Takbum Ohn

Valproic acid promotes neuronal differentiation by induction of neuroprogenitors in human bone-marrow mesenchymal stromal cells.

Recent studies have shown that the inhibition of histone deacetylases (HDACs) induces the differentiation of diverse cancer and stem cells, which suggests HDAC inhibitors may be good candidates for the induction of stem cell differentiation. In this study, we investigated the effects of a HDAC inhibitor, valproic acid (VPA), for the neuronal differentiation of human bone marrow-mesenchymal stromal cells (hBM-MSCs). VPA-treated MSCs had significant increases in their expression of the neuro-progenitor marker Nestin, Musashi, CD133, and GFAP, as measured by real-time PCR and immunoblot analysis. When VPA-pretreated MSCs were differentiated with neuronal induction media (VPA-dMSCs), they exhibited a cell body and dendritic morphology similar to neurons. The number and neurite length of these VPA-dMSCs significantly increased compared to differentiated MSCs (dMSCs). The VPA-dMSCs and dMSCs had significantly increased transcripts of neuronal-specific marker genes, including Nestin, Musashi, CD133, GFAP, NeuN, MAP-2, NF-M, KCNH1, and KCNH5. The cells also showed a higher expression of the neuronal marker proteins Nestin and NF-M from immunocytochemical staining and immunoblot analysis. This study has shown that VPA pretreatment of hBM-MSCs, following their incubation with neuronal induction media, effectively stimulates neuronal cell differentiation to BM-MSCs.

Ischemic brain extract increases SDF-1 expression in astrocytes through the CXCR2/miR-223/miR-27b pathway.

Ischemic cerebral stroke is one of the leading global causes of mortality and morbidity. Ischemic preconditioning (IPC) refers to a sublethal ischemia and resulting in tolerance to subsequent severe ischemic injury. Although several pathways are reportedly involved in IPC-mediated neuroprotection, the functional role of astrocytes is not fully understood. Stromal cell-derived factor-1 (SDF-1), a CXC chemokine produced mainly in astrocytes, is a ligand for chemokine receptor CXCR4. SDF-1 is reported to play a critical role in neuroprotection after stroke by mediating the migration of neuronal progenitor cells. We hypothesized that stimuli derived from ischemic brain were involved in the protective effects of IPC. To investigate this hypothesis, the mechanism in which ischemic brain extract (IBE) induced SDF-1 expression was investigated in C6 astrocytoma cells. IBE treatment of C6 cells increased SDF-1 expression compared to that in untreated or normal brain extract (NBE)-treated cells by downregulating SDF-1 targeting miRNA, miR-27b. MiR-223 was inversely upregulated in IBE-treated cells; overexpression of miR-223 decreased the expression of miR-27b by suppressing IKKα expression. Analysis of cytokine array data revealed an IBE associated enhanced expression of CINC-1 (CXCL1) and LIX1 (CXCL5). Knockdown or inhibition of their receptor, CXCR2, abolished IBE-mediated increased expression of SDF-1. These results were confirmed in primary cultured astrocytes. Taken together, the data demonstrate that IBE-elicited signals increase SDF-1 expression through the CXCR2/miR-223/miR-27b pathway in C6 astrocytoma cells and primary astrocytes, supporting the view that increased expression of SDF-1 by ischemic insults is a possible mechanism underlying therapeutic application of IPC.

Splicing factor SRSF3 represses the translation of programmed cell death 4 mRNA by associating with the 5′-UTR region

Serine/arginine-rich splicing factor 3 (SRSF3), a member of the serine/arginine (SR)-rich family of proteins, regulates both alternative splicing of pre-mRNA and export of mature mRNA from the nucleus. Although its role in nuclear mRNA processing is well understood, the mechanism by which it alters the fate of cytoplasmic mRNA molecules remains elusive. Here, we provide evidence that SRSF3 not only regulates the alternative splicing pattern of programmed cell death 4 (PDCD4) mRNA, but also modulates its translational efficiency in the cytoplasm by lowering translation levels. We observed a marked increase in PDCD4 mRNA in translating polysome fractions upon silencing of SRSF3, and, conversely, ectopic overexpression of SRSF3 shifted PDCD4 mRNA into non-translating ribosomal fractions. In live cells, SRSF3 colocalized with PDCD4 mRNA in P-bodies (PBs), where translationally silenced mRNAs are deposited, and this localization was abrogated upon SRSF3 silencing. Furthermore, using two different reporter systems, we showed that SRSF3 interacts directly with PDCD4 mRNA and mediates translational repression by binding to the 5′-untranslated region (5′-UTR). In summary, our data suggest that the oncogenic potential of SRSF3 might be realized, in part, through the translational repression of PDCD4 mRNA.

