From the Department of Pediatrics, Division of Pediatric Endocrinology and Metabolism, Rhode Island Hospital and Brown University, Providence, Rhode Island 02903
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ABSTRACT |
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Stimulation of cell proliferation by mitogens
involves tyrosine phosphorylation of proteins at the cell membrane by
receptor tyrosine kinases. This promotes formation of multi-protein
complexes that can activate the small G-protein, Ras. Activation of
Ras, in turn, leads to sequential activation of the following three serine-threonine kinases: Raf, extracellular signal-regulated kinase
kinase (MEK), and members of the family of mitogen-activated protein
(MAP) kinases. Prior studies have shown that intraperitoneal injection
of epidermal growth factor (EGF) leads to rapid activation of hepatic
MAP kinases in adult rats but not in late gestation (E19) fetal rats
(Boylan, J. M., and Gruppuso, P. A. (1996) Cell Growth
& Differ. 7, 1261-1269). The present studies were undertaken to
determine the mechanism for this "uncoupling" of the MAP kinase pathway. E19 fetal rats and adult male rats were injected with EGF (0.5 µg/g body weight, intraperitoneally) or with saline. After 15 min,
livers were removed and prepared for kinase analyses. EGF injection led
to a rapid and marked activation of hepatic Raf and MEK in both fetal
and adult rats, whereas MAP kinase activation was minimal in fetal as
opposed to adult rats. Examination of the ontogeny of this dissociation
of MAP kinase activation from MEK activation showed gradual acquisition
of intact signaling as an adult hepatocyte phenotype was attained
during the first 4 postnatal weeks. Over this period, MAP kinase
content as determined by Western immunoblotting was constant.
Recombination experiments using partially purified fetal and adult rat
liver MEK and MAP kinase showed intact MAP kinase activation in
vitro, indicating that neither enzyme was irreversibly altered in
the fetus. In studies using primary cultures of E19 fetal rat
hepatocytes, uncoupling of MAP kinase activation from MEK activation
could be induced by incubation of fetal hepatocytes for 24 h with
a potent fetal hepatocyte mitogen, transforming growth factor-.
These findings indicate that a novel negative feedback mechanism for
MAP kinase regulation may be active in developing rat hepatocytes.
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INTRODUCTION |
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A major signaling pathway through which virtually all known mitogens exert intracellular responses is the mitogen-activated protein (MAP)1 kinase cascade (1, 2). A primary mechanism by which growth factor receptor tyrosine kinases activate MAP kinases involves tyrosine phosphorylation of an adaptor protein, Shc. Shc binds to a second adaptor protein, Grb2, by SH2/3 interactions. This complex incorporates a guanyl nucleotide exchange factor, Sos, which interacts with and activates Ras. The GTP-bound Ras recruits Raf kinase to the cell membrane and initiates a protein kinase cascade that phosphorylates and activates a MAP kinase kinase termed MEK (MAP kinase or extracellular signal-regulated kinase kinase). MEK in turn phosphorylates MAP kinases on both tyrosyl and threonyl sites. Active MAP kinases translocate to the nucleus where they phosphorylate other serine-threonine kinases and transcription factors which can mediate immediate early gene induction.
The growth and development of the fetus during late gestation is an
intricate process dependent upon multiple regulatory mechanisms. Changes in hepatic growth tend to parallel effects on fetal somatic growth (3, 4), making liver an appropriate organ for studying fetal
growth control. When we embarked on the present studies we expected
that the proliferation of fetal rat hepatocytes, both in
vivo and in vitro, would require the action of fetal
growth factors. However, late gestation (19 day; E19) rat fetal
hepatocytes cultured without serum, growth factors, or insulin were
found to be highly proliferative (5, 6). By using a variety of approaches, we were able to find no direct evidence for the production of autocrine growth factors. Thus, we went on to employ MAP kinase activity as an indirect indicator of mitogenic signaling. Studies showed that the MAP kinases present in cultured fetal hepatocytes and
in fetal liver were minimally active, although MAP kinase could be
activated in cultures of primary fetal hepatocytes by at least two
mitogens, transforming growth factor- (TGF-
) and hepatocyte
growth factor (7).
