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On the mechanism of hormone action
ARCHIVES
OF
BIOCHEMISTRY
On
AND
BIOPHYSICS
106,
the Mechanism II. Ecdysone
of Hormone
and Protein
C. E. SEKERIS From
the Physiologisch-chemisches Received
(1964)
483-487
AND Institut
Action’
Biosynthesis
P. KARLSON der Universitat
November
Miinchen,
Germany
1, 1963
In the present series of papers, a special hypothesis on the mode of action of hormones will be tested: The hormone is supposed to activate some genes to produce messenger ribonucleic acid which in turn will lead to the synthesis of specific proteins. Inhibitors of RNA and protein synthesis will then abolish the effect of hormones. This has been demonstrated for the insect hormone ecdysone. Pupation of blowfly larvae, which is controlled by ecdysone, can be delayed for considerable time by the injection of streptomycin, chloromycetin, erythromycin, puromycin, mitomycin, actinomycin, or fluoro-deoxy-cytidin, Also, the induction of the enzyme, DOPA-decarboxylase, is inhibited by these metabolic inhibitors. The results are discussed in relation to our knowledge of the mode of action of the steroid hormones of vertebrates.
Based on the findings of Clever and Karlson (2) that the hormone ecdysone induces specific gene activities in Chironomus, we have presented a new hypothesis on the mechanism of action of hormones (3, 4). According to this hypothesis, hormones express their action by regulating, in the cell, the activities of certain genes. In the first paper of this series (1) we have followed the distribution of the hormone in tissues and cell compartments. The present paper describes the influence of inhibitors which are known to act on the chain between gene (DNA) and protein in the following sites
examined this question using ecdysone, the metamorphosis hormone of the insects. In previous work it was shown that ecdysone induces certain enzyme activities, especially that of the DOPA decarboxylase (12), an enzyme involved in the sclerotization metabolism (13). MATERIALS
: (Proposed
DNA T Mitomycin
activation
)
mess.
RNA
ribosomes
act. I Actinomycin
Puromycin Chloromvcetin Streptomycin Erythromycin
(7)
INJECTION Third
I, see (1).
erythrocephala, 483
Protein
amino
acid
T
(5)
If our hypothesis is correct, these inhibitors must block hormone action. We have Paper
METHODS
by hormones) 1
5-Fluorodeoxycytidine (6)
‘For
AND
5-Fluorodeoxy-cytidine and 5-fluorodeoxyuridine were obtained from Hoffmann La Roche through the kindness of Drs. Duschinsky and Fox; Puromycin from Prof. Wacker, Mitomycin C from Dr. Kersten; and Actinomycin C from Bayer. Streptomycin sulfate, Erythromycin, and Chloromycetin were commercial products.
instar
AND larvae were
(8) (9)
(ioj (11)
BIOLOGIC~~L of the blow fly, used throughout.
ACTION CalZipho,w The sub-
IOOso60. 70. ,
60-
streptomycin
.s F so39 $ 40-/ 30. 20. IO-
20 Hours
50
60
30 offer
40 injection
70 Hours
50
60 offer
60
d
injection
so
100
FIG. 1. Delay of pupation after injection of different antibiotics. the percentage of the pupated animals; the abscissa the time after stances were injected (a) 8, (b) 20, and (c) 69 hours before pupation. mitomycin,-@-O-a-; actinomycin,- - -; Chloromycetin,-X-X-X-; tin,-O-O-O-; erythromycin,-A-A-A-; and puromycin,-X-X-X-. 484
110
---+
The ordinate shows injection. The subControls,-----. streptomyl
ON
THE
?(IECHANIS?UI
OF
stances were dissolved in water or buffer and injected with a Desaga microinjection syringe. The concentrations were chosen so as to permit a maximum of action with a minimum of lethality: streptomycin, 300; Chloromycetin, 250; erythromycin, 400; puromycin, G; mitomycin, 0.1; actinomycin, 0.02; and 5-Fluorodeoxy-cytidine, 5 y. The substances were given at different intervals before pupation, and the delay in pupation was noted. Forty to 80 animals were used for each series. Animals of the same batch which were either injected with buffer or not at all (there was no difference between the two) were used as controls. To enable the comparison between the different series, the time at which 5Oc/;, of the animals had pupated was taken as “pupation time.”
