Kwang Jin Baek

Involvement of p38 mitogen-activated protein kinase and apoptosis signal-regulating kinase-1 in nitric oxide-induced cell death in PC12 cells

Although nitric oxide (NO) plays key signaling roles in the nervous systems, excess NO leads to cell death. In this study, the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and apoptosis signal-regulating kinase-1 (ASK1) in NO-induced cell death was investigated in PC12 cells. NO donor transiently activated p38 MAPK in the wild type parental PC12 cells, whereas the p38 MAPK activation was abolished in NO-resistant PC12 cells (PC12-NO-R). p38 MAPK inhibitors protected the cells against NO-induced death, whereas the inhibitors were not significantly protective against the cytotoxicity of reactive oxygen species. Stable transfection with dominant negative p38 MAPK mutant reduced NO-induced cell death. Stable transfection with dominant negative mutant of ASK1 attenuated NO-stimulated activation of p38 MAPK and decreased NO-induced cell death. These results suggest that p38 MAPK and its upstream regulator ASK1 are involved in NO-induced PC12 cell death.

Leucine-rich glioma inactivated 3 associates with syntaxin 1.

Leucine-rich glioma inactivated 3 (LGI3) is a member of LGI/epitempin (EPTP) family. The biological function of LGI3 and its association with disease are not known. We previously reported that mouse LGI3 was highly expressed in brain in a developmentally and transcriptionally regulated manner. In this study, we identified syntaxin 1, a SNARE component in exocytosis, as a candidate functional target of LGI3. Western blot analysis of mouse brain extract with LGI3 antibodies detected multiple protein forms (75-, 60-, 35- and 25-kDa). Proteomic analysis, pull-down and coimmunoprecipitation experiments identified syntaxin 1 as an LGI3-associated protein. LGI3 colocalized with syntaxin 1 in processes of cortical neurons with punctate synaptic pattern and was enriched in synaptosomal fraction. Coimmunoprecipitation showed that LGI3-syntaxin 1 complex did not contain other SNARE components, SNAP25 and VAMP2. Recombinant LGI3 attenuated Ca(2+)-evoked glutamate release from digitonin-permeabilized synaptosomes and transfection of PC12 cells with LGI3 decreased K(+)-induced secretion of human growth hormone. Thus, LGI3 may play a regulatory role in neuronal exocytosis via its interaction with syntaxin 1.

Leucine-rich glioma inactivated 3 induces neurite outgrowth through Akt and focal adhesion kinase.

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein that belongs to LGI/epitempin family. LGI3 is highly expressed in brain in a transcriptionally and developmentally regulated manner. Here we found that LGI3 induced neurite outgrowth in Neuro-2a cells and dorsal root ganglia explants. LGI3 treatment or overexpression increased neurite outgrowth and knockdown of LGI3 by siRNA had opposite effect. LGI3 treatment increased phosphorylation of Akt and a 125-kDa protein. Immunoprecipitation identified the 125-kDa protein as focal adhesion kinase (FAK). LGI3 overexpression increased phospho-Akt, phospho-FAK and FAK protein. Inhibition of Akt activation by PI3 kinase inhibitor attenuated LGI3-induced FAK phosphorylation and neurite outgrowth. Taken together, we propose that LGI3 is a neuritogenic factor whose signaling pathway involves Akt-mediated FAK activation.

ERK Activation by Fucoidan Leads to Inhibition of Melanogenesis in Mel-Ab Cells

Fucoidan, a fucose-rich sulfated polysaccharide derived from brown seaweed in the class Phaeophyceae, has been widely studied for its possible health benefits. However, the potential of fucoidan as a possible treatment for hyperpigmentation is not fully understood. This study investigated the effects of fucoidan on melanogenesis and related signaling pathways using Mel-Ab cells. Fucoidan significantly decreased melanin content. While fucoidan treatment decreased tyrosinase activity, it did not do so directly. Western blot analysis indicated that fucoidan downregulated microphthalmia-associated transcription factor and reduced tyrosinase protein expression. Further investigation showed that fucoidan activated the extracellular signal-regulated kinase (ERK) pathway, suggesting a possible mechanism for the inhibition of melanin synthesis. Treatment with PD98059, a specific ERK inhibitor, resulted in the recovery of melanin production. Taken together, these findings suggest that fucoidan inhibits melanogenesis via ERK phosphorylation.

