Biochimic~ et Biopl~vsica Acta, 1082 (1991) 27-32
27
e. 1991 ElsevierScience PublishersB.V. 0005-2760/91/$03.50 ADONIS 000527609100099P
BBALIP 53580
The expression of lipoprotein lipase activity and mRNA in mesenchymal rat heart cell cultures is modulated by bFGF G . F r i e d m a n t, M . B e n - N a i m 2, O. H a l i m i ~, J. E t i e n n e 3 0 .
S t e i n 2 a n d Y. S t e i n
t Lipid Research Laborato~'. Department of Medicine B. Hadc~sah Unicersity Hospital, Jerusalem (Israel). " Department of Experimental Medicine and Cancer Research, Hebrew Unwersi(v-Itadassah Medical School. Jerusalem (Israel) and 3 Laboratoire de Biochimie. Faculte de Medicine. St. Antoine. Paris (France)
(Received 17 August 1990)
Key words: Growth factor: LPL mRNA: Heparin: Lipoprotein lipase; (Rat mesenchymal heart cell)
The effect of basic fibroblast growth factor (FGF) on lipoprotein lipase (LPL) production was studied in mesenchymal rat heart cell cultures. Addition of FGF to culture medium containing 20% senun resulted in a 3-fold increase in LPL activity. The minimal effective dose of FGF was 10 n g / m l and the increase occurred after exposure for 48 IL Addition of FGF w.~s effective during the first week in culture, when enzyme activity was increasing, but not after !! days when the cultures were superconfluent and ~he enzyme activity wa¢ high. Addition of FGF to serum-poor medium was able to replace serum required to sustain LPL activity, in FGF-treatod cultures, more LPL activity was present in the functional pool, but not in the medium, than in the controls. The increase in enzymic activity was accompanied by an increase in enzyme mass and in LPL mRNA.
Introduction Lipoprotein lipase (LPL) activity in cultured mesenchymal cells derived from new-born rat heart increases during the first week after plating and plateaus thereafter [1,21. Optimal conditions for the expression of LPL require the presence of fetal bovine and horse serum and enzyme activity decreases markedly in serum-poor medium [3,4]. The quest for a serum-free medium which will support cell growth and differentiation prompted the use of various compounds, among them fibroblast growth factor (FGF), which became an important ingredient of such media [51. F G F has been isolated and purified from brain and pituitary [6], but it appears that it is more ubiquitous [7]. In a recent study, concerned with the expression of m R N A of basic F G F in cells derived from the heart, it was shown that preplates which are analogous to our mesenchymal cells, are the main site of F G F m R N A expression [8]. Therefore, it seemed of interest to determine whether supple-
Abbre, ations: FGF, fibroblast growth factor; LPL, lipoprotein lipase; m R N A , messenger RNA.
Correspoaoence: Y. Stein, Lipid ResearchLaboratory, Departmentof Medicine ~. Hadassah University Hospital. p.o. Box 12000, Jerusalem 91120. ~rael.
mentation of heart cell cultures with exogenous F G F would enhance the increase in LPL activity and if so, to define the level of such a putative regulation. Materials and Methods
Cell cultures F I heart cell cultures which consisted mainly of nonbeating mesenchymal cells were prepared from 2-4day-old rat hearts, as described [1,2]. Cultures -vere used between 5 - 7 days after their isolation.
Determination of lipoprotein lipase activity The enzyme activity was determined on aliquots of medium and homogenates of cells which had been released from the petri dish with a rubber policeman in 1 ml 0.025 M N H 3 / N H 4 C I buffer (pH 8.1) containing 1 u n i t / m l of heparin. The assa~ system consisted of 0.1 ml of medium, of 0.1 ml of cell homogenate (50-70 ~g protein) and 0.1 mi of substrate containing labeled triolein, prepared according to the method of NilssonEhle and Schotz [9]. Incubations were carried out at 37°C for 45 min. The reaction was stopped by addition of m e t h a n o l / c h l o r o f o r m / h e p t a n e (1.4:1.25 : 1, v / v ) and the extraction of fatty acids was performed according to the methods of Belfrage and Vaughan [10] as modified by Nilsson-Ehle and Schotz [9]. Enzyme activ-
28 ity was calculated according to the formula of NilssonEhle and Schotz [9] and was expressed per mg cell protein/h. To meas~Jre the effect of 1 M NaCI on the lipolytic activity, cell homogenates or medium samples were preincubated in the presence of 1 M NaC1 for 10 min at 27°C. For determination of heparin releasable lipoprotein lipase activity, the medium was collected at the end of the incubation and cultures were incubated for 3 min at room temperature with 0.5 ml F~o medium containing 20% serum and 5 u / m l of heparin. 0.1 ml of the medium was assayed for lipoprotein lipase activity.
Inhibition of lipoprotein lipase by antiserum Goat antiserum to a highly purified rat adipose tissue lipoprotein lipase was prepared as described [11]. To measure inhibition of lipoprotein lipase activity by antibody, the tubes containing the medium or homogenate of cells were preincubated in 0.2 M NaC1, 30% glycerol and 0.005 M sodium veronal (pH 7.0) at 4°C for 30 rain, with antiserum to lipoprotein lipzse or nonimmunized goat serum, diluted 1:50. The tubes were centrifuged at 28 000 x g for 30 min at 4°C and enzyme activity was determined on the supernatant [12].
Partial purification of lipoprotein lipase Partial purification of lipoprotein lipase from heart cell cultures was performed by heparin sepharose column chromatography as described previously [13]. Briefly, heart cell cultures were washed extensively, and homogenized in phosphate buffered saline (PBS). The cell homogenate was extracted with cold diethyl ether. The resultant powder was dissolved in 10 mM Tris-HC! buffer (pH 7.4)/0.1% Triton X-100/20% glycerol and sonicated. The sonicate was centrifuged for 30 rain at 4°C at 12000 rpm in an SS-34 rotor. The supernatant was collected, applied to a heparin-Sepharose column and eluted at 4°C with 2 M NaCI as described [13].
Gel electrophoresis and immunoblotting Electrophoresis was carried out at 50 mA/slab gel for 3 h at 23°C. Gels were calibrated with molecular weight standards (Sigma). Immunoblotting of lipoprorein lipase was performed as described [13,14]. Briefly, proteins were transferred from the SDS gels onto nitrocellulose paper in a transblot cell apparatus (Bio-Rad). Electroblotting was carried out at 400 mA for 4 h at 23°C. After incubation of the nitrocellulose paper with goat antiserum to lipoprotein lipase for 3 h and subsequently with rabbit antigoat antiserum (Bio-Yeda, Israel), diluted 1:50 for 1 h, the paper was washed extensively and incubated with 125I-labeled protein A (spec.act.30 /tCi/mg, Amersham Intl., U.K.), followed by washing and air drying. Immunoreactive proteins on nitrocellulose paper were visualized by exposure to Agfa (Curix RP2) film for 4-7 days.
Nucleic acid probes Hybridization probes were prepared using plasmid pBSLPL which includes a 213 bp fragment of genomic sequences from exon 5 of the mouse LPL gene. This fragment was isolated from a 1.3 kb XbaI fragment of LPL cDNA subcloned into pBS vector (Stratagene). Sequence analysis confirmed that the clones were identical to those published [15]. A cloned fl-tubulin cDNA insert [16] was used as the control probe.
