• Bulletin of the Korean Chemical Society
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• 제30권9호
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• pp.1981-1984
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• 2009

Subcellular localization of protein kinase often plays an important role in determining its activity and specificity. Protein kinase C (PKC), a family of multi-gene protein kinases has long been known to be translocated to the particular cellular compartments in response to DAG or its analog phorbol esters. We used C-terminal green fluorescent protein (GFP) fusion proteins of PKC isoforms to visualize the subcellular distribution of individual PKC isoforms. Intracellular localization of PKC-GFP proteins was monitored by fluorescence microscopy after transient transfection of PKC-GFP expression vectors in the HeLa cells. In unstimulated HeLa cells, all PKC isoforms were found to be distributed throughout the cytoplasm with a few exceptions. PKC$\theta$ was mostly localized to the Golgi, and PKC$\gamma$, PKC$\delta$ and PKC$\eta$ showed cytoplasmic distribution with Golgi localization. DAG analog TPA induced translocation of PKC-GFP to the plasma membrane. PKC$\alpha$, PKC$\eta$ and PKC$\theta$ were also localized to the Golgi in response to TPA. Only PKC$\delta$ was found to be associated with the nuclear membrane after transient TPA treatment. These results suggest that specific PKC isoforms are translocated to different intracellular sites and exhibit distinct biological effects.

• Molecules and Cells
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• 제24권1호
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• pp.9-15
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• 2007

To investigate the effects of chitosan on the redifferentiation of dedifferentiated chondrocytes, we used chondrocytes obtained from a micromass culture system. Micromass cultures of chick wing bud mesenchymal cells yielded differentiated chondrocytes, but these dedifferentiated during serial monolayer subculture. When the dedifferentiated chondrocytes were cultured on chitosan membranes they regained the phenotype of differentiated chondrocytes. Expression of protein kinase $C{\alpha}$ ($PKC{\alpha}$) increased during chondrogenesis, decreased during dedifferentiation, and increased again during redifferentiation. Treatment of the cultures with phorbol 12-myristate 13-acetate (PMA) inhibited redifferentiation and down-regulated $PKC{\alpha}$. In addition, the expression of p38 mitogen-activated protein (MAP) kinase increased during redifferentiation, and its inhibition suppressed redifferentiation. These findings establish a culture system for producing chondrocytes, point to a new role of chitosan in the redifferentiation of dedifferentiated chondrocytes, and show that $PKC{\alpha}$ and p38 MAP kinase activities are required for chondrocyte redifferentiation in this model system.

• Korean Journal of Acupuncture
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• 제30권4호
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• pp.212-219
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• 2013

Objectives : Cirsium japonicum is a traditional Korean medicine that has been used in the treatment of inflammatory diseases such as appendicitis, hepatitis, pulmonary abscess and tumor. The aim of study was to elucidate anti-migratory activity of CJP(Cirsium japonicum pharmacopuncture) through regulation of inflammatory mediators in C6 glioma cell. Methods : Nitric oxide(NO) production was determined by using nitrite assay. The cell migration was analyzed by wound-healing assay and Boyden chamber assay. The expression levels of iNOS, and protein kinase C(PKC)-${\alpha}$ were measured by western blotting assay. Results : CJP showed a significant decrease on NO production. Moreover, glioma cell migration was effectively suppressed by CJP. Furthermore, CJP inhibited the expressions of iNOS and PKC-${\alpha}$ in C6 glioma cells. Conclusions : These results suggest that CJP inhibits glioma cell migration and iNOS expression through regulation of PKC-${\alpha}$. Therefore, it is expected that CJP could be an effective agents for blocking malignant progression of glioma.