INFLUENCE
ON THE DOPA DEC~RBOXYLASE
The different inhibitors were injected in several series of the same group of animals, and the activity of the DOPA decarboxylase was followed in these animals as well as in the controls. Eight animals were used for every measurement. The activity was measured by a radiochemical test previously described (12). The activity was expressed as percentage transformation of DOPA to dopamine.
HORMONE
ACTION.
485
II
80.
90
80
70
60 50 Hours before
40 30 pupation
20 -
10
0
FIG. 2. Influence of injection of antibiotics the pupation of Calliphora larvae. The delay pupation in hours is plotted against the time injection of the antibiotics before pupation. Mitomycin, -O-O-@-; actinomycin, - - Chloromycetin,-X-X-X-; streptomycin,-O-OO-; erythroymcin, -&a-&; and puromycin, -x-x-x-.
on of of -;
The inhibitors injected are probably degraded and thus cannot act at the time of secretion of the hormone. The third larval instar in our CccZZipho~~ From these experiments a critical phase of stock usually lasts 5 days. The larvae feed the primary action of ecdysone can be demainly in the first 2 days of the last instar; termined. In Fig. 2, the delay of pupation of they then leave the food and prepare for the injected animals is plotted against the pupation.” time interval between injection and pupation Injection of the inhibitors a short time of the controls. It is evident that all six before pupation leads only to a small delay inhibitors show a maximum of action at the of pupation (Fig. la). The pupation proc- same time, i.e., 20-30 hours before pupation. essesare so advanced that the inhibitors do Only streptomycin is the exception; its not interfere. However, if the injection is specific action is masked by its great toxicity performed 20-30 hours before pupation of so that the curve shows no maximum. the controls, a delay of 30 hours or more is INHIBITION OF ENZYME INDUCTIOS observed (Fig. lb). The biological action of the hormone is greatly inhibited. That pupaParallel to the delay of pupation, a delay tion eventually occurs is probably due to in the appearance of DOPA decarboxylase degradation and/or excretion of the inhibiactivity is seen (Fig. 3 a-d). In every series t’ors. This is clearly seenif the injections are of experiments the curve of the decarboxyperformed very early at 70 hours before lase activity of the controls is shown for pupation, i.e., before ecdysone is secreted. comparison. The experiments differ in that The delay in pupation is very small (Fig. lc). the inhibitors were given at different times before pupation. The general appearance of *The formation of the dark and hard pupal the curves, however, is similar, and the cut,icle will be called, for simplicity’s sake, “pupation.” action of actinomycin in Figs. 3a and 3d, RESULTS
DELAY
IN PUPATION
486
SEKERIS
AND
KARLSON
ON
THE
MECHANISM
OF
as well as that of Chloromycetin in Figs. 3b and 3d, are believed to be within the experimental error, The time of injection of mitomycin seems to play a great role. Early injection has a greater effect than a late one (Fig. 3c). DISCUSSION
Our hypothesis of hormone action predicts that inhibitors of nucleic acid and protein biosynthesis will inhibit hormone action. This prediction has been proved to be correct in our case: The biological action of ecdysone, i.e., puparium formation of the adult larvae of Calliphora, is delayed, especially if the inhibitors are given 25 hours before pupation. Some hours later a sharp increase in hormone concentration is seen which continues up to the newly formed pupa.3 WC believe that the inhibitors interfere with the early effects of the hormone. The analysis of Fig. 2 is of special interest. The fact that all substances tested show the maximum of action at the same time (30-20 hours before pupation) points to a common site of action, i.e., the stimulation of biosynthesis of specific proteins (enzymes). Jlitomycin shows a second peak 50 hours before pupation. It interferes with the first step of the postulated mechanism, i.e., the production of messenger RNA. That production of messenger R?jA is one of the first effects of a hormone has recently been shown by Liao and WilliamsAshman (21) in the case of testosterone. It is also known that the action of other hormones can be abolished by inhibitors of protein biosynthesis. This is true for thyroxine (14) and for the steroids estradiol (15) and cortisol (16). This inhibition cannot be explained on the basis of previous theories of hormone action (influence of cell permeability, coenzyme function (17, 18), etc.). Steroid hormones especially act by influencing protein biosynthesis. In this connection it seems to us interesting that ecdysone has been shown to be a steroid (19). In this connection, the steroid ring possibly has a special function which remains to be elucidated. The inhibitors tested also influence the increase of DOPA decarboxylase activity, 3 Unpublished
experiments
of E. Shaaya.
