EXPERIMENTAL

CELL

RESEARCH

191,

105-114

(1990)

Interleukin-I -induced Suppression of Type II Collagen Gene Transcription Involves DNA Regulatory Elements SRINIVASANCHANDRASEKHAR,~ANITA Department

K. HARVEY,JILL

of Connective Tissue and Monoclonal Eli Lilly and Company, Corporate

Antibody Research, Center, Indianapolis,

INTRODUCTION Cartilage is a highly specialized tissue that is composed of a complex extracellular matrix of collagens, proteoglycans, and several noncollagenous glycoproteins. The collagens of cartilage include types II, VI, IX, and XI, of which type II collagen is the most abundant [5, 32, 34, 371. Type II collagen exists as thin, randomly

National 21224.

Lilly Research Indiana 46285

E. HORTON~

Laboratories,

distributed fibrils as a consequence of its multiple interactions with other matrix molecules, including proteoglycans, type IX collagen, and several matrix glycoproteins [4,6,7,19,42,49]. This arrangement of extracellular matrix facilitates the function of cartilage as a shock-absorbing cushion by redistributing the tensional forces throughout the matrix and allowing compressibility of the tissue. In degenerative joint diseases, the integrity of the cartilage is lost because of structural and functional alterations in the constituent connective tissue molecules [3, 28, 311. The mechanism of cartilage destruction in these disease conditions is not fully understood, but it is presumed that certain inflammatory signals induce chondrocytes of the articular cartilage to secrete metalloproteases that cause the destruction of matrix macromolecules [la, 13, 15, 20, 36, 38, 431. The best studied inflammatory factor is interleukin-1,3 a polypeptide molecule of M, = 17,600 Da that is synthesized and secreted by a variety of cell types including inflammatory monocytes, fibroblasts, and chondrocytes [reviewed in 14, 411. IL-l exists in two biochemically distinct forms, namely IL-la and IL-l@. Both the forms are indistinguishable in several biological activities and share a common receptor [17, 29, 331. IL-l plays a dual role in cartilage metabolism; it causes proteoglycan degradation by inducing the production of metalloproteases such as stromelysin, but it is also believed to prevent cartilage repair by causing an inhibition of proteoglycan synthesis [9, 45-471. While the effects of IL-l on proteoglycan synthesis and degradation have been well studied, very little is known about the effects of IL-l on collagen metabolism. Recent studies have suggested that IL-l may suppress the synthesis of cartilage type collagens and facilitate the phenotypic expression of noncartilage type collagens [16, 21, 481. Because the recovery and repair of cartilage require a return to a normal synthetic capacity

Interleukin-1 is a proinflammatory polypeptide that influences cartilage macromolecular degradation and synthesis. Since previous studies have suggested that interleukin-1 may inhibit type II collagen synthesis, we have studied the mechanism of inhibition of type II collagen synthesis by interleukin-1. When rabbit articular chondrocytes were treated with purified recombinant interleukinl/3 or macrophage-conditioned medium, the synthesis and assembly of type II collagen into the extracellular matrix were greatly reduced. The inhibition was concentration-dependent and occurred within 10 h of treatment with interleukin-1, with greater inhibition occurring at 30 h. The reduced level of collagen synthesis correlated with a reduction in the steadystate mRNA levels coding for type II collagen, as measured by a Northern blot analysis. This further correlated with a reduction in the transcription of type II collagen gene, as determined by nuclear run-on experiments. Finally, transfection studies using plasmid constructs containing DNA regulatory sequences from the type II gene, coupled to a reporter gene (CAT), revealed that in comparison to control chondrocytes, interleukin-l treated cells showed a reduced level of CAT activity. These studies demonstrate that the inhibition of collagen type II synthesis by interleukin-1 is due to a reduction in the transcription of the type II collagen gene and that the reduction in gene transcription involves DNA regulatory sequences that determine type II collagen gene expression. M 1990 Academic press, Inc.

’ To whom reprint requests should be addressed. * Current address: Gerontology Research Center, tute of Aging, 4940 Eastern Avenue, Baltimore, MD

D. HIGGINBOTHAM,AND~ALTER

3 Abbreviations used: IL-l, interleukin-1; DMEM, Dulbecco’s modified Eagle’s medium; MCM, macrophage-conditioned medium; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CAT, chloramphenicol acetyltransferase; FGF, fibroblast growth factor.

Insti-

105

0014.4827/90

Copyright All rights

0 1990 of reproduction

by Academic in any form

$3.00

Press, Inc. reserved.

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CHANDRASEKHAR

of chondrocytes that should include type II collagen, and since type II collagen is important in maintaining the integrity of cartilage, we wanted to know the mechanism of inhibition of type II collagen synthesis by IL-l. Our results confirm that rabbit articular chondrocytes exposed to IL-l exhibit a reduced synthesis of type II collagen and its subsequent incorporation into the extracellular matrix. This reduction in protein level correlates with a reduction in the steady-state level of type II collagen messenger RNA. Quantitation of run-on nuclear transcripts further showed that the transcription of type II collagen gene was greatly reduced after IL-1 treatment. Finally, transfection of chondrocytes with plasmid DNA constructs containing DNA regulatory sequences for the type II collagen gene suggests that IL-l-treated chondrocytes also show a reduced ability to transcribe the type II collagen gene. We conclude that the inhibitory effects of IL-1 on type II collagen synthesis may occur via mechanisms involving DNA regulatory elements that control the transcription of the type II collagen gene. MATERIALS