• BMB Reports
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• 제42권8호
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• pp.516-522
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• 2009

SH2D4A, comprising a single SH2 domain, is a novel protein of the SH2 signaling protein family. We have previously demonstrated SH2D4A is expressed ubiquitously in various tissues and is located in the cytoplasm. In this study we investigated the function of SH2D4A in human embryonic kidney (HEK) 293 cells using interaction analysis, cell proliferation assays, and kinase activity detection. SH2D4A was found to directly bind to estrogen receptor $\alpha$ (ER$\alpha$), and prevent the recruitment of phospholipase C-$\gamma$ (PLC-$\gamma$) to ER$\alpha$. Moreover, we observed its inhibitory effects on estrogen-induced cell proliferation, involving the protein kinase C (PKC) signaling pathway. Together, these findings suggested that SH2D4A inhibited cell proliferation by suppression of the ER$\alpha$/PLC-$\gamma$/PKC signaling pathway. SH2D4A may be useful for the development of a new anti-cancer drug acting as an ER signaling modulator.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제23권4호
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• pp.295-305
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• 2001

구강은 흡연이나 음주와 같은 화학적 발암물질이 쉽게 접촉할 수 있는 화학적 발암물질의 표적장기이며 구강암을 포함한 대부분의 암 발생의 근원이 되는 세포는 상피세포이다. 따라서 본 연구는 인체상피세포를 화학적 발암물질인 MNING에 노출시켜 발암화를 유도하고 이에 따른 작용 기전을 분석함으로써 구강암과 같은 상피세포 기원의 종양 발생기전을 이해하는 데 기여하고자 하였다. 인체 상피세포에 $0.001{\mu}g/ml$에서 $1{\mu}g/ml$ 용량의 MNNG를 투여한 결과 용량 의존적인 세포발암성을 나타내었으며 $0.01{\mu}g/ml$ 투여군이 가장 높은 암세포의 지표를 보였다. MNNG투여후 TPA를 처리한 결과 발암세포의 지표인 saturation density, soft agar colony formation, cell aggregation 등에서 MNNG의 단독 투여시보다 높은 발암성을 나타내었으며 최초의 foci출현시기도 단축되었다. 이와같은 결과는 Phorbol ester binding assay에서도 나타나 세포 발암화 촉진에 PKC활성이 관여함을 추정할 수 있다. PKC translocation 현상은 세포외 칼슘이 있을 경우에만 나타나 MNNG에 의한 PKC활성에 classical PKC가 관여함을 추정할 수 있었다. MNNG에 대한 초기반응으로 cPKC의 경우 $PKC-{\alpha}$와 $PKC-{\gamma}$가 고농도에서 활성의 증가를 보였으며 nPKC의 경우 $PKC-{\varepsilon}$가 뚜렷한 활성을 보여 이들 isoform이 MNNG에 의한 발암화 초기단계에 관여함을 암시하였다. 반면 aPKC는 어느 형태도 MNNG에 반응하지 않아 화학적 발암화 과정에 isoform의 특이성이 존재함을 입증하였다. MNNG에 의해 발암화 특성을 나타낸 세포는 $PKC-{\alpha}$ 및 $PKC-{\gamma}$의 지속적인 활성증가를 나타내어 발암의 초기단계부터 지속적이 활성을 유지하고 있는 isoform으로 추정된다. 본 연구결과 인체상피 세포의 모든 PKC isoform에 대한 발현을 분석하고 화학적 발암화에 관여하는 isoform을 선별해냄으로써 특정한 inhibitor 등을 상요한 발암화 억제제의 개발에 필요한 기초자료를 제공하였을 뿐만 아니라 구강암과 같은 상피세포 기원의 암발생 기전을 이해하는 데 기여할 것으로 사료된다.

• The Korean Journal of Physiology and Pharmacology
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• 제6권1호
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• pp.33-39
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• 2002

