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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

Department of Zoology, University of Hong Kong, Hong Kong, Peoples Republic of China

Address all correspondence and requests for reprints to: Dr. Anderson 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{at}hkucc.hku.hk.

	Abstract

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Calmodulin (CaM), the Ca²⁺ sensor in living cells, is essential for biological functions mediated by Ca²⁺-dependent mechanisms. However, modulation of CaM gene expression at the pituitary level as a means to regulate pituitary hormone synthesis 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 establish the structural identity of CaM expressed in the grass carp, a CaM cDNA, CaM-L, was isolated from the carp pituitary using 3'/5' 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 ubiquitously expressed in the grass carp. In carp pituitary cells, IGF-I and IGF-II induced CaM mRNA expression with a concurrent drop in GH transcript levels. These stimulatory effects 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 5' promoter activity 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 expression at the pituitary level.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
CALMODULIN (CaM) IS a Ca²⁺-binding protein serving as an intracellular Ca²⁺ sensor in living cells. Upon Ca²⁺ 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), apoptosis (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 hypothalamic 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 stimulatory 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), CaM antagonism has little or no effect on GH gene expression 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 influence 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 secretion (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 activity through phosphotidylinositol 3-kinase-mediated signaling 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 (including 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. Recently, IGF has been shown to induce LH and PRL release in Coho salmon (32) and striped bass (33), respectively. These 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-releasing factors, e.g. GnRH and dopamine, can be blocked by CaM antagonists and CaM kinase II inhibitors (35, 36). These results indicate that CaM and its downstream signaling pathways 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 hypothesis 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 established by molecular cloning using 5'/3' rapid amplification 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 expression 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

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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 acquired 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 µU/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 µM. 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.

Molecular cloning of grass carp CaM cDNA
Total RNA (5 µg) 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 conserved 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 Technologies, Inc., Carlsbad, CA) with pituitary total RNA (5 µg) 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 sequencing using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, 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 µg) 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 transblotted 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 0.5x standard saline citrate with 0.1% sodium dodecyl sulfate, and hybridization signals were detected using a DIG Lumin Detection Kit (Roche). In these studies, a Northern blot of ß-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 x 10⁶ cells/pituitary, and cell viability was always 95% or greater. After cell dispersion, 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 x 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 treatment was routinely fixed at 48 h. For the evaluation of CaM transcript stability, cells were pretreated with actinomycin D (8 µM) and incubated with or without IGF treatment for the duration as indicated in individual experiments. After drug treatment, total RNA (2.5 µg) 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 hybridization, the membranes were washed, and signal development was carried out as described for the Northern blot. Measurement of hybridization 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 trapping was performed using genomic DNA as a template to pull out introns 1–5 of the grass carp CaM gene (GenBank no. AY656698). Primers 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 µg) 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 transcripts 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 melting 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 prepared 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 construct 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 µl Lipofectamine (Invitrogen Life Technologies, Inc.), 0.05 µg pCaM(–1369).Luc, 0.01 µg pEGFP-N1 (EGFP, enhanced green fluorescence protein), and 0.44 µg 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 x g at 4 C for 15 min. For measurement of Luc activity, a 50-µl volume of cleared lysate was mixed with 100 µl luciferase 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 µl 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 subcloned 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 µl Lipofectamine, 0.18 µg pGH.Luc, 0.01 µg pEGFP-N1, and 0.05–0.25 µg 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 overexpression 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 phenylmethylsulfonylfluoride (150 µg/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 membrane 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 substrate. 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 ß-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 analysis (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 (t_1/2) 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 fluorescence 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. Differences were considered significant at P < 0.05.

	Results

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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 polyadenylation 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 mammals (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 protozoa (e.g. trypanosome), only a low level of a.a. substitutions were noted, and most of them were conserved mutations (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 conserved, 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).

View larger version (70K): [in this window] [in a new window]   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.

View larger version (121K): [in this window] [in a new window]   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.

View larger version (50K): [in this window] [in a new window]   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 ß-actin mRNA was used as an internal control.

