0021-972x/92/7502-0459$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 15, No. 2 Prrnted
in U.S.A.
Role of Protein Kinase-C in Regulation of Insulin-Like Growth Factor-Binding Protein- 1 Production by HepG2 Cells* PHILLIP
D. K. LEE,
Department
of
Pediatrics,
LAURA
S. ABDEL-MAGUID,
Baylor College of Medicine,
Houston,
AND
MARK
B. SNUGGS
Texas 77030 did not inhibit PMA stimulation. The transient PKC activator diCs had no effect, while studies with the PKC inhibitors sphinganine and H-7 were limited by solvent vehicle cytotoxicity. Staurosporine (STS), a potent PKC inhibitor, stimulated IGFBP-1 production 2- to 4-fold and augmented the stimulatory effect of PMA. Concanavalin-A, an inhibitor of PMA-stimulated PKC translocation and down-regulation, inhibited the effects of PMA and STS. Our findings indicate that PKC is involved in the regulation of hepatic IGFBP-1 production. The effects of PMA, which causes rapid activation, followed by membrane translocation and down-regulation of PKC, are similar to those of STS and are countered by Concanavalin-A. These data suggest that PKC activity may mediate tonic inhibition of IGFBP-1 production, while PKC downregulation stimulates the production of this regulatory protein. (J Clin Endocrinol Metab 75: 459-464, 1992)
ABSTRACT Insulin-like growth factor binding protein-l (IGFBP-1) is a liverderived protein that modulates the mitogenic actions of the insulinlike growth factors (IGFs). IGFBP-1 production is potently inhibited by insulin both in uiuo and in HepG2 human hepatoma cells. To further define the pathways of IGFBP-1 regulation, we studied the effects of modulators of nrotein kinase-C (PKC) on HenG2 cell IGFBP-1 nroduction. Phorbbl 12-myristate li-acetate (PM& stimulated IGFiP-1 production in a timeand dose-dependent manner, with maximal stimulation occurring at lo-100 nmol/L. The degree of stimulation was dependent on cell density, ranging from about 2-fold in confluent to more than IO-fold in sparse cultures. Preincubation with PMA abolished the inhibitory effect of insulin, while preincubation with insulin
I
NSULIN -like growth factor-binding protein-l (IGFBP-1) is one of several structurally related specific BPS for insulin-like growth factor-I and -11(IGF-I and -11)(l-3). In nonpregnant humans, IGFBP-1 is produced primarily by the liver and ovarian granulosa cells (4, 5). Plasma IGFBP-1 levels may vary more than lo-fold in response to normal daily meal patterns, an effect that is mediated by insulin (6). Since IGFBP-1 has been shown to inhibit IGF-mediated mitogenesis in vitro (7-9), we have postulated that insulin regulation of IGFBP-1 production may play a role in modulating IGF action in relation to substrate availability (6). We have previously characterized the HepG2 human liver cell line, a minimal change continuous line derived from a hepatoma, as a model for studiesof IGFBP-1 production (10, 11). HepG2 cells produce IGFBP-1, but none of the other IGFBPs. Since phorbol 12-myristate 13-acetate (PMA) has been shown to increase IGFBP-1 production by ovarian granulosa cells (12), we studied its effect on HepG2 IGFBP1 production and its possibleinteractions with insulin. Our resultsdemonstrate that PMA is a potent stimulant of HepG2 IGFBP-1 production, and that the mechanism of this stimulation may be complex.
Materials
and Methods
Materials The HepG2 human hepatoma-derived cell line, originally reported by Knowles et al. (13), was obtained from the American Type Culture Collection (Rockville, MD). Pure IGFBP-I was prepared from HepG2 cell-conditioned medium or human amniotic fluid, as previously described (14). Pure recombinant DNA-derived human insulin was obtained from Lilly (Indianapolis, IN). PMA and o-erythro-sphingosine (SPH) were obtained from Calbiochem (San Diego, CA); 1,2dioctanoylsn-glycerol (diQ, staurosporine (STS), 1-(5.isoquinolinylsulfonyl)2methyl piperazine (H-7), and Concanavalin-A (type IV; Con-A) were obtained from Sigma (St. Louis, MO). Stock solutions of 1 mmol/L PMA, 100 mmol/L diCs, and 1 mmol/L STS were prepared in dimethylsulfoxide. SPH was prepared to 100 mmol/L in absolute ethanol, then diluted to 1 mmol in 17% RIA grade, insulin-free BSA (Miles Pentex, Kankakee, IL) (15). Stock solutions were stored in aliquots at -70 C and diluted to appropriate concentrations in serum-free medium (SFM) for experiments.
Cell culture HepG2 cells were cultivated in Dulbecco’s Minimum Essential Medium, 2 mmol/L L-glutamine, 1% penicillin/streptomycin, and 10% fetal bovine serum (Gibco, Grand Island, NY), passaged at confluence after trypsin detachment, and plated into 24-well multiwell plates (2 cm’/ well) for the investigations. After l-3 days, the complete medium was removed, monolayers were washed with sterile saline, and the medium was replaced with serum-free RPMI-1640 medium with 2 mmol/L L-glutamine and experimental additives. The conditioned medium was removed at the specified time intervals and stored in siliconized tubes at -70 C until assay. HepG2 cells tend to aggregate in suspension, and direct cell counting was not routinely feasible. Therefore, cell number was measured using a DNA minifluorometer (Hoeffer Scientific Instruments, San Francisco, CA) after dissolution in 0.1% sodium dodecyl sulfate and staining with bisbenzimide (Hoechst dye 33258) (16). Standards were calf thymus
Received August 26, 1991. Address all correspondence and requests for reprints to: Phillip D. K. Lee, M.D., Texas Children’s Hospital, Clinical Care Center, MC 3-2351, 6621 Fannin Street, Houston, Texas 77030-2399. * Portions of these investigations were presented at the 73rd Annual Meeting of The Endocrine Society, Washington, D.C., 1991 (Abstract 1797). This work was supported by a Feasibility Grant from the American Diabetes Association, with funds contributed by the Colorado Chapter of the ADA (to P.D.K.L.), and by the Baylor College of Medicine Summer Medical and Research Training program (to L.S.A.).
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LEE, ABDEL-MAGUID,
460
AND SNUGGS 600
DNA (Clontech, Palo Alto, CA). With single cell suspensions of HepG2 cells, we found a direct correlation of cell number by hemocytometer and DNA by fluorometry (r = 0.98; P = 0.0004). Total protein in the conditioned medium was determined using the Bio-Rad Protein Assay Kit (Bio-Rad, Richmond, CA).
