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Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: Robert A. Frost, Ph.D., Department of Cellular and Molecular Physiology, Hershey Medical Center: H166, Hershey, Pennsylvania 17033.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alterations in the GH-insulin-like growth factor I (IGF-I) axis are commonly associated with muscle wasting. In response to infection, the plasma concentration of IGF-I is markedly reduced (9), and the loss of the anabolic action of IGF-I is thought to negatively impact muscle protein metabolism via changes in protein synthesis and degradation (10). Qualitatively similar decreases have also been observed in response to various inflammatory stimuli (11, 12).
Essentially all of the IGF-I in blood and tissues is bound to IGF-binding proteins (IGFBPs) (13). IGFBP-1 (BP-1) is the most dynamically regulated of the IGFBPs, and circulating levels of this binding protein are believed to control the free or bioavailable fraction of IGF-I (14, 15). BP-1 may also influence IGF activity within skeletal muscle, as muscle BP-1 content is dramatically increased after injection of Gram-negative endotoxin or proinflammatory cytokines (11, 12).
Numerous studies suggest that there is a molecular interaction between
IGF system components and various members of the integrin family
of receptors. For example, ligand occupancy of the
vß3 receptor by the extracellular matrix
protein vitronectin is required for IGF-I to stimulate migration of
porcine aortic smooth muscle cells (pSMC) (16). The integrin antagonist
echistatin blocks the ability of IGF-I to stimulate protein synthesis,
DNA synthesis, and IGF signaling in pSMCs (17). BP-1 also contains a
fibronectin-like arginine, glycine, and glutamic acid (RGD) sequence.
This sequence is essential for cell migration in response to BP-1 and
the ability of an IGF-I/BP-1 complex to promote wound healing (18).
Although IGFBPs have been shown to inhibit IGF binding to rat L6 myoblasts (19), little is known about the effect of BP-1 on protein synthesis and degradation in human myoblasts. It is possible that BP-1 could bind to IGF-I and thereby impair ligand receptor interaction or, alternatively, BP-1 could interact with cell surface integrin receptors. The aim of the present study was to examine the ability of BP-1 to regulate IGF-I-dependent processes in human myoblasts. Secondly, we examined the ability of BP-1 to alter protein degradation independent of IGF-I via integrin binding.
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Materials and Methods |
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Protein synthesis
Protein synthesis was determined as previously described (20).
Human myoblasts were treated with serum-free MEM alone or one of the
following reagents: recombinant human IGF-I (Genentech, Inc., San Francisco, CA), IGFBP-1 (Sigma),
echistatin (Sigma), or anti-IGF-I receptor antibody
-IR-3 (Calbiochem, San Diego, CA). Alternatively, cells
received anti-vß3 or -ß1
integrin antibodies (Chemicon, Temecula, CA) or various combinations of
the above for 6 h. Control cells received either an equal volume
of medium containing 0.1% BSA and/or DMSO. Cells were labeled with 2
µCi/well [3H]phenylalanine (132 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) for the entire period.
Cells were washed, isolated in trypsin-EDTA, and precipitated overnight
at 4 C with 10% trichloroacetic acid (TCA). After washing,
TCA-precipitable radioactivity was solubilized in 1 N
sodium hydroxide and liquid scintillation cocktail (Scintsafe I,
Fisher Scientific, Springfield, NJ) and counted in a
liquid scintillation counter (Wallac, Inc., Gaithersburg,
MD).
Protein degradation
Myoblasts were subcultured to 24-well cluster plates as
described above and after 24–48 h prelabeled with
[3H]tyrosine (70 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) for 72 h in MEM with 5%
NBCS. Cells were washed with serum-free medium and treated with
hormones, antibodies, or echistatin as described above. Tyrosine
release was measured over 72 h as soluble counts in the
conditioned medium after precipitation of secretory proteins in the
presence of 1 mg/ml BSA and 10% TCA, as described previously (21).
Protein degradation was linear over a 96-h period in control and
IGF-I-treated cells. BP-1 that was added to skeletal muscle cells and
subsequently recovered from the medium was neither degraded nor
dephosphorylated over this time period. In experiments using
nonphosphorylated BP-1 (npBP-1), the phosphorylated protein was
purified from HepG2-conditioned medium and treated with alkaline
phosphatase to obtain the nonphosphorylated form. These preparations
are greater than 90% pure (Sigma Chemical Co.). We have
confirmed that these preparations contain mostly npBP-1 and pBP-1,
respectively, by nondenaturing PAGE. We have also obtained identical
results with preparations of BP-1 purified from amniotic fluid and
supplied by an independent source (Calbiochem; data not
shown).
