Jung-Hoon Pyo

Mechanism of metformin: inhibition of DNA damage and proliferative activity in Drosophila midgut stem cell.

Age-related changes in stem cells could have a profound impact on tissue aging and the development of age-related diseases such as cancer. However, the effects of metformin, a recently recognized anti-cancer drug, on stem cell aging remain largely unknown. In the present study, an experiment was set up to investigate the underlying mechanism of metformins beneficial effects on age-related changes in intestinal stem cells (ISCs) derived from Drosophila midgut. Results showed that metformin reduced age- and oxidative stress-related accumulation of DNA damage marked by Drosophila γH2AX foci and 8-oxo-dG in ISCs and progenitor cells. Metformin also inhibited age and- oxidative stress-related ISC hyperproliferation as well as intestinal hyperplasia. Our study further revealed that the inhibitory effects of metformin on DNA damage accumulation may be due to the down-regulation of age-related and oxidative stress-induced AKT activity. These data indicate that metformin has beneficial effects on age-related changes in ISCs derived from Drosophila midgut. Further, our results suggest a possible impact of DNA damage on stem cell genomic instability, which leads to the development of age-related diseases. Additionally, our study suggests that Drosophila midgut stem cells can be a suitable model system for studying stem cell biology and stem cell aging.

Slug, mammalian homologue gene of Drosophila escargot, promotes neuronal-differentiation through suppression of HEB/daughterless

At the neuron developmental stage, neuron-precursor cells can be differentiated into neuron or glia cells. However, precise molecular mechanism to determine the cell fate has not been clearly demonstrated. In this study, we reveal that Drosophila esgarcot and its mammalian homologue genes, Snail and Slug, play a key role in neuronal differentiation. In Drosophila model system, overexpression of Esg, like as Wingless, suppresses the bristle formation. In contrast, elimination of Esg though RNAi promotes double bristle phenotype. We can also observe the similar phenotype in Snail-overexpression system. In mammalian system, overexpression of Slug or Snail can induce neuronal differentiation. Esg and its mammalian homologue gene Slug directly interact with Daughtherless and its mammalian homologue HEB and eliminate them through siah-1 mediated protein degradation. Thus, overexpression of siah-1 can promote neuron cell differentiation, whereas si-siah-1 blocks the Slug-induced HEB suppression. In fact, Drosophila SINA, Siah-1 homologue, has been also known to be involved in bristle formation and Neuronal differentiation. In addition, it has been revealed that CK1 is involved in Esg or Snail stability and Neuronal differentiation. However, Snail is regulated only by CK1 but not by Siah. Considering the fact that Slug mutations have been found in human genetic disease, waardenberg syndrome, major symptoms of which is loss of hearing neuron and odd eye, our result implies that Slug/Snail system is required for proper neuronal differentiation, like as Esg in Drosophila.

Functional Modification of Drosophila Intestinal Stem Cells by Ionizing Radiation

Although the diverse effects of ionizing radiation on biological and pathological processes at various levels ranging from molecular to whole body are well studied, the effects on adult stem cells by ionizing radiation remain largely unknown. In this study, we characterized the functional modifications of adult Drosophila midgut intestinal stem cells after ionizing radiation treatment. A dose of 10 Gy of radiation decreased the proliferative capacity of intestinal stem cells. Interestingly, after irradiation at 2 Gy, the intestinal stem cells exhibited increased proliferative activity, misdifferentiation and γH2AvD and 8-oxo-dG levels. In addition, the guts irradiated with 2 Gy showed increased JNK and AKT activities. Furthermore, we showed that 2 Gy of ionizing radiation induced centrosome amplification in intestinal stem cells of adult midguts. Our data gives molecular insights into the effects of ionizing radiation on functional modifications of stem cells. The adult Drosophila midgut intestinal stem cells offer a potentially rich new system for the exploration of the biological effects of ionizing radiation.

Deficiency of Atg6 impairs beneficial effect of metformin on intestinal stem cell aging in Drosophila

Age-related changes of adult stem cell are crucial for tissue aging and age-related diseases. Thus, clarifying mechanisms to prevent adult stem cell aging is indispensable for healthy aging. Metformin, a drug for type 2 diabetes, has been highlighted for its anti-aging and anti-cancer effect. In Drosophila intestinal stem cell (ISC), we previously reported the inhibitory effect of metformin on age-related phenotypes of ISC. Here, we showed that knockdown of Atg6, a crucial autophagy-related factor, in ISC induces age-related phenotypes of ISC such as hyperproliferation, centrosome amplification, and DNA damage accumulation. Then, we revealed that metformin inhibits ISC aging phenotypes in Atg6-dependent manner. Taken together, our study suggests that Atg6 is required for the inhibitory effect of metformin on ISC aging, providing an intervention mechanism of metformin on adult stem cell aging.

Drosophila PEBP1 inhibits intestinal stem cell aging via suppression of ERK pathway

The intestine is a high cellular turnover tissue largely dependent on the regenerative function of stem cell throughout life, and a signaling center for the health and viability of organisms. Therefore, better understanding of the mechanisms underlying the regulation of intestinal stem cell (ISC) regenerative potential is essential for the possible intervention of aging process and age-related diseases. Drosophila midgut is a well-established model system for studying the mechanisms underlying ISC regenerative potential during aging. Here, we report the requirement of Drosophila phosphatidylethanolamine binding protein 1 (PEBP1) in ISC regenerative potential. We showed that PEBP1 was strongly expressed in enterocytes (ECs) of guts and its decrease with age and oxidative stress. Furthermore, the downregulation of PEBP1 in ECs accelerates ISC aging, as evidenced by ISC hyper-proliferation, γH2AX accumulation, and centrosome amplification, and intestinal hyperplasia. The decrease in PEBP1 expression was associated with increased extracellular signal-regulated kinase (ERK) activity in ECs. All these phenotypes by EC-specific depletion of PEBP1 were rescued by the concomitant inhibition of ERK signaling. Our findings evidence that the age-related downregulation of PEBP1 in ECs is a novel cause accelerating ISC aging and that PEBP1 is an EC-intrinsic suppressor of epidermal growth factor receptor (EGFR)/ERK signaling. Our study provides molecular insights into the tight regulation of EGFR/ERK signaling in niches for stem cell regenerative potential.

