From the Departments of Microbiology and
§ Biochemistry and Molecular Biology, Mount Sinai School of
Medicine, New York, New York, 10029 and the ¶ Dairy Science Group,
AgResearch, Private Bag 3123, Hamilton New Zealand
Received for publication, February 2, 2001
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ABSTRACT |
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Previously, by a yeast 2-hybrid screen, we
identified signal transducer and activator of transcription 5b (Stat5b)
as a substrate of the insulin receptor (IR). We demonstrated that
refeeding of fasted mice leads to rapid activation of Stat5 proteins in
liver, skeletal muscle, and fat, suggesting that Stat5b is a
physiological target of insulin. Here, we show that injection of
glucose or insulin into fasted mice leads to robust activation of both
Stat5a and Stat5b in skeletal muscle. In C2C12 myotubes, we find that insulin stimulates tyrosine phosphorylation of Stat5a and Stat5b by
3-5-fold. This degree of Stat5 activation in vitro is
significantly lower than what we observe in vivo and
inversely correlates with IRS-1/2 levels. We can recapitulate robust
insulin activation of Stat5 in C2C12 cells by stable overexpression of
the human IR (hIR). To identify insulin-activated genes that are Stat5
targets, we also overexpressed an IR mutant (LA-hIR) that signals
normally for mitogen-activated protein kinase- and phosphatidylinositol 3-kinase-dependent pathways but is deficient in Stat5
signaling in response to insulin. We demonstrate that insulin induces
the expression of SOCS-2 mRNA in the wild type hIR but not in the LA-hIR-overexpressing cells. The induction of SOCS-3 by insulin is
reduced but not lost in the LA-hIR cells. Therefore, our results suggest that insulin induction of SOCS-2, and in part SOCS-3 mRNA expression, is mediated by Stat5 and can be independent of
mitogen-activated protein kinase and phosphatidylinositol
3-kinase-signaling pathways.
Insulin plays a pivotal role in the regulation of glucose
homeostasis and exerts numerous metabolic and proliferative responses in insulin-sensitive tissues (1). These effects are mediated by the
binding of insulin and subsequent activation of the insulin receptor
(IR)1 tyrosine kinase (1).
The activated receptor phosphorylates itself as well as several other
intracellular substrates. Unlike several members of the receptor
tyrosine kinase superfamily, which directly recruit and phosphorylate
signaling/adapter proteins, the IR mainly signals by recruiting and
phosphorylating members of the insulin receptor substrate family,
IRS-1, -2, -3, and -4, Gab1 and -2, and p62dok-1, -2, and -3 (2, 3).
These proteins then serve as multivalent docking sites for the
recruitment of other Src homology domain 2-containing signaling
proteins. Subsequently, at least two major signal transduction cascades
are initiated including the mitogen-activated protein kinase- and
phosphatidylinositol 3'-kinase-signaling pathways, which propagate the
signal to various cytoplasmic and nuclear effectors (1, 4).
Unlike the IR, cytokine receptors do not possess intrinsic tyrosine
kinase activity. Instead, they depend upon receptor-associated Janus
kinase tyrosine kinases to initiate signaling after ligand binding
(5-7). These Janus kinases become activated and phosphorylate themselves, the cytokine receptor to which they are bound, and Src
homology domain 2-containing signaling/adapter proteins, including Stat
transcription factors (6, 7). This family of transcription factors
includes Stats1, -2, -3, -4, -5a, -5b, and -6, where Stat5a and Stat5b
are two separate but homologous gene products (95% amino acid
identity) (8). Stat transcription factor proteins are found latent in
the cytoplasm. Phosphorylation of the activating tyrosine residue in
the Stat proteins results in the nuclear translocation of Stat dimers
and confers the ability of the Stat dimers to specifically bind to
promoter sequences of target genes and to transactivate those genes
(9). Like receptor tyrosine kinases, cytokine receptors also activate
the mitogen-activated protein kinase- and phosphatidylinositol 3'-kinase-signaling pathways and in some instances through Janus kinase
phosphorylation of IRS family members (2, 6). Activation of Stats is
not limited to cytokine receptors, as several receptor tyrosine
kinases, such as the epidermal growth factor receptor and the
platelet-derived growth factor receptor, have been shown to activate
one or more Stat family members through Janus kinase-independent and
Janus kinase-dependent pathways (10-12).
Recently, by yeast two hybrid screen analysis using the kinase active
cytoplasmic domain of the IR as a probe, we (13) and others (14)
identified Stat5b as a direct substrate of the IR. We then provided the
first evidence that insulin can stimulate the activation of Stats both
in vitro and in vivo (13). We demonstrated that
Stats (1, 3, 5a, and 5b) can be rapidly activated by insulin in two
different IR-overexpressing cell lines and that perfusion of mouse
livers with insulin selectively activates Stat5 proteins. Furthermore,
we observed that refeeding of fasting mice, which causes post-prandial
secretion of insulin, leads to the rapid activation of Stat5 proteins
in liver, skeletal muscle, and fat, all physiologic target tissues of
insulin action. Van Obberghen and co-workers (14) also find that
insulin could stimulate tyrosine phosphorylation of Stat5b when
co-expressed with the human IR in 293 cells. The potential importance
of this pathway in insulin signaling is further supported by recent
studies suggesting that Stat5b can play a role in the activation of
glucokinase and suppressor of cytokine signaling-3 (SOCS-3) gene
transcription by insulin (15, 16).
