- Email: [email protected]
Lactosylceramide stimulates aortic smooth muscle cell proliferation
Vol. 181, No. 2, 1991 December 16, 1991
BIOCHEMICAL
Lactosylceramide
stimulates aortic cell proliferation Subroto
Department
Received
October
23,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 554-561
smooth muscle
Chatterjee
of Pediatrics, Johns Hopkins University, Baltimore, Maryland 21205 1991
Summary: We have investigated the effects of various sphingolipids on aortic smooth muscle cell proliferation employing viable cell counting, [3H] thymidine incorporation into DNA and the release of lactate dehydrogenase. Assays for UDP Gal: GlcCer Bl-4 galactosyltransferase (GalT-2) in control and treated cells were pursued simultaneously. Lactosylceramide stimulated cell proliferation in the order of 5 fold. Antibody against LacCer but not GbOsejCer blocked the proliferative effects of LacCer in these cells. This phenomena was specific for aortic smooth muscle cells as LacCer decreased cell viability of aortic endothelial cells and had no effect on pulmonary endothelial cells. D-PDMP inhibited the activity of GalT-2 in smooth muscle cells and markedly prevented cell proliferation. In contrast, L-PDMP stimulated the activity of GalT-2 in smooth muscle cells and stimulated cell proliferation. Antibody against GalT-2 inhibited cell proliferation. Our findings suggest that the activation of GalT-2 leads to increased LacCer levels, which in turn, may be involved in aortic smooth muscle cell proliferation. 0 1991Academic Press,Inc. Glycosphingolipids (GSL) are important constituents of the cell membrane and have been accorded several biochemical functions (1, 2). The presence of large amounts of glucosylceramide (Glccer) and lactosylceramide (LacCer) in affected intima but not in unaffected intima in patients who died of myocardial infarction has been shown (3). Although proliferation of aortic smooth muscle cells is a hallmark in the pathogenesis of atherosclerosis, the biochemical mechanisms The abbreviations used in this manuscriut are: GSL, glycosphingolipids; Glccer, glucosylceramider Laccer, lactosylceramide; The glyoosphingolipids are abbreviated GM3, monosialo ganglioside. according to the recommendations of the IUPAC-IUB commission on Biochemical Nomenclature (1977) Lipids 12: 455-463. D-PDMP, l-phenyl2-decanoylamino-3-morpholino-1-propanol; PBS, phosphate buffered saline: LDH, lactate dehydrogenase. 0006-291X/91 Copyright All rights
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
554
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
involved in this process and the role of GSL is not known. In the present work, we show that the activation of UDP-Gal; GlcCer Bl-4 galactosyltransferase (GalT-2) leads to LacCer production involved in aortic smooth muscle cell proliferation. A preliminary account of this work has been published (4). Materials
and Methods
Isotones and Chemicals: 6-r3H] thymidine (specific activity 1.055 Bg/mmole) and UDP galactose (specific activity 1.04 GBg/mmol) were purchased from Amersham Corporation. D- f DMP and L-threo PDMP were gifts from Dr. Norman Radin, and N, N dimethylsphingosine was from Dr. S.I. Hakomori. Glycosphingolipids and antibodies against LacCer and GbOse3Cer were prepared and characterized in our laboratory (5, 6). Other chemicals were purchased from Sigma Chemical Company. Cells: Rabbit aortic smooth muscle cells were prepared in our laboratory according to the procedure of Ross (7). Bovine aortic smooth muscle cells and bovine aortic endothelial cells were Bovine obtained from Dr. Gerard Leutty and Carol Merges. pulmonary endothelial cells were purchased from ATCC. Cells x 104were Incubation of cells with various sohinsolioids: seeded in.24 well micro titer plates and grown in Eagles's minimum essential medium. On the day of the experiment, medium was removed, cells were washed with PBS and incubated with Ham's F-10 medium containing sphingolipids for various time periods. Following incubation, cell proliferation assays were pursued. All assays were performed in duplicate from three separate dishes The and results from a typical experiment is presented. variation in the cell proliferation values was within 5% - 10%. Measurement of Cell Proliferation: Cell proliferation was measured by viable cell counting (8). c3H] thymidine (5 uCi/ml) incorporation (8) and lactate dehydrogenase assays (9). Incubation of cells with D-PDMP and L-POMP and measurement of the activitv of GalT-2: Appropriate dilution of D-PDMP and L-POMP stock solutions were made in Ham's F-10 medium and added directly to culture dishes. Cell proliferation assays and GalT-2 assays (l.O~bw;;~np~ued subsequently. f cells with antibodies: Suitable aliguots of LacCer Inc a were mixed with various dilutions of antibodies against LacCer, GbOsegCer and preimmune rabbit serum IgG. Following incubation for 4 h at 4OC suitable aliguots of the mixture was pipeted into the culture dishes. After incubation for 24 h, cell proliferation assays were performed. Similarly, cells were incubated with antibody against GalT-2, raised in rabbits (11). Results Effects of various snhinaolinids on rabbit aortic smooth muscle cell nroliferatioq As shown in Fig. 1, sphingosine, N-N'dimethylsphingosine and galactosylsphingosine decreased cell proliferation in the order of 77.3%, 269% and 44.1% compared to control. Glucosylsphingosine, sulfatide (GalSO4Cer) and GM3 marginally increased cell proliferation ( 115% compared to control). In contrast, 555
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Sphingolipid Ficx.
