Liver collagen hydroxylation in murine schistosomiasis

328

Biochimica et Biophysica Acta, 538 (1978) 328--333

© Elsevier/North-Holland Biomedical Press

BBA

28390

L I V E R COLLAGEN H Y D R O X Y L A T I O N IN MURINE SCHISTOSOMIASIS

MICHAEL A. DUNN, MICHAEL E. MARAGOUDAKIS and PENELOPE K. HAIT Liver Research Center, Albert Einstein College o f Medicine, Bronx, N.Y. 10461 (U.S.A.)

(Received May 27th, 1977)

Summary The activity of procollagen prolyl hydroxylase was measured in fibrotic liver obtained from mice with hepatosplenic schistosomiasis, an animal model of the most prevalent form of human liver fibrosis. Measurable activity of prolyl hydroxylase in fibrotic liver supernatants was 47-fold higher than that of normal liver. The effect of prolyl hydroxylase inhibition on collagen synthesis in fibrotic liver slices was studied, using 8,9-dihydroxy-7-methyl benzo[b]quinolizinium bromide (GPA 1734). This c o m p o u n d was shown in other systems to inhibit prolyl and lysyl hydroxylations by iron chelation at concentrations which did not affect total protein synthesis. The formation of nondialyzable labelled hydroxyproline was inhibited by GPA 1734, 40, 70 and 95% at 30, 50 and 100 ffM, respectively. Incorporation of proline into total liver protein was unaffected at 30 and 50 pM, b u t was inhibited 20% at 100 pM GPA 1734. Underhydroxylated collagen synthesized by liver slices with GPA 1734 was extracted with neutral salt solution and was subsequently hydroxylated with partiallypurified prolyl hydroxylase to the same extent as control material synthesized in the absence of GPA 1734.

Introduction In hepatic fibrosis, excessive deposition of liver collagen leads to permanent distortion of liver architecture and circulation [ 1 ]. Mice infected with S c h i s t o s o m a m a n s o n i cercariae provide an experimental model of liver collagen deposition [2--4] which resembles human hepatosplenic schistosomiasis, the most prevalent form of liver fibrosis in man [5]. We have previously described increased collagen synthesis by fibrotic liver slices obtained from mice with experimental schistosomiasis [ 6 ]. Liver fibrosis in animals and man has been associated with increased activity of procollagen prolyl hydroxylase [7--14]. Hydroxylation of prolyl residues

329 is considered to be important for helical stability, secretion and deposition of collagen [15--17]. In the present study, we report an approximately 47-fold increase in the activity of procollagen prolyl hydroxylase in the livers of mice with hepatosplenic schistosomiasis. We also report studies with a newly-described inhibitor of prolyl hydroxylase, 8,9 dihydroxy-7-methyl benzo [b ] quinolizinium bromide (GPA 1734), an iron-chelating c o m p o u n d [18--20]. Methods

Materials GPA 1734 was prepared and purified as described by Watthey et al. [21]. Basal Medium Eagle was obtained from Grand Island Biological Co. Other reagents and isotopes were obtained as described previously [6,20]. Female CF1 mice, 20--25 g, from Carworth Farms, Inc., were injected subcutaneously [22] with a strain of Puerto Rican S. mansoni in the laboratory of Dr. K.S. Warren, Case-Western Reserve University, Cleveland, Ohio. Underhydroxylated 3Hlabelled procollagen was prepared from rat parietal yolk sacs by a modification [20] of the m e t h o d described by Jiminez et al. [23]. Procollagen prolyl hydroxylase from newborn rat skin was purified through the DEAE-Sephadex stage as described by Rhoads and Udenfriend [24]. Procollagen prolyl hydroxylase assay Fibrotic livers were obtained from five mice 9 weeks after infection with 50 S. mansoni cercariae. A portion, 500 mg wet weight, of each liver was homogenized in 5 ml 0.25 M sucrose containing 0.1 mM dithiothreitol and 0.1 mM EDTA, using an ice-jacketed glass homogenizer, for 6 s. Homogenates were centrifuged at 16 000 × g for 30 min at 4°C. Aliquots of 5 and 10 ~l of each supernatant were assayed for procollagen prolyl hydroxylase activity as described by Rhoads and Udenfriend [24]. The reaction mixture contained 5.5 • 10 s dpm [3,4-3H]prolyl procollagen substrate in 1 ml. Protein nitrogen content of each supernatant was determined by a Kjeldahl m e t h o d [25]. Normal livers from six uninfected mice were also assayed as described. Incubation o f liver slices Fibrotic liver slices were incubated with labelled proline as described previously [6] except that Basal Medium Eagle was used rather than Earle's Balanced Salt Solution, and no additional unlabelled proline was added to the medium. The modification permitted use of lower levels of radioactivity than had been used in the earlier studies, in which measurements of labelled collagen production were made at or near the saturating concentration of 0.4 mM proline required for maximal collagen synthesis b y the fibrotic liver slices [6]. Duplicate 200 mg portions of liver slices from the same mouse were incubated with 50 pCi L-[3H]proline, randomly labelled, and GPA 1734, at concentrations of 30, 50 or 100 pM. GPA 1734 was added as a solution in 40% {v/v) aqueous dimethylsulfoxide. Control flasks contained the same a m o u n t of solvent without GPA 1734. No differences in proline incorporation into total protein, or production of labelled hydroxyproline, were noted in control

