Muscle-specific overexpression of the type 1 IGF receptor results in myoblast-independent muscle hypertrophy via PI3K, and not calcineurin, signaling

The insulin-like growth factors (IGF-I and IGF-II), working through the type 1 IGF receptor (IGF-1R), are key mediators of skeletal muscle fiber growth and hypertrophy. These processes are largely dependent on stimulation of proliferation and differentiation of muscle precursor cells, termed myoblasts. It has not been rigorously determined whether the IGFs can also mediate skeletal muscle hypertrophy in a myoblast-independent fashion. Similarly, although the phosphatidylinositol 3-kinase (PI3K) and calcineurin signaling pathways have been implicated in skeletal muscle hypertrophy, these pathways are also involved in skeletal myoblast differentiation. To determine whether the IGFs can stimulate skeletal muscle hypertrophy in a myoblast-independent fashion, we developed and validated a retroviral expression vector that mediated overexpression of the human IGF-1R in rat L6 skeletal myotubes (immature muscle fibers), but not in myoblasts. L6 myotubes transduced with this vector accumulated significantly higher amounts of myofibrillar proteins, in a ligand- and receptor-dependent manner, than controls and demonstrated significantly increased rates of protein synthesis. Stimulation of myotube hypertrophy was independent of myoblast contributions, inasmuch as these cultures did not exhibit increased levels of myoblast proliferation or differentiation. Experiments with PI3K and calcineurin inhibitors indicated that myoblast-independent myotube hypertrophy was mediated by PI3K, but not calcineurin, signaling. This study demonstrates that IGF can mediate skeletal muscle hypertrophy in a myoblast-independent fashion and suggests that muscle-specific overexpression of the IGF-1R or stimulation of its signaling pathways could be used to develop strategies to ameliorate muscle wasting without stimulating proliferative pathways leading to carcinogenesis or other pathological sequelae.     

Regulation of cell growth by redox-mediated extracellular proteolysis of platelet-derived growth factor receptor beta

Redox-regulated processes are important elements in various cellular functions. Reducing agents, such as N-acetyl-l-cysteine (NAC), are known to regulate signal transduction and cell growth through their radical scavenging action. However, recent studies have shown that reactive oxygen species are not always involved in ligand-stimulated intracellular signaling. Here, we report a novel mechanism by which NAC blocks platelet-derived growth factor (PDGF)-induced signaling pathways in hepatic stellate cells, a fibrogenic player in the liver. Unlike in vascular smooth muscle cells, we found that reducing agents, including NAC, triggered extracellular proteolysis of PDGF receptor-beta, leading to desensitization of hepatic stellate cells toward PDGF-BB. This effect was mediated by secreted mature cathepsin B. In addition, type II transforming growth factor-beta receptor was also down-regulated. Furthermore, these events seemed to cause a dramatic improvement of rat liver fibrosis. These results indicated that redox processes impact the cell's response to growth factors by regulating the turnover of growth factor receptors and that "redox therapy" is promising for fibrosis-related disease.     

Growth factors in skeletal muscle regeneration

Adult skeletal muscles are able to regenerate after injury. This process is due to the activation of quiescent muscle precursor cells, also called satellite cells, which proliferate and differentiate to form new myotubes. In this regeneration process, several growth factors which come from the muscle and/or from the motor nerve and inflammatory cells have been shown to play key roles. However, most of our knowledge comes from in vitro studies, where, during myogenesis, proliferation of satellite cells is regulated by FGFs, TGFβs, PDGF, IGF-I and II, while differentiation appears to be promoted mainly by IGFs. During regeneration in vivo, most of these factors have been shown to operate and interact. Other factors also appear to condition the regeneration process, such as LIF, which acts predominantly as a proliferative factor; and HARP/PTN/HB-GAM and other neurotrophic factors, which may be necessary for the formation of new neuromuscular junctions. TGFβ has a major influence on the reorganisation of the extracellular matrix. This review presents a critical summary of the known effects of growth factors on skeletal muscle regeneration.     

Involvement of Ras and Ral in chemotactic migration of skeletal myoblasts

In skeletal myoblasts, Ras has been considered to be a strong inhibitor of myogenesis. Here, we demonstrate that Ras is involved also in the chemotactic response of skeletal myoblasts. Expression of a dominant-negative mutant of Ras inhibited chemotaxis of C2C12 myoblasts in response to basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1), key regulators of limb muscle development and skeletal muscle regeneration. A dominant-negative Ral also decreased chemotactic migration by these growth factors, while inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase (MEK) showed no effect. Activation of the Ras-Ral pathway by expression of an activated mutant of either Ras, the guanine-nucleotide dissociation stimulator for Ral, or Ral resulted in increased motility of myoblasts. The ability of Ral to stimulate motility was reduced by introduction of a mutation which prevents binding to Ral-binding protein 1 or phospholipase D. These results suggest that the Ras-Ral pathway is essential for the migration of myoblasts. Furthermore, we found that Ras and Ral are activated in C2C12 cells by bFGF, HGF and IGF-1 and that the Ral activation is regulated by the Ras- and the intracellular Ca(2+)-mediated pathways. Taken together, our data indicate that Ras and Ral regulate the chemotactic migration of skeletal muscle progenitors.     

