Atherosclerosis, 97 (1992) 2 l-28 0 1992 Elsevier Scientific Publishers Printed and Published in Ireland
ATHERO
21 Ireland,
Ltd. All rights reserved.
0021-9150/92/$05.00
04909
Transforming
Catriona
‘Department
growth factor-& and interleukin- 1p stimulate LDL receptor activity in Hep G2 cells
D. Moorby”,
Ermanno
Gherardib, Lynda Doveya, Cheryl Godliman” and David E. Bowyera
of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 lQP and bI. C.R.F. Laboratory, AddenbrookeS Hospital, Hills Road, Cambridge CB2 2QH (UK)
Cell Interactions
(Received 28 October, 1991) (Revised, received 26 June, 1992) (Accepted 22 July, 1992)
Summary
The effect of transforming growth factor-pi (TGF-P,) and interleukin-lp (IL-l,) on LDL receptor in Hep G2 cells was investigated. A greater than two-fold stimulation of the binding and internalisation of [1251]-labelled LDL at 37°C was observed after an 18-h incubation of the cells with TGF-& at 50 @ml and IL-l, at 11 700 units/ml compared with control cells. Scatchard analysis of the binding of [‘251]labelled LDL at 4°C after an 18-h incubation of the cells with 1170 units/ml IL-l, and 5 rig/ml TGF-PI showed that they were both acting primarily by increasing LDL receptor number. The increase in LDL receptor activity could not be attributed to an increase in cell proliferation as TGF-/3t at concentrations from 0.05 ng/ml to 50 t&ml had no significant effect on either cell number or [3H]thymidine incorporation into DNA whilst IL-l, inhibited DNA synthesis by more than 80% at a concentration of 11 700 units/ml but had significant effect on cell number. Cholesterol biosynthesis from [ 14C]acetate, in contrast to the stimulation of LDL receptor activity, was inhibited by approximately two-fold by incubation with TGF-0, at 50 rig/ml and IL-1~ at 11 700 units/ml,
Key words: LDL receptor; TGF-0,; IL-l; Hep G2
Introduction
Correspondence to: Dr. D.E. Bowyer, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 IQP, UK. Tel.: (0223) 33 37 22; Fax: (0223) 33 33 46.
Uptake of low density lipoprotein (LDL) by the LDL receptor in the liver is a major determinant of the plasma cholesterol level [ 11. It is, therefore, important to understand what controls the activity of the hepatic LDL receptor. Previous investi-
22
gators have shown that the level of the LDL receptor in hepatocytes and/or Hep G2 cells can be modulated by the lipoproteins LDL and HDL and the hormones insulin, 17&estradiol, dexamethasone and triiodo+thyronine [2-41. However, little attention has focused on the potential role of paracrine factors in regulating LDL receptor activity in the hepatocyte. That paracrine factors may be important in controlling LDL receptor activity in the liver is suggested by two lines of evidence. Firstly, there is a growing number of reports of cytokines modulating LDL receptor activity in extrahepatic cell types, for example PDGF has been shown to increase LDL receptor activity in fibroblasts [5]. Secondly, we have found that conditioned medium from porcine smooth muscle cells (PC SMC) potently stimulates the binding and internalisation of [1251]-labelled LDL at 37°C by Hep G2 cells [6]. The mediator of this stimulation has not been identified, but preliminary characterisation studies suggest that it is a protein(s) of low molecular weight. To investigate further the role of paracrine factors in regulating the activity of the hepatic LDL receptor we investigated the effect of two cytokines, transforming growth factor-pi (TGF-Pi) and interleukin-1 (IL-Is) on the binding and internalisation of [ 1251]-labelled LDL by Hep G2 cells. As serum contains several factors which are known to modulate LDL receptor the Hep G2 cells used in these studies were cultured in a serumfree chemically defined medium (CDM) [7]. During the preparation of this manuscript Grove et al. have reported TGF-/3 at 200 &ml and IL-l at 25 @ml increase LDL uptake, respectively by 24% and 27% by Hep G2 cells after a 20-h incubation compared with cells incubated in RPM1 containing 5% LPDS [8]. The studies reported here confirm and extend these findings by establishing the concentration range at which TGF-/3 and IL-l are active and their effects on LDL receptor number ([ ‘251]-labelled LDL binding at 4”C), cholesterol synthesis and cell proliferation. Methods Materials
TGF-Pi, prepared from human platelets, was obtained from British Biotechnology Limited. Recombinant IL-l s was from Bachem UK
Limited. TGF-Pi (1 pg) was reconstituted in 1 ml of 4 mM HCI. IL-l, was reconstituted in PBS. Further dilutions of the cytokines were made in RPM1 1640 containing 1 mg/ml defatted BSA as indicated in the figure legends immediately prior to adding to the cells. Cell culture
Hep G2 cells (ATCC) were routinely passaged in RPM1 1640 (Flow laboratories) supplemented with 1.19 8/l Hepes, 2 g/l sodium bicarbonate (BDH), 0.06 g/l penicillin G (Sigma), 0.1 g/l streptomycin sulphate (Sigma) and 10% v/v FCS (Biological Industries) in 95% sir/5% CO2 at 37°C. For experiments the cells were plated at a density of 75 000 cells/cm2 in 2-cm2 microwell dishes (Falcon) in 1.5 ml of CDM. The CDM consisted of RPM1 1640 (Flow laboratories) containing 2 g/l sodium bicarbonate, 1.19 g/l Hepes, 0.06 g/l penicillin G, 0.1 g/l streptomycin sulphate, 10 I.U./l prolactin (Sigma), 5 x 10m7 M insulin (Sigma), 5 x 10m8 M glucagon (Sigma), 1 x 10e8 M hydrocortisone (Sigma), 1 x 10V9 M triiodo+thyronine (Sigma), 10 mg/ml LDL, 5 x 10e5 M inosine (Sigma), 1 x low7 M copper sulphate, 5 x lo-” M zinc sulphate, 1 x 10e7 M manganese sulphate, 5 x lo-l5 M nickel chloride, 1 x 10m7M iron sulphate and 3 x 10m9 M sodium selenite. Two days after plating the cells were fed with 1 ml of CDM. When the cells had reached confluence, day 4, they were washed 2 times with PBS before incubating for 18 h with either RPM1 1640 or RPM1 1640 containing the indicated concentrations of cytokines. The binding and internalisation of [ 1251]-labelled LDL at 37°C cholesterol synthesis or DNA synthesis by the cells was then measured. Measurement of [‘“I+labelled LDL binding and internalisation LDL, d 1.019-1.063 g/ml, was isolated from
freshly prepared human serum by sequential density gradient ultracentrifugation [9]. The LDL was labelled with Na”‘I (Amersham) using lactoperoxidase (Sigma) conjugated to Sepharose 4B (Pharmacia) [lo]. The binding of [1251]-labelled LDL at 4°C and the binding and internalisation of [ ‘251]-labelled
23
LDL at 37°C was then measured essentially as described by Goldstein and Brown [1 11. For measurement of the binding of [12?]labelled LDL at 4°C the cells were placed on ice for 30 min, washed with PBS and then incubated with RPM1 1640 containing 1% BSA and the indicated concentrations of [ ‘251]-labelled LDL for 2 h in the presence (to measure low affinity binding and internalisation) or absence (to measure total binding and internalisation) of 0.5 mg/ml of unlabelled LDL at 4°C. The cells were then washed three times with 0.05 M Tris 0.15 M NaCl pH 7.4 containing 2 mg/ml BSA and once with 0.05 M Tris 0.15 M NaCl pH 7.4. The cells were harvested with 1% SDS and the radioactivity associated with them measured by gamma counting. High affinity or receptor-mediated binding was calculated by subtracting the low affinity binding from the total binding. Results were standardised in terms of the protein/dish. The binding and internalisation of [1251]labelled LDL at 37°C was determined by incubating the cells for 4 h with RPM1 1640 containing 1% BSA and a non-saturating concentration of 5 pg/ml of [‘251]-labelled LDL with a specific activity of 100 counts/min in the presence or absence of 0.5 mg/ml native LDL. The cells were then placed on ice and washed and harvested as described above for the measurement of [ ‘251]-labelled LDL binding at 4°C.
