Joseph A. Covi

Conserved role of cyclic nucleotides in the regulation of ecdysteroidogenesis by the crustacean molting gland

Molting processes in crustaceans are regulated by ecdysteroids produced in the molting gland (Y-organ), and molting is indirectly controlled by circulating factors that inhibit the production of these polyhydroxylated steroids. Two of these regulatory factors are the neuropeptides molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). CHH appears to inhibit ecdysteroidogenesis in the Y-organ through the activation of a receptor guanylyl cyclase. The signaling pathway activated by MIH, however, remains a subject of controversy. It is clear that neuropeptides inhibit ecdysteroidogenesis by simultaneously suppressing ecdysteroid biosynthetic processes, protein synthesis, and uptake of high density lipoproteins. Data demonstrate that cAMP is the primary regulator of critical catabolic, anabolic, and transport processes, which ultimately support the capacity for ecdysteroid production by the Y-organ. While cAMP also regulates acute ecdysteroidogenesis to some extent, data indicate that cGMP is the primary signaling molecule responsible for acute inhibition by neuropeptides. It is clear that the regulatory roles filled by cAMP and cGMP are conserved among decapod crustaceans. It is unknown if these complementary second messengers are linked in a single signaling pathway or are components of independent pathways activated by different factors present in extracts of eyestalk ganglia.

Metabolic Restructuring during Energy-Limited States: Insights from Artemia franciscana Embryos and Other Animals

Many life history stages of animals that experience environmental insults enter developmental arrested states that are characterized by reduced cellular proliferation, with or without a concurrent reduction in overall metabolism. In the case of the most profound metabolic arrest reported in invertebrates, i.e., anaerobic quiescence in Artemia franciscana embryos, acidification of the intracellular milieu is a major factor governing catabolic and anabolic downregulation. Release of ions from intracellular compartments is the source for approximately 50% of the proton equivalents needed for the 1.5 unit acidification that is observed. Recovery from the metabolic arrest requires re-sequestration of the protons with a vacuolar-type ATPase (V-ATPase). The remarkable facet of this mechanism is the ability of embryonic cells to survive the dissipation of intracellular ion gradients. Across many diapause-like states, the metabolic reduction and subsequent matching of energy demand is accomplished by shifting energy metabolism from oxidative phosphorylation to aerobic glycolysis. Molecular pathways that are activated to induce these resilient hypometabolic states include stimulation of the AMP-activated protein kinase (AMPK) and insulin signaling via suite of daf (dauer formation) genes for diapause-like states in nematodes and insects. Contributing factors for other metabolically depressed states involve hypoxia-inducible factor-1 and downregulation of the pyruvate dehydrogenase complex. Metabolic similarities between natural states of stasis and some cancer phenotypes are noteworthy. Reduction of flux through oxidative phosphorylation helps prevent cell death in certain cancer types, similar to the way it increases viability of dauer stages in Caenorhabditis elegans. Mechanisms that underlie natural stasis are being used to pre-condition mammalian cells prior to cell biostabilization and storage.

Molt cycle regulation of protein synthesis in skeletal muscle of the blackback land crab, Gecarcinus lateralis, and the differential expression of a myostatin-like factor during atrophy induced by molting or unweighting

SUMMARY In decapod crustaceans, claw muscle undergoes atrophy in response to elevated ecdysteroids while thoracic muscle undergoes atrophy in response to unweighting. The signaling pathways that regulate muscle atrophy in crustaceans are largely unknown. Myostatin is a negative regulator of muscle growth in mammals, and a myostatin-like cDNA is preferentially expressed in muscle of the land crab, Gecarcinus lateralis (Gl-Mstn). Contrary to prediction, levels of Gl-Mstn mRNA decreased dramatically in both the claw closer and weighted thoracic muscles when molting was induced by either eyestalk ablation (ESA) or multiple limb autotomy (MLA). However, the effect of molt induction was greater in the claw muscle. By late premolt, Gl-Mstn mRNA in the claw muscle decreased 81% and 94% in ESA and MLA animals, respectively, and was negatively correlated with ecdysteroids. Gl-Mstn mRNA in thoracic muscle decreased 68% and 82% in ESA and MLA animals, respectively, but was only weakly correlated with ecdysteroid. Claw and thoracic muscles also differed to varying extents in the expression of ecdysteroid receptor (Gl-EcR and Gl-RXR), elongation factor-2 (Gl-EF-2), and calpain T (Gl-CalpT) in response to molt induction, but levels of the four transcripts were not correlated with ecdysteroid. The downregulation of Gl-Mstn expression in premolt claw muscle coincided with 11- and 13-fold increases in protein synthesis in the myofibrillar and soluble protein fractions, respectively. Furthermore, the rate of the increase in the synthesis of soluble proteins was greater than that of myofibrillar proteins during early premolt (1.4:1, soluble:myofibrillar), but the two were equivalent during late premolt. By contrast, Gl-Mstn mRNA increased 3-fold and Gl-CalpT mRNA decreased 40% in unweighted thoracic muscle; there was little or no effect on Gl-EF-2, Gl-EcR, and Gl-RXR mRNA levels. These data indicate that Gl-Mstn expression is negatively regulated by both ecdysteroids and load-bearing contractile activity. The downregulation of Gl-Mstn in claw muscle may induce the elevated protein turnover associated with remodeling of the contractile apparatus during molt-induced atrophy. The upregulation of Gl-Mstn in unweighted thoracic muscle suggests that this factor is also involved in disuse atrophy when hemolymph ecdysteroid levels are low.

