. Vol. 189, No. 2, 1992 December
BIOCHEMICAL
AND BIOPHYSICAL
15, 1992
INSULIN-LIKE
RESEARCH COMMUNICATIONS Pages 1177-1183
EPIDERMAL GROWTH FACTOR ENHANCES GROWTH FACTOR BINDING PROTEIN-l IN HUMAN HEPATOMA CELLS
SYNTHESIS
Maarit Anget-vo
Departments I and II of Obstetrics and Gynecology, Helsinki University Central Hospital, SF-00290 Helsinki, Finland Received
November
2,
1992
Epidermal growth factor (EGF) was found to induce a rapid 2-fold increase in the amount of insulin-like growth factor binding protein-l (IGFBP-1) mRNA in human hepatoma Hep2G cells, and this was accompanied by a 2-fold increase in IGFBP-1 secretion. A protein synthesis inhibitor cycloheximide (CHX) caused a 2-3-fold increase in the amount of IGFBP-1 n-RNA, which could be accounted for the observed stabilization in decay of IGFBP-1 mRNA after CHX treatment. In nuclear run-on transcription experiments neither EGF nor CHX affected the transcription rate of the IGFBP-1 gene. It is concluded that EGF increases IGFBP-1 secretion rapidly by enhancing IGFBP-1 mRNA accumulation, and the addition of a protein synthesis inhibitor results in a specific increment of IGFBP-1 mRNA, suggesting that a labile 0 1992 Academic protein repressor protein is involved in the turnover IGFBP- 1 mRNA. Press,
Inc.
The stability, availibility and bioactivity of insulin-like growth factors (IGF-I and IGF-II) are regulated by their receptors and six binding proteins (IGFBPs) (1). The liver is the primary source of IGFs and IGFBP-1 (2,3). Human IGFBP-1 is a nonglycosylated 25 kDa protein translated from a single 1.6 kb mRNA transcript of the IGFBP-1 gene (4). The liver-derived IGFBP-1 is involved in rapid metabolic adaptation to nutritional stimuli. Thus the IGFBP-1 levels are increased in response to hypoglycemia and fasting (5). The circulating IGFBP- 1 levels are suppressed during a euglycemic insulin clamp (6), and studies on Hep2G cells indicate that insulin potently suppresses hepatic IGFBP- 1 production by inhibiting IGFBP- 1 gene transcription (7). Epidermal growth factor (EGF) is a polypeptide growth factor produced by glands of the gastrointestinal tract (8). EGF has a role in liver regeneration, and it also exerts biological activity on carbohydrate metabolism in the liver (9). EGF increases IGFBP1 secretion in human granulosa-luteal cells (10) and in an endometrial cancer cell line (11). Considering that the liver is an important site of IGFBP-1 synthesis and an important target for EGF action (8), it was of interest to study the effects of EGF on IGFBP-1 gene regulation in HepG2 cells that exhibit most of the characteristics of normal hepatocytes (12,13). MATERIALS
AND METHODS
HepG2 cells (American Type Culture Collection, ATCC, Rockville, Md) were cultured in Earle’s minimal essential medium (Whittaker Bioproducts Inc.,
Cell culture.
