Estrogen and EGFR Pathways Regulate Notch Signaling in Opposing Directions for Multi-Ciliogenesis in the Fallopian Tube
Round 1
Reviewer 1 Report
Very interesting work and nicely carried out. The studies of the Estrogen and EGFR pathways in parallel are nicely done and convincing. They lead to an overall picture of how ciliogenesis is controlled in the fallopian tube epithelial cells, which is of high interest especially since the estrogen and the Notch pathway shown to be involved here are also known to be implicated in carcinogenesis. The results obtained are summarized and displayed very clearly on a schematic which I really appreciated and the discussion opens to exciting future work on the interplay between the pathways dissected her and cancer.
A brief summary
In this paper the authors show that differentiation of fallopian tube epithelial cells (FTEC) into multiciliated cells, thus ciliogenesis is dually regulated by estrogen (E2) and EGFR, which both act on Notch signaling but in opposite directions. Using agonists and antagonists of its receptor ERbeta, they demonstrate that E2 inhibits Notch through suppression of its ligand DLL1, leading to downregulation of Notch signaling by DAPT and decreased expression of NCID (Notch Intracellular Domain) and Notch downstream genes HES1 and HEY1. In parallel, FOXJ1 expression is increased resulting in an upregulation of cilia-related genes which leads to ciliogenesis. On the contrary, in this paper, EGF is shown to activate Notch through the MEK and ERK pathway and thus suppress ciliogenesis. Indeed, when the author use gefitinib, an inhibitor of the ERK-MEK pathway, ciliogenesis is restored while NCID decreases and HES1 is downregulated Finally, since exposing the cells to E2 and gefitinib at the same time had a higher effect on DLL1 expression and inhibition of Notch than E2 alone, a synergistic effect of the two pathways – E2 and EGF mediated – has been suggested. Taken together, the authors successfully manage to untangle how ciliogenesis is controlled in FTEC and give an overall picture of this phenomenon. The fact that this regulation goes through estrogen and Notch signaling is of particular importance since these pathways are known to be involved in carcinogenesis.
Broad comments
This work is build up logically and nicely lead and the experiments and conclusions drawn convincing and of real relevance for the research on how epithelial cells differentiation is regulated. Finally, the discussion opens to interesting questions and leaves the reader in expectation of a follow-up paper on the effects of estrogen and Notch signaling on carcinogenesis in ovarian cancer. Thus I don’t see any big changes to be made.
Specific comments
Some minor points in this part:
P2, L49, “Taking the factors into account that Notch signaling is involved in carcinogenesis, elucidation of the molecular mechanism in Notch and estrogen would be important.” – could you rephrase into “molecular mechanisms linking Notch to estrogen signaling” P3, L111: “After dehydration, samples were substituted in butyl alcohol, as is a medium for freeze-drying” – please substitute “as” for “which” or “as it is” P3, L118: “A movie of the ciliary beat was recorded by with a high-speed camera” – please remove either “by” or “with” P6, L200: “We assumed that EGF may attenuate the effects of E2 in ciliogenesis, as basal medium includes EGF”.” Could you change into “as the basal medium used in the previous experiments”, just to help the readers understanding? P12, L352, “It well known that ERα and EGF play an important role in breast cancer”, the verb “is” is missing.Author Response
Response to Reviewer 1 Comments
Specific comments
Some minor points in this part:
P2, L49, “Taking the factors into account that Notch signaling is involved in carcinogenesis, elucidation of the molecular mechanism in Notch and estrogen would be important.” – could you rephrase into “molecular mechanisms linking Notch to estrogen signaling”
Response 1: Thank you for carefully checking our manuscript and gave us valuable comments.
P2, L48: As you have suggested, we have rephrased “Taking the factors into account that Notch signaling is involved in carcinogenesis, elucidation of the molecular mechanism in Notch and estrogen would be important” into “Taking the factors into account that Notch signaling is involved in carcinogenesis, elucidation of the molecular mechanisms linking Notch to estrogen signaling would be important”.
P3, L111: “After dehydration, samples were substituted in butyl alcohol, as is a medium for freeze-drying” – please substitute “as” for “which” or “as it is”
Response 2: P3, L117: In the sentence “After dehydration, samples were substituted in butyl alcohol, as is a medium for freeze-drying”, “as” has been substituted by “which”.
