Direct effect of parathyroid hormone on the proliferation of osteoblast-like cells; A possible involvement of cyclic AMP

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DIRECT EFFECT OF PARATHYROID HORMONE ON THE PROLIFERATION OF OSTEOBLAST-LIKE CELLS; A POSSIBLE INVOLVEMENT OF CYCLIC AMP

Arie van der Plas, Jean H.H. Feyen and Peter J. Nijweide Laboratory for University Received

Cell Biology and Histology, of Leiden, 2333 AA Leiden,

Rijnsburgerweg The Netherlands

10,

May 9, 1985

SUMHART: Serum-starved

chick osteoblast-like cells (08 cells) and periosteal fibroblasts (PF cells) were used to study the proliferative effects of parato 10-8 M) thyroid hormone (PTH) and prostaglandin E2 (PGE2). Both PTH (lo-l1 dose-related effect on the de novo and PGE2 (low9 to 10m5 M) had a direct, synthesis of DNA in 08 cells. The PF cells only showed a dose-dependent effect in the presence of PGE2 (lo-9 to 10-5 M). The hormonally induced proliferation of these cells was shown to be dependent on cell density and stimulation time, An optimal response for both cell types was observed in the cell density range 1.5 to 3.5 pg DNA / 2 cm2, when stimulated for 18 hours. As CAMP-enhancing substances (N6-dBcAMP, forskolin and IBMX) could mimic the PTH- and PGE2the increased DNA synthesis was concluded induced proliferation in 08 cells, B 1985 Academic Press, Inc. to be mainly caused by enhanced CAMP concentrations.

INTRODUCTION: A carefully

controlled balance between osteoblastic bone formation and osteoclastic bone resorption is a prerequisite in bone modelling (growth of the skeleton) and remodelling of the adult skeleton. This balance is maintained by regulation (hormonally or otherwise) of cellular activities of bone-forming and bone-resorbing cells and by regulation of the number of active cells, Hormones and factors regulating bone formation have been classified into four groups: (a) systemic hormones, including the calcium regulatory hormones , (b) systemic growth factors, e.g. insulin-like growth factor I and II (IGF-I and II), platelet-derived growth factor (PDGF), epidermal growth factor and fibroblast growth factor C5--91, (c) local factors like prostaglandins [2,31 and bone-derived growth factors (BDGF-1, 2) L41 and (d) ions (see Raisz and Kream 1983, for a review Cl]). It. is known from cell culture and tissue culture studies that the classical calcium regulatory hormones, parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) have a dedifferentiating effect on osteoblasts, leading to a decreased expression of the osteoblastic phenotype: a decreased alkaline phosphatase activity [lOI, citrate decarboxylation [11J and collagen synthesis within 24 hours after administration [121. Gaillard [13,141 reported a loss of cytoplasmic basophilia in the osteoblasts of mouse radii rudiments, prior to their transformation into spindle shaped cells. Despite this effect of PTH, it has been reported that PTH can stimulate bone formation. Tam et al. Cl51 showed a dose-dependent stimulation of the bone apposition rate in vivo by bPTH(l-84) and hPTH(1-34). This effect was independent of the resorptive effects of the hormone. Explanted mouse radii in vitro showed a stimulated osteoblastic activity in the pre0006-291X/85 $1.50 CoWright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

