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Tripeptide analysis of collagen synthesized by silica-exposed fibroblast cultures
Life Sciences, Vol. 32, pp. 2379-2384 Printed in the U.S.A.
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TRIPEPTIDE ANALYSIS OF COLLAGEN SYNTHESIZED BY SILICA-EXPOSED FIBROBLAST CULTURES William H. P a i l e s 1 a n d H a r o l d R e s n i c k 1 ' 2 1Biochemistry Section, Appalachian Laboratory for Occupational Safety and Health Morgantown, ,Vest Virginia 26505, U . S . A . and 2Department of Biochemistry, West Virginia U n i v e r s i t y , Medical Center, Morgantown, West Virginia 26506, U.S.A. (Received in final form February 23, 1983) Summary
The effect of silica on collagen biosynthesis by confluent monolayers of Wl-38 fibroblast cultures was examined by a more comprehensive method of analysis. The presence of the particulates had no direct effect on protein (collagen) synthesis, proline incorpora'don or prolyl hydroxylase a c t i v i t y ; the latter as determined by the degree of hydroxylation. Silica however, was highly toxic to the cells. Silica-induced fibrogenesis has been extensively studied in animal tissues and cell cultures (1,2, 3, 4, 5). For the most part the studies have focused on collagen biosynthesis with hydroxyproline and prolyl hydroxylase (E.C. 1.14.11.2) a c t i v i t y as markers of fibrogenic a c t i v i t y . The presence of hydroxyproline is due to the unique post-translational enzymic hydroxylation of specific proline residues that have already been incorporated into the collagen polypeptide c~-chains (6). The level of a c t i v i t y of prolyl hydroxylase is known to be high in tissues actively synthesizing collagen. However, enzyme a c t i v i t y does not necessarily correlate with rates of collagen synthesis (7). For example, in lung tissue from silicotic rats, prolyl hydroxylase activity increased approximately 250% and the increase preceded any demonstration of fibrosis based on histological staining or h y d r o x y proline analyses (8). It should also be noted that prolyl hydroxylase a c t i v i t y has been demonstrated in many nonfibroblastic cells, all of which presumably may have the capability of synthesizing collagen (9,10). In cell cultures, hydroxyproline formation has been used as an index of silica -induced fibrosis. The studies indicate that exposure to silica dust alone does not stimulate fibroblasts to synthesize collagen (11,12). However, extracts from silica-exposed macrophages when added to fibroblast cultures have been shown to induce fibrogenesis (13, 14) although there have been some conflicting results reported (12, 15). It becomes apparent however from these studies that h y d r o x y lation, i . e . , hydroxyproline formation, becomes limiting when investigating collagen synthesis. While hydroxylation of proline residues in polypeptide c~chains is not random it is not absolute (16, 17). Furthermore, collagen concentrations based on hydroxyproline assays do not probe the insoluble collagenous material when the protein is extracted from cell cultures (18). Also the extent to which collagen synthesis and subsequent secretion is dependent on hydroxylation is not well understood (19,20,21). In a review by Reiser and Last (22) many of the discrepancies noted in the literature are discussed as well as the difficulties 0024-3205/83/202379-06503.00/0
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in making comparisons on presumably similar types of studies. The purpose of the p r e s e n t s t u d y was to examine the f i b r o g e n i c a c t i v i t y of a f i b r o b l a s t c u l t u r e g r o w n in d i r e c t contact w i t h silica particles by t r i p e p t i d e analyses. The method developed by Paz and Gallop (23,24) examines p r o l y l h y d r o x y lase a c t i v i t y both in terms of u n d e r h y d r o x y l a t e d and n o r m a l l y - h y d r o x y l a t e d collagenous p r o t e i n s y n t h e s i s , as well as p r o l i n e i n c o r p o r a t i o n and h y d r o x y p r o l i n e formation. T h e method makes use of the r e p e t i t i v e amino acid sequence ( G I y - X - Y ) _ , f o u n d in all collagen p o l y p e p t i d e c~-chains, where X and Y are v a r i able aminor~cids (17). S p e c i f i c a l l y , analyses are made for the t r i p e p t i d e s , G l y - P r o - H y p and G l y - P r o - P r o , both obtained by collagenase ( B i o f a c t u r e s , I n c . ) digestion of insoluble and soluble collagen f r a c t i o n s ; the digests are q u a n t i t a t e d by i o n - e x c h a n g e c h r o m a t o g r a p h y . Consideration is also made for the fact that the t r i p e p t i d e , G l y - P r o - A l a , f o u n d in r e l a t i v e l y h y g h c o n c e n t r a t i o n s a f t e r colla genase d i g e s t i o n , is c h r o m a t o g r a p h i c a l l y eluted w i t h G l y - P r o - P r o . A normally - h y d r o x y l a t e d collagen would t h e r e f o r e have a high level of the t r i p e p t i