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Endocrinology, doi:10.1210/en.2007-0694
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Endocrinology Vol. 149, No. 2 783-792
Copyright © 2008 by The Endocrine Society

3', 5'-Cyclic Adenosine 5'-Monophosphate Response Element-Dependent Transcriptional Regulation of the Secretogranin II Gene Promoter Depends on Gonadotropin-Releasing Hormone-Induced Mitogen-Activated Protein Kinase Activation and the Transactivator Activating Transcription Factor 3

Jianjun Xie and Mark S. Roberson

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

Address all correspondence and requests for reprints to: Mark S. Roberson Ph.D., T3-004d Veterinary Research Tower, Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853. E-mail: msr14{at}cornell.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies demonstrated that GnRH-induced secretogranin II (SgII) promoter regulation required a consensus cAMP response element (CRE) and protein kinase A/CRE binding protein. The present studies examined the role of additional components of the GnRH signaling network on SgII promoter activity with particular attention devoted to CRE-dependent gene regulation. Disruption of the SgII CRE by mutagenesis resulted in inhibition of GnRH agonist (GnRHa) induction of this promoter in {alpha}T3-1 cells. Pharmacological and dominant-negative inhibition of the ERK and c-Jun N-terminal kinase (JNK) signaling pathways revealed that GnRHa-induced SgII promoter activity required functional JNK and ERK modules. Combined inhibition of both pathways nearly abolished GnRHa-induced SgII promoter activity. Specific induction of the ERK cascade alone using overexpression of Raf-CAAX was not sufficient to activate the SgII gene promoter. In contrast, overexpression of the catalytic domain of the more pleiotropic MAPK activator, MAPK/ERK kinase-1, was sufficient to induce SgII promoter activity. The effect(s) of mitogen-activated protein/ERK kinase-1 on SgII promoter activity was CRE dependent and was reversed by the combined pharmacological inhibition of both JNK and ERK modules. CRE DNA binding studies demonstrated the recruitment of activating transcription factor (ATF)-3 and c-Jun to the CRE after administration of GnRHa to {alpha}T3-1 cells. Specific small interfering RNA knockdown of ATF3 reduced ATF3 DNA binding and the effect of GnRHa on the SgII promoter. These studies support the conclusion that MAPK signaling and ATF3 action are essential for full SgII promoter activation by GnRHa through a canonical CRE. Moreover, we suggest that within the GnRH signaling network, CRE-dependent gene regulation in general may be mediated primarily through the immediate early response gene ATF3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APPROPRIATELY COORDINATED secretion of gonadotropic hormones is central to regulation of normal reproductive function in mammals. This requires biosynthesis and packaging of LH and FSH into secretory granules in anticipation of a secretory event induced by a pulse of GnRH or a surge of GnRH associated with the preovulatory surge of gonadotropins. Stabilization and putative sorting of secretory granules within the pituitary as well as other neuroendocrine cell types may involve a family of proteins referred to as granins. The chromogranin and secretogranin gene family includes a cohort of glycosylated and sulfated acidic proteins that are organized into secretory granules along with peptide hormones such as LH (for recent review see Ref. 1). The family of granins is made up of seven members including chromogranin A, chromogranin B, and secretogranins II–VI. With specific reference to the anterior pituitary and gonadotropes, several groups have investigated the role of GnRH and secretogranin II (SgII) in gonadotrope cell function and secretion of gonadotropins (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Originally identified as a major secretory product of the anterior pituitary gland, GP-87 (later referred to as SgII) was shown to be synthesized and secreted from the anterior pituitary after administration of GnRH. Immunohistochemical studies suggested that SgII resides within secretory granules in gonadotropes and is secreted along with LH after GnRH stimulation (7). Most recently SgII was used as a marker for the kinetics of secretory vesicle recovery after pharmacological stimulation with leuprorelin in rat gonadotropes containing LHβ immunoreactivity (13). SgII likely plays an important role in the assembly of secretory granules subject to regulated secretion; however, once secreted, the physiological function of SgII or proteolytically cleaved peptides from SgII remains to be determined.

Early studies provided clear evidence that in vivo, rat SgII was modulated by GnRH at the mRNA level in the anterior pituitary gland (12, 14). SgII mRNA was increased along with LHβ subunit mRNA after ovariectomy, suggesting that increased pulsatile GnRH stimulation may mediate SgII mRNA accumulation. Moreover, increased steady-state levels of SgII and LHβ mRNAs subsequent to ovariectomy were blocked with administration of a GnRH receptor antagonist (12), suggesting a direct effect of the GnRH receptor on SgII biosynthesis. Estradiol may also influence the regulation of SgII mRNA levels both indirectly (presumably through hypothalamic GnRH secretion) and directly at the level of the anterior pituitary gland (15, 16). Cloning of the SgII gene promoters from rat and human identified a consensus cAMP response element (CRE) conserved within the 5' flanking sequences of the SgII promoter that was critical for transcriptional regulation by the cAMP/protein kinase A (PKA) pathway in several neuroendocrine cell types (17, 18, 19, 20, 21, 46).

