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The uptake and release of ponasterone a by the Kc cell line of Drosophila melanogaster
Molecular and Cellular Endocrinology, 17 (1980) 5 l-59 0 Elsevier/North-Holland Scientific Publishers, Ltd.
51
THE UPTAKE AND RELEASE OF PONASTERONE OF Drosophila melanogaster
A BY THE K, CELL LINE
Christoph BECKERS ‘!*, Peter MAR6Y 2, Roger DENNIS 3, John D. O’CONNOR and Hans EMMERICH ’ ’ FB 10 Zoologie, Technische Hochschule, D-6100 Darmstadt, Schnittspahnstr. IO (West Germany), 2 Institute of Genetics, Hungarian Academy of Science, Szeged (Hungary), 3 Physiologisch-Chemisches Institut, Deutschhausstr. I-2, D-3550 Marburg (West Germany) and 4 Department of Biology, University of California, Los Angeles, CA 90024 (U.S.A.)
Received 22 June 1979; accepted 18 October 1979
The association of ponasterone A (PNA) and 20-hydroxyecdysone with Kc cells is commensurate with their biological activity on this Kc cell line, the physiological activity ratio for PNA, 20-hydroxyecdysone and ecdysone is 1 : 50 : 2000, resp. Both association and release of [3H]PNA are temperature-dependent, the activation energy was calculated as 16.7 caJ (Arrhenius analysis). This association is compatible with unlabelled PNA and various ecdysteroids. The KD for PNA (Scatchard analysis) was estimated as 3.6 X 10e9 M, giving the number of binding sites as approx. 1800 per cell. Keywords:
ponasterone A; 20-hydroxyecdysone;
uptake-release.
The present paradigm of the activation of vertebrate cells by steroid hormones follows the temporal sequence: binding of the hormone by cytoplasmic receptors, their resultant transformation and translocation to the nucleus, and the synthesis of specific RNA species (Reviews: Baulieu, 1975; Liao, 1976). Since none of the steroid specific receptors demonstrated until recently were detected on the cell surface, the step prior to steroid-receptor binding is the transfer of the hormone into the cell. The mechanism of internalisation of steroid hormones by cells is obscure. It was originally thought to be by simple diffusion (peck et al., 1973) or diffusion involving cytoplasmic receptors of the hormone (Jackson and Chalkley, 1974). However, evidence for the presence of saturable components of the plasma membrane, that are involved in the internalisation of steroid hormones by an energydependent process, is accumulating (Milgrom et al., 1973; Rao et al., 1976, 1977). The mechanism of uptake of ecdysteroids by insect cells is unknown. Maroy et al. (1978) and Yund et al. (1978) have demonstrated that the concentrating of PNA by K, cells and imaginal discs can be accounted for by the presence of receptors. * To whom requests for reprints should be sent.
52
C. Beckers et al.
Similarly, the association of 20-hydroxyecdysone by the former tissue shows properties characteristic of receptor binding, i.e. saturation, temperature dependency and specificity of uptake and retention (Yund and Fristrom, 1975). In this paper we have investigated the nature of association and release of PNA from K, cells. The results have served to define the problem by indicating that the parameters of association are caused by those of cytoplasmic and nuclear receptors of ecdysteroids.