Baicalin-induced Akt activation decreases melanogenesis through downregulation of microphthalmia-associated transcription factor and tyrosinase

Scutellaria baicalensis has been used topically to treat inflammatory skin diseases in traditional East Asian medicine. Because post-inflammatory hyperpigmentation of the skin is difficult to manage, we investigated the effects of baicalin, a major component of S. baicalensis, on melanin synthesis in Mel-Ab cells. Our data showed that baicalin significantly inhibited melanin production and tyrosinase activity in a dose-dependent fashion, but it did not directly influence tyrosinase activity. Moreover, baicalin treatment triggered decreases in both mRNA and protein levels of microphthalmia-associated transcription factor (MITF) and tyrosinase. Although AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) activation were induced in baicalin-treated Mel-Ab cells, they were not responsible for baicalin-induced hypopigmentation. Because the Akt pathway is also known to be involved in regulation of melanogenic protein expression and melanin synthesis, we examined the effects of baicalin on the Akt pathway. Our results showed that baicalin treatment stimulated Akt activation. Treatment with LY294002, a specific Akt inhibitor, restored baicalin-induced melanogenesis inhibition and abolished MITF and tyrosinase downregulation by baicalin. Taken together, our data suggest that Akt activation by baicalin inhibits melanin production via downregulation of MITF and tyrosinase in Mel-Ab cells.

Leucine-rich glioma inactivated 3 regulates adipogenesis through ADAM23.

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein and a member of LGI/epitempin family. We previously showed that LGI3 was highly expressed in brain and played regulatory roles in neuronal exocytosis and differentiation. Besides the nervous system, LGI3 was shown to be expressed in diverse tissues. In this study, we found that LGI3 and its receptor candidate ADAM23 were expressed in adipose tissues and 3T3-L1 cells. 3T3-L1 preadipocytes secreted a 60-kDa protein, a major secreted form of LGI3, which declined with adipocyte differentiation. LGI3 was also expressed in adipose tissue macrophages in the ob/ob mice and in macrophage cell line. The 60-kDa LGI3 protein was selectively increased in the ob/ob adipose tissues comparing with the lean mice. Pull-down experiments, coimmunoprecipitation and immunocytochemistry indicated that LGI3 associated with ADAM23 in adipose tissues and 3T3-L1 cells. Knockdown of LGI3 or ADAM23 by siRNA increased adipogenesis in 3T3-L1 cells. Treatment with LGI3 protein did not affect preadipocyte proliferation but attenuated adipogenesis and this effect was reversed by siRNA-mediated knockdown of ADAM23. Taken together, we propose that LGI3 may be a candidate adipokine that is perturbed in obesity and suppresses adipogenesis through its receptor, ADAM23.

Nitric oxide mediates membrane depolarization-promoted survival of rat neuronal PC12 cells

Membrane depolarization promotes neuronal survival through increases in intracellular calcium. Nitric oxide (NO) is a signaling molecule involved in many neuronal activity-dependent events. Since neuronal NO is generated by NO synthase (NOS) in a calcium-dependent manner and was shown to promote cell survival, we tested whether NO is involved in depolarization-promoted survival in neuronally differentiated PC12 cells. NOS inhibitor attenuated depolarization-promoted survival and NO donors promoted survival. This effect was partially cGMP-dependent as a guanylyl cyclase inhibitor decreased NO-promoted survival. Ras inhibitor, Erk blocker or phosphatidylinositol 3-kinase inhibitor decreased depolarization- or NO donor-promoted survival. Depolarization-induced Ras activation was blocked by NOS inhibitor. Inducible expression of dominant negative Ras or S-nitrosylation-defective Ras attenuated depolarization- or NO donor-promoted survival. Thus, NO might be a mediator via Ras and cGMP pathways in depolarization-promoted survival in neuronal PC12 cells.

