Intracellular signaling molecules involved in an inhibitory factor–induced decrease in fetal‐type AChR expression

The innervation‐induced down‐regulation of fetal‐type acetylcholine receptor (AChR) expression in developing muscle fibers has largely been attributed to nerve‐evoked muscle activity; however, there is increasing evidence that a neural trophic factor also contributes to this receptor down‐regulation. Previous studies from this laboratory have shown that neural extracts contain a factor which decreases fetal‐type AChR expression in skeletal muscle cell lines and therefore may account for the proposed inhibitory neurotrophic influence. The current study investigated possible intracellular signaling molecules involved in this receptor down‐regulation and demonstrated that activation of protein kinase C and p70^S6k appeared to be important in receptor down‐regulation. Decreases in AChR density were independent of myogenin. In addition, the receptor down‐regulation was independent of neuregulin, which also induces p70^S6k activity. These studies demonstrate that neural extracts contain an inhibitory factor which can down‐regulate fetal‐type AChR expression independently of nerve‐evoked muscle activity through intracellular signaling molecules which are known to regulate AChR expression. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 190–201, 2000     

Heregulin-stimulated acetylcholine receptor gene expression in muscle: requirement for MAP kinase and evidence for a parallel inhibitory pathway independent of electrical activity.

Binding of heregulin (HRG) to its receptor, ErbB3, results in a dimerization with ErbB2/neu and activation of their intrinsic tyrosine kinases, initiating a cascade of events resulting in the stimulation of acetylcholine receptor (AChR) genes in muscle. Here we have examined the signalling downstream of the HRG receptor. We show that phosphatidylinositol 3'-kinase (PI3K) and SHC bind to the HRG-activated ErbB3 in myotubes. Subsequently, p70S6 kinase (p70S6k), and MAP kinase ERK2 and thereby p90rsk are activated. However, inhibition of PI3K and p70S6k by wortmannin and rapamycin, respectively, failed to antagonize AChR alpha-subunit gene expression stimulated by HRG, despite the fact that the activities of the kinases were inhibited. In contrast, these inhibitors elevated AChR alpha-subunit mRNA levels, by themselves, independently of muscle electrical activity. On the other hand, the 17mer antisense oligonucleotide, EAS1, caused a specific depletion of ERK2 and eliminated the ability of HRG to stimulate AChR alpha-subunit gene expression. These results indicate that HRG stimulates expression of AChR genes via ERK2 activation, and provide a physiological example of neurotrophic factor-associated repression of AChR genes by stimulation of p70S6k activity which may contribute to the expression of adult type AChR genes at the neuromuscular junction.     

Identification of agrin, a synaptic organizing protein from Torpedo electric organ

Extracts of the electric organ of Torpedo californica contain a proteinaceous factor that causes the formation of patches on cultured myotubes at which acetylcholine receptors (AChR), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) are concentrated. Results of previous experiments indicate that this factor is similar to the molecules in the synaptic basal lamina that direct the aggregation of AChR and AChE at regenerating neuromuscular junctions in vivo. We have purified the active components in the extracts 9,000-fold. mAbs against four different epitopes on the AChR/AChE/BuChE-aggregating molecules each immunoprecipitated four polypeptides from electric organ extracts, with molecular masses of 150, 135, 95, and 70 kD. Gel filtration chromatography of electric organ extracts revealed two peaks of AChR/AChE/BuChE-aggregation activity; one comigrated with the 150-kD polypeptide, the other with the 95-kD polypeptide. The 135- and 70-kD polypeptides did not cause AChR/AChE/BuChE aggregation. Based on these molecular characteristics and on the pattern of staining seen in sections of muscle labeled with the mAbs, we conclude that the electric organ-aggregating factor is distinct from previously identified molecules, and we have named it "agrin."     

