Vol. 147, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

September 15, 1987 Pages 550-555

INSULIN STIMULATES SECRETION OF A COLLAGENASE INHIBITOR BY

SWARM RAT CHONDROSARCOMA CHONDROCYTES

Judith Harper and Elvin Harper

Department of Chemistry, D-006,

University of California, San Diego,

La Jolla, CA 92093

Received July 20, 1987

Swarm rat chondrosarcoma chondrocytes produce an inhibitor of collagenase similar to that found in bovine articular chondrocytes and extracts of bovine scapular cartilage. These cells syn- thesize normal levels of cartilage type proteoglycana when cultured in serum free medium with insulin. Collagen synthesis is also increased when insulin is added to chondrosarcoma chondro- cytes. We have demonstrated that insulin stimulates collagenase inhibitor production by these chondrocytes. Enhancement of inhibitory activity occurs over the range of 10 to 1000 rig/ml. A 3.2 fold stimulation was observed at a concentration of 1 pg/ml. There was a lag period of 24 to 48 hours before the insulin effect became evident. Latent or active collagenaze was not detectable under these conditions. These results suggest that the hormone insulin controls the levels of col- lagen in this tumor by stimulating synthesis of collagen and inhibitors of collagenase. 0 1987 Academic Press. Inc.

We have previously demonstrated that minced tumor cultures from the Swarm rat chondro-

sarcoma secrete an inhibitor of human skin and rabbit skin collagenases (1). Thii inhibitor was

purified, partially characterized, and its properties appear to be similar to those described for an

inhibitor from extracts of bovine scapular cartilage (2). Since serum contains inhibitors of col-

lagenase (3), an evaluation of the levels of collagenase and its inhibitors produced by cells is com-

plicated by the presence of serum components. We avoided this problem by studying collagen

catabolism in chondrocytes isolated from the Swarm rat chondrosarcoma, because these cells can

be cultured in the absence of serum for as long as 2 weeks. When insulin is added to these cells,

they synthesize proteoglycans at levels comparable to those obtained with serum supplementation

(4). Insulin is important for growth of the Swarm rat chondrosarcoma and physiological concen-

trations of the hormone have been shown to enhance both proteoglycan and collagen synthesis in

primary cultures of chondrocytes isolated from thii tumor (4,5).

0006-291X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved. 550

Vol. 147, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The regulation of collagenase inhibitor production may play an important role in maintain-

ing the large tumor mass, since type II collagen is one of the major protein components (6) of the

tumor matrix. We have examined the effect of insulin on the level of collagenaze inhibitor that is

produced by primary cultures of chondrosarcoma chondrocytes.

MATERIALS AND METHODS

Female Sprague-Dawley rats were obtained from Simonsen Laboratories, Gilroy, CA. Swarm rat chondrosarcoma tissue was kindly supplied by Dr. Karl Piez of the Collagen Corpora tion, Palo Alto, CA, and passaged into rats in our laboratory. HEPES, TES, BES and porcine insulin were purchased from Calbiochem-Behring, La Jolla, CA. Tissue culture media, trypsin solution, serum and antibiotics were obtained from the UCSD Tissue Culture Facility. Bacterial collagenase was obtained from Worthington Biochemicala, Freehold, New JerBey, and NITEX screens from Tetko, Elmsford, New York. Sephacryl S-200 wsz purchased from Pharmacia Inc., Piscataway, New Jersey. All other chemicals were reagent grade.