1.
slnoo
gffects
ccl
Proliferatio_n Rabbit
aortic smooth muscle cells (x 104) were seeded in 24 well plates in medium containing 10% fetal bqvine serum. The Next, fresh medium cells were washed with phosphate buffered saline. plus various sphingolipids (10 p) were added and cells incubated for 24 h in a CO2 incubator. Thymidine (2 +/ml containing 0.z pg of cold thymidine) was added and incubation cogtinued for 2 h at 37 C. Medium was removed and the incorporation of [ Ii] thymidine was measured as described (8). The control value was 3.5 X 10 dpm/dish.
microtiter
ceramide, galactosylceramide, glucosylceramide, globotriosylceramide and globotetraosylceramide increased cell proliferation in the order of 200-350% compared to control. The most profound increase in cell proliferation occurred in cells incubated with LacCer; this was in the order of 558% compared to control. Complex GSL such as GMl, GDla, and GT suppressed cell proliferation in the order of 50% compared to control. Effects of time of incubation and concentration of LacCer on the proliferation of bovine aortic and Pulmonary cells Incubation for 2 days with 5, 10 and 20 uM LacCer increased bovine aortic smooth muscle cell proliferation in the order of 125%, 157% and 105%, respectively (Fig. 2A). Maximum stimulation of cell proliferation (230% compared to control) occurred upon Incubation incubation of cells with 10 uM LacCer for three days. (20 uM) did not of cells with higher concentrations of LacCer increase cell proliferation further. Incubation of cells with 5, 556
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A
0 2 day
4-
I
20
Loctosylceromide fkkll
Effects ceramide
(FM)
of time of incubation and concentration OS on the viabilitv of bovine aortic and Dulmonarv
cells
Bovine cells (x 104) were seeded as described in the legend for Fig. 1. Medium was changed and cells incubated with 5, 10 and 20 p LacCer . Following incubation for 2, 3 and 7 days, cells were trypsinfzed and viable cell count was Performed.
10 and 20 uM LacCer for seven days resulted in 129%, 184% and 36% cell proliferation compared to control. Incubation of aortic endothelial cells with 5, 10 and 20 uM LacCer for 2 days had no effects on cell proliferation. In contrast, incubation of such cells with 5, 10 and 20 uM LacCer resulted in a concentration-dependent marked decrease in cell proliferation in the order of 31%, 21% and 27%, respectively compared to control (Fig. 2B). Aortic endothelial cells did not survive upon incubation with LacCer (5-20 mM) for three days (data not shown). Incubation of pulmonary endothelial cells with LacCer for 2, 3 and 7 days had no effect on cell proliferation compared to control (Fig. 2C). Only at a high concentration (20 uM) of LacCer and incubation for seven days suppressed cell proliferation. Taken together, these studies suggest that LacCer mediated stimulation of cell proliferation is specific for aortic smooth muscle cells. Effects of incubation with D-PDMP and L-PDMP on aortic smooth muscle cell oroliferation and the activitv of GalT-2 Compared to control, in cells incubated with D-PDMP for 24 h, aortic smooth muscle cell viability was decreased to 42%, C3H]
Vol. 181, No. 2, 1991
Table I.