AND

METHODS

Cells and Cell Culture Rabbit articular chondrocytes. With two exceptions (steady-state mRNA levels (Fig. 6) and nuclear run-on (Table 3)), all other studies reported here were conducted using rabbit articular chondrocytes maintained as monolayer cultures [8, 131. In brief, articular cartilage was removed from both tibia and femur of 2- to 3-lb New Zealand white rabbits, using sterile techniques, and was dissected into very small pieces. The pieces were initially treated for 20 min in sequence with 2 mg/ml each of hyaluronidase (Sigma) and TPCK trypsin (Worthington), followed by a 4- to 5-h treatment with collagenase (Worthington). All incubations were performed at 37°C in an atmosphere of 5% CO, + 95% air. The cells released after digestion were seeded at a density of 2 X lo4 cells/cm’ in Ham’s F-12 medium containing 10% fetal bovine serum plus glutamine and antibiotics and were grown to confluency in an atmosphere of 5% CO, + 95% air. For all the experiments described, only primary cultures were used. Rat costal chondrocytes. In order to measure the steady-state levels of mRNA for type II collagen, rib cages of 19.day rat embryos were used as the source of chondrocytes. The cells, isolated as above, were maintained in culture for 3 days in F-12 medium + 10% serum and were treated with IL-1 for additional periods of time in serum-free DMEM. Macrophage

Conditioned

Medium

Rabbit peritoneal MCM was prepared as before [13]. Cells influxed into the peritoneum 96 h after an intraperitoneal injection of light mineral oil were harvested and plated in DMEM. The attached cells (mostly macrophages) were treated with 30 ag/ml lipopolysaccharide from Escherichia coli (Sigma) for 24 h. In some experiments the conditioned medium was used as the source of IL-l. Interleukin-

I /Growth

Factors

The human IL-10 (provided by Dr. Lee Bobbit, Lilly Research Laboratories) used in these studies was of recombinant origin and was prepared by cloning and expressing a synthetic gene [23]. The endotoxin concentration, measured by a Limulus amebocyte lysate assay,

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AL.

was found to be in the range of 0.3 ng/mg. Recombinant blast growth factor was purchased from AMGEN Corp. Oaks, CA). Biosynthesis

basic fibro(Thousand

of Collagen

The effect of IL-1 on type II collagen synthesis was studied by labeling confluent monolayers of rabbit articular chondrocytes with [3H]proline (ICN; sp act, 100 Ci/mmol). Cells seeded into a loo-mm dish (6 X lo6 cells per plate) were washed with serum-free DMEM and treated with either MCM (1:20 dilution with DMEM, an amount that showed the peak activity in inducing protease activity [13,44]) or IL-la (O-100 rig/ml) for indicated time intervals (10, 30, or 48 h) in the presence of ascorbic acid (50 pg/ml). During the last 4 h of incubation, [aH]proline (100 FCi/ml) was also added to the cells. The final volume of the media was maintained at 5 ml. The media were collected, the cell layers were rinsed twice with serum-free DMEM, and the rinsings were added to the media. The cell layers were subsequently extracted with 5 ml of 4 M guanidinium chloride + 0.01 M CHAPS (Calbiochem) for 48 h at 4°C by constant shaking. Both the media and extracts of cell layers were extensively dialyzed against distilled water and an aliquot of the nondialyzable fraction was counted for radioactive incorporation. The rest of the samples were lyophilized and suspended in equal volumes of 2X sample buffer. The samples (approximately 1 X lo5 cpm) were examined by SDS-PAGE followed by fluorography (see below). In order to determine the collagenous nature of the radioactive material, the media and the matrix samples (2 X 105cpm) were solubilized in 0.5 M acetic acid, pH 2.0, and digested with pepsin (final concentration of pepsin = 100 ag/ml) at 4°C for 18 h, by constant shaking. The samples were lyophilized and the pepsin-resistant proteins were analyzed by SDS-PAGE. Some of these samples were also treated with bacterial collagenase (Advanced Biofactures, NY; results not shown). The relative levels of the pepsin-resistant collagenous hand were estimated by scanning the autoradiograms with UltroScan XL densitometer (LKB, Bromma, Sweden) interfaced with an IBM PC and with the 2400 Gel Scan XL software (LKB) capable of integrating peak areas or heights and performing data analyses [51]. SUS-PAGEIFluorography The conditioned media and the extracellular matrix of chondrocytes labeled with [3H]proline were examined by SDS-PAGE [30] using a 4% stacking gel and a 7.5% resolving gel. High-molecularweight protein standards from Bio-Rad and “‘C-labeled rat tail tendon type I collagen were included for comparison of electrophoretic mobility. The protein bands were initially visualized by staining with Coomassie blue R-250. For fluorographic detection of the radiolabeled bands, the gel was equilibrated in Enlightning (NEN), dried under vacuum, and exposed to X-ray film (X-Omat AR, Kodak) at -70°C. Measurement Blot Analysis

of Steady-State

Messenger

RNA

Levels

by Northern

The effect of II,-1 on type II collagen mRNA levels was measured by treating rat costal chondrocytes with IL-1 (100 rig/ml) or MCM (1:ZO dilution) for 10, 30, or 48 h in serum-free DMEM and then analyzing the levels of steady-state mRNA. At the end of the treatment period, total cellular RNA was prepared using a single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction [ll]. Equal amounts of RNA (10 pg per lane) were fractionated on a 1% agarose-formaldehyde gel 1241 and transferred to GeneScreen (NEN). The filter was prehybridized with denatured salmon sperm DNA (2 mg/ml) and was hybridized with a cDNA probe for rat procollagen II [50]. The prehybridization and hybridization were performed at 42’C for 18 h using a solution of 5X SSC (0.75 M NaCl, 0.075 M sodium citrate, pH 7.0) containing 5X Denhardt’s

