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Modulation of Calmodulin Gene Expression as a Novel
Mechanism for Growth Hormone Feedback Control by
Insulin-like Growth Factor in Grass Carp Pituitary Cells
Longfei Huo, Guodong Fu, Xinyan Wang, Wendy K. W. Ko, and Anderson O. L. Wong
Department of Zoology, University of Hong Kong, Hong Kong, Peoples Republic of China
Calmodulin (CaM), the Ca2ⴙ sensor in living cells, is essential
for biological functions mediated by Ca2ⴙ-dependent mecha-
nisms. However, modulation of CaM gene expression at the
pituitary level as a means to regulate pituitary hormone syn-
thesis has not been characterized. In this study we examined
the functional role of CaM in the feedback control of GH by
IGF using grass carp pituitary cells as a cell model. To estab-
lish the structural identity of CaM expressed in the grass carp,
a CaM cDNA, CaM-L, was isolated from the carp pituitary
using 3ⴕ/5ⴕ rapid amplification of cDNA ends. The open reading
frame of this cDNA encodes a 149-amino acid protein sharing
the same primary structure with CaMs reported in mammals,
birds, and amphibians. This CaM cDNA is phylogenetically
related to the CaM I gene family, and its transcripts are ubiq-
C 2⫹
ALMODULIN (CaM) IS a Ca2⫹-binding protein serving
as an intracellular Ca2⫹ sensor in living cells. Upon
Ca binding, conformational changes occur in the CaM
molecule with the exposure of hydrophobic domains for
subsequent association with CaM target proteins (1). The
interactions of CaM with its target proteins activate a variety
of cellular processes, including gene expression (2), protein
synthesis (3), hormone secretion (4, 5), cell motility (6), ap-
optosis (7, 8), and cell proliferation (9, 10). In mammals, a
high level of CaM expression was noted in the brain-pituitary
axis (11, 12). This finding is consistent with the reports that
CaM is involved in the synthesis and/or secretion of hypo-
thalamic factors [e.g. somatostatin (SRIF) (13) and GHRH
(14)] as well as pituitary hormones [e.g. prolactin (PRL) (4, 15)
and LH (16)]. In GH3 cells, modulation of GH release by CaM
has been implicated (5). In this case, CaM antagonists, by
altering phosphodiesterase activity, can induce a biphasic
effect on GH release, being inhibitory at low doses and stim-
ulatory at high doses. Although CaM can activate PRL gene
expression both in vivo (17) and in vitro (18) by elevating PRL
promoter activity through a proximal enhancer element (15),
First Published Online June 2, 2005
Abbreviations: a.a., Amino acid; Ca2⫹/CaM kinase II, calmodulin-
dependent protein kinase II; CaM, calmodulin; DIG, digoxigenin; EGFP,
enhanced green fluorescence protein; FBS, fetal bovine serum; LSD, least
significance difference; Luc, luciferase; ORF, open reading frame; PRL,
prolactin; RACE, rapid amplification of cDNA ends; Sp1, specificity
protein-1; SRIF, somatostatin; Tm, melting temperature; 3⬘UTR, 3⬘-un-
translated region.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
3821
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Endocrinology 146(9):3821–3835
Copyright © 2005 by The Endocrine Society
doi: 10.1210/en.2004-1508
uitously expressed in the grass carp. In carp pituitary cells,
IGF-I and IGF-II induced CaM mRNA expression with a con-
current drop in GH transcript levels. These stimulatory ef-
fects on CaM mRNA levels were not mimicked by insulin and
appeared to be a direct consequence of IGF activation of CaM
gene transcription without altering CaM transcript stability.
CaM antagonism and inactivation of calcineurin blocked the
inhibitory effects of IGF-I and IGF-II on GH gene expression,
and CaM overexpression also suppressed the 5ⴕ promoter ac-
tivity of the grass carp GH gene. These results, as a whole,
provide evidence for the first time that IGF feedback on GH
gene expression is mediated by activation of CaM gene ex-
pression at the pituitary level. (Endocrinology 146: 3821–3835,
2005)
CaM antagonism has little or no effect on GH gene expres-
sion in these reports. Given that functional studies of CaM
production have not been fully characterized, direct evidence
for modulation of CaM gene expression at the pituitary level
as a means to regulate pituitary functions is still lacking.
IGF, a polypeptide with structural similarity to proinsulin,
is produced mainly in the liver under the stimulatory influ-
ence of GH. In general, IGF serves as a mediator for GH
actions in regulating somatic growth, body metabolism, and
cell proliferation and differentiation (19, 20). IGF can also
exert a long-loop feedback to inhibit GH synthesis and se-
cretion (21), and this negative feedback is well documented
in humans (22) and nonprimate mammals (23, 24). The site
of action for IGF long-loop feedback appears to be species
specific. Direct actions of IGF at the pituitary level without
a hypothalamic component have been reported in sheep (25),
whereas central actions to modify GHRH and SRIF secretion
from the hypothalamus have been clearly demonstrated in
the rat (26). In somatotroph cell lines, e.g. MtT/S cells, IGF
inhibits GH gene expression by reducing GH promoter ac-
tivity through phosphotidylinositol 3-kinase-mediated sig-
naling events (24). Although no information is available to
date in lower vertebrates on IGF regulation of GH-releasing
factors at the hypothalamic level, the long-loop feedback by
IGF appears to be conserved in fish. This idea is supported
by the findings that 1) the molecular structure of IGFs (in-
cluding IGF-I and IGF-II) is highly conserved from fish to
mammals (27); 2) GH stimulates IGF production in the liver
of fish species (28, 29); and 3) IGF inhibits GH secretion (30,
31) and synthesis (30, 31) in fish pituitary cells in vitro. Re-
cently, IGF has been shown to induce LH and PRL release in
Coho salmon (32) and striped bass (33), respectively. These
3822 Endocrinology, September 2005, 146(9):3821–3835
findings suggest that IGF may also act on pituitary cells other
than somatotrophs in lower vertebrates.
In fish models, e.g. goldfish, the availability of extracellular
Ca2⫹ and its entry via voltage-sensitive Ca2⫹ channels are
essential for both basal and stimulated GH release (34). In
goldfish pituitary cells, GH secretion induced by GH-releas-
ing factors, e.g. GnRH and dopamine, can be blocked by CaM
antagonists and CaM kinase II inhibitors (35, 36). These re-
sults indicate that CaM and its downstream signaling path-
ways are involved in the GH-releasing actions of these GH
regulators. At present, however, it is still unclear whether
GH gene expression can be regulated by CaM at the pituitary
level, and to our knowledge, the functional role of CaM in
GH feedback by IGF has not been previously examined. In
this study, using grass carp as a model, we tested the hy-
pothesis that IGF inhibits GH gene expression directly at the
pituitary level via modulation of CaM gene expression. As a
first step, the structural identity of grass carp CaM was es-
tablished by molecular cloning using 5⬘/3⬘ rapid amplifica-
tion of cDNA ends (RACE). The expression profile of CaM
mRNA was examined in various tissues of the grass carp by
Northern blot. Based on the CaM sequence obtained, a slot-
blot assay was set up for CaM mRNA measurement. Using
this assay system, the effects of IGF-I and IGF-II on CaM
mRNA expression were tested in grass carp pituitary cells
and correlated with the corresponding changes in GH
mRNA levels. In parallel experiments, the effects of IGF-I and
IGF-II on CaM transcript stability, CaM primary transcript
expression, and 5⬘ promoter activity of the CaM gene were
also examined. The functional role of CaM in GH gene ex-
pression was also evaluated by testing the effects of CaM
antagonism on GH mRNA levels and the effects of CaM
overexpression on GH promoter activity.
Materials and Methods
Animals
One-year old (1⫹) Chinese grass carp (Ctenopharyngodon idellus) were
purchased from the local markets in Hong Kong and housed in 200-liter
aquaria at 18 ⫾ 2 C for at least 3 d before tissue collection and/or
pituitary cell preparation. On the day of the experiments, the fish were
killed by anesthesia in MS222 (tricaine methanesulfonate; Sigma, St.
Louis, MO), followed by spinosectomy according to the regulations for
animal use at University of Hong Kong.
Reagents and test substances
Human IGF-I and IGF-II were purchased from Sigma-Aldrich Corp.
(St. Louis, MO). TRIzol, medium 199, Opti-MEM, Ham’s F-10, and fetal
bovine serum (FBS) were obtained from Invitrogen Life Technologies,
Inc. (Grand Island, NY). Calmidazolium and human insulin were ac-
quired from Sigma-Aldrich Corp., and actinomycin D was obtained
from Calbiochem (La Jolla, CA). Insulin was first dissolved in double-
distilled deionized water to prepare a working stock with an activity
level of 109 ␮U/ml. The stock solution was then aliquoted into small
volume and stored frozen at ⫺80 C until used. IGF-I and IGF-II were
prepared in a similar manner, except that these peptide hormones were
dissolved in 10 nm HCl to give a stock concentration at 100 ␮m. Given
that calmidazolium has a low solubility in aqueous solution, the stock
solution at 10 mm was prepared in dimethylsulfoxide. On the day of
experiments, test substances were diluted to appropriate concentrations
with culture medium 20 min before drug treatment. In these studies, the
final dilutions of dimethylsulfoxide and HCl were always 0.1% (vol/vol)
or less of the original levels and did not affect basal expression of CaM
and GH mRNA in grass carp pituitary cells.
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Huo et al. • IGF Feedback on GH Expression
Molecular cloning of grass carp CaM cDNA
Total RNA (5 ␮g) was extracted from grass carp pituitaries using
TRIzol (Invitrogen Life Technologies, Inc.), and first strand cDNA was
synthesized using a SuperScript II first strand synthesis kit (Invitrogen
Life Technologies, Inc.). A pair of primers designed based on the con-
served regions of mammalian CaM cDNAs were used to isolate a 463-bp
fragment of grass carp CaM cDNA by PCR using the first strand cDNA
as the template. Based on the sequence obtained, new primers were
designed for 5⬘/3⬘ RACE using a GeneRacer kit (Invitrogen Life Tech-
nologies, Inc., Carlsbad, CA) with pituitary total RNA (5 ␮g) as the
template. PCRs were conducted according to the instructions of the
manufacturer. PCR products were gel-purified and subcloned into the
pGEM-T Easy vector (Promega Corp., Madison, WI) for DNA sequenc-
ing using a BigDye Terminator Cycle Sequencing Kit (Applied Biosys-
tems, Foster City, CA). The full-length cDNA of CaM (GenBank no.
AY627883) was reconstructed from its 5⬘ and 3⬘ sequences using
MacVector 6.5 (Oxford Molecular, Madison, WI) and MacDNASIS PRO
3.5 programs (Hitachi, San Bruno, CA). After that, a digoxigenin (DIG)-
labeled cDNA probe for grass carp CaM mRNA was prepared for
subsequent Northern blot and slot-blot assays using a PCR DIG Probe
Synthesis Kit (Roche, Mannheim, Germany).
Northern blot of CaM transcripts
Total RNA (25 ␮g) was extracted with TRIzol from selected tissues of
the grass carp, including brain, pituitary, muscle, liver, kidney, spleen,
heart, intestine, and gills. After that, mRNA was purified using a
PolyATract mRNA Isolation System (Promega Corp.). These mRNA
samples were denatured, size fractionated in 1% agarose gel, and trans-