JCE & M. 1992 Vol75’NoZ
r
1000 900
IGFBP-1 RIA IGFBP-1 levels were measured by specific RIA, as previously described (17). Briefly, 100 PL conditioned medium [diluted if necessary in assay buffer (phosphate-buffered saline, pH 7.4; 0.05% Tween-20; and 0.1% BSA)] were preincubated for 1 h with 100 PL polyclonal rabbit antihuman IGFBP-1 (final titer, 1:2500), followed by the addition of 5OpL (-10,000 cpm) [iZ51]IGFBP-1. [‘2’I]IGFBI’-1 was prepared using the Iodobead method (Pierce Chemical Co., Rockford, IL). After a 16-h incubation at 4 C, bound and free counts were separated using agaroseimmobilized goat antirabbit immunoglobulin (Bio-Rad), and bound counts were measured in an automatic y-counter. Inter- and intraassay coefficients of variation at 7.86 ng/mL were estimated to be 17.3 rt 1.4% and 13.4 f 3.8%, respectively, with assay limits of approximately 1 and 200 ng/mL. Samples from each study were grouped to minimize the effect of interassay variability. RIA data were analyzed using the IBMPC RIA Data Reduction Program (M. L. Jaffe, Silver Spring, MD).
Immunoprecipitation Mouse ascites fluid containing monoclonal antibody D5 to human IGFBP-1 was prepared as previously described (1 l), coupled to Affi-Gel 10 agarose beads (Bio-Rad) according to manufacturer’s instructions, and resuspended in PBST (Dulbecco’s phosphate-buffered saline, pH 7.4, and 0.05% Tween-20) binding buffer. HepG2 cells were grown in 24-well plates, and at confluence, the medium was changed to serumfree methionine-free Minimum Essential Medium (Gibco) containing TRAN3S-LABEL (ICN Biomedicals, Costa Mesa, CA; 50 &i/O.5 mL. well), with or without PMA (100 nM). The medium was removed after specified time periods. Twenty-five microliters of the monoclonal antibody D5-Affi Gel 10 suspension were incubated with 5-10 FL of the labeled medium overnight at 4 C, then washed three times in cold PBST. The final pellet was resuspended in 25 FL PBS and 25 FL 2 X Laemmli sample buffer containing 50 mmol/L dithiothreitol (final concentration) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% separating gel). The gel was fixed in 40% ethanol-lo% acetic acid overnight at 4 C, permeated with EN3HANCE (New England Nuclear Research Products, Wilmington, DE) for 30 min, washed with distilled water, dried, and autoradiographed using Kodak XAR film (Eastman Kodak Co., Rochester, NY). Bands were quantified by densitometry (Bio-Rad).
Results As previously reported, exposure to PMA (0.1-100 nmol/ L) led to distinct changesin HepG2 cell morphology (18, 19), including a more flattened and sharply delineated cell morphology. These changes were evident within 2 h of PMA addition and served asa visual marker for PMA action. PMA concentrations above l-10 pmol/L were cytotoxic, leading to decreasedIGFBP-1 and total protein production, morphological changes, and nonviability after refeeding with complete medium. PMA causeddose- and cell density-dependent stimulation of IGFBP-1 production by HepG2 cells. Figure 1 compares dose responsesin confluent and sparsecultures after a 14-h incubation. For this experiment, the DNA content was 22 f 2 and 8 + 1 pg/well in confluent and sparse cultures, respectively. Maximal PMA stimulation was noted between 100-1000 nmol/L. PMA (100 nmol/L) stimulated IGFBP-1 2-fold and more than 7-fold over the control (no additives)
600
z
700 600
zi ;
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z mm
200
200
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iii a--
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(nM)
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1OOK
FIG. 1. IGFBP-1 levels in response to PMA. Sparse (0) and confluent (0) cultures were incubated with SFM alone or SFM with PMA in doses between 0.1 nmol/L and 100 Fmol/L for 14 h. IGFBP-1 levels are plotted against the left y-axis as the mean + SE for triplicate wells. The percent increase over SFM-only cultures for sparse (0) and confluent (0) cultures is plotted against the right y-axis.
value in confluent and sparsecultures, respectively, a pattern confirmed in several similar studies. For subsequentexperiments, cells were studied at near confluence. Figure 2 (upper panel) shows a typical time-course study of IGFBP-1 production with PMA (1000 nmol/L). In several experiments a significant stimulatory effect of PMA over the control value was noted within 4-6 h and continued for at least 48 h. As shown in the inset graph in Fig. 2, PMA had no effect on total DNA over a 20-h time period. In similar experiments, total soluble protein was also unaffected by PMA (data not shown). By immunoprecipitation, PMA increasedthe synthesis of IGFBP-1 within 1 h of exposure (Fig. 2, lower panel). For most experiments, the conditioned medium was sampled at 14 h. The interactions of PMA with insulin over a 14-h time period are shown in Fig. 3. In the upper panel, increasing amounts of insulin were added with or without PMA (100 nmol/L). PMA increasedIGFBP-1 levels from 175 + 9 to 709 f 78 ng/mL (400%) in the absenceof insulin. With 10 nmol/ L insulin, IGFBP-1 levels were 33 -I 17 ng/mL without PMA and 462 + 116 ng/mL with PMA, a 14-fold stimulation. The PMA effect was negligible at insulin dosesof 100 and 200 nmol/L. In other experiments, a stimulatory effect of PMA was noted at the higher insulin doses, but the degree of stimulation was inhibited by insulin. The bottom panel of Fig. 3 shows the converse effect. PMA concentrations of 100 nmol/L or greater eliminated the inhibitory effect of insulin (100 nmol/L) on IGFBP-1 production. The relative interactions of insulin and PMA are likely to depend on the relative rates of binding, internalization, and intracellular action for these reagents when they are added simultaneously. To minimize the effects of these variables and examine the relative priority of PMA VS.insulin action, preincubation studies were performed. Cells were incubated in SFM with or without either PMA (100 nmol/L) or insulin (100 nmol/L) at 37 C for 1 h. The medium was then removed and replaced with graded dosesof insulin or PMA. As shown in Fig. 4 (upper panel), preexposure to PMA led to a 6-fold
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PKC EFFECT
= E s
700
5 7 iii '
500
600
400
4
2
6
time
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(hr)
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-
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-
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FIG. 2. Time course of IGFBP-1 response to PMA. Upper panel, At near confluence, cells were incubated with SFM (I@ or SFM plus PMA (1000 nmol/L; El). Data are shown as the mean f SE of triplicate wells. An asterisk indicates a significant increase (P < 0.05) for the PMAtreated samples compared to SFM-only cultures at that time point. The inset chart shows the DNA content per well for the same experiment. Total DNA was significantly higher than baseline at 20 h, but there was no difference between SFM and SFM plus PMA treatments at any time point. Lower panel, Immunoprecipitation of ?‘S-labeled medium at 1, 3, and 6 h in the presence and absence of PMA (100 nmol/L). The first lane contains pure [‘251]IGFBP-1 standard. Densitometric readings at 1, 3, and 6 h were 0, 0.43, and 1.64 for control and 0.26, 0.93, and 2.49 for PMA lanes, respectively (values expressed as area under the curve, OD X mm).