View larger version (37K): [in this window] [in a new window]   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 expressed as the mean ± SEM (n = 4) and are the pooled results of four separate experiments. Groups denoted by the same letterrepresent 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.

View larger version (18K): [in this window] [in a new window]   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 µU/ml). After that, RNA samples were extracted and assayed 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 between CaM and GH mRNA expression. Pearson analysis was performed 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.

View larger version (34K): [in this window] [in a new window]   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, respectively (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 immunoreactivity was monitored using Western blot as described in Materials and Methods. Western blot of ß-actin was also conducted to serve as an internal control, and in these studies, no significant changes in ß-actin expression were noted after IGF-I treatment. Data presented are expressed as the mean ± SEM (n = 3) and are the pooled results of three separate experiments. Groups denoted by the same letterrepresent a similar level of CaM content or CaM mRNA expression (P > 0.05, by ANOVA, followed by Fisher’s LSD test).

View larger version (65K): [in this window] [in a new window]   FIG. 7. Spatial correlation between CaM and GH mRNA expression 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 somatotrophs (84%) was observed compared with the freshly dispersed pituitary cells (inset). B, Differential effects of IGF on CaM and GH mRNA expression in carp somatotrophs. Enriched somatotrophs 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 performed 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).

View larger version (27K): [in this window] [in a new window]   FIG. 8. Temporal correlation between CaM and GH mRNA expression 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).

View larger version (20K): [in this window] [in a new window]   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, Pituitary cells were exposed to IGF-I (100 nM) and IGF-II (100 nM) for 48 h 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 µM) or the calcineurin 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 letterrepresent a similar level of GH mRNA expression (P > 0.05, by ANOVA, followed by Fisher’s LSD test).

View larger version (49K): [in this window] [in a new window]   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 µM) 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).

Using grass carp pituitary cells pretreated with the transcription inhibitor actinomycin D (8 µM), 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 t_1/2 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 (data not shown), confirming that IGF-induced CaM gene expression in carp pituitary cells does not involve modification of CaM transcript stability.

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 endogenous expression of Pit-1, a pituitary-specific transcription factor, to support basal GH promoter activity. Overexpression 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 µg) 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 virus 40 promoter) compared with that in the group transfected with the promoterless pGL3.Basic, confirming that the reporter 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 inhibition of GH promoter activity, CaM overexpression was conducted 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-responsive element(s) is located within the proximal promoter region before position –115.

View larger version (29K): [in this window] [in a new window]   FIG. 11. CaM overexpression on GH promoter activity. A, CaM overexpression at the protein level in GH4C1 cells. After transfection with the CaM expression vector pcDNA.CaM, GH4C1 cells were incubated 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 transfected 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 preparation. 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 letterindicate 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 represents 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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Using static incubation of grass carp pituitary cells as a model, IGF-I and -II have been demonstrated for the first 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 reduced 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 confirms 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 stability, because the t_1/2 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, indicating that CaM gene transcription might have been activated. 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 induction 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 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 approach, 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 receptors. 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-dependent mechanisms. Although our results are in agreement with the general observations that IGF inhibition of GH release and GH gene expression tends to have a slow onset (12 h) and requires de novo protein synthesis (24), the situation 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 expression (18). In somatotroph cell lines, e.g. MtT/S cells, IGF inhibits GH promoter activity via activation of the phosphotidylinositol 3-kinase, but not MAPK or p70^S6K cascades (24). To our knowledge, the involvement of CaM gene expression in the postreceptor signaling mechanisms for IGF receptors 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 phylogenetically 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 transcription. In parallel experiments, we have shown that CaM antagonism and calcineurin inactivation were effective in blocking IGF-inhibited GH gene expression, whereas CaM overexpression significantly suppressed grass carp GH promoter 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 provides direct evidence that modulation of CaM gene expression at the pituitary level can serve as the site of regulation 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.

	Footnotes

 
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.

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'-untranslated region.

Received November 22, 2004.

Accepted for publication May 25, 2005.

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