Localization of collagen prolyl hydroxylase to the hepatocyte

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Experimental

LOCALIZATION

Cell Research 123 (1979) 269-279

OF COLLAGEN

PROLYL

HYDROXYLASE

TO THE HEPATOCYTE Studies

in Primary

Monolayer

Cultures

Cells from Adult PHILIP S. GUZELIAN’ Division

of Parenchymal

Rat Liver

and ROBERT F. DIEGELMANN

of Plastic Surgery and ‘The Liver Study Unit, Departments of Surgery and Medicine, Medical College of Virginia, Richmond, VA 23298, USA

SUMMARY Significant levels of prolyl hydroxylase activity (prolyl-glycyl-peptide, 2-oxoglutarate : oxygen oxidoreductase; EC 1.14.11.2) have been found in freshly isolated hepatocvtes oreoared from norma1or regenerated adult rat liver and primary non-proliferating monolayer culiures of these cells. Four days after partial hepatectomy, the intact regenerated liver contained two times the normal level of prolyl hydroxylase activity. Freshly isolated hepatocytes contained 24% of the total prolyl hydroxylase activity in normal liver and 47 % of that in regenerated liver. Upon incubation of hepatocytes for 24 h in a chemically defined culture medium containing insulin, prolyl hydroxylase activity rose 2- to 3-fold, and gradually declined during the next 48 h. The rise in prolyl hydroxylase activity was blocked by addition of cycloheximide to the culture medium. The presence of prolyl hydroxylase activity in hepatocyte cultures was not likely due to contamination with non-parenchymal liver cells. The latter cells contained less than 20% of the total enzyme activity recovered in all cells isolated from the liver. Furthermore. nrolvl hvdroxvlase was localized by immunofluorescence uniformly to the hepatocytes in culture’. Cultured hepatocytes converted [‘4Cjproline to [‘4C]hydroxyproline at rates comparable to those reported for whole liver. However, only a small portion of the hydroxyproline containing product was present as collagen protein, suggesting its rapid degradation in culture. We conclude that the liver parenchymal cell may actively participate in collagen synthesis and possibly in collagen degradation.

Hepatic fibrosis is a common sequela of diverse forms of liver injury and is characterized by abnormal deposition of collagen. The pathogenesis of hepatic fibrosis is believed to involve accumulation of newly formed collagen fibers [l] either by stimulation of pre-existing fibroblasts [l] or by transformation of the perisinusoidal cells (of Ito) into fibroblasts [2, 31. However, the number of fibroblasts and the amount of fibrosis in the liver often correlate poorly, and therefore, it has been suggested that collagen may arise from cells other than 18-791813

fibroblasts, possibly the hepatic parenchymal cell [4]. Consistent with the concept of multicellular fibrogenesis are recent findings that fibrotic liver contains several forms of collagen and collagen-like proteins

[5, 81. Investigation of the cellular origin of hepatic collagen and the possible role of the hepatocyte in this process has been impeded by lack of suitable techniques. Electron microscopic examinations of normal liver and of progressive parenchymal fibrosis have shown that hepatocytes are ExpCellRes 123 (1979)

270

Guzelian and Diegelmann

contiguous with reticular fibers in the space of Disse and at times appear to envelop these collagenous structures [9, lo]. Moreover, deposition of collagen or collagen-like material in the sinusoids proximate to the hepatocytes is characteristic of many forms of advanced cirrhosis [ 11. Unfortunately, morphologic techniques do not distinguish between collagen synthesis and degradation and because of possible artifacts due to sectioning of tissue, interpretation of results is complicated. Cell cultures provide an alternative approach and several investigators have demonstrated synthesis of collagen in ‘epithelial’ cell lines derived from liver [ 11, 121.However, culture systems of proliferatit’& cells