In addition to being activated after refeeding, Stat5b has been shown
to be activated by insulin in pmi28 mouse muscle cells and Kym-1
rhabdomyosarcoma cells (17, 18). The ability to detect
insulin-stimulated Stat5b activation in both skeletal muscle in
vivo and in vitro suggested that Stat5 might play an
important role in insulin-regulated gene expression in muscle. Here, we show that injection of glucose or insulin into mice leads to robust activation of both 5a and 5b in skeletal muscle. To develop a model
system to study the role of Stat5 in insulin signaling in skeletal
muscle cells, we examined the activation of Stat5a and Stat5b by
insulin in the myogenic C2C12 cell line. We demonstrate that insulin
treatment results in a rapid 3-5-fold increase in tyrosine
phosphorylation of both Stat5a and Stat5b in C2C12 myotubes but not in
undifferentiated myoblasts.
Interestingly, Sawka-Verhelle et al. (14) mapped the site of
interaction of Stat5 with the human IR to Y972 located in the juxtamembrane region of the IR by yeast two-hybrid analysis. This site
contains a motif, 972pYLSA (pY is phosphotyrosine),
that is similar to other Stat5 recruitment sites in cytokine receptors
(19-21). Overlapping this site is an NPEpY (972) motif that recruits
IRS-1 and Shc (22). In addition, by yeast 2-hybrid analysis, SOCS-3
competes with Stat5 for binding to this site as a part of the negative
regulation of IR signaling via Stat5 (16). Because IR levels are
equivalent in C2C12 myoblasts and myotubes, we speculate that the
ability to detect Stat5 activation only in the myotubes is a
consequence of reduced levels of IRS-1 and IRS-2 expression and
increased levels of Stat5 expression compared with the myoblasts. In
support of this hypothesis, the expression levels of IRS-1 and IRS-2 in isolated skeletal muscle are dramatically less than those observed in
C2C12 cells. In contrast, IR expression is essentially equivalent in
skeletal muscle and C2C12 cells. In lieu of significantly reducing the
IRS-1 and IRS-2 content in C2C12 cells, we stably overexpressed the
human IR (hIR) in C2C12 cells and obtained cell lines with an IRS:IR
ratio that more closely approximates the ratio that exists in skeletal
muscle. These cells respond to insulin with potent activation of Stat5
proteins and induction of putative Stat target genes such as CIS,
SOCS1, SOCS2, and SOCS3 (23, 24).
To identify insulin-activated genes that are specific Stat5 targets, we
took advantage of an IR mutant (LA-hIR) that signals normally for
mitogen-activated protein kinase- and phosphatidylinositol 3'-kinase-dependent pathways but is deficient in Stat5
signaling in response to
insulin.2 We established cell
lines expressing equivalent levels of either WT-hIR or LA-hIR, and we
have demonstrated that insulin induces the expression of SOCS-2
mRNA in the WT-hIR but not in the LA-hIR-overexpressing cells.
Interestingly, the induction of SOCS-3 by insulin is lost in LA-hIR
myotubes but is only reduced in LA-hIR myoblasts. Therefore, our
results suggest that in hIR cells Stat5 mediates the insulin induction
of SOCS-2 and, in part, SOCS-3 mRNA expression.
Animals Reagents and Antibodies--
Insulin was a kind gift from Eli
Lily. Recombinant IGF-1 was obtained from GroPep (Adelaide, Australia).
Bovine serum albumin and water-soluble dexamethasone were obtained from
Sigma. Matrigel was purchased from Collaborative Biomedical Products
(Bedford, MA). Chick embryo extract, HEPES, and TRIzol were obtained
from Life Technologies. Luciferase assay reagent was obtained from Promega (Madison, WI). The LHRR construct was a kind gift from Fabrice
Gouilleux. The constructs containing the cDNAs for the SOCS/CIS
genes were kind gifts from Douglas Hilton. The anti-phosphotyrosine monoclonal antibody 4G10 and anti-IRS-1 polyclonal antibody were obtained from Upstate Biotechnology (Lake Placid, NY). The polyclonal antibodies recognizing the C terminus of mouse Stat5A (L20) or mouse
Stat5B (C17) used for immunoprecipitations were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). For immunoblot analysis, the
monoclonal antibody S21520 recognizing both Stat5a and Stat5b was
obtained from Transduction Laboratories (Los Angeles, CA). Generation
and characterization of the monoclonal antibody recognizing the
activated Stat5 (18E5) will be described
elsewhere.3 Generation and
characterization of polyclonal antisera recognizing the IR Cell Culture and Transfections--
Mouse skeletal muscle C2C12
myoblasts were cultured in growth medium: Dulbecco's modified Eagle's
medium containing 15% heat-inactivated fetal bovine serum, 0.5% chick
embryo extract, 25 mM HEPES, and 0.2% gentamicin. For
myoblast cultures, C2C12 cells were grown to 80% of confluence on
tissue culture dishes, washed with phosphate-buffered saline (PBS), and
placed in placed in serum starvation media: Dulbecco's modified
Eagle's medium supplemented with 0.2% bovine serum albumin and 25 mM HEPES for 16 h before treatment with insulin or
diluent (PBS). For the generation of myotubes, C2C12 cells were grown
to confluence on tissue culture dishes coated with Matrigel, washed
with PBS, and placed in differentiation medium (DM): Dulbecco's
modified Eagle's medium containing 2% heat-inactivated horse serum
and 25 mM HEPES for 3 days. Well differentiated cultures (>60% of the plate multinucleated myotubes) were then washed with PBS
and placed in serum starvation media for 16 h before treatment with insulin or diluent (PBS). For generation of the WT-hIR and LA-hIR
cell lines, C2C12 cells were plated at 1.5 × 105
cells/35-mm tissue culture dish. After 16 h, the cells were
transfected with 2.0 µg of pEF/hIR/Neo or pEF/LA-hIR/Neo. Twenty-four
h after transfection, the cells were trypsinized and plated at low
density. Cells were selected for 14 days in neomycin (G418) before
screening isolated colonies by analysis of lysates for the expression
of hIR. For transient transfections, WT-IR cells were plated at 3 × 105 cells/60-mm tissue culture dish. After 16 h,
the cells were washed and transfected with 3.0 µg of LHRR luciferase
reporter plasmid, 1.5 µg of CMV- Immunoprecipitation and Western Blotting--
Tissue or cells
were lysed in radioimmune precipitation buffer (200 mM
NaCl, 50 mM NaF, 50 mM Tris, pH 7.5, 1% Triton
X-100, 1% sodium deoxycholate, 1 mM EDTA, 1 mM
sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride,
5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM benzamidine,
5 µg/ml pepstatin A, 5 nM microcystin) for 20 min at
4 °C. Lysates were cleared of insoluble material by centrifugation.
