1.1 Endoplasmic Reticulum Stress Pathway
The Endoplasmic Reticulum (ER) is a membrane-bound, intracellular organelle responsible for the proper folding, posttranslational modifications, and trafficking of transmembrane and extracellular proteins as well as maintenance of calcium homeostasis. Perturbations of the specialized environment of the ER due to numerous stressors, including glucose deprivation, aberrant calcium regulation, or increased protein flux lead to an increase in unfolded proteins. The increase in unfolded proteins activate a series of signaling transduction cascades to return the ER to equilibrium known as the unfolded protein response (UPR) or ER stress pathway . Overall, these signals lead to a global decrease in protein synthesis, increase in chaperone proteins, ER proliferation, and degradation of proteins. These adaptations attempt to alleviate the stress; however, if it cannot be resolved, the UPR can also lead to apoptosis.
There are three major sensors for ER stress: inositol requiring kinase 1 (IRE1), activating transcription factor 6 (ATF6), and double-stranded RNA activated protein kinase-like endoplasmic reticulum kinase (PERK). Each of these sub-pathways provide a specialized role for resolving stress and normal cellular function, especially in cells with large secretory roles such as hepatocytes, cells, and osteocytes.
Each of the three sensors are associated with the molecular chaperone BiP in normal state. Following stress, an increase in unfolded proteins sequesters BiP to the ER lumen, and away from the sensors, allowing activation of the sensors. A summary of ER stress signaling is found in Figure 1.
1.1.a The IRE1 pathway:
IRE1 is present in all species from yeast to mammals, and is the most evolutionary conserved branch of the UPR. IRE1 is a type I ER transmembrane protein with both endoribonuclease and kinase activity. There are two homologues: the ubiquitous IRE1 and intestinal epithelia-specific IRE1. Following BiP release from IRE1, IRE1 oligomerizes and trans-autophosphorylates the C-terminal kinase domain at S724, leading to activation of both kinase and endoribonuclease activity.
The endonuclease activity of IRE1 leads to the splicing of a 26-base intron from the X-box Protein 1 (XBP1)-mRNA. Spliced XBP1 (sXBP1) is a transcription factor that regulates ER chaperone and protein degradation-related gene expression. IRE1’s kinase domain leads to the activation of cJun NH2-terminal kinase (JNK). JNK activates of the pro-apoptotic Bim protein and the inhibition of Bcl2, and is therefore a pro-apoptotic signal. The kinase domain of IRE1 also regulates extracellular signal-regulated kinases (ERKs) and nuclear factor B (NF-B). However, the role of this is not particularly well-understood.
1.1.b The ATF6 pathway:
ATF6 is a transmembrane protein with two homologues, ATF6 is thought responsible for the transcriptional regulation of pro-survival genes. Following dissociation of BiP, ATF6 is translocated to the golgi apparatus where two proteolytic events release the transcription factor domain of ATF6.
Cleaved ATF6 leads to increased ER chaperones and unfolded protein degradation by increasing the expression of BiP, protein disulfide isomerase, and ER degradation-enhancing alpha-mannosidase-like protein 1. ATF6 also induces the expression of XBP1, which following IRE1 processing, leads to chaperone transcription. ATF6 signaling leads to a decrease in pro-apoptotic factors, thus it is cytoprotective.
1.1.c The PERK Pathway:
The main function of the PERK pathway is to modulate translation, though it does transduce both pro-survival and pro-apoptotic signals. PERK is a type I transmembrane protein, composed of an ER luminal stress sensor and a cytosolic protein kinase domain. BiP dissociation from PERK, leads to dimerization and trans-autophosphorylation of the kinase domain to activate PERK. Active PERK phosphorylates the eukaryotic translation initiation factor 2 (eIF2); eIF2 is required for translation start in eukaryotic cells. Phosphorylation of eIF2 leads to global attenuation of translation initiation which decreases the protein flux through the ER. Paradoxically, mRNAs containing internal ribosomal entry site sequences are upregulated, the most well-characterized being activating transcription factor 4 (ATF4).
ATF4 is a bZIP transcription factor that drives expression of amino acid transport and synthesis, redox reactions, and protein secretion to transduce pro-survival signals. However, ATF4 can also lead to apoptosis through upregulation of the transcription factor C/EBP homologous protein (CHOP). While CHOP does not directly cause apoptosis, it does sensitize cells to damage by reducing the pro-survival protein Bcl-2 and the antioxidant glutathione, and increasing the apoptotic genes Death Receptor 5, BIM, and PUMA.
PERK’s kinase activity also leads to the activation of NF-E2-related-factor 2 (NRF2) as a pro-survival signal and to help maintain glutathione levels as a buffer for the many free radicals produced during the UPR. NRF2 is also pro-survival through the downregulation of CHOP.