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amount of knowledge sence of bPTH(l-34) L161. Although there is a considerable about the hormonal regulation of calcium metabolism, little is known about the mitogenic effects of hormones on different types of bone cells. In this study we examined the effects of PTH on cell proliferation, i.e. [3H]-thymidine incorporation and DNA content of the cultures, in secondary cultures of chick OB cells. To study the specificity of the PTH effects we compared OB cells with PF cells, and PTH with PGE2. MATERIALS AND CIETHODS Cell culture: OB cells and PF cells were isolated according to the method of Nijweide et al. [171 with a minor modification according to Hefley et al. [181. In brief, calvaria were dissected from 18-day-old chick embryos and the outer fibrous layer of the periostea was separated from the OB-rich inner layer and the calcified matrix. OB cells: The inner layer of the periosteum together with the calcified matrix was treated with collagenase (1 mg crude collagenas@ / ml, Sigma) in Mg2+-free isolation medium supplemented with 7.5 FM N"-tosyl-L-lysylchloromethane (Calbiochem Behring Corp.), once for 3 min (wash fraction, discarded) and twice for 45 min at 37 oC in a shaking waterbath. The isolation medium consisted of 25 mM HEPES (pH 7.41, IO mM NaHC03, 100 mM NaCl, 3 mM K2HP04, 1 mM CaC12, 30 mM KCl, 1 mg / ml bovine serum albumin (BSA, fraction V, Sigma) and 5 mg/ml glucose. The combined isolated cells in the 45 min fractions were washed and resuspended in culture medium. PF cells: The PF cells were isolated from the outer fibrous layer of the periosteum using the isolation method as described above. After cell isolation, the isolated fibroblasts were separated from residual fibrous tissue by means of a Cellector tissue sieve (stainless steel screen of 94 pm opening size, Bellco Glass Inc., USA). Both cell types were cultured in 150 cm2 Costar tissue culture flasks in a C02-incubator ( 5 % CO2 in air) (Forma Scientific, model 3029). The culture medium consisted of otMEM (GIBCO) supplemented with heat-inactivated cock serum (10 % (v/v)), embryonic extract (5 X (v/v)), 200 pg /ml L-glutamine, 50 pg /ml gentamycin, 50 pg / ml ascorbic acid and 1 mg / ml glucose. The embryonic extract was prepared from lo-day-old chick embryos. After 3 days' culture half the culture medium (12,5 ml) was replaced by fresh medium leaving out embryonic extract. [3H]-thymidine incorporation: The [3Hl-thymidine incorporation ([3Hl-TdR) was performed using secondary cultures of osteoblast-like cells. After 5-7 days' culture, the primary cell cultures were incubated (15 min, 37 oC) with 1 mg/ml collagenase in isolation medium in the presence of NW-tosyl-L-lysylchloromethane,, The harvested cells were centrifuged (5 min, 200 x g), washed in Hanks' balanced salt solution (BSS), supplemented with fresh cock serum (IO $ (v/v)), resuspended in culture medium and seeded in 24-well-plates. In order to determine the optimal cell density, cells were plated at increasing cell densities ( 0.4 x 105 - 1 x 107 cells per well of 2 cm2). After attachment (5 h) cells were washed twice withciMEM to remove the non-adhering cells and residual serum. To each well 350 ~1 of fresh medium, in the presence or absence of this medium of the stimuli to be investigated, was added . The composition was virtually the same as that for the culture medium, but cock serum and embryonic extract were omitted while 0.5 X (w/v) BSA was added. After a preincubation period of 16 h, the cells were pulse-incubated for 2 h in the presence of 37 KBq / ml [3H]-thymidine (total incubation volume 365 ~1). The pulse incubation was stopped by placing the cells on ice and washing three times with ice-cold Hanks' BSS. To remove free intracellular radioactive label, cells were lysed in aqua dest (15 min. 000. In order to determine the [3H]-TdR into DNA and the total amount of DNA after stimulation, the cell were trypsinized (I mg trypsine / ml, Difco 1:250) and sonicated for residues IO set (Brandson Sonic Power, model No R75, position 4). Aliquots were taken 919

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for direct according

liquid scintillation to the method of Karsten

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counting and for total and Wollenberger [191.

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DNA determination

Chemicals: C6-3H]-thymidine (spec. act. 74 GBq / mmol) was obtained from Amersham (U.K.). Bovine parathyroid hormone (PTHl-84) was a generous gift from the Medical Research Council (U.K.). N6-02-dibutyryladenosine 3’:5’-cyclic monophosphate (N6-dBcAMP), the calcium ionophore A23187, prostaglandin E2 (PGE2), and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) were obtained from Sigma, and forskolin from Calbiochem-Behring Corp. Statistical

method: Values are expressed as percentages of the control values + S.E.M. and statistical analysed applying the Student’s t-test, Values represent the average of quadruplicate measurements in two separate experiments. RESULTS Influence cultures:

of cell

density

on [3HI-TdR into DNA and total

DNAcontentofthe

In figure 1A the effect of PTH and PGE2 on the proliferation of secondary cultures of respectively 08 cells and PF cells is shown. The cells were incubated for 16 h in the presence of the stimulus and then exposed to [3H]-thymidine for 2 h. At low cell densities PTH (10-8 M) caused a proportional increase in the C3H1-TdR into the DNA. An optimal stimulation was observed at 1.5 pg DNA / 2.0 cm2. At seeding densities higher than 1.5 pg DNA of radioactivity into the DNA gradually decreased. / 2 cm2 the incorporation At very high cell densities (25 pg DNA / well) no detectable amount of [3H3TdR could be observed above control values. As PF cells do not respond to PTH

E/( :

,

( 100%

400 300 200

100

140 120.