ANG II activates effectors of mTOR via PI3-K signaling in human coronary smooth muscle cells

We have previously shown that the vasoconstrictive peptide angiotensin II (ANG II) is a hypertrophic agent for human coronary artery smooth muscle cells (cSMCs), which suggests that it plays a role in vascular wall thickening. The present study investigated the intracellular signal transduction pathways involved in the growth response of cSMCs to ANG II. The stimulation of protein synthesis by ANG II in cSMCs was blocked by the immunosuppressant rapamycin, which is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway that includes the 70-kDa S6 kinase (p70(S6k)) and plays a key role in cell growth. The inhibitory effect of rapamycin was reversed by a molar excess of FK506; this indicates that both agents act through the common 12-kDa immunophilin FK506-binding protein. ANG II caused a rapid and sustained activation of p70(S6k) activity that paralleled its phosphorylation, and both processes were blocked by rapamycin. In addition, both of the phosphatidylinositol 3-kinase inhibitors wortmannin and LY-294002 abolished the ANG II-induced increase in protein synthesis, and wortmannin also blocked p70(S6k) phosphorylation. Furthermore, ANG II triggered dissociation of the translation initiation factor, eukaryotic initiation factor-4E, from its regulatory binding protein 4E-BP1, which was also inhibited by rapamycin and wortmannin. In conclusion, we have shown that ANG II activates components of the rapamycin-sensitive mTOR signaling pathway in human cSMCs and involves activation of phosphatidylinositol 3-kinase, p70(S6k), and eukaryotic initiation factor-4E, which leads to activation of protein synthesis. These signaling mechanisms may mediate the growth-promoting effect of ANG II in human cSMCs.     

Factors released by ciliary neurons and spinal cord explants induce acetylcholine receptor mRNA expression in cultured muscle cells

The nuclei of cultured noninnervated muscle cells are heterogeneous with respect to production of mRNA for the nicotinic acetylcholine receptor (AChR). Some nuclei actively express AChR mRNA while others have a low level of activity or are inactive. To determine if innervation, or a factor released by neurons, influences nuclear expression of AChR mRNA, we examined mRNA at a single cell level via in situ hybridization and autoradiography with an alpha-subunit AChR genomic probe. Four days after plating, we co-cultured chicken primary muscle cells with spinal cord explants, ciliary neurons, or dorsal root ganglia (DRG) cells. In situ hybridization of the spinal-cord and muscle-cell co-cultures with the AChR alpha-subunit probe revealed a high density of silver grains on muscle cells, which were within two explant diameters of the spinal cord explant, and a graded decrease in silver grain density as the distance from the explant increased, as well as the appearance of a strikingly nonhomogenous distribution of active and inactive muscle cell nuclei. When ciliary neurons were uniformly distributed over the muscle cells, a high level of AChR mRNA was induced, but no gradients appeared. Neither an increased mRNA level nor a gradient was observed when DRG cells were co-cultured with muscle cells. When ciliary neurons are cultured within Costar permeable inserts, which prevent any contact between the neurons and the underlying muscle cells, AChR messenger RNA is still induced, showing that diffusible factors are responsible. Our results indicate that molecules released by cholinergic neurons regulate the expression of AChR mRNA in the myotubes and raise the possibility that AChR expression depends on both neuronal signals and on intracellular information from the muscle cell.     

Novel role for cyclin-dependent kinase 2 in neuregulin-induced acetylcholine receptor epsilon subunit expression in differentiated myotubes

Cyclin-dependent kinases (CDKs) are a family of evolutionarily conserved serine/threonine kinases. CDK2 acts as a checkpoint for the G(1)/S transition in the cell cycle. Despite a down-regulation of CDK2 activity in postmitotic cells, many cell types, including muscle cells, maintain abundant levels of CDK2 protein. This led us to hypothesize that CDK2 may have a function in postmitotic cells. We show here for the first time that CDK2 can be activated by neuregulin (NRG) in differentiated C2C12 myotubes. In addition, this activity is required for expression of the acetylcholine receptor (AChR) epsilon subunit. The switch from the fetal AChRgamma subunit to the adult-type AChRepsilon is required for synapse maturation and the neuromuscular junction. Inhibition of CDK2 activity with either the specific CDK2 inhibitory peptide Tat-LFG or by RNA interference abolished neuregulin-induced AChRepsilon expression. Neuregulin-induced activation of CDK2 also depended on the ErbB receptor, MAPK, and PI3K, all of which have previously been shown to be required for AChRepsilon expression. Neuregulin regulated CDK2 activity through coordinating phosphorylation of CDK2 on Thr-160, accumulation of CDK2 in the nucleus, and down-regulation of the CDK2 inhibitory protein p27 in the nucleus. In addition, we also observed a novel mechanism of regulation of CDK2 activity by a low molecular weight variant of cyclin E in response to NRG. These findings establish CDK2 as an intermediate molecule that integrates NRG-activated signals from both the MAPK and PI3K pathways to AChRepsilon expression and reveal an undiscovered physiological role for CDK2 in postmitotic cells.     