Effects actiVitV
BIOCHEMICAL
of D-PDWP of GalT-2 in aortic
Cell Viability (x104/dish) Control D-PDMP L-PDMP
1.2 0.7 1.7
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
on cell proliferation smooth muscle cells
and the
r3H] Thymidine LDIi GalT-2 Incorporation Activity Activity (cpm/dish) (unite/ml/min)(nmole/2h/mg protein) 711 386 2,568
60 160 ND
4.5 3.9 5.3
Rabbit aortic smooth muscle cells were grown as described in the legend for Fig. 1. Next; cells were incubated with 10 pM D-PDMPor LPDMPfor 24 h and Cell proliferation assays performed. Cells incubated for 4 h with these compounds were used in GalT-2 assay8 (lo). thymidine incorporation was decreased 50%, and the release of LDH in the medium increased in the order of 260% (Table I). This was accompanied by a 20% decrease in the activity of GalT-2. In Contrast, incubation of cells with L-PDMP increased cell viability and [3H] thymidine incorporation in the order of 40% and 360% respectively compared to control. This compound also increased the activity of GalT-2 in the order of 20% compared to control. To assess the effects of L-PDMP concentration on proliferation further, cells were incubated with 1.5 mM, 7.5 mM and 15 mM L-PDMP for 24 h and cell viability and [3H] thymidine incorporation was measured. We found that 1.5, 7.5 and 15 uM LPDMP increased cell viability in the order of 106%, 202% and 380% and increased [3H] thymidine incorporation in the order of 104%, 400% and 300%, respectively, compared to control. Effects of incubation of cells with LacCer and LacCer antibodv on cell proliferation As shown in Table II following incubation for 24 h, LacCer increased aortic smooth muscle cell viability in the order of 180%, and the incorporation of [3H] thymidine in the order of 140% compared to control. LacCer decreased the release of LDH activity in the culture medium in the order of 30% compared to control. Antibody against LacCer but not GbOse3Cer (data not shown) compromised the proliferative effects of LacCer in these cells. Effects of antibodv aaainst GalT-2 on cell aroliferation Incubation of cells with 1:lOO dilution and 1:200 dilution of GalT-2 antibody decreased cell proliferation in the order of 44% and 24% respectively compared to control (Table III).
Vol.
181, No. 2, 1991
Table
II.
Effects
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of lactosvlceramide and corresnondina cell vroliferation
Addition
Cell Count (x104/dish)
Control
1.6
LacCer
2.9
LacCer + LacCer antibody
1.6
r3H] Thymidige poration (xl0
incordpm/dish)
antibodv
on
LDH Activity (units/min)
843
250
1,225
80
899
60
In these experiments, cells were incubated with LacCer (10 pM) k LacCer antibody at a dilution of 1:lO for 24 h and cell proliferation assays performed.
Discussion
of incubation of cells with GSLs have been addressed previously. These include inhibition of cell growth through extension of the Gl phase of the cell cycle by globotetraosylceramide (12). Incubation of HL-60 cells with GM3 resulted in their differentiation into monocytes/raacrophages However, when these cells were incubated with (13). sialylparagloboside they differentiated into granulocytes (14). In another study, GM3 decreased cell proliferation whereas GlcCer was found to stimulate cell proliferation in cells (15). In one report LacCer was found to inhibit cell proliferation (16) and in another to stimulate cell proliferation (17). The reasons for the discrepancy in these two studies are not known. We found The consequences
Table
III.
