help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Skrtic, S.
Right arrow Articles by Jansson, J.-O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Skrtic, S.
Right arrow Articles by Jansson, J.-O.
Endocrinology Vol. 138, No. 11 4683-4689
Copyright © 1997 by The Endocrine Society

ARTICLES

Insulin-Like Growth Factors Stimulate Expression of Hepatocyte Growth Factor But Not Transforming Growth Factor ß1 in Cultured Hepatic Stellate Cells1

S. Skrtic, V. Wallenius, S. Ekberg, A. Brenzel, A. M. Gressner and J.-O. Jansson

Research Center for Endocrinology and Metabolism (S.S., V.W., S.E., J.-O.J.), Sahlgrenska University Hospital, Göteborg S-413 45, Sweden; and Department of Clinical Chemistry (A.B., A.M.G.), Philipps-University, Marburg D-35033, Germany

Address all correspondence and requests for reprints to: John-Olov Jansson, Research Center for Endocrinology and Metabolism, Endocrine Division, Gröna Stråket 8, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hepatic stellate cells (HSC) are located adjacent to hepatocytes and produce hepatocyte growth factor (HGF) in the normal liver, whereas transformed HSC in fibrotic livers produce transforming growth factor ß1 (TGFß1), an inhibitor of hepatocyte proliferation. In addition to the endocrine actions of hepatic insulin-like growth factor-I (IGF-I), it also stimulates the proliferation of HSC. In this study we found that addition of IGF-1 (20–500 ng/ml) for 48 h to 2- to 7-day-old primary cultures of rat HSC resulted in a time- and dose-dependent increase by 50–190% of the concentrations of immunoreactive HGF in the medium. The levels of HGF as well as DNA synthesis measured as thymidine incorporation were also enhanced by IGF-II and des(1–3)IGF-I, which has reduced binding to IGF binding proteins. There was no consistent effect of the IGFs on the levels of immunoreactive TGFß1 or on the total DNA content of the cultures. There was no effect of human GH on medium levels of HGF or TGFß1, thymidine incorporation, or total DNA content. IGF-I increased the abundance of HGF messenger RNA, as measured by the RNase protection/solution hybridization technique, whereas there was no effect on TGFß1 or glyceraldehyde phosphate dehydrogenase messenger RNA. The results suggest that IGFs stimulate the production of HGF but not TGFß1 by HSC in vitro.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT is well recognized that liver-derived insulin-like growth factor-I (IGF-I) contributes a large part of circulating IGF-I (1), and that it exerts endocrine effects on peripheral tissues in conjunction with locally produced IGF-I (2, 3, 4). On the other hand, few studies have addressed the possibility of local effects of IGFs in the liver. Although IGF-I was originally identified by its ability to stimulate proliferation of various cell types in vitro (4), hepatocytes in vitro do not proliferate in response to IGF-I (5). Moreover, the hepatocytes of the normal liver have few IGF-I receptors (1, 6), arguing against autocrine effects of hepatocyte-produced IGF. Instead, several reports indicate that IGF-I can stimulate DNA synthesis of hepatic stellate cells (HSC; also called Ito cells, lipocytes, and fat-storing cells) in vitro (7, 8, 9, 10). It has also been shown that HSC in vitro have specific IGF-I binding sites, immunoreactive type 1 IGF receptor ß-subunits, and express type 1 IGF receptor messenger RNA (mRNA) (11).

The HSC are parasinusoidal cells adjacent to the hepatocytes (12, 13, 14). If the liver is damaged, the HSC are believed to transform to myofibroblast-like cells that produce collagen and extracellular matrix. Therefore, these cells may be of pathophysiological importance in the development of liver cirrhosis (13, 14). The transformed HSC or myofibroblast-like cells produce transforming growth factor ß1 (TGFß1), which contributes to the production of extracellular matrix by an autocrine effect. TGFß1 also exerts paracrine effects on hepatocytes, including growth inhibition and induction of apoptosis (13, 14, 15, 16).

In the intact liver, HSC contain a large part of the retinoic acid stores of the body (12). The untransformed HSC may also exert local paracrine effects in the intact liver, because they produce hepatocyte growth factor (HGF) (17, 18, 19). HGF can exert substantial mitogenic, motogenic, and morphogenic effects, mainly on cells of epithelial origin such as hepatocytes (20, 21) and biliary epithelial cells (22, 23). The aim of the present study was to investigate whether IGF-I can affect the production and secretion of HGF and TGFß1 from HSC in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and culture of HSC
Isolation and culture of rat HSC have previously been described in detail (24). Briefly, 1-yr-old male Sprague-Dawley rats (BW 500–700 g, Lippische Versuchtierzucht, Extertal, Germany) were used. The animal studies were conducted in accordance with rules for the care and use of laboratory animals, and the protocol was approved by the local committee on animal care. Nonparenchymal liver cells were obtained by the pronase-collagenase method, and HSC were further purified by a single-step density gradient centrifugation with Nycodenz (analytical grade; Nyegaard Co. AS, Oslo, Norway) as described previously (24). The HSC were identified by their typical light microscopic appearance, transmission electron microscopy, immunofluorescent staining for desmin and vimentin, vitamin A-specific autofluorescence, by staining of fat droplets with oil red O and negatively, by their inability to phagocytose latex beads, to stain for peroxidase and to express Fc receptors (25). The cell viability, checked by trypan blue exclusion, was higher than 95%, and the mean purity of freshly isolated HSC was 90 ± 5%.

