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Dexamethasone Inhibits Insulin-Like Growth Factor Signaling and Potentiates Myoblast Apoptosis

Department of Neurology (J.R.S.), Howard Hughes Medical Institute (B.L.B.), and Huntsman Cancer Institute (A.T.), University of Utah Medical School, Salt Lake City, Utah 84132

Address all correspondence and requests for reprints to: J. Robinson Singleton, M.D., Department of Neurology, University of Utah Medical School, Room 3R-152, 50 North Medical Drive, Salt Lake City, Utah 84132. E-mail: rob.singleton{at}hsc.utah.edu

	Abstract

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In the critically ill, glucocorticoids induce myopathy, combining profound protein catabolism and mild myotubular death. Insulin-like growth factors (IGFs) inhibit muscle catabolism through activation of phosphatidylinositol 3-kinase (PI3K). Using rat L6 myoblasts, we show that IGF-I also acts through PI3K to inhibit apoptosis induced by hyperosmolar metabolic stress with 300 mM mannitol. We find that the glucocorticoid dexamethasone inhibits this antiapoptotic effect of IGF-I by impairing PI3K signaling. Dexamethasone induces overexpression of the PI3K subunit p85, which, in turn, competes with the complete PI3K heterodimer for binding at insulin receptor substrate-1, inhibiting PI3K activation. Dexamethasone blocks IGF-I-induced phosphorylation of Akt, a PI3K-dependent process. Increased cellular p85 abundance, induced by either 10 µM dexamethasone or transient transfection with a plasmid coding for p85, significantly inhibits IGF-I rescue from apoptosis induced by mannitol, as indicated by both loss of cell viability and increased activity of caspase-3 by fluorogenic assay. Conversely, constitutively active PI3K inhibits death induced by mannitol, even in the presence of dexamethasone. These findings may have particular relevance in the pathogenesis of acute steroid myopathy in critical illness, in which catabolic glucocorticoid effects combine with acute metabolic stressors, including sepsis, fasting, and chemical denervation.

	Introduction

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GLUCOCORTICOID USE is associated both with chronic atrophic myopathy and, in the setting of critical illness, with acute quadriplegic myopathy (AQM) (1, 2, 3, 4). The acute myopathy is recognized clinically as rapidly progressive weakness of both limbs and diaphragm, and often is considered in patients with difficulty weaning from ventilatory support. Patients are often septic, intubated, and receiving nondepolarizing neuromuscular junction-blocking agents for paralysis to aid ventilation (4). Metabolic stressors may include a variable combination of sepsis, starvation, endocrine dysfunction, and hepatic or renal insufficiency with electrolyte imbalance. Histological evaluation of muscle biopsy shows sarcomeric disruption with loss of myosin thick filaments (5). In contrast to myopathy associated with chronic steroid use, myotubular fragmentation and lysis often occur in AQM, as indicated by elevated creatine phosphokinase, abnormal spontaneous activity on electromyography, and evidence of scattered myotubular necrosis on biopsy (4, 6).

The striking muscle atrophy and protein loss associated with AQM reflect the potent catabolic effects of glucocorticoids in synergy with other metabolic stressors in critical illness. One likely mechanism for this catabolic effect is inhibition of hormones that normally act to maintain anabolic homeostasis in skeletal muscle. Insulin-like growth factor I (IGF-I) is one of a family of hormones that have recognized proliferative and anabolic effects on skeletal muscle cells. IGF-I binds to and activates type I IGF receptor (IGF-IR) on the surface of cells (7). Intracellular signaling derived from IGF-IR activation occurs through two recognized pathways: activation of the mitogen-activated protein kinase cascade potentiates proliferation of myoblasts and satellite cells, and signaling through phosphatidylinositol 3-kinase (PI3K) is responsible for the anabolic effects of IGF-I on muscle (increased glucose uptake, protein synthesis, and inhibition of protein breakdown) (8, 9). In addition, in many cells, signaling through PI3K increases the resistance of cells to programmed cell death (10, 11).