100, 80, #

I

I

,rlll,

0.5

I

I

,,“I,,

I

5

1

10 pg

,

r,

50 DNA/2cm2

Fig. 1: Effect of cell density on hormonally induced proliferation, as measured by the [3HI-TdR into DNA and total DNA, in secondary cultures of 08 cells stimulated by PTH (0) and PF cells stimulated by PGE2 (0). The cells were incubated for 18 h at 37 OC, the last 2 h in the presence of [3Hl-thymidine at different cell densities. The results are expressed as [E/C x 100 %I against the mean values of DNA content of the control group after an 18 h stimulation period. Panel A, [3HI-TdR into DNA: panel B, total DNA of the cultures (*, p < 0.05;**, p < 0.02; ***, p < 0.001). 920

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(see fig. 3) these cells were stimulated with 10e5 M PGE2. The proliferative effect of PGE2 on the PF cells showed a similar pattern as was seen in the case of PTH stimulation of OB cells (fig. 1A). Optimal stimulation for [3HlTdR incorporation was observed at + 3.5 pg DNA / 2 cm2. In fig. lB, the effects are shown on the total DNAcontent of the cultures after stimulation. The proliferative effect of PTH on the OB cells was optimal at 3.5 pg DNA / 2 cm2. The PF cells showed an optimal effect at the cell density 1.5 pg DNA/ well. From these results it was concluded that the optimal cell density for both [3H:I-TdR into DNAand total DNA content is 1.5 - 3.5 ttg DNA / 2 cm2 for OB cells as well as for PF cells.

Influence of stimulation time on [3H]-TdR into DNA and total DNAcontent of the cultures: To study the effect of the stimulation time on the proliferative response of the OB cells to PTH, cells were seeded in the optimal cell density range (see above) and were incubated with 10-8 M PTH for respectively 2, 3, 5, 7, 18 and 36 h. During the last two hours of stimulation, the cells were exposed to [3H1-thymidine. The results (fig. 2) showed a time-dependent increase in [3H1-TdR. At 18 h of incubation, an optimal incorporation was observed, which decreased at longer stimulation periods. In contrast to the effect of PTH on the incorporation of labelled thymidine into the DNA, where a significant increase could be observed at 5 h incubation, a first significant effect on total DNA content was observed after 7 h stimulation. An optimal response of PF cells to PGE2was also observed when the cells were stimulated for 18 h (results not shown). Dose dependence of the cell proliferation: To define the concentration ranges of PTH and PGE2action on the bone-derived cells, cells were incubated with increasing concentrations of PTH (10-" - 10-8 MI and PGE2(10-g - 10-5 M) for 16 h and pulse-incubated for the last 2 hours with [3H1-thymidine (fig. 3). PTH showed a dose-dependent stimulation of the incorporation of radioactivity into the DNAand total DNAcontent of OBcells, and remained at a plateau of 10-g - 10-8 M PTH. No proliferative response was observed in the PF cells after stimulation with PTH. When the cells were stimulated with PGE2, both OB cells ansdPF cells showed a dose-dependent response to PGE2.OB cells are more responsive to PGE2; 10v6 M PGE2resulted in an increase of 300 X. At the same concentration, PGE2 enhanced the C3H1-TdR into DNAto only 115 X (fig. 3A). As illustrated in fig. 38, the effects of PTH and PGE2 on the total DNA content in both OB cell and PF cell cultures showed the same characteristics as in the case for the [3HI-TdR. Effect of forskolin, IBHX, NC-dBcAUP and A23187 on the proliferation of OB cells: Agents known to modify the intracellular CAMPconcentrations or the penetration of Ca2+ were used in determining whether the OB cell proliferation was dependent on a rise in CAMPconcentrations or Ca2+ changes (table 1). The diterpene forskolin, an a-specific activator of adenylate cyclase, caused a dose-dependent rise in the [3H]-TdR into the DNAand total DNAcontent in the M. An enhanced proliferation in the preconcentration range of 10-7 - 10-5 nucleotide analosence of the phosphodiesterase inhibitor IBMX, or the cyclic gue N64BcAMP, only occurred at relatively high concentrations of respectively 10-3 and 10m4 M. Administration of the calcium ionophore A23187 (10-g - 10-8 921