d e G l y - P r o - H y p ; u n d e r h y d r o x y l a t e d collagen would have a h i g h e r c o n c e n t r a t i o n of G l y - P r o - P r o ; the c a l c u l a t e d amount of collagenous p r o t e i n formed is corrected for the degree of u n d e r h y d r o x y l a t i o n (24). As a c o n t r o l , h y d r o x y l a t i o n is made l i m i t i n g by a d d i t i o n or deletion of a necessary cofactor, i . e . , L-ascorbic acid, a r e d u c t a n t for p r o l y l h y d r o x y l a s e a c t i v i t y (25,26), w i t h o u t i n t e r f e r r i n g w i t h p r o tein s y n t h e s i s . For more details, see Ref. 24, pp. 304-305. Methods The human embryonic lung f i b r o b l a s t s t r a i n WI-38 (American T y p e ~ : u l t u r e Collection) was used in t h i s s t u d y . The cells were g r o w n in T-150 c m ~ p l a s t i c c u l t u r e flasks ( C o m i n g ) c o n t a i n i n g 30 ml BME (basal medium Eagle, Earle's Salts; Gibco) supplemented w i t h 10% fetal calf serum and a n t i b i o t i c s (24). Cell c u l t u r e s were g r o w n at 37°C in an atmosphere of 5% CO_ and 95% a i r and s p l i t at a 1:4 ratio at weekly i n t e r v a l s u n t i l s u f f i c i e n t flasksZfor the t h r e e g r o u p s (10 f l a s k s / g r o u p ) of c o n f l u e n t c u l t u r e s were obtained. The s u f f i c i e n t number of flasks c o n t a i n i n g WI-38 cells were at the 26th passage level when held for one week at c o n f l u e n c y . One g r o u p received complete BME medium w i t h 50 ~ g l m l of L - a s c o r bic acid, a n o t h e r g r o u p received complete i3ME w i t h 50 ~ g l m l of L-ascorbic acid and 100 ~ g l m l of silica particles (Duke S t a n d a r d s ; 0 . 2 - 0 . 5 ~ ) ; c o n c e n t r a t i o n of silica selected based on p r e l i m i n a r y studies w i t h consideration of cell v i a b i l i t y o v e r time-frame of e x p e r i m e n t , as well as, c o n c e n t r a t i o n s used by o t h e r i n v e s t i gators (11,12). The t h i r d g r o u p received complete i3ME medium w i t h o u t L - a s c o r bic acid or silica p a r t i c l e s . D u r i n g the one week at c o n f l u e n c y for all g r o u p s , the respective media were changed and collected on a l t e r n a t e days. T w e n t y - f o u r hours before h a r v e s t i n g the medium was collected and the cells refed w i t h th~jir r e s p e c t i v e supplement for each g r o u p and " p u l s e d " w i t h 1.0 1=Ci/ml of 2 , 3 - L - [ H ] - p r o l i n e (New England N u c l e a r ) . A f t e r 24 h o u r s of labelling the cells and media were separated, pooled w i t h t h e i r respective g r o u p s and analyses were made. Cell v i a b i l i t y counts were performed on separate flasks c o n t a i n i n g c o n f l u e n t cell monolayers by the p r o c e d u r e of t r y p a n blue e x c l u s i o n . Procedures used for total p r o t e i n and DNA, performed when cells reached c o n f l u e n c y and a f t e r one week at c o n f l u e n c y , are the same as those reported by o t h e r i n v e s t i g a t o r s (24). Collagen c o n c e n t r a t i o n s were based on h y d r o x y p r o l i n e determination (27) of collagenase-digested material ( b o t h soluble and insoluble) a f t e r h y d r o l y s i s w i t h 6 N HCI @ 110°C for 24 h o u r s ; c o n c e n t r a t i o n s were corrected for degree of u n d e r h y d r o x y l a t i o n (24). Separations were obtained u s i n g an amino acid a n a l y z e r (Beckman 120C) e q u i p ped w i t h a physiological ion exchange column [ 0 . 9 x 56 cm; PA-28 resin,
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Beckman) and the sodium c i t r a t e g r a d i e n t b u f f e r system s t a r t i n g w i t h 0.2 N sodium c i t r a t e at pH 3.18. T h e column was c a l i b r a t e d w i t h reference tripeptide--s and an amino acid m i x t u r e so that peak (A) ( G l y - P r o - H y p ) eluted s l i g h t l y ahead of methionine while peak (B) ( G l y - P r o - P r o and G l y - P r o - A l a ) eluted between leucine and t y r o s i n e . Lypholozed aliquots dissolved in 0.5 ml of 0.2 N sodium c i t r a t e b u f f e r , pH 2.2, were applied to the column from 4 to 18 mg f o ~ c e l l - r e l a t e d d i gests and 15-45 mg for digests from medium. The f r a c t i o n s collected were 2.5 ml at a flow rate of 50 m l l h r and counted in a l i q u i d s c i n t i l l a t i o n c o u n t e r as DPM ( d i s i n t e g r a t i o n s per m i n u t e ) . Results
F i g u r e I shows the elution profiles of labelled t r i p e p t i d e s a f t e r collagenase digestion of collagen found in the medium and collagen associated w i t h the cells and cell l a y e r . The peaks labelled (A) are G l y - P r o - H y p while peaks labelled (13) Cells
Medium
? o
and
IQyer
cell
"7 B
K ~E
B = 4
i0
161
~o
)0
40 50 Fraction
60
70
60
90
i00
ii0
number
number
Fraction
Clll$
Medium
and
cell
layer
? A
ff
B
90 Fraction
~4
+
A$corbate
100 II0
number
Fraction
Cells a n d
Medium
17 "7
cell
number
layer
?