Cleared lysates were immunoprecipitated overnight at 4 °C with
either anti-Stat5A or anti-Stat5B antibodies, incubated with
protein-A-Sepharose, washed four times in radioimmune precipitation buffer, boiled in SDS-PAGE sample buffer containing 100 mM
dithiothreitol, and subjected to SDS-PAGE (7.5% acrylamide).
Alternatively for Western blotting of whole cell lysates, cleared
radioimmune precipitation buffer lysates containing equal protein were
boiled in SDS-PAGE sample buffer and subjected to SDS-PAGE (7.5%
acrylamide). After electrophoretic transfer of proteins to
nitrocellulose, the membranes were blocked overnight at 4 °C in
either bovine serum albumin blocking buffer (3% bovine serum albumin
in PBS, 0.1% Tween 20) for anti-phosphotyrosine Western blots or milk
blocking buffer (4% nonfat dried milk in PBS, 0.1% Tween 20) for all
other Western blots. Blocked membranes were incubated with primary
antibodies in the appropriate blocking buffer for 2 h at room
temperature. The membranes were washed extensively, incubated with
anti-mouse IgG2b-horseradish peroxide or anti-rabbit IgG-horseradish
peroxide in PBS, 0.1% Tween 20, 4% nonfat dried milk for 1 h,
washed extensively, developed with Supersignal ECL reagent from Pierce,
and exposed to film (Kodak X-Omat AR).
Electrophoretic Mobility Shift Analysis--
Whole cell
extracts were prepared as described previously (26). Electrophoretic
mobility shift analysis was performed on the whole cell extracts as
described previously (26) with 32P-radiolabeled
double-stranded oligonucleotide probes containing high affinity binding
sites for Stat5 ( RNA Extraction and Northern Analysis--
After treatment with
insulin, C2C12 or hIR64 cells were rinsed twice with ice-cold PBS, and
total RNA was extracted using TRIzol (Life Technologies). Total RNA was
fractionated by electrophoresis on agarose-formaldehyde gel,
transferred to Hybond XL membrane (Amersham Pharmacia Biotech) using
the Northern Max kit (Ambion), and fixed by UV cross-linking. Membranes
were hybridized at 68 °C overnight (except for SOCS-1 at 75 °C)
with 32P-radiolabeled antisense riboprobe derived from
full-length cDNA inserts encoding CIS, SOCS-1, SOCS-2, or SOCS-3
(27) prepared with the Strip-EZ RNA kit (Ambion). Membranes were washed
at high stringency (0.1× SSC (1× SSC = 0.15 M NaCl
and 0.015 M sodium citrate), 0.1% SDS at 68 °C)
and exposed to Kodak X-Omat AR-5 film with an intensifying screen at
Insulin Activates Stat5a and Stat5b in Skeletal Muscle in
Vivo--
We have shown that refeeding of fasting mice leads to
increases in the tyrosine phosphorylation of Stat5b in liver, adipose tissue, and skeletal muscle (13). Although these data suggest that
physiological levels of insulin released after feeding are capable of
stimulating Stat5 activation, we considered the possibility that
peptides secreted by the gut after feeding might be responsible for
stimulating the tyrosine phosphorylation of Stat5 proteins. We
therefore bypassed the feeding response and increased circulating insulin levels by direct injection of glucose into fasted mice. We
detect strong increases in the tyrosine phosphorylation of both Stat5a
and Stat5b in skeletal muscle within 15 min of glucose injection (Fig.
1). As expected, direct injection of
insulin into fasted mice also leads to rapid increases in the tyrosine
phosphorylation of both Stat5a and Stat5b in skeletal muscle (Fig. 1).
Similar results were obtained in fat and
liver.4
Insulin Activates Stat5a and Stat5b in C2C12 Myotubes but Not
Myoblasts--
Tyrosine phosphorylation of Stat5b in response to
insulin is detectable in several in vitro cell models
including Kym-1 rhabdomyosarcoma (18) and pmi28 mouse muscle cells (17)
as well as in 3T3-L1 adipocytes (16) and HepG2 cells (15) when these
cells are pretreated with protein tyrosine phosphatase inhibitors.
Stat5b, but not Stat5a, is activated in Kym-1 rhabdomyosarcoma cells
(18), which is in contrast to the in vivo activation of both
Stat5a and Stat5b in the skeletal muscle that we observe. To determine whether insulin activates both Stat5 proteins in skeletal muscle cells
in vitro, we performed experiments with the myogenic C2C12 cell line. These cells are maintained in culture as myoblasts in the
presence of high concentrations of serum. When C2C12 cells are grown to
confluence and high serum-containing medium was removed, the myoblasts
withdraw from the cell cycle and begin a myogenic program of
differentiation. After 3 days in low serum, the myoblasts have already
fused to form multinucleated myotubes that express muscle-specific
genes at high levels. To determine whether insulin stimulates Stat5a or
Stat5b either as proliferating myoblasts or at any point during the
differentiation of myoblasts to myotubes, we treated C2C12 myoblasts or
C2C12 cultures at 1, 2, 3, or 4 days in low serum media with or without
insulin. We analyzed the control and insulin-stimulated lysates by
immunoprecipitation with antibodies recognizing either Stat5a or Stat5b
followed by blotting with a phosphorylation state-specific monoclonal
antibody that recognizes both Stat5a and Stat5b phosphorylated on the
activating tyrosine. We observed reproducible insulin-induced 3-5-fold
increases in the tyrosine phosphorylation of both Stat5a and Stat5b in
C2C12 myotubes with little or no effect detected in the
undifferentiated myoblasts (Fig. 2).