PI3K is a heterodimer. The 85-kDa regulatory subunit (p85) contains two Src homology-2 (SH-2) domains that allow it to bind to the IGF-IR-associated protein insulin receptor substrate-1 (IRS-1) and approximate the catalytic 110-kDa (p110) subunit to the cell membrane surface, where it can initiate phosphoinositide conversion (12, 13). Two closely related 85-kDa isoforms, p85 and p85ß, have been characterized (14, 15). In skeletal muscle, glucocorticoid signaling has been shown to induce transcriptional up-regulation and increased cellular abundance of the p85 subunit (16, 17). Overabundant p85 monomer competes for IRS-1 binding with p110/p85 PI3K heterodimers, inhibiting approximation of the p110 catalytic subunit to the membrane surface and thus retarding PI3K activity (16). The pathogenetic relevance of this observation to disease states associated with endogenous or exogenous glucocorticoid excess has not been explored.

IGF-I signaling often acts to protect cells from apoptosis due to metabolic stress (18). We hypothesize that dexamethasone contributes to myopathy in the setting of critical illness in part by inhibiting the PI3K-mediated, anabolic, and antiapoptotic effects of IGF-I. The L6 rat myoblast cell line is a recognized model for evaluation of myogenesis and response to myocyte injury (19). We have examined the response of L6 myoblasts in culture to metabolic hyperosmolar stress with mannitol, modified by IGF-I and the glucocorticoid dexamethasone in combination.

	Materials and Methods

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Recombinant human IGF-I was a gift from Dr. Eva Feldman (Ann Arbor, MI). Plasmids encoding PI3K subunits p85 and p110 CAAX were gifts from Dr. Julian Downward (Imperial Cancer Research Fund, London, UK). L6 rat myoblasts were obtained from American Type Culture Collection (Manassas, VA). Monoclonal antibodies to PI3K subunit p85 (sc-1637) and to IRS-1 (sc-559) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antibodies to total Akt and phosphorylated Akt were obtained from New England Biolabs, Inc. (Beverly, MA), CaspACE fluorometric assay kit for caspase-3 (G3540) was purchased from Promega Corp. (Madison, WI), and Lipofectamine reagents were obtained from Life Technologies, Inc.(Gaithersburg, MD). FCS and horse serum (HS) were purchased from HyClone Laboratories, Inc. (Logan, UT), and matched lot numbers were used throughout these experiments. The PI3K inhibitor LY-294002 and the mitogen-activated protein kinase inhibitor PD-98059 were purchased from Calbiochem (San Diego, CA). Dexamethasone sodium phosphate was obtained through the University of Utah in-patient pharmacy from Elkins-Sinn, Inc. (Cherry Hills, NJ).

L6 rat myoblasts were cultured at 37 C in 10% CO₂ in DMEM containing 1.0 mg/ml glucose, 20% FCS, penicillin, and streptomycin. Myoblasts were passaged or used for experiments at 80% confluence and were detached from plates using 0.25% trypsin and 1 mM EDTA. To minimize apoptosis due to serum withdrawal, all experimental conditions were performed in a background of DMEM containing 1% HS.

For green fluorescence protein (GFP) counting experiments, cells were cultured on 3.5-cm, 6-well plates on which 2 x 3 grid lines had been etched to facilitate reproducibly identifying and following specific cell populations longitudinally. Myoblasts in log phase growth at 60–80% confluence were transfected with plasmid encoding GFP behind the cytomegalovirus constitutive promoter or in separate experiments were cotransfected with GFP plus plasmids encoding either the PI3K p85 subunit or the p110 subunit linked to a CAAX sequence, targeting it to the cell membrane. Transfection using Lipofectamine yielded 1–5% efficiency. Transfected myoblasts were returned to growth medium for a period of 16 h, then a microscope field of GFP-positive cells was counted at 6 intersections of the grid (yielding 400–800 cells) before treating the cells with differentiation medium containing combinations of 300 mM mannitol, 10 µM dexamethasone, and 10 nM IGF-I. For each condition, GFP-positive cells at established grid sites were recounted at 12, 24, and 48 h after treatment. All GFP experiments were counted and tabulated in a blinded fashion after treatment conditions were independently randomized and coded.