More recent studies investigated the effects of GnRH directly in the SgII gene promoter in cell lines derived from the gonadotrope lineage (22). These studies suggested that the CRE was instrumental in the regulation of SgII gene promoter activity by GnRH via PKA and CRE binding protein (CREB). However, these studies did not consider alternative regulation via GnRH-induced cell signaling mediated by MAPK modules. Activation of the GnRH receptor in pituitary gonadotropes induces a complex signaling network that includes activation of at least three MAPK pathways or modules (23, 24, 25, 26, 27, 28). These MAPK family members consist of ERKs, c-Jun N-terminal kinases (JNKs) and p38 MAPK (for review see Refs. 29, 30, 31). The present studies provide evidence that the SgII CRE is central to regulation of the SgII gene promoter via GnRH-mediated activation of the JNK and ERK modules. The effects of GnRH-induced MAPK activity were highly correlated to up-regulation of the immediate early genes activating transcription factor (ATF)-3 and the specific JNK substrate, c-Jun. Specific inhibition of ATF3 using small interfering RNA (siRNA) strategies reduced the binding of ATF3 on the SgII CRE and consequently the ability of GnRH to increase CRE-dependent SgII promoter activity, suggesting a central role of ATF3 on GnRH-mediated SgII gene regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture, plasmids, and transient transfection
The 5' deletion series of the rat SgII promoter, SgII –1129, –723, –471, –317, –149, –101, –87, –56, and –42 was graciously provided by Dr. Richard Maurer (Oregon Health Sciences University, Portland, OR). The mutations within the SgII CRE were constructed by replacing the CRE site (TGACGTCA) with a NotI enzyme restriction site (GCGGCCGC) using a PCR-based strategy as previously described (32, 33). The mutation was confirmed by nucleotide sequence analysis. The human glycoprotein hormone-{alpha} subunit (wild-type and CRE mutant) luciferase reporter constructs have been reported previously (33). The c-Fos luciferase reporter has been described previously (26). The expression vector for Raf-CAAX was a gift from Dr. Linda Van Aelst (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The expression vector for the catalytic domain of MAPK/ERK kinase (MEKK)-1 (referred to as MEKK{Delta}) was a gift from Dr. Carol Lange (University of Minnesota Cancer Center, Minneapolis, MN). The expression vector for JNK-interacting peptide (JIP) was a gift from Dr. Roger Davis (University of Massachusetts, Medical School, Worcester, MA).

{alpha}T3-1 cells were generously provided by Dr. Pamela Mellon (University of California, San Diego, San Diego, CA) and were grown in DMEM (Sigma, St. Louis, MO) containing 10% fetal bovine serum and 100 IU/ml penicillin-streptomycin. Cells were plated at a density of 65% in 60-mm culture dishes and transfected the next day by calcium phosphate transfection reagents (Invitrogen, Carlsbad, CA; following the manufacturer’s instructions) for 18 h with 1 µg of reporter gene [the series of deletion of the SgII promoter, c-Fos luciferase, or human {alpha}-subunit luc, (wild-type or CRE mutant)]. For the SgII deletion studies, the cells were treated with either control solution (saline) or 10 nM GnRH agonist (GnRHa; buserelin; Sigma, St. Louis, MO), and luciferase activity was determined 6 h later. For pharmacological studies, PD98059 [MAPK/ERK (MEK)-1/MEK2 inhibitor, used at 50 µM] or SP600125 (JNK inhibitor, used at 25 µM) or the combination of both was administrated 30 min before GnRHa or saline administration. For Raf-CAAX, MEKK{Delta}, and JIP overexpression studies, the expression vectors were cotransfected at the doses provided with the SgII-1129 reporter gene. For MEKK{Delta} studies using PD98059 and SP600125, drugs were administered 3 h after the application of the calcium-phosphate DNA precipitates. The media were changed on all cells the following morning, and the drugs (or control solution) were readministered 8 h before lysis and collection for luciferase activity. All data are presented as mean ± SEM for luciferase activity standardized by total cellular protein as determined by Bradford assay. The figures shown are representative experiments performed on at least three separate occasions.

CRE pull-down assay
Nuclear extracts (NE) were prepared from {alpha}T3-1 cells as described previously (33). The CRE pull-down assay has been described in detail previously (33, 34). Briefly, biotinylated oligonucleotides representing the single consensus SgII CRE were bound to streptavidin agarose (SA) beads and combined with {alpha}T3-1 cell NEs in the presence of nonspecific competitor DNA (poly dI-dC). Specific NEs obtained from unstimulated (control) or GnRHa-stimulated (2 h treatment) {alpha}T3-1 cells were used. In other studies, NEs were derived from unstimulated (control) or GnRHa-stimulated (2 h treatment) {alpha}T3-1 cells stabling expressing a control siRNA and siRNAs specifically directed at ATF3 as described below. Binding reactions were incubated for 2 h at 4 C with constant rocking. Control reactions included a pool of control and GnRHa-treated NE (1:1) and SA beads alone (without the CRE) as a negative control. Additional binding reactions included 20-fold molar excess of nonbiotinylated SgII single-consensus CRE or CCAAT binding site as homologous and heterologous competitors, respectively. After binding reactions reached equilibrium and were washed extensively, complexes were resolved using SDS-PAGE, and Western blot analyses were used to determine the presence of ATF3 (antibody titer at 1:500) and c-Jun (antibody titer at 1:500) in Western blots using commercially available antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). These DNA binding studies were carried out on three independent occasions using three different sets of Nes, and the studies presented are from a representative experiment.