Another gene that is positively regulated by ATF4 and CHOP is the pseudokinase Tribbles-related protein 3 (Trb3). Trb3 has been shown to bind directly to CHOP, to downregulate CHOP-dependent transcription, including its own transcription (Ohoka, EMBO, 2005). Knockout of Trb3 leads to resistance to ER stress-dependent apoptosis, suggesting Trb3 is also pro-apoptotic (Ohoka, EMBO, 2005). Additionally, Trb3 has been shown to inhibit the positive protein-synthesis regulator Protein Kinase B (PKB/Akt) in the liver (Keyong Du, Science, 2003) and skeletal muscle (Liu, AJP Endocrinol Metab, 2010), by binding directly to Akt. Trb3 also blocks the translocation of the glucose transporter GLUT4 in response to insulin in skeletal muscle (Liu, AJP, 2010). Thus, aberrant signaling of Trb3 may lead to disruption of insulin homeostasis. However, Trb3 knockout animals show no changes in body composition, serum glucose, insulin and lipid levels, and glucose or insulin tolerance (Okamoto Diabetes 2007) and Trb3 is induced in skeletal muscle by exercise. In fact, Trb3 overexpression in the muscle lead to an increase muscle mass and exercise capacity without an effect on insulin signaling or glucose uptake (An, AJP Ref Inte Comp Physiol 2014).
ATF4 also leads to the upregulation of the pro-apoptotic gene CHAC1.
1.2 Cation transport regulator-like protein 1 (CHAC1)
The name CHAC1 is derived from the chaC bacterial protein, which is part of the cha operon [3-5]. The function of chaC is not defined in bacteria, but may have an auxiliary function with chaA in its antiport function of Na+, K+, Ca2+, and H+. Human CHAC1 has about 30% amino acid identity to bacterial chaC, and homologs in other organisms, including S. cerevisiae and M. musculus, have been identified. The modelled structure of CHAC1 matches a known protein fold, known as BtrG/-GCT fold , that is found in the enzyme -glutamylcyclotransferase (C7orf24, -GCT) despite no sequence similarity. The structures align well (Figure X) leading to the identification of E115 as the catalytic residue. This family of enzymes act on -glutamyl amino acids to yield 5-oxoproline and amino acids .
CHAC1 (also known as MGC4504, Botch, glutathione-specific -GCT) was identified as a gene of interest in a systems biology study of pathways involved in the inflammatory response of a population of primary human aortic endothelial cells (HAEC) treated with the oxidized phospholipid oxidized palmitoyl arachidonyl phosphatidylcholine (Ox-PAPC) . This genetics and genomics approach successfully created a network map encompassing 15 modules of highly correlated genes that are functionally linked based on their population-wide mRNA co-expression. Information from the network map was used to generate hypotheses and elucidate function of the uncharacterized gene CHAC1, which clustered with genes involved in the ER stress pathway. Treatment of multiple cell types with the ER stress agonists Tunicamycin (Tm), Thapsigargin (Th), and Dithiothreitol (DTT) increased CHAC1 mRNA, whereas other stressors like ultraviolet light and heat shock did not induce CHAC1 [8-10].
The data on CHAC1 distinguish two different roles for CHAC1 at different developmental time points. CHAC1 is largely pro-apoptotic, potentially due to regulation of glutathione, in differentiated cells. However, during development, CHAC1 appears to regulate Notch signaling, thereby regulating differentiation decisions.
1.2.a CHAC1 and Programmed Cell Death
CHAC1 overexpression has been shown to induce apoptosis as measured by increases in Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, cleaved poly(ADP-ribose) polymerase 1 (PARP), and Apoptosis Inducing Factor (AIF) nuclear translocation, which occurs as a result of ER stress agonist treatment, which CHAC1 siRNA knockdown reduced apoptosis with no alteration in the levels of ATF3, BiP, or CHOP, indicating that the CHAC1 target likely lies downstream of CHOP in the pathway. Tumor necrosis factor receptor superfamily member 6B (TNFRSF6B) was identified and tested as a potential downstream target for CHAC1, and showed decreased mRNA levels following CHAC1 overexpression and increased mRNA levels following CHAC1 siRNA treatment, consistent with a role for CHAC1 in regulating TNFRSF6B . Another report defined a role for CHAC1 in affecting Nisin induced apoptosis, assayed by DNA-fragmentation in human UM-SCC-17b cells. Here, siRNA mediated CHAC1 knockdown prevented DNA fragmentation .
CHAC1 has also been shown to be highly upregulated in response to erastin, an inhibitor of xc- (a Na+-independent cystine-glutamate antiporter) and inducer of ferroptosis. Inhibition of the xc- system by erastin leads to a depletion of intracellular cysteine, activation of the eIF2alpha-ATF4 branch of ER stress, a depletion of intracellular glutathione, and finally cell death via ferroptosis. Ferroptosis is a non-apoptotic mechanism of regulated cell death, which is dependent on iron and oxidative injury. Inactivation of GSH-dependent antioxidant systems leads to an accumulation of oxidized lipids within the cells and triggers ferroptosis. Thus, CHAC1’s regulation of glutathione may make it a regulator of ferroptosis in addition to apoptosis.
1.2.b CHAC1 and Glutathione
CHAC1 has been shown to have -glutamylcyclotransferase (GGCT) activity; one known substrate is glutathione (GSH) in a yeast and cell-free model . GSH is a ubiquitous, antioxidant tripeptide (glu-cys-gly) synthesized in the cytosol. The main function of GSH is as a reducing agent to protect vulnerable thiol moieties from oxidative damage, with other roles in signal transduction, gene expression, apoptosis, and nitric oxide metabolism. The intracellular concentration of GSH is in the millimolar range, with the reduced GSH being the major form with its concentration being from 10-100 times that of the disulfide species (GSSG). Known mechanisms for GSH depletion are oxidation, conjugation to electrophiles, and plasma membrane efflux.
