The Effects of Baicalein on Osteoclast Differentiation from Bone Marrow Derived Macrophage / 체질인류학회지

As prediction of rapidly aging society, bone health is considered increasingly important and received more attention than ever. Bone health is regulated by balancing between bone resorptive osteoclasts and bone formative osteoblasts. Disruption of balance between bone-resorbing osteoclasts and bone-forming osteoblasts results in bone disease. Natural products have recently received much attention as an alternative tool for the development of novel therapeutic strategy. Baicalein is reported it has anti-cancer, anti-inflammatory and antioxidant effects. Baicalein also has been known that it has both promotive effect on MC3T3-E1 cell line and inhibitory effect on RAW 264.7 cell line. However, the inhibitory mechanism of baicalein using bone marrow derived macrophages (BMMs) on osteoclast differentiation remains not clear. In this study, the suppressive mechanism by baicalein on osteoblast differentiation was evaluated. Bicalein inhibited receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclast differentiation in BMMs in a dose dependent manner without any toxicity. Baicalein suppressed phosphorylation of protein kinaseB (Akt), c-Jun N-terminal kinases (JNK) and phosphoinositide-specific phospholipaseCgamma2 (PLCgamma2). Furthermore, Baicalein suppressed the induction of RANKL-induced c-Fos and Nuclear factor of activated T cell c1 (NFATc1), essential genes on osteoclastogenesis. In BMMs, Bicalein inhibited the mRNA expression of tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), cathepsinK, dendritic cell-specific transmembrane protein (DC-STAMP). Moreover, baicalein promoted differentiation of osteoblast on bone marrow stromal cells (BMSCs). Taken together, these results suggest that baicalein has a potential for treating bone lytic diseases, such as osteoporosis, periodontitis, and rheumatoid arthritis.

Preeclampsia serum-induced collagen I expression and intracellular calcium levels in arterial smooth muscle cells are mediated by the PLC-gamma1 pathway

In women with preeclampsia (PE), endothelial cell (EC) dysfunction can lead to altered secretion of paracrine factors that induce peripheral vasoconstriction and proteinuria. This study examined the hypothesis that PE sera may directly or indirectly, through human umbilical vein ECs (HUVECs), stimulate phospholipase C-gamma1-1,4,5-trisphosphate (PLC-gamma1-IP3) signaling, thereby increasing protein kinase C-alpha (PKC-alpha) activity, collagen I expression and intracellular Ca2+ concentrations ([Ca2+]i) in human umbilical artery smooth muscle cells (HUASMCs). HUASMCs and HUVECs were cocultured with normal or PE sera before PLC-gamma1 silencing. Increased PLC-gamma1 and IP3 receptor (IP3R) phosphorylation was observed in cocultured HUASMCs stimulated with PE sera (P<0.05). In addition, PE serum significantly increased HUASMC viability and reduced their apoptosis (P<0.05); these effects were abrogated with PLC-gamma1 silencing. Compared with normal sera, PE sera increased [Ca2+]i in cocultured HUASMCs (P<0.05), which was inhibited by PLC-gamma1 and IP3R silencing. Finally, PE sera-induced PKC-alpha activity and collagen I expression was inhibited by PLC-gamma1 small interfering RNA (siRNA) (P<0.05). These results suggest that vasoactive substances in the PE serum may induce deposition in the extracellular matrix through the activation of PLC-gamma1, which may in turn result in thickening and hardening of the placental vascular wall, placental blood supply shortage, fetal hypoxia-ischemia and intrauterine growth retardation or intrauterine fetal death. PE sera increased [Ca2+]i and induced PKC-alpha activation and collagen I expression in cocultured HUASMCs via the PLC-gamma1 pathway.

