Presence of activating transcription factor 4 (ATF4) in the porcine anterior pituitary

Molecular and Cellular Endocrinology 154 (1999) 151 – 159 www.elsevier.com/locate/mce

Presence of activating transcription factor 4 (ATF4) in the porcine anterior pituitary Yukio Kato a,*, Yoshiko Koike a, Kyoko Tomizawa a, Satoshi Ogawa a, Kohei Hosaka b, Susumu Tanaka b, Takako Kato a a

Biosignal Research Center, Institute for Molecular and Cellular Regulation, 3 -39 -15 Showa-machi, Maebashi, Gunma 371 -8512, Japan b School of Medicine, Gunma Uni6ersity, Maebashi, Gunma 371 -8512, Japan Received 8 February 1999

Abstract The two-hybrid system that identifies protein–protein interactions in a yeast expression system was used to investigate porcine anterior pituitary transcription factors. Four cDNA clones of a protein interacting with the leucine zipper domain of porcine cJun were obtained. Their nucleotide sequences revealed that they encode activating transcription factor 4 (ATF4). A full-length cDNA of porcine ATF4 was obtained by the polymerase chain reaction, and its deduced amino acid sequence showed 88 and 83% identity to human and mouse ATF4, respectively. Reverse transcription-polymerase chain reaction analysis of mRNAs prepared from 11 porcine tissues demonstrated that ATF4 is ubiquitous. Immunohistochemistry showed that ATF4 is present in the hormone producing cells of the anterior pituitary, but absent in some cells of the anterior pituitary. Further binding analysis revealed that ATF4 also interacts with itself and cFos. This evidence of ATF4 homodimerization, as well as heterodimerization with cJun and cFos in the anterior pituitary suggests a novel mechanism for the regulation of gene expression in this tissue. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Activating transcription factor 4; cJun; Transcription factor; Two-hybrid cloning; Porcine anterior pituitary; Gonadotropin

1. Introduction Transcription factors modulate gene expression by interaction with regulatory elements of the target gene (Maniatis et al., 1987; Mitchell and Tjian, 1989; Ptashne and Gann, 1990). One common type of transcription factor is a family of basic leucine zipper proteins, bZIP (Turner and Tjian, 1989), consisting of three major subfamilies, cJun/cFos, CREB/ATF and C/EBP. The consensus recognition sequences of CREB/ ATF and cJun/cFos are the cAMP-response element (CRE; TGACGTCA) and AP1 protein binding site (TGAC/GTCA), respectively. Although the symmetric sequence of a binding element is the preferred recognition site for homodimers of bZIP proteins, recent studies have shown that members of the bZIP family can also form various types of heterodimers. These heterodimers bind alternative asymmetric recognition se* Corresponding author. Tel: + 81-27-220-8866; fax: +81-27-2208897.

quences and are involved in novel regulation of target genes (Hai et al., 1989; Benbrook and Jones, 1990; Hai and Curran, 1991; Benbrook and Jones, 1994). Pituitary hormone genes are regulated through interactions between specific extracellular signals and their respective specific receptors that activate an intracellular signal cascade, which in turn activates several transcription factors. There are several putative response elements such as the CRE and AP1 protein binding sites in pituitary hormone genes (Kato et al., 1991; Peers et al., 1991; Steinfelder et al., 1992; Shepard et al., 1994; Kraus and Hollt, 1995), as well as in the gene for the pituitary transcription factor, Pit-1 (Chen et al., 1990). Thus, a thorough understanding of the transcription factors that regulate CRE and AP1 protein binding sites provides valuable information about the mechanisms that regulate hormone production in the anterior pituitary. We previously reported that a nuclear extract of the porcine anterior pituitary contains several DNA binding proteins capable of forming alternative heterodimers that recognize CRE or AP1 protein binding

0303-7207/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 9 9 ) 0 0 0 7 8 - 7