Fffects of antibodv on cell vroliferation
Addition
auainst
GalT-2
[3H] thymidine incorporation (x 10 dpm/dish)
Control
1223
GalT-2 antibody (1:lOO dilution)
689
GalT-2 antibody (1:200 dilution)
930
Cells were incubated 24 h and the incorporation
with Qntibody against GalT-2 for of [ ?I] thymidine was measured. 559
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
that LacCer specifically stimulated aortic smooth muscle cell proliferation. The ability of both aortic endothelial and pulmonary endothelial cells to resist the adverse effects of LacCer at low concentration (less than 10 uM) may explain why such cells are selectively chosen to serve as a barrier between circulating GSL including LacCer on lipoproteins (18). On the other hand, it is tempting to speculate that the destruction of the aortic endothelial layer in the presence of high concentrations of LacCer may permit the entry of LacCer into the smooth muscle cell layer and promote their proliferation which, in turn, could contribute to the pathogenesis of atherosclerosis. The role of endogenous GSL and corresponding biosynthetic enzymes has been assessed recently because of the availability of D-PDMP and L-PDMP. The former compound inhibits the synthesis of GlcCer by suppressing the activity of UDP-Glucose: ceramide Bl, 4 glucosyltransferase and inhibited the proliferation of cultured cells (15). Moreover, D-PDMP enhances the synthesis of N, Nldimethylsphingosine, a potent inhibitor of cell proliferation and (19) ' We also found that D-PDMP, N, N1-dimethylsphingosine sphingosine inhibited aortic smooth muscle cell proliferation. In addition, we found that D-PDMP inhibited the activity of GalT2. Perhaps this may have been due to a decrease in the cellular levels of GlcCer. Previous studies with El6 melanoma cells have revealed that L-PDMP increases the cellular levels of GlcCer and LacCer but not other GSLs (14). We found that L-PDMP increased the activity of GalT-2 and increased aortic smooth muscle cell proliferation. Thus our studies suggest that activation of GalT2 leading to increased cellular LacCer levels leads to aortic smooth muscle cell proliferation. This tenet is supported further by our observation that i) antibody against GalT-2 exerted a concentration-dependent decrease in cell proliferation. ii) moreover antibody against LacCer but not GbOsegCer compromised the proliferative effects of Laccer. Whether LacCer directly increases cell proliferation or indirectly does so by activating various cytokines and/or one or more compounds/enzymes in the signal transduction pathway is not known. Our studies reported here are provocative and require further investigations to ascertain the role of LacCer in cell proliferation. Acknowledaments their
I would like to thank Mr. Mike Shin and Dr. Nupur Ghosh for assistance during various phases of this work. Ms. Kat 560
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Lovejoy's assistance in preparing this manuscript is greatly appreciated. This work was partially supported by a NIH grant RO-l-DK-31722. References Sphingolipid Biochemistry (Kanfer, J.N. 1. Hakomori, S.I. (1983) In: and Hakomori, S.I. eds) pp. 381-436, Plenum Publishing Corp. New York. 2. Karlsson, K.A. (1989) Ann. Rev. Biochem. 58: 309-350. 3. Prokarova, N.V., Mukhin, D.N., Orekhov, A.N., Gladkoya, V.L., Dushew, V.N., Bergelson, L.D. (1989) Eur. J. Biochem. 180: 167172. 4. Chatterjee, S. (1991) pp. 108 Int. Union of Biochem. Meeting, Jerusalem, Israel. 5. Chatterjee, S., Yanni, S. (1987) LC GC 5: 571-574. 6. Chatterjee, S., Kwiterovich, P.O., Gupta, P.K., Erozan, Y., Alving, C.A., Richards, R. (1983) Proc. Natl. Acad. Sci. USA 1,313-1,318.
7. Ross, R. J. Cell. Biol. 50: 172-175. 8. Chatterjee, S., Sekerke, C.S., Kwiterovich, P.O. (1982) Eur. J. Biochem. 120: 435-441. 9. Bergmeyer, H.V.: In: Principles of enzymatic analysis. (1978) Verlag Chemie, New York. lO.Chatterjee, S., Castiglione, E. (1987) Biochim. Biophys. Acta 923: 136-142, ll.Chatterjee, S., Ghosh, N., Khurana, S. (1991) J. Biol. Chem. Manuscript submitted for publication. 12.Laine R.A., Hakomori, S.I. (1973) Biochem. Biophys. Res. Comm. 54: 1,049-1,053. 13.Noijiri, H., Takahu, F., Terui, Y., Miara, Y. and Saito, Y. (1986) Proc. Natl. Acad. Sci. USA 83: 782-786. 14.Noijiri, H., Kitagama, S., Nakamura, M., Kirato, K., Enomoto, Y. and Saito, M. (1988) J. Biol. Chem. 263: 7,443-7,446. 15.Inokuchi, J., Monrosaki, U., Shimeno, H., Nagamatsu, A., Radin, N.S. (1989) J. Cell.. Physiol. 141: 573-585. 16.Handa, S., Taki, T., Rokukawa, C., Ogura, K., Hara, T., Kawakita, M. (1990) J. Glycosphingolipid Res. Abst. #24. 17.0gura, K., Sweeley, C.C. (1991) Glycoconjugate J. 8: 160. 18.Chatterjee. S., Kwiterovich, P.O. Jr. (1976) Lipids 11: 462-466. lg.Felding-Habermann, B., Igarashi, Y., Fenderson, B.A., Park, L.S., Radin, N.S., Inokuchi, J., Strassman, G., Harda, K. and Hakomori, S.I. (1990) Biochemistry 29: 6,314-6,322.
