• BMB Reports
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• v.32 no.2
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• pp.119-126
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• 1999

The SH3 domain of PLC-${\gamma}1$ has been known to induce DNA synthesis. However, little is known about the putative effector proteins that associate with the domain. In this report, we provide evidence that the SH3 domain of PLC-${\gamma}1$ associates with Shc, which has been implicated in the activation of p21Ras in response to many growth factors. The association between Shc and PLC-${\gamma}1$ is enhanced either by v-Src-induced transformation or EGF-stimulation in vivo and in vitro. Furthermore, from transient expression studies with COS-7 cells, we show that the SH3 domain of PLC-${\gamma}1$ is required for association with Shc in vivo, whereas tyrosyl phosphorylation of PLC-${\gamma}1$ is not. Taken together, we suggest that Shc might be involved in the PLC-${\gamma}1$-mediated signaling pathway.

• Journal of Yeungnam Medical Science
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• v.9 no.2
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• pp.288-301
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• 1992

The objective of the present study was to identify the characteristics of phospholipase C (PLC) isozymes purified from bovine brain and to investigate their interrelationship with G protein. The purified PLC isozymes ${\beta}$, ${\gamma}$ and ${\delta}$ were obtained and the characteristics of PLC activity on various concentrations of free $Ca^{2+}$ were observed. The activity of PLC was increased with increasing $Ca^{2+}$ concentration and the activity PLC ${\delta}$ was increased higher in the presence of phosphatidyl choline(PC) than in the abscence of PC. For vesicle formation as the structure of cell membrane, cholic acid and deoxycholic acid as detergent on phosphatidylinositol bisphosphate($PIP_2$) substrate containing PC were used, and then the activity of PLC isozymes were increased with increasing concentration of cholate, from 0.2% to 1% and were increased slightly in deoxycholate. In the $PIP_2$ containing phospholipid and glycolipid as brain extract, the activity of PLC isozymes were checked in 0.2%-1% cholic acid. The activities of PLC isoyzmes were continuously increased up to 1% cholic acid. The quantitation of PLC isozymes from several bovine organs by radioimmunoassay was made. Brain was the most sufficient organ in terms of amount of PLC ${\beta}$and ${\delta}$. A large amount of PLC ${\delta}$ was existed in adrenal gland. The binding capacity of GTPrS and G protein was observed and other observations of the binding effect of GTPrS-G protein and PLC monoclonal Ab-Protein A from tissue homogenate with PLC were made. From the observation the binding capacity was revealed the range of 0.11%-1.49%. The effects of each type of G protein on the percent activity of purified PLC isozymes were observed. From the observation, activities of isozymes were increased in $Go{\alpha}$ & Gmix, and the activities of PLC ${\beta}$ and ${\delta}$ were increased in $G{\beta}{\gamma}$ and $Gi{\alpha}$. Activities of PLC ${\beta}$ and ${\gamma}$ were decreased in $Gt{\alpha}$ but PLC ${\delta}$ increased.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.186-186
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• 1994

Many agonists have been known to activate the hydrolysis of membrane phospholipids through the bindings with corresponding receptors on the various cells. Diacylglycerol and inositol 1,4,5-trisphosphate(IP3) generated by the action of phosphoinositide-specific phospholipase C (PI-PLC) are well known second messengers for the activation of protein kinase C and the mobilization of Ca2+ in many cells. Three types of PI-PLC isozyme (${\alpha}$,${\gamma}$, and $\delta$) and several subtrpes for each type have been identified from mammalian sources by purification of enzymes and cloning of their cDNAs. Each type PI-PLC isozyme is coupled to different receptors and mediators, for example, ${\beta}$-types are coupled to the seven-transmembrane-receptors via Gq family of G-proteins and ${\beta}$-types directly to the receptor tyrosine kinases. Specific modulators for the signaling pathway through each type of PI-PLC should be very useful as potential potential candidates for lend substances in developing novel drugs. To establish the sensitive and convenient screening systems for searching modulators on PI-PLC mediated signaling, two kinds of approaches have been tried. (1) Establishment of in vitro assay condition for each type of PI-PLC isozyme: Overexpression by using vaccinia virus and purification of each isozyme was carried out for the preparation of large amounts of enaymes. Optimum and sensitive assay condition for the measurements of PI-ELC activities were established. (2) Development of the cell lines in which each type of PI-PLC is permanently overexpressed: A fibroblast cell line (3T3${\gamma}$1-7) in which PI-PLC-${\gamma}$1 was overexpressed by using pZip-neo expression vector was developed and used for the measurement of PDGF-induced IP3 formation. The responses for IP3 formed in 3T3${\gamma}$1-7 cells by the treatment of PDGF is 8 times more sensitive than those in control cells. 3T3${\gamma}$l-7 cell is useful for the screening of the inhibitors on the PDGF-induced cellular responses from large number of samples in a small volume(50 ${\mu}$l) and short time(5-15 min). Using these systems, we screened hundreds of herb-extracts for the inhibition of PDGF-induced IP3 formation and selected several extracts that showed the inhibition as the candidates for isolation and characterization of active substances. The determination of the acting point of selected extracts or fractions in the PDGF signaling pathway has been analyzing.

