Role of mechanistic Target of Rapamycin (mTOR) signaling in the crustacean molting gland

2012 Fall.Includes bibliographical references.Regulation of the molt cycle in decapod crustaceans is mainly controlled by the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). Molt-inhibiting hormone (MIH), secreted by the XO/SG complex, suppresses production of molting hormone (ecdysteroids) by a pair of YOs. In the blackback land crab, Gecarcinus lateralis, molting can be induced by eyestalk ablation (ESA) or autotomy of 5 or more walking legs (multiple leg autotomy or MLA). During the molt cycle, the YO transitions through four physiological states: "basal" state at postmolt and intermolt; "activated" state at early premolt; "committed" state at mid premolt and "repressed" state at late premolt. The basal to activated state transition is triggered by a transient reduction in MIH; the YOs hypertrophy, but remain sensitive to MIH. The main hypothesis is that up-regulation of mechanistic Target of Rapamycin (mTOR) signaling, which controls global translation of mRNA into protein, is necessary for YO hypertrophy and ecdysteroidogenesis. cDNAs encoding mTOR, Rheb, Akt (protein kinase B) and p70 S6 kinase (S6k) were cloned from blackback land crab, G. lateralis, and green shore crab, Carcinus maenas. All four genes were expressed in all tissues examined. mTOR appears to be involved in YO activation in early premolt, as rapamycin inhibited YO ecdysteroidogenesis in vivo and in vitro. In addition, the expression of Gl-elongation factor 2 (EF2), Gl-mTOR, and Gl-Akt increased significantly in YOs from premolt, suggesting that an increase in protein synthetic capacity is necessary for YO activation. A putative transforming growth factor-beta (TGFâ) appeared to be involved in the transition of the YO from the activated to committed state, as SB431542, an Activin receptor antagonist, lowered hemolymph ecdysteroid titers in mid premolt animals and abrogated the premolt increases in Gl-EF2, Gl-mTOR, and Gl-Akt mRNA levels. By contrast, molting had no effect on Cm-EF2, Cm-mTOR, Cm-Rheb, Cm-Akt, and Cm-S6k expression in C. maenas YOs. Unlike G. lateralis, adult C. maenas was refractory to ESA. ESA caused a small increase in hemolymph ecdysteroid titers, but animals did not immediately enter premolt. Some ES-ablated animals molted after many months, but most failed to molt at all. We hypothesized that other regions of the nervous system, specifically the brain and/or thoracic ganglion, were secondary source(s) of MIH. Nested endpoint RT-PCR showed that MIH transcript was present in brain and thoracic ganglion of intermolt crabs. Sequencing of the PCR product confirmed its identity as MIH. Real time PCR was used to quantify the effects of ESA on MIH expression in brain and thoracic ganglion on C. maenas red and green color morphs. ESA had little effect on MIH transcript levels, indicating that MIH was not regulated transcriptionally by the loss of the eyestalks. The data suggest that MIH secreted by neurons in the brain and thoracic ganglion is sufficient to prevent molt induction when the primary source of MIH is removed by ESA. There was also no effect of ESA on the expression of Gl-EF2 and mTOR signaling components in C. maenas YOs

Mechanistic target of rapamycin (mTOR) signaling genes in decapod crustaceans: cloning and tissue expression of mTOR, Akt, Rheb, and S6 kinase in the green crab, Carcinus maenas, and blackback land crab, Gecarcinus lateralis

Mechanistic target of rapamycin (mTOR) controls global translation of mRNA into protein by phosphorylating p70 S6 kinase (S6K) and eIF4E-binding protein-1. Akt and Rheb, a GTP-binding protein, regulate mTOR protein kinase activity. Molting in crustaceans is regulated by ecdysteroids synthesized by a pair of molting glands, or Y-organs (YOs), located in the cephalothorax. During premolt, the YOs hypertrophy and increase production of ecdysteroids. Rapamycin (1 μM) inhibited ecdysteroid secretion in Carcinus maenas and Gecarcinus lateralis YOs in vitro, indicating that ecdysteroidogenesis requires mTOR-dependent protein synthesis. The effects of molting on the expression of four key mTOR signaling genes (mTOR, Akt, Rheb, and S6K) in the YO was investigated. Partial cDNAs encoding green crab (C. maenas) mTOR (4031 bp), Akt (855 bp), and S6K (918 bp) were obtained from expressed sequence tags. Identity/similarity of the deduced amino acid sequence of the C. maenas cDNAs to human orthologs were 72%/81% for Cm-mTOR, 58%/73% for Cm-Akt, and 77%/88% for Cm-S6K. mTOR, Akt, S6K, and elongation factor 2 (EF2) in C. maenas and blackback land crab (G. lateralis) were expressed in all tissues examined. The two species differed in the effects of molting on gene expression in the YO. In G. lateralis, Gl-mTOR, Gl-Akt, and Gl-EF2 mRNA levels were increased during premolt. By contrast, molting had no effect on the expression of Cm-mTOR, Cm-Akt, Cm-S6K, Cm-Rheb, and Cm-EF2. These data suggest that YO activation during premolt involves up regulation of mTOR signaling genes in G. lateralis, but is not required in C. maenas

