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Research Center for Endocrinology and Metabolism (S.S., K.W., J.-O.J.), Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden; and Department of Clinical Chemistry and Pathobiochemistry (A.M.G.), Rheinisch-Westfalische Technische Hochschule-University Hospital, D-52057 Aachen, Germany
Address all correspondence and requests for reprints to: Stanko Skrtic, Research Center for Endocrinology and Metabolism, Endocrine Division, Gröna Stråket 8, Sahlgrenska, University Hospital, SE-413 45 Göteborg, Sweden. E-mail: stanko{at}medic.gu.se
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Abstract |
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Both LY 294002 [a phosphatidylinositol 3-kinase (PI3-K) inhibitor], and rapamycin [a blocker of activation of the serine/threonine p70 S6 kinase (p70S6K), a molecule downstream from PI3-K] completely reversed the IGF-1-induced stimulation of DNA synthesis. Mitogen-activated protein kinase (MAPK) inhibition by PD 98059 had a less pronounced suppressory effect, although the used PD 98059 dose was fully effective in inhibiting MAPK phosphorylation. Both LY 294002 and PD 98059 lowered the IGF-1-induced increase of HGF in the medium by about 40%, but LY 294002 was 10 times more potent than PD 98059. Inhibition of p70S6K activation by rapamycin blocked IGF-1-induced DNA synthesis but not the increase in HGF.
In conclusion, PI3-K (and, to some extent, MAPK) signaling pathways seem to be important for IGF-1-stimulated DNA synthesis and HGF production. DNA synthesis also seems to be dependent on rapamycin-sensitive activation of the PI3-K effector p70S6K.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HGF is a pleiotropic factor with mitogenic, motogenic, and morphogenic effects on cells of epithelial origin, such as hepatocytes (13, 14). HSC seem to be the main producers of HGF in the intact liver in vivo (15), and freshly-seeded HSC also produce HGF in vitro (16, 17). The HGF messenger RNA (mRNA) levels of HSC in vitro are then decreased during transformation (16, 17). HSC, synonymously termed Ito cells, lipocytes, and fat-storing cells, are nonparenchymal cells located adjacent to the hepatocytes in the perisinusoidal space of Disse (18, 19, 20). Their main physiologic role in the intact liver is the storage and metabolism of retinoids (18). When the liver is injured, the HSC are activated to proliferate and transform to myofibroblast-like cells, producing different matrix proteins (20). This transformation also occurs when HSC are cultured on plastic (19).
IGF-1 signaling pathways in different cell types have many features in common, although it has been shown that the relative importance of the different pathways can vary between cell types. The IGF-1 receptor is a heterotetrameric protein with ligand-stimulated tyrosine kinase activity (21). Because the IGF-1 receptor has large homology with the insulin receptor, it is not surprising that insulin in high doses can bind to the IGF-1 receptor and mimic the effects of IGF-1 (21). The activated IGF-1 receptor phosphorylates insulin receptor substrate-1. Phosphorylated insulin receptor substrate-1 serves as a multisite docking protein by binding to numerous SH2 domain-containing proteins, such as phosphatidylinositol 3-kinase (PI3-K) and Grb-2 (21). Grb-2 can also associate with IGF-1 receptor-phosphorylated Shc, which leads to the activation of the ras-raf-MAPK pathway (21). Activated PI3-K has multiple downstream effector molecules. One of them is the serine/threonine p70 S6 kinase (p70S6K), which, in turn, phosphorylates the ribosomal S6 protein (22). Another effector is protein kinase B (PKB) (23).
IGFs are unusual among growth factors, in that they affect both the proliferation and differentiation of cells. It is well known that different effects of IGF-1 may employ different intracellular pathways. Special attention has been paid to the MAPK and PI-3K pathways, where MAPK has been implicated in the mediation of the mitogenic action of IGF-1 and PI-3K on the more differentiated effects of IGF-1 in myoblasts (24). However, in the human breast cancer cell line MRC-7, PI-3K is more crucial than MAPK for IGF-1’s mitogenic signaling (25).
One preliminary study of transformed myofibroblast-like cells indicates that IGF-1 and insulin stimulate mitogenesis via a PI-3K-dependent pathway (26). Few studies have been performed on IGF signaling in untransformed HSC. The aim of this study was to identify the intracellular pathways that mediate the effects of IGF-1 on DNA synthesis and HGF production in primary, untransformed HSC cultures.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cells were seeded, on day 0, with a density of 20 x 103 cells/cm2 in 24-well cell culture plates and were maintained as monolayers with DMEM containing L-glutamine (4 mM), penicillin (100 IU/ml), streptomycin (100 µg/ml), 10% FCS (all from Roche Molecular Biochemicals, Mannheim, Germany), and insulin (0.02 U/ml, from bovine pancreas; Sigma, St. Louis, MO) in a humidified atmosphere of 5% CO2-95% air at 37 C. On day 1, the medium was changed about 16 h after seeding. The purity was then higher than 97%. About 10 h later, medium was changed to a serum-deprived one, with 0.2% FCS, for 24 h. On day 2, the cells were exposed to recombinant human IGF-1 in a medium with 0.2% FCS or a control medium with 0.2% FCS alone. The cultures were kept in these media for 48 h before collecting the media and harvesting the cells. Twenty-four hours before harvest, 1 µCi/mL 3H-thymidine was added to all groups. The experiments were repeated, and representative data are shown.
