Sandhu Harkirat Singh, Bhanwer A J S, Puri Sanjeev
Department of Human Genetics, Guru Nanak Dev University, Amritsar, Punjab, India.
Centre for Stem Cell Tissue Engineering and Biomedical Excellence, Panjab University, Chandigarh, India.
PLoS One. 2017 Mar 14;12(3):e0173031. doi: 10.1371/journal.pone.0173031. eCollection 2017.
The cell differentiation can be exploited as a paradigm to evaluate the effects of noxious chemicals, on human health, either alone or in combinations. In this regard, the effect of a known cell differentiation agent, retinoic acid (RA) was analyzed in the presence of a noxious chemical chlorpyrifos (CPF), an organophosphate (OP), the receptors of which have recently been localized to mesenchymal stem cells (MSCs). The observed imbalance of adipogenic to skeletal differentiation by CPF together with conundrum about adipogenic potential of RA prompted us to delineate their combinatorial effects on C3H10T½MSC-like undifferentiated cells. Based on MTT assay, the cellular viability was retained by CPF at concentrations ranging from 0.01-50μM, beyond which it caused cytotoxicity. These non-toxic concentrations also mildly interfered with adipogenesis of C3H10T½ cells following exposure to adipogenic cocktail. However, upon exposure to RA alone, these MSCs adopted elongated morphology and accumulated lipid vesicles, by day 20, as discerned by phase-contrast and transmission electron microscopy (TEM), in concert with enhanced Oil Red O stained cells. This effect got strongly augmented upon exposure to combination of CPF and RA in a dose-dependent manner. Simultaneous up-regulation in perilipin-1 (PLIN1) and adipsin (ADN) genes, additionally reiterated the adipogenic differentiation. Mechanistically, GSK3β pathway was found to be a major player, whereby inhibiting it with lithium chloride (LiCl) resulted in complete blockage of lipid accumulation, accompanied by complete down regulation of PLIN1 and ADN gene expression. In conclusion, these observations for the first time, lend evidence that exposure of CPF accompanied by RA directs commitment of C3H10T½ cells to adipogenic differentiation through a process involving a crosstalk at GSK3β signaling.
细胞分化可作为一种范例，用于评估有害化学物质单独或联合作用对人类健康的影响。在这方面，我们分析了已知的细胞分化剂视黄酸（RA）在有害化学物质毒死蜱（CPF，一种有机磷酸酯（OP））存在时的作用，CPF的受体最近已定位到间充质干细胞（MSC）。CPF导致的脂肪生成与骨骼分化的失衡，以及RA脂肪生成潜力的难题，促使我们描绘它们对C3H10T½ MSC样未分化细胞的联合作用。基于MTT分析，CPF在0.01 - 50μM浓度范围内可保持细胞活力，超过该浓度则会导致细胞毒性。这些无毒浓度在暴露于脂肪生成混合物后也会轻微干扰C3H10T½细胞的脂肪生成。然而，单独暴露于RA时，这些MSC在第20天时呈现出细长形态并积累脂质小泡，通过相差显微镜和透射电子显微镜（TEM）可观察到，同时油红O染色细胞增多。暴露于CPF和RA的组合时，这种效应以剂量依赖方式强烈增强。 perilipin - 1（PLIN1）和脂肪酶（ADN）基因的同时上调，进一步证实了脂肪生成分化。从机制上讲，发现GSK3β途径是主要参与者，用氯化锂（LiCl）抑制它会导致脂质积累完全受阻，同时PLIN1和ADN基因表达完全下调。总之，这些观察首次证明，CPF与RA共同暴露通过涉及GSK3β信号通路串扰的过程，引导C3H10T½细胞向脂肪生成分化。