[ 3H]cholesterol (Amersham) as an internal standard were added sequentially to 450 ~1 of the cell sonicate in a glass tube and incubated at 40°C for 1 h. The extracts were Folch-washed with 1.4 ml of 0.73% saline for 18 h at 4°C [ 131. The aqueous layer was removed by aspiration and the lower phase evaporated to dryness in a Buchler vortex evaporator. The lipids were saponified by adding 200 ~1 of 0.5 M NaOH in 96% ethanol (BDH) to each tube and incubating at 60°C for 90 min. Water (400 ~1) was added to each tube and the non-saponified lipids extracted with 500-~1 aliquots of hexane (BDH). The pooled extracts were washed with 800 ~1 of 0.25 M NaOH in 48% ethanol and then evaporated to dryness under N2 gas. The sample was redissolved in 20 ~1 of chloroform/methanol 2:1 v/v and applied to 14 cm2 Alugram Sil G thin layer chromatography plates (Macherey-Nagel, Duren). The plates were run in hexanelheptaneldiethyletheriacetic acid solvent 60:20:20:1 by vol. until the solvent front was 0.5 cm from the top of the plate. The lipids on the plate were visualised with iodine vapour (BDH). Pieces of plate containing spots corresponding to cholesterol, as judged by marker cholesterol, were cut out and placed in scintillation vials. Radioactivity associated with them was measured after the addition of 5 ml of scintillation fluid (Hi-safe II containing 10%)w/v water) in a Nuclear Chicago MK2 scintillation counter.
Measurement of cholesterol synthesis
The rate of cholesterol synthesis by Hep G2 cells was determined by measuring the incorporation of [ 14C]acetate into non-saponifiable cholesterol as follows. The preincubation medium was removed and the cells incubated with 0.5 ml of RPM1 1640 containing 1 mM [14C]acetate with a specific activity of 2 mCi/mmol for 4 h at 37°C. The cells were washed three times with 1 ml of PBS at room temperature, detached with 2 mg/ml trypsin and collected into Eppendorf tubes using two 0.3-ml aliquots of PBS. The cell suspensions were sonicated on ice at 20 pm amplitude for 20 s (Soniprep 150, MSE). Fifty microlitres were removed for protein determination [12], and 3 ml of methanol (BDH), 6 ml of chloroform (BDH) and 10 000 counts/min of
Measurement of DNA synthesis
The rate of DNA synthesis was estimated by measuring the rate of incorporation of [3H]thymidine into trichloroacetic acid precipitable material. The cells were incubated for 2 h with 5 x 10e6 M [3H]thymidine with a specific activity of 2 mCi/mmol at 37°C. The cells were washed two times with PBS, placed on ice and incubated with 0.5 ml of ice-cold 50 g/l trichloroacetic acid for 30 min. The cells were washed two times with icecold water and then harvested with 0.6 ml of 0.2 M NaOH and placed in scintillation vials. Radioactivity associated with them was measured after the addition of 5 ml of scintillation fluid (Hisafe II) in a Nuclear Chicago MK2 scintillation counter.
24
Results
As an initial approach to see whether IL-I, and TGF-& could modulate LDL receptor activity in Hep G2 cells, dose response curves were constructed for the effect of these cytokines on the
binding and internalisation of [ 1251]-labelled LDL at 37°C after an 18-h incubation with the cells. ILI, produced a significant stimulation of LDL receptor activity at concentrations above 117 units/ml (P < 0.01, Student-Newman Keuls test). After incubation for 18 h with 11 700 units/ml IL(4
(a) IL-1 !a-
**I
II
.
??
117ouIwnllIL-1
**
L
0
11.7
117
Concentration
1170
0
10
11700
Concent&n
(units/ml)
20
30
40
[ 125[I-IDL (nmoleyl)
04
(b) TGF-I3
**
[ 12’ II_LDLtx3ui-d (Mrng 0.5
Concentration
5
50
(ng/ml)
Fig. 1. Effect of TGF-6, and IL-l0 on LDL receptor activity. Hep G2 cells were grown to confluence in CDM. The cells were washed two times with PBS and then incubated for 18 h at 37°C with the indicated concentrations of IL-l0 (a) and TGF-0, (b) in RPM1 1640 containing I mgknl defatted BSA. LDL receptor activity was then measured by incubating the cells with 0.5 ml of RPM1 1640 containing 1 mg/ml BSA and 5 pg [‘251]labelled LDL in the presence (to measure low affinity binding and internalisation) or absence (to measure total binding and internalisation) of 0.25 mg/ml unlabelled LDL for 4 h. The cells were then washed extensively and the radioactivity associated with them determined by gamma counting. Results represent the high affinity binding and intemalisation of [‘ZSI]-labe.lled LDL calculated by subtracting the low affinity binding and intemalisation from the total binding and internalisation and are given as the mean f SD. of triplicate observations. **Significant difference to the level observed in cells incubated in RPM1 1640 containing 1 mg/ml defatted BSA alone (P c 0.01, Student-Newman Keuls test).
cell protein)
Fig. 2. Effect of TGF-fl, and IL-I, on [‘2SI]-labelled LDL binding at 4°C. Hep G2 cells were grown to confluence in CDM. The cells were washed two times with PBS and then incubated for 18 h at 37OC with 1170 units/ml IL-Js, 15 ng/ml TGF-0, or RPM1 1640 containing 1 mg/ml defatted BSA alone. The cells were then placed on ice for 30 mitt before incubating the cells with 0.5 ml of RPM1 1640 containing 1mgknl BSA and the indicated concentrations of [‘251]-labelled LDL in the presence (to measure low affinity binding) or absence (to measure total binding) of 0.5 mgknl unlabelled LDL for 2 h at 4°C. The cells were then washed extensively as described in the Methods and the radioactivity associated with them measured by gamma counting. (a) High affinity binding of [‘2SI]-labelled LDL at 4°C at each [‘251]-labelled LDL concentration calculated by subtracting the low affinity binding from the total binding. In experiments performed in parallel the binding and intemalisation of [‘2SI]-labelled LDL at 37°C was increased by 186% after incubation with 1170 units/ml IL-$ and by 95% after incubation with 15 ngml TGF-B, compared to control cells. (b) Scatchard plot of the data presented in (a) to enable the LDL receptor number and affinity to be calculated.
