ZFP36L1 Regulates Fgf21 mRNA Turnover and Modulates Alcoholic Hepatic Steatosis and Inflammation in Mice

Zinc finger protein 36 like 1 (ZFP36L1) enhances the turnover of mRNAs containing AU-rich elements (AREs) in their 3\u27-untranslated regions (3\u27UTR). The physiological and pathological functions of ZFP36L1 in liver, however, remain largely unknown. Liver-specific ZFP36L1-deficient (Zfp36l1/Cre; L1) mice were generated to investigate the role of ZFP36L1 in liver physiology and pathology. Under normal conditions, the L1 mice and their littermate controls (Zfp36l1/Cre; L1) appeared normal. When fed a Lieber-DeCarli liquid diet containing alcohol, L1 mice were significantly protected from developing alcohol-induced hepatic steatosis, injury, and inflammation compared with L1 mice. Most importantly, fibroblast growth factor 21 (Fgf21) mRNA was significantly increased in the livers of alcohol diet-fed L1 mice compared with the alcohol diet-fed L1 group. The Fgf21 mRNA contains three AREs in its 3\u27UTR, and Fgf21 3\u27UTR was directly regulated by ZFP36L1 in luciferase reporter assays. Steady-state levels of Fgf21 mRNA were significantly decreased by wild-type ZFP36L1, but not by a non-binding zinc finger ZFP36L1 mutant. Finally, wild-type ZFP36L1, but not the ZFP36L1 mutant, bound to the Fgf21 3\u27UTR ARE RNA probe. These results demonstrate that ZFP36L1 inactivation protects against alcohol-induced hepatic steatosis and liver injury and inflammation, possibly by stabilizing Fgf21 mRNA. These findings suggest that the modulation of ZFP36L1 may be beneficial in the prevention or treatment of human alcoholic liver disease

Arsenic, cadmium and neuron specific enolase (ENO2, γ-enolase) expression in breast cancer

Abstract Background Neuron specific enolase (ENO2, γ-enolase) has been used as a biomarker to help identify neuroendocrine differentiation in breast cancer. The goal of the present study was to determine if ENO2 expression in the breast epithelial cell is influenced by the environmental pollutants, arsenite and cadmium. Acute and chronic exposure of MCF-10A cells to As+3 and Cd+2 sufficient to allow colony formation in soft agar, was used to determine if ENO2 expression was altered by these pollutants. Results It was shown that both As+3 and Cd+2 exposure caused significant increases in ENO2 expression under conditions of both acute and chronic exposure. In contrast, ENO1, the major glycolytic enolase in non-muscle and neuronal cells, was largely unaffected by exposure to either As+3 or Cd+2. Localization studies showed that ENO2 in the MCF-10A cells transformed by As+3 or Cd+2 had both a cytoplasmic and nuclear localization. In contrast, ENO1 was localized to the cytoplasm. ENO2 localized to the cytoplasm was found to co-localized with ENO1. Conclusion The results are the first to show that ENO2 expression in breast epithelial cells is induced by acute and chronic exposure to As+3 or Cd+2. The findings also suggest a possible link between As+3 and Cd+2 exposure and neuroendocrine differentiation in tumors. Overall, the results suggest that ENO2 might be developed as a biomarker indicating acute and/or chronic environmental exposure of the breast epithelial cell to As+3 and Cd+2.</p

Cadherin Expression, Vectorial Active Transport, and Metallothionein Isoform 3 Mediated EMT/MET Responses in Cultured Primary and Immortalized Human Proximal Tubule Cells

<div><p>Background</p><p>Cultures of human proximal tubule cells have been widely utilized to study the role of EMT in renal disease. The goal of this study was to define the role of growth media composition on classic EMT responses, define the expression of E- and N-cadherin, and define the functional epitope of MT-3 that mediates MET in HK-2 cells.</p><p>Methods</p><p>Immunohistochemistry, microdissection, real-time PCR, western blotting, and ELISA were used to define the expression of E- and N-cadherin mRNA and protein in HK-2 and HPT cell cultures. Site-directed mutagenesis, stable transfection, measurement of transepithelial resistance and dome formation were used to define the unique amino acid sequence of MT-3 associated with MET in HK-2 cells.</p><p>Results</p><p>It was shown that both E- and N-cadherin mRNA and protein are expressed in the human renal proximal tubule. It was shown, based on the pattern of cadherin expression, connexin expression, vectorial active transport, and transepithelial resistance, that the HK-2 cell line has already undergone many of the early features associated with EMT. It was shown that the unique, six amino acid, C-terminal sequence of MT-3 is required for MT-3 to induce MET in HK-2 cells.</p><p>Conclusions</p><p>The results show that the HK-2 cell line can be an effective model to study later stages in the conversion of the renal epithelial cell to a mesenchymal cell. The HK-2 cell line, transfected with MT-3, may be an effective model to study the process of MET. The study implicates the unique C-terminal sequence of MT-3 in the conversion of HK-2 cells to display an enhanced epithelial phenotype.</p></div

The effect of forced overexpression of E-cadherin on the expression of N-cadherin and on dome formation in HK-2 cells.

<p>E-cadherin was stably transfected into the HK-2 human proximal tubule cell line and individual clones were isolated and assessed for E-cadherin and N-cadherin expression and the formation of domes. A) Expression of E-cadherin mRNA in five individual clones, HK-2 cells and the MT-3 stably transfected HK-2 line, HK-2 (MT-3) assessed quantitatively with real-time PCR and normalized to the levels of transcripts of glyceraldehyde phosphate dehydrogenase; B) Expression of N-cadherin mRNA; C) Expression of E- and N-cadherin protein in each isolated clone, D) and E) Morphology of the highest expressing E-cadherin clone (E) in comparison to the parental HK-2 cells (D) showing the absence of dome formation.</p

Connexin 32 expression in HK-2, HPT, and HK-2 cells expressing MT3.

<p>Messenger RNA of connexin 32 was assessed with real-time PCR and expressed as a fold increase of the HK-2 cells stably transfected with the blank vector. The change in connexin 32 expression was normalized to the change in β-actin expression. Western analysis of connexin 32 is shown below the graph.</p

Effect of altered domains of metallothionein-3 on the formation of domes in HK-2 cells.

<p>To assess the domain of MT-3 that is responsible for the ability to confer dome formation, site-directed mutants of MT-3 were produced and the two unique domains of MT-3 were inserted into the non-doming MT isoform, MT-1E, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120132#pone.0120132.g001" target="_blank">Fig. 1</a>. Each expression construct was stably transfected into HK-2 cells, and expressing clones were assessed for the ability to form domes. A) wild-type MT-3 showing dome formation when stably transfected, B) MT-3-ΔNT where the prolines in the N-terminal domain that confers growth inhibitory activity were converted to threonines, show that the ability to form domes was not compromised, C) MT-3ΔCT where the C-terminal EAAEAE sequence unique to the third isoform of metallothionein was deleted, shows the lack of dome formation, D) MT-1E, wild-type human metallothionein 1E, commonly expressed at high levels in many cell types exhibits no domes when stably transfected, E) 1E-NT, the N-terminal unique sequence of MT-3 was inserted into the corresponding position of MT-3 shows no conference of dome formation, and F) 1E-CT, the EAAEAE sequence of MT-3 was inserted in the corresponding position of MT-1E and when stably transfected into HK-2 cells confers dome formation.</p

Transepithelial resistance and dome formation in HK-2 cells overexpressing MT-3 with altered protein domains.

<p>Transepithelial resistance and dome formation in HK-2 cells overexpressing MT-3 with altered protein domains.</p