In experiments to determine the activity of caspase-3, L6 myoblasts were grown to 70% confluence in 10-cm dishes, then treated with combinations of mannitol, dexamethasone, and IGF-I. At various time points, treatment medium was collected from plates, adherent cells were washed with ice-cold PBS, then lysed with ice-cold RIPA buffer (1 x PBS, 1% Nonidet-40, 0.5% sodium deoxycholate, and 0.1% SDS with protease inhibitors: 1 mM PMSF, 1 mM Na₃VO₄, 10 µg/ml aprotinin, and 0.5 µg/ml leupeptin) for 15 min. Detached cells in the medium were isolated by centrifugation, washed once with PBS, then combined with adherent cells in the lysis buffer. Lysed cells were scraped from plates with a rubber policeman and sonicated (half-power for 15 sec twice), and supernatants were separated from membrane debris by microcentrifugation 12,000 rpm for 5 min. Aliquots of cell lysates containing 75 µg protein were assayed in triplicate for caspase-3 activity using the kit according to the manufacturer’s instructions.

Immunoblotting for total p85, total Akt, and phospho-Akt was performed using modification of methods previously described (10). Cell lysates were collected as described for the caspase-3 assay. For each condition, equal amounts of lysate protein (usually 75 µg) were separated on a 12% SDS-polyacrylamide minigel along with mol wt markers, transblotted to a polyvinylidene difluoride membrane, blocked in 1x Tris-buffered saline with Tween-20 with 5% milk overnight, and blotted with primary and appropriate secondary antibodies at concentrations recommended by the manufacturers. Blots were evaluated using enhanced chemiluminescence according to the manufacturer’s manual (Amersham Pharmacia Biotech, Arlington Heights, IL).

Statistical analysis for differences in GFP-positive cell viability and caspase-3 activity, were performed using one-tailed Student’s t test. Unless otherwise stated in the figure legends, graphs represent the mean ± SEM for three to six independent determinations for each data point.

	Results

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Mannitol is a chemically inert sugar that has been used to generate hyperosmolar conditions as a model of acute and chronic metabolic stress in several cell systems. Osmolar gradients in the ranges used in this study are not sufficient to cause physical disruption of cell membranes, but instead induce apoptosis. Cultured L6 myoblasts respond to mannitol in a dose-dependent fashion by undergoing cell death, as indicated by progressive loss of GFP-positive cells on grid counting (Fig. 1A). In contrast, control GFP-positive myoblasts treated with 1% HS alone slowly increase in number over 48 h, indicating some continued proliferation.

View larger version (19K): [in this window] [in a new window]   Figure 1. L6 myoblasts respond to hyperosmolar stress with mannitol in a dose-dependent fashion by undergoing programmed cell death. A, Time-course experiment using GFP-transfected L6 myoblasts plated and counted on gridded plates before and 12–48 h after treatment with 1% HR alone or together with 30–300 mM mannitol, as described in Materials and Methods. The number of GFP-positive cells for each condition is expressed as a percentage of cells at time zero. Results are the mean ± SEM for three to five separate determinations for each data point. B, Caspase-3 activity mirrors and precedes loss of viability after mannitol treatment. An assay for caspase-3 activity was performed on cell lysates as described in Materials and Methods. For each time point, results are expressed as the ratio of caspase-related fluorescence from each mannitol dose to fluorescence from cells treated with 1% HS alone. Caspase data were obtained in triplicate, and each data point represents the results (±SEM) of three to five separate experiments.

To demonstrate that glucocorticoids induce overexpression and intracellular accumulation of the p85 subunit of PI3K in our system, we performed Western blot analysis of L6 cell lysates 6 h after treatment with increasing doses of dexamethasone (Fig. 2A). Probing with mouse monoclonal antibody directed at p85 demonstrates a dose-dependent increase in cellular p85 abundance above baseline, beginning with 100 nM dexamethasone and reaching maximal abundance at 30 µM.

View larger version (24K): [in this window] [in a new window]   Figure 2. Dexamethasone increases p85 abundance, but does not induce cell death of L6 myoblasts. A, Western blot analysis of L6 myoblast lysates 6 h after treatment with increasing doses of dexamethasone. Probing with mouse monoclonal antibody directed at p85 demonstrates a dose-dependent increase in cellular p85 abundance above baseline, beginning with 100 nM dexamethasone and reaching maximal abundance at 30 µM. B, Time-course experiment using GFP-transfected L6 myoblasts plated and counted on gridded plates as described in Materials and Methods 48 h after treatment with 1% HS alone or together with increasing doses of dexamethasone. The number of GFP-positive cells for each condition are expressed as a percentage of cells at time zero, just before adding experimental conditions. L6 myoblasts treated with dexamethasone for 48 h show no significant death compared with 1% HS alone at dexamethasone doses below 100 µM.