blotted onto a positively charged nylon membrane (Roche) using a
VacuGene Vacuum Blotting System (Pharmacia Biotech, Piscataway,
NJ). The membrane was then UV cross-linked, prehybridized for 3 h, and
incubated with the DIG-labeled CaM cDNA probe for 15 h at 42 C. On
the following day, the membrane was washed twice at 68 C in 0.5⫻
standard saline citrate with 0.1% sodium dodecyl sulfate, and hybrid-
ization signals were detected using a DIG Lumin Detection Kit (Roche).
In these studies, a Northern blot of ␤-actin was used as an internal
control.
Measurement of steady-state CaM and GH
mRNA expression
Primary cultures of pituitary cells were prepared from 1-yr-old grass
carp by a trypsin/deoxyribonuclease digestion method as described
previously (37). The average cell yield was approximately 8 ⫻ 106 cells/
pituitary, and cell viability was always 95% or greater. After cell dis-
persion, pituitary cells were cultured overnight (⬎15 h) with 5% FBS in
24-well culture plates precoated with poly-d-lysine (Sigma-Aldrich
Corp.) at a seeding density of about 3 ⫻ 106 cells/ml/well. After that,
test substances at appropriate concentrations were prepared in carp
MEM (38) and gently overlaid onto pituitary cells after removal of old
culture medium. For dose-response studies, the duration of drug treat-
ment was routinely fixed at 48 h. For the evaluation of CaM transcript
stability, cells were pretreated with actinomycin D (8 ␮m) and incubated
with or without IGF treatment for the duration as indicated in individual
experiments. After drug treatment, total RNA (2.5 ␮g) was extracted
with TRIzol, heat-denatured at 70 C for 15 min, and vacuum-blotted onto
nylon membranes in duplicate using a Bio-Dot microfiltration unit (Bio-
Rad Laboratories, Hercules, CA). The membranes were UV cross-linked,
prehybridized for 3 h, and hybridized overnight with DIG-labeled CaM
probe and grass carp GH cDNA probe (38), respectively. After hybrid-
ization, the membranes were washed, and signal development was
carried out as described for the Northern blot. Measurement of hybrid-
ization signals was conducted in a Kodak 440 Image Station (Kodak
Digital Science, Rochester, NY). In this study, slot blots of 18S RNA were
also conducted to serve as an internal control.
Real-time PCR of mature CaM mRNA and CaM
primary transcript
Based on the sequence of the newly cloned CaM cDNA, intron trap-
ping was performed using genomic DNA as a template to pull out
Huo et al. • IGF Feedback on GH Expression
introns 1–5 of the grass carp CaM gene (GenBank no. AY656698). Prim-
ers flanking the junction between intron 4 and exon 5 from positions
10,408 –10,789 were used for real-time PCR of CaM primary transcripts.
To serve as a parallel control, real-time PCR of mature CaM mRNA was
also performed using primers flanking intron 4 from position 10,209 (in
exon 4) to 10,789 (in exon 5). In this study, pituitary cells were incubated
for 48 h with increasing levels of IGF-I and IGF-II. Total RNA (5 ␮g) was
isolated and digested with deoxyribonuclease I to remove genomic DNA
contamination. After that, RT was carried out, and RT samples were
subjected to PCR using a LightCycler-DNA Master SYBR Green I Kit
(Roche) in a RotorGene 2000 Real-Time PCR System (Corbett Research,
Eight Mile Plains, Australia). PCRs for CaM mRNA and primary tran-
scripts were conducted for 35 cycles at 94 C for 30 sec for denaturing,
69 C for 30 sec for annealing, and 72 C for 30 sec for primer extension.
Based on our validation, the primers for CaM primary transcripts were
ineffective in PCR amplification of mature CaM mRNA. Furthermore,
PCR of CaM primary transcripts in the nuclear fraction of grass carp
pituitary cells consistently produced a 381-bp PCR product with a melt-
ing temperature (Tm) at 92.1 C, whereas the mature CaM mRNA in the
cytosolic fraction would generate a 181-bp PCR product with Tm at 89.8
C. In these experiments, serial dilutions of plasmid DNA with CaM
cDNA or the amplicon covering the junction between intron 4 and exon
5 of the CaM gene were used as the standards for real-time PCR of
mature CaM mRNA and CaM primary transcripts, respectively.
Measurement of CaM promoter activity
Based on the sequence of grass carp CaM cDNA, nested primers
covering the 5⬘-untranslated region (5⬘UTR) were designed to pull out
the 5⬘ promoter of CaM gene (GenBank no. AY656698) using a Universal
GenomeWalker Kit (BD Clontech, Palo Alto, CA). Genomic DNA pre-
pared from whole blood was used as the template, and PCRs were
conducted according to the instructions of the manufacturer. The 5⬘
promoter obtained (⫺1369 to ⫹49) was subcloned into the NheI and XhoI
sites of the pGL3.Basic vector (Promega Corp.) to generate the reporter
construct pCaM(⫺1369).luciferase (Luc). This luciferase-expressing con-
struct was used to examine IGF action on CaM promoter activity using
the mouse pituitary cell line ␣T3-1 as the host cells. The cells were
maintained in DMEM with 44 mm NaHCO3, 10% FBS, and 1% (vol/vol)
antibiotics-antimycotics (pH 7.4). Transfection was conducted for 6 h in
0.5 ml Opti-MEM with 1.16 ␮l Lipofectamine (Invitrogen Life Technol-
ogies, Inc.), 0.05 ␮g pCaM(⫺1369).Luc, 0.01 ␮g pEGFP-N1 (EGFP, en-
hanced green fluorescence protein), and 0.44 ␮g pBluescript (Stratagene)
as carrier DNA. After transfection, ␣T3-1 cells were cultured in DMEM
with 10% FBS for 6 h, followed by serum starvation for 6 h before the
initiation of IGF treatment. Based on our validation, the optimal duration
of drug treatment was fixed at 12 h. After IGF treatment, ␣T3-1 cells were
dissolved in lysis buffer (Promega Corp.), and the lysate was cleared by
centrifugation at 20,000 ⫻ g at 4 C for 15 min. For measurement of Luc
activity, a 50-␮l volume of cleared lysate was mixed with 100 ␮l lucif-
erase assay reagent (Promega Corp.), and luminescence signal was
quantified using a Lumat LB9507 luminometer (EG&G, Gaithersburg,
MD). In these experiments, cotransfection with pEGFP-N1 was used as
an internal control, and GFP expression was monitored by measuring
the fluorescence signal in 100 ␮l lysate using a Cytofluor series 4000
Multiple-Well Plate Reader (Applied Biosystems, Foster City, CA).
CaM overexpression on GH promoter activity
To examine the functional role of CaM in GH gene transcription, the
coding sequence of grass carp CaM cDNA was PCR isolated and sub-
cloned into the expression vector pcDNA3.1 to produce pcDNA.CaM.
The effects of CaM overexpression were examined with cotransfection
of pGH.Luc constructs (39) carrying decreasing lengths (⫺986 to ⫺115)
of the grass carp GH gene promoter (GenBank no. X30419) using the rat
pituitary cell line GH4C1 as the host cells. The cells were maintained in
Ham’s F-10 with 14 mm NaHCO3, 10% FBS, and 1% (vol/vol) antibiotics-
antimycotics (pH 7.4). Transfection was conducted for 6 h in 0.5 ml
Opti-MEM with 1.16 ␮l Lipofectamine, 0.18 ␮g pGH.Luc, 0.01 ␮g
pEGFP-N1, and 0.05– 0.25 ␮g pcDNA.CaM (with pBluescript as carrier
DNA). After transfection, GH4C1 cells were cultured in Ham’s F-10 for
18 h, dissolved in lysis buffer, and assayed for luciferase activity and GFP
expression as described in the preceding section. To confirm CaM over-
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Endocrinology, September 2005, 146(9):3821–3835 3823
expression at the protein level, Western blot was also performed in
GH4C1 cells after pcDNA.CaM transfection. Cell lysate was prepared by
three cycles of repeated freezing and thawing in phenylmethylsulfo-
nylfluoride (150 ␮g/ml) with EDTA (1.5 mm), cleared by centrifugation,
and resolved in a 15% gel by SDS-PAGE for 1 h at 200 V. After that,
protein samples were transferred onto a polyvinylidene difluoride mem-
brane by low current electroblotting at 65 V for 2.5 h. The membrane was
then subjected to blocking with 3% BSA and incubation with sheep
antibovine CaM antibody (1:1000; Chemicon International, Temecula,
CA) for 2 h. After washing, rabbit antisheep IgG-horseradish peroxidase
(1:5000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added, and
signal development was carried out in a Kodak 440 Image Station with
SuperSignal WestPico (Pierce Chemical Co., Rockford, IL) as the sub-
strate. Using a similar approach, Western blot was also performed in
carp pituitary cells to examine the effects of IGF treatment on CaM
expression at the protein level. In this case, parallel probing of ␤-actin
using an Actin Ab-1 Kit (Calbiochem) was also conducted to serve as an
internal control.
Data transformation and statistical analysis
For quantitation of CaM gene expression, CaM mRNA levels were
measured in terms of arbitrary density unit and normalized against 18S
rRNA expressed in the same sample. Given that no significant changes
in 18S rRNA levels were noted, these data were simply transformed into
a percentage of the mean value in the control group for statistical anal-
ysis (as percentage of control). The transformation was conducted to
allow for data pooling from separate experiments without increasing the
overall variability of the results. The half-life (t1/2) of CaM mRNA,
defined as the time required for CaM mRNA to decrease to half its
original level, was deduced by the one-phase exponential decay model
using PRISM 3.02 (GraphPad, Inc., San Diego, CA). For real-time PCR,
a standard curve with a dynamic range of 105 or greater and a correlation
coefficient of 0.95 or more were used for data calibration. Mature CaM
mRNA and primary transcripts were measured in terms of femtomoles
per million cells and transformed as a percentage of the control for
statistical analysis. For luciferase activity measurement, the raw data in
arbitrary light units were normalized against the level of GFP expression
in the same sample expressed in arbitrary fluorescence units. These data
were expressed as the ratio of arbitrary light units/arbitrary fluores-
cence units or transformed as a percentage of the control as described
in the preceding section. The transformed data for CaM mRNA and
luciferase activity were analyzed by Student’s t test or ANOVA (two-
way), followed by Fisher’s least significance difference (LSD) test. Dif-
ferences were considered significant at P ⬍ 0.05.
Results
Molecular cloning of grass carp CaM cDNA
Using RT-PCR coupled to 5⬘/3⬘ RACE, a full-length CaM
cDNA, CaM-L, with a size of 1.55 kb was isolated from the
grass carp pituitary (Fig. 1). In parallel studies, a shorter
version of CaM cDNA, CaM-S, with a size of 0.73 kb was also
obtained with CaM-L using RT samples prepared from the
brain, gills, heart, and kidney. Nucleotide sequence analysis
revealed that CaM-S is a truncated form of CaM-L with a
shorter 3⬘UTR probably produced by differential use of poly-
adenylation signals. The 447-bp ORF of these newly cloned
CaM cDNAs encodes a 149-amino acid (a.a.) protein with
primary sequence identical with the CaMs reported in mam-
mals (e.g. human, bovine, mouse, and rat), birds (e.g. chicken
and duck), and amphibian (e.g. Xenopus) as well as in the
representative species of modern bony fish (e.g. medaka and
perch). Compared with CaMs reported in early evolved bony
fish (e.g. eel), cyclostome (e.g. hagfish), urochordate (e.g.
ciona), cephalochordate (e.g. brachiostoma), mollusca (e.g.
aplysia), nematode (e.g. C. elegans), porifera (e.g. sponge), and
3824 Endocrinology, September 2005, 146(9):3821–3835 Huo et al. • IGF Feedback on GH Expression