The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

Background

Postsynaptic enrichment of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction (NMJ) depends on the activation of the muscle receptor tyrosine MuSK by neural agrin. Agrin-stimulation of MuSK is known to initiate an intracellular signaling cascade that leads to the clustering of AChRs in an actin polymerization-dependent manner, but the molecular steps which link MuSK activation to AChR aggregation remain incompletely defined.

Methodology/Principal Findings

In this study we used biochemical, cell biological and molecular assays to investigate a possible role in AChR clustering of cortactin, a protein which is a tyrosine kinase substrate and a regulator of F-actin assembly and which has also been previously localized at AChR clustering sites. We report that cortactin was co-enriched at AChR clusters in situ with its target the Arp2/3 complex, which is a key stimulator of actin polymerization in cells. Cortactin was further preferentially tyrosine phosphorylated at AChR clustering sites and treatment of myotubes with agrin significantly enhanced the tyrosine phosphorylation of cortactin. Importantly, forced expression in myotubes of a tyrosine phosphorylation-defective cortactin mutant (but not wild-type cortactin) suppressed agrin-dependent AChR clustering, as did the reduction of endogenous cortactin levels using RNA interference, and introduction of the mutant cortactin into muscle cells potently inhibited synaptic AChR aggregation in response to innervation.

Conclusion

Our results suggest a novel function of phosphorylation-dependent cortactin signaling downstream from agrin/MuSK in facilitating AChR clustering at the developing NMJ.     

Amino acids and insulin control autophagic proteolysis through different signaling pathways in relation to mTOR in isolated rat hepatocytes

Autophagy, a major bulk proteolytic pathway, contributes to intracellular protein turnover, together with protein synthesis. Both are subject to dynamic control by amino acids and insulin. The mechanisms of signaling and cross-talk of their physiological anabolic effects remain elusive. Recent studies established that amino acids and insulin induce p70 S6 kinase (p70(S6k)) phosphorylation by mTOR, involved in translational control of protein synthesis. Here, the signaling mechanisms of amino acids and insulin in macroautophagy in relation to mTOR were investigated. In isolated rat hepatocytes, both regulatory amino acids (RegAA) and insulin coordinately activated p70(S6k) phosphorylation, which was completely blocked by rapamycin, an mTOR inhibitor. However, rapamycin blocked proteolytic suppression by insulin, but did not block inhibition by RegAA. These contrasting results suggest that insulin controls autophagy through the mTOR pathway, but amino acids do not. Furthermore, micropermeabilization with Saccharomyces aureus alpha-toxin completely deprived hepatocytes of proteolytic responsiveness to RegAA and insulin, but still maintained p70(S6k) phosphorylation by RegAA. In contrast, Leu(8)-MAP, a non-transportable leucine analogue, did not mimic the effect of leucine on p70(S6k) phosphorylation, but maintained the activity on proteolysis. Finally, BCH, a System L-specific amino acid, did not affect proteolytic suppression or mTOR activation by leucine. All the results indicate that mTOR is not common to the signaling mechanisms of amino acids and insulin in autophagy, and that the amino acid signaling starts extracellularly with their "receptor(s)," probably other than transporters, and is mediated through a novel route distinct from the mTOR pathway employed by insulin.     

A role for acetylcholine receptors in their own aggregation on muscle cells

Both neurotrophic factors and activity regulate synaptogenesis. At neuromuscular synapses, the neural factor agrin released from motor neuron terminals stimulates postsynaptic specialization by way of the muscle specific kinase MuSK. In addition, activity through acetylcholine receptors (AChRs) has been implicated in the stabilization of pre- and postsynaptic contacts on muscle at various stages of development. We show here that activation of AChRs with specific concentrations of nicotine is sufficient to induce AChR aggregation and that this induction requires the function of L-type calcium channels (L-CaChs). Furthermore, AChR function is required for agrin induced AChR aggregation in C2 muscle cells. The same concentrations of nicotine did not induce observable tyrosine phosphorylation on either MuSK or the AChR beta subunit, suggesting significant differences between the mechanisms of agrin and activity induced aggregation. The AChR/L-CaCh pathway provides a mechanism by which neuromuscular signal transmission can act in concert with the agrin-MuSK signaling cascade to regulate NMJ formation.     