Based on these experiments, a dexamethasone dose of 10 µM was selected for further investigation. We found that at this dose, dexamethasone acts to potentiate death in response to mannitol primarily by inhibiting IGF-I signaling. Figure 3A shows GFP counting results at 24 and 48 h for L6 myocytes treated with various combinations of 300 mM mannitol, 1 µM dexamethasone, and 10 nM IGF-I. Mannitol causes a 60% loss of viability compared with treatment with 1% HS. IGF-I exerts a potent trophic effect on these myocytes and largely prevents the loss of cell viability in response to mannitol. Dexamethasone does not cause cell death alone and results in only a modest increase in cell death when added to mannitol. However, dexamethasone significantly inhibits the ability of IGF-I to rescue myocytes from death induced by mannitol. Caspase-3 activity measured in L6 lysates after 12 h of treatment with these conditions (Fig. 3B) closely mirrors the results of GFP counting. Mannitol induced a 3-fold increase in active caspase-3 over baseline, indicating initiation of apoptosis, an effect that IGF-I inhibits. In the presence of dexamethasone, protection from mannitol-induced apoptosis by IGF-I was inhibited.

View larger version (19K): [in this window] [in a new window]   Figure 3. Dexamethasone acts to potentiate L6 myocyte death in response to mannitol, primarily by inhibiting IGF-I signaling through PI3K. A, GFP counting results 24 and 48 h after treatment of L6 myoblasts with various combinations of 300 mM mannitol (M), 10 µM dexamethasone (D), and 10 nM IGF-I (I) in the background of 1% HS. Dexamethasone does not induce cell death alone and does not significantly decrease cell viability when added to mannitol (DM). Addition of dexamethasone in combination with mannitol and IGF-I (DMI) significantly reduces GFP-positive cell number at both 24 and 48 h compared with mannitol and IGF-I alone (MI; *, P < 0.05, by one tailed Student’s t test). B, Loss of cell viability measured by GFP-positive cell counting corresponds to induction of apoptosis in L6 myocytes. Assay for active caspase-3 in L6 cell lysates obtained 12 h after treatment with the same dose and combinations of mannitol, dexamethasone, and IGF-I as in A. Results are expressed as arbitrary fluorescence units and represent the mean ± SEM for four separate determinations. Addition of dexamethasone in the presence of mannitol and IGF-I (DMI) significantly increases caspase-3 activity compared with mannitol and IGF-I alone (MI; *, P < 0.05, by one-tailed Student’s t test). C, Immunoblot analysis of total Akt (Akt) and phosphorylated Akt (pAkt). All conditions were on a background of 1% HS. L6 myoblasts were treated with 1% HS alone for 1 h or with 10 µM dexamethasone for 1 or 5 h before lysis, or dexamethasone was added either together with (0 h) or 1 h (1h) or 5 h (5h) before addition of 10 nM IGF-I for 1 h. As a control, L6 cells were pretreated for 4 h with the specific PI3K inhibitor Ly-294002 before addition of IGF-I for 1 h (Ly). Equal amounts of L6 lysate proteins were separated by PAGE and immunoblotted for total and phosphorylated Akt, as described in Materials and Methods. Results are representative of separate experiments performed twice.

Experiments using constitutively active or dominant negative PI3K subunits were performed to further evaluate disruption of IGF-I signaling through PI3K. L6 myoblasts were cotransfected with GFP plus either a plasmid coding for the p85 subunit behind a constitutive cytomegalovirus promoter or a plasmid coding for the catalytic 110-kDa subunit to which a CAAX sequence was appended to localize the subunit to the plasma membrane. As no interaction with the p85 subunit or with IRS-I was necessary for activation, transfection with p110 CAAX produced a constitutively active PI3K.