METHODS The K, cell line was provided by C. Wyss (Zurich) and maintained as a suspension culture in a modified D 22 media (wyss, 1977) at 23°C. Ponasterone A (PNA: 2P,3P,1~,20R,22R-pentahydroxy-5P-cholest-7-en-6~ne) was a gift of K. Nakanishi (New York), 20-hydroxyecdysone (2P,3/3,14a,20R,22R,25-hexahydroxy-V-cholest7-en-6-one), ecdysone (2P,3P,14cu,22R,25-pentahydroxy-5P-cholest-7-en-6-one), 5-hydroxyponasterone A (2P,3B,58,14a,20R,22R-hexahydroxy-5P-cholest-7en-6one), makisterone A (2P,3P,14a,20R,22R,25-hexahydroxy-24-methyl-5P-cholest-7poststerone (21 -nor-2/3,3/3,14o-trihydroxy-5/3-pregn-7en-6,20-dione) end-one), were purchased from Simes Corp. (Milan), inokosterone (2/3,3/3,14~,2OR,22R,26hexahydroxy-5@cholest-7en-6-one) from Rhoto Pharmaceutical Co. (Osaka), cortisol (4-pregnene-1 l&l 7o,2 1-trihydroxy3,20dione) and cholesterol (5-cholesten-3/301) from Serva (Heidelberg). Trio1 (2/3,3&14o-trihydroxy-5/3-cholest-7en-6-one) and 5,20dihydroxyecdysone (2P,3P,5P,1~,20R,22R,25-heptahydroxy-5~-cholest-7un6-one) were generous gifts of D.H.S. Horn (Melbourne). [3H]PNA in a limited quantity was available for this study. This compound was obtained as previously reported (Maroy et al., 1978) with a specific activity of 122 Ci/mmol. The proliferation test of Wyss (1976) was used with minor modifications: K, cells at 2.5 X 10’ cells/ml were inoculated into gently shaking erlenmeyer flasks and grown in the presence of various concentrations of ecdysteroids at 25’C. Cell density was determined 72 h later by means of a hemocytometer. Uptake and release studies were performed according to Munck and Wira (1975) using [3H]PNA (5 X lo-” M) incubated for various times in the presence of 1 X 10’ cells/O.5 ml of modified D 22 media. This [3H]PNA concentration was chosen due to the limited quantity of labelled PNA available for this study. Incubation was terminated by a 40-fold dilution with ice-cold media, the cells pelleted after 5 min on ice by centrifugation for 5 min at 800 g at 4°C and counted, following resuspension in 0.5 ml distilled water and 5 ml Aqualuma scintillation cocktail (Lumac, Munich, Germany). Nonspecifically bound [3H]PNA to the entire pellet is almost completely removed by this dilution technique: only 3.8% of the total radioactivity taken up can be washed off by 5 subsequent washes in 20 ml icecold media.
53
RESULTS Proliferation of Kc cells The effect on the multiplication of K, cells is concentration dependent for each
ecdysteroid tested (Fig. l), but is without qualitative differences, the response curve is bimodal: low ecdysteroid concentrations (PNA: 5 X 10-12 M, 20-hydroxyecdysone 2 X 10-l* M, ecdysone 10m8M) stimulating proliferation, whilst high concentrations cause in~bition. The approximate activity ratios at the 10% inhibition level are 1 : SO : 2000. Uptake of PNA The kinetics of [3H]PNA uptake indicates a two-stage process with an initial
rapid phase being succeeded by a slower component, whilst the equilibrium state is reached after ca. 60 min (data not shown). The rapid component shows secondorder kinetics. The initial rate of association at 20°C is linear and gives an initial velocity of 8.5 X lo-‘* Mfmin (Fig. 2). An Arrhenius analysis of the initial association velocities at different temperatures (0-3O*C) gives a linear curve (Fig. 3). The activation energy was calculated from the slope as 16.7 cal. The saturability of [3H]PNA association is shown in Fig. 4, in a competition curve using unlabelled PNA as competitor. Scatchard analysis (Scatchard, 1949) gives a linear relation~p in the concentration range tested and estimated KD for PNA of 3.6 X lo9 M with the con~entratjon of binding sites as 0.86 nM. This corresponds to approx. 1800 binding sites per cell.
so-
Pig. 1. Effect of ecdysteroids on the proliferation of K, cells following 72-h incubation at varying concentrations of (A) PNA, (e) 20-hydroxyecdysone and (a) ecdysone, every symbol shows a single incubation. Results plotted as relative cell number expressed as percent of control (cultures without hormone) vs. the molar concentration of ecdysteroids. The dotted line represents the 10% inhibition level.
C. Beckers et al.
54
3.0
3
\ 2,5
0 2
0/
; 0 u 5
,/ ;1 0 ‘i
.’
E
.*
c E
J
$
\
2,o
‘\ 1,:
i-
/
,o
20
40
min
60
1.t )-3.2
‘\
,
3.4
l/TxlO-3
Fig. 2. Association of 13H]PNA (5
X
\ . ,
I
3.8
3.6 (OK)
10-l i M) with 1Oa cells at 2O’C.
Fig. 3. Arrhenius plot of association of 13H]PNA with Kc cells. The activation energy was calculated from the slope as 16.7 cal.
-0
lo-
28P M 6-
:
n
42-
0
0,5 l,o nM bound
0;
ng PNA
;
Fig. 4. Competition of [3H]PNA 0.5 ml) were incubated for 1 h at son of different experiments the total f3H]PNA bound vs. ng PNA.
lb
associated with Kc cells by unlabelled PNA. Cells (5 X 107/ 25°C with various concentrations of PNA. For the compariradioactivity associated with cells was plotted as percent of Inset: Scatchard analysis of the PNA competition data.
