Corticotropin (ACTH) induces a characteristic retraction of rat adrenocortical cells in culture. Densitometric analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell lysates showed that ACTH induced a 20% decrease in actin content. These results were fully confirmed by quantitation of actin by the DNase 1 inhibition assay. The decrease in actin is hormone-specific, concentration-dependent, and correlates temporally with the morphological change induced by ACTH. The change in actin content and the reorganization of microfilament ultrastructure may together mediate hormone regulation of cell shape at the cytoskeletal level

Cheitlin, R.

Ramachandran, J.

English

Publications of the IAS Fellows

Communication Vol. 256, No. 7, Issue of April 10, pp. 3156-3158, 1981 Printed in U.S.A THE JOURNAL OF BIOLOGICAL CHEMISTRY 
Regulation of Actin  in Rat 
Adrenocortical  Cells by 
Corticotropin* 
(Received for publication, December 15, 1980, and in revised form, 
January 20, 1981) 
Roger Cheitlin and J. Ramachandran 
From  the Hormone Research  Laboratory, University of 
California, Sun Francisco,  California 94143 
Corticotropin (ACTH) induces a characteristic re- 
traction of rat adrenocortical cells in culture. Densito- 
metric analysis of s o d h m  dodecyl sulfate-polyacryl- 
amide gel electrophoresis gels of cell  lysates  showed 
that ACTH induced a 20% decrease in actin content. 
These results  were  fully  confirmed  by  quantitation of 
actin  by the DNase 1 inhibition  assay. The decrease in 
actin is hormone-specific,  concentration-dependent, 
and correlates  temporally  with the morphological 
change induced by ACTH. The change in actin content 
and the reorganization of microfilament ultrastructure 
may  together  mediate  hormone  regulation of cell shape 
at the  cytoskeletal level. 
Nonmuscle actin is a major structural protein in many cell 
types and is  a principal component of the cytoskeleton. Actin 
has been  localized in a wide spectrum of organelles including 
pseudopodia, microvilli, and the mitotic spindle. It is the 
major component of microfilaments, which  comprise the so- 
called “stress cables” seen in cells by phase contrast micros- 
copy (1). Due t o  the many physicochemical properties in 
common with muscle actin, nonmuscle actin has been impli- 
cated in the movement of these organelles as well as the 
movement of the entire celI and its  attachment  to  the sub- 
stratum in culture (1). Furthermore, the ability of nonmuscle 
actin to polymerize  reversibly has led to  the proposal that  the 
organization of stress cables determines the cytoskeletal ar- 
chitecture of the cell to  a large extent. 
The shapes of many  cell types are altered by specific exter- 
nal stimuli. Lutropin and dibutyryl-CAMP  influence the mor- 
phology of bovine luteal cells in culture (2) and thrombin 
affects the shape of human platelets (3) .  FoUitropin induces 
retraction of Sertoli cells in culture (4) and corticotropin 
produces a characteristic retraction of rat adrenocortical cells 
in culture (5, 6). 
Immunofluorescent staining of the cytoskeleton has re- 
vealed that such shape changes are accompanied by dramatic 
alterations in microfilament organization. It has been sug- 
gested that  the regulation of cell shape may be mediated by 
the organization of actin ( 7 ) .  We have examined the effects of 
ACTH’ on rat adrenocortical cell actin. Quantitative meas- 
urements of the actin content of cells showed that ACTH 
causes a significant decrease in the amount of actin. In this 
* This work was supported by Grant CA-16417 awarded by the 
National Cancer Institute, United States Department of Health, 
Education and Welfare. The costs of publication of this article were 
defrayed in part by the payment of page charges. This article must 
therefore be hereby marked “aduertisement” in accordance with 18 