152

Y. Kato et al. / Molecular and Cellular Endocrinology 154 (1999) 151–159

sites (Ezashi et al., 1992). The abundant presence of factors binding to CRE and AP1 protein binding sites in gonadotropin producing cells has also been demonstrated by in situ DNA binding assays (Kato et al., 1993). Recently, we found that cJun and cFos are present in the porcine anterior pituitary and that their mRNA levels are modulated by hypothalamic factors (Chung et al., 1996, 1998). Thus, clarification of the role of transcription factors is indispensable to an understanding of the molecular mechanisms that regulate pituitary hormone genes. There is some evidence for the involvement of the bZIP family in the anterior pituitary. CREMo, a protein that acts to repress target gene transcription was cloned from aT3-1 cells established from mouse pituitary (Brindle et al., 1993). The presence of CREB (Bertherat et al., 1995) and JunB (Passegue et al., 1994) is supported by immunohistochemistry and Northern blot analysis, respectively. Additional information regarding transcription factors in the anterior pituitary is clearly required. The development of the two-hybrid system now allows the identification of partners in protein–protein interactions (Fields and Song, 1989; Chien et al., 1991). Since factors containing a bZIP domain can form heterodimers with other members of the family (Hai et al., 1989; Benbrook and Jones, 1990; Hai and Curran, 1991; Benbrook and Jones, 1994), this method provides a novel approach to search for unknown transcription factors. In the present study, we attempted to identify pituitary nuclear factors that interact with cJun, a protein whose presence in the anterior pituitary was previously confirmed (Chung et al., 1996). Two-hybrid expression cloning in yeast cells was performed using the leucine zipper domain of porcine cJun against a cDNA library from porcine anterior pituitary. A clone encoding the activator transcription factor 4 (ATF4 (Hai et al., 1988), TAXREB67 (Tsujimoto et al., 1991), CREB-2 (Karpinski et al., 1992) and C/ATF (Vallejo et al., 1993) are the same protein) was, for the first time, identified in the anterior pituitary.

2. Materials and methods

2.1. Yeast and culture medium Yeast strain YRG-2 (Mata ura3 -52 his3 -200 ade2 -101 lys2 -801 trp1 -901 leu2 -3 112 gal4 -542 gal80 -538 LYS2:: UASGAL1-TATAGAL1-HIS3 URA3::UASGAL4 17mer(x3) -TATACYC1-lacZ) was purchased from Stratagene (La Jolla, CA). The following media were used for propagation of the yeast cells: YPD medium (1% (w/v) yeast extract, 2% (w/v) polypeptone, 2% (w/v) glucose), and synthetic dropout medium (SD): (0.67% (w/v) yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI) and 2% (w/v) glucose containing the ap-

propriate dropout solution) (Sherman et al., 1979). SD media depleted of selective amino acid(s) were prepared: SD-Trp, SD-Leu, SD-His, SD-Trp-Leu and SD-Trp-Leu-His media (amino acids indicated at the upper right were depleted from the SD medium). Agar plates were prepared by adding 2% (w/v) agar to the media described above.

2.2. Construction of plasmids carrying genes of the target proteins The DNA fragment encoding amino acids numbers 270–331 that includes the leucine zipper region of porcine cJun (porcine cJun bZIP; amino acid numbers 280 to 303 (Chung et al., 1996)) was prepared by polymerase chain reaction (PCR), using the following primers: 5%-ATTCCCGGGGCGGAAAAGGAAGCTGGAGAGGATC-3% and 5%-GGCTGCAGGTCGACGGATCCTGCAACTGCTGCGTTAGCATG-3%. PCR was performed in a reaction mixture (100 ml) containing two required primers (50 pmol each) and 2 units Tth DNA polymerase (TOYOBO, Osaka, Japan) with 28 cycles of denaturation (94°C, 1 min), annealing (52°C, 1.5 min) and extension (72°C, 3 min) using a programmable thermal cycler (MJ Research, Watertown, MA). Amplified DNA was digested with SmaI and PstI, and ligated to SmaI and PstI digested pBD-GAL4 to make a hybrid protein with a GAL4 DNA binding domain. The clone pBD-GAL4/Jun was obtained by transformation of Escherichia coli, and the nucleotide sequence and the reading frame were confirmed by nucleotide sequence analysis. pBD-GAL4/Fos that contains the bZIP domain (amino acids numbers 165–193) of porcine cFos was also constructed.

2.3. Porcine anterior pituitary cDNA library A cDNA library from the porcine anterior pituitary was constructed in HybriZAP phage containing phagemid pAD-GAL4 using the HybriZAP Two-Hybrid cDNA Gigapack Cloning Kit (Stratagene, La Jolla, CA), according to the instruction manual. The library consisting of 2×106 independent plaques was amplified, and single stranded phagemids were excised with helper phage, followed by infection of E. coli XLOLR to obtain the double stranded phagemid cDNA library (pAD-GAL4/cDNAs), according to the instruction manual. A porcine anterior pituitary cDNA library in the lZAP vector (Stratagene) was also constructed and the double stranded phagemid cDNA library was obtained (pBluescript/cDNAs).

