OBJECTIVETo study the effect of PC-1 gene knockdown on the biological action of prostate cancer cell line C4-2.

METHODSRecombinant plasmids of expressing short hairpin RNA targeting PC-1 mRNA were constructed using DNA recombinant technology and transfected into C4-2 cells via liposome. The positive cell clones were selected by G418. The expression of PC-1 gene was analyzed by RT-PCR and Western blotting technology. MTT and soft agar cloning formation were applied to observe the changes of the growth rate and independent anchor ability of C4-2 cells.

RESULTSPC-1 RNA interference severely affected the expression of PC-1 gene and reduced the growth and colony formation ability of C4-2 cells.

CONCLUSIONRNA interference-mediated PC-1 gene knockdown can decrease the growth and cloning formation ability of C4-2 cells.

Cell Line, Tumor ; Down-Regulation ; Gene Expression ; Humans ; Male ; Phosphoric Diester Hydrolases ; biosynthesis ; genetics ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Pyrophosphatases ; biosynthesis ; genetics ; RNA Interference ; RNA, Messenger ; genetics ; Transfection

BACKGROUNDEctonucleotide pyrophosphatase/phosphodiesterase (ENPP)-1 is a membrane-bound protein that catalyzes the hydrolysis of extracellular nucleoside triphosphates to monophosphate and extracellular inorganic pyrophosphate (ePPi). Mechanical stimulation regulates ENPP-1 expression. This study sought to investigate the changes in ENPP-1 expression after stimulation using cyclic mechanical tension (CMT).

METHODSRat end-plate chondrocytes were cultured and subjected to CMT (at 3%, 6%, and 9% elongation) for 20, 40, and 60 minutes to observe changes in the expression of ENPP-1. To investigate the pathway, end-plate chondrocytes were exposed to 10 ng/ml of transforming growth factor beta 1 (TGF-β1), TGF-β1 siRNA, or a specific extracellular signalregulated kinase (ERK)1/2 inhibitor, U0126, in addition to CMT. Changes in ENPP-1 expression were measured by reverse transcription PCR (RT-PCR) and Western blotting.

RESULTSWe observed the largest increase in ENPP-1 expression following 3% elongation CMT stimulation. ENPP-1 expression was also increased when end-plate chondrocytes were exposed to 10 ng/ml of TGF-β1, but decreased after TGF-β knockdown with siRNA. ERK1/2 phosphorylation was activated after 3% elongation for 40 minutes, and the stimulatory effect of TGF-β1 on ENPP-1 mRNA and protein expression was inhibited by the suppression of the ERK1/2 pathway using U0126.

CONCLUSIONCMT increases the expression of ENPP-1 in end-plate chondrocytes in a manner likely dependent on TGF-β induction by the ERK1/2 signaling pathway.

Animals ; Blotting, Western ; Cells, Cultured ; Chondrocytes ; metabolism ; Phosphoric Diester Hydrolases ; genetics ; metabolism ; Pyrophosphatases ; genetics ; metabolism ; RNA, Small Interfering ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Stress, Mechanical ; Transforming Growth Factor beta1 ; genetics ; metabolism

Ectonucleotide pyrophosphatase/phosphodiestrase 2 (Enpp2) isolated from the supernatant of human melanoma cells is a lysophospholipase D that transforms lysophosphatidylcholine into lysophospatidic acid. Although multiple analyses have investigated the function of Enpp2 in the hypothalamus, its role in the uterus during the estrous cycle is not well understood. In the present study, rat uterine Enpp2 was analyzed by RT-PCR, Western blotting, and immunohistochemistry. Quantitative PCR analysis demonstrated that uterine Enpp2 mRNA was decreased during estrus compared to proestrus and diestrus. To determine whether uterine Enpp2 expression is affected by sex steroid hormones, immature rats were treated with 17beta-estradiol (E2), progesterone, or both on postnatal days 14 to 16. Interestingly, the expression of Enpp2 mRNA and protein were down-regulated by E2 in the uterus during estrus but not during proestrus or diestrus, suggesting that Enpp2 may play a role in uterine function during estrus. Enpp2 is primarily localized in the stromal cells of the endometrium during proestrus and estrus. During diestrus, Enpp2 was highly expressed in the epithelial cells of the endometrium. Taken together, these results suggest that uterine Enpp2 may be regulated by E2 and plays a role in reproductive functions during female rat development.
Animals ; Estradiol/pharmacology ; Estrous Cycle/*physiology ; Female ; Gene Expression Regulation/*physiology ; Immunohistochemistry ; Mifepristone/pharmacology ; Phosphoric Diester Hydrolases/genetics/*metabolism ; Progesterone/pharmacology ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Uterus/*metabolism

OBJECTIVETo evaluate the effect of Xuefu Zhuyu Capsule ()-containing serum (XFZY-CS) on EphB4/ephrinB2 and its reverse signal in human microvascular endothelial cell-1 (HMEC-1).

