Molecular and Cellular Endocrinology 399 (2015) 69–77

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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

Induction of insulin-like growth factor 1 splice forms by subfragments of myofibrillar proteins Irina V. Kravchenko a, Vladimir A. Furalyov a, Spyros Chatziefthimiou b, Matthias Wilmanns b, Vladimir O. Popov a,c,* a

Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskiy Prospect 33, 119071 Moscow, Russia European Molecular Biology Laboratory, Hamburg Unit, c/o DESY, Notkestraße 85, 22603 Hamburg, Germany c Kurchatov NBIC Centre, Russian National Research Centre “Kurchatov Institute”, Akademika Kurchatova sq. 1, 123182 Moscow, Russia b

A R T I C L E

I N F O

Article history: Received 7 April 2014 Received in revised form 19 August 2014 Accepted 19 August 2014 Available online 22 August 2014 Keywords: Insulin-like growth factor 1 Mechano-growth factor Titin Myomesin Fn type III domain Ig-like domain

A B S T R A C T

Expression of insulin-like growth factor 1 (IGF-1) mRNAs splice forms was recently shown to be stimulated by myofibrillar proteins released from the damaged muscle. In this study, we report that individual subfragments of titin and myomesin composed of Fn type III and Ig-like domains can activate expression of two IGF-1 splice forms in cultured myoblasts, both at protein and mRNA levels. Competition studies showed that each of the domain-types interacts with its own receptor. Induction of IGF-1 expression caused by domains of different types showed dissimilar sensitivity to inhibitors of regulatory cascades. The effect of Fn type III domains was more sensitive to inhibition of Ca2+/calmodulin dependent protein kinase, whereas the effect of Ig-like domains showed greater sensitivity to the inhibition of the adenylyl cyclase–cAMP– PKA pathway. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Insulin-like growth factor 1 (IGF-1) is one of the most important physiological regulators affecting various cellular, tissue and organ functions. IGF-1 is known as an endocrine hormone stimulating overall somatic growth, including skeletal muscle (Frank, 2007). In skeletal muscle it also acts as a paracrine and autocrine factor, particularly in response to trauma and mechanical strain (Glass, 2003; Rotwein, 2003). There are a number of different protein products encoded by the Igf1 gene. IGF-1 mRNA is translated as a long precursor that is further cleaved forming the N-terminal peptide, mature IGF-1 and the C-terminal fragment known as the E-peptide (Musaro et al., 2001). IGF-1 pre-mRNA can undergo alternative splicing that is responsible for generation of several forms of E-peptide including Ea, Eb and Ec in humans or Ea and Eb in rodents. The major IGF-1 mRNA splice form present both in skeletal muscle and liver is an mRNA bearing exons 4 and 6. The other splice variant of the gene contains exon 4, initial part of the exon 5 and the exon 6 that is translated in a reading frame different from that of IGF-1Ea (Chew et al., 1995; Yang et al.,

* Corresponding author. Address: Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskiy Prospect 33, Moscow 119071, Russia. Tel./fax: +7 495 952 34 41. E-mail address: [email protected] (V.O. Popov). http://dx.doi.org/10.1016/j.mce.2014.08.010 0303-7207/© 2014 Elsevier Ireland Ltd. All rights reserved.

1996). This form is classified as IGF-1Eb in the rat and IGF-1Ec in humans. The latter form attracted special interest of muscle biochemists because its expression was reported to drastically increase in response to mechanical stimuli and tissue damage (Hameed et al., 2003; McKoy et al., 1999; Yang et al., 1996) and to peak before IGF1Ea maximum (Hill and Goldspink, 2003). 2.5 hours after intensive muscle exercise the level of this mRNA rises 4–8 fold, thus the respective encoded protein was named mechano-growth factor (MGF). A confusion exists concerning the terminology: in some papers the name MGF is attributed solely to the C-terminal part of the protein, the 24 amino acid E-peptide, while the others use the name MGF for full-size protein product (for example, refer to a review of Dai et al., 2010). In this paper we will use the term MGF to designate the full-size protein product comprising IGF-1 and C-terminal peptide, and MGF E-peptide to refer exclusively to its C-terminal part. The mechanisms of induction of IGF-1 synthesis in muscle tissue and how a shift of splicing pattern from a normal major form of IGF1Ea toward IGF-1Ec is affected are still insufficiently understood. Growth hormone, the classical inductor of IGF-1 expression, activated IGF-1 (Frost et al., 2002) as well as MGF synthesis (Imanaka et al., 2008) in myoblast cell line C2C12. The mechanical load also is a well known stimulator of IGF-1 and MGF (Li et al., 2009; McKoy et al., 1999) expression. Some cellular stress factors including hyperthermia and acidification (Kravchenko et al., 2008) induced MGF expression in myoblasts in culture. A second messenger, cAMP, was