,0
o
e +
Ascorbate and silica
B
! =
A
1o
Fraction
number
~o
~
~
~o
Fraction
~o
~o
8o
~o
1oo
.0
number
FIG. I Elution profiles of t r i p e p t i d e s obtained from collagenase-digested, T C A - s o l u b l e collagen e x t r a c t e d from medium and c o r r e s p o n d i n g cells and and cell l a y e r .
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consist o f G l y - P r o - P r o and G l y - P r o - A l a . The position of the peaks e l u t i n g from the i o n - e x c h a n g e column was established with commercially a v a i l a b l e t r i p e p t i d e s (Bachem and Sigma Chemical). The ratio of peak (A) to peak (B) as determined from the total DPM f o r each peak was used to calculate the degree of h y d r o x y l ation (23, 24); it also reflects the degree of u n d e r h y d r o x y l a t i o n . The ratio has been d e f i n e d as the h y d r o x y l a t i o n i n d e x [ H I . It has an e x p e r i m e n t a l v a lu e o f 3.5 for n o r m a l l y - h y d r o x y l a t e d collagen produced by WI-38 cells. The h y d r o x y l ation i n d e x is used to determine the degree (percentage) of h y d r o x y l a t i o n in the collagen f i b e r (24). The decrease in a c t i v i t y , i . e . , peak heights o b s e r v e d f o r cells grown in the presence of silica reflects the c y t o t o x i c i t y of the p a r t i c l e s . T_he cell counts f or each g r o u p at s t a r t of c o n f l u e n c y was a p p r o x i m a t e l y 180 x I0 b cells. A f t e r one week, the g r o u p maintained in the presence of silica, showed a decrease in cell count by a p p r o x i m a t e l y o n e - t h i r d . The decrease in cells is also indicated by a decrease in total p r o t e i n and DNA as shown in Table I. TABLE I Summary of Data Obtained for Collagen Associated with Cells and Cell L a y e r Ascorbic Acid
-
+
+
Silica
-
-
+
Total Protein (mg)
105
115
71
Total DNA (mg)
2.4
2.9
1.8
Total Collagen (rag)
0.4
1.4
0.3
mg C o l l a g e n l m g DNA
0.2
0.5
0.2
2
9
3
62
98
93
Collagen (cells) Total Collagen
(%)
Degree H y d r o x y l a t i o n (%)
T A B L E II Summary of Data Obtained for Collagen Found in Medium Ascorbic Acid
+
Silica
-
Total Collagen (mg)
-
16.6
14.6
10.6
mg Collagen/mg DNA
6.9
5.0
5.9
Collagen (medium) Total Collagen
98
91
97
52
100
97
(%)
Degree H y d r o x y l a t i o n
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T h e data obtained for the three g r o u p s mentioned above are summarized in Tables I and I I . A l t h o u g h absolute q u a n t i t i e s do v a r y , the t r e n d in the data f o r cells g r o w n in the absence and presence of ascorbate ( w i t h o u t silica) are similar to those reported by Paz and Gallop (23,24). More total collagen w a s p r o duced by cells in the absence of the vitamin but the p r o t e i n found in the medium was only 52% h y d r o x y l a t e d (Table II) while collagen associated w i t h the cells was 62% h y d r o x y l a t e d (Table I ) . T h e r e was more collagen associated w i t h the cells g r o w n in the presence of ascorbate than those g r o w n in absence of the reductant~ F u r t h e r m o r e , collagen for both cells and medium was n o r m a l l y - h y d r o x y l a t e d (Tables I and I I ) . An examination of the data obtained for cells grown in the presence of silica p a r ticles plus ascorbate indicates that the particles had no effect on p r o l y l h y d r o x y l a s e a c t i v i t y ; the degree of h y d r o x y l a t i o n was 93% and 97% for cells and medium r e s p e c t i v e l y . F u r t h e r m o r e , it is q u i t e apparent that the p a r t i c u l a t e s had no effect on protein (collagen) s y n t h e s i s . The decrease in total collagen - as mentioned above - was due to less than t w o - t h i r d s of viable cells remaining a f t e r one week at c o n f l u e n c y in the presence of silica. The r e l a t i v e l y high ratio of collagen to DNA found in the medium (Table II) reflects partial l y s i s of cells (in presence of silica) r e s u l t i n g in solubilization (in medium) of collagen normally associated w i t h cell l a y e r ; c o n v e r s e l y a decrease in the ratio (mg Collagen/mg D N A ) is seen in cells and cell layer (Table I ) . Discussion T h e r e has been considerable use of cell c u l t u r e s in biochemical studies related to pulmonary f i b r o s i s . Studies on the effects of o c c u p a t i o n a l l y - r e l a t e d d u s t s on the p r o