1177
0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
. Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Walkersville, Md.) supplemented with 10% fetal calf serum (Flow Laboratories, Strathclyde, U.K.), 2mM L-glutamine, 100 III/ml penicillin and 100 kg/ml streptomycin (Gibco, Grand Island, NY) and incubated in 95% air-5% C02at 37°C. For IGFBP-1 protein analysis 105 cells were plated on 48-well dishes (Costar, Cambridge, NY), and 5x106 cells were plated on 100 mm-dishes (Falcon, Los Angeles, CA) for the RNA studies. The cells were maintained in growth medium for 48 h, after which the subconfluent monolayer cultures were washed twice with phosphate-buffered saline and refed with 0.5 ml and 10 ml of serum-free medium containing 100 j.tg/ml EGF (Sigma, St. Louis, MO) and 0.1% bovine serum albumin for various times up to 24 h. To study the effect of a protein synthesis inhibitor on hormonal induction, 2 kg/ml CHX (Sigma) was added to the cultures 1 h before the addition of EGF. The chosen concentration of CHX did not affect cell viability during the 24 h incubation period as estimated by total cellular protein mass, but it inhibited protein synthesis by 88% as previously described (14). Assays. The concentration of IGFBP-1 was measured by a specific time-resolved immunofluorometric assay essentially as described (11). The sensitivity of the assay was 0.1 p.gJl.The interassay variation was 12% and the intra-assay variation was 6.5% at the level of 4.5 pg/l. To study the possible effect of EGF or CHX on cellular growth, protein content of the cell lysates was measured by the Bradford method (Biorad, Richmond, CA). Extraction of RNA. Total cellular RNA was extracted using the method described by Chomczynski and Sacchi (15). RNA was quantitated by measuring absorbance at 260 %&A probes. The entire 1.5 kb long human IGFBP-1 cDNA probe was used (4). As an internal control for the amount of RNA loaded the rat glyceraldehyde-3phosphate dehydrogenase (GAPDH) cDNA probe (16) was used. Northern hybridization. Northern blot analysis was performed by electrophoresis of total RNA, 5 pg/lane, on 1.2% agarose formaldehyde denaturating gels and transfered onto nitrocellulose filters (Amersham International plc, Amersham, UK) according to the manufacturer’s instructions. To verify integrity of the RNA and equivalence of the RNA loads per lane, ribosomal RNAs (28s and 18s) were visualized by ethidium bromide staining. The blots were prehybridized at 42’C for 2-6 h in a buffer containing 50% formamide, 5 x SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 5 x Denhardt’s solution, 0.5% sodium dodecyl sulfate (SDS), and 10 mg/ml heterologous herring sperm DNA (Sigma). The probes were labeled with [32P]dCTP (3000 Ci/mmol; Amersham) by random priming according to the manufacturer’s instructions (Multiprime DNA Labelling Systems, RPN. 1601Y, Amersham). Hybridization was performed overnight at 42°C in the same buffer containing the radioactive probes at a final concentration of 1 x 106cpm/ml. The blots were washed as described (15) and autoradiographed by exposing on KODAK-XOMAT AR film (Eastman Kodak, Rochester, NY) at -7O’C. The signal intensity of the autoradiograms was analyzed quantitatively by using an LKB Ultro Scan 2202 laser densitometer. The amount of IGFBP-1 mRNA was expressed in relation to GAPDH mRNA. In vitro nuclear run-on transcription assay. The nuclear transcription analyses were performed using minor modifications of the method described by Powell (8). 