Inhibitory effects of phospholipase D on chondrogenesis in vitro

EXPERIMENTAL

CELL

RESEARCH

195,

551-555 (1991)

SHORT NOTE Inhibitory Effects of Phospholipase D on Chondrogenesis in Vitro PANAGIOTIS A. TSONIS,**’ KATIA DEL RIo-TsoNIs,*St JAMES ROTHROCK,* JESUSDOMINGUEZ,+ DENISENGLISH,~KIMBERLYGLADE,§ANDPAUL F. GOETINCK~ *Department

of Biology, University of Dayton, Dayton, Ohio 45469.2320; $VA Medical Center and t Walther Oncology Center, Department Medicine, Indiana

during the above process are not known. However, production of second messengers involving G-proteins, such as inositol trisphosphate (InP,), are known to mobilize intracellular calcium [9]. Phospholipase D converts phospholipids to phosphatidic acid (PA) which subsequently increases InP, and elevates intracellular calcium and in fact acts as a growth factor in A431 carcinoma cells [lo]. Such elevation of calcium is very rapid and probably released from intracellular stores. In order to study the possible mechanisms involved during the mesenchymal cell differentiation to chondrocytes we undertook, as a first step, the examination of the effects of exogenously supplied phospholipase D in high density cultures of limb bud mesenchymal cells. Such an experiment allows us to clarify whether or not an increase in calcium through the action of phospholipase D plays a role in chondrocyte differentiation.

Mesenchymal cells from the wing buds of stage 24 chick embryos undergo differentiation to cartilage when plated at high density. Treatment of these cultures with phospholipase D resulted in inhibition of chondrogenesis. Phospholipase D treatment (which produces phosphatidic acid from membrane phospholipids) was found to affect cell proliferation and to dramatically increase intracellular free calcium levels and inositol phosphate production. Intracellular free Ca’+, mobilized as a result of phosphatidylinositol phosphate hydrolysis, may therefore inhibit chondrogenesis in em:c‘ issi Academic press, IIIC. bryonic mesenchymal cells.

INTRODUCTION Mesenchymal cells from the wing bud of stage 24 chick embryos are capable of differentiating to chondrocytes in vitro when cultured at high density. The high density may stimulate intercellular interaction which may play a role in initiating cartilage differentiation [l-4]. Factors other than cell density, however, seem to be important for chondrogenesis to take place. For example, before stage 20, mesenchymal cells cultured at high density do not differentiate unless treated with dibutyryl CAMP [5, 61. The mechanism of the action of CAMP is not known. It has been suggested that the aggregation of limb bud cells, which is a necessary step before differentiation, is dependent on extracellular calcium [7]. Bee and Jeffries [8] have shown that intracellular calcium levels can affect chondrogenesis. It seems that a certain concentration of calcium is required for normal chondrogenesis. Relatively lower or higher concentrations could result in inhibition of chondrogenesis. The mechanisms involved in cell calcium elevation

‘To whom dressed.

correspondence

of

University, Indianapolis, Indiana 46226; and QLa Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La Jolla, California 92037

and reprint

requests

should

MATERIALS

AND METHODS

Animals. Fertilized eggs were purchased from McIntyre Poultry Farm (Lakeside, CA) and Frank Conway Hatchery (Indianapolis, IN). Usually 100 embryos were used for each experiment described in this paper. Wing buds from stage 24 chick embryos were Cells and Cultures. dissociated into single cell suspensions. High density cell cultures ( lo6 cells/l.6 c m plate) were treated with 1 U phospholipase D (from Streptomyces chromofuscus, Sigma). This product contains 50% protein; therefore, the use of units is preferred over w/v. This is the same product used in other studies where the growth factor action of the enzyme was shown [lo]. It is unknown whether or not this product contains any proteases that could affect the results. Cultures were maintained for 4 days and then analyzed for the presence of chondrocyte nodules. For this, cultures were stained with alcian blue and metanil yellow [ll]. Phospholipase D was added to the medium (F-12 supplemented with 10% fetal calf serum, 50 pg/ml ascorbic acid, penicillin/streptomycin, and fungizone). Cells from parallel plates were collected in order to isolate RNA to quantify chondrogenesis by measuring cartilage-specific gene expression. RNA isolation, probes, and slot-blot hybridizations were performed as described before [12-151. Intracellular calcium measurement-aequorin loading procedures. Cells were washed and centrifuged at 100 g for approximately 2 min in a “GKN” solution containing: glucose 11.1 mM; KH,PO,, 1.5 mM; K,HPO,, 0.5 mM; KC1 25 mM; NaCl 137 mM. These washings re-

be ad-

551

All

Copyright 0 1991 rights of reproduction

0014-4827/91 $3.00 by Academic Press, Inc. in any form reserved.

