0013-7227/90/1275-2343$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 5 Printed in U.S.A.
Secretion of Insulin-Like Growth Factor-I and Binding Proteins by Rat Liver Fat-Storing Cells: Regulatory Role of Platelet-Derived Growth Factor* MASSIMO PINZANI, HANNA E. ABBOUDf, AND DAVID C. ARON Department of Medicine, V.A. Medical Center and Case Western Reserve University, Cleveland, Ohio 44106
ABSTRACT. Insulin-like growth factor (IGF-I) is synthesized in multiple organs, including the liver, and may play a role in tissue growth and repair. We report that liver fat-storing cells (FSC) secrete IGF-I immunoreactivity in the culture medium. Secretion of IGF-I immunoreactivity was blocked in the presence of cycloheximide, suggesting de novo synthesis. Culture medium conditioned by FSC was concentrated and applied to a Sephadex G100 column equilibrated in a denaturing buffer. Two major species with apparent mol wts of 7.5 and greater than 25 k were identified by IGF-I RIA. Reverse phase HPLC of the 7.5 kilodalton species (the size of IGF-I) showed that it eluted in a single peak. To determine whether the higher mol wt species possessed IGF-I binding activity, appropriate fractions were desalted, incubated with [125I]IGF-I for 2 h at 30 C and applied
I
NSULIN-like growth factor-I (IGF-I) or somatomedin C is a polypeptide mitogen structurally homologous to proinsulin. IGF-I mediates many of the effects of GH on target tissues. Although the liver has been considered the primary site of IGF-I synthesis, in situ hybridization and Northern blot hybridization studies have demonstrated the presence of IGF-I mRNA in a wide variety of tissues, and several studies indicate that IGF-I immunoreactivity is present in other organs and tissues (1, 2). In addition, IGF-I production has been shown in a variety of cell types in vitro, particularly cells of mesenchymal origin (3-5). The production of IGF-I by many cell types suggests that IGF-I may act not only in an endocrine manner but also as a paracrine or autocrine factor and that biologically active IGF-I levels can be regulated locally at the site of action (6-8). IGF-I circulates in Received April 26, 1990. Address correspondence and requests for reprints to: David C. Aron, M.D., Endocrinology Section 151W, Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, Ohio 44106. * Portions of this work were presented in abstract form at the annual meeting of the American Association for the Study of Liver Diseases, Chicago, IL, October 28-31,1989. These studies were supported by the Medical Research Service of the Department of Veterans Affairs, grants-in-aid from the American Heart Association (H.E.A. and D.C.A.) and by NIH Grants DK-33665 and DK-41527. t Established Investigator of the American Heart Association.
to a Sephadex G100 column equilibrated in a nondissociating buffer. The major peak of radioactivity was confined to a high mol wt region. Western blot ligand analysis revealed the presence of two insulin-like growth factor binding proteins of approximately 28 and 31 kilodaltons. Platelet-derived growth factor, a potent mitogen for FSC, resulted in a 230% increase in release of IGF-I immunoreactivity that could be accounted for by an increase in IGF-I binding activity. In addition IGF-I increased DNA synthesis in FSC and this effect was additive to that of platelet-derived growth factqr. IGF-I treatment also resulted in an increase in cell number. IGF-I and insulin-like growth factor binding proteins secreted by FSC may play a role in the hepatic tissue response to injury via autocrine and/or paracrine mechanisms. (Endocrinology 127: 2343-2349, 1990)
plasma tightly bound to specific binding proteins. Several IGF-binding proteins (IGFBP) have been identified not only in serum but also in culture medium conditioned by a variety of cell types (9-14). These proteins may be involved in the regulation of the biological actions of IGF-I (15), including mitogenic activity as well as regulation of extracellular matrix components (16, 17). While IGF-I and IGFBP secretion by primary hepatocyte cultures and established cell lines of presumed hepatocyte origin has been shown (18-22), recent studies in fetal human liver have localized IGF-I mRNA to perisinusoidal cells (1). Liver fat-storing cells (FSC), perisinusoidal cells of mesenchymal origin, have been shown to play a key role in the production of several matrix components, including collagen (23, 24) and proteoglycans (25) and are potential sources of IGF-I. In the present study we examined if liver FSC are able to secrete IGF-I and/or IGFBP and we explored the effect of platelet-derived growth factor (PDGF), a potent mitogen for FSC (26, 27), on the release of IGF-I and IGFBP. Our results indicate that FSC secrete a 7.5 kilodalton (kDa) IGF-I immunoreactive species as well as two small binding species of approximately 28 and 31 kDa. PDGF increases the release of IGFBP from FSC. In addition, IGF-I is a mitogen for FSC and its effects are additive to those of PDGF.
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IGF-I AND IGFBP IN LIVER FAT-STORING CELLS
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Materials and Methods Growth factors Recombinant human IGF-I (hIGF-I) and recombinant multiplication stimulating activity [(MSA) rat IGF-II) were obtained from Collaborative Research Inc. (Lexington, MA). Recombinant PDGF (PDGFv.9is) was obtained from Amgen Inc. (Thousand Oaks, CA). Recombinant human GH was obtained from Eli Lilly Co. (Indianapolis, IN). Cell isolation and culture Liver FSC were isolated from the liver of male SpragueDawley rats weighing 450-500 g (Charles River Breeding Laboratories, Inc., Wilmington, MA) by the method of Friedman and Roll (28) with minor modifications (27). Primary cultures of the cells were allowed to grow until confluent in plastic culture dishes or flasks (Costar, Cambridge, MA) in Waymouth's MB 752/1 medium (Gibco Laboratories, Grand Island, NY), supplemented with 15 mM HEPES, 0.6 U/ml insulin (Sigma, Chemical Co., St. Louis, MO), 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, antibiotic-antifungal solution, 10% horse serum (7-globulin free; Gibco Laboratories), and 10% calf serum (HyClone Laboratories, Inc., Logan, UT). For further passage, confluent cells were washed with Hank's balanced salt solution (HBSS) without calcium and magnesium and removed with 0.025% trypsin/0.5 mM EDTA in calcium- and magnesium-free Dulbecco's PBS (Gibco Laboratories) and plated in Waymouth's medium. Identity and purity of rat liver FSC was confirmed by detailed morphological criteria as previously reported (27). Experiments included in this study were performed on cells between first and third passage from two different cell lines. Release of IGF-I immunoreactivity by FSC Confluent FSC were washed twice with serum-free/insulinfree (SFIF) Waymouth's medium and incubated overnight in the same medium. This medium was then discarded and fresh SFIF medium was added with or without cycloheximide (Cx) at a final concentration of 10~5 M. Conditioned medium was collected at 8, 24, and 48 h, centrifuged and supernatants stored in polypropylene tubes at —70 C for the RIA. Cells in each well were trypsinized and counted using a Coulter counter (Coulter Electronics, Hialeah, FL). The antiserum UBK487 used for the RIA was a gift of Drs. L. Underwood and J. J. Van Wyk, distributed by the Hormone Distribution Program of the NIDDK. This antiserum has a 0.5% cross-reactivity with IGFII and minimal cross-reactivity with insulin at 10"6. It was used at a final dilution of 1:18,000. The RIA was performed in polypropylene tubes using a protamine-containing phosphate buffer. Recombinant hIGF-I was used as a standard. Standards and unknown were incubated in duplicate with antibody for 2 h at room temperature before the addition of [125I]Thr59-IGF-I (Amersham Co., Arlington Heights, IL). After overnight incubation, the antibody-bound [125I] IGF-I was precipitated using goat antirabbit 7-globulin and normal rabbit serum as a carrier as previously reported (29).
