JOURNAL OF CELLULAR PHYSIOLOGY 1 4 2 5 - 6 9 (1990)

Forskolin Has Biphasic Effects on Osteoprogenitor Cell Differentiation In Vitro KURSAD TURKSEN,* AGAMEMNON E. GRIGORIADIS, JOHAN N.M. HEERSCHE, AND JANE E. AUBIN Medical Research ( ouncil Group in I’criodontal Physiology, Faculty of Dentistry, Univervty of Toronto, Toronto, Ontario M55 JA8, Canada Cells isolated from fetal rat calvarid (RC) and maintained in vitro in medium containing ascorbic acid and B-glycerophosphate form three-dimensional, mineralized nodules having the histological, immunohistological, and ultrastructural characteristics of woven bone. We have studied the eiiects of forskolin (FSK), a diterpene that activates adenylate cyclase, in this system. While lO-’-lO M FSK significantly stimulated cAMP levels in RC cells, lower concentrations did not. cAMP levels with lo-‘ M tSK rcachcd a maximum by 30 min at 37°C and returned to basal level in 2-13 hr. Changes in cAMP levels correlated with changes in cellular shape: cells treated with l o - “ M FSK assumed a stellate morphology, lost microfilament bundles, and reduced their substrate adhesiveness, while cells treated with lo-” M were not affected. Exponential growth and saturation densities of FSK-treated cultures were similar to untreated cultures, indicating that FSK was neither toxic nor stiniulatory to the population. The effect on bone nodule formation of FSK present continuously depended on concentration: 10 M FSK significantly inhibited the number of nodules formed, while M FSK significantly stimulated bone nodule formation. Single short treatments with either 1 OP5 M or .I 0-‘ M FSK had no effect on nodule formation, but repeated short duration treatments (1 hr every 2 days for 21 days) gave results similar to continuous exposure. These results indicate that intermittent elevations in intracellular cAMP have an inhibitory effect on bone formation. In addition, our work indicates that low concentrations of FSK stiniiilale differentiation of osteoprogenitor cells possibly through a non-CAMP-dependent process.



Several hormones which influence bone turnover have direct effects on osteoblastic cells (for review, see Nijweide et al., 1986; Martin et al., 1987). Parathyroid hormone (PTH), a n important physiological regulator of Ca’+ homeostasis, elevates intracellular cAMP and increases CAMP-dependent protein kinase activity in osteoblast-enriched primary cell cultures and osteosarcoma-derived cell lines (Luben et al., 1976; Partridge et al., 1981). However, although alkaline phosphatase activity (Beresford et al., 1984; Majeska and Rodan, 1982) and collagen synthesis (Kream et al., 1980) have been shown to be decreased in PTH-treated cells, suggesting that PTH may inhibit osteoblastic activity and bone formation, there is also evidence that PTH may increase collagen synthesis and alkaline phosphatase activity (Hakeda e t al., 1985; Martin et al., 1985). Prostaglandin E, (PGE,) also markedly elevates cAMP in osteoblastic cell cultures and in some, but not all, osteosarcoma cell lines (for review, see Martin et al., 1987). PGE,, like PTH, has complex effects on osteoblastic cells, i.e., PGE, has been shown both to inhibit and to stimulate collagen synthesis under some conditions in osteoblastic cell and organ cultures (for review, see Martin et al., 1987). PGEz also stimulated alkaline phosphatase activity in the osteoblastic clonal cell line MC3T3-El (Hakeda et al., 1986). Because of the effects 6