Immunolocalization of steroidogenic acute regulatory protein-related lipid transfer (START) domain-containing proteins in the developing cerebellum of normal and hypothyroid rats.

Cholesterol transport proteins are a prerequisite for neurosteroidogenesis. Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain-containing proteins, such as StAR and START domain-containing 6 (StarD6), are known to be distributed in the brain. Since perinatal hypothyroidism affects cerebellar development, we examined postnatal changes in StAR and StarD6 immunolocalization in the developing cerebellum of control and hypothyroid rats. Pregnant Sprague-Dawley rats were given 0.05% 6-propyl-2-thiouracil (PTU) or water from gestation day 11 until postnatal day (P) 28, and were then killed together with age-matched control rats. As shown by calbindin D-28k immunostaining, the developing cerebellar cytoarchitecture and Purkinje cells were affected by PTU-induced hypothyroidism as compared to control rats. The immunolocalization of StAR and StarD6 generally followed the maturation pattern of Purkinje cells from the vermis to the cerebellar hemisphere. StAR immunostaining first appeared in the Purkinje cells of the vermis at P7 in both control and hypothyroid rats. In control rats, a few StarD6 immunoreactive cells were seen at birth and a nuclear localization of StarD6 in Purkinje cells was obvious at P14. PTU-induced hypothyroidism delayed the appearance of StarD6 immunopositive cells until P7. Moreover, the nuclear localization of StarD6 in PTU-treated rats was not obvious at P14. An adult-like distribution of StAR and StarD6 was achieved by P21 in control and hypothyroid rats. These results suggest that StarD6 may affect the development of Purkinje cells during the first and second postnatal weeks, a known period of thyroid hormone action.

Inactivating UBE2M Impacts the DNA Damage Response and Genome Integrity Involving Multiple Cullin Ligases

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

Proteomic Analysis of the Copper Ion-Induced Stress Response in a Human Embryonic Carcinoma Cell Line

Excessive exposure to copper, a redox-active metal, generates free radicals, which can cause cellular damage. In this study, we aim to identify the proteins that are up- or downregulated by copper exposure in human embryonic carcinoma (NCCIT) cells and to understand the mechanisms that play a role in the copper-induced stress response. After exposure to copper ions, the cells showed upregulated levels of 78 kDa glucose-regulated protein, fibrillin 1, CWC22 spliceosome-associated protein (KIAA1604), heat shock protein (HSP) 60, and HSP70, while the tumor necrosis factor receptor-associated factor 6, vimentin, 14-3-3 protein zeta, and RAC-beta (AKT2) serine/threonine protein kinase were downregulated. The GeneGo Process Networks of the proteins upregulated by copper ions were analyzed, and the 3 highest-scoring networks from the proteins upregulated by copper ions are presented here. In particular, the increased level of HSP70 in response to copper ions occurred in a dose-dependent manner, indicating that HSP70 could be a potential biomarker for copper toxicity in mammalian cells.

Splicing inhibition of U2AF65 leads to alternative exon skipping

Significance Transcription is a biological procedure in which DNA is transcribed to an RNA molecule. However, only fragments of this RNA are needed for protein synthesis. These fragments are exons that are interrupted by introns. Introns are removed by so-called RNA splicing process. Some exons could be alternatively included or excluded from the final RNA molecule. In this study, we have found that U2 snRNP auxiliary factor 65 kDa (U2AF65), a general splicing regulator, can significantly promote the exclusion of alternative exons. Strikingly, U2AF65 suppresses flanking intron splicing of alternative exons, and even constitutive intron splicing. We deduce that the stimulatory effects of U2AF65 on alternative exon exclusion are induced by the splicing inhibitory effects of U2AF65. U2 snRNP auxiliary factor 65 kDa (U2AF65) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF65 stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5′ splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF65. U2AF65 overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF65 inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF65 effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF65 even inhibits general splicing of adenovirus major late (Ad ML) or β-globin pre-mRNA. Thus, we conclude that U2AF65 possesses a splicing Inhibitory function that leads to alternative exon skipping.