l i f e r a t i o n of collagen f i b e r s by f i b r o b l a s t cells however, have been conflicting~ The discrepancies have been a t t r i b u t e d to (i) phase of cell g r o w t h (logarithmic v e r s u s s t a t i o n a r y ) (12,28), (ii) cell species (29), and ( i i i ) concentration and physical state of the p a r t i c u l a t e (12). A n o t h e r consideration has been macrophage involvement in f i b r o g e n e s i s ; the effects of e x t r a c t s from silica-exposed macrophages on f i b r o b l a s t c u l t u r e s (12,28,30,31). A u n i f y i n g concept has also been proposed. The concept relates to excessive p r o l i f e r a t i o n of cells and t u r n o v e r of c e l l u l a r m a t r i x as c o n t r i b u t i n g s i g n i f i c a n t l y to the pathogenesis of several diseases i n c l u d i n g pulmonary f i b r o s i s (32). The present r e p o r t does not specifically address i t s e l f to questions that may have been raised w i t h regard to earlier studies. Instead, it focuses on a method of analysis that is s u f f i c i e n t l y comprehensive and o f f e r s a more s a t i s f a c t o r y basis for e v a l u a t i n g the effects of p a r t i c u l a t e s on collagen s y n t h e s i s . The purpose of t h i s s t u d y was to evaluate and possibly refine an in v i t r o t e c h n i q u e s u f f i c i e n t l y to be able to forecast the p r o b a b i l i t y of a p a r t i c u l a t e involvement in collagen syl~lthesis and an eventual u n d e r s t a n d i n g of pulmonary f i b r o s i s . The method of t r i peptide analysis goes beyond analysis for h y d r o x y p r o l i n e formation or the i n c o r poration of radioactive p r o l i n e and the subsequent h y d r o x y l a t i o n of t h i s residue; it i n v o l v e s o t h e r phases of collagen s y n t h e s i s . The results of the present s t u d y however, confirms p r e v i o u s studies w i t h cell c u l t u r e s that would seem to indicate that silica has no d i r e c t effect on the s y n t h e s i s of f i b r o u s protein by f i b r o b l a s t cells. References
I. 2. 3.
M. C H V A P I L and E. HOLECKOVA, B r . J. I n d . Med. 16, 286-289 (1959). A . C . ALLISON, J.S. HARINGTON and N. B I R B E C K , J. Exp. Med. 124, 141-153 (1966). M. C H V A P I L , J. HURYCH, B. CMUCHALOVA, R. HOLUSA and E. EHRLICHOVA, E n v i r o n . Res. I, 89-101 (1967).
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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
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T . A . K I L R O E - S M I T H , I. WEBSTER, M. VAN DRIMELEN and L. MARASAS, E n v i r o n . Res. 6, 298-305 (1973). S. AHO and E. KULONEN, Exp. Cell Res. 104, 31-38 (1977). R . L . MILLER and S. UDENFRIEND, A r c h . 13iochem. B i o p h y s . 139, 104-113 (1970). C . I . LEVENE and C . J . B A T E S , in Biology of F i b r o b l a s t , (Eds. E. Kulonen and J. Pikkaraein) pp. 397-410, Academic Press, New Y o r k (1973). J. HALME, J. U l T T O , K. KAHANPP, P. KARHUNEN and S. L I N D Y , J. Lab. C l i n . Med. 75, 535-541 (1970). U. LANGNESS and S, UDENFRIEND, Proc. Nat'l. Acad. Sci. U . S . A . 71, 50-51 (1974). R . L . CHURCH, M . L . TANZER and S.E. PFEIFFER, Proc. Nat'l. Acad. Sci. U . S . A . 70, 1943-1946 (1973). R . J . RICHARDS, F.S. WUSTEMAN and K . S . DODGSON, Life Sci. 10, 1149-1159 (1971). R.J. RICHARDS and F.S. WUSTEMAN, Life Sci. 14, 355-364 (1974). A . G . HEPPLESTON and J . A . STYLES, N a t u r e (London) 214, 521-522 (1967). R. BURRELL and M. ANDERSON, E n v i r o n . Res, 6, 389-394 (1973). J.S. H A R I N G T O N , M. R I T C H I S , P.C. KING and K. MILLER, J. Pathol. 109, 21-37 (1973). P. BORNSTEIN, B i o c h e m i s t r y 6, 3082-3093 (1967). P.M. GALLOP, O.O. BLUMENF-ELD and S. SEIFTER, A n n . Rev. Biochem. 41, 617-672 (1972). B. FARIS, R. SNIDER, A. LEVlNE, R. MOSCARITOLO, L. SALCEDO and C. F R A N Z B L A U , In V i t r o 14, 1022-1027 (1978). R . L . MARGOLIS and L . N . LUKENS, A r c h . Biochem. B i o p h y s . 147, 612-618 (1971). M . J . BARNES and E. KODICEK, Vitam. Horm. 30, 1-43 (1972). K . H . B R A D L E Y , S . D . McCONNELL and R.G. C R Y S T A L , J. Biol. Chem. 249, 2674-2683 (1974). K . M . REISER and J . A . L A S T , T o x i c o l o g y 13, 51-72 (1979). P.M. GALLOP and M . A . PAZ, A d v . Exp. Med. Biol. 53, 329-338 (1975). M . A . PAZ and P.M. GALLOP, In V i t r o 11, 302-312 (1975). K . I . K I V l R I K K O and D.J. PROCKOP, A r c h . Biochem. B i o p h y s . 