The results of GFP counting after treatment of transfected myocytes with mannitol and/or IGF-I are shown in Fig. 4. Overexpression of p85 by transfection recapitulated the effect of dexamethasone (Fig. 4A). IGF-I inhibited myocyte death induced by mannitol. However, in cells overexpressing p85, this inhibitory effect was retarded. In contrast, L6 myoblasts transfected with constitutively active p110 CAAX showed inhibition of apoptosis induced by mannitol. Significantly, cotreatment of p110 CAAX-transfected cells with IGF-I did not further increase rescue from mannitol-induced death, suggesting that the constitutively active p110 and IGF-I act through a common pathway to mediate their antiapoptotic effects. Cotreatment of L6 cells transiently transfected with p110 CAAX with 10 µM dexamethasone and 300 mM mannitol did not decrease their viability compared with that of similarly transfected myoblasts treated with mannitol alone (Fig. 4B). This result is consistent with the concept that constitutively active p110 acts downstream of the inhibitory effects of increased IRS-1 occupancy by p85 monomer induced by dexamethasone.

View larger version (17K): [in this window] [in a new window]   Figure 4. GFP-positive cell counting after genetic manipulation of PI3K signaling. L6 myoblasts were transfected with GFP alone or cotransfected with either a plasmid inducing overexpression of p85 or a plasmid coding for a constitutively active form of the catalytic 110-kDa subunit (p110CAAX), as described in Materials and Methods. A, GFP-positive cotransfectants 48 h after treatment with 1% HS alone, 300 mM mannitol (M), or mannitol plus 10 nM IGF-I (MI). B, L6 myoblasts transfected with GFP alone or in combination with p110CAAX and treated for 48 h with HS alone, mannitol (M), 10 µM dexamethasone (D), or mannitol and dexamethasone in combination (DM). Results are expressed as a percentage of GFP-positive cells at time zero. Significant differences (P < 0.05, as measured by one-tailed Student’s t test) in survival between p85 and p110 CAAX-transfected cells (a) or between GFP alone vs. GFP plus p110 CAAX (b) are indicated (*).

	Discussion

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We find that dexamethasone increases the L6 myocyte abundance of the regulatory p85 subunit of PI3K severalfold, as measured by immunoblotting, in agreement with previous studies (16, 17). Giorgiono et al. showed that dexamethasone induced a dramatic increase in undimerized p85 subunit occupancy on IRS-1, with consequent reduction of IRS-1-associated p110 (by immunoblotting) and PI3K activity (by metabolic labeling experiments) (16). Undimerized p85 may exert inhibitory effects on PI3K activation out of proportion to its stochiometric excess compared with the p110/p85 heterodimer. p85 interacts with IRS-1 at sites distinguished by phosphorylated tyrosine residues (26). Free p85 has been reported to bind to these tyrosine-phosphorylated motifs more avidly than do p110/p85 heterodimers, magnifying the IRS-1 occupancy of a modest p85 monomer excess (27, 28). Moreover, phosphoinositol 3,4,5-triphosphate, the product of PI3K phosphoconversion, may act to displace PI3K from IRS-1 in a highly localized feedback inhibition loop by interacting with the p85 SH2 domain (29). Catalytically inactive p85 might thus enjoy relatively prolonged occupancy on IRS-1 while at the same time preferentially binding to IRS-1 sites abandoned by more rapidly cycling p85/p110 heterodimers.

Our results indicate that this dexamethasone-induced inhibition of PI3K signaling has dire functional consequences for the ability of IGF-I to protect myocytes from apoptosis induced by metabolic stress. Increased p85 abundance, induced by either dexamethasone or transient transfection with a plasmid coding for p85, inhibits IGF-I rescue from apoptosis, as indicated by both loss of cell viability and increased activity of caspase-3. Three of our results directly support the concept that dexamethasone’s effect on L6 myoblasts is mediated specifically by inhibition of PI3K. 1) Dexamethasone ameliorates IGF-I-induced Akt phosphorylation. The time course of this inhibition over 5 h is consistent with a requirement for new p85 gene transcription and translation in response to dexamethasone. 2) Introduction of PI3K catalytic subunit p110 made constitutively active by a cell membrane localization sequence prevents dexamethasone effects on L6 viability. 3) Dexamethasone alone does not induce myoblast death even at suprapharmacological doses.