IL-l-INDUCED reagent, 0.1% SDS, and 50% formamide. The filters were washed three times at 56°C with 1X SSC plus 0.1% SDS at 10 min intervals and were exposed to X-ray film (Kodak, X-Omat). The relative intensity of the bands was determined by scanning the X rays in a laser densitometer (Helena Labs) and comparing the relative heights of the peaks. The control values were considered to be 100% while the other values were expressed as a percentage of control. In vitro

Transcription

In Isolated

10h

Nuclei

Nuclei were isolated from fetal costal chondrocytes treated with IL-l and control cells, and elongation of nascent transcripts was carried out as described [lZa]. Approximately 1 X lo7 cells were lysed in a solution containing 0.5 ml of 0.32 M sucrose, 3 mM MgCl, 1 mA4 Hepes, pH 6.8, and 0.5% (v/v) Triton X-100. The nuclei were pelleted by centrifugation, resuspended in 2.1 M sucrose, 1 mM MgCl, 1 mM Hepes, and Triton X-100, and centrifuged for 60 min at 20,000 rpm (SW 50.2 rotor). The nuclear pellet was washed in 0.25 M sucrose, 1 mM MgCl, 1 mM Hepes and stored in liquid nitrogen. Approximately 1 X lo6 nuclei were labeled with [(?P]UTP for 5 min. Radioactive nuclei were hybridized to 1-2 pg of type II cDNA linearized by boiling and immobilized onto the GeneScreen. The hybridization was performed according to the manufacturer’s instructions. The radioactivity in the individual spots was quantitated using a Betagen analyzer. Transfection

107

SUPPRESSION

e 4 3

Medium

and CA 7’ Assa.)

In order to test the effect of IL-l on type II collagen gene transcription, rabbit articular chondrocytes were transfected with plasmid DNAs containing various regulatory sequences for the rat type II collagen gene [25]. In brief, rabbit chondrocytes seeded at a density of lo4 cells/cm* were grown to about 80% confluency. The final density at the time of transfection was approximately 1 X IO5 cells/cm’. One day before transfection, cells were fed with fresh F-12 + 10% serum, rinsed with DMEM, and transfected in serum-free DMEM with 10 pg of plasmid DNA for 3 h by the calcium phosphate precipitation method [22]. Four different DNA constructs coupled to the CAT gene were used for transfection: (a) one containing a 315.bp promoter sequence from the rat type II collagen gene plus a 1500.bp fragment of the first intron that includes the enhancer sequence, (b) the 315.bp promoter sequence alone, and (c), the Rous sarcoma virus LTR (RSV LTR) as a strong promoter that served as a control to determine the efficiency of transfection. After transfection with each of these constructs, cells were rinsed extensively to remove the DNA-calcium phosphate precipitate and treated with DMEM, IL-l (30 rig/ml), or MCM (1:20 dilution) for 48 h. All treatments were done under serumfree conditions in duplicate. The cells were lysed in 0.2 M Tris-HCl, pH ‘i.9, by sonication and the lysates were assayed for CAT activity using [i4C]chloramphenicol as the substrate. RESULTS

Effect of IL-1 on Type II Collagen Synthesis Articular Chondrocytes

by Rabbit

Initially, confluent monolayers of rabbit articular chondrocytes at various passages were tested for their ability to maintain their phenotypic stability with reference to the expression of type II collagen molecule. This was studied by treating the primary, secondary, or tertiary cultures with [3H]proline for 24 h and then analyzing the protein products released into the medium by SDS-PAGE followed by Auorography. The results (data not shown) indicated that under these conditions, both primary and secondary passage cells contained a

Matrix

Total

Treatments FIG. 1. Effect of IL-l/MCM on [3H]proline incorporation. Rabbit articular chondrocytes were grown to confluency and treated with IL-1 (30 rig/ml) or MCM (1:20 dilution in DMEM) in the presence of ascorbic acid (50 pg/ml) for the indicated times (10 or 30 h). During the last 4 h of treatment, cells were also incubated with [3H]proline (10 &i/ml). The media were collected, and the cell layer was rinsed and extracted with 4 M GuHCl + 0.01 MCHAPS for 48 h at 4°C. Both the media and the cell/matrix extracts were dialyzed against distilled water and the radioactive incorporation in the nondialyzable fractions was determined.

band that comigrated with an authentic a,(I)-collagen band but did not synthesize any detectable levels of the 01~(1) band. In contrast, the tertiary cultures synthesized both type I and II collagens (data not shown). These results were in confirmation of the previous observations indicating the loss of chondrocytic phenotype with increasing in vitro age [Z, 351. Therefore, for all the subsequent studies, only primary cells maintained no more than a week in culture were used. The effect of IL-l on collagen synthesis was examined by pretreating the primary chondrocytes with human recombinant IL-10 (100 rig/ml) or 120 dilution of MCM (as a source of IL-l) for 10 or 30 h in the presence of [3H]proline (10 &i/ml) and ascorbic acid (50 pg/ml). Previous studies have shown that MCM at 1:20 dilution showed maximal activity in inducing neutral protease [13, 44, and unpublished data]. The conditioned media and the extracellular matrix + cell layer, extracted with 4 M GuHCl + 0.010 CHAPS, were evaluated either quantitatively (Fig. 1) or qualitatively (Figs. 2 and 3). Quantitative comparison of the relative incorporation of [3H]proline in the nondialyzable fractions showed