Expression of alternatively spliced transcripts for a myostatin-like protein in the blackback land crab, Gecarcinus lateralis

Three complete cDNAs for the first myostatin-like gene identified in a crustacean species were cloned from the land crab, Gecarcinus lateralis. Sequence analysis demonstrates a high degree of conservation with myostatin orthologs from vertebrates. The furin cleavage site is identical to that of human myostatin, and all nine cysteines critical to the structure/function of mature myostatin peptides are conserved. Message levels for transcripts encoding the complete crustacean preproprotein were highest in skeletal muscle and heart. Lower levels of expression were observed in nervous tissue, gill, gonad, and hepatopancreas. This expansive distribution is similar to that observed for teleost myostatin, vertebrate GDF-11, and amphioxus GDF8/11, and indicates a potentially broad functional repertoire for the land crab ortholog. In addition to one cDNA encoding a complete preproprotein, two cDNAs encoding C-terminal truncated proteins lacking a mature peptide domain were identified. Expression of these truncated splice variants was restricted to skeletal muscle and heart. Myostatin is a potent negative regulator of muscle mass in mammals, and strong expression of this TGF-beta factor in skeletal muscle during intermolt indicates that a myostatin-like gene product could regulate muscle mass in crustaceans when growth is physically restricted by a calcified exoskeleton.

Myostatin from the American lobster, Homarus americanus: Cloning and effects of molting on expression in skeletal muscles

A cDNA encoding a myostatin (Mstn)-like gene from an astacuran crustacean, Homarus americanus, was cloned and characterized. Mstn inhibits skeletal muscle growth in vertebrates and may play a role in crustacean muscle as a suppressor of protein synthesis. Sequence analysis and three-dimensional modeling of the Ha-Mstn protein predicted a high degree of conservation with vertebrate and other invertebrate myostatins. Qualitative polymerase chain reaction (PCR) demonstrated ubiquitous expression of transcript in all tissues, including skeletal muscles. Quantitative PCR analysis was used to determine the effects of natural molting and eyestalk ablation (ESA) on Ha-Mstn expression in the cutter claw (CT) and crusher claw (CR) closer muscles and deep abdominal (DA) muscle. In intermolt lobsters, the Ha-Mstn mRNA level in the DA muscle was significantly lower than the mRNA levels in the CT and CR muscles. Spontaneous molting decreased Ha-Mstn mRNA during premolt, with the CR muscle, which is composed of slow-twitch (S₁) fibers, responding preferentially (82% decrease) to the atrophic signal compared to fast fibers in CT (51% decrease) and DA (69% decrease) muscles. However, acute increases in circulating ecdysteroids caused by ESA had no effect on Ha-Mstn mRNA levels in the three muscles. These data indicate that the transcription of Ha-Mstn is differentially regulated during the natural molt cycle and it is an important regulator of protein turnover in molt-induced claw muscle atrophy.