RNA preparation and blot hybridization analysis Total cellular RNA was extracted from cultured rat heart cells with guanidine thiocyanate and sedimented through a cesium chloride cushion according to Chirgwin et al. [17]. The integrity of the RNA preparation was verified by denaturing gel electrophoresis [18]. Dot blot analysis was performed with a template manifold apparatus (Sehleicher & Schueii, Keene NH). To assure uniform dot size, total cellular RNA was employed at different concentrations (5, 10, 15/Lg) and supplemented with yeast tRNA to provide a total of 15 pg of RNA per sample. The RNA samples were denatured by 1 M formaldehyde and heating at 60°C for 15 min, then diluted into 20 voi. of 3 M NaCI containing 0.3 M trisodium citrate and applied to nitrocellulose filter under vacuum. The filter was washed with additional diluent, baked at 800C for 2 h and then hybridized. Stringencies employed for filter hybridization and washing were as described [19]. DNA probes were labeled by random hexanucleotide priming by using 32p-labeled nucleotide triphosphates [20]. Autoradiography was done with standard techniques.
Analytical procedures Protein was determined according to the methods of Lowry et al. [21], using bovine serum albumin as standard. Radioactivity was determined in a r-scintillation spectrometer (Tri-carb 2660). The scintillation fluid used was 20% Triton X-100/0.005% 1A-bis(2-(5-phenyioxazolyl)benzene (POPOP)/0.4%, 2,5-diphenyl-oxazole (PP0) in toluene.
Materials Partially purified basic fibroblast growth factor (bFGF) isolated from bovine brain was kindly provided by Dr. Vlodavsky, Hadassah University Hospital, Jerusalem, Israel. Glycerol tri[9,10(n)-3H]oleate, specific activity 1 C i / m m o l was obtained from Amersham International, U.K. 32P-adCTP, specific activity 3000 Ci/mmol was obtained from New England Nuclear, U.S.A. Heparin, thrombolique was obtained from Organon, Oss, The Netherlands. Culture media and sera were obtained from Gibco, Grand Island, N.Y.
29 TABLE I
Results
T o s t u d y the effect of basic fibroblast g r o w t h f a c t o r ( b F G F ) o n the activity of lipoprotein lipase in r a t m e s e n c h y m a l h e a r t cell cultv, res, various c o n c e n t r a t i o n s o f the g r o w t h f a c t o r were a d d e d to c o m p l e t e culture m e d i u m . A s seen in Fig. 1, the increase in l i p o p r o t e i n lipase activity w a s d e p e n d e n t o n the c o n c e n t r a t i o n o f the effector; a 60% increase in cellular l i p o p r o t e i n lipase activity w a s seen a f t e r 72 h of i n c u b a t i o n with 10 n g / m l o f b F G F a n d a m o r e t h a n 3-fold increase o c c u r r e d at a c o n c e n t r a t i o n o f 100 n g / m l . N o c h a n g e w a s o b s e r v e d in the l i p o p r o t e i n lipase activity in the c u l t u r e m e d i u m , a n d n o c h a n g e in p r o t e i n c o n t e n t w a s o b s e r v e d b e t w e e n c o n t r o l a n d c u l t u r e s to w h i c h b F G F w a s a d d e d u p to 72 h before t e r m i n a t i o n o f the e x p e r i m e n t (0.65 + 0.07 nag cell p r o t e i n in c o n t r o l vs. 0.67 + 0.1 m g in t r e a t e d dishes). T o verify t h a t the e n z y m i c activity w h i c h w a s increased aeter t r e a t m e n t with b F G F is l i p o p r o t e i n lipase, the activity w a s d e t e r m i n e d a f t e r a d d i t i o n o f a specific a n t i s e r u m , o r 1 M N a C I . A l m o s t c o m p l e t e inhibition o f e n z y m i c activity o c c u r r e d in c o n t r o l s a n d b F G F t r e a t e d h e a r t cell cultures, i n d i c a t i n g t h a t the i n c r e a s e d lipolytic activity is i n d e e d l i p o p r o t e i n iipase (Table I). T h e t i m e - c o u r s e o f the i n d u c t i o n o f cellular l i p o p r o tein lipase activity w a s studied in the p r e s e n c e o f 200 n g / m l o f b F G F (Table II). U p to 24 h o f i n c u b a t i o n , there w a s 'almost n o c h a n g e in cellular l i p o p r o t e i n lipase activity in the b F G F t r e a t e d cultures; 1.6-fold increase in cellular l i p o p r o t e i n lipase activity w a s f o u n d a f t e r 48 h o f i n c u b a t i o n a n d a m a x i m a l value of 3.3-fold increase o c c u r r e d a f t e r 72 h. D u r i n g t h a t time, n o t w i t h -
Characteri:ation of the increased lipolytic actiritv in b F G F treated rat hfart cell cultures
Conditions: as in legend to Fig. I. To determine inhibition by zntiserum to LPL and I M NaCL aliquots of medium or of cell homogenates were incubated with 1 : 50 dilution of antiserum at 4°C for 30 min or with l M NaCI at 27°C for 10 rain prior to assay of LPL. Preincubation with nonimmune serum did- not affect enzymic activity. Results are means± S.E. of triplicate dishes. Addition to assay medium
LPL activity (nmol fatty acid released/h per mg cell protein)
None Antiserum to LPL I M NaCI
control
bFGF treated
820 + 26 96± 7 56+ 8
3052 ± 64 110:t: 9 63+ 5
s t a n d i n g the steep rise in cellular e n z y m i c activity, there w a s n o increase in m e d i u m lipoprotein lipase activity. L i p o p r o t e i n lipase in the c u l t u r e d m e s e n c h y m a l rat h e a r t cells is p r e s e n t in a n intraceUular a n d in a surface-related, f u n c t i o n a l c o m p a r t m e n t , f r o m w h i c h it c a n be released b y b e p a r i n [22]. In the next experiment, rat h e a r t cell c u l t u r e s w e r e e x p o s e d to 200 n g / m l of b F G F for 72 h. T h e r e a f t e r , h e p a r i n w a s a d d e d for 3 m i n to release t h e cell s u r f a c e l i p o p r o t e i n lipase activity, w h i c h r e p r e s e n t s the f u n c t i o n a l pool o f the enzyme. T h e d a t a in T a b l e I11 d e m o n s t r a t e d t h a t b F G F t r e a t m e n t resulted in a 5-fold increase in the f u n c t i o n a l p o o l of the e n z y m e a n d a 3-fold increase in residual cellular e n z y m e activity. T h e d a t a p r e s e n t e d so far h a v e s h o w n t h a t b F G F t r e a t m e n t induces a p r o n o u n c e d increase in lipoprotein lipase activity in r a t h e a r t cell cultures in the intraTABLE 11 Increase in cellular lipoprotein lipase activity by b F G F as a func:~~ qf time
,~001/ 5OO
o
0
J ~'--~:~ 50 ioo 500 bFGF (r~l/ml)
Fig. I. Effect of increasing concentrations of basic fibroblast growth factor (bFGF) on lipoprutein lipase activity of rat heart cell cultures. Conditions: The cells were cultured in Fw medium supplemented with 10% fetal bovine serum and 10~ horse serum for 3 days. On day 3, the medium was changed to 2 ml of fresh medium containing varying concentrations of bFOF and incubation was carried out for 72 h. At the end of the experiment, the medium was collected, the cell layer washed with phosphate-buffered saline scraped with 1 ml 0.025 M NH3/NH4CI buffer (pH 8.1) comaining I unit/ml heparin and homogenized at 0"C. 0.1 ml of cell homogenate or medium was assayed for lipoprotein lipase activity. Values are means±S.E, of triplicate dishes. O, cellular; o medium.