METHODSXFZY-CS and the blank control serum were collected. HMEC-1 cells were randomly assigned to 6 groups including the concentration 1.25%, 2.5%, and 5% XFZY-CS groups and their blank serum control ones. The angiogenesis effect of XFZY-CS was tested with an in vitro tube formation assay and the best condition of pro-angiogenesis was determined. The effect of XFZY-CS on EphB4/ephrinB2 and the reverse signal were determined by Western blot and real-time quantitative polymerase chain reaction, respectively; we also confifirmed the results through activating and inhibiting the reverse signal by EphB4/fc and pyrophosphatase/ phosphodiesterase2 (PP2).

RESULTSXFZY-CS promoted angiogenesis at the concentration of 2.5% corresponding serum after being cultured for 48 h, while inhibited angiogenesis at the concentration of 5% after culturing for 48 and 72 h. Under the 2.5% serum concentration, XFZY up-regulated the expression of EphB4-mRNA at 12 h (P<0.05), and down-regulates its expression at 24 h (P<0.01). Protein expression of EphB4 was apparently up-regulated at 12 h and down-regulated at 24 h. The phosphorylation of ephrinB2 increased at 9 h (P<0.05). In addition, 2.5% XFZY-CS played a similar role as the reverse signaling activator EphB4/Fc ranging from 0.5 to 5 μg/mL (P>0.05). XFZY-CS also reduced the inhibitive effect of PP2 in limited periods.

CONCLUSIONSEphB4/ephrinB2 was the upstream signal in the process of angiogenesis and its reverse signaling was responsible for XFZY's effect on promoting angiogenesis.

Adult ; Capsules ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; Ephrin-B2 ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; Male ; Microvessels ; pathology ; Middle Aged ; Neovascularization, Physiologic ; drug effects ; genetics ; Phosphoric Diester Hydrolases ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptor, EphB4 ; genetics ; metabolism ; Serum ; metabolism ; Time Factors ; Young Adult

OBJECTIVETo investigate the molecular mechanism of human ether-a-go-go-related gene (HERG) potassium channels regulated by protein kinase A (PKA) in a human cell line.

METHODSHERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, and currents were measured with the patch clamp technique. The direct phosphorylation of HERG channel proteins expressed heterologously in Xenopus laevis oocytes was examined by (32)P labeling and immunoprecipitation with an anti-HERG antibody.

RESULTSElevation of the intracellular cAMP-concentration by incubation with the adenylate cyclase activator, forskolin (10 micromol/L), and the broad range phosphodiesterase inhibitor, IBMX (100 micromol/L), caused a HERG tail current reduction of 83.2%. In addition, direct application of the membrane permeable cAMP analog, 8-Br-cAMP (500 micromol/L), reduced the tail current amplitude by 29.3%. Intracellular application of the catalytic subunit of protein kinase A (200 U/ml) led to a tail current decrease by 56.9% and shifted the activation curve by 15.4 mV towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of approximately 155 kDa and a lower band with a molecular mass of approximately 135 kDa, indicating that both the core- and the fully glycosylated forms of the protein were phosphorylated.

CONCLUSIONSPKA-mediated phosphorylation of HERG channels causes current reduction in a human cell line. The coupling between the repolarizing cardiac HERG potassium current and the protein kinase A system could contribute to arrhythmogenesis under pathophysiological conditions.

1-Methyl-3-isobutylxanthine ; pharmacology ; 8-Bromo Cyclic Adenosine Monophosphate ; pharmacology ; Adenylyl Cyclases ; metabolism ; Animals ; Anti-Arrhythmia Agents ; pharmacology ; Cation Transport Proteins ; Cell Line ; Colforsin ; pharmacology ; Cyclic AMP ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; DNA-Binding Proteins ; ERG1 Potassium Channel ; Enzyme Activation ; drug effects ; Ether-A-Go-Go Potassium Channels ; Female ; Humans ; Membrane Potentials ; drug effects ; Microinjections ; Oocytes ; Patch-Clamp Techniques ; Phenethylamines ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; Phosphoric Diester Hydrolases ; drug effects ; metabolism ; Phosphorylation ; Potassium Channels ; genetics ; metabolism ; physiology ; Potassium Channels, Voltage-Gated ; RNA, Complementary ; administration & dosage ; genetics ; Sulfonamides ; pharmacology ; Trans-Activators ; Transcriptional Regulator ERG ; Xenopus laevis