siRNA and Western blot analyses
Control and ATF3 hairpin loop siRNAs were cloned using the expression vector pSUPER-Retro-Neo. Gene-specific siRNA hairpin loops targeting a 19-nucleotide sequences within mouse ATF3 were obtaining using the siRNA design algorithm from Dharmacon (Dharmacon.com). Originally, three ATF3 siRNAs were tested to identify an optimal ATF3 siRNA(s). Only one of three siRNA sequences (5'-GCGGCGAG AAAGAAATAAA) proved useful for ATF3 knockdown. An additional siRNA vector used as a negative control has been described previously (33). The ATF3 and control siRNA sequences contained BglII and HindIII restriction sites/sticky ends included in the forward and reverse strands, respectively. The synthesized oligonucleotides were phosphorylated, annealed, and inserted into pSUPER-Retro-Neo by using the BglII and HindIII sites. The hairpin siRNA sequences were confirmed by nucleotide sequence analysis. {alpha}T3-1 cells were then transfected with pSUPER-Retro-Neo-ATF3 or -control siRNAs using FuGENE6 transfection reagent (Roche Applied Science, Indianapolis, IN) for 18 h. Stably transfected cells were then selected using neomycin (500 µg/ml). No effort was made to isolate clonal populations of cells. After approximately 3 wk of neomycin resistance selection, the cell lines were ready for use in experiments. The siRNA cell lines were plated at a density of 60% in six-well plates and the following day were transiently transfected with luciferase reporters for 3 h followed by either control solution or GnRHa. The cells were harvested 6 h after hormone treatment and assayed for luciferase as previously described (33). For Western blot analyses, the control and ATF3 siRNA cell lines were cultured in 60-mm plates at a density of 60–70%. The following day, cells were serum starved for 2 h, followed by administration of 10 nM GnRHa for 0, 30, 60, and 120 min. Whole-cell lysates were prepared as described previously (33), and Western blot analyses were used to determine ATF3 and actin (as lane loading control) protein expression. ATF3 (antibody titer at 1:500) and actin (antibody titer at 1:500) antibodies were purchased from Santa Cruz Biotechnology.

Statistical analysis
Data sets were subjected to an ANOVA, followed by individual pairwise comparisons using the Tukey honestly significant difference test. P < 0.05 was deemed significant. Data are reported as mean ± SEM for representative studies. All studies were carried out at least three times on separate occasions. Means reported in transfection studies reflect three replicates within each experiment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRHa action on the rat SgII gene promoter is orchestrated through a consensus CRE
Initially we examined transcriptional responses of a series of deletions of the 5' flanking sequences of the SgII gene promoter (Fig. 1AGo). For these studies, basal and GnRHa stimulated SgII promoter-luciferase reporter gene activity were examined in transfection studies using {alpha}T3-1 cells, a model for pituitary gonadotropes (35, 36). The 5' deletions extended from –1129 through –42 relative to the transcription start site. Whereas some variability was observed in basal and GnRHa-induced expression of this deletion series of luciferase reporter genes, the greatest loss of basal and GnRHa-inducible activity was observed with the deletion from –87 to –56. This region is characterized by a consensus CRE (TGACGTCA) (22). Using the –1129 nucleotide promoter fragment, we prepared a substitution mutation disrupting the consensus CRE. Comparing the wild-type and CRE mutant (–1129) SgII gene promoters, the CRE mutation substantially reduced GnRHa-induced SgII promoter-luciferase activity in {alpha}T3-1 cells (Fig. 1BGo). Consistent with previous reports (22), the ability of GnRH signaling to couple with the SgII gene promoter requires the presence of an intact, consensus CRE.


Figure 1
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  		FIG. 1. GnRH-induced SgII promoter activity depends on a single CRE. A, Serial 5' deletions of an SgII promoter fragment were constructed with upstream limits at –1129, –723, –471, –317, –149, –101, –87, –56, and –42 and were cloned into a luciferase reporter plasmid. The black box in the schematic of the 5' flanking sequence represents the position of a consensus CRE. {alpha}T3-1 cells were transiently transfected with the luciferase reporter genes using calcium phosphate transfection. The following day, transfected cells received saline control or GnRHa (the GnRH agonist buserelin; 10 nM) for a 6-h period. Cells were then harvested and luciferase activity determined and standardized using total cell protein. B, Wild-type and CRE mutant (hatched box in promoter schematic) SgII-Luc –1129 reporter genes were transfected into {alpha}T3-1 cells, treated with GnRHa, and luciferase activity determined as described for A. All data are presented as mean ± SEM for relative luciferase activity from a representative experiment carried out in triplicate. These studies were completed on three separate occasions with similar results. The fold changes for these studies are reported in the column adjacent to the histograms. a and b, Differences (P < 0.05) in pairwise comparisons of 5' deletions or mutations between control vs. GnRHa treatment groups. **, Differences (P < 0.05).