Inhibition of signal transduction pathways
The cells were exposed to different inhibitors for 48 h of
culture after 24 h of serum starvation. Inhibitors PD 98059 1–10
µM final [0.1–1 mM stock in 10%
dimethylsulfoxide (DMSO)], LY 294002 1–10 µM final
(0.1–1 mM stock in 10% DMSO), and rapamycin 0.1–1
µg/liter final (0.1–1 mg/liter in 10% DMSO) came from
Calbiochem (La Jolla, CA). Control cultures received the
same amount of solvent.
Determination of DNA synthesis and protein content
On day 4, the medium was collected. The cells were washed three
times in PBS, trypsinized, and aliquoted in two tubes. One sample was
used for either measurements of 3H-thymidine incorporated
into DNA, as described before (27), or DNA measurements with
bisbenzimidazole, as described (28). The protein content of the
supernatant was measured using the DC Protein Assay, according to the
manufacturer’s instructions (Bio-Rad Laboratories, Inc.,
Hercules, CA).
Measurement of immunoreactive rat HGF
When the cell cultures were stopped, the medium was aspirated
and spun at 3,300 x g, for 20 min, at 4 C. The
supernatant was collected and frozen at -70 C. An enzyme-linked
immunosorbent assay was used to measure immunoreactive rat HGF
(Institute of Immunology, Tokyo, Japan). Briefly, 50 µl of sample
medium in duplicate was dispensed to a 96-well plate precoated with
monoclonal antibodies against human HGF, as described (29). Rat HGF
standard solution was provided by the manufacturer for a standard
curve. The plates were then incubated overnight at room temperature
and, thereafter, washed and incubated with an antirat HGF rabbit
polyclonal antibody. Peroxidase-labeled goat antirabbit Ig, together
with a chromogenic substrate, was added. The absorbance was monitored
by an enzyme-linked immunosorbent assay reader at 490 nm.
In most experiments, HGF content was normalized to DNA content in the cells as an indicator of cell number. See above for the DNA content measurements.
Measurement of phosphorylated MAPK
HSC (cultured in 6-well plates) were washed in PBS, 50 µl SDS
sample buffer was added, and the cells were scraped and frozen in -70
C. The samples were then sonicated (two times; 15 sec each time) and
centrifuged (12,000 x g; 10 min). For each sample,
equal amounts (15 µl) were separated on one-dimensional
SDS-polyacrylamide gels (NuPage gels; Novex, San Diego,
CA). The proteins were transferred to a polyvinyldifluoride membrane
(Novex) using a blotting device (Xcell II;
Novex). The membranes were then incubated with monoclonal
antibodies against phoshporylated p44/42 MAPK (Thr202/Tyr204)
(New England Biolabs, Inc., Beverly, MA). Immunoreactive
protein was visualized by chemiluminescence using a horseradish
peroxidase-conjugated second antibody with an enhanced
chemiluminescence detection system (Amersham Pharmacia Biotech, Uppsala, Sweden). The filters were exposed to
enhanced chemiluminescence film (Amersham Pharmacia Biotech) at room temperature for 2–5 min, and the films were
subsequently developed. The relative levels of p44 and p42
immunoreactive MAPK were estimated by scanning with a Shimadzu CS-9000
densitometer (Shimadzu, Kyoto, Japan). Protein levels are
expressed as a percent of the mean value of the untreated control
cultures (percent of control).
Statistical analysis
Values are given as the mean ± SEM.