have

the disadvantage

Ihat

bio-

chemical studies are carried out in cells which

are

may

generations

descended

from the intact liver. Consequently, the identity of the progenitor of these cultures is uncertain. Moreover, expression of genes often is altered in continuously replicating cells and this leaves open to question the relevance of findings in such culture systerns to the liver in vivo. To circumvent some of the difficulties encountered with traditional cell culture techniques, we have examined collagen synthesis in primary monolayer cultures of parenchymal cells from adult rat liver [13]. One advantage of this system is that the starting material for cultures are preparations of freshly isolated hepatocytes free of blood cells and other non-parenchymal cells of the liver. Furthermore, the cells are viable for many days in a non-proliferating state and exhibit an impressive array of differentiated functions characteristic of the liver in vivo [13-161. Finally, because the hepatocytes are only hours or days removed from the parent liver, proximate comparisons of cultures with the intact liver are possible. As markers for collagen synExp Cell Rrs 123 (19791

thesis, we have measured (1) the activity of prolyl hydroxylase which often parallels-the rate of collagen formation in many tissues [ 171including liver [18]; and (2) the formation of hydroxyproline. The results reveal that the hepatocyte contains a significant portion of liver prolyl hydroxylase and may function in collagen synthesis and possibly in its degradation. MATERIALS

AND METHODS

Materials Adult male Sprague-Dawley rats (200-250 g) obtained from Flow Laboratories (Dublin, Va) and housed individuallv with free access to food and water were used for all-studies. Collagenase (type l), pronase, and amino acids were purchased from Sigma Chemical Co. (st Louis, MO); Eagle’s Minimal Essential Vitamin Mixture from Gibco (Grand Island, N.Y.); ~-[3,4-~H]proline and @C]proline from New England Nuclear Corporation (Boston, Mass.). Impurities in [‘“clproline were removed by Dowex column chromatography (see Methods). Other chemicals were of the highest purity available commercially. Experimental

Procedure

Primary culture of adulr rat liver parenchymal cells in monolayer. Hepatocyte cultures were prepared by a

previously described method with minor modifications [13]. In brief, either normal or regenerated liver (4 days following a two-thirds hepatectomy) was perfused in situ with calcium-free salt solution followed by culture medium containing 0.03 % collagenase. Hepatocytes were separated cleanly from non-parenchymal cells of the liver by repeated centrifugation and resuspension in culture medium. The hepatocytes were placed in 60 mm plastic Petri dishes precoated with rat tail collagen [16] in a final volume of 3.0 ml of chemically defined culture medium. Inoculation of plates with 3.0-3.5 million hepatocytes leads to formation of a confluent monolayer firmly adherent to the bottom of the dish after 4-8 h of incubation at 35°C in humidified air with 5 % CO*. Approx. 85 % of the seeded cells remained attached to the culture dish after 24 h of incubation as judged by recovery of DNA. Viability of the attached cells is greater than 95% based on exclusion of Trypan blue. The control medium was changed every 24 h in these experiments and consisted of Medium 199(Microbiological Associates, Bethesda, Md) modified as follows: arginine, hydroxyproline, Fe(NO,),, and all ‘other components’ were omitted, and NaHCO, was reduced to 700 mg/l. Eagle’s Vitamin Mixture was used with added glucose (200 mg/l), insulin (4x lo6 M), and omithine (20 mg/l). Preparation of isolated non-parenchymal cells. Non-parenchymal cells were isolated from collagenase-dispersed liver by treating the supematant

