Seung Hwan Hong

Adiponectin Increases Fatty Acid Oxidation in Skeletal Muscle Cells by Sequential Activation of AMP-Activated Protein Kinase, p38 Mitogen-Activated Protein Kinase, and Peroxisome Proliferator–Activated Receptor α

Adiponectin has recently received a great deal of attention due to its beneficial effects on insulin resistance and metabolic disorders. One of the mechanisms through which adiponectin exerts such effects involves an increase in fatty acid oxidation in muscle and liver. In the present study, we demonstrate that 5′–AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (MAPK) are involved in the activation of peroxisome proliferator–activated receptor (PPAR)α by adiponectin in muscle cells. Adiponectin increases the transcriptional activity of PPARα and the expression of its target genes, including ACO, CPT1, and FABP3 in C2C12 myotubes. These effects were suppressed by the overexpression of a dominant-negative form of AMPK. Moreover, chemical inhibitors of AMPK and p38 MAPK potently repressed fatty acid oxidation and the induction of PPARα target gene expression by adiponectin. Interestingly, araA, an AMPK inhibitor, prevented the activation of p38 MAPK, whereas SB203580, a p38 MAPK inhibitor, did not affect AMPK activation, suggesting that p38 MAPK is a downstream signaling factor of AMPK. Taken together, these results suggest that adiponectin stimulates fatty acid oxidation in muscle cells by the sequential activation of AMPK, p38 MAPK, and PPARα.

Sonic hedgehog and FGF8 collaborate to induce dopaminergic phenotypes in the Nurr1-overexpressing neural stem cell

Neural stem cells are self-renewing cells capable of differentiating into all neural lineage cells in vivo and in vitro. In the present study, coordinated induction of midbrain dopaminergic phenotypes in an immortalized multipotent neural stem cell line can be achieved by both overexpression of nuclear receptor Nurr1, and fibroblast growth factor-8 (FGF-8), and sonic hedgehog (Shh) signals. Nurr1 overexpression induces neuronal differentiation and confers competence to respond to extrinsic signals such as Shh and FGF-8 that induce dopaminergic fate in a mouse neural stem cell line. Our findings suggest that immortalized NSCs can serve as an excellent model for understanding mechanisms that regulate specification of ventral midbrain DA neurons and as an unlimited source of DA progenitors for treating Parkinson disease patients by cell replacement.

Epithelial Cell-Specific Laminin 5 Is Required for Survival of Early Thymocytes

The gene LamC2 encoding the γ2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the α3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75–90%) was seen in the CD25+CD44+ and CD25+CD44−subsets of the CD4−CD8− double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin α3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.

8-Cl-cAMP induces cell cycle-specific apoptosis in human cancer cells

8‐Cl‐cyclic adenosine monophosphate (8‐Cl‐cAMP) has been known to induce growth inhibition and differentiation in a variety of cancer cells by differential modulation of protein kinase A isozymes. To understand the anticancer activity of 8‐Cl‐cAMP further, we investigated the effect of 8‐Cl‐cAMP on apoptosis in human cancer cells. Most of the tested human cancer cells exhibited apoptosis upon treatment with 8‐Cl‐cAMP, albeit with different sensitivity. Among them, SH‐SY5Y neuroblastoma cells and HL60 leukemic cells showed the most extensive apoptosis. The effect of 8‐Cl‐cAMP was not reproduced by other cAMP analogues or cAMP‐elevating agents, showing that the effect of 8‐Cl‐cAMP was not caused by simple activation of protein kinase A (PKA). However, competition experiments showed that the binding of 8‐Cl‐cAMP to the cAMP receptor was essential for the induction of apoptosis. After the treatment of 8‐Cl‐cAMP, cells initially accumulated at the S and G2/M phases of the cell cycle and then apoptosis began to occur among the population of cells at the S/G2/M cell cycle phases, indicating that the 8‐Cl‐cAMP‐induced apoptosis is closely related to cell cycle control. In support of this assumption, 8‐Cl‐cAMP‐induced apoptosis was blocked by concomitant treatment with mimosine, which blocks the cell cycle at early S phase. Interestingly, 8‐Cl‐cAMP did not induce apoptosis in primary cultured normal cells and non‐transformed cell lines, showing that 8‐Cl‐cAMP‐induced apoptosis is specific to transformed cells. Taken together, our results show that the induction of apoptosis is one of the mechanisms through which 8‐Cl‐cAMP exerts anticancer activity.