Comparisons between two groups were made using Student’s t
test. Comparisons between more than two groups were made using a
one-way ANOVA, followed by Student’s-Newman-Keul’s multiple-range
test for pairwise contrasts among a group of means.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effects of MAPK inhibition on IGF-1-stimulated DNA synthesis and
HGF production
To investigate the effects of IGF-1 on DNA synthesis and HGF
production and to define the intracellular signals that mediate these
effects, we added different signal transduction inhibitors to the HSC
cultures. We investigated the importance of the ras-raf-MAPK cascade by
addition of the specific MAP kinase kinase or ERK kinase
inhibitor PD 98059 to HSC cultures treated with IGF-1 (100 ng/ml). As
shown in Fig. 1A
, IGF-1 induced a 4-fold
increase in DNA synthesis in HSC, as measured by
3H-thymidine incorporation. However, after addition of the
MEK inhibitor PD 98059, there was no significant stimulatory effect of
IGF-1 on DNA synthesis. IGF-1 increased the levels of immunoreactive
HGF in the cell culture medium 2-fold (Fig. 1B). Addition of the MEK
inhibitor PD 98059 to the cultures reversed the stimulatory effect of
IGF-1 on the HGF levels. Compared with cultures treated with IGF-1
only, the levels were decreased by about 30% and did not differ from
those of untreated controls (Fig. 1B). PD 98059 alone had no effect on
either DNA synthesis or HGF content in the control cultures (Fig. 1, A
and B).
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). IGF-1-induced stimulation of HGF
production was also reversed after treatment with LY 294002. LY 294002
alone had no effect on either DNA synthesis or HGF content in the
control cultures (Fig. 2, A and B).
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). In contrast, rapamycin had no effect
on the HGF levels after IGF-1 treatment (Fig. 3B). Rapamycin alone had
no effect on either DNA synthesis or HGF content in the control
cultures (Fig. 3, A and B).
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, PD 98059
(given in doses of 1 and 3 µM) decreased the
IGF-1-induced DNA synthesis by about 45%, whereas 10 µM
had no significant effect (Fig. 4A). In control cultures not treated
with IGF-1, PD 98059 had no effect on DNA synthesis (Fig. 4A). Only the
highest dose (10 µM) of PD 98059 suppressed the
immunoreactive HGF content in the HSC cultures (Fig. 4B). The PI-3
kinase inhibitor LY 294002 suppressed the IGF-1-induced DNA synthesis
in the HSC cultures, with a maximum decrease (about 80%) at 3–10
µM (Fig. 4C). The addition of LY 294002 to control
cultures not given IGF-1 at doses of 3 and 10 µM
decreased DNA synthesis by 30%–45%, whereas a dose of 1
µM had no effect (Fig. 4C). As shown in Fig. 4D, the
IGF-1-induced increase in the HGF content in the culture medium was
also significantly inhibited by about 40% by LY 294002 at doses of
1–10 µM. As shown in Fig. 4E, inhibition of activation
of the p70S6K by rapamycin suppressed IGF-1-induced HSC
proliferation by about 70% at doses of 0.1–1 ng/ml. As can be seen in
Fig. 4F, the levels of IGF-1-stimulated HGF content in the culture
medium were reduced by rapamycin at doses of 0.1–0.3 ng/ml but not by
1 ng/ml. Rapamycin alone had no significant inhibitory effects on
either DNA synthesis or HGF content (Fig. 4, E–F). The inhibitory
effects of PD 98059, LY 294002, and rapamycin on HGF content were
specific, given that none of these compounds affected total protein
content in the medium (data not shown).
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, A–D), we wanted to verify that the used doses of PD 98059 were able to
completely inhibit MAPK activity. As shown in Fig. 5, both 10% FCS (the positive control)
and 100 ng/ml IGF-1 were able to induce phosphorylation of bands
corresponding to the size of p44/42 MAPK. The highest used dose of PD
98059 in the previous experiments was 10 µM, and this
dose completely inhibited the IGF-1-induced MAPK phosphorylation. The
stimulatory effect of IGF-1 on MAPK phosphorylation and its inhibition
by PD 98059 were confirmed by densitometric measurements of the bands
(Table 1). IGF-1 also increased DNA synthesis, measured as
3H-thymidine incorporation in parallel cultures from the
same HSC isolation, whereas 10 µM PD 98059 was not able
to significantly inhibit the IGF-1-induced DNA synthesis in this
experiment. Immunoreactive HGF in these parallel cultures was increased
by IGF-1, and this increase was reversed by the addition of PD 98059
(Table 1), in agreement with previous experiments (e.g. Fig. 1B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported (3) that IGF-1 can enhance the production of HGF from cultured HSC. This was shown also in this study, where we further investigated the signal transduction pathways for IGF-1 on HGF production. There was a clear inhibitory effect of LY 294002 on the IGF-1-induced HGF content in the culture medium. MAPK inhibition by PD 98059 also attenuated the IGF-1-stimulation on HGF but at a 10-times higher dose. A preliminary report of cultured human myofibroblast-like cells, i.e. HSC that have been transformed by prolonged culture in vitro, where IGF-1-increased collagen type I production, could be inhibited both by MAPK blockade and by PI3-K blockade (26). These similarities with our results are somewhat surprising, because we have studied untransformed HSC that may differ in many ways from transformed myofibroblast-like cells. For instance the IGF-1 responsiveness in HSC is much higher than that of myofibroblast-like cells (6). Moreover, HGF mRNA levels have been reported to be higher in untransformed HSC (16, 17), whereas collagen type I is mainly produced in myofibroblast-like cells (19).