Conditions: as in legend to Fig. I. On day 3, the medium was changed and the experiment was continued for an additional 96 h. bFGF 200 ng/ml was added for 6-96 h prior to termination of the experiment. At the end of the incubation, the medium was collected and LPL activity was determined on medium aliquot and cell homogenates. Values are means + S.E. of triplicate dishes. Addition
None bFGF
Time (h)
LPL activity (nmol fatty acid released/h per mg cell protein) medium
total cellular
96
115+ 6
6
121+ 4
24 48 72 96
129± 8 127+11 119+ 3 132-t- 7
896:t:12 899:t=16 953+24 a 1496+31 2944+22 3021 +18
30 TABLE III
TABLE V
Effect of bFGF on the functional (heparir. releasable) LPL activity in rat heart cell cultures
Effect of addition of bFGF to culture medium on lipoprotein lipase activity in overconfluent rat heart cell cultures
Conditions: as in legend to Fig. 1. On day 3, the medium was changed and the cells were incubated for 72 h with 2 ml of fresh medium containing 200 ng/ml of bFGF (where appropriate). Thereafter, the medium was removed and lipoprolein lipase activity was determined on aliquots of medium and cell homogenates. 3-min heparin-releasable lipoprotein lipa~e activity was determined as described under Materials and Methods. Values are means ± S.E. of triplicate dishes.
Conditions: Cells were cultured in F~o medium supplemented with 10~ fetal bovine serum and 10% horse serum for 11 days. The medium was changed every 3 days. On day ll, the medium was changed and bFGF 200 ng/ml was added where appropriate. Incubation was carried out for 72 h. At the end of the incubation, lipoprotein lipase activity was determined on aliquots of medium and of cell homogenates. Values are means + S.E. of triplicate dishes.
Exp. No.
Addilions
Exp. No.
Treatment
medium
cellular
heparin releasable
medium
cellular
I
None bFGF
1054-7 112+8
592±10 1768+17
198± 4 958+11
1
None bFGF
135 ± 16 139 ± 21
1 190 + 65 1 181 ± 54
11
None bFGF
99±5 89±4
610± 8 1791+14
2044- 6 991+16
11
None bFGF
152 4-26 161 ± 31
1 101 +96 1 198 ± 66
Lipoprotein lipase activity (nmol fatty acid released/h per mg cell protein)
cellular a n d in the surface related f u n c t i o n a l c o m p a r t m e n t . However, a l m o s t n o c h a n g e w a s o b s e r v e d in the lipoprotein lipase activity in the c u l t u r e m e d i u m . In the next experiment, the effect o f multiple a d d i t i o n s o f b F G F o n lipoprotein iipase activity w a s studied. Since the increase in l i p o p r o t e i n lipase activity with time in culture is m o s t p r o n o u n c e d u p to 6 d a y s a n d t e n d s to p l a t e a u thereafter [1,2], the effect of b F G F w a s studied between d a y s 0 - 6 . A 3-fold increase in cellular l i p o p r o rein lipase activity o c c u r r e d w h e n a single d o s e o f b F G F w a s a d d e d o n d a y 3. W h e n a d d i t i o n o f b F G F w a s started o n d a y 0 a n d r e p e a t e d o n d a y s 3 a n d 5, the s a m e increase w a s o b s e r v e d (Table IV). W h e n b F G F w a s a d d e d only o n d a y 5, the increase in l i p o p r o t e i n lipase activity w a s 6 7 ~ (Table II, 48 h). S u p p l e m e n t a tion of o v e r c o n f l u e n t r a t m e s e n c h y m a l h e a r t cell c u l t u r e with b F G F did n o t increase l i p o p r o t e i n lipao¢ activity (Table V). N e x t , a n a t t e m p t w a s m a d e to s t u d y the effect of b F G F in the presence of s e r u m - p o o r m e d i u m . In the two e x p e r i m e n t s s h o w n in T a b l e VI, the e n z y m e
LPL activity (nmol fatty acid relcascd/h per mg cell protein)
activity w a s low ( 2 0 ~ ) w h e n the c u l t u r e m e d i u m w a s s u p p l e m e n t e d w i t h 0 . 5 ~ fetal c a l f s e r u m only. H o w e v e r , the a d d i t i o n o f 200 n g / m l o f b F G F to the s e r u m p o o r m e d i u m resulted in a l m o s t c o m p l e t e recovery o f the cellular l i p o p r o t e i n h p a s e activity. T o a s c e r t a i n w h e t h e r the increase in cellular l i p o p r o rein lipase activity in b F G F t r e a t e d c u l t u r e s p a r a l l e l e d the i n c r e a s e in l i p o p r o t e i n lipase p r o t e i n , the cellular l i p o p r o t e i n lipase c o n t a i n i n g fraction, eluted f r o m heparin-Sepharose column with 2 M NaCI, was subj e t t e d to gel d e c t r o p h o r e s i s a n d i m m u n o b l o t t i n g . A 3.2-fold i n c r e a s e in the d e n s i t y o f the b a n d identified as i i p o p r o t e i n iipas¢ is seen in c u l t u r e s t r e a t e d w i t h b F G F for 72 h (Fig. 2). T o e x a m i n e f u r t h e r the m e c h a n i s m o f the effect o f b F G F o n l i p o p r o t e i n lipas¢ p r o d u c t i o n , we c o m p a r e d cellular L P L m R N A levels b y d o t b l o t hyb r i d i z a t i o n a n a l y s i s in b F G F t r e a t e d a n d in c o n t r o l cultures. T h e c h a n g e s in L P L m R N A in t r e a t e d cells as c o m p a r e d to n o n t r e a t e d cells a r c s h o w n in Fig. 3. L P L m R N A levels w e r e 2.9-fold h i g h e r in c u l t u r e s t r e a t e d TABLE Vi
TABLE IV Effect of addition of bFGF to culture medium at early stages of growth on lipoprotein lipase activity in rat heart cell cultures
Effect of addition of bFGF on lipoprotein lipase actir~ity in rat heart cell cultures grown in serum poor medium
Conditions: Cells were grown in medium supplemented with 20% serum, bFGF 200 ng/ml was added to the culture medium as indicated. LPL activity was delermined on day 6. Values are means 4-S.E. of triplicate dishes.
Conditions: Cells were grown in culture medium containing 20~ serum for 3 days. On day 3, the medium was changed and mediumpoor serum was added where indicated. On day 5, bFGF 200 ng/ml was added where appropriate. The experiment was terminated on day 7. Values are means + S.E. of triplicate dishes.
Addition to culture
Addition to serum free medium
None bFGF bFGF bFGF
Day of addition
3 3, 5 0. 3, 5
LPL activity (nmol fatty acid released/h per mg cell protein) medium
cellular
96 + 15 III +14 101 ± 9 121 ± 18
860 + 14 2501+10 2610+16 2691 ± 12
10% FCS + 10% horse serum 0.5% FCS 0.5% FCS + bFGF
Cellular LPL activity (nmol fatty acid released/h per mg cell protein) Exp. I
Exp. I!
870 ± 12 160± 7 763 ± 14
901 + 22 189+18 851 ± I l
31
II6-
LPL-.-4,6Fig. 2. lmmunoblot of lipoprotein lipase from rat heart cell cultures. Heart cells were cultured for 3 days in Fi0 medium supplemented with 107o fetal bovine serum and 10% horse serum. On day 3, the medium was changed and bFGF 200 ng/ml was added for 72 h (where appropriate). At the end of incubation, the medium was collected, the cells washed six times with phosphate-buffered saline (PBS) and homogenized. Acetone ether powder of the homogenates was extracted and loaded on a heparin-Sepharose column. The fraction eluted with 2 M NaCI was concentrated and subjected to electro phoresis and immunoblotted with goat antiserum to lipoprotein lipase (see Materials and Methods). Lipoprotein lipase trom control cells, lane I; cells treated with bFGF, lane If. with b F G F than in the control cells cultured for the same time. The same R N A samples were analyzed by dot blot hybridization using a # - t u b u l i n e D N A probe to assess the specificity of L P L m R N A response. As shown in Fig. 3, almost no change in # - t u b u l i n m R N A was noted with b F G F treatment.