108

CHANDRASEKHAR

TABLE Effect

of IL-l

on Type

1 II Collagen Type

Treatment Control IL-1 MCM

II collagen (% control)

Levels levels

10 h

30 h

100 38 i 5 37 f 3

100 33 + 6 0

Note. The protein bands corresponding to lanes 2,4, and 6 (10 h), to lanes 8, 10, and 12 (30 h) (Fig. 2), and from two other similar experiments were scanned using a laser densitometer. The relative peak heights were obtained by a software designed to integrate the absorbance (Materials and Methods). The average peak height of the 10-h control was 0.814 absorbance unit, whereas the peak height of the 30-h control was 1.183 absorbance units. The peak heights of the controls in each category were considered to be 100% and the other values are expressed as a percentage of control f mean deviations.

that (a) IL-l or MCM treatment of chondrocytes for 10 h resulted in an increase in the proline incorporation in the conditioned media and the matrix; (b) however, after 30 h of treatment with IL-l or MCM, the proline incorporation was decreased. Since proline incorporation may reflect the synthesis of both collagenous and noncollagenous proteins, we next analyzed these molecules qualitatively by SDSPAGE. The results are shown in Figs. 2 and 3. Rabbit chondrocytes were treated with IL-l or MCM for either 10 or 30 h; the conditioned media proteins were treated initially with pepsin for 18 h at 4°C (to remove noncollagenous extension peptides and other noncollagenous proteins) and were examined by SDS-PAGE/fluorography (Fig. 2 and Table 1). As expected, pepsin treatment of all conditioned media samples resulted in the processing of the procollagens into pepsin-resistant collagenous bands that comigrate with the a,(I) band of the purified type I collagen (Fig. 2, lanes 2,4, and 6). Within 10 h of treatment with either IL-l (lanes 3 and 4) or MCM (lanes 5 and 6) chondrocytes showed a reduction in the amount of type II collagen. By 30 h after treatment, both IL-l- and MCM-treated cells exhibited a further reduction of type II collagen synthesis, with MCM showing a greater inhibitory effect. Quantitative comparison of the pepsin-resistant collagen bands after densitometric scanning (Table 1) indicated that within 10 h of treatment with either IL-l or MCM, type II collagen synthesis was reduced to 38% of the control values. IL-l treatment for 30 h resulted in a reduction of type II collagen to 33% of control, while MCM treatment resulted in complete loss of synthesis of collagenous protein. The inhibitory effect of IL-l/MCM on type II collagen was also reflected in the matrix + cell layer of cells treated with IL-l/MCM. Control chondrocyte matrix

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AL.

contained a major band that comigrated with standard a,(I) type I collagen and is likely to be type II collagen. A 30-h IL-l treatment (Fig. 3, lane 2) resulted in a greatly reduced amount of a band corresponding to type II collagen, while MCM (Fig. 3, lane 3)-treated cells show virtually no detectable levels of the type II band. These results confirm that IL-l-treated chondrocytes contained greatly reduced amounts of type II collagen in the matrix. We next tested the effect of various concentrations of IL-l on type II collagen synthesis. IL-l (O-100 rig/ml) was added to chondrocytes for 30 h and the [14C]proline-labeled procollagens released into the conditioned media were examined after pepsinization, as before. The results (Fig. 4) show that the minimal effective concentration was 0.3 rig/ml and a dose-dependent inhibition of collagen synthesis was observed. However, even at 100 nglml, complete inhibition was not observed within 30 h of treatment with IL-l. In both the medium and the matrix (Figs. 2 and 3), MCM-treated cells showed a more pronounced reduction in the type II collagen levels than IL-l-treated cells. Because MCM contains several other growth factors that modulate the synthesis of degradative enzymes, the greater reduction in collagen level after MCM treatment may reflect cooperativity among these factors on collagen synthesis and/or degradation [ 10, 39, 431. In order to distinguish between these possibilities, we

123456

7 6

9 10 1112

FIG. 2. Effect of IL-l/MCM on type II collagen synthesis and release into the conditioned media. Cells were pretreated with either IL-1 (30 rig/ml) or MCM (1:20 dilution in DMEM) for either 10 or 30 h in serum-free DMEM. During the last 4 h of treatment, cells were also incubated with [3H]proline (10 &i/ml). The media were dialyzed against 0.5 M acetic acid, pH 2.0, and divided into two equal halves. One half was treated with pepsin (100 fig/ml) for 16 h at 4°C. The samples were lyophilized and analyzed by SDS-PAGE (7.5% resolving/4% stacking)/fluorography. Type I collagen was included as a standard. Lanes 1 and 7, Control media; lanes 2 and 8, control media + pepsin; lanes 3 and 9, IL-l-treated chondrocyte media; lanes 4 and 10, IL-l-treated chondrocyte media + pepsin; lanes 5 and 11, MCMtreated chondrocyte media; and lanes 6 and 12, MCM-treated chondrocyte media + pepsin.

