Rat hepatic lipocytes synthesize and secrete transin (Stromelysin) in early primary culture

GASTROENTEROLOGY 1995;109:889-898

LIVER, PANCREAS, AND BILIARY TRACT

Rat Hepatic Lipocytes Synthesize and Secrete Transin (Stromelysin) in Early Primary Culture

SAMIR K. VYAS, HELEN LEYLAND, JANET GENTRY, and MICHAEL J. P. ARTHUR University Medicine, University of Southampton, Southampton, Hampshire, England

Background & Aims: Hepatic lipocyte proliferation and activation are pivotal in liver fibrosis. Disruption of normal lipocyte-matrix interactions may contribute to this process. The synthesis of transin, which degrades normal liver matrix, by culture-activated hepatic lipocytes was investigated. Methods: Lipocytes were isolated by pronase/collagenase perfusion, density gradient centrifugation, and centrifugal elutriation. Transin messenger RNA in lipocytes was analyzed by Northern blotting. Transin activity was analyzed by zymography, West- ern blotting, immunocytochemistry, and quantitative [14C]13-casein degradation assay. Results: Transin messenger RNA was detected in early primary culture (3 - 5 days) but not in freshly isolated lipocytes or late primary culture. Zymography of lipocyte medium showed caseinolytic activity (relative molecular weight, 57 kilodaltons and 60 kilodaltons) inhibited by ethylenediaminetetraacetic acid but not thiol or serine protease inhibitors. Immunoblotting and immunocytochemistry confirmed the presence of transin in media and cells. Quantitative transin activity decreased progressively with increasing duration of primary lipocyte culture and myoflbroblastic transformation. Conclu- sions: Rat hepatic lipocytes express the transin gene and secrete its product during the early phase of lipocyte activation in primary culture. Because this enzyme degrades a wide spectrum of normal basement membrane proteins and activates progelatinase B and interstitial collagenase, it may have an important role in liver injury and fibrosis.

L iver fibrosis is characterized by proliferation and myofibroblastic transformation of the normally qui-

escent lipocytes (perisinusoidal, stellate, fat-storing, or Ito cells) present in the space of Disse. 1'2 In studies of cultured lipocytes, ~':' experimental fibrosis, 4 and human disease, 5 lipocytes have now been determined as the major cellular source of collagens I and III, 6-S proreogly- cans, 9 laminin, m fibronectin, 5'6 entactin, and hyaluro-

11 nan. In normal liver, lipocytes are the major storage site of

vitamin A ~2')5 with dendritic processes that encircle the

hepatic sinusoid. 14 As much as 80% of total liver retinoids (vitamin A) are stored in lipocytes, mainly as retinyl esters 15 in large cytoplasmic vacuoles distinguished

by autofluorescence under UV excitation. In response to liver injury, lipocytes proliferate and adopt a myofibroblastic phenotype, showing loss of their retinoid content, enlargement of their rough endoplasmic reticulum, and expression of smooth muscle Ot-actin, desmin, and cell membrane-associated platelet-derived growth factor (PDGF) and transforming growth factor (TGF) [3 receptors. 2 These observations have led to the concept of lipocyte activation, which is similar in many respects to the spontaneous phenotypic changes observed in cell culture on uncoated plastic in serum-containing media.

The factors that initiate and perpetuate lipocyte activation are complex. Although soluble mediators, including cytokines (e.g., TGF-~I, 1~' PDGF, Iv interleukin l, and tumor necrosis factor [TNF] O~ is) and retinoids, 19 e, have a role, cell-cell lv'22 and cell-matrix interactions may be of equal importance. ~9'>'24 The normal space of Disse contains a basement membrane-like matrix comprising type IV collagen, fibronectin, laminin, and proteoglycans. 2~ Hepatic lipocytes cultured on a model basement membrane (derived from the Engelbreth-Holm-Swarm murine sarcoma; EHS matrix) remain in a quiescent, nonproliferative, nonfibrogenic phenotype. 26 Differenti- ated functional integrity of hepatocytes (e.g., albumin or cytochrome P450 isoenzyme expression) is also maintained for prolonged periods in culture when these cells are plated on EHS matrix. 25 In contrast, hepatocytes cultured on plastic or type I collagen progressively deterio- rate, indicating that the integrity of the normal matrix is fundamental to normal liver function. This suggests that degradation of normal liver matrix may be an ira-

Abbreviations used in this paper: DMEM, Dulbecco's modified Ea- gle medium; LCM, lipocyte-conditioned media; PDGF, platelet-derived growth factor; TGF, transforming growth factor; TNF, tumor necrosis factor.