stimulation of IGFBP-1 production, which was not inhibited by subsequentaddition of insulin. Conversely, preexposure to insulin failed to significantly suppress dose-dependent stimulation by PMA (Fig. 4, bottom panel). Several other compounds known to affect the protein kinase-C (PKC) pathway were investigated for their influence on IGFBP-1 production. diC, is a rapidly metabolized, transient PKC stimulant (20). In several studies diCs (O.Ol100 nmol/L) had no effect on IGFBP-1 levels and no effect on insulin suppression or PMA stimulation of IGFBP-1 at time points ranging from 2-48 h (data not shown). Higher doseswere limited by the solvent dimethylsulfoxide, which was toxic at concentrations of 0.1% or more. SPH (0.1-100 pmol/L), a PKC inhibitor (15), did not influence IGFBP-1 production in the presence or absenceof either insulin or PMA. Preincubation (1 h) with SPH also had no influence on the effects of insulin or PMA. Low doses (0.1-10 pmol/L) of SPH alone had a slight stimulatory effect
ON IGFBP-1
461
on IGFBP-1 levels, but this was not consistently observed in all studies (data not shown). SPH dosesabove 100 pmol/L were limited by cytotoxicity secondary to the high concentration of BSA required for preparation of this reagent. In the absenceof SPH, BSA concentrations greater than 0.17% causedmorphologically evident cell death, with no recovery after refeeding. STS, another PKC inhibitor (21), increased IGFBP-1 production 2- to 4-fold in concentrations ranging from 0.1-10 nmol/L. As shown in Fig. 5 (upper panel), this effect was observed in both sparseand confluent cultures. As shown in Fig. 5 (lower panel), STS augmented PMA stimulation of IGFBP-1 production at both 16 and 24 h. Control IGFBP-1 levels at 16 and 24 h in the absence of PMA or STS are shown with the bars in Fig. 5. STS (l-10 nmol/L) also inhibited the ability of insulin to suppressIGFBP-1 levels by approximately 50%. STS concentrations of 100 nmol/L or more causedmorphologically evident cytotoxicity, rapid cell death, and failure to recover viability with complete medium. This effect was apparently due to the STS and not the dimethylsulfoxide solvent, which was maintained at lessthan 0.1%. H-7, reported to be a more specific PKC inhibitor than SPH or STS (22, 23), was also apparently cytotoxic. Ten and 100 mmol/L concentrations caused adverse morphological changes, including cell rounding and detachment; higher doses led to cell death and decreased IGFBP-1 levels. At these same H-7 doses, however, PMA (100 nmol/L) was capable of stimulating IGFBP-1 levels, suggesting that the effect of PMA was not inhibited in surviving cells. At doses below 10 mmol/L, H-7 had no independent effect on IGFBP1 levels, but caused a slight, not statistically significant, augmentation of PMA stimulation. Con-A has been reported to inhibit PMA-stimulated PKC translocation and degradation (24). As shown in Fig. 6, a lh preincubation with Con-A (250 cLg/mL.well) had no independent effect on IGFBP-1 production. However, Con-A completely inhibited the stimulatory effect of PMA (100 nmol/L) and suppressedthe effects of STS (1 nmol/L). As observed in a previous experiment (Fig. 5), simultaneous addition of PMA and STS had an additive effect to stimulate IGFBP-1 production, and this was also suppressedby ConA. Discussion Our results provide additional information regarding the effects of the PKC pathway on human IGFBP-1 production. Jalkanen et al. (12) found that PMA stimulates IGFBP-1 production by primary cultures of ovarian granulosa-luteal cells, with a maximal responsenoted at a PMA concentration of 1 ng/mL (-2 nmol/L) after 48-h exposure. The degree of stimulation was extremely variable, ranging from 3- to 30fold, and the earliest sampling time point was 24 h. The researchersconcluded that the PKC pathway probably has a role in regulating ovarian IGFBP-1 production. Lewitt et al. (25) reported that PMA stimulates HepG2 cell IGFBP-1 production; however, full details of this effect have not been published.
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LEE, ABDEL-MAGUID,
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AND SNUGGS
JCE & M. 1992 Vol’75.No2
FIG. 3. Interactions of PMA and insulin. The upper panel shows the effect of adding increasing concentrations of insulin in the presence (0) or absence (0) of PMA (100 nmol/L). The lower panel shows the effect of increasing concentrations of PMA in the presence (0) or absence (0) of insulin (100 nmol/L). All additions were made at time zero, with sampling after 14-h incubation. IGFBP1 levels are shown as the mean f SE of triplicate wells.
+I- preincubation: FIG. 4. Effects of preincubation with PMA or insulin. The upper panel shows the effects of adding increasing concentrations of insulin after a l-h preincubation with SFM (H) or SFM plus PMA (1000 nmol/L; 0). Asterisks indicate a significant change (P c 0.05) from the no insulin control. Plus PMA bars are significantly higher than the paired no PMA bar at each insulin concentration; however, there was no difference between the plus PMA bars. The lower panel shows the effects of adding increasing concentrations of PMA after a l-h incubation with SFM (I@ or insulin (100 nmol/L; 0). PMA caused a significant stimulation of IGFBP-1 production over the no-PMA controls, but there was no difference between the paired bars at each PMA concentration. Preincubations were begun at -1 h; preincubation medium was then removed and replaced with test conditions for 14 h. Data are the mean f SE of triplicate wells.
-
PMA
1 OOOnM
T
1
T 1
T
’
*
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*
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(nM)
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T +/- preincubation:
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1 OOnM
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Our results show that HepG2 IGFBP-1 production is also potently stimulated by PMA. Stimulation of IGFBP-1 synthesis was apparent within 1 h of PMA exposure. We also found that PMA has a greater relative stimulatory effect on IGFBP-1 levels in sparsecompared to confluent cell cultures. This latter observation may be related to the density dependence of HepG2 differentiation (26). In our studies, we used several known inhibitors of PKC to further define the specificity of the PMA effect. All three inhibitors, STS, SPH, and H-7, were used at concentrations
1000
(nM)
reported to cause PKC inhibition (15, 21, 22). We hypothesized that if basal or stimulated PKC activity actually enhanced IGFBP-1 production, PKC inhibitors should diminish this effect. However, the PMA effect was not inhibited by any of these compounds. At the higher dosestested, all were cytotoxic, possibly due to the effects of the solvent vehicle. At lower doses, STS stimulated IGFBP-1 production, while SPH and H-7 had no independent effect. However, STS and, to a lesserextent, SPH and H-7 caused an apparent paradoxical augmentation of the PMA stimulation.
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PKC EFFECT ON IGFBP-1
463
FIG. 5. Effects
of STS on HepG2 IGFBP-1 production. The upper panel shows the effects of increasing doses of SFM plus STS alone in sparse (0) and confluent (0) cultures after a 14-h incubation. The lower pane2 shows the effects of increasing amounts of STS in the presence of PMA (10 nmol), with wells sampled at 16 h (0) and 24 h (0) in a single experiment. The bars show IGFBP-1 levels in the absence of PMA and STS at 16 h (I@ and 24 h (0). Data are presented as the mean + SE for triplicate wells.
1 staurosporine
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j@jj#
STS STS+ConA PMA+STS PMA +STS+ConA 0
1000
2000
3000
4000
IGFBP-1 (rig/ml)
6. Effects of Con-A on IGFBP-1 production. Cells were preincubated with or without (control; C) Con-A (250 pg/mL.well) for 1 h, then exposed to experimental additives for 14 h. IGFBP-1 levels are presented as the mean + SE for triplicate wells.
FIG.