118, 611-618 (1967). J . J . H U T T O N , A . L . TAPPEL and S. UDENFRIEND, A r c h . Biochem. B i o p h y s . 118, 231-240 (1967). K . I . K I V I R I K K O , O. L A I T I N E N and D.J. PROCKOP, Anal. Biochem. 19, 249-255 (1967). L . D . NOURSE, P.N. NOURSE, H. BATES and H.M. SCHWARTZ, E n v i r o n . Res. 9, 115-127 (1975). J . S . H A R I N G T O N , E n v i r o n . Health Perspectives 9, 271-279 (1974). A . C . A L L I S O N , in B i o c h e m i s t r y of Silicon and ReTated Problems, Nobel Symposium, Vol. 40, (Eds. G Bendz and I. L i n d q u i s t ) pp. 337-356, Plenum Press, New Y o r k / L o n d o n (1977). A . G , HEPPLESTON, in B i o c h e m i s t r y of Silicon and Related Problems, Nobel Synposium, Vol. 40, (Eds. G. Bendz and I. L i n d q u i s t ) pp. 357-359, Plenum Press, New Y o r k / L o n d o n (1977). M . B . SPORN and E.D. HARRIS, Am. J. Med. 70, 1231-1236 (1981).
Pergamon Pres
TRIPEPTIDE ANALYSIS OF COLLAGEN SYNTHESIZED BY SILICA-EXPOSED FIBROBLAST CULTURES William H. P a i l e s 1 a n d H a r o l d R e s n i c k 1 ' 2 1Biochemistry Section, Appalachian Laboratory for Occupational Safety and Health Morgantown, ,Vest Virginia 26505, U . S . A . and 2Department of Biochemistry, West Virginia U n i v e r s i t y , Medical Center, Morgantown, West Virginia 26506, U.S.A. (Received in final form February 23, 1983) Summary
The effect of silica on collagen biosynthesis by confluent monolayers of Wl-38 fibroblast cultures was examined by a more comprehensive method of analysis. The presence of the particulates had no direct effect on protein (collagen) synthesis, proline incorpora'don or prolyl hydroxylase a c t i v i t y ; the latter as determined by the degree of hydroxylation. Silica however, was highly toxic to the cells. Silica-induced fibrogenesis has been extensively studied in animal tissues and cell cultures (1,2, 3, 4, 5). For the most part the studies have focused on collagen biosynthesis with hydroxyproline and prolyl hydroxylase (E.C. 1.14.11.2) a c t i v i t y as markers of fibrogenic a c t i v i t y . The presence of hydroxyproline is due to the unique post-translational enzymic hydroxylation of specific proline residues that have already been incorporated into the collagen polypeptide c~-chains (6). The level of a c t i v i t y of prolyl hydroxylase is known to be high in tissues actively synthesizing collagen. However, enzyme a c t i v i t y does not necessarily correlate with rates of collagen synthesis (7). For example, in lung tissue from silicotic rats, prolyl hydroxylase activity increased approximately 250% and the increase preceded any demonstration of fibrosis based on histological staining or h y d r o x y proline analyses (8). It should also be noted that prolyl hydroxylase a c t i v i t y has been demonstrated in many nonfibroblastic cells, all of which presumably may have the capability of synthesizing collagen (9,10). In cell cultures, hydroxyproline formation has been used as an index of silica -induced fibrosis. The studies indicate that exposure to silica dust alone does not stimulate fibroblasts to synthesize collagen (11,12). However, extracts from silica-exposed macrophages when added to fibroblast cultures have been shown to induce fibrogenesis (13, 14) although there have been some conflicting results reported (12, 15). It becomes apparent however from these studies that h y d r o x y lation, i . e . , hydroxyproline formation, becomes limiting when investigating collagen synthesis. While hydroxylation of proline residues in polypeptide c~chains is not random it is not absolute (16, 17). Furthermore, collagen concentrations based on hydroxyproline assays do not probe the insoluble collagenous material when the protein is extracted from cell cultures (18). Also the extent to which collagen synthesis and subsequent secretion is dependent on hydroxylation is not well understood (19,20,21). In a review by Reiser and Last (22) many of the discrepancies noted in the literature are discussed as well as the difficulties 0024-3205/83/202379-06503.00/0