 
The JNK and ERK signaling modules are essential for GnRH-induced SgII promoter activity
GnRH-induced MAPK activity is central to GnRH action in pituitary gonadotropes. For the ERK and JNK modules, pharmacological inhibitors are available to disrupt these pathways with reasonable levels of specificity (33, 37). At the doses used in these experiments, PD98059 and SP600125 were effective in specifically blocking GnRHa-induced ERK phosphorylation and phosphorylation of c-Jun (a specific substrate of JNK catalytic activity), respectively (data not shown and (33). Independent blockade of the ERK and JNK modules resulted in marked reductions in GnRHa-induced SgII promoter activity (Fig. 2AGo). To confirm the results with SP600125 (the JNK inhibitor), we also examined the effects of overexpression JIP. JIP has been reported to physically interact with JNK isoforms (38, 39) and when overexpressed serves as a dominant-negative inhibitor of the JNK module by putatively causing cytoplasmic retention of JNK and/or uncoupling protein-protein interactions within the JNK module (38). Overexpression of JIP blocked GnRHa-induced SgII promoter activation in a dose-dependent manner (Fig. 2BGo) consistent with the studies using SP600125. This suggested that full activation of the JNK module or nuclear translocation of activated JNK was necessary for GnRHa action on the SgII gene promoter. The combined pharmacological inhibition of JNK and ERK signaling nearly abolished the action of GnRHa on SgII promoter activity (Fig. 2AGo), leading to the conclusion that activation of both pathways is essential for SgII gene promoter regulation by GnRHa.


Figure 2
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  		FIG. 2. GnRHa activation of the SgII gene promoter is ERK and JNK dependent. A, Using the SgII –1129 luciferase reporter construct, transient transfection studies were carried out in {alpha}T3-1 cells using a specific pharmacological agent to block the ERK (PD98059; 50 µM) and JNK (SP600125; 25 µM) signaling modules. Cells were transfected with the SgII reporter. The following day, cells were pretreated with dimethylsulfoxide (vehicle control), PD98059, SP600125, or the combination of PD98059 and SP600125 30 min before application of saline or GnRHa. Cells were collected 6 h after GnRHa administration and luciferase activity was determined as described in Fig. 1Go. B, {alpha}T3-1 cells were cotransfected with the SgII –1129 luciferase reporter and either control plasmid DNA (pcDNA3 parent vector) or pcDNA3-JIP expression plasmid (2.5 or 5.0 µg). JIP is a JNK module interacting protein known to disrupt JNK signaling. Twenty-four hours later, cells were harvested and assayed for luciferase activity as described in Fig. 1Go. All data are presented as mean ± SEM for relative luciferase activity from a representative experiment carried out in triplicate. These studies were completed on three separate occasions with similar results. The fold changes for these studies are reported in the column adjacent to the histograms. a and b, Differences (P < 0.05) in pairwise comparisons between control and GnRHa treatment groups using the pharmacology inhibitors or JIP overexpression. **, Differences (P < 0.05).

 
A pleiotropic activator of MAPK pathways is sufficient to activate the SgII gene promoter in a CRE-dependent manner
The ERK and JNK modules characteristically contain three kinases coupling an upstream signaling event (such as activation of the GnRH receptor) to transcriptional responses after activation and nuclear translocation of the terminal MAPK. For the ERK pathway, the canonical module consists of c-Raf kinase, MEK1/MEK2, and ERK1/ERK2. For the JNK pathway, one variant of the signaling module includes MEKK1, MAPK kinase 4/7, and JNK1/ JNK2 (40). Cross talk between these two modules is possible through MEKK1 activation of MEK1 and MEK2; however, the reciprocal activation of MAPK kinase 4/7 by Raf kinase does not appear to occur (41). Specific activation of the ERK module is possible through overexpression of a form of c-Raf kinase that is specifically targeted to the plasma membrane through a fusion of c-Raf kinase with the CAAX sequences from the low-molecular-weight G protein, ras (42, 43). Addition of the CAAX sequences to c-Raf kinase provides a mechanism for the translocation of c-Raf kinase to the membrane without putative modulation by other signaling intermediates. In contrast to Raf-CAAX, combined activation of the ERK and JNK modules is possible through overexpression of the catalytic domain of MEKK1 (referred to as MEKK{Delta}) (44).

Overexpression of Raf-CAAX alone was not sufficient to increase expression of the SgII luciferase reporter, whereas overexpression of MEKK{Delta} induced SgII promoter activity to a level consistent with GnRHa administration (Fig. 3AGo). Overexpression of the combination of Raf-CAAX and MEKK{Delta} did not result in SgII promoter activation beyond what was observed with MEKK{Delta} alone. This was not particularly surprising because MEKK{Delta} is capable of JNK and ERK activation through shared signaling intermediates. At an equivalent dose, Raf-CAAX overexpression was clearly capable of up-regulating a c-Fos gene promoter-luciferase reporter in control experiments, providing evidence for the effective action of the Raf-CAAX expression vector in this experimental paradigm (Fig. 3BGo). These studies suggested that the discrete activation of the JNK and ERK modules by MEKK{Delta} but not the ERK module alone was sufficient to mediate a transcriptional response of the SgII gene promoter.