Indeed, the ability of insulin to activate Stat5 appears to be
correlated with the level of differentiation in the C2C12 myotubes
(Fig. 2A).
To determine whether the activation of Stat5 in skeletal muscle occurs
at doses of insulin that do not result in activation of the IGF-1
receptor, we treated C2C12 myotubes with varying concentrations of
insulin for 10 min. We detected insulin activation of Stat5a and Stat5b
in C2C12 myotubes at doses of insulin below levels that activate the
IGF-1 receptor (Fig. 2B). To confirm this result, we treated
C2C12 myotubes with 200 ng/ml recombinant insulin growth factor-1 for
10 min. Analysis of recombinant IGF-1-stimulated lysates with
anti-phospho-Stat5 antibodies showed no detectable increases in Stat5
phosphorylation in response to IGF-1 (data not shown). To determine the
kinetics of Stat5 activation in response to insulin, we treated C2C12
myotubes with insulin for several time points and assayed cell lysates
for Stat5 tyrosine phosphorylation. As shown in Fig. 2B, we
detected Stat5 activation in myotubes at the earliest time point tested
(2 min). This response peaked at 10 min, and the decline was evident by
20 min. These results suggest that the Stat5 activation by insulin in
myotubes is rapid and transient.
Insulin Activation of Stat5 in C2C12 Cells Stably Overexpressing
the hIR--
We were surprised that the Stat5 activation in response
to insulin in C2C12 cells was relatively weak compared with the
response we observed in skeletal muscle in vivo (Fig. 1).
Since Stat5 and IRS-1 are recruited to the same phosphorylated residue
on the IR (Tyr(P)-972) (14, 22), we speculated that IRS-1 and
Stat5 might be competing for recruitment to limited amounts of IR in the C2C12 cells. We therefore performed biochemical analysis of C2C12
myoblasts and myotubes with regard to the expression levels and
insulin-stimulated tyrosine phosphorylation levels of several IR
substrates (Fig. 3). Although IR
Based on these observations, we hypothesized that increasing the IR
content in these cells by stable overexpression of the IR would result
in cell lines in which the activation of Stat5 approaches the levels we
observe in vivo. To test our hypothesis, we generated
several clones of C2C12 cells that stably overexpress the hIR that are
capable of differentiating into myotubes. As shown in Fig.
3B, in a representative clone (hIR64), insulin activates Stat5 proteins 25-40-fold in both hIR myoblasts and myotubes, approaching the response to insulin we observed in skeletal muscle in vivo. Further biochemical analysis in regard to other IR
substrates revealed that, compared with C2C12 myoblasts, the hIR
myoblasts display increased IRS-1 tyrosine phosphorylation in response
to insulin despite reduced IRS-1 expression. In addition,
insulin-stimulated mitogen-activated protein kinase activation is also
increased in the hIR myoblasts, as evidenced by increases in the
amounts of activated extracellular-regulated kinase-1 and -2. In
contrast, there is little augmentation of insulin-stimulated
phosphatidylinositol 3'-kinase activation, as shown by nearly
equivalent levels of activated protein kinase B in hIR64 cells compared
with C2C12 cells.
Insulin Induces Stat5 DNA Binding Activity and Transactivates a
Stat5 Reporter Gene in hIR Cells--
To verify that the
insulin-stimulated tyrosine phosphorylation of Stat5 results in
functional DNA binding, we performed electrophoretic mobility shift
analysis with insulin-treated whole cell extracts incubated with a
Stat5 DNA binding site (
The glucocorticoid receptor (GR) has been shown to physically interact
with both Stat5a and Stat5b and augment the relatively weak
transcriptional activation potential of Stat5 proteins (28-31). We
therefore tested whether the potent synthetic glucocorticoid, dexamethasone (DEX) has any effect in our system. DEX alone does not
stimulate expression of the Stat5 reporter gene because there is no
functional GRE in this construct. When DEX and insulin were added
together, however, a synergistic induction of luciferase was observed.
Consistent with the idea that the Stat5-GR interaction recruits potent
transactivation domains to Stat5, the addition of DEX allows the
Stat5a/5b Insulin Induces the mRNA Expression of Several Members of the
SOCS/CIS Family of Signaling Proteins in hIR64 Myoblasts and
Myotubes--
Recently two genes were described whose regulation by
insulin is mediated in part by Stat5, glucokinase, and SOCS-3 (15, 16).
SOCS-3 is a member of a novel family of cytokine-inducible genes, the
CIS/SOCS genes, which were initially isolated as suppressors of
cytokine signaling (24, 27). Emanuelli et al. (16)
demonstrate that the insulin induction of SOCS-3 in COS-7 cells is
enhanced by Stat5B expression. To determine whether insulin induces the expression of SOCS-3 or other members of the SOCS family in hIR myoblasts or myotubes, we isolated total RNA from serum-starved hIR
myoblasts or myotubes stimulated for 0, 0.5, 1, 2, and 4 h with
100 nM insulin and performed Northern analysis with
antisense riboprobes for CIS, SOCS-1, SOCS-2, and SOCS-3. As shown in
Fig. 5, insulin strongly induces all four
SOCS/CIS family members in hIR myoblasts and induces SOCS-1, SOCS-2,
and SOCS-3 but not CIS in hIR myotubes. As reported previously, the
kinetics of activation among the family members differ as SOCS-1 and
SOCS-3 are rapidly induced, whereas CIS and SOCS-2 show slower
induction kinetics (33).