Changes in expression of phospholipase C-gamma1(tyr783) in young rat condylar cartilage during functional mandibular protraction / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the changes in the expression of phospholipase C-gamma1tyr783 (PLC-γ1tyr783) in the condylar cartilage of a young rat during functional mandibular protraction. This work also explores the function of PLC-γ1tyr783 in the rat mandibular condylar cartilage bone remodeling, which could provide experimental evidence for clinical bone ortho- pedic work.</p><p><b>METHODS</b>A total of 60 four-week-old male Sprague-Dawley (SD) rats were used in this study. The rats were divided equally and randomly into experimental group and control group. The functional appliances that were fitted to the upper incisors of the animals in the experimental group were worn 24 h a day after the rats were fed for 7 d with homemade pellet feed. The animals in the experimental group, along with their matched controls, were sacrificed after 1, 3, 7, 14, 21, and 28 d. The bilateral condylar was fixed, decalcified, dehyded, and then conventional paraffin embedded. Immunohisto- chemistry of PLC-γ1tyr783 was applied to observe its express distribution and variation.</p><p><b>RESULTS</b>The expression of PLC-γ1tyr783 decreased gradually in the control group, which showed age-related changes (P > 0.05). On the 14th day, PLC-γ1tyr783 expres- sion in the experimental group was significantly higher than that in the control group. PLC-γ1tyr783 expression began to appear statistically and significantly different between the two groups (P < 0.01).</p><p><b>CONCLUSION</b>PLC-γ1tyr783 is involved in the bone remodeling process of the rat condylar cartilage after functional mandibular-protraction.</p>

The role and clinical significance of 12-LOX passway in arachidonic acid metabolism induced by phospholipase Cgamma-2 in laryngeal squamous cell carcinoma / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To explore the expression of phospholipase Cgamma-2 (PLCgamma-2), lipoxygenase-12 (12-LOX) and arachidonic acid (AA) in laryngeal squamous cell carcinoma and to study the the relationship between lipid metabolism and laryngeal squamous cell carcinoma.@*METHOD@#In 30 cases of carcinoma tissue and peritumoral laryngeal mucosa tissues (confirmed to be normal laryngeal tissues by pathology), immunohistochemical method (Streptavidin-peroxidase method, SP method) was used for the detection of expression of PLCgamma-2 and 12-LOX, and gas chromatography/mass spectrometry (GC/MS) for the content of the arachidonic acid in carcinoma tissue and peritumoral normal laryngeal mucosa tissues.@*RESULT@#The positive rates of PLCgamma-2 and 12-LOX in carcinoma tissue were higher than in peritumoral normal laryngeal mucosa tissues with statistically significance differences (P 0.05). Both the expression of PLCgamma-2 and 12-LOX and the content of arachidonic acid had no statistically significant correlation with age (P > 0.05).@*CONCLUSION@#PLCgamma-2, AA and 12-LOX play important roles in laryngeal squamous cell carcinoma. It may be meaningful to the treatment of laryngeal carcinoma by suppressing this passway.

Effects of theanine on monoamine neurotransmitters and related genes in cerebral ischemia-reperfusion injury rats / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the effects of theanine on dopamine (DA), 5-hydroxy tryptamine (5-TH) and glutamate receptor 2 (GluR2) mRNA, phospholipase-γ1 (PLC-γ1) mRNA in cerebral ischemia-reperfusion injury rats and explore the mechanism of protective effects of theanine on the induced brain injury by ischemia-reperfusion in rats.</p><p><b>METHODS</b>According to random number table, a total of 56 sprague-dawley rats in SPF grade about six-week old and 100 - 120 grams weighting were divided into five groups according to the body weight levels: model group (n = 12), sham-operation group (n = 8), low theanine group (10 mg/kg), middle theanine group (30 mg/kg) and high theanine group (90 mg/kg). There were 12 rats in each of the theanine group. The rats in model group and sham-operation groups were given distilled water, and the rats in theanine groups were given corresponding theanine solution intragastrically for fifteen days. Then the cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion (MCAO). The score of neurological behavior was evaluated at the 3rd and 24th hours after reperfusion. Rats were sacrificed at 24 hours after reperfusion, the concentrations of DA, 5-HT and theanine in rats brain following ischemia-reperfusion were determined. At the same time, we determined the levels of reactive oxygen species (ROS) and activities of catalase (CAT) in mitochondria of brain. The expressions of GluR2 mRNA and PLC-γ1 mRNA in rat brain were examined by reverse transcription polymerase chain reaction (RT-PCR) technique.</p><p><b>RESULTS</b>The score of neurological behavior of rats in model group, theanine-low, middle, high dose groups at the 3rd hour was 6.000 ± 0.926, 4.100 ± 0.738, 3.444 ± 0.726 and 2.250 ± 0.886 respectively (F = 29.70, P < 0.01), and the score at the 24th hour in these groups was 6.625 ± 0.916, 5.000 ± 0.817, 3.667 ± 0.707 and 2.625 ± 0.916 respectively(F = 34.68, P < 0.01). The concentration of DA in model group, theanine-low, middle, high dose groups and sham-operation group was (10.26 ± 1.12), (12.48 ± 1.09), (14.55 ± 0.94), (15.97 ± 0.92) and (11.98 ± 0.63) µg/g respectively (F = 43.76, P < 0.01). The concentration of 5-HT in these groups was (1.091 ± 0.160), (0.818 ± 0.101), (0.571 ± 0.050), (0.453 ± 0.111) and (0.863 ± 0.063) µg/g respectively (F = 48.68, P < 0.01). The level of ROS was (3.072 ± 0.503), (1.331 ± 0.268), (1.295 ± 0.061), (0.804 ± 0.200) and (2.158 ± 0.218) U×min⁻¹×mg⁻¹ (F = 80.82, P < 0.01) respectively and the activities of CAT in these groups were (4.880 ± 1.121), (8.405 ± 1.356), (9.535 ± 2.511), (15.090 ± 4.054) and (21.260 ± 6.054) U/g respectively (F = 28.58, P < 0.01). The expressions of GluR2 mRNA were 0.842 ± 0.020, 1.063 ± 0.100, 1.170 ± 0.152, 1.254 ± 0.131 and 1.012 ± 0.056 respectively (F = 9.23, P < 0.01). The expressions of PLC-γ1 mRNA in these groups were 0.737 ± 0.090, 0.887 ± 0.045, 0.963 ± 0.025, 0.991 ± 0.049 and 0.867 ± 0.079 respectively(F = 10.24, P < 0.01).</p><p><b>CONCLUSION</b>Theanine has a protective effect on the induced brain injury by ischemia-reperfusion in rats, which might be associated with its interaction with monoamine neurotransmitters and up-regulating the expressions of GluR2 mRNA and PLC-γ1 mRNA.</p>