Figure 3
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  		FIG. 3. Activation of the MAPK modules is sufficient to activate the SgII luciferase reporter gene in {alpha}T3-1 cells. A, {alpha}T3-1 cells were transiently transfected with the SgII –1129 luciferase reporter and either treated with saline or GnRHa or cotransfected with expression vectors for the control (parent plasmid), a constitutively active form of c-Raf kinase (Raf-CAAX; 5 µg), the catalytic domain of MEKK1 (MEKK{Delta}; 2.5 µg), or the combination of Raf-CAAX and MEKK{Delta}. Cells were harvested 6 h after the GnRHa administration and assayed for luciferase activity as described in Fig. 1Go. B, To confirm Raf-CAAX catalytic activity in transfection studies, SgII –1129 or the c-Fos promoter luciferase reporters were cotransfected with control plasmid (parent vector) or Raf-CAAX (5 µg). Twenty-four hours later, luciferase activity was assayed as described in Fig. 1Go. All data are presented as mean ± SEM for relative luciferase activity from a representative experiment carried out in triplicate. These studies were completed on three separate occasions with similar results. The fold changes for these studies are reported in the column adjacent to the histograms. a and b, Differences (P < 0.05) in pairwise comparisons between control and GnRHa, Raf-CAAX, or MEKK{Delta} overexpression treatment groups.

 
Consistent with the actions of GnRHa, up-regulation of the SgII promoter by overexpression of MEKK{Delta} required the consensus CRE present within the SgII gene promoter. Either CRE-deficient promoter deletion mutants (Fig. 4AGo) or a specific mutation within the CRE in the context of the SgII –1129 gene promoter (Fig. 4BGo) blocked MEKK{Delta}-dependent up-regulation of luciferase activity. The combined roles of the ERK and JNK modules in MEKK{Delta} action was also confirmed in studies using PD98059 and SP600125 (data not shown and Fig. 5AGo) or JIP overexpression (Fig. 5BGo) to block the actions of MEKK{Delta} on SgII promoter activity. Thus, reconstitution of specific portions of the GnRH signaling network with Raf-CAAX and MEKK{Delta} provided a means to determine whether ERK and/or ERK and JNK pathway activation was sufficient for SgII promoter regulation. Consistent with our earlier pharmacological approach (Fig. 2Go), these studies provide additional evidence for the role of the MAPK modules in CRE-dependent regulation of the SgII gene promoter.


Figure 4
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  		FIG. 4. The effects of MEKK{Delta} on SgII promoter activity require the consensus CRE within the SgII gene promoter. A, {alpha}T3–1 cells were transiently cotransfected with a subset of the 5'deletion series (described in Fig. 1Go) with either control or the MEKK{Delta} expression plasmid (2.5 µg). Cells were harvested and assayed for luciferase activity 24 h later as described in Fig. 1Go. B, To examine the direct impact of the SgII CRE on MEKK{Delta}-induced transcriptional activation, {alpha}T3-1 cells were cotransfected with the SgII –1129 wild-type luciferase reporter or the SgII –1129 luciferase reporter with the mutant CRE (SgII CRE Mut) and either control plasmid (parent vector) or expression vector for MEKK{Delta} (2.5 µg). Some cells cotransfected with SgII –1129 luciferase wild-type and CRE mutant vectors and control plasmid were also treated with saline or GnRHa for a 6 h period as described in Fig. 1Go. After transfection, cells were harvested and assayed for luciferase activity as described in Fig. 1Go. All data are presented as mean ± SEM for relative luciferase activity from a representative experiment carried out in triplicate. These studies were completed on three separate occasions with similar results. The fold changes for these studies are reported in the column adjacent to the histograms. a and b, Differences (P < 0.05) in pairwise comparisons between control and MEKK{Delta} or GnRHa treatment groups using specific 5' deletions or the CRE mutant. **, Differences (P < 0.05).

 

Figure 5
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  		FIG. 5. The effects of MEKK{Delta} on SgII gene promoter activity is reduced by JNK inhibition. A, To examine the role of the ERK and JNK modules, {alpha}T3-1 cells were transfected with the SgII –1129 luciferase reporter with either control plasmid (parent vector) or MEKK{Delta} (2.5 µg) expression vector. Pharmacological inhibitors PD98059 (PD) and SP600125 (SP) were administered together initially approximately 3 h after the start of transfection and then again (with a media change) approximately 8 h before collection of lysates for luciferase assay. B, To examine the role of the JNK module in MEKK{Delta}-mediated SgII promoter activity, {alpha}T3-1 cells were cotransfected with the SgII –1129 luciferase reporter and control vector (parent vector) or MEKK{Delta} (2.5 µg) expression vector. Some cells also received either control plasmid (parent vector) or expression plasmid for JIP (2.5 µg). Twenty-four hours after transfection, cells were harvested and assays for luciferase activity as described in Fig. 1Go. All data are presented as mean ± SEM for relative luciferase activity from a representative experiment carried out in triplicate. This study was completed on three separate occasions with similar results. The fold changes for this study are reported in the column adjacent to the histograms. a and b, Differences (P < 0.05) in pairwise comparisons between control and MEKK{Delta} treatment groups using PD98059 or JIP overexpression. **, Differences (P < 0.05).

