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Department of Pediatrics, Rhode Island Hospital and Brown University, Providence, Rhode Island 02903
Address all correspondence and requests for reprints to: Charlotte M. Boney, M.D., Department of Pediatrics, MPS-2, Rhode Island Hospital, 593 Eddy Street, Providence, Rhode Island 02903. E-mail: charlotte_boney{at}brown.edu
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Abstract |
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Studies of proximal signaling revealed Shc phosphorylation and Shc/Grb2 complex formation in IGF-I-treated proliferating, but not differentiating, cells. Insulin receptor substrate-1 phosphorylation after IGF-I treatment was present in proliferating, quiescent, and differentiating preadipocytes. Shc phosphorylation and Grb2 association after EGF treatment were present throughout all phases of growth. The change in IGF-I signaling via Shc phosphorylation and MAPK activity during 3T3-L1 differentiation indicates that loss of IGF-I mitogenic signaling via the MAPK pathway is part of the differentiation process.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although insulin receptor signaling in differentiated 3T3-L1 adipocytes has been the focus of intensive study, there has been relatively little attention to IGF-I signaling during preadipocyte proliferation and differentiation. The biological actions of IGF-I, EGF, and insulin are mediated through specific receptors that are members of the tyrosine kinase family of growth factor receptors (9). Propagation of the signal occurs when the activated receptor tyrosine kinase phosphorylates substrates that act downstream to mediate diverse actions, including mitogenesis, cell differentiation, and metabolic events. An important substrate of both the insulin and IGF-I receptors is insulin receptor substrate-1 (IRS-1), a 185-kDa protein containing at least 20 tyrosine phosphorylation sites in sequence motifs that recognize src homology 2 (SH2) domains (10). The tyrosine-phosphorylated form of IRS-1 acts as a docking protein by associating with various SH2-domain containing proteins involved in multiple signaling pathways.
The most well understood mitogenic signaling pathway used by receptor tyrosine kinases, including IGF-I, insulin, and EGF, is the mitogen-activated protein kinase (MAPK) pathway (11). This pathway involves a cascade of three serine-threonine protein kinases leading to MAPK. Activation of the MAPK cascade by receptor tyrosine kinases can occur by multiple mechanisms, all involving the formation of multiprotein signaling complexes. One adapter protein for which a role in MAPK activation is established is Shc. Shc is an SH2 domain containing protein that is a direct substrate for receptor tyrosine kinases. The majority of studies characterizing Shc have focused on EGF and platelet-derived growth factor signaling, but recent investigation has confirmed the direct interaction of Shc with the IGFR (12). After tyrosine phosphorylation of Shc by an activated receptor tyrosine kinase, Shc becomes an adapter protein that binds growth factor receptor-bound protein 2 (Grb2). Grb2 is associated with Sos, a guanine nucleotide exchange protein; Shc, Grb2, and Sos thereby create a signaling complex that activates the small G protein Ras (13). IRS-1 can also bind Grb2 and Sos and activate Ras, giving the IGFR a second potential mechanism of MAPK activation (11, 14). Ras, in turn, activates the first kinase in the three-kinase cascade leading to MAPK. The best described target of Ras is the protooncogene product Raf, a serine-threonine kinase that phosphorylates and activates MEK, a MAPK kinase (15). MEK phosphorylates and activates multiple MAPK isoforms, including ERK1 and ERK2 (16). These two MAPKs mediate the mitogenic effects of many growth factors through activation of transcriptional regulators in the nucleus (11).