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Fig. 1. Scheme of the domain organization of the myomesin molecule and of the fragment of the titin molecule used in the present study. Colour coding: red – individual domains able to activate IGF-1Ea/IGF-1 and IGF-1Ec/MGF synthesis; pink – group of domains able to activate IGF-1Ea/IGF-1 and IGF-1Ec/MGF synthesis; blue – individual domains unable to activate IGF-1Ea/IGF-1 and IGF-1Ec/MGF; white – not tested. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

shown to be implicated in the synthesis of both splice forms of IGF-1 mRNA, IGF-1Ea and IGF-1Ec as well as IGF-1 and MGF proteins, in human and murine myoblasts as well as in differentiated myotubes (Kravchenko et al., 2011). Regulatory cascades associated with protein kinases A and C have been shown to be implicated in signal transduction (Kravchenko et al., 2011). Recently, it was also shown that the damaged muscle releases certain myofibrillar proteins identified as titin, myomesin and myosin-binding protein C that stimulate IGF-1Ea and IGF-1Ec as well as IGF-1 and MGF protein expression in normal myoblasts and myotubes Kravchenko et al., 2012). It is well known that titin and myomesin are among the major scaffolding proteins of muscle tissues (titin is the third most abundant muscle protein after actin and myosin) and function as filaments and filament bridges respectively. These proteins have a modular structure and are mainly composed of long arrays of immunoglobulin-like (Ig) and fibronectin type III (Fn III) domains (Kenny et al., 1999; Li et al., 2002; Obermann et al., 1996; Tskhovrebova and Trinick, 2003; Williams et al., 2003). For the understanding of the way by which these gigantic, up to several millions of daltons in molecular weight in the case of titin, proteins can transduce a signal inside the myocyte, it is required to study a response of MGF synthesis against the subfragments of these proteins. To enable this research we capitalized on the use of recombinant fragments of titin and myomesin composed of one or more adjacent Ig and/or Fn III domains (Fig. 1). The aim of this work was to study the ability of the individual Fn type III and Ig domains of titin and myomesin to induce synthesis of IGF-1 splice forms, as well as respective proteins, and to investigate the signaling pathways involved. In this study, we provide experimental evidence on how protein fragments comprising individual Fn III and Ig domains could stimulate the induction process. Moreover, we were able to show that these two structural domains transduce signals through two different types of receptors. 2. Materials and methods 2.1. Cell culture Primary human myoblast cultures were obtained as earlier described (Kravchenko et al., 2011). All experiments were performed using cells from 5 to 15 passages. Purity of the myoblast cultures (>95%) was evaluated by anti-desmin immunostaining. The experiments on the induction of IGF-1 splice form synthesis in cell cultures were performed as follows. 2 × 105 primary myoblasts were seeded at 60-mm Petri dish (Corning-Costar) in 5 ml of a growth medium (DMEM with 10% fetal calf serum) and left overnight. The cells were subjected to the tested proteins with or without regulatory enzyme

inhibitors (all from Sigma) samples dissolved in 0.2 ml of phosphate buffered saline (PBS) for 24 hours in the experiments with mRNA or 48 hours in the experiments with protein. The following inhibitors were used (their targets are denoted in parentheses): GF109203X (protein kinase C and glycogen synthase kinase-3), SP600125 (JNK), manumycin A (Ras farnesyltransferase), dideoxyadenosine (adenylyl cyclase), Rp-cAMPS (protein kinase A) and KN93 (Ca2+/calmodulin-dependent protein kinase II). 2.2. Recombinant proteins The DNA sequences (Titin_HUMAN, Q8WZ42 and MYOM1_ HUMAN, P52179) encoding titin domains (TA166-167 with a. a. 31648–31848, TA170 with a. a. 32047–32143 and TM4 with amino acid residues 33294–33395) and myomesin domains (My2 with a. a. 273–374, My3 with a. a. 409–502, My4 with a. a. 503–605, My5 with a. a. 635–731, My7-My9 with a. a. 935–1227, My11-My13 with a. a. 1352–1666) respectively were amplified by PCR from cDNA libraries. The PCR products were cloned into the pETM11 vector (EMBL), a vector carrying an N-terminal hexa-histidine tag and a tobacco etch virus cleavage site N-terminal to the titin and myomesin encoding sequence. The constructs were expressed in Escherichia coli strain BL21 (DE3) CodonPlus-RIL. The purification protocol included the Ni-NTA affinity chromatography step, removal of the hexa-histidine tag by 6–8 h of incubation with tobacco etch virus protease and a final purification step of size exclusion chromatography (GE, Superdex 75 16/60). 2.3. Quantitation of mRNA of IGF-1 splice forms For measurements of mRNA of IGF-1 splice forms total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. The integrity of RNA was confirmed by visual inspection of ethidium bromide stained 18S and 28S rRNA under UV light. 1.5 μg of each sample of RNA was reverse-transcribed in 25 μl using RT kit with oligo-dT primer (Sileks, Russia). Levels of expression of IGF-1 splice forms were determined by real-time PCR (QRT-PCR) performed with CFX96 system (Bio-Rad) using a reagent kit (Syntol, Russia) containing the fluorescence dye SYBR green. Betaactin was used as a reference gene. Amplification of cDNA samples was carried out according to the following protocol: activation of HotTaq polymerase at 95 °C for 5 min; denaturation at 95 °C for 15 sec, annealing at 60 °C for 30 sec and elongation at 72 °C for 45 sec, 40 cycles. The following PCR primers were used: human IGF-1Ec, forward, 5′-ACCAACAAGAACACGAAGTC-3′, reverse, 5′CAAGGTGCAAATCACTCCTA-3′ (Kim et al., 2005); human IGF-1Ea, forward, 5′-GCCTGCTCACCTTCACCAGC-3, reverse, 5′-TCAAATGTACTTC