U.S.C. Section 1734 solely to indicate this fact. 
’ The abbreviation used  is: ACTH, corticotropin. 
communication we present biochemical  evidence that ACTH 
regulates rat adrenocortical cell actin. 
MATERIALS AND METHODS 
Highly purified porcine ACTH (8),  synthetic corticotropin-inhibit- 
ing peptide (9),  and  synthetic endorphin (10) were prepared in this 
laboratory by the published procedures. Deoxyribonuclease I from 
bovine pancreas, calf thymus DNA, type I, lima bean trypsin inhibitor, 
and phenylmethylsulfonyl fluoride were obtained from Sigma. All  gel 
electrophoresis reagents were obtained from Bio-Rad. Purified rabbit 
skeletal muscle actin was a gift of Dr. Roger Cooke. 
Cell Culture-Adrenocortical cells were isolated from the decap- 
sulated adrenals of adult male Sprague-Dawley rats  as previously 
described (6) and plated in medium 199-D-valine containing 10% 
dialyzed fetal bovine serum and garamycin (80 pg/ml) at a density of 
5 X lo5 cells/well(Il).  The medium was changed every other  day  and 
cultures were used 7-14 days after plating. 
DNase I Inhibition Assay for Actin-The assay was performed 
essentially as described by Blikstad et al. (12). Cells were treated with 
hormone or other  agents  and at specified times, the cells were washed 
3 times with ice-cold phosphate-buffered saline and lysed mechani- 
cally by scraping in 150 pl of lysis buffer (5 mM phosphate, pH 7.6, 
containing 150 mM NaCl, 2 mM MgCL, 0.2 mM NaZATP, 0.2 mM 
dithiothreitol, 0.5% (v/v)  Triton X-100, 10 p~ phenylmethylsulfonyl 
fluoride, and 0.001% lima bean trypsin inhibitor. A 50-pl aliquot was 
immediately removed, mixed with 50 pl of dissociating buffer (20 mM 
Tris-HC1, pH 7.6, 1 M sodium acetate, 1 mM CaC12, 1 mM NaZATP, 1.5 
M guanidine-HCl), and incubated on ice for 15 min. Then 20 pl of 
DNase 1 (0.1 mg/ml in 50 mM Tris-HC1, pH 7.5, 0.5 mM CaCI2,  10 p~ 
phenylmethylsulfonyl fluoride) were added. Sixty pl of this mixture 
were removed and added to 3 rnl of DNA at a concentration of  50 pg/ 
ml in 100 mM Tris-HC1, pH 7.5, 4 mM MgSO,, 1.8 mM CaCL  The 
change in absorbance at 260 nm was recorded for a t  least 5 min to 
determine V,,,,,. Standard curves were generated in exactly the same 
way  using purified rabbit  skeletal muscle actin. 
DNA was determined by the method of Ceriotti (13) using the 
modifications of Short et al. (14). 
Polyacrylamide Gel Electrophoresis-Cells were washed 5 times 
with ice-cold phosphate-buffered saline and then lysed in 58 mM Tris- 
HCl, pH 6.8, containing 2% sodium dodecyl sulfate, 10% glycerol, 0.4% 
dithiothreitol,  and 0.1% bromphenol blue. The lysates were heated 
for 5 min at  90 “C, cooled to room temperature,  and 25-50 pl were 
applied per lane of  gel and subjected to electrophoresis according to 
Laemmli (15) on 10% polyacrylamide gels (1.5 mm thickness). Gels 
were stacked at 18 mA for 15 min and  run at  35 mA for 4 h. The gels 
were stained with 0.025% Coomassie brilliant blue (R-250) overnight 
and destained first in 25% isopropyl alcohol, 10% acetic acid, 0.0025% 
Coomassie blue, and then in 10% acetic acid. The gels  were air-dried 
between dialysis membranes and scanned in a GCA/McPherson 
Model EU 205-11 scanning densitometer at 550 nm at a rate of 5 cm/ 
rnin at 1 mA sensitivity. 
RESULTS 
Rat adrenocortical cells in culture formed a confluent mono- 
layer of flattened cells. A marked retraction of the cells  was 
observed after 6 h in the presence of 0.22 nM ACTH. The 
morphological changes were identical with those described 
previously (6) except that they were produced by physiological 
concentrations of the hormone. Maximal retraction was ob- 
served in the presence of 2.2 nM ACTH. Other basic polypep- 
tides such as insulin, endorphin, or corticotropin-inhibiting 
peptide (ACTH (7-38)) had no effect on rat adrenocortical 
cell  morphology. 