P3, L118: “A movie of the ciliary beat was recorded by with a high-speed camera” – please remove either “by” or “with”
Response 3: P3, L124: In the sentence “A movie of the ciliary beat was recorded by with a high-speed camera”, “with” has been deleted.
P6, L200: “We assumed that EGF may attenuate the effects of E2 in ciliogenesis, as basal medium includes EGF”.” Could you change into “as the basal medium used in the previous experiments”, just to help the readers understanding?
Response 4: P6, L207: “We assumed that EGF may attenuate the effects of E2 in ciliogenesis, as basal medium includes EGF” has been changed in to “We assumed that EGF may attenuate the effects of E2 in ciliogenesis, as basal medium used in the previous experiments includes EGF”.
P12, L352, “It well known that ERα and EGF play an important role in breast cancer”, the verb “is” is missing.
Response 5: P12, L361: “It well known that ERα and EGF play an important role in breast cancer” has been changed into “It is well known that ERα and EGF play an important role in breast cancer”.
Reviewer 2 Report
Differentiation of multiciliated cells is a challenging topic to study, and the manuscript presented by Zhu, Iwano, and Takeda is a useful contribution to the growing literature on this topic. The authors provide a thorough description of their primary culture system to differentiate fallopian tube epithelial cultures, derived from reasonably accessible porcine tissues. The cellular and molecular characterization of the derived cells, before, during, and after differentiation, is somewhat limited, but sufficient to show that the culture conditions are working well. The authors then use this system to address the respective roles of estrogen and EGF for the differentiation process, using acetylated tubulin immunoreactivity as a marker of multiciliated cells to address timing and frequency of their appearance. These two stimuli converge on the Notch pathway, which has been previously implicated multiciliated cell differentiation.
The report is a solid and well-written contribution that both supports previous findings and makes new connections. Above all, it provides detailed information about how to establish their culture system that will likely help others to do the same, or to adapt the method for other animal models.
One critical point is the heavy reliance on chemical inhibitors to make their conclusions. Genetic tools (siRNAs, shRNAs, CRISPR sgRNAs) to specifically target components of estrogen, EGF, and Notch signaling, would have added strength to the conclusions (i.e. if the same result is obtained using chemical and genetic inhibition, it is more likely to be true). However, use of chemical inhibitors is somewhat justified since porcine primary cell cultures are used. Off-the-shelf genetic tool resources for models other than mouse and human are limited, and all require challenging techniques (e.g. electroporation or viral transduction) to introduce these reagents into primary cultured cells. Also, due to the incubation times (1-2 weeks or more), use of siRNAs/shRNAs would be difficult.
In addition to the reliance on inhibitors, there is also an assumption that these compounds, all of which were likely isolated and characterized using human target proteins, work equally well and maintain the same specificity in the porcine model. This was only addressed with DAPT (Notch inhibitor) by monitoring NICD and HES1/HEY1 target gene expression. Any information that can be provided for the other inhibitors used (references or additional controls to be added in supplementary material) would strengthen the report. Somewhere in the text (left to the authors’ discretion), the reviewer would like to see a qualifying sentence(s) stating that use of inhibitors is based on the assumption that they work on the respective pathways in porcine cells, or something similar to alert readers of this caveat.
Additional points from the text/figures:
Section 2.4. A bigger font size is used – this will likely be corrected in final proofs.
General comment: Reviewer is used to seeing in vitro and in vivo italicized, but depends on journal policy.
Line 166: “at least in vitro culture” should be “at least in in vitro culture”
Figure 1E legend: “Thirsty-two ciliated cells” should be “Thirty-two”
Figure 2: This figure shows effect of ERbeta agonist DPN and ERbeta antagonist PHTPP. It would useful for these and other compounds to briefly state in the text their known or proposed mechanism of action for non-experts. This would be helpful to understand why the action of PHTPP is dominant over DPN.
Figure 3C. As authors do in Figure3A with +/- EGF and ALI Day 5/8/13, it would be useful to show more information in Figure3C, to show that 0, 100nM, 500nM, 1 µM refers to “Gefitinib” (to avoid searching in legend) and that images represent “ALI Day 10”.
Figure 3E. This panel shows treatment of FTECs with various inhibitors. The authors should specify passage # and culture condition for these particular FTECs (# of passages after thawing, in collagen-coated wells, in ALI system?)