~edyste~oid bi~ldi~g in ~rosophi~ff
oml
55
1
aol OS p M competitor
o ponasteroneA l 5-OH-ponasterone A e 5.2~dihydroxyecdysone A 20.OH-ecdysone
Fig. 5. Competition
A o v +
makisterone A inokosterone ecdysone trio1
10
lii0
l posterone 9 cortisol (r chdesterd
of 13H]PNA association with K, cells (lOs/ml) by various steroids.
The specificity of [3H]PNA uptake was tested by using various ecdysteroids as competitors (Fig. 5). The steroids inhibiting PNA uptake seem to fit into 4 different classes of decreasing competition activity: ecdysteroids without the terminal hydroxyl function, analogs based on 20-hydroxyecdysone, ecdysone and compounds with a strongly modified side-chain.
I
1
05
1
i
1.5
1
2
hours Fig. 6. Kinetics of release of [3H]PNA from cells. Cells were incubated with 13H]PNA (5 X 10-l 1 M, 60 min, 25”C), the incubation terminated by a 40-fold dilution of hormone-free medium. At the times indicated cell suspensions were centrifuged and the radioactivity associated with the pellet counted. a, 20°C; o, 0°C.
56
C. Beckers et al.
Fig. 7. Kinetics of dissociation at 20°C of 13H]PNA from cells. 13H]PNA bound to cells was exchanged as described in the text. Results are plotted as M [‘H]PNA associated vs. time of exchange.
Release of PNA Simple dilution of the incubation mixture results in a composite curve (Fig. 6), where reiease from the cells is temperature dependent with the rate being 25 times greater at 2I’C than at 0°C. At 2I’C the dissociation consists of a fast and slow component, whilst at O’C the slow component predominates. The release at 21*C does not show first-order kinetics. A more satisfying approach in estimating the release kinetic of ecdysteroids by K, cells is: following normal incubation, the media is replaced with fresh media containing unlabelled PNA at the same concentration as that of f3H]PNA not taken up. The resultant dissociation velocity decreases at 2O*C in a first-order reaction of 5.8 X 10e3 min-’ (Fig. 7). Using the association and dissociation constant an estimate of the equilibrium dissociation constant at 20°C is 4.2 X 10” M.
DISCUSSION Ecdysteroids induce both stimulation and inhibition of multiplication of K, cells, but at different concentrations (Cherbas et al., 19’77; Courgeon, 1972,1975; Wyss, 1976, 1977). The 3 ecdysteroids tested show quantitative differences only, with decreasing order of effectiveness: PNA < ZO-hydroxyecdysone < ecdysone; i.e. a distinctive ecdysteroid concentration does not exhibit stimulation and inhibition after different times of hormone treatment (O’Connor et al., 1979), indicating an identical induction mechanism. PNA was used in this study, because it is
Ecd~stero~d binding in drosophila
57
required in the lowest concentrations possible, to induce proliferative changes in K, cells or evagination of imaginal discs of D. melanogaster (Yund et al., 1978). Attempts to demonstrate a specific uptake of 20-hydroxyecdysone or ecdysone in insect tissues have been mostly unsatisfactory, due to the low specific activity of the ecdysteroids then available and the low concentrations of hormone specifically bound (Yund and Fristrom, 1975; Maroy et al., 1978). Tritiated PNA with high specific activity therefore should be useful as a probe for studying the uptake, binding and release of ecdysteroids in insect tissues, even though this ecdysteroid has not yet been isolated from insect tissues. [3H]PNA uptake and release studies using established Drosophila cell lines provide two more advantages: K, cells grown in artificial media lack endogenous ecdysteroids, and do not metabolise PNA or 20hydroxyecdysone (Mardy et al., 1978, O’Connor et al., 1979). Most of the i3H]PNA (70%) taken up by K, cells is located in the nucleus after a 60-min period of incubation, whilst approx. 25% of the radioactivity inco~orated can be assayed in the high speed cytosol (Marby et af., 1978; O’Connor et al., 1979). This pattern of intracellularly distributed t3H]PNA clearly shows that most of the [3H]PNA associated with the cells is really internalised. The KD for the association of whole cells with PNA (3.6 X lo-’ M, Scatchard analysis) approximates the concentration for the biological response of the cells to the ecdysteroid, i.e. inhibition of cell proliferation. The saturation data for nuclear binding of E3H]PNA in K, cells is of the same order of magnitude as the concentration of these ecdysteroids for this biological response (Maroy et al., 