8-Chloro-Cyclic AMP–Induced Growth Inhibition and Apoptosis Is Mediated by p38 Mitogen-Activated Protein Kinase Activation in HL60 Cells

8-Chloro-cyclic AMP (8-Cl-cAMP), which is known to induce growth inhibition, apoptosis, and differentiation in various cancer cell lines, has been studied as a putative anticancer drug. However, the mechanism of anticancer activities of 8-Cl-cAMP has not been fully understood. Previously, we reported that the 8-Cl-cAMP-induced growth inhibition is mediated by protein kinase C (PKC) activation. In this study, we found that p38 mitogen-activated protein kinase (MAPK) also plays important roles during the 8-Cl-cAMP-induced growth inhibition and apoptosis. SB203580 (a p38-specific inhibitor) recovered the 8-Cl-cAMP-induced growth inhibition and apoptosis, whereas other MAPK inhibitors, such as PD98059 (an extracellular signal-regulated kinase-specific inhibitor) and SP600125 (a c-Jun NH2-terminal kinase-specific inhibitor), had no effect. The phosphorylation (activation) of p38 MAPK was increased in a time-dependent manner after 8-Cl-cAMP treatment. Furthermore, SB203580 was able to block PKC activation induced by 8-Cl-cAMP. However, PKC inhibitor (GF109203x) could not attenuate p38 activation, indicating that p38 MAPK activation is upstream of PKC activation during the 8-Cl-cAMP-induced growth inhibition. 8-Chloro-adenosine, a metabolite of 8-Cl-cAMP, also activated p38 MAPK and this activation was blocked by adenosine kinase inhibitor. These results suggest that 8-Cl-cAMP exerts its anticancer activity through p38 MAPK activation and the metabolite(s) of 8-Cl-cAMP mediates this process.

Upstream regulatory regions controlling the expression of the yeast maltase gene.

The expression of the maltase (MALS) and the maltose permease (MALT) genes in Saccharomyces species is coregulated at the transcriptional level; they are coordinately induced by maltose in the presence of a positively acting regulatory (MALR) gene and carbon catabolite repressed by glucose. We generated a series of deletions in the upstream region of the MAL6S gene to examine the regulatory elements in detail. The results showed that inducible expression by maltose was lost when the region between 320 and 380 base pairs upstream of the translation initiation codon was deleted. This region contained an imperfect inverted repeat sequence (-361 to -327) or four copies of short direct repeats that might serve as components of the upstream activation site (UASM) for the maltase gene, or both. When a stretch of T-rich sequence (-253 to -237) was deleted, the susceptibility of the maltase gene to carbon catabolite repression was affected.

Participation of type II protein kinase A in the retinoic acid-induced growth inhibition of SH-SY5Y human neuroblastoma cells.

To examine the role of protein kinase A (EC 2.7.1.37) isozymes in the retinoic acid‐induced growth inhibition and neuronal differentiation, we investigated the changes of protein kinase A isozyme patterns in retinoic acid–treated SH‐SY5Y human neuroblastoma cells. Retinoic acid induced growth inhibition and neuronal differentiation of SH‐SY5Y cells in a dose‐ and time‐dependent manner. Neuronal differentiation was evidenced by extensive neurite outgrowth, decrease of N‐Myc oncoprotein, and increase of GAP‐43 mRNA. Type II protein kinase A activity increased by 1.5‐fold in differentiated SH‐SY5Y cells by retinoic acid treatment. The increase of type II protein kinase A was due to the increase of RIIβ and Cα subunits. Since type II protein kinase A and RIIβ have been known to play important role(s) in the growth inhibition and differentiation of cancer cells, we further investigated the role of the increased type II protein kinase A by overexpressing RIIβ in SH‐SY5Y cells. The growth of RIIβ‐overexpressing cells was slower than that of parental cells, being comparable to that of retinoic acid‐treated cells. Retinoic acid treatment further increased the RIIβ level and further inhibited the growth of RIIβ‐overexpressing cells, showing strong correlation between the level of RIIβ and growth inhibition. However, RIIβ‐overexpressing cells did not show any sign of neuronal differentiation and responded to retinoic acid in the same way as parental cells. These data suggest that protein kinase A participates in the retinoic acid–induced growth inhibition through the up‐regulation of RIIβ/type II protein kinase A. J. Cell. Physiol. 182:421–428, 2000.