Based on these observations, we postulate that the stimulation of IGFBP-1 levels by PMA may be mediated through down-regulation, rather than stimulation, of PKC. PMA is known to potently stimulate PKC action (18, 27), followed by a rapid translocation of the enzyme and consequent down-regulation. In 3T3-Ll fibroblasts, this effect has been shown to occur within minutes of PMA exposure, and recovery of intracellular PKC activity may take several hours due to the long intracellular half-life of PMA (27). In HepG2 cells, exposure to 100 nmol/L PMA caused a 50% reduction in PKC activity within 24 h of exposure (19); earlier time points were not investigated. Our hypothesis predicts that PKC inhibitors will independently stimulate IGFBP-1 production, an effect noted for STS. Studies of the other inhibitors were dose-limited due to cytotoxicity, although the slight potentiation of the effect of
10
100 (nM)
PMA by low doses of H-7 and SPH agrees with our hypothesis that IGFBP-1 is stimulated by PKC inhibition. Furthermore, diCs, a PKC stimulant with a relatively short half-life which does not cause PKC down-regulation, did not stimulate IGFBP-1 synthesis. Finally, Con-A, which inhibits the down-regulation of PKC by PMA, inhibited the stimulatory effect of PMA on IGFBP-1 synthesis. All three PKC inhibitors tested also inhibit CAMP-dependent protein kinase-A (15, 21, 22). However, it is unlikely that this action accounts for our observations. Stimulation of CAMP has been found to enhance IGFBP-1 production by fetal liver explants (28, 29), and we have confirmed this finding in HepG2 cells in separate ongoing studies (unpublished data). Therefore, inhibition of CAMP, would also be expected to cause a decrease in IGFBP-1 production. The interactions of insulin with the PKC pathway appear to be complex. Simultaneous addition of insulin and PMA caused a dose-dependent inhibition of the effect of PMA, such that the effect of 100 nmol/L PMA was negligible in the presence of 100 nmol/L insulin. Conversely, PMA doses of 100 nmol/L or greater were capable of overriding the insulin effect. Although insulin potently inhibits HepG2 IGFBP-1 production, this effect was countered by preincubation with PMA, and preincubation with insulin had no effect on subsequent PMA stimulation. None of the PKC inhibitors had an independent effect on insulin action, suggesting that insulin-mediated suppression of IGFBP-1 production is not directly dependent on the PKC pathway. This latter finding was not unexpected, since other cellular effects of insulin are not mediated by the PKC system (30). More detailed studies will be required to define the interactions of insulin and PMA in regulation of IGFBP-1 production. It has been shown, for instance, that phorbol esters can alter insulin
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LEE, ABDEL-MAGUID,
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binding, cause phosphorylation of insulin receptor serine and threonine residues, and diminish insulin receptor-mediated events (18, 31). Our findings in HepG2 cells may have implications for understanding hepatocyte production of IGFBP-1. The HepG2 cell line has been a reliable model for numerous hepatocyte metabolic functions, including hepatic protein production, cholesterol metabolism, production of acute phase reactants, and insulin regulation of IGFBP-1 production (10, 26, 32, 33). Based on our data, the PKC system, or inhibition of this system, may play a role in maintaining basal and stimulated hepatic IGFBP-1 production. The mechanism(s) of this action is not defined and could include a number of known PKC-mediated intracellular actions, including effects on insulin/IGF receptor phosphorylation, ion channels, protooncogene induction, mitogenesis, and cell differentiation (18-20, 27, 30). Moreover, since the in viva regulation and function of the PKC system are poorly understood, further speculation regarding the interactions of PKC, insulin, IGFBP-1, and the IGFs will depend on additional investigations. Acknowledgments The authors acknowledge and C. A. Conover.
the thoughtful
advice
of Drs. D. R. Powell
References 1. Baxter RC, Martin
JL. 1989 Binding proteins for the insulin-like growth factors: structure, regulation and function. Prog Growth Factor Res. 1:49-68. On the nomenclature of 2. Drop SLS, Hintz RL. 1989 Introduction. the IGF binding proteins. In: Drop SLS, Hintz RL, eds: Insulin-like growth factor binding proteins. Amsterdam: Excerpta Medica; vvii. 3. Lee YL, Hintz RL, James PM, Lee PDK, Shively JE, Powell DR. 1988 Insulin-like growth factor (IGF) binding protein complementary deoxyribonucleic acid from HEP G2 hepatoma cells: predicted protein sequence suggest an IGF-binding domain different from those of the IGF-I and IGF-II receptors. Mol Endocrinol. 2:404-11. 4. Julkunen M, Koistinen R, Aalto-Setala K, Seppili M, Janne OA, Kontula K. 1988 Primary structure of human insulin-like growth factor binding protein/placental protein 12 and tissue-specific expression of its mRNA. FEBS Lett. 236:295-302. 5. Suikkari AM, Jalkanen J, Koistinen R, et al. 1989 Human granulosa cells synthesize low molecular weight insulin-like growth factor binding protein. Endocrinology. 124:1088-90. 6. Conover CA, Butler PC, Wang M, Rizza RA, Lee PDK. 1990 Lack of growth hormone effect on insulin-associated suppression of insulinlike growth factor binding protein 1 in humans. Diabetes. 39:1251-6. growth factor 7. Ritvos 0, Ranta T, Jalkanen J, et al. 1988 Insulin-like (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells. Endocrinology. 122:2150-7. 8. Burch WM, Correa J, Shively JE, Powell DR. 1990 The 25kilodalton insulin-like growth factor (IGF)-binding protein inhibits both basal and IGF-I-mediated growth of chick embryo cartilage in vitro. J Clin Endocrinol Metab. 70:173-80. an insulin-like 9. Liu L, Brinkman A, Blat C, Hare1 L. 1991 IGFBP-1, growth factor binding protein, is a cell growth inhibitor. Biochem Biophys Res Commun. 174:673-9. 10. Conover CA, Lee PDK. 1990 Insulin regulation of insulin-like growth factor binding protein production in cultured HepG2 cells.
AND J Clin 11.
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Powell DR, Suwanichkul A, Cubbage ML, DePaolis LA, Snuggs MB, Lee PDK. 1991 Insulin inhibits transcription of the human gene for insulin-like 266~18868-76.
growth
factor
binding
12.
Jalkanen J, Suikkari
29.
