Hello,
Does anyone know about any compounds that would increase ERK1/2 phosphorylation in fibroblasts, but that do not involve agonizing any membrane receptors? Most of the recommended strategies for increasing ERK phosporylation involve growth factors, but I want to avoide that as I suspect that I have problems with these receptors in my cells. All that I could find so far was a mysterious 'compound A' from a study published by Novartis, L-ascorbic acid, diallyl sulphide and hydrogenperoxide. The problem with the second two is that I read that they have different effects on different cell types with respect to ERK and I am afraid of DNA damage in case of H2O2. Any suggestions?
Cheers!
inhibit phosphatases, though that will increase P of all molecules. orthovanadate is a widely used option but w/H2O2, there are other phosphatase inhibitors (NaF) but I do not know if they have been used in culture, the concentration or side effects.
https://www.lifetechnologies.com/mx/es/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/protease-phosphatase-inhibitors.html
As suggested above arsenite is a good option. It will activate all 3 classical MAPKs in MEFs. Phorbol esters (PMA/TPA- 100-400ng/ml) will also activate ERK signaling with less prominent activation of p38/JNK. You can have a look at the link for reference
http://nar.oxfordjournals.org/content/39/7/2503/F2.expansion.html
Thank you for all the suggestions!
So far it looks like Phorbol 12-myristate 13-acetate seems like a nice candidate. I want to look at cell migration and it looks like PMA has some reports of enhancing it. I am still open to further suggestions.
Cheers!
Are you able to say what your cells are, so we can understand why you have concerns about their growth-factor receptors?
Do they respond to serum (by proliferating)? In which case you could use low vs high serum for low vs high MAPK, even if you don't want to use an individual growth factor.
Or yes, TPA would be good; it stimulates protein kinase C which feeds into the ERK pathway among others.
Depending on the cell type some G-protein-coupled receptors also talk to MAPKs, so in some cases small-molecule ligands like glutamate would work.
I know that my experimental cells have impaired proliferation and there were reports of cells with similar phenotypes that responded poorly to growth factors. The cells are fibroblasts and the controls do respond nicely to serum.
I am trying to check whether the specific defects that I see could be rescued by ERK stimulation, although I know already that one of the cell types has elevated PKC, but still diminished ERK phosphorylation...
OK so perhaps you are talking about defects in GF signalling upstream of MAPKs.
Something else that might be worth a try then (if it is proliferation as such that you'd want to improve), would be cAMP agonists, which can feed into proliferation controls via CREB downstream of MAPKs -- to AP1 which acts on MYC and cyclin D1. Sorry don't have primary ref to hand, but pathways are in Weinberg's Biology of Cancer. However growth response to cAMP agents is biphasic (in our experience) , so you'd probably need to do a dose-response curve to find the low/medium levels (if any) that are mitogenic for your cells. 100-200 pM cholera toxin for instance.
Thanks Dorothy!
First I want to check if improving ERK signalling will improve my cell migration, as it is meant to play a role in cell polarization. But yes, then I will look at cell proliferation. Thank you for your help!
We saw (in fish cells) nice ERK activation with hypoosmolarity (and also with Cu2+, but that is also toxic of course), see: http://www.ncbi.nlm.nih.gov/pubmed/17337716 (free article). With gentle and brief hyposmotic exposure you will not damage the cells, if this is your concern. Good luck.
PMA works well, but if you want to stimulate tyrosine phosphorylation of ERKs (and everything else!!) try using the global tyrosine phosphatase inhibitor pervanadate. Mix equal volumes of 10 mM sodium orthovanadate (fresh) with 10 mM hydrogen peroxide to give a 5 mM solution of pervanadate. Use at between 1-100 micromolar final concentration depending on cell type and assay. Would be interesting to look at the effects on cell polarization - do you use a wound healing/scratch assay for this?
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