Periodic mechanical stress activates MEK1/2-ERK1/2 mitogenic signals in rat chondrocytes through Src and PLCγ1

The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively but the mechanisms whereby chondrocytes sense and respond to periodic mechanical stress remain a matter of debate. We explored the signal transduction pathways of chondrocyte proliferation and matrix synthesis under periodic mechanical stress. In particular, we sought to identify the role of the MEK1/2-ERK1/2 signaling pathway in chondrocyte proliferation and matrix synthesis following cyclic physiologic mechanical compression. Under periodic mechanical stress, both rat chondrocyte proliferation and matrix synthesis were significantly increased (P < 0.05) and were associated with increases in the phosphorylation of Src, PLCγ1, MEK1/2, and ERK1/2 (P < 0.05). Pretreatment with the MEK1/2-ERK1/2 selective inhibitor, PD98059, and shRNA targeted to ERK1/2 reduced periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis (P < 0.05), while the phosphorylation levels of Src-Tyr418 and PLCγ1-Tyr783 were not inhibited. Proliferation, matrix synthesis and phosphorylation of MEK1/2-Ser217/221 and ERK1/2-Thr202/Tyr204 were inhibited after pretreatment with the PLCγ1 inhibitor U73122 in chondrocytes in response to periodic mechanical stress (P < 0.05), while the phosphorylation site of Src-Tyr418 was not affected. Inhibition of Src activity with PP2 and shRNA targeted to Src abrogated chondrocyte proliferation and matrix synthesis (P < 0.05) and attenuated PLCγ1, MEK1/2 and ERK1/2 activation in chondrocytes subjected to periodic mechanical stress (P < 0.05). These findings suggest that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis in part through the Src-PLCγ1-MEK1/2-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade.

Phosphatidylinositol phosphates directly bind to neurofilament light chain (NF-L) for the regulation of NF-L self assembly

Phosphatidylinositol phosphates (PtdInsPs) are ubiquitous membrane phospholipids that play diverse roles in cell growth and differentiation. To clarify the regulation mechanism acting on neurofilament light chain (NF-L) self assembly, we examined the effects of various PtdInsPs on this process. We found that PtdInsPs, including PI(4,5)P2, directly bind to the positively charged Arg54 of murine NF-L, and this binding promotes NF-L self assembly in vitro. Mutant NF-L (R53A/R54A) proteins lacking binding affinity to PtdInsPs did not have the same effect, but the mutant NF-L proteins showed greater self assembly than the wild-type in the absence of any PtdInsP. These results collectively suggest that Arg54 plays a pivotal role in NF-L self assembly by binding with PtdInsPs.