Discussion In this study we have shown that addition of basic F G F to rat heart cell cultures results in a 3-fold increase in lipoprotein lipase activity. This effect is concentration and time d e p e n d e n t and is seen already with 10 n g / m l of F G F , b u t only after 48 h of exposure. It seems pertinent to add that F G F was effective when added to the culture m e d i u m c o n t a i n i n g 20% serum. F G F was also able to sustain LPL activity u n d e r conditions of serum deprivation, which leads to a rapid fall in enzyme activity [3,4]. As in our previous studies [1,2],
A RNA
I
,GO
B II
I
•
O0
mOO
II
O0
Fig. 3. Dot blot analysis of LPL mRNA levels from rat heart cell cultures. Conditions: Heart cells were cultured as described in legend to Fig, l), in the presence or absence of 200 ng/ml bFGF for 72 h. Total RNA was isolated and applied to nitrocellulose membrane. LPL mRNA was detected using azP-labeled mouse LPL ganomic fragment as described under Materials and Methods. Lane i, control; Lane II, bFGF treated (A). After exposure, the blot was stripped and rehybridized with a probe for a constitutively expressed form of/t'-tubulin to confirm that approximately equal amounts of RNA were loaded in each dot (B). Lane t-control; lane II-bFGF treated. Data are from one experiment typical of three performed.
the presently used cultured cells are of the F t type (preplates of 3 0 - 4 5 rain) which consist mainly of nonheating cells. The LPL activity in these cultures increases d u r i n g the first week and later plateaus. The effect of F G F could be demonstrated only when added d u r i n g that phase, but not at the later time interval, even though the maximal enzymic activity in these cultures was still below that observed in younger cultures exposed to F G F . F G F was shown to be mitogenic for more than 20 different cell types [7]. W e hypothesize that in F t cultures F G F might have been mitogenic for a small fraction of mesenchymal cells which synthesize LPL, as there was no significant increase in the cellular protein c o n t e n t / d i s h . F G F is mainly considered as an inhibitor of differentiation of myogenic cells [7,23], even though in a recent study a s u b p o p u l a t i o n of cells derived from chick wing muscle was shown to require F G F for differentiation in culture [24]. Divergent results were obtained by addition of F G F to these cells on their differentiation. Thus, in culture of 3"1"3cells, F G F when a d d e d to serum c o n t a i n i n g m e d i u m inhibited their conversion into adipocytes, with little if any effect on cell multiplication [25]. Similarly, Spizz et al. [26] have shown that F G F can inhibit myogenic differentiation through a m e c h a n i s m independent of cell proliferation. On the other hand, differentiation of sheep preadipocytes was inhibited by removal of F G F from a serum free culture m e d i u m [27]. G r o w t h hormone is another effector of differentiation of precursor cells into adipocytes [28,29]. However, it is m a i n l y required for the expression of later markers of differentiation, such as glycerol-3-phosp h a t e dehydrogenase, but apparently not for LPL, which is considered to b e a n early marker [29] and the expression of which is coupled with growth arrest [30]. The presently described effect of F G F on LPL activity was accompanied also by a similar increase in enzyme protein and in LPL m R N A , indicating regulation at transcriptional level. The ability of F G F to control gene expression was d e m o n s t r a t e d also d u r i n g regulation of myogenesis [26]. A d d i t i o n of F G F resulted in a decline in muscle creatine kinase m R N A , which was independent of cell proliferation [26]. In analogy to our previous findings with effectors of adenylate cyclase [12,22], the distribution of cellular LPL in F G F treated cells was changed with an increase in enzyme activity in the functional pool, released by brief e.,posure to heparin. A t the same time, there was no change in L P L activity in the medium, contrary to the effect of Hepes buffer [31], which in the same system increases cellular and m e d i u m LPL activity. The effect of F O F on L P L activity in the heart cell cultures resembles to some extent that described in cultures of rabbit costal chondrocytes [32]. In that system, F G F caused an increase in the rate of incorporation of 35Ssulfate into proteoglycans and resulted in an expression
32 of c h o n d r o c y t e p h e n o t y p e . Moreover, similar to o u r findings, the F G F h a d to be present in the cultures p r i o r to c o n f l u e n c e a n d w a s not effective w h e n a d d e d at a later phase. It seems plausible t h a t the increase in the functional pool o f L P L b y F G F , w i t h o u t a c o n c o m i t a n t increase in the s p o n t a n e o u s release i n t o the c u l t u r e m e d i u m c o u l d b e d u e to an increase in cell surface h e p a r a n sulfate p r o t e o g l y c a n , w h i c h b i n d s L P L . T h e physiological relevance o f the i n d u c t i o n o f L P L b y F G F c o u l d be related to w o u n d repair. U n d e r these circ u m s t a n c e s , F G F is m o s t active a n d L P L m i g h t be required to p r o v i d e free fatty a c i d s as a source of e n e r g y for the p r o l i f e r a t i n g cells. Acknowledgement W e t h a n k Dr. J a n Breslow, Rockefeller University, N . Y . for his g e n e r o u s gift of the g e n o m i c m o u s e L P L DNA fragment. This s t u d y w a s s u p p o r t e d in p a r t b y the M a r i o S h a p i r o F u n d , T h e H e b r e w University, J e r u s a l e m to Y.S., a n d f r o m the S a l o m o n a n d F a n i G o t t e s f e l d F o u n d a t i o n to G . F . T h e excellent assistance o f Ms. L. N o e with the p r e p a r a t i o n of the anti s e r u m to L P L is gratefully a c k n o w l e d g e d . References 1 Chajek, T., Stein, O. and Stein. Y. (1978) Biochim. Biophys. Acta 528, 456-465. 2 Chajek, T., Stein, O. and Stein, Y. (1978) Biochim. Biophys. Acta 528, 466-474. 3 Friedman, G., Stein, O. and Stein, Y. (1978) Biochim. Biophys. Acta 531,222-232. 4 Friedman, G., Stein, O. and Stein, Y. (1980) Biochim. Biophys. Acta 619, 650-659. 5 Darmon, M., Serrero, G., Rizzino, A. and Sato, G. (1981) Exp. Cell Res. 132, 313-327. 6 Gospodarowicz, D. (1975) J. Biol. Chem. 250, 2515-2520. 7 Gospodarowicz, D., Ferrara, N., Schweigerer, L. and Nenfeld, G. (1987) Endocrine Revs. 8, 95-114.