CHAC1 is the first described protein to directly degrade GSH in the cytosol. Yeast (YER163C) and mouse (Chac1) homologues were shown to contain the BtrG/GGCT fold with a highly conserved catalytic site. This catalytic site was validated by lack of GGCT activity when this residue was mutated. The GGCT activity of CHAC1 family proteins was specific for glutathione and yielded 5-oxoproline and a cysteine-glycine dipeptide in yeast and a cell-free model. The authors postulate that the pro-apoptotic function of CHAC1 is due to degradation of GSH, leading to a permissive state for apoptosis (Kumar, Embo Reports). Further work, using a ER-sequestered CHAC1 construct, showed that CHAC1’s enzymatic activity is selective for reduced GSH over GSSG (Tsunoda, eLIFE, 2014). A degradation of reduced glutathione should increase oxidative stress and injury, further suggesting that CHAC1’s activity on glutathione is responsible for its pro-apoptotic activity. Paradoxically, a CHAC2 homologue in Arabidopsis thaliana, which also has GGCT activity towards glutathione, protects the plant from heavy metal toxicity by recycling the glutamate and allowing for increased synthesis of glutathione (Paulose, The Plant Cell, 2013). Thus, the overall effect of this degradation of glutathione may actually be protective by allowing for increased oxidative stress buffering.
1.2.c CHAC1 and Notch
CHAC1 has also been defined as a regulator of neural cell development, through a direct effect on Notch 29, a highly conserved developmental pathway. Notch is a pleiotropic, conserved, developmental receptor that can influence differentiation, proliferation and apoptotic cell fates in a context-dependent manner through lateral specification events. Generally, Notch signaling inhibits differentiation. This signaling pathway links the fate of one cell with that of its neighbor 30. In the canonical pathway, one of four Notch receptors (Notch 1-4) is trafficked through the Golgi, where maturation requires a furin-like cleavage 31 and O-linked glycosylation at several sites 32. The mature receptor is then deposited on the cell membrane where it can interact to one of five ligands (Delta-like ligands 1,3, 4, or Jagged ligands J1, J2). Ligand binding leads to two sequential proteolytic cleavages by an Adam family metalloproteinase and the presenillin-containing gamma-secretase. These cleavages result in the release of an intracellular transcription factor that associates with the DNA binding protein CSL (CBF-1, Supressor of Hairless, Lag-1; aka RBP-J), which bind and regulate the transcription of Notch-responsive genes, which generally influence differentiation decisions 30. In the absence of the Notch intracellular domain, CSL binds to co-repressors Notch-responsive genes and inhibits their transcription. In the brain, Notch-induced transcription inhibits neuronal differentiation and maintains cells in the progenitor fate 33. A known inhibitor of Notch signaling is the Numb protein, through effects of endocytosis. Asymmetric division of Numb in daughter cells helps determine Notch activity and thus, cell fate of the daughter cells.
CHAC1 was found to be spatiotemporally expressed at the same time as Notch and to antagonize Notch signaling leading to increased neurogenesis. This finding led the authors to rename CHAC1 to Botch (Blocks Notch). Overexpression of CHAC1 drove neural stem cell exit of the proliferating zone, while down-regulation led to stem cell retention in the proliferating zone. Confocal microscopy and sucrose gradient fractionation suggest CHAC1 is located in the trans-Golgi network as it co-localizes with the marker TGN38. These In vitro furin cleavage assays data suggest that CHAC1 prevents the furin-like cleavage of Notch, in the trans-Golgi, thereby preventing the maturation of Notch. This finding led the authors to rename CHAC1 to Botch (Blocks Notch) 29.
Further work demonstrated that the Notch receptor is a substrate of CHAC1’s GGCT activity, with the same active site moiety (E115 in humans) as is required for CHAC1’s activity toward glutathione. E1669 in the Notch receptor has a post-translationally added -linked glycine, which is required for the furin-like cleavage which occurs in the golgi apparatus. CHAC1 de-glycinates the Notch receptor through its GGCT activity, thus blocking the necessary furin-like cleavage and the Notch receptor’s presentation at the membrane. Thus, CHAC1 regulates neuronal cell fate during embryogenesis via GGCT activity on the Notch receptor.
1.3 Skeletal System
The skeletal system has five main functions: support, leverage for skeletal muscle, protection of internal organs, storage of minerals (calcium and phosphorous) and lipids, and hematopoiesis . Osseous tissue is a specialized connective tissue that contains a calcium matrix, osteocytes, osteoblasts, and osteoclasts.
The bone matrix is formed from hydroxyapatite and collagen. Hydroxyapatite is a crystal of calcium phosphate and calcium hydroxide, which incorporates ions and other calcium salts into the matrix as they crystallize. These crystals are very hard, yet they are brittle. Thus, the hydroxyapatite allows bones to withstand compression. However, resistance to bending, twisting, and sudden impacts is provided by collagen.