Prolyl hydroxylase in cultured hepatocytes fraction of the initial sedimentation plus first wash of the parenchymal cells with 0.04% pronase [19]. The final non-parenchymal cell isolate was separated from the pronase digest by flotation on albumin. The cells were counted with a standard hemocytometer. Fluorescent-labelled antibody technique. Hepatocyte cultures were studied after the first 24 h of incubation. The medium was removed and the monolayers were washed twice with iced phosphate-buffered saline (pH 7.4) and then fixed for 5 min in 95 % ethanol. The fixed cultures were air-dried and then incubated at 37°C in a humidified chamber for 30 min with either normal goat serum or goat antiserum directed against purified rat skin prolyl hydroxylase. Both sera had been absorbed with washed rat ervthrocvtes. The immune serum (a generous gift from Drs G. Cardinale and S. Udenfriend from the Roche Institute of Molecular Biology) is highly specific for the enzyme. Extensive studies documenting specificity of the globulin for prolyl hydroxylase have been described elsewhere [20]. After removing the sera, the monolayer cultures were rinsed four times in phosphate-buffered saline and then exuosed to fluorescein-labelled rabbit antigoat globulin (Meloy Laboratories, Inc., Springfield, Va) for 30 min at 37°C. The excess labelled antiserum was removed and the cultures were washed four times with buffer, twice with water, and allowed to dry. The monolayers were examined for direct immunofluorescence using a Zeiss Photomicroscope III with FITC exciter filter and barrier filter 53. Analytical Procedures For measurement of prolyl hydroxylase, radiolabelled unhydroxylated collagen substrate was prepared by incubating L-[3,4JH]proline with embryo calvaria in the presence of 0.5 mM a&-dipyridyl [2l]. Liver enzyme was extracted in 0.5 ml of freshly prepared 0.5 M Tris buffer (pH 7.2), containing 1X 10e4 M dithiothreitol, 1x 10m5M EDTA, and 0.1% Triton X-100, from the following sources: the cellular material in five monolayer cultures; an equivalent number of freshly isolated hepatocytes; three-quarters of the total yield of non-parenchymal cells from a single liver: or 10% homogenate of intact liver perfused with iced phosphate-buffered saline (pH 7.4). Homogenates were made in an iced Teflon-glass homogenizer at 1500 rom (10 strokes (whole liver) or 16 strokes (cells)), and were centrifuged at 30000 g for 30 min. The clear portion of the supematant contained the extracted prolyl hydroxylase and 50-55 % of the total protein in cells or intact liver (7-8 mglml). In a typicai reaction, 55000 dpm of radiolabelled, heat-treated substrate was combined with 50 ul of liver extract in a total volume of 100 ~1, containing the following cofactors (final concentration): Tris-HCI, pH 7.6, 40 mM; dithiothreitol, 0.5 mM; cY-ketoglutarate, 1.0 mM; ascorbic acid, 1.0 mM; ferrous ammonium sulfate, 0.2 mM; catalase, 0.4 mglml; bovine serum albumin, 2.0 mg/ml. The incubation was carried out for 15 min at 37°C and was terminated by addition of trichloroacetic acid (TCA) [21]. The blank was formed by substituting buffer for liver extract. The tritiated water formed was separated from other soluble radiolabelled material by passage through small Dowex 50 columns directly into count-

271

ing vials for measurement by liquid scintillation spectrometry [21]. The results were expressed as dpm of tritium released per 15 minlmg of 30000 g supernatant protein. In preliminary experiments with extracts of intact liver, freshly isolated hepatocytes, or cultured hepatocytes, tritium release was directly proportional to time for 15-20 min and to the amount of added protein in the range of loo-400 pg and was inhibited by more than 98 % by addition of a,a’dipyridyl(2.0 mM) or by the omission of a-ketoglutarate. The release of tritium agreed within 5% of the expected formation of ~4 [3H]hydroxyproline when the latter was assayed directly in hydrolysed TCA precipitates of the incubated substrate using an amino acid analyser (see below, method II). In contrast to previous reports, we found no inhibitory effect of 1.0 mM a-ketoglutarate in the assay with liver enzyme [22], and no superiority of Non Idet-40 over Triton X-100 in extraction of enzyme activity [23].

Measurement of collagen synthesis in cultured hepatocytes (see table 3) I. The radiolabelled cells plus medium from culture dishes were scraped into a single tube on ice and precipitated in 5 % TCA (final concentration). The insoluble material was centrifuged and washed repeatedly with 5 % TCA to remove non-protein radioactivity. The precipitated proteins were dissolved in 0.2 M NaOH and digested with urotease-free bacterial collagenase [24]: Non-collagenous protein was reprecipitated by addition of tannic acid/TCA followed by centrifugation. Relative collagen synthesis was calculated from the radioactivity in the supematant (collagenous protein) and in the precipitate assuming collagen is 5.4 times enriched in imino acids as compared to general proteins [25]. Method II. (A) The radiolabelled material from 11 dishes was precipitated and washed with TCA as in method I. The precipitated oroteins and the first TCA supematant were separately-hydrolysed in 6 N HCI for 14 h at 15 psi, 120°C. The hydrolysate of each sample was decolorized with NORIT-A and the HCI was evaporated under vacuum. The residue was dissolved in 1.N HCI and was placed on a Dowex AG50W-X8 column (1 x30 cm) oreviouslv equilibrated with 1 N HCI according to the procedure‘of Hirs et al. [26]. Upon elution with 1 N HCI, two peaks of radioactivity were recovered which co-chromatomaohed with standard L-4-hydroxyproline (peak A) and L-proline (peak B). Althouah oeak B consisted solelv of lYlproline, peak A Contained large quantities of radioactive amino acids in addition to hydroxyproline. Peak A was combined with unlabelled L-4-hydroxyproline as carrier and the mixture was placed on a JELL 5AH amino acid analyser equipped with stream splitter and fraction collector. The L-4-[i4C]hydroxyproline was cleanly separated from all other radiolabelled amino acids and was counted in a Beckman LS-9000 liquid scintillation spectrometer. A blank formed by adding [r4C]proline after cultures had been incubated for 48 h in unlabelled culture medium showed less than 0.5 % of the L-4-[r4C]hydroxyproline Method