FIG. 1. Nucleotide and deduced a.a. sequences of grass carp CaM. The full-length cDNA sequences of grass carp CaM-L and CaM-S were
constructed using MacVector 6.5.3. The a.a. sequence deduced from the ORF is presented along with the corresponding DNA sequences. The
Ca2⫹-binding EF domains, namely, EF-I, EF-II, EF-III, and EF-IV, are marked by solid lines. Multiple polyadenylation signals (AATAAA or
AACAAA) identified in the 3⬘UTR are marked by dotted lines. An asterisk represents the stop codon at the end of the coding sequence.

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Huo et al. • IGF Feedback on GH Expression Endocrinology, September 2005, 146(9):3821–3835 3825

FIG. 2. Alignment of grass carp CaM with the corresponding a.a. sequences of CaM reported in other species. Alignment was conducted using
the Clustal W algorithm with MacVector 6.5.3. The conserved a.a. residues in these protein sequences are shaded for identification. The four
EF domains (i.e. EF-I, EF-II, EF-III, and EF-IV) are marked by arrows, and the Ca2⫹-binding sites in the helix-loop-helix structure of individual
EF hands are boxed. The a.a. sequences of CaM in representative species were downloaded from GenBank, and the accession numbers are listed
as follows: human, MCHU; bovine, MCBO; mouse, CAA43674; rat, MCRT; duck, I51292; chicken, MCCH; Xenopus, AAA49668; medaka, JC1305;
perch, AAC63306; hagfish, AAD56955; electric eel, MCEE; Aplysia, CAA40207; brachiostoma, CAA68327; Caenorhabditis elegans, CAA10601;
ciona, CAA73906; sponge, CAA77069; and trypanosoma, CAA 36316.

protozoa (e.g. trypanosome), only a low level of a.a. substi-
tutions were noted, and most of them were conserved mu-
tations (Fig. 2). Sequence alignment at the a.a. level also
revealed that the four helix-loop-helix EF hands (EF-I to
EF-IV), the functional motifs of CaM for Ca2⫹ binding, are
structurally conserved in vertebrates from modern bony fish
to mammals. Although the structure of CaM is highly con-
served, multiple copies of CaM genes, namely CaM I, CaM
II, and CaM III, have been reported in birds and mammals
(40, 41). Using unrooted analysis by PHYLIP (version 3.6),
grass carp CaM can be clustered in the same clade with the
CaM I genes reported in the rat, mouse, human, and chicken
and is distally related to CaM II and CaM III gene subfamilies
(Bootstrap values, 998 –1000).
Tissue distribution of CaM transcripts
A DIG-labeled cDNA probe covering the ORF of CaM-L
and -S was used to examine the tissue distribution of CaM
expression by Northern blot (Fig. 3). mRNA samples were
prepared from various tissues of the grass carp, including
spleen, pituitary, muscle, liver, gut, kidney, heart, gills, and
brain. The highest levels of CaM transcripts were noted in
brain and gut; to a lesser extent in spleen, muscle, and pi-

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3826 Endocrinology, September 2005, 146(9):3821–3835 Huo et al. • IGF Feedback on GH Expression

ognized. These results, as a whole, indicate that different

forms of CaM mRNA are expressed in the grass carp in a
tissue-specific manner.