There are three main types of cells within the bone: osteoblasts, osteocytes, and osteoclasts. Osteoblasts are immature cells that produce new bone matrix. First, they deposit the proteins of the extracellular matrix, mainly collagen type I, which acts as the template for mineralization. A layer of osteoid, or unmineralized bone matrix, is also deposited on the surface under osteoblasts. Mineralization is then stimulated by an increase in local calcium phosphate concentration. Prior to mineralization, osteoblasts form cytoplasmic connections, via gap-junctions with one another to be used to share nutrients, metabolites, and communication between osteocytes. Once mineralization has occurred, osteoblasts mature to form osteocytes. Osteocytes maintain the bone matrix around themselves by both synthesis and (limited) resorptions of matrix.
Osteoclasts are cells that participate in bone remodeling by osteolysis, or resorption of bone matrix. These large cells form from the fusion of mononuclear precursors from hematopoietic tissues, and so are multinucleated and have a high density of lysosomes. Osteoblasts positively regulate osteoclast differentiation, survival, fusion, and function via release of RANKL, and negatively regulate osteoclasts via osteoprotegrin, which is a soluble, decoy receptor for RANKL.
1.3.a Bone Formation
There are two mechanisms for the formation of bone: intramembranous ossification and endochondral ossification. Endochondral ossification occurs when cells in the center of mesenchymal condensations differentiate to chondrocytes. These chondrocytes then go through a process of proliferation, maturation, and apoptosis to form the skeleton. During intramembranous ossification, mesenchymal cells give rise to pre-osteoblasts, which then differentiate to form functional osteoblasts through the action of the transcription factors Runx2 and Osterix.
Bone remodeling, a process by which old matrix is resorped and neosynthesis of new matrix occurs, happens continuously in post-natal life to control bone quality and structure. The processes of resorption and synthesis are coupled to ensure the maintenance of healthy bone mass. Osteoblasts can regulate osteoclastogenesis through the release of the negative regulator osteoprotegrin, or the positive regulators RANKL and MSCF. Dysregulation of either osteoblast or osteoclast function can cause severe effects on bone, leading to clinical disease such as osteoporosis (decreased bone density) and osteopetrosis (increased bone density).
1.3.b Osteoblast Development
Osteoblasts arise from mesenchymal stem cells, which are found in the bone marrow stroma. Mesenchymal stem cells are multipotent stem cells that may form bone, cartilage, marrow adipocytes, and fibrous tissue (Friedenstein 1990, Owen 1998, Prockop 1997). Runt related transcription factor 2 (Runx2) is a major regulatory factor that can induce an osteoblastic fate. Ectopic expression of Runx2 in non-osteoblastic cells leads to expression of osteocalcin, an osteoblastic-specific marker (Banerjee 1997, and Ducy 1997), 1(I) collagen, the major collagen in bone, osteopontin, and bone sialoprotein (Ducy 1997). Opposingly, Runx2 knockout mice have a skeleton solely made of cartilage – no bone formation occurs in these animals due to maturational defects in osteoblasts (Komori 1997). Even Runx2-heterozygote animals have skeletal abnormalities, such as delay in the suture of fontanelles, which suggests that Runx2 gene dosage is critically important. While Runx2 is a requirement for differentiation of osteoblasts, it is not sufficient (Lee, 1999; Wang 1999).
1.3.c Notch Signaling in Bone
Notch receptors 1,2 and their receptors are expressed in osteoblasts, while Notch 3 receptor is found in specific osteoblast lineage cells at low levels (Pereira 2002, JCell biochem; Nobta, 2005, JBC; Schnabel, 2002, J. Mol. Med). Notch has recently been shown to have multifunctional effects during development and postnatal life in the bone. Notch signaling is required for proper somitogenesis and patterning of the embryo. Beyond Notch’s role in embryogenesis, Notch plays an integral role in osteoblastogenesis, though this role has been shown to be both pro-differentiation (Tezuka, 2002, J. Bone Miner Res; MCLarren, 2000, JBC) and anti-differentiation (Deregowski, 2006, JBC; Sciaudone, 2003, Endocrinology; Zamurovic, 2004, JBC), osteoclastogenesis (Yamada, 2003, Blood), and chondrogenesis (Hayes 2003, J Anat; Fujimaki, 2006, J Bone Miner Metab; Watanabe, 2003, J Bone Miner Metab; Nakanishi, 2007, Mech Dev; Oldershaw, 2008, Stem Cells; Crowe, 1999, Development).
