Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture

28

Biochimica et Biophysica Acta, 499 (1977) 28--35 © Elsevier/North-Holland Biomedical Press

BBA 28300

E F F E C T S OF CALCITONIN AND P A R A T H Y R O I D HORMONE ON THE METABOLISM OF CHONDROCYTES IN C U L T U R E

KALINDI DESHMUKH *, W.G. KLINE and B.D. SAWYER

Lilly Research Laboratories, Indianapolis, Ind. 46206 (U.S.A.) (Received December 7th, 1976)

Summary Rabbit articular chondrocytes in suspension culture synthesize Type II collagen [3al(II)] in the absence of extraceUular Ca 2÷ and Type I collagen [ 2 a t (I) • a2] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca 2÷ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaC12. If added directly to the suspension culture medium containing no CaC12, calcitonin stimulates an active efflux of Ca 2÷ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.

Rabbit articular chondrocytes synthesize cartilage-specific Type II collagen [3al(II)] in vivo. In monolayer cultures, these cells switch their synthesis to a more ubiquitous form of collagen, Type I collagen [ 2 a l ( I ) - a 2 ] [1,2]. Upon transfer from monolayer to suspension cultures the phenotypic expression of chondrocytes mainly depends on the presence or absence of Ca 2÷ in the extracellular medium. In the absence of Ca 2÷ the cells synthesize Type II collagen, while in the complete medium the major product is Type I collagen [2]. Treatment of chondrocytes in monolayer cultures with ionophore A23187 increases the transmembrane flux of Ca 2., elevates intracellular levels of cyclic AMP and allows the cells to synthesize Type I collagen in suspension culture in the absence of Ca 2÷ in the medium. Addition of dibutyryl cyclic AMP to the medium mimics to a certain extent the effect of extracellular Ca 2÷ [3]. The role of parathyroid hormone and calcitonin in maintaining the calciam balance of bone cells is well known. Although these hormones exhibit opposite mechanisms of action in vivo, in the cultures of isolated bone cells both the * To whom

the correspondence

should he addressed.

29 hormones activate cellular adenyl cyclase and cause the changes in Ca 2. flux as well as distribution of calcium among the cell organelles [4--8]. Distinct populations of bone cells which respond to either calcitonin or parathyroid hormone and which differ in their biochemical characteristics have been isolated [9,10]. In this communication we report the effects of calcitonin and parathyroid hormone on chondrocytes and their collagen synthesis in suspension cultures. Materials and Methods Ham's F-12 medium, Dulbecco's complete medium (Dulbecco's Modified Eagle's medium containing 4.5 g glucose/liter) and Dulbecco's special medium (same medium without CaC12) were obtained from GIBCO; highly purified preparations of porcine calcitonin (100 units/mg) and bovine parathyroid hormone (1000 units/mg) were made at Eli Lilly and Company; and 2,3 [3H]proline (spec. act. 30--50 Ci/mmol) and 4SCa (spec. act. I Ci/mmol) were purchased from New England Nuclear Corp.

Cell culture The chondrocytes were obtained from articular cartilage of knee and hip joints of male rabbits (2--3 months old} b y digestion with hyaluronidase, trypsin and collagenase (Worthington} [11]. The cells were grown to confluency in monolayer culture flasks with Ham's F-12 nutrient mixture containing 10% (v]v) dialyzed fetal calf serum, antibiotics and in the atmosphere of 5% CO2 in air [ 2 ] . At this stage, the cells were treated in two different ways: (A) In monolayer cultures, the cells were incubated in Dulbecco's complete medium containing various concentrations of calcitonin or parathyroid hormone for 2 h. The cells were then isolated by treatment with trypsin for about 10 min and transferred to suspension culture flasks with Dulbecco's special medium containing fetal calf serum and antibiotics. (B) The cells were isolated from monolayer culture flasks b y treatment with trypsin, transferred to suspension culture flasks containing special medium, fetal calf serum and antibiotics, and various concentrations of calcitonin or parathyroid hormone were added directly to this medium. In all the cases stock solutions of the hormones were prepared in 10% ethanol. To attain final concentrations, these stock solutions were diluted 1000 fold or more with culture medium. After the incubation in suspension culture for 24 h, 10 pCi/ml of [3H]proline, 50 pg/ml of ascorbic acid and 50 pg/ml of ~-aminopropionitrile were added to the medium. Next day, the medium was centrifuged and the cell pellet was extracted with 0.5 M acetic acid. Isolation and characterization o f collagen synthesized by chondrocytes The medium and cell extract were dialysed, lyophilized, dissolved in 0.5 M acetic acid and treated with pepsin (100 #g/ml) at 4°C for 48 h. The enzyme was inactivated by adjusting the pH to 8--8.5. Rat skin acid-soluble collagen (4--6 mg) was added as the carrier and the labelled and unlabelled collagen was precipitated with 20% NaC1 at pH 7.5. The precipitate was dissolved in 0.5 M acetic acid, dialyzed against 0.06 M sodium acetate buffer, pH 4.8 and chromatographed on a CM-cellulose column (0.9 × 10 cm) at 42°C [2].