5 x 10 cells were treated with 100 rig/ml EGF, or 2 /.tg/ml of CHX, for 2 h. The nuclei were prepared by lysis in 0.5% Nonidet-40 buffer (17) at 4’C. The pelleted nuclei were suspended in 100 l.tl glycerol buffer (40% glycerol, 5 mM MgCl , 0.1 mM EDTA, 50 mM Hepes, pH 7.4 ) frozen in liquid nitrogen and stored at -70 ‘3 until use. The nuclei (100 l.tl) were incubated for 15 min at room temperature in 400 ~1 reaction buffer containing 2 l,tl RNAse inhibitor (Boehringer Mannheim, Mannhein, Germany), 100 l.tCi of P2P]UTP (3000 Ci/mmol)(Amersham) and 250 l.tM each of ATP, GTP and CTP; 150 mM KCL, 5 mM MgCl , 1 mM MnCl , 2.5 mM dithiotreitol. The reaction was terminated by the addition 28 pg of DNase T for 5 min, followed by addition of 0.2 mg proteinase-K at 37’C for 30 min. The nascent RNA was purified and extracted as described (7). The labeled RNA was hybridized to optimized excess of IGFBP-1 cDNA (3 pg plasmid pGem3 containing full-length IGFBP-1 cDNA) denatured and immobilized onto a nitrocellulose membrane using a slot-blot apparatus (Biorad) (17). Each membrane also contained a dot of pGem3 as a negative control and 3 Vg PU13 containing full-length GAPDH cDNA as a positive and quantitative hybridization control. Typical incorporation ranged between 2-6 x 106/cpm. The samples in each 1178
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
hybridization experiment were equalized to contain the same final cpm of [32Pl RNA/ml (= lx 106/m& Prehybridization and hybridization were performed at 42°C for 72 h in buffers described above. After hybridization the filters were washed and autoradiographed for 4-6 days. IGFBP-1 mRNA decay. HepG2 cells were incubated with CHX for 6 h, i.e., the time of maximal induction of IGFBP-I mRNA and 5 pg/ml actinomycin D, an inhibitor of DNA transcription, was added to the CHX-treated and control cultures. IGFBP-1 mRNAs isolated up to 24 h incubation were quantified by Northern blot analysis. Statistical analyses. The experiments were repeated at least three times in each case. Statistical analyses were performed by the Student’s unpaired t-test.
RESULTS In serum-free Hep2G cultures EGF significantly increased the release of IGFBP-1 at 6 and 24 h incubation. The addition of 100 @ml EGF resulted in 1.5-fold (at 6h) and 2-fold (at 24 h) increase in secreted IGFBP-1 (Fig.1). In these serum-free cultures cellular growth was not affected by EGF, as the total protein content of cell lysates remained the same after each experiment (data not shown). Stimulation of Hep2G cells with 100 rig/ml EGF resulted in rapid and specific accumulation of IGFBP- 1 mRNA. The induction of IGFBP- 1 mRNA was apparent at 1.5 h, it reached the maximum at 3-6 h and declined at 24 h. The maximal increase in the amount of IGFBP-1 mRNA was 2-fold (Fig. 2A). The level of internal control, GAPDH mRNA, did not change in response to EGF. CHX inhibited the accumulation of IGFBP-1 in medium by 75%, but a rapid and transient increase in the amount of steady-state IGFBP-1 mRNA was observed in cells treated with CHX. The relative amount of IGFBP-1 mRNA increased 2-fold at 1.5 h, 3-fold at 3-6 h and there was no effect at 24 h (Fig. 2C). When both EGF and CHX were added to the cultures, a consistent 2-3-fold rise in accumulation of steady-state IGFBP-1 mRNA was seen at each time point (Fig. 2B). This effect was not additive to the effects induced by either EGF and CHX alone at 1.5-6 h. At 24 h, when CHX had no effect and EGF had a small effect, the presence of both substances brought about a 3-fold increment in IGFBP-1 mRNA. Again, the level of internal control, GAPDH mRNA, did not change in response to EGF or CHX. Changes in the level of steady state IGFBP-1 mRNA could be due to alterations in the transcription rate of the IGFBP- 1 gene, altered stability of IGFBP- 1 mRNA, or both. The effect of CHX on the rate of IGFBP-1 mRNA decay was tested, and IGFBP-1 mRNA was found to be stabilized by CHX through the time course of 24 h studied (Fig.3).
0
3
6 24 Time (h)
Fig. 1. Time-dependent effect of 100 @ml EGF on IGFBP-1 secretion by HepG2
cells cultured in serum-freemedium. The meansf SEM reflect the oooled data from. six separateexperiments.The averageIGFBP-I concentrationwitho& added EGF was taken as 100% at each time point. *** , P < 0.001 compared to the cultures without added EGF. 1179
Vol.
189,
No.