L

.

272

Guzelian and Diegelmann

formed in cultures incubated for 24 h in the presence of the isotope. From the values for total radioactive hydroxyproline formed and radioactive proline (peak B) incorporated in protein, relative collagen synthesis was calculated as described above (method A), assuming the ratio of hydroxyproline to proline in collagenous protein equals 1.1 [24]. (B) The radiolabelled material from 11 dishes was dialysed against 1 1of water at 4°C for 48 h with fresh water substituted at 24 h. The retentate and the combined dialysates, concentrated by lyophilization, were separately hydrolysed and analysed for radioactive hydroxyproline and proline as described for II(A). (C) The medium from 11 dishes was removed and pooled. The monolayers were rinsed once with iced phosphate-buffered saline and the cells were scraped and washed twice with buffer. The cells and medium were separately hydrolysed and measurements of radioactive hydroxyproline and proline were made as described in II(A). Since the value for cell-associated [‘4C]proline represents only 60% of the total peptide bound W.Zlnroline in the cells plus medium, the latter figures-(obtained from HA and-IIB) were used to calculate relative collagen synthesis. Protein was measured by the procedure of Schaterlee & Pollak [27] using crystalline bovine albumin as standard.

RESULTS

1000i 1

0

6, I

2

I

I

4

I

I

6

I

I

I

8

Fig. 1. Abscissa: time of incubation (hours); or&are: nrolvl hvdroxvlase act. (% of value in freshly isolated .hepatocytes from normal liver). Prolyl hydroxylase activity in rat liver following partial hepatectomy. Rats were subjected to a two-thirds hepatectomy or sham hepatectomy and, at the indicated times thereafter, were killed by decapitation. The livers were nerfused with iced phosphate-buffered saline (pH 7.4) and were excised. Prolyl hydroxylase activity was measured in extracts of homogenates of the livers as described in Materials and Methods. Normal liver prolyl hydroxylase activity was determined from the average & S.D. of 18 values from the excised lobes of the hepatectomized rats. Brackets denote the S.E.M. (or range) of each value and figures in parentheses indicate the number of animals. Each data point for ‘sham operation’ represents a single rat.

Prolyl hydroxylase activity in intact liver and in freshly isolated hepatocytes and non-parenchymal cells

Freshly isolated hepatocytes obtained from slightly lower than in sham-operated conthe ‘regenerated’ liver 4-6 days after partial trols. Thereafter, enzyme activity rose hepatectomy in rats have been recom- steadily to a peak of twice the normal at mended as the starting material for mono- 4 days after surgery, and subsequently layer cultures [13]. We have adopted this declined into the normal range (fig. 1). method as routine for previous studies of Sham hepatectomy was without signifimicrosomal function in cultured hepato- cant effect on prolyl hydroxylase activity cytes [28, 291because the reliability of suc- throughout 9 days of observation (fig. 1). cessful formation of monolayer cultures in Because maximal stimulation of prolyl serum-free medium is improved with cells hydroxylase activity and the optimal time prepared from regenerated liver. Although for preparing isolated hepatocytes for culthe levels of several microsomal enzymes in ture both occurred 4 days after partial heparegenerated liver 4-6 days after hepatec- tectomy, the operation provided a simple tomy are in the same range as those in rest- and convenient means for comparing the ing liver [29], we found in preliminary ex- intercellular distribution of the normal and periments that partial hepatectomy exerted induced enzyme in the following studies profound effects on prolyl hydroxylase ac- with isolated hepatocytes. tivity. The level of prolyl hydroxylase acProlyl hydroxylase activity was readily tivity 24 h after partial hepatectomy was detectable in freshly isolated hepatocytes Exn CdRes

123 (1979)

Prolyl hydroxylase

in cultured hepatocytes

273

of prolyl hydroxylase

in the

Table 1. Effect of partial hepatectomy on the specific activity liver and in freshly isolated hepatocytes

F’rolyl hydroxylase spec. act.