30 The elution was carried o u t with a linear gradient between 0 and 0.1 M NaC1 (80 ml each). The effluent was monitored at 230 nm and the aliquots of the fractions were counted for radioactivity. Further characterization of a-chains of the labelled collagen was done b y treatment with CNBr [12] and chromatography on a CM-cellulose column [2]. The CNBr-peptides were dissolved in 0.02 M sodium citrate buffer, pH 3.6, containing 0.02 M NaC1, applied to the column (0.9 × 10 cm) and eluted with a linear gradient between 0.02 M and 0.15 M NaC1 (100 ml each). The effluent was monitored at 234 nm and the fractions were subjected to estimation of radioactivity. The elution patterns of the CNBr-peptides of labelled a-chains synthesized by chondrocytes under different conditions were compared with those of the peptides of ~ 1(I) and ~2 of chains of rat skin acid soluble collagen and a l(II) chains of rabbit cartilage collagen.

4SCa-uptake by chondrocytes The cells were incubated with Dulbecco's complete medium containing calcitonin or parathyroid hormone in monolayer or suspension cultures for various time periods in presence of 4SCa (5 pCi/ml) in the medium. At the end of each time period, the cells were isolated from monolayer culture by trypsin-treatment and from suspension culture by centrifugation. The pellet was washed with fresh medium, sonicated and the radioactivity of the aliquots was estimated. In some experiments, the cells were transferred from monolayer to suspension culture and incubated for 2 h in the complete medium containing 4SCa (5/aCi/ml). The mixture was centrifuged, cell pellet was washed twice with special medium and transferred to suspension culture flasks with special medium containing calcitonin or parathyroid hormone. The aliquots of the medium were used for estimation of the label released from the cells into the medium at various time intervals. Estimation of intracellular cyclic AMP The change in the levels of cyclic AMP of chondrocytes as a result of treatment with calcitonin or parathyroid hormone under various conditions was followed b y Gilman procedure [13] using commercial protein kinase {Sigma). Results and Discussion

(A ) Treatment of the cells with calcitonin or parathyroid hormone in monolayer cultures with complete medium Table I reveals the effects of ealcitonin and parathyroid hormone on the intracellular cyclic AMP content, uptake of 4SCa by the cells in monolayer cultures and the type of collagen synthesized b y them when transferred from monolayer to suspension cultures with special medium. The presence of calcitonin (10 ng/ml or 1 ng/ml) and parathyroid hormone (10 -7 M or 10 -s M) led to a 2--3 fold increase in cyclic AMP levels as well as in the uptake of 4SCa, as compared to the untreated cells. When transferred to suspension cultures with special medium, the treated cell synthesized mainly Type I collagen as judged b y the elution pattern on CM-ceUulose column (Fig. la). The ratio of the labelled a t : a 2 chains varied between 2 and 3 in different experiments. Further

31

TABLE 1 C H A N G E IN C Y C L I C AMP C O N T E N T , 4 S C a - U P T A K E A N D C O L L A G E N S Y N T H E S I S BY C H O N D R O C Y T E S T R E A T E D W I T H C A L C I T O N I N O R P A R A T H Y R O I D H O R M O N E IN M O N O L A Y E R C U L TURE T h e cells w e r e t r e a t e d w i t h c a l c i t o n i n or p a r a t h y r o i d h o r m o n e in m o n o l a y e r c u l t u r e w i t h c o m p l e t e m e d i u m f o r 2 h a n d t h e n t r a n s f e r r e d t o s u s p e n s i o n c u l t u r e w i t h special m e d i u m . Cyclic AMP a n d 4 5 C a u p t a k e e s t i m a t i o n s w e r e c a r r i e d o u t in m o n o l a y e r c u l t u r e . T h e t y p e of c o l l a g e n w a s assessed a f t e r t h e t r a n s f e r t o s u s p e n s i o n c u l t u r e as d e s c r i b e d in t e x t . Treatment

