Moomin said:
Well I think there is some regulation of the pathway by some tyrosine kinases but I think the wnt pathway has a much greater importance in regulating beta-catenin levels and there is no tyrosine kinase involved in this signalling pathway as far as I can tell. If you see otherwise please let me know.
I don't know how you can say that, Wnt works on Tyrosine Kinase and GSk-3 which
communicate downstream to upregulate beta catenin adhesion and neutralise/limit phosphorylation of beta catenin, respectively. Its not a case that Wnt upregulates beta-catenin all by itself as if by magic but works via intermediaries. This information is not esoteric, it is plastered all of the internet.
No. Wnt inhibits the action of Gsk3-Beta which is not a tyrosine kinase. As far as I am aware I have not seen anything about a tyrosine kinase in the wnt signalling pathway. If you find something please post a link to it.
Here is a link showing that at least one non-canonical wnt ligand does activate a tyrosine kinase. But this has nothing to do with beta-catenin.
http://www.ncbi.nlm.nih.gov/pubmed/1861 ... d_RVDocSum
At any rate none of this is really important for the reason that I gave before -
there are different tyrosine kinases. Even if what you were saying were true
it doesnt mean that an EGFR inhibitor that interfered directly with the EGFR tyrosine kinase would affect beta-catenin levels.
[quote:2i9y1nmg]This quote is not saying that EGCG inhibits tyrosine kinase - EGCG "directly blocks EGF binding to the EGFR" and it is only subsequently ie. indirectly that the EGFR mediated tyrosine kinase inhibitor is blocked. So the mechanism of EGCG inhibiting EGFR is not related to EGFR tyrosine kinase.
I do not understand what you mean by this. If Tyrosine Kinase has an an EGFR site and ECGC blocks EGF from binding to those sites, and EGF - EGFR binding is necessary, at least in part, for beta catenin binding/adhesion then wouldn't BCGC negatviely affect beta catenin production if TK played any role in that production (which it does).
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EGF is not necessary for functional levels of beta-catenin. But again
there are many types of tyrosine kinase. Stop referring to them as if they are all the one protein and it might become clearer as to why what you are proposing is not the case.
[quote:2i9y1nmg]I dont recall saying how EGCG works but as the quote above shows it works as an EGFR inhibitor by directly stopping EGF from docking at the receptor.
No you said how it didn't work, and also I already explained how EGCG worked in my post prior to this. But repetition never harmed anyone.
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OK I see that I didnt say how EGCG works as an EGFR inhibitor before but I did say how it "doesnt work" in the sense of not working out well for us as an inhibitor of wnt signalling.
Anyway as to EGCG inhibiting wnt signalling via the way I proposed
and the way you proposed:
Read the below abstract/study - EGCG blocks wnt signalling but has no effect on beta-catenin levels. Because it blocks the signal downstream of beta-catenin.
J Biol Chem. 2006 Apr 21;281(16):10865-75. Epub 2006 Feb 22.Click here to read Links
Suppression of Wnt signaling by the green tea compound (-)-epigallocatechin 3-gallate (EGCG) in invasive breast cancer cells. Requirement of the transcriptional repressor HBP1.
Kim J, Zhang X, Rieger-Christ KM, Summerhayes IC, Wazer DE, Paulson KE, Yee AS.
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Genetic and biochemical de-regulation of Wnt signaling is correlated with breast and other cancers. Our goal was to identify compounds that block Wnt signaling as a first step toward investigating new strategies for suppression of invasive and other breast cancers. In a limited phytonutrient screen, EGCG ((-)-epigallocatechin 3-gallate), the major phytochemical in green tea, emerged as an intriguing candidate. Epidemiological studies have associated green tea consumption with reduced recurrence of invasive and other breast cancers. Wnt signaling was inhibited by EGCG in a dose-dependent manner in breast cancer cells. The apparent mechanism targeted the HBP1 transcriptional repressor, which we had previously characterized as a suppressor of Wnt signaling. EGCG treatment induced HBP1 transcriptional repressor levels through an increase in HBP1 mRNA stability, but not transcriptional initiation. To test functionality, DNA-based short hairpin RNA (shRNA) was used to knockdown the endogenous HBP1 gene. Consistently, the HBP1 knockdown lines had reduced sensitivity to EGCG in the suppression of Wnt signaling and of a target gene (c-MYC). Because our ongoing studies clinically link abrogation of HBP1 with invasive breast cancer, we tested if EGCG also regulated biological functions associated with de-regulated Wnt signaling and with invasive breast cancer. EGCG reduced both breast cancer cell tumorigenic proliferation and invasiveness in an HBP1-dependent manner.
