Peutz-Jeghers LKB1 mutants fail to activate GSK-3β, preventing it from inhibiting Wnt signaling

Peutz-Jeghers syndrome (PJS) is caused by germline mutations in the LKB1 gene, which encodes a serine-threonine kinase that regulates cell proliferation and polarity. This autosomal dominant disorder is characterized by mucocutaneous melanin pigmentation, multiple gastrointestinal hamartomatous polyposis and an increased risk of developing various neoplasms. To understand the molecular pathogenesis of PJS phenotypes, we used microarrays to analyze gene expression profiles in proliferating HeLa cells transduced with lentiviral vectors expressing wild type or mutant LKB1 proteins. We show that gene expression is differentially affected by mutations that impair the kinase activity (K78I) or alter the cellular localization of the LKB1 protein. However, both mutations abrogate the ability of LKB1 to up-regulate the transcription of several genes involved in Wnt signaling, including DKK3, WNT5B and FZD2. In addition—and in contrast to the wild type protein—these LKB1 mutants fail to activate the GSK-3β kinase, which otherwise phosphorylates β-catenin. The increase in β-catenin phosphorylation that occurs upon expression of wild-type LKB1 results in transcriptional inhibition of a canonical Wnt reporter gene. This suggests that pathogenic LKB1 mutations that lead to activation of the Wnt/β-catenin pathway could contribute to the cancer predisposition of PJS patient

LKB1 interacts with and phosphorylates PTEN: a functional link between two proteins involved in cancer predisposing syndromes

Germline mutations of the LKB1 (STK11) tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS) and predisposition to cancer. LKB1 encodes a serine/threonine kinase generally inactivated in PJS patients. We identified the dual phosphatase and tumor suppressor protein PTEN as an LKB1-interacting protein. Several LKB1 point mutations associated with PJS disrupt the interaction with PTEN suggesting that the loss of this interaction might contribute to PJS. Although PTEN and LKB1 are predominantly cytoplasmic and nuclear, respectively, their interaction leads to a cytoplasmic relocalization of LKB1. In addition, we show that PTEN is a substrate of the kinase LKB1 in vitro. As PTEN is a dual phosphatase mutated in autosomal inherited disorders with phenotypes similar to those of PJS (Bannayan-Riley-Ruvalcaba syndrome and Cowden disease), our study suggests a functional link between the proteins involved in different hamartomatous polyposis syndromes and emphasizes the central role played by LKB1 as a tumor suppressor in the small intestin

A systematic enhancer screen using lentivector transgenesis identifies conserved and non-conserved functional elements at the olig1 and olig2 locus

Finding sequences that control expression of genes is central to understanding genome function. Previous studies have used evolutionary conservation as an indicator of regulatory potential. Here, we present a method for the unbiased in vivo screen of putative enhancers in large DNA regions, using the mouse as a model. We cloned a library of 142 overlapping fragments from a 200 kb-long murine BAC in a lentiviral vector expressing LacZ from a minimal promoter, and used the resulting vectors to infect fertilized murine oocytes. LacZ staining of E11 embryos obtained by first using the vectors in pools and then testing individual candidates led to the identification of 3 enhancers, only one of which shows significant evolutionary conservation. In situ hybridization and 3C/4C experiments suggest that this enhancer, which is active in the neural tube and posterior diencephalon, influences the expression of the Olig1 and/or Olig2 genes. This work provides a new approach for the large-scale in vivo screening of transcriptional regulatory sequences, and further demonstrates that evolutionary conservation alone seems too limiting a criterion for the identification of enhancers

Protéine "La" et terminaison de la transcription des gènes de classe III

La protéine La (48 kDa), cible d'anticorps dans les maladies autoimmunes telles le Lupus érythémateux ou le syndrome de Sjörgen, s'associe avec l'extrémité 3' riche en résidus uridines des transcrits primaires synthétisés par l'ARN polymérase III (Pol III). Cette protéine a été impliquée à la fois dans la terminaison de la synthèse et dans le relâchement des ARNs naissants. En éliminant la protéine La d'un extrait cellulaire S-100 à l'aide d'anticorps spécifiques, nous avons montré que La n'est pas le facteur de terminaison de la transcription par Pol III. Nos résultats suggèrent plutôt que La protège les ARNs précurseurs d'une dégradation 3' exonucléasique. Nous avons cloné chez "Saccaromyces cerevisiae", le gène LAH1 (non essentiel à la viabilité cellulaire) qui code la protéine Lah1 homologue à La. Les anticorps anti-La dirigés contre la protéine de "Xenopus" ne réagissent pas avec Lah1. Cependant, ils détectent une telle activité dans les extraits bruts de levure suggérant que d'autres homologues de La pourraient exister chez la levure

Peutz-Jeghers LKB1 mutants fail to activate GSK-3beta, preventing it from inhibiting Wnt signaling

Peutz-Jeghers syndrome (PJS) is caused by germline mutations in the LKB1 gene, which encodes a serine-threonine kinase that regulates cell proliferation and polarity. This autosomal dominant disorder is characterized by mucocutaneous melanin pigmentation, multiple gastrointestinal hamartomatous polyposis and an increased risk of developing various neoplasms. To understand the molecular pathogenesis of PJS phenotypes, we used microarrays to analyze gene expression profiles in proliferating HeLa cells transduced with lentiviral vectors expressing wild type or mutant LKB1 proteins. We show that gene expression is differentially affected by mutations that impair the kinase activity (K78I) or alter the cellular localization of the LKB1 protein. However, both mutations abrogate the ability of LKB1 to up-regulate the transcription of several genes involved in Wnt signaling, including DKK3, WNT5B and FZD2. In addition-and in contrast to the wild type protein-these LKB1 mutants fail to activate the GSK-3beta kinase, which otherwise phosphorylates beta-catenin. The increase in beta-catenin phosphorylation that occurs upon expression of wild-type LKB1 results in transcriptional inhibition of a canonical Wnt reporter gene. This suggests that pathogenic LKB1 mutations that lead to activation of the Wnt/beta-catenin pathway could contribute to the cancer predisposition of PJS patients

Targeted RNA-sequencing identifies FBXW4 instead of MGEA5 as fusion partner of TGFBR3 in pleomorphic hyalinizing angiectatic tumor

Pleomorphic hyalinizing angiectatic tumor (PHAT) is a rare mesenchymal tumor of intermediate malignancy. PHAT, and the related hemosiderotic fibrolipomatous tumor, show a recurrent t(1;10)(p22;q24). Fluorescence in situ hybridization (FISH) and BAC (bacterial artificial chromosome) clones have previously identified TGFBR3 and MGEA5 as fusion partners. However, targeted RNA-sequencing allowed for the correct identification of FBXW4 and not MGEA5 as the fusion partner of TGFBR3 in a subcutaneous PHAT, a finding further confirmed by RT-PCR. FBXW4 and MGEA5 share a common cytogenetic location at 10q24.32, thereby suggesting that the use of less precise technology may have led to inaccurate gene identification. The study of additional cases is however required

Autologous bone marrow-derived cell transplantation in decompensated alcoholic liver disease: what is the impact on liver histology and gene expression patterns?

Abstract Background Liver stem cell therapy (SCT) has been suggested as a promising means to improve liver regeneration in advanced liver disease. However, data from trials are heterogeneous, with no systematic histological evaluation. The aim of this study is to specifically analyze the effect of autologous SCT on liver regeneration and on gene expression changes. Methods Individuals in the randomized controlled trial of SCT in alcoholic hepatitis with paired liver biopsies were included (n = 58). Immunohistochemistry (Ki67, K7, and CD68), in situ hybridization (SPINK1), and global gene expression analysis were performed on liver biopsies (30 control patients and 28 patients with transarterial administration of bone marrow-derived stem cells) both at baseline and after 4 weeks of follow-up. Results No difference between the two groups could be observed regarding the proliferative hepatocyte number, proliferative K7-positive cells, or total K7-positive cells at the 4-week follow-up liver biopsy. However, patients who received SCT showed a more important liver macrophagic expansion as compared to standard treatment. Transcriptome data revealed changes in genes linked with inflammation (CD68 and SAA), regeneration (SPINK1 and HGF), fibrosis (COL1A1), and stem cells (CD45). No changes in gene pathways involved in liver growth and cell cycle proteins were evident. SPINK1 mRNA was present by in situ hybridization at week 4 in SCT patients in the liver parenchyma areas adjacent to macrophage recruitment and liver cell proliferation. Conclusions The analysis of liver tissue after SCT demonstrated an expansion of macrophages concurrent with an upregulated expression of genes involved in inflammatory and regenerative pathways. With the negative results from the clinical trial, the impact of the SCT has to be interpreted as weak, and it is not able to modify the clinical course of this severe liver disease

High liver expression of SPINK-1 is associated with progenitor cell and hepatocyte proliferation and determines MELD score improvement in decompensated alcoholic liver disease

Background and aims: The prognostic significance of liver progenitor cell (LPC) and macrophage expansion in the regeneration of decompensated alcoholic liver disease (ALD) remain ill defined. In a well-characterized population of patients with acutely decompensated ALD (Hepatology 2011, A62), we analysed macrophage infiltration, proliferative LPC and differential expression of hepatic genes at baseline in relation to outcome at 3 months follow up. Methods: Fifty-eight patients (MELD 20) were included. Liver biopsy at inclusion was used for (1) immunohistological analysis of proliferative LPC (MIB1+/CK7+), proliferative hepatocytes (MIB1+/CK7- parenchymal cells), morphometric analysis of macrophage infiltration (CD68) and LPC expansion (CK7), and (2) transcriptome profiling using Affymetrix GeneChip Human arrays. Serum levels of HGF were determined by immunoassay. A ≥ 3 points decrease in MELD at 3 months as compared to baseline defined the improvers. Fifteen abstinent cirrhotics served as controls. CD68 and SPINK3 mRNA expression was determined in various mice models of liver injury. Results: At baseline, patients with decompensated ALD presented a significant expansion of CD68+ macrophages and CK7+ cells compared to abstinent cirrhotics. Patients who will improve (n=34) were characterized at baseline by a higher number of CK7+/MIB1+ cells (1.9 ± 1.5 versus 0.9 ± 0.9 cells/field, p<0.01), MIB1+ hepatocytes (4.1 ± 3.6 versus 1.8 ± 1.4 cells/field, p<0.01), an increased expansion of liver macrophages (4.4% versus 3.3% of surface area, p<0.05) and a higher level of serum HGF (p<0.05), compared to those who will not (n=24). Transcriptome analysis revealed that the first pathways upregulated in improvers were related to cell cycle and a 7-fold increase of liver serine peptidase inhibitor Kazal type I (SPINK1) compared with non-improvers (p=0.005). SPINK1 liver expression positively correlated with CD68 (r=0.46) and cyclinE1 (r=0.6). In mice, a 20-fold increase in liver SPINK3 expression, the homolog of human SPINK1, was evidenced following partial hepatectomy, concurrent with hepatocyte proliferation. Conclusions: Baseline markers of liver macrophages and liver cell proliferation predict the clinical outcome in decompensated ALD. This study reveals an unexpected implication of SPINK1, an acute phase reactant, in liver regeneration and human ALD

LKB1 interacts with and phosphorylates PTEN: a functional link between two proteins involved in cancer predisposing syndromes

Germline mutations of the LKB1 (STK11) tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS) and predisposition to cancer. LKB1 encodes a serine/threonine kinase generally inactivated in PJS patients. We identified the dual phosphatase and tumor suppressor protein PTEN as an LKB1-interacting protein. Several LKB1 point mutations associated with PJS disrupt the interaction with PTEN suggesting that the loss of this interaction might contribute to PJS. Although PTEN and LKB1 are predominantly cytoplasmic and nuclear, respectively, their interaction leads to a cytoplasmic relocalization of LKB1. In addition, we show that PTEN is a substrate of the kinase LKB1 in vitro. As PTEN is a dual phosphatase mutated in autosomal inherited disorders with phenotypes similar to those of PJS (Bannayan-Riley-Ruvalcaba syndrome and Cowden disease), our study suggests a functional link between the proteins involved in different hamartomatous polyposis syndromes and emphasizes the central role played by LKB1 as a tumor suppressor in the small intestine