A B C D

Normal intact liver Isolated hepatocytes from normal liver Regenerated intact liver Isolated hepatocytes from regenerated liver

(dpm/mg protein)

(% of specific activity in intact liver)

2 560f370, n=5 65lk196 4 810+552, n=6 2 347k455

100 24.4k5.5 100 47.Ok5.9

Isolated hepatocytes were prepared % h after rats were subjected to a two-thirds hepatectomy (C, D), and also from normal animals (A, B). Enzyme activity was measured in the isolated cells as described in Materials and Methods, and was compared with the activity measured in one lobe of intact liver taken from the donor organ just prior to its perfusion with collagenase. Results are expressed as dpm of tritium released per mg of extracted liver protein. Data are given as mean f S.E.M.

A vsC,p
prepared from either normal or regenerated liver (table 1). The fraction of the total enzyme in the liver residing in the hepatocyte may be conveniently estimated by comparing the specific activities of prolyl hydroxylase in freshly isolated hepatocytes and in the parent liver. The validity of this Table 2. Distribution and non-parenchymal

of prolyl cells

hydroxylase

approach is based on the knowledge that hepatocytes contain 93 % of total liver protein [30]. As calculated from the data in table 1, hepatocytes contain 24% of the prolyl hydroxylase activity in normal liver and nearly twice this value in regenerated liver. The latter observation suggests that activity

between freshly

isolated hepatocytes

Prolyl hydroxylase Specific activity Total act. recovered Expt

Source of liver

Isolated cell fraction

(dpm/mg protein)

(dpm/ 105 cells)

(dpm x 10e3) %

I

Normal

II

Normal

III

Normal

IV

Regenerated

V

Regenerated

Hepatocyte non-parenchymal Hepatocyte non-parenchymal Hepatocyte non-parenchymal Hepatocyte non-parenchymal Hepatocyte non-parenchymal

1 377 1 553 1% 1 523 270 1 838 4204 1 100 4 087 4 731

89.0 3.3 12.3 5.1 32.0 4.5 259.2 5.7 186.9 8.1

98.0 5.1 13.0 2.8 48.1 8.0 285.2 3.1 300.9 3.8

95.1 4.9 82.3 17.7 85.7 14.3 99.0 1.0 98.8 1.2

In each experiment, isolated hepatocytes and non-parenchymal cells were prepared from a single liver by collagenase and pronase treatments as described in Materials and Methods. Rats for expts IV and V were subjected to a two-thirds hepatectomy 4 days prior to cell isolation. Exp CellRes 123 (1979)

274

Guzelian and Diegelmann

0

24

48

72

Fig. 2. Abscissa: time following laparotomy (days); ordinate: prolyl hydroxylase act (dpm/mg protein).

F’rolyl hydroxylase in cultured hepatocytes. Freshly isolated hepatocytes were prepared 96 h after partial hepatectomy (regenerated liver) or from normal animals, and were mcubated in control medium in the presence or absence of cycloheximide (1 x 10m5M). At the indicated times, cells from five plates were’harvested and extracts were prepared for measurement of prolyl hydroxylase as described in Materials and Methods. The results were expressed as the percent of the average value in freshly isolated hepatocytes derived from normal liver. Brackets indicate the S.E.M. (or range) with the number of experiments given in parentheses.