IL-l-INDUCED

109

SUPPRESSION

ZOOK *n,(ll) 116K\_ 93K -

i

68K -

,

-3 ,= ‘::

Procollagenase 43K -

1

2

3

4

5

678

PepsIn,

123

4

FIG. 3. Effect of IL-l/MCM on type II collagen incorporation into the cell layer/matrix. Extracts of matrix/cell layers after a 30-h treatment with either IL-l or MCM (fig. 2), were analyzed by SDSPAGE (4% stacking ge1/7.5% resolving), followed by fluorography. Each lane contained 50,000 cpm. Lane 1, type I collagen; lane 2, control chondrocytes; lane 3, IL-l-treated cells; and lane 4, MCM-treated cells.

chose to examine whether FGF, a known component of MCM [39], will modulate the effects of IL-l on type II collagen synthesis. We have previously demonstrated

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IL-1 Cont.,

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FIG. 4. Effect of various concentrations of IL-l on type II collagen synthesis. Rabbit chondrocytes were treated (see Fig. 2) with various IL-l concentrations (O-100 rig/ml) for 30 h. During the last 4 h of treatment, cells were also treated with [i4C]proline (10 PCilml). The conditioned media proteins were examined by SDS-PAGE, after pepsinization. The bands were quantitated by densitometry and are expressed as a percentage of control.

+ ,

FIG. 5. Effect of FGF on IL-l-induced inhibition of collagen synthesis. Chondrocytes were initially treated with DMEM, IL-l (30 ng/ ml), FGF (10 rig/ml), or IL-1 + FGF for 10 h and during the last 4 h, [3H]proline (10 &i/ml) was also incubated. The media were dialyzed against 0.5 M acetic acid, pH 2.0, and one-half of the media was subjected to pepsin digestion as in Fig. 4. The samples were analyzed by SDS-PAGE. Lanes 1 and 5, control media; lanes 2 and 6, IL-l-treated media; lanes 3 and 7, FGF-treated media; lanes 4 and 8, IL-l and FGF media; lanes l-4, nonpepsinized; and lanes 5-8, pepsinized samples of lanes l-4.

that induction of degradative enzymes by IL-l was potentiated by FGF [lo]. The [3H]proline-labeled media of the chondrocytes treated with IL-l, FGF, or a combination thereof, for 10 h were examined by SDS-PAGE. The bands corresponding to iV, = 57 kDa and 53 kDa have previously been identified as procollagenase/prostromelysin [lo]. In confirmation of our previous observations, IL-l alone induced the synthesis of procollagenase (Fig. 5, lane 2), while a combination of IL-l plus FGF (lane 4) induced a greater level of procollagenase synthesis. Control (lane 1) and FGF-treated cells (lane 3) did not synthesize any detectable level of procollagenase. Examination of the conditioned media after pepsin treatment (Fig. 5, lanes 5-8) shows that the extent of inhibition of type II collagen synthesis by IL-l (lane 6) and that by IL-l + FGF (lane 8) were comparable. FGF alone caused no discernible effect on collagen synthesis after this treatment time (lane 7). These studies suggest that at the earlier time point (10 h), FGF neither potentiates nor reverses the inhibitory effect of IL-l on collagen synthesis although it has a dramatic effect on the induction of degradative enzyme, namely, collagenase. Thus, it is likely that the greater reduction in the level of type collagen after MCM treatment may

110

CHANDRASEKHAR IOh r-w

30h

ab

ab

48h

10h

30h

r-

5.2 kb,

ab

IL.1

ab

ab MCM

FIG. 6. Northern blot analysis of type II mRNA levels after ILl/MCM treatment. Rat costal chondrocytes were plated at high density (2 X lo5 cells per cm’) into a loo-mm dish and maintained in F-12 medium + 10% fetal bovine serum for 3 days. The media were removed and cells were rinsed and incubated with IL-I (100 rig/ml) or MCM (1:50 dilution) for indicated times. All dilutions were made in serum-free DMEM. Control cells were similarly treated with DMEM alone. At the end of the treatment, total cellular RNA was isolated, electrophoresed in a 1% agarose-formaldehyde gel, and transferred onto GeneScreen. The filters were prehybridized with 2% denatured salmon sperm DNA dissolved in a hybridization buffer (see Materials and Methods) for 16 h at 42°C and then hybridized with a rat type II procollagen cDNA probe for 16 h at 42°C. The filters were thoroughly washed at 56°C (refer to Materials and Methods) and exposed to an X-ray film. Lane a, control (DMEM) cells; lane b, (IL-l or MCM) treated cells.

be a consequence of increased degradation rather than greater inhibition of synthesis. Effect of IL-1 on the Steady-State Collagen mRNA

Level of Type II

The reduced synthesis of type II collagen after IL-l treatment could be either due to a reduction in the steady-state level of the type II collagen transcripts or due to inefficient translation of normal type II collagen mRNA level or both. Therefore, we measured the steady-state levels of type II collagen mRNA from control, IL-l-, and MCM-treated cells. Since the rat type II cDNA did not recognize the rabbit RNA, for this experiment, costal chondrocytes isolated from rat embryos were used. Initially, we confirmed that rat cells treated with IL-l (30 or 100 rig/ml) showed a reduction in type II collagen synthesis (52% of control values at 100 ng/ ml of IL-l). In order to measure the steady-state mRNA levels, chondrocytes were treated with IL-l (100 rig/ml) or MCM (1:20 dilution in DMEM) for 10, 30, or 48 h in serum-free medium. At the end of the incubation period, total cellular RNAs were prepared by a single-step extraction procedure, electrophoresed, transferred to GeneScreen, and hybridized with a cDNA probe for the rat type II procollagen gene [50]. Analysis of mRNA by Northern blot showed that control chondrocytes synthesized a prominent band of 5.2 kb (Fig. 6, lane 1) that is consistent with the reported size for the type II collagen