1990 WILEY-LISS. INC

of PTH and PGE, on expression of differentiated properties of osteoblastic cells, and because they also increase intracellular cAMP in most osteoblastic cells, cAMP has been suggested to be a n important mediator of osteoblast differentiation and function. One problem in elucidating the effects that changes in cAMP concentration may have on osteoblasts and bone formation is that most of the assays used (e.g., alkaline phosphatase activity or collagen synthesis) are indirect measures of differentiation. It has been suggested that at least some of the diversity of ef’fects measured in response to PTH, PGE,, or cAMP has resulted from the fact that these agents may elicit different effects on cells depending on their stage of differentiation or maturation (Heersche and Aubin, 1989). Recently, we have developed a n assay in which osteoprogenitor cells differentiate in vitro, allowing a quantitative analysis of bone nodule formation. When enzymatically digested fetal rat calvaria cells are grown in vitro in the presence of ascorbic acid and Bglycerophosphate, discrete three-dimensional mineralized nodular areas form with the histological and imReceived February 28, 1989; accepted July 20, 1989.

*To whom reprint requestsicorrespondence should be addressed.

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munohistological (Bellows et al., 1986) and ultrastructural (Bhargava et al., 1988) appearance of woven bone. The number of bone nodules that form is directly related to the number of cells plated and limiting dilution analysis has indicated that a nodule arises by differentiation of a discrete class of osteoprogenitor cells present a t a low frequency in the osteoblastic populations (Bellows and Aubin, 1989). Earlier we have shown that formation of nodules can be regulated by additives to the culture medium, such as glucocorticoids (Bellows et al., 1986,1987).We decided to use this assay to determine directly whether changes in cAMP levels affect differentiation of osteoprogenitor cells. To elevate CAMP,we exposed the cultures to the diterpene forskolin (FSK), a n activator of adenylate cyclase (AC) in most cell types (Seamon and Daly, 1986). Although the precise mechanism by which FSK activates AC is not yet clear, FSK does cause transient increases in cAMP to levels comparable to those caused by hormones coupled to the AC system (for review, see Ammon and Muller, 1985; Seamon and Daly, 1986). FSK has been shown to raise cAMP levels in bone organ cultures (Herrmann-Eh-lee et al., 1988; Lerner et al., 1986) and in various osteoblastic cell lines such a s the clonal mouse MC3T3-El cell line (Hakeda et al., 1985), the human osteosarcoma cell line MG-63 (van Valen and Keck, 1988) and in early passage chick and human osteoblast-enriched cells (van Valen and Keck, 1988). We report here that FSK elevates intracellular cAMP in primary fetal rat calvaria (RC) osteoblastic cells in a dose-dependent manner. Interestingly, it had complex effects on osteoprogenitor differentiation and bone formation, either inhibiting or stimulating bone nodule formation, depending on its concentration. MATERIALS AND METHODS Cell culture Cells were enzymatically isolated from 21 day fetal Wistar rat calvariae (RC) as described previously (Rao et al., 1977; Aubin et al., 1982; Bellows et al., 1986). Cells obtained from five sequential digestions (populations I-V) were plated separately into T-75 culture flasks (Falcon 3024; Becton Dickinson Labware, Lincoln Park, NJ) in alpha-minimal essential medium (aMEM) containing 15%heat inactivated (30 min, 56°C) fetal bovine serum (FBS) (Flow Laboratories, McLean, VA, lot #29161020) and antibiotics (100 pg/ml penicillin B [Sigma Chemical Co., St. Louis, MO], 50 pgiml gentamicin sulfate [Sigmal, and 0.3 pgiml fungizone [Flow]). After a 24 hr incubation, floating cells and debris were washed out, and the attached cells were trypsinized and counted on a Coulter Counter (Model Z,; Coulter Electronics, Hialeah, FL). Population I cells were not used in these experiments. Pooled RC cell populations (11-V) were plated into 35 mm tissue culture dishes (Falcon 3001; Becton Dickinson Labware) a t a density of 3 x 104-105 cells per dish in standard growth medium as above (day 0). The next day (day 11, the medium was changed t o standard medium supplemented with 50 pgiml ascorbic acid (AA, Fisher Scientific Comp., Fair Lawn, NJ) and 10 mM sodium-R-glycerophosphate (P-GP, BDH Chemicals, Toronto, Ont.) with or without other additives as outlined below. The medium was changed completely every two days. Cultures were maintained in a humidified atmosphere