Given the abundance of these growth factors in the late gestation fetal rat, we hypothesized that the relatively low activity of hepatic MAP kinases in vivo might be the result of negative feedback inhibition. In our initial studies (8), we demonstrated that intraperitoneal injection of epidermal growth factor (EGF)2 into E19 fetuses or adult rats resulted in the activation of the proximal portion of the EGF signaling pathway in the liver. This was based on the EGF-induced tyrosine phosphorylation of Shc and Shc-Grb2 complex formation. When the downstream activation of MAP kinases was examined, EGF was shown to result in the marked (~50-fold) activation of MAP kinase in the adult liver. However, minimal MAP kinase activation occurred in the fetus (8).
The present studies were performed following our initial observation of uncoupling of the MAP kinase signaling cascade in E19 fetal liver. We have determined when in development uncoupling of the hepatic MAP kinase cascade occurs as well as the site in this cascade where the actual uncoupling of MAP kinase activation takes place. We proceeded to characterize the constituents of the MAP kinase pathway to better understand this physiologic mechanism for down-regulation of mitogenic signaling.
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EXPERIMENTAL PROCEDURES |
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Animals-- Pregnant Sprague-Dawley rats (Charles River Breeding Laboratory, Wilmington, MA), of known gestation (term specified as 21 days), were delivered by cesarean section under pentobarbital anesthesia (50 mg/kg, intraperitoneally). Male Sprague-Dawley rats (150-175 g) were used for adult liver preparation. Rats were injected intraperitoneally with EGF (0.5 µg/g body weight) or phosphate-buffered saline and sacrificed 15 min later.
Hepatocyte Isolation and Primary Culture-- Fetal hepatocytes were isolated on day 19 of gestation by collagenase digestion as described previously (6). Cell suspensions were diluted to 3 × 106 cells per 100-mm Primaria tissue culture plate with supplemented minimum essential medium (6) containing 5% fetal bovine serum. After a 2-h attachment period, the medium was removed and replaced with supplemented minimum essential medium without fetal bovine serum. Media contained 0.1 mg/ml bovine serum albumin to avoid nonspecific binding of growth factors.
Immune Complex Kinase Assay--
Rat liver homogenates were
prepared as described previously for MAP kinase activity (7).
Homogenates corrected for protein (4 mg) were precleared with Protein
A-Sepharose CL-4B (Pharmacia Biotech Inc.). MAP kinase
immunoprecipitation was accomplished with a peptide antibody against
the rat ERK-1 sequence (anti-MAP kinase R2, Upstate Biotechnology, Lake
Placid, NY) that had been covalently coupled to Protein A-Sepharose
CL-4B using dimethyl pimelimidate (20 mM in 0.1 M sodium borate, pH 9.0). The immunoprecipitates were
washed 4 × in 10 mM Tris, pH 7.6, 5 mM
EDTA, 50 mM NaCl, 50 mM NaF, 100 µM Na3VO4, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 25 µg/ml
phenylmethylsulfonyl fluoride, and then 1 × with 25 mM -glycerophosphate, 50 µM
Na3VO4, 10 mM MgCl2,
0.5 mM EGTA, 10 mM NaF, 0.2% Triton X-100,
plus the above protease inhibitors. The immunoprecipitated kinase was
resuspended in 25 µl of this buffer. MAP kinase was assayed by adding
an equal volume of 2 × reaction mixture containing 50 mM
-glycerophosphate, pH 7.2, 100 µM
Na3VO4, 20 mM MgCl2, 1 mM EGTA, 200 µM [
-32P]ATP
(0.05 µCi/nmol), and 0.667 mg/ml MBP and incubating for 20 min at
30 °C. The assays were terminated by adding 10 µl of 250 mM EDTA. MBP was separated on a 12% polyacrylamide gel
under reducing conditions. The dried gel was exposed to Kodak XAR-5 film at
70 °C in the presence of intensifying screens, and
phosphate incorporation into MBP was quantified with a Hoefer model
300S scanning densitometer connected to a Hewlett-Packard model 3390A integrator.
Mono Q Fractionation and Kinase Assays-- Rat liver homogenates and hepatocyte lysates were prepared for MAP kinase activity determinations as described previously (7). Where indicated, samples were fractionated using Mono Q fast protein liquid chromatography. MAP kinase activity was determined using myelin basic protein (MBP) as substrate (7).