Cyclic A M P ( p m o l / 1 0 6 cells/2 h)

45Ca u p t a k e ( e p m / 1 0 6 cells/2 h)

Collagen Type I (%)

None

5.3 +- 0 . 3 *

T y p e II C%)

1 0 4 0 +- 42

0

100

90 I00 15

I0 0 85

87 90 20

13 10 80

Calcitonin i0 ng/ml 1 ng/ml 100pg/ml

1 3 . 5 +- 1.2 13.2 + 1.0 8.1 -+ 0 . 2

2456 + 102 2 2 5 2 +- 91 1265+- 4 7

Parathyroid hormone 10 - T M 10 - 8 M 10 - 9 M

16.2+- 1.0 16.6 +- 1.1 7.4 +- 0 . 3

2671 + 2577 + 1275 +

85 73 39

* M e a n +- S.D.

characterization of labelled a-chains was done by examining their CNBr-cleavage products. The elution patterns of CNBr-peptides of labelled a, and a2 chains coincided closely with the peptides of ~ 1(I) and ~2 chains of rat skin acid soluble collagen (Fig. 2a and 2b). The collagen synthesized by untreated cells

al

0.8-

(a)

li/~l Bll

06-

- 40

~2 12.

-30

0.4-

20 3"

0.2-

[~

~

}

10

al i%

0.8=a == 0.6-

I

(b) t

11

~l

40 30

~12 ~2

0.2 -

10 i

10

20

.

.

.

.

i.

.

.

.

=

30 40 50 60 70 80 FRACTION NO. Fig. 1. CM-cellulose chromatogram o f collagen - - , rat skin acid soluble collagen; . . . . . . , collagen s y n t h e s i z e d b y (a) e h o n d x o c y t e s p r e - t r e a t e d w i t h e a l e i t o n i n or p a r a t h y r o i d h o r m o n e in c o m p l e t e m e d i u m f o r 2 h a n d t h e n t r a n s f e r r e d t o s u s p e n s i o n cultttres in special m e d i u m a n d (b) u n t r e a t e d c h o n d r o c y t e s in s u s p e n s i o n c u l t u r e s in special m e d i u m .

32

0.5

(a)

0.4 0.3

4

0.2

II

01-

-8

7 /"~

6

8

;;,', il!l IU/",\ '"

I

I

I

I

I

-6 -4 '

. . . . . . . . . . .

I

I

I

0.5-

--2

0.3-

A I I

I

(b)

E 0.4, c i--

-10

10 j_ Z

6 ~

345

0.2-

z 0.1

l.,

,,,v,-

I 2 ~---

.......... I

I

I

I

I

I

i

I

"" 0.5

(¢1

0.40.3~

6(97 )7

. l

.

I

3

I

I

20

40

4 (8,.)A

I

I

!

I

I

×

I

8 ~1~)

8 6

t,,

4

I

60 80 100 120 t40 EFFLUENTVOLUME (ml)

I

I

160 180 200

F i g . 2. Elution pattern ot C N B r p e p t i d e s o f ( a ) - - , ~i(I) chains of rat skin acid soluble collagen; ...... , ~I chains synthesized by chondrocytes pre-treated with calcitonin or parathyroid hormone. (b) - - , ~2 chains of rat skin acid soluble collagen; ...... , ~2 chains synthesized by treated chondrocytes. (c) , a' i (1) c h a i n s o f r a t s k i n a c i d s o l u b l e c o l l a g e n ; . . . . . . , a I chains synthesized by untreated chondrocytes.

eluted as a single peak in the area where a l chains elute (Fig. lb). Furthermore, the elution pattern of CNBr-peptides of this collagen did not coincide with that of a,(I) chains (Fig. 2c). We have previously characterized these a-chains as a l(II) [2]. Thus, pre-treatment of the cells in complete medium with parathyroid hormone or calcitonin increased the influx of Ca 2÷ into the cells. These cells had the capacity to synthesize Type I collagen in suspension cultures in the absence of extracellular Ca 2+. When the ratio of al : a2 chains was greater than 2, a mixture of Type I and Type II collagen was observed. The changes produced by these hormones were similar to those seen with ionophore A23187 [3].

