Daina Z. Ewton

The Mitogenic and Myogenic Actions of Insulin-like Growth Factors Utilize Distinct Signaling Pathways

It is well established that mitogens inhibit differentiation of skeletal muscle cells, but the insulin-like growth factors (IGFs), acting through a single receptor, stimulate both proliferation and differentiation of myoblasts. Although the IGF-I mitogenic signaling pathway has been extensively studied in other cell types, little is known about the signaling pathway leading to differentiation in skeletal muscle. By using specific inhibitors of the IGF signal transduction pathway, we have begun to define the signaling intermediates mediating the two responses to IGFs. We found that PD098059, an inhibitor of mitogen-activated protein (MAP) kinase kinase activation, inhibited IGF-stimulated proliferation of L6A1 myoblasts and the events associated with it, such as phosphorylation of the MAP kinases and elevation of c-fos mRNA and cyclin D protein. Surprisingly, PD098059 caused a dramatic enhancement of differentiation, evident both at a morphological (fusion of myoblasts into myotubes) and biochemical level (elevation of myogenin and p21 cyclin-dependent kinase inhibitor expression, as well as creatine kinase activity). In sharp contrast, LY294002, an inhibitor of phosphatidylinositol 3-kinase, and rapamycin, an inhibitor of the activation of p70 S6 kinase (p70S6k), completely abolished IGF stimulation of L6A1 differentiation. We found that p70S6k activity increased substantially during differentiation, and this increase was further enhanced by PD098059. Our results demonstrate that the MAP kinase pathway plays a primary role in the mitogenic response and is inhibitory to the myogenic response in L6A1 myoblasts, while activation of the phosphatidylinositol 3-kinase/p70S6k pathway is essential for IGF-stimulated differentiation. Thus, it appears that signaling from the IGF-I receptor utilizes two distinct pathways leading either to proliferation or differentiation.

Effects of the somatomedins and insulin on myoblast differentiation in vitro

Abstract The effects of insulin and the somatomedins on differentiation of rat myoblasts were investigated in experiments on cells cloned from Yaffes L6 line. Incubation for 48 hr with either insulin or Temins multiplication stimulating activity (MSA), a member of the somatomedin family, caused a dramatic increase in myoblast fusion. This stimulation of differentiation is not a simple consequence of the increased cell density resulting from the effects of these hormones on myoblast proliferation, and the increase in fusion is not an effect common to all mitogens (FGF inhibits the process). Other somatomedins (human somatomedin C and insulin-like growth factor I), were as effective as MSA in stimulating differentiation. The somatomedins were active at concentrations in the range of their levels in fetal blood, in contrast to insulin, which was inactive at concentrations below 10−7, M. Growth hormone (GH) had no effect on muscle differentiation. In serum-free medium MM-1 (in which myoblasts maintain apparently normal morphology and metabolic activity), the very high levels of insulin required to stimulate differentiation could be replaced entirely by physiological levels (1.0 μg/ml) of MSA, further supporting our view that insulin at high concentrations serves primarily as an analogue of the somatomedins in stimulating the growth and development of muscle cells.

Modulation of insulin-like growth factor actions in L6A1 myoblasts by insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5: A dual role for IGFBP-5