1978). This is consistent with the more extensive kinetic data for imaginal discs (Yund and Fristrom, 1975; Yund et al., 1978) where morphogenetic induction and KD values coincide. The dissociation constant obtained by Scatchard analysis of whole cells and PNA agrees with the data for imaginal discs (Yund et al., 1978) and for cytoplasmic and nuclear preparations of K,.cells (Marby et al., 1978). However, it is to be noted that the dissociation constant computed from kinetical data shows a different value from steady-state experiments, as was found by Hansen et al. (1976) for the chickoviduct receptor of progesterone. PNA uptake in K, cells demonstrates the classical properties of hormone receptors: saturability, high affinity and specificity. The ecdysteroids tested were found to have widely different activities, but it is not possible using systemic in vivo assay systems to determine whether high or low competition activity of ecdysteroids is due to intrinsic properties of the tested analogue or to the rate of conversion of the analogue to more or less active metabolites. Under conditions in which no metabolism of PNA or 20-hydroxyecdysone occurs (Ma&y et al., 1978; O’Connor et al., 1979), E3H]PNA uptake is competed more effectively by PNA analogues than by 20-hydroxyecdysone analogues. Ecdysone competes about IOO-fold less effectively than 20-hydroxyecdysone, a value similar to the activity ratio in several biological assay systems (Ashburner, 1971; Chihara et al., 1972). Compounds with dramatic changes in the side-chain, vertebrate steroid hormones like cortisol and cholesterolx
58
C. Be&m
et al.
do not compete in physiological concentrations. The uptake of PNA by whole cells is temperature-dependent, with an increase at 20°C of 12 times over the rate at 0°C. The temperature dependency can be interpreted by at least 4 possible mechanisms. The active transport of the ecdysteroid into the cell; permeability changes in the plasma membrane; viscosity changes in the cytoplasm and the temperature activation of a specific hormone receptor. As yet, the association shows no indication of the mediation of hormone transport by carrier protein(s), as in cortisol uptake by liver cells (Rao et al., 1976) because both Arrhenius and Scatchard analyses show a functionally homogeneous class of binding sites in whole cells, as opposed to a multicomponent system such as that for liver cells and cortisol reception. The mechanism of an active transport of the hormone into the cell is excluded by a number of additional observations not presented here. The uptake of labelled PNA is not saturable by increasing concentrations of ligand at preequilibrium times, e.g. 5 min (O’Connor et al., 1979). Additionally the uptake of ecdysteroid is unaffected by KCN. It is more likely that the dramatic temperature dependency reflects a change in the permeability of the cell membrane or a temperature activation of the specific hormone receptor. The results suggest a specific uptake of the ecdysteroid PNA by K, cells, but the mechanism by which this occurs remains to be elucidated in more detail. Considering that there can be demonstrated only one functionally homogeneous class of binding sites in whole cells, exhibiting identical properties to cytoplasmic and nuclear receptors of ecdysteroids, probably PNA uptake will be regulated by the properties of these receptors.
REFERENCES
Ashburner, M. (1971) Nature (London) 230,222-224. Baulieu, E.-E. (1975) Mol. Cell. Biochem. 7,7.57-774. Cherbas, P., Cherbas, L., and Williams, CM. (1977) Science 197,275-277. Chihara, C.J., Petri, W.H., Fristrom, J.W., and King, D.S. (1972) J. Insect. Physiol. 18, 11151123. Courgeon, A. (1972) Exp. Cell Res. 74,327-336. Courgeon, A. (1975) Exp. Cell Res. 94,283-291. Hansen, P.E., Johnson, A., Schrader, W.T., and O’Malley, B.W. (1976) J. Steroid Biochem. 7, 723-732. Jackson, V., and Chalkley, R. (1974) J. Biol. Chem. 249,1627-1636. Liao, S. (1976) Int. Rev. Cytol. 41,87-172. Mar&y, P., Dennis, R., Beckers, C., Sage, B., and O’Connor, J.D. (1978) Proc. Natl. Acad. Sci. (U.S.A.) 75,6035-6038. Milgrom, E., Atger, M., and Baulieu, E.-E. (1973) Biochim. Biophys. Acta 320,267-283. Munck, A., and Wira, C. (1975) Methods Enzymol. 36, 255-264. O’Connor, J.D., Maroy, P., Beckers, C., Dermis, R., Alvarez, M.C., and Sage, B.A. (1979) Proc. Meadowbrook Conf. Steroid Action, in press. Peck, E.J., Bungner, J., and Clark, J.H. (1973) Biochemistry 12,4596-4603.