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AL.

mRNA [50]. Cells treated with IL-l or MCM (at all time points) showed the presence of transcripts of identical size, indicating that IL-l/MCM treatment resulted in no major qualitative differences in the steady-state mRNA. In general, a time-dependent reduction of mRNA level was observed of the control cells maintained in serum-free medium for various times (10 > 30 > 48 h). Further, quantitative differences in the type II transcript levels were observed between control and treated cells at each time point. Within 10 h of treatment with either IL-l (Fig. 6, lane 2) or MCM (Fig. 6, lane 3), the treated cells displayed a reduction in the steady-state levels of type II collagen mRNA. The inhibition was more complete in cells treated with IL-l for 48 h. Densitometric scanning of the relative levels of mRNA after a 10-h treatment of cells with either IL-l or MCM (Table 2) revealed that the treated cells exhibited only 39-41% of the control mRNA levels. These results demonstrate that IL-l/MCM treatment of chondrocytes resulted in reduced levels of type II mRNA and may, at least in part, account for the reduction in the protein products. Inhibition of Transcriptional Collagen Gene by IL-1

Elongation of Type II

In order to determine if the reduction in mRNA level was due to a reduction in type II gene transcription, nuclear run-on experiments were carried out on nuclei isolated from rat costal chondrocytes that were treated with IL-l (30 rig/ml) for 30 h. Labeled nascent transcripts were hybridized to rat type II cDNA or actin cDNA. Quantitation of the slot blot (Table 3) shows that IL-l treatment of chondrocytes resulted in a significant reduction in transcription of type II collagen. In contrast, under identical conditions, the actin gene transcription was enhanced after IL-l treatment, indicating that the inhibitory effect of IL-l on type II collagen gene transcription was specific.

TABLE Effect

of IL-l

2

on Steady-State

Type

II

Collagen mRNA Levels Type Treatment Control IL-1 MCM

II collagen

mRNA

levels

(% control)

10 h

30 h

48 h

100 38 41

100 39 37

100 0 determined

Not

Note. The values are representative of a typical experiment. The relative levels of mRNA obtained after treatments with IL-l or MCM for indicated times (from Fig. 6) are shown. The bands corresponding to the transcript size of 5.2 kb were scanned by a densitometer. In each case, the control values were considered to be 100%.

IL-l-INDUCED TABLE Nuclear

Run-on

111

SUPPRESSION

3

Analysis of Type II Collagen After IL-1 Treatment Transcript

Transcription

levels (% control)

Treatment

Type II collagen

Actin

Control

100

100

44

151

a

IL-l

Note. Rat chondrocytes were treated (30 rig/ml, 30 h), the nuclei were isolated transcripts were labeled using [32P]UTP. bridized to rat type II cDNA or actin intensity was quantitated by exposing lyzer.

Inhibition by IL-l

b

c

a

b

c

with DMEM (control) or IL-1 as described, and the nuclear The labeled RNA was hycDNA. The hybridized signal the filter to a Betagen ana-

of Type II Collagen Gene Transcription

Previous studies have established the presence of two DNA regulatory elements for the rat type 11 collagen gene, namely, (a) a promoter sequence in the 5’ flanking region and (b) an enhancer element in the first intron [25]. These studies have also shown that the activity of the DNA elements correlates with the transcriptional activity of the cellular type 11collagen gene. In order to determine whether the inhibitory effects of IL-1 occurred at the level of type 11collagen gene transcription, we next examined whether IL-1 treatment of chondrocytes resulted in reduced activity of the type 11collagen gene regulatory elements. This was determined by transfection of chondrocytes with various plasmid DNA constructs containing the regulatory sequences for the type 11collagen gene coupled to a reporter gene (CAT) and then treating the cells with IL-1 or MCM for 48 h. The cells were lysed and the lysates were analyzed for the transcription of the CAT gene by assaying for CAT activity using [14C]chloramphenicol as the substrate. Both control and IL-1 (or MCM)-treated rabbit articular chondrocytes showed no detectable CAT activity when they were transfected with CAT-type 11promoter (Fig. 7) or CAT-type 11enhancer (Horton and Chandrasekhar, unpublished data). A moderate level of CAT activity was observed in rabbit chondrocytes only when they were transfected with a construct containing both the type 11 collagen gene promoter and enhancer sequences in the same construct (Fig. 7 and Table 3). This is in agreement with previous observations of rat and chick chondrocytes [25, 261. IL-l/MCM treatment of cells abolished the expression of type 11promoter + enhancer coupled CAT gene, but not RSV-CAT expression. Quantitative comparison of the CAT activity by densitometric scan of acetylated products (Fig. 7) confirmed that IL-l- or MCM-treated cells showed only 5-8s of control level of activity (Table 4). These studies

b

c

b

c

FIG. 7. Effect of IL-1 on type II collagen promoter activity. Rabbit articular chondrocytes were cultured as monolayers. When the cells reached approximately 80% confluency, cultures were transfected in duplicate, with plasmid constructs containing (a) type II promoter alone, (b) type II promoter + enhancer and (c) RSV-CAT, as described under Materials and Methods. After transfection for 3 h, cells were washed and treated with DMEM, IL-1 (30 rig/ml), or MCM (120 dilution in DMEM) for another 48 h at 37°C. All treatments were performed using serum-free DMEM. At the end of the treatments, cell lysates were analyzed for CAT activity using [“‘Clchloramphenicol as substrate. The acetylated products were separated by thin-layer chromatography and identified by exposure to an X-ray film.