© 1995 by the American Gastroenterological Association 0016-5085/95/$3.00

890 VYAS ET AL. GASTROENTEROLOGY Vol. 109, No. 3

portant aspect of the pathogenesis of liver injury and fibrosis.

The major proteolytic enzymes involved in extracellu-

lar matrix remodeling are the metalloproteinases. These are a family of closely related enzymes that contain zinc

at the active site and operate at neutral pH. They can be broadly classified by their substrate profile: interstitial collagenases that degrade fibrillar collagens (types I, II,

and III), type IV collagenases/gelatinases that degrade

native (type IV) collagen and denatured collagens (gela-

tins), and stromelysins that degrade a much broader spec-

t rum of matrix (and other) proteins, including proteogly-

cans, laminin, fibronectin, type IV collagen, and casein. In previous studies, we have identified release by rat

hepatic lipocytes of gelatinase A (72-kilodahon type IV collagenase/gelatinase) 27 and shown that rat Kupffer cells

release gelatinase B (95-kilodalton collagenase/gelatinase). 28 Expression of transin (the rat homologue of

human stromelysin) in liver is of particular interest be-

cause it is capable of degrading the noncollagenous com- ponents of the normal matrix (namely, fibronectin, lami-

nin, and proteoglycans) in addition to type IV collagen. Another important function of this enzyme is its ability to activate procollagenase 29-31 and progelatinase B. 32 In

this study, we report transin messenger RNA expression, transin synthesis, and release of transin activity in primary cultures of purified lipocytes derived from normal

rat liver.

Mater ia ls and Methods

Cell Isolation and Culture

Lipocytes were isolated from normal rat liver (male Sprague-Dawley rats that had been treated with humane care; 450-600 g) by sequential in situ perfusion with collagenase and pronase as described previously. 27 Buoyant lipocytes were separated from the resulting cell suspension over a discontinu- ous two-layer Nycodenz (Nyegard, Birmingham, England) gradient prepared as described by Hendriks et al. 33 This con- sisted of a lower layer of 17.2% Nycodenz (wt/vol) in Gey's balanced salt solution without NaCI (Gibco, Paisley, Scotland) and an upper layer of cell suspension in Hank's balanced salt solution (Gibco) containing 11.5 % Nycodenz and 0.1% deoxy- ribonuclease carefully overlaid in 15-mL centrifuge tubes (Fal- con; Beckton Dickinson UK Ltd., Plymouth, England) and covered with 1.5 mL of Hank's balanced salt solution. After centrifugation at 1400g for 17 minutes in a Mistral 6 L centrifuge (MSE Scientific Instruments, Crawley, Sussex, England), the rotor was allowed to stop with the brake off. Lipocytes were recovered from the top of the 11.5% layer and Kupffer cells from the 11.5%-17.2% interface and further purifed by centrifugal elutriation (Beckman JE2-21 centrifuge fitted with a JE6-B rotor; Beckman Instruments, Geneva, Switzerland). Purified lipocytes were collected at a rotor speed of 1500 rpm

with a flow rate of 18 mL/min in 200 mL of elutriated Hank's balanced salt solution; Kupffer cells were obtained at a rotor speed of 2500 rpm with a flow rate of 45 mL/min in 200 mL of elutriated Hank's balanced salt solution. Purified lipocytes and Kupffer cell fractions were centrifuged and resuspended in Dulbecco's modified Eagle medium (DMEM; Gibco) containing 20% fetal calf serum, penicillin (10 mU/mL), strepto- mycin (10 Hg/mL), and gentamicin (32 gtg/mL). Cell viability and total yield in this suspension were calculated on a hemocy- tometer using trypan blue dye exclusion (final concentration, 0.05%) in DMEM supplemented with 20% fetal calf serum and antibiotics. Cells were seeded at a plating density of 2 × 106 cells per well on uncoated 35-ram plastic wells (Costar, Cambridge, MA) in 1.5 mL. The culture medium was changed every 48 hours.