the rise in prolyl hydroxylase activity in the intact regenerated liver may be accounted for almost entirely by increased specific activity of the enzyme in the hepatocyte (fig. 1, table 1). It is unlikely that contamination with non-parenchymal cells accounts for the prolyl hydroxylase activity in freshly isolated hepatocytes because the latter preparations obtained from either normal or regenerated liver contained l-2% nonparenchymal cells and, thus, are purified lo-20-fold over intact liver which contains 30-40 % non-parenchymal cells [30]. Hence, the observed specific activity of prolyl Exp Cell Res 123 (1979)

hydroxylase in freshly isolated hepatocytes prepared from regenerated liver is 5-10 times higher than the value that would be expected if the enzyme resided exclusively in non-parenchymal cells. Furthermore, when isolated hepatocytes and non-parenchymal cells were prepared from the same liver and were compared for prolyl hydroxylase activity, hepatocytes from normal or regenerated liver accounted for greater than 80 or 98 %, respectively, of the total recovered enzyme activity (table 2). Similar results were obtained when the data were normalized for the number of recovered cells (table 2). The specific activity of prolyl hydroxylase was variable among these experiments, but tended to be higher in non-parenchymal cells than in hepatocytes (table 2). This variability may be attributable in part to the presence of hepatocytes (O-3 %) in the preparations of isolated non-parenchymal cells. Prolyl hydroxylase activity in cultured hepatocytes Isolated hepatocytes were incubated in arginine-free culture medium and in the absence of serum to discourage survival of any possible contaminating non-parenchyma1cells [3 11.Prolyl hydroxylase activity in the cultured hepatocytes derived from either normal or regenerated liver rose 2to 3-fold during the first 24 h of incubation (fig. 2). This change was blocked in cells incubated in the presence of cycloheximide (1 x lop5 M) (fig. 2). Omission of insulin from the culture medium reduced or abolished the increase in prolyl hydroxylase activity [32]. The specific activity of the enzyme gradually declined during the second and third days in culture to a value slightly lower than the initial level in freshly isolated cells (fig. 2).

Prolyl hydroxylase in cultured hepatocytes

275

Fig. 3. Exposure of monolayer cultures to (lefr) antiprolyl hydroxylase antiserum or (right) control serum. (Left) Hepatocytes show intense cytoplasmic immuno-

reaction;

Immunofluorescent techniques confirmed the presence of prolyl hydroxylase in cultured hepatocytes. Antibody directed against highly purified prolyl hydroxylase prepared from rat skin adhered to the cytoplasm of the hepatocytes, sparing the nuclei (fig. 3). Fluorescence from cultures incubated with immune serum was 4-5 times greater than from those exposed to control globulin. No evidence for a subpopulation of hepatocytes containing prolyl hydroxylase was obtained as the antibody stained the parenchymal cells uniformly throughout the cultures.

revealed a relative rate of collagenous protein synthesis of 0.1% (table 3 (I)). A similar value was obtained when radiolabelled proteins in cultures incubated with L-['~C]proline were precipitated with TCA and, after hydrolysis, total L-[14C]proline and L-4-[14C]hydroxyproline was measured with an amino acid analyser (table 3 (II, A)). Limited accumulation of radiolabelled collagenous protein in culture appeared to be due to rapid degradation of this material. Thus, the majority of [14C]hydroxyproline was present as dialysable or TCA-soluble material (table 3 (II, A, B)). Moreover, when cultures were incubated with L-['~C]proline and the cells and medium were separated and hydrolysed, the medium contained greater than 97% of the L-4[14C]hydroxyproline (table 3 (II, C)). From these results (table 3 (II, A, B, C)), the calculated rate of relative collagen synthesis is approx. l%, assuming that total ~-4-[l~C]hydroxyproline in the cultures reflects collagen.

Collagen synthesis in cultured hepatocytes We detected only small quantities of collagenous protein synthesis in cultured hepatocytes using standard assay methods. Digestion of TCA-precipitable material from cultures labelled with L-[14C]proline using protease free-bacterial collagenase

x200.

(righf)

non-specific staining only is detected.