It has been suggested that $Ca^{2+}$ sensitization mechanisms might contribute to myogenic tone, however, specific mechanisms have not yet been fully identified. Therefore, we investigated the role of protein kinase C (PKC)- or RhoA-induced $Ca^{2+}$ sensitization in myogenic tone of the rabbit basilar vessel. Myogenic tone was developed by stretch of rabbit basilar artery. Fura-2 $Ca^{2+}$ signals, contractile responses, PKC immunoblots, translocation of PKC and RhoA, and phosphorylation of myosin light chains were measured. Stretch of the resting vessel evoked a myogenic contraction and an increase in the intracellular $Ca^{2+}$ concentration $([Ca^{2+}]_i)$ only in the presence of extracellular $Ca^{2+}$. Stretch evoked greater contraction than high $K^+$ at a given $[Ca^{2+}]_i.$ The stretch-induced increase in $[Ca^{2+}]_i$ and contractile force were inhibited by treatment of the tissue with nifedipine, a blocker of voltage-dependent $Ca^{2+}$ channel, but not with gadolinium, a blocker of stretch-activated cation channels. The PKC inhibitors, H-7 and calphostin C, and a RhoA-activated protein kinase (ROK) inhibitor, Y-27632, inhibited the stretch-induced myogenic tone without changing $[Ca^{2+}]_i.$ Immunoblotting using isoform-specific antibodies showed the presence of $PKC_{\alpha}$ and $PKC_{\varepsilon}$ in the rabbit basilar artery. $PKC_{\alpha},$ but not $PKC_{\varepsilon},$ and RhoA were translocated from the cytosol to the cell membrane by stretch. Phosphorylation of the myosin light chains was increased by stretch and the increased phosphorylation was blocked by treatment of the tissue with H-7 and Y-27632, respectively. Our results are consistent with important roles for PKC and RhoA in the generation of myogenic tone. Furthermore, enhanced phosphorylation of the myosin light chains by activation of $PKC_{\alpha}$ and/or RhoA may be key mechanisms for the $Ca^{2+}$ sensitization associated with myogenic tone in basilar vessels.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2003년도 Annual Meeting of KSAP : International Symposium on Pharmaceutical and Biomedical Sciences on Obesity
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• pp.78-78
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• 2003

Inulin, an active component of Chicorium intybus root, has been shown to stimulate the growth of bifidobacteria, and inhibit colon carcinogenesis. NO mediates a number of the host-defense functions of activated macrophages, including antimicrobial and tumoricidal activity. We examined the effect of inulin on the synthesis of NO in RAW 264.7 cells. Inulin alone had no effect, whereas inulin with IFN-ν synergistically increased the NO production and inducible NO synthase (iNOS) expression in RAW 264.7 cells. Synergy between IFN-ν and inulin was mainly dependent on inulin-induced TNF-${\alpha}$ secretion. Also, protein kinase C (PKC)-${\alpha}$ was involved in the inulin-induced NO production. Inulin-mediated NO production was inhibited by the protein tyrosine kinase (PTK) inhibitor, tyrphostin AG126. Since iNOS gene transcriptions have been shown to be under the control of the NF -$\kappa$B/Rel family of transcription factors, we assessed the effect of inulin on NF -$\kappa$B/Rel using an EMSA. Inulin produced strong induction of NF-$\kappa$B/Rel binding, whereas AP-l binding was slightly induced in RAW 264.7 cells. Inulin stimulated phosphorylation and degradation of I$\kappa$B-${\alpha}$. These results suggest that in IFN-ν-primed RAW 264.7 cells inulin might stimulate NO synthesis via activation of PKC-${\alpha}$ and PTK, resulting in the activation of NF-$\kappa$B.

• 대한방사선기술학회지:방사선기술과학
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• 제26권1호
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• pp.91-97
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• 2003

