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Department of Physiology (T.-W.L.G., J.L., G.S.C., X.W., C.C.-S., J.S.) and Program in Cellular and Molecular Biology (D.J.M., C.L.H., C.C.-S., J.S.), University of Michigan Medical School, Ann Arbor, Michigan 48109-0622; and Hagedorn Research Laboratory (N.B.), Gentofte, Denmark
Address all correspondence and requests for reprints to: Jessica Schwartz, Ph.D., Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622. E-mail: jeschwar{at}umich.edu
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P) abrogated all of these responses to GH,
indicating that box 1, a site of association of GHR with the tyrosine
kinase JAK2, is crucial for these GH-stimulated responses. As the
C-terminal half of the cytoplasmic domain of GHR is required for
GH-stimulated calcium flux and for stimulation of
spi-2.1 transcription, GHR lacking this sequence
(GHR1–454) were examined. Not only did
GHR1–454 mediate stimulation of c-fos and
egr-1 expression and 2-deoxyglucose uptake, but they
also mediated GH-stimulated transcriptional activation via Elk-1, a
transcription factor associated with the c-fos Serum
Response Element. Thus, the C-terminal half of the cytoplasmic domain
of GHR is not required for GH-stimulated c-fos
transcription, suggesting that increased calcium is not required for
GH-stimulated c-fos expression. In CHO cells lacking all
but five N-terminal residues of the cytoplasmic domain
(GHR1–294), GH did not induce c-fos or
egr-1 expression or stimulate 2-deoxyglucose uptake.
Further, in 3T3-F442A fibroblasts with endogenous GHR, GH-stimulated
c-fos expression and 2-deoxyglucose uptake were reduced
by the tyrosine kinase inhibitors herbimycin A, staurosporine, and P11.
Herbimycin A and staurosporine inhibit JAK2 and tyrosyl phosphorylation
of all proteins stimulated by GH, whereas P11 inhibits the GH-dependent
tyrosyl phosphorylation of only some proteins, including extracellular
signal regulated kinases ERK1 and -2, but not JAK2. Taken together,
these results implicate association of GHR with JAK2 and GH-stimulated
tyrosyl phosphorylation of an additional cellular protein in
GH-stimulated glucose transport and c-fos and
egr-1 expression. These studies also indicate that, in
contrast to spi-2.1, the N-terminal half of the
cytoplasmic domain of GHR is sufficient to mediate stimulation of
c-fos and egr-1 expression and Elk-1
activation, supporting multiple mechanisms for GH signaling to the
nucleus. |
Introduction |
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The present study examines the ability of mutated and truncated GHR to
mediate GH responses thought to be representative of its growth and
metabolic effects. GH-regulated early response genes such as
c-fos have been implicated in growth regulatory events.