C2C12 Cells Overexpressing the LA-hIR Mutant Are Deficient in Stat5
Signaling in Response to Insulin--
To examine the role of Stat5 in
insulin-regulated transcription in C2C12 muscle cells, we overexpressed
a mutant hIR (LA-hIR) that is deficient in Stat5 signaling. We
generated this mutant as part of a detailed analysis of the amino acid
residues critical for Stat5 recruitment to the YLSA motif in the
juxtamembrane region of the IR.2 A schematic of the
mutations is shown in Fig. 6A.
We screened several WT-hIR and LA-hIR myoblast clones and identified a
matched pair that expresses equivalent amounts of the WT-hIR (hIR18) or the mutant LA-hIR (LA2E). Compared with the WT-hIR18 myoblasts, the
LA-hIR clone LA2E myoblasts display a greater than 50-fold reduction in
Stat5 activation in response to insulin (Fig. 6B). In
contrast, insulin-stimulated tyrosine phosphorylation of IRS-1 and
activation of extracellular-regulated kinase-1 and -2 and protein
kinase B are essentially equivalent. These results suggest that we can
use these cell lines as a tool to examine the role of Stat5 in
insulin-regulated transcription in C2C12 cells either in proliferating
myoblasts or in differentiated myotubes.
Insulin Induction of SOCS-2 and SOCS-3 Is Mediated by
Stat5--
To determine whether Stat5 is involved in insulin
stimulation of SOCS-2 or SOCS-3 mRNA expression, we isolated total
RNA from serum-starved WT-hIR(hIR18) myoblasts and myotubes or
LA-hIR(LA2E) myoblasts and myotubes stimulated for 0, 0.5, 1, 2, and
4 h with 100 nM insulin. For comparative purposes, we
also isolated total RNA from serum-starved C2C12 or WT-hIR (hIR64)
myoblasts and myotubes stimulated for the same time points with 100 nM insulin. We performed Northern analysis on each set of
RNAs with either an antisense riboprobe for SOCS-2 or SOCS-3, and the
results are shown in Fig. 7. We detected
similar patterns of SOCS-2 induction in response to insulin in both
WT-hIR clones, although the basal levels in the myotubes of hIR18 was
slightly higher. In contrast, the SOCS-2 induction in response to
insulin in the LA-hIR (LA2E) clone was reduced to levels observed in
C2C12 cells. These results provide compelling evidence that SOCS-2
mRNA induction by insulin in hIR cells involves Stat5
activation.
Like SOCS-2, we observed similar patterns of insulin-stimulated SOCS-3
induction in the two WT-hIR clones, although again, basal levels in the
myoblasts of hIR18 were slightly higher. Unlike SOCS-2, the
insulin-stimulated SOCS-3 induction in response to insulin in the
LA-hIR (LA2E) myoblasts was reduced to levels observed in C2C12 cells
only in the myotubes. The insulin-stimulated SOCS-3 induction in the
LA-hIR myoblasts was blunted (~50%) but not reduced completely,
suggesting that transcription factors other than Stat5 are required for
insulin-stimulated SOCS-3 transcription in myoblasts. Nevertheless,
these results show that Stat5 is involved in SOCS-3 mRNA induction
by insulin especially in the LA-hIR myotubes.
Insulin exerts its pleiotrophic effects on metabolism,
proliferation, and differentiation by several mechanisms (1, 3, 4).
These include changing the activity and the intracellular localization
of its effector proteins as well as regulating gene expression at the
transcriptional level (1, 3, 4). Recent progress in identifying the
factors involved in insulin regulation of gene expression indicate that
the Forkhead (FKHD)- and sterol-regulated enhancer-binding protein
(SREBP) families of transcription factors play critical roles in the
regulation of key metabolic enzymes such as fatty acid synthase,
phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and
glucokinase (34-39). We and others have identified a novel signaling
pathway involving activation of Stat transcription factors by insulin
(13, 14). Injection of insulin into mice activates Stat5a and Stat5b in
skeletal muscle, suggesting that Stat5 proteins are physiological
substrates of insulin-stimulated signaling pathways. Nevertheless, the
specific functional roles of Stats in insulin action remain to be
determined. To address this question we have engineered an in
vitro model system for skeletal muscle such that we can now
identify potential insulin-regulated genes that require Stat5 function.
We show evidence that induction of SOCS-2 mRNA expression by
insulin in muscle cells overexpressing the IR is mediated by Stat5.
Thus, we have identified an insulin target gene in IR-overexpressing
muscle cell lines that is clearly regulated by a Stat protein. We also
confirm that the induction of SOCS-3 mRNA expression by insulin in
these cells is mediated in part by Stat5.
The level of insulin stimulation of Stat5a and Stat5b tyrosine
phosphorylation in skeletal muscle is quite robust. Consistent with
this observation, insulin activation of Stat5b has been reported in two
muscle cell models, Kym-1 rhabdomyosarcoma (18) and pmi28 mouse muscle
cells (17), although at significantly lower levels than we have
observed in vivo. Similarly, the insulin activation of Stat5
proteins in C2C12 cells is relatively weak and observed only in
myotubes. We explored potential reasons for the differential ability of
insulin to stimulate Stat5 activation in skeletal muscle versus cell culture models. Although the expression levels
of the IR In the hIR cell lines, insulin stimulates the expression of four
putative Stat target genes from the SOCS/CIS family of proteins: SOCS-1, SOCS-2, SOCS-3, and CIS (24, 27). Insulin activation of a SOCS
gene is not novel, as Emanuelli et al. (16) have recently demonstrated that insulin induces SOCS-3 mRNA expression in 3T3-L1 adipocytes. In these cells, however, they did not observe insulin induction of either SOCS-2 or CIS mRNA expression, suggesting the
possibility of tissue-specific insulin regulation of SOCS/CIS genes.