Lipocytes were identified as more than 95% pure by their characteristic lipid droplets on phase-contrast microscopy, endogenous vitamin A autofluorescence, and immunostaining for desmin as previously described. 34-36 In addition, contaminat- ing Kupffer cells were identified by staining for endogenous peroxidase. 37 The results presented in this report originate from 16 separate rat lipocyte preparations.

RNA Extraction and Northern Blotting

Total RNA was extracted from rat lipocyte cultures after lysis in guanidium isothiocyanate and purified by the method of Chomczynski and Saachi.38 Expression of messenger RNA for transin was analyzed by Northern blotting. Equal amounts of total RNA were subjected to electrophoresis on a 1% denaturing agarose gel, transferred to a Hybond N membrane (Amersham), and hybridized with a 1.9-kilobase complementary DNA probe for transin (pTR1; kindly donated by Professor R. Breathnach, INSERM, Strasbourg, France) labeled with [32P]adenosine triphosphate by random priming using the Klenow reagent (Amersham). After hybridization, membranes were washed at 30°C in 0.2% sodium doceyl sulfate/0.2×SSC for 30 minutes (I×SSC = 0.15 mol/L NaCl/0.015 mol/L sodium citrate) and exposed to Kodak X-Omat S film (Eastman Kodak, Rochester, NY) for 48 hours at -70°C. A ribosomal $14 complementary DNA probe was used to control for quan- tity of loading and integrity of total RNA in each lane.

Media Collection for Analysis of Proteinase Activity

Cells maintained in serum-containing media for the required incubation period were washed threefold in serum- free DMEM with antibiotic supplements and preincubated for 4 hours in this medium. This was then discarded, and the cells were placed in fresh serum-free DMEM for 24 hours before harvest. The media were clarified by centrifugation and stored at -20°C until analysis.

Rat Skin Fibroblast Preparation and Culture

Passaged rat skin fibroblasts (kindly donated by Dr. J. P. Iredale, University Medicine, University of Southampton,

September 3.995 RAT HEPATIC LIPOCYTES SECRETE TRANSIN 891

Southampton, Hampshire, England) were maintained in culture on uncoated plastic in DMEM containing 20% fetal calf serum. Before collection of serum-free conditioned medium as described above, these cells were stimulated for 24 hours with 3% (wt/vol) partially purified cytokines. 39

Substrate Gel Analysis

Proteinase activities in crude unconcentrated lipocyte- conditioned media (LCM) were shown by ~-casein-substrate sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under nonreducing conditions as previously described 4° with a single modification. A final concentration of 5 mg/mL ~-casein (Sigma Chemical Co., Poole, England) was incorporated in a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel to improve visualization of casein-degrading activity. 2v For some experiments, gels were incubated in the presence of proteinase inhibitors (as described in Re- sults).

Western Blotting

Serum-free conditioned medium from either confluent (after 21 days) rat lipocyte or skin fibroblast cultures were concentrated 104old before electrophoresis on a nonreducing 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to the method of Laemmli. 41 Tenfold concentrated serum-free DMEM was used as a negative control. The gel was presoaked for 30 minutes in transfer buffer (0.025 mol/L Tris, 0.192 mol/L glycine plus 20% methanol) and transferred electrophoretically onto a Hybond C nitrocellulose filter (Amersham) that was then blocked for 1 hour at room temperature in Tris-buffered saline plus Tween 20 containing 2.5% dried milk. The filter was incubated overnight at 4°C with a 1:1000 dilution of rabbit antitransin antiserum (kindly donated by Dr. L. M. Matrisian, Nashville, TN) as previously described. 42 After rinsing the filter threefold in Tris-buffered saline plus Tween 20, immunoreactive bands were visualized using an alkaline phosphatase-conjugated sheep anti-rabbit immunoglobulin G.