• Radiation Oncology Journal
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• v.12 no.3
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• pp.271-284
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• 1994

Purpose : Phospholipase C(PLC) isozymes play significant roles in transmembrane signal transduction. PLC-${\gamma}1$ acts as the intracellular effector in signal transduction for cellular proliferation and differentiation. Ras oncoprotein is also involved in cell growth. We determined the biological significance of PLC and ras oncoprotein in regeneration following radiation and the effect of different modes of administration of 5-FU. Materials and Methods : To determine the effect of the administration mode of 5-FU on the regeneration of intestinal mucosa of rats following radiation, we compared the expression of PLC and ras oncoprotein in six groups. Group I had no treatment. Group II received radiation(8 Gy) only. Group III received radiation(8 Gy) and 5-FU(150mg/kg) continuous intravenous (iv) infusion for 12 hours. Group IV received radiation(8 Gy) and 5-FU(750mg/kg) iv bolus injection. Group V received only 5-FU(150mg/kg) continuous iv infusion for 12 hours, Group VI received only 5-FU (150mg/kg) iv bolus injection. Through immunoblotting and immunohistochemistry, we examined the expression of PLC and ras oncoprotein in rat jejunum at 96 hours after radiation or 5-FU administration and at 120 hours after radiation and 5-FU adminstration. We also investigated the histological findings using hematoxylin and eosin stain. Results : In the immunohistochemistry study, PLC-${\gamma}1$ expression was the highest in group III followed by groups II and VI in that order and was weakly positive in groups V and VI. PLC-${\gamma}1$ was hardly detected in the control group. The expression of ras oncoprotein was the same as the PLC-${\gamma}1$ expression for all groups. These results were confirmed by the histological findings regarding the mucosal regeneration. In the immunoblotting analysis, PLC-${\gamma}1$ expression was the highest in group III followed by group IV and II in that order. This difference between the immunoblotting and immunohistochemistry study was due to the high expression of PLC-${\gamma}1$ on the damaged surface epithelium rather than to its expression in the regeneration region as observed in the immunohistochemistry study for group IV. The expression of PLC-${\delta}1$ was positive only in group V and VI, which received both radiation and 5-FU, and the expression of PLC-${\beta}1$ was negligible for all groups. Conclusion : These results suggest that PLC-${\gamma}1$ mediated signal transduetion and ras oncoprotein may have a significant role in mucosal regeneration after radiation, and that continuous iv infusion of 5-FU may induce active regeneration in intestinal mucosa following radiation. In addition, the expression of PLC-${\delta}1$ in combined group of radiation and 5-FU implies that PLC-${\delta}1$ may be involved in signal transduction mediated by concerted action between radiation and 5-FU.

• BMB Reports
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• v.41 no.6
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• pp.415-434
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• 2008

Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-$\beta$, -$\gamma$, -$\delta$, -$\varepsilon$, -$\zeta$ and -$\eta$. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.

• International Journal of Oral Biology
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• v.45 no.1
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• pp.15-24
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• 2020

The fruit of Chaenomeles sinensis (Thouin) Koehne (Chaenomelis Fructus) known as "Mo-Gua" in Korea has been commonly used in traditional medicine to treat inflammatory diseases, such as sore throat. However, its effect on bone metabolism has not been elucidated yet. Here, we examined the effect of Chaenomelis Fructus ethanol extract (CF-E) on receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated osteoclast differentiation and formation. CF-E considerably inhibited osteoclast differentiation and tartrate-resistant acid phosphatase-positive multinuclear cell formation from bone marrow-derived macrophages and osteoclast precursor cells in a dose-dependent manner. In addition, the formation of actin rings and resorption pits were significantly suppressed in CF-E-treated osteoclasts as compared with the findings in non-treated control cells. Consistent with these phenotypic inhibitory results, the expressions of osteoclast differentiation marker genes (Acp5, Atp6v0d2, Oscar, CtsK, and Tm7sf4) and Nfatc1, a pivotal transcription factor for osteoclastogenesis, were markedly decreased by CF-E treatment. The inhibitory effect of CF-E on RANKL-induced osteoclastogenesis was associated with the suppression of NFATc1 expression, not by regulation of mitogen-activated protein kinases and NF-κB activation but by the inactivation of phospholipase C gamma 1 and 2. These results indicate that CF-E has an inhibitory effect on osteoclast differentiation and formation, and they suggest the possibility of CF-E as a traditional therapeutic agent against bone-resorptive diseases, such as osteoporosis, rheumatoid arthritis, and periodontitis.