This dimorphic effect of Notch is illustrated by a set of transgenic mice in which the Notch intracellular domain (NICD) was under control of either the early osteoblast promoter, 3.6kb Col1a1, or the late osteoblast promoter, 2.3kb Col1a1. In the mice with NICD overexpressed in early osteoblasts, differentiation was inhibited and they had an osteopenic phenotype (Zanotti, 2008, Endocrinology). In vitro studies illustrate that Notch suppresses Wnt/beta-catenin signaling to prevent differentiation (Deregowski, 2006, JBC). However, in mice where NICD was overexpressed later during differentiation, under control of the 2.3kb Col1a1 promoter, there was an increase in osteoblast number, proliferation, and formation, leading to an osteosclerotic phenotype (Engin, 2008, Nature Medicine). The loss of Notch in the two different developmental periods also show the dimorphic effects of Notch. In osteoblast progenitors in which Notch signaling is ablated early in development, by the deletion of Presenilins, there is a high bone mass, though as these mice age, they lose bone mass, likely due to the loss of osteoblast progenitor cells (Hilton, 2008, Nature Medicine). However, if Notch is ablated in mature osteoblasts, mice have low bone density due to low osteoprotegrin, which increases osteoclastogenesis and no direct effect on osteoblast number or bone formation (Engin, 2008, Nature Medicine).
Notch may also regulate bone density in mature osteocytes. Knockout of Notch-1 and -2 in osteocytes, via control of the osteocyte specific promoter dentin matrix protein 1 (DMP1), leads to an increase in trabecular bone volume due to increased osteoblasts, and decreased osteoclasts (Canalis, 2013, JBC). Activation of Notch-1 and -2 in osteocytes, via control of the DMP1 promoter, lead to an increase in trabecular bone via decreased bone resorption, and increased cortical bone via increased bone formation (Canalis, 2013, JBC). Thus, it is clear from these studies that Notch plays an important role in all stages of bone development and maintenance, and perturbations in this pathway may lead to one of many different phenotypes depending on the developmental stage.
Notch signaling likely regulates osteoblastogenesis through its downstream-effector gene Hes1 and Hes1’s regulation of Runx2 (McLarren, 2000, JBC).
1.3.d ER stress in Bone:
ATF4, the transcription factor that controls CHAC1 gene expression, has been shown to be extremely important for bone biology. ATF4-/- mice are runted and die perinatally (Tanaka, 1998; Hettman 2000; Masuok 2002), suggesting that ATF4 is involved in osteoblastogenesis. Further work showed ATF4-/- embryos have decreased mineralization as compared to control embryos. However, this change in skeletal elements in ATF4-/- embryos is similar to wild-type on embryonic day 13 and earlier, so ATF4 must be involved in later skeletonogenesis (Yang, 2004, Cell). A clear delay in osteoblastogenesis was seen in E15 embryos where trabeculae were absent in ATF4-/- embryos (Yang, 2004, Cell). This defect is explained by the requirement of ATF4 for the expression of osteocalcin – a regulator of terminal osteoblast differentiation (Yang, 2004, Cell). ATF4 also regulates trabecular number and thickness postnatally; ATF4-/- mice continue to have trabecular deficiencies throughout their lives, as they never terminally differentiate as evidenced by a decrease in osteocalcin expression (Yang 2004, Cell). The importance of ATF4 to osteoblast differentiation is further illustrated by the fact that ATF4 is able to induce osteoblastic differentiation in non-osteoblastic cell lineages via induction of osteocalcin expression (Yang and Karsenty, 2004 JBC).
Additionally, ATF4 may regulate bone density via transport of amino acids. ATF4-/- embryos (E16) show a defect in protein synthesis, as suggested by the decrease in cell size and collagen type I protein expression, though mRNA expression of alpha1 (I) Collagen is unchanged compared to wildtype (Yang 2004).
These data suggest that ATF4 is required for the terminal differentiation of osteoblasts by directing late osteoblast gene transcription, but it does not affect early differentiation and osteogenesis. Additionally, since ATF4 is a transcription factor, it is likely that a downstream effector may also be involved separate from its effects on osteocalcin, and that downstream effector may be CHAC1.
1.4 Skeletal Muscle
Skeletal muscle is the predominant tissue in the body, making up ~50% of body mass. Skeletal muscles attach to the skeleton and produce movement via nerve-induced contraction.
Somites, which give rise to the myotome, dermatome, and sclerotome, are formed from the mesoderm during embryonic development and are located on either side of the notochord. Cells of the dorsal part of the somite, via transcription factor Pax3 and Pax7 expression, become the dermomyotome. The dermomyotome forms the dermis and musculature . Progenitor cells from the dermomyotome migrate to the limb buds, via regulation by Pax3 and c-Met (a tyrosine kinase receptor), to form the myotome from which the skeletal muscle will develop [14-16]. The myoblast developmental program is then turned on by the down-regulation of Pax3 and the expression of myogenic transcription factors Myf5, Mrf4, and MyoD. Terminal differentiation of myoblasts is controlled by two families of transcription factors: the basic Helix-Loop-Helix myogenic regulatory factors (MRFs), such as MyoD, myogenin, and Mrf4, and myocyte enhancer factor 2 (MEF2) proteins. These two families of proteins work together to lead to myogenic differentiation by cooperatively binding to target DNA sequences. Each MRF is able to initiate myogenesis in any cell type, but it requires the function of MEF2 proteins; but, MEF2 proteins are not sufficient to induce myogenesis. The muscle is then formed as myocytes fuse to form multinucleated myofibers and the myotome.