Adult and fetal rat liver samples were prepared for determination of Raf or MEK activity by homogenizing liver in Raf/MEK homogenization buffer (10 mM Tris, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 0.1 mM Na3VO4, 1% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 25 µg/ml phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 100,000 × g for 30 min and frozen atIn Vitro Activation of Rat Liver MAP Kinase--
Livers from
fetal and adult rats injected with EGF were homogenized in 50 mM Hepes, pH 7.2, 50 mM NaCl, 1 mM
Na3VO4, 20 mM NaF, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, and 25 µg/ml phenylmethylsulfonyl fluoride. After centrifugation at 100,000 × g for 1 h,
250 µl was loaded onto a Superose 6 gel filtration column
(Pharmacia), run at 0.5 ml/min with the above buffer. Fifty 0.5-ml
fractions were collected to separate active MEK. The reaction was
started by mixing 10 µl of each fraction with 5 µl of either
control fetal rat liver MAP kinase partially purified by Mono Q
chromatography (7), recombinant human ERK-1 (0.4 µg), or MAP kinase
Mono Q buffer A as control (7). After incubation for 3 min at 30 °C, 5 µl of 20 mM magnesium acetate plus 800 µM
ATP was added. This mixture was incubated for 20 min at 30 °C, after
which was added 80 µl of 50 mM Tris, pH 7.4, 0.125 mM EGTA, 0.125 mM
Na3VO4, 12.5 mM magnesium acetate,
250 µM [-32P]ATP (0.05 µCi/nmol), and
0.4 mg/ml MBP. After an additional 10 min incubation, the reaction was
stopped with 10 µl of 250 mM EDTA. Proteins were
separated on a 10% polyacrylamide gel and analyzed as above.
Partial Purification of Activated MEK--
Liver from an adult
rat injected with EGF was fractionated on a Mono Q column. For these
experiments, Triton X-100 was not added to Mono Q buffers. Fractions
containing MEK activity were pooled and then precipitated by adding a
volume of 3.6 M ammonium sulfate (2 M final
concentration). The pellet was resuspended in 25 mM Tris,
pH 7.4, 1 mM EDTA, 5% glycerol (v/v), 0.02% Brij 35 (w/v), 25 mM NaF, 0.1% -mercaptoethanol, and 1 mM benzamidine. The solution was dialyzed overnight using
the same buffer plus 50% glycerol.
Western Immunoblotting-- For detection of immunoreactive kinases, samples were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting with the MAP kinase R2 antibody or antibodies to either MEK-1 or MEK-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Western immunoblots were also performed using an antibody to the active dually phosphorylated (Thr/Tyr) MAP kinase (Promega, Madison, WI), as well as an antibody to active dually phosphorylated (Ser-217/221) MEK-1/2 (New England Biolabs, Beverly, MA). In all cases, detection employed chemiluminescence (ECL, Amersham Corp.).
To examine the subcellular localization of immunoreactive MAP kinases, nuclei were separated from cytosol using sucrose density centrifugation (10). MAP kinase immunoprecipitation was carried out on rat liver cytosol and nuclear extracts. The immunoprecipitates were analyzed by Western immunoblotting as described above. ![]() |
RESULTS |
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MAP Kinase Activation in Developing Rat Liver-- To confirm our previous observations that MAP kinase activation in fetal liver is uncoupled (8), we immunoprecipitated MAP kinase in fetal liver homogenates from E19 fetal and adult rats given an intraperitoneal injection of phosphate-buffered saline (control) or EGF. MAP kinase activity was then measured directly on immunoprecipitated MAP kinase. Results showed that control fetal and adult rats showed minimal MAP kinase activity (Fig. 1). Upon EGF administration, there was a slight increase in fetal MAP kinase activity, whereas adult MAP kinase was seen to increase approximately 15-fold (Fig. 1). Western immunoblotting of parallel immunoprecipitates (Fig. 1, bottom) showed similar MAP kinase content in fetal and adult samples, indicating that the diminished ability of EGF to stimulate MAP kinase activity in the fetal rat was not due to a decrease in MAP kinase content.
