A Novel Glycerophosphodiester Phosphodiesterase, GDE5, Controls Skeletal Muscle Development via a Non-enzymatic Mechanism

Norihisa Kato,Yuichiro Fujioka,Keiko Okamoto,Yasutomi Kamei,Ikumi Yoshizawa,Noriyuki Yanaka,Noriyasu Ohshima,Masahiro Maeda,Takashi Izumi,Yuri Okazaki,Masanobu Wada,Yoshitsugu Shiro,Daniela Corda,Yoshihiro Ogawa,Yoshihito Nogusa,Stefania Mariggiò

doi:10.1074/jbc.m110.106708

Abstract

Mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) have been identified recently and shown to be implicated in several physiological functions. This study isolated a novel GP-PDE, GDE5, and showed that GDE5 selectively hydrolyzes glycerophosphocholine (GroPCho) and controls skeletal muscle development. We show that GDE5 expression was reduced in atrophied skeletal muscles in mice and that decreasing GDE5 abundance promoted myoblastic differentiation, suggesting that decreased GDE5 expression has a counter-regulatory effect on the progression of skeletal muscle atrophy. Forced expression of full-length GDE5 in cultured myoblasts suppressed myogenic differentiation. Unexpectedly, a truncated GDE5 construct (GDE5DeltaC471), which contained a GP-PDE sequence identified in other GP-PDEs but lacked GroPCho phosphodiesterase activity, showed a similar inhibitory effect. Furthermore, transgenic mice specifically expressing GDE5DeltaC471 in skeletal muscle showed less skeletal muscle mass, especially type II fiber-rich muscle. These results indicate that GDE5 negatively regulates skeletal muscle development even without GroPCho phosphodiesterase activity, providing novel insight into the biological significance of mammalian GP-PDE function in a non-enzymatic mechanism.

Highlights

Mammalian glycerophosphodiester phosphodiesterases (GPPDEs) have been identified recently and shown to be implicated in several physiological functions
We focused on a cDNA termed GDE5, showing that GDE5 is a novel glycerophosphocholine (GroPCho) phosphodiesterase and that GDE5 has an inhibitory role in skeletal muscle differentiation that is carried out independently of its GroPCho enzymatic activity
We have shown that a novel cytosolic Glycerophosphodiester phosphodiesterases (GP-PDEs), GDE5, selectively hydrolyzes GroPCho

Summary

EXPERIMENTAL PROCEDURES

Materials—Restriction endonucleases and DNA-modifying enzymes were purchased from TaKaRa Bio (Kyoto, Japan). A cDNA encoding full-length mouse GDE5 was subcloned into the SalI (made blunt with T4 DNA polymerase) and NotI sites of the mammalian expression vector pEF/myc/cyto (Invitrogen), generating pEF-GDE5. To obtain a deletion mutant of GDE5 (amino acids 1– 470, GDE5⌬C471), cDNA was applied using a PCR primer set (5Ј-GGGGATCCTATCCATCCCTGTGTTGGCAAA-3Ј and 5Ј-GCGCGGCCCGGACCGGCAGAAATCC-3Ј) designed according to the nucleotide sequences of mouse GDE5 and subcloned into pEF/myc/cyto, which generated pEF-GDE5⌬C471. The transgene construct contained from Ϫ2000 to ϩ200 base pairs of the human ␣-skeletal actin promoter, 2.0 kb of complete mouse GDE5 cDNA, and a polyadenylation signal encoded by bovine growth hormone. DNA Microarray—Total RNA derived from the skeletal muscle (gastrocnemius) of WT and GDE5⌬C471 mice at 12 weeks of age was isolated using an RNeasy kit and RNase-Free DNase set (Qiagen) and subjected to cRNA synthesis for a DNA.

RESULTS

DISCUSSION

63 Ϯ 9 65 Ϯ 11 69 Ϯ 15 73 Ϯ 11

98 Ϯ 2 100 Ϯ 7 mg