The cells were seeded on day 0 at a density of 40 x 103 cells/cm2 in either 6-well 10-cm2 plates or 75-cm2 flasks (for determination of HGF and TGFß1 mRNA). The cells were maintained as monolayers on plastic with DMEM containing L-glutamine (4 mmol/l), penicillin (100 IU/ml), streptomycin (100 µg/ml), 10% FCS (all from Boehringer Mannheim GmbH, Mannheim, Germany), and insulin (0.02 U/ml from bovine pancreas; Sigma, St. Louis, MO) and in a humidified atmosphere of 5% CO2-95% air at 37 C. On day 1, about 16 h after seeding, the medium was changed to 10% FCS. The purity was then higher than 97%. During the experiments, the cells were exposed to IGFs or GH in medium with 0.2% FCS or control medium with 0.2% FCS only. The cultures were maintained in these media for 48 h before harvesting of the media and the cells. Before the experiments, the cells had mostly been serum starved in medium with 0.2% FCS for 24 h. The cells exposed to IGF-I on days 1–3 had been exposed to 0.2% FCS for 8 h before the experiment.

Determination of HSC proliferation
Cells were seeded on day 0 at a density of 40 x 103 cells/2 cm2/ml medium (same medium as above) in 24-well plates. After 24 h on day 1, the medium was changed from 10% to 0.2% FCS. On day 2 (24 h later) IGF-I and other factors were added. Control cultures received 0.2% or 10% FCS. After a further 24 h (on day 3), 1 µCi/ml [3H]thymidine was added to all groups. On day 4, medium was collected, cells were washed three times in PBS, trypsinized, and aliquoted into two tubes, one for measurement of incorporated [3H]thymidine into DNA as previously described (26) and one for DNA measurement with bisbenzimidazole as previously described (27).

Probe synthesis
A 646-bp complementary DNA (cDNA) fragment (corresponding to nt 1698–2343) of rat HGF transcript (28, 29) in a transcription vector pGEM-3Z (Promega, Madison, WI) was used for antisense RNA probe synthesis (29). The vector was linearized, and radiolabeled antisense transcript was synthesized in vitro using T3 RNA polymerase and [33P]UTP (2000 Ci/mmol, Amersham Int., Buckinghamshire, England). The full-length RNA probe was purified from unincorporated radioactivity on a G-50 Sephadex spin column (Pharmacia, Uppsala, Sweden). A 318-bp cDNA fragment of glyceraldehyde phosphate dehydrogenase (GAPDH) corresponding to nt 367–685 in pBluescript SK (Stratagene, La Jolla, CA) was used for antisense RNA probe synthesis. A 200-bp cDNA fragment of rat (nt 1003–1203) was generated by RT-PCR and TA cloned into pCRII (In Vitrogen Co., San Diego, CA), then linearized with XbaI, and an antisense probe was synthesized in vitro using Sp6 RNA polymerase.

RNA isolation and RNase protection assay
To stop the assays, medium was aspirated, the cell layer washed twice with HBSS, the flasks put on ice, and guanidine thio-cyanate solution 2 ml/flask was added. A rubber policeman was used to collect the cells, which were then transferred to a 15-ml Falcon tube and frozen in liquid nitrogen. Total RNA was isolated as previously described (30). For the RNase protection assay (kit 1440; Ambion, Austin, TX), samples of 20 µg of total cellular RNA were hybridized at 45 C overnight with 5 x 105 cpm of the rat HGF RNA antisense probe, then digested with RNase. The RNA:RNA hybrids were precipitated, resuspended, and separated on an 8% polyacrylamide/7 M urea gel. The signal from protected fragments was quantified on a PhosphoImager (Molecular Dynamics, Sunnyvale, CA). The areas of the bands were quantified by the PhosphoImager software.

Measurement of immunoreactive HGF and TGFß1
When the cell culture was stopped, the medium was aspirated and centrifuged at 3300 x g for 20 min at 4 C. The supernatant was collected and frozen at -70 C. For the measurement of immunoreactive rat HGF, an ELISA from the Institute of Immunology Co. Ltd., Tokyo, Japan was used (31). Briefly, 50 µl sample medium, in duplicate, was dispensed into a 96-well plate, precoated with a monoclonal antibody against human HGF (31). Rat HGF standard solution was provided by the manufacturer for a standard curve. The plate was then incubated overnight at room temperature, washed thereafter, and incubated with an antirat HGF rabbit polyclonal Ab, which was then visualized by a peroxidase-labeled antirabbit goat Ig. Absorbance was monitored with an ELISA spectrophotometer at 490 nm. For the measurement of total content of TGFß1, an ELISA (G1230; Promega) was used. The ELISA is specific for the TGFß1 isoform, and the antibodies in the ELISA cross-react between rat and human. Samples were diluted 1:1 in DPBS and transiently acidified with 1 N HCl. Neutral pH was restored with 1 N NaOH. Thereafter, 100 µl sample in duplicate was dispensed into a 96-well plate that was coated in advance with a TGFß1 monoclonal antibody. Recombinant human TGFß1 standard solution was provided by the manufacturer for a standard curve. The plate was then incubated at room temperature for 90 min, washed, and incubated with anti-TGFß1 polyclonal antibody for another 2 h at room temperature. After one more wash, a peroxidase-labeled anti-TGFß1 polycolonal antibody immunoglobulin was added, followed by incubation with a chromogenic substrate and subsequent measurement in an ELISA spectrophotometer at 450 nm.