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In Vivo Activation of Raf and MEK-- We hypothesized that the attenuation of growth factor-mediated phosphorylation and activation of MAP kinase were due to the failure to activate the kinases upstream of this enzyme. The activity of Raf (MEK kinase), the proximal kinase in the signaling pathway, was examined to determine indirectly if Ras activation was intact. Liver homogenates were fractionated by Mono Q ion exchange chromatography. Individual fractions were assayed for MEK kinase activity using recombinant, kinase-deficient MEK-1 as substrate. Fetuses injected with EGF showed marked activation of MEK kinase activity, present in multiple peaks (Fig. 5). EGF-inducible MEK kinase activity was also present in the adult, albeit at a lower level than in the fetus and as a single chromatographic peak (Fig. 5). Although we have not yet characterized the multiple MEK kinase activity peaks present in liver from EGF-injected fetuses, the response to EGF was taken as evidence for intact signaling at this step.
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In Vitro Activation of Fetal MAP Kinase-- The results to this point were interpreted as indicating that the ability of EGF to only minimally activate hepatic MAP kinase in vivo resided at the level of MAP kinase phosphorylation by MEK. The first hypothetical mechanism that was investigated was that MAP kinase and/or MEK were altered in fetal liver such that they did not interact normally. This possibility had not been addressed in the experiments utilizing recombinant kinases as substrates.
Active fetal or adult liver MEKs obtained from EGF-injected animals and fractionated by high pressure liquid chromatography gel filtration chromatography were examined for their ability to activate rat hepatic MAP kinases. Inactive fetal rat liver MAP kinase from an uninjected animal, partially purified by Mono Q chromatography, and recombinant human ERK-1 were used as substrates. Results (Fig. 6) showed that both MAP kinases were potently activated by both fetal and adult rat liver MEK that had been activated in vivo by EGF injection. This was interpreted as indicating that there was no significant alteration in fetal MAP kinases that could account for their resistance to in vivo activation by MEK. Western immunoblotting of the gel filtration fractions that contained MEK activity (fractions 30-37) using antibodies to either MEK-1 or MEK-2 revealed that both proteins were present in approximately equal amounts in the fetus and adult. Immunoblotting with an antibody against the active (Ser-217/221 phosphorylated) MEK-1 and -2 proteins demonstrated immunoreactive proteins with Mr ~47,000 in fractions coinciding with MAP kinase kinase activity (Fig. 7). The presence of these immunoreactive proteins was interpreted as supporting activation of MEK-1 and/or MEK-2, although we were not able to discern which of the MEKs was activated due to the similarity of their molecular weights. Nonetheless, these data were interpreted as supporting the conclusion that MEK activation was intact in fetal liver, whereas MAP kinase activation was markedly attenuated.
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Uncoupling of MAP Kinase Activation in Fetal Hepatocytes--
We
had shown previously that 24 h exposure of fetal hepatocytes in
primary culture to TGF- or hepatocyte growth factor resulted in
desensitization of MAP kinase to activation by either growth factor
(11). We hypothesized that this desensitization might be analogous to
the uncoupling seen in vivo. To examine this, E19 fetal
hepatocytes were cultured in the absence or presence of TGF-
for
24 h and then stimulated with additional TGF-
for 10 min. Shc
immunoprecipitation showed an increase in tyrosine phosphorylation as
well as Shc-Grb2 complex formation upon acute TGF-
stimulation. This
was not affected by 24 h preincubation with TGF-
(Fig.
9). This was interpreted as indicating
that tonic growth factor exposure for 24 h had not resulted in
sufficient down-regulation of EGF receptor number or tyrosine kinase
activity to cause an attenuation of proximal signaling. Given that this result was consistent with the in vivo findings, we went on
to examine downstream signaling.
