Tissue inhibitor of metalloproteases-4 (TIMP-4) modulates adipocyte differentiation in vitro.

Tissue inhibitors of metalloproteases (TIMPs) are multifunctional proteins that inhibit matrix metalloproteases (MMPs). The latest described member of the family, TIMP-4, is expressed mainly in adipose tissue, with detectable levels in the brain and heart. Besides its high expression in fat, the role of this inhibitor in adipose tissue is unknown. In order to study the role of TIMP-4 during adipogenesis in vitro, 3T3-L1 cells were stably transfected with a TIMP-4 specific shRNA or a control shRNA. Unexpectedly, upon TIMP-4 knockdown, 3T3-L1 cells differentiated faster into mature adipocytes. To get better insight of TIMP-4's role in adipogenesis, microarray expression analyses were performed. Network enrichment analyses uncovered 25 significant upstream signaling pathways, among which the NFκB cascade was found. Previous works have shown that NFκB is a key regulator of adipogenesis. In accordance, we found that TIMP-4 knockdown decreased NFκB activity during adipogenesis. The present work suggests that TIMP-4 might act as a negative regulator of adipogenesis through NFκB cascade modulation.

Autocrine/paracrine erythropoietin signalling promotes JAK/STAT-dependent proliferation of human cervical cancer cells.

Erythropoietin (Epo) regulates erythropoiesis by binding to its receptor (EpoR) and promoting cell proliferation, differentiation and inhibition of apoptosis. Epo is widely used to treat cervical cancer-related anaemia. However, there are data suggesting that administration of Epo is associated with an increment in recurrence rate, and decreased disease-free and overall survival. In the present study, we investigated the expression of Epo and EpoR on cervical cancer cell lines. We observed that both EpoR and extracellular Epo are constitutively expressed in cervical cancer cells. Inhibition of either Epo or EpoR expression with siRNA attenuated cell proliferation, whereas addition of exogenous Epo led to a significant increase in cell growth, both in vitro and in vivo. Epo-induced proliferation was associated with the activation of JAK2, JAK3, STAT3 and STAT5 but not JAK1 and STAT1. Our results are consistent with the existence of a functional, endogenous Epo/EpoR system in cervical cancer with the capacity to activate the transduction of signals resulting in an increased proliferation potential.

Binding of human papillomavirus type 16 to heparan sulfate is inhibited by mucosal antibodies from patients with low-grade squamous intraepithelial lesions but not from cervical cancer patients.

Mucosal antibodies against human papillomavirus type 16 (HPV16) capsids have been detected in infected women. To determine whether these antibodies recognize and block the receptor site mediating attachment of HPV16 to heparan sulfate, mucus samples from 126 HPV16-associated low-grade squamous intraepithelial lesion (LSIL) and 85 cervical cancer patients, previously found to react to HPV16 virus-like particles (VLP), and 101 normal controls were tested in an inhibition assay, using HPV16 VLP and heparan sulfate proteoglycan-coated plates. Inhibition levels of 9.3-67.2% were mediated by type-specific antibodies in 94.4% of LSIL patients. Cervical cancer cases showed significantly lower levels of inhibition than LSIL samples (P < 0.0001). The potential of antibodies to inhibit infection was explored in a pseudoinfection system using HPV16 pseudovirions. Inhibition of pseudoinfection by LSIL samples was significantly higher than that observed in the controls (P < 0.001) and cervical cancer cases (P < 0.005). These results indicate that mucosal antibodies inhibiting binding of VLP to heparan sulfate are developed in most LSIL patients, but are hardly present in cervical cancer patients.

Co-Expression of Ezrin-CLIC5-Podocalyxin Is Associated with Migration and Invasiveness in Hepatocellular Carcinoma.

BACKGROUND AND AIM: Prognostic markers are important for predicting the progression and staging of hepatocellular carcinoma (HCC). Ezrin (EZR) and Podocalyxin (PODXL) are proteins associated with invasion, migration and poor prognosis in various types of cancer. Recently, it has been observed that chloride intracellular channel 5 (CLIC5) forms a complex with EZR and PODXL and that it is required for podocyte structure and function. In this study, we evaluated the overexpression of EZR, PODXL and CLIC5 in HCC. METHODS: The modified resistant hepatocyte model (MRHR), human biopsies and HCC cell lines (HepG2, Huh7 and SNU387) were used in this study. Gene and protein expression levels were evaluated in the MRHR by qRT-PCR, Western blot and immunohistochemistry analyses, and protein expression in the human biopsies was evaluated by immunohistochemistry. Protein expression in the HCC cell lines was evaluated by immunofluorescence and Western blot, also the migration and invasive abilities of Huh7 cells were evaluated using shRNA-mediated inhibition. RESULTS: Our results indicated that these genes and proteins were overexpressed in HCC. Moreover, when the expression of CLIC5 and PODXL was inhibited in Huh7 cells, we observed decreased migration and invasion. CONCLUSION: This study suggested that EZR, CLIC5 and PODXL could be biological markers to predict the prognosis of HCC and that these proteins participate in migration and invasion processes.