Lewitt MS, Baxter RC. 1990 Inhibitors
protein
1. J Biol Chem.
AM, Koistinen R, et al. 1989 Regulation of insulin-like growth factor-binding protein-l production in human granulosa-luteal cells. J Clin End&&o1 Metab. 69:1174-9. 13. Knowles BB. Howe CC, Aden DP. 1980 Human heoatocellular carcinoma cell lines secrete the maior plasma proteins and hepatitis B surface antigen. Science. 2093497-9: 14. Powell DR. Lee PDK. Shivelv IE. Eckenhausen M. Hintz RL. 1987 Method for purification oiah insulin-like growth’factor binding protein produced by human HEP-G2 hepatoma cells. J Chromatogr. 420:163-70. 15. Lambeth JD, Burnham DN, Tyagi SR. 1988 Sphinganine effects on chemoattractant-induced diacylglycerol generation, calcium fluxes, superoxide production, and on cell viability in the human neutrophil. J Biol Chem. 263:3818-22. 16. Labarca C, Paigen K. 1980 A simple, rapid and sensitive DNA assay procedure. Anal Biochem. 102:344-52. 17. Lee PDK, Hintz RL, Sperry JB, Baxter RC, Powell DR. 1989 Serum insulin-like growth factor binding proteins in growth-retarded children with chronic renal failure. Pediatr Res. 36:308-15. 18. Blake AD, Strader CD. 1986 Potentiation of specific association of insulin with HepG2 cells by phorbol esters. Biochem J. 236:227-34. 19. Duronio V, Huber BE, Jacobs S. 1990 Partial down-regulation of protein kinase C reverses the growth inhibitory effect of phorbol esters on HepG2 cells. J Cell Physiol. 145:381-9. 20. Nishizuka Y. Studies and perspectives of protein kinase C. 1986 Science. 233:305-12. 21. Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F. 1986 Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase. Biochem Biophys Res Commun. 135:397402. 22 Hidaka H, Inagaki M, Kawamoto S, Sasaki Y. 1984 Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 23:5036-41. 23. Felipo V, Miftana MD, Grisolia S. 1990 A specific inhibitor of protein kinase C induces differentiation of neuroblastoma cells. J Biol Chem. 265:9599-601. 24. Pate1 J, Kassis S. 1987 Concanavalin A prevents phorbol-mediated redistribution of protein kinase C and fl-adrenergic receptors in rat glioma C6 cells. Biochem Biophys Res Commun. 144:1265-72. 25. Lewitt MS, Ballesteros M, Baxter RC. Inhibition of glucose uptake by Hep G2 cells stimulates insulin-like growth factor binding protein-l (IGFBP-1) mRNA levels and protein synthesis [Abstract #1798]. Proc of the 73rd Annual Meet of The Endocrine Sot. 1991. 26. Kelly JH, Darlington GJ. 1989 Modulation of the liver specific phenotype in the human hepatoblastoma line HEP G2. In Vitro Cell Dev Biol. 25:217-22. 27. Blackshear PJ. 1988 Approaches to the study of protein kinase C involvement in signal transduction, Am J Med Sci. 296:231-40. 28. Lewitt MS, Baxter RC. 1989 Reeulation of erowth hormone-independent insulin-like growth fact&-binding Grotein (BP-28) in cultured human fetal liver explants. J Clin Endocrinol Metab. 69:246E? of glucose uptake stimulate of insulin-like growth factor-binding protein (IGFBPfetal liver. Endo&inology. 126:1527-u3’3. ’ Blackshear PJ. 1988 Insulin-stimulated protein biosynthesis as a paradigm of protein kinase C-independent growth factor action. Clin Res. OOO:OOO-00. Mudd LM, Raizada MK. 1989 Regulation of growth factor receptors by protein kinase C. In: LeRoith D, Raizada MK, eds. Molecular and cellular biology of insulin-like growth factors and their receptors. New York: Plenum Press; 315-25. Baumann H. 1989 Hepatic acute phase reaction in vivo and in vitro. In Vitro Cell Dev Biol. 25:115-26. Javitt NB. 1990 HepG2 cells as a resource for metabolic studies: lipoprotein, cholesterol and bile acids, FASEB J. 4:161-8. the production 1) by human
30.
31.
32. 33.
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Vol. 15, No. 2 Prrnted
in U.S.A.
Role of Protein Kinase-C in Regulation of Insulin-Like Growth Factor-Binding Protein- 1 Production by HepG2 Cells* PHILLIP
D. K. LEE,
Department
of
Pediatrics,
LAURA
S. ABDEL-MAGUID,
Baylor College of Medicine,
Houston,
AND
MARK
B. SNUGGS
Texas 77030 did not inhibit PMA stimulation. The transient PKC activator diCs had no effect, while studies with the PKC inhibitors sphinganine and H-7 were limited by solvent vehicle cytotoxicity. Staurosporine (STS), a potent PKC inhibitor, stimulated IGFBP-1 production 2- to 4-fold and augmented the stimulatory effect of PMA. Concanavalin-A, an inhibitor of PMA-stimulated PKC translocation and down-regulation, inhibited the effects of PMA and STS. Our findings indicate that PKC is involved in the regulation of hepatic IGFBP-1 production. The effects of PMA, which causes rapid activation, followed by membrane translocation and down-regulation of PKC, are similar to those of STS and are countered by Concanavalin-A. These data suggest that PKC activity may mediate tonic inhibition of IGFBP-1 production, while PKC downregulation stimulates the production of this regulatory protein. (J Clin Endocrinol Metab 75: 459-464, 1992)
ABSTRACT Insulin-like growth factor binding protein-l (IGFBP-1) is a liverderived protein that modulates the mitogenic actions of the insulinlike growth factors (IGFs). IGFBP-1 production is potently inhibited by insulin both in uiuo and in HepG2 human hepatoma cells. To further define the pathways of IGFBP-1 regulation, we studied the effects of modulators of nrotein kinase-C (PKC) on HenG2 cell IGFBP-1 nroduction. Phorbbl 12-myristate li-acetate (PM& stimulated IGFiP-1 production in a timeand dose-dependent manner, with maximal stimulation occurring at lo-100 nmol/L. The degree of stimulation was dependent on cell density, ranging from about 2-fold in confluent to more than IO-fold in sparse cultures. Preincubation with PMA abolished the inhibitory effect of insulin, while preincubation with insulin
I
NSULIN -like growth factor-binding protein-l (IGFBP-1) is one of several structurally related specific BPS for insulin-like growth factor-I and -11(IGF-I and -11)(l-3). In nonpregnant humans, IGFBP-1 is produced primarily by the liver and ovarian granulosa cells (4, 5). Plasma IGFBP-1 levels may vary more than lo-fold in response to normal daily meal patterns, an effect that is mediated by insulin (6). Since IGFBP-1 has been shown to inhibit IGF-mediated mitogenesis in vitro (7-9), we have postulated that insulin regulation of IGFBP-1 production may play a role in modulating IGF action in relation to substrate availability (6). We have previously characterized the HepG2 human liver cell line, a minimal change continuous line derived from a hepatoma, as a model for studiesof IGFBP-1 production (10, 11). HepG2 cells produce IGFBP-1, but none of the other IGFBPs. Since phorbol 12-myristate 13-acetate (PMA) has been shown to increase IGFBP-1 production by ovarian granulosa cells (12), we studied its effect on HepG2 IGFBP1 production and its possibleinteractions with insulin. Our resultsdemonstrate that PMA is a potent stimulant of HepG2 IGFBP-1 production, and that the mechanism of this stimulation may be complex.