demonstrate that the IL-1 inhibition of type 11collagen synthesis is likely to be a direct consequence of the inhibition of the type 11collagen gene transcription and that the effects of IL-1 were specific to type 11 collagen promoter and not to other unrelated promoters. DISCUSSION

Previous studies have suggested that IL-1 may decrease the synthesis of type 11 collagen [16, 21, 481. In

TABLE

4

Effect of IL-l/MCM on Type II Collagen + Enhancer CAT Activity Relative Treatment Control IL-1 MCM

(DMEM)

Type

levels

II promoter 100 8k3 5f2

of CAT

activity

+ enhancer

Promoter

(% control) RSV promoter 100 94 + 8 90 + 7

Note. After transfection with various constructs, the CAT activity was assayed as in Fig. 7. The spots, visualized by exposure to X rays, were quantitated by densitometric scan. The control CAT activity in each case was considered to be 100%. The values represent the averages of three independent experiments (*SD), with each treatment conducted in duplicate.

112

CHANDRASEKHAR

this report, we have examined the mechanism by which IL-l inhibited the synthesis of type II collagen. This was studied by testing the effects of IL-l at three different levels of collagen metabolism: (1) analyses of [3H]proline-labeled protein products, (2) measurement of the steady-state levels of type II collagen mRNA by Northern blot analysis, (3) determination of nuclear run-on transcription, and (4) measurement of the transient activity of type II collagen gene regulatory elements in chondrocytes. Our results indicate that chondrocytes exposed to IL-l show a reduction in type II collagen synthesis because of reduced type II collagen gene transcription. The collagenous nature of the proteins was demonstrated on the following criteria: (a) the ability of the proteins to incorporate [3H]proline in the presence of ascorbic acid, (b) limited susceptibility to pepsin treatment, (c) electrophoretic comigration of pepsinized conditioned media proteins with the ai collagen band of purified rat-tail tendon collagen, and (d) digestibility of the proteins with bacterial collagenase (data not shown). The decrease in the synthesis of type II collagen as a result of IL-l treatment was reflected both in the amount of collagen synthesized/secreted into the medium and in that incorporated into the extracellular matrix/cell layer of chondrocyte cultures. The inhibition of collagen synthesis was dose-dependent (Fig. 4). The maximal inhibition occurred at 1 ng/ ml of IL-l, with no further inhibition up to 100 rig/ml. Our previous studies on the induction of protease activity and receptor saturation suggested a maximal response occurring at lo-20 rig/ml [lo]. An interpretation of these results is that IL-l may be more effective in inhibiting type II collagen synthesis, with minimal receptor occupancy. However, it is also worth noting that within 30 h of treatment, complete inhibition of collagen synthesis was not observed (up to 100 rig/ml of IL-l). The cause for this observation is not known at present. It is possible that cells become refractory, because IL-l receptors become down-regulated. Alternatively, it may reflect the need for other factors that may work in concert with IL-l in causing the inhibition of type II collagen synthesis. The inhibition of type II synthesis was observed within 10 h of treatment with IL-l or MCM (Fig. 3 and Table l), was more pronounced after a 30-h treatment, and was complete by 48 h. This was particularly evident in MCM-treated cultures (Figs. l-3). Both MCM and IL-l showed comparable inhibitory effects after a 10-h treatment at all levels of analysis (protein, RNA, and gene transcription). However, after a 30-h treatment period, MCM-treated cells contained no collagenous bands (Figs. 2 and 3) although both IL-l- and MCMtreated cells showed comparable inhibition of the steady-state type II collagen RNA levels (38%; Fig. 6 and Table 2). The greater inhibitory effect of MCM on

ET

AL.

