14 depletion. Altogether, our work reveals that SOX9 is a moonlighting protein which modulates either transcription or splicing of distinct sets of targets.

Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells.

UNLABELLED: BACKGROUND& AIMS: Embryonic biliary precursor cells form a periportal sheet called the ductal plate, which is progressively remodeled to generate intrahepatic bile ducts. A limited number of ductal plate cells participate in duct formation; those not involved in duct development are believed to involute by apoptosis. Moreover, cells that express the SRY-related HMG box transcription factor 9 (SOX9), which include the embryonic ductal plate cells, were proposed to continuously supply the liver with hepatic cells. We investigated the role of the ductal plate in hepatic morphogenesis. METHODS: Apoptosis and proliferation were investigated by immunostaining of mouse and human fetal liver tissue. The postnatal progeny of SOX9-expressing ductal plate cells was analyzed after genetic labeling, at the ductal plate stage, by Cre-mediated recombination of a ROSA26RYFP reporter allele. Inducible Cre expression was induced by SOX9 regulatory regions, inserted in a bacterial artificial chromosome. Livers were studied from mice under normal conditions and during diet-induced regeneration. RESULTS: Ductal plate cells did not undergo apoptosis and showed limited proliferation. They generated cholangiocytes lining interlobular bile ducts, bile ductules, and canals of Hering, as well as periportal hepatocytes. Oval cells that appeared during regeneration also derived from the ductal plate. We did not find that liver homeostasis required a continuous supply of cells from SOX9-expressing progenitors. CONCLUSIONS: The ductal plate gives rise to cholangiocytes lining the intrahepatic bile ducts, including its most proximal segments. It also generates periportal hepatocytes and adult hepatic progenitor cells.

A classification of ductal plate malformations based on distinct pathogenic mechanisms of biliary dysmorphogenesis.

UNLABELLED: Ductal plate malformations (DPMs) are developmental anomalies considered to result from lack of ductal plate remodeling during bile duct morphogenesis. In mice, bile duct development is initiated by the formation of primitive ductal structures lined by two cell types, namely ductal plate cells and hepatoblasts. During ductal plate remodeling, the primitive ductal structures mature to ducts as a result from differentiation of the ductal plate cells and hepatoblasts to cholangiocytes. Here, we report this process is conserved in human fetal liver. These findings prompted us to evaluate how DPMs develop in three mouse models, namely mice with livers deficient in hepatocyte nuclear factor 6 (HNF6), HNF1ß, or cystin-1 (cpk [congenital polycystic kidney] mice). Human liver from a patient with a HNF1B/TCF2 mutation, and from fetuses affected with autosomal recessive polycystic kidney disease (ARPKD) were also analyzed. Despite the epistatic relationship between HNF6, HNF1ß, and cystin-1, the three mouse models displayed distinct morphogenic mechanisms of DPM. They all developed biliary cysts lined by cells with abnormal apicobasal polarity. However, the absence of HNF6 led to an early defect in ductal plate cell differentiation. In HNF1ß-deficient liver, maturation of the primitive ductal structures was impaired. Normal differentiation and maturation but abnormal duct expansion was apparent in cpk mouse livers and in human fetal ARPKD. CONCLUSION: DPM is the common endpoint of distinct defects initiated at distinct stages of bile duct morphogenesis. Our observations provide a new pathogenic classification of DPM.

The TRRAP transcription cofactor represses interferon-stimulated genes in colorectal cancer cells.