Elegant studies by other investigators suggest that Ras (17) and Raf (18) are part of the signaling mechanism mediating 3T3-L1 preadipocyte differentiation. Expression of transfected ras or raf oncogenes led to differentiation of cells in the absence of added IGF-I or insulin; however, MAPK was not activated. We hypothesized that MAPK is involved in the mitogenic, but not the differentiating, effects of IGF-I in 3T3-L1 preadipocyte differentiation. In this paper, we analyze MAPK activity in proliferating, quiescent, and differentiating cells after treatment with IGF-I or EGF, two growth factors with mitogenic, but otherwise diverse, actions in 3T3-L1 cells. Proximal signaling through phosphorylation of Shc and IRS-1 is also investigated. Our data demonstrate dramatic changes in IGF-I-stimulated Shc phosphorylation and MAPK activity during 3T3-L1 differentiation. We interpret these data as suggesting that uncoupling of IGF-I mitogenic signaling via the MAPK pathway is part of the differentiation program.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture conditions
The murine preadipocyte line 3T3-L1 was obtained from the
American Type Culture Collection (Rockville, MD). Cells were grown in
DMEM with L-glutamine and 1 g/liter glucose and
supplemented with 50 µg/ml penicillin, 100 µg/ml streptomycin, 0.25
µg/ml amphotericin B, and 10% FBS. Cultures were maintained in an
atmosphere of 5% CO2-95% humidified air at 37 C.
Serum-containing medium (SCM) was replaced every 3 days. To study
proliferating cells, monolayers were used at 
80% confluence.
Quiescent or growth-arrested cells were obtained by growing monolayers
to confluence, then maintained in SCM for 3 days postconfluence. Cells
were stimulated to differentiate after the 3-day period of growth
arrest by adding a cocktail of 0.05 µM dexamethasone, 0.5
mM methylisobutylxanthine, and 7 nM IGF-I (DMI)
in SCM for 3 days (5). Early differentiating cells were studied after
this 72-h exposure to DMI. We and others have previously demonstrated
that expression of early markers of adipocyte differentiation,
e.g. glycerol-3-phosphate dehydrogenase, occurs at this
stage before significant lipid accumulation (5, 19, 20). Later
differentiating adipocytes were studied after 3 additional days in SCM,
at which time more than 50% had intracellular lipid. Cell monolayers
were placed in serum-free medium (SFM) with 0.1% BSA overnight before
treatment the next day.
Preparation of cell lysates for in vitro analyses
Soluble and membrane fractions for analysis of kinase activity
and signaling complexes were prepared as previously described (21). For
the preparation of soluble fractions containing kinases, cell
monolayers were washed twice in cold PBS and then lysed in 100
mM NaCl, 25 mM ß-glycerophosphate (pH 7.2),
10 mM NaF, 0.2 mM sodium orthovanadate, 10
mM MgCl2, 1 mM EGTA, 1
mM dithiothreitol, 0.2% Triton X-100, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, and 25 µg/ml
phenylmethylsulfonylfluoride. Cells were removed by scraping, and the
lysate was sonicated and centrifuged at 40,000 x g for 20
min. For preparation of cell extracts containing membrane-associated
signaling molecules, monolayers were washed in cold PBS, and cells were
lysed in 10 mM Tris (pH 7.6) with 1% Triton X-100, 5
mM EDTA, 50 mM NaCl, 30 mM sodium
pyrophosphate, 50 mM NaF, 0.1 mM sodium
orthovanadate, and protease inhibitors (as in the MAPK lysis buffer)
followed by centrifugation as described above. Cell lysates were stored
at -80 C until use.
Anion exchange chromatography
Cells incubated with or without hormone were lysed in kinase
lysis buffer followed by fractionation on a Mono-Q fast protein liquid
chromatography (FPLC) HR5/5 column (Pharmacia Biotech, Uppsala,
Sweden). One milligram of cell lysate protein was loaded and, after a
brief wash, eluted from the column with a linear salt gradient (up to 1
M NaCl) (21). Column fractions were collected and assayed
immediately for kinase activity. Aliquots of fractions containing MAPK
activity were frozen for Western blotting to determine the MAPK
content.
MAPK assay
MAPK activity was determined in crude cell lysates using MBP as
substrate. Two micrograms of cell extract protein or a 20-µl aliquot
from each column fraction were incubated for 60 min at 30 C with 20
µl 50 mM ß-glycerophosphate (pH 7.2), 0.1
mM sodium orthovanadate, 20 mM
MgCl2, 1 mM EGTA, 200 µM
[
-32P]ATP (0.25 µCi/nmol; DuPont-New England Nuclear
Research Products, Boston, MA), and 13.3 µg MBP. The assay was
stopped with 10 µl 250 mM EDTA, and 12.5 µl 5 x
Laemmli sample buffer were added to each sample and boiled for 10 min.