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Characterization of the immunoreactiue species by gel filtration chromatography and HPLC In order to concentrate IGF-I moieties, SFIF-conditioned medium from FSC was applied to a 500-mg octadecylsilane silica column (29, 30). The column was then washed with 1% trifluoroacetic acid, and retained species were eluted with 90% acetonitrile in 0.1% aqueous trifluoroacetic acid. The dried eluates were dissolved in 6 M guanidine-HCl and applied to a 0.7 x 45-cm Sephadex G-100 column equilibrated in 6 M guanidine HCl-0.03% Brij. The column was calibrated with a series of standards, including blue dextran, cytochrome-c (mol wt 12,400) and [125I]Thr59-IGF-I. The recovery of the applied radioactivity was greater than 90%. For HPLC analysis, gel filtration fractions (200 ^1) were injected onto a C18 analytical column (Vydac, The Separations Group, Hesperia, CA) and were eluted using an acetonitrile-water gradient containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. One milliliter fractions were collected and dried, and the residues dissolved in RIA buffer. Charcoal adsorption assay for IGF-I binding activity IGF-I binding activity was measured using a modification of the method of Martin and Baxter (31). Duplicate or triplicate aliquots of conditioned media were incubated with 125I-IGF-I (10,000 cpm) in a volume of 500 n\ 50 mM Tris-HCl, 0.1% BSA (Sigma), pH 7.4. After 2-h incubation at room temperature, 1% activated charcoal (Sigma) without protamine was added to the tubes followed by incubation for 15 min and centrifugation for 30 min, both at 4 C. Aliquots of supernatant containing protein bound [125I]IGF-I were counted in a 7-counter. Nonconditioned medium was assayed in parallel and was subtracted from total bound radioactivity to determine specific IGF-I binding activity, expressed as a percentage of total counts per min. Doseresponse curves were generated by assaying three concentrations (4-20 /il) of culture medium. Displacement curves were generated by coincubation with multiple dilutions of unlabeled hIGF-I, rat IGF-II, or bovine insulin. In some experiments, the IGF-I binding activity was measured in the absence and presence of PDGF. Western blot analysis of IGF-I binding protein(s) Unreduced samples of conditioned medium from FSC and from Buffalo rat liver cells (kindly provided by Dr. N. Ross, Case Western Reserve University) were electrophoresed through a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. Separated proteins were electroblotted onto nitrocellulose filters (0.45 /*m pore size). Filters were washed and then incubated with [125I]IGF-I overnight at 4 C and visualized by autoradiography according to the method of Hassenlopp et al. (32). Effect of IGF-I and PDGF on DNA synthesis in FSC DNA synthesis was measured as the amount of [methyl-3H] thymidine ([3H]TdR) incorporated into trichloroacetic acid (TCA)-precipitable material. Cells were plated in 24-well dishes at a density of 2 x 104 cells per well and incubated with
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IGF-I AND IGFBP IN LIVER FAT-STORING CELLS Waymouth's medium containing 10% horse serum and 10% calf serum until they became confluent (density of about 1.0 X 105 cells per well). Confluent cells were made quiescent by placing them for 48 h in SFIF Waymouth's medium containing 1% Zeta serum (AMF, Meriden, CT), a low mitogenicity serum. Cells were then incubated with or without the conditions to be tested for 20 h and then pulsed for 4 h with 1.0 /uCi/ml [3H] TdR (6.7 Ci/mmol; New England Nuclear, Boston, MA). At the end of the pulsing period medium was carefully aspirated, ice-cold 5% TCA was added, and dishes were kept on ice for 15 min. After two additional washes with 5% TCA, cells were solubilized by adding 750 fil 0.25 N NaOH, 0.1% SDS. Five tenths ml of the solubilized cells solution was then neutralized with 50 MI 6 N HC1 and counted in a scintillation counter. Cell number was determined in three separate wells from each dish after trypsinization and counting in a Coulter counter. Cell proliferation assay 4
Rat FSC were plated in 12-well dishes at a density of 4 x 10 in Waymouth's medium containing serum. After 24 h (day 0), the cells were washed and placed in fresh SFIF Waymouth's medium containing 1% Zeta-serum with or without synthetic IGF-I (100 ng/ml) and allowed to grow for 3 or 6 days. Fresh medium with or without IGF-I was added to the remaining wells at day 3. Cells were trypsinized and counted using a Coulter counter.
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I5OO-
5
1000-
5OO-
FIG. 1. Time course for IGF-I immunoreactivity in SFIF medium conditioned (CM) by rat liver FSC. Confluent FSC were washed twice with Waymouth's SFIF medium and incubated overnight in the same medium. This medium was then discarded and fresh SFIF medium was added with or without Cx at a final concentration of 10~5 M. Conditioned medium was collected at indicated time points and frozen at —70 C until assayed. Data, expressed as mean ± SE, are from two experiments done in triplicate. Changes were statistically significant at 8 h (P < 0.01), 24 and 48 h (P < 0.001). 5.0
Vi
Statistical analysis Data, expressed as mean ± SD or SE, were analyzed by analysis of variance.
Results The time course for IGF-I immunoreactivity in SFIF medium conditioned by rat FSC is illustrated in Fig. 1. In the absence of Cx, levels of IGF-I immunoreactivity were readily detectable after 8 h of incubation with a further time-dependent increase after 24 and 48 h. When the cells were incubated with 10~5 M Cx (a concentration which did not affect cell viability as assessed by both cell counting and Trypan blue exclusion) a marked reduction of IGF-I immunoreactivity compared to control wells was observed at each time point, indicating that the presence of IGF-I immunoreactivity in the media is likely due to de nouo protein synthesis. To characterize further the IGF-I immunoreactive species, SFIF culture medium conditioned by FSC was concentrated by passage over an octadecylsilane silica column and applied to a 0.7 x 45 cm Sephadex G-100 column equilibrated in a 6 mM guanidine-HCl denaturing buffer. As shown in Fig. 2, the gel filtration fractions demonstrated two major peaks of apparent IGF-I immunoreactivity. One peak coeluted with [125I]Thr59IGFI, indicating a mol wt of approximately 7.5-8.0 k, the size of native IGF-I. A second peak of IGF-I immunoreactivity had an apparent mol wt greater than 25 k. The
o
jS
4.0
p O
25 kD) region, suggesting the presence of IGF-I binding activity. In the presence of excess unlabeled IGF-I, radioactivity eluted with radioiodinated IGF-I suggesting that the binding activity was specific for IGF-I. IGF-I binding activity in conditioned medium was then characterized by Western blot analysis. As shown in Fig. 5, two IGF-I binding species of approximately 28 and 31 kDa were observed in conditioned medium from rat FSC while a single species of 31 kDa was observed from medium conditioned by Buffalo rat liver cells. IGF-I binding in conditioned medium from FSC was further studied using a charcoal adsorption assay. Serial dilutions of conditioned medium ranging from 0.8-4% resulted in a linear dose-dependent increase in specific binding of [125I]IGFI (data not shown). The competitive binding characteristics of FSC conditioned medium are shown in Fig. 6. Displacement of [125I]IGF-I by hIGF-I and rat IGF-II were similar. There was no displacement by bovine insulin. This pattern of displacement is characteristic of
FlG. 4. Sephadex G100 gel filtration profile (PBS pH 7.4) of [125I]IGFI binding to greater than 25 kDa IGF-I immunoreactive species from FSC-conditioned medium (48 h). The elution position of mol wt markers are indicated by the arrows. Vo, Void volume (blue dextran); Vi, 125I volume. In the absence of excess unlabeled IGF-I, radioactivity elutes at a high mol wt (>25 kD). Addition of excess unlabeled IGF-I resulted in elution of radioactivity at the mol wt of radioiodinated IGF-I.
97.466.2-
FIG. 5. Western blot analysis of IGF-I binding protein(s) in FSC conditioned medium (24 h). Samples of culture medium conditioned by FSC (15 /xl) or Buffalo rat liver cells (1 ft\) were subjected to electrophoresis through a SDS-polyacrylamide gel (10%). Separated proteins were electroblotted onto nitrocellulose filters and then incubated with [125I]IGF-I overnight at 4 C and visualized by autoradiography. Molecular weights in kDa were determined by comparison to a series of standards. Species of 28 and 31 kDa were observed in conditioned medium from FSC (lane 2) and 31 kDa in conditioned medium from Buffalo rat liver cells (lane 1).