2,
1992
BIOCHEMICAL
III, EGF CHX
-
1.5 + -
AND
BIOPHYSICAL
3 + +
+
-
+ -
RESEARCH
6 + +
+
-
+ -
COMMUNICATIONS
24 + +
+
IGFBP-1 GAPDH
-
+ -
BIOCHEMICAL
AND BIOPHYSICAL
15, 1992
INSULIN-LIKE
RESEARCH COMMUNICATIONS Pages 1177-1183
EPIDERMAL GROWTH FACTOR ENHANCES GROWTH FACTOR BINDING PROTEIN-l IN HUMAN HEPATOMA CELLS
SYNTHESIS
Maarit Anget-vo
Departments I and II of Obstetrics and Gynecology, Helsinki University Central Hospital, SF-00290 Helsinki, Finland Received
November
2,
1992
Epidermal growth factor (EGF) was found to induce a rapid 2-fold increase in the amount of insulin-like growth factor binding protein-l (IGFBP-1) mRNA in human hepatoma Hep2G cells, and this was accompanied by a 2-fold increase in IGFBP-1 secretion. A protein synthesis inhibitor cycloheximide (CHX) caused a 2-3-fold increase in the amount of IGFBP-1 n-RNA, which could be accounted for the observed stabilization in decay of IGFBP-1 mRNA after CHX treatment. In nuclear run-on transcription experiments neither EGF nor CHX affected the transcription rate of the IGFBP-1 gene. It is concluded that EGF increases IGFBP-1 secretion rapidly by enhancing IGFBP-1 mRNA accumulation, and the addition of a protein synthesis inhibitor results in a specific increment of IGFBP-1 mRNA, suggesting that a labile 0 1992 Academic protein repressor protein is involved in the turnover IGFBP- 1 mRNA. Press,
Inc.
The stability, availibility and bioactivity of insulin-like growth factors (IGF-I and IGF-II) are regulated by their receptors and six binding proteins (IGFBPs) (1). The liver is the primary source of IGFs and IGFBP-1 (2,3). Human IGFBP-1 is a nonglycosylated 25 kDa protein translated from a single 1.6 kb mRNA transcript of the IGFBP-1 gene (4). The liver-derived IGFBP-1 is involved in rapid metabolic adaptation to nutritional stimuli. Thus the IGFBP-1 levels are increased in response to hypoglycemia and fasting (5). The circulating IGFBP- 1 levels are suppressed during a euglycemic insulin clamp (6), and studies on Hep2G cells indicate that insulin potently suppresses hepatic IGFBP- 1 production by inhibiting IGFBP- 1 gene transcription (7). Epidermal growth factor (EGF) is a polypeptide growth factor produced by glands of the gastrointestinal tract (8). EGF has a role in liver regeneration, and it also exerts biological activity on carbohydrate metabolism in the liver (9). EGF increases IGFBP1 secretion in human granulosa-luteal cells (10) and in an endometrial cancer cell line (11). Considering that the liver is an important site of IGFBP-1 synthesis and an important target for EGF action (8), it was of interest to study the effects of EGF on IGFBP-1 gene regulation in HepG2 cells that exhibit most of the characteristics of normal hepatocytes (12,13). MATERIALS
AND METHODS
HepG2 cells (American Type Culture Collection, ATCC, Rockville, Md) were cultured in Earle’s minimal essential medium (Whittaker Bioproducts Inc.,
Cell culture.