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E/C x 100 %

E/C, x 100 % 500

. . 160 -

300 400

-

200

-

100

*

.. A

P

. . 1 &-P-

140 .

120 -

..,Y

g:g-?;

5

1

02

20

10

40

/

J tool &-f I ; , , 16”

03

HOURS

,o-“0 10-g 10-*&j

10-g 10-T 10-5 M

LPTH~

LPGE+

Fig. 2: Effect of stimulation time on PTH-induced proliferation, as measured by3Hl-TdR into DNA (01 and total DNA ($r) in secondary cultures of OB cells. The cells were incubated with 10-8 M PTH for respectively 2, 3, 5, 7, 18 and 36 h, the last 2 h in the presence of C3Hl-thymidine. The results are expressed as [E/C x 100 Xl against time (h) (*, p < 0.02;**, p < 0.001).

3: Dose-dependency of hormonally (PTH, PGE2) induced proliferation, as measured by [3HI-TdA into DNA and total DNA, on secondary cultures of OB cells (0) and PF cells (0). Cells were incubated for 18 h at 37 OC, the last 2 h in the presence of [3Hl-thymidine with different concentrations of the stimuli. The results are expressed as [E/C x 100 II against hormone concentration, Panel A, [3HI-TdR into DNA; panel B, total DNA content of the cultures.

Fig.

Table

the

1: Effects incorporation

of of

CAMP-enhancing C3Hl-thymidine OB cells after

drugs

and

[3Hl-TdR

addition

(E/C

x

the

calcium

ionophore

(C3Hl-TdR) and 18 h of stimulation Pd

total (E/C

100%)

x

total

A23187

DNA

on

content

DNA 100%)

Pd

in

n

2 26.9

< 0.001 < 0.001

106.9 134.1 140.4

+ T 4.6 3.8 T- 5.1

< 0.002 < 0.001 < 0.001

10 10 10

92.8 122.4

+ 8.6 z 20.4

< i::;

107.7 112.5

6.6 ++ 6.2

< 0.02 < 0.001

10 10

N6-dBcAMP "

214.2 95.2

z+

10.8 37.1


100.4 129.7

+ +

< n.s. 0.001

10 10

M M

A2318711

110.5 97.7

+

13.7 13.2

104.6 101.8

M

11

30.0

+2

3.3

+T 4.7 4.8 z 4.8

10-7 10-6 10-5

M M M

1O-4 10-3

M M

1O-4 10-5

M

IO-9 10-8 10-7

Forskolin

100.0

11 w

229.1 302.2

IBMX 11

9.0 + 20.0

n.s.

n.s. < 0.001

922

1

102.3

3.7 7.0

n.s. n.s. n.s.

8 8 8

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M) had no effect on the proliferative of very high concentrations of A23187

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of the OB cells. In the presence M) the DNA synthesis decreased.