E.rp Cd Res 123 (1979)

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Guzelian and Diegelmann

Table 3. kfeasurement

of collagen synthesis PwYdroxY-

Method

Sample

I Digestion with bacterial collagenase II [WlHydroxyproline by amino acid analysis

Cells+medium TCA-insoluble

(total dpm)

[14C]Rroline (total dpm)

Collagen synthesis (% total protein synthesis) 0.07; 0.13; 0.02

A

Cells+medium TCA-insoluble TCA-soluble

4 430 90090

4.91x1@

0.033 0.706

10 010 92 415

5.05x 106

0.198 0.673

-

0.024 1.076

B

Cells+medium Non-dialysable Dialysable C Cells Medium

3 210+994 148 314+14 185

Freshly isolated hepatocytes were prepared from regenerated liver as usual and were incubated in control culture medium for 24 h. The medium was then replaced with control medium supplemented with ferrous sulfate (1 X lo-’ M), ascorbic acid (1.4~ 10e3M), 1 mM proline, and [W]proline at 1 &i/ml. Incubation of the cells with the radioisotope was carried out for 4 h (I) or for 24 h (II). The cultures were then terminated and analysis for collagen synthesis was made using the indicated method as described in Materials and Methods. Results for II(A) and II(B) reflect a single experiment from the same batch of cells. Results for II(C) reflect mean k S.E.M. for the total [‘*C]hydroxyproline in hydroxylates of the cells or medium in three separate batches of cells. Relative collagen synthesis was calculated from this value and from the average value for [Wlproline in II(A) and II(B).

DISCUSSION It has been asserted that the hepatocyte either plays no role in collagen metabolism [3] or serves only passively as an anchor for attachment of collagen fibrils synthesized by fibroblasts in the liver [ 11. However, the present report presents biochemical and immunochemical evidence that parenchymal cells contain functional prolyl hydroxylase and, thus, actively participate in hepatic collagen synthesis. This complements recent immunohistochemical studies in which prolyl hydroxylase was localized to the hepatocyte in neonatal rat liver [33]. Detectable prolyl hydroxylase activity in freshly isolated adult rat hepatocytes has been reported previously [34]. However, some workers have questioned the validity of this finding ascribing the activity to contamination of isolated hepatocyte preparations with non-parenchymal cells [35]. This Exp Cell Res 123 (19791

criticism now appears untenable because isolated non-parenchymal cells contained minimal prolyl hydroxylase activity (table 2). Moreover, isolated hepatocytes derived from regenerated liver are contaminated at most by 5 % of the non-parenchymal cells in the liver, and yet these preparations contained almost 50% of the total hepatic prolyl hydroxylase activity. Whereas these data and immunological studies (fig. 3) establish that prolyl hydroxylase is a constituent of the hepatocyte, the identity of the other hepatic cell(s) containing the remaining prolyl hydroxylase activity was not resolved by fractionation of liver into two major populations of cells. It is possible that the enzyme is contained in cells in the vasculature of the liver and these may not be separated from the organ by collagenase perfusion. Supporting this idea are immunohistochemical studies demonstrating high

Prolyl hydroxylase

concentrations of prolyl hydroxylase in the terminal hepatic veinules [33]. An alternative possibility is that the postulated cell may be destroyed by the enzymes used in the isolation procedures. One candidate would be the perisinusoidal cell (of Ito) which is believed capable of synthesizing collagen [2], and hence, would likely contain prolyl hydroxylase. In unpublished studies, we have found the expected number of perisinusoidal cells among the cells initially isolated by collagenase perfusion of the liver, but less than 3 % of the perisinusoidal cells were recovered in the final isolates of hepatocytes and non-parenchyma1 cells. The perisinusoidal cells may be sensitive to pronase, as failure to find perisinusoidal cells in freshly isolated ‘sinusoidal’ cells has been noted by others [36, 371. An important observation was that prolyl hydroxylase activity in parenchymal cells was higher following partial hepatectomy than in normal liver. This finding, recently observed by others [38], suggests that prolyl hydroxylase activity in the hepatocyte may be linked to proliferation of this cell. In line with this interpretation, prolyl hydroxylase activity is abnormally high in the serum of patients with hepatoma [39]. Furthermore, in several lines of Morris hepatomas, the enzyme activity is present in concentrations inversely related to the doubling time of the tumor [39]. Stimulation of prolyl hydroxylase activity in replicating hepatocytes may explain the rise in enzyme activity in the liver of rats treated with carbon tetrachloride [18, 22, 401, thioacetamide [41], dimethylnitrosamine [42], or ethanol [43], or in the liver of patients with primary biliary cirrhosis [44] because common to many forms of liver injury is regeneration of hepatocytes as part of the healing process. Partial hepatectomy should prove useful for