Transcription is essential for cells to respond to signaling cues and involves factors with multiple distinct activities. One such factor, TRRAP, functions as part of two large complexes, SAGA and TIP60, which have crucial roles during transcription activation. Structurally, TRRAP belongs to the phosphoinositide 3 kinase-related kinases (PIKK) family but is the only member classified as a pseudokinase. Recent studies established that a dedicated HSP90 co-chaperone, the triple T (TTT) complex, is essential for PIKK stabilization and activity. Here, using endogenous auxin-inducible degron alleles, we show that the TTT subunit TELO2 promotes TRRAP assembly into SAGA and TIP60 in human colorectal cancer cells (CRCs). Transcriptomic analysis revealed that TELO2 contributes to TRRAP regulatory roles in CRC cells, most notably of MYC target genes. Surprisingly, TELO2 and TRRAP depletion also induced the expression of type I interferon genes. Using a combination of nascent RNA, antibody-targeted chromatin profiling (CUT&RUN), ChIP, and kinetic analyses, we propose a model by which TRRAP directly represses the transcription of IRF9, which encodes a master regulator of interferon-stimulated genes. We have therefore uncovered an unexpected transcriptional repressor role for TRRAP, which we propose contributes to its tumorigenic activity.

Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.

BACKGROUND & AIMS: A number of diseases are characterized by defective formation of the intrahepatic bile ducts. In the embryo, hepatoblasts differentiate to cholangiocytes, which give rise to the bile ducts. Here, we investigated duct development in mouse liver and characterized the role of the SRY-related HMG box transcription factor 9 (SOX9). METHODS: We identified SOX9 as a new biliary marker and used it in immunostaining experiments to characterize bile duct morphogenesis. The expression of growth factors was determined by in situ hybridization and immunostaining, and their role was studied on cultured hepatoblasts. SOX9 function was investigated by phenotyping mice with a liver-specific inactivation of Sox9. RESULTS: Biliary tubulogenesis started with formation of asymmetrical ductal structures, lined on the portal side by cholangiocytes and on the parenchymal side by hepatoblasts. When the ducts grew from the hilum to the periphery, the hepatoblasts lining the asymmetrical structures differentiated to cholangiocytes, thereby allowing formation of symmetrical ducts lined only by cholangiocytes. We also provide evidence that transforming growth factor-beta promotes differentiation of the hepatoblasts lining the asymmetrical structures. In the absence of SOX9, the maturation of asymmetrical structures into symmetrical ducts was delayed. This was associated with abnormal expression of CCAAT/Enhancer Binding Protein alpha and Homolog of Hairy/Enhancer of Split-1, as well as of the transforming growth factor-beta receptor type II, which are regulators of biliary development. CONCLUSIONS: Our results suggest that biliary development proceeds according to a new mode of tubulogenesis characterized by transient asymmetry and whose timing is controlled by SOX9.

MiR-495 and miR-218 regulate the expression of the Onecut transcription factors HNF-6 and OC-2.

MicroRNAs are small, non-coding RNAs that posttranscriptionally regulate gene expression mainly by binding to the 3'UTR of their target mRNAs. Recent data revealed that microRNAs have an important role in pancreas and liver development and physiology. Using cloning and microarray profiling approaches, we show that a unique repertoire of microRNAs is expressed at the onset of liver and pancreas organogenesis, and in pancreas and liver at key stages of cell fate determination. Among the microRNAs that are expressed at these stages, miR-495 and miR-218 were predicted to, respectively, target the Onecut (OC) transcription factors Hepatocyte Nuclear Factor-6 (HNF-6/OC-1) and OC-2, two important regulators of liver and pancreas development. MiR-495 and miR-218 are dynamically expressed in developing liver and pancreas, and by transient transfection, we show that they target HNF-6 and OC-2 3'UTRs. Moreover, when overexpressed in cultured cells, miR-495 and miR-218 decrease the endogenous levels of HNF-6 and OC-2 mRNA. These results indicate that the expression of regulators of liver and pancreas development is modulated by microRNAs. They also suggest a developmental role for miR-495 and miR-218.

Epithelial expression of angiogenic growth factors modulate arterial vasculogenesis in human liver development.

