Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway

Adrenomedullin 2 plays diverse physiological roles such as regulating cardiovascular functions and blood pressure. It was reported that adrenomedullin 2 can activate protein kinase C in murine ventricular myocytes to augment cardiomyocyte contractile function. Using a protein kinase Cα knockout mouse model, we show here that adrenomedullin 2 activates extracellular-signal-regulated kinase in a protein kinase Cα-independent mechanism in endothelial cells. Michael Simons ( ) Corresponding author: Michael.simons@yale.edu Guo X, Ju R, Cha C and Simons M. How to cite this article: Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway [version 1; referees: 2 approved with reservations] F1000Research 2016, :26 (doi: ) 5 10.12688/f1000research.2420.1 © 2016 Guo X . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the (CC0 1.0 Public domain dedication). Creative Commons Zero "No rights reserved" data waiver This work is supported by OHSE fund (Department of Surgery, Yale School of Medicine) funding to XG. Grant information: Competing interests: There is no disclosure of competing interests. 06 Jan 2016, :26 (doi: ) First published: 5 10.12688/f1000research.2420.1 1 2 1

Introduction Adrenomedullin 2 (ADM2), also known as intermedin, is a secreted peptide that belongs to the calcitonin gene-related-peptide family 1,2 .It has been reported that ADM2 regulates intracellular calcium levels and contractile function in protein kinase C (PKC) -and protein kinase A (PKA) -dependent mechanisms in cardiomyocytes 3 .ADM2 activates the cAMP/PKA signaling pathway, which mediates inactivation of contractility and strengthening of cell-cell adhesion in endothelial cells 4 .ADM2 activates extracellularsignal-regulated kinase (ERK), a key signaling molecule for cell proliferation in endothelial cells 5 .To investigate whether ADM2 activates ERK through PKCα, which is a major upstream activator of ERK in endothelial cells we examined the effect of phosphorylation of ERK on ADM2 stimulation in endothelial cells isolated from PKCα null mice or wild type (wt) counterpart mice.

Methods
Animal care and experimental procedures were performed under protocol # CC0004 approved by the Institutional Animal Care and Use Committees of Yale University.Endothelial cells were isolated from wild type (C57BL/6J, The Jackson Laboratory, Cat # 000664) and PKCα-/-mice (Prkca tm1Jmk , The Jakson Laboratory, Cat # B6;129-Prkcatm1Jmk/J) and maintained as previously described 6 .
Briefly, the arteries of both wild-type and knockout mice were harvested, finely minced with scissors, and digested with 25 ml collagenase (2 mg/ml) at 37°C for 45 min under gentle agitation.The crude preparation was triturated, passing it 12 times through a cannula needle, and was then filtered on a 70-μM sterile cell strainer.The filtered preparation was spun at 400 × g, and the pellet was resuspended in 2 ml of 0.1% BSA.Magnetic beads (Invitrogen) coated with anti-mouse CD31 (BD Biosciences) were added to the cell suspension and incubated with rotation at room temperature for 15 min.The bead-bound cells were recovered with a magnetic separator and washed with DMEM containing 20% FBS.Cells were suspended in 10 ml of complete DMEM and seeded on cell culture plates (Catalog # 353003, Corning Inc., Corning, NY).Subconfluent cells were serum-starved for 16h followed by incubation with 10 ng/ml ADM2 peptide (Pheonix Pharmaceuticals, Burlingame, CA) for the indicated time length: 0, 5, and 30min As shown in Figure 1, ADM2 increased phosphorylation of ERK in endothelial cells.However, there was no difference in ERK phosphorylation levels in wt versus PKCα null endothelial cells (Figure 1).
Our results indicate that ADM2 activates ERK in endothelial cells via a PKCα -independent pathway.

Open Peer Review
Current Referee Status: The purpose of the current study was to investigate whether ADM2 activates ERK through PKCα, which is a major upstream activator of ERK in endothelial cells.The authors examined the effect of phosphorylation of ERK on ADM2 stimulation in endothelial cells isolated from PKCα null mice or wild type (wt) counterpart mice.The findings obtained in their experiments were clear.However, they studied by only a single method.They need to study, for example, by siRNA method to certify the independency on PKCα.Another concern is that the authors are expected to, at least, discuss the crucial signal pathway to activate ERK in this cell system.In this context, they need to study Ras-Raf-MAPK/ERK kinase (MEK).

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
No competing interests were disclosed.

Competing Interests:
19 February 2016 Referee Report doi:10.5256/f1000research.2633.r12575James G. Taylor VI Genomic Medicine Section, Hematology Branch, NHLBI, National Institutes of Health, Bethesda, MD, 20892-1476, USA The manuscript "Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway" presents an elegant mutational analysis of cellular activation by the enigmatic adrenomedullin 2. Endothelial cells harvested from PKC-α knock out mice still demonstrate ERK phosphorylation, suggesting that the current paradigm of ADM2 intracellular activation (ADM2 signalling occurring through a PKC receptor pathway) is not completely understood.While this work is limited in scope, it does set a foundation for needed work in this field.There are some technical and context specific elements which would significantly strengthen this research note.

Major points:
Which specific ADM2 peptide was used?It would be helpful to mention this in the methods; ADM2-47 is believed to be the more potent agonist compared to the -40 peptide.Perhaps it would also be appropriate to test both ADM2 peptides (both -47 and -40) for comparison. 2.

3.
also be appropriate to test both ADM2 peptides (both -47 and -40) for comparison.
The abstract and introduction sets the context for this work by describing ADM2 signalling in cardiomyocytes, but this research note presents data for arterial endothelial cells.The addition of the same phospho-Westerns for the same PKC-α knock out mouse cardiomyocytes would address this criticism.
The results section is clearly presented, but a brief discussion and conclusion to put these results in a physiologic context would again enhance the impact of this research note.Specifically, this reviewer could cite a review by where it is stated that the receptors that mediate Hong (2012) et al.ADM2 action are not completely understood (i.e. the authors results suggest that perhaps the calcitonin like receptor pathway for ADM2 might activate endothelial cells independent of protein kinase).Presenting an updated version of Figure 4 from this review would also further work in this field and put the results in context.
I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
No competing interests were disclosed.Competing Interests:

Figure 1 .
Figure 1.ERK, but not PKCα, mediates ADM2 signaling in endothelial cells.Representative immunoblot showing that ADM2 increased phosphorylation of ERK, via a PKCα-independent pathway, in endothelial cells.Mouse endothelial cells (mEC) were isolated from wild type (wt) and Protein kinase Cα knockout mice (PKCα -/-).Cells were serum-starved overnight followed by stimulation with ADM2 synthetic peptide (10ng/ml) for indicated time and cell lysates were analyzed by immunoblotting for ERK activation.
of Respiratory Medicine, School of Medicine, Kyorin University, Mitaka, Japan