Identification of the DNA damage-responsive elements of therhp51+ gene, arecA andRAD51 homolog from the fission yeastSchizosaccharomyces pombe

TheSchizosaccharomyces pombe rhp51+ gene encodes a recombinational repair protein that shares significant sequence identities with the bacterial RecA and theSaccharomyces cerevisiae RAD51 protein. Levels ofrhp51+ mRNA increase following several types of DNA damage or inhibition of DNA synthesis. Anrhp51::ura4 fusion gene was used to identify the cis-acting promoter elements involved in regulatingrhp51+ expression in response to DNA damage. Two elements, designated DRE1 and DRE2 (fordamage-responsiveelement), match a decamer consensus URS (upstream repressing sequence) found in the promoters of many other DNA repair and metabolism genes fromS. cerevisiae. However, our results show that DRE1 and DRE2 each function as a UAS (upstream activating sequence) rather than a URS and are also required for DNA-damage inducibility of the gene. A 20-bp fragment located downstream of both DRE1 and DRE2 is responsible for URS function. The DRE1 and DRE2 elements cross-competed for binding to two proteins of 45 and 59 kDa. DNase I footprint analysis suggests that DRE1 and DRE2 bind to the same DNA-binding proteins. These results suggest that the DRE-binding proteins may play an important role in the DNA-damage inducibility ofrhp51+ expression.

Insulin‐Induced Oxidative Neuronal Injury in Cortical Culture: Mediation by Induced N‐Methyl‐D‐aspartate Receptors

While effectively attenuating neuronal apoptosis in mouse cortical culture, insulin paradoxically induced neuronal necrosis with 48 h of exposure. The insulin neurotoxicity was blocked by an antioxidant but not by caspase inhibitors. Exposure to insulin led to tyrosine phosphorylation of the insulin receptor and the insulin‐like growth factor‐1 (IGF‐1) receptor and activation of protein kinase C (PKC) and phosphoinositide 3‐kinase (PI3‐kinase). Inhibitors of tyrosine kinase and PKC, but not PI3‐kinase, attenuated the insulin neurotoxicity. Conversely, the inhibitor of PI3‐kinase but not PKC reversed the antiapoptotic effect of insulin. Suggesting that the gene activity‐dependent emergence of excitotoxicity contributed to insulin neurotoxicity, macromolecule synthesis inhibitors and N‐methyl‐D‐aspartate (NMDA) antagonists blocked it. Consistently, exposure to insulin increased the level of the NR2A subunit of the NMDA receptor without much altering NR1 or NR2B levels. The present study suggests that insulin can be both neuroprotective and neurotoxic in the same cell system but by way of different signaling cascades.

Protein synthesis-dependent but Bcl-2-independent cytochrome C release in zinc depletion-induced neuronal apoptosis

Previously, we reported that chelation of intracellular zinc with N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN)‐induced macromolecule synthesis‐dependent apoptosis of cultured cortical neurons. According to the current theory of apoptosis, release of mitochondrial cytochrome C into the cytosol is required for caspase activation. In the present study, we examined whether cytochrome C release is dependent on macromolecule synthesis. Exposure of cortical cultures to 2 μM TPEN for 24 hr induced apoptosis as previously described. Fluorescence immunocytochemical staining as well as immunoblots of cell extracts revealed the release of cytochrome C into the cytosol 18–20 hr after the exposure onset. The cytochrome C release was completely blocked by the addition of cycloheximide or actinomycin D. Addition of the caspase inhibitor zVAD‐fmk did not attenuate the cytochrome C release, whereas it blocked TPEN‐induced apoptosis. Because Bcl‐2 has been shown to block cytochrome C release potently, we exposed human neuroblastoma cells (SH‐SY5Y) to TPEN. Whereas Bcl‐2 overexpression completely blocked both cytochrome C release and apoptosis induced by staurosporine, it attenuated neither induced by TPEN. The present results suggest that, in neurons, macromolecule synthesis inhibitors act upstream of cytochrome C release to block apoptosis and that, in addition to the classical Bcl‐2 sensitive pathway, there may exist a Bcl‐2‐insensitive pathway for cytochrome C release. J. Neurosci. Res. 61:508–514, 2000.