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Investigation of a Potential Role for MAP Kinase Phosphatases in
MAP Kinase/MEK Uncoupling--
Recently cloned dual specificity
phosphatases like MKP-1 (MAP kinase phosphatase 1, encoded by the
murine gene 3CH134 (12)) or CL100, the human homologue of
MKP-1 (13), exhibit dual catalytic activity toward phosphotyrosine and
phosphothreonine, thus providing a mechanism for the inactivation of
MAP kinase. Based on the above studies, we treated cultured (E19) fetal
hepatocytes incubated with or without TGF- for 24 h with either
the tyrosine phosphatase inhibitor, sodium orthovanadate, or the
serine-threonine phosphatase inhibitor, okadaic acid. Phosphatase
inhibitors were added for 30 min prior to restimulation with TGF-
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In both cases, suppression of MAP kinase activation remained intact,
that is MEK activation was unaffected by growth factor preincubation
while MAP kinase activation was attenuated in a manner
indistinguishable from that shown in Fig. 10.
Investigation into the Presence of an Inhibitory MAP Kinase Binding Protein-- An alternative hypothesis to explain the dissociation of MAP kinase and MEK activation in fetal liver could involve a protein which binds to MAP kinase, thereby preventing its activation in vivo. To obtain an indicator of the relative size of fetal and adult liver MAP kinases, fetal and adult rat liver homogenates, prepared using conditions aimed at minimizing disruption of protein-protein interactions, were analyzed using a Superose 6 gel filtration column (Pharmacia). Results (not shown) were consistent with the elution of MAP kinases from both fetal and adult rat liver as soluble monomers.
Gel filtration chromatographic analysis was performed on lysates derived from fetal hepatocytes preincubated for 24 h with or without TGF- ![]() |
DISCUSSION |
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Our prior observation that intraperitoneal injection of late gestation fetal rats in situ with EGF showed only minimal activation of hepatic MAP kinases (8) was initially interpreted as indicating a probable block at the level of Ras activation. This was based on observations from several laboratories indicating that growth factor activation of the MAP kinase pathway can lead to phosphorylation of the Ras guanyl nucleotide exchange protein SOS, thereby down-regulating the MAP kinase pathway (14-19). Such a mechanism might pertain to negative feedback inhibition of hepatic MAP kinase activation in the growth factor-rich fetal environment. The ability of EGF to activate hepatic MAP kinases in vivo was recovered during the early phase of postnatal development, a period when hepatocyte proliferation is slowing and fetal hepatocytes are making the transformation to an adult hepatocyte phenotype. However, the observation that Raf and MEK activation were intact in the absence of MAP kinase activation indicated an alternative regulatory mechanism.
The conclusion that in situ EGF injection of a late gestation fetal rat could produce MEK activation with minimal MAP kinase activation is consistent with the entirety of the experiments we performed. In addition to activity measurements, Western immunoblotting confirmed the presence of MAP kinase but the absence of its phosphorylation in fetal liver. Furthermore, the lack of activation was accompanied by the lack of nuclear translocation. Gonzalez et al. (20) have reported that the majority of the MAP kinase in quiescent cells is in the cytoplasm. Upon activation of the cells with serum, cytoplasmic MAP kinase translocates into the nucleus. Similar translocation could be detected in subcellular fractions from adult, but not fetal, liver. The evidence for intact MEK activation included not only activity measurements but also Western immunoblotting for the active forms of MEK-1 and -2.
The physiologic in vivo uncoupling of MAP kinase activation at the level of MAP kinase phosphorylation by MEK has not been reported previously. However, several reports have suggested similar uncoupling of MAP kinase activation in vitro. By using a mouse myoblast cell line, Campbell et al. (21) showed that following withdrawal from serum and bFGF for 3 h bFGF stimulated MAP kinase kinase activity but that MAP kinase and S6 peptide kinase activities were not detected. When serum and bFGF were withdrawn for 10 h, the activities of MAP kinase kinase, MAP kinase, and S6 peptide kinase were coordinately stimulated by bFGF. Similar results were seen using EGF. Furthermore, it was demonstrated that incubation with the tyrosine phosphatase inhibitor, sodium orthovanadate, restored MAP kinase activation. Microcystin, a serine-threonine phosphatase inhibitor, had little effect.