Glucose uptake
Glucose uptake was determined as described by Steele-Perkins
et al. (22). Briefly, myoblasts were subcultured into
six-well cluster dishes and grown to confluence. Cells were washed with
buffer containing 140 mM NaCl, 2.7 mM KCl, 1
mM CaCl2, 1.5 mM
KH2PO4, 8 mM
Na2PO4, 0.5 mM MgCl2,
and 0.1% BSA. The assay was initiated by the addition of hormone for
30 min, followed by the addition of
deoxy-D-[14C] glucose (1 µCi) in 0.1
mM 2-deoxy-D-glucose. After 12 min, the cells
were washed and isolated in 400 µl 2 N sodium
hydroxide.
Western blots
Conditioned media and cell extracts were electrophoresed on
7.5% denaturing or nondenaturing polyacrylamide gels and
electrophoretically transferred to nitrocellulose with a semidry
blotter (Bio-Rad Laboratories, Inc., Melville, NY). The
resulting blots were blocked with 5% nonfat dry milk for 1.5 h
and incubated with either antibodies against human BP-1 (Upstate Biotechnology, Inc., Lake Placid, NY) or p70 S6 kinase
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Unbound
primary antibody was removed with Tris-buffered saline containing 0.5%
Tween-20, and blots were incubated with antirabbit Ig conjugated with
horseradish peroxidase. Blots were briefly incubated with the
components of an enhanced chemiluminescent detection system
(Amersham Pharmacia Biotech). Dried blots were used to
expose x-ray film for 1–3 min.
Statistics
Values are the mean ± SEM. Unless otherwise
noted, each experimental condition was tested in sets of six, and each
experiment was repeated three times. Data were analyzed by ANOVA
followed by Student-Newman-Keuls test. Statistical significance was set
at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, top panel). This
response was completely inhibited by either a nonphosphorylated
(npBP-1) form or a mixture of phosphorylated (pBP-1) forms of BP-1.
Neither npBP-1 nor pBP-1 altered the basal level of protein synthesis
on its own (data not shown). Furthermore, the ability of IGF-I to
stimulate protein synthesis was inhibited by BP-1 in a dose-dependent
manner (Fig. 1, bottom panel). Half-maximal inhibition
occurred at a BP-1 concentration of 120 ng/ml or a molar ratio of
1.5:1. Maximal inhibition of IGF-stimulated protein synthesis occurred
at a BP-1 concentration of 400 ng/ml or more. Phosphorylated BP-1 was
slightly more potent than nonphosphorylated BP-1 at inhibiting protein
synthesis, but this difference only reached statistical significance at
a high molar ratio of BP-1/IGF-I (data not shown). In contrast, BP-1
failed to inhibit the ability of a truncated form of IGF-I (desIGF-I)
to stimulate protein synthesis (Fig. 1, bottom panel).
Similarly, IGF-I, but not desIGF-I, formed a complex with a mixture of
BP-1 phosphovariants (from Hep G2 cells) as demonstrated by
nondenaturing PAGE and Western blot analysis (Fig. 1, bottom
panel, inset, compare lanes 2 and 3).
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, top panel). This
response was almost completely inhibited by a 5-fold excess of npBP-1.
The ability of IGF-I to stimulate glucose uptake was inhibited dose
dependently by BP-1 with a maximal inhibition similar to that observed
for protein synthesis (Fig. 2, bottom panel). BP-1 did not
alter the basal level of glucose uptake.
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, top panel). npBP-1 alone, in the absence of IGF-I, also
inhibited protein degradation dose dependently (Fig. 3, middle
panel). Significant inhibition of protein degradation occurred
with as little as 125 and 500 ng/ml IGF-I and BP-1, respectively.
Phosphorylated BP-1 inhibited protein degradation to a similar extent
as npBP-1 (data not shown). Figure 3 (bottom panel)
indicates that BP-1 did not prevent or attenuate IGF-I-dependent
inhibition of protein breakdown. Likewise, desIGF-I inhibited
proteolysis to the same extent as IGF-I, and BP-1 was again unable to
prevent this response. IGFBP-1 in the medium was not degraded or
dephosphorylated during the time frame of these experiments.
Nonphosphorylated and phosphorylated BP-1 added to skeletal muscle
cells and recovered in the conditioned medium after either 30 sec or
48 h migrated identically by nondenaturing PAGE (Fig. 4).