561
BIOCHEMICAL
Lactosylceramide
stimulates aortic cell proliferation Subroto
Department
Received
October
23,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 554-561
smooth muscle
Chatterjee
of Pediatrics, Johns Hopkins University, Baltimore, Maryland 21205 1991
Summary: We have investigated the effects of various sphingolipids on aortic smooth muscle cell proliferation employing viable cell counting, [3H] thymidine incorporation into DNA and the release of lactate dehydrogenase. Assays for UDP Gal: GlcCer Bl-4 galactosyltransferase (GalT-2) in control and treated cells were pursued simultaneously. Lactosylceramide stimulated cell proliferation in the order of 5 fold. Antibody against LacCer but not GbOsejCer blocked the proliferative effects of LacCer in these cells. This phenomena was specific for aortic smooth muscle cells as LacCer decreased cell viability of aortic endothelial cells and had no effect on pulmonary endothelial cells. D-PDMP inhibited the activity of GalT-2 in smooth muscle cells and markedly prevented cell proliferation. In contrast, L-PDMP stimulated the activity of GalT-2 in smooth muscle cells and stimulated cell proliferation. Antibody against GalT-2 inhibited cell proliferation. Our findings suggest that the activation of GalT-2 leads to increased LacCer levels, which in turn, may be involved in aortic smooth muscle cell proliferation. 0 1991Academic Press,Inc. Glycosphingolipids (GSL) are important constituents of the cell membrane and have been accorded several biochemical functions (1, 2). The presence of large amounts of glucosylceramide (Glccer) and lactosylceramide (LacCer) in affected intima but not in unaffected intima in patients who died of myocardial infarction has been shown (3). Although proliferation of aortic smooth muscle cells is a hallmark in the pathogenesis of atherosclerosis, the biochemical mechanisms The abbreviations used in this manuscriut are: GSL, glycosphingolipids; Glccer, glucosylceramider Laccer, lactosylceramide; The glyoosphingolipids are abbreviated GM3, monosialo ganglioside. according to the recommendations of the IUPAC-IUB commission on Biochemical Nomenclature (1977) Lipids 12: 455-463. D-PDMP, l-phenyl2-decanoylamino-3-morpholino-1-propanol; PBS, phosphate buffered saline: LDH, lactate dehydrogenase. 0006-291X/91 Copyright All rights
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
554
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
involved in this process and the role of GSL is not known. In the present work, we show that the activation of UDP-Gal; GlcCer Bl-4 galactosyltransferase (GalT-2) leads to LacCer production involved in aortic smooth muscle cell proliferation. A preliminary account of this work has been published (4). Materials
and Methods
Isotones and Chemicals: 6-r3H] thymidine (specific activity 1.055 Bg/mmole) and UDP galactose (specific activity 1.04 GBg/mmol) were purchased from Amersham Corporation. D- f DMP and L-threo PDMP were gifts from Dr. Norman Radin, and N, N dimethylsphingosine was from Dr. S.I. Hakomori. Glycosphingolipids and antibodies against LacCer and GbOse3Cer were prepared and characterized in our laboratory (5, 6). Other chemicals were purchased from Sigma Chemical Company. Cells: Rabbit aortic smooth muscle cells were prepared in our laboratory according to the procedure of Ross (7). Bovine aortic smooth muscle cells and bovine aortic endothelial cells were Bovine obtained from Dr. Gerard Leutty and Carol Merges. pulmonary endothelial cells were purchased from ATCC. Cells x 104were Incubation of cells with various sohinsolioids: seeded in.24 well micro titer plates and grown in Eagles's minimum essential medium. On the day of the experiment, medium was removed, cells were washed with PBS and incubated with Ham's F-10 medium containing sphingolipids for various time periods. Following incubation, cell proliferation assays were pursued. All assays were performed in duplicate from three separate dishes The and results from a typical experiment is presented. variation in the cell proliferation values was within 5% - 10%. Measurement of Cell Proliferation: Cell proliferation was measured by viable cell counting (8). c3H] thymidine (5 uCi/ml) incorporation (8) and lactate dehydrogenase assays (9). Incubation of cells with D-PDMP and L-POMP and measurement of the activitv of GalT-2: Appropriate dilution of D-PDMP and L-POMP stock solutions were made in Ham's F-10 medium and added directly to culture dishes. Cell proliferation assays and GalT-2 assays (l.O~bw;;~np~ued subsequently. f cells with antibodies: Suitable aliguots of LacCer Inc a were mixed with various dilutions of antibodies against LacCer, GbOsegCer and preimmune rabbit serum IgG. Following incubation for 4 h at 4OC suitable aliguots of the mixture was pipeted into the culture dishes. After incubation for 24 h, cell proliferation assays were performed. Similarly, cells were incubated with antibody against GalT-2, raised in rabbits (11). Results Effects of various snhinaolinids on rabbit aortic smooth muscle cell nroliferatioq As shown in Fig. 1, sphingosine, N-N'dimethylsphingosine and galactosylsphingosine decreased cell proliferation in the order of 77.3%, 269% and 44.1% compared to control. Glucosylsphingosine, sulfatide (GalSO4Cer) and GM3 marginally increased cell proliferation ( 115% compared to control). In contrast, 555
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Sphingolipid Ficx.