552

SHORT

moved extracellular calcium. The cells were then resuspended in the above solution with EGTA, pH 7.4, to further bind extracellular calcium. From this point all tubes and pipet tips used were washed with 10 m M EDTA and rinsed with chelexed water which has been previously run through a column containing chelex 100 (Bio-Rad, Torrance, CA) to remove all traces of calcium. Once rinsed with the EGTA solution the supernatant is aspirated and the cells are resuspended in a chelexed “loading solution” containing 140 m M KC1 and 3 m M Hepes, pH 7.4. Ten to fifteen microliters of aequorin (Mayo Foundation, Rochester, MN) was added and the cell suspension was centrifuged three times at 250g for 30 s with pipet mixing in between each centrifugation. Following this loading procedure, the supernatant was aspirated, and the cells were resuspended in a 1:l mixture of 3.0% type VII agarose (Sigma) and a solution giving the final concentrations OE NaCl, 150 mM; Hepes, 10 mM; KCI, 4 mM; MgSO,, 1 mM; KH,PO,, 1 mM; CaCl,, 1.3 mM. The agarose has the properties of being a liquid at 37°C and a gel at lower temperatures. The agarose/balanced salt solution with cells is slowly injected through a 50. c m length of 0.04 in. i.d. polyethylene tubing which is coiled in a beaker of ice. The agarose gel as it passes through the tubing is collected as a single-gelled “thread” with evenly dispersed cells. The thread is placed in a perfusion cuvette containing 1 ml of Krebs-Henseleit bicarbonate buffer (KHB). The cuvette is then housed in a luminescence photometer where the temperature can be kept at 37°C while a constant perfusion of KHB (1 ml/min) can occur. Aequorin emits photons of light while monitoring intracellular calcium. The signal is amplified and recorded in nanoamperes on a chart recorder at the termination of the perfusion of 5% Triton X-100 (Sigma) in KHB. This results in the release of all light stored in the cells. The amount of light released is recorded as an integration value in microamperes. These values can later be used in the calculation of actual intracellular calcium levels at any given point during the experiment [16, 171. Measurement of inositol phosphates. Mesenchymal cells were labeled with 50 &i/ml [3H]myoinositol (DuPont) immediately after plating for 24 h. After washing, the cells were treated with phospholipase D for 10 min. The reaction was stopped by adding 10% ice-cold trichloroacetic acid (TCA). The supernatants were extracted with ether to remove TCA, neutralized, and analyzed with high-performance liquid chromatography. Samples were applied to Partisil lS A X anion exchange column and eluted with a gradient of ammonium formate as described by Hawkins et al. [18]. Fractions were collected and assayed for radioactivity in a liquid scintillation counter. The elution positions of inositol phosphates were confirmed using standard procedures from New England Nuclear. [3H]Thymidine incorporation. For proliferation assay we determined the incorporation of [3H]thymidine into DNA. For this 140,000 cells were inoculated per 6-mm plate (equivalent of lo6 cells/l6-mm plate). At Days 1,2,3, and 4 the cells were harvested on filters and the radioactivity was measured. [3H]Thymidine (5 &i/ml) was added to the medium 18 h before the cell harvesting for each time point.

RESULTS

AND

DISCUSSION

When mesenchymal cells isolated from Day 4 chick limb bud are plated at high cell density in culture they differentiate spontaneously to chondrocytes. The process takes a few days and it is marked by two major events: (1) The rapid increase in cell proliferation especially at Days 2 and 3 and (2) the appearance of cartilage nodules after 48 h in culture which coincides with the

NOTE

TABLE

Incorporation

1

of [3H]Thymidine by Limb Bud Mesenchymal Cells Plated at High Cell Density Day

C PD

1

2

3

4

6331 8299

33494 13739

20580 12459

19831 6140

Note. C, control cultures; PD, phospholipase D-treated cultures. Figures are given as mean value of counts per minute per plate of three different experiments.