Ecdysteroid
binding in Drosophila
59
Rao, G.S., Schulze-Hagen, V., Rao, M.L., and Breuer, H. (1976) J. Steroid Biochem. 7, 11231129. Rao, M.L., Rao, G.S., and Breuer, H. (1977) Biochim. Biophys. Res. Commun. 77,566-573. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51,660-672. Wyss, C. (1976) Experientia 32,1272-1274. Wyss, C. (1977) J. Insect. Physiol. 23, 739-748. Yund, MA., and Fristrom, J.W. (1975) Develop. Biol. 43, 287-298. Yund, MA., King, D.S., and Fristrom, J.W. (1978) Proc. Nat]. Acad. Sci. (U.S.A.) 75,60396043.
51
THE UPTAKE AND RELEASE OF PONASTERONE OF Drosophila melanogaster
A BY THE K, CELL LINE
Christoph BECKERS ‘!*, Peter MAR6Y 2, Roger DENNIS 3, John D. O’CONNOR and Hans EMMERICH ’ ’ FB 10 Zoologie, Technische Hochschule, D-6100 Darmstadt, Schnittspahnstr. IO (West Germany), 2 Institute of Genetics, Hungarian Academy of Science, Szeged (Hungary), 3 Physiologisch-Chemisches Institut, Deutschhausstr. I-2, D-3550 Marburg (West Germany) and 4 Department of Biology, University of California, Los Angeles, CA 90024 (U.S.A.)
Received 22 June 1979; accepted 18 October 1979
The association of ponasterone A (PNA) and 20-hydroxyecdysone with Kc cells is commensurate with their biological activity on this Kc cell line, the physiological activity ratio for PNA, 20-hydroxyecdysone and ecdysone is 1 : 50 : 2000, resp. Both association and release of [3H]PNA are temperature-dependent, the activation energy was calculated as 16.7 caJ (Arrhenius analysis). This association is compatible with unlabelled PNA and various ecdysteroids. The KD for PNA (Scatchard analysis) was estimated as 3.6 X 10e9 M, giving the number of binding sites as approx. 1800 per cell. Keywords:
ponasterone A; 20-hydroxyecdysone;
uptake-release.
The present paradigm of the activation of vertebrate cells by steroid hormones follows the temporal sequence: binding of the hormone by cytoplasmic receptors, their resultant transformation and translocation to the nucleus, and the synthesis of specific RNA species (Reviews: Baulieu, 1975; Liao, 1976). Since none of the steroid specific receptors demonstrated until recently were detected on the cell surface, the step prior to steroid-receptor binding is the transfer of the hormone into the cell. The mechanism of internalisation of steroid hormones by cells is obscure. It was originally thought to be by simple diffusion (peck et al., 1973) or diffusion involving cytoplasmic receptors of the hormone (Jackson and Chalkley, 1974). However, evidence for the presence of saturable components of the plasma membrane, that are involved in the internalisation of steroid hormones by an energydependent process, is accumulating (Milgrom et al., 1973; Rao et al., 1976, 1977). The mechanism of uptake of ecdysteroids by insect cells is unknown. Maroy et al. (1978) and Yund et al. (1978) have demonstrated that the concentrating of PNA by K, cells and imaginal discs can be accounted for by the presence of receptors. * To whom requests for reprints should be sent.
52
C. Beckers et al.
Similarly, the association of 20-hydroxyecdysone by the former tissue shows properties characteristic of receptor binding, i.e. saturation, temperature dependency and specificity of uptake and retention (Yund and Fristrom, 1975). In this paper we have investigated the nature of association and release of PNA from K, cells. The results have served to define the problem by indicating that the parameters of association are caused by those of cytoplasmic and nuclear receptors of ecdysteroids.