Stimulation of c-fos occurs in response to a wide variety of
agents and can be mediated by various upstream regulatory sequences,
including those regulated via ERKs, Stat1 and -3, or calcium (23, 24),
making it useful for assessing GH signaling and potentially identifying
JAK2-dependent or -independent mechanisms. Analysis of GHR requirements
for induction of c-fos is also revealing by comparison with
the spi-2.1 gene, which is stimulated by GH through
-activated sequence-like elements (GLE) that bind Stat5; such
stimulation requires both N- and C-terminal regions of the cytoplasmic
domain of GHR (4). In c-fos, the Serum Response Element
(SRE) can mediate induction by GH (25), and SRE-associated
transcription factors Elk-1 and serum response factor are required for
such induction (26). The evaluation presented herein of the ability of
GHR to mediate expression of SRE-containing genes such as
c-fos and egr-1 (27, 28, 29, 30), and transcriptional
activation via Elk-1 can thus provide insight into whether GHR
requirements are similar for regulating different genes that use
different transcription factors. As a representative metabolic response
to GH, the regulation of glucose transport, a rate-limiting step in
cellular carbohydrate metabolism and an important determinant in the
ability of GH to regulate carbohydrate and lipid metabolism (31, 32),
was examined. Responses to GH were assessed in Chinese hamster ovary
(CHO) cells expressing wild-type or mutated GHR. Signaling mechanisms
elicited by the endogenous GHR were also probed using 3T3-F442A
fibroblasts treated with a panel of tyrosine kinase inhibitors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture and treatment of cells expressing GHR
GHR variants were assessed in CHO cells stably transfected with
mammalian expression vectors containing the complementary DNA (cDNA)
encoding the full-length rat liver GHR (GHR1–638)
sequence, truncated GHR sequences with stop codons at positions 455
(GHR1–454) or 295 (GHR1–294), as described
previously (15, 33). The same full-length GHR cDNA was mutated to
delete box 1 by deleting the codons for amino acids at positions
297–311 (GHRP). CHO cells expressing full-length,
truncated, or mutated GHR were maintained as described previously (10, 34). The relative levels of [125I]human GH binding among
the cell lines were comparable to those reported previously (8, 10, 15). Confluent CHO cells were deprived of serum overnight by incubating
cells in Ham’s F-12 medium containing 1% BSA. Cells were then treated
with or without 500 ng/ml (22 nM) GH for 30 min, unless
indicated otherwise, and used for analysis of GH activity.
To assess the function of endogenous GHR, 3T3-F442A preadipocyte fibroblasts were grown to confluence as described previously (35). In experiments using tyrosine kinase inhibitors, 3T3-F442A fibroblasts were deprived of serum overnight in DMEM containing 1% BSA. Cells were preincubated during the deprivation period with one of the inhibitors, herbimycin A (1.8 µM; for 18–22 h), P11 (100 µM; for the final 1 h), staurosporine (500 nM; for the final 1 h or 10 min), or vehicle (DMSO). The conditions used were those established previously to interfere with GH-dependent tyrosyl phosphorylation of cellular proteins (12), except that when glucose uptake was measured, cells were preincubated with staurosporine for 1 h instead of 10 min. Immunoblotting of whole cell lysates with antiphosphotyrosine antibody confirmed that the inhibitors reduced tyrosyl phosphorylation of JAK2 and other cellular proteins as described previously (12). GH (500 ng/ml; 22 nM) was added in the presence of inhibitors for 30 min, unless indicated otherwise, and cells were used for analysis of c-fos expression or measurement of 2-deoxyglucose uptake.
Preparation of total RNA and Northern blot analysis
After treatment, the 3T3-F442A or CHO cells were washed with
PBS, and RNA was prepared by the acid phenol/guanidine isothiocyanate
method (36). Total RNA was fractionated on a 1% agarose-formaldehyde
gel and transferred to nitrocellulose or Nytran (Schleicher and
Schuell, Keene, NH). The membranes were hybridized with mouse
c-fos (35) or mouse egr-1 (37) probes labeled
with [32P]deoxy-CTP by random priming (35). The
egr-1 cDNA was provided by Dr. L. Lau (University of
Illinois, Chicago, IL).
Elk-1-mediated transcriptional activation
For analysis of transcriptional activation via Elk-1, the
expression plasmid Gal4/ElkC (38), encoding the transcriptional
activation domain of Elk-1 fused to the Gal4 DNA-binding domain, and
the reporter plasmid 5X Gal/Luc (39), containing five consensus Gal4
DNA-binding sites upstream of the luciferase gene, were provided by Dr.
C. Der (University of North Carolina, Chapel Hill, NC). CHO cells
expressing the indicated GHR (2 x 105 cells/35 mm
well) were cotransfected with 5 µg/well Gal4/ElkC and Gal/Luc DNA,
using calcium phosphate (40). Forty-four to 48 h after
transfection, cells were deprived of serum and treated with or without
GH for 4 h. Cells were then lysed in reporter lysis buffer (100
mM potassium phosphate, 0.2% Triton X-100, and 1
mM dithiothreitol), and luciferase activity was measured
using an Autolumat Luminometer (Wallac-Berthold, Gaithersberg, MD).