Emanuelli et al. (16) suggest that the insulin induction of
SOCS-3 is mediated in part by Stat5 because there are Stat5 binding
sites present in the SOCS-3 promoter, and insulin-stimulated expression
of SOCS-3 in COS cells is enhanced by co-transfection of Stat5b. To
determine whether insulin activation of SOCS-2 and SOCS-3 in hIR cells
is mediated by Stat5, we compared the induction of these genes in a
cell line that expresses WT-IR with a cell line expressing the mutant
LA-hIR that does not activate Stat5 but is otherwise equivalent to the
WT-hIR. Insulin stimulation of SOCS-2 mRNA expression is not
observed in either the LA-hIR myoblasts or myotubes. These results
demonstrate that insulin induction of SOCS-2 mRNA expression in the
hIR cells is mediated by Stat5. Insulin stimulation of SOCS-3 is
reduced in LA-hIR myoblasts and not observed at all in LA-hIR myotubes.
Thus, the insulin-stimulated expression of SOCS-3 in hIR cells is also
mediated, at least in part, through Stat5 proteins. Our results support
and extend those of Emanuelli et al. (16), who showed that
co-transfection of Stat5b enhances the ability of insulin to stimulate
SOCS-3 gene expression in COS cells transiently transfected with the
IR. Furthermore, SOCS-3 was also found to directly interact with
Tyr(P)-972 in the IR and prevent activation of Stat5b in
co-transfection assays (16). SOCS-2 has also been reported to interact
with the IR (43). These data are provocative, as hyperinsulinemia
clearly leads to insulin resistance in vivo (4). The overall
importance of SOCS-2 and SOCS-3 in normal regulation of insulin action
remains unclear. The gigantism phenotype of the SOCS-2 knockout mice
suggests that SOCS-2 functions as an inhibitor of the growth
hormone/IGF-1 axis (44), whereas the early lethality in the SOCS-3
knockout due to excessive fetal liver hematopoiesis (45) precludes an evaluation of its role as a physiological inhibitor of insulin action.
Several studies suggest that CIS is a Stat5 target gene. A tandem pair
of high affinity Stat5 binding sites is present in the proximal
promoter of the CIS gene and is required for activation (46, 47). We
therefore suspect that this will also be the case for insulin
activation of CIS mRNA expression. In contrast, the expression of
SOCS-1 is most potently stimulated by cytokines that predominantly
activate Stat1 and Stat3 (24). Therefore, the insulin induction of
SOCS-1 is less likely to be mediated by Stat5. We are now in the
process of determining the role of Stat5 in the induction of these and
other insulin target genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
6-week-old male C57/Black mice were fasted for
48 h before a bolus intraperitoneal injection of either glucose,
insulin, or vehicle (phosphate-buffered saline). 10 min (glucose) or 25 min (insulin) after the injection, the mice were euthanized, and
tissues were removed and frozen in liquid nitrogen. Tissue lysates were prepared as described previously (13).
-subunit
was described previously (25). Horseradish peroxide-conjugated
secondary antibodies were obtained from CALTAG (Burlingame, CA).
Monoclonal anti-phospho-p44/42 mitogen-activated protein kinase
(Thr-202/Tyr-204) antibody, polyclonal antibody recognizing phospho-Akt
(Ser473), and polyclonal antibody recognizing phospho-Stat3 (Tyr-705)
were obtained from New England Biolabs (Beverly, MA).
gal, and either 1.5 µg of pRK5
empty vector or 0.75 µg of pRK5Stat5aFLAG and pRK5Stat5bFLAG with 18 µl of Fugene (Roche Molecular Biochemicals) according to the
manufacturer directions. Twenty-four h after transfection, the
transfected cells were split onto Matrigel-coated 6-well plates. Six h
later, the cells were placed in serum starvation media for 16 h
before treatment with dexamethasone and/or insulin or diluent (PBS).
After stimulation with insulin for 6 h, cells were harvested in
1× reporter lysis buffer from Promega. After normalization for
-galactosidase activity, lysates were assayed for luciferase
activity using the luciferase assay kit from Promega.
-casein gene promoter (8)).
70 °C. For reprobing, membranes were stripped according to
manufacturer directions for the Strip-EZ RNA kit (Ambion).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (50K):
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Fig. 1.
Injection of insulin or glucose increases
tyrosine phosphorylation of Stat5 in skeletal muscle. Mice were
fasted for 48 h and intraperitoneally injected with either PBS,
1.5 mg/kg of glucose, or 0.5 units of insulin. After 15 min (glucose)
or 25 min (insulin), the mice were sacrificed, and the skeletal muscle
was rapidly excised and frozen in liquid nitrogen. Lysates were
prepared, and tyrosine phosphorylation of the Stat5 proteins was
measured by immunoprecipitation (IP) with antibodies
recognizing the C terminus of either Stat5a or Stat5b followed by
immunoblotting with anti-phosphotyrosine ( -pY) antibodies
as indicated. The blots were then stripped and reprobed with antibodies
recognizing Stat5a or Stat5b.
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Fig. 2.
Physiological doses of insulin rapidly
activate Stat5 in C2C12 myotubes. A, ability of insulin
(Ins) to activate Stat5 increases during differentiation.
For day 0 (d0), 24 after plating, C2C12 cells were
serum-starved for 3 h and treated with 100 nM insulin
for 20 min before lysis. For d1-d4, the cells were switched
to DM for 24 h (d1), 48 h (d2), 72 h (d3), or 96 h (d4) before serum starvation,
stimulation, and lysis. Lysates were subjected to immunoprecipitation
(IP) with antibodies recognizing either Stat5a or Stat5b
followed by immunoblotting with an antibody recognizing Stat5
phosphorylated at the activating tyrosine. B, insulin
activates Stat5 at physiological doses in C2C12 myotubes. After 4 days
in DM, C2C12 myotubes were serum-starved for 3 h and treated with
different doses of insulin as indicated for 20 min before lysis.