Line 230-231, Fig S2A: The negative result of absent NOTCH4 expression is actually not shown in Fig S2A, so perhaps “(Fig. S2A)” should be changed to “(Fig. S2A and data not shown)”, if journal policy allows it.
Figure 4F and 4G: To match the flow of results in the text (NICD change is presented before FOXJ1 data), it would make sense to switch the order of these two panels.
Figure 6 legend: “While there is no definitive evidence by this study,” should be “in this study”.
Discussion:
Line 336: “are known to be key molecular for cell…” doesn’t make sense and should be “are known to be key regulators for cell…” or similar.
Line 351: “In the case of homeostasis involving these molecules,” doesn’t make sense to this reviewer and should be rephrased.
Author Response
Response to Reviewer 2 Comments
Additional points from the text/figures:
Thank you for carefully checking our manuscript and gave us valuable comments.
Section 2.4. A bigger font size is used – this will likely be corrected in final proofs.
Response 1: The bigger font size comes from the different magnification of the figure in the word format. This may be unified in the final version.
General comment: Reviewer is used to seeing in vitro and in vivo italicized, but depends on journal policy.
Response 2: We have changed both “in vivo” and “in vitro” by italicized style throughout the text.
Line 166: “at least in vitro culture” should be “at least in in vitro culture”
Response 3: P4 Line 172: “at least in vitro culture” has been changed into “at least in in vitro culture”
Figure 1E legend: “Thirsty-two ciliated cells” should be “Thirty-two”
Response 4: P5 Line 181: We have corrected this typo, “Thirsty-two” was corrected into “Thirty-two”
Figure 2: This figure shows effect of ERbeta agonist DPN and ERbeta antagonist PHTPP. It would useful for these and other compounds to briefly state in the text their known or proposed mechanism of action for non-experts. This would be helpful to understand why the action of PHTPP is dominant over DPN.
Response 5: P2, Line 70-78: According to your suggestion, we have added more information of the drugs in the Materials & Methods.
Figure 3C. As authors do in Figure3A with +/- EGF and ALI Day 5/8/13, it would be useful to show more information in Figure3C, to show that 0, 100nM, 500nM, 1 µM refers to “Gefitinib” (to avoid searching in legend) and that images represent “ALI Day 10”.
Response 6: We have added the detailed information including the name of drug, gefitinib and ALI Day 10 in the figure 3C.
Figure 3E. This panel shows treatment of FTECs with various inhibitors. The authors should specify passage # and culture condition for these particular FTECs (# of passages after thawing, in collagen-coated wells, in ALI system?)
Response 7: P7 Line 232-234: As you suggested, more detailed information on the culture of FTECs in has been added in the figure legend. FTECs P1 was applied to thaw, when they reached confluency, passaged to a normal 12-well plate for drugs treatment.
Line 230-231, Fig S2A: The negative result of absent NOTCH4 expression is actually not shown in Fig S2A, so perhaps “(Fig. S2A)” should be changed to “(Fig. S2A and data not shown)”, if journal policy allows it.
Response 8: P8 Line 239: We have added the information of “data not shown” in the text.
Figure 4F and 4G: To match the flow of results in the text (NICD change is presented before FOXJ1 data), it would make sense to switch the order of these two panels.
Response 9: We have rearranged the order of Figure 4F and 4G in the new figure 4.
Figure 6 legend: “While there is no definitive evidence by this study,” should be “in this study”.
Response 10: P11, Line 306: we have changed “by this study” into “in this study”.
Discussion:
Line 336: “are known to be key molecular for cell…” doesn’t make sense and should be “are known to be key regulators for cell…” or similar.
Response 11: P12, L344: In the revised version, “In general, EGF, Notch and estrogen pathways are known to be key regulators for cell proliferation and carcinogenesis”, which we have changed “key molecular” into “key regulators”.
Line 351: “In the case of homeostasis involving these molecules,” doesn’t make sense to this reviewer and should be rephrased.
Response 12: P12, Line 359-361: we have rephrased the sentence “In the case of homeostasis involving these molecules” to“Considering the roles of E2, EGF, and Notch involved in the regulation of FTE homeostasis, dysfunction of these factors may transform the FTECs into tumor cells that are the basis for HGSC”