HORMONE
ACTION.
II
487
which is the biochemical manifestation of action of ecdysone. We have previously described this effect and interpreted it as enzyme induction, i.e., a de novo synthesis of enzyme protein (13, 20). The results described above have given experimental verification to this assumption. A simple reaction of hormone and enzyme molecule could not be blocked so uniformly by such different inhibitors. The action of ecdysone fits well in the picture of action of other steroids. A review about enzyme induction as a principle of hormone action has recently appeared elsewhere (4). REFERENCES 1. K.\RLSON, I?., SEKERIS, C. E., AND MAURER, It., 2. Physiol. Chem. 336, 100 (1964). 2. CLEVER, U., AND K.IRLSON, P., Exptl. Cell Res. 20, 623 (1960). 3. KARLSON, P., Dtsch. Med. Wochschr. 86, 668 (1961). 4. KIRLSON, P., Persp. Biol. Med. 6, 203 (1963). *5 KERSTEN, H., AND R~UEN, H. M., !Vature 190, 1195 (1961). L., RICH, M. A., .~ND EIDINOFF, M. 6. CHEONG, L., Cancer Res. 20, 1602 (1960). E., FR.INKLIN, R. M., SHSTKIN, A. J., 7. REICH, AND T,\TI.M, E. L., Science 134, 556 (1961). M. B., .ZND 1>ELaH~sa, G., 8. YARMOLINSKY, Proc. b’utl. Bead. Sci. 46, 1721 (1959). 0, . NATH~NS, D., AND LIPM.ZNN, F., Proc. Xatl. Acad. Sci. 47,497 (1961). F. E., C.\rs, J., WOLFE, A. I)., HIRT10. HAHN, UN, R. E., ALLISON, J. I,., .\ND HAR’I’M.\N, R. J., Biochim. Biophys. Acta 61, 741 (1962). 11. BROCK, T. D., Bacterial. Rev. 26,41 (1961). C. E., AND KARLSON, P., Biochim. 12. SEKERIS, Biophys. Acta 62, 103 (1962). P., AND SEKERIS, C. E., Biochim. 13. K.~RLSON, Biophys. Acta 63, 489 (1962). 197, 1167 (1963). 14. T.IT.~, J. R., Nature 15. MITELLER, G. C., GORSKI, J., AND AIZAWA, Y., Proc. Natl. Acad. fki. 47, 164 (1961). 16. GREENOARD, O., :\ND Acs, G., Biochim. Biophys. Acta 61, 652 (1962). O., AND LESTER, G., Recent Progr. 17. HECHTER, Harm. Res. 16, 139 (1960). 18. T~L.ILAY, P., HLJRLOCK, B., AND WILLL4MSASHMAN, H. G., Proc. Natl. Acad. Sci. 44, 862 (1958). 19. KARLSON, P., HOFFMEISTER, H., HOPPE, W., A4~~ HUBER, R., Lieb. Ann. 662, 1 (1963). 20. SEKERIS, C. E., International Atomic Energy Agency, Vienna 1953, p. 281. 21. LIAO, S., AND WILLIAMS-ASHMAN, H. G., Proc. .Vatl. Acad. Sci. 48, 1956 (1962).