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CTTCTGGGTCTTG-3′ (Aperghis et al., 2009), human β-actin, forward 5′-GGCGGCACCACCATGTACCCT-3′, reverse 5′-AGGGGCCGGACT CGTCATACT-3′ (Abrahamsen et al., 2003). Relative expression of IGF-1Ec and IGF-1Ea was calculated using 2−ΔΔCT method. Four independent experiments were performed for each experimental condition and the averages (±SEM) of the results are presented in the figures and in the table. The quantitative values were normalized to the samples of mRNA from the untreated cells taken as a control. To assess PCR specificity, melting curves in the 55–95 °C interval with measurement of fluorescence were generated at the end of each PCR run. A single melting peak and a single band on 2% agarose gel electrophoresis were detected for each gene product. 2.4. Quantitation of MGF and IGF-1 proteins In order to determine secreted IGF-1 protein concentration, culture supernatants from cells on a 60-mm Petri dish were taken, and IGF-1 levels were measured with Quantkine ELISA kit (R&D Systems) according to the manufacturer’s instructions. MGF concentration measurements were performed in cell lysates because the sensitivity of the test system used was insufficient for determining MGF concentrations in culture supernatants. Cells on a 60-mm Petri dish were lysed by pipetting in 0.2 ml PBS containing 0.05% Tween 20 (PBS-T) and a mixture of protease inhibitors (Sigma-Aldrich). The MGF concentration was estimated with a sandwich ELISA assay based on monoclonal antibodies to full-length MGF protein as described earlier (Kravchenko et al., 2006) using the amplification system. The assay is specific to MGF and allows to discriminate it from IGF-1 protein. Microtiter plates (CorningCostar) were coated with 8B9 monoclonal antibody (specific to C-terminal part of MGF molecule, the MGF E-peptide) solution (10 μg/ml) overnight at 4 °C. After washing with PBS-T plates were incubated with 100 μl of cell lysates for 1 h at 37 °C. After washing with PBS-T, the conjugate of alkaline phosphatase with 4F3 monoclonal antibody (specific to N-terminal part of MGF molecule, the IGF-1 moiety) (1:10,000 dilution) was added for 1 h at 37 °C, and then washed away. Plates were developed using ELISA amplification system (Invitrogen) according to the manufacturer’s instructions. The absorbance was measured at 492 nm by Multiscan microplate photometer (Thermo Labsystems). For comparative purposes IGF-1 protein concentrations in cell lysates were also measured. Cells on a 60-mm Petri dish were lysed by pipetting in 0.2 ml PBS-T with a mixture of protease inhibitors. Cell lysates were diluted 50 times by PBS-T with protease inhibitors. IGF-1 levels in diluted lysates were measured with Quantkine ELISA kit (R&D Systems) according to the manufacturer’s instructions. Four independent experiments were performed for each experimental condition and the averages (±SEM) of the results are presented on the figures. MGF concentrations and cell lysate IGF-1 concentrations were normalized to mg of total protein. Total protein concentration was determined using the Lowry method with the Bio-Rad DC Protein Assay kit. 2.5. Iodine labeling and competition experiments 125 I labeling of proteins was performed with chloramine T (Sigma) as usual (Hunter, 1970). Briefly, the reagents were added as follows: 5 μl of 0.2 M sodium phosphate buffer, pH 7.5; 1 μl of protein (1 mg/ ml in the same buffer), 20 μl of Na125I (Isotope, Russia), containing 20 MBq; 5 μl of chloramine T (5 mg/ml); 50 μl of sodium thiosulfate (1.2 mg/ml); 50 μl of NaI (20 mg/ml); 4 μl of bovine serum albumin (BSA, 10 mg/ml). The reaction was carried out at 4 °C in glass tubes, and a few seconds elapsed before the addition of each