METHODS The K, cell line was provided by C. Wyss (Zurich) and maintained as a suspension culture in a modified D 22 media (wyss, 1977) at 23°C. Ponasterone A (PNA: 2P,3P,1~,20R,22R-pentahydroxy-5P-cholest-7-en-6~ne) was a gift of K. Nakanishi (New York), 20-hydroxyecdysone (2P,3/3,14a,20R,22R,25-hexahydroxy-V-cholest7-en-6-one), ecdysone (2P,3P,14cu,22R,25-pentahydroxy-5P-cholest-7-en-6-one), 5-hydroxyponasterone A (2P,3B,58,14a,20R,22R-hexahydroxy-5P-cholest-7en-6one), makisterone A (2P,3P,14a,20R,22R,25-hexahydroxy-24-methyl-5P-cholest-7poststerone (21 -nor-2/3,3/3,14o-trihydroxy-5/3-pregn-7en-6,20-dione) end-one), were purchased from Simes Corp. (Milan), inokosterone (2/3,3/3,14~,2OR,22R,26hexahydroxy-5@cholest-7en-6-one) from Rhoto Pharmaceutical Co. (Osaka), cortisol (4-pregnene-1 l&l 7o,2 1-trihydroxy3,20dione) and cholesterol (5-cholesten-3/301) from Serva (Heidelberg). Trio1 (2/3,3&14o-trihydroxy-5/3-cholest-7en-6-one) and 5,20dihydroxyecdysone (2P,3P,5P,1~,20R,22R,25-heptahydroxy-5~-cholest-7un6-one) were generous gifts of D.H.S. Horn (Melbourne). [3H]PNA in a limited quantity was available for this study. This compound was obtained as previously reported (Maroy et al., 1978) with a specific activity of 122 Ci/mmol. The proliferation test of Wyss (1976) was used with minor modifications: K, cells at 2.5 X 10’ cells/ml were inoculated into gently shaking erlenmeyer flasks and grown in the presence of various concentrations of ecdysteroids at 25’C. Cell density was determined 72 h later by means of a hemocytometer. Uptake and release studies were performed according to Munck and Wira (1975) using [3H]PNA (5 X lo-” M) incubated for various times in the presence of 1 X 10’ cells/O.5 ml of modified D 22 media. This [3H]PNA concentration was chosen due to the limited quantity of labelled PNA available for this study. Incubation was terminated by a 40-fold dilution with ice-cold media, the cells pelleted after 5 min on ice by centrifugation for 5 min at 800 g at 4°C and counted, following resuspension in 0.5 ml distilled water and 5 ml Aqualuma scintillation cocktail (Lumac, Munich, Germany). Nonspecifically bound [3H]PNA to the entire pellet is almost completely removed by this dilution technique: only 3.8% of the total radioactivity taken up can be washed off by 5 subsequent washes in 20 ml icecold media.
53
RESULTS Proliferation of Kc cells The effect on the multiplication of K, cells is concentration dependent for each
ecdysteroid tested (Fig. l), but is without qualitative differences, the response curve is bimodal: low ecdysteroid concentrations (PNA: 5 X 10-12 M, 20-hydroxyecdysone 2 X 10-l* M, ecdysone 10m8M) stimulating proliferation, whilst high concentrations cause in~bition. The approximate activity ratios at the 10% inhibition level are 1 : SO : 2000. Uptake of PNA The kinetics of [3H]PNA uptake indicates a two-stage process with an initial
rapid phase being succeeded by a slower component, whilst the equilibrium state is reached after ca. 60 min (data not shown). The rapid component shows secondorder kinetics. The initial rate of association at 20°C is linear and gives an initial velocity of 8.5 X lo-‘* Mfmin (Fig. 2). An Arrhenius analysis of the initial association velocities at different temperatures (0-3O*C) gives a linear curve (Fig. 3). The activation energy was calculated from the slope as 16.7 cal. The saturability of [3H]PNA association is shown in Fig. 4, in a competition curve using unlabelled PNA as competitor. Scatchard analysis (Scatchard, 1949) gives a linear relation~p in the concentration range tested and estimated KD for PNA of 3.6 X lo9 M with the con~entratjon of binding sites as 0.86 nM. This corresponds to approx. 1800 binding sites per cell.
so-
Pig. 1. Effect of ecdysteroids on the proliferation of K, cells following 72-h incubation at varying concentrations of (A) PNA, (e) 20-hydroxyecdysone and (a) ecdysone, every symbol shows a single incubation. Results plotted as relative cell number expressed as percent of control (cultures without hormone) vs. the molar concentration of ecdysteroids. The dotted line represents the 10% inhibition level.
