From the Centre for Animal Biotechnology, Four members (SOCS-1, SOCS-2, SOCS-3, and CIS) of
a family of cytokine-inducible, negative regulators of cytokine
receptor signaling have recently been identified. To address whether
any of these genes are induced in response to growth hormone (GH), serum-starved 3T3-F442A fibroblasts were incubated with GH for various
time points, and the expression of the SOCS gene family was analyzed by
Northern blotting. GH stimulated the rapid, transient induction of
SOCS-3 mRNA, peaking 30 min after the initiation of GH
exposure and declining to basal levels by 2 h. Expression of the
other SOCS genes (SOCS-1, SOCS-2,
CIS) was also up-regulated by GH, although to a lesser
extent than SOCS-3 and with differing kinetics.
SOCS-3 expression was also strongly induced in 3T3-F442A cells treated with leukemia-inhibitory factor (LIF), with weaker induction of SOCS-1 and CIS being observed. The
preferential induction of SOCS-3 mRNA was also observed
in hepatic RNA isolated from the livers of mice that had received a
single supraphysiological dose of GH intraperitoneally. Co-transfection
studies revealed that constitutive expression of SOCS-1 and SOCS-3, but
not SOCS-2 or CIS, blocked GH-induced transactivation of the
GH-responsive serine protease inhibitor 2.1 gene promoter.
The elucidation of the pivotal role played by the tyrosine kinase
Jak2 in initiating signal transduction from the
GH1 receptor has led to the
identification of a number of intracellular pathways that mediate the
cellular response to GH (1). However, the mechanism(s) by which
signaling from GH receptor-activated Jak2 is attenuated is unclear.
Ligand-induced tyrosine phosphorylation/activation of Jak2 by the
erythropoietin (EPO) receptor, a member of the cytokine receptor
superfamily that includes the GH receptor, is followed by the binding
of the protein-tyrosine phosphatase SHP-1 to the cytoplasmic domain of
the receptor (2). The recruitment of SHP-1 is accompanied by the
dephosphorylation/inactivation of Jak2 and subsequent termination of
EPO-induced cellular proliferation.
A similar role for SHP-1 in mediating the down-regulation of Jak2
following stimulation of cells with GH has been proposed (3), although
whether SHP-1 can directly associate with the GH receptor remains to be
established. Recently, a novel family of cytokine-inducible genes has
been identified that appear to function as negative regulators of the
JAK signaling pathway (4-7). Constitutive expression of one member,
SOCS-1 (also referred to as SSI-1 and JAB) in the murine myeloid
leukemia M1 cell line blocked growth factor-induced differentiation and
apoptosis and inhibited interleukin-6 (IL-6)-mediated tyrosine
phosphorylation of the cell-surface receptor component, gp130, and the
transcription factor, Stat3 (5, 6). SOCS-1 can interact with all four members of the JAK family of tyrosine kinases (6, 7), suppressing kinase activity and the subsequent tyrosine phosphorylation/activation of STAT factors (7). As a diverse array of cytokines appears to be able
to induce expression of one or more members of this gene family (5), we
sought to establish which, if any, of these genes might be a target for
transcriptional activation by GH in vivo and in the
classically GH-responsive cell line, 3T3-F442A fibroblasts (8).
Materials--
Recombinant human GH was the generous gift of Dr.
Ken Ho (Garvan Institute for Medical Research, Sydney, NSW); human
insulin was obtained from Novo Nordisk Pharmaceuticals Pty Ltd. (North Rocks, NSW); leukemia-inhibitory factor (LIF) was obtained from AMRAD
Pharmacia Biotech (Melbourne, Victoria). Radionucleotide [ Cell Culture--
3T3-F442A fibroblasts were maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum, 2 mM glutamine, 50 IU/ml penicillin, and
50 mg/ml streptomycin in a humidified atmosphere of 5% CO2
at 37 °C. For hormone induction experiments, cells were grown to
confluence and serum-starved overnight in DMEM containing 1% (w/v)
bovine serum albumin. Human GH (500 ng/ml), LIF (104
units/ml), or insulin (56 ng/ml) were added, and cell culture was
maintained for different time intervals.
Animals and GH Administration--
Female C57BL/6 mice (7 weeks
old) were injected intraperitoneally with GH (10 µg/10 g of body
weight) and were subsequently sacrificed at different time points.
Livers were removed, snap frozen in liquid nitrogen, and stored at
RNA Extraction and Northern Blotting--
Cell cultures were
rinsed twice with ice-cold phosphate-buffered saline; total RNA was
extracted from cell cultures and liver samples using guanidinium
thiocyanate-acid phenol (9). Total RNA was fractionated by
electrophoresis through agarose-formaldehyde gels and transferred and
subsequently fixed onto Hybond-N+ membranes as described
(10). Membranes were hybridized at 63 °C overnight with random
primed, radiolabeled probes derived from full-length cDNA inserts
encoding SOCS-1, SOCS-2, SOCS-3, or CIS (5). Membranes were then washed
at high stringency (0.1 × SSC, 0.1% SDS at 60 °C) and exposed
to Kodak BioMax MS film with an intensifying screen at Cell Transfection and Reporter Gene Assays--
Chinese hamster
ovary (CHO) cells were maintained in Ham's F-12 medium supplemented
with 10% fetal calf serum, 100 IU/ml penicillin, and 100 mg/ml
streptomycin. The cells were transfected as described previously (11).