The sodium dodecyl sulfate gel patterns of cells incubated 
with or without 2.2 nM ACTH are shown in Fig. 1. The major 
protein band in control as well as ACTH-treated cells corre- 
sponded to M ,  = 43,000. That this band is indeed actin was 
3 156 
Regulation of Adrenal  ctin by ACTH 3157 
43K 
r) 1 * ‘ I  
FIG. 1. Densitometric analysis of sodium dodecyl sulfate- 
polyacrylamide gel electrophoresis gels. Cells were incubated 
with (botforn) or  without (top) 2.2 nM ACTH for 24 h and processed 
as described under “Materials and Methods” for sodium dodecyl 
sulfate-polyacrylamide gel electrophoresis. The  actin  band  is  denoted 
by the molecular  weight (43K. 43,000). 
‘ O r  
0.4 0.8 1.2 1.6 2.0 
ACTIN ( f i g  1 
FIG. 2. Standard  curve for actin  measurement by the DNase 
1 inhibition  assay. Purified rabbit  skeletal muscle actin was  used to 
inhibit DNase 1 .  Details of the  assay  are  outlined  under  “Materials 
and  Methods.”  Each point represents  the  mean  of  three  determina- 
tions. The correlation coefficient is 0.992. 
c o n f i i e d  by amino acid  analysis of the  protein  eluted  from 
the gels and DNase 1-Sepharose affinity chromatography 
(data  not  shown). By weighing the  actin peak as well as the 
other  protein peaks, it was  found that  actin  amounted  to 10- 
12% of the  total cellular protein in control cells. The  actin 
band was significantly  decreased in ACTH-treated cells. The 
decrease was estimated  to  be 25% compared  to  the  control 
cells. 
In order to c o n f i i  and quantitate this change in actin 
content  more  accurately, we resorted  to  the  DNase 1  inhibi- 
tion assay for actin (12). This  assay is  based  on the fact that 
monomeric actin (G-actin) binds tightly and irreversibly to 
DNase 1 and inhibits its enzymic activity. The presence of 
guanidine-HC1 at  a final concentration of  0.75 M ensures 
complete depolymerization of actin in cell lysates. The degree 
of inhibition  is therefore a direct  measure of actin  content. A 
standard curve of inhibition using purified rabbit skeletal 
muscle actin is  shown in  Fig. 2. It is known that  both muscle 
and nonmuscle actins  inhibit  DNase 1 in a comparable  manner 
(12). It is evident from Fig. 2 that in the range 0.3 to 1.5  pg  of 
actin  the inhibition is linear. Cell lysates were  diluted appro- 
priately  to  produce  inhibitions within this range. 
Using this  assay,  the ffects of ACTH on rat adrenocortical 
cell actin were  investigated. The results in Table I show that 
ACTH caused a selective dose-dependent decrease in the 
actin  content which cannot be attributed  to  changes in cell 
number. During the 12-h incubation with the hormone the 
DNA content of the cells was  unchanged. The kinetics of the 
action of ACTH  on  actin  content  are shown in Fig. 3. It is 
apparent  that a maximal  decrease in actin  content is  produced 
within 12 h after  the cells are exposed to  the hormone. 
The specificity of the  change in actin  content induced by 
ACTH  are  shown in Fig. 4. Other  peptide  hormones which do 
not  stimulate  adrenocortical function  did not  alter  the  actin 
content of these cells. o-Nitrophenylsulfenyl-Trp“-ACTH, 
which  is a weak ACTH agonist  (16),  also  produced  a  concen- 
tration-dependent  decrease in actin  but only at higher  concen- 
trations. The effect of ACTH on the  actin  content of adreno- 
TABLE I 
Effect of ACTH on rat  adrenocortical cell  actin 
Adrenocortical cells (5 X lO”/well) were incubated with or  without 
ACTH for 12 h, 7 days after plating. The cells were processed as 
described under “Materials and Methods” and actin content was 
determined by the  DNase 1 inhibition assay  and DNA content 
according to  Short et al. (14). Values are  mean k S.E. of the  number 
of separate  determinations  shown in parentheses. 
Hormone Actin DNA  Actin/DNA czA 