330 incubations with or without comparable amounts of added dimethylsulfoxide. After 6 h of incubation, the reaction was stopped by addition of 5 ml of cold ethanol. Nondialyzable labelled proline and hydroxyproline were determined as described previously [6].

Hydroxylation of labelled collagen Labelled collagen was synthesized by fibrotic liver slices incubated with L[U-14C]proline, 20 gCi per 200 mg liver, in the presence of 1.0 mM ~-aminopropionitrile, as described previously [6]. The neutral salt extractable material from 6 such incubations with 50 gM GPA 1734 was pooled, as was the material from six control incubations without the inhibitor. Both samples were dialyzed at 4°C, initially against 0.1 N acetic acid with 10 mM N-ethylmaleimide/20 mM EDTA/20 mM mercaptoethanol, and subsequently against 6 changes of 0.1 N acetic acid. The dialyzed samples were evaporated to dryness, taken up in 5 ml of the reaction mixture for prolyl hydroxylase as described by Rhoads and Udenfriend [24] and divided into two equal portions. Half of each sample was then incubated for 18 h at 25°C with partially-purified procollagen prolyl hydroxylase, corresponding to 260 gg protein. The remaining half was incubated without enzyme under the same conditions. Incubation was terminated by addition of HC1 to a final concentration of 6 N and the samples were hydrolyzed at l l 0 ° C for 18 h. Labelled proline and hydroxyproline were then determined chromatographically as described previously [6]. Results

The activity of procollagen prolyl hydroxylase in fibrotic and normal liver supernatants is shown in Table I. The fibrotic livers contained approximately 47-fold higher measurable enzyme activity than the normal livers under the assay conditions used. The effect of GPA 1734 on protein-bound hydroxyproline formation by fibrotic liver slices in a representative experiment is shown in Fig. 1. Formation of labelled hydroxyproline was inhibited by GPA 1734, 40, 70 and 95% at

TABLE I PROCOLLAGEN PROLYL LIVER SUPERNATANTS

HYDROXYLASE

ACTIVITY

IN

FIBROTIC

AND

NORMAL

MOUSE

E n z y m e a c t i v i t y w a s m e a s u r e d in liver s u p e r n a t a n t s i n c u b a t e d w i t h [ 3 , 4 - 3 H ] p r o l y l p r o c o l l a g e n s u b s t r a t e as d e s c r i b e d under M e t h o d s . Prolyl h y d r o x y l a s c activity

F i b r o t i c (5 m i c e ) N o r m a l (6 m i c e ) • -+ 1 S . D .

dpm X 10-5/mg liver s u p e r n a t a n t protein

dpm × lO-6/liver

2.22 _+ 0 . 3 5 * 0 . 0 8 -+ 0 . 0 5

3 9 . 1 2 _+ 6 . 6 5 0 . 8 3 _+ 0 . 5 6

331 8

6

•

[3i ~ 4-

•

6 × •

2:2

'

'

3'0

'

~o

. . . .

40

GPA 1734 (pM) Fig. 1. I n h i b i t i o n o f f o r m a t i o n o f p r o t e i n - b o u n d [ 3 H I h y d r o x y p r o l i n e b y G P A 1 7 3 4 . F i b r o t i c liver s l i c e s f r o m t h e s a m e a n h n a l w e r e i n c u b a t e d in d u p l i c a t e w i t h 50 pCi L-[3 H ] p r o l i n e p e r 2 0 0 m g liver a n d O, 30, 50 a n d 1 0 0 p M G P A 1 7 3 4 , as d e s c r i b e d u n d e r M e t h o d s .