Chondrosarcomas were grown in 6 to 8 week old Sprague-Dawley rats by subcutaneous injec- tions of 0.5 to 1 g tumor mince in 0.5 ml to 1.0 ml of phospate buffered saline, pH 7.2. After 6 to 8 weeks, chondrosarcoma tumors (20-40 g) were removed and the chondrocytes isolated according to the method of Kimura et al (7), modified by Stevens et al. (4). Briefly, the cells were isolated by mechanical disruption using a 1 mm2 stainless steel sieve. The resulting mixture was then incubated with 0.16% trypsin for 40 minutes at 37 ’ followed by a 90 minute digestion with 0.2% bacterial collagenase (Worthington, Class I). Cells were then filtered through nylon screens (NITEX). An initial filtration was performed with a 70 ~1 screen followed by a 20 p screen to yield single cell suspensions. The cells were counted in a hemacytometer, and plated at a final density of 1-2 x 10s cells /ml. Trypan blue exclusion showed 85 to 90% viability. Chondrocytes were cultured in Dulbecco’s modified Eagle’s medium with 4.5 g glucose/liter containing 15 mM HEPES, 10 mM TES, 10 mM BES, pH 7.2. Penicillin (100 units/ml) and streptomycin (100 ug/ml) were also included. The chondrocytes were initially plated for 18 hours in medium con- taining 20% fetal calf serum to allow for cell attachment (day 0). After the cells attached, the serum containing medium was removed, the cells washed three times with serum free medium, and insulin added (day 1). Media were harvested daily and replaced with fresh media containing either insulin or the same dilution of the vehicle (0.01 N HCL). Protein was determined by the method of Bradford (8).

Partial purification of collagenase inhibitor was performed on media from cultures of both whole minced tumor and isolated tumor chondrocytes. The culture media was concentrated five fold by ultrafiltration on an Amicon YMlO membrane. Samples were dialyzed against 50 mM Tris-HCl, pH 7.4, 5 mM CaC12, 500 mM NaCl, 0.01% Brij-35 (Buffer A) and applied to a column (1.5 cm x 7.5 cm) of ConA-Sepharose equilibrated in the same buffer. Fractions (3 ml) were col- lected at a flow rate of 30 ml/hour. Bound inhibitor was eluted with buffer A containing 2% cy- methyl mannoside. The pooled inhibitor was concentrated on a YMlO membrane, dialyzed against buffer A and fractionated on a Sephacryl S-290 column (1.5 cm x 95 cm). Fractions (1.8 ml) were collected at a flow rate of 12 ml/hour. All chromatography procedures were carried out at 4’C.

Collagenases were prepared from cultures of human facial skin and rabbit back skin (10,ll). Media with activity were pooled, concentrated by lyophilization, and partially purified by precipi- tation between 30% and 60% (NH1)2SOI. The coltagenase is fully active, and no increase in activity was observed upon treatment with trypsin.

Collagenase inhibition assays were conducted az described previously using the “C-glycine peptide releese. assay (9). Thii assay employs neutral salt soluble guinea pig skin collagen purified

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after in Go labelling with “C-(U) glycine (4). Briefly, 50 ~1 (1200-1600 cpm) of a solution (4 mg/ml) of collagen in 0.4 M NaCl were added to 400 ~1 capacity tubes and fibrils allowed to form by incubation for 18 to 24 hrs in a 37 * C water bath. An aliquot of medium (RI-100 ~1) was preincubated with collagenase (100 ~1; 0.06-0.08 unit) in the presence of 50 mM Tris-HCl, pH 7.4, 5 mM CaClz, 500 mM NaCl, 0.01% Brij-35 for 15 minutes at room temperature (20-22 ’ C). The reaction was terminated by centrifugation in a Beckman 152 microfuge for 5 min at room tem- perature. Aliquots of the supernatant were counted in Biofluor (New England Nuclear) on a Beckman LS-3133T scintillation counter. A trypsin control was included to correct for counts released by any denatured or noncollagenous labelled to correct for counts released by any dena- tured or noncollagenous labelled protein cleavage, and collagenase activity expressed as cpm above the trypsin blank. One unit of collagenase degrades 1 pg of collagen per minute at 37 l C.

Percent inhibition was determined by this formula:

1 _ cpm (collagenase + inhibitor )] cpm (collagenase + buffer )

x 100 = % inhibition

One unit of inhibitor inactivates one unit of eollagenase.

RESULTS AND DISCUSSION

We had previously isolated an inhibitor of collagenase from cultures of minced tumors (1).