Neuropeptide signaling mechanisms in crustacean and insect molting glands

Growth in arthropods requires the periodic synthesis of a new exoskeleton and shedding, or molting, of the old exoskeleton. Both processes are initiated and coordinated by ecdysteroid molting hormones synthesized and secreted by a pair of molting glands: Y-organ (YO) in decapod crustaceans and the prothoracic gland (PG) in insects. The primary regulation of arthropod molting glands is mediated by brain neuropeptides that either stimulate (prothoracicotropic hormone – PTTH – in insects) or inhibit (molt-inhibiting hormone – MIH – in crustaceans) ecdysteroidogenesis. Both neuropeptide signaling pathways involve Ca2+/calmodulin (CaM) and cyclic nucleotides. In the crustacean YO, the MIH signaling pathway is proposed to have two phases: a cAMP/CaM-dependent “triggering” phase that activates a NO/cGMP-dependent “summation” phase to repress ecdysteroidogenesis during most of the intermolt period. A model is presented that incorporates the following data: (1) there is a pulsatile release of MIH from the eyestalk neurosecretory center; (2) MIH has a short half-life (5–10 min) in the hemolymph; and (3) there is a decrease in sensitivity of the YO to MIH that occurs during the premolt period. Molting is initiated by a temporary reduction in MIH titers due to a decrease in the amount and/or frequency of MIH released. During premolt, an increase in MIH release that is linked to a cAMP-dependent and cGMP-independent stimulation of protein synthesis drives an increase in molting hormone synthesis. By contrast, PTTH stimulates PG ecdysteroidogenesis by a Ca2+-dependent activation of cAMP and mitogen-activated protein (MAP) kinase pathways. A hypothetical scheme of how these two diametrical regulatory mechanisms may have originated and evolved is presented. It assumes that both systems evolved from a common ecdysozoan ancestor that had a cAMP-dependent stimulatory pathway antagonized by a NO/cGMP-dependent inhibitory pathway. This “parallel” arrangement of the two pathways regulates ecdysteroidogenesis in the gonad of adult insects. In insect PG, cAMP-dependent signaling became the dominant pathway and NO/cGMP-dependent signaling was lost. MAP kinase signaling was later co-opted as an ancillary pathway. This dual pathway occurs in lepidopteran species (Manduca sexta and Bombyx mori), but MAP kinase signaling is the dominant pathway in Drosophila melanogaster. In crustacean YO, the cAMP- and NO/cGMP-dependent pathways became arranged in series such that a transient increase in cAMP triggers a large and sustained increase in cGMP, which leads to prolonged repression of ecdysteroidogenesis during the intermolt period. It is now recognized that the control of molting involves a constant communication between the nervous system and the molting gland mediated by a variety of endocrine factors. Both the YO and PG undergo changes in sensitivity to MIH and PTTH, respectively, that are associated with the commitment to molt. It is not surprising, however, that the changes in sensitivities of the two glands to their respective neuropeptides are opposite: the PG is the most sensitive to PTTH, whereas the YO is least sensitive to MIH, prior to and during the large peak in hemolymph ecdysteroid titers that triggers molting. As more research is conducted, we anticipate that more similarities in the neuropeptide signaling pathways of crustacean and insect molting glands will be revealed.

V-ATPase inhibition prevents recovery from anoxia in Artemia franciscana embryos: quiescence signaling through dissipation of proton gradients.

SUMMARY The metabolic downregulation critical for long-term survival of Artemia franciscana embryos under anoxia is mediated, in part, by a progressive intracellular acidification. However, very little is known about the mechanisms responsible for the pH transitions associated with exposure to, and recovery from, oxygen deprivation. In the present study, we demonstrate with 31P-NMR that incubation of intact embryos with the V-ATPase inhibitor bafilomycin A1 severely limits intracellular alkalinization during recovery from anoxia without affecting the restoration of cellular nucleotide triphosphate levels. Based on these data, it appears that oxidative phosphorylation and ATP resynthesis can only account for the first 0.3 pH unit alkalinization observed during aerobic recovery from the 1 pH unit acidification produced during 1 h of anoxia. The additional 0.7 pH unit increase requires proton pumping by the V-ATPase. Aerobic incubation with bafilomycin also suggests that V-ATPase inhibition alone is not enough to induce an acute dissipation of proton gradients under anoxia. In intact embryos, the dissipation of proton gradients and uncoupling of oxidative phosphorylation with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) leads to an intracellular acidification similar to that seen after 1 h of anoxia. Subsequent exposure to anoxia, in the continued presence of CCCP, yields little additional acidification, suggesting that proton gradients are normally dissipated under anoxia. When combined with protons generated from net ATP hydrolysis, these data show that the dissipation of proton chemical gradients is sufficient to account for the reversible acidification associated with quiescence in these embryos.

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

SUMMARY 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.