Together, the emerging mechanism is that EGCG blocks Wnt signaling by inducing the HBP1 transcriptional repressor and inhibits aspects of invasive breast cancer. These studies provide a framework for considering future studies in breast cancer treatment and prevention.
From the study itself
"HBP1 Contributes to the Suppression of Wnt Signaling and Gene Expression by EGCG—The experiments of Fig. 1 suggested two testable mechanisms for EGCG blocking Wnt signaling: 1) EGCG might trigger a reduction in beta-catenin levels, or 2) EGCG might increase the levels of HBP1. Previously, we showed that HBP1 is a suppressor of Wnt signaling by inhibiting the LEF/TCF/beta-catenin transcriptional activation complex (7).
As shown in Fig. 2A, endogenous beta-catenin levels were unchanged with EGCG treatment. In addition, both the phosphorylation of beta-catenin by GSK-3beta, as well as total, cytoplasmic and nuclear beta-catenin levels were unchanged (Fig. 2B), further supporting the lack of change in beta-catenin levels with EGCG. However, we observed that in 293T cells (Fig. 2A) and in MDA-MB-231 cells (Fig. 4A), endogenous HBP1 protein levels increased with EGCG treatment. These results were extended with the observation that EGCG treatment increased HBP1 mRNA, as measured by real-time RT-PCR (Fig. 3A). Thus, EGCG increased expression of HBP1, suggesting that HBP1 may be a plausible target of EGCG action in the inhibition of Wnt signaling."
So EGCG did not effect beta-catenin levels despite being a potent inhibitor of EGFR.
I feel as though we are just going back and forth here.
Fundamentally I believe TK plays an important role in Beta-Catenin binding as do other proteins. Further you speak as though EGF only affects certain TK but not others, which isn't the case. You also don't seem to appreciate the difference between Wnt Proteins and signalling, as TK as well as GSK-3 all play their part in Wnt signalling, making it impossible to say TK nor GSK isn't important for our purposes. Finally, on this matter it is because EGF affects TK and GSK-3, as well as others, that so far their hasn't been a single suggestion of an EGF inhibitor that doesn't affect Wnt signalling. GO FIGURE.
So you are implying that the EGFR is functionally linked to every type of tyrosine kinase? Can you provide some evidence for this?
I appreciate the comment that I dont understand the difference between wnt proteins and wnt signalling but so far you havent shown a single link between wnt proteins and beta-catenin via a tyrosine kinase. ie you havent shown how they are relevant to the discussion of the canonical wnt pathway. Which is the type of wnt signalling that we are interested in. And even if you did show this then it wouldnt be important unless you could show that these same tyrosine kinases would be effected by gefitinib or whatever other EGFR inhibitor we are talking about. And even then your central concern is not important since Follica showed that EGFR inhibitors increased both hair follcile density and size.
And quercetin was more or less hown not to effect wnt signalling.
Nevertheless, as much as I enjoyed and learnt from this conversation, it clearly isn't going to be developed any futher. My reason for this discussion was to see if any practical information could be garnered from the theory and I believe there has been some. Has anyone considered upregulating Wnt signalling over an extended period of time. As in applying Lithium Chloride for a month prior to the wounding, during the wounding and then applying an EGFR inhibitor and lithium chloride 3 days prior to the wounding. My thinking is that the ability for beta-catenin to bind and the lack/limitation of it being phosphorylated would make the beta-catenin more potent in its ability to pair with Lef1.
...how would it become "more potent in its ability to pair with Lef1"? It either binds or it doesn't - having elevated levels for a long time is not going to make a difference anymore than holding your keys in your hand for a few days is going to make them any more likely to open your car when you try. Besides which there is a critical period where wnt signalling is required to be upregulated outside of which it is unimportant. There is also a critical period in which you probably want it downregulated also in order to create pigmented hairs.
This should obviate the affect of the EGFR-inhibitor on Wnt signalling, or at least it should limit the effect. Also the mice that over expressed Wnt signalling, were they genetically engineered (i seem to remember it was) to do this or was it done through some sort of feed and were they overexpressing Wnt before wounding or only prior to wounding? Of course this is just a theory. I'll be trying this out on myself soon.
They were genetically engineered to overexpress wnt7a. They overexpressed wnt7a all the time but later Follica work has further identified the existence of a critical post-wounding period where wnt signalling is important (though the original work hinted at this).
hh