Regulation of CaM mRNA expression in grass carp

FIG. 3. Tissue distribution of CaM transcripts in the grass carp.
Northern blot of CaM mRNA was performed in selected tissues of the
grass carp, including brain, pituitary, liver, gills, gut, kidney, spleen,
heart, and muscle. After size fractionation in 1% agarose gel and
transblotting onto a nylon membrane, CaM mRNAs were detected by
hybridization with a DIG-labeled CaM cDNA probe. In this study,
parallel blotting of ␤-actin mRNA was used as an internal control.
tuitary; and to a lower level in heart, kidney, liver, and gills.
In this study, three CaM transcripts (1.3, 1.7, and 3.0 kb,
respectively) were identified, and all of them were expressed
in brain, spleen, kidney, heart, intestine, and muscle. In the
gills, only the 1.3- and 1.7-kb, not the 3.0-kb, transcripts were
detected. Interestingly, the 3.0- and 1.7-kb, but not the 1.3-kb,
transcripts were expressed in the liver. In the case of the
pituitary, however, only the 1.7-kb transcript could be rec-
pituitary cells
Because Northern blot using purified mRNA is not ideal
for quantitative studies with a large number of samples, a
slot-blot assay using total RNA was established to study
steady-state CaM mRNA expression in grass carp pituitary
cells. To examine IGF effects on CaM gene expression at the
pituitary level, pituitary cells were incubated for 48 h with
increasing doses (0.1–100 nm) of IGF-I (Fig. 4A) and IGF-II
(Fig. 4B). In these experiments, IGF-I and IGF-II were both
effective in elevating CaM mRNA levels in a dose-dependent
manner, with a concurrent drop in GH mRNA expression.
The differential effects of IGFs on CaM and GH mRNA
expression, however, were not mimicked by increasing doses
of insulin (102–106 ␮U/ml). Unlike IGFs, insulin inhibited
CaM mRNA levels (Fig. 5A), with a concurrent rise in GH
mRNA expression (Fig. 5B). In these experiments, a negative
correlation between CaM and GH mRNA levels was noted
after IGF and insulin treatment (Fig. 5B), suggesting that
modulation of CaM expression at the pituitary level may be
involved in GH gene regulation. Because multiple transcripts
of CaM are expressed in the grass carp, the possibility of IGF
induction of CaM gene expression via differential expression

FIG. 4. Differential effects of IGF on

CaM and GH mRNA expression in
grass carp pituitary cells. Pituitary
cells were exposed to increasing doses
(0.1–100 nM) of IGF-I (A) and IGF-II (B)
for 48 h under static incubation. After
that, RNA samples were extracted and
assayed for CaM mRNA (upper panels)
and GH mRNA levels (lower panels),
respectively. In these studies, parallel
blotting of 18S RNA was used as an
internal control. Data presented are ex-
pressed as the mean ⫾ SEM (n ⫽ 4) and
are the pooled results of four separate
experiments. Groups denoted by the
same letter represent a similar level of
transcript expression (P ⬎ 0.05, by
ANOVA, followed by Fisher’s LSD test).
Representative results of slot blots are
also included for reference.

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Huo et al. • IGF Feedback on GH Expression
FIG. 5. Opposite trends in CaM and GH mRNA expression in grass
carp pituitary cells. A, Differential effects of insulin treatment on
CaM mRNA (upper panel) and GH mRNA expression (lower panel).
Pituitary cells were incubated for 48 h with increasing doses of insulin
(102–106 ␮U/ml). After that, RNA samples were extracted and as-
sayed for CaM and GH mRNA levels. In these experiments, parallel
blotting of 18S RNA was used as the internal control. Data presented
are expressed as the mean ⫾ SEM (n ⫽ 4) and are the pooled results
of four separate experiments. Groups denoted by the same letter
represent a similar level of transcript expression (P ⬎ 0.05, by
ANOVA, followed by Fisher’s LSD test). B, Negative correlation be-
tween CaM and GH mRNA expression. Pearson analysis was per-
formed with GraphPad PRISM 3.02 using the pooled data from Figs.
4 and 5A, and the correlation between CaM and GH mRNA expression
was considered to be significant at P ⬍ 0.05. The deduced equation
describing the negative correlation between CaM and GH mRNA
levels as well as the correlation coefficient (as Pearson r value) are also
included for reference.
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Endocrinology, September 2005, 146(9):3821–3835 3827
of various forms of CaM mRNA was examined by Northern
blot (Fig. 6A). In this case, IGF-I (100 nm) and IGF-II (100 nm)
significantly increased the expression level of the 1.7-kb CaM
mRNA in grass carp pituitary cells (Fig. 6B). The 1.3- and
3.0-kb CaM transcripts, however, remained undetectable in
this study. Using Western blot, a time-dependent increase in
CaM protein content was noted from 12– 48 h in carp pitu-
itary cells after the initiation of IGF-I treatment (100 nm; Fig.
6C). The protein levels of ␤-actin, the internal control of these
experiments, remained unchanged during the period of drug
treatment. Similar results were also obtained using IGF-II
(100 nm) as the stimulant for CaM expression (data not
shown).
To evaluate the role of CaM in IGF inhibition of GH gene
expression, spatial and temporal correlations between CaM
and GH gene expression after IGF treatment were examined
to test whether 1) the opposite changes in CaM and GH
mRNA levels indeed occurred in the same cell type (i.e. in
carp somatotrophs), and 2) the response in CaM mRNA
expression was initiated before and/or during the corre-
sponding changes in GH mRNA levels. For the study of
spatial correlation, enriched somatotrophs were prepared
from mixed populations of pituitary cells using Percoll gra-
dient centrifugation (Fig. 7A). After a 48-h incubation with
IGF-I (100 nm) and IGF-II (100 nm), CaM mRNA levels were
increased in this somatotroph preparation, with a concurrent
drop in GH mRNA expression (Fig. 7, B and C). In parallel
time-course studies, basal levels of CaM mRNA were sig-
nificantly increased (P ⬍ 0.05) in carp pituitary cells after 6-h
incubation with IGF-I (100 nm) and IGF-II (100 nm), respec-
tively, and remained elevated from 12– 48 h (Fig. 8A). The
corresponding drop in GH mRNA levels (P ⬍ 0.05), however,
was noted only after 24-h incubation with IGF-I and IGF-II.
After that, GH mRNA levels were reduced gradually from 24
to 48 h in a time-dependent manner (Fig. 8B). To establish the
functional role of CaM in GH feedback by IGF, the effects of
calmidazolium, a CaM antagonist, on both basal and IGF-
inhibited GH mRNA expression were tested at the pituitary
level. Calmidazolium is known to bind CaM tightly with
nanomolar affinity to perturb a wide range of Ca2⫹-depen-
dent mechanisms by immobilizing the methionine residues
in the Ca2⫹-binding sites of helix-loop-helix EF motifs (42).
In this case, GH mRNA levels were elevated in a dose-related
fashion by increasing doses of calmidazolium (0.1 nm to 1
␮m; Fig. 9A). In the presence of calmidazolium (5 nm), the
inhibitory actions of IGF-I (100 nm) and IGF-II (100 nm) on
GH mRNA expression were totally abolished (Fig. 9B). To
elucidate the downstream signaling events that occurred
after CaM activation, the functional roles of CaM kinase II
and calcineurin in IGF inhibition of GH mRNA expression
were also examined (Fig. 9C). Similar to calmidazolium, a
48-h incubation with the calcineurin inhibitor cyclosporin A
(100 nm) increased basal levels of GH mRNA and blocked the
inhibitory effect of IGF-I (100 nm) on GH transcript expres-
sion. The CaM kinase II inhibitor KN62 (5 ␮m), in contrast,
reduced GH mRNA levels in grass carp pituitary cells and
did not modify the inhibitory action of IGF-I (100 nm) on GH
gene expression. Similar results were obtained by substitut-
ing IGF-II for IGF-I in these experiments (data not shown).
3828 Endocrinology, September 2005, 146(9):3821–3835 Huo et al. • IGF Feedback on GH Expression

FIG. 6. Activation of CaM synthesis by IGF in grass

carp pituitary cells. A, Effects of IGF treatment on CaM
mRNA expression. Pituitary cells were incubated for
48 h with IGF-I (100 nM) and IGF-II (100 nM). After
that, a Northern blot was performed to examine the
pituitary expression of various isoforms of CaM mRNA.
In this case, only the 1.7-kb transcript, not the 1.3- or
3.0-kb transcript, could be detected (left panel). The
levels of the 1.7-kb CaM transcript were significantly
increased after treatment with IGF-I and IGF-II, re-
spectively (right panel). B, Effects of IGF treatment on
CaM expression at the protein level. Pituitary cells
were incubated with IGF-I (100 nM) for the duration
indicated. Cell lysate was prepared, and CaM immu-
noreactivity was monitored using Western blot as de-
scribed in Materials and Methods. Western blot of ␤-
actin was also conducted to serve as an internal control,
and in these studies, no significant changes in ␤-actin
expression were noted after IGF-I treatment. Data pre-
sented are expressed as the mean ⫾ SEM (n ⫽ 3) and are
the pooled results of three separate experiments.
Groups denoted by the same letter represent a similar
level of CaM content or CaM mRNA expression (P ⬎
0.05, by ANOVA, followed by Fisher’s LSD test).