Analysis of the lysates was identical to panel A. C,
activation of Stat5 by insulin in C2C12 myotubes is rapid and
transient. After 4 days in DM, C2C12 myotubes were serum-starved for
3 h and treated with 100 nM insulin for the indicated
time points before lysis. Analysis of the lysates was identical to
panel A.
-subunit expression and insulin-stimulated tyrosine phosphorylation
are equivalent in C2C12 myoblasts and myotubes, IRS-1 protein
expression is extremely high in the C2C12 myoblasts, which could block
efficient recruitment of Stat5 proteins to the IR in the myoblasts.
Consistent with this observation, in C2C12 myotubes, where
insulin-stimulated Stat5 activation is detected, IRS-1 expression is
reduced 3-5-fold, and Stat5 expression is increased 4-5-fold.
Nevertheless, the level of Stat5 activation by insulin in C2C12
myotubes is significantly less than that observed in skeletal muscle.
We therefore performed a direct comparison of the levels of IR, IRS-1,
IRS-2, and Stat5 proteins in lysates from isolated skeletal muscle,
C2C12 myoblasts, and C2C12 myotubes (Fig. 3A). Isolated
skeletal muscle, C2C12 myoblasts, and C2C12 myotubes express equivalent
levels of IR protein. In contrast, the expression of IRS-1 and IRS-2 is
dramatically lower in skeletal muscle compared with the immortalized
C2C12 cells. These data are consistent with our hypothesis that the
high expression levels of IRS proteins prevent efficient recruitment
and activation of Stat5 proteins by limiting amounts of IR.
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Fig. 3.
A lower ratio of IRS-1 and/or -2 to IR
correlates with the ability of insulin to activate Stat5.
A, the ratio of IRS-1,-2 to IR is dramatically less in
isolated skeletal muscle compared with C2C12 cells. Lysates were
prepared from either skeletal muscle excised from two different fasted
mice or from C2C12 myoblasts (MB) or myotubes
(MT) that were serum-starved for 3 h. 60 µg of
lysates were analyzed directly by immunoblotting with the indicated
antibodies. B, decreasing the ratio of IRS-1 and IRS-2 to IR
by stable overexpression of the IR in C2C12 cells results in high
levels of insulin-stimulated Stat5 activation. C2C12 cells were stably
transfected with an expression plasmid for the human insulin receptor
and selected in neomycin. One positive clone that differentiates to a
similar extent as C2C12 cells, hIR64, was further characterized and
compared with C2C12 in its response to insulin. Twenty-four h after
plating, C2C12 or hIR64 myoblasts were serum-starved for 3 h and
treated with 100 nM insulin for 0, 2.5, or 10 min before
lysis. For myotubes, C212 or hIR64 cells were switched to DM 24 h
after plating. After 4 days in DM, C2C12 or hIR64 myotubes were
serum-starved for 3 h and treated with 100 nM insulin
for 0, 2.5, or 10 min before lysis. 75 µg of lysates were analyzed
directly by immunoblotting with the indicated antibodies. The
asterisk (*) next to the Stat5 Tyr(P) (pY)
indicates that the blot was exposed significantly longer in order to
visualize the Stat5 Tyr(P) in the C2C12 cells. ERK,
extracellular-regulated kinase; PKB, protein kinase B.
-casein promoter (8)) (Fig.
4A). Stat5-specific DNA
complexes were detected in both hIR myoblasts and myotubes, confirming
the results obtained in Fig. 3. To investigate whether
insulin-activated Stat5 proteins are capable of transactivation in
hIR-overexpressing cells, we performed transient transfections with a
luciferase reporter gene construct driven by six copies of the Stat5
binding site located upstream of a minimal thymidine kinase promoter.
Insulin stimulates Stat5 reporter gene expression in hIR myoblasts by
up to 3-fold with endogenous levels of Stat5 and by 35-fold when Stat5
proteins are co-transfected (Fig. 4B). In contrast,
C-terminal truncation mutants of Stat5 (Stat5a/5b
CT), which lack the
transactivation domain, do not support insulin stimulation of the Stat5
reporter.
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Fig. 4.
Insulin activates Stat5 DNA binding and
transactivation. A, insulin (Ins) activates
Stat5 DNA binding activity in both hIR myoblasts and myotubes. HIR64
myoblasts or myotubes were serum-starved for 3 h and treated with
100 nM insulin for 15 min before lysis. 18 µg of native
whole cell lysates (Stat5) were assayed for Stat5 DNA binding activity
by electrophoretic mobility shift analysis as described under
"Experimental Procedures." B, insulin activation of a
Stat5 reporter gene is potentiated by dexamethasone (DEX)
and requires the Stat5 C-terminal transactivation domain. WT-hIR
(hIR64) myoblasts were transiently co-transfected with 0.5 µg of
LHRR, a Stat5 luciferase reporter construct, 0.25 µg of CMV- gal,
and either 0.75 µg of empty vector (pRK5), 0.75 µg of Stat5 (0.375 Stat5a and 0.375 µg Stat5b), or 0.75 µg of Stat5
CT (0.375 µg
of 5a and 0.375 µg of 5b) as indicated below the graph. Twenty-four h
after transfection, the myoblasts were serum-starved for 16 h,
treated with either vehicle, 100 nM insulin, 100 nM dexamethasone, or both for 6 h as indicated, and
then assayed for
-galactosidase and luciferase activity.
CON, control.
CT proteins to activate the Stat5 reporter gene in an
insulin-stimulated fashion (29). The synergistic effect of DEX is also
apparent in the absence of co-transfected Stat5 expression vectors.
Therefore, a significant fraction of the endogenous Stat5 proteins are
likely to be interacting with the GR, and this may be important for
genes that are synergistically regulated by insulin and
glucocorticoids, such as glucokinase (32). Although the Stat5-GR
interaction may lead to significant increases in the activation of
Stat5 target genes, this interaction has also been shown to lead to a
decreased ability of the GR to transactivate a simple GRE-containing
reporter gene (28). Therefore, it is possible that insulin-stimulated
Stat5 could play a role in the inhibitory effect of insulin on several
glucocorticoid-inducible genes in vivo.