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new reagent. 125I-labeled proteins were purified at Sephadex G-25 column. For investigation of the binding of 125I-labeled proteins to the cell surface, myoblasts were seeded at 24-well plates in a growth medium (5 × 104 cells per well). 24 h later, the plates were washed 3 times with ice-cold PBS, and 0.4 ml of ice-cold solutions containing the labeled proteins in PBS with 5 mg/ml BSA were added to the wells. The plates were incubated at 4 °C for 2 hours, in order to avoid internalization of the receptor and/or ligand, and were quickly washed 7 times with ice-cold PBS. The proteins were solubilized with 0.5 ml of 0.1 M NaOH, solutions were neutralized with HCl, and mixed with 10 ml of dioxane scintillator. Samples were counted for radioactivity in a Beckman liquid-scintillation in 125I channel. For investigation of competition between different domains, 125Ilabeled recombinant proteins at 50 ng/ml concentration were added to wells with or without unlabeled ligands at 20 μg/ml. For binding constant measurement myoblasts were incubated with increasing amounts of 125I-labeled domains with (for nonspecific binding) or without (for total binding) a 400-fold excess unlabeled ones. The specific binding was obtained after subtracting the nonspecific binding from the total binding. The dissociation constants (Kd) were determined using the Scatchard plot, from the saturation-binding experiment, using the program Prism (version 4). 2.6. Statistical analysis Data are presented as mean ± standard error of the mean of four separate experiments. Statistical significance of difference between each experimental group and the control was determined using twotailed Student’s t-test. The difference among the means of multiple groups was analyzed by one-way analysis of variance followed by the Tukey test. A difference was defined as significant at p < 0.05. 3. Results 3.1. Ability of different domains to induce IGF-1 splice forms and related proteins Several subfragments of titin and myomesin protein molecules were used in this study (Fig. 1, Table 1). We have tested constructs of these two proteins, containing either Ig (My2, My3, My11-13, TM4) or Fn III (My4, My5, TA166-167, TA170), or both types of domains (My7-9). From the tested constructs, My2, My3, My4, My7-9 and TA166167 were not able to stimulate either IGF-1 or MGF synthesis, whereas My5 and TA170 (composed of Fn III domains) and My1113, TM4 (Ig domains) were shown to activate the expression of IGF1Ea and IGF-1Ec as well as IGF-1 and MGF proteins (Table 1), thus manifesting effect on both mRNA and protein levels and for both splice forms of IGF-1. The pattern of IGF-1 protein expression was the same irrespective of the method used (quantification in the supernatant or cell lysates) and correlated with that for MGF. Thus, the data show that the effect is specific, and not all the generic Ig or Fn III folds are able to induce IGF-1/MGF synthesis. They also show that stimulation of IGF-1 and MGF synthesis is linked, since the structural fragments that are able to induce IGF-1Ec and MGF also stimulate IGF-1Ea and IGF-1 synthesis, while the fragments that do not stimulate MGF synthesis also failed to induce IGF1. Stimulation of IGF-1Ec was always slightly higher than that of IGF1Ea by a factor of ~1.4 on the edge of statistical significance. It could also be noted that the fragments comprising Fn III domains were always slightly more potent inductors compared to Ig containing fragments. This difference was manifested both on mRNA and protein levels.

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Table 1 Effects of individual domains of myofibrillar proteins on IGF-1Ea/IGF-1 and IGF-1Ec/MGF expression in human myoblasts. Protein fragment

Domain(s)

Relative IGF-1Ec levels, fold

Relative IGF-1Ea mRNA levels, fold

MGF, pcg/mg of total protein

Secreted IGF-1, pcg/ml

IGF-1, pcg/mg of total protein

My2 My3 My11-13 TM4 My4 TA166-167 My5 TA170 My7-9

Ig-like

1.1 ± 0.2 0.9 ± 0.1 8.6 ± 1.2** 7.0 ± 1.3** 1.0 ± 0.2 1.4 ± 0.4 13 ± 1.3** 9.8 ± 1.4** 1.1 ± 0.3

0.9 ± 0.2 1.0 ± 0.2 5.4 ± 0.6** 5.6 ± 0.6** 1.0 ± 0.2 1.2 ± 0.3 9.2 ± 0.8** 6.9 ± 0.7** 1.1 ± 0.2

6±4 4±3 61 ± 5** 54 ± 8** 4±2 7±6 84 ± 6** 79 ± 7** 5±3

48 ± 15 41 ± 18 221 ± 17** 202 ± 17** 33 ± 14 39 ± 16 305 ± 24** 258 ± 21** 41 ± 17

429 ± 113 450 ± 101 2434 ± 207** 2852 ± 184** 417 ± 95 405 ± 108 3910 ± 219** 3015 ± 201** 493 ± 111

Fn type III

Ig-like and Fn type III

Primary human myoblasts were incubated with 5 μg/ml recombinant proteins for 24 hours (mRNA measurements) or 48 hours (protein measurements). Once total RNA has been isolated and reverse-transcribed, the relative amount of IGF-1Ea and IGF-1Ec was determined by QRT-PCR (the reference gene was β-actin). The quantitative values were normalized to the samples of mRNA from the untreated cells. Secreted IGF-1 concentrations and IGF-1 concentrations in cell lysates were measured with Quantkine ELISA kit. MGF protein concentrations were measured in cell lysates by sandwich ELISA with signal amplification. Shown is the mean ± s.e. of results from 4 separate experiments. Significantly different from control values: **p < 0.01.