IrR  IrR rk 
None 18.84 f 0.64 (4) 4.05 f 0.02 (3) 4.65 f 0.16 
ACTH 16.70 f 0.00 (8) 4.08 f 0.07 (3) 4.09 f 0.07 12 
ACTH (2.2 14.85 f 0.07 (6) 3.98 f 0.04 (3) 3.67 f 0.04 21 
(0.22 nM) 
nM) 
2 I/’ 
V I I I I I 
0 IO 20 30 40 50 
TIME (hrs.) 
FIG. 3. Time course of the decrease in actin induced by 
ACTH. Cells  were incubated  with  and  without 2.2 nM ACTH  and a t  
specified times  the cells  were  processed as described under  “Materials 
and  Methods” for the  determination of actin by the  DNase 1 inhibi- 
tion assay. DNA was  also measured a t  each  time point.  Hesults are 
from  four  experiments.  Each  point  represents  the  mean f S.E. of a t  
least  three  experiments.  In  each  experiment cells  were incubated  in 
triplicate with or  without  ACTH.  Percentage of decrease was  calcu- 
lated from micrograms of actin/pg of DNA  values a t  each  time point. 
3158 Regulation of Adrenal  Actin by ACTH 
- s 
W 
v) 9 20- 
LT u 
W n 
z 
a L 10- 
0- 
ng/ml- Io 
28.8 f 3.1 
T 
12+0 
NPS-ACTH I] Nf 
CIP p-ENDORPHIN 
100 1000 10 10 
FIG. 4. Specificity of ACTH-induced decrease in actin. Cells 
were incubated with the various peptides for 24 h  and then processed 
for actin determination by DNase 1 inhibition assay. Values are  the 
means 2 S.E. of triplicate incubations. CIP, corticotropin-inhibiting 
peptide; NPS, o-nitrophenyl sulfenyl. 
cortical cells was reversed when the hormone was removed. 
Cells incubated in the absence of ACTH for 20 days had 5.38 
1.23 pg  of actin/pg of DNA compared to a value of 2.45 & 
0.43 for cells incubated with ACTH. Cells treated with ACTH 
for 14 days and incubated for 6 more days  in the absence of 
ACTH had 6.35 & 1.12 pg  of actin/pg of DNA. 
DISCUSSION 
Actin is a major protein  in  a  variety of cell types. In  rat 
adrenocortical cells, actin  represents at  least 10% of the  total 
cellular protein. Both densitometric  scans of sodium dodecyl 
sulfate gels and  the DNase 1 inhibition assay show that ACTH 
induces a 20-25% decrease in actin. Therefore, at  least 2% of 
the  total cellular protein is lost in response to the hormone. 
Such  a major change in  protein  must have tremendous con- 
sequences for cell function and shape. 
Changes in actin  content  in response to  hormonal  stimuli 
have been reported (17, 18). Sidhu (19) found a fall in total 
actin to approximatelv 20% of unstimulated levels during the 
maturation of 3T3-Ll mouse preadipocytes into functional 
adipocytes by treatment with dexamethasone. This decrease 
was accompanied by a morphological transformation from 
flattened to spherical shape. Bragina et al. (20) used the 
DNase 1 inhibition assay to demonstrate  a fall in actin content 
in mouse myeloma cells in response to dexamethasone. 
Immunofluorescent techniques for visualizing  specific com- 
ponents of the cytoskeleton have implicated microfilaments 
in defining cell shape. Weber et al. (21) have shown that 
agents  such as cytochalasin B which disrupt microfilaments 
cause both  retraction and rounding of cultured 3T3 cells and 
a  dramatic depolymerization of actin  stress cables. 
Direct measurements of monomeric and polymeric actin 
pools in cells c o n f i  the results  obtained by immunofluores- 
cent methods. Carlsson et al. (3) utilized the DNase 1 inhibi- 
tion assay to demonstrate  a  rapid depolymerization of micro- 
filaments in human platelets stimulated with thrombin to 
change shape from flattened discs to spheres.  Swanston-Flatt 
et al. (22) used a similar approach to show microfilament 
formation in pancreatic p-cells during glucose stimulation of 
insulin release. These observations, taken together with the 
immunofluorescence data  and measurements of actin content, 
strongly suggest that changes in  both  actin content  and or- 
ganization define and possibly regulate cell shape.  Preliminary 