C. Beckers et al.
54
3.0
3
\ 2,5
0 2
0/
; 0 u 5
,/ ;1 0 ‘i
.’
E
.*
c E
J
$
\
2,o
‘\ 1,:
i-
/
,o
20
40
min
60
1.t )-3.2
‘\
,
3.4
l/TxlO-3
Fig. 2. Association of 13H]PNA (5
X
\ . ,
I
3.8
3.6 (OK)
10-l i M) with 1Oa cells at 2O’C.
Fig. 3. Arrhenius plot of association of 13H]PNA with Kc cells. The activation energy was calculated from the slope as 16.7 cal.
-0
lo-
28P M 6-
:
n
42-
0
0,5 l,o nM bound
0;
ng PNA
;
Fig. 4. Competition of [3H]PNA 0.5 ml) were incubated for 1 h at son of different experiments the total f3H]PNA bound vs. ng PNA.
lb
associated with Kc cells by unlabelled PNA. Cells (5 X 107/ 25°C with various concentrations of PNA. For the compariradioactivity associated with cells was plotted as percent of Inset: Scatchard analysis of the PNA competition data.
~edyste~oid bi~ldi~g in ~rosophi~ff
oml
55
1
aol OS p M competitor
o ponasteroneA l 5-OH-ponasterone A e 5.2~dihydroxyecdysone A 20.OH-ecdysone
Fig. 5. Competition
A o v +
makisterone A inokosterone ecdysone trio1
10
lii0
l posterone 9 cortisol (r chdesterd
of 13H]PNA association with K, cells (lOs/ml) by various steroids.
The specificity of [3H]PNA uptake was tested by using various ecdysteroids as competitors (Fig. 5). The steroids inhibiting PNA uptake seem to fit into 4 different classes of decreasing competition activity: ecdysteroids without the terminal hydroxyl function, analogs based on 20-hydroxyecdysone, ecdysone and compounds with a strongly modified side-chain.
I
1
05
1
i
1.5
1
2
hours Fig. 6. Kinetics of release of [3H]PNA from cells. Cells were incubated with 13H]PNA (5 X 10-l 1 M, 60 min, 25”C), the incubation terminated by a 40-fold dilution of hormone-free medium. At the times indicated cell suspensions were centrifuged and the radioactivity associated with the pellet counted. a, 20°C; o, 0°C.
56
C. Beckers et al.
Fig. 7. Kinetics of dissociation at 20°C of 13H]PNA from cells. 13H]PNA bound to cells was exchanged as described in the text. Results are plotted as M [‘H]PNA associated vs. time of exchange.
Release of PNA Simple dilution of the incubation mixture results in a composite curve (Fig. 6), where reiease from the cells is temperature dependent with the rate being 25 times greater at 2I’C than at 0°C. At 2I’C the dissociation consists of a fast and slow component, whilst at O’C the slow component predominates. The release at 21*C does not show first-order kinetics. A more satisfying approach in estimating the release kinetic of ecdysteroids by K, cells is: following normal incubation, the media is replaced with fresh media containing unlabelled PNA at the same concentration as that of f3H]PNA not taken up. The resultant dissociation velocity decreases at 2O*C in a first-order reaction of 5.8 X 10e3 min-’ (Fig. 7). Using the association and dissociation constant an estimate of the equilibrium dissociation constant at 20°C is 4.2 X 10” M.