Immunolocalization of Transin

Rat lipocytes were isolated and grown in DMEM containing 20% fetal calf serum as described above for 4 days and then washed threefold in DMEM to remove serum before immunostaining for transin. Each subsequent step in the ensu- ing protocol was followed by a threefold wash in phosphate- buffered saline. After washing, the cells were incubated in DMEM with monensin (5 [imol/L) for 4 hours, fixed in 4% paraformaldehyde in phosphate-buffered saline for 5 minutes, and incubated in 0.1% Triton X-100 and phosphate-buffered saline for 5 minutes to permeabilize the cells and then in 5% normal pig serum diluted in phosphate-buffered saline and 5% bovine serum albumin to block nonspecific labeling. This was followed by addition of primary antiserum (rabbit antitransin, 1:200 in phosphate-buffered saline and 5% bovine serum albumin; gift from Dr. L. M. Matrisian) or nonimmune rabbit serum for 1 hour. Specific transin staining was detected

by addition of a secondary antibody (fluorescein isothiocyanate-conjugated pig anti-rabbit immunoglobulin G, 1:50 in phosphate-buffered saline and 5 % bovine serum albumin). Cy- toskeletal actin filaments were stained by incubation in a 1:100 dilution of rhodamine-phalloidin (Sigma Chemical Co.) or phosphate-buffered saline alone (negative control) for 5 minutes. Ceils were mounted in Citifluor (City University, London, England) and viewed and photographed (Kodak Elite ASA 200) using a Leica TCS4D confocal laser microscope (Heidel- berg, Germany).

Quantitative Analysis of Casein Degrading Activity

Quantitative p-casein degrading activity was determined using ~4C-labeled p-casein, which was prepared by acet- ylating 13-casein with [1J4C]acetic anhydride according to the method of Cawston et al. 43 Unmodified serum-free LCM (150 btL) was incubated with 100 ~lg of [14C]~-casein (2 mg/mL; sp act, 1 × 105 cpm/mg) and 50 gL Tris assay buffer (200 mmol/L Tris/HC1, pH 7.9, 60 mmol/L CaC12, 0.02% azide) with and without 10 mmol/L aminophenylmercuric acetate in a final volume of 250 gtL at 37°C. Samples were assayed in duplicate, and the reaction was stopped after a recorded incubation period by the addition of 50 gL of ice-cold 100% trichloroacetic acid (final concentration, 18% wt/vol), which precipitated remaining intact (nondegraded) ~-casein. After centrifugation at 10,000g for 15 minutes, 200 BL of the super- natant was counted for '4C radioactivity in 2 mL of Ecolume scintillation fluid (ICN, Costa Mesa, CA). One unit of caseinase activity degrades 1 ~g of ~-casein per minute at 37°C to peptides soluble in 18% (wt/vol) trichloroacetic acid.

Analysis of Cellular DNA Content

After collection of media, the corresponding cell cultures were washed once with phosphate-buffered saline, stored in sterile water at -20°C, and subsequently lysed by sonication before assaying for DNA. The total DNA content was assayed using a spectrofluorimetric method described by LaBarca and Paigen. 44 Total DNA content was used to normalize the quantitative [14C]l]-caseinase activity, which was expressed per mi- crogram of cellular DNA.

Results

Detection of Messenger RNA for Transin

By Nor thern analysis, total R N A from cultured

rat lipocytes was found to contain a single hybridizat ion

signal of 1.9 kilobases when probed with a complementary D N A probe for transin. This signal was strongest

at 3 days in culture (Figure 1) but could not be detected

at 7, 14, or 21 days after autoradiographic exposure for

48 hours (n = 3). A more detailed t ime course analysis

for transin messenger R N A expression was therefore un-

dertaken dur ing the early phase of lipocyte culture up to 7 days. In lipocytes from six separate isolations, no


messenger RNA for transin was detected in freshly isolated cells (even after prolonged autoradiograph exposure for 7 days). However, in cultured lipocytes, expression of transin messenger RNA relative to $14 messenger RNA (control) was first detectable and maximal at 3 days but declined to being barely detectable by 5 days in culture (Figure 2A and B).

Substrate Gel Analysis of LCM

All LCM were found to contain caseinolytic activity, but the pattern and intensity varied with duration of culture. A time course experiment showed two major bands of ~-caseinolytic activity migrating to relative molecular weights of 57 and 60 kilodaltons were present in the media from lipocytes cultured for 3, 7, 14, and 21 days (Figure 3). This activity was present in pure lipocyte cultures containing <2% Kupffer cells and seemed to be strongest in LCM from 3-day-old cells. Some lower relative molecular weight bands (36 and 25 kilodaltons) were present in LCM from cultures at 7 and 14 days, and a higher relative molecular weight band (approxi- mately 120 kilodaltons) was present in LCM from 3-day- old cells. Negligible casein degrading activity was found in 98% pure Kupffer cell cultures at 2, 4, and 6 days consistent with the degree of lipocyte contamination (ap- proximately 2%; Figure 3).