30, 50 and 100 gM, respectively. The incorporation of labelled proline into total protein {data not shown) was 100-fold greater than the formation of labelled hydroxyproline, as described previously [6]. In these experiments, incorporation of labelled proline into total protein was unaffected by 30 and 50 ~M GPA 1734, but was decreased by 20% at 100 ~M concentration of the compound. The effect of 50 pM GPA 1734 on the system was abolished by addition of an equimolar concentration of FeSO4 to incubation flasks. When tested at 50 pM concentration in this system, other iron-chelating compounds, EDTA, deferoxamine and a,a'-dipyridyl were not effective inhibitors of labelled hydroxyT A B L E II H Y D R O X Y L A T I O N BY P R O L Y L H Y D R O X Y L A S E O F P R O T E I N L I V E R BY E X T R A C T I O N W I T H N E U T R A L S A L T S O L U T I O N

OBTAINED FROM

FIBROTIC

L i v e r slices w e r e i n c u b a t e d w i t h o r w i t h o u t 50 pM G P A 1 7 3 4 in t h e p r e s e n c e o f 1.0 m M f l - a m i n o p r o p i o . nitrfle. Neutral-salt soluble m a t e r i a l w a s s u b s e q u e n t l y d i a l y z e d a n d i n c u b a t e d w i t h p a r t i a l l y - p u r i f i e d pro. collagen prolyl hydroxylase. F i b r o t i c liver slices incubated with [ 14C]prolin e

Control

G P A 1 7 3 4 , 50 DM

Subsequent incubation with:

No e n z y m e Prolyl h y d r o x y l a s e No e n z y m e Prolyl h y d r o x y l a s e

N o n d i a l y z a b l e 14 C r a d i o a c t i v i t y ( d p m X 10 - S ) Hydroxyproline

Proline

1.7 1.2 0.2 1.0

14.1 14.6 14.9 15.0

332 proline formation. L-DOPA, a catechol which inhibits hydroxylation of basement membrane collagen [18] was ineffective at 50 gM in this system. Extraction of labelled protein with neutral salt solution is shown in Table II, for liver slices incubated with and without 50 gM GPA 1734. Approximately 30% of the [14C]hydroxyproline was extracted by this procedure. Formation of neutral salt extractable protein-bound hydroxyproline was strongly inhibited, but incorporation of labelled proline into total extractable protein was unaffected by the compound. After dialysis, the extracted material was incubated with procollagen prolyl hydroxylase. Additional protein-bound hydroxyproline was formed only when the GPA 1734-treated material was used as substrate; the yield of hydroxyproline was comparable to that found with the control extract. Discussion

In murine schistosomiasis an immunologic host response results in a 20-fold increase in liver collagen; fibrotic liver slices obtained 9 weeks after infection synthesize approximately 16-fold more collagen than normal [6]. Type I collagen obtained from this system by extraction with neutral salt solution is fully h y d r o x y l a t e d [6]. The present study shows an approximately 47-fold increase in activity of liver procollagen prolyl hydroxylase in murine schistosomiasis. That result is consistent with the increased activity of hydroxylase reported in fibrotic livers of animals after administration of liver cell toxins [7--9, 11--13], and in livers of persons with fibrosis [10,14]. Increased hydroxylating activity preceded the onset of liver collagen accumulation in the earlier animal studies [7--9,11--13] b u t there is no direct evidence that increased activity of this enzyme stimulates collagen synthesis per se. The data reported by Risteli et al. [13] in rats treated with dimethylnitrosamine were consistent with the explanation that the increased enzyme activity was due to activation of pre-existing enzyme protein. GPA 1734 is a p o t e n t inhibitor of the hydroxylation of interstitial and basement membrane collagen in several systems [18,20]. Its mechanism of action is probably related to its high affinity for ferrous iron, an essential cofactor for prolyl hydroxylase. In the present study, formation of labelled protein-bound hydroxyproline was inhibited more strongly by 50 gM GPA 1734 than by equimolar concentrations of other c o m p o u n d s tested. On the other hand, 50 pM GPA 1734 did not affect the incorporation of proline into total liver protein, similar to observations in other systems [18,20]. The major effect of GPA 1734 on the formation of labelled collagen in this system was inhibition of hydroxylation: evidence for an inhibitory effect at this step was provided by extraction of underhydroxylated protein from GPA 1734-treated incubation mixtures and subsequent hydroxylation with prolyl hydroxylase. These studies show increased activity of procollagen prolyl hydroxylase in murine schistosomiasis. The possibilities that this enzyme may be rate-controlling in collagen deposition, and that its inhibition may be therapeutically useful in liver fibrosis, warrant further study.

333

Acknowledgments We are grateful to Dr. J.W.H. Watthey of CIBA-GEIGY Corp., Ardsley, N.Y., for preparation of GPA 1734 and to Dr. K.S. Warren for infected mice. We acknowledge with appreciation the encouragement of Drs. I.M. Arias, J. Betheil, M. Rojkind, and S. Seifter. This work was supported by the Edna McConnell Clark Foundation, by a U.S. Public Health Service Postdoctoral Research Fellowship to M.A.D. (F22 AI 01373) and by National Institutes of Health Grant AM 17702. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

21 22 23 24 25

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