IGFBP (33). The effects of PDGF on levels of IGF-I immunoreactivity and IGF-I binding activity were then studied. With PDGF treatment, IGF-I immunoreactivity increased significantly, 2.3-fold (mean of three experiments, P < 0.005). Since samples of conditioned medium were not extracted to eliminate interference with the RIA by IGFBP, we then assessed the effect of PDGF on IGF-I
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IGF-I AND IGFBP IN LIVER FAT-STORING CELLS
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120
T
2800 100
~ c CD
S o
2600
• IGF-I
80
D MSA •
Insulin
60
2400 -
m °
600
40
20
:
j
T
400 0.5
1.0
5.0 10.0
50.0 100
500 1000
5000 10000
nq Peptide/ml 128
FIG. 6. Competition curves for [ I]IGF-I binding displacement by human IGF-I, MSA (rat IGF-II) and insulin. SFIF medium conditioned by FSC was harvested as stated in Materials and Methods. IGF-I binding activity was assessed by the charcoal adsorption assay. Nonconditioned medium was assayed in parallel and subtracted to determine specific IGF-I binding activity. Samples were incubated with serial dilutions of IGF-I, IGF-II, or insulin. Basal binding in this representative experiment was 28.1% of added counts. Results are expressed as percentage of basal binding. The points represent the means of triplicate determinations.
binding activity. IGF-I binding activity also increased significantly, 2.1-fold (P < 0.01). In parallel experiments, incubation of FSC monolayers with GH (100 ng/ml) for 24 or 48 h did not result in any significant increase of IGF-I immunoreactivity in the conditioned medium. Quiescent rat FSC in culture were assayed for their ability to incorporate [3H]TdR into DNA after exposure to IGF-I alone, to PDGF alone, or to a combination of IGF-I plus PDGF for a total of 24 h. As illustrated in Fig. 7, the addition of IGF-I induced a significant increase in DNA synthesis when compared to 1% Zeta serum alone. However, this effect was small compared to the effect of a submaximal dose of PDGF. The dose of IGF-I used (100 ng/ml) appeared to be maximal since higher doses (up to 500 ng/ml) did not induce additional effects on [3H]TdR incorporation. The combination of IGF-I and PDGF induced an additive increase in [3H] TdR incorporation compared with PDGF alone, but the effect was additive not synergistic. To confirm that the enhanced [3H]TdR incorporation induced by IGF-I is associated with cell proliferation, we performed cell counts on cultures incubated with this growth factor. Basal cell number was 4.4 ± 0.17 x 104. The number of cells did not change when maintained in 1% Zeta serum alone, but cell counts significantly increased to 5.5 ± 0.33 x 104 and 6.1 ± 0.48 x 104 after 3 and 6 days of exposure to IGF-I, respectively ( P < 0.05 for both values). Discussion IGF-I or somatomedin C is a biologically active growth factor with diverse properties (8). The liver is a major
T
200
(19)
(21)
(8)
(8)
1%ZS -Ins
+IGF-I +PDGF 100ng/ml 2.5ng/ml
+PDGF 2.5ng/ml + IGF-I 100ng/ml
FlG. 7. Effect of synthetic IGF-I on [3H]thymidine incorporation into DNA of quiescent rat FSC. Confluent cells were made quiescent by incubation in insulin-free Waymouth's medium containing 1% Zeta serum for 48 h. Cells were then incubated with IGF-I, PDGF, or IGFI plus PDGF for 20 h and then pulsed for 4 h with 1.0 /uCi/ml [3H] TdR. Data are mean ± SD. The numbers shown on the bars indicate the number of wells tested. *, P < 0.01 vs. 1% Zeta serum alone; **, P < 0.01 vs. PDGF alone.
source of circulating IGF-I. High levels of IGF-I mRNA have been demonstrated in adult rat liver (34) and in primary cultures of rat liver cells (20). Production of IGF-I and IGFBP in adult rat hepatocytes in primary culture appears to be under the control of GH (18, 19). Recent studies in fetal human liver have localized IGF-I mRNA to perisinusoidal cells (1). Several recent studies demonstrated that cells of mesenchymal origin such as fibroblasts (5, 35), smooth muscle cells (5), and glomerular mesangial cells (29) synthesize or secrete IGF-I-like peptides and IGFBP with potential implication in local tissue hypertrophy and repair. These observations raise the possibility that liver FSC, liver-specific pericytes (capillary smooth muscle cells), produce and release IGFI and/or IGFBP. Our results indicate that liver FSC secrete an IGF-I immunoreactive species which has the same size and hydrophobicity as human IGF-I suggesting that it is IGFI protein. In addition, these cells also secrete two small IGFBP of 28 and 31 k mol wt. The release of IGF-I immunoreactivity from FSC appears to be under the control of PDGF but not GH. Since the increase in IGFI binding activity, as assessed by the charcoal adsorption assay, is of the same magnitude (2-fold) as that of IGFI immunoreactivity, it is likely that this increase in IGFI binding accounts for most if not all of the increase in apparent IGF-I immunoreactivity, indicating that, in FSC, the effect of PDGF is mainly directed to the regu-
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IGF-I AND IGFBP IN LIVER FAT-STORING CELLS
lation of IGFBP secretion. The 1:1 ratio of IGFBP to the 7.5 kDa IGF-I immunoreactive species in conditioned medium based upon denaturing gel filtration chromatography is an underestimate due to the low recovery of IGFBP from reverse phase chromatography (30). The identification of various types of IGFBP has generated several hypotheses concerning their origin and role. While large mol wt GH-dependent IGFBP may be important for increasing the half-life of circulating IGF-I and protecting tissues against the insulin-like effects of free IGF-I, smaller GH-independent IGFBP, with a mol wt of about 30 kDa, may have a role in modulating the local effects of IGF-I. Purified IGFBP have been shown to enhance (36, 37) or to attenuate IGF-I effects (38). However, the factors that determine whether the modulating effects of IGFBP are positive or negative are still largely unknown. IGFBP with similar mol wt and structure, could exert different functional effects in different cell types under different conditions. At present, three rat IGFBP (rIGFBP-1, 2, and 3) have been identified (22). While rIGFBP-3 has a mol wt of 40 kDa and it is thought to be the glycosylated binding subunit of the circulating 150 kDa species, rIGFBP-1 and 2 have a mol wt of approximately 30 k and are not glycosylated. The displacement of [125I]IGF-I by IGF-I and IGF-II but not by insulin, observed in experiments using the charcoal adsorption assay of nonreduced conditioned medium, is characteristic of IGFBP (33). It is likely that at least one of two IGF-I binding species found in FSC conditioned media corresponds to rIGFBP-1 or 2, although further characterization will be required. We and others have recently demonstrated the presence of PDGF receptors on FSC (26, 27). PDGF as well as other polypeptide growth factors have been shown to increase secretion of IGF-I immunoreactivity in cultured human fibroblasts (39, 40) and smooth muscle cells (36), leading to the concept of a local regulation of IGF-I release. Multiple IGFBP with mol wt ranging from 41.523 k have been detected in media conditioned by cultured human fibroblasts and their secretion appears to be under the control of GH whereas it is unaffected by PDGF (14). Our findings indicate that PDGF induces a significant increase in the secretion of IGF-I binding activity and, therefore, the hormonal regulation of IGFI binding species secreted by FSC differs from that present in other cell types. Several studies have also suggested that IGF-I and PDGF interact in regulating growth of smooth muscle cells, fibroblasts, glomerular mesangial cells, and other mesenchymal cell types (4144). PDGF has been thought to modulate cell growth in response to IGF-I, this latter acting as a "progression factor" (42,44). In our experimental conditions, however, we found that IGF-I and PDGF interact additively on DNA synthesis in FSC suggesting that the two growth
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factors act through different pathways. These results are in agreement with recent studies showing additive effects of IGF-I and PDGF in the induction of the protooncogene c-myc and cell proliferation in smooth muscle cells (45). While hepatocytes constitute the major source of circulating IGF-I, the results of the present study suggest that FSC are another potential source of this peptide. The precise biological role of IGF-I and IGFBP synthesized by FSC remains to be determined. IGF-I, like PDGF, is a mitogen for FSC. IGF-I, in concert with other cytokines released by activated macrophages (46, 47), may contribute to both proliferative and fibrotic responses during liver inflammation. As already shown for other mesenchymal cells (16, 17), IGF-I could also influence the production of matrix components by FSC, currently considered the predominant source of collagen and proteoglycan within the liver (23-25). The secretion by FSC of IGFBP, which are regulated by PDGF, may be important in regulating the local effects of IGF-I. Acknowledgments We thank Christina N. Nye and Marina Karam for expert technical assistance.