increased rate of proliferation [l, 2,131. Usually, at Day 4 (termination of the experiment) a considerable area of the plate is occupied by the nodules. Under these conditions differentiation does not continue beyond this time point and eventually the cell sheet starts detaching from the plate. In the present study this sequence of events was clearly shown in the control untreated cultures (Table 1, Fig. 1). In phospholipase D-treated cultures, however, the sequence of events was different than that seen in the control cultures. First, the rate of proliferation in the treated cultures never achieved that received for the control. We also observed somewhat of an increase in the [3H]thymidine incorporation at Days 2 and 3 but it was much less when compared to the control cultures (Table 1). At Day 4 the rate was much reduced. Cartilage nodules were not detected in the treated cultures at the end of Day 3. By Day 4 when cultures were terminated none or only a few cartilage nodules were present and occupied much less area in the plate than the control (Fig. 1B). By that time proliferation has declined in the treated cultures mainly because the cells failed to differentiate and, therefore, detachment has started. The [3H]thymidine incorporation assay suggests that phospholipase D is not toxic to the cells since at Day 1 there was no significant difference in the incorporation between the control and the treated cultures and since the rate of proliferation actually increased at Days 2 and 3. The possibility of toxicity was further assessed by staining the cells with trypan blue, which indicated no differences in the number of dead cells between the control and the treated cultures. Due to these data we suggest that phospholipase D treatment inhibits differentiation of the high cell density limb bud mesenchymal cells to chondrocytes through regulation of cell proliferation. For further quantitation of chondrogenesis we probed total RNA extracted from treated and untreated cultures at the end of the 4 days in culture with cDNA probe for collagen type II. The results shown in Fig. 2 indicate that the transcripts of the cartilage-specific gene were considerably lower in treated cultures reflect-

SHORT

NOTE

553

FIG. 1. Cultures stained with alcian blue and metanil yellow at Day 4 after inoculation. This stain gives a green color of the chondrocyte nodules and a yellow of the undifferentiated fibroblastic mesenchymal cells; however, cartilage nodules in a black and white photograph are shown as black dots and the rest of the cells are seen as white area. One million cells were plated at Day 0 and the experiment was terminated at Day 4. At that time in both control and treated cultures all the area of the plate was uniformly covered with cells. (A) control culture; (B) PD-treated culture showing greatly reduced levels of chondrogenesis.

ing the greatly reduced levels of chondrogenesis (Fig. 2). This kind of quantitation is very accurate in analyzing chondrogenesis [6, 12-141. Treatment of mesenchymal cells with phospholipase D resulted in an immediate and dramatic increase of intracellular calcium. This increase persisted even after withdrawal of phospholipase D (Fig. 3) and removal of extracellular calcium by EGTA did not lower the high signal (not shown), indicating that the calcium was released from intracellular stores. The highest signal was 100 nA (the dark current was only 5 nA). Calculations

FIG. 2. Slot-blot RNA hybridization pholipase D (PD)-treated cultures made Day 4. Total RNA hybridized to a cDNA collagen type II is shown. Three different of RNA were used.

from control (C) and phosfrom cultures collected at probe for cartilage-specific concentrations (3,6, 15 ag)

from experiments, such as the one presented in Fig. 3, show that the increase in intracellular calcium concentration ranges from 0.1 to 0.2 PM. These values cannot be compared with those of Bee and Jeffries [8], who measured relative amounts of calcium. The possible release of intracellular calcium from intracellular stores implicated the production of inositol phosphates (IP) [9]. This possibility was examined by measuring the inositol phosphate levels in control versus treated cultures. In our preliminary experiment it was found that IP, levels were dramatically increased in mesenchymal cells treated with phospholipase D (Fig. 4). In this experiment we did not detect IP, or IP,. It is possible that IP, and IP, were produced but converted to IP, by phosphatase action [ 18,191. Usually IP, production is rapid after the stimulus is added [ 191. In other systems it has been shown that only a few seconds are necessary to induce IP, production [20]. However, treatment with phospholipase D in the present experiment was for 10 min, and this time difference could explain the presence of IP, and the absence of IP,. The present study clearly demonstrated that phospholipase D increases intracellular calcium levels and inhibits chondrocyte differentiation of limb bud mesenchymal cells. It has been shown before that high cell density, a key condition that promotes chondrogenesis, correlates with intracellular calcium levels [7,8]. Inhibition of chondrogenesis can also result from the action of the tumor promoter, 12-0-tetradecanoyl-l3-acetate (TPA). That action of TPA was not found to be associated with intracellular calcium increase [13]. However, both agents had an effect on cell density and it is