Molecular mechanisms for IGF induction of CaM
gene expression
To shed light on the molecular mechanisms for IGF
induction of CaM gene expression, the effects of IGF on
CaM gene transcription and CaM transcript stability were
examined. As a first step, real-time PCR of CaM primary
transcripts was performed in grass carp pituitary cells
after a 48-h incubation with increasing doses (0.1–100 nm)
of IGF-I and IGF-II. As a parallel control, mature CaM
mRNA levels were also monitored in these experiments.
Calibration of primary transcript levels was conducted by
linear regression of critical thresholds of the standard
curve (Fig. 10A). In the same experiment, a separate stan-
dard curve was used for calibration for mature CaM
mRNA (data not shown). In this study, IGF-I and IGF-II
consistently induced a dose-dependent increase in CaM
primary transcripts and mature mRNA (Fig. 10B). The
specificity of PCRs was also confirmed by melting curve
analysis and ethidium bromide staining of PCR products.
In this case, a single 381-bp (Tm,92.1 C) and a single 181-bp
PCR product (Tm, 89.8 C) were detected for CaM primary
transcripts and mature CaM mRNA, respectively.
To provide additional evidence that IGF increases CaM
mRNA levels via activation of CaM gene transcription, a
reporter gene approach was used to examine the effects of
IGF treatment on CaM promoter activity expressed in ␣T3-1
cells. Compared with the time-matched controls, a signifi-
cant increase in luciferase activity (P ⬍ 0.05) was noted after

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Huo et al. • IGF Feedback on GH Expression Endocrinology, September 2005, 146(9):3821–3835 3829

FIG. 7. Spatial correlation between CaM and GH mRNA expres-

sion after IGF treatment. A, Enriched somatotrophs prepared by
discontinuous Percoll gradient centrifugation. Somatotrophs were
identified by immunostaining using GH antiserum (1:8000). After
gradient purification, a significant enrichment of grass carp so-
matotrophs (⬃84%) was observed compared with the freshly dis-
persed pituitary cells (inset). B, Differential effects of IGF on CaM
and GH mRNA expression in carp somatotrophs. Enriched soma-
totrophs were incubated for 48 h with IGF-I (100 nM) and IGF-II
(100 nM). After that, slot blots for CaM and GH mRNA were per-
formed as described in Materials and Methods. In these studies,
parallel blotting of 18S RNA was used as an internal control. C,
Normalized data for the experiments with enriched somatotrophs.
CaM and GH mRNA data were normalized as a ratio of 18S RNA
level expressed in the same sample. Data presented are expressed
as the mean ⫾ SEM (n ⫽ 4) and are the pooled results of four
separate experiments. Groups denoted by the same letter represent
a similar level of transcript expression (P ⬎ 0.05, by ANOVA,
followed by Fisher’s LSD test).

exposure to IGF-I (100 nm) for 6 h (Fig. 10C). The maximal

difference in luciferase activity expressed in the control
groups vs. the treatment groups was observed 12 h after the
initiation of IGF-I treatment. Therefore, the duration of drug
treatment was fixed at 12 h for our dose-response studies
with IGF-I and IGF-II, respectively. In this case, increasing
levels of IGF-I and IGF-II (0.01–100 nm) were both effective
in stimulating luciferase activity expression in a dose-de-
pendent manner (Fig. 10D). Although the maximal stimu-
latory effects on luciferase activity were similar, IGF-I ap-
peared to be more potent than IGF-II in inducing luciferase
expression, especially in subnanomolar doses.
Using grass carp pituitary cells pretreated with the tran-
scription inhibitor actinomycin D (8 ␮m), the clearance
curves of CaM mRNA were constructed in the presence or
absence of IGF-I (100 nm; Fig. 10E). In these experiments, the
two curves were found to be overlapping, and the t1/2 values
deduced for CaM mRNA (73.4 h for the control vs. 73.2 h for
IGF treatment) were also similar (P ⬎ 0.05). Comparable
results were obtained in parallel experiments with IGF-II
FIG. 8. Temporal correlation between CaM and GH mRNA expres-
sion after IGF treatment. Pituitary cells were incubated with IGF-I
(100 nM) and IGF-II (100 nM) for the duration indicated. Total RNA
samples were harvested at the respective time points for slot blots of
CaM mRNA (A) and GH mRNA (B), respectively. In these studies,
parallel probing of 18S RNA was used as the internal control. Data
presented are expressed as the mean ⫾ SEM (n ⫽ 4) and are the pooled
results from four experiments. A treatment group marked with an
asterisk represents a significant difference (P ⬍ 0.05) compared with
the time-matched control (by Student’s t test).
(data not shown), confirming that IGF-induced CaM gene
expression in carp pituitary cells does not involve modifi-
cation of CaM transcript stability.

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3830 Endocrinology, September 2005, 146(9):3821–3835
FIG. 9. Functional role of CaM in basal and IGF-inhibited GH mRNA
expression. A, Pituitary cells were incubated for 48 h with increasing
doses of the CaM antagonist calmidazolium (0.1–1000 nM). B, Pitu-
itary cells were exposed to IGF-I (100 nM) and IGF-II (100 nM) for 48 h
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Huo et al. • IGF Feedback on GH Expression
CaM overexpression on GH gene promoter activity
The rat somatotroph cell line GH4C1 was used for the
promoter studies of grass carp GH gene, because it has en-
dogenous expression of Pit-1, a pituitary-specific transcrip-
tion factor, to support basal GH promoter activity. Overex-
pression of CaM at the protein level was tested by Western
blot in GH4C1 cells. Compared with the control transfected
with the blank vector pcDNA 3.1, a significant increase in
CaM immunoreactivity (16.8 kDa) was detected in the cell
lysate prepared from the treatment group transfected with
pcDNA.CaM (Fig. 11A). In GH4C1 cells cotransfected with
pGH(⫺986).Luc carrying the ⫺986 to ⫹13 promoter of the
grass carp GH gene, CaM overexpression was effective in
reducing luciferase activity in a time-dependent manner (Fig.
11B). Apparently, this inhibitory effect was specific to the GH
promoter, and the expression levels of GFP, the internal
control, were not affected by CaM overexpression. In parallel
experiments, transfection with increasing levels of pcDNA.
CaM (0.05– 0.25 ␮g) also suppressed luciferase activity in
GH4C1 cells in a dose-dependent manner (Fig. 11C). In these
studies, transfection of pGL3.Control and pGL3.Basic were
used as the positive and negative controls, respectively. A
high level of luciferase activity was consistently detected in
the group transfected with pGL3.Control (with a simian vi-
rus 40 promoter) compared with that in the group transfected
with the promoterless pGL3.Basic, confirming that the re-
porter system was appropriate for quantitative analysis of
promoter activity. In our validation studies, the blank vector
pGL3.Basic was also found to be nonresponsive to CaM
overexpression (data not shown). To map out the coarse
location of the promoter region responsible for CaM inhibi-
tion of GH promoter activity, CaM overexpression was con-
ducted in GH4C1 cells transfected with pGH.Luc constructs
carrying decreasing lengths of GH promoter from ⫺986 to
⫺115. In all pGH.Luc constructs examined, basal expression
of luciferase activity was significantly suppressed by CaM
overexpression (Fig. 11D), suggesting that the CaM-respon-
sive element(s) is located within the proximal promoter re-
gion before position ⫺115.
Discussion
In the present study, two CaM cDNAs, CaM-L and CaM-S,
have been isolated in the grass carp. CaM-S is a truncated
form of CaM-L, and both of them share a common ORF
encoding a 149-a.a. protein with primary sequence identical
with CaMs in mammals, birds, and amphibians. In mam-
mals, nonallelic CaM genes, namely CaM I, CaM II, and CaM
III, encoding the same CaM protein have been reported (40,
41). In general, these isoforms of CaM genes are believed to
in the presence or absence of calmidazolium (5 nM). C, Pituitary cells
were incubated with IGF-I (100 nM) with or without simultaneous
treatment of the CaM kinase II inhibitor KN62 (5 ␮M) or the cal-
cineurin inhibitor cyclosporine A (100 nM). After drug treatment, total
RNA was prepared for GH mRNA measurement, and parallel probing
of 18S RNA was used as the internal control. Data presented are
expressed as the mean ⫾ SEM (n ⫽ 4) and are the pooled results of four
experiments. Groups denoted by the same letter represent a similar
level of GH mRNA expression (P ⬎ 0.05, by ANOVA, followed by
Fisher’s LSD test).
Huo et al. • IGF Feedback on GH Expression Endocrinology, September 2005, 146(9):3821–3835 3831

FIG. 10. Molecular mechanisms for IGF induction of CaM gene expression. A, Effects of IGF treatment on basal expression of mature CaM
mRNA and CaM primary transcripts in grass carp pituitary cells. Standard curves for real-time PCR of CaM primary transcripts, including
1) the PCR profiles with increasing levels of the standard, and 2) linear regression of critical thresholds (CT) with respect to template
concentrations, are presented (left panels). Mature CaM mRNA and primary transcripts were monitored in pituitary cells after a 48-h incubation
with increasing doses (0.1–100 nM) of IGF-I and IGF-II, respectively (right panels). B, Time course of IGF-I treatment on CaM promoter activity.
After transfection with pCaM(⫺1369).Luc, ␣T3-1 cells were incubated with IGF-I (100 nM) for the duration indicated. Cell lysate was prepared
at the respective time points for the measurement of luciferase activity. C, Dose dependence of IGF treatment on CaM promoter activity. After
transfection, ␣T3-1 cells were exposed to increasing doses (0.01–100 nM) of IGF-I and IGF-II for 12 h, and cell lysate was prepared for luciferase
activity measurement. In these promoter studies, parallel measurement of GFP expression was used as the internal control. D, Effects of IGF
treatment on CaM transcript stability. Pituitary cells were incubated with actinomycin D (8 ␮M) in the presence or absence of IGF-I (100 nM)
for the duration indicated. Total RNA samples were isolated at the respective time points for the construction of CaM mRNA clearance curves.
The t1/2 of CaM mRNA, defined as the time required for CaM mRNA to drop to 50% of its initial values, was deduced by the one-phase exponential
decay model using GraphPad PRISM 3.02. The t1/2 values for the control and treatment groups were 73.4 and 73.2 h, respectively. Data presented
are expressed as the mean ⫾ SEM (n ⫽ 4) and are the pooled results of four experiments. Groups denoted by the same letter represent a similar
level of transcript expression or promoter activity (P ⬎ 0.05, by ANOVA, followed by Fisher’s LSD test).