Indole-3-carbinol inhibits prostate cancer cell migration via degradation of beta-catenin.

We determined whether indole-3-carbinol (I3C) could affect DU145 human prostate carcinoma cell migration to prevent the development and progression of prostate cancer. Although previous studies have shown anticancer properties of I3C in various cancer cell lines, it has not been determined how I3C regulates epidermal growth factor (EGF)-induced migration and related signaling pathways. DU145 cells were treated with I3C (100 microM) in the absence or presence of EGF (10 ng/ml). Our results showed that I3C significantly inhibited DU145 cell migration with and without EGF stimulation. It has been reported that the beta-catenin signaling pathway controls androgen receptor (AR)-mediated prostate cancer progression, which plays a key role in the metastasis of prostate cancer. Western blot analysis demonstrated that I3C led to the phosphorylation of beta-catenin and subsequent degradation of beta-catenin in the absence and presence of EGF. In contrast, I3C did not have any effect on the expression of beta-catenin mRNA. From these results, we suggest that I3C inhibits EGF (dependent or independent)-induced DU145 cell migration through beta-catenin degradation.

Involvement of mTOR signaling in sphingosylphosphorylcholine-induced hypopigmentation effects

BackgroundSphingosylphosphorylcholine (SPC) acts as a potent lipid mediator and signaling molecule in various cell types. In the present study, we investigated the effects of SPC on melanogenesis and SPC-modulated signaling pathways related to melanin synthesis.MethodsMelanin production was measured in Mel-Ab cells. A luciferase assay was used to detect transcriptional activity of the MITF promoter. Western blot analysis was performed to examine SPC-induced signaling pathways.ResultsSPC produced significant hypopigmentation effects in a dose-dependent manner. It was found that SPC induced not only activation of Akt but also stimulation of mTOR, a downstream mediator of the Akt signaling pathway. Moreover, SPC decreased the levels of LC3 II, which is known to be regulated by mTOR. Treatment with the mTOR inhibitor rapamycin eliminated decreases in melanin and LC3 II levels by SPC. Furthermore, we found that the Akt inhibitor LY294002 restored SPC-mediated downregulation of LC3 II and inhibited the activation of mTOR by SPC.ConclusionsOur data suggest that the mTOR signaling pathway is involved in SPC-modulated melanin synthesis.

The regulatory mechanism of melanogenesis by FTY720, a sphingolipid analogue

Abstract:  We previously reported that sphingosine‐1‐phosphate (S1P) decreases melanin synthesis via extracellular signal‐regulated protein kinase (ERK) activation and microphthalmia‐associated transcription factor (MITF) degradation. Although FTY720 is an S1P structural analogue, the effects of FTY720 on melanogenesis are not completely understood. Thus, we investigated the influence of FTY720 on melanin synthesis in a spontaneously immortalized mouse melanocyte cell line (Mel‐Ab). FTY720 inhibited melanin synthesis in a concentration‐dependent manner. Further, FTY720 has a different signal transduction mechanism to regulate melanogenesis from the S1P‐induced signalling pathway. Our results showed that FTY720 down‐regulated MITF and tyrosinase expression without ERK activation. MITF, the master regulator of pigmentation, is a target for the Wnt signalling pathway, including glycogen synthase kinase 3β (GSK3β) and β‐catenin. Thus, the influence of FTY720 on GSK3β and β‐catenin was further investigated. Decreased MITF and tyrosinase were associated with a reduction of β‐catenin protein and mRNA levels. Decreased β‐catenin expression by FTY720 may down‐regulate expression of MITF, which finally reduces melanin synthesis.