OF
BIOCHEMISTRY
On
AND
BIOPHYSICS
106,
the Mechanism II. Ecdysone
of Hormone
and Protein
C. E. SEKERIS From
the Physiologisch-chemisches Received
(1964)
483-487
AND Institut
Action’
Biosynthesis
P. KARLSON der Universitat
November
Miinchen,
Germany
1, 1963
In the present series of papers, a special hypothesis on the mode of action of hormones will be tested: The hormone is supposed to activate some genes to produce messenger ribonucleic acid which in turn will lead to the synthesis of specific proteins. Inhibitors of RNA and protein synthesis will then abolish the effect of hormones. This has been demonstrated for the insect hormone ecdysone. Pupation of blowfly larvae, which is controlled by ecdysone, can be delayed for considerable time by the injection of streptomycin, chloromycetin, erythromycin, puromycin, mitomycin, actinomycin, or fluoro-deoxy-cytidin, Also, the induction of the enzyme, DOPA-decarboxylase, is inhibited by these metabolic inhibitors. The results are discussed in relation to our knowledge of the mode of action of the steroid hormones of vertebrates.
Based on the findings of Clever and Karlson (2) that the hormone ecdysone induces specific gene activities in Chironomus, we have presented a new hypothesis on the mechanism of action of hormones (3, 4). According to this hypothesis, hormones express their action by regulating, in the cell, the activities of certain genes. In the first paper of this series (1) we have followed the distribution of the hormone in tissues and cell compartments. The present paper describes the influence of inhibitors which are known to act on the chain between gene (DNA) and protein in the following sites
examined this question using ecdysone, the metamorphosis hormone of the insects. In previous work it was shown that ecdysone induces certain enzyme activities, especially that of the DOPA decarboxylase (12), an enzyme involved in the sclerotization metabolism (13). MATERIALS
: (Proposed
DNA T Mitomycin
activation
)
mess.
RNA
ribosomes
act. I Actinomycin
Puromycin Chloromvcetin Streptomycin Erythromycin
(7)
INJECTION Third
I, see (1).
erythrocephala, 483
Protein
amino
acid
T
(5)
If our hypothesis is correct, these inhibitors must block hormone action. We have Paper
METHODS
by hormones) 1
5-Fluorodeoxycytidine (6)
‘For
AND
5-Fluorodeoxy-cytidine and 5-fluorodeoxyuridine were obtained from Hoffmann La Roche through the kindness of Drs. Duschinsky and Fox; Puromycin from Prof. Wacker, Mitomycin C from Dr. Kersten; and Actinomycin C from Bayer. Streptomycin sulfate, Erythromycin, and Chloromycetin were commercial products.
instar
AND larvae were
(8) (9)
(ioj (11)
BIOLOGIC~~L of the blow fly, used throughout.
ACTION CalZipho,w The sub-
IOOso60. 70. ,
60-
streptomycin
.s F so39 $ 40-/ 30. 20. IO-
20 Hours
50
60
30 offer
40 injection
70 Hours
50
60 offer
60
d
injection
so
100
FIG. 1. Delay of pupation after injection of different antibiotics. the percentage of the pupated animals; the abscissa the time after stances were injected (a) 8, (b) 20, and (c) 69 hours before pupation. mitomycin,-@-O-a-; actinomycin,- - -; Chloromycetin,-X-X-X-; tin,-O-O-O-; erythromycin,-A-A-A-; and puromycin,-X-X-X-. 484
110
---+
The ordinate shows injection. The subControls,-----. streptomyl
ON
THE
?(IECHANIS?UI
OF
stances were dissolved in water or buffer and injected with a Desaga microinjection syringe. The concentrations were chosen so as to permit a maximum of action with a minimum of lethality: streptomycin, 300; Chloromycetin, 250; erythromycin, 400; puromycin, G; mitomycin, 0.1; actinomycin, 0.02; and 5-Fluorodeoxy-cytidine, 5 y. The substances were given at different intervals before pupation, and the delay in pupation was noted. Forty to 80 animals were used for each series. Animals of the same batch which were either injected with buffer or not at all (there was no difference between the two) were used as controls. To enable the comparison between the different series, the time at which 5Oc/;, of the animals had pupated was taken as “pupation time.”