Proteins were resolved on 15% SDS-polyacrylamide gels, dried, and
exposed to Kodak X-Omat film at -70 C for 1 h. A Hoefer model
300S scanning densitometer (Hoefer, San Francisco, CA) connected to a
Hewlett-Packard model 3390A integrator (Hewlett-Packard, Palo Alto, CA)
was used to measure phosphate incorporation into MBP. Fractions from
Mono-Q chromatography were assayed similarly, except that aliquots of
the assay mixture were spotted on Whatman P81 filter paper (Whatman,
Clifton, NJ), washed extensively in 75 mM
H3PO4 buffer, dried, and counted in Opti-fluor
scintillation fluid (Packard, Downers Grove, IL) in a Packard Tri-Carb
counter.
Immunoprecipitation and Western blotting
Immunoprecipitation of signaling proteins was performed using
protein A-Sepharose CL-4B beads (Pharmacia Biotech, Uppsala, Sweden) to
which specific antibody had been covalently bound using
dimethylpemilimidate (22). Cell extracts (an aliquot containing 800
µg total protein) were incubated with immobilized antibodies on beads
overnight at 4 C and then washed extensively in Tris lysis buffer
containing protease and phosphatase inhibitors as described above.
Immunoprecipitates were eluted from the beads by boiling in SDS-Laemmli
sample buffer. The eluate was resolved by SDS-PAGE, transferred to
Hybond C nitrocellulose (Amersham Life Science, Arlington Heights, IL),
and probed with various antisera, including 0.1 µg/ml
antiphosphotyrosine and 2 µg/ml anti-IRS-1, anti-Shc, and anti-Grb2
antibodies. Western blotting of MAPK isoforms in Mono-Q column
fractions was performed by resolving MAPK species on 15% SDS-PAGE,
transferring to nitrocellulose, and probing with 1 µg/ml antirat MAPK
R2 antibodies that recognize mouse ERK1 and ERK2. Specific binding was
visualized using enhanced chemiluminescence (Amersham Life Science,
Arlington Heights, IL) and Hyperfilm ECL (Amersham, Aylesbury, UK)
followed by quantification using scanning densitometry as described
above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). However, MAPK activation was greatly
attenuated in growth-arrested and early differentiating cells. Time
courses of EGF and insulin activation of MAPK in proliferating cells
were similar, with a peak activation that occurred at 5 min (Fig. 1B).
Although both IGF-I and EGF stimulate a rapid peak and decline of MAPK
activity, the change in slope after the MAPK peak following EGF
stimulation suggests a second phase of activation with EGF and not
IGF-I. In addition, EGF was much more potent than IGF-I or insulin at
comparable molar concentrations. Dose-response curves comparing EGF,
IGF-I, and insulin after 5-min exposure were generated for
proliferating 3T3-L1 cells (Fig. 2). EGF
generated the greatest MAPK activity even at low concentrations and was
much more potent than IGF-I, with a maximal effective dose of 0.1
nM. IGF-I was more potent than insulin, with maximal
effective doses of 7 and 170 nM respectively, but large
doses of insulin approached the level of MAPK activation seen with
IGF-I.
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).
EGF-stimulated MAPK activity was maximal in proliferating cells and
declined modestly in differentiating cells. IGF-I activation of MAPK
declined to a much greater degree in differentiating cells. MAPK
activity in adipocytes (late differentiating cells) treated with 8
nM insulin was greater than that in IGF-I-treated cells,
but less than that EGF-treated cells.
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). MAPK isoforms ERK1 and ERK2 were
identified by Western blot analysis of column fractions containing MAPK
activity. For each stage, MAPK content was similar in column fractions
regardless of IGF-I or EGF stimulation; therefore, only the
IGF-I-stimulated fractions are shown. As preadipocytes progress from
proliferation through growth arrest to differentiation, MAPK activation
of both isoforms by EGF declined, but IGF-I activation declined
disproportionately (Fig. 4, left). Comparison of areas under
the curve for proliferating and differentiating cells treated with EGF
or IGF-I revealed a 40% decrease in EGF-stimulated MAPK activity, but
an 80% decrease in IGF-I-stimulated MAPK activity. Western blotting of
fractions using specific antiserum for ERK1 (p42) and ERK2 (p44)
revealed a significant decrease in ERK1 content compared with ERK2
content in differentiating cells (Fig. 4, right). Activation
of these two MAPK isoforms in lysates from differentiating cells did
not parallel their abundance: loss of IGF-I activation of ERK2 was
greater despite its abundance compared with ERK1 content. Clearly,
IGF-I activation of ERK1 decreases during growth arrest and
differentiation, but activation of ERK2 decreases dramatically in early
differentiating cells compared with the effects of EGF.