Statistical analysis
Values are given as means ± SEM. Comparisons between two groups were made by Student’s t test. Comparisons between more than two groups were made by one-way ANOVA followed by Student-Newman-Keuls multiple range test for pairwise contrasts among a group of means. Overall significance between groups was calculated by two-way ANOVA. Significances between groups were also calculated using the nonparametric tests. The Kruskal-Wallis test was followed by Mann-Whitney’s U test for pairwise comparison between groups. When the nonparametrical calculations gave similar results as the parametric tests, only results from the latter are reported.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of IGF-I on HGF production
It is known that the properties of cultures of HSC on a plastic surface may vary between cell isolations. In Fig. 1Go, HSC from three separate isolations were exposed to either IGF-I (100 ng/ml) or control medium during days 2–4 of culture. The concentrations of HGF, as measured by ELISA, in the culture media were normalized to the DNA content in each culture to eliminate the possible influence of cell number on the HGF levels. The levels of the control cultures varied between 7 and 450 pg/µg DNA. We also observed that the stimulatory effect of IGF-I varied between different experiments. In Exp 1, the increase was significant when using the nonparametric Mann-Whitney U test for pairwise comparison between groups, but not when using the parametric Student’s t test. In Exps 2 and 3, the stimulatory effect was significant with both parametric and nonparametric tests. There was an overall significant effect of IGF-I in all three experiments as calculated by two-way ANOVA (P < 0.001). In the experiment shown in Fig. 2Go, A and B, doses of 100 and 500 ng/ml of IGF-I enhanced the HGF content in the medium significantly (P < 0.01), whereas there was no significant effect of 20 ng/ml (Fig. 2AGo). The thymidine incorporation was stimulated significantly by 20 (P < 0.05), 100, and 500 ng/ml (P < 0.01) of IGF-I (Fig. 2BGo). In this and other experiments, there were no marked or consistent differences between the dose-response curves for these two parameters.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 1. Content of immunoreactive rat HGF (nanogram per microgram DNA) in supernatant of cultured HSC that were incubated with IGF-I (100 ng/ml) or control medium on days 2–4 of culture. Bars represent cultures. Data from three different experiments from three separate HSC isolations and each treatment group were run in triplicate. Means ± SEM of three wells are shown. *, P < 0.05; **, P < 0.01 compared with control cultures (Student’s t test).

 


View larger version (13K):
[in this window]
[in a new window]
  		Figure 2. Effects of incubation of HSC on days 2–4 of culture with different doses of IGF. HSC were incubated with control medium or IGF-I (20–500 ng/ml) at 2–4 days of age. A, Content of immunoreactive rat HGF in supernatant of HSC. B, [3H]Thymidine incorporation into DNA as a determinant of DNA synthesis in HSC cultures. Each point represents mean ± SEM for three culture wells. **, P < 0.01 compared with control cultures (ANOVA followed by Student-Newman-Keuls multiple range test).

 
HGF stimulation by IGF-I: effects of culture age and incubation time
In the next experiment, we tested whether culture age is a determinant of the effect of IGF-I on the content of HGF in the culture medium of HSC. For comparison, the content of immunoreactive TGFß1 was measured in the medium with an ELISA, and cell number was estimated by measurement of DNA content in the cultures. Exposure of HSC to IGF-I (100 ng/ml) for 2 days caused a significant increase in the release of immunoreactive HGF in cell cultures that were 2–4, 3–5, 4–6, or 5–7 days of age. In this experiment, no effect of IGF-I was seen in a 1- to 3-day-old HSC culture (Fig. 3AGo). In other experiments, however, we saw a stimulatory effect of IGF-I also in these newly seeded cells (not shown). To verify that the stimulatory effect of IGF-I on HGF is specific and not due to a nonspecific overall increase in protein production, the content of total TGFß1, another important growth factor released from HSC, was measured in the culture medium with an ELISA. As shown in Fig. 3BGo, there was no such stimulatory effect of IGF-I on TGFß1 content at different culture ages, except at days 2–4, when a significant effect (P = 0.0495) was calculated with Student’s t test (Fig. 3BGo). However, there was no significant overall effect of IGF-I at the different culture ages according to two-way ANOVA. There was no significant effect of IGF-I on total DNA content in the cultures (Fig. 3CGo), suggesting that the effect of IGF-I on immunoreactive HGF in the medium was not due to an increased number of HSC capable of HGF production. Calculations with two-way ANOVA indicated that the culture content of HGF and TGFß1 as well as DNA varied as a function of culture age (P < 0.001 for all three parameters). Comparisons between individual groups showed that the levels of HGF, TGFß1, and DNA per culture generally increased with increasing culture age.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 3. Effect of IGF-I on HSC of different ages. Numbers under panels indicate culture age at which cells were incubated with or without IGF-I (100 ng/ml). A, Content of immunoreactive rat HGF in supernatants of HSC. B, Content of immunoreactive TGFß1 in supernatant of cultures. C, Total content of DNA in HSC cultures. Each bar represents mean ± SEM for three culture wells. *, P < 0.05; **, P < 0.01 compared with control cultures (Student’s t test).