consisting of 95% air and 5% COz at 37°C (Forma Scientific, Model 33325-118 Marietta, OH) for up to 21 days and fixed and stained as described below. Forskolin (7P-acetoxy-8,13-epoxy-la,6B,9a-trihydroxy-labd-14-ene-11-one) (C2,H,,O7) (Calbiochem, La Jolla, CA, lots #405268 and #507242) (FSK) and the FSK analogue 1,9-dideoxy-forskoIin (Calbiochem, lot #703385) stock solutions were prepared a t concentrations of lop2M in absolute ethanol and stored a t -20°C for up to 4 months. For use, the stocks were diluted in standard growth medium. The concentration of ethanol in treated cultures did not exceed 0.1%; vehicle controls indicated that this amount of ethanol had no significant effect on either the cAMP levels, cell growth or the number of bone nodules. Histological procedures Cultures were terminated by fixing them in neutral buffered formalin (NBF) and they were stained by the von Kossa technique for calcium mineral deposits. Bone nodules were counted with a dissecting microscope a s described by Bellows et al. (1986). Alkaline phosphatase activity Alkaline phosphatase activity was measured in samples of cell homogenates using the method of Lowry (1955) as described by Grigoriadis et al. (1985).

cAMP a s s a y The effects of FSK on cAMP levels were determined in cultured cells using a modification of the method of Shimizu et al. (1969), as described by Grigoriadis et al. (1986). Statistical analyses A test for significance between the means of control and test groups within each experiment was done with the two-tailed Student’s t-test.

Fluorescence microscopy To visualize F-actin with rhodamine-conjugated phalloidin (Barak et al., 1980) (Molecular Probes, Junction City, OR), the cells were fixed and processed as described (Opas et al., 1985). Interference reflection microscopy To visualize the cell substratum adhesion patterns of cells grown on coverslips, interference reflection microscopy (IRMI was performed with a Zeiss Antiflex Neofluar 63i1.25 oil objective a t a n illuminating numerical aperture of 0.94 as described (Opas and Kalnins, 1984; Opas, 1985). RESULTS Effects of FSK on rat calvaria cell c A M P levels Cultures of RC 11-V cells in the exponential phase of M growth were treated for varying times with FSK, a concentration effective in many cell types, and the cAMP levels were determined. The cAMP levels of the RC cells increased steadily and reached maximum values by 20-30 min a t 37°C (Fig. 1) and by 1h r a t room temperature (data not shown). Thereafter, the cAMP levels decreased slowly to values slightly above basal level by 2-3 h r (Fig. 1).The vehicle (0.1% ethanol) had no effect on cAMP levels. Whether the de-

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FOKSKOLIN EFFECTS ON OSTEOPROGENITOR CELLS IN VITRO

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MI effects on cAMP accumulation Fig. 1. Time course of FSK in RC 11-V cells. Exponentially growing RC 11-V cells were incubated with M FSK and the cAMP levels were determined at the indicated times. Intracellular cAMP levels increased steadily, reaching a maximum value by 20-30 min at 37°C. Thereafter, the CAMPlevels decreased and reached basal levels by 2-3 hr. Neither vehicle (0.1% ethanol j nor M of the forskolin analogue, 1,9-dideoxy-forskolin had any measurable effect on cAMP levels. The results are means tSEM for 3 dishes. ( + ) 10-5M FSK; ( C ) vehicle, the same as 10 M 1,9-dideoxy-forskolin.Values for FSK are significantly greater than control values !P

Forskolin has biphasic effects on osteoprogenitor cell differentiation in vitro.

Cells isolated from fetal rat calvaria (RC) and maintained in vitro in medium containing ascorbic acid and B-glycerophosphate form three-dimensional, ...
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