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IR-3)
inhibited the basal level of protein synthesis in human myoblasts by
8% (Fig. 5, top panel).
Furthermore, IR-3 also completely blocked the ability of IGF-I to
stimulate protein synthesis. The same antibody diminished the ability
of IGF-I to inhibit protein degradation by 60% (Fig. 5, bottom
panel). The IR-3 antibody exhibited a slight IGF-I receptor
agonist activity on its own for inhibiting protein degradation, but
this did not reach statistical significance.
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IIß3, vß1,
and 5ß1 integrins (27). In this
experimental series, IGF-I inhibited protein degradation by 14%.
Incubation of cells with echistatin (10-7 M)
did not significantly alter this response (Fig. 6, top panel). In contrast,
the ability of BP-1 to inhibit proteolysis was completely prevented by
the same concentration of echistatin (Fig. 6, bottom
panel).
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, top
panel). This response was blocked by a vitronectin receptor
(vß3) antibody. These data indicated that
the vitronectin receptor may be involved in protein turnover in muscle
cells. Therefore, we attempted to block the biological effect of BP-1
by preincubating cells with vß3-specific
antibodies. BP-1 inhibited protein degradation to a similar extent as
vitronectin. This effect, however, was not prevented by the
vß3 antibody (Fig. 7). The
anti-vß3 integrin antibody also failed to
block the ability of insulin to inhibit protein degradation (data not
shown). As BP-1 has previously been shown to bind to the fibronectin
receptor in Chinese hamster ovary cells (23), we examined the role of
the ß1 integrin in mediating the ability of BP-1 to
inhibit protein degradation. A ß1 integrin antibody
completely blocked the ability of BP-1 to inhibit protein degradation
(Fig. 7, bottom panel).
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, top panel) and stimulated
the phosphorylation of p70 S6 kinase (Fig. 8, top panel,
inset, lane 2). The effect of IGF-I on protein synthesis was
inhibited by rapamycin in a dose-dependent manner (Fig. 8, top
panel). Rapamycin also inhibited the ability of IGF-I to stimulate
the phosphorylation of p70 S6 kinase (Fig. 8, inset, lane
3). By comparison, rapamycin at a dose that inhibited protein synthesis
(25 ng/ml) had no effect on the basal rate of protein degradation in
myoblasts and failed to block the ability of IGF-I to inhibit protein
degradation (Fig. 8, bottom panel). In contrast, the ability
of BP-1 to inhibit protein degradation was completely blocked by
rapamycin (Fig. 8, bottom panel).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As little as 10-7 M echistatin blocked BP-1
from inhibiting protein degradation. This is similar to the
concentration of echistatin needed to inhibit vitronectin-mediated
migration of porcine aortic smooth muscle cells (pSMC) and DNA
synthesis in response to IGF-I (16). Vitronectin inhibits protein
degradation in skeletal muscle cells, and the effect of vitronectin is
blocked by anti-vß3 antibody. Thus,
changes in protein metabolism may be a general response to changes in
the occupancy of various integrin receptors. In skeletal muscle cells,
echistatin did not block the ability of IGF-I to inhibit protein
degradation, suggesting that IGF-I and BP-1 inhibit protein degradation
through different receptors.
Although echistatin inhibits binding to the vitronectin
(vß3) receptor, and vitronectin inhibits
protein degradation to a similar extent as BP-1, our data imply that
BP-1 does not signal through the vß3
receptor. A specific antibody to this receptor failed to block the
ability of BP-1 to inhibit protein degradation, although it blocked the
ability of vitronectin to inhibit protein degradation. This suggests
that BP-1 signals through another integrin receptor, such as the
fibronectin receptor (5ß1 integrin), as
previously demonstrated in Chinese hamster ovary cells (23). Indeed, an
antibody to the ß1 integrin subunit is a potent inhibitor
of the effect of BP-1 on protein degradation. Myoblasts have previously
been shown to express a tissue specific isoform of the ß1
integrin receptor subunit (29).
It is noteworthy that BP-1 inhibited protein synthesis in the same concentration range as that found in the plasma of patients with thermal injury (30) and children with uncontrolled diabetes (31). BP-1 is also elevated to a similar extent in patients with AIDS wasting (32), and these patients show a muscle-specific resistance to GH/IGF-I at the level of protein synthesis (33, 34). Hence, it is possible that the impairment in muscle protein synthesis observed during these catabolic conditions is in part a result of an increase in the plasma concentration of BP-1. Furthermore, BP-1 can be detected in human muscle by immunohistochemistry, and muscle BP-1 content is dramatically elevated in rats injected with either endotoxin or proinflammatory cytokines (11, 12, 35).