INFLUENCE
ON THE DOPA DEC~RBOXYLASE
The different inhibitors were injected in several series of the same group of animals, and the activity of the DOPA decarboxylase was followed in these animals as well as in the controls. Eight animals were used for every measurement. The activity was measured by a radiochemical test previously described (12). The activity was expressed as percentage transformation of DOPA to dopamine.
HORMONE
ACTION.
485
II
80.
90
80
70
60 50 Hours before
40 30 pupation
20 -
10
0
FIG. 2. Influence of injection of antibiotics the pupation of Calliphora larvae. The delay pupation in hours is plotted against the time injection of the antibiotics before pupation. Mitomycin, -O-O-@-; actinomycin, - - Chloromycetin,-X-X-X-; streptomycin,-O-OO-; erythroymcin, -&a-&; and puromycin, -x-x-x-.
on of of -;
The inhibitors injected are probably degraded and thus cannot act at the time of secretion of the hormone. The third larval instar in our CccZZipho~~ From these experiments a critical phase of stock usually lasts 5 days. The larvae feed the primary action of ecdysone can be demainly in the first 2 days of the last instar; termined. In Fig. 2, the delay of pupation of they then leave the food and prepare for the injected animals is plotted against the pupation.” time interval between injection and pupation Injection of the inhibitors a short time of the controls. It is evident that all six before pupation leads only to a small delay inhibitors show a maximum of action at the of pupation (Fig. la). The pupation proc- same time, i.e., 20-30 hours before pupation. essesare so advanced that the inhibitors do Only streptomycin is the exception; its not interfere. However, if the injection is specific action is masked by its great toxicity performed 20-30 hours before pupation of so that the curve shows no maximum. the controls, a delay of 30 hours or more is INHIBITION OF ENZYME INDUCTIOS observed (Fig. lb). The biological action of the hormone is greatly inhibited. That pupaParallel to the delay of pupation, a delay tion eventually occurs is probably due to in the appearance of DOPA decarboxylase degradation and/or excretion of the inhibiactivity is seen (Fig. 3 a-d). In every series t’ors. This is clearly seenif the injections are of experiments the curve of the decarboxyperformed very early at 70 hours before lase activity of the controls is shown for pupation, i.e., before ecdysone is secreted. comparison. The experiments differ in that The delay in pupation is very small (Fig. lc). the inhibitors were given at different times before pupation. The general appearance of *The formation of the dark and hard pupal the curves, however, is similar, and the cut,icle will be called, for simplicity’s sake, “pupation.” action of actinomycin in Figs. 3a and 3d, RESULTS
DELAY
IN PUPATION
486
SEKERIS
AND
KARLSON
ON
THE
MECHANISM
OF
as well as that of Chloromycetin in Figs. 3b and 3d, are believed to be within the experimental error, The time of injection of mitomycin seems to play a great role. Early injection has a greater effect than a late one (Fig. 3c). DISCUSSION
Our hypothesis of hormone action predicts that inhibitors of nucleic acid and protein biosynthesis will inhibit hormone action. This prediction has been proved to be correct in our case: The biological action of ecdysone, i.e., puparium formation of the adult larvae of Calliphora, is delayed, especially if the inhibitors are given 25 hours before pupation. Some hours later a sharp increase in hormone concentration is seen which continues up to the newly formed pupa.3 WC believe that the inhibitors interfere with the early effects of the hormone. The analysis of Fig. 2 is of special interest. The fact that all substances tested show the maximum of action at the same time (30-20 hours before pupation) points to a common site of action, i.e., the stimulation of biosynthesis of specific proteins (enzymes). Jlitomycin shows a second peak 50 hours before pupation. It interferes with the first step of the postulated mechanism, i.e., the production of messenger RNA. That production of messenger R?jA is one of the first effects of a hormone has recently been shown by Liao and WilliamsAshman (21) in the case of testosterone. It is also known that the action of other hormones can be abolished by inhibitors of protein biosynthesis. This is true for thyroxine (14) and for the steroids estradiol (15) and cortisol (16). This inhibition cannot be explained on the basis of previous theories of hormone action (influence of cell permeability, coenzyme function (17, 18), etc.). Steroid hormones especially act by influencing protein biosynthesis. In this connection it seems to us interesting that ecdysone has been shown to be a steroid (19). In this connection, the steroid ring possibly has a special function which remains to be elucidated. The inhibitors tested also influence the increase of DOPA decarboxylase activity, 3 Unpublished
experiments
of E. Shaaya.