25
1, in RPM1 1640 LDL receptor activity was approximately 2.4-fold higher than in cells incubated in RPM1 1640 alone (Fig. la). TGF-/3i also stimulated the binding and internalisation of [‘251]-labelled LDL by Hep G2 cells (Fig. lb). A significant increase in LDL receptor activity was detected after an 18-h incubation with TGF-/3i concentrations as low as 5 @ml (P < 0.01, Student-Newman Keuls test). After an 18-h incubation with 50 ng/ml TGF-Pi, the highest concentration studied, LDL receptor activity was 2.2-fold higher than in cells incubated in RPM1 1640 alone. To determine the mechanism by which these increases in the binding and internalisation of [1251]-labelled LDL at 37°C occurred Scatchard analysis [ 141 of the binding of [ ‘251]-labelled LDL at 4°C after an 18-h incubation of the cells with 1170 units/ml IL-l,, 15 rig/ml TGF-Pi or RPM1 1640 alone (control cells) was performed (Fig. 2). There was only a small difference in the affinity of the receptor for LDL in cells incubated with IL-l, (& = 10.5 nmol/l) or TGF-Pi (& = 12.1 nmol/l) to that observed in control cells (& = 15.2 nmol/l). In contrast, B,,, was increased to 56.3 x lo-l5 molimg after incubation with IL-I, and to 25.5 x IO-l5 mol/mg after incubation with TGF-/3, from 15.6 x IO-l5 mol/mg observed in control cells. Thus the increase in [‘251]-labelled LDL binding could be accounted for almost entirely by an increase in LDL receptor number on the cell surface. An increase in LDL receptor activity is often a result of an increase in mitotic activity of the cell [ 151. To see whether the rise in LDL receptor activity could be accounted for by an increase in cell proliferation, the effect of these cytokines on cell number and DNA synthesis was measured. There was no significant difference in cell number between cells incubated with IL-l, at 1170 units/ml (210 000 f 12 500 cells/cm2) or TGF-Pi at 5 rig/ml (192 000 f 50 000 cells/cm2) to that observed in cells incubated in RPM1 1640 alone (215 000 f 22 500 cells/cm2). Similarly, TGF-Pi at doses from 0.05 ngml to 50 ng/ml had no significant effect on DNA synthesis as measured by the incorporation of [3H]thymidine into TCA precipitable material (Fig. 3b). IL- 1@, however, produced a significant inhibition of DNA syn-
0
11.7
117
11TJ
11703
Concentration (units/ml) a)TGF-D
J
0
T
_
0.05 Concentration(@ml)
Fig. 3. Effect of IL-Is and TGF-8, on DNA synthesis in Hep G2 cells. Hep G2 cells were grown to confluence in CDM. The cells were washed two times with PBS and then incubated for 18 h at 37°C with the indicated concentrations of IL-Is (a) and TGF-0, (b) in RPM1 1640 containing 1 mg/ml defatted BSA. The cells were then incubated with 5 x 10m6M [3H]thymidine with a specific activity of 2 mCi/mmol for 2 h at 37°C. The TCA precipitable material was then isolated as described in the Methods. Results are given as the counts/mm of [3H]thymidine incorporated into TCA precipitable material per well and represent the mean f SD. of triplicate observations. **Significant difference to the level observed in cells incubated in RPM1 1640 alone (P < 0.01, Student-Newman Keuls test).
thesis at doses as low as 11.7 units/ml and after incubation with 11 700 units/ml IL-l, for 18 h DNA synthesis was only 17% of that observed in control cells (Fig. 3a). Cholesterol synthesis and LDL receptor activity are often regulated co-ordinately [16]. We therefore looked at the effect of these cytokines on the [ 14C]acetate incorporation into cholesterol. In contrast to their effect on LDL receptor activity
26
60000
, _
ICY
1 0
11.7
** 1 Discussion
(a) IL-1
~
117
Concentration
1170
11700
(units/ml)
(bl TGF-O
0
0.05
0.5
Concentration
5
50
(ng/ml)
Fig. 4. Effect of IL-l@ and TGF-& on cholesterol synthesis in Hep G2 cells. Hep G2 cells were grown to confluence in CDM. The cells were washed two times with PBS and then incubated for 18 h at 37°C with the indicated concentrations of IL-lg (a) and TGF-j3, (b) in RPM1 1640 containing 1 mg!ml defatted BSA. The cells were then incubated with 1 mM [t4C]acetate with a specific activity of 2 mCiknmo1 for 4 h at 37°C. The lipids were extracted from the cells and saponified. Cholesterol was then isolated by thin layer chromatography and the radioactivity associated with it determined by scintillation counting. Results are given as the counts/min of [ 14C]acetate incorporated into cholesterol per mg cell protein and represent the mean f SD. of triplicate observations. ** Significant difference to the level observed in cells incubated in RPM1 1640 alone (P < 0.01, Student-Newman Keuls test).
cholesterol synthesis was inhibited by IL-l, and TGF-8, (Fig. 4). Thus after an 18-h incubation with 1170 units/ml IL-l, cholesterol synthesis was 42% and with 5 r&ml TGF-/3i cholesterol synthesis was 47% of the levels observed in cells incubated in RPM1 1640 containing 2 mg/ml defatted BSA alone.