Glucose transport assay
Glucose transport was assayed by measurement of
2-deoxy-D-glucose uptake. Where specified, cells were
incubated with the indicated inhibitor or corresponding vehicle in 1%
BSA-Ham’s F-12 medium (CHO cells) or with 1% BSA in DMEM (3T3-F442A
fibroblasts), followed by 30-min incubation with vehicle (control) or
with GH (500 ng/ml) in Krebs-Ringer phosphate buffer (KRP) containing
1% BSA. The glucose transport assay was initiated by incubating cells
at 37 C with fresh KRP-BSA plus 100 µM unlabeled
2-deoxyglucose and 3.4 µM
[14C]2-deoxyglucose. In 3T3-F442A fibroblasts, the assay
was terminated after 5 min by aspiration of the 2-deoxyglucose
solution. In CHO cells, the time of incubation with radioactive tracer
was extended to 20 min because the amount of Glut1 glucose transporter
present in these cells is very low (41). Cells were washed twice with
ice-cold KRP containing phloretin (200 µM) to block
further uptake of 2-deoxyglucose and were scraped in 0.1% SDS.
Aliquots of the cell lysates were used to assess radioactivity and
protein content (42). Glucose transport was calculated as picomoles of
2-deoxyglucose transported per mg protein/min. Each condition was
tested in triplicate in each experiment, and values for the increments
due to GH are presented as mean ± SE for replicate
experiments. Data were analyzed by Student’s t test. As
established previously for 3T3-F442A cells (32, 43), GH-stimulated
2-deoxyglucose uptake was inhibited by cytochalasin B and was linear
for the duration of the assay in the CHO cells (not shown). In
addition, 2-deoxyglucose uptake was stimulated independently by insulin
(1 µg/ml) and phorbol dibutyrate (500 ng/ml), an activator of protein
kinase C (PKC),8 providing
evidence for a functional glucose transport mechanism in CHO cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, lane 4, top; and Fig. 2B, lane 2), whereas the parent CHO cells not expressing GHR failed to
respond to GH (Fig. 2A, lane 2). The expression of egr-1,
another early response gene, was also stimulated via the full length
GHR (Fig. 2A, lane 4, middle; Fig 2C, lane 2). The fact that
CHO cells expressing GHR exhibit these responses to GH indicates that
the intracellular components essential in GH signaling to stimulate
early response gene expression are present in CHO cells. A GH-induced
increase in egr-1 was also mediated by endogenous GHR in
3T3-F442A cells (Fig. 3), which showed a
time course and dose dependence similar to that for c-fos
(35).
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P) interfered with its ability to mediate
c-fos or egr-1 induction in response to GH
compared with the full-length receptor (Fig. 2A
, lane 6). The lack of
response with GHRP supports the necessity for the
association of JAK2 with GHR for stimulation of c-fos and
egr-1 expression by GH. GHR in which all but five residues
of the cytoplasmic domain were deleted (GHR1–294) failed
to mediate induction of c-fos or egr-1 expression
in response to GH (Fig. 2, B and C, lane 6), as it failed to elicit
other early signaling events, including activation of JAK2 (16).
The C-terminal half of the cytoplasmic domain of GHR is not
required for GH-stimulated early response gene expression
To examine whether the C-terminal region of GHR is required to
mediate stimulation of c-fos and egr-1
expression, as it is for spi-2.1 expression and for
increased intracellular calcium, cells expressing GHR in which the
C-terminal half of the cytoplasmic domain was deleted
(GHR1–454) were studied. Truncation of the C-terminal half
of the cytoplasmic domain of GHR did not interfere with induction of
c-fos or egr-1 expression by GH (Fig. 2, B and C,
lane 4). Stimulation of c-fos and egr-1 was
consistently observed with both GHR1–638 and
GHR1–454, although the relative intensity of the
stimulation via each GHR was variable. Thus, the N-terminal half of the
cytoplasmic domain of GHR appears to be sufficient for GH signaling to
regulate c-fos and egr-1 expression,
distinguishing induction of c-fos and egr-1 from
that of spi-2.1, and suggesting that increased intracellular
calcium is not required for their expression.