References 1. Han VKM, D'Ercole AJ, Lund PK 1987 Cellular localization of somatomedin (Insulin-like growth factor) messenger RNA in the human fetus. Science 236:193-197 2. Lund PK, Moats-Staats BM, Haynes MA, Simmons JG, Jansen D'Ercole AJ, Van Wyk JJ 1986 Somatomedin-C/Insulin-like growth factor-I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues. J Biol Chem 261:14539-14544 3. Atkinson PR, Weidman ER, Bhaumick B, Bala RM 1980 Release of somatomedin-like activity by cultured WI-38 human fibroblasts. Endocrinology 106:2006-2012 4. Clemmons DR, Underwood LE, Van Wyk JJ 1981 Hormonal control of immunoreactive somatomedin production by cultured human fibroblasts. J Clin Invest 67:10-19 5. Clemmons DR, Van Vyk JJ 1985 Evidence for a functional role of endogenously produced somatomedin-like peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells. J Clin Invest 75:1914-1918 6. D'Ercole AJ, Stiles AD, Underwood LE 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 7. Underwood LE, D'Ercole AJ, Clemmons DR, Van Wyk JJ 1986 Paracrine functions of somatomedins. Clin in Endocrinol Metab 15:59-77 8. Holly JMP, Wass JAH 1989 Insulin-like growth factors; autocrine, paracrine or endocrine? New perspectives of the somatomedin hypothesis in the light of recent developments. J Endocrinol 122:611-618 9. Baxter RC, Martin JL 1986 Radioimmunoassay of growth-hormone-dependent insulin-like growth factor binding protein in human plasma. J Clin Invest 78:1504-1512 10. Koistinen R, Kalkkinen N, Huhtala M, Seppala M, Bohn H, Rutanen E 1986 Placental protein 12 is a decidual protein that bind somatomedins and has identical N-terminal amino acid sequence with somatomedin-binding protein from human amniotic fluid. Endocrinology 118:1375-1378
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IGF-I AND IGFBP IN LIVER FAT-STORING CELLS 11. Clemmons DR, Elgin RG, Han VKM, Casella SJ, D'Ercole AJ, Van Wyk JJ 1986 Cultured fibroblasts monolayer secrete a protein that alters the cellular binding of somatomedin-C/insulinlike growth factor I. J Clin Invest 77:1548-1553 12. DeVroede MA, Tseng LY-H, Katsoyannis G, Nissley SP, Rechler MM 1986 Modulation of insulinlike growth factor I binding to human fibroblasts monolayer cultures by insulin-like growth factor carrier proteins released to the incubation media. J Clin Invest 77:602-613 13. Baxter RC, Martin JL, Wood MH 1987 Two immunoreactive binding proteins for insulin-like growth factors in human amniotic fluid: relationship to fetal maturity. J Clin Endocrinol Metab 65:423-431 14. Conover CA, Liu F, Powell D, Rosenfeld RG, Hintz RL 1989 Insulin-like growth factor binding proteins from cultured human fibroblasts. Characterization and hormonal regulation. J Clin Invest 83:852-859 15. Baxter RC, Martin JL 1989 Binding proteins for the insulin-like growth factors: structure, regulation and function. Progress in growth factor research. 1:49-68 16. Bar RS, Dake BL, Stueck S 1987 Stimulation of proteoglycans by IGF-I and II in microvessel and large vessel endothelial cells. Am J Physiol 253:E21-E27 17. Goldstein RH, Poliks CF, Pilch PF, Smith BD, Fine A 1989 Stimulation of collagen formation by insulin and insulin-like growth factor I in cultures of human lung fibroblasts. Endocrinology 124:964-970 18. Scott CD, Martin JL, Baxter RC 1985 Production of insulin-like growth factor I and its binding protein by adult rat hepatocytes in primary culture. Endocrinology 116:1094-1101 19. Scott CD, Martin JL, Baxter RC 1985 Rat hepatocyte insulin-like growth factor and binding protein: effect of growth hormone in vitro and in vivo. Endocrinology 116:1102-1107 20. Norstedt G, Moller C 1987 Growth hormone induction of insulinlike growth factor I messenger RNA in primary cultures of rat liver cells. J Endocrinology 15:135-139 21. Lee Y-L, Hintz RL, James PM, Lee PDK, Shively JE, Powell DR 1988 Insulin-like growth factor (IGF) binding protein complementary deoxyribonucleic acid from human HEP G2 hepatoma cells: predicted protein sequence suggest an IGF binding domain different from those of IGF-I and IGF-II receptors. Mol Endocrinol 2:404-411 22. Yang YWH, Brown AL, Orlowski CC, Graham DE, Tseng LYH, Romanus JA, Rechler MM 1990 Identification of rat cell lines that preferentially express insulin-like growth factor binding proteins rIGFBP-1,2, or 3. Mol Endocrinol 4:29-38 23. Friedman SL, Roll FJ, Boyles J, Bissell DM 1985 Hepatic lipocytes: the principal collagen-producing cells of normal liver. Proc Natl Acad Sci USA 82:8681-8685 24. Maher JJ, Bissell DM, Friedman SL, Roll FJ 1988 Collagen measured in primary cultures of normal rat hepatocytes derives from lipocytes within the monolayer. J Clin Invest 82:450-459 25. Arenson DM, Friedman SL, Bissell DM 1988 Formation of extracellular matrix in normal rat liver: lipocytes as a major source of proteoglycan. Gastroenterology 95:441-447 26. Friedman SL, Arthur MJP 1989 Activation of cultured rat hepatic lipocytes by Kuppfer cell conditioned medium. Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. J Clin Invest 84:1780-1785 27. Pinzani M, Gesualdo L, Sabbah GM, Abboud HE 1989 Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells. J Clin Invest 84:1786-1793 28. Friedman SL, Roll FJ 1987 Isolation and culture of hepatic lipo-
29. 30. 31. 32.
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35. 36. 37.
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46. 47.