Mitochondrial heat shock protein 70, a molecular chaperone for proteins encoded by mitochondrial DNA

The protracted absence of muscle activation initiates complex cellular and molecular reactions aimed at restoring functional neuromuscular transmission and preventing degenerative processes. A central aspect of these reactions is the sprouting of intramuscular nerves in the vicinity of inactivated muscle fibers. Sprouts emerging from terminal nerve branches and nodes of Ranvier can reestablish functional contacts with inactive muscle fibers, and this is an essential restorative process in pathological conditions of the neuromuscular system. Due to their rapid upregulation in inactive skeletal muscle fibers and their ability to induce nerve sprouting in adult muscle, insulin-like growth factors (IGFs) are candidate signaling molecules to promote restorative reactions in the neuromuscular system. In this study we have exploited the high affinity and specificity of IGF-binding protein 4 (IGF-BP4) and IGF-BP5 for IGF1 and IGF2 to determine whether these growth factors are involved in the nerve sprouting reaction in paralyzed skeletal muscle. In tissue culture experiments with sensory- and motoneurons we demonstrate that the neurite promoting activity of IGF1 is blocked by IGF-BP4, and that a similar IGF-BP-sensitive activity is detected in muscle extracts from paralyzed, but not from control muscle. In in vivo experiments, we show that local delivery of IGF-BP4 to Botulinum toxin A-paralyzed skeletal muscle effectively prevents nerve sprouting in that muscle. Our findings indicate that muscle IGFs play an essential role in intramuscular nerve sprouting. In addition, these findings suggest that IGFs are major signaling factors from inactivated muscle to promote local restorative reactions, including interstitial cell proliferation and nerve sprouting.     

Cellular actions of insulin-like growth factor binding proteins.

The insulin-like growth factors (IGFs), insulin-like growth factor binding proteins (IGFBPs), and the IGFBP proteases are involved in the regulation of somatic growth and cellular proliferation both in vivo and in vitro. IGFs are potent mitogenic agents whose actions are determined by the availability of free IGFs to interact with the IGF receptors. IGFBPs comprise a family of proteins that bind IGFs with high affinity and specificity and thereby regulate IGF-dependent actions. IGFBPs have recently emerged as IGF-independent regulators of cell growth. Various IGFBP association proteins as well as cleavage of IGFBPs by specific proteases modulate levels of free IGFs and IGFBPs. The ubiquity and complexity of the IGF axis promise exciting discoveries and applications for the future.     

Dual control of muscle cell survival by distinct growth factor-regulated signaling pathways

In addition to their ability to stimulate cell proliferation, polypeptide growth factors are able to maintain cell survival under conditions that otherwise lead to apoptotic death. Growth factors control cell viability through regulation of critical intracellular signal transduction pathways. We previously characterized C2 muscle cell lines that lacked endogenous expression of insulin-like growth factor II (IGF-II). These cells did not differentiate but underwent apoptotic death in low-serum differentiation medium. Death could be prevented by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we analyze the signaling pathways involved in growth factor-mediated myoblast survival. PDGF treatment caused sustained activation of extracellular-regulated kinases 1 and 2 (ERK1 and -2), while IGF-I only transiently induced these enzymes. Transient transfection of a constitutively active Mek1, a specific upstream activator of ERKs, maintained myoblast viability in the absence of growth factors, while inhibition of Mek1 by the drug UO126 blocked PDGF-mediated but not IGF-stimulated survival. Although both growth factors activated phosphatidylinositol 3-kinase (PI3-kinase) to similar extents, only IGF-I treatment led to sustained stimulation of its downstream kinase, Akt. Transient transfection of a constitutively active PI3-kinase or an inducible Akt promoted myoblast viability in the absence of growth factors, while inhibition of PI3-kinase activity by the drug LY294002 selectively blocked IGF- but not PDGF-mediated muscle cell survival. In aggregate, these observations demonstrate that distinct growth factor-regulated signaling pathways independently control myoblast survival. Since IGF action also stimulates muscle differentiation, these results suggest a means to regulate myogenesis through selective manipulation of different signal transduction pathways.     

Variation among cell types in the signaling pathways by which IGF-I stimulates specific cellular responses.