(B) Effect o f calcitonin or parathyroid hormone on chondrocytes in suspension culture In these experiments, the hormones were added directly to the suspension culture medium. Table II indicates the change in intracellular cyclic AMP levels and 4SCa-uptake by chondrocytes maintained in the complete medium in presence of calcitonin or parathyroid hormone. We have observed that the transfer

33 T A B L E II E F F E C T O F C A L C I T O N I N O R P A R A T H Y R O I D H O R M O N E O N C Y C L I C A M P L E V E L S A N D 4SCao U P T A K E BY C H O N D R O C Y T E S I N C O M P L E T E M E D I U M IN S U S P E N S I O N C U L T U R E Treatment

Cyclic A M P ( p m o l / 1 0 6 cells)

4 5 C a - U p t a k e ( c p m / 1 0 6 cells)

15 rain

15 m i n

None

2 8 . 2 -+ 1.2 *

Calcitonin 1 ng/ml 100 pg/ml Parathyroid hormone 10 - 8 M 10 - 9 M

4 h

2 h

8.7 +- 0 . 6

2 2 3 5 +- 26

6 5 4 5 +- 9 8

4 2 . 3 +- 2.4 2 9 . 4 +- 2.1

1 2 . 4 -+ 0.9 8.5 +- 1.0

4 3 3 2 -+ 78 2 5 8 8 -+ 12

13,411 ± 208 9249 ± 75

4 4 . 0 • 1.9 2 7 . 3 + 1.4

10.9 +- 0 . 7 8.0 + 0 . 5

3 5 2 0 -+ 51 2 5 0 1 +- 2 0

1 6 , 2 5 0 -~ 5 4 8 10.252 ± 107

* Mean-+ S.D.

of chondrocytes from monolayer to suspension cultures activates the cells and causes an increase in their cyclic AMP levels during the first 15 min of incubation, after which there is a decrease to the basal levels within 4--6 h. This phen o m e n o n is observed to the same extent with cells maintained in special medium or complete medium, i.e. the presence or absence of extracellular calcium has little or no effect on the cellular cyclic AMP levels in suspension culture (Deshmukh et al. manuscript in preparation). Treatment with parathyroid hormone or calcitonin increased the cyclic AMP levels significantly above those of untreated cells during the initial incubation period. These values, however, reached almost the basal levels within 4 h. The uptake of 4SCa by chondrocytes treated with calcitonin {1 ng/ml) or parathyroid hormone (10 -8 M) was also increased significantly as compared to the untreated cells {Table II). In the complete medium, the collagen-phenotype of chondrocytes was Type I and was n o t affected by addition of calcitonin or parathyroid hormone to the medium. In suspension cultures with special medium, the chondrocytes synthesized primarily Type II collagen [2]. If calcitonin was added at a concentration of 1 ng/ml, the cells produced mainly Type I collagen. Addition of parathyroid hormone did not cause any change in the phenotype {Table III). These observations raised possibilities that calcitonin activates certain sites on the cell membrane that are usually activated by extracellular Ca2+directly or indirectly, leading to a change in the phenotypic expression of cells; or in the absence of extracellular Ca 2., calcitonin stimulates an efflux of calcium from the cells into the medium which in turn activates proper sites on the membrane. An indirect evidence was obtained from the observation that in the presence of EGTA (10 -410 -s M) in the medium, the effect of calcitonin on switching of collagen-phenotype from Type II to Type I was n o t seen {Table III), indicating t h a t calcitonin causes efflux of Ca 2÷ from the cells and leads them to synthesize Type I collagen. More direct data on the efflux of Ca2+was obtained by incubating the chondrocytes, prelabelled with 4SCa, in special medium {Table IV). It is evident that addition of calcitonin to the medium enhances the efflux of 4SCa from the cells. Parathyroid h o r m o n e caused a slow and transient efflux, which may not be sufficient to change the synthesis of collagen-phenotype.

34 TABLE III T Y P E O F C O L L A G E N S Y N T H E S I Z E D BY C H O N D R O C Y T E S IN S U S P E N S I O N C U L T U R E I N P R E S ENCE OF C A L C I T O N I N OR P A R A T H Y R O I D H O R M O N E .

.