GH-stimulated transcriptional activation mediated by Elk-1 does not
require the C-terminal half of the cytoplasmic domain of GHR
In c-fos, the SRE can mediate induction by GH (25), and
SRE-associated transcription factors Elk-1 and Serum Response Factor
are required for such induction (26). For insight into how GHR might
mediate transcriptional activation via the c-fos SRE, the
ability of GHR lacking the C-terminal half of the cytoplasmic domain to
mediate activation of transcription via Elk-1 was examined.
Elk-1-mediated transcriptional activation was doubled by GH in CHO
cells expressing either full-length GHR or GHR1–454 (Fig. 4), indicating that the C-terminal half
of the cytoplasmic domain of GHR is not required for transcriptional
activation via Elk-1 in response to GH. Basal transcription via Elk-1
was slightly lower with GHR1–454 for reasons that are not
clear. GHR1–294 failed to mediate transcriptional
activation via Elk-1 (Fig. 4) in response to GH. Thus, the N-terminal
half of the cytoplasmic domain of GHR is sufficient to mediate
GH-promoted c-fos expression and activation of Elk-1,
consistent with a critical role for Elk-1 in GH-stimulated
c-fos expression.
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). GH
stimulated 2-deoxyglucose uptake via the full-length GHR
(GHR1–638) in CHO cells. The relatively modest stimulation
of glucose uptake by GH most likely reflects the low abundance of Glut1
and the absence of Glut4 glucose transporters in CHO cells (40, 41).
The increment in glucose uptake in response to GH was statistically
significant and reproducible and was clearly in contrast to the lack of
response to GH treatment in untransfected parental CHO cells.
Importantly, the stimulation of glucose transport by GH was not evident
in cells expressing GHRP, which lacks box 1, indicating
the importance of the association of GHR and JAK2 for GH-stimulated
glucose uptake. CHO cells expressing GHR1–454 showed an
increase in 2-deoxyglucose uptake in response to GH that was comparable
to that with GHR1–638, indicating that the N-terminal half
of the cytoplasmic domain of the GHR is sufficient to mediate the
stimulation of glucose transport by GH. GHR1–294 failed to
mediate the stimulation of 2-deoxyglucose uptake by GH. These results
indicate that the ability of GH to stimulate glucose transport, like
induction of c-fos or egr-1 expression,
corresponds to the retention in GHR of the ability to associate with
JAK2. They also indicate that the N-terminal half of the cytoplasmic
domain of GHR is sufficient to mediate GH-stimulated glucose
transport.
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, lane 2) was absent when herbimycin A,
P11, or staurosporine was present (Fig. 6, lanes 4, 6, and 8,
respectively), indicating that all three tyrosine kinase inhibitors
interfered with GH-stimulated c-fos expression.
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). Preincubation with herbimycin
A, staurosporine, or P11 diminished the stimulation by GH to similar
extents (79%, 84%, and 62%, respectively; Fig. 7). In the adipocyte
form of 3T3-F442A cells, degrees of inhibition comparable to those
observed in the fibroblast form were obtained when GH-stimulated
glucose transport was measured in the presence of the inhibitors (data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P lacking box 1, the site of association with JAK2,
fails to mediate SHC, MAPK, IRS, or Stat activation (15, 16, 17, 48, 49).
However, JAK2 is not implicated in all GHR-mediated events. GH-induced
calcium oscillations are reported to occur in cells expressing
GHRP and GHRP>A lacking a functional box 1
(4), suggesting a JAK2-independent GH signaling pathway for this
response.