CD40 Co-stimulation Inhibits Sustained BCR-induced Ca2+ Signaling in Response to Long-term Antigenic Stimulation of Immature B Cells

Regulation of B cell receptor (BCR)-induced Ca2+ signaling by CD40 co-stimulation was compared in long-term BCR-stimulated immature (WEHI-231) and mature (Bal-17) B cells. In response to long-term pre-stimulation of immature WEHI-231 cells to alpha-IgM antibody (0.5~48 hr), the initial transient decrease in BCR-induced [Ca2+]i was followed by spontaneous recovery to control level within 24 hr. The recovery of Ca2+ signaling in WEHI-231 cells was not due to restoration of internalized receptor but instead to an increase in the levels of PLCgamma2 and IP3R-3. CD40 co-stimulation of WEHI-231 cells prevented BCR-induced cell cycle arrest and apoptosis, and it strongly inhibited the recovery of BCR-induced Ca2+ signaling. CD40 co-stimulation also enhanced BCR internalization and reduced expression of PLCgamma2 and IP3R-3. Pre-treatment of WEHI-231 cells with the antioxidant N-acetyl-L-cysteine (NAC) strongly inhibited CD40-mediated prevention of the recovery of Ca2+ signaling. In contrast to immature WEHI-231 cells, identical long-term alpha-IgM pre-stimulation of mature Bal-17 cells abolished the increase in BCR-induced [Ca2+]i, regardless of CD40 co-stimulation. These results suggest that CD40-mediated signaling prevents antigen-induced cell cycle arrest and apoptosis of immature B cells through inhibition of sustained BCR-induced Ca2+ signaling.

A double point mutation in PCL-gamma1 (Y509A/F510A) enhances Y783 phosphorylation and inositol phospholipid-hydrolyzing activity upon EGF stimulation

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-gamma1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-gamma1, abolished interactions with translational elongation factor 1-alpha. Here, we report that the Y509A/F510A mutant PLC-gamma1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-gamma1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-gamma1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-gamma1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-gamma1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-gamma1 activation in vivo.

VEGF165-induced angiogenesis by regulating intracellular free Mg2+ in HUVECs / 中国应用生理学杂志

<p><b>AIM</b>The mechanism of vascular endothelial growth factor165 (VEGF165) on intracellular free magnesium ([Mg2+]i) in human umbilical vein endothelial cells (HUVECs) was investigated.</p><p><b>METHODS</b>[Mg2+]i in HUVECs loaded with fluorescent magnesium indicator mag-fura-2 were quantitatively detected the use of intracellular cation measurement system.</p><p><b>RESULTS</b>VEGF165 significantly increased [Mg2+]i in the extracellular Mg2+ and this effect could be blocked by pretreatment with tyrosine kinase inhibitors (tyrphostin A23 and genistein), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY294002) and phospholipase Cgamma (PLCgamma) inhibitor (U73122). In contrast, phospholipase Cgamma (PLCgamma) inhibitor analog (U73343), mitogen-activated protein kinase inhibitors (SB202190 and PD98059) had no effect on the VEGF165-induced [Mg2+]i increase.</p><p><b>CONCLUSION</b>The increase of [Mg2+]i by VEGF165 originates from intracellular Mg2+ pool through tyrosine kinase/ PI3K/PLCgamma-dependent signaling pathways.</p>

Polymorphisms in RAS Guanyl-releasing Protein 3 are Associated with Chronic Liver Disease and Hepatocellular Carcinoma in a Korean Population

RAS guanyl-releasing protein 3 (RasGRP3), a member of the Ras subfamily of GTPases, functions as a guanosine triphosphate (GTP)/guanosine diphosphate (GDP)-regulated switch that cycles between inactive GDP- and active GTP-bound states during signal transduction. Various growth factors enhance hepatocellular carcinoma (HCC) proliferation via activation of the Ras/Raf-1/ extracellular signal-regulated kinase (ERK) pathway, which depends on RasGRP3 activation. We investigated the relationship between polymorphisms in RasGRP3 and progression of hepatitis B virus (HBV)-infected HCC in a Korean population. Nineteen RasGRP3 SNPs were genotyped in 206 patients with chronic liver disease (CLD) and 86 patients with HCC. Our results revealed that the T allele of the rs7597095 SNP and the C allele of the rs7592762 SNP increased susceptibility to HCC (OR=1.55, p=0.04 and OR=1.81~2.61, p=0.01~0.03, respectively). Moreover, patients who possessed the haplotype (ht) 1 ( A-T-C-G) or diplotype (dt) 1 ( ht1/ht1) variations had increased susceptibility to HCC (OR=1.79 ~2.78, p=0.01~0.03). In addition, we identified an association between haplotype1 (ht1) and the age of HCC onset; the age of HCC onset are earlier in ht1 +/+ than ht1 +/- or ht1 -/- (HR=0.42~0.66, p=0.006~0.015). Thus, our data suggest that RasGRP3 SNPs are significantly associated with an increased risk of developing HCC.