3.2. Competition for receptor binding of protein fragments containing domains of different types Experiments with radioactively labeled recombinant protein fragments containing domains of different types showed that human myoblasts bound 125I-labeled proteins in a time-dependent manner, reaching equilibrium in about 2 h (shown in Appendix SA). Binding of the labeled proteins with the cells was quite specific and was strongly inhibited by the initial addition of their unlabeled form. As shown in Fig. 2, the addition of a 400-fold excess of unlabeled myomesin 5 (Fn III) or unlabeled titin M4 (Ig) resulted in a 6.7-

times and 12.6-times reduction of binding of the labeled protein respectively. Binding of 125I-labeled myomesin 11–13 and titin A170 decreased only slightly in the presence of excess of unlabeled form, so the experiments with them were discontinued. To find out if protein fragments containing different structural protein domains compete for the same or different receptors, the following set of competition binding experiments was performed: binding of the 125I-labeled myomesin 5 fragment (Fn III) with myoblast membrane in the presence or absence of unlabeled myomesin 11–13 (Ig), titin A170 (Fn III) and titin M4 (Ig) was investigated. It was shown that the fragments containing the other domain type, i.e. Ig, did not compete with My5, since neither My11-13 nor TM4 at 20 μg/ml (0.56 μM and 1.76 μM, respectively) had any effect on binding of the 125I-labeled My5 at 50 ng/ml (4.83 nM) with membrane receptors. On the contrary, a protein fragment containing the same domain type, TA 170, was a potent inhibitor of My5 binding. The unlabeled TA170 (Fn III) at the same concentration inhibited binding 4.8-fold (Fig. 2A). The same inhibition pattern was obtained with the protein fragments comprising Ig domains. Binding of the 125I-labeled titin M4 fragment (Ig domain) with myoblast membrane was inhibited 10.5times by My11-13 (same Ig domain) but was not affected by the fragments containing Fn III domains – My5 and TA170 (Fig. 2B). 3.3. Measurement of dissociation constants and EC50 for myomesin 5 and titin M4 protein fragments

Fig. 2. Binding studies of 125I-labeled recombinant proteins to human myoblasts in the presence of unlabeled ones. 125I-labeled myomesin 5 (125I-My5) (A) or 125Ilabeled titin M4 (125I-TM4) (B) at 50 ng/ml was added to the cells at 4°C with or without myomesin 5 (My5) or myomesin 11–13 (My11-13) or titin M4 (TM4) at 20 μg/ ml. The cells were harvested 2 h later. Graph represents the mean ± s.e. of 4 measurements. Significantly different from control values: **p < 0.01.

To determine the affinity constants of myomesin 5 and titin M4 protein fragments toward myoblasts, cells were titrated with increasing concentrations of 125I-labeled recombinant proteins. An account was taken for nonspecific binding (as defined by binding in the presence of 400-fold excess of the corresponding unlabeled protein), that was less than 15% of the total binding in the case of labeled myomesin 5 and less than 8% of the total binding in the case of labeled titin M4 (data not shown). Linear Scatchard plots of specific binding were obtained for both My5 (Fn III) (Fig. 3A) and TM4 (Ig) (Fig. 3B) (correlation coefficients 0.943 and 0.952, respectively), suggesting thus a unique class of binding sites for each recombinant protein. My5 binding was characterized by an apparent dissociation constant (Kd) of 71 ± 38 nM, whereas the Kd for the TM4 was of similar value (56 ± 31 nM). The effect of the concentration of the respective recombinant protein fragments on the synthesis of IGF-1Ec was investigated and relevant EC50 parameters for My5 (2.2 μg/ml or 213 nM) and TM4 (1.6 μg/ml or 157 nM) were measured (Fig. 3C, 3D). Concentration

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Fig. 3. Concentration dependence of binding with myoblasts and MGF-inducing activity of myomesin 5 (A, C) and titin M4 (B, D). (A) and (B) Scatchard plot analysis of 125I-labeled myomesin 5 (A) and 125I-labeled titin M4 (B) receptor binding sites in intact myoblasts. Each point is the mean of quadruplicate determinations. Binding data were analyzed with GraphPad Prism 4. (C) and (D) Concentration dependence of IGF-1Ec expression from concentrations of myomesin 5 (C) and titin M4 (D). Graph represents the mean ± s.e. of 4 measurements.

dependencies of IGF-1Ea mRNA induction were quite similar with close EC50 parameters (data not shown). 3.4. Sensitivity of the effects of different protein fragments toward inhibitors of regulatory enzyme cascades To investigate a possible difference in the mechanisms of intracellular signaling via two signaling pathways employing Ig and Fn III domain types, the effects of several inhibitors of regulatory protein kinases and Ras protein on IGF-1Ec and IGF-1Ea induction were studied. None of the inhibitors studied exhibited cytotoxic effect on myoblasts at the concentrations used (cell viability determined with Trypan blue was not less than 95% in all the cases). Protein kinase C inhibitor GF109203X at 1 μM, JNK inhibitor SP600125 at 30 μM and farnesyltransferase inhibitor manumycin A, at a concentration of 10 μM, had no influence on IGF-1Ea and IGF1Ec synthesis activation induced by either the Ig domain TM4 or the Fn III domain My5 (data not shown).