1.
slnoo
gffects
ccl
Proliferatio_n Rabbit
aortic smooth muscle cells (x 104) were seeded in 24 well plates in medium containing 10% fetal bqvine serum. The Next, fresh medium cells were washed with phosphate buffered saline. plus various sphingolipids (10 p) were added and cells incubated for 24 h in a CO2 incubator. Thymidine (2 +/ml containing 0.z pg of cold thymidine) was added and incubation cogtinued for 2 h at 37 C. Medium was removed and the incorporation of [ Ii] thymidine was measured as described (8). The control value was 3.5 X 10 dpm/dish.
microtiter
ceramide, galactosylceramide, glucosylceramide, globotriosylceramide and globotetraosylceramide increased cell proliferation in the order of 200-350% compared to control. The most profound increase in cell proliferation occurred in cells incubated with LacCer; this was in the order of 558% compared to control. Complex GSL such as GMl, GDla, and GT suppressed cell proliferation in the order of 50% compared to control. Effects of time of incubation and concentration of LacCer on the proliferation of bovine aortic and Pulmonary cells Incubation for 2 days with 5, 10 and 20 uM LacCer increased bovine aortic smooth muscle cell proliferation in the order of 125%, 157% and 105%, respectively (Fig. 2A). Maximum stimulation of cell proliferation (230% compared to control) occurred upon Incubation incubation of cells with 10 uM LacCer for three days. (20 uM) did not of cells with higher concentrations of LacCer increase cell proliferation further. Incubation of cells with 5, 556
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A
0 2 day
4-
I
20
Loctosylceromide fkkll
Effects ceramide
(FM)
of time of incubation and concentration OS on the viabilitv of bovine aortic and Dulmonarv
cells
Bovine cells (x 104) were seeded as described in the legend for Fig. 1. Medium was changed and cells incubated with 5, 10 and 20 p LacCer . Following incubation for 2, 3 and 7 days, cells were trypsinfzed and viable cell count was Performed.
10 and 20 uM LacCer for seven days resulted in 129%, 184% and 36% cell proliferation compared to control. Incubation of aortic endothelial cells with 5, 10 and 20 uM LacCer for 2 days had no effects on cell proliferation. In contrast, incubation of such cells with 5, 10 and 20 uM LacCer resulted in a concentration-dependent marked decrease in cell proliferation in the order of 31%, 21% and 27%, respectively compared to control (Fig. 2B). Aortic endothelial cells did not survive upon incubation with LacCer (5-20 mM) for three days (data not shown). Incubation of pulmonary endothelial cells with LacCer for 2, 3 and 7 days had no effect on cell proliferation compared to control (Fig. 2C). Only at a high concentration (20 uM) of LacCer and incubation for seven days suppressed cell proliferation. Taken together, these studies suggest that LacCer mediated stimulation of cell proliferation is specific for aortic smooth muscle cells. Effects of incubation with D-PDMP and L-PDMP on aortic smooth muscle cell oroliferation and the activitv of GalT-2 Compared to control, in cells incubated with D-PDMP for 24 h, aortic smooth muscle cell viability was decreased to 42%, C3H]
Vol. 181, No. 2, 1991
Table I.