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cytes, Kuppfer cells, and sinusoidal endothelial cells by density gradient centrifugation with stractan. Anal Biochem 161:207-218 Aron DC, Rosenzweig JL, Abboud HE 1989 Synthesis and binding of insulin-like growth factor I by human glomerular mesangial cells. J Clin Endocrinol Metab 68:585-591 Daughaday WH, Kapadia M, Mariz I 1987 Serum somatomedin binding proteins: physiological significance and interference in radioligand assay. J Lab Clin Med 109:355-363 Martin JL, Baxter RC 1986 Insulin-like growth factor-binding protein from human plasma: purification and characterization. J Biol Chem 261:8754-8760 Hassenlopp P, Sevrin D, Segovia-Quinson B, Hardouin S, Binoux M 1986 Analysis of serum insulin-like growth factor binding proteins using Western blotting. Use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 154:138-143 Rosenfeld RG, Pham H, Oh Y, Ocrant I 1989 Characterization of insulin-like growth factor-binding proteins in cultured rat pituitary cells. Endocrinology 124:2867-2874 Norstedt G, Levinovitz A, Moller C, Eriksson LC, and Andersson G 1988 Expression of insulin-like growth factor I (IGF-I) and IGFII mRNA during hepatic development, proliferation and carcinogenesis in the rat. Carcinogenesis 9:209-213 Clemmons DR, Shaw DS 1986 Purification and biologic properties of fibroblast somatomedin. J Biol Chem 261:10293-10298 Elgin RG, Busby WH, Clemmons DR 1987 An insulinlike growth factor £IGF-I) binding protein enhances the biologic response to IGF-I. Proc Natl Acad Sci USA 84:3254-3258 Blum WF, Jenne EW, Reppin F, Kietzmann K, Ranke MB, Bierich JR 1989 Insulin-like growth factor I (IGF-I)binding protein complex is a better mitogen than free IGF-I. Endocrinology 125:766772 Knaver DJ, Smith GL 1980 Inhibition of biological activity of multiplication-stimulating activity by binding to its carrier proteins. Proc Natl Acad Sci USA 77:7252-7257 Clemmons DR, Shaw DS 1983 Variables controlling somatomedin production by cultured human fibroblasts. J Cell Physiol 115:137142 Clemmons DR 1984 Multiple hormones stimulate the production of somatomedin by cultured human fibroblasts. J Clin Endocrinol Metab 58:850-856 Clemmons DR 1985 Exposure to platelet-derived growth factor modulates the porcine aortic smooth muscle cell response to somatomedin-C. Endocrinology 117:77-83 Stiles CD, Capone GT, Scher CD, Antoniades HN, Van Wyk JJ, Pledger WJ 1979 Dual control of cell growth by somatomedins and platelet-derived growth factor. Proc Natl Acad Sci USA 76:12791283 Clemmons DR, Van Wyk JJ 1981 Somatomedin-C and plateletderived growth factor stimulate human fibroblasts replication. J Cell Physiol 106:361-367 Doi T, Striker LJ, Elliot SJ, Conti FG, Striker GE 1989 Insulinlike growth factor-1 is a progression factor for human mesangial cells. Am J Pathol 134:395-404 Banskota NK, Taub R, Zellner K, King GL 1989 Insulin, insulinlike growth factor I and platelet-derived growth factor interact additively in the induction of the protooncogene c-myc and cellular proliferation in cultured bovine aortic smooth muscle cells. Mol Endocrinol 3:1183-1190 Rom WN, Bassett P, Fells GA, Nukiwa T, Trapnell BC, Crystal RG 1988 Alveolar macrophages release an insulin-like growth factor I-type molecule. J Clin Invest 82:1685-1693 Nagoaka I, Trapnell BC, Crystal RG 1990 Regulation of insulinlike growth factor I gene expression in the human macrophage-like cell line U937. J Clin Invest 85:448-455
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Vol. 127, No. 5 Printed in U.S.A.
Secretion of Insulin-Like Growth Factor-I and Binding Proteins by Rat Liver Fat-Storing Cells: Regulatory Role of Platelet-Derived Growth Factor* MASSIMO PINZANI, HANNA E. ABBOUDf, AND DAVID C. ARON Department of Medicine, V.A. Medical Center and Case Western Reserve University, Cleveland, Ohio 44106
ABSTRACT. Insulin-like growth factor (IGF-I) is synthesized in multiple organs, including the liver, and may play a role in tissue growth and repair. We report that liver fat-storing cells (FSC) secrete IGF-I immunoreactivity in the culture medium. Secretion of IGF-I immunoreactivity was blocked in the presence of cycloheximide, suggesting de novo synthesis. Culture medium conditioned by FSC was concentrated and applied to a Sephadex G100 column equilibrated in a denaturing buffer. Two major species with apparent mol wts of 7.5 and greater than 25 k were identified by IGF-I RIA. Reverse phase HPLC of the 7.5 kilodalton species (the size of IGF-I) showed that it eluted in a single peak. To determine whether the higher mol wt species possessed IGF-I binding activity, appropriate fractions were desalted, incubated with [125I]IGF-I for 2 h at 30 C and applied
I
NSULIN-like growth factor-I (IGF-I) or somatomedin C is a polypeptide mitogen structurally homologous to proinsulin. IGF-I mediates many of the effects of GH on target tissues. Although the liver has been considered the primary site of IGF-I synthesis, in situ hybridization and Northern blot hybridization studies have demonstrated the presence of IGF-I mRNA in a wide variety of tissues, and several studies indicate that IGF-I immunoreactivity is present in other organs and tissues (1, 2). In addition, IGF-I production has been shown in a variety of cell types in vitro, particularly cells of mesenchymal origin (3-5). The production of IGF-I by many cell types suggests that IGF-I may act not only in an endocrine manner but also as a paracrine or autocrine factor and that biologically active IGF-I levels can be regulated locally at the site of action (6-8). IGF-I circulates in Received April 26, 1990. Address correspondence and requests for reprints to: David C. Aron, M.D., Endocrinology Section 151W, Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, Ohio 44106. * Portions of this work were presented in abstract form at the annual meeting of the American Association for the Study of Liver Diseases, Chicago, IL, October 28-31,1989. These studies were supported by the Medical Research Service of the Department of Veterans Affairs, grants-in-aid from the American Heart Association (H.E.A. and D.C.A.) and by NIH Grants DK-33665 and DK-41527. t Established Investigator of the American Heart Association.
to a Sephadex G100 column equilibrated in a nondissociating buffer. The major peak of radioactivity was confined to a high mol wt region. Western blot ligand analysis revealed the presence of two insulin-like growth factor binding proteins of approximately 28 and 31 kilodaltons. Platelet-derived growth factor, a potent mitogen for FSC, resulted in a 230% increase in release of IGF-I immunoreactivity that could be accounted for by an increase in IGF-I binding activity. In addition IGF-I increased DNA synthesis in FSC and this effect was additive to that of platelet-derived growth factqr. IGF-I treatment also resulted in an increase in cell number. IGF-I and insulin-like growth factor binding proteins secreted by FSC may play a role in the hepatic tissue response to injury via autocrine and/or paracrine mechanisms. (Endocrinology 127: 2343-2349, 1990)
plasma tightly bound to specific binding proteins. Several IGF-binding proteins (IGFBP) have been identified not only in serum but also in culture medium conditioned by a variety of cell types (9-14). These proteins may be involved in the regulation of the biological actions of IGF-I (15), including mitogenic activity as well as regulation of extracellular matrix components (16, 17). While IGF-I and IGFBP secretion by primary hepatocyte cultures and established cell lines of presumed hepatocyte origin has been shown (18-22), recent studies in fetal human liver have localized IGF-I mRNA to perisinusoidal cells (1). Liver fat-storing cells (FSC), perisinusoidal cells of mesenchymal origin, have been shown to play a key role in the production of several matrix components, including collagen (23, 24) and proteoglycans (25) and are potential sources of IGF-I. In the present study we examined if liver FSC are able to secrete IGF-I and/or IGFBP and we explored the effect of platelet-derived growth factor (PDGF), a potent mitogen for FSC (26, 27), on the release of IGF-I and IGFBP. Our results indicate that FSC secrete a 7.5 kilodalton (kDa) IGF-I immunoreactive species as well as two small binding species of approximately 28 and 31 kDa. PDGF increases the release of IGFBP from FSC. In addition, IGF-I is a mitogen for FSC and its effects are additive to those of PDGF.
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IGF-I AND IGFBP IN LIVER FAT-STORING CELLS
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Materials and Methods Growth factors Recombinant human IGF-I (hIGF-I) and recombinant multiplication stimulating activity [(MSA) rat IGF-II) were obtained from Collaborative Research Inc. (Lexington, MA). Recombinant PDGF (PDGFv.9is) was obtained from Amgen Inc. (Thousand Oaks, CA). Recombinant human GH was obtained from Eli Lilly Co. (Indianapolis, IN). Cell isolation and culture Liver FSC were isolated from the liver of male SpragueDawley rats weighing 450-500 g (Charles River Breeding Laboratories, Inc., Wilmington, MA) by the method of Friedman and Roll (28) with minor modifications (27). Primary cultures of the cells were allowed to grow until confluent in plastic culture dishes or flasks (Costar, Cambridge, MA) in Waymouth's MB 752/1 medium (Gibco Laboratories, Grand Island, NY), supplemented with 15 mM HEPES, 0.6 U/ml insulin (Sigma, Chemical Co., St. Louis, MO), 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, antibiotic-antifungal solution, 10% horse serum (7-globulin free; Gibco Laboratories), and 10% calf serum (HyClone Laboratories, Inc., Logan, UT). For further passage, confluent cells were washed with Hank's balanced salt solution (HBSS) without calcium and magnesium and removed with 0.025% trypsin/0.5 mM EDTA in calcium- and magnesium-free Dulbecco's PBS (Gibco Laboratories) and plated in Waymouth's medium. Identity and purity of rat liver FSC was confirmed by detailed morphological criteria as previously reported (27). Experiments included in this study were performed on cells between first and third passage from two different cell lines. Release of IGF-I immunoreactivity by FSC Confluent FSC were washed twice with serum-free/insulinfree (SFIF) Waymouth's medium and incubated overnight in the same medium. This medium was then discarded and fresh SFIF medium was added with or without cycloheximide (Cx) at a final concentration of 10~5 M. Conditioned medium was collected at 8, 24, and 48 h, centrifuged and supernatants stored in polypropylene tubes at —70 C for the RIA. Cells in each well were trypsinized and counted using a Coulter counter (Coulter Electronics, Hialeah, FL). The antiserum UBK487 used for the RIA was a gift of Drs. L. Underwood and J. J. Van Wyk, distributed by the Hormone Distribution Program of the NIDDK. This antiserum has a 0.5% cross-reactivity with IGFII and minimal cross-reactivity with insulin at 10"6. It was used at a final dilution of 1:18,000. The RIA was performed in polypropylene tubes using a protamine-containing phosphate buffer. Recombinant hIGF-I was used as a standard. Standards and unknown were incubated in duplicate with antibody for 2 h at room temperature before the addition of [125I]Thr59-IGF-I (Amersham Co., Arlington Heights, IL). After overnight incubation, the antibody-bound [125I] IGF-I was precipitated using goat antirabbit 7-globulin and normal rabbit serum as a carrier as previously reported (29).