Ligand-regulated binding of FAP68 to the hepatocyte growth factor receptor

We have used the yeast two-hybrid system to identify proteins that interact with the intracellular portion of the hepatocyte growth factor (HGF) receptor (Met). We isolated a human cDNA encoding a novel protein of 68 kDa, which we termed FAP68. This protein is homologous to a previously described FK506-binding protein-associated protein, FAP48, which derives from an alternative spliced form of the same cDNA, lacking an 85-nucleotide exon and leading to an early stop codon. Here we show that epithelial cells, in which the HGF receptor is naturally expressed, contain FAP68 and not FAP48 proteins. FAP68 binding to Met requires the last 30 amino acids of the C-terminal tail, which are unique to the HGF receptor. Indeed, FAP68 does not interact with related tyrosine kinases of the Met and insulin receptor families. FAP68 interacts specifically with the inactive form of HGF receptor, such as a kinase-defective receptor or a dephosphorylated wild type receptor. In vivo, endogenous FAP68 can be coimmunoprecipitated with the HGF receptor in the absence of stimuli and not upon HGF stimulation. Thus, FAP68 represents a novel type of effector that interacts with the inactive HGF receptor and is released upon receptor phosphorylation. Free FAP68 exerts a specific stimulatory activity toward the downstream target p70 S6 protein kinase (p70S6K). Significantly, nonphosphorylated HGF receptor prevents FAP68 from stimulating p70S6K. These data suggest a role for FAP68 in coupling HGF receptor signaling to the p70S6K pathway.     

Selective androgen receptor modulator,S42 has anabolic and anti-catabolic effects on cultured myotubes

We previously identified a novel selective androgen receptor modulator, S42, that does not stimulate prostate growth but has a beneficial effect on lipid metabolism. S42 also increased muscle weight of the levator ani in orchiectomized Sprague–Dawley rats. These findings prompted us to investigate whether S42 has a direct effect on cultured C2C12 myotubes. S42 significantly lowered expression levels of the skeletal muscle ubiquitin ligase (muscle atrophy-related gene), atrogin1 and Muscle RING-Finger Protein 1(MuRF1) in C2C12 myotubes, as determined by real time PCR. Phosphorylation of p70 S6 kinase (p70S6K), an essential factor for promoting protein synthesis in skeletal muscle, was significantly increased by S42 to almost the same extent as by insulin, but this was significantly prevented by treatment with rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). However, phosphorylation of Akt, upstream regulator of mTORC1, was not changed by S42. S42 did not increase insulin-like growth factor 1 (Igf1) mRNA levels in C2C12 myotubes. These results suggest that S42 may have an anabolic effect through activation of mTORC1–p70S6K signaling, independent of IGF-1-Akt signaling and may exert an anti-catabolic effect through inhibition of the degradation pathway in cultured C2C12 myotubes.     

A potential role for extracellular signal-regulated kinases in prostaglandin F2alpha-induced protein synthesis in smooth muscle cells

To understand the mechanisms of prostaglandin F2alpha (PGF2alpha)-induced protein synthesis in vascular smooth muscle cells (VSMC), we have studied its effect on two major signal transduction pathways: mitogen-activated protein kinases and phosphatidylinositol 3-kinase (PI3-kinase) and their downstream targets ribosomal protein S6 kinase (p70(S6k)) and eukaryotic initiation factor eIF4E and its regulator 4E-BP1. PGF2alpha induced the activities of extracellular signal-regulated kinase 2 (ERK2) and Jun N-terminal kinase 1 (JNK1) groups of mitogen-activated protein kinases, PI3-kinase, and p70(S6k) in a time-dependent manner in growth-arrested VSMC. PGF2alpha also induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and basic fibroblast growth factor-2 (bFGF-2) expression in VSMC. Whereas inhibition of PI3-kinase by wortmannin completely blocked the p70(S6k) activation, it only partially decreased the ERK2 activity, and had no significant effect on global protein synthesis and bFGF-2 expression induced by PGF2alpha. Rapamycin, a potent inhibitor of p70(S6k), also failed to prevent PGF2alpha-induced global protein synthesis and bFGF-2 expression, although it partially decreased ERK2 activity. In contrast, inhibition of ERK2 activity by PD 098059 led to a significant loss of PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and bFGF-2 expression. PGF2alpha-induced phosphorylation of eIF4E and 4E-BP1 was also found to be sensitive to inhibition by both wortmannin and rapamycin. These findings demonstrate that 1) PI3-kinase-dependent and independent mechanisms appear to be involved in PGF2alpha-induced activation of ERK2; 2) PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation appear to be mediated by both ERK-dependent and PI3-kinase-dependent rapamycin-sensitive mechanisms; and 3) ERK-dependent eIF4E phosphorylation but not PI3-kinase-dependent p70(S6k) activation correlates with PGF2alpha-induced global protein synthesis and bFGF-2 expression in VSMC.     