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, left).
Insulin-stimulated IRS-1 tyrosine phosphorylation in adipocytes (late
differentiating cells) was comparable to IGF-I-stimulated IRS-1
phosphorylation. IRS-1 protein content was determined by Western
blotting with anti-IRS-1 antibodies. Although IRS-1 protein content per
mg cell lysate decreased substantially with differentiation, there was
an increase in total protein content per cell that occurred with
differentiation, and equal amounts of cell lysate protein, not lysate
per equal number of cells were analyzed per condition. Densitometry of
phosphorylated IRS-1 and IRS-1 content in three independent experiments
revealed that IRS-1 phosphorylation by IGF-I was maintained as cells
differentiated, although a modest decrease in IRS-1 activation was seen
in late differentiating cells (Fig. 5, right). Western
blotting of IRS-1 immunoprecipitates failed to detect coprecipitated
Grb2 in samples obtained at all stages of cell differentiation (data
not shown).
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).
Western blotting of phosphotyrosine in Shc immunoprecipitates revealed
phosphorylation of the 52-kDa Shc isoform in IGF-I-treated
proliferating, but not differentiating, cells (Fig. 6, left). Repeated experiments could not detect any
phosphorylated Shc in IGF-I-treated differentiating cells, nor was Shc
phosphorylated in response to 8 nM insulin in adipocytes
(late differentiating cells). Phosphorylated Shc was variably detected
in growth-arrested cells treated with IGF-I (data not shown).
Sequential Western blotting revealed associated Grb2 in Shc
immunoprecipitates of IGF-I-treated proliferating cells, consistent
with Shc activation. In contrast, Shc phosphorylation of multiple
isoforms in response to EGF was present in proliferating,
growth-arrested, and differentiating cells. Although Shc
phosphorylation in response to EGF declined during differentiation,
several replicate experiments consistently demonstrated phosphorylation
of the 52-kDa Shc by EGF in differentiating cells. Shc content
determined by Western blotting using anti-Shc antibodies revealed that
the levels of all three Shc isoforms (66, 52, and 46 kDa) remained
relatively constant during the three phases of cell growth.
Densitometry comparing phosphorylated 52-kDa Shc to Shc content in
IGF-I- and EGF-treated 3T3-L1 cells from four independent experiments
confirmed that stimulation of Shc phosphorylation persisted through
differentiation only in response to EGF (Fig. 6, right).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The dramatic decrease in IGF-I-stimulated MAPK activity during early differentiation suggests that IGF-I signaling changes after growth arrest of 3T3-L1 cells. Unlike the differentiation-dependent expression of the insulin and hybrid receptors, IGFR expression is relatively unchanged (5), so loss of IGFR expression does not account for the decline in IGF-I-stimulated MAPK activity in differentiating 3T3-L1 cells. The MAPK protein content decreases during differentiation. This decrease in relative abundance may reflect an increase in total protein content per cell that occurs as differentiation proceeds. However, it does not explain the more significant decrease in ERK1 abundance, which may indicate differential regulation of the expression of ERK1 vs. ERK2. A change in mitogenic signaling via the MAPK pathway has also been suggested to occur in IGF-I-mediated myoblast differentiation (27) and high dose insulin-induced differentiation of rat preadipocytes (28), indicating that down-regulation of the MAPK pathway may be an important component of IGF-I signal transduction pathways mediating differentiation.