investigations of microfilament organization in cultured rat 
adrenocortical cells by indirect immunofluorescence show a 
depolymerization of actin stress cables in response to ACTH. 
The results  presented  here  demonstrate  a hormonally me- 
diated decrease in actin in cultured rat adrenocortical ce&. 
The observation that both the loss of actin and  the retraction 
of cells in response to ACTH are hormone-specific, concentra- 
tion-dependent, and follow similar time courses is suggestive 
evidence that these effects are in some way related. The 
ACTH-induced changes in cell shape  and actin  content are 
both observable 6 h after  the addition of the hormone. This 
lag period is very similar to  that observed for other trophic 
actions of ACTH, namely, inhibition of DNA synthesis (23), 
induction of 3P-hydroxysteroid dehydrogenase-isomerase (11) 
and ectokinase activity (24). It is tempting  to  speculate that 
all of these effects are interrelated manifestations of the 
pleiotropic response of the adrenocortical cell to ACTH. 
Whether these effects are regulated via some common media- 
tor and how reorganization of the cytoskeleton might be 
related  to growth control are  important questions that remain 
to be elucidated. 
Acknowledgments-We wish to thank Professor C. H. Li for his 
interest  and  Dr. Roger Cooke for the gift of purified rabbit skeletal 
muscle actin  and access to his scanning densitometer. 
REFERENCES 
1. Lindberg, U., Carlsson, L., Markey, F., and Nystrom, L. E. (1979) 
2. Gospadarowicz, D., and Gospodarowicz,  F. (1975) Endocrinology 
3. Carlsson, L., Markey, F., Blikstad, I., Persson, T., and Lindberg, 
U. (1979) Proc.  Natl.  Acad. Sci. U. S. A .  76,6376-6380 
4. Tung, P. S., Dorrington, J. H., and Fritz, I. B. (1975) Proc. Natl. 
Acad. Sci. U. S. A .  72, 1838-1842 
5. O’Hare, M. J., and Neville, A. M. (1973) J. Endocrinol. 56, 529- 
553 
6. Ramachandran, J., and Suyama, A. T. (1975) Proc. Natl.  Acad. 
Sci. U. S. A .  72, 113-117 
7. Goldman, R. D., Pollard, T. D., and Rosenbaum, J., eds (1976) 
Cell Motility, Book B, Cold Spring Harbor Laboratory, New 
York 
8. Canova-Davis, E., and  Ramachandran, J. (1976) Biochemistry 15, 
9. Li,  C. H., Chung, D., Yamashiro, D., and Lee,  C. Y. (1978) Proc. 
10. Li, C. H., Yamashiro, D., Tseng, L-F., and Loh, H. H. (1977) J. 
11. Liles, S., and Ramachandran, J. (1977) Biochem. Biophys. Res. 
12. Blikstad, I., Markey, F., Carlsson, L., Persson, T., and Lindberg, 
13. Ceriotti, G. (1952) J. Biol. Chem. 198, 297-303 
14. Short, E. C., Jr., Warner, H. R., and Koerner, J. F. (1968) J.  Biol. 
15. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685 
16. Moyle, W. R., Kong, Y.  C., and  Ramachandran, J. (1973) J.  Biol. 
17. Hoffman-Liebermann, B., and Sachs, L. (1978) Cell 14,825-834 
18. Riddle, V. G. H., Dubrow, R., and Pardee, A. G. (1979) Proc. 
19. Sidhu, R. S. (1979) J. Biol. Chem. 254,11111-11118 
20. Bragina, E. E., Vasilier, Ju. M., and Gelfand, I. M. (1976) EXP. 
21. Weber, K., Rathke, P. C., Osborn, M., and Franke, W. w. (1976) 
22. Swanston-Flatt, S. K., Carlsson, L., and Gylfe, E. (1980) FEBS 
23. McPherson, M., and Ramachandran, J. (1980) J. Cell Biol. 86, 
24. McPherson, M., and Ramachandran, J. (1980) Biochem. Biophys. 
Methods Achieu. Exp.  Pathol. 8, 143-170 
96,458-467 
921-927 
Natl.  Acad. Sci. U. S. A.  75,4306-4309 
Med. Chem. 20,325-328 
Cornrnun. 79, 226-233 
U. (1978) Cell 15,935-943 
Chem. 243, 3342-3344 
Chem. 248,2409-2417 
Natl.  Acad. Sci. U. S. A .  76,1298-1302 
Cell.  Res. 97, 241-248 
Exp. Cell Res. 102,285-297 
Lett. 117, 299-302 
129-134 
Res. Cornrnun. 94, 1057-1065 


Regulation of actin in rat adrenocortical cells by corticotropin

http://repository.ias.ac.in/40876/1/54-Pub.pdf

Regulation of actin in rat adrenocortical cells by corticotropin

Abstract

Similar works

Full text

Available Versions

Publications of the IAS Fellows