8 Weiner, H.L. and Swain, J.L. (1989) Proc. Natl. Acad. Sci. USA 86, 2683-2687. 9 Nilsson-Ehle, P. and Schotz, M.C. (1976) J. Lipid Res. 17, 536-541. 10 Belfrage, P. and Vaughan, M. (1969) J. Lipid Res. 10, 341-344. 11 Videlingum, N., Drake, R.L., Etienne, J. and Kissebah, A.N. (1983) J. Am. Phys. 121-131. 12 Knobler, H., Chajek-Shaul, T., Stein, O. and Stein, Y. (1984) Biochim. Biophys. Acta 795, 363-371. 13 Friedman, G., Gallily, R., Chajek-Shaul, T., Stein, O., Shiloni, E., Etienne, J. and Stein, Y. (1988) Biochim. Biophys. Acta 960, 220-228. 14 Friedman, G., Chajek-Shaul, T., Etienne, J., Stein, O. and Stein, Y. (1986) Biochim. Biophys. Acta 875, 397-399. 15 Kirchgessner, T.G., Svenson, K.L., Lusis, A.J. and Schotz, M.C. (1987) J. Biol. Chem. 262, 8463-8466. 16 Warner, S.J.C., Friedman. G.B. and Libby, P. (1989) Arteriosclerosis 9, 279-299. 17 Chirgwin, M.J., Przybyla, EA., MacDonald, J.R. and Rutter, J.W. (1979) Biochemistry 18, 5294-5299. 18 Bailey, J.M. and Davidson, N. (1976) Anal. Biochem. 70, 75-85. 19 Detainer, L.A., Levin, M.S., Elovson, J., Reuban, M.A., Lusis, A.J. and Gordon, J.l. (1986) Proc. Natl. Acad. Sei. USA 83, 8102-8106. 20 Feinberg, A.P. and Vogelstein, M. (1983) Anal. Biochem. 132, 6-13. 21 Lowry, O.H., Rosebrough, N.J., Fan', A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 22 Friedman, G., Chajek-Shaul, T., Stein, O., Etienne, J. and Stein, Y. (1986) Bioehim. Biophys. Acta 877, 112-120. 23 Florini, J.R. and Magri, K.A. (1989) Am. J. Physiol. 256, C701C711. 24 Seed, J. and Hauschka, S.D. (1988) Developmental Biol. 128, 40-49. 25 Hayashi, I., Nixon, 1"., Morikawa, M. and Green, H. (1981) Proe. Natl. Acad. Sci. USA, 78, 3969-3972. 26 Spizz, G., Roman, D., Strauss, A. and Olson, E.N. (1986) J. Biol. Chem. 261, 9483-9488. 27 Broad, T.E. and Ham, R.H. (1983) Eur. J. Biochcm. 135, 33-39. 28 Nixon, T. and Green, H. (1984) Endocrinology 114, 527-532. 29 Doglio, A., Dani, C., Grimaldi, P. and Ailhaud, G. (1986) Biochem. J. 238, 123-129. 30 Amri, E.-Z., Dani, C., Doglio, A., Grimaldi, P. and Ailhaud, G. (1986) Biochem. Biophys. Res. Commun., 137, 903-910. 31 Friedman, G., Chajek-Shaul, T., Etienne, J., Stein, O. and Stein, Y. (1987) Biochim. Biophys. Acta 919, 1-12. 32 Kato, Y. and Gospodarow!ez, D. (1985) J. Cell Biol. 100, 477-485.
27
e. 1991 ElsevierScience PublishersB.V. 0005-2760/91/$03.50 ADONIS 000527609100099P
BBALIP 53580
The expression of lipoprotein lipase activity and mRNA in mesenchymal rat heart cell cultures is modulated by bFGF G . F r i e d m a n t, M . B e n - N a i m 2, O. H a l i m i ~, J. E t i e n n e 3 0 .
S t e i n 2 a n d Y. S t e i n
t Lipid Research Laborato~'. Department of Medicine B. Hadc~sah Unicersity Hospital, Jerusalem (Israel). " Department of Experimental Medicine and Cancer Research, Hebrew Unwersi(v-Itadassah Medical School. Jerusalem (Israel) and 3 Laboratoire de Biochimie. Faculte de Medicine. St. Antoine. Paris (France)
(Received 17 August 1990)
Key words: Growth factor: LPL mRNA: Heparin: Lipoprotein lipase; (Rat mesenchymal heart cell)
The effect of basic fibroblast growth factor (FGF) on lipoprotein lipase (LPL) production was studied in mesenchymal rat heart cell cultures. Addition of FGF to culture medium containing 20% senun resulted in a 3-fold increase in LPL activity. The minimal effective dose of FGF was 10 n g / m l and the increase occurred after exposure for 48 IL Addition of FGF w.~s effective during the first week in culture, when enzyme activity was increasing, but not after !! days when the cultures were superconfluent and ~he enzyme activity wa¢ high. Addition of FGF to serum-poor medium was able to replace serum required to sustain LPL activity, in FGF-treatod cultures, more LPL activity was present in the functional pool, but not in the medium, than in the controls. The increase in enzymic activity was accompanied by an increase in enzyme mass and in LPL mRNA.
Introduction Lipoprotein lipase (LPL) activity in cultured mesenchymal cells derived from new-born rat heart increases during the first week after plating and plateaus thereafter [1,21. Optimal conditions for the expression of LPL require the presence of fetal bovine and horse serum and enzyme activity decreases markedly in serum-poor medium [3,4]. The quest for a serum-free medium which will support cell growth and differentiation prompted the use of various compounds, among them fibroblast growth factor (FGF), which became an important ingredient of such media [51. F G F has been isolated and purified from brain and pituitary [6], but it appears that it is more ubiquitous [7]. In a recent study, concerned with the expression of m R N A of basic F G F in cells derived from the heart, it was shown that preplates which are analogous to our mesenchymal cells, are the main site of F G F m R N A expression [8]. Therefore, it seemed of interest to determine whether supple-
Abbre, ations: FGF, fibroblast growth factor; LPL, lipoprotein lipase; m R N A , messenger RNA.
Correspoaoence: Y. Stein, Lipid ResearchLaboratory, Departmentof Medicine ~. Hadassah University Hospital. p.o. Box 12000, Jerusalem 91120. ~rael.
mentation of heart cell cultures with exogenous F G F would enhance the increase in LPL activity and if so, to define the level of such a putative regulation. Materials and Methods
Cell cultures F I heart cell cultures which consisted mainly of nonbeating mesenchymal cells were prepared from 2-4day-old rat hearts, as described [1,2]. Cultures -vere used between 5 - 7 days after their isolation.
Determination of lipoprotein lipase activity The enzyme activity was determined on aliquots of medium and homogenates of cells which had been released from the petri dish with a rubber policeman in 1 ml 0.025 M N H 3 / N H 4 C I buffer (pH 8.1) containing 1 u n i t / m l of heparin. The assa~ system consisted of 0.1 ml of medium, of 0.1 ml of cell homogenate (50-70 ~g protein) and 0.1 mi of substrate containing labeled triolein, prepared according to the method of NilssonEhle and Schotz [9]. Incubations were carried out at 37°C for 45 min. The reaction was stopped by addition of m e t h a n o l / c h l o r o f o r m / h e p t a n e (1.4:1.25 : 1, v / v ) and the extraction of fatty acids was performed according to the methods of Belfrage and Vaughan [10] as modified by Nilsson-Ehle and Schotz [9]. Enzyme activ-
28 ity was calculated according to the formula of NilssonEhle and Schotz [9] and was expressed per mg cell protein/h. To meas~Jre the effect of 1 M NaCI on the lipolytic activity, cell homogenates or medium samples were preincubated in the presence of 1 M NaC1 for 10 min at 27°C. For determination of heparin releasable lipoprotein lipase activity, the medium was collected at the end of the incubation and cultures were incubated for 3 min at room temperature with 0.5 ml F~o medium containing 20% serum and 5 u / m l of heparin. 0.1 ml of the medium was assayed for lipoprotein lipase activity.
Inhibition of lipoprotein lipase by antiserum Goat antiserum to a highly purified rat adipose tissue lipoprotein lipase was prepared as described [11]. To measure inhibition of lipoprotein lipase activity by antibody, the tubes containing the medium or homogenate of cells were preincubated in 0.2 M NaC1, 30% glycerol and 0.005 M sodium veronal (pH 7.0) at 4°C for 30 rain, with antiserum to lipoprotein lipzse or nonimmunized goat serum, diluted 1:50. The tubes were centrifuged at 28 000 x g for 30 min at 4°C and enzyme activity was determined on the supernatant [12].
Partial purification of lipoprotein lipase Partial purification of lipoprotein lipase from heart cell cultures was performed by heparin sepharose column chromatography as described previously [13]. Briefly, heart cell cultures were washed extensively, and homogenized in phosphate buffered saline (PBS). The cell homogenate was extracted with cold diethyl ether. The resultant powder was dissolved in 10 mM Tris-HC! buffer (pH 7.4)/0.1% Triton X-100/20% glycerol and sonicated. The sonicate was centrifuged for 30 rain at 4°C at 12000 rpm in an SS-34 rotor. The supernatant was collected, applied to a heparin-Sepharose column and eluted at 4°C with 2 M NaCI as described [13].