protein level, but not RNA level, at the 30-h period could be due to differential effects of IL-l and MCM on synthesis versus degradation. Our previous studies have shown that MCM was much more potent than IL-l in inducing matrix-degrading protease activity which would lead to the degradation of collagen [lo, 441. This is because MCM contains IL-l and several other growth factors that can potentiate the ability of IL-l to induce procollagenase activity [lo, 39, 431. Thus, the greater reduction in collagen level after MCM treatment is likely to be due to the cumulative effects on synthesis plus degradation. The studies in which a combination of IL-l + FGF was able to induce high levels of procollagenase but not alter the IL-l-induced reduction in type II collagen levels (Ref. [lo] and Fig. 5) further support that FGF, a known component of MCM, may modulate the IL-l activity primarily on degradation of collagen but not the effect of IL-l on collagen synthesis, at the earlier time point. However, since MCM contains other growth factors such as TGF-0 and PDGF [39], the cumulative effect is likely to be influenced by those factors as well. The reduction in the collagen synthesis appears to be a direct consequence of a reduced level of steady-state mRNA coding for type II collagen. In order to measure the levels of mRNA, we used a cDNA probe from rat chondrosarcoma [50]. Since this probe did not recognize rabbit articular chondrocytes (unpublished data; Horton and Chandrasekhar), we used rat embryonic costal chondrocytes and have confirmed that these cells also showed reduced type II collagen synthesis in response to IL-l (52% of control values at 100 rig/ml of IL-l). Northern blot analysis using RNA preparations from these cells revealed that within 10 h of treatment with IL-1 or MCM, cells synthesized only 40% of the control levels of the steady-state type II transcripts. The inhibition was virtually complete after 48 h of treatment with IL-l. These studies suggest that the reduction in the type II collagen levels is at least in part due to a reduced level of mRNA coding for type II collagen. The reduction in the mRNA level may be due to (a) reduced transcription of collagen II gene or (b) a reduction in the stability of the type II collagen mRNA or both. That the reduced level of mRNA was at least in part due to an inhibition of transcription of the type II collagen gene was confirmed by (a) determination of transcription of nascent chains in vitro and (b) by transfection experiments using a recombinant plasmid containing DNA regulatory sequences (promoter + enhancer) coupled to a marker gene, CAT, and then monitoring the expression of CAT gene activity in response to IL-l. Previous studies have indicated that the 5’ flanking region of the type II collagen gene contains the cell-specific promoter for the initiation of type II collagen transcription and that the first intron contains an enhancer sequence [25]. Those studies have also indicated that the transcription

IL-l-INDUCED

of the type II gene required a cooperative interaction between the two regulatory elements. If the effects of IL-l were at the level of gene transcription, it is likely to involve one or more of the regulatory sequences. Our studies confirm that the expression of type II collagen in rabbit chondrocytes requires both promoter + enhancer elements and was inhibited by IL-l/MCM treatment. However, this is relatively specific effect, since IL-l/ MCM did not affect the expression of RSV promoter. As in previous studies using these constructs, the expression of type II gene requires a cooperation between both the promoter and the enhancer sequences. Transfection of cells with CAT constructs containing either type II promoter or enhancer alone resulted in no detectable CAT activity [ 251 and IL-l had no influence in this process (data not shown). We do not know the precise mechanism(s) of the inhibitory effects of IL-l on the regulatory sequences. It could potentially involve various transacting elements that may be responsive to various signal transduction mechanisms. Whether IL-l affects the promoter or enhancer or both remains to be studied. The reduction in collagen synthesis was relatively selective, since the general protein synthesis ( [35S]methionine incorporation) was either unaffected or slightly increased after IL-l/MCM treatment [lo]. Further, the total proline incorporation after a 10-h treatment (Fig. 1) reflects an increase, whereas a qualitative analysis of type II collagen band in an SDS gel (Figs. 2 and 3) revealed a decrease in type II collagen synthesis. This would imply that while IL-l/MCM may reduce type II collagen synthesis, it may stimulate the incorporation of proline into noncollagenous proteins such as procollagenase (Fig. 2; Ref. [31]) and prostromelysin (Chandrasekhar, Hrubey, and Harvey, unpublished data). This is further supported by our observations where IL1 increases the mRNA levels for prostromelysin/collagenase (manuscript in preparation), whereas the mRNA levels for type II collagen were greatly reduced (Fig. 5). It is difficult to determine whether the reduction in type II collagen synthesis occurs because of the increase in the synthesis of other noncollagenous proteins. In addition to its effect on collagen synthesis, IL-1 has been shown to inhibit the synthesis of cartilage proteoglycan [9, 15, 45-471 and link proteins (Harvey and Chandrasekhar, unpublished observations). It would therefore appear that IL-l selectively down-regulates the synthesis of matrix macromolecules, but stimulates the expression of enzymes that cause the degradation of cartilage. Thus, IL-l may play a dual role in cartilage metabolism by coordinately regulating the expression of several cartilage genes and may influence the biology and pathology of cartilage extracellular matrix. The in vivo significance of these studies remains to be established. Cartilage damage is a common feature of rheumatoid arthritis and osteoarthritis. IL-l is proba-

113

SUPPRESSION

bly one of the potent inflammatory mediators and is likely to play an important role in the etiology of cartilage degenerative diseases [la, 13, 15, 20, 36, 38, 431. Although studies on human osteoarthritic cartilage have indicated that chondrocyte repair can occur, it is clear that the repair process is inadequate and does not result in a proper redistribution of chondrocytes throughout the tissue probably because of an extracellular matrix that is inhospitable to the cells [3, 28, 311. Previous studies have suggested that in degenerative joint diseases (such as osteoarthritis), the major collagen of the cartilage is type I collagen rather than type II collagen [40], although this has been disputed [ 181. While this discrepancy remains to be resolved, it is clear that IL-l induces a phenotypic alteration in the expression of collagen types by chondrocytes and causes the synthesis of noncartilage type collagen [al]. Thus, IL-l may interfere with cartilage repair not only by causing a reduction in cartilage-specific molecules, but also by inducing qualitative changes in the type of extracellular matrix synthesized and incorporated by chondrocytes. Ultimately, whether or not a proper repair of cartilage occurs is likely to depend on the ability of chondrocytes to synthesize and organize various connective tissue macromolecules even in the presence of inflammatory signals such as IL-l. The authors are grateful to Dr. Lee Bobbitt and Mr. Joe Manetta (Biochemistry department, Lilly Research Labs) for providing us with recombinant IL-/3 and to Dr. Steven Zuckerman for help in densitometric scanning of the protein gels and for critical evaluation of the manuscript.

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