1177
0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
. Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Walkersville, Md.) supplemented with 10% fetal calf serum (Flow Laboratories, Strathclyde, U.K.), 2mM L-glutamine, 100 III/ml penicillin and 100 kg/ml streptomycin (Gibco, Grand Island, NY) and incubated in 95% air-5% C02at 37°C. For IGFBP-1 protein analysis 105 cells were plated on 48-well dishes (Costar, Cambridge, NY), and 5x106 cells were plated on 100 mm-dishes (Falcon, Los Angeles, CA) for the RNA studies. The cells were maintained in growth medium for 48 h, after which the subconfluent monolayer cultures were washed twice with phosphate-buffered saline and refed with 0.5 ml and 10 ml of serum-free medium containing 100 j.tg/ml EGF (Sigma, St. Louis, MO) and 0.1% bovine serum albumin for various times up to 24 h. To study the effect of a protein synthesis inhibitor on hormonal induction, 2 kg/ml CHX (Sigma) was added to the cultures 1 h before the addition of EGF. The chosen concentration of CHX did not affect cell viability during the 24 h incubation period as estimated by total cellular protein mass, but it inhibited protein synthesis by 88% as previously described (14). Assays. The concentration of IGFBP-1 was measured by a specific time-resolved immunofluorometric assay essentially as described (11). The sensitivity of the assay was 0.1 p.gJl.The interassay variation was 12% and the intra-assay variation was 6.5% at the level of 4.5 pg/l. To study the possible effect of EGF or CHX on cellular growth, protein content of the cell lysates was measured by the Bradford method (Biorad, Richmond, CA). Extraction of RNA. Total cellular RNA was extracted using the method described by Chomczynski and Sacchi (15). RNA was quantitated by measuring absorbance at 260 %&A probes. The entire 1.5 kb long human IGFBP-1 cDNA probe was used (4). As an internal control for the amount of RNA loaded the rat glyceraldehyde-3phosphate dehydrogenase (GAPDH) cDNA probe (16) was used. Northern hybridization. Northern blot analysis was performed by electrophoresis of total RNA, 5 pg/lane, on 1.2% agarose formaldehyde denaturating gels and transfered onto nitrocellulose filters (Amersham International plc, Amersham, UK) according to the manufacturer’s instructions. To verify integrity of the RNA and equivalence of the RNA loads per lane, ribosomal RNAs (28s and 18s) were visualized by ethidium bromide staining. The blots were prehybridized at 42’C for 2-6 h in a buffer containing 50% formamide, 5 x SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 5 x Denhardt’s solution, 0.5% sodium dodecyl sulfate (SDS), and 10 mg/ml heterologous herring sperm DNA (Sigma). The probes were labeled with [32P]dCTP (3000 Ci/mmol; Amersham) by random priming according to the manufacturer’s instructions (Multiprime DNA Labelling Systems, RPN. 1601Y, Amersham). Hybridization was performed overnight at 42°C in the same buffer containing the radioactive probes at a final concentration of 1 x 106cpm/ml. The blots were washed as described (15) and autoradiographed by exposing on KODAK-XOMAT AR film (Eastman Kodak, Rochester, NY) at -7O’C. The signal intensity of the autoradiograms was analyzed quantitatively by using an LKB Ultro Scan 2202 laser densitometer. The amount of IGFBP-1 mRNA was expressed in relation to GAPDH mRNA. In vitro nuclear run-on transcription assay. The nuclear transcription analyses were performed using minor modifications of the method described by Powell (8). 5 x 10 cells were treated with 100 rig/ml EGF, or 2 /.tg/ml of CHX, for 2 h. The nuclei were prepared by lysis in 0.5% Nonidet-40 buffer (17) at 4’C. The pelleted nuclei were suspended in 100 l.tl glycerol buffer (40% glycerol, 5 mM MgCl , 0.1 mM EDTA, 50 mM Hepes, pH 7.4 ) frozen in liquid nitrogen and stored at -70 ‘3 until use. The nuclei (100 l.tl) were incubated for 15 min at room temperature in 400 ~1 reaction buffer containing 2 l,tl RNAse inhibitor (Boehringer Mannheim, Mannhein, Germany), 100 l.tCi of P2P]UTP (3000 Ci/mmol)(Amersham) and 250 l.tM each of ATP, GTP and CTP; 150 mM KCL, 5 mM MgCl , 1 mM MnCl , 2.5 mM dithiotreitol. The reaction was terminated by the addition 28 pg of DNase T for 5 min, followed by addition of 0.2 mg proteinase-K at 37’C for 30 min. The nascent RNA was purified and extracted as described (7). The labeled RNA was hybridized to optimized excess of IGFBP-1 cDNA (3 pg plasmid pGem3 containing full-length IGFBP-1 cDNA) denatured and immobilized onto a nitrocellulose membrane using a slot-blot apparatus (Biorad) (17). Each membrane also contained a dot of pGem3 as a negative control and 3 Vg PU13 containing full-length GAPDH cDNA as a positive and quantitative hybridization control. Typical incorporation ranged between 2-6 x 106/cpm. The samples in each 1178
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
hybridization experiment were equalized to contain the same final cpm of [32Pl RNA/ml (= lx 106/m& Prehybridization and hybridization were performed at 42°C for 72 h in buffers described above. After hybridization the filters were washed and autoradiographed for 4-6 days. IGFBP-1 mRNA decay. HepG2 cells were incubated with CHX for 6 h, i.e., the time of maximal induction of IGFBP-I mRNA and 5 pg/ml actinomycin D, an inhibitor of DNA transcription, was added to the CHX-treated and control cultures. IGFBP-1 mRNAs isolated up to 24 h incubation were quantified by Northern blot analysis. Statistical analyses. The experiments were repeated at least three times in each case. Statistical analyses were performed by the Student’s unpaired t-test.