be the result of gene duplication that occurred 1400 million netic redundancy of CaM genes has been attributed to the
yr ago (43). Using unrooted analysis, it has been shown that important function of CaM as the intracellular Ca2⫹ sensor
the newly cloned CaM cDNAs are phylogenetically related in living cells (40). This idea is also in agreement with our
to the CaM I gene subfamily. In higher vertebrates, the ge- findings that CaM transcripts are ubiquitously expressed in

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3832 Endocrinology, September 2005, 146(9):3821–3835
FIG. 11. CaM overexpression on GH promoter activity. A, CaM over-
expression at the protein level in GH4C1 cells. After transfection with
the CaM expression vector pcDNA.CaM, GH4C1 cells were incubated
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Huo et al. • IGF Feedback on GH Expression
the grass carp. In this case, the highest levels of CaM tran-
scripts can be found in the brain and intestine. In the rat, a
high level of CaM (up to 0.5% of total protein) can be detected
in the brain (44), which may be related to the functions of
CaM in modulating neuronal excitability (45, 46) and exo-
cytosis of neurotransmitters (47, 48). In this study, CaM tran-
scripts of 1.3, 1.7, and 3.0 kb have been identified, and the
expression pattern of these isoforms appears to be tissue
specific. In the case of the pituitary, only the 1.7-kb transcript
could be recognized. The presence of a single transcript for
CaM in the pituitary is consistent with the results of our
cloning studies, in which only CaM-L (corresponding to the
1.7-kb transcript), but not CaM-S, could be isolated from the
RT samples prepared from the pituitary. Because both
CaM-L and -S were pulled out from the RT samples of the
gills, in which only the 1.3- and 1.7-kb transcripts could be
detected, it would be logical to assume that the 1.3-kb tran-
script is the mRNA template responsible for the cloning of
CaM-S. Given that our 3⬘/5⬘ RACE using RT samples pre-
pared from brain, heart, and kidney did not yield additional
cDNAs for CaM, the sequence identity of the 3.0-kb tran-
script is still unknown. Although nucleotide sequence align-
ment of CaM-L and -S indicates that these two transcripts are
generated probably by differential use of polyadenylation
signals, we do not exclude the possibility that the variants of
CaM mRNA may be caused by alterations in promoter usage
or differential expression of multiple CaM genes. At present,
the physiological relevance of tissue-specific expression of
different isoforms of CaM transcripts is unclear.
Using static incubation of grass carp pituitary cells as a
model, IGF-I and -II have been demonstrated for the first
for 18 h, and cell lysate was prepared for Western blot using the
antibody for bovine CaM (1:1000). Parallel transfection with the blank
vector pcDNA3.1 was used as the control, and bovine CaM was used
as the standard for CaM immunoreactivity. B, Time course of CaM
overexpression on GH promoter activity. GH4C1 cells were trans-
fected with pcDNA.CaM and pGH(⫺986).Luc and incubated for the
duration indicated. Cell lysate was prepared at the respective time
points for luciferase activity measurement (upper panel). In these
studies, parallel measurement of GFP expression was also performed
to serve as an internal control (lower panel). The group denoted by an
asterisk represents a significant difference compared with the time-
matched control (P ⬍ 0.05, by Student’s t test). C, Dose dependence
of CaM overexpression on GH promoter activity. Similar to the time-
course experiments, GH4C1 cells were transfected with increasing
levels of pcDNA.CaM and incubated for 24 h before cell lysate prep-
aration. Parallel transfections with pGL3.Control and pGL3.Basic
were used as the positive and negative controls, respectively. In this
study, groups marked by the same letter indicate a similar level of
promoter activity (P ⬎ 0.05, by ANOVA, followed by Fisher’s LSD
test). D, 5⬘ Deletion of GH promoter to map the responsive sequence
for CaM overexpression. In this case, GH4C1 cells were transfected
with pGH.Luc constructs carrying decreasing lengths (⫺986 to ⫺115)
of the grass carp GH promoter with or without the cotransfection of
pcDNA.CaM. After incubation for 24 h, cell lysate was prepared for
luciferase activity measurement. In these experiments, parallel
transfection with the blank vector pcDNA3.1 was used as the control
treatment. The numerical values presented with the bar graph are the
fold induction of luciferase activity for individual constructs induced
by CaM overexpression. The group denoted with an asterisk repre-
sents a significant difference compared with the parallel control
transfected with pcDNA.3.1 (P ⬍ 0.05, by Student’s t test). Data
presented are expressed as the mean ⫾ SEM (n ⫽ 4) and are the pooled
results of four experiments.
Huo et al. • IGF Feedback on GH Expression
time to stimulate CaM mRNA expression at the pituitary
level. These stimulatory effects were specific to the 1.7-kb
CaM transcript and did not involve the expression of 1.3- and
3.0-kb CaM transcripts. This increase in CaM transcripts also
paralleled a rise in CaM content in pituitary cells, indicating
that IGF can induce CaM synthesis by up-regulation of CaM
gene expression. Based on spatial and temporal correlation
studies, these CaM mRNA responses occurred in grass carp
somatotrophs preceding the inhibitory actions of IGF-I and
IGF-II on GH mRNA expression. Apparently, IGF-induced
CaM gene expression could not be due to a cross-reactivity
with insulin receptors, because insulin did not mimic the
effects of IGF-I and IGF-II in the present study. Unlike IGF,
insulin treatment increased GH mRNA expression, but re-
duced CaM mRNA levels, in grass carp pituitary cells. In
mammals, it is well documented that IGF can exert a long-
loop feedback at the pituitary level to suppress GH release
and GH gene expression (49). The present finding of IGF
inhibition on GH mRNA expression in the grass carp con-
firms that the role of IGF as a negative regulator of GH
synthesis is conserved during vertebrate evolution. Similar
findings have been reported in other fish species, including
the tilapia (31), striped bass (33), and turbot (50). It is worth
mentioning that insulin can induce CaM gene transcription
(51) by activation of specificity protein-1 (Sp1)/Sp3 binding
to the Sp1 sites in the CaM promoter (52). In rat pituitary cells
(53) or pituitary cell lines transfected with GH promoter (54),
insulin treatment can also suppress GH release and GH gene
transcription. In fish models, the actions of insulin on GH
release tend to be more variable. Insulin has no effect on
GH secretion in trout pituitary cells (55) but can suppress GH
release in pituitary cells prepared from striped bass (33) and
turbot (50). In this study, insulin, by acting directly at the
pituitary level, was shown to activate GH gene expression in
a carp species. These findings as a whole suggest that the
functional role of insulin on GH regulation may be species
specific in lower vertebrates. Given that 1) both mammalian
and fish insulin can induce bioactivity with similar potency
in fish hepatocytes (56); and 2) mammalian insulin does not
bind IGF receptors in fish models (57), the possibility of
differential actions of insulin on CaM and GH transcript
expression caused by the use of heterologous insulin (i.e.
human insulin) is rather unlikely.
Apparently, IGF induction of CaM mRNA expression is
not mediated through modulation of CaM transcript stabil-
ity, because the t1/2 of CaM mRNA in carp pituitary cells was
not affected by IGF treatment. In contrast, the expression
levels of CaM primary transcripts were elevated in a dose-
dependent manner after IGF-I and IGF-II stimulation, indi-
cating that CaM gene transcription might have been acti-
vated. This idea is consistent with the results of transfection
studies in ␣T3-1 cells, in which IGF-I and IGF-II could up-
regulate grass carp CaM promoter activity. Although cis-
acting elements, including activator protein-1, Pit-1, insulin
receptor substrate, and Sp1 binding sites, can be identified in
the grass carp CaM promoter (Huo, L., and A. O. L. Wong,
unpublished observations), their functional role in IGF in-
duction of CaM gene expression remains to be determined.