본 연구를 통해 X선에 의한 연골세포 분화 억제 작용경로를 조사하고자 하였다. 계배 limb bud 간충직세포를 배양하면서 여러 가지 선량(1-10Gy)의 X선을 조사하고 연골분화도를 조사한 결과, X선은 연골세포에 특이하게 발현되어 분화척도로 이용되는 typeII collagen의 발현과 proteoglycan의 축적을 저해하였다. 또한 세포내 신호전달 과정에서 중요한 매개자 역할을 하는 것으로 알려진 PKC 동위효소중 $PKC{\alpha}$의 발현을 저해하였다. 그러나 $PKC{\lambda}({\iota}),\;{\varepsilon}$ 등 다른 동위효소의 발현에는 별다른 영향을 미치지 못하였다. X선 조사에 의한 연골세포 분화 억제가 $PKC{\alpha}$의 downstream으로 알려져 있는 Erk-1을 통하여 이루어지는지 알아보기 위하여 Erk-1의 발현 및 인산화를 조사한 결과 X선은 그 발현에는 영향을 미치지 못했으나 인산화는 증가시켰다. 연골세포 분화 저해 효과가 Erk-1의 활성 변화에 의한 것인가를 확인하기 위하여 Erk-1을 인산화하는 MEK의 저해제인 PD98059를 처리하여 Erk-1의 인산화를 저해한 결과 X선 조사에 의한 연골분화 억제효과를 극복하는 것으로 나타났다. 또한 X선 조사가 분화 초기의 세포응집 과정에 어떤 영향을 미치는 지 알아보기 위하여 PNA 염색으로 조사한 결과 보선 조사는 세포응집을 저해하였다. 본 연구 결과를 종합하면 X선 조사는 분화 초기에 세포 응집을 억제하며 $PKC{\alpha}$의 발현을 저해하고 Erk-1의 인산화를 촉진하여 연골세포 분화를 억제하는 것으로 사료된다.

• Environmental Analysis Health and Toxicology
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• 제22권3호
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• pp.279-285
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• 2007

The present study attempted to analyze the mechanism of PCB-induced neurotoxicity with respect to the PKC signaling. Since the developing neuron is particularly sensitive to PCB-induced neurotoxicity, we isolated cerebellar granule cells derived from 7-day old SD rats and grew cells in culture for additional 7 days to mimic PND-14 conditions. Only non-coplanar PCBs at a high dose showed a significant increase of total PKC activity at $[^3H]PDBu$ binding assay, indicating that non-coplanar PCBs are more neuroactive than coplanar PCBs in neuronal cells. PKC isoforms were immunoblotted with respective monoclonal antibodies. PKC-alpha and-epsilon were activated with non-coplanar PCB exposure. The result suggests that coplanar PCBs have a PKC pathway different from non-coplanar PCBs. Activation of PKC with exposure was dampened with treatment of high molecular weight of chitosan. Chilean (M.W. > 1,000 kDa) inhibited the total activity of PKC induced by the non-coplanar PCBs. Translocation of PKC isoforms was also inhibited by the high molecular weight of chitosan. The study demonstrated that non-coplanar PCBs are more potent neurotoxic congeners than coplanar PCBs and the alteration of PKC activities by PCB exposure can be blocked with the treatment of chitosan. The results suggest a potential use of chitosan as a means of nutritional intervention to prevent the harmful effects of pollutant-derived diseases.

• Archives of Pharmacal Research
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• 제23권5호
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• pp.518-524
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• 2000

We examined the modulation of protein kinase C (PKC) subtypes during apoptosis induced by ginsenoside Rh2 (G-Rh2) in human neuroblastoma SK-N-Bl(2) and rat glioma C6Bu-1 cells. Apoptosis induced by C-Rh2 in both cell lines was confirmed, as indicated by DNA fragmentation and in situ strand breaks, and characteristic morphological changes. During apoptosis induced by G-Rh2 in SK-N-BE(2) cells, PKC subtypes $\alpha$, $\beta$ and $\gamma$ were progressively increased with prolonged treatment, whereas PKC $\delta$ increased transiently at 3 and 6 h and PKC $\varepsilon$ was gradually down-regulated after 6 h following the treatment. On the other hand, PKC subtype $\beta$ markedly increased at 24 h when maximal apoptosis was achieved. In C6Bu-l cells, no significant changes in PKC subtypes $\alpha$, $\gamma$, $\delta$, $\varepsilon$ and $\beta$ were observed during apoptosis induced by G-Rh2. These results suggest the evidence for a possible role of PKC subtype in apoptosis induced by G-Rh2 in SK-N-BE(2) cells but not in C6Bu-1 cells, and raise the possibility that G-Rh2 may induce apoptosis via different pathways interacting with or without PKC in different cell types.