UNLABELLED: Intrahepatic bile ducts maintain a close anatomical relationship with hepatic arteries. During liver ontogenesis, the development of the hepatic artery appears to be modulated by unknown signals originating from the bile duct. Given the capability of cholangiocytes to produce angiogenic growth factors and influence peribiliary vascularization, we studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, and their cognate receptors (VEGFR-1, VEGFR-2, Tie-2) in fetal human livers at different gestational ages and in mice characterized by defective biliary morphogenesis (Hnf6(-/-)). The results showed that throughout the different developmental stages, VEGF was expressed by developing bile ducts and angiopoietin-1 by hepatoblasts, whereas their cognate receptors were variably expressed by vascular cells according to the different maturational stages. Precursors of endothelial and mural cells expressed VEGFR-2 and Tie-2, respectively. In immature hepatic arteries, endothelial cells expressed VEGFR-1, whereas mural cells expressed both Tie-2 and Angiopoietin-2. In mature hepatic arteries, endothelial cells expressed Tie-2 along with VEGFR-1. In early postnatal Hnf6(-/-) mice, VEGF-expressing ductal plates failed to incorporate into the portal mesenchyma, resulting in severely altered arterial vasculogenesis. CONCLUSION: The reciprocal expression of angiogenic growth factors and receptors during development supports their involvement in the cross talk between liver epithelial cells and the portal vasculature. Cholangiocytes generate a VEGF gradient that is crucial during the migratory stage, when it determines arterial vasculogenesis in their vicinity, whereas angiopoietin-1 signaling from hepatoblasts contributes to the remodeling of the hepatic artery necessary to meet the demands of the developing epithelium.

IL-24 contributes to skin inflammation in Para-Phenylenediamine-induced contact hypersensitivity.

Para-Phenylenediamine (PPD) is an aromatic amine used in hair dyes and in temporary black henna tattoos, which is a frequent cause of allergic contact dermatitis (ACD). ACD is a skin inflammatory reaction characterized by modifications such as spongiosis, exocytosis and acanthosis. The aim of this study is to characterize the expression and the role of IL-20-related cytokines, including IL-19, IL-20, IL-22 and IL-24, in ACD. The expression of IL19, IL20, IL22 and IL24 is increased in affected skin from PPD allergic patients compared with uninvolved skin. In addition, the expression of these cytokines positively correlates with clinical symptoms. To assess their role in ACD, we set up a mouse model of PPD-induced allergic contact dermatitis and we showed that, in contrast to Il22-deficient mice, Il22ra1-, Il20rb- and Il24-deficient mice are partially protected against development of PPD-induced contact hypersensitivity. These mice have decreased ear thickening and less acanthosis compared with WT mice after PPD treatment. In addition, the absence of IL-22R, IL-20R2 or IL-24 affects the recruitment of neutrophils into the skin but not the total IgE production. Taken together, these results demonstrate the implication of IL-24 via the IL-20R type II receptor in the inflammatory process of ACD.

Ensa controls S-phase length by modulating Treslin levels.

The Greatwall/Ensa/PP2A-B55 pathway is essential for controlling mitotic substrate phosphorylation and mitotic entry. Here, we investigate the effect of the knockdown of the Gwl substrate, Ensa, in human cells. Unexpectedly, Ensa knockdown promotes a dramatic extension of S phase associated with a lowered density of replication forks. Notably, Ensa depletion results in a decrease of Treslin levels, a pivotal protein for the firing of replication origins. Accordingly, the extended S phase in Ensa-depleted cells is completely rescued by the overexpression of Treslin. Our data herein reveal a new mechanism by which normal cells regulate S-phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.The Greatwall/Ensa/PP2A-B55 pathway controls mitotic substrate phosphorylation and mitotic entry. Here the authors show that cells regulate S phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.

Applying ecological and evolutionary theory to cancer: a long and winding road.