RESULTS In serum-free Hep2G cultures EGF significantly increased the release of IGFBP-1 at 6 and 24 h incubation. The addition of 100 @ml EGF resulted in 1.5-fold (at 6h) and 2-fold (at 24 h) increase in secreted IGFBP-1 (Fig.1). In these serum-free cultures cellular growth was not affected by EGF, as the total protein content of cell lysates remained the same after each experiment (data not shown). Stimulation of Hep2G cells with 100 rig/ml EGF resulted in rapid and specific accumulation of IGFBP- 1 mRNA. The induction of IGFBP- 1 mRNA was apparent at 1.5 h, it reached the maximum at 3-6 h and declined at 24 h. The maximal increase in the amount of IGFBP-1 mRNA was 2-fold (Fig. 2A). The level of internal control, GAPDH mRNA, did not change in response to EGF. CHX inhibited the accumulation of IGFBP-1 in medium by 75%, but a rapid and transient increase in the amount of steady-state IGFBP-1 mRNA was observed in cells treated with CHX. The relative amount of IGFBP-1 mRNA increased 2-fold at 1.5 h, 3-fold at 3-6 h and there was no effect at 24 h (Fig. 2C). When both EGF and CHX were added to the cultures, a consistent 2-3-fold rise in accumulation of steady-state IGFBP-1 mRNA was seen at each time point (Fig. 2B). This effect was not additive to the effects induced by either EGF and CHX alone at 1.5-6 h. At 24 h, when CHX had no effect and EGF had a small effect, the presence of both substances brought about a 3-fold increment in IGFBP-1 mRNA. Again, the level of internal control, GAPDH mRNA, did not change in response to EGF or CHX. Changes in the level of steady state IGFBP-1 mRNA could be due to alterations in the transcription rate of the IGFBP- 1 gene, altered stability of IGFBP- 1 mRNA, or both. The effect of CHX on the rate of IGFBP-1 mRNA decay was tested, and IGFBP-1 mRNA was found to be stabilized by CHX through the time course of 24 h studied (Fig.3).
0
3
6 24 Time (h)
Fig. 1. Time-dependent effect of 100 @ml EGF on IGFBP-1 secretion by HepG2
cells cultured in serum-freemedium. The meansf SEM reflect the oooled data from. six separateexperiments.The averageIGFBP-I concentrationwitho& added EGF was taken as 100% at each time point. *** , P < 0.001 compared to the cultures without added EGF. 1179
Vol.
189,
No.
2,
1992
BIOCHEMICAL
III, EGF CHX
-
1.5 + -
AND
BIOPHYSICAL
3 + +
+
-
+ -
RESEARCH
6 + +
+
-
+ -
COMMUNICATIONS
24 + +
+
IGFBP-1 GAPDH
-
+ -