Given that 1) a negative correlation between CaM and GH
mRNA expression was noted; and 2) the rise in CaM mRNA
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Endocrinology, September 2005, 146(9):3821–3835 3833
occurred before the drop in GH gene expression induced by
IGF treatment, the involvement of CaM in GH long-loop
feedback by IGF is suspected. Using a pharmacological ap-
proach, suppressing the functionality of endogenous CaM by
CaM antagonism was found to be effective in raising basal
GH mRNA levels as well as blocking the inhibitory effects of
IGF on GH gene expression. Besides, lowering of GH mRNA
levels by IGF treatment could be abolished by inactivation of
calcineurin, but not CaM kinase II, suggesting that the CaM/
calcineurin cascades may be acting downstream of IGF re-
ceptors. In GH4C1 cells, GH promoter activity was inhibited
by CaM overexpression, and the responsive element(s) could
be mapped to the proximal promoter of GH gene before
position ⫺115. Our sequence analysis based on TESS site
search has revealed that a consensus sequence of nuclear
factor-AT (NF-AT) binding site can be located in this region
(Wong, A. O. L., unpublished observations), which raises the
possibility that CaM by acting through calcineurin-induced
dephosphorylation, and nuclear translocation of (NF-AT)
can modulate GH gene expression at the transcriptional
level. Our results as a whole have two implications regarding
the function and regulation of CaM in the carp pituitary: 1)
the basal level of GH gene transcription may be under the
negative regulation of endogenous CaM; and 2) IGF-I and
IGF-II may up-regulate CaM gene expression to inhibit GH
synthesis at the pituitary level via CaM/calcineurin-depen-
dent mechanisms. Although our results are in agreement
with the general observations that IGF inhibition of GH re-
lease and GH gene expression tends to have a slow onset
(ⱖ12 h) and requires de novo protein synthesis (24), the sit-
uation in the fish model appears to be different from that in
mammalian counterparts. In mammals, especially in the rat,
CaM is involved in GH secretion (5), but not GH gene ex-
pression (18). In somatotroph cell lines, e.g. MtT/S cells, IGF
inhibits GH promoter activity via activation of the phos-
photidylinositol 3-kinase, but not MAPK or p70S6K cascades
(24). To our knowledge, the involvement of CaM gene ex-
pression in the postreceptor signaling mechanisms for IGF re-
ceptors has not been previously demonstrated.
In summary, we have established the structural identity of
grass carp CaM by molecular cloning and confirmed that
multiple transcripts of CaM are ubiquitously expressed in the
grass carp. The newly cloned CaM cDNAs are phylogeneti-
cally related to the CaM I gene subfamily. Based on the
sequence obtained, a slot-blot system was set up to quantify
the expression of CaM mRNA in grass carp pituitary cells. In
this case, we have demonstrated for the first time that IGF-I
and IGF-II induced CaM mRNA expression at the pituitary
level, and this stimulatory effect could be correlated both
spatially and temporally with the drop in GH mRNA levels.
The stimulatory action of IGFs on CaM transcript expression
was not mediated by modulation of CaM mRNA stability,
but was caused by a direct activation of CaM gene transcrip-
tion. In parallel experiments, we have shown that CaM an-
tagonism and calcineurin inactivation were effective in
blocking IGF-inhibited GH gene expression, whereas CaM
overexpression significantly suppressed grass carp GH pro-
moter activity. These results, taken together, indicate that
IGF inhibits GH synthesis in carp pituitary cells through
up-regulation of CaM gene expression. The present study
3834 Endocrinology, September 2005, 146(9):3821–3835
provides direct evidence that modulation of CaM gene ex-
pression at the pituitary level can serve as the site of regu-
lation for GH synthesis. Our studies in fish also provide a
comparative model for mammalian research to revisit the
signal transduction of IGF long-loop feedback at the pituitary
level. Given that IGF is involved in the transformation and
metastasis of tumor cells (53), the identification of CaM gene
expression as a novel signaling component for IGF action
may have clinical implications in cancer research.
Acknowledgments
Special thanks are given to Dr. P. C. Leung for the supply of CaM
antiserum and bovine CaM. We are also indebted to Drs. W. K. K. Ho
and W. W. M. Lee for their support in setting up the assay systems for
GH mRNA and CaM primary transcripts, respectively.
Received November 22, 2004. Accepted May 25, 2005.
Address all correspondence and requests for reprints to: Dr. Ander-
son O. L. Wong, Room 4S-12, Kadoorie Biological Sciences Building,
Department of Zoology, University of Hong Kong, Pokfulam Road,
Hong Kong, SAR, Peoples Republic of China. E-mail: olwong@hkucc.
hku.hk.
This work was supported by RGC (H.K.) and CRCG grants (H.K.U.)
to A.O.L.W. Financial support from the Department of Zoology
(H.K.U.) to L.H., G.F., and X.W. in the form of postgraduate studentships
is also acknowledged.
References
1. Weinstein H, Mehler EL 1994 Ca2⫹-binding and structural dynamics in the
functions of calmodulin. Annu Rev Physiol 56:213–236
2. Means AR 2000 Calcium and calmodulin-mediated regulatory mechanisms.
In: Conn PM, Means AR, eds. Principles of molecular regulation. Totowa, NJ:
Humana Press; 207–231
3. Kumar RV, Panniers R, Wolfman A, Henshaw EC 1991 Inhibition of protein
synthesis by antagonists of calmodulin in Ehrlich ascites tumor cells. Eur
J Biochem 195:313–319
4. Schettini G, Florio T, Meucci O, Landolfi E, Cronin MJ, MacLeod RM 1987
Calmodulin modulates prolactin secretion in vitro: studies with calmodulin
containing liposomes. Life Sci 41:2437–2444
5. Sletholt K, Haug E, Gordeladze J, Sand O, Gautvik KM 1987 Effects of
calmodulin antagonists on hormone release and cyclic AMP levels in GH3
pituitary cells. Acta Physiol Scand 130:333–343
6. Bonafe NM, Sellers, JR 1998 Calmodulin-binding protein of the cytoskeleton.
In: Van Eldik L, Watterson DM, eds. Calmodulin and signal transduction. San
Diego: Academic Press; 348 –396
7. Schmalzigaug R, Ye Q, Berchtold MW 2001 Calmodulin protects cells from
death under normal growth conditions and mitogenic starvation but plays a
mediating role in cell death upon B-cell receptor stimulation. Immunology
103:332–342
8. Takano H, Fukushi H, Morishima Y, Shirasaki Y 2003 Calmodulin and
calmodulin-dependent kinase II mediate neuronal cell death induced by de-
polarization. Brain Res 962:41– 47
9. Prostko CR, Zhang C, Hait WN 1997 The effects of altered cellular calmodulin
expression on the growth and viability of C6 glioblastoma cells. Oncol Res
9:13–17
10. Luk SC, Ngai SM, Tsui SK, Fung KP, Lee CY, Waye MM 1999 In vivo and
in vitro association of 14 –3-3 epsilon isoform with calmodulin: implication for
signal transduction and cell proliferation. J Cell Biochem 73:31–35
11. Walker SW, Wark JD, MacNeil S, Mellersh H, Brown BL, Tomlinson S 1984
Isolation, purification and cell-free synthesis of calmodulin from the pig an-
terior pituitary gland. Biochem J 217:827– 832
12. Kakiuchi S, Yasuda S, Yamazaki R, Teshima Y, Kanda K, Kakiuchi R, Sobue
K 1982 Quantitative determinations of calmodulin in the supernatant and
particulate fractions of mammalian tissues. J Biochem 92:1041–1048
13. Aguila MC, McCann SM 1988 Calmodulin dependence of somatostatin re-
lease stimulated by growth hormone-releasing factor. Endocrinology 123:305–
309
14. Honegger J, D’Urso R, Besser GM, Grossman AB 1991 Calcium and calmod-
ulin mediation of growth hormone-releasing hormone release from the rat
hypothalamus in vitro. Endocrinology 129:11–16
15. Davis JR, Hoggard N, Wilson EM, Vidal ME, Sheppard MC 1991 Calcium/
calmodulin regulation of the rat prolactin gene is conferred by the proximal
enhancer region. Mol Endocrinol 5:8 –12
16. Izumi S, Iwashita M, Makino T, Saito S, Sakamoto S, Takeda Y, Nozawa S
Downloaded from endo.endojournals.org on October 14, 2005
Huo et al. • IGF Feedback on GH Expression
1991 Phorbol ester-induced LH release in pituitary gonadotrophs: effects of
antagonists of calmodulin and GnRH. Endocrinol Jpn 38:195–204
17. Martinez-Campos A, Hernandez RP, Forsbach G, Barrera-Saldana HA 1991
The stimulatory effect of estradiol 17-␤ on prolactin mRNA is inhibited by