The activity of the LDL receptor in hepatocytes and/or Hep G2 cells has been shown to be modulated by the presence of hormones and lipoproteins in the medium. During the preparation of this manuscript Grove et al. have reported that several different cytokines (25 @ml IL- 1, 100 ng/ml IL-6,200 @ml TGF-0) give minor increases in LDL uptake (respectively 27%, 28% and 24% above control cells in RPM1 containing 5% LPDS), and that another cytokine Oncostatin M is a potent stimulator of LDL uptake suggesting that cytokines may also have a role in regulating the LDL receptor in the liver [8,17]. The studies reported here confirm that the cytokines IL-l, and TGF-& can increase LDL receptor activity in Hep G2 cells. Indeed by utilising a CDM rather than medium containing LPDS in which the LDL receptors will be in an up-regulated state we suggest that TGF-0, and IL-l, may be more potent stimulators of LDL receptor activity in Hep G2 cells than is suggested by the studies of Grove et al. [8]. Thus in our hands LDL receptor uptake was increased by more than two-fold by both TGF-fir and IL-l, compared with control cells. Further, we have been able to show that IL-10 at concentrations as low as 117 units/ml (1.17 r&ml) and TGF-& at concentrations as low as 5 rig/ml can produce a significant stimulation of LDL receptor activity. TGF-@ has also been reported to increase specific LDL binding in smooth muscle cells by approximately two-fold [ 181. In contrast, Hotta and Baird demonstrated that TGF-/3 inhibited the binding and internalisation of [ ‘251]-labelled LDL by bovine adrenocortical cells [ 191. It thus appears that TGF-/3 may have tissue-specific effects on LDL receptor activity. This is of interest as although it has been known for several years that there is differential uptake of LDL by the liver and extrahepatic tissues in vivo the mechanism for this differential uptake remains unclear [ 11. An ability of cytokines to stimulate specifically LDL receptor activity in hepatocytes and not other cell types could account for such differential uptake. Scatchard analysis of the binding of [12511labelled LDL at 4°C demonstrated that the increase in LDL binding and internalisation at 37°C
21
could almost entirely be accounted for by a change in LDL receptor number. Further work will be necessary, however, to establish whether this change is mediated at the level of transcription of the LDL receptor mRNA. Previous studies have shown that stimulation of LDL receptor activity can generally be attributed to one of two sets of circumstances. Firstly, a rise in LDL receptor activity is observed if the demand of the cell for cholesterol is increased; for example, by mitogenic stimulation [ 141. TGF-/3, had no significant effect on cell proliferation as measured by either DNA synthesis (Fig. 3b) or cell number. IL-l, decreased DNA synthesis (Fig. 3a) but had no effect on cell number. Although the effects of IL-10 on cell number and DNA synthesis appear at first sight inconsistent it should be remembered that the cells were confluent when the incubation with the cytokines was commenced and that although the relatively short incubation time of 18 h was sufficient to reveal a decrease in DNA synthesis it is unlikely that it was sufficient to reveal a change in the number of cells in the dish. We would predict from the [3H]thymidine data that if the cells were grown up in the presence of IL-Is at a concentration of cytokine that inhibits DNA synthesis over a period of days then a decrease in cell number compared with control cells without cytokine would be detectable. Inhibition of cell proliferation would be expected to lead to an inhibition rather than the stimulation of LDL receptor activity observed. Further, when the demand of the cell for cholesterol is increased it is generally found that cholesterol synthesis as well as LDL receptor activity is enhanced [15]. TGF-P, and IL-l,, however, were found to inhibit cholesterol synthesis (Fig. 4). A second set of circumstances in which LDL receptor activity is enhanced is when cholesterol synthesis is inhibited so that the cell must rely on uptake of lipoproteins by the LDL receptor as its major source of cholesterol. This is the mechanism by which certain HMG CoA reductase inhibitors such as Compactin raise LDL receptor levels [20]. Inhibition of cholesterol synthesis, therefore, may be the primary target of TGF-& and IL-l@. It cannot be ruled out at this stage, however, that TGF-& and IL-l8 have a direct effect on LDL
receptor activity. Of interest in this respect are the recent observations by Grove and co-workers who have shown that, similarly to TGF-PI and IL-I,, Oncostatin M stimulates LDL receptor activity and inhibits cholesterol synthesis [17]. Further, it has been shown that the fall in cholesterol is subsequent to the rise in LDL receptor activity suggesting that inhibition of cholesterol synthesis is not the primary target of Oncostatin M (171. Further work will be necessary to determine whether the primary target of TGF-PI and IL-l, is the inhibition of cholesterol synthesis or, if like Oncostatin M, they have direct effects on both cholesterol synthesis and LDL receptor activity. Production of TGF-P and IL-l has been demonstrated by a wide variety of cell types both in vivo and in vitro [2 1,221. Although mRNAs for both TGF-/3 and IL-1 have been demonstrated by in situ hybridisation techniques to be present in liver sinusoidal cells, detailed information about the local concentration of these cytokines in the liver is lacking [23,24]. It is, therefore, not. known whether in vivo the concentrations of TGF-P, and IL-l are within the range found to stimulate LDL receptor activity in vitro. In support of a role for TGF-/3 and IL-I in modulating LDL receptor activity in vivo, in at least some circumstances, is the observation that during the acute phase response, which is associated with increased plasma levels of TGF-P and IL-l, there is a fall in plasma cholesterol [25]. Whilst the mechanism for this fall is not known one possibility consistent with the studies reported here is that the elevated circulating levels of IL-l and TGF-0 raise the hepatic LDL receptor in vivo as they do in vitro. In summary, we have shown that TGF-PI and IL-l, can potently stimulate LDL receptor activity in Hep G2 cells. Further work will be necessary, however, to determine the precise mechanisms by which these cytokines act and whether they have a role in vivo in regulating LDL receptor activity in the liver. Acknowledgements
We are grateful to the SERC and RhonePoulenc Rorer for their funding of this work. We would like to thank Dr. C. Fitzsimmons and Dr. D. Riddell for their advice during the course of this work.
28
References 1
Spady, D.K., Turley, S.D. and Dietschy, J.M., Receptorindependent low density lipoprotein transport in the rat in vivo. Quantitation, characterization and metabolic consequences, J. Clin. Invest., 76 (1985) 1113. 2 Havekes, L.M., Schouten, D., de Wit, E.C., Cohen, L.H., GrifIioen, M., van Hinsbergh, V.W. and Princen, H.M., Stimulation of the LDL receptor activity in the human hepatoma cell line Hep G2 by high-density serum fractions, B&him. Biophys. Acta, 875 (1986) 236. 3 Salter, A.M., Fisher, SC. and Brindley, D.N., Interactions of triiodothyronine, insulin and dexamethasone on the binding of human LDL to rat hepatocytes in monolayer culture, Atherosclerosis, 71 (1988) 77. 4 Semenkovich, C.F. and Ostlund, R.E., Estrogens induce low-density lipoprotein receptor activity and decrease intracellular cholesterol in human hepatoma cell line Hep G2, Biochemistry, 26 (1987) 4987. 5 Chait, A., Ross, R., Albers, J.J. and Bierman, E.L., Platelet derived growth factor stimulates activity of low density lipoprotein receptors, Proc. Natl. Acad. Sci. USA, 77 (1980) 4084. 6 Moorby, C.D., Gherardi, E., Riddell, D. and Bowyer, D.E., Porcine smooth muscle cell conditioned medium stimulates LDL receptor activity in Hep G2 cells, J. Cell Sci., 103 (1992). 7 Gherardi, E., Thomas, K., Le Cras, T.D., Moorby, C.D., Fitzsimmons, C. and Bowyer, D.E., Growth requirements and expression of LDL receptor and HMG CoA reductase in Hep G2 hepatoblastoma cells cultured in a chemically defined medium, J. Cell Sci., 103 (1992). 8 Grove, RI., Mazzucco, C., Allegretto, N., Kiener, P.A., Spitalny, S.F., Radka, S.F., Shoyab, M., Antonaccio, M. and Warr, G.A., Macrophage-derived factors increase low density lipoprotein uptake and receptor number in cultured human liver cells, J. Lipid Res., 32 (1991) 1889. Have], R.J., Eder, H.A. and Bragdon, J.H., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. Marchalonis, J.J., An enzymic method for the trace iodination of immunoglobulins and other proteins, Biochem. J., 113 (1969) 299. Brown, M.S. and Goldstein, J.L., Familial hypercholesterolemia: Defective binding of lipoproteins to cultured tibroblasts associated with impaired regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, Proc. Natl. Acad. Sci. USA, 71 (1974) 788. Markwell, M.A., Haas, SM., Bieber, H:L. and Tolbert, N.E., A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples, Anal. Biochem., 87 (1978) 206.