Materials
and Methods
Materials The HepG2 human hepatoma-derived cell line, originally reported by Knowles et al. (13), was obtained from the American Type Culture Collection (Rockville, MD). Pure IGFBP-I was prepared from HepG2 cell-conditioned medium or human amniotic fluid, as previously described (14). Pure recombinant DNA-derived human insulin was obtained from Lilly (Indianapolis, IN). PMA and o-erythro-sphingosine (SPH) were obtained from Calbiochem (San Diego, CA); 1,2dioctanoylsn-glycerol (diQ, staurosporine (STS), 1-(5.isoquinolinylsulfonyl)2methyl piperazine (H-7), and Concanavalin-A (type IV; Con-A) were obtained from Sigma (St. Louis, MO). Stock solutions of 1 mmol/L PMA, 100 mmol/L diCs, and 1 mmol/L STS were prepared in dimethylsulfoxide. SPH was prepared to 100 mmol/L in absolute ethanol, then diluted to 1 mmol in 17% RIA grade, insulin-free BSA (Miles Pentex, Kankakee, IL) (15). Stock solutions were stored in aliquots at -70 C and diluted to appropriate concentrations in serum-free medium (SFM) for experiments.
Cell culture HepG2 cells were cultivated in Dulbecco’s Minimum Essential Medium, 2 mmol/L L-glutamine, 1% penicillin/streptomycin, and 10% fetal bovine serum (Gibco, Grand Island, NY), passaged at confluence after trypsin detachment, and plated into 24-well multiwell plates (2 cm’/ well) for the investigations. After l-3 days, the complete medium was removed, monolayers were washed with sterile saline, and the medium was replaced with serum-free RPMI-1640 medium with 2 mmol/L L-glutamine and experimental additives. The conditioned medium was removed at the specified time intervals and stored in siliconized tubes at -70 C until assay. HepG2 cells tend to aggregate in suspension, and direct cell counting was not routinely feasible. Therefore, cell number was measured using a DNA minifluorometer (Hoeffer Scientific Instruments, San Francisco, CA) after dissolution in 0.1% sodium dodecyl sulfate and staining with bisbenzimide (Hoechst dye 33258) (16). Standards were calf thymus
Received August 26, 1991. Address all correspondence and requests for reprints to: Phillip D. K. Lee, M.D., Texas Children’s Hospital, Clinical Care Center, MC 3-2351, 6621 Fannin Street, Houston, Texas 77030-2399. * Portions of these investigations were presented at the 73rd Annual Meeting of The Endocrine Society, Washington, D.C., 1991 (Abstract 1797). This work was supported by a Feasibility Grant from the American Diabetes Association, with funds contributed by the Colorado Chapter of the ADA (to P.D.K.L.), and by the Baylor College of Medicine Summer Medical and Research Training program (to L.S.A.).
459
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LEE, ABDEL-MAGUID,
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AND SNUGGS 600
DNA (Clontech, Palo Alto, CA). With single cell suspensions of HepG2 cells, we found a direct correlation of cell number by hemocytometer and DNA by fluorometry (r = 0.98; P = 0.0004). Total protein in the conditioned medium was determined using the Bio-Rad Protein Assay Kit (Bio-Rad, Richmond, CA).
JCE & M. 1992 Vol75’NoZ
r
1000 900
IGFBP-1 RIA IGFBP-1 levels were measured by specific RIA, as previously described (17). Briefly, 100 PL conditioned medium [diluted if necessary in assay buffer (phosphate-buffered saline, pH 7.4; 0.05% Tween-20; and 0.1% BSA)] were preincubated for 1 h with 100 PL polyclonal rabbit antihuman IGFBP-1 (final titer, 1:2500), followed by the addition of 5OpL (-10,000 cpm) [iZ51]IGFBP-1. [‘2’I]IGFBI’-1 was prepared using the Iodobead method (Pierce Chemical Co., Rockford, IL). After a 16-h incubation at 4 C, bound and free counts were separated using agaroseimmobilized goat antirabbit immunoglobulin (Bio-Rad), and bound counts were measured in an automatic y-counter. Inter- and intraassay coefficients of variation at 7.86 ng/mL were estimated to be 17.3 rt 1.4% and 13.4 f 3.8%, respectively, with assay limits of approximately 1 and 200 ng/mL. Samples from each study were grouped to minimize the effect of interassay variability. RIA data were analyzed using the IBMPC RIA Data Reduction Program (M. L. Jaffe, Silver Spring, MD).
Immunoprecipitation Mouse ascites fluid containing monoclonal antibody D5 to human IGFBP-1 was prepared as previously described (1 l), coupled to Affi-Gel 10 agarose beads (Bio-Rad) according to manufacturer’s instructions, and resuspended in PBST (Dulbecco’s phosphate-buffered saline, pH 7.4, and 0.05% Tween-20) binding buffer. HepG2 cells were grown in 24-well plates, and at confluence, the medium was changed to serumfree methionine-free Minimum Essential Medium (Gibco) containing TRAN3S-LABEL (ICN Biomedicals, Costa Mesa, CA; 50 &i/O.5 mL. well), with or without PMA (100 nM). The medium was removed after specified time periods. Twenty-five microliters of the monoclonal antibody D5-Affi Gel 10 suspension were incubated with 5-10 FL of the labeled medium overnight at 4 C, then washed three times in cold PBST. The final pellet was resuspended in 25 FL PBS and 25 FL 2 X Laemmli sample buffer containing 50 mmol/L dithiothreitol (final concentration) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% separating gel). The gel was fixed in 40% ethanol-lo% acetic acid overnight at 4 C, permeated with EN3HANCE (New England Nuclear Research Products, Wilmington, DE) for 30 min, washed with distilled water, dried, and autoradiographed using Kodak XAR film (Eastman Kodak Co., Rochester, NY). Bands were quantified by densitometry (Bio-Rad).
Results As previously reported, exposure to PMA (0.1-100 nmol/ L) led to distinct changesin HepG2 cell morphology (18, 19), including a more flattened and sharply delineated cell morphology. These changes were evident within 2 h of PMA addition and served asa visual marker for PMA action. PMA concentrations above l-10 pmol/L were cytotoxic, leading to decreasedIGFBP-1 and total protein production, morphological changes, and nonviability after refeeding with complete medium. PMA causeddose- and cell density-dependent stimulation of IGFBP-1 production by HepG2 cells. Figure 1 compares dose responsesin confluent and sparsecultures after a 14-h incubation. For this experiment, the DNA content was 22 f 2 and 8 + 1 pg/well in confluent and sparse cultures, respectively. Maximal PMA stimulation was noted between 100-1000 nmol/L. PMA (100 nmol/L) stimulated IGFBP-1 2-fold and more than 7-fold over the control (no additives)
600
z
700 600
zi ;
500
z
400 a
300
Sk -&
z mm
200
200
100
iii a--
100 0 0
0.1
1
10
100
PMA
(nM)
1000
10K
1OOK
FIG. 1. IGFBP-1 levels in response to PMA. Sparse (0) and confluent (0) cultures were incubated with SFM alone or SFM with PMA in doses between 0.1 nmol/L and 100 Fmol/L for 14 h. IGFBP-1 levels are plotted against the left y-axis as the mean + SE for triplicate wells. The percent increase over SFM-only cultures for sparse (0) and confluent (0) cultures is plotted against the right y-axis.