Since the mid 1970s, cancer has been described as a process of Darwinian evolution, with somatic cellular selection and evolution being the fundamental processes leading to malignancy and its many manifestations (neoangiogenesis, evasion of the immune system, metastasis, and resistance to therapies). Historically, little attention has been placed on applications of evolutionary biology to understanding and controlling neoplastic progression and to prevent therapeutic failures. This is now beginning to change, and there is a growing international interest in the interface between cancer and evolutionary biology. The objective of this introduction is first to describe the basic ideas and concepts linking evolutionary biology to cancer. We then present four major fronts where the evolutionary perspective is most developed, namely laboratory and clinical models, mathematical models, databases, and techniques and assays. Finally, we discuss several of the most promising challenges and future prospects in this interdisciplinary research direction in the war against cancer.

Biliary differentiation and bile duct morphogenesis in development and disease.

The biliary tract consists of a network of intrahepatic and extrahepatic ducts that collect and drain the bile produced by hepatocytes to the gut. The bile ducts are lined by cholangiocytes, a specialized epithelial cell type that has a dual origin. Intrahepatic cholangiocytes derive from the liver precursor cells, whereas extrahepatic cholangiocytes are generated directly from the endoderm. In this review we discuss the mechanisms of cholangiocyte differentiation and bile duct morphogenesis, and describe how developing ducts interact with the hepatic artery. We also present an overview of the mechanisms of biliary dysgenesis in humans.

Notch signaling controls liver development by regulating biliary differentiation.

In the mammalian liver, bile is transported to the intestine through an intricate network of bile ducts. Notch signaling is required for normal duct formation, but its mode of action has been unclear. Here, we show in mice that bile ducts arise through a novel mechanism of tubulogenesis involving sequential radial differentiation. Notch signaling is activated in a subset of liver progenitor cells fated to become ductal cells, and pathway activation is necessary for biliary fate. Notch signals are also required for bile duct morphogenesis, and activation of Notch signaling in the hepatic lobule promotes ectopic biliary differentiation and tubule formation in a dose-dependent manner. Remarkably, activation of Notch signaling in postnatal hepatocytes causes them to adopt a biliary fate through a process of reprogramming that recapitulates normal bile duct development. These results reconcile previous conflicting reports about the role of Notch during liver development and suggest that Notch acts by coordinating biliary differentiation and morphogenesis.

Four promoters direct expression of the calpastatin gene.

Calpastatin is a specific endogenous protein inhibitor of the ubiquitous calcium dependent proteinases mu- and m-calpain. The calpain-calpastatin system is involved in various physiological and pathological processes. In the present study, we determined the bovine calpastatin gene structure and demonstrated that four promoters direct its expression. The gene harbours 35 exons spanning at least 130kb on genomic DNA. Its structure is similar to that of mouse, pig, and human gene. Transient transfection assays in both C2C12 and COS7 cell lines demonstrated that the putative promoter regions situated 5' to exon 1xa, 1xb, 1u, and 14t were functional. We also established that the region situated upstream exon 14t is subjected to a tissue specific regulation. The implication of numerous high-scoring cis acting transcriptional motifs which are present in these regions will need to be determined. The existence of four promoters suggests differential expression patterns which must have a physiological significance.

Correlation between bovine calpastatin mRNA transcripts and protein isoforms.

Calpastatin is a specific calpain protease inhibitor: calpains are a family of calcium-activated neutral proteases, which have been implicated in various processes. Despite all the available data concerning calpastatin, little is known about how this gene is regulated, particularly in bovine. The existence of four types of transcripts differing at their 5' ends (Type I, II, III, and IV) has been demonstrated. Here, we show that the Type I, II, and III transcripts are ubiquitous while Type IV is testis-specific. In addition, a Northern blot analysis revealed that the Type III transcript may have three different 3' termini. Using specific anti-peptide anti-sera, a correspondence between a 145 and a 125 kDa isoforms, and Type I and/or II and III transcripts, respectively, has been established. Finally, we discuss the origin of a 70 kDa isoform, recognized by anti-sera directed against the N-terminal region.