Adenylyl cyclase inhibitor dideoxyadenosine (DDA) and protein kinase A inhibitor Rp-cAMPS were more potent inhibitors of both the IGF-1Ea/IGF-1 and IGF-1Ec/MGF induction processes (both on the protein and mRNA levels) by Ig domains than by Fn III containing protein fragments. The inhibitory effects were always 2–2.8 times more profound in the case of Ig domains compared to Fn III domains (Fig. 4). The opposite phenomenon was observed with Ca2+/calmodulin dependent protein kinase II inhibitor KN93. This inhibitor was more effective in retarding activation processes induced by Fn III domains, compared to the constructs comprising Ig domains. In this case, the difference varied in the same range (1.7–2.9 times). In the absence of the inducing protein fragments KN93 by itself as well as DDA and Rp-cAMPS did affect neither IGF-1, nor MGF levels (data not shown). Additivity effects of My5 (Fn type III domain) and TM4 (Ig domain) were also studied. The simple additivity of effects of both domains was observed at low concentrations up to 1.67 μg/ml, whereas there

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Fig. 4. Effects of several regulatory protein inhibitors on the IGF-1Ec/MGF (A, B) and IGF-1Ea/IGF-1 (C, D) induction by Fn III and Ig domains of myofibrillar proteins. Primary human myoblasts were treated or not with 10 μM dideoxyadenosine (DDA) or 100 μM Rp-cAMPS or 10 μM KN93 for 30 min and then incubated with 5 μg/ml recombinant proteins for 24 hours for subsequent mRNA measurements (A, C) or 48 hours for MGF and secreted IGF-1 protein measurements (B). Once total RNA has been isolated and reverse-transcribed, the relative amount of IGF-1Ec and IGF-1Ea was determined by QRT-PCR (the reference gene was β-actin). The quantitative values were normalized to the samples of mRNA from the cells treated with corresponding recombinant protein without the addition of inhibitors. MGF protein concentrations were measured in cell lysates by sandwich ELISA with signal amplification. Secreted IGF-1 concentrations were measured in culture supernatants with Quantkine ELISA kit. Protein concentrations were normalized to the samples from the cells treated with corresponding recombinant protein without the addition of inhibitors. All values are presented in percentage. Graph represents the mean ± s.e. of 4 measurements. Significantly different from values of samples treated with corresponding recombinant protein without inhibitors: *p < 0.05, **p < 0.01. Significantly different from values of samples treated with corresponding recombinant protein + KN93: #p < 0.05, ##p < 0.01.

was no additivity at high concentrations of 15 μg/ml (data are shown in Appendix SB). Finally, no synergism of effects between fragments comprising different types of domains, at any concentration, was observed. 4. Discussion All the three myofibrillar proteins previously shown to stimulate IGF-1 and MGF synthesis, namely gigantic titin, myomesin and myosin-binding protein C (Kravchenko et al., 2012), are mainly composed of a number of specific structural units. For example, some titin isoforms have a molecular mass of about 4 MDa, 90% of which consists of repeating Ig and Fn III structural domains (112 and 132 units respectively). Both these domains are known to provide binding sites to a number of proteins including signaling molecules (Kontrogianni-Konstantopoulos et al., 2009). Thus, to understand further the structural basis of the stimulating effect of titin and other similar myofibrillar proteins on IGF-1 and MGF synthesis it was essential to work with isolated recombinant protein fragments composed of individual structural domains. In this study, fragments of two proteins – titin and myomesin based on individual, either Ig or Fn III domains, have been used to study MGF and IGF-1 synthesis.

We tested in total nine recombinant protein constructs, consisting of two different domain-types for the ability to activate IGF1Ea/IGF-1 and IGF-1Ec/MGF expression. It was found that some of the Ig and Fn III studied domains were able to induce IGF-1 and MGF expression both on mRNA and protein levels. IGF-1Ea vs IGF-1Ec and IGF-1 vs MGF showed qualitatively the same induction pattern. Those domains that were able to induce IGF-1Ec/MGF were also active toward IGF-1Ea/IGF-1, while the fragments that have not stimulated synthesis of the former also failed to induce synthesis of the latter. Both alternative splice forms, IGF-1Ea and IGF-1Ec, were substantially (5.5- to 13-fold) induced against the background upon stimulation. However we did not observe any dramatic preference of one splice form over the other under our experimental conditions – 24 h period of induction for RNAs and 48 h for proteins. In the previous work (Kravchenko et al., 2011) it was shown that over these periods IGF-1Ec and MGF reached their maximum values respectively when induced by the second messengers. The prevalence of IGF-1Ec over IGF-1Ea was nevertheless always slightly higher by a factor of about 1.3–1.6 that could reflect a minor shift in splicing in favor of IGF-1Ec and MGF. Taking into account a known dominance of the major splice form, the IGF-1Ea, over the IGF-1Ec under normal conditions that reaches several orders of magnitude (Aperghis