Briefly, serum-starved cells were transfected using the calcium
phosphate procedure with 1.5 µg of rat GH receptor-encoding plasmid,
3 µg of GH Preferentially Induces SOCS-3 mRNA Expression in 3T3-F442A
Fibroblasts--
In the absence of GH, Northern blotting of total RNA
isolated from serum-starved 3T3-F442A cells detected low levels of
SOCS-3 and SOCS-1 mRNA (Fig.
1). Transcripts corresponding to
SOCS-2 and CIS were only observed upon prolonged
exposure. Upon the addition of GH, induction of SOCS-3
expression was apparent at the first time point examined (15 min),
peaking at 30 min before declining rapidly over subsequent time points
(Fig. 1). This pattern of induction has been confirmed independently
using both total and poly(A)+ mRNA (data not shown).
The induction of SOCS-1, by comparison, was much less
pronounced although with similar kinetics (Fig. 1). Both CIS
and SOCS-2 mRNA were up-regulated in response to GH
treatment (Fig. 1); with respect to CIS, induction peaked 60 min after hormonal exposure, and although the abundance of transcript had declined at time points assayed thereafter, expression remained significantly above basal levels. Similarly, while the induction of
SOCS-2 mRNA exhibited delayed kinetics compared with
other SOCS genes, elevated levels of the SOCS-2 message
persisted to the final time point assayed (4 h).
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]dCTP (3000 Ci/mmol) was from DuPont NEN (AMRAD
Pharmacia Biotech), Hybond-N+ membrane from Amersham
Australia Pty Ltd (Melbourne), and GIGA prime DNA Labeling Kits from
Bresatec Ltd. (Adelaide, SA). Cell culture media and reagents were from
Life Technologies Pty Ltd. (Melbourne).
75 °C.
70 °C.
Where blots were reprobed, membranes were first stripped in boiling
0.1% SDS prior to prehybridization.
-galactosidase internal control reporter plasmid, 1.5 µg
of the GH-responsive, serine protease inhibitor (Spi) 2.1 promoter-chloramphenicol acetyltransferase (CAT) construct, and 1 µg
of Flag epitope-tagged SOCS-1, SOCS-2, SOCS-3, or CIS expression vectors. After overnight culture in the absence or presence of GH (25 nM), cell extracts were prepared, and CAT and
-galactosidase activities were measured. Equivalent expression of
the various SOCS and CIS poteins was confirmed by Western blotting
portions of extracts from transfected cells with the Flag-specific
monoclonal antibody, M2.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Northern blot analysis of SOCS gene family
expression in 3T3-F442A cells following GH stimulation. Total RNA
(15 µg/lane) isolated from serum-starved 3T3-F442A cells stimulated
with human GH (500 ng/ml) for the indicated time points was
sequentially hybridized with cDNA probes for the different SOCS
genes. The integrity of RNA samples and efficiency of transfer onto
nylon membranes was monitored by ethidium bromide staining. The
autoradiograph exposure times are 24 h.
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SOCS-3 Is Induced by GH in Vivo-- Strikingly, analysis of hepatic RNA isolated from the livers of mice injected with a single dose of GH confirmed the preferential activation of SOCS-3 mRNA expression by GH (Fig. 3). A marked elevation of SOCS-3 expression was observed around 60 min following GH injection, declining thereafter. Of the other SOCS family genes, clear induction of CIS expression was found 30 min after hormone injection, peaking at 60 min, and remaining elevated even after 24 h (Fig. 3). In contrast, SOCS-1 and SOCS-2 were only weakly induced by GH.
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SOCS-1 and -3 Block Transactivation of a GH-responsive Promoter Element-- To explore the functional consequences of GH-induced expression of the different SOCS family genes, we determined the ability of GH to transactivate the GH-responsive Spi 2.1 promoter, linked to a CAT reporter gene, in CHO cells co-transfected with the various SOCS-encoding plasmids. In cells that were not transfected with SOCS expression constructs, GH induced a 3.4-fold increase in CAT activity from the Spi 2.1 promoter (Fig. 4). While constitutive expression of CIS had no effect on the hormonal responsiveness of the Spi 2.1 promoter, expression of either SOCS-1 or SOCS-3 ablated the ability of GH to transactivate reporter gene activity from the same promoter. Paradoxically, co-expression of SOCS-2 resulted in the superinduction of CAT activity in response to GH, giving a 7-9-fold increase above basal levels of activity.