 
In another experiment, 4-day-old HSC were incubated with or without IGF-I at days 2–4 (Fig. 4Go). During this period, samples were taken from the supernatant after 0, 12, 24, 36, and 48 h of incubation. HGF accumulated during the incubation period in both the IGF-I-stimulated cultures and the control cultures. In this experiment, the levels were not significantly higher in the IGF-I-treated cultures until after 36–48 h of incubation with IGF-I (Fig. 4Go). In another experiment, there was a 2-fold difference between the IGF-I-treated cultures and control cultures already after 24 h (data not shown).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 4. Accumulation of immunoreactive rat HGF in medium from HSC incubated with or without IGF-I (100 ng/ml) on days 2–4 of culture. Samples were taken from supernatant at 0, 12, 24, 36, and 48 h after start of experiment. Each point represents mean ± SEM for three medium samples. **, P < 0.01 compared with control cultures (ANOVA followed by Student-Newman-Keuls multiple range test).

 
Effects of different IGF forms and GH on HGF secretion
In the next experiment, we tested the effects of incubation of 100 ng/ml of different IGF variants on the medium content of HGF and TGFß1 and DNA synthesis and total DNA content in 2- to 4-day-old cultures of HSC. IGF-I caused a 2-fold increase in the HGF content in the medium (Fig. 5AGo). A similar effect was obtained by adding desIGF-I (100 ng/ml). IGF-II (100 ng/ml) also significantly increased HGF concentration but to a smaller degree than IGF-I and desIGF-I. In contrast, there was no significant effect of human GH (hGH) (50 ng/ml) on HGF secretion. The total level of immunoreactive TGFß1 in the culture medium was not significantly affected by the various forms of IGF or by hGH (Fig. 5BGo). Therefore, the borderline significant stimulation (P = 0.0495) of TGFß1 by IGF-I on days 2–4 seen in Fig. 3BGo could not be repeated in this experiment. The incorporation of [3H]thymidine into DNA was also measured to evaluate DNA synthesis (Fig. 5CGo). There was a significant increase in DNA synthesis, measured as [3H]thymidine incorporation, in the cultures that were treated with IGF-I and desIGF-I. In contrast, the DNA content (Fig. 5DGo) was not affected by the different forms of IGF within the time period of exposure, indicating that the stimulatory effects on accumulation of immunoreactive HGF (Fig. 5AGo) are not due to an increased number of cells that can express HGF. There was no significant effect of hGH (50 ng/ml) on DNA synthesis (Fig. 5CGo) or total DNA content (Fig. 5DGo).



View larger version (57K):
[in this window]
[in a new window]
  		Figure 5. Effects of incubation of HSC on days 2–4 of culture with different forms of IGF and with GH. HSC were incubated with control medium, IGF-I (100 ng/ml), desIGF-I (100 ng/ml), IGF-II (100 ng/ml), or hGH (50 ng/ml) of 2–4 days of age. A, Content of immunoreactive rat HGF in supernatant of HSC. B, Content of total TGFß1 in supernatant of HSC. C, [3H]Thymidine incorporation into DNA as a determinant of DNA synthesis in HSC cultures. D, Total DNA content in HSC. Each bar represents mean ± SEM for three culture wells. ***, P < 0.001 compared with control cultures (ANOVA followed by Student-Newman-Keuls multiple range test).

 
Effects of IGF-I on rat HGF mRNA levels
We next wanted to investigate whether the stimulatory effect of IGF-I on HGF content in medium of HSC was accompanied by an increase in steady state levels of mRNA, which could facilitate production and secretion of HGF. Therefore, an RNase protection assay with a 650-bp rat-specific complementary RNA probe was used for determination of HGF mRNA. A typical RNase protection assay gel is shown in Fig. 6AGo. In 5- to 7-day-old HSC cultures incubated with IGF-I (100 ng/ml) for 48 h, there was a marked increase in the intensity of the HGF mRNA band compared with HSC exposed to control medium. In the latter cells, the intensity of the band was close to the detection limit (Fig. 6AGo). The determinations of HGF mRNA were repeated in three different experiments with similar results, i.e. more intense bands after incubation with IGF-I than in control cultures (Table 1Go). To verify that the effect of IGF-I on HGF mRNA is specific, we measured TGFß1 and GAPDH transcripts with 200- and 145-bp rat-specific probes, respectively, in a RNase protection assay. As shown in Fig. 6BGo, there were no marked differences between the IGF-I-treated HSC cultures and the control cultures.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 6. PhosphoImage scans of RNase protection assays of total RNA from a representative culture of HSC incubated with control medium (C) or IGF-I (100 ng/ml) for 48 h on days 2–4. A, Measurement of HGF mRNA with a 650- bp unprotected rat-specific probe. B, Measurement of TGFß1 mRNA with a 200-bp rat-specific TGFß1 probe and of GAPDH mRNA with a 145-bp rat GAPDH probe.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Results of quantification of PhosphoImage scans of RNase protection assays of total RNA from three cultures of HSC incubated with control medium or IGF-1 (100 ng/ml) for 48 h on days 2–4