Gel electrophoresis and immunoblotting Electrophoresis was carried out at 50 mA/slab gel for 3 h at 23°C. Gels were calibrated with molecular weight standards (Sigma). Immunoblotting of lipoprorein lipase was performed as described [13,14]. Briefly, proteins were transferred from the SDS gels onto nitrocellulose paper in a transblot cell apparatus (Bio-Rad). Electroblotting was carried out at 400 mA for 4 h at 23°C. After incubation of the nitrocellulose paper with goat antiserum to lipoprotein lipase for 3 h and subsequently with rabbit antigoat antiserum (Bio-Yeda, Israel), diluted 1:50 for 1 h, the paper was washed extensively and incubated with 125I-labeled protein A (spec.act.30 /tCi/mg, Amersham Intl., U.K.), followed by washing and air drying. Immunoreactive proteins on nitrocellulose paper were visualized by exposure to Agfa (Curix RP2) film for 4-7 days.
Nucleic acid probes Hybridization probes were prepared using plasmid pBSLPL which includes a 213 bp fragment of genomic sequences from exon 5 of the mouse LPL gene. This fragment was isolated from a 1.3 kb XbaI fragment of LPL cDNA subcloned into pBS vector (Stratagene). Sequence analysis confirmed that the clones were identical to those published [15]. A cloned fl-tubulin cDNA insert [16] was used as the control probe.
RNA preparation and blot hybridization analysis Total cellular RNA was extracted from cultured rat heart cells with guanidine thiocyanate and sedimented through a cesium chloride cushion according to Chirgwin et al. [17]. The integrity of the RNA preparation was verified by denaturing gel electrophoresis [18]. Dot blot analysis was performed with a template manifold apparatus (Sehleicher & Schueii, Keene NH). To assure uniform dot size, total cellular RNA was employed at different concentrations (5, 10, 15/Lg) and supplemented with yeast tRNA to provide a total of 15 pg of RNA per sample. The RNA samples were denatured by 1 M formaldehyde and heating at 60°C for 15 min, then diluted into 20 voi. of 3 M NaCI containing 0.3 M trisodium citrate and applied to nitrocellulose filter under vacuum. The filter was washed with additional diluent, baked at 800C for 2 h and then hybridized. Stringencies employed for filter hybridization and washing were as described [19]. DNA probes were labeled by random hexanucleotide priming by using 32p-labeled nucleotide triphosphates [20]. Autoradiography was done with standard techniques.
Analytical procedures Protein was determined according to the methods of Lowry et al. [21], using bovine serum albumin as standard. Radioactivity was determined in a r-scintillation spectrometer (Tri-carb 2660). The scintillation fluid used was 20% Triton X-100/0.005% 1A-bis(2-(5-phenyioxazolyl)benzene (POPOP)/0.4%, 2,5-diphenyl-oxazole (PP0) in toluene.
Materials Partially purified basic fibroblast growth factor (bFGF) isolated from bovine brain was kindly provided by Dr. Vlodavsky, Hadassah University Hospital, Jerusalem, Israel. Glycerol tri[9,10(n)-3H]oleate, specific activity 1 C i / m m o l was obtained from Amersham International, U.K. 32P-adCTP, specific activity 3000 Ci/mmol was obtained from New England Nuclear, U.S.A. Heparin, thrombolique was obtained from Organon, Oss, The Netherlands. Culture media and sera were obtained from Gibco, Grand Island, N.Y.
29 TABLE I
Results
T o s t u d y the effect of basic fibroblast g r o w t h f a c t o r ( b F G F ) o n the activity of lipoprotein lipase in r a t m e s e n c h y m a l h e a r t cell cultv, res, various c o n c e n t r a t i o n s o f the g r o w t h f a c t o r were a d d e d to c o m p l e t e culture m e d i u m . A s seen in Fig. 1, the increase in l i p o p r o t e i n lipase activity w a s d e p e n d e n t o n the c o n c e n t r a t i o n o f the effector; a 60% increase in cellular l i p o p r o t e i n lipase activity w a s seen a f t e r 72 h of i n c u b a t i o n with 10 n g / m l o f b F G F a n d a m o r e t h a n 3-fold increase o c c u r r e d at a c o n c e n t r a t i o n o f 100 n g / m l . N o c h a n g e w a s o b s e r v e d in the l i p o p r o t e i n lipase activity in the c u l t u r e m e d i u m , a n d n o c h a n g e in p r o t e i n c o n t e n t w a s o b s e r v e d b e t w e e n c o n t r o l a n d c u l t u r e s to w h i c h b F G F w a s a d d e d u p to 72 h before t e r m i n a t i o n o f the e x p e r i m e n t (0.65 + 0.07 nag cell p r o t e i n in c o n t r o l vs. 0.67 + 0.1 m g in t r e a t e d dishes). T o verify t h a t the e n z y m i c activity w h i c h w a s increased aeter t r e a t m e n t with b F G F is l i p o p r o t e i n lipase, the activity w a s d e t e r m i n e d a f t e r a d d i t i o n o f a specific a n t i s e r u m , o r 1 M N a C I . A l m o s t c o m p l e t e inhibition o f e n z y m i c activity o c c u r r e d in c o n t r o l s a n d b F G F t r e a t e d h e a r t cell cultures, i n d i c a t i n g t h a t the i n c r e a s e d lipolytic activity is i n d e e d l i p o p r o t e i n iipase (Table I). T h e t i m e - c o u r s e o f the i n d u c t i o n o f cellular l i p o p r o tein lipase activity w a s studied in the p r e s e n c e o f 200 n g / m l o f b F G F (Table II). U p to 24 h o f i n c u b a t i o n , there w a s 'almost n o c h a n g e in cellular l i p o p r o t e i n lipase activity in the b F G F t r e a t e d cultures; 1.6-fold increase in cellular l i p o p r o t e i n lipase activity w a s f o u n d a f t e r 48 h o f i n c u b a t i o n a n d a m a x i m a l value of 3.3-fold increase o c c u r r e d a f t e r 72 h. D u r i n g t h a t time, n o t w i t h -
Characteri:ation of the increased lipolytic actiritv in b F G F treated rat hfart cell cultures
Conditions: as in legend to Fig. I. To determine inhibition by zntiserum to LPL and I M NaCL aliquots of medium or of cell homogenates were incubated with 1 : 50 dilution of antiserum at 4°C for 30 min or with l M NaCI at 27°C for 10 rain prior to assay of LPL. Preincubation with nonimmune serum did- not affect enzymic activity. Results are means± S.E. of triplicate dishes. Addition to assay medium
LPL activity (nmol fatty acid released/h per mg cell protein)
None Antiserum to LPL I M NaCI
control
bFGF treated
820 + 26 96± 7 56+ 8
3052 ± 64 110:t: 9 63+ 5
s t a n d i n g the steep rise in cellular e n z y m i c activity, there w a s n o increase in m e d i u m lipoprotein lipase activity. L i p o p r o t e i n lipase in the c u l t u r e d m e s e n c h y m a l rat h e a r t cells is p r e s e n t in a n intraceUular a n d in a surface-related, f u n c t i o n a l c o m p a r t m e n t , f r o m w h i c h it c a n be released b y b e p a r i n [22]. In the next experiment, rat h e a r t cell c u l t u r e s w e r e e x p o s e d to 200 n g / m l of b F G F for 72 h. T h e r e a f t e r , h e p a r i n w a s a d d e d for 3 m i n to release t h e cell s u r f a c e l i p o p r o t e i n lipase activity, w h i c h r e p r e s e n t s the f u n c t i o n a l pool o f the enzyme. T h e d a t a in T a b l e I11 d e m o n s t r a t e d t h a t b F G F t r e a t m e n t resulted in a 5-fold increase in the f u n c t i o n a l p o o l of the e n z y m e a n d a 3-fold increase in residual cellular e n z y m e activity. T h e d a t a p r e s e n t e d so far h a v e s h o w n t h a t b F G F t r e a t m e n t induces a p r o n o u n c e d increase in lipoprotein lipase activity in r a t h e a r t cell cultures in the intraTABLE 11 Increase in cellular lipoprotein lipase activity by b F G F as a func:~~ qf time
,~001/ 5OO
o
0
J ~'--~:~ 50 ioo 500 bFGF (r~l/ml)
Fig. I. Effect of increasing concentrations of basic fibroblast growth factor (bFGF) on lipoprutein lipase activity of rat heart cell cultures. Conditions: The cells were cultured in Fw medium supplemented with 10% fetal bovine serum and 10~ horse serum for 3 days. On day 3, the medium was changed to 2 ml of fresh medium containing varying concentrations of bFOF and incubation was carried out for 72 h. At the end of the experiment, the medium was collected, the cell layer washed with phosphate-buffered saline scraped with 1 ml 0.025 M NH3/NH4CI buffer (pH 8.1) comaining I unit/ml heparin and homogenized at 0"C. 0.1 ml of cell homogenate or medium was assayed for lipoprotein lipase activity. Values are means±S.E, of triplicate dishes. O, cellular; o medium.