Inhibition or down-regulation of protein kinase C attenuates late phase p70s6k activation induced by epidermal growth factor but not by platelet-derived growth factor or insulin.

The late phase of the time-dependent epidermal growth factor (EGF)-induced biphasic activation of the p70s6k is selectively attenuated by the specific PKC inhibitor, CGP 41,251, a staurosporine derivative. At a 40-fold lower concentration than CGP 41,251, staurosporine inhibits both phases of S6 kinase activation to the same extent, whereas the inactive staurosporine derivative CGP 42,700 shows no effect on either phase. Platelet-derived growth factor (PDGF) and insulin also induce biphasic S6 kinase activation, but in neither case is either phase of activation affected by the presence of CGP 41,251. This finding was unexpected in the case of PDGF, which is a potent activator of PKC and whose receptor directly interacts with phospholipase C gamma 1. However, similar results were obtained following down-regulation of PKC by prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Therefore, even though EGF and PDGF induce PKC activation, PDGF, unlike EGF, does not appear to use this signaling pathway for late phase p70s6k activation.     

Temporal and Spatial Requirements of unplugged/MuSK Function during Zebrafish Neuromuscular Development

One of the earliest events in neuromuscular junction (NMJ) development is the accumulation of acetylcholine receptor (AChR) at the center of muscle cells. The unplugged/MuSK (muscle specific tyrosine kinase) gene is essential to initiate AChR clustering but also to restrict approaching growth cones to the muscle center, thereby coordinating pre- and postsynaptic development. To determine how unplugged/MuSK signaling coordinates these two processes, we examined the temporal and spatial requirements of unplugged/MuSK in zebrafish embryos using heat-shock inducible transgenes. Here, we show that despite its expression in muscle cells from the time they differentiate, unplugged/MuSK activity is first required just prior to the appearance of AChR clusters to simultaneously induce AChR accumulation and to guide motor axons. Furthermore, we demonstrate that ectopic expression of unplugged/MuSK throughout the muscle membrane results in wildtype-like AChR prepattern and neuromuscular synapses in the central region of muscle cells. We propose that AChR prepatterning and axonal guidance are spatio-temporally coordinated through common unplugged/MuSK signals, and that additional factor(s) restrict unplugged/MuSK signaling to a central muscle zone critical for establishing mid-muscle synaptogenesis.     

Involvement of p120 catenin in myopodial assembly and nerve-muscle synapse formation

At developing neuromuscular junctions (NMJs), muscles initially contact motor axons by microprocesses, or myopodia, which are induced by nerves and nerve-secreted agrin, but it is unclear how myopodia are assembled and how they influence synaptic differentiation at the NMJ. Here, we report that treatment of cultured muscle cells with agrin transiently depleted p120 catenin (p120ctn) from cadherin junctions in situ, and increased the tyrosine phosphorylation and decreased the cadherin-association of p120ctn in cell extracts. Whereas ectopic expression of wild-type p120ctn in muscle generated myopodia in the absence of agrin, expression of a specific dominant-negative mutant form of p120ctn, which blocks filopodial assembly in nonmuscle cells, suppressed nerve- and agrin-induction of myopodia. Significantly, approaching neurites triggered reduced acetylcholine receptor (AChR) clustering along the edges of muscle cells expressing mutant p120ctn than of control cells, although the ability of the mutant cells to cluster AChRs was itself normal. Our results indicate a novel role of p120ctn in agrin-induced myopodial assembly and suggest that myopodia increase muscle-nerve contacts and muscle's access to neural agrin to promote NMJ formation.