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Characterization of the immunoreactiue species by gel filtration chromatography and HPLC In order to concentrate IGF-I moieties, SFIF-conditioned medium from FSC was applied to a 500-mg octadecylsilane silica column (29, 30). The column was then washed with 1% trifluoroacetic acid, and retained species were eluted with 90% acetonitrile in 0.1% aqueous trifluoroacetic acid. The dried eluates were dissolved in 6 M guanidine-HCl and applied to a 0.7 x 45-cm Sephadex G-100 column equilibrated in 6 M guanidine HCl-0.03% Brij. The column was calibrated with a series of standards, including blue dextran, cytochrome-c (mol wt 12,400) and [125I]Thr59-IGF-I. The recovery of the applied radioactivity was greater than 90%. For HPLC analysis, gel filtration fractions (200 ^1) were injected onto a C18 analytical column (Vydac, The Separations Group, Hesperia, CA) and were eluted using an acetonitrile-water gradient containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. One milliliter fractions were collected and dried, and the residues dissolved in RIA buffer. Charcoal adsorption assay for IGF-I binding activity IGF-I binding activity was measured using a modification of the method of Martin and Baxter (31). Duplicate or triplicate aliquots of conditioned media were incubated with 125I-IGF-I (10,000 cpm) in a volume of 500 n\ 50 mM Tris-HCl, 0.1% BSA (Sigma), pH 7.4. After 2-h incubation at room temperature, 1% activated charcoal (Sigma) without protamine was added to the tubes followed by incubation for 15 min and centrifugation for 30 min, both at 4 C. Aliquots of supernatant containing protein bound [125I]IGF-I were counted in a 7-counter. Nonconditioned medium was assayed in parallel and was subtracted from total bound radioactivity to determine specific IGF-I binding activity, expressed as a percentage of total counts per min. Doseresponse curves were generated by assaying three concentrations (4-20 /il) of culture medium. Displacement curves were generated by coincubation with multiple dilutions of unlabeled hIGF-I, rat IGF-II, or bovine insulin. In some experiments, the IGF-I binding activity was measured in the absence and presence of PDGF. Western blot analysis of IGF-I binding protein(s) Unreduced samples of conditioned medium from FSC and from Buffalo rat liver cells (kindly provided by Dr. N. Ross, Case Western Reserve University) were electrophoresed through a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. Separated proteins were electroblotted onto nitrocellulose filters (0.45 /*m pore size). Filters were washed and then incubated with [125I]IGF-I overnight at 4 C and visualized by autoradiography according to the method of Hassenlopp et al. (32). Effect of IGF-I and PDGF on DNA synthesis in FSC DNA synthesis was measured as the amount of [methyl-3H] thymidine ([3H]TdR) incorporated into trichloroacetic acid (TCA)-precipitable material. Cells were plated in 24-well dishes at a density of 2 x 104 cells per well and incubated with
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IGF-I AND IGFBP IN LIVER FAT-STORING CELLS Waymouth's medium containing 10% horse serum and 10% calf serum until they became confluent (density of about 1.0 X 105 cells per well). Confluent cells were made quiescent by placing them for 48 h in SFIF Waymouth's medium containing 1% Zeta serum (AMF, Meriden, CT), a low mitogenicity serum. Cells were then incubated with or without the conditions to be tested for 20 h and then pulsed for 4 h with 1.0 /uCi/ml [3H] TdR (6.7 Ci/mmol; New England Nuclear, Boston, MA). At the end of the pulsing period medium was carefully aspirated, ice-cold 5% TCA was added, and dishes were kept on ice for 15 min. After two additional washes with 5% TCA, cells were solubilized by adding 750 fil 0.25 N NaOH, 0.1% SDS. Five tenths ml of the solubilized cells solution was then neutralized with 50 MI 6 N HC1 and counted in a scintillation counter. Cell number was determined in three separate wells from each dish after trypsinization and counting in a Coulter counter. Cell proliferation assay 4
Rat FSC were plated in 12-well dishes at a density of 4 x 10 in Waymouth's medium containing serum. After 24 h (day 0), the cells were washed and placed in fresh SFIF Waymouth's medium containing 1% Zeta-serum with or without synthetic IGF-I (100 ng/ml) and allowed to grow for 3 or 6 days. Fresh medium with or without IGF-I was added to the remaining wells at day 3. Cells were trypsinized and counted using a Coulter counter.
2345
2OOO-
I5OO-
5
1000-
5OO-
FIG. 1. Time course for IGF-I immunoreactivity in SFIF medium conditioned (CM) by rat liver FSC. Confluent FSC were washed twice with Waymouth's SFIF medium and incubated overnight in the same medium. This medium was then discarded and fresh SFIF medium was added with or without Cx at a final concentration of 10~5 M. Conditioned medium was collected at indicated time points and frozen at —70 C until assayed. Data, expressed as mean ± SE, are from two experiments done in triplicate. Changes were statistically significant at 8 h (P < 0.01), 24 and 48 h (P < 0.001). 5.0
Vi
Statistical analysis Data, expressed as mean ± SD or SE, were analyzed by analysis of variance.
Results The time course for IGF-I immunoreactivity in SFIF medium conditioned by rat FSC is illustrated in Fig. 1. In the absence of Cx, levels of IGF-I immunoreactivity were readily detectable after 8 h of incubation with a further time-dependent increase after 24 and 48 h. When the cells were incubated with 10~5 M Cx (a concentration which did not affect cell viability as assessed by both cell counting and Trypan blue exclusion) a marked reduction of IGF-I immunoreactivity compared to control wells was observed at each time point, indicating that the presence of IGF-I immunoreactivity in the media is likely due to de nouo protein synthesis. To characterize further the IGF-I immunoreactive species, SFIF culture medium conditioned by FSC was concentrated by passage over an octadecylsilane silica column and applied to a 0.7 x 45 cm Sephadex G-100 column equilibrated in a 6 mM guanidine-HCl denaturing buffer. As shown in Fig. 2, the gel filtration fractions demonstrated two major peaks of apparent IGF-I immunoreactivity. One peak coeluted with [125I]Thr59IGFI, indicating a mol wt of approximately 7.5-8.0 k, the size of native IGF-I. A second peak of IGF-I immunoreactivity had an apparent mol wt greater than 25 k. The
o
jS
4.0
p O
25 kD) region, suggesting the presence of IGF-I binding activity. In the presence of excess unlabeled IGF-I, radioactivity eluted with radioiodinated IGF-I suggesting that the binding activity was specific for IGF-I. IGF-I binding activity in conditioned medium was then characterized by Western blot analysis. As shown in Fig. 5, two IGF-I binding species of approximately 28 and 31 kDa were observed in conditioned medium from rat FSC while a single species of 31 kDa was observed from medium conditioned by Buffalo rat liver cells. IGF-I binding in conditioned medium from FSC was further studied using a charcoal adsorption assay. Serial dilutions of conditioned medium ranging from 0.8-4% resulted in a linear dose-dependent increase in specific binding of [125I]IGFI (data not shown). The competitive binding characteristics of FSC conditioned medium are shown in Fig. 6. Displacement of [125I]IGF-I by hIGF-I and rat IGF-II were similar. There was no displacement by bovine insulin. This pattern of displacement is characteristic of
FlG. 4. Sephadex G100 gel filtration profile (PBS pH 7.4) of [125I]IGFI binding to greater than 25 kDa IGF-I immunoreactive species from FSC-conditioned medium (48 h). The elution position of mol wt markers are indicated by the arrows. Vo, Void volume (blue dextran); Vi, 125I volume. In the absence of excess unlabeled IGF-I, radioactivity elutes at a high mol wt (>25 kD). Addition of excess unlabeled IGF-I resulted in elution of radioactivity at the mol wt of radioiodinated IGF-I.