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DISCUSSION |
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The identification of SOCS-3 as a major
transcriptional target of GH action raises a number of questions, in
particular how might SOCS-3 act to regulate signaling by the GH
receptor. SOCS-1 can bind directly to Jak2 and Tyk2 (6, 7) and is
capable of inhibiting the activity of all four members of the JAK
kinase family (6, 7). As Jak2 is the principal effector of GH action at
a cellular level, it would be anticipated that enforced expression of
SOCS-1 in transfected CHO cells would block GH-mediated transactivation of the Spi 2.1 promoted CAT reporter construct, as observed in the
present study (Fig. 4). To what extent SOCS-1 actually participates to
down-regulate signaling from the ligand-activated GH receptor-Jak2 complex in vivo is not known, particularly given the very
low level of SOCS-1 mRNA expressed in hepatic tissue
following a GH pulse. In a contrasting mechanism of action, CIS,
presumably via its SH2 domain, binds directly to the
tyrosine-phosphorylated cytoplasmic domains of the EPO receptor and
-chain of the interleukin-3 receptor (4). While CIS has little
effect on the activity of Jak2 (6), the principal effector molecule
activated by EPO, it appears to act by preventing the docking of Stat5
(and other potential substrates?) onto the tyrosine-phosphorylated
cytoplasmic domain of the EPO receptor (13). The absence of a
discernible effect of constitutively expressed CIS on the ability of GH
to transactivate the Spi 2.1 promoter is an interesting result. Three tyrosines in the cytoplasmic carboxyl terminus of the GH receptor (at
positions 534, 566, and 627) can independently mediate GH-induced transcription from the Spi 2.1 promoter (11). Deletion/mutation of all
three blocks activation of this promoter by GH. Recently, we have
established that, in their phosphorylated state, each of these
tyrosines can directly interact with Stat5 (14), thus representing a
critical first step leading toward the transactivation of the Spi 2.1 promoter by GH. The inability of CIS overexpression to block
transactivation would suggest that CIS does not bind to any of the
three phosphotyrosines in the cytoplasmic tail of the GH receptor that
serve as Stat5 binding sites, implying there may be specificity in
cytokine receptor-CIS interaction. What mechanism is employed by SOCS-3
in down-regulating signaling from the ligand-activated GH receptor, or
any other cytokine receptor, is not known at this point in time.
Similarly, how co-expression of SOCS-2 facilitates the superinduction
of reporter gene activity in response to GH has not been elucidated. It
is conceivable that, in transfected cells, overexpression of SOCS-2
might somehow result in a dominant-negative phenotype by interfering
with the capacity of GH to activate endogenous regulators
(i.e. SOCS-3) of hormone-receptor signal transduction.
GH activates Jak2 in 3T3-F442A cells, while LIF activates both Jak1 and -2; both hormones induce the tyrosyl phosphorylation of Stats1, -3, and -5 in these cells (12). Clearly, one or more of these factors may be involved in the transcriptional induction of SOCS-3 and other gene family members following hormonal exposure. This is supported by the observation that expression of a dominant-negative Stat5 blocks IL-3 induction of CIS in Ba/F3 cells (13) while a dominant-negative Stat3 blocks IL-6/LIF induction of SOCS-1 mRNA in M1 cells (7). Interestingly, only Stat5 is activated in the livers of normal rats in response to a GH pulse (15). If STAT transcription factors assume a dominant role in the transcriptional activation of the SOCS gene family, then this last observation would imply that Stat5 is a critical element in the activation of SOCS-3 and CIS transcription by GH in vivo.
The preferential induction of SOCS-3 mRNA by GH, both in vivo and in vitro, contrasts with results obtained using IL-6 (5). All four SOCS gene transcripts are elevated in mouse liver following the injection of IL-6, although the kinetics of induction/persistence of individual mRNAs differs. However, only the induction of SOCS-1 and CIS mRNA is found in M1 cells treated with IL-6 (5). Whether the association between GH and SOCS-3 occurs in other tissues, or other GH-responsive cell lines, remains to be established.
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ACKNOWLEDGEMENTS |
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We thank Lisa Robertson for preparing this manuscript and Ken Snibson for photography.
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FOOTNOTES |
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* This work was supported by the Anti-Cancer Council of Victoria, Melbourne, Australia, AMRAD Operations Pty Ltd, Melbourne, Australia, The National Health and Medical Research Council, Canberra, Australia, The J. D. and L. Harris Trust, National Institutes of Health Grant CA-22556, and the Australian Federal Government Cooperative Research Centers Programme.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.
The first two authors contributed equally to this work.
§ To whom correspondence should be addressed: CSIRO Division of Molecular Science, 343 Royal Parade, Parkville Victoria 3052, Australia. Tel.: 61-3-9662-7317; Fax: 61-3-9662-7101; E-mail: Tim.Adams{at}molsci.csiro.au.
Supported by the Danish Research Academy.
1 The abbreviations used are: GH, growth hormone; LIF, leukemia-inhibitory factor; EPO, erythropoietin; IL, interleukin; STAT, signal transducer and activator of transcription; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary; Spi, serine protease inhibitor; CAT, chloramphenicol acetyltransferase.
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REFERENCES |
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