13 Folch, J., Lees, A.M., Meath, J.A. and LeBaron, F.N., Preparation of lipide extracts from brain tissue, J. Biol. Chem., 191 (1951) 833. 14 Scatchard, G., The attraction of proteins for small molecules and ions, Ann. NY Acad. Sci., 51 (1949) 660. 15 Mazzone, T., Basheeruddin, K., Ping, L., Frazer, S. and Getz, G.S., Mechanism of the growth related activation of the low density lipoprotein receptor pathway, J. Biol. Chem., 264 (1989) 1787. 16 Brown, M.S. and Goldstein, J.L., A receptor-mediated pathway for cholesterol homeostasis, Science, 232 (1986) 34. 17 Grove, RI., Mazzucco, C.E., Radka, S.F., Shoyab, M. and Kiener, P.A., Oncostatin M up-regulates low density lipoprotein receptors in Hep G2 cells by a novel mechanism, J. Biol. Chem., 266 (1991) 18194. 18 Nicholson, AC., Stopeck, A.T. and Hajjar, D.P., Cytokine regulation of LDL binding and LDL receptor mRNA steady state levels in arterial cells, FASEB J., 5 (1991) A1246. 19 Hotta, M., Baird, A., The inhibition of low density lipoprotein metabolism by transforming growth factor-p mediates its effects on steroidogenesis in bovine adrenocortical cells in vitro, Endocrinology, 121 (1987) 150. 20 Cohen, L.H., Grifftoen M., Havekes, L., Schouten, D., van Hinsbergh, V. and Kempen H.J., Effects of compactin, mevalonate and low-density lipoprotein on 3hydroxy-3-methylglutaryl-coenzyme A reductase activity and low-density-lipoprotein receptor activity in the human hepatoma cell line Hep G2, Biochem. J., 222 (1984) 35. 21 Pfeilschifter, J., Transforming growth factor-p. In: A. Habenicht (Ed.), Growth Factors, Differentiation Factors and Cytokines, Springer-Verlag, Berlin, 1990. 22 Schindler, R. and Dinarello, C.A., Interleukin I. In: A. Habenicht (Ed.), Growth Factors, Differentiation Factors and Cytokines, Springer-Verlag, Berlin, 1990, pp. 88- 102. 23 Takacs, L., Kovacs, E.J., Smith, M.R., Young, H.A. and Durum, S.K., Detection of IL-l, and IL-Is gene expression by in situ hybridisation. Tissue localisation of IL-l mRNA in the normal C57B/6 mouse, J. Immunol., 141 (1988) 3081. 24 Braun, L., Mead, J.E., Panzica, M., Mikumo, R. and Bell, G.I., Transforming growth factor @mRNA increases during liver regeneration: A possible paracrine mechanism of growth regulation, 85 (1988) 1539. 25 Olpin, S.E. and Price, C.P., High density lipoprotein and apolipoprotein A-l in acute phase response, Atherosclerosis, 69 (1988) 93.
ATHERO
21 Ireland,
Ltd. All rights reserved.
0021-9150/92/$05.00
04909
Transforming
Catriona
‘Department
growth factor-& and interleukin- 1p stimulate LDL receptor activity in Hep G2 cells
D. Moorby”,
Ermanno
Gherardib, Lynda Doveya, Cheryl Godliman” and David E. Bowyera
of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 lQP and bI. C.R.F. Laboratory, AddenbrookeS Hospital, Hills Road, Cambridge CB2 2QH (UK)
Cell Interactions
(Received 28 October, 1991) (Revised, received 26 June, 1992) (Accepted 22 July, 1992)
Summary
The effect of transforming growth factor-pi (TGF-P,) and interleukin-lp (IL-l,) on LDL receptor in Hep G2 cells was investigated. A greater than two-fold stimulation of the binding and internalisation of [1251]-labelled LDL at 37°C was observed after an 18-h incubation of the cells with TGF-& at 50 @ml and IL-l, at 11 700 units/ml compared with control cells. Scatchard analysis of the binding of [‘251]labelled LDL at 4°C after an 18-h incubation of the cells with 1170 units/ml IL-l, and 5 rig/ml TGF-PI showed that they were both acting primarily by increasing LDL receptor number. The increase in LDL receptor activity could not be attributed to an increase in cell proliferation as TGF-/3t at concentrations from 0.05 ng/ml to 50 t&ml had no significant effect on either cell number or [3H]thymidine incorporation into DNA whilst IL-l, inhibited DNA synthesis by more than 80% at a concentration of 11 700 units/ml but had significant effect on cell number. Cholesterol biosynthesis from [ 14C]acetate, in contrast to the stimulation of LDL receptor activity, was inhibited by approximately two-fold by incubation with TGF-0, at 50 rig/ml and IL-1~ at 11 700 units/ml,
Key words: LDL receptor; TGF-0,; IL-l; Hep G2
Introduction
Correspondence to: Dr. D.E. Bowyer, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 IQP, UK. Tel.: (0223) 33 37 22; Fax: (0223) 33 33 46.
Uptake of low density lipoprotein (LDL) by the LDL receptor in the liver is a major determinant of the plasma cholesterol level [ 11. It is, therefore, important to understand what controls the activity of the hepatic LDL receptor. Previous investi-
22
gators have shown that the level of the LDL receptor in hepatocytes and/or Hep G2 cells can be modulated by the lipoproteins LDL and HDL and the hormones insulin, 17&estradiol, dexamethasone and triiodo+thyronine [2-41. However, little attention has focused on the potential role of paracrine factors in regulating LDL receptor activity in the hepatocyte. That paracrine factors may be important in controlling LDL receptor activity in the liver is suggested by two lines of evidence. Firstly, there is a growing number of reports of cytokines modulating LDL receptor activity in extrahepatic cell types, for example PDGF has been shown to increase LDL receptor activity in fibroblasts [5]. Secondly, we have found that conditioned medium from porcine smooth muscle cells (PC SMC) potently stimulates the binding and internalisation of [1251]-labelled LDL at 37°C by Hep G2 cells [6]. The mediator of this stimulation has not been identified, but preliminary characterisation studies suggest that it is a protein(s) of low molecular weight. To investigate further the role of paracrine factors in regulating the activity of the hepatic LDL receptor we investigated the effect of two cytokines, transforming growth factor-pi (TGF-Pi) and interleukin-1 (IL-Is) on the binding and internalisation of [ 1251]-labelled LDL by Hep G2 cells. As serum contains several factors which are known to modulate LDL receptor the Hep G2 cells used in these studies were cultured in a serumfree chemically defined medium (CDM) [7]. During the preparation of this manuscript Grove et al. have reported TGF-/3 at 200 &ml and IL-l at 25 @ml increase LDL uptake, respectively by 24% and 27% by Hep G2 cells after a 20-h incubation compared with cells incubated in RPM1 containing 5% LPDS [8]. The studies reported here confirm and extend these findings by establishing the concentration range at which TGF-/3 and IL-l are active and their effects on LDL receptor number ([ ‘251]-labelled LDL binding at 4”C), cholesterol synthesis and cell proliferation. Methods Materials
