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

Xiaojia Guo; Rong Ju; Charles Cha; Michael Simons

doi:10.12688/f1000research.2420.1

Home Browse Adrenomedullin 2 activates extracellular-signal-regulated kinase in...

ALL Metrics

-

Views

-

Downloads

Get PDF

Get XML

Export

▬

✚

Research Note

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

[version 1; peer review: 2 approved with reservations]

Xiaojia Guo¹, Rong Ju², Charles Cha¹, Michael Simons ²

PUBLISHED 06 Jan 2016

Author details Author details

¹ Section of Surgical Oncology, Department of Surgery, Yale University School of Medicine, New Haven, USA
² Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, USA

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

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.

Keywords

adrenomedullin 2 (ADM2), intermedin (IMD), protein kinase C (PKC), endothelial cells, signal transduction

Corresponding author: Michael Simons

Competing interests: There is no disclosure of competing interests.

Grant information: This work is supported by OHSE fund (Department of Surgery, Yale School of Medicine) funding to XG.

Copyright: © 2016 Guo X et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which 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 Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

How to cite: Guo X, Ju R, Cha C and Simons M. Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway [version 1; peer review: 2 approved with reservations]. F1000Research 2016, 5:26 (https://doi.org/10.12688/f1000research.2420.1) First published: 06 Jan 2016, 5:26 (https://doi.org/10.12688/f1000research.2420.1) Latest published: 06 Jan 2016, 5:26 (https://doi.org/10.12688/f1000research.2420.1)

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 cardiomyocytes³. ADM2 activates the cAMP/PKA signaling pathway, which mediates inactivation of contractility and strengthening of cell-cell adhesion in endothelial cells⁴. ADM2 activates extracellular-signal-regulated kinase (ERK), a key signaling molecule for cell proliferation in endothelial cells⁵. 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 described⁶. 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 . Cells were lysed in RIPA buffer (Catalog # R0278, Sigma-Aldrich, St Louis, MI), supplemented with protease inhibitor cocktail (Catalog # 11 873 580 001, Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitor cocktails (Catalog # P0044 and P5726, Sigma-Aldrich, St Louis, MI) as instructed by manufactures. Total cell lysates were subjected to immunoblotting analysis as described previously². The membranes were hybridized with antibodies recognizing phospho-ERK (at 1:2,000 dilution of Catalog # 4370, Cell Signaling Technologies, Danvers, MA), total ERK (at 1:1,000 dilution of Catalog # 4695, Cell Signaling Technologies, Danvers, MA), PKCα (at 1:500 dilution of Catalog # 610108, BD BioSciences, San Jose, CA), and β-actin (at 1:10,000 dilution of Catalog # sc-47778, Santa Cruz Biotechnology Inc., Dallas, Texas). Following incubation with horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG (Zymed Laboratories Inc., San Francisco, CA). Western signals were visualized with enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA).

Results and conclusion

Dataset 1.Gel images for ‘Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway’ by Guo X., et al..

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.

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.

Data availability

F1000Research: Dataset 1. Gel images for ‘Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway’ by Guo X., et al., 10.5256/f1000research.2420.d110689⁷

Author contributions

XG cultured and treated cells and performed Western analyses, and prepared the manuscript; RJ isolated endothelial cells from mice, CC and MS directed the study.

Competing interests

There is no disclosure of competing interests.

Grant information

This work is supported by OHSE fund (Department of Surgery, Yale School of Medicine) funding to XG.

Faculty Opinions recommended

References

1. Kobayashi Y, Liu YJ, Gonda T, et al.: Coronary vasodilatory response to a novel peptide, adrenomedullin 2. Clin Exp Pharmacol Physiol. 2004; 31(Suppl 2): S49–50. PubMed Abstract | Publisher Full Text
2. Guo X, Schmitz JC, Kenney BC, et al.: Intermedin is overexpressed in hepatocellular carcinoma and regulates cell proliferation and survival. Cancer Sci. 2012; 103(8): 1474–1480. PubMed Abstract | Publisher Full Text
3. Dong F, Taylor MM, Samson WK, et al.: Intermedin (adrenomedullin-2) enhances cardiac contractile function via a protein kinase C- and protein kinase A-dependent pathway in murine ventricular myocytes. J Appl Physiol (1985). 2006; 101(3): 778–784. PubMed Abstract | Publisher Full Text
4. Aslam M, Pfeil U, Gündüz D, et al.: Intermedin (adrenomedullin2) stabilizes the endothelial barrier and antagonizes thrombin-induced barrier failure in endothelial cell monolayers. Br J Pharmacol. 2012; 165(1): 208–222. PubMed Abstract | Publisher Full Text | Free Full Text
5. Smith RS, Gao L, Bledsoe G, et al.: Intermedin is a new angiogenic growth factor. Am J Physiol Heart Circ Physiol. 2009; 297(3): H1040–1047. PubMed Abstract | Publisher Full Text | Free Full Text
6. Corti F, Finetti F, Ziche M, et al.: The syndecan-4/protein kinase Cα pathway mediates prostaglandin E2-induced extracellular regulated kinase (ERK) activation in endothelial cells and angiogenesis in vivo. J Biol Chem. 2013; 288(18): 12712–12721. PubMed Abstract | Publisher Full Text | Free Full Text
7. Guo X, Ju R, Cha C, et al.: Dataset 1 in: Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway. F1000Research. 2015. Data Source

Comments on this article Comments (0)

Version 1

VERSION 1 PUBLISHED 06 Jan 2016

Author details Author details

¹ Section of Surgical Oncology, Department of Surgery, Yale University School of Medicine, New Haven, USA
² Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, USA

Competing interests

There is no disclosure of competing interests.