This review compares the signaling pathways leading to cellular responses (primarily proliferation and differentiation) of cells to the insulin-like growth factors (IGFs). Although some systems (such as myoblasts and adipocytes) clearly employ the Ras-Raf-Mitogen Activated Protein (MAP) kinase pathway in signaling for cell proliferation, others (such as MCF-7 mammary tumors and brain capillary cells) proliferate in response to signals mediated by phosphatidylinositol-3 kinase and p70 S6 kinase. Similarly, most of the systems surveyed use a phosphatidylinositol-3 kinase pathway in differentiating in response to IGFs, but others (such as SH-SY5Y neuroblastoma cells) differentiate in response to the MAP kinase pathway. Thus, it seems that there are no simple generalizations that can be used to forecast the signaling pathway that will be involved in any response to the IGFs.     

Pancreatic β-cell regeneration: From molecular mechanisms to therapy

Pancreatic β cells are a type of cells that are present in the islets of Langerhans. These cells are highly specialized for the secretion of insulin in response to low increasing of blood glucose levels. Hence, pancreatic β cells could contribute to maintaining systemic glucose homeostasis. Increasing evidence has revealed that a variety of internal (ie, genetic and epigenetic factors) and external factors (ie, radical-oxidative stress) are involved in the protection and/or regeneration of pancreatic β cells. The pathways regulating β-cell replication have been intensely investigated. Glucose has an important role in cell cycle entry of quiescent β cells, which exerts its effect via glucose metabolism and unfolded proteins. A variety of growth factors, hormones, and signaling pathways (ie, calcium-calcineurin nuclear factor of activated T cells) are others factors that could affect β-cell replication under different conditions. Therefore, a greater understanding of the underlying pathways involved in the regeneration and protection of pancreatic β cells could lead to finding and developing new therapeutic approaches. Utilization of stem cells and various phytochemical agents have provided new aspects for preventing β-cell degeneration and stimulating the endogenous regeneration of islets. Thus, these therapeutic platforms could be used as potential therapies in the treatment of insulin-dependent diabetes mellitus. Here, we summarized the various mechanisms involved in pancreatic β-cell regeneration. Moreover, we highlighted different therapeutic approaches which could be used for the regeneration of pancreatic β cells.     

From planarians to mammals - the many faces of regeneration

This report presents the history of the involvement of the Department of Cytology in studies of different aspects of regeneration. It can be divided into two major phases; the first focused on the regeneration of Turbellarians and the second on the regeneration of rat skeletal muscles including the differentiation of satellite cells in vitro. Regeneration of Turbellarians was investigated both at the cellular and molecular levels including the role of the protein kinase C (PKC) in this process. Studies on skeletal muscle regeneration initially focused on factors involved in regulation of signal transduction pathways. Next, we explored the influence of growth factors on the modulation of the regeneration process. Another important aspect of our studies was investigating of the distribution and function of different proteins involved in adhesion and fusion of myoblasts. Finally, we are also conducting research on the role of stem cells from other tissues in the regeneration of skeletal muscle.     

Role of insulin-like growth factor binding protein (IGFBP)-3 in TGF-beta- and GDF-8 (myostatin)-induced suppression of proliferation in porcine embryonic myogenic cell cultures

Both transforming growth factor (TGF-beta) and growth and development factor (GDF)-8 (myostatin) affect muscle differentiation by suppressing proliferation and differentiation of myogenic cells. In contrast, insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cells. In vivo, IGFs are found in association with a family of high-affinity insulin-like growth factor binding proteins (IGFBP 1-6) that affect their biological activity. Treatment of porcine embryonic myogenic cell (PEMC) cultures with either TGF-beta(1) or GDF-8 suppressed proliferation and increased production of IGFBP-3 protein and mRNA (P < 0.005). An anti-IGFBP-3 antibody that neutralizes the biological activity of IGFBP-3 reduced the ability of either TGF-beta(1) or GDF-8 to suppress PEMC proliferation (P < 0.005). However, this antibody did not affect proliferation rate in the presence of both TGF-beta(1) and GDF-8. These data show that IGFBP-3 plays a role in mediating the activity of either TGF-beta(1) or GDF-8 alone but not when both TGF-beta(1) and GDF-8 are present. In contrast to findings in T47D breast cancer cells, treatment of PEMC cultures with IGFBP-3 did not result in increased levels of phosphosmad-2. Since TGF-beta and GDF-8 are believed to play a significant role in regulating proliferation and differentiation of myogenic cells, our current data showing that IGFBP-3 plays a role in mediating the activity of these growth factors in muscle cell cultures strongly suggest that IGFBP-3 also may be involved in regulating these processes in myogenic cells.