The results of this study are consistent with JAK2-dependent events
mediating induction of c-fos and egr-1 and
stimulation of glucose uptake in response to GH. This role is supported
by the observation that GHRP failed to mediate these
responses, indicating that the site of association of JAK2 and GHR is
required for the ability of GH to elicit these responses. Furthermore,
the tyrosine kinase inhibitors staurosporine and herbimycin, which are
known to inhibit JAK2, blocked all three responses. It is recognized
that although the simplest interpretation of these findings is that
JAK2 is involved, the alternative explanation, that conformational
changes in GHR secondary to deletion of box 1 are involved in these
responses cannot be ruled out. Furthermore, involvement of tyrosine
kinases in addition to JAK2 is suggested below.
Signaling pathways mediating GH stimulated c-fos expression
Stimulated c-fos can be mediated by various upstream
regulatory sequences, including those regulated via ERKs, Stat1 and -3,
or calcium (23, 24). Several signaling pathways converge on the SRE
(23), which is known to mediate stimulation of c-fos by GH
(25, 26, 50, 51). Several lines of evidence in the present studies
suggest a role for regulation of the SRE by MAPK in GH-stimulated
c-fos expression. First, GHR that activate MAPK
(GHR1–638 and GHR1–454) activate
c-fos expression, whereas GHR that do not activate MAPK
(GHR1–294, GHRP) do not activate
c-fos. Second, the tyrosine kinase inhibitor P11 inhibits
GH-stimulated c-fos expression under conditions identical to
those in which it inhibits tyrosine phosphorylation of ERK1 and -2, but
not JAK2 (12, data not shown). Thus, the inhibition of GH-stimulated
c-fos expression by P11 may reflect the contribution of a
P11-sensitive kinase downstream of JAK2 but upstream of MAPK. Third, in
cells expressing GHR1–638 and GHR1–454, GH
stimulated Elk-1-mediated transcriptional activation, whereas cells
expressing GHR1–294 failed to show such stimulation.
Phosphorylation of Elk-1 and subsequent Elk-1-mediated transcription of
c-fos via the SRE are well characterized events dependent on
ERKs (52, 53, 54, 55). ERK activation of GH-stimulated Elk-1 transcription is
consistent with recent observations that GH stimulates the
phosphorylation and activation of Elk-1, facilitating SRE-mediated
transcriptional activation of c-fos in response to GH
(26).
Fourth, the GHR that mediate GH-stimulated c-fos expression also mediate stimulation of egr-1. The present studies document for the first time that GH stimulates egr-1 expression, both via endogenous GHR in 3T3-F442A cells and via expressed GHR in CHO cells. A preliminary report is consistent with GH-stimulated egr-1 (56). Regulation of egr-1 by growth factors involves core SRE sequences and Ternary Complex Factor (29, 30, 57), similar to c-fos, and would be predicted to be regulated similarly to c-fos. In fact, expression of egr-1 and c-fos in response to insulin has been reported to involve MAPK activation (58). Whether the mechanism for GH-promoted egr-1 expression is the same as that for c-fos remains to be determined. Inhibition of c-fos expression by staurosporine also raises the possibility that PKC could contribute to regulation of c-fos, consistent with previous observations (35, 59, 60, 61). Possible interactions among these signaling pathways in GH-stimulated c-fos or egr-1 expression remain to be determined.
GHR uses multiple mechanisms to mediate transcriptional regulation
in response to GH
As discussed, Elk-1-mediated transcription in response to GH was
observed in cells expressing only the N-terminal half of the
cytoplasmic domain of GHR (GHR1–454). Consistent with
this, stimulation of SRE-mediated luciferase expression by GH (26) was
evident in CHO cells expressing GHR1–638 or
GHR1–454, but not GHR1–294 (data not shown).