Our results support the concept that dexamethasone, by virtue of its competitive mechanism, is a relatively weak and incomplete inhibitor of PI3K. In contrast to LY294002, dexamethasone pretreatment only partially inhibits Akt phosphorylation by IGF-I. Similarly, we find that dexamethasone alone does not induce L6 myoblast death even at suprapharmacological doses. Potent inhibitors of PI3K, including LY294002 and the hydroxymethylglutaryl coenzyme A inhibitor simvastatin, have been reported to directly decrease viability of L6 myoblasts (19). Taken together, these observations suggest that dexamethasone instead plays a permissive role, by inhibiting the PI3K-mediated antiapoptotic effects of IGF-I in response to a separate metabolic stressor, hyperosmolar injury with mannitol.

Myopathic damage due to glucocorticoids forms a clinical and pathological spectrum, from the subtle weakness, mild myosin loss, and type II fiber atrophy associated with chronic, moderate dose steroid use to the severe paresis, sarcomeric disarray, profound atrophy, and myotube lysis associated with AQM (4, 5, 6, 30, 31). AQM pathogenesis is distinguished from chronic steroid myopathy not only by the large doses of glucocorticoid involved, but by synergism with catabolic effects of other metabolic stressors confronting the critically ill, including sepsis, starvation, inactivity, and chemical denervation. Many of these metabolic concomitants of critical illness have been shown to decrease signaling at steps along the GH-IGF-I anabolic pathway in skeletal muscle.

Our results define one mechanism for inhibition of IGF signaling downstream of IGF-IR binding. However, other postreceptor defects in IGF signaling associated with critical illness probably exist. Sepsis is characterized by overexpression of the immune cytokine tumor necrosis factor- (TNF). Exogenous TNF induces cachexia in experimental animals, and in L6 myoblasts it inhibits muscle protein synthesis induced by IGF-I in a dose-dependent manner (32), suggesting that TNF may inhibit IGF signaling at or beyond the level of IGF-IR activation on skeletal muscle. Similarly, inactivity after hindlimb unweighting in rats results in a transient decrease in muscle sensitivity to both IGF-I and insulin (33), suggesting inhibition of intracellular signal transduction enzymes common to both pathways.

Metabolic stressors most often affect serum IGF abundance. IGF-I messenger RNA and serum IGF-I decrease during acute and chronic fasting, and rebound with refeeding (34). Sepsis is also associated with decreased production and serum concentration of IGF-I (35). Recent experiments indicate that high dose methylprednisolone or triamcinolone reduces IGF-I and IGF-II expression in the rat liver and in diaphragm and gastrocnemius muscles (36), suggesting that PI3K inhibition may not be the only effect of glucocorticoids on IGF signaling in skeletal muscle. Overall, it is plausible that additive or synergistic effects of IGF-I inhibition due to multiple metabolic stressors contribute to the severe protein catabolism and muscle apoptosis observed in AQM.

These results do not provide a direct explanation for the profound loss of strength that defines AQM clinically. Although severe myotubular necrosis has been reported in AQM, in most patients myosin loss and sarcomeric disarray, suggestive of catabolic effects on muscle, are accompanied by only modest evidence of myotubular death (5, 6). Murine models of AQM combining high dose iv steroids with acute focal denervation confirm the observation in human patients of electrical inexcitability of many myofibers in this condition (37, 38). Although the steroid/denervation model does not reproduce the systemic stressors induced in the critically ill patient by sepsis, starvation, and organ failure, this model predicts that weakness in AQM results in large part from reversible changes in sodium and chloride channels that inhibit depolarization (38). The mechanism by which steroids may induce these changes in myofiber membrane conductance is not known.

In summary, we suggest that glucocorticoids antagonize the trophic effects of IGF-I signaling through PI3K and may synergize with other stressors inhibiting IGF-I signaling to induce myopathy in the critically ill. Our results indicate that in severe cases of steroid-associated myopathy, apoptosis may also occur as a result of cumulative IGF-I inhibition. These results have uncertain implications for the use of IGF-I in the treatment or prophylaxis of AQM. Intravenous treatment with IGF-I has recently been shown to prevent muscle atrophy due to chronic administration of glucocorticoids in rats (39). However, our results suggest that reversing the catabolic or apoptotic effects of glucocorticoids on muscle in the setting of acute critical illness may be difficult with supplementation of IGF-I.

Received December 10, 1999.

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