97.466.2-
FIG. 5. Western blot analysis of IGF-I binding protein(s) in FSC conditioned medium (24 h). Samples of culture medium conditioned by FSC (15 /xl) or Buffalo rat liver cells (1 ft\) were subjected to electrophoresis through a SDS-polyacrylamide gel (10%). Separated proteins were electroblotted onto nitrocellulose filters and then incubated with [125I]IGF-I overnight at 4 C and visualized by autoradiography. Molecular weights in kDa were determined by comparison to a series of standards. Species of 28 and 31 kDa were observed in conditioned medium from FSC (lane 2) and 31 kDa in conditioned medium from Buffalo rat liver cells (lane 1).
IGFBP (33). The effects of PDGF on levels of IGF-I immunoreactivity and IGF-I binding activity were then studied. With PDGF treatment, IGF-I immunoreactivity increased significantly, 2.3-fold (mean of three experiments, P < 0.005). Since samples of conditioned medium were not extracted to eliminate interference with the RIA by IGFBP, we then assessed the effect of PDGF on IGF-I
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IGF-I AND IGFBP IN LIVER FAT-STORING CELLS
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120
T
2800 100
~ c CD
S o
2600
• IGF-I
80
D MSA •
Insulin
60
2400 -
m °
600
40
20
:
j
T
400 0.5
1.0
5.0 10.0
50.0 100
500 1000
5000 10000
nq Peptide/ml 128
FIG. 6. Competition curves for [ I]IGF-I binding displacement by human IGF-I, MSA (rat IGF-II) and insulin. SFIF medium conditioned by FSC was harvested as stated in Materials and Methods. IGF-I binding activity was assessed by the charcoal adsorption assay. Nonconditioned medium was assayed in parallel and subtracted to determine specific IGF-I binding activity. Samples were incubated with serial dilutions of IGF-I, IGF-II, or insulin. Basal binding in this representative experiment was 28.1% of added counts. Results are expressed as percentage of basal binding. The points represent the means of triplicate determinations.
binding activity. IGF-I binding activity also increased significantly, 2.1-fold (P < 0.01). In parallel experiments, incubation of FSC monolayers with GH (100 ng/ml) for 24 or 48 h did not result in any significant increase of IGF-I immunoreactivity in the conditioned medium. Quiescent rat FSC in culture were assayed for their ability to incorporate [3H]TdR into DNA after exposure to IGF-I alone, to PDGF alone, or to a combination of IGF-I plus PDGF for a total of 24 h. As illustrated in Fig. 7, the addition of IGF-I induced a significant increase in DNA synthesis when compared to 1% Zeta serum alone. However, this effect was small compared to the effect of a submaximal dose of PDGF. The dose of IGF-I used (100 ng/ml) appeared to be maximal since higher doses (up to 500 ng/ml) did not induce additional effects on [3H]TdR incorporation. The combination of IGF-I and PDGF induced an additive increase in [3H] TdR incorporation compared with PDGF alone, but the effect was additive not synergistic. To confirm that the enhanced [3H]TdR incorporation induced by IGF-I is associated with cell proliferation, we performed cell counts on cultures incubated with this growth factor. Basal cell number was 4.4 ± 0.17 x 104. The number of cells did not change when maintained in 1% Zeta serum alone, but cell counts significantly increased to 5.5 ± 0.33 x 104 and 6.1 ± 0.48 x 104 after 3 and 6 days of exposure to IGF-I, respectively ( P < 0.05 for both values). Discussion IGF-I or somatomedin C is a biologically active growth factor with diverse properties (8). The liver is a major
T
200
(19)
(21)
(8)
(8)
1%ZS -Ins
+IGF-I +PDGF 100ng/ml 2.5ng/ml
+PDGF 2.5ng/ml + IGF-I 100ng/ml
FlG. 7. Effect of synthetic IGF-I on [3H]thymidine incorporation into DNA of quiescent rat FSC. Confluent cells were made quiescent by incubation in insulin-free Waymouth's medium containing 1% Zeta serum for 48 h. Cells were then incubated with IGF-I, PDGF, or IGFI plus PDGF for 20 h and then pulsed for 4 h with 1.0 /uCi/ml [3H] TdR. Data are mean ± SD. The numbers shown on the bars indicate the number of wells tested. *, P < 0.01 vs. 1% Zeta serum alone; **, P < 0.01 vs. PDGF alone.
source of circulating IGF-I. High levels of IGF-I mRNA have been demonstrated in adult rat liver (34) and in primary cultures of rat liver cells (20). Production of IGF-I and IGFBP in adult rat hepatocytes in primary culture appears to be under the control of GH (18, 19). Recent studies in fetal human liver have localized IGF-I mRNA to perisinusoidal cells (1). Several recent studies demonstrated that cells of mesenchymal origin such as fibroblasts (5, 35), smooth muscle cells (5), and glomerular mesangial cells (29) synthesize or secrete IGF-I-like peptides and IGFBP with potential implication in local tissue hypertrophy and repair. These observations raise the possibility that liver FSC, liver-specific pericytes (capillary smooth muscle cells), produce and release IGFI and/or IGFBP. Our results indicate that liver FSC secrete an IGF-I immunoreactive species which has the same size and hydrophobicity as human IGF-I suggesting that it is IGFI protein. In addition, these cells also secrete two small IGFBP of 28 and 31 k mol wt. The release of IGF-I immunoreactivity from FSC appears to be under the control of PDGF but not GH. Since the increase in IGFI binding activity, as assessed by the charcoal adsorption assay, is of the same magnitude (2-fold) as that of IGFI immunoreactivity, it is likely that this increase in IGFI binding accounts for most if not all of the increase in apparent IGF-I immunoreactivity, indicating that, in FSC, the effect of PDGF is mainly directed to the regu-
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2348
IGF-I AND IGFBP IN LIVER FAT-STORING CELLS
lation of IGFBP secretion. The 1:1 ratio of IGFBP to the 7.5 kDa IGF-I immunoreactive species in conditioned medium based upon denaturing gel filtration chromatography is an underestimate due to the low recovery of IGFBP from reverse phase chromatography (30). The identification of various types of IGFBP has generated several hypotheses concerning their origin and role. While large mol wt GH-dependent IGFBP may be important for increasing the half-life of circulating IGF-I and protecting tissues against the insulin-like effects of free IGF-I, smaller GH-independent IGFBP, with a mol wt of about 30 kDa, may have a role in modulating the local effects of IGF-I. Purified IGFBP have been shown to enhance (36, 37) or to attenuate IGF-I effects (38). However, the factors that determine whether the modulating effects of IGFBP are positive or negative are still largely unknown. IGFBP with similar mol wt and structure, could exert different functional effects in different cell types under different conditions. At present, three rat IGFBP (rIGFBP-1, 2, and 3) have been identified (22). While rIGFBP-3 has a mol wt of 40 kDa and it is thought to be the glycosylated binding subunit of the circulating 150 kDa species, rIGFBP-1 and 2 have a mol wt of approximately 30 k and are not glycosylated. The displacement of [125I]IGF-I by IGF-I and IGF-II but not by insulin, observed in experiments using the charcoal adsorption assay of nonreduced conditioned medium, is characteristic of IGFBP (33). It is likely that at least one of two IGF-I binding species found in FSC conditioned media corresponds to rIGFBP-1 or 2, although further characterization will be required. We and others have recently demonstrated the presence of PDGF receptors on FSC (26, 27). PDGF as well as other polypeptide growth factors have been shown to increase secretion of IGF-I immunoreactivity in cultured human fibroblasts (39, 40) and smooth muscle cells (36), leading to the concept of a local regulation of IGF-I release. Multiple IGFBP with mol wt ranging from 41.523 k have been detected in media conditioned by cultured human fibroblasts and their secretion appears to be under the control of GH whereas it is unaffected by PDGF (14). Our findings indicate that PDGF induces a significant increase in the secretion of IGF-I binding activity and, therefore, the hormonal regulation of IGFI binding species secreted by FSC differs from that present in other cell types. Several studies have also suggested that IGF-I and PDGF interact in regulating growth of smooth muscle cells, fibroblasts, glomerular mesangial cells, and other mesenchymal cell types (4144). PDGF has been thought to modulate cell growth in response to IGF-I, this latter acting as a "progression factor" (42,44). In our experimental conditions, however, we found that IGF-I and PDGF interact additively on DNA synthesis in FSC suggesting that the two growth
Endo • 1990 Vol 127 • No 5
factors act through different pathways. These results are in agreement with recent studies showing additive effects of IGF-I and PDGF in the induction of the protooncogene c-myc and cell proliferation in smooth muscle cells (45). While hepatocytes constitute the major source of circulating IGF-I, the results of the present study suggest that FSC are another potential source of this peptide. The precise biological role of IGF-I and IGFBP synthesized by FSC remains to be determined. IGF-I, like PDGF, is a mitogen for FSC. IGF-I, in concert with other cytokines released by activated macrophages (46, 47), may contribute to both proliferative and fibrotic responses during liver inflammation. As already shown for other mesenchymal cells (16, 17), IGF-I could also influence the production of matrix components by FSC, currently considered the predominant source of collagen and proteoglycan within the liver (23-25). The secretion by FSC of IGFBP, which are regulated by PDGF, may be important in regulating the local effects of IGF-I. Acknowledgments We thank Christina N. Nye and Marina Karam for expert technical assistance.
