Rheb, an activator of target of rapamycin, in the blackback land crab, Gecarcinus lateralis: cloning and effects of molting and unweighting on expression in skeletal muscle.

Molt-induced claw muscle atrophy in decapod crustaceans facilitates exuviation and is coordinated by ecdysteroid hormones. There is a 4-fold reduction in mass accompanied by remodeling of the contractile apparatus, which is associated with an 11-fold increase in myofibrillar protein synthesis by the end of the premolt period. Loss of a walking limb or claw causes a loss of mass in the associated thoracic musculature; this unweighting atrophy occurs in intermolt and is ecdysteroid independent. Myostatin (Mstn) is a negative regulator of muscle growth in mammals; it suppresses protein synthesis, in part, by inhibiting the insulin/metazoan target of rapamycin (mTOR) signaling pathway. Signaling via mTOR activates translation by phosphorylating ribosomal S6 kinase (s6k) and 4E-binding protein 1. Rheb (Ras homolog enriched in brain), a GTP-binding protein, is a key activator of mTOR and is inhibited by Rheb-GTPase-activating protein (GAP). Akt protein kinase inactivates Rheb-GAP, thus slowing Rheb-GTPase activity and maintaining mTOR in the active state. We hypothesized that the large increase in global protein synthesis in claw muscle was due to regulation of mTOR activity by ecdysteroids, caused either directly or indirectly via Mstn. In the blackback land crab, Gecarcinus lateralis, a Mstn-like gene (Gl-Mstn) is downregulated as much as 17-fold in claw muscle during premolt and upregulated 3-fold in unweighted thoracic muscle during intermolt. Gl-Mstn expression in claw muscle is negatively correlated with hemolymph ecdysteroid level. Full-length cDNAs encoding Rheb orthologs from three crustacean species (G. lateralis, Carcinus maenas and Homarus americanus), as well as partial cDNAs encoding Akt (Gl-Akt), mTOR (Gl-mTOR) and s6k (Gl-s6k) from G. lateralis, were cloned. The effects of molting on insulin/mTOR signaling components were quantified in claw closer, weighted thoracic and unweighted thoracic muscles using quantitative polymerase chain reaction. Gl-Rheb mRNA levels increased 3.4-fold and 3.9-fold during premolt in claw muscles from animals induced to molt by eyestalk ablation (ESA) and multiple leg autotomy (MLA), respectively, and mRNA levels were positively correlated with hemolymph ecdysteroids. There was little or no effect of molting on Gl-Rheb expression in weighted thoracic muscle and no correlation of Gl-Rheb mRNA with ecdysteroid titer. There were significant changes in Gl-Akt, Gl-mTOR and Gl-s6k expression with molt stage. These changes were transient and were not correlated with hemolymph ecdysteroids. The two muscles differed in terms of the relationship between Gl-Rheb and Gl-Mstn expression. In thoracic muscle, Gl-Rheb mRNA was positively correlated with Gl-Mstn mRNA in both ESA and MLA animals. By contrast, Gl-Rheb mRNA in claw muscle was negatively correlated with Gl-Mstn mRNA in ESA animals, and no correlation was observed in MLA animals. Unweighting increased Gl-Rheb expression in thoracic muscle at all molt stages; the greatest difference (2.2-fold) was observed in intermolt animals. There was also a 1.3-fold increase in Gl-s6k mRNA level in unweighted thoracic muscle. These data indicate that the mTOR pathway is upregulated in atrophic muscles. Gl-Rheb, in particular, appears to play a role in the molt-induced increase in protein synthesis in the claw muscle.

Effects of elevated ecdysteroid on tissue expression of three guanylyl cyclases in the tropical land crab Gecarcinus lateralis: possible roles of neuropeptide signaling in the molting gland.

Two eyestalk (ES) neuropeptides, molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), increase intracellular cGMP levels in target tissues. Both MIH and CHH inhibit ecdysteroid secretion by the molting gland or Y-organ (YO), but apparently through different guanylyl cyclase (GC)-dependent pathways. MIH signaling may be mediated by nitric oxide synthase (NOS) and NO-sensitive GC. CHH binds to a membrane receptor GC. As molting affects neuropeptide signaling, the effects of ecdysteroid on the expression of the land crab Gecarcinus lateralis beta subunit of a NO-sensitive GC (Gl-GC-Ibeta), a membrane receptor GC (Gl-GC-II) and a NO-insensitive soluble GC (Gl-GC-III) were determined. Gl-GC-Ibeta isoforms differing in the absence or presence of an N-terminal 32-amino acid sequence and Gl-GC-III were expressed at higher mRNA levels in ES ganglia, gill, hepatopancreas, ovary and testis, and at lower levels in YO, heart and skeletal muscle. Three Gl-GC-II isoforms, which vary in the length of insertions (+18, +9 and +0 amino acids) within the N-terminal ligand-binding domain, differed in tissue distribution. Gl-GC-II(+18) was expressed highly in striated muscle (skeletal and cardiac muscles); Gl-GC-II(+9) was expressed in all tissues examined (ES ganglia, YO, gill, hepatopancreas, striated muscles and gonads); and Gl-GC-II(+0) was expressed in most tissues and was the dominant isoform in ES and thoracic ganglia. ES ablation, which increased hemolymph ecdysteroid, increased Gl-GC-II(+18) mRNA level in claw muscle. Using real-time RT-PCR, ES ablation increased Gl-GC-Ibeta, Gl-GC-III and ecdysone receptor mRNA levels in the YOs approximately ten-, approximately four- and approximately twofold, respectively, whereas Gl-GC-II mRNA level was unchanged. A single injection of 20-hydroxyecdysone into intact animals transiently lowered Gl-GC-Ibeta in hepatopancreas, testis and skeletal muscle, and certain Gl-GC-II isoforms in some of the tissues. These data suggest that YO and other tissues can modulate responses to neuropeptides by altering GC expression.