Cellular Signalling 28 (2016) 60–73 Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig FHL3 differentially regulates the expression of MyHC isoforms through interactions with MyoD and pCREB Yunxia Zhang, Wentao Li, Mingfei Zhu, Yuan Li, Zaiyan Xu ⁎,BoZuo⁎ Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture and Key Lab of Agricultural Animal Genetics and Breeding, Ministry of Education, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, PR China article info abstract Article history: In skeletal muscle, muscle fiber types are defined by four adult myosin heavy chain (MyHC) isoforms. Four Received 10 June 2015 and a half LIM domain protein 3 (FHL3) regulates myoblasts differentiation and gene expression by acting Received in revised form 9 October 2015 as a transcriptional co-activator or co-repressor. However, how FHL3 regulates MyHC expression is currently Accepted 19 October 2015 not clear. In this study, we found that FHL3 down-regulated the expression of MyHC 1/slow and up-regulated Available online 22 October 2015 the expression of MyHC 2a and MyHC 2b, whereas no significant effect was found on MyHC 2x expression. Keywords: MyoD and phosphorylated cAMP response element binding protein (pCREB) played important roles in Muscle fiber type the regulation of MyHC 1/slow and MyHC 2a expression by FHL3, respectively. FHL3 could interact with FHL3 MyoD, CREB and pCREB in vivo. pCREB had stronger interaction with the cyclic AMP-responsive elements MyoD (CRE) of the MyHC 2a promoter compared with CREB, and FHL3 significantly affected the binding capacity MyHC of pCREB to CRE. We established a model in which FHL3 promotes the expression of MyHC 2a through CREB- CREB mediated transcription and inhibits the expression of MyHC 1/slow by inhibiting MyoD transcription activity during myogenesis. Our data support the notion that FHL3 plays important roles in the regulation of muscle fiber type composition. © 2015 Elsevier Inc. All rights reserved. 1. Introduction larger in diameter. The high proportion of MyHC 2b contributes to an in- crease in muscle mass [5]. However, the overall proportion of MyHC 2b Skeletal muscle comprises different types of muscle fibers. Tradition- has been shown to correlate with the occurrence of pig PSE (pale, soft ally, fiber types are classified as slow-oxidative (1 type), fast oxidative- and exudative) meat, which could lead to a fast pH decline after glycolytic (2a type) and fast-glycolytic (2b type) fibers, according to slaughter [6,7]. In human beings, fast-dominant skeletal muscle their contraction and metabolic characteristics [1]. Fiber types are con- induced by denervation easily leads to skeletal muscle atrophy com- vertible in adult skeletal muscle in response to exercise training [2]. pared with type 1 fibers [8]. Oxidative enzymatic activities in human Four postnatal fiber types exist in skeletal muscle, each defined by the skeletal muscle are correlated with fiber type composition [9].The presence of dominant MyHC isoforms (MyHC 1/slow, MyHC 2a, MyHC fastest, most powerful muscle fiber type (type 2b fibers) tends to 2b, MyHC 2x) [3]. In animal production, meat quantity and quality are be lost in the elderly population [10,11], suggesting that the compo- significantly affected by the composition of the four MyHC isoforms [4]. sition of skeletal muscle is associated with aging-related loss of mus- Skeletal muscle with a higher composition of type 2b fibers tends to be cle function and muscle disease. To date, several signal transduction pathways, including myogenic regulatory transcription factors (MRFs), insulin-like growth factors (IGFs), calcineurin-nuclear factor of acti- Abbreviations: MyHC, myosin heavy chain; FHL, four and half LIM domain protein; vated T cells (CaN-NFAT), CCAAT enhancer binding protein δ (C/EBP-δ) CRE, cyclic AMP-responsive element; CREB, cAMP-response-element-binding protein; and peroxisome proliferator-activated receptor-γ coactivator (PGC)- PKA, cAMP-activited protein kinase A; MyoG, myogenin; MyoD, myogenic differentiation 1α,havebeenreportedinregulatingfiber type-specific gene expressions 1; SDS, Sodium Dodecyl Sulfate; cDNA, complementary DNA; siRNA, small hairpin RNA; – PCR, polymerase chain reaction; MRFs, myogenic regulatory transcription factors; IGFs, [12 15]. insulin-like growth factors; CaN-NFAT, calcineurin-nuclear factor of activated T cells; The LIM domain has one or more cysteine-rich zinc fingers and C/EBP-δ, CCAAT enhancer binding protein δ; PGC-1α, peroxisome proliferator-activated regulates gene transcription [16]. The LIM domain is a zinc-binding receptor-γ coactivator-1α; CDS, coding sequence; bp, base pair; PVD, polyvinylidene fluo- structural motif, of which the consensus