BP-1 may also serve to minimize protein loss during the recovery phase of catabolic conditions by preventing further protein degradation. This would be in agreement with the ability of BP-1 to promote wound healing (18), and the observation that BP-1 can remain in muscle even after it has been cleared from the circulation (36). Retention of BP-1 in muscle may result in a relatively high local concentration of BP-1 at the cell membrane similar to that used in our skeletal muscle cell cultures.
BP-1 exists in multiple forms, including at least five phosphoisoforms (37, 38). Phosphorylated BP-1 has a higher affinity for IGF-I than its nonphosphorylated counterpart, and BP-1 in human plasma is predominantly in a highly phosphorylated state (39, 40). Both forms of BP-1 inhibited the ability of IGF-I to stimulate protein synthesis in myoblasts, and we only observed a significant difference in their inhibitory action at a high molar ratio of BP-1 to IGF-I. The two isoforms also inhibited protein degradation to a similar extent, and both failed to block the ability of IGF-I to inhibit protein degradation. These data suggest that BP-1 functional activity is independent of its phosphorylation state in this model system. In addition, BP-1 that was added to the cells could be recovered from the conditioned medium. It was neither degraded nor dephosphorylated over a 48-h period. BP-1 phosphovariants have previously been shown not to differ in their ability to inhibit IGF-I-stimulated DNA synthesis in human endometrial stromal cells (24) and to either enhance (41) or inhibit IGF action (42) in pSMC. Therefore, BP-1 effects may be either cell type or culture condition specific (43).
Our data also indicate that IGF-I requires the IGF-I receptor, but not
an integrin receptor, to mediate its effects on protein synthesis and
degradation. Inhibition of receptor binding with an IGF-I receptor
antibody (IR-3) prevented IGF-I-mediated protein synthesis and
blocked IGF-I from inhibiting protein degradation. Protein synthesis
was stimulated at a lower concentration of IGF-I than was necessary to
inhibit protein degradation. This may be due to the time frame of the
proteolysis assay. Protein degradation is measured over a 48-h period
to detect the breakdown of long-lived proteins. IGFBPs accumulate in
the medium during this period, and they may inhibit IGF action (R.
A. Frost, unpublished observation). Administration of GH to healthy
control subjects stimulates muscle protein synthesis to the same extent
(25%) as we have observed in myoblasts treated with IGF-I (33), and it
is likely that this effect is mediated by IGF-I. These findings suggest
that IGF-I uses predominantly the IGF-I receptor rather than the
insulin receptor for these two processes. The IR-3 antibody also
inhibited protein synthesis on its own, suggesting the presence of an
IGF-I or IGF-II autocrine loop in these cells. Indeed, endogenous IGF-I
peptide is detectable in human myoblasts by RIA (R. A. Frost,
unpublished observation).
Although IGF-I uses the IGF-I receptor for stimulation of protein synthesis, insulin and IGF-I share intracellular signal transduction pathways. Insulin is thought to stimulate protein synthesis through a protein kinase B (Akt) signaling pathway. Overexpression of a dominant negative form of this protein can inhibit protein synthesis (44, 45, 46). Both PI-3 kinase and p70 S6 kinase are part of this signal transduction pathway, based on the finding that insulin-stimulated protein synthesis can be prevented by pretreatment with either wortmannin or rapamycin (47). It is likely that IGF-I also uses this pathway. Indeed, we found that rapamycin is a potent inhibitor of both p70S6 kinase phosphorylation and the ability of IGF-I to stimulate protein synthesis. In contrast, rapamycin, at a dose that inhibited protein synthesis, had no effect on either the basal level of protein degradation or the ability of IGF-I to inhibit protein degradation.