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et al., 2009), the observed slight preference for IGF-1Ec upon stimulation is unable to result in any sizable burst of MGF protein against the background IGF-1. MGF stimulates myoblast proliferation, but there is contradictory data about its influence on cell differentiation. In an early work MGF (as well as its E-peptide) was described to attenuate myoblast differentiation and fusion with myotube formation (Yang and Goldspink, 2002), but recent investigation revealed its activity as an activator of differentiation of satellite cells (Kandalla et al., 2011). There is an ongoing discussion about the actual active moiety of MGF. Recently it was reported that mature IGF-1 and full-length MGF stimulated myoblast proliferation and differentiation (Fornaro et al., 2014), whereas MGF E-peptide had no effect on these processes. However this observation is inconsistent with some other earlier results (Ates et al., 2007; Qin et al., 2012; Yang and Goldspink, 2002). Thus, MGF E-peptide biological function is called into question now (Rotwein, 2014) despite its being widely debated quite recently (for example, Dai et al., 2010). Whatever the situation really is, the presence of the biological activity of full-length MGF whose expression is studied by us in the present and earlier works is generally accepted. The possibility to use MGF or fragments thereof for the improvement of muscle activity indicators of the aged people and sportsmen is the subject of wide speculation (Goldspink et al., 2008; Mavrommatis et al., 2013). Competition studies between various domains able to stimulate IGF-1 and MGF expression for binding with myoblast membrane showed that different Ig domains compete with each other for a common receptor/binding site, but did not compete with Fn III domains. In an analogous manner, different Fn III domains did not compete with Ig domains but competed with each other. These data allow us to conclude that two different receptor types recognizing fragments (individual structural domains) of myofibrillar proteins exist on myoblast membrane. One type of receptor binds Fn III domains, whereas receptors of the second type interact with Ig domains. Dissociation constants determined from titration studies appear to be quite similar for both receptor types – 71 ± 38 nM for Fn III domain and 56 ± 31 nM for Ig domain and correlate with respective EC50 values. Binding constants of Fn III as well as Ig domains are relatively modest. However, both titin and myomesin contain several domains able to bind to myocyte membrane and to stimulate expression of IGF-1 and MGF. The presence of multiple binding domains increases the probability of forming a contact of myofibrillar protein with a cell surface, the life-time of such a complex and, therefore, the intensity of physiological response. Furthermore the additive effects of domains belonging to different types observed in the present communication would provide the summation of responses of the cell to each of the signals. The recognition of the putative agonist(s) by both types of receptors is most probably spatially governed. The extensive treatment of the protein fragments able to induce MGF, either containing Ig or Fn III domains, with proteases (trypsin or chymotrypsin) resulted in the complete loss of their ability to stimulate MGF synthesis (unpublished results). Thus, the receptor binding site should recognize the structural, not the linear (short peptide sequence) determinant of the interacting protein fragment(s). The two signaling cascades originating from two receptor types seem to have a different organization. Activation of IGF-1 and MGF expression by Ig domains is rather sensitive to the inhibitors of the adenylyl cyclase–cAMP–PKA system, whereas Ca2+/calmodulin dependent protein kinase inhibition attenuates it to a lesser extent. On the contrary, stimulation of expression of both IGF-1 splice forms by Fn III domains demonstrates lower sensitivity to both adenylyl cyclase and PKA inhibitors compared to Ca2+/calmodulin dependent protein kinase inhibitor (Fig. 4). The inhibition effect is the most pronounced in the case of secreted IGF-1 protein. Perhaps, inhibi-

