Implication of Protein Kinase Cα in PAF-Stimulated Phospholipase D Activation in Chinese Hamster Ovary (CHO) Cells Expressing PAF Receptor

1995 ◽  
Vol 214 (2) ◽  
pp. 418-423 ◽  
Author(s):  
B. Liu ◽  
S. Nakashima ◽  
T. Takano ◽  
T. Shimizu ◽  
Y. Nozawa
Keyword(s):  
Protein Kinase ◽  
Phospholipase D ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Protein Kinase Cα ◽  
Paf Receptor

scholarly journals Prolonged activation of phospholipase D in Chinese hamster ovary cells expressing platelet-activating-factor receptor lacking cytoplasmic C-terminal tail

1997 ◽  
Vol 327 (1) ◽  
pp. 239-244 ◽  
Author(s):  
Bo LIU ◽  
Shigeru NAKASHIMA ◽  
Takahito ADACHI ◽  
Yuzuru ITO ◽  
Tomoko TAKANO ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Platelet Activating Factor ◽  
Chinese Hamster ◽  
Mrna Levels ◽  
Primary Role ◽  
Ovary Cells ◽  
Protein Kinase Cα ◽  
Paf Receptor

The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Cα (PKCα) to membrane was similar to that of DG formation. In WT-H cells, PKCα was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCα was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCα. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that sustained PLD activation in turn leads to chronic activation and membrane translocation of PKCα, which might play an important role in the expression of c-fos and c-jun.

scholarly journals Functional coupling of adenosine A2a receptor to inhibition of the mitogen-activated protein kinase cascade in Chinese hamster ovary cells

1996 ◽  
Vol 316 (1) ◽  
pp. 81-86 ◽  
Author(s):  
Daisuke HIRANO ◽  
Yoshiko AOKI ◽  
Hiroyuki OGASAWARA ◽  
Hisashi KODAMA ◽  
Iwao WAGA ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Activation ◽  
A2a Receptor ◽  
Mitogen Activated Protein

Activation of Gs-coupled receptors enhances the increase in cyclic AMP mediated by adenylate cyclases. As it has been shown that cyclic AMP inhibits the epidermal growth factor-activated mitogen-activated protein kinase (MAPK) signalling pathway, stimulation of Gs-coupled receptors may lead to the inhibition of MAPK activation. To investigate the effect of a Gs-coupled receptor on the MAPK cascade, we cloned the adenosine (Ado) A2a receptor from a guinea-pig leucocyte cDNA library, and established Chinese hamster ovary (CHO) cells stably expressing the receptor (CHOAdoA2R). The [3H]5´-N-ethylcarbamoyladenosine (NECA) binding characteristics (Kd = 91.0±5.4 nM, Bmax = 707±11 fmol/mg of protein, n = 3) and NECA-induced cyclic AMP production indicate that the cloned Ado A2a receptor was functionally expressed in the cells. In CHO cells, thrombin induced intracellular Ca2+ increase and MAPK activation through the intrinsic G-coupled receptor. In CHOAdoA2R cells, NECA partially inhibited thrombin-elicited MAPK activation. When combining NECA-treatment with 1,2-bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) loading, a nearly complete inhibition of the MAPK activation occurred. Forskolin also partially inhibited the MAPK activation and synergized with BAPTA-AM, suggesting that partial inhibition of MAPK activation by NECA results from cyclic AMP production via Ado A2a receptor activation. The same synergism of MAPK inhibition between wortmannin and BAPTA-AM was observed, but not between wortmannin and NECA. These results suggest that cyclic AMP production through Ado A2a receptor inhibits thrombin-elicited MAPK activation by a Ca2+-independent/ wortmannin-sensitive pathway in CHO cells.

scholarly journals Concerted action of cytosolic Ca2+ and protein kinase C in receptor-mediated phospholipase D activation in Chinese hamster ovary cells expressing the cholecystokinin-A receptor

1999 ◽  
Vol 337 (2) ◽  
pp. 263-268 ◽  
Author(s):  
Remko R. BOSCH ◽  
Rolf L. L. SMEETS ◽  
Frank SLEUTELS ◽  
Anjana M. P. PATEL ◽  
Sjenet E. Van EMST-De VRIES ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Kinase C ◽  
Peptide Amide ◽  
Sustained Increase

Receptor-mediated activation of phosphatidylcholine phosphatidohydrolase or phospholipase D (PLD) was studied in Chinese hamster ovary (CHO) cells expressing the cholecystokinin-A (CCK-A) receptor. Cells were labelled with [3H]myristic acid for 24 h and PLD-catalysed [3H]phosphatidylethanol formation was measured in the presence of 1% (v/v) ethanol. Cholecystokinin-(26–33)-peptide amide (CCK8) increased PLD activity both time- and dose-dependently. Maximal activation of protein kinase C (PKC) with 1 µM PMA or sustained elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) with 1 µM thapsigargin increased PLD activity to 50% and 70% of the maximal value obtained with CCK8 respectively. The stimulatory effects of CCK8, PMA and thapsigargin were abolished in cells in which PKC was downregulated or inhibited by chelerythrine. PMA/Ca2+-stimulated PLD activity was absent in a homogenate of PKC-downregulated cells but could be restored upon addition of purified rat brain PKC. CCK8-induced PLD activation was inhibited by 90% in the absence of external Ca2+, demonstrating that receptor-mediated activation of PKC in itself does not significantly add to PLD activation but requires a sustained increase in [Ca2+]i. Taken together, the results presented demonstrate that, in CHO-CCK-A cells, receptor-mediated PLD activation is completely dependent on PKC, but that the extent to which PLD becomes activated depends largely, if not entirely, on the magnitude and duration of the agonist-induced increase in [Ca2+]i.

Review of the current methods of Chinese Hamster Ovary (CHO) cells cultivation for production of therapeutic protein

2020 ◽  
Vol 17 ◽  
Author(s):  
Shazid Md. Sharker ◽  
Md. Atiqur Rahman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mass Production ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Therapeutic Protein ◽  
Specific Productivity ◽  
Ovary Cells ◽  
Further Development ◽  
Interesting Area

Most of clinical approved protein-based drugs or under in clinical trial have a profound impact in the treatment of critical diseases. The mammalian eukaryotic cells culture approaches, particularly the CHO (Chinese Hamster Ovary) cells are mainly used in the biopharmaceutical industry for the mass-production of therapeutic protein. Recent advances in CHO cell bioprocessing to yield recombinant proteins and monoclonal antibodies have enabled the expression of quality protein. The developments of cell lines are possible to upgrade specific productivity. As a result, it holds an interesting area for academic as well as industrial researchers around the world. This review will concentrate on the recent progress of the mammalian CHO cells culture technology and the future scope of further development for the mass-production of protein therapeutics.

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