Our data are in agreement with that of Dardevet et al. (28) and suggests that the IGF-I signal transduction pathway diverges at a point before the mammalian target of rapamycin (mTOR) such that protein synthesis, but not degradation, is sensitive to inhibition by rapamycin. The PI3 kinase inhibitor wortmannin inhibited the basal rate of protein synthesis and prevented IGF-I from stimulating protein synthesis above that found in cells grown in serum-free medium. Yet, this effect required a relatively high concentration of wortmannin (1 µM). Shigemitsu et al. (48) have shown that the sensitivity of p70 S6 kinase activity in cultured cells to inhibition by wortmannin is dependent on the prevailing concentration of amino acids. Under conditions where protein synthesis is measured, amino acid levels are high, and a corresponding high concentration of wortmannin (1 µM) is needed to inhibit protein synthesis. Wortmannin and LY294002 have previously been shown to inhibit PI3-kinase and PI3-kinase homologs, such as mTOR, at this concentration (49). Therefore, a definitive role for PI3-kinase in protein synthesis in human skeletal muscle cells requires a more specific inhibitor.
Although integrin receptor binding has previously been shown to stimulate the mitogen-activated protein kinase (50), PI3 kinase (51), and p70 S6 kinase (52) pathways, there have been no previous reports on the pathway(s) that BP-1 uses to mediate its effects. Our data suggest BP-1 signals through a rapamycin-sensitive pathway. Rapamycin blocked the ability of BP-1 to inhibit protein degradation. However, as noted above, rapamycin had no effect on the ability of IGF-I to inhibit proteolysis. We have been unable to demonstrate a shift in the mobility of p70 S6 kinase in cells treated with BP-1, and this response suggests BP-1 signals through a rapamycin-sensitive protein other than mTOR. Rapamycin binds to FKBP-12, and this protein interacts with multiple partners, including calcineurin (53) and the ryanodine receptor (54).
In conclusion, the results of the present study indicate that IGF-I stimulates protein synthesis through the IGF-I receptor, and this effect can be blocked equally well by either phosphorylated or nonphosphorylated BP-1. IGF-I also stimulates glucose uptake in myoblasts, and this can be prevented by BP-1. In contrast, BP-1 does not prevent IGF-I from inhibiting protein degradation. Indeed, BP-1 inhibits protein degradation independently of IGF-I. BP-1 uses a rapamycin-sensitive pathway to inhibit protein degradation and the effect of BP-1 on protein degradation is mediated by a ß1 integrin. Thus, BP-1 has a unique role in regulating muscle protein metabolism based on its ability to bind IGF-I and interact with integrin receptors.
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Acknowledgments |
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Footnotes |
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Received December 14, 1998.
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References |
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5ß3 integrin receptor inhibits insulin-like growth factor I
signaling in vascular smooth muscle cells. Proc Natl Acad Sci USA 95:11217–112225ß1 integrin by means of its
Arg-Gly-Asp sequence. Proc Natl Acad Sci USA 90:10553–10557IIbß3, Vß3
and 5ß1 and in cell adhesion inhibition. Cell Adhes Commun 2:491–501[Medline]
vß3 requirement for sustained mitogen-activated protein
kinase activity during angiogenesis. J Cell Biol 140:1255–1263This article has been cited by other articles:
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R. A. Frost, G. J. Nystrom, and C. H. Lang Stimulation of Insulin-Like Growth Factor Binding Protein-1 Synthesis by Interleukin-1{beta}: Requirement of the Mitogen-Activated Protein Kinase Pathway Endocrinology, September 1, 2000; 141(9): 3156 - 3164. [Abstract] [Full Text] [PDF]
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C. H. Lang, X. Liu, G. J. Nystrom, and R. A. Frost Acute response of IGF-I and IGF binding proteins induced by thermal injury Am J Physiol Endocrinol Metab, June 1, 2000; 278(6): E1087 - E1096. [Abstract] [Full Text] [PDF]
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K. Sakai, W. H. Busby Jr., J. B. Clarke, and D. R. Clemmons Tissue Transglutaminase Facilitates the Polymerization of Insulin-like Growth Factor-binding Protein-1 (IGFBP-1) and Leads to Loss of IGFBP-1's Ability to Inhibit Insulin-like Growth Factor-I-stimulated Protein Synthesis J. Biol. Chem., March 16, 2001; 276(12): 8740 - 8745. [Abstract] [Full Text] [PDF]
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20 ng/ml) and an increasing concentration of pBP-1. The ability of BP-1
to form a complex with IGF was assessed by nondenaturing PAGE of either
BP-1 alone (from HepG2 cells; inset, lane 1), IGF-I and
BP-1 (lane 2), or desIGF-I and BP-1 (lane 3) followed by Western blot
analysis for BP-1. Free BP-1 (F) and IGF-I/BP-1 complex (C),
respectively. Values are the mean ± SE (n = 6).
Groups with different letters are significantly different from each
other (P < 0.05). Groups with the same letter are
not significantly different.