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results showed that IGFs, including IGF-I, increased the content of immunoreactive HGF in the medium of primary cultures of HSC. Moreover, IGF-I enhanced the levels of HGF mRNA in the HSC. Taken together, these results suggest that IGF-I stimulates the cellular HGF synthesis at a pretranslatory level and that the enhanced HGF levels in the medium after IGF treatment are due to enhanced secretion from the cells. The stimulation of HGF by IGF is probably not only due to the stimulatory effect on DNA synthesis in HSC shown in this study and reported previously (7, 8, 9, 13), because the amount of DNA per culture had virtually not yet been affected during the comparatively short 2-day incubation of this study. In line with this, IGF-I treatment also increased the ratio between immunoreactive HGF and DNA content in HSC cultures. Our finding that IGF-I did not consistently affect the medium concentration of TGFß1 or the abundance of TGFß1 or GAPDH mRNA indicates that the increases in HGF production and release are specific.

It is well known that incubation of HSC on plastic in vitro results in spontaneous transformation to myofibroblast-like cells (13, 14). This transformation is accompanied by decreased HGF mRNA levels (17, 18) and also increased production of TGFß1 (13, 14, 32). There is no evidence that the stimulatory effect of IGF on HGF production observed in this study was due to inhibited transformation, because the TGFß1 expression was unchanged. Moreover, the HSC used in this study may have been too young to have been substantially transformed. It has been reported previously that the HSC have reached an early stage of transformation by days 5–7, i.e. the oldest HSC culture age in this study (32). The lack of transformation in this study may gain support from the finding that the levels of HGF immunoreactivity were markedly (10- to 25-fold) higher than the TGFß1 immunoreactivity even at a culture age of 7 days, i.e. the latest time studied. There was a culture age-dependent increase in the levels of DNA and probably also the number of HSC before day 7. However, proliferation of HSC has been observed before morphological signs of cell transformation occur (32).

There are some previous reports on factors that can regulate HGF production in HSC as well as fibroblasts. TGFß1 decreases the levels of HGF mRNA in HSC (18) and HGF mRNA and protein levels in fibroblasts (21). TGFß inhibitory elements have been shown to be present together with several other regulatory elements in the promotor sequence of the HGF gene (33, 34, 35). Therefore, IGF-I and TGFß1 may have opposite effects on HGF expression in HSC. The levels of IGFBP-3 mRNA in endothelial cells have also been reported to be enhanced by IGF-I and suppressed by TGFß1 (36). In fibroblasts, there are several as yet unidentified factors besides TGFß1 that influence HGF expression (21, 37). However, it is uncertain to what extent these results apply to the liver. IGFs have been shown to regulate the expression of several genes (see Ref. 38 and references therein). There are few reports so far about elements with connections to IGF effects in the HGF gene promotor region (33, 34, 35).

It has been shown in several reports that IGFs can stimulate DNA synthesis of HSC in vitro during the first week after seeding (7, 8, 9) and after several passages when the cells are more myofibroblast-like (10). Moreover, newly seeded and transformed HSC express IGF-I receptors (11). The present results show that IGFs stimulate both HGF production, i.e. a differentiated effect of HSC, and proliferation of HSC, and that these effects were exerted at approximately the same doses. In myoblasts, the stimulatory effects of IGF-I on proliferation and markers of differentiation are exerted at markedly different doses (39). In cultured HSC, TGFß1 seems to suppress both HGF expression (18) and DNA synthesis (32, 40), giving another example of parallel regulation of these two parameters.

It is incompletely investigated whether the stimulatory effect of IGFs on HGF production in vitro shown in this study reflects a similar effect in vivo, although we have reported such a stimulatory effect of IGF-I in regenerating liver in one study (29). The HSC seem to contribute the major part of the HGF produced by the intact liver (17, 18, 19), whereas Maher (19) reported that the endothelial cells may cause the increased HGF production after hepatectomy or liver injury. The IGF-producing hepatocytes are located in close proximity to the HSC in the liver (12) and this may facilitate a paracrine interaction between these two cell types. In fact, the HSC could be exposed to very high levels of IGF-I in vivo, raising the possibility that there may be additional modulatory influences that counteract IGF in the intact liver. IGF-binding proteins produced by various cell types in the liver (10, 41, 42, 43, 44) could modulate the effects of IGF on HSC in vivo.

In vitro, it has been reported by Pinzani et al. (10) that rat HSC transformed by two to three passages in vitro produce IGF-binding proteins, but no IGF-binding proteins were found in medium of human HSC (45). The present results do not support a modulatory influence of IGF-binding proteins on IGF-stimulated HGF expression and DNA synthesis in comparatively young, untransformed rat HSC. The effects of desIGF-I and IGF-I on these parameters were of similar magnitude, despite the fact that desIGF has markedly impaired affinity to IGF-binding proteins (46).