TGF-Pi, prepared from human platelets, was obtained from British Biotechnology Limited. Recombinant IL-l s was from Bachem UK
Limited. TGF-Pi (1 pg) was reconstituted in 1 ml of 4 mM HCI. IL-l, was reconstituted in PBS. Further dilutions of the cytokines were made in RPM1 1640 containing 1 mg/ml defatted BSA as indicated in the figure legends immediately prior to adding to the cells. Cell culture
Hep G2 cells (ATCC) were routinely passaged in RPM1 1640 (Flow laboratories) supplemented with 1.19 8/l Hepes, 2 g/l sodium bicarbonate (BDH), 0.06 g/l penicillin G (Sigma), 0.1 g/l streptomycin sulphate (Sigma) and 10% v/v FCS (Biological Industries) in 95% sir/5% CO2 at 37°C. For experiments the cells were plated at a density of 75 000 cells/cm2 in 2-cm2 microwell dishes (Falcon) in 1.5 ml of CDM. The CDM consisted of RPM1 1640 (Flow laboratories) containing 2 g/l sodium bicarbonate, 1.19 g/l Hepes, 0.06 g/l penicillin G, 0.1 g/l streptomycin sulphate, 10 I.U./l prolactin (Sigma), 5 x 10m7 M insulin (Sigma), 5 x 10m8 M glucagon (Sigma), 1 x 10e8 M hydrocortisone (Sigma), 1 x 10V9 M triiodo+thyronine (Sigma), 10 mg/ml LDL, 5 x 10e5 M inosine (Sigma), 1 x low7 M copper sulphate, 5 x lo-” M zinc sulphate, 1 x 10e7 M manganese sulphate, 5 x lo-l5 M nickel chloride, 1 x 10m7M iron sulphate and 3 x 10m9 M sodium selenite. Two days after plating the cells were fed with 1 ml of CDM. When the cells had reached confluence, day 4, they were washed 2 times with PBS before incubating for 18 h with either RPM1 1640 or RPM1 1640 containing the indicated concentrations of cytokines. The binding and internalisation of [ 1251]-labelled LDL at 37°C cholesterol synthesis or DNA synthesis by the cells was then measured. Measurement of [‘“I+labelled LDL binding and internalisation LDL, d 1.019-1.063 g/ml, was isolated from
freshly prepared human serum by sequential density gradient ultracentrifugation [9]. The LDL was labelled with Na”‘I (Amersham) using lactoperoxidase (Sigma) conjugated to Sepharose 4B (Pharmacia) [lo]. The binding of [1251]-labelled LDL at 4°C and the binding and internalisation of [ ‘251]-labelled
23
LDL at 37°C was then measured essentially as described by Goldstein and Brown [1 11. For measurement of the binding of [12?]labelled LDL at 4°C the cells were placed on ice for 30 min, washed with PBS and then incubated with RPM1 1640 containing 1% BSA and the indicated concentrations of [ ‘251]-labelled LDL for 2 h in the presence (to measure low affinity binding and internalisation) or absence (to measure total binding and internalisation) of 0.5 mg/ml of unlabelled LDL at 4°C. The cells were then washed three times with 0.05 M Tris 0.15 M NaCl pH 7.4 containing 2 mg/ml BSA and once with 0.05 M Tris 0.15 M NaCl pH 7.4. The cells were harvested with 1% SDS and the radioactivity associated with them measured by gamma counting. High affinity or receptor-mediated binding was calculated by subtracting the low affinity binding from the total binding. Results were standardised in terms of the protein/dish. The binding and internalisation of [1251]labelled LDL at 37°C was determined by incubating the cells for 4 h with RPM1 1640 containing 1% BSA and a non-saturating concentration of 5 pg/ml of [‘251]-labelled LDL with a specific activity of 100 counts/min in the presence or absence of 0.5 mg/ml native LDL. The cells were then placed on ice and washed and harvested as described above for the measurement of [ ‘251]-labelled LDL binding at 4°C.
[ 3H]cholesterol (Amersham) as an internal standard were added sequentially to 450 ~1 of the cell sonicate in a glass tube and incubated at 40°C for 1 h. The extracts were Folch-washed with 1.4 ml of 0.73% saline for 18 h at 4°C [ 131. The aqueous layer was removed by aspiration and the lower phase evaporated to dryness in a Buchler vortex evaporator. The lipids were saponified by adding 200 ~1 of 0.5 M NaOH in 96% ethanol (BDH) to each tube and incubating at 60°C for 90 min. Water (400 ~1) was added to each tube and the non-saponified lipids extracted with 500-~1 aliquots of hexane (BDH). The pooled extracts were washed with 800 ~1 of 0.25 M NaOH in 48% ethanol and then evaporated to dryness under N2 gas. The sample was redissolved in 20 ~1 of chloroform/methanol 2:1 v/v and applied to 14 cm2 Alugram Sil G thin layer chromatography plates (Macherey-Nagel, Duren). The plates were run in hexanelheptaneldiethyletheriacetic acid solvent 60:20:20:1 by vol. until the solvent front was 0.5 cm from the top of the plate. The lipids on the plate were visualised with iodine vapour (BDH). Pieces of plate containing spots corresponding to cholesterol, as judged by marker cholesterol, were cut out and placed in scintillation vials. Radioactivity associated with them was measured after the addition of 5 ml of scintillation fluid (Hi-safe II containing 10%)w/v water) in a Nuclear Chicago MK2 scintillation counter.
Measurement of cholesterol synthesis
The rate of cholesterol synthesis by Hep G2 cells was determined by measuring the incorporation of [ 14C]acetate into non-saponifiable cholesterol as follows. The preincubation medium was removed and the cells incubated with 0.5 ml of RPM1 1640 containing 1 mM [14C]acetate with a specific activity of 2 mCi/mmol for 4 h at 37°C. The cells were washed three times with 1 ml of PBS at room temperature, detached with 2 mg/ml trypsin and collected into Eppendorf tubes using two 0.3-ml aliquots of PBS. The cell suspensions were sonicated on ice at 20 pm amplitude for 20 s (Soniprep 150, MSE). Fifty microlitres were removed for protein determination [12], and 3 ml of methanol (BDH), 6 ml of chloroform (BDH) and 10 000 counts/min of
Measurement of DNA synthesis
The rate of DNA synthesis was estimated by measuring the rate of incorporation of [3H]thymidine into trichloroacetic acid precipitable material. The cells were incubated for 2 h with 5 x 10e6 M [3H]thymidine with a specific activity of 2 mCi/mmol at 37°C. The cells were washed two times with PBS, placed on ice and incubated with 0.5 ml of ice-cold 50 g/l trichloroacetic acid for 30 min. The cells were washed two times with icecold water and then harvested with 0.6 ml of 0.2 M NaOH and placed in scintillation vials. Radioactivity associated with them was measured after the addition of 5 ml of scintillation fluid (Hisafe II) in a Nuclear Chicago MK2 scintillation counter.