amino acid sequence is ride; EMSA, Electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation. CX2CX16–23HX2CX2CX2CX16–21CX2-3 (C/H/D) [17]. The LIM domain ⁎ Corresponding authors. E-mail addresses: [email protected] (Z. Xu), [email protected] lacks DNA-binding ability and is involved in gene expression and cell (B. Zuo). differentiation through protein–protein interactions [16]. The four and http://dx.doi.org/10.1016/j.cellsig.2015.10.008 0898-6568/© 2015 Elsevier Inc. All rights reserved. Y. Zhang et al. / Cellular Signalling 28 (2016) 60–73 61 a half LIM domain (FHL) proteins, including FHL1, FHL2, FHL3, FHL4 listed in Table 1. The mutants of CREB binding sites were generated and ACT, are a family of LIM-only proteins with four-and-a-half LIM using the overlapping extension PCR and mutagenic primers. domains and can act as transcriptional coactivators [18]. Fhl3 is high- ly expressed in skeletal muscle [18-20] and interacts with other pro- 2.2. Cell culture and differentiation of C2C12 myoblasts teins, including FHL2, CREB, Sox15, MZF-1, BKLF, Ang and MT-1X, as a transcriptional co-activator or co-repressor [18,21–26].FHL3has Mouse C2C12 (ATCC) myoblasts were cultured in 10% (v/v)fetal been reported to play important roles in cell growth, development, bovine serum (Gibco, Australia) in DMEM (high-glucose Dulbecco's tumorigenesis and cancer [27–30].Inskeletalmuscle,FHL3 modified Eagle's medium) (Hyclone, USA) under humidified air con- localizes to the nucleus and focal adhesions and is a significant regu- taining 5% CO2 at 37 °C, and were differentiated at confluence in lator of actin cytoskeletal dynamics in skeletal muscle [31].FHL3 DMEM with 2% horse serum (Gibco, USA). contributes to the regulation of MyoD dependent transcription of muscle specific genes during myogenesis [32]. In pig production, a 2.3. Transfection of plasmid DNA or siRNA oligonucleotides polymorphism within the coding region of pig Fhl3 is also signifi- cantly associated with meat mass and quality traits [19,20].Based Transfections were performed using plasmid (4 μg) or siRNA on these results, we speculat that FHL3 may regulate the expression (100 pmol) by Lipofectamine 2000 (9 μl) (Invitrogen, USA) after of MyHC isoforms of different muscle fiber types, thereby regulating seeded C2C12 myoblasts had been allowed to settle for 12–18 h, the fiber type composition of skeletal muscle. However, no studies according to the manufacturer's instruction. Fhl3 small hairpin RNA regarding the regulation of MyHC gene expression by FHL3 have (siRNA) oligonucleotides were synthesized according to Mm_FHL3_2_HP been reported thus far. In this study, we present a novel molecular siRNA (SI01002890, Qiagen, Germany) and transfected into C2C12 mechanism for the regulation of MyHC expression by FHL3, which myoblasts as previously described [32]. Creb siRNA oligonucleotides may contribute to meat quality control and may benefitmuscledisease (S: GGACCUUUACUGCCACAAATT; A: UUUGUGGCAGUAAAGGUCCTT) therapy in the future. and MyoD siRNA oligonucleotides (S: CCCCAAUGCGAUUUAUCATT; A: UGAUAAAUCGCAUUGGGGTT) were designed and synthesized by 2. Material and methods GenePharma (China, Shanghai). 2.1. Plasmid constructs 2.4. Quantitative real-time PCR The coding sequence (CDS) of the Fhl3 gene (870 bp) was obtained Total RNA from C2C12 myoblasts was extracted using TRIzol reagent by polymerase chain reaction (PCR) using cDNA (complementary (Invitrogen, USA). The concentration and quality of RNA were assessed DNA) of C2C12 myoblasts as a template. The 5′ and 3′ ends of the with a NanoDrop 2000 (Thermo, USA) and agarose gel electrophoresis. primers contain BamH IorXba Ienzymesites(Table 1). The ampli- One microgram of total RNA was used for reverse transcription with the fied CDS was digested with BamH IandXba I and was then ligated PrimeScript RTreagent kit with gDNA Eraser (Takara, Japan). The quan- into pcDNA3.1 using T4 DNA Ligase (Takara, Japan) to generate titative real-time PCR reaction was performed in a LightCycler 480 II pcDNA3.1-FHL3. (Roche, Switzerland) system using the THUNDERBIRD™ probe qPCR The CDS of Creb (1026 bp) was obtained by PCR using cDNA of Mix or SYBR®Green Real-time PCR Master Mix (Toyobo, Japan). The C2C12 myoblasts as a template. The 5′ and 3′ ends of the primers sequence of TaqMan probes and primers can be found in Table 2 and ΔΔ contain Kpn IorXho I enzyme sites (Table 1). The amplified CDS was were determined according to the literature [33].TheCt(2- Ct) digested with Kpn IandXho I and was then ligated into pcDNA3.1 method was used to analyze the relative gene expression data according using T4 DNA Ligase to generate pcDNA3.1-CREB. to the literature [34]. Five deletion fragments of the