Thus, these caseinolytic bands produced by lipocytes

are consistent with the molecular weights of the glyco- sylated (60 kilodaltons) and unglycosylated (57 kilodaltons) proenzyme forms of the metalloproteinase transin. The activity was inhibited by 10 mmol/L ethylenediaminetetraacetic acid added to the substrate gel

A 0 3 4 5 7 Days in culture

1.9kb Transin

S14

B1.6

1.4

1,2

0 ,8

3 7 14 21 Days in culture

1.9Kb --> ~ Transin

S14

Figure 1. Transin messenger RNA detection by Northern blotting. Equal quantities (5 pg) of total RNA were extracted from lipocytes from an identical isolation after 3, 7, 14, and 21 days in culture, electrophoresed on a 1% agarose gel, transferred onto a nylon membrane, and probed sequentially with [32P]adenosine tr iphosphate-labeled complementary DNA probes for transin messenger RNA (1.9 kilobases) and S14 messenger RNA as described in Materials and Methods. A single 1.9-kilobase hybridization signal was detected with the complementary DNA probe for transin in total RNA from 3-day-old lipocytes but not from those cultured for 7, 14, or 21 days.

0.6

0.4

0.2

0 - - -

I I

I

0 3 4 5 7

Days in culture

Figure 2. (A) Transin messenger RNA detection by Northern blotting in freshly isolated lipocytes. A representative Northern blot is shown of total RNA extracted from freshly isolated lipocytes and after 3, 4, 5, and 7 days in culture sequentially probed with [32p]adenosine tr iphosphate-labeled complementary DNA probes for transin (1.9 ki- Iobases) and ribosomal S14 as described in Materials and Methods. No hybridization signal could be detected using the transin complementary DNA probe in total RNA from freshly isolated lipocytes, but messenger RNA for transin relative to S14 was present in lipocytes cultured for up to 5 days, becoming negative again by day 7. (B) Band intensity for transin messenger RNA was measured by laser densitometry and expressed as a ratio of that for ribosomal S14 messenger RNA (control). This figure shows that peak transin messenger RNA expression by lipocytes in primary culture as described in Materials and Methods occurs on day 3, declining to undetectable levels after 7 days. Furthermore, no transin messenger RNA expression is found in freshly isolated lipocytes from normal rat liver.

September 1995 RAT HEPATIC LIPOCYTES SECRETE TRANSIN 893

Rat Lipocytes

1

3 7 14

Rat Kupffer cells

21' i 4 Days in culture

1 2 3

66kD

45kD 29kD 14.2kD

Rgure 3. Substrate gel analysis of serum-free culture media. Primary lipocyte and Kupffer cell cultures were established after seeding at an equal plating density from identical preparations. Serum-free cell conditioned media were harvested during a period of 24 hours from 3-, 7-, 14-, and 21-day-old lipocytes and 2-, 4-, and &day-old Kupffer cells as described in Materials and Methods. Conditioned media were subjected to electrophoresis on 10% polyacrylamide gels incorporat- ing I~-casein (5 mg/mL) incubated in 0.05 mol/L Tris, pH 8.0, 5 mmol / L CaCI2, and 0.02% azide for 16 hours before being fixed and stained. Two major bands of caseinolytic activity with relative molecular weights of 57 and 60 kilodaltons were present in the media from lipocytes cultured for 3, 7, 14, and 21 days, but negligible activity was present in Kupffer cell-conditioned media (98% pure by endogenous peroxidase staining). Lower relative molecular weight bands of caseinolytic activity in LCM migrating to 36 and 25 kilodaltons were inhibited when the gels were incubated in the presence of 10 mmol/ L ethylenediaminetetraacetic acid but not 2 mmol/L N-ethylmaleimide or 1 mmol/L PMSF (data not shown).

incubation buffer but was not affected by either 2 mmol/L phenylmethylsulfonyl fluoride (Sigma Chemi- cal Co.) or 1 mmol/L N-ethylmaleimide (BDH, Poole, England) (Figure 4). These results indicate that the ~- casein degrading activity is a metalloproteinase with the properties of transin.