 
ATF3 appears to be a central regulator of CRE-dependent SgII promoter activity
Once we had clarified the signaling intermediates that were essential for GnRHa action on the SgII gene promoter, we sought to establish potential transcriptional regulators that were capable of conveying putative ERK/JNK responsiveness through the SgII CRE. We previously demonstrated a role for the immediate early response gene, ATF3, in GnRHa action on CRE-dependent regulation of the human glycoprotein hormone {alpha}-subunit gene promoter (33). In those studies, the combined actions of the ERK and JNK modules were essential for up-regulation of ATF3 protein by GnRHa. Using a similar approach, the present studies demonstrated that after GnRHa treatment, the complex of proteins capable of binding to the SgII CRE include ATF3 and c-Jun (Fig. 6AGo). These studies were carried out using nuclear extracts purified from {alpha}T3-1 cells in binding reactions using biotinylated oligonucleotides encoding the SgII CRE. The pull-down studies were carried out using nuclear extracts from control-treated cells and {alpha}T3-1 cells treated with GnRHa for 2 h. Regardless of hormone treatment, CRE pull-down studies demonstrated the constitutive presence of CREB (Fig. 6AGo). However, with a 2-h GnRHa treatment, ATF3 and c-JUN were specifically recruited to the SgII CRE (Fig. 6AGo). Competition studies with homologous (nonbiotinylated CRE) and heterologous competitor (a nonbiotinylated consensus CCAAT box element) oligonucleotides confirmed the specificity of the interaction. Streptavidin beads in the absence of the biotinylated CRE was used as a negative control. Consistent with our previous observation with the dual CREs present within the {alpha}-subunit gene promoter (33), ATF3 and c-Jun appeared to bind the SgII CRE after administration of GnRHa. Collectively, these studies support a potential role for an ATF3 homodimer or an ATF3-c-Jun heterodimer in the regulation of the SgII gene promoter.


Figure 6
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  		FIG. 6. ATF3 and c-Jun bind specifically to the SgII CRE. A, DNA binding studies using a CRE pull-down approach were used to determine the identity and specificity of CRE binding complexes in {alpha}T3-1 nuclear extracts. {alpha}T3-1 cell NEs were purified from unstimulated (control) or GnRHa-treated (2 h) cells and used in binding reactions containing the SgII CRE (biotinylated) coupled to SA agarose beads. Some binding reactions contained 20-fold molar excess of nonbiotinylated CRE as a homologous competitor; other reactions contained 20-fold molar excess of nonbiotinylated CCAAT box binding site as a heterologous competitor. Binding complexes were allowed to reach equilibrium, washed extensively, and resolved using SDS-PAGE followed by Western blot analyses for ATF3 and c-Jun. DNA binding studies were carried out on at least three separate occasions with similar results. The blot shown is representative of these studies. B, Transient cotransfection studies in {alpha}T3-1 cells examined the possibility that overexpression of ATF3 (epitope tagged with the HA epitope; HA-ATF3; 2.5 µg), c-Jun (2.5 µg), or the combination of HA-ATF3 and c-Jun would increase expression of the SgII gene promoter. Twenty-four hours after transfection, cells were harvested for luciferase activity as described in Fig. 1Go. All data are presented as mean ± SEM for relative luciferase activity from a representative experiment carried out in triplicate. This study was completed on three separate occasions with similar results. The fold changes for this study are reported in the column adjacent to the histograms. a and b, Differences (P < 0.05) in pairwise comparisons between control and HA-ATF3 or c-Jun overexpression.

 
To examine this issue further, we overexpressed epitope-tagged forms of ATF3 (HA-ATF3), c-Jun, or the combination of HA-ATF3 and c-Jun along with the SgII –1129 luciferase reporter gene in transient transfection studies in {alpha}T3-1 cells. These studies revealed that HA-ATF3 or c-Jun alone were sufficient to induce SgII promoter activity (Fig. 6BGo). We previously demonstrated that ATF3 and c-Jun physically interact in {alpha}T3-1 cells after GnRHa treatment (33). The combined effect(s) of HA-ATF3 and c-Jun were approximately additive, suggesting that both ATF3 and c-Jun may be important in the regulation of the SgII gene promoter.

These overexpression studies provided evidence that ATF3 and or c-Jun was sufficient for the transactivation of the SgII promoter in {alpha}T3-1 cells in the absence of GnRH receptor stimulation. To examine a potential requirement for ATF3 in this mechanism, we established a group of {alpha}T3-1 cell lines stably expressing putative hairpin loop siRNAs directed against ATF3. One of these lines provided excellent siRNA-mediated knockdown of ATF3 (Fig. 7AGo). An {alpha}T3-1 cell line stably expressing a nonsense siRNA was used as a control in these experiments demonstrating the specificity of the ATF3 siRNA. Furthermore, neither control nor ATF3 siRNAs caused an appreciable reduction in actin expression levels, again suggesting that the actions of the ATF3 siRNA were specific. To confirm that the siRNA-mediated knockdown also perturbed ATF3 binding the SgII CRE, we performed additional CRE pull-down studies using NE from control and ATF3 siRNA cell lines. These studies demonstrated a specific loss of ATF3 binding to the SgII CRE in the ATF3 siRNA cell line, compared with the stable cell line expressing the control siRNA (Fig. 7BGo). Importantly, CREB and c-Jun binding in the ATF3 siRNA cell line was not appreciably perturbed, providing additional evidence for the specificity of these siRNA lines. Moreover, these cell lines provided a critical model to assess the specific effect of ATF3 loss on the functional consequences of GnRHa-induced SgII promoter activity. Using these cell lines, we transfected the SgII –1129 luciferase reporter to determine whether the specific loss of GnRHa-induced up-regulation of ATF3 would interfere with SgII gene promoter activity. In the control siRNA cell line, the SgII reporter gene was induced by GnRHa approximately 5-fold (Fig. 7CGo). Similar results were obtained with the human {alpha}-subunit gene promoter reporter. In the ATF3 knockdown cell line, loss of ATF3 resulted in a 40–60% loss in GnRHa inducibility of the SgII and {alpha}-subunit reporters. These studies provided direct evidence that GnRHa-induced ATF3 contributes to the mechanism(s) regulating GnRH responsiveness of the SgII gene promoter.