Little is known about the possible role of HGF production by HSC in vivo. The HGF produced by HSC of intact livers (17, 18, 19) probably does not exert endocrine effects, because the levels of circulating HGF are very low in these animals (20, 21, 37, 47). In the liver, HGF has been shown to stimulate proliferation and morphogenesis of biliary epithelial cells (22, 23) in addition to its well-known effects on hepatocytes (20, 21, 37). In contrast, HGF does not seem to affect the proliferation of parasinusoidal cells such as endothelial or Kupffer cells or the HSC themselves (19). The bioactivity of HGF in vivo is in all probability also regulated at a posttranslatory level by several factors in vivo. These factors include binding to extracellular matrix, and bioactivation of prepro HGF by proteases that may be produced by target cells of HGF action including the hepatocytes (48). In addition, the responsiveness of the target cells may be modulated at the HGF receptor or postreceptor level (20, 21, 48).

We found no effects of treatment of HSC with human GH on HGF accumulation or thymidine incorporation. These results are in line with the finding that there are much fewer GH binding sites on nonparenchymal than on parenchymal cells of the rat liver (44, 49).

It has previously been shown that HSC, a cell type located adjacent to the hepatocytes in the liver (12), have IGF receptors and proliferate in response to IGF (8, 9, 11). It is generally assumed that HSC are the main producers of the potent hepatocyte mitogen HGF in the intact liver (17, 19). The results of the present study suggest that IGF can also stimulate HGF production and release by HSC in vitro.


   Acknowledgments
 
Excellent technical assistance was provided by Brigitte Heitmann, Birgit Lahme, and Helén Rawet.


   Footnotes
 
1 This work was supported by the Swedish Medical Research Council (NOREF>9894), Deutsche Forschungsgesellschaft (g 463/9–2), Söderberg Foundation, Lundberg Foundation, Nordisk Insulin Foundation, Swedish Medical Society and Göteborg Medical Society. It was presented in part at the 10th International Congress of Endocrinology in San Francisco, California, June 12–15, 1996 (Abstract P2–232). Back