Grant information

This work is supported by OHSE fund (Department of Surgery, Yale School of Medicine) funding to XG.

Article Versions (1)

version 1

Published: 06 Jan 2016, 5:26

https://doi.org/10.12688/f1000research.2420.1

Copyright

© 2016 Guo X et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which 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 Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Download

Export To

metrics

	Views	Downloads
F1000Research	-	-
PubMed Central Data from PMC are received and updated monthly.	-	-

Citations

0

SEE MORE DETAILS

CITE

how to cite this article

Guo X, Ju R, Cha C and Simons M. Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway [version 1; peer review: 2 approved with reservations]. F1000Research 2016, 5:26 (https://doi.org/10.12688/f1000research.2420.1)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

track

receive updates on this article

Track an article to receive email alerts on any updates to this article.

Open Peer Review

Version 1

VERSION 1

PUBLISHED 06 Jan 2016

Views

14

Reviewer Report 04 May 2016

Hajime Takizawa, Department of Respiratory Medicine, School of Medicine, Kyorin University, Mitaka, Japan

Approved with Reservations

https://doi.org/10.5256/f1000research.2633.r13696

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 ... Continue reading

CITE

Report a concern

Respond or Comment

Views

9

Reviewer Report 19 Feb 2016

James G. Taylor VI, Genomic Medicine Section, Hematology Branch, NHLBI, National Institutes of Health, Bethesda, MD, 20892-1476, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.2633.r12575

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 ... Continue reading

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.
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 Hong et al. (2012) where it is stated that the receptors that mediate 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.

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and 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.

CITE

Report a concern

Respond or Comment

Comments on this article Comments (0)

Version 1

VERSION 1 PUBLISHED 06 Jan 2016

Open Peer Review

Reviewer Status

Reviewer Reports

	Invited Reviewers
	1	2
Version 1 06 Jan 16	read	read

James G. Taylor VI, National Institutes of Health, Bethesda, USA
Hajime Takizawa, Kyorin University, Mitaka, Japan

Comments on this article

All Comments(0)

Add a comment

Sign up for content alerts

Browse by related subjects

Back to all reports

Reviewer Report

14 Views

04 May 2016 | for Version 1

Hajime Takizawa, Department of Respiratory Medicine, School of Medicine, Kyorin University, Mitaka, Japan

14 Views Cite this report Responses(0)

Approved With Reservations

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).

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and 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.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

9 Views

19 Feb 2016 | for Version 1

James G. Taylor VI, Genomic Medicine Section, Hematology Branch, NHLBI, National Institutes of Health, Bethesda, MD, 20892-1476, USA

9 Views Cite this report Responses(0)

Approved With Reservations

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.
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 Hong et al. (2012) where it is stated that the receptors that mediate 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.

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and 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.

Respond to this report

Responses (0)

[1] 1. Kobayashi Y, Liu YJ, Gonda T, et al.: Coronary vasodilatory response to a novel peptide, adrenomedullin 2. Clin Exp Pharmacol Physiol. 2004; 31(Suppl 2): S49–50. PubMed Abstract | Publisher Full Text

[2] 2. Guo X, Schmitz JC, Kenney BC, et al.: Intermedin is overexpressed in hepatocellular carcinoma and regulates cell proliferation and survival. Cancer Sci. 2012; 103(8): 1474–1480. PubMed Abstract | Publisher Full Text

[3] 3. Dong F, Taylor MM, Samson WK, et al.: Intermedin (adrenomedullin-2) enhances cardiac contractile function via a protein kinase C- and protein kinase A-dependent pathway in murine ventricular myocytes. J Appl Physiol (1985). 2006; 101(3): 778–784. PubMed Abstract | Publisher Full Text

[4] 4. Aslam M, Pfeil U, Gündüz D, et al.: Intermedin (adrenomedullin2) stabilizes the endothelial barrier and antagonizes thrombin-induced barrier failure in endothelial cell monolayers. Br J Pharmacol. 2012; 165(1): 208–222. PubMed Abstract | Publisher Full Text | Free Full Text

[5] 5. Smith RS, Gao L, Bledsoe G, et al.: Intermedin is a new angiogenic growth factor. Am J Physiol Heart Circ Physiol. 2009; 297(3): H1040–1047. PubMed Abstract | Publisher Full Text | Free Full Text

[6] 6. Corti F, Finetti F, Ziche M, et al.: The syndecan-4/protein kinase Cα pathway mediates prostaglandin E2-induced extracellular regulated kinase (ERK) activation in endothelial cells and angiogenesis in vivo. J Biol Chem. 2013; 288(18): 12712–12721. PubMed Abstract | Publisher Full Text | Free Full Text

[7] 7. Guo X, Ju R, Cha C, et al.: Dataset 1 in: Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway. F1000Research. 2015. Data Source

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

Abstract

Keywords

Introduction

Methods

Results and conclusion

Figure 1. ERK, but not PKCα, mediates ADM2 signaling in endothelial cells.

Data availability

Author contributions

Competing interests

Grant information

References

Comments on this article Comments (0)

Open Peer Review

Comments on this article Comments (0)

Open Peer Review

Reviewer Status

Reviewer Reports

Comments on this article

Browse by related subjects

The problem

How to fix it

Competing Interests Policy

Stay Updated