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in making comparisons on presumably similar types of studies. The purpose of the p r e s e n t s t u d y was to examine the f i b r o g e n i c a c t i v i t y of a f i b r o b l a s t c u l t u r e g r o w n in d i r e c t contact w i t h silica particles by t r i p e p t i d e analyses. The method developed by Paz and Gallop (23,24) examines p r o l y l h y d r o x y lase a c t i v i t y both in terms of u n d e r h y d r o x y l a t e d and n o r m a l l y - h y d r o x y l a t e d collagenous p r o t e i n s y n t h e s i s , as well as p r o l i n e i n c o r p o r a t i o n and h y d r o x y p r o l i n e formation. T h e method makes use of the r e p e t i t i v e amino acid sequence ( G I y - X - Y ) _ , f o u n d in all collagen p o l y p e p t i d e c~-chains, where X and Y are v a r i able aminor~cids (17). S p e c i f i c a l l y , analyses are made for the t r i p e p t i d e s , G l y - P r o - H y p and G l y - P r o - P r o , both obtained by collagenase ( B i o f a c t u r e s , I n c . ) digestion of insoluble and soluble collagen f r a c t i o n s ; the digests are q u a n t i t a t e d by i o n - e x c h a n g e c h r o m a t o g r a p h y . Consideration is also made for the fact that the t r i p e p t i d e , G l y - P r o - A l a , f o u n d in r e l a t i v e l y h y g h c o n c e n t r a t i o n s a f t e r colla genase d i g e s t i o n , is c h r o m a t o g r a p h i c a l l y eluted w i t h G l y - P r o - P r o . A normally - h y d r o x y l a t e d collagen would t h e r e f o r e have a high level of the t r i p e p t i d e G l y - P r o - H y p ; u n d e r h y d r o x y l a t e d collagen would have a h i g h e r c o n c e n t r a t i o n of G l y - P r o - P r o ; the c a l c u l a t e d amount of collagenous p r o t e i n formed is corrected for the degree of u n d e r h y d r o x y l a t i o n (24). As a c o n t r o l , h y d r o x y l a t i o n is made l i m i t i n g by a d d i t i o n or deletion of a necessary cofactor, i . e . , L-ascorbic acid, a r e d u c t a n t for p r o l y l h y d r o x y l a s e a c t i v i t y (25,26), w i t h o u t i n t e r f e r r i n g w i t h p r o tein s y n t h e s i s . For more details, see Ref. 24, pp. 304-305. Methods The human embryonic lung f i b r o b l a s t s t r a i n WI-38 (American T y p e ~ : u l t u r e Collection) was used in t h i s s t u d y . The cells were g r o w n in T-150 c m ~ p l a s t i c c u l t u r e flasks ( C o m i n g ) c o n t a i n i n g 30 ml BME (basal medium Eagle, Earle's Salts; Gibco) supplemented w i t h 10% fetal calf serum and a n t i b i o t i c s (24). Cell c u l t u r e s were g r o w n at 37°C in an atmosphere of 5% CO_ and 95% a i r and s p l i t at a 1:4 ratio at weekly i n t e r v a l s u n t i l s u f f i c i e n t flasksZfor the t h r e e g r o u p s (10 f l a s k s / g r o u p ) of c o n f l u e n t c u l t u r e s were obtained. The s u f f i c i e n t number of flasks c o n t a i n i n g WI-38 cells were at the 26th passage level when held for one week at c o n f l u e n c y . One g r o u p received complete BME medium w i t h 50 ~ g l m l of L - a s c o r bic acid, a n o t h e r g r o u p received complete i3ME w i t h 50 ~ g l m l of L-ascorbic acid and 100 ~ g l m l of silica particles (Duke S t a n d a r d s ; 0 . 2 - 0 . 5 ~ ) ; c o n c e n t r a t i o n of silica selected based on p r e l i m i n a r y studies w i t h consideration of cell v i a b i l i t y o v e r time-frame of e x p e r i m e n t , as well as, c o n c e n t r a t i o n s used by o t h e r i n v e s t i gators (11,12). The t h i r d g r o u p received complete i3ME medium w i t h o u t L - a s c o r bic acid or silica p a r t i c l e s . D u r i n g the one week at c o n f l u e n c y for all g r o u p s , the respective media were changed and collected on a l t e r n a t e days. T w e n t y - f o u r hours before h a r v e s t i n g the medium was collected and the cells refed w i t h th~jir r e s p e c t i v e supplement for each g r o u p and " p u l s e d " w i t h 1.0 1=Ci/ml of 2 , 3 - L - [ H ] - p r o l i n e (New England N u c l e a r ) . A f t e r 24 h o u r s of labelling the cells and media were separated, pooled w i t h t h e i r respective g r o u p s and analyses were made. Cell v i a b i l i t y counts were performed on separate flasks c o n t a i n i n g c o n f l u e n t cell monolayers by the p r o c e d u r e of t r y p a n blue e x c l u s i o n . Procedures used for total p r o t e i n and DNA, performed when cells reached c o n f l u e n c y and a f t e r one week at c o n f l u e n c y , are the same as those reported by o t h e r i n v e s t i g a t o r s (24). Collagen c o n c e n t r a t i o n s were based on h y d r o x y p r o l i n e determination (27) of collagenase-digested material ( b o t h soluble and insoluble) a f t e r h y d r o l y s i s w i t h 6 N HCI @ 110°C for 24 h o u r s ; c o n c e n t r a t i o n s were corrected for degree of u n d e r h y d r o x y l a t i o n (24). Separations were obtained u s i n g an amino acid a n a l y z e r (Beckman 120C) e q u i p ped w i t h a physiological ion exchange column [ 0 . 9 x 56 cm; PA-28 resin,