*- Tmnsin (57kD)

Rgure 5. Characterization of the caseinolytic activity in LCM by West- ern blotting. Lipocytes were grown to confluence (21 days), and serum- free medium was conditioned during a period of 24 hours as described in Materials and Methods. Serum-free conditioned medium from confluent cytokine-stimulated rat skin fibroblasts stimulated with partially purified cytokines was used as a positive control, and cell-free culture medium was used as a negative control. Media were concentrated lO-fold and electrophoresed on a 10% polyacrylamide gel, transferred to a nitrocellulose filter, and probed with a polyclonal rabbit antitransin antibody. Hybridization was visualized using an alkaline phosphatase- conjugated secondary antibody. Lane 1, negative control; lane 2, positive control; lane 3, lipocyte conditioned medium.

E D T A P M S F N E M CONTROL

~ ' i ~7 ~ ~

* - 66 k D

*-45 liD

,--29 kD

Figure 4. Characterization by substrate gel analysis of the caseinolytic activity in LCM. Replicate samples from a single lipocyte preparation were electrophoresed in four lanes of the same ][~asein substrate gel. After electrophoresis, individual lanes were excised and incubated in 0.05 mol/L Tris, pH 8.0, 5 mmol/L CaCl2, 0.02% azide containing no protease inhibitors (control), N-ethylmaleimide (2 mmol/L), PMSF (1 mmol/L), or ethylenediaminetetraacetic acid (10 mmol/L) before being fixed and stained.

Western Blotting

Characterization of the secreted metalloproteinase was undertaken by determining its immunoreactivity in a Western blot against a rabbit antitransin antibody to a COOH-terminal peptide of rat transin 42 (Figure 5). The major proenzyme bands (57 and 60 kilodaltons) were found to cross-react with this antibody, which recognizes both transin 1 and transin 2. These immunoreactive bands produced by unstimulated lipocyte cultures on uncoated plastic comigrated with transin in serum-free media conditioned by cytokine-stimulated rat skin fibroblasts but were not detectable in cell-free culture medium.

Immunostaining for Transin

Transin was immunolocalized in 4-day-old primary rat lipocyte cultures (Figure 6A and B). The immunofluorescence for transin (fluorescein isothiocy-

894 VYAS ET AL. GASTROENTEROLOGY Vol, 109, No. 3

C D

Flgure 6. Localization of transin in cultured lipocytes by immunocytochemistry. Lipocytes were isolated and cultured on plastic for 4 days and then exposed to 5 ~mol/L monensin for 4 hours. Cells were then immunostained for transin (detected by fluorescein isothiocyanate-conjugated secondary antibody) and cytoskeletal actin filaments (detected by rhodamine isothiocyanate-conjugated phalloidin) as described in Materials and Methods using appropriate nonimmune and phosphate-buffered saline diluent controls. This figure shows dual-channel confocal laser photomicrographs of lipocytes stained for (A) transin and cytoskeletal actin, (B) transin with phosphate-buffered saline diluent only (negative control for cytoskeletal actin), (C) dual negative controls for transin and cytoskeletal actin filaments, and (D) cytoskeletal actin and nonimmune rabbit serum (negative control for transin).

anate-labeled) was strongest in the perinuclear cyto- plasm, which is the characteristic distribution of the cellular Golgi. Interestingly, some binding to the nu- cleus was also evident and specific. Cells incubated with nonimmune rabbit or mouse immunoglobulin G were uniformly negative (Figure 6C and D). Cytoskele- tal actin filaments stained directly using rhodamine- phalloidin are also shown (Figure 6A and D) with a

negative control in which cells were incubated with phosphate-buffered saline diluent only (Figure 6B and C).