Effects of lipid rafts on signal transmembrane transduction mediated by c-Met / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effects of lipid rafts on cell signal transmembrane transduction mediated by c-Met.</p><p><b>METHODS</b>After HepG2Cells were treated with MbCD to disrupt the lipid rafts and were treated with artificial recombination hepatocyte growth factor to activate c-Met, the activities of PLCr1/PKC, PI3K/Akt and MAPK signaling pathways in HepG2 cells were analyzed using Western blot.</p><p><b>RESULTS</b>(1) After disruption of lipid rafts with MbCD, phosphorylation of PLCr1 decreased by 35% (P = 0.022); the content of PLCr in the cytoplasm increased by 1.75 fold (P = 0.017); PLCr1 conjugated with membrane decreased by 30% (P = 0.037). (2) The content of PKB in the cytosol decreased by 38% (P = 0.028), and the phosphorylation level of PKB conjugated with membrane decreased by 14% (P = 0.041). At the same time, PDK translocation from cytosol to the plasma membrane and its activation were inhibited by treatment with MbCD. (3) Treatment with MbCD had no significant effect on ErK/MAPK, p38/MAPK and JNK/MAPK signaling pathways.</p><p><b>CONCLUSION</b>Disruption of lipid rafts with MbCD inhibits the activation of PLCr1/PKC and PI3K/PKB signaling pathways by HGF/cMet, but has no effect on MAPK signaling pathway.</p>

Role of phospholipase C-gamma1 signaling pathway in H(2)O(2)-induced apoptosis of PC12 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of phospholipase C-gamma1 (PLC-gamma1) signaling pathway in H(2)O(2)-induced apoptosis of PC12 cells.</p><p><b>METHODS</b>PC12 cells were exposed to 50 micromol/L H(2)O(2) after pretreatment with 10 micromol/L U73122, a specific PLC-gamma1 inhibitor. Hoechst/PI double staining was performed to observe the morphological changes of the cells under light microscope. MTT assay was used to evaluate the cell viability, and the percentage of apoptotic cells was analyzed by flow cytometry. DNA fragmentation assay was carried out to characterize the cell apoptosis.</p><p><b>RESULTS</b>After inhibition of the PLC-gamma1 signaling pathway with 10 micromol/L U73122, PC12 cells showed obvious apoptotic morphology, the viable cells decreased significantly, and the percentage of apoptotic cells rose to 35.7%. PC12 cells treated with U73122 presented with a distinct DNA ladder on electrophoresis resulting from DNA cleavage in the apoptotic cells.</p><p><b>CONCLUSION</b>PLC-gamma1 signaling pathway plays an important protective role in H(2)O(2)-induced PC12 cell apoptosis.</p>

Antitumour effects on human colorectal carcinomas cells by stable silencing of phospholipase C-gamma 1 with lentivirus-delivered siRNA / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>In most colorectal carcinomas, the level of phospholipase C (PLC)-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells.</p><p><b>METHODS</b>Recombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed.</p><p><b>RESULTS</b>Stable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined. Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis (30%-40%) of LoVo cells.</p><p><b>CONCLUSIONS</b>PLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas.</p>

Exploration of the alternative splicing variants of rat phospholipase C-gamma 1 pre-mRNA / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the expression of phospholipase C-gamma 1 (PLC-gamma1) alternative splicing variants in rats.</p><p><b>METHODS</b>According to the sequence of human PLCG1 splicing variant, specific primers for rat PLC-gamma1 were designed and synthesized. The rat RNA was reverse transcribed into cDNA, which was amplified using the specific primers, and the PCR products were sequenced and analyzed using BLAST and bioinformatics methods. Totally 21 rat tissue samples were examined, including the heart, liver, lung, kidney, eyeball, and brain obtained in 3 different embryonic stages, 7 different early postnatal stages, and in adulthood.</p><p><b>RESULTS</b>The result did not show that rat PLC-gamma1 had the same splicing variant (PLC-gamma1a, NM_002660) as human does.</p><p><b>CONCLUSIONS</b>The same splicing variant of PLC-gamma1 detectable in human may not exist in rats, and the pre-mRNA may undergo splicing resulting predominantly in PLC-gamma1b mRNA. Very likely, the alternative splicing site of rat PLC-gamma1 is not identical to that of human.</p>