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Silica and Collagen Synthesis
2381
Beckman) and the sodium c i t r a t e g r a d i e n t b u f f e r system s t a r t i n g w i t h 0.2 N sodium c i t r a t e at pH 3.18. T h e column was c a l i b r a t e d w i t h reference tripeptide--s and an amino acid m i x t u r e so that peak (A) ( G l y - P r o - H y p ) eluted s l i g h t l y ahead of methionine while peak (B) ( G l y - P r o - P r o and G l y - P r o - A l a ) eluted between leucine and t y r o s i n e . Lypholozed aliquots dissolved in 0.5 ml of 0.2 N sodium c i t r a t e b u f f e r , pH 2.2, were applied to the column from 4 to 18 mg f o ~ c e l l - r e l a t e d d i gests and 15-45 mg for digests from medium. The f r a c t i o n s collected were 2.5 ml at a flow rate of 50 m l l h r and counted in a l i q u i d s c i n t i l l a t i o n c o u n t e r as DPM ( d i s i n t e g r a t i o n s per m i n u t e ) . Results
F i g u r e I shows the elution profiles of labelled t r i p e p t i d e s a f t e r collagenase digestion of collagen found in the medium and collagen associated w i t h the cells and cell l a y e r . The peaks labelled (A) are G l y - P r o - H y p while peaks labelled (13) Cells
Medium
? o
and
IQyer
cell
"7 B
K ~E
B = 4
i0
161
~o
)0
40 50 Fraction
60
70
60
90
i00
ii0
number
number
Fraction
Clll$
Medium
and
cell
layer
? A
ff
B
90 Fraction
~4
+
A$corbate
100 II0
number
Fraction
Cells a n d
Medium
17 "7
cell
number
layer
?
,0
o
e +
Ascorbate and silica
B
! =
A
1o
Fraction
number
~o
~
~
~o
Fraction
~o
~o
8o
~o
1oo
.0
number
FIG. I Elution profiles of t r i p e p t i d e s obtained from collagenase-digested, T C A - s o l u b l e collagen e x t r a c t e d from medium and c o r r e s p o n d i n g cells and and cell l a y e r .
2382
Silica and Collagen Synthesis
Vol. 32, No. 20, 1983
consist o f G l y - P r o - P r o and G l y - P r o - A l a . The position of the peaks e l u t i n g from the i o n - e x c h a n g e column was established with commercially a v a i l a b l e t r i p e p t i d e s (Bachem and Sigma Chemical). The ratio of peak (A) to peak (B) as determined from the total DPM f o r each peak was used to calculate the degree of h y d r o x y l ation (23, 24); it also reflects the degree of u n d e r h y d r o x y l a t i o n . The ratio has been d e f i n e d as the h y d r o x y l a t i o n i n d e x [ H I . It has an e x p e r i m e n t a l v a lu e o f 3.5 for n o r m a l l y - h y d r o x y l a t e d collagen produced by WI-38 cells. The h y d r o x y l ation i n d e x is used to determine the degree (percentage) of h y d r o x y l a t i o n in the collagen f i b e r (24). The decrease in a c t i v i t y , i . e . , peak heights o b s e r v e d f o r cells grown in the presence of silica reflects the c y t o t o x i c i t y of the p a r t i c l e s . T_he cell counts f or each g r o u p at s t a r t of c o n f l u e n c y was a p p r o x i m a t e l y 180 x I0 b cells. A f t e r one week, the g r o u p maintained in the presence of silica, showed a decrease in cell count by a p p r o x i m a t e l y o n e - t h i r d . The decrease in cells is also indicated by a decrease in total p r o t e i n and DNA as shown in Table I. TABLE I Summary of Data Obtained for Collagen Associated with Cells and Cell L a y e r Ascorbic Acid
-
+
+
Silica
-
-
+
Total Protein (mg)
105
115
71
Total DNA (mg)
2.4
2.9
1.8
Total Collagen (rag)
0.4
1.4
0.3
mg C o l l a g e n l m g DNA
0.2
0.5
0.2
2
9
3
62
98
93
Collagen (cells) Total Collagen
(%)
Degree H y d r o x y l a t i o n (%)
T A B L E II Summary of Data Obtained for Collagen Found in Medium Ascorbic Acid
+
Silica
-
Total Collagen (mg)
-
16.6
14.6
10.6
mg Collagen/mg DNA
6.9
5.0
5.9
Collagen (medium) Total Collagen
98
91
97
52
100
97
(%)
Degree H y d r o x y l a t i o n
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Silica and Collagen Synthesis
2383