Cells expressing Pax3 and Pax7, but no myogenic markers, migrate from dermomyotome to the myotome and proliferate extensively to create the skeletal muscle tissue. These Pax3+/Pax7+ cells are also the most likely source of satellite cells, which emerge towards the end of embryogenesis. There is also evidence that satellite cells go through a Pax7+/MyoD+ state. Satellite cells are the stem cells of the muscle, and play a major role in postnatal growth and development.
1.4.b Satellite Cells
In 1961, Alexander Mauro detected a mononucleated cell between the muscle fiber and basement membrane in electron micrographs of skeletal muscle from frogs, which he hypothesized could represent a muscle progenitor cell (Mauro 1961). A series of studies using tritiated thymidine confirmed that satellite cells are mitotically quiescent in healthy muscle and are the source of regenerating muscle nuclei (Reznik, 1969, J Cell Biol; Schultz 1978, J. Exp. Zool; Snow, 1977, Anat. Rec). Additionally, it has been shown that daughter cells from satellite cells both add to the regenerating muscle and re-populate the stem cell niche (Lipton and Schultz, 1979, Science; Moss and Leblond, 1970 J. Cell Biol; Moss and Leblond, 1971, Anat. Rec.; Schultz, 1996, Dev. Biol.). Thus, it has been confirmed that satellite cells are bona fide skeletal muscle stem cells with the ability self-renew. This makes satellite cells unique among other tissue stem cells in that it was found histologically before functionally.
Immunotypic analysis identified the paired box 7 (Pax7) transcription factor as a marker of satellite cells (Seale, 2000, Cell). In a quiescent state, satellite cells also express Myf5, but lack MyoD (Charge, 2004, Physiol. Rev.)
Satellite cells are stem cells, as such they must be able to self-renew. Two proposed mechanisms for this self-renewal have been proposed: asymmetric division to one activated daughter cell and one quiescent daughter cell, or symmetric division where some cells retain Pax7 expression and return to quiescence. So long as only one daughter of the symmetric cell division is instructed to differentiate, the stem cell pool can be maintained and regeneration can occur. Evidence about the asymmetric inheritance of Numb (Conboy and Rando, 2002, Dev. Cell; Shinin 2006, Nat. Cell Biol), MyoD (Zammit 2004, J. Cell biol), and Dek (Cheung 2012, Nature) suggests asymmetric cell division. Most recently, evidence in zebrafish demonstrates asymmetric division of stem cells during muscle injury repair (Gurevich 2016, Science), validating the previous in vitro studies and confirming that satellite cells exhibit asymmetric division to maintain the satellite cell pool.
The satellite cell niche, a depression in the sarcolemma, provides inhibitory mitogenic signals (Bischoff, 1986; Dev Biol “Proliferation of muscle stem cell…”; Orford and Scadden 2008, Nat Rev Genet.). This suggests an elegant model for the activation of satellite cells following injury: following injury in which the sarcolemma is damaged, the satellite cell is no longer receives inhibitory signals, and therefore proliferates quickly. The inhibitory signals that the niche uses to maintain satellite cell quiescence has not yet been identified.
1.4.c Postnatal Growth
Postnatal growth of skeletal muscle mass generally occurs through hypertrophy, or enlargement of muscle fibers. This growth occurs in response to mechanical overload (strength training) or anabolic hormonal stimulation (-adrenergic agonist or testosterone). The regulation of muscle cell size is dependent on the balance between protein synthesis and protein degradation. The two pathways that regulate protein synthesis are the positive regulator Insulin-like growth factor 1 – phosphoinositide kinase, protein kinase B/Akt, mammalian target of rapamycin (IGF1-PI3K-Akt-mTOR) pathway [17, 18] (which will be explored in more detail below), and the negative regulator myostatin-Smad2/3 pathway. Hypertrophy requires an excess of protein synthesis over protein degradation. Multiple different stimuli, including nutrient availability, androgens, and loading, all integrate to cause changes in muscle mass, which depend on the muscle and muscle fiber type.
Myostatin, a member of the TGF superfamily, negatively regulates protein synthesis; myostatin mutations in mammals lead to muscle hypertrophy (Lee, 2004, Annu Rev Cell Dev Biol). Further studies in vitro strengthen this idea: addition of purified myostatin to myotubes lead to decreased protein synthesis and myotube size (Taylor, 2001, Am J Physiol Endocrinol Metab). Finally, induction of muscle atrophy in mice occurs following systemic administration of myostatin (Lee, 2004, Annu Rev Cell Dev Biol). Myostatin forms a heterodimeric receptor with Activin A to signal through phosphorylation and nuclear translocation of the Smad-2 or -3 transcription factors, and dimerization with Smad-4. It is still not known the exact molecular targets downstream of Smad-2/-4 or Smad-3/-4 dimers, though it is possible that they interfere with Akt-mTOR signaling. For example, inhibition of Smad3 activity is required for follistatin-induced muscle growth and mTOR activation (Remy, 2004, Nat Cell Biol).
It has been shown that skeletal muscle hypertrophy occurs without activation of satellite cells. Satellite cell ablation does not affect muscle fiber size during a two-week hypertrophic stimulus. Though, longer periods of hypertrophy may require satellite cells as there is an accretion of nuclei, in addition to size.