value in confluent and sparsecultures, respectively, a pattern confirmed in several similar studies. For subsequentexperiments, cells were studied at near confluence. Figure 2 (upper panel) shows a typical time-course study of IGFBP-1 production with PMA (1000 nmol/L). In several experiments a significant stimulatory effect of PMA over the control value was noted within 4-6 h and continued for at least 48 h. As shown in the inset graph in Fig. 2, PMA had no effect on total DNA over a 20-h time period. In similar experiments, total soluble protein was also unaffected by PMA (data not shown). By immunoprecipitation, PMA increasedthe synthesis of IGFBP-1 within 1 h of exposure (Fig. 2, lower panel). For most experiments, the conditioned medium was sampled at 14 h. The interactions of PMA with insulin over a 14-h time period are shown in Fig. 3. In the upper panel, increasing amounts of insulin were added with or without PMA (100 nmol/L). PMA increasedIGFBP-1 levels from 175 + 9 to 709 f 78 ng/mL (400%) in the absenceof insulin. With 10 nmol/ L insulin, IGFBP-1 levels were 33 -I 17 ng/mL without PMA and 462 + 116 ng/mL with PMA, a 14-fold stimulation. The PMA effect was negligible at insulin dosesof 100 and 200 nmol/L. In other experiments, a stimulatory effect of PMA was noted at the higher insulin doses, but the degree of stimulation was inhibited by insulin. The bottom panel of Fig. 3 shows the converse effect. PMA concentrations of 100 nmol/L or greater eliminated the inhibitory effect of insulin (100 nmol/L) on IGFBP-1 production. The relative interactions of insulin and PMA are likely to depend on the relative rates of binding, internalization, and intracellular action for these reagents when they are added simultaneously. To minimize the effects of these variables and examine the relative priority of PMA VS.insulin action, preincubation studies were performed. Cells were incubated in SFM with or without either PMA (100 nmol/L) or insulin (100 nmol/L) at 37 C for 1 h. The medium was then removed and replaced with graded dosesof insulin or PMA. As shown in Fig. 4 (upper panel), preexposure to PMA led to a 6-fold
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PKC EFFECT
= E s
700
5 7 iii '
500
600
400
4
2
6
time
20
(hr)
4
6
time
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-
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+
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-
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+
-
+
6
FIG. 2. Time course of IGFBP-1 response to PMA. Upper panel, At near confluence, cells were incubated with SFM (I@ or SFM plus PMA (1000 nmol/L; El). Data are shown as the mean f SE of triplicate wells. An asterisk indicates a significant increase (P < 0.05) for the PMAtreated samples compared to SFM-only cultures at that time point. The inset chart shows the DNA content per well for the same experiment. Total DNA was significantly higher than baseline at 20 h, but there was no difference between SFM and SFM plus PMA treatments at any time point. Lower panel, Immunoprecipitation of ?‘S-labeled medium at 1, 3, and 6 h in the presence and absence of PMA (100 nmol/L). The first lane contains pure [‘251]IGFBP-1 standard. Densitometric readings at 1, 3, and 6 h were 0, 0.43, and 1.64 for control and 0.26, 0.93, and 2.49 for PMA lanes, respectively (values expressed as area under the curve, OD X mm).
stimulation of IGFBP-1 production, which was not inhibited by subsequentaddition of insulin. Conversely, preexposure to insulin failed to significantly suppress dose-dependent stimulation by PMA (Fig. 4, bottom panel). Several other compounds known to affect the protein kinase-C (PKC) pathway were investigated for their influence on IGFBP-1 production. diC, is a rapidly metabolized, transient PKC stimulant (20). In several studies diCs (O.Ol100 nmol/L) had no effect on IGFBP-1 levels and no effect on insulin suppression or PMA stimulation of IGFBP-1 at time points ranging from 2-48 h (data not shown). Higher doseswere limited by the solvent dimethylsulfoxide, which was toxic at concentrations of 0.1% or more. SPH (0.1-100 pmol/L), a PKC inhibitor (15), did not influence IGFBP-1 production in the presence or absenceof either insulin or PMA. Preincubation (1 h) with SPH also had no influence on the effects of insulin or PMA. Low doses (0.1-10 pmol/L) of SPH alone had a slight stimulatory effect
ON IGFBP-1
461
on IGFBP-1 levels, but this was not consistently observed in all studies (data not shown). SPH dosesabove 100 pmol/L were limited by cytotoxicity secondary to the high concentration of BSA required for preparation of this reagent. In the absenceof SPH, BSA concentrations greater than 0.17% causedmorphologically evident cell death, with no recovery after refeeding. STS, another PKC inhibitor (21), increased IGFBP-1 production 2- to 4-fold in concentrations ranging from 0.1-10 nmol/L. As shown in Fig. 5 (upper panel), this effect was observed in both sparseand confluent cultures. As shown in Fig. 5 (lower panel), STS augmented PMA stimulation of IGFBP-1 production at both 16 and 24 h. Control IGFBP-1 levels at 16 and 24 h in the absence of PMA or STS are shown with the bars in Fig. 5. STS (l-10 nmol/L) also inhibited the ability of insulin to suppressIGFBP-1 levels by approximately 50%. STS concentrations of 100 nmol/L or more causedmorphologically evident cytotoxicity, rapid cell death, and failure to recover viability with complete medium. This effect was apparently due to the STS and not the dimethylsulfoxide solvent, which was maintained at lessthan 0.1%. H-7, reported to be a more specific PKC inhibitor than SPH or STS (22, 23), was also apparently cytotoxic. Ten and 100 mmol/L concentrations caused adverse morphological changes, including cell rounding and detachment; higher doses led to cell death and decreased IGFBP-1 levels. At these same H-7 doses, however, PMA (100 nmol/L) was capable of stimulating IGFBP-1 levels, suggesting that the effect of PMA was not inhibited in surviving cells. At doses below 10 mmol/L, H-7 had no independent effect on IGFBP1 levels, but caused a slight, not statistically significant, augmentation of PMA stimulation. Con-A has been reported to inhibit PMA-stimulated PKC translocation and degradation (24). As shown in Fig. 6, a lh preincubation with Con-A (250 cLg/mL.well) had no independent effect on IGFBP-1 production. However, Con-A completely inhibited the stimulatory effect of PMA (100 nmol/L) and suppressedthe effects of STS (1 nmol/L). As observed in a previous experiment (Fig. 5), simultaneous addition of PMA and STS had an additive effect to stimulate IGFBP-1 production, and this was also suppressedby ConA. Discussion Our results provide additional information regarding the effects of the PKC pathway on human IGFBP-1 production. Jalkanen et al. (12) found that PMA stimulates IGFBP-1 production by primary cultures of ovarian granulosa-luteal cells, with a maximal responsenoted at a PMA concentration of 1 ng/mL (-2 nmol/L) after 48-h exposure. The degree of stimulation was extremely variable, ranging from 3- to 30fold, and the earliest sampling time point was 24 h. The researchersconcluded that the PKC pathway probably has a role in regulating ovarian IGFBP-1 production. Lewitt et al. (25) reported that PMA stimulates HepG2 cell IGFBP-1 production; however, full details of this effect have not been published.
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LEE, ABDEL-MAGUID,
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AND SNUGGS
JCE & M. 1992 Vol’75.No2
FIG. 3. Interactions of PMA and insulin. The upper panel shows the effect of adding increasing concentrations of insulin in the presence (0) or absence (0) of PMA (100 nmol/L). The lower panel shows the effect of increasing concentrations of PMA in the presence (0) or absence (0) of insulin (100 nmol/L). All additions were made at time zero, with sampling after 14-h incubation. IGFBP1 levels are shown as the mean f SE of triplicate wells.