anti-calmodulin drugs. Life Sci 48:2475–2485
18. White BA 1985 Evidence for a role of calmodulin in the regulation of prolactin
gene expression. J Biol Chem 260:1213–1217
19. Le Roith D 2003 The insulin-like growth factor system. Exp Diabetes Res
4:205–212
20. Svensson J, Lonn L, Johannsson G, Bengtsson BA 2003 Effects of GH and
insulin-like growth factor-I on body composition. J Endocrinol Invest 26:823–
831
21. Dupont J, Khan J, Qu BH, Metzler P, Helman L, LeRoith D 2001 Insulin and
IGF-I induce different patterns of gene expression in fibroblast NIH-3T3 cells:
identification by cDNA microarray analysis. Endocrinology 142:4969 – 4975
22. Jaffe CA, Pan W, Brown MB, DeMott-Friberg R, Barkan AL 2001 Regulation
of GH secretion in acromegaly: reproducibility of daily GH profiles and at-
tenuated negative feedback by IGF-I. J Clin Endocrinol Metab 86:4364 – 4370
23. Weber MM, Melmed S, Rosenbloom J, Yamasaki H, Prager D 1992 Rat
somatotroph insulin-like growth factor-II (IGF-II) signaling: role of the IGF-I
receptor. Endocrinology 131:2147–2153
24. Niiori-Onishi A, Iwasaki Y, Mutsuga N, Oiso Y, Inoue K, Saito H 1999
Molecular mechanisms of the negative effect of insulin-like growth factor-I on
growth hormone gene expression in MtT/S somatotroph cells. Endocrinology
140:344 –349
25. Fletcher TP, Thomas GB, Dunshea FR, Moore LG, Clarke IJ 1995 IGF feed-
back effects on growth hormone secretion in ewes: evidence for action at the
pituitary but not the hypothalamic level. J Endocrinol 144:323–331
26. Becker K, Stegenga S, Conway S 1995 Role of insulin-like growth factor I in
regulating growth hormone release and feedback in the male rat. Neuroen-
docrinology 61:573–583
27. Moriyama S, Ayson FG, Kawauchi H 2000 Growth regulation by insulin-like
growth factor-I in fish. Biosci Biotechnol Biochem 64:1553–1562
28. Tse MC, Vong QP, Cheng CH, Chan KM 2002 PCR-cloning and gene ex-
pression studies in common carp insulin-like growth factor-II. Biochim Bio-
phys Acta 1575:63–74
29. Vong QP, Chan KM, Cheng CH 2003 Quantification of common carp IGF-I
and IGF-II mRNA by real-time PCR: differential regulation of expression by
GH. J Endocrinol 178:513–521
30. Fruchtman S, Gift B, Howes B, Borski R 2001 Insulin-like growth factor-I
augments prolactin and inhibits growth hormone release through distinct as
well as overlapping cellular signaling pathways. Comp Biochem Physiol B
129:237–242
31. Kajimura S, Uchida K, Yada T, Hirano T, Aida K, Gordon Grau E 2002 Effects
of insulin-like growth factors (IGF-I and -II) on growth hormone and prolactin
release and gene expression in euryhaline tilapia, Oreochromis mossambicus.
Gen Comp Endocrinol 127:223–231
32. Baker DM, Davies B, Dickhoff WW, Swanson P 2000 Insulin-like growth
factor I increases follicle-stimulating hormone (FSH) content and gonado-
tropin-releasing hormone-stimulated FSH release from Coho salmon pituitary
cells in vitro. Biol Reprod 63:865– 871
33. Fruchtman S, Jackson L, Borski R 2000 Insulin-like growth factor I disparately
regulates prolactin and growth hormone synthesis and secretion: studies using
the teleost pituitary model. Endocrinology 141:2886 –2894
34. Chang JP, Johnson JD, Van Goor F, Wong CJ, Yunker WK, Uretsky AD,
Taylor D, Jobin RM, Wong AOL, Goldberg JI 2000 Signal transduction
mechanisms mediating secretion in goldfish gonadotropes and somatotropes.
Biochem Cell Biol 78:139 –153
35. Chang JP, Abele JT, Van Goor F, Wong AOL, Neumann CM 1996 Role of
arachidonic acid and calmodulin in mediating dopamine D1- and GnRH-
stimulated growth hormone release in goldfish pituitary cells. Gen Comp
Endocrinol 102:88 –101
36. Jobin RM, Neumann CM, Chang JP 1996 Roles of calcium and calmodulin in
the mediation of acute and sustained GnRH-stimulated gonadotropin secre-
tion from dispersed goldfish pituitary cells. Gen Comp Endocrinol 101:91–106
37. Wong AOL, Ng S, Lee EK, Leung RC, Ho WK 1998 Somatostatin inhibits
(d-Arg6, Pro9-NEt) salmon gonadotropin-releasing hormone- and dopamine
D1-stimulated growth hormone release from perifused pituitary cells of Chi-
nese grass carp, Ctenopharyngodon idellus. Gen Comp Endocrinol 110:29 – 45
38. Zhou H, Wang X, Ko WK, Wong AOL 2004 Evidence for a novel intrapituitary
autocrine/paracrine feedback loop regulating growth hormone synthesis and
secretion in carp pituitary cells by functional interactions between gonado-
trophs and somatotrophs. Endocrinology 145:5548 –5559
39. Zhou H, Jiang Y, Ko WK, Li W, Wong AOL 2005 Paracrine regulation of
growth hormone gene expression by gonadotrophin release in grass carp
pituitary cells: functional implications, molecular mechanisms and signal
transduction. J Mol Endocrinol 34:415– 432
40. Toutenhoofd SL, Strehler EE 2000 The calmodulin multigene family as a
unique case of genetic redundancy: multiple levels of regulation to provide
spatial and temporal control of calmodulin pools? Cell Calcium 28:83–96
41. Berchtold MW, Egli R, Rhyner JA, Hameister H, Strehler EE 1993 Localization
of the human bona fide calmodulin genes CALM1, CALM2, and CALM3 to
Huo et al. • IGF Feedback on GH Expression
chromosomes 14q24 – q31, 2p21.1-p21.3, and 19q13.2-q13.3. Genomics 16:461–
465
42. Reid DG, MacLachlan LK, Gajjar K, Voyle M, King RJ, England PJ 1990 A
proton nuclear magnetic resonance and molecular modeling study of calmi-
dazolium (R24571) binding to calmodulin and skeletal muscle troponin C.
J Biol Chem 265:9744 –9753
43. Nojima H 1987 Molecular evolution of the calmodulin gene. FEBS Lett 217:
187–190
44. Saimi Y, Kung C 2002 Calmodulin as an ion channel subunit. Annu Rev
Physiol 64:289 –311
45. Sola C, Barron S, Tusell JM, Serratosa J 1999 The Ca2⫹/calmodulin signaling
system in the neural response to excitability. Involvement of neuronal and glial
cells. Prog Neurobiol 58:207–232
46. Sola C, Barron S, Tusell JM, Serratosa J 2001 The Ca2⫹/calmodulin system
in neuronal hyperexcitability. Int J Biochem Cell Biol 33:439 – 455
47. Gill KD, Gupta V, Sandhir R 2003 Ca2⫹/calmodulin-mediated neurotrans-
mitter release and neurobehavioural deficits following lead exposure. Cell
Biochem Funct 21:345–353
48. Zhang J, Suneja SK, Potashner SJ, Rameau GA, Chiu LY, Ziff EB 2004 Protein
kinase A and calcium/calmodulin-dependent protein kinase II regulate gly-
cine and GABA release in auditory brain stem nuclei: bidirectional regulation
of neuronal nitric-oxide synthase phosphorylation at serine 847 by the N-
methyl-d-aspartate receptor. J Neurosci Res 75:361–370
49. Le Roith D, Bondy C, Yakar S, Liu JL, Butler A 2001 The somatomedin
hypothesis: 2001. Endocr Rev 22:53–74
Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the
endocrine community.
Downloaded from endo.endojournals.org on October 14, 2005
Endocrinology, September 2005, 146(9):3821–3835 3835
50. Duval H, Rousseau K, Elies G, Le Bail PY, Dufour S, Boeuf G, Boujard D
2002 Cloning, characterization, and comparative activity of turbot IGF-I and
IGF-II. Gen Comp Endocrinol 126:269 –278
51. Solomon SS, Palazzolo MR, Smoake JA, Raghow RS 1995 Insulin-stimulated
calmodulin gene expression in rat H-411E cells can be selectively blocked by
antisense oligonucleotides. Biochem Biophys Res Commun 210:921–930
52. Solomon SS, Palazzolo MR, Takahashi T, Raghow R 1997 Insulin stimulates
rat calmodulin I gene transcription through activation of Sp1. Proc Assoc Am
Physicians 109:470 – 477
53. Yamashita S, Melmed S 1986 Effects of insulin on rat anterior pituitary cells.
Inhibition of growth hormone secretion and mRNA levels. Diabetes 35:440 –
447
54. Prager D, Melmed S 1988 Insulin regulates expression of the human growth
hormone gene in transfected cells. J Biol Chem 263:16580 –16585
55. Weil C, Carre F, Blaise O, Breton B, Le Bail PY 1999 Differential effect of
insulin-like growth factor I on in vitro gonadotropin and growth hormone
secretions in rainbow trout (Oncorhynchus mykiss) at different stages of the
reproductive cycle. Endocrinology 140:2054 –2062
56. Pierce AL, Fukada H, Dickhoff WW 2005 Metabolic hormones modulate the
effect of growth hormone (GH) on insulin-like growth factor-I (IGF-I) mRNA
level in primary culture of salmon hepatocytes. J Endocrinol 184:341–349
57. Castillo J, Le Bail PY, Paboeuf G, Navarro I, Weil C, Fauconneau B, Gutierrez
J 2002 IGF-I binding in primary culture of muscle cells of rainbow trout:
changes during in vitro development. Am J Physiol 283:R647–R652