Effects actiVitV
BIOCHEMICAL
of D-PDWP of GalT-2 in aortic
Cell Viability (x104/dish) Control D-PDMP L-PDMP
1.2 0.7 1.7
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
on cell proliferation smooth muscle cells
and the
r3H] Thymidine LDIi GalT-2 Incorporation Activity Activity (cpm/dish) (unite/ml/min)(nmole/2h/mg protein) 711 386 2,568
60 160 ND
4.5 3.9 5.3
Rabbit aortic smooth muscle cells were grown as described in the legend for Fig. 1. Next; cells were incubated with 10 pM D-PDMPor LPDMPfor 24 h and Cell proliferation assays performed. Cells incubated for 4 h with these compounds were used in GalT-2 assay8 (lo). thymidine incorporation was decreased 50%, and the release of LDH in the medium increased in the order of 260% (Table I). This was accompanied by a 20% decrease in the activity of GalT-2. In Contrast, incubation of cells with L-PDMP increased cell viability and [3H] thymidine incorporation in the order of 40% and 360% respectively compared to control. This compound also increased the activity of GalT-2 in the order of 20% compared to control. To assess the effects of L-PDMP concentration on proliferation further, cells were incubated with 1.5 mM, 7.5 mM and 15 mM L-PDMP for 24 h and cell viability and [3H] thymidine incorporation was measured. We found that 1.5, 7.5 and 15 uM LPDMP increased cell viability in the order of 106%, 202% and 380% and increased [3H] thymidine incorporation in the order of 104%, 400% and 300%, respectively, compared to control. Effects of incubation of cells with LacCer and LacCer antibodv on cell proliferation As shown in Table II following incubation for 24 h, LacCer increased aortic smooth muscle cell viability in the order of 180%, and the incorporation of [3H] thymidine in the order of 140% compared to control. LacCer decreased the release of LDH activity in the culture medium in the order of 30% compared to control. Antibody against LacCer but not GbOse3Cer (data not shown) compromised the proliferative effects of LacCer in these cells. Effects of antibodv aaainst GalT-2 on cell aroliferation Incubation of cells with 1:lOO dilution and 1:200 dilution of GalT-2 antibody decreased cell proliferation in the order of 44% and 24% respectively compared to control (Table III).
Vol.
181, No. 2, 1991
Table
II.
Effects
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of lactosvlceramide and corresnondina cell vroliferation
Addition
Cell Count (x104/dish)
Control
1.6
LacCer
2.9
LacCer + LacCer antibody
1.6
r3H] Thymidige poration (xl0
incordpm/dish)
antibodv
on
LDH Activity (units/min)
843
250
1,225
80
899
60
In these experiments, cells were incubated with LacCer (10 pM) k LacCer antibody at a dilution of 1:lO for 24 h and cell proliferation assays performed.
Discussion
of incubation of cells with GSLs have been addressed previously. These include inhibition of cell growth through extension of the Gl phase of the cell cycle by globotetraosylceramide (12). Incubation of HL-60 cells with GM3 resulted in their differentiation into monocytes/raacrophages However, when these cells were incubated with (13). sialylparagloboside they differentiated into granulocytes (14). In another study, GM3 decreased cell proliferation whereas GlcCer was found to stimulate cell proliferation in cells (15). In one report LacCer was found to inhibit cell proliferation (16) and in another to stimulate cell proliferation (17). The reasons for the discrepancy in these two studies are not known. We found The consequences
Table
III.