+I- preincubation: FIG. 4. Effects of preincubation with PMA or insulin. The upper panel shows the effects of adding increasing concentrations of insulin after a l-h preincubation with SFM (H) or SFM plus PMA (1000 nmol/L; 0). Asterisks indicate a significant change (P c 0.05) from the no insulin control. Plus PMA bars are significantly higher than the paired no PMA bar at each insulin concentration; however, there was no difference between the plus PMA bars. The lower panel shows the effects of adding increasing concentrations of PMA after a l-h incubation with SFM (I@ or insulin (100 nmol/L; 0). PMA caused a significant stimulation of IGFBP-1 production over the no-PMA controls, but there was no difference between the paired bars at each PMA concentration. Preincubations were begun at -1 h; preincubation medium was then removed and replaced with test conditions for 14 h. Data are the mean f SE of triplicate wells.
-
PMA
1 OOOnM
T
1
T 1
T
’
*
0
*
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*
100
insulin
T
1000
(nM)
c
T +/- preincubation:
0
insulin
1 OOnM
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100
PMA
Our results show that HepG2 IGFBP-1 production is also potently stimulated by PMA. Stimulation of IGFBP-1 synthesis was apparent within 1 h of PMA exposure. We also found that PMA has a greater relative stimulatory effect on IGFBP-1 levels in sparsecompared to confluent cell cultures. This latter observation may be related to the density dependence of HepG2 differentiation (26). In our studies, we used several known inhibitors of PKC to further define the specificity of the PMA effect. All three inhibitors, STS, SPH, and H-7, were used at concentrations
1000
(nM)
reported to cause PKC inhibition (15, 21, 22). We hypothesized that if basal or stimulated PKC activity actually enhanced IGFBP-1 production, PKC inhibitors should diminish this effect. However, the PMA effect was not inhibited by any of these compounds. At the higher dosestested, all were cytotoxic, possibly due to the effects of the solvent vehicle. At lower doses, STS stimulated IGFBP-1 production, while SPH and H-7 had no independent effect. However, STS and, to a lesserextent, SPH and H-7 caused an apparent paradoxical augmentation of the PMA stimulation.
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PKC EFFECT ON IGFBP-1
463
FIG. 5. Effects
of STS on HepG2 IGFBP-1 production. The upper panel shows the effects of increasing doses of SFM plus STS alone in sparse (0) and confluent (0) cultures after a 14-h incubation. The lower pane2 shows the effects of increasing amounts of STS in the presence of PMA (10 nmol), with wells sampled at 16 h (0) and 24 h (0) in a single experiment. The bars show IGFBP-1 levels in the absence of PMA and STS at 16 h (I@ and 24 h (0). Data are presented as the mean + SE for triplicate wells.
1 staurosporine
PM,PMAtConA
j@jj#
STS STS+ConA PMA+STS PMA +STS+ConA 0
1000
2000
3000
4000
IGFBP-1 (rig/ml)
6. Effects of Con-A on IGFBP-1 production. Cells were preincubated with or without (control; C) Con-A (250 pg/mL.well) for 1 h, then exposed to experimental additives for 14 h. IGFBP-1 levels are presented as the mean + SE for triplicate wells.
FIG.
Based on these observations, we postulate that the stimulation of IGFBP-1 levels by PMA may be mediated through down-regulation, rather than stimulation, of PKC. PMA is known to potently stimulate PKC action (18, 27), followed by a rapid translocation of the enzyme and consequent down-regulation. In 3T3-Ll fibroblasts, this effect has been shown to occur within minutes of PMA exposure, and recovery of intracellular PKC activity may take several hours due to the long intracellular half-life of PMA (27). In HepG2 cells, exposure to 100 nmol/L PMA caused a 50% reduction in PKC activity within 24 h of exposure (19); earlier time points were not investigated. Our hypothesis predicts that PKC inhibitors will independently stimulate IGFBP-1 production, an effect noted for STS. Studies of the other inhibitors were dose-limited due to cytotoxicity, although the slight potentiation of the effect of
10
100 (nM)
PMA by low doses of H-7 and SPH agrees with our hypothesis that IGFBP-1 is stimulated by PKC inhibition. Furthermore, diCs, a PKC stimulant with a relatively short half-life which does not cause PKC down-regulation, did not stimulate IGFBP-1 synthesis. Finally, Con-A, which inhibits the down-regulation of PKC by PMA, inhibited the stimulatory effect of PMA on IGFBP-1 synthesis. All three PKC inhibitors tested also inhibit CAMP-dependent protein kinase-A (15, 21, 22). However, it is unlikely that this action accounts for our observations. Stimulation of CAMP has been found to enhance IGFBP-1 production by fetal liver explants (28, 29), and we have confirmed this finding in HepG2 cells in separate ongoing studies (unpublished data). Therefore, inhibition of CAMP, would also be expected to cause a decrease in IGFBP-1 production. The interactions of insulin with the PKC pathway appear to be complex. Simultaneous addition of insulin and PMA caused a dose-dependent inhibition of the effect of PMA, such that the effect of 100 nmol/L PMA was negligible in the presence of 100 nmol/L insulin. Conversely, PMA doses of 100 nmol/L or greater were capable of overriding the insulin effect. Although insulin potently inhibits HepG2 IGFBP-1 production, this effect was countered by preincubation with PMA, and preincubation with insulin had no effect on subsequent PMA stimulation. None of the PKC inhibitors had an independent effect on insulin action, suggesting that insulin-mediated suppression of IGFBP-1 production is not directly dependent on the PKC pathway. This latter finding was not unexpected, since other cellular effects of insulin are not mediated by the PKC system (30). More detailed studies will be required to define the interactions of insulin and PMA in regulation of IGFBP-1 production. It has been shown, for instance, that phorbol esters can alter insulin
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LEE, ABDEL-MAGUID,
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binding, cause phosphorylation of insulin receptor serine and threonine residues, and diminish insulin receptor-mediated events (18, 31). Our findings in HepG2 cells may have implications for understanding hepatocyte production of IGFBP-1. The HepG2 cell line has been a reliable model for numerous hepatocyte metabolic functions, including hepatic protein production, cholesterol metabolism, production of acute phase reactants, and insulin regulation of IGFBP-1 production (10, 26, 32, 33). Based on our data, the PKC system, or inhibition of this system, may play a role in maintaining basal and stimulated hepatic IGFBP-1 production. The mechanism(s) of this action is not defined and could include a number of known PKC-mediated intracellular actions, including effects on insulin/IGF receptor phosphorylation, ion channels, protooncogene induction, mitogenesis, and cell differentiation (18-20, 27, 30). Moreover, since the in viva regulation and function of the PKC system are poorly understood, further speculation regarding the interactions of PKC, insulin, IGFBP-1, and the IGFs will depend on additional investigations. Acknowledgments The authors acknowledge and C. A. Conover.
the thoughtful
advice
of Drs. D. R. Powell
References 1. Baxter RC, Martin
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