Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model

Keren Ettinger; Shimon Lecht; Hadar Arien-Zakay; Gadi Cohen; Shlomit Aga-Mizrachi; Nurit Yanay; H. Uri Saragovi; Hinyu Nedev; Cezary Marcinkiewicz; Yoram Nevo; Philip Lazarovici

doi:10.1016/j.cellsig.2012.08.008

Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75^NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model

Keren Ettinger, Shimon Lecht, Hadar Arien-Zakay, Gadi Cohen, Shlomit Aga-Mizrachi, Nurit Yanay, H. Uri Saragovi, Hinyu Nedev, Cezary Marcinkiewicz, Yoram Nevo^*, Philip Lazarovici

^*Corresponding author for this work

School of Pharmacy

Research output: Contribution to journal › Article › peer-review

20 Scopus citations

Abstract

The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75^NTR, α9β1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and βNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9β1 integrin; and (c) p75^NTR antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of βNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and βNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75^NTR and α9β1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9β1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.

Original language	English
Pages (from-to)	2378-2388
Number of pages	11
Journal	Cellular Signalling
Volume	24
Issue number	12
DOIs	https://doi.org/10.1016/j.cellsig.2012.08.008
State	Published - Dec 2012

Bibliographical note

Funding Information:
P.L. holds Jacob Gitlin chair in physiology and is affiliated and acknowledges the financial support of David R. Bloom Center of Pharmacy and Dr. Adolf and Klara Brettler Center for Research in Molecular Pharmacology and Therapeutics at The Hebrew University of Jerusalem, Israel. K.E. acknowledges a travel grant from the Alex Grass Center for Drug Design and Synthesis of Novel Therapeutics at the Hebrew University, to present this study at ASPET meeting, EB2011 conference, Washington DC. C.M. acknowledges NIH NCI support, grant R01CA133262 . We would like to acknowledge Prof. David Yaffe, The Weizmann Institute of Science, Rehovot, Israel for providing us the C2 clones and Mrs. Zehava Cohen for the help with the graphics.

Keywords

C2C12 cells
ERK1/2
Myoprotection
NGF
P75
Skeletal muscle
α9β1 integrin

Access to Document

10.1016/j.cellsig.2012.08.008

Cite this

Ettinger, K., Lecht, S., Arien-Zakay, H., Cohen, G., Aga-Mizrachi, S., Yanay, N., Saragovi, H. U., Nedev, H., Marcinkiewicz, C., Nevo, Y., & Lazarovici, P. (2012). Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75^NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model. Cellular Signalling, 24(12), 2378-2388. https://doi.org/10.1016/j.cellsig.2012.08.008

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title = "Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model",

abstract = "The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75NTR, α9β1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and βNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9β1 integrin; and (c) p75NTR antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of βNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and βNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75NTR and α9β1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9β1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.",

keywords = "C2C12 cells, ERK1/2, Myoprotection, NGF, P75, Skeletal muscle, α9β1 integrin",

author = "Keren Ettinger and Shimon Lecht and Hadar Arien-Zakay and Gadi Cohen and Shlomit Aga-Mizrachi and Nurit Yanay and Saragovi, {H. Uri} and Hinyu Nedev and Cezary Marcinkiewicz and Yoram Nevo and Philip Lazarovici",

note = "Funding Information: P.L. holds Jacob Gitlin chair in physiology and is affiliated and acknowledges the financial support of David R. Bloom Center of Pharmacy and Dr. Adolf and Klara Brettler Center for Research in Molecular Pharmacology and Therapeutics at The Hebrew University of Jerusalem, Israel. K.E. acknowledges a travel grant from the Alex Grass Center for Drug Design and Synthesis of Novel Therapeutics at the Hebrew University, to present this study at ASPET meeting, EB2011 conference, Washington DC. C.M. acknowledges NIH NCI support, grant R01CA133262 . We would like to acknowledge Prof. David Yaffe, The Weizmann Institute of Science, Rehovot, Israel for providing us the C2 clones and Mrs. Zehava Cohen for the help with the graphics. ",

year = "2012",

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doi = "10.1016/j.cellsig.2012.08.008",

language = "אנגלית",

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Ettinger, K, Lecht, S, Arien-Zakay, H, Cohen, G, Aga-Mizrachi, S, Yanay, N, Saragovi, HU, Nedev, H, Marcinkiewicz, C, Nevo, Y & Lazarovici, P 2012, 'Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75^NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model', Cellular Signalling, vol. 24, no. 12, pp. 2378-2388. https://doi.org/10.1016/j.cellsig.2012.08.008

Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75^NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model. / Ettinger, Keren; Lecht, Shimon; Arien-Zakay, Hadar et al.
In: Cellular Signalling, Vol. 24, No. 12, 12.2012, p. 2378-2388.

Research output: Contribution to journal › Article › peer-review

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AU - Saragovi, H. Uri

AU - Nedev, Hinyu

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AU - Nevo, Yoram

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N2 - The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75NTR, α9β1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and βNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9β1 integrin; and (c) p75NTR antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of βNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and βNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75NTR and α9β1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9β1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.

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