1.4.d Notch Signaling in Muscle
Notch has been shown to play a role in both embryonic development and postnatal regeneration. During embryogenesis, Notch regulates the development of the somites, which give rise to the myotome. More specifically, activation of Notch signaling leads to inhibition of differentiation both in vitro and in vivo. Muscle injury is known to lead to the activation of Notch receptor, as evidenced by increased cleaved Notch1. Additionally, the Notch-target gene Hey1 inhibits the MyoD-dependent induction of Mef2C and myogenin by compromising the recruitment of MyoD to its target promoters. Other Notch-target genes, Hes1 and 2, has also been shown to prohibit MyoD-induced myogenesis. Constitutive active Notch signaling leads to an increase in the stem cell marker Pax3 and a decrease in Myf-5, MyoD, and desmin, but no change in Pax7, while blocking Notch, through upregulation of the Notch-inhibitor, leads to an increase in Myf-5 and desmin proteins and a decrease in Pax3. These biochemical changes also lead to changes I cell fate. Notch overexpression also leads to increased cell proliferation, as evidenced by increased BrdU incorporation; while Numb overexpression leads to decreased cell proliferation. However, overexpression of Notch1 decreases myotube formation, while overexpression of Numb increases myotube formation. Thus, Notch signaling promotes progenitor cell proliferation while the blockade of Notch signaling by Numb is required for these progenitors to exit the cell cycle and express MRFs, ultimately leading to terminal differentiation. It has also been shown that NICD overexpression can rescue Pax7-deficient satellite cells from cell death and lead to proliferation of these cells (Pasut et al, Cell Reports 16, 1-11). However, these rescued satellite cells do not differentiate to MyoD expressing myotubes, but rather to brown adipocytes (Pasut).
Notch can also block myogenesis through a CSL-independent mechanism
The Notch-inhibitor Numb is also known to be asymmetrically divided between two daughter cells, allowing for differential cell fate decisions. Numb expression leads to differentiation of daughter cells down the myogenic fate.
Muscle injury induces the activation of Notch1 and the proliferation of satellite cells. Within twenty-four hours, the Notch ligand, Delta, increases in expression, along with the activated form of the Notch receptor. At this point, Notch promotes the proliferation of satellite cells and progenitors which is required for proper muscle regeneration. This is supported by the studies that show that deletion of CSL leads to the activation and ectopic differentiation of satellite cells that skip the progenitor stage. This skipping of the progenitor stage leads to the loss of the satellite cell pool. Following this, a decrease in Notch is required for progenitors to become fusion competent myoblasts; this decrease in Notch occurs by increased expression and polar localization of the Notch inhibitor in a subset of progenitors, Numb. The presence of Numb in cells promotes differentiation of progenitors down the myogenic pathway. Further inhibition of Notch, potentially through Numb, is required for the fusion of myoblasts to become myotubes.
The role of Notch in hypertrophy is not well understood, as hypertrophy usually does not involve a large amount of satellite cell activation. But, a recent publication illustrates that Notch-1 and myogenin were co-localized in hypertrophied muscle (Akiho M, Life Sciences 2009).
As an organism ages, the regenerative potential of satellite cells decrease. This may be due to a decrease in satellite cell number or a decrease in the ability of satellite cells to proliferate. Conboy et al. demonstrated the Notch signaling can improve the ability of aged muscle to regenerate following injury. Additionally, inhibition of Notch signaling can also decrease the regenerative capacity in young animals.
Thus, it is clear that the dosage and timing of Notch signaling is critically important for the proper regeneration of injured muscles.
1.4.e ER Stress and Muscle:
The muscle is unique in its stressors that cause ER stress; muscle has a restricted secretory function but the extensive sarcoplasmic reticulum plays a major role in calcium homeostasis, which is critical for proper muscle contraction. Thus, any stress in the sarcoplasmic reticulum, can have major effects on muscle contraction. While it has been known that ER stress occurs in muscle pathologies, such as myotonic dystrophy Type I (Ikezoe et al 2007) and autoimmune myositis (Nagaraju, 2005; Vitadello 2010), it had been unclear whether ER stress occurred in non-pathological states. However, more recently, multiple studies showed ER stress during myogenesis (Nakanishi 2005, 2007), aging (Ogata, 2009), exercise (Kim, 2011; Wu et al, 2011, Cell Metab), and unloading (Alibegovic, 2010).
Use of the ER stress agonists tunicamycin and thapsigargin can lead to increased formation of contracting myofibers and apoptosis (Nakanishi, 2007). These changes have been shown to be due to increased CHOP and BiP in response to increased ATF6 activity (Nakanishi 2005). These data suggest that ER stress is required for proper development on muscles, though this mechanism needs to be verified in vivo. Interestingly, aging leads to increased ER stress signaling (Ogata, 2009); this is likely due to decreased chaperones causing cellular stress (Naidoo, 2009).