Fffects of antibodv on cell vroliferation
Addition
auainst
GalT-2
[3H] thymidine incorporation (x 10 dpm/dish)
Control
1223
GalT-2 antibody (1:lOO dilution)
689
GalT-2 antibody (1:200 dilution)
930
Cells were incubated 24 h and the incorporation
with Qntibody against GalT-2 for of [ ?I] thymidine was measured. 559
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
that LacCer specifically stimulated aortic smooth muscle cell proliferation. The ability of both aortic endothelial and pulmonary endothelial cells to resist the adverse effects of LacCer at low concentration (less than 10 uM) may explain why such cells are selectively chosen to serve as a barrier between circulating GSL including LacCer on lipoproteins (18). On the other hand, it is tempting to speculate that the destruction of the aortic endothelial layer in the presence of high concentrations of LacCer may permit the entry of LacCer into the smooth muscle cell layer and promote their proliferation which, in turn, could contribute to the pathogenesis of atherosclerosis. The role of endogenous GSL and corresponding biosynthetic enzymes has been assessed recently because of the availability of D-PDMP and L-PDMP. The former compound inhibits the synthesis of GlcCer by suppressing the activity of UDP-Glucose: ceramide Bl, 4 glucosyltransferase and inhibited the proliferation of cultured cells (15). Moreover, D-PDMP enhances the synthesis of N, Nldimethylsphingosine, a potent inhibitor of cell proliferation and (19) ' We also found that D-PDMP, N, N1-dimethylsphingosine sphingosine inhibited aortic smooth muscle cell proliferation. In addition, we found that D-PDMP inhibited the activity of GalT2. Perhaps this may have been due to a decrease in the cellular levels of GlcCer. Previous studies with El6 melanoma cells have revealed that L-PDMP increases the cellular levels of GlcCer and LacCer but not other GSLs (14). We found that L-PDMP increased the activity of GalT-2 and increased aortic smooth muscle cell proliferation. Thus our studies suggest that activation of GalT2 leading to increased cellular LacCer levels leads to aortic smooth muscle cell proliferation. This tenet is supported further by our observation that i) antibody against GalT-2 exerted a concentration-dependent decrease in cell proliferation. ii) moreover antibody against LacCer but not GbOsegCer compromised the proliferative effects of Laccer. Whether LacCer directly increases cell proliferation or indirectly does so by activating various cytokines and/or one or more compounds/enzymes in the signal transduction pathway is not known. Our studies reported here are provocative and require further investigations to ascertain the role of LacCer in cell proliferation. Acknowledaments their
I would like to thank Mr. Mike Shin and Dr. Nupur Ghosh for assistance during various phases of this work. Ms. Kat 560
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Lovejoy's assistance in preparing this manuscript is greatly appreciated. This work was partially supported by a NIH grant RO-l-DK-31722. References Sphingolipid Biochemistry (Kanfer, J.N. 1. Hakomori, S.I. (1983) In: and Hakomori, S.I. eds) pp. 381-436, Plenum Publishing Corp. New York. 2. Karlsson, K.A. (1989) Ann. Rev. Biochem. 58: 309-350. 3. Prokarova, N.V., Mukhin, D.N., Orekhov, A.N., Gladkoya, V.L., Dushew, V.N., Bergelson, L.D. (1989) Eur. J. Biochem. 180: 167172. 4. Chatterjee, S. (1991) pp. 108 Int. Union of Biochem. Meeting, Jerusalem, Israel. 5. Chatterjee, S., Yanni, S. (1987) LC GC 5: 571-574. 6. Chatterjee, S., Kwiterovich, P.O., Gupta, P.K., Erozan, Y., Alving, C.A., Richards, R. (1983) Proc. Natl. Acad. Sci. USA 1,313-1,318.
7. Ross, R. J. Cell. Biol. 50: 172-175. 8. Chatterjee, S., Sekerke, C.S., Kwiterovich, P.O. (1982) Eur. J. Biochem. 120: 435-441. 9. Bergmeyer, H.V.: In: Principles of enzymatic analysis. (1978) Verlag Chemie, New York. lO.Chatterjee, S., Castiglione, E. (1987) Biochim. Biophys. Acta 923: 136-142, ll.Chatterjee, S., Ghosh, N., Khurana, S. (1991) J. Biol. Chem. Manuscript submitted for publication. 12.Laine R.A., Hakomori, S.I. (1973) Biochem. Biophys. Res. Comm. 54: 1,049-1,053. 13.Noijiri, H., Takahu, F., Terui, Y., Miara, Y. and Saito, Y. (1986) Proc. Natl. Acad. Sci. USA 83: 782-786. 14.Noijiri, H., Kitagama, S., Nakamura, M., Kirato, K., Enomoto, Y. and Saito, M. (1988) J. Biol. Chem. 263: 7,443-7,446. 15.Inokuchi, J., Monrosaki, U., Shimeno, H., Nagamatsu, A., Radin, N.S. (1989) J. Cell.. Physiol. 141: 573-585. 16.Handa, S., Taki, T., Rokukawa, C., Ogura, K., Hara, T., Kawakita, M. (1990) J. Glycosphingolipid Res. Abst. #24. 17.0gura, K., Sweeley, C.C. (1991) Glycoconjugate J. 8: 160. 18.Chatterjee. S., Kwiterovich, P.O. Jr. (1976) Lipids 11: 462-466. lg.Felding-Habermann, B., Igarashi, Y., Fenderson, B.A., Park, L.S., Radin, N.S., Inokuchi, J., Strassman, G., Harda, K. and Hakomori, S.I. (1990) Biochemistry 29: 6,314-6,322.
561