554

SHORT

possible that both could have inhibitory effects through the action of protein kinase C, which is activated both upon TPA treatment and by second messengers such as IPs and diacylglycerol. Phosphatidic acid produced by the action of phospholipase D has been found to cause protein kinase C-mediated activation effects [lo]. Such a mechanism could possibly account for the requirement for high cell density. It is possible that the high cell density necessary for cartilage differentiation is required for receptor-mediated cell-cell interactions. In this respect, it could be that activated protein kinase C phosphorylates receptor(s) involved in such a process, with an effect on their function. For example, it is known that protein kinase C phosphorylation of EGF receptor appears to inhibit its tyrosine kinase activity and to decrease binding affinity of the receptor for EGF [al]. Similar events in the limb bud cells could affect intercellular interaction, leading to inhibition of differentiation. Related to this, it has recently been found that increased levels of PI hydrolysis in 3T3 cells when they become confluent are due to the action of a PIphospholipase C at the cell surface [22]. Such an observation suggests that PI hydrolysis may play a role in density-dependent cell growth, a condition that is applied to the differentiation of the limb bud cells. Taken together, the present data provide evidence

iz 4 8

-L16nln

NOTE

30007 2000-

5 0 IOOO-

I

2

3

4

5

6

I

7

I

8

I

9

,

1

IO II

I

1

I

I

I

12 13 14 I5 16

FRACTIONS

FIG. 4. Elution profiles of inositol phosphates from control (0) and phospholipase D-treated (0) wing mesenchymal cell culture.

that inhibition of aggregation-dependent differentiation of wing bud mesenchymal cells to chondrocytes by the action of phospholipase D could be driven by PI signaling and intracellular calcium elevation. Such possibilities could be studied in the present system and expand our understanding of molecular mechanisms involved during chondrogenesis in the developing wing or limb. Supported by the Veterans Administration and the Walther Oncology Center, and by NIH Grants DK 39655 to J.D., HD22016 to P.F.G., and HD 27684, and by an Arthritis Award to P.A.T. P.A.T. thanks Dr. H. Broxmyer for his support.

REFERENCES 1. 2. 3. 4. 5. FIG. 3. Changes in intracellular calcium after the addition of phospholipase D to the wing bud mesenchymal cells. First arrow (PD) is the time when PD was added. Second (KHB) indicates removal of PD.

6.

Ahrens, P. B., Solursh, M., and Reiter, R. S. (1977) Dew. Biol. 60,69-82. Caplan, A. (1970) Exp. Cell Res. 62, 341-355. Kosher, R. A., Kulyk, W. M., and Gay, S. W. (1986) J. Cell Biol. 102, 1151-1156. Gay, S. W., and Kosher, R. A. (1984) J. Exp. Zool. 232,3177326. Solursh, M., Reiter, R. S., Ahrens, P. B., and Vertel, B. M. (1981) Deu. Bid. 83, 9-19. Kosher, R. A., Gay, S. W., Kanaitz, J. R., Kulyk, W. M., Rodgers, B. J., Sai, S., Tanaka, T., and Tanzer, M. (1986) Deu. Biol. 118, 112-118.

SHORT 7.

San Antonio,

J. D., and Tuan,

R. S. (1986) Dew. Biol. 115, 313-

16. Borle, A. B., and Snowdowne,

324. 8. 9. 10. 11. 12.

Bee, J. A., and Jeffries, R. (1987) Development 100, 73-81. Berridge, M. J., and Irvine, R. F. (1984) Nature 312, 315-321. Moolenaar, W. H., Kruijer, W., Tilly, B. C., Verlaan, I., Bierman, A. J., and de Laat, S. W. (1986) Nature 323, 171-173. Ham, R. C., andsutler, G. L. (1968) J. CellPhysiol. 72,1099114. Tsonis, P. A., and Goetinck, P. F. (1988) Exp. Eye Res. 46,753-

764. 13. Tsonis, P. A., and Goetinck, P. F. (1990) Exp. Cell Res. 190, 247-253. 14. Tsonis, P. A. (1991) Deu. Biol. 143, 130-134. 15. Tsonis, P. A., and Walker, E. (1991) &o&em. Biophys. Res. Commun. 174,688-695. Received November 14, 1990 Revised version received April

2, 1991

555

NOTE

17. 18. 19.

20. 21. 22.

K. W. (1986) in Methods in Enzymology (Conn, P. M., Ed.), Vol. 124, pp. 90-116, Academic Press, San Diego. Borle, A. B., Frendenrich, C. C., and Snowdowne, K. W. (1986) Am. J. Physiol. C. 251, 323-326. Hawkins, P. T., Stephends, L., and Downes, C. P. (1986) Biothem. J. 238, 507-516. Majerus, P. W., Connolly, T. M., Deckmyn, H., Ross, T. S., Bross, T. E., Ishii, H., Bansal, V. S., and Wilson, D. B. (1986) Science 234, 1519-1526. Leitz, T., and Muller, W. A. (1987) Deu. Biol. 121, 82-89. Sibley, D. R., Benovic, J. I,., Caron, M. G., and Lefkowitz, F. J. (1987) Cell 48, 913-922. Ting, A. E., and Pagano, R. E. (1990) J. Biol. Chem. 265,5337-

5340.