in cultured hepatocytes

277

studying the control of hepatic prolyl hydroxylase and collagen metabolism because the factors of cell necrosis and inflammation associated with most models of experimental hepatic fibrosis are excluded and because the change in enzyme activity appears to be confined largely to the hepatocytes (table 2). The specific activity of prolyl hydroxylase in cultured hepatocytes spontaneously rose during the first 24 h of incubation in culture medium containing insulin. The sensitivity of this increase to cycloheximide suggests the change represents de novo synthesis of the enzyme rather than its activation [17]. It should be emphasized that change in prolyl hydroxylase activity in cultured hepatocytes occurs selectively since the levels of other microsomal enzymes such as glucose-6-phosphatase [ 13],p-nitroanisole 0demethylase [13, 291 remain unchanged whereas others such as cytochrome P-450 decline dramatically [28, 291. The explanation for these phenotypic changes in primary hepatocyte culture currently is under investigation; nevertheless, this system appears useful for defining the metabolic and cellular factors which regulate prolyl hydroxylase activity in liver parenchymal cells. Demonstration of prolyl hydroxylase can no longer be regarded as sufficient evidence of collagen formation in a given type of cell. The enzyme has been found recently in a variety of cultured cells of diverse origins [45], and yet collagen synthesis was not detected in some of these cultures [20]. Synthesis of collagenous protein by cultured hepatocytes was measurable although only at the lower limits of sensitivity of several standard radiometric assays (table 3). The apparent explanation is that the newly synthesized proteins containing hydroxyproline (probably collagenous proteins) are Exp Cell Res 123 (1979)

278

Guzelian and Diegelmann

rapidly degraded in cultured hepatocytes. This assumes, (1) the generally accepted principle that hydroxyproline is formed in cells only by oxidation of proline incorporated in a polypeptide [46]; and (2) that hydroxyproline is a specific marker for collagen. With respect to the latter assumption, hydroxyproline has been found in the collagen-like regions of the non-connective tissue protein, Clq [47], however, synthesis of this protein is not detectable in the liver [48]. Further, despite the presence of prolyl hydroxylase in hepatomas [39], these cells do not synthesize the first component of complement [49]. Thus, based on measurements of total hydroxyproline formation (table 3), the cultured hepatocytes appear to synthesize collagen relative to non-collagenous protein at a rate slightly lower than that reported in liver slices (2-7%) [50], and slightly higher than the rate found in the liver in vivo (0.3 to 0.7%) [51, 521. The reasons for rapid degradation of collagen in hepatocyte cultures are unknown. A similar phenomenon has been reported in cultured fibroblasts [53], and in lung explants in which 30% of newly synthesized collagen was degraded within minutes to dialysable products containing hydroxyproline [54]. This may also be true for hepatocytes which contain lysosomal enzymes capable of degrading collagen [55]. We have recently found that freshly isolated hepatocytes retain some of the crude bacterial collagenase used in their isolation [56]. This could explain the more extensive degradation (greater than 90 %) of the newly synthesized collagenous proteins in the present culture system. In line with this idea, we have found that as the age of hepatocyte cultures increases, the apparent fractional rate of degradation of the hydroxyproline containing product decreases, coincident with loss of contaminating clostridial Exp Cell Res 123 (1979)

collagenase [Guzelian, P S & Diegelmann, R F, unpublished observations]. Ultimate proof of collagen formation and breakdown by the hepatocyte will require modification of the conditions in the hepatocyte culture system to prevent collagen degradation and appropriate characterization of the hydroxyproline-containing proteins. The authors thank Nelsen Niehaus and Suzanne Fernandez for technical assistance and Carol Nance for performing amino acid analyses. We are grateful to Dr I. K. Cohen, Chairman of the Division of Plastic Surgery, for support and for encouragement in initiating these studies. This project was supported by a grant from the NIH (AM 18976). P. S. G. is the recipient of a Clinical Investigator Award in Gastroenterology from the NIH (5K08-AM-00128). Portions of this work have been published in abstract form in Gastroenterology 74 (1978) 1165.

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Exp CrllRes 123 (1979)