줄기 세포의 분화를 조절하는 새로운 인자들의 규명 및 기능 연구

Abstract

Stem cells are an attractive model not only for investigating molecular mechanisms of cell growth and differentiation under normal and pathological conditions but also for use in cell-based therapies for regenerative medicine. To maintain homeostasis, a precise balance between self-renewal and differentiation of stem cells is essential. Loss of this balance tends to uncontrolled cell growth or pre-maturation and thus results in cancers or tissue defects. In this respect, the identification of molecules that control stem cell self-renewal and differentiation is crucial for future therapeutic applications in regenerative medicine. However, although the significance of factors that regulate homeostasis between self-renewal and differentiation, many of factors have not been identified yet. Therefore, I have tried to identify the novel factors which are involved in self-renewal and differentiation in human mesenchymal stem cells and mouse embryonic stem cells. First, I demonstrated the novel role of glucosylceramide synthase (GCS) as a regulator on adipogenesis and osteogenesis in human adipose tissue-derived mesenchymal stem cells (hAMSCs). GCS is the key enzyme that catalyzes the transfer of glucose to ceramide in glycosphingolipid biosynthesis. GCS has been implicated in many biological phenomena, including brain maturation, multidrug resistance in cancer and insulin resistance in diabetes. However, very little is known about the roles of GCS in stem cell differentiation. Here, I observed that the mRNA and protein levels of GCS were increased during adipogenesis in hAMSCs. To examine the role of GCS in adipocyte differentiation, I treated cells with D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of GCS, during adipogenesis. PDMP suppressed lipid droplet formation and the expression of adipocyte-specific markers such as peroxisome proliferator-activated receptor-gamma (PPAR-γ) and CCAAT/Enhancer binding protein-alpha (C/EBP-α). Knockdown of GCS also inhibited lipid accumulation and downregulated adipocyte-specific markers. At the molecular level, overexpression of GCS increased PPAR-γ activity, an essential transcriptional factor for adipogenesis, and exogenous treatment with glycosphingolipids, metabolites of GCS, also enhanced PPAR-γ activity. Interestingly, I found that knockdown of GCS promotes osteogenesis. These data reveal an important role for GCS as a differentiation regulator between adipogenesis and osteogenesis.Second, I suggested the effect of ochratoxin A (OTA), one of fungi metabolites, on adipogenesis in hAMSCs. OTA is a ubiquitous fungal metabolite with nephrotoxic, carcinogenic and apoptotic potential. Although the toxic effects of OTA in various cell types are well characterized, it is not known whether OTA has an effect on stem cell differentiation. In this study, I demonstrate that OTA inhibits adipogenesis in hAMSCs, as indicated by decreased accumulation of intracellular lipid droplets. Furthermore, OTA significantly reduces the expression of adipocyte-specific markers, including PPAR-γ, C/EBP-α, lipoprotein lipase (LPL) and aP2. At the molecular level, OTA phosphorylates PPAR-γ through extracellular signal-regulated kinase 1/2 (ERK1/2) activation and inhibits PPAR-γ activity. I also found that treatment with the MEK inhibitor, PD98059, significantly blocked the OTA-induced inhibition of adipogenesis. These results indicate that OTA suppresses adipogenesis in an ERK1/2-dependent manner. Taken together, our results suggest a novel effect of OTA on adipocyte differentiation in hAMSCs and the possibility that OTA might affect the differentiation of other types of stem cells. Third, I proved that lysophosphatidic acid (LPA) controls self-renewal and differentiation in mouse embryonic stem cells (mES cells). LPA involves various cellular events such as proliferation, differentiation and migration. However, very little is known about the presence of LPA receptors and the roles of LPA in ES cells. Here, I investigated the effect of LPA on self-renewal and differentiation in mES cells. Undifferentiated mES cells expressed six LPA receptor subtypes (LPA1-6) and activation of these receptors increased [Ca+2] i mobilization and activated Akt and ERK1/2. I found that LPA suppressed self-renewal of mES cells in vitro even in the presence of LIF. The expression levels of undifferentiated markers, such as Oct3/4 and Nanog were also reduced by LPA treatment. Furthermore, RT-PCR analysis and pharmacological study showed that LPA induces the differentiation of mES cells into endoderm and mesoderm lineage through Gαi/o-ERK1/2 pathway. Taken together, this study demonstrates the novel role of LPA on self-renewal and differentiation in mES cells. In conclusion, I identified several molecules to modulate stem cell differentiation. These finding provide the better understanding about the regulation mechanisms on stem cell differentiation and self-renewal.