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tors of both types can affect somehow the processes of maturation of IGF-1Ea proprotein and/or secretion of mature IGF-1. The observed additivity of the effects induced by Fn and Ig type of domains at low concentrations and a lack of additivity at higher (saturating) concentration may be explained by convergence of both signaling pathways and existence of a common “rate limiting interaction” governing the overall response. At low concentrations of the effectors, the rate limiting interaction is not saturated and could be affected by both signaling pathways, thus the additivity is observed. However, at higher concentration of any of the effectors leading to saturation of the rate limiting interaction the latter becomes non-responsive to the other signaling pathway. Meanwhile, incomplete inhibition of the stimulation of both IGF-1 and MGF synthesis by both types of inhibitors (affecting either PKA signaling or Ca2+ signaling systems) may point to a substantial crosstalk and shunting of a signal transduction between two pathways. Further down the integration point a signal is further transmitted to the nucleus leading to an increase in the overall level of transcription of the Igf1 gene over background values. Alterations of the functioning of splicing and/or mRNA degrading machinery may lead to a preference toward IGF-1Ec with concomitant synthesis of respective protein products. It is evident that protein kinase A and cAMP play an essential role in the signal transduction, at least via one of the pathways (employing Ig specific receptor as shown in the present study). It was described earlier that such a physiological activator of cAMP synthesis as prostaglandin E2 is able to stimulate expression of both IGF-1 splice forms (Kravchenko et al., 2011). Our present results corroborate an important role of cAMP level elevation in the course of MGF and IGF-1 induction. The role of Ca2+ ion in the regulation of MGF synthesis is somewhat controversial. Calcium ionophore A23187 was shown earlier to inhibit both MGF and IGF-1Ea expressions (Kravchenko et al., 2011). However, this ionophore produces a constant build-up of calcium level in the cytosol, the situation being quite different from the physiological one when calcium is provided in short pulses during e.g. muscle contraction (physical exercise). Our present results showed the implication of Ca2+/calmodulin dependent protein kinase both in the IGF-1 and MGF syntheses. The effect was most pronounced for the induction provided by Fn III domains. It could be speculated that the treatment of cells with the protein fragments comprising these domains leads to the pulsed increase of Ca2+ ion concentration that can cause quite different physiological response than its long term permanent elevation produced by calcium ionophore (the role of dynamics of concentration change of regulatory molecules in shaping of the physiological response was reviewed by Purvis and Lahav, 2013). It should be noted that intense muscle exercise is accompanied by continuous pulse elevations of cytosolic calcium concentration which hypothetically can induce the start-up of MGF synthesis. In a previous work we showed that protein kinase C could be somehow implicated in the induction of different IGF-1 splice form synthesis (Kravchenko et al., 2011). However, this response on a time scale lagged considerably behind that mediated by PKA. In this study we were unable to verify direct implication of PKC in IGF-1 and MGF stimulation as the inhibitor of PKC failed to affect induction of these growth factors by both Ig and Fn III domains. It favors the proposal that PKC is not the major regulatory hub stimulating IGF-1 biosynthesis but could perform some subordinate shunting role. Lack of effect of a JNK inhibitor (Jun kinase being downstream to PKC) on IGF-1 induction (present work) further corroborates this proposal. On the other hand, GF109203X at 1 μM concentration effectively inhibits only a part of PKC isoform spectra (Martiny-Baron et al., 1993). It may be proposed that some atypical PKCs less sensitive to this inhibitor are involved in the regulation of IGF-1 gene expression by myofibrillar proteins.

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The potent trigger of the start-up of MGF induction and thus initiation of the muscle repair process is considered to be a release of the essential scaffolding myofibril associated proteins like titin (Kravchenko et al., 2012). It is not probably by chance that these huge multidomain proteins act as initiators of the repair process. Release of these gigantic proteins or parts thereof is a direct manifestation of the far-reaching process of the muscle damage/deterioration that requires urgent regenerative counteraction. The known effect of such stress factors, as heating or acidification (Kravchenko et al., 2008), to induce MGF can also be regarded as a manifestation of muscle injury/deterioration process. It is well known that muscle training stimulates MGF production (based on this fact, this splice form of IGF-1 has received its trivial name), however physical exercise is also associated with local temperature increase and/or acidification that can result in muscle damage and release of scaffolding proteins triggering in the long run a change of the splicing pattern. Some parts of the scaffolding myofibril proteins, comprising both individual Ig and Fn III domains, can interact with receptors expressed on the myocyte membrane. In this study at least two types of such receptors individual for each of the domains (Ig and Fn III) have been shown to be implicated in signal transduction inside the cell. Engagement of multiple receptors during the recognition step with the multidomain agonist and their further mobilization could be an advantage for initial signal amplification. Further investigation of the structural motives of the Ig and Fn III domains of the protein fragments recognized by the respective receptors is required as well as identification of the receptors themselves. Understanding of the details of the recognition process will pave way for devising low-molecular weight molecules able to bind with aforementioned receptors, to activate them and thus endogenously induce both IGF-1 and MGF and facilitate muscle repair process. In conclusion, titin and myomesin and/or their fragments thereof released from the damaged muscle can stimulate the expression of IGF-1 splice forms initiating muscle repair process. The signal is transmitted inside the cell via two different receptors. One receptor type recognizes Ig domains of myofibrillar proteins and acts via activation of cAMP synthesis and PKA. The other receptor type interacts with Fn III domains, and its signaling implicates stimulation of Ca2+/calmodulin dependent protein kinase. The two signaling pathways converge at some stage transmitting the integrated signal to the nucleus biosynthesis machinery. Endogenous stimulation of the synthesis of IGF-1 splice forms in response to specific external stimuli could be a viable alternative to therapeutic administration of exogenous recombinant proteins or their fragments. The present study may serve as a step toward this ultimate goal. Acknowledgements These studies were supported by the grants from the Russian Foundation for Basic Research (12-04-01090a), the Molecular and Cell Biology Program of the Russian Academy of Sciences, grant N 6P, and the Bundesministerium für Bildung und Forschung (BMBF) research grant SYNC-LIFE (Contract 05K10YEA). Appendix: Supplementary Material Supplementary data to this article can be found online at doi:10.1016/j.mce.2014.08.010. References Abrahamsen, H.N., Steiniche, T., Nexo, E., Hamilton-Dutoit, S.J., Sorensen, B.S., 2003. Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients. J. Mol. Diagn. 5, 34–41.

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