Journal of Biochemistry Advance Access originally published online on June 11, 2009
Journal of Biochemistry 2009 146(3):407-415; doi:10.1093/jb/mvp090
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Subtilisin-like Proprotein Convertase PACE4 is Required for Skeletal Muscle Differentiation
Department of Biological Science and Technology, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan
*To whom correspondence should be addressed. Tel: +81-88-656-7526, Fax: +81-88-655-3161, E-mail: tsuji{at}bio.tokushima-u.ac.jp
Received April 25, 2009; Accepted May 27, 2009
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Abstract |
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Most growth factors stimulate myoblast proliferation and prevent differentiation, whereas insulin-like growth factors (IGFs) promote myoblast differentiation through the phosphatidylinositol 3-kinase (PI3K) pathway. Subtilisin-like proprotein convertases (SPCs) are involved in cell growth and differentiation via activation of pro-growth factors. However, the role of SPCs in myogenesis remains poorly understood. Here we show that PACE4, a member of the SPC family, plays a critical role in myogenic differentiation of C2C12 cells. PACE4 mRNA levels increased markedly during myogenesis, whereas the expression of other member of SPC family, furin and PC6, remained unchanged. The expression pattern of pro-IGF-II, which is processed extracellularly by SPCs, was similar to that of PACE4. The expression of shRNA targeting PACE4, but not furin, suppressed the expression of the muscle-specific myosin light chain (MLC). Interestingly, reduced expression of MLC was restored following treatment with recombinant mature IGF-II. Finally, we demonstrated that the PI3K inhibitor LY294002 blocked the induction of PACE4 mRNA, a result not observed when another myogenic differentiation inhibitor, SB203580 (p38 MAP kinase inhibitor), was employed, indicating the presence of a positive feedback loop regulating PACE4 expression. These results suggest that PACE4 plays an important role in myogenic differentiation through its association with the IGF-II pathway.
Key Words: insulin-like growth factor (IGF), myogenic differentiation, PACE4, processing, subtilisin-like proprotein convertase (SPC)
Abbreviations:
1-AT, 1-antitrypsin; 1-PDX, 1-antitrypsin variant Portland; bHLH, basic helix-loop-helix; dec-RVKR-CMK, decanoyl-Arg-Val-Lys-Arg-chloromethylketone; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FoxO, forkhead box protein O; GFP, green fluorescent protein; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; IGFBP, insulin-like growth factor-binding protein; IGF(s), insulin-like growth factor(s); MAP kinase, mitogen-activated protein kinase; MHC, myosin heavy chain; MLC, myosin light chain; MMP11, matrix metalloproteinase 11; PI3K, phosphatidylinositol 3-kinase; RT-PCR, reverse transcriptase-PCR; SEAP, secreted alkaline phosphatase; shRNA, short hairpin RNA; SPC(s), subtilisin-like proprotein convertase(s); TGF-β, transforming growth factor β