Quantitative Analysis of [140]]3-Casein Degrading Activity Released by Lipocytes

Total transin activity (measured after aminophenylmercuric acetate activation) was highest in serum-free


~2s r~

E e. v 1 5

#. U q= 10 m o s

o = 5 ? ¢.1

~" 0

• APMA-treated

[ ] No APMA

3 7 14 21 Time in Culture (days)

Rgure 7. Quantitative analysis of [14C]lE~-caseinase activity LCM de- pendent on duration of culture. Primary lipocyte cultures from six separate isolations were established after seeding at an equal plating density. Serum-free LCM were harvested from lipocytes after 3, 7, 14, and 21 days in culture, and duplicate samples were assayed for [14C]l[~caseinase activity as described in Materials and Methods with and without treatment with 10 mmol/L aminophenylmemuric acetate to measure release of total transin activity (aminophenylmercuric ace- tare-treated) and spontaneously activated transin activity (no aminophenylmercuric acetate), respectively. Results are expressed per mi- cregram DNA, and error bars represent the SEM for each variable.

LCM obtained from 3-day-old lipocyte cultures. With increasing duration of lipocyte culture up to 21 days, total transin activity decreased progressively, reducing to more than fivefold less than in the 3-day-old cells (Figure 7). At every time point, at least 50% of the released transin was already in the activated form. With increasing duration of lipocyte culture, the amount of spontaneously active transin (non-aminophenylmercuric acetate- treated) in lipocyte medium initially remained constant (or reduced minimally at days 7 and 14). However, by day 21 of primary culture, the amount of active transin also decreased dramatically, but again there was a high ratio of active/total transin activity in LCM.

Discussion

We have shown that highly purified hepatic lipocytes isolated from normal rat liver synthesize and secrete a protein with the biochemical properties of transin. Fur- ther evidence that this protein was transin was provided by showing its immunoreactivity against a specific antiserum 42 by Western blotting. This same antibody was used to coimmunolocalize transin to desmin-positive lipocytes in primary culture. We were also able to detect transin messenger RNA in total RNA extracted from these highly pure lipocyte cultures by Northern analysis using a 1.9-kilobase transin complementary DNA

probe. 45 Taken together, these data indicate that cultured rat hepatic lipocytes can express the gene for transin, synthesize transin, and release this enzyme activity into the extracellular space.

Both transin messenger RNA content and release of total transin activity were greatest in the early phases of primary culture (days 3-5). Isolated lipocytes require up to 48 hours to become adherent to uncoated plastic in primary culture, 46 which precluded accurate study of their release of transin activity during this phase. How- ever, we repeatedly found that transin messenger RNA could not be detected by Northern hybridization in total RNA extracted from freshly isolated cells (Figure 2A and B), indicating that transin gene expression is switched on during early primary culture. With increasing duration of lipocyte culture, there was a progressive decrease in the total released transin activity. This was associated with a reduction in lipocyte transin messenger RNA content in later cultures, which provides the most likely explanation for this finding. However, we have previously shown that culture-activated lipocytes also synthesize both O~2-macroglobulin and tissue inhibitor of metalloproteinase 1,47'48 which can inhibit transin activity at the protein level and may therefore have contributed to this observation.

The stromelysin subclass includes three enzymes with high homology: stromelysin 1, stromelysin 2, and stromelysin 3. In addition, a smaller related enzyme that lacks the nonproteolytic hemopexin domain, 49 named matrilysin, has a similar substrate specificity except that it also degrades elastin. 49 Although the antiserum used in our immunologic studies cross-reacts with both transin 1 and transin 2, 42 the detection of a 1.9-kilobase signal using the pTR1 complementary DNA probe and the electrophoretic mobility of the caseinolytic bands on zymography confirm the detection of transin 1 in this study. We cannot exclude the possibility that there may also be some synthesis of transin 2. The detection of smaller relative molecular weight caseinolytic bands by zymography at some time points in culture is consistent with the presence of minor activated species of transin because they were inhibited by ethylenediaminetetraacetic acid but not by N-ethylmaleimide or PMSF (data not shown). Alternatively, these bands could represent other unidentified caseinolytic metalloproteinases coexpressed with transin.

Previous studies of the origin and expression of stromelysin in the liver are limited. A proteinase with similar properties has been reported to be released by cells with a fibroblastic phenotype (but uncertain origin) derived from murine Schistosoma granuloma explants. 5° Messen- ger RNA for transin has also been located in CC14-treated


rat liver by in situ hybridization 51 or by Northern analysis of messenger RNA from regenerating whole liver ho- mogenates) 2 By both techniques, transin expression was minimal or undetectable in normal rat liver. By in situ hybridization, messenger RNA transcripts were initially detectable over hepatocytes 4 - 6 hours after injury, but maximal detection occurred after 24 hours predomi- nantly over sinusoidal cells that were not further characterized but were most likely lipocytes. The studies we report here provide conclusive evidence that cultured lipocytes are a cellular source of transin.