Construction of lentivirus producing PLC-gamma1 siRNA and its effect on apoptosis of human colorectal carcinomas cell lines / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct recombinant lentivirus that stably suppresses phospholipase C (PLC) gamma1 expression in human colorectal carcinoma LoVo cells to obtain LoVo cell lines deficient in PLC gamma1 for investigation of the role of PLC gamma1 gene.</p><p><b>METHODS</b>Recombinant lentivirus producing PLC gamma1 siRNA were constructed to infect LoVo cells, and the stably transduced cells were selected with blasticidin. The protein and mRNA expression of PLC gamma1 was examined by Western blotting and RT-PCR, and the effect of the lentivirus on cell apoptosis was analyzed by flow cytometry.</p><p><b>RESULTS AND CONCLUSION</b>PLC gamma1 siRNA significantly suppressed PLC gamma1 expression in LoVo cells, suggesting high efficiency of gene silencing induced by the siRNA produced by the recombinant lentivirus. Concomitantly, cell apoptosis induced by 5-FU was significantly increased.</p>

Pleckstrin homology domain of phospholipase C-gamma1 directly binds to 68-kDa neurofilament light chain

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.

Overexpression of PEMT2 inhibits the phosphorylation and translocation of PLC gamma 1 / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To explore the mechanism of CBRH-7919 cell proliferation inhibition by transfecting phosphatidylethanolamine N-methyltransferase 2 gene (PEMT2).</p><p><b>METHODS</b>The effects of PEMT2 transfection on phosphorylation and translocation from cytosol to plasma membrane of PLC gamma 1 in cells were studied using SDS-PAGE and Western blot techniques. The phosphorylation and activity of c-Met were determined.</p><p><b>RESULTS</b>After transfection of pemt2, the PLC gamma 1 and phosphorylated PLC gamma 1 conjugated with plasma membrane were decreased by 45% and 27% of that of control cells respectively, and the phosphorylated c-Met was decreased to 32% of that of control cells.</p><p><b>CONCLUSION</b>Transfection of phosphatidylethanolamine N-methyltransferase 2 gene can inhibit the phosphorylation and translocation from cytosol to plasma membrane of PLC gamma 1 in cells. At the same time, the autophosphorylation of c-Met was decreased, which suggests that transfection of phosphatidylethanolamine N-methyltransferase 2 gene can downregulate the c-Met/PLC gamma 1 signaling pathway in CBRH-7919 cells.</p>

Immunocytochemical study of phospholipase C-gamma1 expression in mouse embryonic tissue / 南方医科大学学报

To investigate the expression of phospholipase C-gamma1 (PLC-gamma1) in mouse embryonic tissues, serial tissue sections were prepared routinely for immunocytochemistry for PLC-gamma1. The results showed that PLC-gamma1 was expressed in the cartilage, skeletal muscles, myocardium, the collecting tubule of the kidney, connective tissues and the brain, suggesting the important role PLC-gamma1 and the related signal pathway may play in the development of mouse embryonic tissues.

Sensitization of human glioma SWO cell line to tumor necrosis factor-induced apoptosis by blocking phospholipase C-gamma1 signaling pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of phospholipase C-gamma1 (PLC-gamma1) in tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis of human glioma SWO cells.</p><p><b>METHODS</b>The PLC-gamma1 pathway was blocked by U73122 in SWO cells, and the inhibitory effect of TNF-alpha on SWO glioma cell proliferation with or without U73122 treatment was investigated by MTT assay. The cell apoptosis induced by TNF-alpha along or in combination with U73122 was detected by flow cytometry with PI staining. The expression of caspase-3 and Bcl-2 was detected by Western blotting.</p><p><b>RESULTS AND CONCLUSION</b>U73122 can sensitize SWO glioma cells to TNF-alpha-induced apoptosis. Blocking the PLC-gamma1 pathway may not induce apoptosis of SWO glioma cells, but can sensitize SWO glioma cells to small-dose TNF-alpha-induced apoptosis, the mechanism of which may involve down-regulation of bcl-2.</p>