Conditions: as in legend to Fig. I. On day 3, the medium was changed and the experiment was continued for an additional 96 h. bFGF 200 ng/ml was added for 6-96 h prior to termination of the experiment. At the end of the incubation, the medium was collected and LPL activity was determined on medium aliquot and cell homogenates. Values are means + S.E. of triplicate dishes. Addition
None bFGF
Time (h)
LPL activity (nmol fatty acid released/h per mg cell protein) medium
total cellular
96
115+ 6
6
121+ 4
24 48 72 96
129± 8 127+11 119+ 3 132-t- 7
896:t:12 899:t=16 953+24 a 1496+31 2944+22 3021 +18
30 TABLE III
TABLE V
Effect of bFGF on the functional (heparir. releasable) LPL activity in rat heart cell cultures
Effect of addition of bFGF to culture medium on lipoprotein lipase activity in overconfluent rat heart cell cultures
Conditions: as in legend to Fig. 1. On day 3, the medium was changed and the cells were incubated for 72 h with 2 ml of fresh medium containing 200 ng/ml of bFGF (where appropriate). Thereafter, the medium was removed and lipoprolein lipase activity was determined on aliquots of medium and cell homogenates. 3-min heparin-releasable lipoprotein lipa~e activity was determined as described under Materials and Methods. Values are means ± S.E. of triplicate dishes.
Conditions: Cells were cultured in F~o medium supplemented with 10~ fetal bovine serum and 10% horse serum for 11 days. The medium was changed every 3 days. On day ll, the medium was changed and bFGF 200 ng/ml was added where appropriate. Incubation was carried out for 72 h. At the end of the incubation, lipoprotein lipase activity was determined on aliquots of medium and of cell homogenates. Values are means + S.E. of triplicate dishes.
Exp. No.
Addilions
Exp. No.
Treatment
medium
cellular
heparin releasable
medium
cellular
I
None bFGF
1054-7 112+8
592±10 1768+17
198± 4 958+11
1
None bFGF
135 ± 16 139 ± 21
1 190 + 65 1 181 ± 54
11
None bFGF
99±5 89±4
610± 8 1791+14
2044- 6 991+16
11
None bFGF
152 4-26 161 ± 31
1 101 +96 1 198 ± 66
Lipoprotein lipase activity (nmol fatty acid released/h per mg cell protein)
cellular a n d in the surface related f u n c t i o n a l c o m p a r t m e n t . However, a l m o s t n o c h a n g e w a s o b s e r v e d in the lipoprotein lipase activity in the c u l t u r e m e d i u m . In the next experiment, the effect o f multiple a d d i t i o n s o f b F G F o n lipoprotein iipase activity w a s studied. Since the increase in l i p o p r o t e i n lipase activity with time in culture is m o s t p r o n o u n c e d u p to 6 d a y s a n d t e n d s to p l a t e a u thereafter [1,2], the effect of b F G F w a s studied between d a y s 0 - 6 . A 3-fold increase in cellular l i p o p r o rein lipase activity o c c u r r e d w h e n a single d o s e o f b F G F w a s a d d e d o n d a y 3. W h e n a d d i t i o n o f b F G F w a s started o n d a y 0 a n d r e p e a t e d o n d a y s 3 a n d 5, the s a m e increase w a s o b s e r v e d (Table IV). W h e n b F G F w a s a d d e d only o n d a y 5, the increase in l i p o p r o t e i n lipase activity w a s 6 7 ~ (Table II, 48 h). S u p p l e m e n t a tion of o v e r c o n f l u e n t r a t m e s e n c h y m a l h e a r t cell c u l t u r e with b F G F did n o t increase l i p o p r o t e i n lipao¢ activity (Table V). N e x t , a n a t t e m p t w a s m a d e to s t u d y the effect of b F G F in the presence of s e r u m - p o o r m e d i u m . In the two e x p e r i m e n t s s h o w n in T a b l e VI, the e n z y m e
LPL activity (nmol fatty acid relcascd/h per mg cell protein)
activity w a s low ( 2 0 ~ ) w h e n the c u l t u r e m e d i u m w a s s u p p l e m e n t e d w i t h 0 . 5 ~ fetal c a l f s e r u m only. H o w e v e r , the a d d i t i o n o f 200 n g / m l o f b F G F to the s e r u m p o o r m e d i u m resulted in a l m o s t c o m p l e t e recovery o f the cellular l i p o p r o t e i n h p a s e activity. T o a s c e r t a i n w h e t h e r the increase in cellular l i p o p r o rein lipase activity in b F G F t r e a t e d c u l t u r e s p a r a l l e l e d the i n c r e a s e in l i p o p r o t e i n lipase p r o t e i n , the cellular l i p o p r o t e i n lipase c o n t a i n i n g fraction, eluted f r o m heparin-Sepharose column with 2 M NaCI, was subj e t t e d to gel d e c t r o p h o r e s i s a n d i m m u n o b l o t t i n g . A 3.2-fold i n c r e a s e in the d e n s i t y o f the b a n d identified as i i p o p r o t e i n iipas¢ is seen in c u l t u r e s t r e a t e d w i t h b F G F for 72 h (Fig. 2). T o e x a m i n e f u r t h e r the m e c h a n i s m o f the effect o f b F G F o n l i p o p r o t e i n lipas¢ p r o d u c t i o n , we c o m p a r e d cellular L P L m R N A levels b y d o t b l o t hyb r i d i z a t i o n a n a l y s i s in b F G F t r e a t e d a n d in c o n t r o l cultures. T h e c h a n g e s in L P L m R N A in t r e a t e d cells as c o m p a r e d to n o n t r e a t e d cells a r c s h o w n in Fig. 3. L P L m R N A levels w e r e 2.9-fold h i g h e r in c u l t u r e s t r e a t e d TABLE Vi
TABLE IV Effect of addition of bFGF to culture medium at early stages of growth on lipoprotein lipase activity in rat heart cell cultures
Effect of addition of bFGF on lipoprotein lipase actir~ity in rat heart cell cultures grown in serum poor medium
Conditions: Cells were grown in medium supplemented with 20% serum, bFGF 200 ng/ml was added to the culture medium as indicated. LPL activity was delermined on day 6. Values are means 4-S.E. of triplicate dishes.
Conditions: Cells were grown in culture medium containing 20~ serum for 3 days. On day 3, the medium was changed and mediumpoor serum was added where indicated. On day 5, bFGF 200 ng/ml was added where appropriate. The experiment was terminated on day 7. Values are means + S.E. of triplicate dishes.