HORMONE
ACTION.
II
487
which is the biochemical manifestation of action of ecdysone. We have previously described this effect and interpreted it as enzyme induction, i.e., a de novo synthesis of enzyme protein (13, 20). The results described above have given experimental verification to this assumption. A simple reaction of hormone and enzyme molecule could not be blocked so uniformly by such different inhibitors. The action of ecdysone fits well in the picture of action of other steroids. A review about enzyme induction as a principle of hormone action has recently appeared elsewhere (4). REFERENCES 1. K.\RLSON, I?., SEKERIS, C. E., AND MAURER, It., 2. Physiol. Chem. 336, 100 (1964). 2. CLEVER, U., AND K.IRLSON, P., Exptl. Cell Res. 20, 623 (1960). 3. KARLSON, P., Dtsch. Med. Wochschr. 86, 668 (1961). 4. KIRLSON, P., Persp. Biol. Med. 6, 203 (1963). *5 KERSTEN, H., AND R~UEN, H. M., !Vature 190, 1195 (1961). L., RICH, M. A., .~ND EIDINOFF, M. 6. CHEONG, L., Cancer Res. 20, 1602 (1960). E., FR.INKLIN, R. M., SHSTKIN, A. J., 7. REICH, AND T,\TI.M, E. L., Science 134, 556 (1961). M. B., .ZND 1>ELaH~sa, G., 8. YARMOLINSKY, Proc. b’utl. Bead. Sci. 46, 1721 (1959). 0, . NATH~NS, D., AND LIPM.ZNN, F., Proc. Xatl. Acad. Sci. 47,497 (1961). F. E., C.\rs, J., WOLFE, A. I)., HIRT10. HAHN, UN, R. E., ALLISON, J. I,., .\ND HAR’I’M.\N, R. J., Biochim. Biophys. Acta 61, 741 (1962). 11. BROCK, T. D., Bacterial. Rev. 26,41 (1961). C. E., AND KARLSON, P., Biochim. 12. SEKERIS, Biophys. Acta 62, 103 (1962). P., AND SEKERIS, C. E., Biochim. 13. K.~RLSON, Biophys. Acta 63, 489 (1962). 197, 1167 (1963). 14. T.IT.~, J. R., Nature 15. MITELLER, G. C., GORSKI, J., AND AIZAWA, Y., Proc. Natl. Acad. fki. 47, 164 (1961). 16. GREENOARD, O., :\ND Acs, G., Biochim. Biophys. Acta 61, 652 (1962). O., AND LESTER, G., Recent Progr. 17. HECHTER, Harm. Res. 16, 139 (1960). 18. T~L.ILAY, P., HLJRLOCK, B., AND WILLL4MSASHMAN, H. G., Proc. Natl. Acad. Sci. 44, 862 (1958). 19. KARLSON, P., HOFFMEISTER, H., HOPPE, W., A4~~ HUBER, R., Lieb. Ann. 662, 1 (1963). 20. SEKERIS, C. E., International Atomic Energy Agency, Vienna 1953, p. 281. 21. LIAO, S., AND WILLIAMS-ASHMAN, H. G., Proc. .Vatl. Acad. Sci. 48, 1956 (1962).