These observations emphasize the difference between SRE-mediated
trans-activation of c-fos and Stat5-mediated
trans-activation of the spi-2.1 gene.
GHR1–454 does not mediate activation of spi-2.1
by GH (3, 4, 7, 45). The promoter of spi-2.1 contains GLEs
that bind tyrosyl-phosphorylated Stat5 in response to GH (2).
Stat5-dependent transcriptional activation via the GLE requires both
box 1 and the C-terminal half of the cytoplasmic domain of GHR (4).
Activation of Stat5 and spi-2.1 transcription thus appear to
require JAK2 as well as a second GH-dependent signaling event involving
C-terminal sequences in GHR, possibly the one mediating GH-stimulated
calcium oscillations (4).
The c-fos promoter contains sequences other than the SRE, including the Sis-inducible element (SIE), an Activating Protein-1 site, and a Calcium/cAMP Response Element, all of which are required for expression of c-fos in vivo (62). The SIE binds Stat1 and -3 (20, 21, 22, 63). Presumably mediated by a JAK2-dependent pathway, Stat1 and -3 are tyrosyl phosphorylated and bind to the SIE in response to GH. The SIE can mediate GH-stimulated reporter expression in cells overexpressing Stat3 (9). The activation and binding of Stats 1 and -3 are reported to occur in cells expressing only the N-terminal half of the cytoplasmic domain (GHR1–454) (8, 9). Thus, c-fos expression in cells expressing GHR1–454 may reflect contributions of the SIE as well as the SRE. The relative contributions of SIE and SRE in GH-regulated c-fos transcription are currently under study. The present findings argue against a role for the Calcium/cAMP Response Element in GH-stimulated c-fos expression based on the requirement for the C-terminal half of the cytoplasmic domain for GH-stimulated calcium oscillations (4), but not for GH-stimulated c-fos expression. This further distinguishes regulation of c-fos and spi-2.1 by GH, supporting the idea that the mechanisms by which GHR mediate c-fos and spi-2.1 expression differ and suggesting two distinct signaling mechanisms for GHR-mediated induction of c-fos and spi-2.1.
Multiple signaling pathways may mediate GH-stimulated glucose
uptake
The ability of GH to stimulate glucose uptake was found to require
box 1 of GHR, did not require the C-terminal half of the cytoplasmic
domain of GHR, and was blocked by tyrosine kinase inhibitors that
inhibit JAK2 as well as an inhibitor that does not. The requirement for
box 1 is consistent with a role for association of GHR and JAK2 in
GH-stimulated glucose uptake. One JAK2-dependent pathway by which GH
might regulate glucose transport that is consistent with these results
involves IRS-1 and -2. The current view of insulin-stimulated glucose
transport involves insulin receptor-mediated tyrosyl phosphorylation of
IRS-1 and -2, leading to association of IRS-1 or -2 with
phosphatidylinositol-3' kinase (PI-3K) and activation of PI-3K, which
is required for recruitment of Glut4 glucose transporters to the plasma
membrane (64). GH, like insulin, stimulates the tyrosyl phosphorylation
of IRS-1 and IRS-2, presumably via JAK2 (17, 65). Like stimulation of
glucose transport, the ability of GHR to transduce the signal for IRS-1
and IRS-2 tyrosyl phosphorylation in response to GH requires box 1, but
does not require the C-terminal half of the cytoplasmic domain of GHR
(17). In response to GH, phosphorylated tyrosines in IRS-1 and IRS-2
bind and activate PI-3K (17, 65). The finding that wortmannin, an
inhibitor of PI-3K, inhibits GH-stimulated lipogenesis (66) supports a
role, either direct or indirect, for PI-3K in the regulation of glucose
transport by GH. Further, GH, like insulin, rapidly stimulates glucose
transport in adipocytes by recruiting Glut1 and Glut4 glucose
transporters to the plasma membrane (67, 68).8 Taken together, the
available data support a JAK2-dependent mechanism for GH-stimulated
glucose uptake potentially mediated via IRS-1 or -2 and PI-3K.