Addition to culture
Addition to serum free medium
None bFGF bFGF bFGF
Day of addition
3 3, 5 0. 3, 5
LPL activity (nmol fatty acid released/h per mg cell protein) medium
cellular
96 + 15 III +14 101 ± 9 121 ± 18
860 + 14 2501+10 2610+16 2691 ± 12
10% FCS + 10% horse serum 0.5% FCS 0.5% FCS + bFGF
Cellular LPL activity (nmol fatty acid released/h per mg cell protein) Exp. I
Exp. I!
870 ± 12 160± 7 763 ± 14
901 + 22 189+18 851 ± I l
31
II6-
LPL-.-4,6Fig. 2. lmmunoblot of lipoprotein lipase from rat heart cell cultures. Heart cells were cultured for 3 days in Fi0 medium supplemented with 107o fetal bovine serum and 10% horse serum. On day 3, the medium was changed and bFGF 200 ng/ml was added for 72 h (where appropriate). At the end of incubation, the medium was collected, the cells washed six times with phosphate-buffered saline (PBS) and homogenized. Acetone ether powder of the homogenates was extracted and loaded on a heparin-Sepharose column. The fraction eluted with 2 M NaCI was concentrated and subjected to electro phoresis and immunoblotted with goat antiserum to lipoprotein lipase (see Materials and Methods). Lipoprotein lipase trom control cells, lane I; cells treated with bFGF, lane If. with b F G F than in the control cells cultured for the same time. The same R N A samples were analyzed by dot blot hybridization using a # - t u b u l i n e D N A probe to assess the specificity of L P L m R N A response. As shown in Fig. 3, almost no change in # - t u b u l i n m R N A was noted with b F G F treatment.
Discussion In this study we have shown that addition of basic F G F to rat heart cell cultures results in a 3-fold increase in lipoprotein lipase activity. This effect is concentration and time d e p e n d e n t and is seen already with 10 n g / m l of F G F , b u t only after 48 h of exposure. It seems pertinent to add that F G F was effective when added to the culture m e d i u m c o n t a i n i n g 20% serum. F G F was also able to sustain LPL activity u n d e r conditions of serum deprivation, which leads to a rapid fall in enzyme activity [3,4]. As in our previous studies [1,2],
A RNA
I
,GO
B II
I
•
O0
mOO
II
O0
Fig. 3. Dot blot analysis of LPL mRNA levels from rat heart cell cultures. Conditions: Heart cells were cultured as described in legend to Fig, l), in the presence or absence of 200 ng/ml bFGF for 72 h. Total RNA was isolated and applied to nitrocellulose membrane. LPL mRNA was detected using azP-labeled mouse LPL ganomic fragment as described under Materials and Methods. Lane i, control; Lane II, bFGF treated (A). After exposure, the blot was stripped and rehybridized with a probe for a constitutively expressed form of/t'-tubulin to confirm that approximately equal amounts of RNA were loaded in each dot (B). Lane t-control; lane II-bFGF treated. Data are from one experiment typical of three performed.
the presently used cultured cells are of the F t type (preplates of 3 0 - 4 5 rain) which consist mainly of nonheating cells. The LPL activity in these cultures increases d u r i n g the first week and later plateaus. The effect of F G F could be demonstrated only when added d u r i n g that phase, but not at the later time interval, even though the maximal enzymic activity in these cultures was still below that observed in younger cultures exposed to F G F . F G F was shown to be mitogenic for more than 20 different cell types [7]. W e hypothesize that in F t cultures F G F might have been mitogenic for a small fraction of mesenchymal cells which synthesize LPL, as there was no significant increase in the cellular protein c o n t e n t / d i s h . F G F is mainly considered as an inhibitor of differentiation of myogenic cells [7,23], even though in a recent study a s u b p o p u l a t i o n of cells derived from chick wing muscle was shown to require F G F for differentiation in culture [24]. Divergent results were obtained by addition of F G F to these cells on their differentiation. Thus, in culture of 3"1"3cells, F G F when a d d e d to serum c o n t a i n i n g m e d i u m inhibited their conversion into adipocytes, with little if any effect on cell multiplication [25]. Similarly, Spizz et al. [26] have shown that F G F can inhibit myogenic differentiation through a m e c h a n i s m independent of cell proliferation. On the other hand, differentiation of sheep preadipocytes was inhibited by removal of F G F from a serum free culture m e d i u m [27]. G r o w t h hormone is another effector of differentiation of precursor cells into adipocytes [28,29]. However, it is m a i n l y required for the expression of later markers of differentiation, such as glycerol-3-phosp h a t e dehydrogenase, but apparently not for LPL, which is considered to b e a n early marker [29] and the expression of which is coupled with growth arrest [30]. The presently described effect of F G F on LPL activity was accompanied also by a similar increase in enzyme protein and in LPL m R N A , indicating regulation at transcriptional level. The ability of F G F to control gene expression was d e m o n s t r a t e d also d u r i n g regulation of myogenesis [26]. A d d i t i o n of F G F resulted in a decline in muscle creatine kinase m R N A , which was independent of cell proliferation [26]. In analogy to our previous findings with effectors of adenylate cyclase [12,22], the distribution of cellular LPL in F G F treated cells was changed with an increase in enzyme activity in the functional pool, released by brief e.,posure to heparin. A t the same time, there was no change in L P L activity in the medium, contrary to the effect of Hepes buffer [31], which in the same system increases cellular and m e d i u m LPL activity. The effect of F O F on L P L activity in the heart cell cultures resembles to some extent that described in cultures of rabbit costal chondrocytes [32]. In that system, F G F caused an increase in the rate of incorporation of 35Ssulfate into proteoglycans and resulted in an expression
32 of c h o n d r o c y t e p h e n o t y p e . Moreover, similar to o u r findings, the F G F h a d to be present in the cultures p r i o r to c o n f l u e n c e a n d w a s not effective w h e n a d d e d at a later phase. It seems plausible t h a t the increase in the functional pool o f L P L b y F G F , w i t h o u t a c o n c o m i t a n t increase in the s p o n t a n e o u s release i n t o the c u l t u r e m e d i u m c o u l d b e d u e to an increase in cell surface h e p a r a n sulfate p r o t e o g l y c a n , w h i c h b i n d s L P L . T h e physiological relevance o f the i n d u c t i o n o f L P L b y F G F c o u l d be related to w o u n d repair. U n d e r these circ u m s t a n c e s , F G F is m o s t active a n d L P L m i g h t be required to p r o v i d e free fatty a c i d s as a source of e n e r g y for the p r o l i f e r a t i n g cells. Acknowledgement W e t h a n k Dr. J a n Breslow, Rockefeller University, N . Y . for his g e n e r o u s gift of the g e n o m i c m o u s e L P L DNA fragment. This s t u d y w a s s u p p o r t e d in p a r t b y the M a r i o S h a p i r o F u n d , T h e H e b r e w University, J e r u s a l e m to Y.S., a n d f r o m the S a l o m o n a n d F a n i G o t t e s f e l d F o u n d a t i o n to G . F . T h e excellent assistance o f Ms. L. N o e with the p r e p a r a t i o n of the anti s e r u m to L P L is gratefully a c k n o w l e d g e d . References 1 Chajek, T., Stein, O. and Stein. Y. (1978) Biochim. Biophys. Acta 528, 456-465. 2 Chajek, T., Stein, O. and Stein, Y. (1978) Biochim. Biophys. Acta 528, 466-474. 3 Friedman, G., Stein, O. and Stein, Y. (1978) Biochim. Biophys. Acta 531,222-232. 4 Friedman, G., Stein, O. and Stein, Y. (1980) Biochim. Biophys. Acta 619, 650-659. 5 Darmon, M., Serrero, G., Rizzino, A. and Sato, G. (1981) Exp. Cell Res. 132, 313-327. 6 Gospodarowicz, D. (1975) J. Biol. Chem. 250, 2515-2520. 7 Gospodarowicz, D., Ferrara, N., Schweigerer, L. and Nenfeld, G. (1987) Endocrine Revs. 8, 95-114.
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