_

C a l e i t o n i n or p a r a t h y r o i d h o r m o n e w e r e a d d e d d i r e c t l y t o the special m e d i u m of s u s p e n s i o n c u l t u r e . Treatment

None

Type I collagen (%)

Type II coUagen (%)

0

100

Calcitonm 1 ng/ml 100 pg]ml

90 15

10 85

Calcitonin 1 n g / m l + E G T A 10 --4 M

25

75

Parathyroid hormone 10 - a M 10 - 9 M

10 0

90 100

0

100

Parathyroid hormone 10 - 8 M + E G T A 1 0 - 4 M

Effects of calcitonin and parathyroid hormone on the Ca 2+flux of bone cells have been studied in detail. Although these hormones act on different cell populations, both increase the influx of calcium and stimulate adenyl cyclase. In addition, parathyroid hormone stimulates the Ca :+ efflux. Calcitonin initially stimulates the efflux, but has a suppressing effect at later stages [4]. Dziak and Brandt [8] showed that calcitonin causes a transient increase in calcium uptake by bone cells, while parathyroid hormone had no consistent effect. Harrell et al. [14,15] demonstrated that either calcitonin or elevated concentration of Ca 2+ in the medium leads to an accumulation of calcium in the bone cells. In the medium with low calcium concentration, there is an efflux of calTABLE IV E F F E C T O F C A L C I T O N I N O R P A R A T H Y R O I D H O R M O N E ON T H E E F F L U X C H O N D R O C Y T E S IN SPECIAL MEDIUM IN SUSPENSION C U L T U R E

OF 45Ca FROM

The cells w e r e i n c u b a t e d in c o m p l e t e m e d i u m e o n t a i n i n g . 4 5 C a f o r 2 h a n d t h e n t r a n s f e r r e d t o u n l a b e l l e d special medium. Treatment

% Release of 45Ca in t h e m e d i u m in 15 rain

1 h

2 h

None

20.2±2.1"

26.4±1.2

29.8±1.0

Calcitonin 1 ng/ml 100 p g / m l

59.6 ± 3.4 3 0 . 3 ± 1.8

6 5 . 4 + 2.8 37.6 + 1.0

78.3±3.7 42.8±2.5

Parathyroidho~one 10-8M 10-9M

34.8±1.0 E9.0±0.7

40.2±2.7 30.5±3.3

4 9 . 7 + 2.3 3 3 . 3 + 2.5

* M e a n + S.D.

35

cium from the cells to the medium which is reversed by parathyroid hormone. Our experiments with articular chondrocytes demonstrate that in the complete medium (containing 1.8 mM CaC12), both parathyroid hormone and calcitonin stimulate an active influx of calcium into the cells, leading to an elevation of intracellular cyclic AMP levels. These chondrocytes synthesize primarily Type I collagen in suspension cultures in special medium (containing no CaC12). If added directly to the suspension culture special medium, calcitonin stimulates an active efflux of calcium from the chondrocytes into the medium and causes a switch in collagen synthesis from Type II to Type I. Such change in the collagen-phenotype was not observed with parathyroid hormone. Acknowledgements We thank Dr. A.H. Tashjian and Dr. D.T. Wong for helpful suggestions and Dr. M.J. Schmidt for the help in cyclic AMP assay. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Layman, D.L., Sokoloff, L. and Miller, E.J. (1972) Exp. Cell Res. 73, 107--112 Deshmukh, K. and Kline, W.G. (1976) Eu~. J. Biochem. 69, 209--215 Deshmukh, K., Kline, W.G. and Sawyer, B.D. (1976) FEBS Lett. 67, 48--51 Rasmussen, H., Goodman, D.B.P. and Tenenhouse, A. (1976) Crit. Rev. Biochem. 1~ 95--148 Chase, L.R. and A~rbach, G.D. (1970) J. Biol. C h e m . 245, 1520--1526 Murad, F., Brewer, Jr., H.B. and Vaughan, M. (1970) Proc. Natl. Acad. Sci. U.S. 65, 446--453 Borle, A.B. (1973) Fed. Proc. 32, 1944--1949 Dziak, R. and Brandt, J.S. (1974) J. Cell Physiol. S4, 85--96 Wong, G.L. and Cohn, D. (1974) Nat. N e w Biol. 252, 713--714 Wong, G.L. and Cohn, D. (1975) Proc. Natl. Aead. Sci. U.S. 72, 3167--3171 Sokoloff, L., Malemud, C.J. and Green, Jr., W.T. (1970) A r t h . R h e u m . 13, 118~124 Deshmukh, K. and Nimni, M.E. (1971) Biochemistry 10, 1640--1647 Gilman, A.G. (1970) Proc. Natl. Aead. Sci. U.S. 67, 305---312 Harell, A., Binderman, I. and Rodan, G.A. (1971) Endocrinology 92, 450~-455 Harell, A., Binderman, I. and Gues, M. (1976) Israel J. Med. Sci. 12, 115---123