We have previously shown that the insulin‐like growth factors (IGFs) stimulate both proliferation and differentiation of skeletal muscle cells in culture, and that these actions in L6A1 muscle cells may be modulated by three secreted IGF binding proteins (IGFBPs), IGFBP‐4, ‐5, and ‐6. Since we found that the temporal expression pattern of IGFBP‐4 and IGFBP‐5 differed dramatically during the transition from proliferating myoblasts to differentiated myotubes, we undertook the current study to examine the effects of purified IGFBP‐4 and IGFBP‐5 on IGF‐ stimulated actions in L6A1 muscle cells. As has been shown for other cell types, we found that IGFBP‐4 had only inhibitory actions, inhibiting IGF‐I and IGF‐II‐ stimulated proliferation and differentiation. In contrast, IGFBP‐5 exhibited both inhibitory and stimulatory actions. When added in the presence of 30 ng/ml IGF‐I, IGFBP‐5 (250 ng/ml) inhibited all markers of the early proliferative response: the tyrosine phosphorylation of the cytoplasmic signaling molecules IRS‐1 and Shc, the activation of the MAP kinases, ERK1 and 2, the elevation of c‐fos mRNA, the early inhibition of the elevation in myogenin mRNA, and the increase in cell number. In contrast, IGFBP‐5 stimulated all aspects of the myogenic response to IGF‐I: the later rise in myogenin mRNA, the elevation of creatine kinase activity, and the fusion of myoblasts into myotubes. This dual response to IGFBP‐5 was greatest when it was added at a molar ratio of IGFBP‐5 to IGF‐I of 2:1. In contrast, when IGFBP‐5 was added in the presence of IGF‐II, it inhibited both proliferation and differentiation. Neither IGFBP had any effect when added in the presence of R3 IGF‐I, an analog with substantially reduced affinity for IGFBPs. Our results suggest that the role of IGFBP‐4 is mainly to sequester excess IGFs, and thus inhibit all actions. IGFBP‐5, however, is capable of eliciting a dual response, possibly due to its unique ability to associate with the cell membrane. J. Cell. Physiol. 177:47–57, 1998.

Mirk/dyrk1B is a Rho-induced kinase active in skeletal muscle differentiation.

The Rho family of small GTPases regulates numerous signaling pathways that control the organization of the cytoskeleton, transcription factor activity, and many aspects of the differentiation of skeletal myoblasts. We now demonstrate that the kinase Mirk (minibrain-related kinase)/dyrk1B is induced by members of the Rho-family in myoblasts and that Mirk is active in skeletal muscle differentiation. Mirk is an arginine-directed serine/threonine kinase which is expressed at elevated levels in skeletal muscle compared with other normal tissues. A Mirk promoter construct was activated when C2C12 myoblasts were switched from growth to differentiation medium and was also activated by the Rho family members RhoA, Cdc42, and to a lesser degree Rac1, but not by MyoD or Myf5. Mirk protein levels increased following transient expression of constitutively active Cdc42-QL, RhoA-QL, or Rac1-QL in C2C12 cells. High concentrations of serum mitogens down-regulated Mirk through activation of the Ras-MEK-Erk pathway. As a result, Mirk transcription was induced by the MEK1 inhibitor PD98059 and by the switch from growth to differentiation medium. Mirk was induced with similar kinetics to another Rho-induced differentiation gene, myogenin. Mirk protein levels increased 10-fold within 24–48 h after primary cultured muscle cells; C2C12 mouse myoblasts or L6 rat myoblasts were induced to differentiate. Thus Mirk was induced following the commitment stage of myogenesis. Stable overexpression of Mirk enabled myoblasts to fuse more rapidly when placed in differentiation medium. The function of Mirk in muscle differentiation was established by depletion of endogenous Mirk by small interfering RNA, which prevented myoblast fusion into myotubes and inhibited induction of markers of differentiation, including myogenin, fast twitch troponin T, and muscle myosin heavy chain. Other members of the dyrk/minibrain/HIPK family of kinases in lower organisms have been shown to regulate the transition from growth to differentiation, and Mirk is now shown to participate in skeletal muscle development.