• BMB Reports
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• v.51 no.5
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• pp.230-235
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• 2018

Bone resorption by multinucleated osteoclasts is a multistep process involving adhesion to the bone matrix, migration to resorption sites, and formation of sealing zones and ruffled borders. Macrophage colony-stimulating factor (M-CSF) and osteopontin (OPN) have been shown to be involved in the bone resorption process by respective activation of integrin ${\alpha}v{\beta}3$ via "inside-out" and "outside-in" signaling. In this study, we investigated the link between signal modulators known to M-CSF- and OPN-induced osteoclast adhesion and spreading. M-CSF- and OPN-induced osteoclast adhesion was achieved via activation of stepwise signals, including integrin ${\alpha}v{\beta}3$, $PLC{\gamma}$, $PKC{\delta}$, and Rac1. Osteoclast spreading induced by M-CSF and OPN was shown to be controlled via sequential activation, consistent with the osteoclast adhesion processes. In contrast to osteoclast adhesion, osteoclast spreading induced by M-CSF and OPN was blocked via activation of $PLC{\gamma}/PKC{\alpha}/RhoA$ signaling. The combined results indicate that osteoclast adhesion and spreading are selectively regulated via $PLC{\gamma}/PKC{\alpha}-PKC{\delta}/RhoA-Rac1$ signaling.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.11a
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• pp.75-84
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• 1996

Platelets from a patient with a mild inherited bleeding disorder and abnormal platelet aggregation and secretion show reduced generation of inositol 1,4,5-trisphosphate (IP$_3$), mobilization of intracellular Ca$\^$2+/, and phosphorylation of pleckstrin in response to several G protein mediated agonists, suggesting a possible defect at the level of phospholipase C (PLC) activation. A procedure was developed that allows quantitation of platelet PLC isozymes. After fractionation of platelet extracts by high-performance liquid chromatography, seven, out often known PLC isoforms were detected by immunoblot analysis. The amount of these isoforms in normal platelets decreased in the order PLC-${\gamma}$2 > PLC-${\beta}$2 > PLC-${\beta}$3 > PLC-${\beta}$l > PLC-${\gamma}$ > PLC-$\delta$1 > PLC-${\beta}$4. Compared with normal platelets, platelets from the patient contained approximately one-third the amount of PLC-${\beta}$2, whereas PLC-${\beta}$4 was increased threefold. These results suggest that the impaired platelet function in the patient in response to multiple G protein mediated agonists is attributable to a deficiency of PLC-${\beta}$2. They document for the first time a specific PLC isozyme deficiency in human platelets and provide an unique opportunity to understand the role of different PLC isozymes in normal platelet function.

• Proceedings of the PSK Conference
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• 2003.10b
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• pp.93.3-94
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• 2003

The signaling components of high affinity IgE receptor (Fc RI) were searched by yeast-hybrid screening of the cDNA library constructed from RBL-2H3 cells. The cytoplasmic part of the Fc RI- chain was found to specifically interact with PLC 2, and further comparatives studies were conducted focusing on the differential regulation of two PLC- isoforms through Fc RI. The inhibitors of Src, Syk, and protein kinase C similarly affected the tyrosine phosporylations of PLC 1 and PLC 2 but the inhibitors of PI3-kinase and p42/44 ERK effectively inhibited the activation of PLC 1 but not PLC 2. (omitted)

• Journal of the Korea Academia-Industrial cooperation Society
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• v.20 no.12
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• pp.298-305
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• 2019

Cudrania tricuspidata has been reported to have many biological activities, including anti-inflammatory, anti-cancer, and antioxidant properties. However, the effects of C. tricuspidata root extract (CTE) on human platelet aggregation induced by collagen as well as the signaling pathways involved remain unknown. In the present study, we investigated the effect of CTE on human platelets. CTE inhibited platelet aggregation via down-regulation of thromboxane A₂ (TXA₂) by blocking cyclooxygenase-1 (COX-1) activity and intracellular Ca²⁺ mobilization in collagen-induced platelets. CTE also reduced the phosphorylation of phospholipase C (PLC) γ2 and syk. CTE regulated platelet aggregation via cyclic guanosine monophosphate (cGMP)-dependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP) Ser²³⁹. In addition, administration of CTE (50 and 100 mg/kg) significantly reduced hyper-aggregated platelet aggregation by collagen (5 ㎍/mL) without hepatotoxicity in HFD (high fat diet)-fed rats. Taken together, these results suggest that CTE has anti-platelet effects both in vitro and in vivo. Therefore, CTE may be an effective therapeutic and preventive agent for cardiovascular disease, and is a safe and natural product.