Repeated contractile activity, such as occurs during exercise, can impair calcium homeostasis. Therefore, it is likely that part of the adaptation to exercise includes improving the ER’s ability to handle this calcium-induced stress. This appears to be the case, as following a 200km race, expression of ATF4 and XBP-1 was increased in humans (Kim, 2011). Studies in mice corroborate these findings; a single-bout of exercise activated the ATF6 branch of the ER stress pathway in weight-bearing muscles, but not the heart or non-weight bearing muscles. Interestingly, these changes were suppressed during long-term training (Wu 2011, Cell Metab). These data suggest that the ER stress pathway leads to physiological changes that allow the muscle to adapt to cellular stress more efficiently during exercise training due to increased mechanical stress. Furthermore, it is interesting to note that muscle unloading, via bed rest, can also induce the ER stress pathway in humans (Alibegovic, 2010). However, these changes are not consistent when applied to a rat model of hindlimb unloading after either 7 (Hunter, 2001) or 14 days (Ogata, 2009). Overall, it is clear that the role of ER stress in muscle physiology requires further investigation.
1.5 Insulin-like Growth Factor-I (IGF-I)
IGF-I is a hormone which is primarily produced by the liver under the control of growth hormone, and regulates growth in children and anabolism in adults (Schoenle E Nature 1982). IGF-I can also signal in autocrine and paracrine methods, and is produced locally in many tissues.
1.5.a IGF-I in Bone:
IGF-I signaling regulates bone size, density, length, and architecture, generally in an autocrine and paracrine manner, though it is likely that endocrine action of IGF-I is also important. Serum levels of IGF-I do correlate to bone mineral density, femur cross-sectional area, and hip fracture risk (Donahue and Rose, 1998, Boonen 1997, Langlois 1998, Nicholas 1994, Sugimoto, 1997, Bauer, 1998, Rosen and Pollak 1999; Garnero, 2000; Kurland, 1997).
The bone is a major source of newly synthesized IGF-I and IGF-II through release from matrix during resorption. IGFs then can act in a paracrine manner to promote growth: IGF-I released from bone matrix stimulates osteoblastic differentiation of mesenchymal stem cells due to activation of the Akt/mTOR pathway (Xian L, Nat Med 2012). Additionally, there is more IGF-I activity in the trabecular bone (Benedict et al 1994). Though, some IGF growth-promoting activity may be derived from IGF-I in the serum. mRNA for the IGFs is found in osteoblasts in trabecular bone during skeletogenesis (Shinar et al 1993, Wang et al, 1995). Transgenic overexpression of IGF-I under control of the osteocalcin promoter leads to significantly higher trabecular and cortical bone mineral density (Zhao, 2000).
Binding of IGF peptides to their plasma-membrane bound receptor is generally anabolic. Stimulation of the type I IGF receptor increases DNA and protein synthesis (Canalix 1993; Jonsson 1993, Raile 1994, Wergedal, 1990) likely through the Akt/mTOR pathway (Xian L, Nat Med 2012).
1.5.b IGF-I in Skeletal Muscle:
The role of IGF1 as a positive regulator of muscle mass has been verified by numerous experiments both in vitro and in vivo. Inactivation of the muscle IGF1 receptor leads to decreased muscle fiber number and size (Mavalli, 2010, JCI), while muscle-specific overexpression of IGF1 leads to hypertrophy (Musaro, 2001, Nat Genet). Downstream, IGF1 activates the mitogen-activated protein kinase (MAPK) and PI3K-Akt pathways. However, the hypertrophic effect of IGF1 is regulated through the PI3K-Akt pathway (Murgia, 2000, Nat Cell Biol), and overactivation of Akt leads to hypertrophy of muscle fibers (Bodine 2001, Nat Cell Biol; Pallafacchina, 2002, PNAS).
Akt stimulates the activation of mTOR; mTOR can form two separate complexes, mTOR complex 1 (mTORC1) containing raptor, and mTOR complex 2 (mTORC2) containing rictor. Muscle specific knockdown of mTOR (Risson, 2009, JCB) and raptor (Bentzinger, 2008, Cell Metab) lead to decreased postnatal growth, due to reduced size of fast fibers (but not slow), and severe myopathy. However, knockdown of rictor (Bentzinger, 2008, Cell Metab) in the muscle does not lead to a phenotype, suggesting that mTORC1 regulates most hypertrophy in the muscle.
Downstream of mTORC1 are eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and S6 kinase 1 (S6K1). 4EBP1 is a negative regulator of translation initiation factor eIF4E, and phosphorylation of 4EBP1 by mTORC1 relieves the inhibition on translation (Fingar DC, 2003 Genes Dev). S6K1 phosphorylation leads to the activation of the activates ribosomal protein S6 (Fingar DC, 2003 Genes Dev). Phosphorylation of both 4EBP1 and S6K1 promote protein synthesis; however, only deletion of S6K1 leads to muscle atrophy (Le Bacquer 2007, JCI; Mounier, 2011, Cell Cycle).
Akt also leads to the phosphorylation of GSK3, thereby preventing the inhibitory effect of GSK3 on eukaryotic initiation factor 2B, which also leads to increased translation (Welsh GI, FEBS Lett. 1998). Thus, Akt increases protein synthesis through mTOR and GSK3. However, Akt can also decrease protein degradation through phosphorylation of the transcription factor FOXO. Phosphorylation of FOXO sequesters it in the cytoplasm, thus it is unable to direct transcription of its target genes: muscle-specific ubiquitin E3 ligases (Stitt TN Mol Cell 2004).
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