24
Results
As an initial approach to see whether IL-I, and TGF-& could modulate LDL receptor activity in Hep G2 cells, dose response curves were constructed for the effect of these cytokines on the
binding and internalisation of [ 1251]-labelled LDL at 37°C after an 18-h incubation with the cells. ILI, produced a significant stimulation of LDL receptor activity at concentrations above 117 units/ml (P < 0.01, Student-Newman Keuls test). After incubation for 18 h with 11 700 units/ml IL(4
(a) IL-1 !a-
**I
II
.
??
117ouIwnllIL-1
**
L
0
11.7
117
Concentration
1170
0
10
11700
Concent&n
(units/ml)
20
30
40
[ 125[I-IDL (nmoleyl)
04
(b) TGF-I3
**
[ 12’ II_LDLtx3ui-d (Mrng 0.5
Concentration
5
50
(ng/ml)
Fig. 1. Effect of TGF-6, and IL-l0 on LDL receptor activity. Hep G2 cells were grown to confluence in CDM. The cells were washed two times with PBS and then incubated for 18 h at 37°C with the indicated concentrations of IL-l0 (a) and TGF-0, (b) in RPM1 1640 containing I mgknl defatted BSA. LDL receptor activity was then measured by incubating the cells with 0.5 ml of RPM1 1640 containing 1 mg/ml BSA and 5 pg [‘251]labelled LDL in the presence (to measure low affinity binding and internalisation) or absence (to measure total binding and internalisation) of 0.25 mg/ml unlabelled LDL for 4 h. The cells were then washed extensively and the radioactivity associated with them determined by gamma counting. Results represent the high affinity binding and intemalisation of [‘ZSI]-labe.lled LDL calculated by subtracting the low affinity binding and intemalisation from the total binding and internalisation and are given as the mean f SD. of triplicate observations. **Significant difference to the level observed in cells incubated in RPM1 1640 containing 1 mg/ml defatted BSA alone (P c 0.01, Student-Newman Keuls test).
cell protein)
Fig. 2. Effect of TGF-fl, and IL-I, on [‘2SI]-labelled LDL binding at 4°C. Hep G2 cells were grown to confluence in CDM. The cells were washed two times with PBS and then incubated for 18 h at 37OC with 1170 units/ml IL-Js, 15 ng/ml TGF-0, or RPM1 1640 containing 1 mg/ml defatted BSA alone. The cells were then placed on ice for 30 mitt before incubating the cells with 0.5 ml of RPM1 1640 containing 1mgknl BSA and the indicated concentrations of [‘251]-labelled LDL in the presence (to measure low affinity binding) or absence (to measure total binding) of 0.5 mgknl unlabelled LDL for 2 h at 4°C. The cells were then washed extensively as described in the Methods and the radioactivity associated with them measured by gamma counting. (a) High affinity binding of [‘2SI]-labelled LDL at 4°C at each [‘251]-labelled LDL concentration calculated by subtracting the low affinity binding from the total binding. In experiments performed in parallel the binding and intemalisation of [‘2SI]-labelled LDL at 37°C was increased by 186% after incubation with 1170 units/ml IL-$ and by 95% after incubation with 15 ngml TGF-B, compared to control cells. (b) Scatchard plot of the data presented in (a) to enable the LDL receptor number and affinity to be calculated.
25
1, in RPM1 1640 LDL receptor activity was approximately 2.4-fold higher than in cells incubated in RPM1 1640 alone (Fig. la). TGF-/3i also stimulated the binding and internalisation of [‘251]-labelled LDL by Hep G2 cells (Fig. lb). A significant increase in LDL receptor activity was detected after an 18-h incubation with TGF-/3i concentrations as low as 5 @ml (P < 0.01, Student-Newman Keuls test). After an 18-h incubation with 50 ng/ml TGF-Pi, the highest concentration studied, LDL receptor activity was 2.2-fold higher than in cells incubated in RPM1 1640 alone. To determine the mechanism by which these increases in the binding and internalisation of [1251]-labelled LDL at 37°C occurred Scatchard analysis [ 141 of the binding of [ ‘251]-labelled LDL at 4°C after an 18-h incubation of the cells with 1170 units/ml IL-l,, 15 rig/ml TGF-Pi or RPM1 1640 alone (control cells) was performed (Fig. 2). There was only a small difference in the affinity of the receptor for LDL in cells incubated with IL-l, (& = 10.5 nmol/l) or TGF-Pi (& = 12.1 nmol/l) to that observed in control cells (& = 15.2 nmol/l). In contrast, B,,, was increased to 56.3 x lo-l5 molimg after incubation with IL-I, and to 25.5 x IO-l5 mol/mg after incubation with TGF-/3, from 15.6 x IO-l5 mol/mg observed in control cells. Thus the increase in [‘251]-labelled LDL binding could be accounted for almost entirely by an increase in LDL receptor number on the cell surface. An increase in LDL receptor activity is often a result of an increase in mitotic activity of the cell [ 151. To see whether the rise in LDL receptor activity could be accounted for by an increase in cell proliferation, the effect of these cytokines on cell number and DNA synthesis was measured. There was no significant difference in cell number between cells incubated with IL-l, at 1170 units/ml (210 000 f 12 500 cells/cm2) or TGF-Pi at 5 rig/ml (192 000 f 50 000 cells/cm2) to that observed in cells incubated in RPM1 1640 alone (215 000 f 22 500 cells/cm2). Similarly, TGF-Pi at doses from 0.05 ngml to 50 ng/ml had no significant effect on DNA synthesis as measured by the incorporation of [3H]thymidine into TCA precipitable material (Fig. 3b). IL- 1@, however, produced a significant inhibition of DNA syn-
0
11.7
117
11TJ
11703
Concentration (units/ml) a)TGF-D
J
0
T
_
0.05 Concentration(@ml)
Fig. 3. Effect of IL-Is and TGF-8, on DNA synthesis in Hep G2 cells. Hep G2 cells were grown to confluence in CDM. The cells were washed two times with PBS and then incubated for 18 h at 37°C with the indicated concentrations of IL-Is (a) and TGF-0, (b) in RPM1 1640 containing 1 mg/ml defatted BSA. The cells were then incubated with 5 x 10m6M [3H]thymidine with a specific activity of 2 mCi/mmol for 2 h at 37°C. The TCA precipitable material was then isolated as described in the Methods. Results are given as the counts/mm of [3H]thymidine incorporated into TCA precipitable material per well and represent the mean f SD. of triplicate observations. **Significant difference to the level observed in cells incubated in RPM1 1640 alone (P < 0.01, Student-Newman Keuls test).
thesis at doses as low as 11.7 units/ml and after incubation with 11 700 units/ml IL-l, for 18 h DNA synthesis was only 17% of that observed in control cells (Fig. 3a). Cholesterol synthesis and LDL receptor activity are often regulated co-ordinately [16]. We therefore looked at the effect of these cytokines on the [ 14C]acetate incorporation into cholesterol. In contrast to their effect on LDL receptor activity
26
60000
, _
ICY
1 0
11.7
** 1 Discussion
(a) IL-1
~
117
Concentration
1170
11700
(units/ml)
(bl TGF-O
0
0.05
0.5
Concentration
5
50
(ng/ml)
Fig. 4. Effect of IL-l@ and TGF-& on cholesterol synthesis in Hep G2 cells. Hep G2 cells were grown to confluence in CDM. The cells were washed two times with PBS and then incubated for 18 h at 37°C with the indicated concentrations of IL-lg (a) and TGF-j3, (b) in RPM1 1640 containing 1 mg!ml defatted BSA. The cells were then incubated with 1 mM [t4C]acetate with a specific activity of 2 mCiknmo1 for 4 h at 37°C. The lipids were extracted from the cells and saponified. Cholesterol was then isolated by thin layer chromatography and the radioactivity associated with it determined by scintillation counting. Results are given as the counts/min of [ 14C]acetate incorporated into cholesterol per mg cell protein and represent the mean f SD. of triplicate observations. ** Significant difference to the level observed in cells incubated in RPM1 1640 alone (P < 0.01, Student-Newman Keuls test).