Received February 17, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Froesch ER, Schmid C, Schwander J, Zapf J 1985 Actions of insulin-like growth factors. Ann Rev Physiol 47:443–467[CrossRef][Medline]
  2. D’Ercole A, Stiles A, Underwood L 1984 Tissue concentrations of somatomedin C: Further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action. Proc Natl Acad Sci USA 81:935–939[Abstract/Free Full Text]
  3. Isaksson OP, Lindahl A, Nilsson A, Isgaard J 1987 Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr Rev 8:426–438[CrossRef][Medline]
  4. Daughaday WH, Rotwein P 1989 Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev 10:68–86[CrossRef][Medline]
  5. Gohda E, Yamasaki T, Tsuboushi H, Kurobe M, Sakiyama O, Aoki H, Niidani N, Shin S, Hayashi K, Hashimoto S, Daikuhara Y, Yamamoto I 1990 Bilogical and immunological properties of hepatocyte growth factor from plasma of patients with fulminant hepatic failure. Biochim Biophys Acta 1053:21–26[Medline]
  6. Caro F, Poulos J, Ittoop O, Pories WJ, Flickinger EG, Sinha MK 1988 Insulin-like growth factor I binding in hepatocytes from human liver, human hepatoma, and normal, regenerating, and fetal rat liver. J Clin Invest 81:976–981
  7. Bachem MG, Meyer D, Schafer W, Riess U, Melchior R, Sell KM, Gressner AM 1993 The response of rat liver perisinusoidal lipocytes to polypeptide growth regulator changes with their transdifferentiation into myofibroblast-like cells in culture. J Hepatol 18:40–52[CrossRef][Medline]
  8. Gressner AM, Brenzel A, Vossmeyer T 1993 Hepatocyte-conditioned medium potentiates insulin-like growth factor (IGF) 1 and 2 stimulated DNA synthesis of cultured fat storing cells. Liver 13:86–94[Medline]
  9. Gressner AM, Lahme B, Brenzel A 1995 Molecular dissection of the mitogenic effect of hepatocytes on cultured hepatic stellate cells. Hepatology 22:1507–1518[CrossRef][Medline]
  10. Pinzani M, Abboud HE, Aron DC 1990 Secretion of insulin-like growth factor-I and binding proteins by rat liver fat-storing cells: regulatory role of platelet-derived growth factor. Endocrinology 127:2343–2349[Abstract]
  11. Brenzel A, Gressner AM 1996 Characterization of insulin-like growth factor (IGF)-I-receptor binding sites during in vitro transformation of rat hepatic stellate cells to myofibroblasts. Eur J Clin Chem Clin Biochem 34:401–409[Medline]
  12. Blomhoff R, Wake K 1991 Perisinusoidal stellate cells of the liver: important roles in retinol metabolism and fibrosis. FASEB J 5:271–277[Abstract]
  13. Pinzani M 1995 Novel insights into the biology and physiology of the Ito cell. Pharmacol Ther 66:387–412[CrossRef][Medline]
  14. Gressner AM 1996 Transdifferentiation of hepatic stellate cells (Ito cells) to myofibroblasts: a key event in hepatic fibrogenesis. Kidney Int 49:39–45
  15. Russell WE, Coffey, Jr, RJ, Ouellette AJ, Moses HL 1988 Type beta transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat. Proc Nat Acad Sci USA 85:5126–5130[Abstract/Free Full Text]
  16. Oberhammer FA, Pavelka M, Sharma S, Tiefenbacher R, Purchio AF, Bursch W, Schulte-Hermann R 1992 Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor beta 1. Proc Nat Acad Sci USA 89:5408–5412[Abstract/Free Full Text]
  17. Schirmacher P, Geerts A, Peitrangelo A, Dienes HP, Rogler CE 1991 Hepatocyte growth factor/hepatopoietin A is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cells. Hepatology 15:5–11
  18. Ramadori G, Neubauer K, Odenthal M, Nakamura T, Knittel T, Schwögler S, Meyer zum Büschenfelde K-H 1992 The gene of hepatocyte growth factor is expressed in fat-storing cells of rat liver and is downregulated during cell growth and by transforming growth factor-beta. Biochem Biophys Res Commun 183:739–742[CrossRef][Medline]
  19. Maher JJ 1993 Cell-specific expression of hepatocyte growth factor in liver. J Clin Invest 91:2244–2252
  20. Zarnegar R, Michalopoulos GK 1995 The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis. J Cell Biol 129:1177–1180[Free Full Text]
  21. Matsumoto K, Nakamura T 1996 Emerging multipotent aspects of hepatocyte growth factor. J Biochem (Tokyo) 119:591–600[Abstract/Free Full Text]
  22. Joplin R, Hishida T, Tsubouchi H, Daikuhara Y, Ayres R, Neuberger JM, Strain AJ 1992 Human intrahepatic biliary epithelial cells proliferate in vitro in response to human hepatocyte growth factor. J Clin Invest 90:1284–1289
  23. Johnson M, Koukoulis G, Matsumoto K, Nakamura T, Iyer A 1993 Hepatocyte growth factor induces proliferation and morphogenesis in nonparenchymal epithelial liver cells. Hepatology 17:1052–1061[CrossRef][Medline]
  24. Schafer S, Zerbe O, Gressner AM 1987 The synthesis of proteoglycans in fat-storing cells of rat liver. Hepatology 7:680–687[Medline]
  25. Chen W, Gendrault JL, Steffan AM, Jeandidier E, Kirn A 1989 Isolation, culture and main characteristics of mouse fat-storing cells: interaction with viruses. Hepatology 9:352–362[Medline]
  26. Zerbe O, Gressner AM 1988 Proliferation of fat-storing cells is stimulated by secretions of Kupffer cells from normal and injured liver. Exp Mol Pathol 49:87–101[CrossRef][Medline]
  27. Labarca C, Paigen K 1980 A simple, rapid and sensitive DNA assay procedure. Anal Biochem 102:344–352[CrossRef][Medline]
  28. Tashiro K, Hagiya M, Nishizawa T, Seki T, Shimonishi M, Shimizu S, Nakamura T 1990 Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues. Proc Natl Acad Sci USA 87:3200–3204[Abstract/Free Full Text]
  29. Ekberg S, Luther M, Nakamura T, Jansson J-O 1992 Growth hormone promotes early initiation of hepatocyte growth factor gene expression in the liver of hypophysectomized rats after partial hepatectomy. J Endocrinol 135:59–67[Abstract/Free Full Text]
  30. Chomczynski P, Sacchi N 1987 Single step method of RNA isolation by acid guanidinum thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  31. Yamada A, Matsumoto K, Iwanari H, Sekiguchi K, Kawata S, Matsuzawa Y, Nakamura T 1995 Rapid and sensitive enzyme-linked immunosorbent assay for measurement of HGF in rat and human tissues. Biomed Res 16:105–114
  32. Bachem MG, Meyer D, Melchior R, Sell KM, Gressner AM 1992 Activation of rat liver perisinusoidal lipocytes by transforming growth factors derived from myofibroblastlike cells. A potential mechanism of self perpetuation in liver fibrogenesis. J Clin Invest 89:19–27