References 1. Han VKM, D'Ercole AJ, Lund PK 1987 Cellular localization of somatomedin (Insulin-like growth factor) messenger RNA in the human fetus. Science 236:193-197 2. Lund PK, Moats-Staats BM, Haynes MA, Simmons JG, Jansen D'Ercole AJ, Van Wyk JJ 1986 Somatomedin-C/Insulin-like growth factor-I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues. J Biol Chem 261:14539-14544 3. Atkinson PR, Weidman ER, Bhaumick B, Bala RM 1980 Release of somatomedin-like activity by cultured WI-38 human fibroblasts. Endocrinology 106:2006-2012 4. Clemmons DR, Underwood LE, Van Wyk JJ 1981 Hormonal control of immunoreactive somatomedin production by cultured human fibroblasts. J Clin Invest 67:10-19 5. Clemmons DR, Van Vyk JJ 1985 Evidence for a functional role of endogenously produced somatomedin-like peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells. J Clin Invest 75:1914-1918 6. D'Ercole AJ, Stiles AD, Underwood LE 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 7. Underwood LE, D'Ercole AJ, Clemmons DR, Van Wyk JJ 1986 Paracrine functions of somatomedins. Clin in Endocrinol Metab 15:59-77 8. Holly JMP, Wass JAH 1989 Insulin-like growth factors; autocrine, paracrine or endocrine? New perspectives of the somatomedin hypothesis in the light of recent developments. J Endocrinol 122:611-618 9. Baxter RC, Martin JL 1986 Radioimmunoassay of growth-hormone-dependent insulin-like growth factor binding protein in human plasma. J Clin Invest 78:1504-1512 10. Koistinen R, Kalkkinen N, Huhtala M, Seppala M, Bohn H, Rutanen E 1986 Placental protein 12 is a decidual protein that bind somatomedins and has identical N-terminal amino acid sequence with somatomedin-binding protein from human amniotic fluid. Endocrinology 118:1375-1378
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IGF-I AND IGFBP IN LIVER FAT-STORING CELLS 11. Clemmons DR, Elgin RG, Han VKM, Casella SJ, D'Ercole AJ, Van Wyk JJ 1986 Cultured fibroblasts monolayer secrete a protein that alters the cellular binding of somatomedin-C/insulinlike growth factor I. J Clin Invest 77:1548-1553 12. DeVroede MA, Tseng LY-H, Katsoyannis G, Nissley SP, Rechler MM 1986 Modulation of insulinlike growth factor I binding to human fibroblasts monolayer cultures by insulin-like growth factor carrier proteins released to the incubation media. J Clin Invest 77:602-613 13. Baxter RC, Martin JL, Wood MH 1987 Two immunoreactive binding proteins for insulin-like growth factors in human amniotic fluid: relationship to fetal maturity. J Clin Endocrinol Metab 65:423-431 14. Conover CA, Liu F, Powell D, Rosenfeld RG, Hintz RL 1989 Insulin-like growth factor binding proteins from cultured human fibroblasts. Characterization and hormonal regulation. J Clin Invest 83:852-859 15. Baxter RC, Martin JL 1989 Binding proteins for the insulin-like growth factors: structure, regulation and function. Progress in growth factor research. 1:49-68 16. Bar RS, Dake BL, Stueck S 1987 Stimulation of proteoglycans by IGF-I and II in microvessel and large vessel endothelial cells. Am J Physiol 253:E21-E27 17. Goldstein RH, Poliks CF, Pilch PF, Smith BD, Fine A 1989 Stimulation of collagen formation by insulin and insulin-like growth factor I in cultures of human lung fibroblasts. Endocrinology 124:964-970 18. Scott CD, Martin JL, Baxter RC 1985 Production of insulin-like growth factor I and its binding protein by adult rat hepatocytes in primary culture. Endocrinology 116:1094-1101 19. Scott CD, Martin JL, Baxter RC 1985 Rat hepatocyte insulin-like growth factor and binding protein: effect of growth hormone in vitro and in vivo. Endocrinology 116:1102-1107 20. Norstedt G, Moller C 1987 Growth hormone induction of insulinlike growth factor I messenger RNA in primary cultures of rat liver cells. J Endocrinology 15:135-139 21. Lee Y-L, Hintz RL, James PM, Lee PDK, Shively JE, Powell DR 1988 Insulin-like growth factor (IGF) binding protein complementary deoxyribonucleic acid from human HEP G2 hepatoma cells: predicted protein sequence suggest an IGF binding domain different from those of IGF-I and IGF-II receptors. Mol Endocrinol 2:404-411 22. Yang YWH, Brown AL, Orlowski CC, Graham DE, Tseng LYH, Romanus JA, Rechler MM 1990 Identification of rat cell lines that preferentially express insulin-like growth factor binding proteins rIGFBP-1,2, or 3. Mol Endocrinol 4:29-38 23. Friedman SL, Roll FJ, Boyles J, Bissell DM 1985 Hepatic lipocytes: the principal collagen-producing cells of normal liver. Proc Natl Acad Sci USA 82:8681-8685 24. Maher JJ, Bissell DM, Friedman SL, Roll FJ 1988 Collagen measured in primary cultures of normal rat hepatocytes derives from lipocytes within the monolayer. J Clin Invest 82:450-459 25. Arenson DM, Friedman SL, Bissell DM 1988 Formation of extracellular matrix in normal rat liver: lipocytes as a major source of proteoglycan. Gastroenterology 95:441-447 26. Friedman SL, Arthur MJP 1989 Activation of cultured rat hepatic lipocytes by Kuppfer cell conditioned medium. Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. J Clin Invest 84:1780-1785 27. Pinzani M, Gesualdo L, Sabbah GM, Abboud HE 1989 Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells. J Clin Invest 84:1786-1793 28. Friedman SL, Roll FJ 1987 Isolation and culture of hepatic lipo-
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