Figure 7
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  		FIG. 7. Knockdown of ATF3 is correlated with reduced responsiveness of the SgII luciferase reporter gene to GnRH stimulation. A, Expression plasmids for hairpin siRNAs (control and siRNAs specific for ATF3) were transfected into {alpha}T3-1 cells. Stable transfected cell lines were selected using neomycin (500 µg/ml). Control and ATF3 siRNA cell lines were then serum starved for a 2-h period followed by administration of GnRHa over a 120-min time course. Whole-cell lysates were prepared and resolved using SDS-PAGE. Western blot analyses were used to determine relative levels of ATF3 and actin in the different siRNA cell lines. These studies were carried out twice using neomycin-selected ATF3 siRNA cell lines with similar results. B, Once established, siRNA cell lines were then transfected with the SgII –1129 or the human glycoprotein hormone {alpha}-subunit luciferase (h {alpha}-subunit-Luc) reporter genes. The following day, transfected cells were administered saline or GnRHa for a 6-h period. Cells were then harvested and luciferase activity was determined as described in Fig. 1Go. All data are presented as mean ± SEM for relative luciferase activity from a representative experiment carried out in triplicate. These studies were completed on three separate occasions with similar results. The fold changes for these studies are reported in the column adjacent to the histograms. a and b, Differences (P < 0.05) in pairwise comparisons between control and GnRHa treatment groups using control siRNA or ATF3 siRNA in stable cell lines. **, Differences (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Granin family members including SgII are involved in vesicular sorting mechanisms in the formation of secretory granules destined for the regulated secretion pathway. Aggregation of peptide hormones (such as gonadotropins) into secretory vesicles may occur optimally in conditions of relatively high intracellular calcium concentrations at acidic pH. Granins coaggregate into these secretory granules under similar conditions within the lumen of the Golgi network (reviewed in Ref. 1). As such, SgII is secreted with gonadotropic hormones during a secretory episode. This has led to speculation that secreted granins may play a systemic role, particularly because granins are subject to proteolytic processing and granin fragments may have some level of biological activity (1). Interestingly, proteolytic processing of SgII leads to the formation of secretoneurin. Secretoneurin plays a role in the modulation of numerous activities including regulation dopamine, serotonin, and melatonin release and alteration of endothelial cell behavior. Additionally, secretoneurin is a potent chemoattractant for monocytes and eosinophils (45). The role of secreted SgII and potentially secretoneurin in the context of GnRH action and the integration of the hypothalamic-pituitary-gonadal axis is currently not known.

Our previous studies suggested that the SgII gene promoter was regulated by GnRH via a consensus CRE present within the 5' flanking region of the gene (22). These studies implicated a role for PKA signaling and CREB activity at the CRE to mediate the effects of GnRH. However, it was unclear from these studies exactly how GnRH coupled to the PKA/CREB mechanism in the cell model used. Because the GnRH-induced signaling network described in clonal gonadotrope cell lines ({alpha}T3-1 and LβT2 cells) and pituitary cells in primary culture encompasses a relatively large number of signaling intermediates, including the MAPK modules (31), we sought to expand our previous studies and examine a role for the ERK and JNK modules in the regulation of SgII promoter activity by GnRH. Interestingly, these studies revealed a probable role for GnRH signaling through the MAPK modules and the immediate early gene, ATF3.

Pharmacological studies provided important evidence that JNK and ERK signaling were required for GnRHa-regulated SgII promoter activity. Given that data interpretation in studies of some pharmacological perturbation of individual MAPK pathways can be limited due to specificity issues, we sought to use parallel approaches for the perturbation of the JNK module. Our studies using both the pharmacological inhibitor SP600125 and overexpression of the putative dominant-negative JNK module inhibitor, JIP, provided similar evidence that GnRHa-induced JNK activity was essential for SgII promoter regulation. From these studies, the combined pharmacological inhibition of GnRHa-induced JNK and ERK signaling together lead to a greater suppression of SgII promoter activity than either inhibitor alone. Equally clear is that simple activation of the ERK cascade alone by overexpression of Raf-CAAX was not sufficient to activate the SgII promoter. This supports the conclusion that ERK activation alone is not sufficient to support transcriptional activation of the SgII gene promoter; however, when combined with increased JNK activity, ERK signaling plays a prominent role in the regulation of SgII promoter activity by GnRH.

In parallel reconstitution studies, combined activation of JNK and ERK signaling using MEKK{Delta} was sufficient to activate the SgII gene promoter. The effects of MEKK{Delta} were reduced by mutation in the SgII CRE and pharmacological and genetic inhibition of the JNK and ERK modules. Collectively these studies provide parallel and overlapping lines of evidence that ERK and JNK activation are both sufficient and necessary for GnRHa-induced SgII promoter activity via the CRE.