  33. Okajima A, Miyazawa K, Kitamura N 1993 Characterization of the promoter region of the rat hepatocyte-growth-factor/scatter-factor gene. Eur J Biochem 213:113–119[Medline]
  34. Liu Y, Michalopoulos GK, Zarnegar R 1994 Structural and functional characterization of the mouse hepatocyte growth factor gene promoter. J Biol Chem 269:4152–4160[Abstract/Free Full Text]
  35. Aravamudan B, Watabe M, Watabe K 1993 Characterization of the 5'-flanking region of the hepatocyte growth factor gene. Biochem Biophys Res Commun 195:346–353[CrossRef][Medline]
  36. Erondu NE, Dake BL, Moser DR, Lin M, Boes M, Bar RS 1996 Regulation of endothelial IGFBP-3 synthesis and secretion by IGF-I and TGF-beta. Growth Regul 6:1–9[Medline]
  37. Rubin JS, Bottaro DP, Aaronson SA 1993 Hepatocyte growth factor/scatter factor and its receptor, the c-met proto-oncogene product. Biochim Biophys Acta 1155:357–371[Medline]
  38. Duan C, Hawes SB, Prevette T, Clemmons DR 1996 Insulin-like growth factor-I (IGF-I) regulates IGF-binding protein-5 synthesis through transcriptional activation of the gene in aortic smooth muscle cells. J Biol Chem 271:4280–4288[Abstract/Free Full Text]
  39. Florini JR, Ewton DZ, Coolican SA 1996 Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev 17:481–517[CrossRef][Medline]
  40. Davis BH 1988 Transforming growth factor beta responsiveness is modulated by the extracellular collagen matrix during hepatic ito cell culture. J Cell Physiol 136:547–553[CrossRef][Medline]
  41. Chin E, Zhou J, Dai J, Baxter RC, Bondy CA 1994 Cellular localization and regulation of gene expression for components of the insulin-like growth factor ternary binding protein complex. Endocrinology 134:2498–2504[Abstract]
  42. Villafuerte BC, Koop BL, Pao CI, Gu L, Birdsong GG, Phillips LS 1994 Coculture of primary rat hepatocytes and nonparenchymal cells permits expression of insulin-like growth factor binding protein-3 in vitro. Endocrinology 134:2044–2050[Abstract]
  43. Scharf JG, Ramadori G, Braulke T, Hartmann H 1996 Synthesis of insulinlike growth factor binding proteins and of the acid-labile subunit in primary cultures of rat hepatocytes, of Kupffer cells, and in cocultures: regulation by insulin, insulinlike growth factor, and growth hormone. Hepatology 23:818–827[CrossRef][Medline]
  44. Skrtic S, Wallenius V, Ekberg S, Brenzel A, Gressner A, Jansson J-O 1997 Expression pattern of growth hormone/insulin-like growth factor associated genes in rat liver. In: Wisse E, Knook D, Balabaud C, eds. Cells of the hepatic sinusoid. Vol 6. Kupffer Cell Foundation, Leyden, The Netherlands
  45. Arany E, Afford S, Strain AJ, Winwood PJ, Arthur MJ, Hill DJ 1994 Differential cellular synthesis of insulin-like growth factor binding protein-1 (IGFBP-1) and IGFBP-3 within human liver. J Clin Endocrinol Metab 79:1871–1876[Abstract]
  46. Carlsson-Skwirut C, Lake M, Hartmanis M, Hall K, Sara VR 1989 A comparison of the biological activity of the recombinant intact and truncated insulin-like growth factor 1 (IGF-1). Biochim Biophys Acta 1011:192–197[Medline]
  47. Lindroos PM, Zarnegar R, Michalopoulos GK 1991 Hepatocyte growth factor (hepatopoietin A) rapidly increases in plasma before DNA synthesis and liver regeneration stimulated by partial hepatectomy and carbon tetrachloride administration. Hepatology 13:743–750[CrossRef][Medline]
  48. Miyazawa K, Shimomura T, Kitamura A, Kondo J, Morimoto Y, Kitamura N 1993 Molecular cloning and sequence analysis of the cDNA for a human serine protease responsible for activation of hepatocyte growth factor. Structural similarity of the protease precursor to blood coagulation factor XII. J Biol Chem 268:10024–10028[Abstract/Free Full Text]
  49. van Neste L, Husman B, Möller C, Andersson G, Norstedt G 1988 Cellular distribution of somatogenic receptors and insulin-like growth factor mRNA in the rat liver. J Endocrinol 119:69–74[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Physiol. Rev.Home page
S. L. Friedman
Hepatic Stellate Cells: Protean, Multifunctional, and Enigmatic Cells of the Liver
Physiol Rev, January 1, 2008; 88(1): 125 - 172.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. Bavik, I. Coleman, J. P. Dean, B. Knudsen, S. Plymate, and P. S. Nelson
The Gene Expression Program of Prostate Fibroblast Senescence Modulates Neoplastic Epithelial Cell Proliferation through Paracrine Mechanisms
Cancer Res., January 15, 2006; 66(2): 794 - 802.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
S Sanz, J B Pucilowska, S Liu, C M Rodriguez-Ortigosa, P K Lund, D A Brenner, C R Fuller, J G Simmons, A Pardo, M-L Martinez-Chantar, et al.
Expression of insulin-like growth factor I by activated hepatic stellate cells reduces fibrogenesis and enhances regeneration after liver injury
Gut, January 1, 2005; 54(1): 134 - 141.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
V. Wallenius, M. Hisaoka, K. Helou, G. Levan, N. Mandahl, J. M. Meis-Kindblom, L.-G. Kindblom, and J.-O. Jansson
Overexpression of the Hepatocyte Growth Factor (HGF) Receptor (Met) and Presence of a Truncated and Activated Intracellular HGF Receptor Fragment in Locally Aggressive/Malignant Human Musculoskeletal Tumors
Am. J. Pathol., March 1, 2000; 156(3): 821 - 829.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Skrtic, K. Wallenius, A. M. Gressner, and J.-O. Jansson
Insulin-Like Growth Factor Signaling Pathways in Rat Hepatic Stellate Cells: Importance for Deoxyribonucleic Acid Synthesis and Hepatocyte Growth Factor Production
Endocrinology, December 1, 1999; 140(12): 5729 - 5735.
[Abstract] [Full Text]

Home page
 Am. J. Pathol.Home page
T. Knittel, C. Dinter, D. Kobold, K. Neubauer, M. Mehde, S. Eichhorst, and G. Ramadori
Expression and Regulation of Cell Adhesion Molecules by Hepatic Stellate Cells (HSC) of Rat Liver : Involvment of HSC in Recruitment of Inflammatory Cells during Hepatic Tissue Repair
Am. J. Pathol., January 1, 1999; 154(1): 153 - 167.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Skrtic, S.
Right arrow Articles by Jansson, J.-O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Skrtic, S.
Right arrow Articles by Jansson, J.-O.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals