WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumors

Wnt family members are critical to many developmental processes, and components of the Wnt signaling pathway have been linked to tumorigenesis in familial and sporadic colon carcinomas. Here we report the identification of two genes, WISP-1 and WISP-2, that are up-regulated in the mouse mammary epithelial cell line C57MG transformed by Wnt-1, but not by Wnt-4. Together with a third related gene, WISP-3, these proteins define a subfamily of the connective tissue growth factor family. Two distinct systems demonstrated WISP induction to be associated with the expression of Wnt-1. These included (i) C57MG cells infected with a Wnt-1 retroviral vector or expressing Wnt-1 under the control of a tetracyline repressible promoter, and (ii) Wnt-1 transgenic mice. The WISP-1 gene was localized to human chromosome 8q24.1-8q24.3. WISP-1 genomic DNA was amplified in colon cancer cell lines and in human colon tumors and its RNA overexpressed (2- to >30-fold) in 84% of the tumors examined compared with patient-matched normal mucosa. WISP-3 mapped to chromosome 6q22-6q23 and also was overexpressed (4- to >40-fold) in 63% of the colon tumors analyzed. In contrast, WISP-2 mapped to human chromosome 20q12-20q13 and its DNA was amplified, but RNA expression was reduced (2- to >30-fold) in 79% of the tumors. These results suggest that the WISP genes may be downstream of Wnt-1 signaling and that aberrant levels of WISP expression in colon cancer may play a role in colon tumorigenesis

Cardiotrophin-1 defends the liver against ischemia-reperfusion injury and mediates the protective effect of ischemic preconditioning

Ischemia-reperfusion (I/R) liver injury occurs when blood flow is restored after prolonged ischemia. A short interruption of blood flow (ischemic preconditioning [IP]) induces tolerance to subsequent prolonged ischemia through ill-defined mechanisms. Cardiotrophin (CT)-1, a cytokine of the interleukin-6 family, exerts hepatoprotective effects and activates key survival pathways like JAK/STAT3. Here we show that administration of CT-1 to rats or mice protects against I/R liver injury and that CT-1-deficient mice are exceedingly sensitive to this type of damage. IP markedly reduced transaminase levels and abrogated caspase-3 and c-Jun-NH2-terminal kinase activation after I/R in normal mice but not in CT-1-null mice. Moreover, the protective effect afforded by IP was reduced by previous administration of neutralizing anti-CT-1 antibody. Prominent STAT3 phosphorylation in liver tissue was observed after IP plus I/R in normal mice but not in CT-1-null mice. Oxidative stress, a process involved in IP-induced hepatoprotection, was found to stimulate CT-1 release from isolated hepatocytes. Interestingly, brief ischemia followed by short reperfusion caused mild serum transaminase elevation and strong STAT3 activation in normal and IL-6-deficient mice, but failed to activate STAT3 and provoked marked hypertransaminasemia in CT-1-null animals. In conclusion, CT-1 is an essential endogenous defense of the liver against I/R and is a key mediator of the protective effect induced by IP

COL11A1 in FAP polyps and in sporadic colorectal tumors

BACKGROUND: We previously reported that the α-1 chain of type 11 collagen (COL11A1), not normally expressed in the colon, was up-regulated in stromal fibroblasts in most sporadic colorectal carcinomas. Patients with germline mutations in the APC gene show, besides colonic polyposis, symptoms of stromal fibroblast involvement, which could be related to COL11A1 expression. Most colorectal carcinomas are suggested to be a result of an activated Wnt- pathway, most often involving an inactivation of the APC gene or activation of β-catenin. METHODS: We used normal and polyp tissue samples from one FAP patient and a set of 37 sporadic colorectal carcinomas to find out if the up-regulation of COL11A1 was associated with an active APC/β-catenin pathway. RESULTS: In this study we found a statistically significant difference in COL11A1 expression between normal tissue and adenomas from one FAP patient, and all adenomas gave evidence for an active APC/β-catenin pathway. An active Wnt pathway has been suggested to involve stromal expression of WISP-1. We found a strong correlation between WISP-1 and COL11A1 expression in sporadic carcinomas. CONCLUSIONS: Our results suggest that expression of COL11A1 in colorectal tumors could be associated with the APC/β-catenin pathway in FAP and sporadic colorectal cancer

Inhibition of CCN6 (WISP3) expression promotes neoplastic progression and enhances the effects of insulin-like growth factor-1 on breast epithelial cells

INTRODUCTION: CCN6/WISP3 belongs to the CCN (Cyr61, CTGF, Nov) family of genes that contains a conserved insulin-like growth factor (IGF) binding protein motif. CCN6 is a secreted protein lost in 80% of the aggressive inflammatory breast cancers, and can decrease mammary tumor growth in vitro and in vivo. We hypothesized that inhibition of CCN6 might result in the loss of a growth regulatory function that protects mammary epithelial cells from the tumorigenic effects of growth factors, particularly IGF-1. METHOD: We treated human mammary epithelial (HME) cells with a CCN6 hairpin short interfering RNA. RESULTS: CCN6-deficient cells showed increased motility and invasiveness, and developed features of epithelial-mesenchymal transition (EMT). Inhibition of CCN6 expression promoted anchorage-independent growth of HME cells and rendered them more responsive to the growth effects of IGF-1, which was coupled with the increased phosphorylation of IGF-1 receptor and insulin receptor substrate-1 (IRS-1). CONCLUSION: Specific stable inhibition of CCN6 expression in HME cells induces EMT, promotes anchorage-independent growth, motility and invasiveness, and sensitizes mammary epithelial cells to the growth effects of IGF-1

Up-regulation of macrophage wnt gene expression in adenoma-carcinoma progression of human colorectal cancer

Defects in the APC-β-catenin pathway are common in colon cancer. We investigated whether aberrant regulation of upstream ligands stimulating this pathway occur in colon cancer. Using RNAase protection analysis, six out of eight wnt genes were expressed in 14 matched cases of normal, adenomatous and malignant colorectal tissues. Wnt 2 and wnt 5a were significantly up-regulated in the progression from normal through adenoma to carcinoma. Transcripts for wnts 4, 7b, 10b and 13, but not wnt 2 and wnt 5a were detected in several colorectal cell lines. In situ hybridization demonstrated that wnt 2 and wnt 5a transcripts were mainly in the lamina propria/stroma region with labelling predominantly in macrophages. Immunostaining with CD68 confirmed the wnt-expressing cells as macrophages. These results show a major difference in wnt expression in colon cancer compared to colon adenomas and suggest stromal wnt expression may play a role in tumour progression. © 1999 Cancer Research Campaig

Domain-and species-specific monoclonal antibodies recognize the Von Willebrand Factor-C domain of CCN5

The CCN family of proteins typically consists of four distinct peptide domains: an insulin-like growth factor binding protein-type (IGFBP) domain, a Von Willebrand Factor C (VWC) domain, a thrombospondin type 1 repeat (TSP1) domain, and a carboxy-terminal (CT) domain. The six family members participate in many processes, including proliferation, motility, cell-matrix signaling, angiogenesis, and wound healing. Accumulating evidence suggests that truncated and alternatively spliced isoforms are responsible for the diverse functions of CCN proteins in both normal and pathophysiologic states. Analysis of the properties and functions of individual CCN domains further corroborates this idea. CCN5 is unique among the CCN family members because it lacks the CT-domain. To dissect the domain functions of CCN5, we are developing domain-specific mouse monoclonal antibodies. Monoclonal antibodies have the advantages of great specificity, reproducibility, and ease of long-term storage and production. In this communication, we injected mixtures of GST-fused rat CCN5 domains into mice to generate monoclonal antibodies. To identify the domains recognized by the antibodies, we constructed serial expression plasmids that express dual-tagged rat CCN5 domains. All of the monoclonal antibodies generated to date recognize the VWC domain, indicating it is the most highly immunogenic of the CCN5 domains. We characterized one particular clone, 22H10, and found that it recognizes mouse and rat CCN5, but not human recombinant CCN5. Purified 22H10 was successfully applied in Western Blot analysis, immunofluorescence of cultured cells and tissues, and immunoprecipitation, indicating that it will be a useful tool for domain analysis and studies of mouse-human tumor models

Inhibition of Fibroblast Growth by Notch1 Signaling Is Mediated by Induction of Wnt11-Dependent WISP-1

Fibroblasts are an integral component of stroma and important source of growth factors and extracellular matrix (ECM). They play a prominent role in maintaining tissue homeostasis and in wound healing and tumor growth. Notch signaling regulates biological function in a variety of cells. To elucidate the physiological function of Notch signaling in fibroblasts, we ablated Notch1 in mouse (Notch1Flox/Flox) embryonic fibroblasts (MEFs). Notch1-deficient (Notch1−/−) MEFs displayed faster growth and motility rate compared to Notch1Flox/Flox MEFs. Such phenotypic changes, however, were reversible by reconstitution of Notch1 activation via overexpression of the intracellular domain of Notch1 (NICD1) in Notch1-deficient MEFs. In contrast, constitutive activation of Notch1 signaling by introducing NICD1 into primary human dermal fibroblasts (FF2441), which caused pan-Notch activation, inhibited cell growth and motility, whereas cellular inhibition was relievable when the Notch activation was countered with dominant-negative mutant of Master-mind like 1 (DN-MAML-1). Functionally, “Notch-activated” stromal fibroblasts could inhibit tumor cell growth/invasion. Moreover, Notch activation induced expression of Wnt-induced secreted proteins-1 (WISP-1/CCN4) in FF2441 cells while deletion of Notch1 in MEFs resulted in an opposite effect. Notably, WISP-1 suppressed fibroblast proliferation, and was responsible for mediating Notch1's inhibitory effect since siRNA-mediated blockade of WISP-1 expression could relieve cell growth inhibition. Notch1-induced WISP-1 expression appeared to be Wnt11-dependent, but Wnt1-independent. Blockade of Wnt11 expression resulted in decreased WISP-1 expression and liberated Notch-induced cell growth inhibition. These findings indicated that inhibition of fibroblast proliferation by Notch pathway activation is mediated, at least in part, through regulating Wnt1-independent, but Wnt11-dependent WISP-1 expression

Identification of Novel Single Nucleotide Polymorphisms in Inflammatory Genes as Risk Factors Associated with Trachomatous Trichiasis

infection, the primary cause of trachoma. Despite control programs that include mass antibiotic treatment, reinfection and recurrence of trachoma are common after treatment cessation. Furthermore, a subset of infected individuals develop inflammation and are at greater risk for developing the severe sequela of trachoma known as trachomatous trichiasis (TT). While there are a number of environmental and behavioral risk factors for trachoma, genetic factors that influence inflammation and TT risk remain ill defined. = 0.001] with the combination of TNFA (-308A), LTA (252A), VCAM1 (-1594C), SCYA 11 (23T) minor allele, and the combination of TNFA (-308A), IL9 (113M), IL1B (5′UTR-T), and VCAM1 (-1594C). However, TT risk increased 13.5 times [odds ratio = 13.5 (95% confidence interval 3.3–22), p = 0.001] with the combination of TNFA (-308G), VDR (intron G), IL4R (50V), and ICAM1 (56M) minor allele.Evaluating genetic risk factors for trachoma will advance our understanding of disease pathogenesis, and should be considered in the context of designing global control programs