ARSENIC INHIBITS P19 STEM CELL DIFFERENTIATION BY ALTERING MICRORNA EXPRESSION AND REPRESSING THE SONIC HEDGEHOG SIGNALING PATHWAY

Arsenic is a naturally-occurring toxicant that exists in bedrock and can be leached into ground water. Humans can be exposed to arsenic via contaminated drinking water, fruit, rice or crops. Epidemiological studies have shown that arsenic is a developmental toxicant, and in utero exposure reduces IQ scores, verbal learning ability, decreases long term memory, and increases the likelihood of dying from a neurological disorder. Arsenic can also reduce birth weight, weight gain, and muscle function after an in utero exposure. Although the mechanism behind these physiological changes is not known, in vitro studies have shown that arsenic can reduce muscle and neuronal cell differentiation. The purpose of this study was to investigate whether arsenic can disrupt signaling pathways or alter the expression of microRNAs that are important in stem cell differentiation. We used P19 murine embryonic stem cells, which were exposed to 0, 0.25, or 0.5 µM arsenite for up to 9 days, to analyze the expression of developmental-related cell signaling pathways and microRNAs by microarray and quantitative PCR. While arsenic does not appear to impact FGF, Bmp nor Notch pathways, it does reduce transcript levels within the sonic hedgehog (Shh) signaling pathway, including the ligand Shh, the key transcription factor Gli2, and its target gene Ascl1, by 2-fold, 3-fold and 5-fold respectively. GLI2 protein expression was also reduced, leading to an inhibition of transcriptional activity. Additionally, exogenous SHH protein rescued the inhibitory effects of arsenic. Based on miRNA microarray data using of 1900 feature mouse miRNAs, Several miRNAs known to be important in development and/or stem cell differentiation, including miR-9, miR-92a, miR-199a, miR-291a and miR-709, were altered by arsenic. Interestingly, a polycomb gene Sfmbt and its hosted miR-466-669 cluster were induced from 1.5-to 3.5 fold by arsenic in a time-dependent manner. Moreover, knockdown of this cluster rescued the arsenic-mediated repression of Gli2 expression and cell differentiation. Taken together, our results imply that arsenic inhibits P19 cell differentiation through repressing the Shh signaling pathway by decreasing Gli2 expression and its activity, and the reduction of Gli2 is directly or indirectly regulated by miR-466-669 cluster. These results suggest two novel mechanisms by which arsenic disrupts cell differentiation