The source of this inhibitor was not known, since host and tumor derived cells may have the

capability to synthesize the inhibitor. We isolated chondrocytes from the tumor and examined

the medium from these cells for collagenase inhibition. Our results indicate that chondrocytes

produce a collagenase inhibitor with properties similar to those described for the inhibitor iso-

lated from cultures of tumor. Both preparations of inhibitor bind to ConcanavalinA-Sepharose

and elute with similar molecular weights from a Sephacryl S-200 column (Figure 1). We

0.200

z

$

1 0.100

x 6

2 cl

0.000 I 80 i5 C 0

'Z fjo :$

.r

0 10 20 30 40 50 60 70 80 90 fraction number

Figure 1. Sephacryl S-200 chromatography of rat chondrosarcoma inhibitor isolated from minced tumor cultures. o-o absorbance at 280 nm; CO collagenaze inhibition per 100 ~1 aliquot. Fractions (1.8 ml) were collected at a Row rate of 12 ml/hour. K,, for minced tumor culture inhibitor = 0.30; K,, for chondrocyte derived inhibitor = 0.31.

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Vol. 147, No. 2. 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o.oL 0

0-Ocontro l ----0insulin

2 4 6 0

days in culture

I

Figure 2. Daily secretion of collagenaee inhibitor by chondrosarcoma chondrocytes in the pres- ence of insulin. o-o control; o---o insulin (1 pg/ml). Duplicate cultures were assayed for each day and the average calculated. Inhibitor is expressed as total units in 2 ml medium (2 x 10’ cells).

estimated the molecular weight to be 35,000 daltons. The chondrocyte-derived inhibitor had a

higher initial specific activity (5-10 units/mg protein) than inhibitor isolated from tumor cultures

(0.2-0.5 units/mg protein). We concluded that Swarm rat chondrosarcoma chondrocytes

represent at least one cellular source of tumor derived inhibitor. We have determined the effect

of insulin on the levels of inhibitor production by these chondrocytes, since insulin has been

shown to promote the growth of this tumor.

The effect of daily additions of insulin on inhibitor production was examined over a seven

day period (Figure 2). Initially, the amount of inhibitor secreted by the chondrocytes was similar

in both control and insulin treated cultures. High levels of inhibitor were observed on day 1 in

both control and experimental cells. This appears to be indicative of a dramatic rise in matrix

synthesis following enzymatic removal of the chondrocytes’ extracellular matrix (12). I f one

quantitates the total units of inhibitor produced in days 2-7, a 2 to 3 fold increment in inhibitor

is obtained from insulin treated chondrocytes (Table 1). Stimulation of inhibitor synthesis is

observed at concentrations of insulin aa low as 10 rig/ml. Medium alone supplemented with insu-

Table 1. Collagenare lnhlbltor Productlon In the Presence ot insulin

Units of Inhibitor (Davs 2-71

COlltTd 1.4 I&n- 10 rig/ml 3.8 I&n - 102 r&ml 3.2 k-E&-l&&J/n-d 5.2

These values were obtained from a typlcal experlment. Duplicate cultures were assayed and the average determined. Total units are expressed per 35 mm dish of chondrocvtes (2 x 106 cells In 2 ml medium).

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lin had no effect on collagenase activity. From these data, we concluded that the effect of insulin

on inhibitor production is similar to that observed for both proteoglycan and collagen synthesis

(4,5).

The regulation of collagenase and its inhibitor by different growth factors has been investi-

gated in skin fibroblasta, synovial fibroblasts and chondrocytes. Phorbol myristate acetate (13),

interleukin 1 (14), platelet derived growth factor, and epidermal growth factor (EGF) (15) stimu-

late collagenase synthesis in fibroblasts. Chondrocytes are also stimulated to produce collagenase

by interleukin 1 (IS), but phorbol myristate acetate causes an enhancement of inhibitor produc-

tion (17). This indicates that collagen catabolism is controlled differently in chondrocytes and

fibroblasts. Our findings that insulin stimulates inhibitor production suggest that phorbol esters

and insulin mediate similar effects in chondrocytes. These stimulatory processes may involve

phosphorylation events that occur following the binding of insulin to cells (18).

ACKNOWLEDGMENTS

We thank the San Diego Chapter of the Arthritis Foundation for their kind support of this

research. We also thank Mrs. Fae Hutzel for her expert typing of the manuscript.

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13. Brinckerhoff, C.E., McMillan, R.M., Fahey, J.V., and Harris, E.D., Jr. (1979) Arthritis Rheum. 22, 11091116.

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