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Fig. 5.
Insulin induces the mRNA expression of
several members of the SOCS/CIS family of signaling proteins.
HIR64 myoblasts or myotubes were serum-starved for 16 h and then
stimulated with either 0 or 100 nM insulin for the
indicated times before harvesting for RNA. 15 µg of total RNA from
each time point was fractionated on a 1% denaturing formaldehyde
agarose gel and transferred to a nylon membrane. The membrane was
hybridized first with a 32P-labeled antisense riboprobe
transcribed from the full-length cDNA for SOCS-3. After exposure to
film, the membrane was stripped and sequentially hybridized and
stripped in this order: SOCS-2, CIS, and then SOCS-1. The exposure
times were 1 h (SOCS-3), 3 h (SOCS-2), 24 h (CIS), and
72 h (SOCS-1).
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Fig. 6.
LA-hIR cell lines are deficient in Stat 5 signaling but are normal for mitogen-activated protein kinase or
phosphatidylinositol 3'-kinase signaling. A, schematic
of IR sequence surrounding Y972 depicts IRS-1 and Stat5 recruitment
sites and LA residue mutations. B, WT-hIR18 or LA-hIR2E
myoblasts were serum-starved for 3 h before stimulation with 100 nM insulin for the indicated time points before lysis. 75 µg of lysates were analyzed directly by immunoblotting with the
indicated antibodies. The asterisk (*) next to the Stat5
Tyr(P) (pY) indicates that the blot was exposed
significantly longer in order to visualize the Stat5 Tyr(P) in the
LA-hIR2E cells. ERK, extracellular-regulated kinase;
PKB, protein kinase B.
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Fig. 7.
Insulin (Ins) induction of
SOCS-2 and SOCS-3 is mediated by Stat5. WT-hIR64, WT-hIR18,
LA-hIR2E, or C2C12 myoblasts or myotubes were serum-starved for 16 h and then stimulated with 100 nM insulin for the
indicated times before harvesting for RNA. 15 µg of total RNA from
each time point was fractionated on a 1% denaturing formaldehyde
agarose gel and transferred to nylon membranes. The membranes were
hybridized with 32P-labeled antisense riboprobe transcribed
from the full-length cDNA for SOCS-2. After exposure to film for
6 h, the membranes were stripped and reprobed with
32P-labeled antisense riboprobe transcribed from the
full-length cDNA for SOCS-3. The exposure time for the SOCS-3
membranes was 3 h.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit are equivalent in isolated skeletal muscle, C2C12 myoblasts, and myotubes, we discovered important differences. First,
the levels of IRS-1 and IRS-2 protein expression are highest in the
C2C12 myoblasts, at least 5-fold lower in the C2C12 myotubes, and
dramatically lower in the skeletal muscle. Second, the expression of
Stat5a and especially Stat5b are significantly higher in the myotubes
compared with the myoblasts. Based on the observation that the
recruitment sites on the IR for IRS-1 and Stat5 are partially overlapping (14, 22), we speculated that these two proteins are
competing for recruitment to the IR. Therefore, high levels of IRS-1
expression in the immortalized C2C12 myoblasts (as well as many other
immortalized cell lines compared with their primary counterparts
(40-42)) could effectively block Stat5 recruitment to the IR. This
model predicts that reducing the IRS:IR ratio either by IRS-1 antisense
or IR overexpression would allow for insulin-stimulated Stat5
activation. Consistent with this model, stable overexpression of the
human insulin receptor in C2C12 cells confers the ability of these
cells to respond to insulin with levels of Stat5 activation that
approach the levels observed in vivo. We have characterized
several other known insulin-signaling pathways in these stable hIR
overexpressing cell lines. Although the ability to augment
insulin-stimulated Stat5 activation is the most dramatic result of
increased levels of IR, the activation of other signaling pathways by
insulin are also enhanced (for example, extracellular-regulated
kinase-1). In contrast, protein kinase B activation in response to
insulin is relatively unaffected. This result suggests that any
transcriptional response above that of C2C12 cells is not likely to be
due to phosphatidylinositol 3'-kinase and more likely due to Stat5 and
extracellular-regulated kinase 1 or other pathways we have not yet identified.
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ACKNOWLEDGEMENTS |
---|
We thank D. Hilton for kindly providing the SOCS/CIS expression plasmids. We are grateful to R. Kohanski for valuable discussions and suggestions. We also thank members of the Wang laboratory for reagents and support.
![]() |
FOOTNOTES |
---|
To whom correspondence should be addressed. Tel.:
212-241-8147; Fax: 212-996-7214; E-mail:
henry.sadowski@mssm.edu.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M101014200
This work was supported by research awards from the American Diabetes Association, National Institutes of Health (NIH) Grant DK53000 (to H. B. S.), and NIH National Research Service Award NS10499 (to C. L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
2 M. Le, R. A. Kohanski, L.-H. Wang, and H. B. Sadowski, submitted for publication.
3 C. L. Sadowski, T. T. Wheeler, L.-H. Wang, and H. B. Sadowski, submitted for publication.
4 T. T. Wheeler and H. B. Sadowski, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: IR, insulin receptor; hIR, human IR; WT, wild type; PBS, phosphate-buffered saline; DM, differentiation medium; PAGE, polyacrylamide gel electrophoresis; Stat, signal transducers and activators of transcription; CIS, cytokine-inducible Src homology domain 2-containing protein; SOCS, suppressor of cytokine signaling; IRS, insulin receptor substrate; IGF-1, insulin-like growth factor-1; GR, glucocorticoid receptor; DEX, dexamethasone.
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REFERENCES |
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