DISCUSSION: Hormonally (PTH, PCE2) stimulated cell proliferation, as measured by incorporation of [3Hl-thymidine into DNA ([3Hl-TdR) and the increase of culture DNA content, is dependent on cell density and stimulation time in both OB cells and PF cells. In the presence of 10-8 M PTH, the OB cells showed a maximal ‘response in [3Hl-TdR into DNA and total DNA content in the cell density range of 1.0 - 3.5 pg DNA / 2 cm2. The optimal cell density of PF cells for PGE2-stimulated cell proliferation was between 1.5 and 3.5 pg DNA / 2 cm2. At high cell densities, OB cells showed a gradual decrease of the PTHinduced proliferative response. This effect was less pronounced for PF cells stimulation of the proliferation at in the presence of the PGE2. The reduced high OB cell densities could be the result of a cell surface mediated contact inhibition. Hare1 et al. [20] reported the presence of diffusible inhibitors of proliferation which accumulate in dense 3T3 cell cultures. Another possible explanation for this effect may be found in our cell culture conditions. During stimulation the cells were kept in 0.5 X BSA to exclude the involvement of growth-inducing factors which might be present in serum and could interfere with the hormonal response to be investigated. At high cell densities medium deprivation could occur in such a relatively llpoorll medium during the 18 h the stimulation lasted. The proliferative effect of PTH on OB cells as function of time showed a significant increase (p < 0.02) of radiolabelled DNA within 5 h. A maximal response was observed after 18 h. From this study it is concluded that PTH can have, besides an effect on osteoblastic activity [21,22,231, a direct effect on the proliferation of OB cells. PTH caused a dose-related increase in [3HI-TdR as well as in total DNA content over the concentration range lo-11 to 10-8 M. In contrast to our findings, Drivdahl et al. [241 reported that PTH, in concentrations ranging from lo-12 to 10-6 M, had no effect on DNA synthesis in cultured chick calvarial cells, although they described the presence of a bone formation stimulaof which synthesis is induced by PTH. Howard et al. [251 suggestion factor, ted the presence of a so-called coupling factor, which is formed at the site of resorption after a PTH-induced stimulation of bone resorption. This factor could stimulate the osteoblasts to form new bone. As expected, the PF cell population did not respond to PTH administration. Our PF cell population showed, with respect to the proliferative response to PTH, the same characteristics as the cultured chick calvarial cells described by Drivdahl L241. The prostanoid PGE2 (10-g - 10v5 M) effected the proliferative state of both OB cells and PF cells. In comparison with the PTH-stimulated proliferation of OB cells, PGE2 in the concentration ranges used in this study, showed a higher [3H]-TdR into the DNA. This effect was not observed for the total DNA content. Secondary cultures of serum-starved OB cells isolated from la-day-old embryonic chick calvaria, as used in this study, showed the same response as primary, confluent cell cultures in respect to the PTH-induced CAMP formation. Parathyroid hormone was still able to stimulate adenylate cyclase. PF cells were insensitive to PTH in this respect (results not shown). PGE2 stimulated CAMP production both in OB and PF cells [171. The increased de novo synthesis 923

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of DNA, as demonstrated in this study, appears to be related to these CAMP responses. The CAMP analogue N6-dBcAMP and the phosphodiesterase inhibitor IBMX, a compound which is known to stimulate the intracellular CAMP concentrations, were capable of mimicking the effects on the proliferation of osteoblasts although at relatively high concentrations. Forskolin, a diterpene stimulating directly the catalytic subunit of adenylate cyclase C263, induced a proliferation of 08 cells at relatively low concentration (10-6 M). In the literature there is no unanimity about the role of cyclic nucleotides in the induction of proliferative processes. Takigawa et al. [27] reported that N6-dBcAMP or 8-BrcAMP increases the differentiated phenotype of cultured chondrocytes. Simultaneously an inhibition of thymidine incorporation could be observed after a stimulation period of 12 h or longer. On the other hand a transient increase of CAMP mediated by PTH did not inhibit chondrocytic cell growth. Mitogenic effects mediated by CAMP were also reported for e.g. thymic lymfocytes 1281, liver cells C291 and rat Schwann cells C301. In our laboratory it has been shown recently,that PTH, besides activation of adenylate cyclase, causes a dose-related increase in cytosolic calcium as measured by the fluorescent calcium indicator QUIN-2 [31I. However, the calcium ionophore A23187, in a concentration which causes a calcium influx (lo-7 M) [311, had an inhibitory effect upon the proliferation of the osteoblasts in the present investigation. Our results are in agreement with the observations that A23187 inhibits DNA synthesis in cultured foetal rat bones [32] and suggest that primarily an increase in cellular CAMP is the mediator for the PTH induced stimulation of proliferation of chick osteoblasts. The apparent absence of a role for intracellular calcium has to be considered with caution. A23187 does not appear to be a suitable parathyromimetic agent for studing the mechanism of PTH action in long-term experiments in vitro [32]. ACKNWLEDCEMEi?T We thank Prof. Dr. J.P. Scherft

for

the helpful

discussions.

REFERENCES

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MP M Tissue