The extent to which GnRHa-induced activation of MAPK modules regulate a GnRH-responsive gene network has focused largely on the role of the ERK module on transcriptional regulation of immediate early genes (IEGs) as well as late genes that depend on IEGs to serve as transcriptional regulators. An excellent example is the regulation of the IEG early growth response (Egr) factor 1. Egr1 mRNA increases dramatically within 1–2 h after a GnRH pulse in gonadotrope cell models and in vivo (47, 48, 49). The effects of GnRH on Egr1 gene transcription appear to require ERK activation via serum response elements within the Egr1 promoter (48, 50). Up-regulated Egr1 in turn is required for the expression of the LHβ gene promoter. The Egr1 knockout mouse is characterized by an infertility phenotype correlated with the specific loss of LHβ mRNA levels (51, 52). A similar mechanism has been defined for Egr1-dependent regulation of the dual-specificity MAPK phosphatase –2 (48, 53). In addition to ERK regulation of the LHβ subunit gene, other groups have provided evidence that the FSHβ subunit gene promoter may also be affected by ERK and JNK signaling (54). However, the effects of ERK and JNK signaling on FSHβ subunit promoter activity may account for only a relative small proportion of transcriptional activation induced by GnRH in the cell models examined. Likely the most important target of direct JNK signaling is the GnRH receptor (GnRHR) gene promoter (55). GnRH-mediated signaling to the GnRHR appears to require JNK (but not ERK) signaling via an activator protein-1 site within a composite enhancer present in the 5' flanking sequence of the GnRHR promoter (56). The common theme here is the MAPK-dependent regulation of IEGs supporting regulation of late gene expression. Our current studies provide important evidence that GnRH-induced SgII gene promoter activity also requires the combined JNK and ERK module likely regulating the key IEGs ATF3 and c-Jun.

After stimulation of the GnRHR, ATF3 and c-Jun were recruited to the SgII CRE (Fig. 6Go). This response was completely consistent with our previous studies examining the role of ATF3 as an IEG regulating the human glycoprotein hormone {alpha}-subunit gene promoter (33). In the studies examining the {alpha}-subunit, two consensus CREs accounted for the target of GnRH action, and specific siRNA-mediated knockdown of ATF3 demonstrated the requirement for the interaction between the dual CREs and ATF3. Based on these studies, it became important to us to determine whether GnRHa-induced CRE-dependent gene expression in general may be mediated by a mechanism(s) commonly requiring ATF3. Our current studies support this view. JNK and ERK signaling through ATF3 have historically been associated with stress responses from cellular insults such as exposure to UV light or treatment with other genotoxic stressors (57). Moreover, in some cases, ATF3 has been reported as a transcriptional repressor (58, 59, 60, 61), whereas in other cases ATF3 enhances gene transcription (62). For example, in differentiating chondrocytes, ATF3 is strongly up-regulated. The putative role of ATF3 in this system was shown to be repression of cyclin A and D1 gene transcription as well as a CRE-dependent reporter. Despite this repression, ATF3 was also shown to increase expression of a Runx2 reporter in the same model system (63). These observations support the conclusion that ATF3 is a versatile integrator of gene transcription that may in fact be cell type, stimulus, and binding partner specific. In our studies, overexpression of ATF3 alone was sufficient to induce SgII promoter activity (Fig. 6Go). Similar observations were made for overexpression of c-Jun. Whereas it is tempting to speculate that ATF3 or c-Jun alone (absent GnRH signaling) may be causal in these overexpression studies, it is not clear that this is the case. All of these studies were carried out in the presence of serum and its related growth factor complement such that overexpression of any one specific factor may be greatly influenced by growth factor signaling and modifications in transactivation potential via phosphorylation. Regardless, our siRNA-mediated ATF3 knockdown studies provide important evidence that the presence of ATF3 at the SgII CRE is essential to mediate the full impact of GnRH signaling on this gene promoter. Given the similarities between ATF3-dependent regulation of the SgII and human {alpha}-subunit gene promoter, it may be reasonable to speculate that CRE-dependent gene regulation in general may be subject to ATF3-dependent regulation.


   Acknowledgments
 
The authors thank Drs. Richard Maurer and Kyoon Kim for providing the rat SgII promoter deletion series. We thank Drs. Roger Davis, Linda Van Aelst, Pamela Mellon, and Carol Lange for providing valuable reagents. We are grateful for the valuable help of Ms. Xia Xu with statistical analyses.


   Footnotes
 
This work was supported by Grant HD34772 from the National Institute of Child Health and Human Development (to M.S.R.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 25, 2007

Abbreviations: ATF, Activating transcription factor; CRE, cAMP response element; CREB, CRE binding protein; Egr, early growth response; GnRHa, GnRH agonist; GnRHR, GnRH receptor; IEG, immediate early gene; JNK, c-Jun N-terminal kinase; JIP, JNK-interacting peptide; MEK, MAPK/ERK kinase; MEKK, MEK kinase; NE, nuclear extract; PKA, protein kinase A; SA, streptavidin agarose; SgII, secretogranin II; siRNA, small interfering RNA.

Received May 23, 2007.

Accepted for publication October 18, 2007.


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J. Xie, K. H. Allen, A. Marguet, K. A. Berghorn, S. P. Bliss, A. M. Navratil, J. L. Guan, and M. S. Roberson
Analysis of the Calcium-Dependent Regulation of Proline-Rich Tyrosine Kinase 2 by Gonadotropin-Releasing Hormone
Mol. Endocrinol., October 1, 2008; 22(10): 2322 - 2335.
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