A MAPK-mediated pathway may also contribute to the events by which GHR mediate stimulation of glucose uptake. P11 blocks GH-induced tyrosyl phosphorylation of ERK1 and -2 but not JAK2 (12, data not shown). The partial inhibition of glucose transport by P11 may reflect the contribution of one or more P11-sensitive kinases downstream of JAK2 but upstream of MAPKs, as discussed for c-fos expression. A possible role for MAPK in regulation of glucose transport is supported by observations that expression of Raf-1, a component of MAPK-mediated pathways, increases glucose uptake in 3T3-L1 cells (69). Activation of MAPK in GH-treated cells may be modulated by PI-3K, as MAPK activation is blocked by the PI-3K inhibitor wortmannin (70)2, which also interferes with translocation of MAPK to the nucleus (71). The present data are thus consistent with involvement of JAK2 and another downstream tyrosine kinase, such as ERK1 or -2, or proteins regulating IRS-1, IRS-2, or PI-3K in the stimulation of glucose uptake by GH.
The inhibition of GH-stimulated glucose uptake by staurosporine observed in 3T3-F442A fibroblasts in the present study is in agreement with the inhibition by staurosporine reported for GH-stimulated lipogenesis in rat adipocytes (66). The concentration of staurosporine (500 nM) used in the present experiments blocks GH-promoted tyrosyl phosphorylation of virtually all GH-responsive cellular proteins (12, 66, data not shown). Herbimycin A, which inhibits tyrosine kinases by a mechanism different from that of staurosporine, inhibited glucose uptake as effectively as staurosporine in the present study. The failure of herbimycin A to interfere with GH responses, including lipogenesis and JAK2 phosphorylation, reported in rat adipocytes (66) most likely reflects the short exposure time (10 min) to the inhibitor relative to the 18- to 22-h exposure used in the present studies. As staurosporine is also an inhibitor of PKC, which has been implicated in GH-induced lipid metabolism in rat adipocytes (72, 73, 74), a role for PKC in GH-stimulated glucose uptake can also be considered. Nevertheless, although each inhibitor cannot be stated to be absolutely specific for individual tyrosine kinases, the present observation that GH-stimulated glucose uptake was inhibited by all three of the diverse tyrosine kinase inhibitors used supports the general conclusion that tyrosine kinases participate in GH-stimulated glucose uptake. Such kinases appear to include a kinase(s) downstream of JAK2 in addition to JAK2.
In summary, responses to GH implicated in growth promotion (early response gene expression) and metabolism (glucose uptake) require association of JAK2 and GHR, distinguishing them from GH-induced calcium oscillations, which are thought to be independent of JAK2. Further, the mechanism for SRE-mediated induction of c-fos transduced by the N-terminal half of the cytoplasmic domain of GHR is distinct from that for Stat5-mediated transcription of spi-2.1, indicating that GH uses multiple mechanisms to signal to the nucleus.
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Acknowledgments |
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Footnotes |
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2 Recipient of Postdoctoral Fellowship DK-08572 from the NIH.
3 Supported by Predoctoral Traineeship in Cellular and Molecular
Biology GM-07315 from the NIH, a Rackham Predoctoral Fellowship from
the University of Michigan, and a Student Research Fellowship from The
Endocrine Society.
4 Supported by Postdoctoral Fellowship DK-09293 from the NIH.
5 Supported by a Minority Graduate Fellowship from the NSF and a
Rackham Merit Fellowship from the University of Michigan.
6 Recipient of Postdoctoral Fellowship GM-14099 from the NIH.
7 Recipient of a predoctoral fellowship from the American Diabetes
Association, MI Affiliate, and a Dissertation Award from Rackham School
of Graduate Studies, University of Michigan.
8 Gong, T. W., and J. Schwartz, unpublished
observations.
