Stimulation of mechano-growth factor expression by second messengers

Abstract

The effect of second messengers on the expression of mechano-growth factor (MGF) synthesis by myoblasts and differentiated myotubes in culture was investigated. cAMP stimulates MGF expression both in murine and human cells. CNG- and HCN-channel blockers slightly activated MGF synthesis, while an activator of Epac protein had no effect. It is assumed that cAMP activates MGF synthesis via protein kinase A. Phorbol ester (PMA) activates MGF synthesis in human myoblasts and myotubes only. The expression of another splice form of IGF-1 gene, IGF-1Ea, was also stimulated in human cells by db-cAMP and PMA and in murine cells by db-cAMP only. Stimulation of MGF expression in human cells by db-cAMP and PMA demonstrated different time dependences but showed additivity when the compounds were applied in a combination. Inhibitors specific to protein kinase A did not affect PMA-mediated activation, while inhibitors specific to protein kinase C did not affect db-cAMP-mediated process. Ca²⁺ ionophore and ROS inductor strongly inhibited synthesis of the growth factor. PGE2 known as physiological stimulator of cAMP synthesis was shown to stimulate MGF expression both in murine and human cells. Implication of protein kinase A and protein kinase C in MGF synthesis stimulation and a cross-talk between two signaling systems is discussed.

Introduction

Insulin-like growth factor 1 (IGF-1) is one of the most important physiological regulators exerting effects on the cellular, tissue and organism levels. Its mRNA is initially translated as a long precursor protein that is subsequently subjected to limited proteolysis yielding N-terminal peptide, mature IGF-1 and the C-terminal fragment known as E-peptide. IGF-1 gene includes six exons and five introns. The first two exons encode different forms of the leader peptide of the N-terminal fragment, exon 3 encodes the C-terminal part of the leader peptide and the N-terminal fragment of the mature IGF-1, exon 4 encodes its C-terminal part and the N-terminal part of the E-peptide, exons 5 and 6 encode different versions of the E-peptide. IGF-1 mRNA is subjected to alternative splicing both in human and in rodent cells.

The dominant form expressed in skeletal muscle and liver is an mRNA that contains exons 4 and 6. Its protein product is cleaved with production of mature IGF-1 and E-peptide variant designated as Ea [1]. Another splice form contains exon 4, initial part of exon 5 and exon 6 that is translated in a reading frame different from that of IGF-1Ea [2], [3]. This splice variant known as IGF-1Eb in rodents and IGF-1Ec in humans is present in both liver and muscles. The expression of the latter form was shown to increase significantly in response to muscle damage and mechanical stimuli and is of particular interest to muscle biochemists [3], [4], [5]. Two and a half hours after intense muscle performance the content of this mRNA increases 4–8-fold, thus the novel translated protein was named mechano-growth factor (MGF).

IGF-1 and MGF exert different physiological actions related to functional muscle hypertrophy and regeneration. MGF activates proliferation of myoblasts but inhibits their differentiation and fusion with myotubes, whereas IGF-1 activates the latter process [6]. MGF acts on target cells through the receptors different from those for IGF-1. Moreover, chemically synthesized 24-aa peptide with the sequence that is present in human MGF but absent in IGF-1 showed the same effect on myoblasts in culture as full-length MGF expression product [6]. The perspectives of MGF use for the improvement of muscle activity indicators of the aged people, patients with some forms of myodystrophia and sportsmen are widely covered in the literature [7], [8].

The mechanisms of induction of MGF synthesis are still poorly understood. Both the mechanical load [9], [10] and treatment with skeletal muscle homogenate [11] induced MGF expression in myoblasts in culture. Mechanical load was also shown to stimulate MGF synthesis in tendon [12]. Growth hormone, the classical inductor of IGF-1 expression, activated MGF synthesis in myoblast cell line C2C12 [13]. Such cellular stress factors as hyperthermia and acidification of culture medium were shown to stimulate MGF expression both in primary myoblasts and in differentiated multinuclear myotubes [14]. At the same time intracellular pathways of signal transduction leading to activation of MGF expression are almost unexplored. The aim of this work was to investigate the possible participation of secondary messengers in the regulation of MGF synthesis in myoblasts and differentiated myotubes in culture and to study the specificity of this response by the measurement of the expression of another splice form, IGF-1Ea.