T h e data obtained for the three g r o u p s mentioned above are summarized in Tables I and I I . A l t h o u g h absolute q u a n t i t i e s do v a r y , the t r e n d in the data f o r cells g r o w n in the absence and presence of ascorbate ( w i t h o u t silica) are similar to those reported by Paz and Gallop (23,24). More total collagen w a s p r o duced by cells in the absence of the vitamin but the p r o t e i n found in the medium was only 52% h y d r o x y l a t e d (Table II) while collagen associated w i t h the cells was 62% h y d r o x y l a t e d (Table I ) . T h e r e was more collagen associated w i t h the cells g r o w n in the presence of ascorbate than those g r o w n in absence of the reductant~ F u r t h e r m o r e , collagen for both cells and medium was n o r m a l l y - h y d r o x y l a t e d (Tables I and I I ) . An examination of the data obtained for cells grown in the presence of silica p a r ticles plus ascorbate indicates that the particles had no effect on p r o l y l h y d r o x y l a s e a c t i v i t y ; the degree of h y d r o x y l a t i o n was 93% and 97% for cells and medium r e s p e c t i v e l y . F u r t h e r m o r e , it is q u i t e apparent that the p a r t i c u l a t e s had no effect on protein (collagen) s y n t h e s i s . The decrease in total collagen - as mentioned above - was due to less than t w o - t h i r d s of viable cells remaining a f t e r one week at c o n f l u e n c y in the presence of silica. The r e l a t i v e l y high ratio of collagen to DNA found in the medium (Table II) reflects partial l y s i s of cells (in presence of silica) r e s u l t i n g in solubilization (in medium) of collagen normally associated w i t h cell l a y e r ; c o n v e r s e l y a decrease in the ratio (mg Collagen/mg D N A ) is seen in cells and cell layer (Table I ) . Discussion T h e r e has been considerable use of cell c u l t u r e s in biochemical studies related to pulmonary f i b r o s i s . Studies on the effects of o c c u p a t i o n a l l y - r e l a t e d d u s t s on the p r o l i f e r a t i o n of collagen f i b e r s by f i b r o b l a s t cells however, have been conflicting~ The discrepancies have been a t t r i b u t e d to (i) phase of cell g r o w t h (logarithmic v e r s u s s t a t i o n a r y ) (12,28), (ii) cell species (29), and ( i i i ) concentration and physical state of the p a r t i c u l a t e (12). A n o t h e r consideration has been macrophage involvement in f i b r o g e n e s i s ; the effects of e x t r a c t s from silica-exposed macrophages on f i b r o b l a s t c u l t u r e s (12,28,30,31). A u n i f y i n g concept has also been proposed. The concept relates to excessive p r o l i f e r a t i o n of cells and t u r n o v e r of c e l l u l a r m a t r i x as c o n t r i b u t i n g s i g n i f i c a n t l y to the pathogenesis of several diseases i n c l u d i n g pulmonary f i b r o s i s (32). The present r e p o r t does not specifically address i t s e l f to questions that may have been raised w i t h regard to earlier studies. Instead, it focuses on a method of analysis that is s u f f i c i e n t l y comprehensive and o f f e r s a more s a t i s f a c t o r y basis for e v a l u a t i n g the effects of p a r t i c u l a t e s on collagen s y n t h e s i s . The purpose of t h i s s t u d y was to evaluate and possibly refine an in v i t r o t e c h n i q u e s u f f i c i e n t l y to be able to forecast the p r o b a b i l i t y of a p a r t i c u l a t e involvement in collagen syl~lthesis and an eventual u n d e r s t a n d i n g of pulmonary f i b r o s i s . The method of t r i peptide analysis goes beyond analysis for h y d r o x y p r o l i n e formation or the i n c o r poration of radioactive p r o l i n e and the subsequent h y d r o x y l a t i o n of t h i s residue; it i n v o l v e s o t h e r phases of collagen s y n t h e s i s . The results of the present s t u d y however, confirms p r e v i o u s studies w i t h cell c u l t u r e s that would seem to indicate that silica has no d i r e c t effect on the s y n t h e s i s of f i b r o u s protein by f i b r o b l a s t cells. References
I. 2. 3.
M. C H V A P I L and E. HOLECKOVA, B r . J. I n d . Med. 16, 286-289 (1959). A . C . ALLISON, J.S. HARINGTON and N. B I R B E C K , J. Exp. Med. 124, 141-153 (1966). M. C H V A P I L , J. HURYCH, B. CMUCHALOVA, R. HOLUSA and E. EHRLICHOVA, E n v i r o n . Res. I, 89-101 (1967).
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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
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