Received August 15, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and leukemia inhibitory factor-promoted tyrosyl
phosphorylation of insulin receptor substrate-1. J Biol Chem 270:14685–14692, and leukemia
inhibitory factor utilize insulin receptor substrate-2 in intracellular
signaling. J Biol Chem 271:29415–29421This article has been cited by other articles:
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G. Piwien-Pilipuk, O. MacDougald, and J. Schwartz Dual Regulation of Phosphorylation and Dephosphorylation of C/EBPbeta Modulate Its Transcriptional Activation and DNA Binding in Response to Growth Hormone J. Biol. Chem., November 8, 2002; 277(46): 44557 - 44565. [Abstract] [Full Text] [PDF]
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C. J. Greenhalgh, P. Bertolino, S. L. Asa, D. Metcalf, J. E. Corbin, T. E. Adams, H. W. Davey, N. A. Nicola, D. J. Hilton, and W. S. Alexander Growth Enhancement in Suppressor of Cytokine Signaling 2 (SOCS-2)-Deficient Mice Is Dependent on Signal Transducer and Activator of Transcription 5b (STAT5b) Mol. Endocrinol., June 1, 2002; 16(6): 1394 - 1406. [Abstract] [Full Text] [PDF]
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 V. Beauloye, B. Willems, V. de Coninck, S. J. Frank, M. Edery, and J.-P. Thissen Impairment of Liver GH Receptor Signaling by Fasting Endocrinology, March 1, 2002; 143(3): 792 - 800. [Abstract] [Full Text] [PDF]
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 K. Miura, S. S. Saini, G. Gauvreau, and D. W. MacGlashan Jr Differences in Functional Consequences and Signal Transduction Induced by IL-3, IL-5, and Nerve Growth Factor in Human Basophils J. Immunol., August 15, 2001; 167(4): 2282 - 2291. [Abstract] [Full Text] [PDF]
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J. Liao, G. Piwien-Pilipuk, S. E. Ross, C. L. Hodge, L. Sealy, O. A. MacDougald, and J. Schwartz CCAAT/Enhancer-binding Protein beta (C/EBPbeta ) and C/EBPdelta Contribute to Growth Hormone-regulated Transcription of c-fos J. Biol. Chem., October 29, 1999; 274(44): 31597 - 31604. [Abstract] [Full Text] [PDF]
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C. Benbassat, L. N. N. Shoba, M. Newman, M. L. Adamo, S. J. Frank, and W. L. Lowe Jr. Growth Hormone-Mediated Regulation of Insulin-Like Growth Factor I Promoter Activity in C6 Glioma Cells Endocrinology, July 1, 1999; 140(7): 3073 - 3081. [Abstract] [Full Text]
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H. Yamashita, J. Xu, R. A. Erwin, A. C. Larner, and H. Rui A Lymphoma Growth Inhibitor Blocks Some but Not All Prolactin-stimulated Signaling Pathways J. Biol. Chem., May 21, 1999; 274(21): 14699 - 14705. [Abstract] [Full Text] [PDF]
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R. W. E. Clarkson, C. A. Shang, L. K. Levitt, T. Howard, and M. J. Waters Ternary Complex Factors Elk-1 and Sap-1a Mediate Growth Hormone-Induced Transcription of Egr-1 (Early Growth Response Factor-1) in 3T3-F442A Preadipocytes Mol. Endocrinol., April 1, 1999; 13(4): 619 - 631. [Abstract] [Full Text]
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C. Hodge, J. Liao, M. Stofega, K. Guan, C. Carter-Su, and J. Schwartz Growth Hormone Stimulates Phosphorylation and Activation of Elk-1 and Expression of c-fos, egr-1, and junB through Activation of Extracellular Signal-regulated Kinases 1 and 2 J. Biol. Chem., November 20, 1998; 273(47): 31327 - 31336. [Abstract] [Full Text] [PDF]
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