Rabbit Anti-HMOX1 monoclonal antibody, clone TQ09-18

Neonatal liver-derived rat epithelial cells (rLEC) from biliary origin are liver progenitor cells that acquire a hepatocyte-like phenotype upon sequential exposure to hepatogenic growth factors and cytokines. Undifferentiated rLEC express several liver-enriched transcription factors, including the hepatocyte nuclear factors (HNF) 3 beta and HNF6, but not the hepatic master regulator HNF4 alpha. In this study, we first investigated the impact of the ectopic expression of HNF4 alpha in rLEC on both mRNA and microRNA (miR) level by means of microarray technology. We found that HNF4 alpha transduction did not induce major changes to the rLEC phenotype. However, we next investigated the influence of DNA methyl transferase (DNMT) inhibition on the phenotype of undifferentiated naive rLEC by exposure to 5 ' azacytidine (AZA), which was found to have a significant impact on rLEC gene expression. The transduction of HNF4 alpha or AZA treatment resulted both in significantly downregulated C/EBP alpha expression levels, while the exposure of the cells to AZA had a significant effect on the expression of HNF3 beta. Computationally, dysregulated miRNAs were linked to target mRNAs using the microRNA Target Filter function of Ingenuity Pathway Analysis. We found that differentially regulated miRNA-mRNA target associations predict ectopic HNF4 alpha expression in naive rLEC to interfere with cell viability and cellular maturation (miR-19b-3p/NR4A2, miR30C-5p/P4HA2, miR328-3p/CD44) while it predicts AZA exposure to modulate epithelial/hepatic cell proliferation, apoptosis, cell cycle progression and the differentiation of stem cells (miR-18a-5p/ESR1, miR-503-5p/CCND1). Finally, our computational analysis predicts that the combination of HNF4 alpha transduction with subsequent AZA treatment might cause changes in hepatic cell proliferation and maturation (miR-18a-5p/ESR1, miR-503-5p/CCND1, miR-328-3p/CD44) as well as the apoptosis (miR-16-5p/BCL2, miR-17-5p/BCL2, miR-34a-5p/BCL2and miR-494-3p/HMOX1) of naive rLEC.

Biosynthesis and degradation of heme, an iron-bound protoporphyrin molecule utilized by a wide variety of metabolic processes, are tightly regulated. Two closely related enzymes, heme oxygenase 1 (HMOX1) and heme oxygenase 2 (HMOX2), degrade free heme to produce carbon monoxide, Fe2+, and biliverdin. HMOX1 expression is controlled via the transcriptional activator, NFE2L2, and the transcriptional repressor, Bach1. Transcription of HMOX1 and other NFE2L2-dependent genes is increased in response to electrophilic and reactive oxygen species. Many tumor-derived cell lines have elevated levels of NFE2L2. Elevated expression of NFE2L2-dependent genes contributes to tumor growth and acquired resistance to therapies. Here, we report a novel role for heme oxygenase activity in melanosphere formation by human melanoma-derived cell lines. Transcriptional induction of HMOX1 through derepression of Bach1 or transcriptional activation of HMOX2 by oncogenic B-RafV600E results in increased melanosphere formation. Genetic ablation of HMOX1 diminishes melanosphere formation. Further, inhibition of heme oxygenase activity with tin protoporphyrin markedly reduces melanosphere formation driven by either Bach1 derepression or B-RafV600E expression. Global transcriptome analyses implicate genes involved in focal adhesion and extracellular matrix interactions in melanosphere formation.