cholesterol synthesis was inhibited by IL-l, and TGF-8, (Fig. 4). Thus after an 18-h incubation with 1170 units/ml IL-l, cholesterol synthesis was 42% and with 5 r&ml TGF-/3i cholesterol synthesis was 47% of the levels observed in cells incubated in RPM1 1640 containing 2 mg/ml defatted BSA alone.
The activity of the LDL receptor in hepatocytes and/or Hep G2 cells has been shown to be modulated by the presence of hormones and lipoproteins in the medium. During the preparation of this manuscript Grove et al. have reported that several different cytokines (25 @ml IL- 1, 100 ng/ml IL-6,200 @ml TGF-0) give minor increases in LDL uptake (respectively 27%, 28% and 24% above control cells in RPM1 containing 5% LPDS), and that another cytokine Oncostatin M is a potent stimulator of LDL uptake suggesting that cytokines may also have a role in regulating the LDL receptor in the liver [8,17]. The studies reported here confirm that the cytokines IL-l, and TGF-& can increase LDL receptor activity in Hep G2 cells. Indeed by utilising a CDM rather than medium containing LPDS in which the LDL receptors will be in an up-regulated state we suggest that TGF-0, and IL-l, may be more potent stimulators of LDL receptor activity in Hep G2 cells than is suggested by the studies of Grove et al. [8]. Thus in our hands LDL receptor uptake was increased by more than two-fold by both TGF-fir and IL-l, compared with control cells. Further, we have been able to show that IL-10 at concentrations as low as 117 units/ml (1.17 r&ml) and TGF-& at concentrations as low as 5 rig/ml can produce a significant stimulation of LDL receptor activity. TGF-@ has also been reported to increase specific LDL binding in smooth muscle cells by approximately two-fold [ 181. In contrast, Hotta and Baird demonstrated that TGF-/3 inhibited the binding and internalisation of [ ‘251]-labelled LDL by bovine adrenocortical cells [ 191. It thus appears that TGF-/3 may have tissue-specific effects on LDL receptor activity. This is of interest as although it has been known for several years that there is differential uptake of LDL by the liver and extrahepatic tissues in vivo the mechanism for this differential uptake remains unclear [ 11. An ability of cytokines to stimulate specifically LDL receptor activity in hepatocytes and not other cell types could account for such differential uptake. Scatchard analysis of the binding of [12511labelled LDL at 4°C demonstrated that the increase in LDL binding and internalisation at 37°C
21
could almost entirely be accounted for by a change in LDL receptor number. Further work will be necessary, however, to establish whether this change is mediated at the level of transcription of the LDL receptor mRNA. Previous studies have shown that stimulation of LDL receptor activity can generally be attributed to one of two sets of circumstances. Firstly, a rise in LDL receptor activity is observed if the demand of the cell for cholesterol is increased; for example, by mitogenic stimulation [ 141. TGF-/3, had no significant effect on cell proliferation as measured by either DNA synthesis (Fig. 3b) or cell number. IL-l, decreased DNA synthesis (Fig. 3a) but had no effect on cell number. Although the effects of IL-10 on cell number and DNA synthesis appear at first sight inconsistent it should be remembered that the cells were confluent when the incubation with the cytokines was commenced and that although the relatively short incubation time of 18 h was sufficient to reveal a decrease in DNA synthesis it is unlikely that it was sufficient to reveal a change in the number of cells in the dish. We would predict from the [3H]thymidine data that if the cells were grown up in the presence of IL-Is at a concentration of cytokine that inhibits DNA synthesis over a period of days then a decrease in cell number compared with control cells without cytokine would be detectable. Inhibition of cell proliferation would be expected to lead to an inhibition rather than the stimulation of LDL receptor activity observed. Further, when the demand of the cell for cholesterol is increased it is generally found that cholesterol synthesis as well as LDL receptor activity is enhanced [15]. TGF-P, and IL-l,, however, were found to inhibit cholesterol synthesis (Fig. 4). A second set of circumstances in which LDL receptor activity is enhanced is when cholesterol synthesis is inhibited so that the cell must rely on uptake of lipoproteins by the LDL receptor as its major source of cholesterol. This is the mechanism by which certain HMG CoA reductase inhibitors such as Compactin raise LDL receptor levels [20]. Inhibition of cholesterol synthesis, therefore, may be the primary target of TGF-& and IL-l@. It cannot be ruled out at this stage, however, that TGF-& and IL-l8 have a direct effect on LDL
receptor activity. Of interest in this respect are the recent observations by Grove and co-workers who have shown that, similarly to TGF-PI and IL-I,, Oncostatin M stimulates LDL receptor activity and inhibits cholesterol synthesis [17]. Further, it has been shown that the fall in cholesterol is subsequent to the rise in LDL receptor activity suggesting that inhibition of cholesterol synthesis is not the primary target of Oncostatin M (171. Further work will be necessary to determine whether the primary target of TGF-PI and IL-l, is the inhibition of cholesterol synthesis or, if like Oncostatin M, they have direct effects on both cholesterol synthesis and LDL receptor activity. Production of TGF-P and IL-l has been demonstrated by a wide variety of cell types both in vivo and in vitro [2 1,221. Although mRNAs for both TGF-/3 and IL-1 have been demonstrated by in situ hybridisation techniques to be present in liver sinusoidal cells, detailed information about the local concentration of these cytokines in the liver is lacking [23,24]. It is, therefore, not. known whether in vivo the concentrations of TGF-P, and IL-l are within the range found to stimulate LDL receptor activity in vitro. In support of a role for TGF-/3 and IL-I in modulating LDL receptor activity in vivo, in at least some circumstances, is the observation that during the acute phase response, which is associated with increased plasma levels of TGF-P and IL-l, there is a fall in plasma cholesterol [25]. Whilst the mechanism for this fall is not known one possibility consistent with the studies reported here is that the elevated circulating levels of IL-l and TGF-0 raise the hepatic LDL receptor in vivo as they do in vitro. In summary, we have shown that TGF-PI and IL-l, can potently stimulate LDL receptor activity in Hep G2 cells. Further work will be necessary, however, to determine the precise mechanisms by which these cytokines act and whether they have a role in vivo in regulating LDL receptor activity in the liver. Acknowledgements
We are grateful to the SERC and RhonePoulenc Rorer for their funding of this work. We would like to thank Dr. C. Fitzsimmons and Dr. D. Riddell for their advice during the course of this work.
28
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