Effect of heterochromatin stability on intestinal stem cell aging in Drosophila

Chromatin change is one of the crucial causes of aging. Specifically, maintenance of heterochromatin stability is critical for cellular integrity, and its loss induces genomic instability and cellular aging. However, the causes and effects of heterochromatin instability in multicellular tissue aging still remain unclear. Here, in the adult Drosophila midgut, we report age-related loss of heterochromatin stability in enterocytes (ECs) due to the loss and dispersion of tri-methylated histone H3 Lys9 (H3K9me3) and heterochromatin protein 1 (HP1). Our study further shows that EC-specific knockdown of Su(var)3-9, histone lysine methyltransferase for H3K9me3 formation, or HP1a leads to intestinal stem cell (ISC) aging through genomic stress, JNK signaling, and apoptotic death in ECs. Our findings revealed the plausible causes of age-related loss of heterochromatin stability in ECs, including oxidative stress and nutrient-sensing AKT/TOR signaling. Taken together, the loss of heterochromatin stability may be the crucial niche aging mechanism for ISC aging which is the prime determinant of intestinal tissue aging. Furthermore, our study provides new clues on the link between heterochromatin and aging.

Overexpression of dJmj differentially affects intestinal stem cells and differentiated enterocytes

Jumonji (Jmj)/Jarid2 is a DNA-binding transcriptional repressor mediated via histone methylation. Nevertheless, the well-known function of Jmj is as a scaffold for the recruitment of various complexes including Polycomb repressive complex 2 (PRC2), and required for mouse embryonic stem cell development. However, PRC2 independent function is suggested for Drosophila Jumonji (dJmj). To clarify the function of dJmj during cell differentiation, we used Drosophila adult intestinal stem cell system that allows to follow stem cell behaviors in vivo. Overexpression of dJmj in intestinal stem cells/enteroblasts (ISCs/EBs) induces cell-autonomous ISC proliferation followed by differentiation, that is controlled by the Notch and EGFR pathway. In contrast, overexpression of dJmj in enterocytes (ECs) resulted in activation of the JNK pathway in ECs followed by the induction of apoptosis. Activated JNK increased the level of Yorkie in ECs and induced the reduction of Upd proteins and EGFR ligands, which activated the JAK/STAT and EGFR pathway in both ISCs and EBs to promote ISC proliferation. The Notch signaling pathway appears to be highly activated to support the differentiation of EBs to ECs. Thus, the combination of these signaling pathways caused by ECs-specific dJmj-overexpression induced non-cell-autonomous ISC proliferation and differentiation. Surprisingly, these effects did not relate to H3K27me3 status, likely represented PRC2 activity, in cells that overexpressed dJmj. Instead of this, the disappearance of H3K27me3 in ISC/EB-specific overexpressed dJmj suggested a possible PRC2-independent role of dJmj in regulating chromatin structure.

Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging in Drosophila

Stem cell dysfunction is closely linked to tissue and organismal aging and age-related diseases, and heavily influenced by the niche cells’ environment. The DNA damage response (DDR) is a key pathway for tissue degeneration and organismal aging; however, the precise protective role of DDR in stem cell/niche aging is unclear. The Drosophila midgut is an excellent model to study the biology of stem cell/niche aging because of its easy genetic manipulation and its short lifespan. Here, we showed that deficiency of DDR in Drosophila enterocytes (ECs) accelerates intestinal stem cell (ISC) aging. We generated flies with knockdown of Mre11, Rad50, Nbs1, ATM, ATR, Chk1, and Chk2, which decrease the DDR system in ECs. EC-specific DDR depletion induced EC death, accelerated the aging of ISCs, as evidenced by ISC hyperproliferation, DNA damage accumulation, and increased centrosome amplification, and affected the adult fly’s survival. Our data indicated a distinct effect of DDR depletion in stem or niche cells on tissue-resident stem cell proliferation. Our findings provide evidence of the essential role of DDR in protecting EC against ISC aging, thus providing a better understanding of the molecular mechanisms of stem cell/niche aging.

Regulation of the Drosophila Fascin by Raf Signaling

Fascin, a cytoskeleton actin binding protein, functions in cell adhesion and cell migration. Fascin is also known as a candidate biomarker for various cancers, however, regulatory mechanisms of fascin expression remains little understood. In this study, we found an abnormal bristle phenotype, which is similar to that of the Drosophila fascin mutant, in Draf mutant flies. Hence, we investigated whether fascin expression is regulated by Raf signaling. RT-PCR and Western blot analysis showed that Drosophila fascin expression was down-regulated in Draf mutant flies and the level was increased in larvae expressing the oncogenic form of Draf (Draf gof ) under the GAL4-UAS system. Immunostaining analysis showed increased fascin in the hemocytes over-expressing Draf gof . Our results indicate that fascin expression is regulated by Raf signaling and suggest that Raf signaling may play an important role in the actin cytoskeleton-associated developmental process and tumor progression via regulation of fascin gene.