The CDK Inhibitor p27kip1 is stabilized in G0 by Mirk/dyrk1B kinase

Elevated levels of the cyclin-dependent kinase (CDK) inhibitor p27 block the cell in G0/G1 until mitogenic signals activate G1 cyclins and initiate proliferation. Post-translational regulation of p27 by different phosphorylation events is critical in allowing cells to proceed through the cell cycle. We now demonstrate that the arginine-directed kinase, Mirk/dyrk1B, is maximally active in G0 in NIH3T3 cells, when it stabilizes p27 by phosphorylating it at Ser-10. The phospho-mimetic mutant p27-S10D was more stable, and the non-phosphorylatable mutant p27-S10A was less stable than wild-type when expressed in G0-arrested cells. Following phosphorylation by Mirk, p27 remains a functional CDK inhibitor, capable of binding to CDK2. Mirk did not induce the translocation of p27 from the nucleus in G0, but instead co-localized with nuclear p27. Depletion of Mirk by RNA interference decreased the phosphorylation of p27 at Ser-10 and the stability of endogenous p27. RNAi to Mirk increased cell entry from G0 into G1 as shown by increased expression of proliferating cell nuclear antigen and decreased expression of p27. These data suggest a model in which Mirk increases the amount of nuclear p27 by stabilizing it during G0 when Mirk is most abundant. Mitogen stimulation then causes cells to enter G1, reduces Mirk levels (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354), and initiates the translocation of p27 to the cytoplasm. In addition, depletion of Mirk by RNAi in postmitotic C2C12 myoblasts decreased protein but not mRNA levels of p27, suggesting that stabilization of p27 by Mirk also occurs during differentiation.

Mirk/Dyrk1B Maintains the Viability of Quiescent Pancreatic Cancer Cells by Reducing Levels of Reactive Oxygen Species

The kinase Mirk/dyrk1B mediated the clonogenic growth of pancreatic cancer cells in earlier studies. It is now shown that Mirk levels increased 7-fold in SU86.86 pancreatic cancer cells when over a third of the cells were accumulated in a quiescent G(0) state, defined by Hoechst/Pyronin Y staining. Depletion of Mirk by a doxycycline-inducible short hairpin RNA increased the G(0) fraction to approximately 50%, suggesting that Mirk provided some function in G(0). Mirk reduced the levels of reactive oxygen species (ROS) in quiescent cultures of SU86.86 cells and of Panc1 cells by increasing transcription of the antioxidant genes ferroxidase, superoxide dismutase (SOD)2, and SOD3. These genes were functional antioxidant genes in pancreatic cancer cells because ectopic expression of SOD2 and ferroxidase in Mirk-depleted cells lowered ROS levels. Quiescent pancreatic cancer cells quickly lost viability when depleted of Mirk because of elevated ROS levels, exhibiting up to 4-fold less colony-forming activity and 4-fold less capability for dye exclusion. As a result, reduction of ROS by N-acetyl cysteine led to more viable cells. Mirk also destabilizated cyclin D1 and D3 in quiescent cells. Thus, quiescent pancreatic cancer cells depleted of Mirk became less viable because they were damaged by ROS, and had increased levels of G(1) cyclins to prime cells to escape quiescence.

INSULIN ACTS AS A SOMATOMEDIN ANALOG IN STIMULATING MYOBLAST GROWTH IN SERUM-FREE MEDIUM

SummaryA serum-free medium that supports the proliferation of myoblasts (but not of fibroblasts) has been developed recently in this laboratory. It is composed of 10−6M insulin, 10−7M dexamethasone, and 10−5M fetuin, and is designated medium MM-1. The latter two components gave optimal stimulation at or near “physiological” concentrations, but insulin was required at levels far in excess of those found in serum. Accordingly, we have now investigated the possibility that insulin acts as a week analog of the somatomedins, as has been suggested in other systems. We found that maximal growth rates were observed when 10−6M insulin was replaced by 0.5 to 1.0 μg/ml multiplication stimulating activity (MSA), indicating that insulin serves a somatomedinlike function of MM-1. We also investigated the possibility that a contaminant of fetuin is responsible for its action in MM-1 but found no evidence to support this suggestion. We conclude that MM-1 is suitable for the study of muscle cell growth and differentiation under rather well-defined conditions, and that insulin probably is serving as a somatomedin analog in this medium.