The effectiveness of the PI-3K inhibitor LY 294002 indicates that both IGF-1 stimulated DNA synthesis and HGF production are dependent on PI-3K. However, the pathways downstream may differ between these two effects. The DNA synthesis seems to be mediated by rapamycin-sensitive activation of the downstream mediator p70S6K, which, in turn, activates ribosomal protein S6 and translation (22). HGF production, on the other hand, seemed to be less dependent on this rapamycin-sensitive pathway. One possibility is that this effect, instead, is mediated by activation of PKB, another downstream effector of PI-3K, which is activated by insulin and IGF-1 (23). Biological effects mediated by PI-3K and PKB have been shown to be inhibited by LY 294002 but not by rapamycin (36).
Our finding that IGF-1 enhances HGF mRNA, as well as HGF protein, suggests that IGF-1 enhances HGF production (3). In the present study, the stimulatory effect of IGF-1 on medium HGF levels was not caused only by a general increase in protein synthesis by HSC, because the HGF/protein ratio in the medium was enhanced by IGF-1. The inhibitory effects of MAPK and PI3-K were not caused by nonspecific inhibition of protein synthesis, although there are reports that IGF-1’s can affect general protein synthesis in some cell systems via both PI3-K and MAPK activation (37). Neither PD 98059 nor LY 294002 decreased medium protein levels. Generally, the signal inhibitors used in this study lowered DNA synthesis more effectively then HGF levels. It is possible that the pathways studied contribute, to a lesser degree, to the HGF-stimulating effect of IGF-1. On the other hand, it is also possible that we were not able to detect major effects on HGF levels by these signal transduction inhibitors, because of low turnover of HGF.
Considerable interest has recently been focused on the issue of comparing mitogenic and differentiating effects of IGF-1. This question has been studied extensively in myoblasts, which seem to differ from HSC in many ways, with regard to the effects of IGF-1. In myoblasts, IGF-1 exerts differentiating effects, i.e. myotubule formation already at low doses of IGF-1, through a PI3-K-dependent pathway. On the other hand, high doses of IGF-1 lead to MAPK activation and subsequently to myoblast proliferation (24). In HSC, the PI3-K pathway seems to be more important than the MAPK pathway, for DNA synthesis. Moreover, dose-response curves showed that similar doses of IGF-1 were effective both on HGF production and on DNA synthesis in cultured HSC (3).
There have been relatively few studies on the intracellular signaling pathways in cultured untransformed HSC (38). However, more is known about transformed HSC, i.e. myofibroblast-like cells, and especially about the signal transduction pathways used by platelet-derived growth factor (PDGF) (38). PDGF has some interesting features in common with IGF, with regard to its effects on HSC. IGF-1 and PDGF are able to act as mitogens, as well as inducers of synthesis of different proteins. PDGF can induce both IGF-1 and the IGF-1 receptor, thereby raising the possibility that IGF-1 acts via an autocrine loop to mediate some of PDGF’s effects on cultured HSC (38). The PDGF receptor has intrinsic tyrosine kinase activity (39) in a way similar to that of the IGF-1 receptor (21). After phosphorylation of the PDGF receptor, signal pathways similar to those in the IGF-1 receptor are activated, including the ras-raf-MAPK and PI3-K-dependent pathways (40, 41). In addition to IGFs, hepatocytes seem to produce other factors that can enhance DNA synthesis and HGF production (11).
The physiological significance in vivo of the effects of IGF on HSC in vitro remains to be determined. DNA synthesis and proliferation of HSC have been associated with their transformation to myofibroblast-like cells, a process that may be associated with development of fibrosis (19, 20). On the other hand, increased HGF production may facilitate liver regeneration, stimulate hepatocyte proliferation, and protect hepatocytes from cell death (13, 14). HGF does not seem to enhance DNA synthesis in untransformed HSC (Skrtic et al., unpublished results). Both IGF and HGF have also been implicated in the development of liver tumors, but their exact roles remain unclear (14, 42). Recent publications have clearly shown, however, that liver-derived IGF-1 is not important for the normal growth of the intact liver during adulthood (2, 43).
In conclusion, the investigation of signaling pathways suggests that PI3-K (and maybe also its downstream effector p70S6K) is important for mediating the stimulatory effect of IGF-1 on DNA synthesis, whereas the MAPK pathway may be less essential for this effect. It is also possible that the PI3-K pathway is more important than the MAPK pathway for IGF-1-stimulated HGF production.
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Acknowledgments |
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Footnotes |
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Received June 18, 1999.
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