DISCUSSION Ecdysteroids induce both stimulation and inhibition of multiplication of K, cells, but at different concentrations (Cherbas et al., 19’77; Courgeon, 1972,1975; Wyss, 1976, 1977). The 3 ecdysteroids tested show quantitative differences only, with decreasing order of effectiveness: PNA < ZO-hydroxyecdysone < ecdysone; i.e. a distinctive ecdysteroid concentration does not exhibit stimulation and inhibition after different times of hormone treatment (O’Connor et al., 1979), indicating an identical induction mechanism. PNA was used in this study, because it is
Ecd~stero~d binding in drosophila
57
required in the lowest concentrations possible, to induce proliferative changes in K, cells or evagination of imaginal discs of D. melanogaster (Yund et al., 1978). Attempts to demonstrate a specific uptake of 20-hydroxyecdysone or ecdysone in insect tissues have been mostly unsatisfactory, due to the low specific activity of the ecdysteroids then available and the low concentrations of hormone specifically bound (Yund and Fristrom, 1975; Maroy et al., 1978). Tritiated PNA with high specific activity therefore should be useful as a probe for studying the uptake, binding and release of ecdysteroids in insect tissues, even though this ecdysteroid has not yet been isolated from insect tissues. [3H]PNA uptake and release studies using established Drosophila cell lines provide two more advantages: K, cells grown in artificial media lack endogenous ecdysteroids, and do not metabolise PNA or 20hydroxyecdysone (Mardy et al., 1978, O’Connor et al., 1979). Most of the i3H]PNA (70%) taken up by K, cells is located in the nucleus after a 60-min period of incubation, whilst approx. 25% of the radioactivity inco~orated can be assayed in the high speed cytosol (Marby et af., 1978; O’Connor et al., 1979). This pattern of intracellularly distributed t3H]PNA clearly shows that most of the [3H]PNA associated with the cells is really internalised. The KD for the association of whole cells with PNA (3.6 X lo-’ M, Scatchard analysis) approximates the concentration for the biological response of the cells to the ecdysteroid, i.e. inhibition of cell proliferation. The saturation data for nuclear binding of E3H]PNA in K, cells is of the same order of magnitude as the concentration of these ecdysteroids for this biological response (Maroy et al., 1978). This is consistent with the more extensive kinetic data for imaginal discs (Yund and Fristrom, 1975; Yund et al., 1978) where morphogenetic induction and KD values coincide. The dissociation constant obtained by Scatchard analysis of whole cells and PNA agrees with the data for imaginal discs (Yund et al., 1978) and for cytoplasmic and nuclear preparations of K,.cells (Marby et al., 1978). However, it is to be noted that the dissociation constant computed from kinetical data shows a different value from steady-state experiments, as was found by Hansen et al. (1976) for the chickoviduct receptor of progesterone. PNA uptake in K, cells demonstrates the classical properties of hormone receptors: saturability, high affinity and specificity. The ecdysteroids tested were found to have widely different activities, but it is not possible using systemic in vivo assay systems to determine whether high or low competition activity of ecdysteroids is due to intrinsic properties of the tested analogue or to the rate of conversion of the analogue to more or less active metabolites. Under conditions in which no metabolism of PNA or 20-hydroxyecdysone occurs (Ma&y et al., 1978; O’Connor et al., 1979), E3H]PNA uptake is competed more effectively by PNA analogues than by 20-hydroxyecdysone analogues. Ecdysone competes about IOO-fold less effectively than 20-hydroxyecdysone, a value similar to the activity ratio in several biological assay systems (Ashburner, 1971; Chihara et al., 1972). Compounds with dramatic changes in the side-chain, vertebrate steroid hormones like cortisol and cholesterolx
58
C. Be&m
et al.
do not compete in physiological concentrations. The uptake of PNA by whole cells is temperature-dependent, with an increase at 20°C of 12 times over the rate at 0°C. The temperature dependency can be interpreted by at least 4 possible mechanisms. The active transport of the ecdysteroid into the cell; permeability changes in the plasma membrane; viscosity changes in the cytoplasm and the temperature activation of a specific hormone receptor. As yet, the association shows no indication of the mediation of hormone transport by carrier protein(s), as in cortisol uptake by liver cells (Rao et al., 1976) because both Arrhenius and Scatchard analyses show a functionally homogeneous class of binding sites in whole cells, as opposed to a multicomponent system such as that for liver cells and cortisol reception. The mechanism of an active transport of the hormone into the cell is excluded by a number of additional observations not presented here. The uptake of labelled PNA is not saturable by increasing concentrations of ligand at preequilibrium times, e.g. 5 min (O’Connor et al., 1979). Additionally the uptake of ecdysteroid is unaffected by KCN. It is more likely that the dramatic temperature dependency reflects a change in the permeability of the cell membrane or a temperature activation of the specific hormone receptor. The results suggest a specific uptake of the ecdysteroid PNA by K, cells, but the mechanism by which this occurs remains to be elucidated in more detail. Considering that there can be demonstrated only one functionally homogeneous class of binding sites in whole cells, exhibiting identical properties to cytoplasmic and nuclear receptors of ecdysteroids, probably PNA uptake will be regulated by the properties of these receptors.
REFERENCES
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