SRSF3-regulated miR-132/212 controls cell migration and invasion by targeting YAP1

ABSTRACT Although SRSF3 (Serine/arginine‐rich splicing factor 3) plays a significant role in various biological processes, many of its functions still remain unclear. More particularly, little is known about SRSF3s involvement in the regulation of miRNA. In this report, we found that invasive and migratory abilities were inhibited in SRSF3‐silenced U2OS and HeLa cells. We also found that a knockdown of SRSF3 results in a decreased expression level of REST (RE1‐silencing transcription factor). The silencing of REST increased the expression of primary miR‐132/212 as well as their mature forms. In particular, miR‐132–3p and miR‐212–3p possess an identical seed sequences and a common target gene. Overexpression of miR‐132–3p and miR‐212–3p suppressed the expression of YAP1 (Yes‐associated protein 1) by directly binding to the 3′UTR of its mRNA. CCND1 (Cyclin D1), which acts downstream of YAP1, was downregulated in both miR‐132–3p and miR‐212–3p‐overexpressed cells, in correlation with diminished YAP1 levels. Taken together, our results reveal that SRSF3 controls the expression of the miR‐132/212 cluster through regulating REST expression, and that the REST‐elicited alteration of miRNA expression is implicated in enabling the migratory and invasive abilities of cancer cells. HIGHLIGHTSREST is involved in the SRSF3‐mediated migration and invasion.SRSF3 regulates the expression of miR‐132/212 through REST.miR‐132/212 suppresses YAP1 expression by directly binding to the 3′UTR of its mRNA.miR‐132/212 inhibits migratory and invasive abilities via downregulation of YAP1/CyclinD1.miR‐132/212 is responsible for the oncogenic function of SRSF3.

A comparison of the steroidogenic acute regulatory protein-related lipid transfer (START) domain-containing 6 on the brain and testes between young and aged rats

The START domain-containing 6 (StarD6) was originally reported to play a role during male germ cell maturation. We have since reported on StarD6 in the developing hypothyroid rat brain. Therefore, we investigated qualitative and quantitative changes of StarD6 in the aging rat brain and testes of male Sprague-Dawley rats. Serum testosterone levels decreased with aging and total protein levels of StarD6 in the testes decreased. While the immunolocalization of StarD6 in the spermatocytes decreased, cytoplasmic localization appeared in the aged testes. Compared with young rats, aged rats showed decreased StarD6 in the cerebrum and cerebellum without changes in immunolocalization in the cortical neurons of the cerebral cortex and Purkinje cells of the cerebellar cortex. Aged rats also showed increases in StarD6 in the hippocampus with changes in its immunolocalization from the Stratum pyramidale to the Stratum radiatum and Stratum lacunosum-moleculare. Taken together, StarD6 decreased with aging in the testes, which implies that StarD6 might play a role in impaired spermatogenesis in the aged rat. StarD6 decreased in the cerebrum and the cerebellum, but slightly increased in the hippocampus, which suggests that StarD6 might also play a role for neurosteroidogenesis in the hippocampus of aged rats.

Resveratrol relieves hydrogen peroxide-induced premature senescence associated with SIRT1 in human mesenchymal stem cells

Cellular senescence of mesenchymal stem cells (MSCs) is often induced during in vitro expansion, by multiple experimental stimuli including oxidative stress. In this study, we investigated expression of senescence-associated proteins including SIRT1after inducing premature senescence of MSCs with hydrogen peroxide (H2O2). We also analyzed the effect of resveratrol (RSV) on premature senescence. We found that H2O2 triggered the recruitment of RCK (p54) to P-bodies in MSCs. Premature senescence of MSCs in response to H2O2 induced a decrease in SIRT1expression and activity (indirectly identified by measuring H3-K9ac). Cellular expression of p21 and phosphorylation of ERK1/2 and p38 kinases were increased in response to H2O2, whereas phosphorylation of pRb was decreased. In contrast, RSV pretreatment resulted in a decrease in the premature senescence of MSCs. In addition, RSV pretreatment before exposing cells to H2O2 not only alleviated changes in the levels of proteins that were sensitive to the H2O2 treatment (SIRT1, p21,ERK1/2 and p38) but also inhibited the decrease of SIRT1 induced by nicotinamide (NAM). Our results suggest that MSCs may exhibit an increased tolerance for H2O2-induced oxidative stress via the senescence-associated proteins that are regulated by RSV.