The Kinase Mirk/Dyrk1B Mediates Cell Survival in Pancreatic Ductal Adenocarcinoma

Ductal adenocarcinoma of the pancreas is almost uniformly lethal as this cancer is invariably detected at an advanced stage and is resistant to treatment. The serine/threonine kinase Mirk/Dyrk1B has been shown to be antiapoptotic in rhabdomyosarcomas. We have now investigated whether Mirk might mediate survival in another cancer in which Mirk is widely expressed, pancreatic ductal adenocarcinoma. Mirk was an active kinase in each pancreatic cancer cell line where it was detected. Mirk knockdown by RNA interference (RNAi) reduced the clonogenicity of Panc1 pancreatic cancer cells 4-fold and decreased tumor cell number, showing that Mirk mediates survival in these cells. Mirk knockdown by synthetic duplex RNAis in Panc1, AsPc1, and SU86.86 pancreatic cancer cells induced apoptosis and enhanced the apoptosis induced by gemcitibine. Mirk knockdown did not increase the abundance or activation of Akt. However, four of five pancreatic carcinoma cell lines exhibited either elevated Mirk activity or elevated Akt activity, suggesting that pancreatic cancer cells primarily rely on Mirk or Akt for survival signaling. Mirk protein was detected by immunohistochemistry in 25 of 28 cases (89%) of pancreatic ductal adenocarcinoma, with elevated expression in 11 cases (39%). Increased expression of Mirk was seen in pancreatic carcinomas compared with primary cultures of normal ductal epithelium by serial analysis of gene expression and by immunohistochemistry. Thus, Mirk is a survival factor for pancreatic ductal adenocarcinoma. Because knockout of Mirk does not cause embryonic lethality, Mirk is not essential for normal cell growth and may represent a novel therapeutic target. (Cancer Res 2006; 66(8): 4149-58)

Polyamine depletion inhibits the differentiation of L6 myoblast cells.

Exposure to alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, inhibited the insulin induced differentiation of L6 myoblast cells. Differentiation was assessed by measuring creatine kinase activity and by determining the percentage of nuclei in myotubes. The levels of putrescine and spermidine increased in stimulated cultures prior to their differentiation and these increases were blocked by alpha-difluoromethylornithine. Provision of exogenous putrescine was able to reverse the inhibitory effect of the drug. The anti-differentiative effect is observed only if alpha-difluoromethylornithine is added within twenty-four hours of insulin stimulation. In the experimental protocol used, alpha-difluoromethylornithine was added as the cultures approached confluence and had no effect on their ultimate DNA content. Therefore, the effect of alpha-difluoromethylornithine on myoblast differentiation is not secondary to an effect on cellular proliferation. These results indicate that polyamines may be involved in the mediation of muscle cell differentiation.

STIMULATION AND INHIBITION OF MYOBLAST DIFFERENTIATION BY HORMONES

SummaryThe growth and differentiation of L6 myoblasts are subject to control by two proteins secreted by cells of the Buffalo rat liver line. The first of these, rat insulinlike growth factor-II (formerly designated multiplication stimulating activity) is a potent stimulator of myoblast proliferation and differentiation, as well as associated processes such as amino acid uptake and incroporation into protein, RNA synthesis, and thymidine incorporation into DNA. In addition, this hormone causes a significant decrease in the rate of protein degradation. All of these actions seem to be attributable to a single molecular species, although their time courses and sensitivity to the hormone differ substantially. The second protein, the differentiation inhibitor (DI), is a nonmitogenic inhibitor of all tested aspects of myoblast differentiation, including fusion and the elevation of creatine kinase. Indirect immunofluorescence experiments demonstrated that DI also blocks accumulation of myosin heavy chain and myomesin. Upon removal of DI after 72 h incubation, all of these effects were reversed and normal myotubes containing the usual complement of muscle-specific proteins were formed. Thus, this system makes it possible to achieve specific stimulation or inhibition of muscle cell differentiation by addition of purified proteins to cloned cells in serum-free medium.