Roles of mechanistic target of rapamycin and transforming growth factor-B signaling in the molting gland (Y-organ) of the blackback land crab, Gecarcinus lateralis

Molting in decapod crustaceans is controlled by molt-inhibiting hormone (MIH), an eyestalk neuropeptide that suppresses production of ecdysteroids by a pair of molting glands (Y-organs or YOs). Eyestalk ablation (ESA) activates the YOs, which hypertrophy and increase ecdysteroid secretion. At mid premolt, which occurs 7–14 days post-ESA, the YO transitions to the committed state; hemolymph ecdysteroid titers increase further and the animal reaches ecdysis ~ 3 weeks post-ESA. Two conserved signaling pathways, mechanistic target of rapamycin (mTOR) and transforming growth factor-β (TGF-β), are expressed in the Gecarcinus lateralis YO. Rapamycin, an mTOR antagonist, inhibits YO ecdysteroidogenesis in vitro. In this study, rapamycin lowered hemolymph ecdysteroid titer in ESA G. lateralis in vivo; levels were significantly lower than in control animals at all intervals (1–14 days post-ESA). Injection of SB431542, an activin TGF-β receptor antagonist, lowered hemolymph ecdysteroid titers 7 and 14 days post-ESA, but had no effect on ecdysteroid titers at 1 and 3 days post-ESA. mRNA levels of mTOR signaling genes Gl-mTOR, Gl-Akt, and Gl-S6k were increased by 3 days post-ESA; the increases in Gl-mTOR and Gl-Akt mRNA levels were blocked by SB431542. Gl-elongation factor 2 and Gl-Rheb mRNA levels were not affected by ESA, but SB431542 lowered mRNA levels at Days 3 and 7 post-ESA. The mRNA level of an activin TGF-β peptide, Gl-myostatin-like factor (Mstn), increased 5.5-fold from 0 to 3 days post-ESA, followed by a 50-fold decrease from 3 to 7 days post-ESA. These data suggest that (1) YO activation involves an up regulation of the mTOR signaling pathway; (2) mTOR is required for YO commitment; and (3) a Mstn-like factor mediates the transition of the YO from the activated to the committed state

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

Molt regulation in green and red color morphs of the crab, Carcinus maenas: gene expression of molt-inhibiting hormone signaling components

In decapod crustaceans, regulation of molting is controlled by the X-organ/sinus gland complex in the eyestalks. The complex secretes molt-inhibiting hormone (MIH), which suppresses production of ecdysteroids by the Y-organ (YO). MIH signaling involves nitric oxide and cGMP in the YO, which expresses nitric oxide synthase (NOS) and NO-sensitive guanylyl cyclase (GC-I). Molting can generally be induced by eyestalk ablation (ESA), which removes the primary source of MIH, or by multiple leg autotomy (MLA). In our work on Carcinus maenas, however, ESA has limited effects on hemolymph ecdysteroid titers and animals remain in intermolt at 7 days post-ESA, suggesting that adults are refractory to molt induction techniques. Consequently, the effects of ESA and MLA on molting and YO gene expression in C. maenas green and red color morphotypes were determined at intermediate (16 and 24 days) and long-term (~90 days) intervals. In intermediate-interval experiments, ESA of intermolt animals caused transient twofold to fourfold increases in hemolymph ecdysteroid titers during the first 2 weeks. In intermolt animals, long-term ESA increased hemolymph ecdysteroid titers fourfold to fivefold by 28 days post treatment, but there was no late premolt peak (\u3e400 pg μl−1) characteristic of late premolt animals and animals did not molt by 90 days post-ESA. There was no effect of ESA or MLA on the expression of Cm-elongation factor 2 (EF2), Cm-NOS, the beta subunit of GC-I (Cm-GC-Iβ), a membrane receptor GC (Cm-GC-II) and a soluble NO-insensitive GC (Cm-GC-III) in green morphs. Red morphs were affected by prolonged ESA and MLA treatments, as indicated by large decreases in Cm-EF2, Cm-GC-II and Cm-GC-III mRNA levels. ESA accelerated the transition of green morphs to the red phenotype in intermolt animals. ESA delayed molting in premolt green morphs, whereas intact and MLA animals molted by 30 days post treatment. There were significant effects on YO gene expression in intact animals: Cm-GC-Iβ mRNA increased during premolt and Cm-GC-III mRNA decreased during premolt and increased during postmolt. Cm-MIH transcripts were detected in eyestalk ganglia, the brain and the thoracic ganglion from green intermolt animals, suggesing that MIH in the brain and thoracic ganglion prevents molt induction in green ESA animals