Akt‐mediated anti‐apoptotic effects of substance P in Anti‐Fas‐induced apoptosis of human tenocytes

Ludvig J Backman,Patrik Danielson

doi:10.1111/jcmm.12059

Abstract

Substance P (SP) and its receptor, the neurokinin-1 receptor (NK-1 R), are expressed by human tenocytes, and they are both up-regulated in cases of tendinosis, a condition associated with excessive apoptosis. It is known that SP can phosphorylate/activate the protein kinase Akt, which has anti-apoptotic effects. This mechanism has not been studied for tenocytes. The aims of this study were to investigate if Anti-Fas treatment is a good apoptosis model for human tenocytes in vitro, if SP protects from Anti-Fas-induced apoptosis, and by which mechanisms SP mediates an anti-apoptotic response. Anti-Fas treatment resulted in a time- and dose-dependent release of lactate dehydrogenase (LDH), i.e. induction of cell death, and SP dose-dependently reduced the Anti-Fas-induced cell death through a NK-1 R specific pathway. The same trend was seen for the TUNEL assay, i.e. SP reduced Anti-Fas-induced apoptosis via NK-1 R. In addition, it was shown that SP reduces Anti-Fas-induced decrease in cell viability as shown with crystal violet assay. Protein analysis using Western blot confirmed that Anti-Fas induces cleavage/activation of caspase-3 and cleavage of PARP; both of which were inhibited by SP via NK-1 R. Finally, SP treatment resulted in phosphorylation/activation of Akt as shown with Western blot, and it was confirmed that the anti-apoptotic effect of SP was, at least partly, induced through the Akt-dependent pathway. In conclusion, we show that SP reduces Anti-Fas-induced apoptosis in human tenocytes and that this anti-apoptotic effect of SP is mediated through NK-1 R and Akt-specific pathways.

Highlights

Studies have shown that the neuropeptide Substance P (SP), originally known for its role in the afferent sensory nervous system, mediates multiple efferent pathways, such as those involved in cell proliferation [1, 2] and apoptosis [3]
All primary tendon cells in culture were immunopositive for Fas Receptor (FasR), albeit to varying degrees, which provides a basis for apoptosis stimulation by Fas ligand (FasL) (Anti-Fas; Fig. 2)
We show that cultured human tenocytes undergo apoptosis following Anti-Fas treatment

Summary

Introduction

Studies have shown that the neuropeptide SP, originally known for its role in the afferent sensory nervous system, mediates multiple efferent pathways, such as those involved in cell proliferation [1, 2] and apoptosis [3]. Expression of SP and the NK-1 R has been observed in human tenocytes in vivo [4]. This is the case for the tenocytes of tendons afflicted by tendinosis, a condition of chronic tendon pain (tendinopathy) and tissue changes such as hypercellularity, angiogenesis and collagen disorganization [5]. Apoptosis is a prominent microscopic feature observed in tendinosis tissues [9], but the role of SP and the NK-1 R in the regulation of apoptosis and cell survival in tenocytes is poorly understood. It has been shown that SP has an anti-apoptotic effect in various cell types [3, 10, 11], either via inhibition of apoptotic pathways and/or activation of cell survival pathways [3, 12]

Objectives

Methods

Results

Conclusion