The role of stromelysins in liver injury or fibrosis is uncertain, but studies of their expression in other tissues both during physiological cellular processes and in disease provide an insight into their diverse potential. Stro- melysin, although not widely expressed normally, is readily induced by growth factors, cytokines, tumor pro- moters, and oncogenes in activated fibroblasts, chondro- cytes, and endothelial cells) 3 In addition to liver fibrosis, fibroblast activation and matrix remodeling are features of other chronic inflammatory diseases, including glo- merulonephritis and rheumatoid arthritis. Studies of stromelysin expression in these diseases and in the c o n -

text of tumor invasion, organ development, and involu- tion have implicated its importance in such diverse roles as tumor metastasis, ~4 cell migration during development, 55 cell proliferation, 56 apoptosis, 5v and activation of other metalloproteinases. 29'31'58

Stromelysin may also have an important interaction with matrix proteins and their bound growth factors. TGF-~I, which plays a significant role in lipocyte transformation to a fibrogenic phenotype, 16'59 is sequestered in its latent form in the extracellular matrix by binding to the core protein of the proteoglycan decor in . 6° The latter has been identified in liver. 61 Stromelysin secretion by lipocytes may release active TGF-~I on degradation from the decorin TGF-I~I complex. 6° In turn, active TGF-~I transcriptionally down-regulates stromelysin expression, 62 thus forming a negative feedback loop. However, the active TGF-~I released by matrix degradation may have other profound effects on hepatic lipocyte fibrogenesis. 6~-65 The extracellular matrix may also act as a reservoir for other cytokines and can either decrease their local effect by sequestration or prolong their local influence by prevention of clearance. Such interactions are shown by the ability of highly negatively charged side chains of heparan sulphate proteoglycan to bind interferon gamma 66 and that of heparin to bind hepatocyte growth factor 6v or basic fibroblast growth factor. 67'68

By degrading proteoglycans and other matrix proteins, stromelysin may thus release cytokines and growth factors, thereby altering their local effects within tissues

such as liver. This is a possible mechanism by which stromelysin can contribute to the pathogenesis of both liver injury and fibrosis.

Another important mechanism is the potential effect of stromelysin on disruption of normal cell-matrix interactions in liver. Our finding that transin is expressed in the early phase of primary lipocyte culture is particularly interesting when lipocytes undergoing similar phenotypic changes are considered in hepatic injury in vivo. Such transitional cells are prominent in areas of active hepatic injury 69-v5 and have been most extensively stud- ied in alcoholic liver disease in humans v2,v3,v6 and experimental animals, v°'v7 The release of stromelysin by such activated lipocytes, coupled to its role in activation of Kupffer cell-derived progelatinase B, 28 may lead to local degradation of proteoglycans, laminin, fibronectin, and type IV collagen. This in turn could, by disrupting cell- matrix interactions, lead to significant hepatocyte dys- function 25'v*-8° and enhanced lipocyte activation] 6 Stro- melysin released by lipocytes could also facilitate, through degradation of liver matrix, their local migration or may, by disturbing the normal cell-matrix interactions, be involved in facilitating their proliferation. Al- though the function of this enzyme in liver remains spec- ulative, our data and that of others clearly indicates that lipocyte stromelysin expression is temporally related to the early phase of culture on plastic and to the initial phases of liver injury in vivo) 1 This raises the intriguing possibility that stromelysin may have a significant bio- logical role in the mechanism(s) of lipocyte proliferation and activation.

References

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Received May 3, 1994. Accepted February 22, 1995. Address requests for reprints to: Samir K. Vyas, M.D., University

Medicine, Level D, South Block, Southampton General Hospital, Tremona Road, Southampton, Hampshire $016 6YD, England. Fax: (44) 170-379-4154.

Supported by the Astra Foundation (United Kingdom) and the Wes- sex Medical Trust. Dr. Vyas is a British Digestive Foundation Training Fellow.

Rat hepatic lipocytes synthesize and secrete transin (Stromelysin) in early primary culture

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