EGF is known to be a potent stimulator of MAPK in these cells (29). Our studies also demonstrate that EGF-stimulated MAPK activity is sustained in proliferating 3T3-L1 cells and that EGF-induced activity does not decrease dramatically in differentiating cells. EGF receptor number decreases as 3T3-L1 cells differentiate (30), so the down-regulation of EGF receptors might contribute to the modest decline in EGF-stimulated MAPK activity. EGF has been shown to inhibit preadipocyte differentiation in vitro (6, 7) and in vivo (8). It is likely that the mechanism of EGF inhibition involves activation of MAPK pathways that have been shown to inhibit crucial adipogenic transcription factors (31). Studies of MAPK activation in PC-12 cells suggest that the duration of MAPK activation by EGF or nerve growth factor determines either mitogenesis or differentiation (32). The sustained MAPK response by EGF in our studies of proliferating 3T3-L1 cells may contribute to its mitogenic potency and antiadipogenic effects. Finally, several studies have compared insulin to EGF activation of MAPK in adipocytes; similar to our results, EGF was more potent than insulin, and the two appeared to activate MAPK through distinct mechanisms (28, 33).
Proximal signaling through Shc correlates with MAPK activity in IGF-I- and EGF-treated 3T3-L1 cells at all stages of growth. This suggests that MAPK activation by both IGF-I and EGF is mediated through the Shc-Grb2-Sos signaling complex that activates Ras and the downstream MAPK cascade. Phosphorylation of the 52-kDa isoform of Shc by IGF-I is similar to insulin, as opposed to phosphorylation of multiple Shc isoforms by EGF (34). Signaling through Shc to activate MAPK has been well described for EGF (35, 36, 37). However, IGF-I, like insulin, has the alternate route via Grb2 binding to IRS-1 for activation of Ras and MAPK pathways (10, 14). We found no association of Grb2 with activated IRS-1 after stimulation with IGF-I, suggesting that the IRS-1-Grb2-Sos signaling pathway may not be a significant mediator of MAPK activation in preadipocytes.
Shc phosphorylation and MAPK activity dramatically decline during IGF-I-mediated differentiation. This switch in IGF-I signaling away from the MAPK pathway appears to occur at the IGFR, as proximal signaling through Shc is uncoupled despite abundant Shc protein. In contrast, EGF stimulation of Shc phosphorylation persists in differentiating cells. IGFR inactivation does not explain the lack of Shc phosphorylation in differentiating cells given that IGF-I treatment results in IRS-1 phosphorylation, an event that requires an activated IGF-I receptor tyrosine kinase (9, 10). Other investigators studying the signaling events proximal to MAPK activation in 3T3-L1 cells treated with 100 nM insulin found that activation of MAPK was Ras dependent in preadipocytes but Ras independent in adipocytes (38), also consistent with a switch in proximal signaling. In our studies, IRS-1 tyrosine phosphorylation is present in all phases of growth, suggesting that IGF-I activation of IRS-1-mediated signaling is important in both mitogenesis and differentiation.
Based on our data, we postulate that down-regulation of the mitogenic pathway involving Shc and leading to MAPK may be necessary for subsequent 3T3-L1 differentiation, although we cannot rule out with certainty that loss of Shc signaling occurs as a result of early differentiation. Porras et al. used a dominant negative Ras mutant to block Raf-1 phosphorylation and MAPK activation in 3T3-L1 preadipocytes (17); in addition, MAPK was not activated in differentiating 3T3-L1 cells despite transfected raf oncogenes inducing differentiation, suggesting that the mechanism of raf-induced differentiation is not via the MAPK pathway. Our data also support the conclusion that IGF-I-mediated 3T3-L1 differentiation is a MAPK-independent process, but that this process involves a switch in signaling at the level of the IGFR substrate, Shc. The loss of Shc, but not IRS-1, phosphorylation by IGF-I in early differentiating cells suggests a specific change in IGFR function. Mechanisms thought to regulate changes in receptor tyrosine kinase function include serine/threonine phosphorylation (39) and interaction of the receptor with other regulatory proteins, e.g. phosphoinositol-3-kinase (40) or Grb10 (41). The down-regulation of mitogenic signaling during IGF-I-mediated 3T3-L1 differentiation suggests that alterations in IGFR function may be part of the differentiation program.
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Acknowledgments |
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Footnotes |
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Received September 22, 1997.
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