Samuels et al. (22), using retroviruses encoding a fusion protein consisting of an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER), have studied MAP kinase regulation in rat1a cells conditionally transformed by hrafER. In these cells, estradiol caused the activation of MAP kinase kinase but not activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. Estradiol-dependent activation of MAP kinase and phosphorylation of p74raf-1 was observed when cells were pretreated with the serine-threonine phosphatase inhibitor okadaic acid. Sodium vanadate alone had little effect. It has also been reported that in v-raf-infected macrophages, MEK activation occurs normally after growth factor treatment, whereas MAP kinase shows no activation correlating with lack of tyrosine phosphorylation of this enzyme (23). Suppression of MAP kinase activation was reversed in these cells by treating with sodium orthovanadate.
We did not observe an effect of phosphatase inhibitors on either the in vivo or in vitro uncoupling of MAP kinase activation from MEK activation. Although these findings do not rule out a fetal hepatic MAP kinase phosphatase as the factor required for uncoupling of MAP kinase activation from MEK activation, they also do not support this hypothesis. A responsible dual function phosphatase would have to be insensitive to vanadate and okadaic acid. In addition, there appears to be a MAP kinase phosphatase expressed in fetal hepatocytes maintained under non-growth factor-stimulated conditions. Thus, induction of a phosphatase would have to modify the effects of one already present. Nonetheless, given that the transcription of the dual specificity phosphatases has been shown to be stimulated by serum and growth factors (12, 24, 25), this remains a viable hypothesis.
Our hypothesis that MAP kinase activation by MEK could be blocked by a MAP kinase binding protein is not without precedent. It has recently been reported that the protein p21WAF1/cip1/Sdi1, a DNA damage-inducible cell cycle inhibitor, acts as an inhibitor of the stress-activated protein kinases, also called the c-Jun amino-terminal kinases (JNKs). Dickens et al. (26) have described an additional mechanism for attenuating effects mediated by the JNK pathway. These investigators have cloned a cytoplasmic inhibitor of the JNK pathway, JIP-1, that binds specifically to JNK. JIP-1 causes cytoplasmic retention of JNK and inhibition of JNK-related gene expression. Given that the JNK enzymes represent a subfamily of the MAP kinases (27), we considered an analogous mechanism. However, we were unable to obtain evidence for fetal hepatic MAP kinases being present in a form other than soluble monomers. In addition, our data indicated a lack of MAP kinase phosphorylation, an effect not attributed to JIP-1.
In summary, our results demonstrate that hepatic, EGF-induced MAP kinase activation is suppressed in late gestation through the early postnatal period in the developing rat. Western immunoblotting indicated that the absence of MAP kinase activity correlates with the absence of MAP kinase phosphorylation. However, activation of MEK, the kinase which phosphorylates and activates MAP kinase, remains intact upon acute stimulation with EGF. Furthermore, the uncoupling of MAP kinase from the signaling cascade can be induced in vitro by the incubation of fetal hepatocytes for 24 h with growth factors. These findings may define a mechanism for attenuating MAP kinase activation under conditions in which cells are subject to tonic growth factor stimulation.
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ACKNOWLEDGEMENTS |
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The histidine-tagged kinase-inactive human MEK-1 construct was kindly provided by G. L. Johnson, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO. The bacteria expressing histidine-tagged human ERK-1 were kindly provided by M. H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX. We thank Theresa C. Bienieki for assistance with the animal experiments and for preparation of fetal hepatocyte primary cultures.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HD24455 and HD11343.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.
To whom correspondence should be addressed: Dept. of Pediatrics,
Rhode Island Hospital, 593 Eddy St., Providence, RI 02903. Tel.:
401-444-5504; Fax: 401-444-8845; E-mail:
Philip_Gruppuso{at}brown.edu.
1
The abbreviations used are: MAP kinase,
mitogen-activated protein kinase; MEK, MAP kinase or extracellular
signal-regulated kinase kinase; EGF, epidermal growth factor; TGF-,
transforming growth factor-
; MBP, myelin basic protein; E, embryonal
day; P, postnatal day; PAGE, polyacrylamide gel electrophoresis; JNK, c-Jun amino-terminal kinases; bFGF, basic fibroblast growth
factor.
2
EGF was used for in vivo experiments
rather than TGF- because of its availability in larger quantities.
Comparative signal transduction experiments using primary cultures of
fetal rat hepatocytes have shown that there are no differences in the
ability of these two growth factors to stimulate phosphorylation of Shc
or activation of MAP kinase (28).
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REFERENCES |
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