2.4. Screening of clones Co-transformation of yeast cells with pBD-GAL4/ Jun and pAD-GAL4/cDNAs was carried out using the

Y. Kato et al. / Molecular and Cellular Endocrinology 154 (1999) 151–159

YRG-2 yeast competent cell kit (Stratagene). YRG-2 competent cells (1 ml) were transformed with 10 mg each of pBD-GAL4/Jun and pAD-GAL4/cDNAs, incubated at 30°C for 30 min, followed by incubation at 42°C for 5 min, according to the instruction manual. The cells were then resuspended in SD-Trp-Leu medium and incubated at 30°C for 2 h in a rotary shaker at 250 rpm. The cells were collected and resuspended in SD-Trp-Leu-His medium. Selection of clones that activate HIS3 gene transcription was carried out by incubation on SD-Trp-Leu-His-agar plates at 30°C for 2 – 4 days.

2.5. b-Galactosidase assay Yeast cells were grown at 30°C for 2 days on a Whatman No. 50 filter paper (Whatman, Maidstone, England) which was placed on a SD-Trp-Leu-agar plate. The filter paper was frozen by soaking in liquid nitrogen and thawed at room temperature. b-Galactosidase activity was detected colorimetorically by incubation at 30°C overnight in 0.1 M sodium phosphate buffer, pH 7.0, containing 10 mM KCl, 1 mM MgSO4, 0.27% (v/v) b-mercaptoethanol and 0.33 mg/ml 5-bromo-4-chloro3-indolyl-b-D-galactopyranoside (X-Gal).

2.6. Western-blot analysis The protein extracts from yeast cells were prepared by the spheroplast method (Becker, 1994). Yeast cells were treated with 50 mM Tris – HCl buffer, pH 7.5, containing 5 mM MgCl2, 3 mM dithiothreitol and 500 mM sorbitol, for 15 min at 37°C, and 10 U of Zymolyase-100T (Seikagaku, Tokyo, Japan) was added. The cells were then incubated for 1 h at 37°C and sonicated three times each for 1 min at 50 W. The proteins were solubilized in 2.5% (w/v) sodium dodecyl sulfate (SDS) and separated on a 12.5% SDS-polyacrylamide gel (Laemmli, 1970), followed by electrophoretic transfer to a nitrocellulose membrane. The enzymelinked immunoassay was carried out by incubation at 4°C overnight with antibody raised against human TAXREB67 (since the TAXREB67 is identical to ATF4, this antibody is referred to as anti-ATF4 antibody in this paper) (Tsujimoto et al., 1991), anti-GAL4 activation domain antibody (CLONTECH Laboratories, Palo Alto, CA) or anti-GAL4 DNA binding domain antibody (CLONTECH Laboratories). This was followed by incubation with horseradish peroxidaseconjugated goat anti-rabbit or goat anti-mouse IgG. The peroxidase activity was detected using the Konica immunostain HRP kit (Konica, Tokyo, Japan).

2.7. Sequence analysis The DNA samples were prepared by the alkaline mini-preparation method. The fluorescence labeled dye-

153

terminator reaction was employed using the FS core system (Perkin-Elmer Cetus, Norwalk, CT), according to the instruction manual. The sequence was determined on a ABI PRISM 310 (Perkin-Elmer Cetus).

2.8. Re6erse transcription polymerase chain reaction (RT-PCR) Poly (A) enriched RNAs were prepared from various porcine tissues by the guanidium thiocyanate method (Chomczynski and Sacchi, 1987). Reverse transcription was carried out using the Ready-to-Go Kit (Pharmacia Biotech, Piscataway, NJ). An aliquot of the reverse transcribed cDNA corresponding to 12.5 ng mRNA was used in the PCR using the same conditions as described above.

2.9. Immunohistochemistry The porcine pituitary glands of a 3-year-old female were fixed in 10% formalin, dehydrated in ethanol, cleared in xylene and embedded in paraffin blocks. Five micrometer thick sections were autoclaved (Shin et al., 1991) and reacted with anti-ATF4 antibody at a dilution of 1:1000 for 30 min. After the sections were reacted with peroxidase-labeled goat anti-mouse IgG (EnVision, DAKO Japan Co.) at a dilution of 1:10 for 30 min, they were visualized with diaminobenzidine (DAB). Fluorescence detection for LHb was performed for the same section previously stained with anti-ATF4 antibody by serial reactions with rabbit anti-LHb serum (NIH, NIAMDD, AFP-2-11-27) at a dilution of 1:2000 for 3 h and fluorescein-conjugated goat anti-rabbit IgG (Organon Teknika, West Chester, MD) at a dilution of 1:200 for 2 h.

3. Results

3.1. Screening of the porcine pituitary cDNA library Co-transformation with pAD-GAL4/cDNAs and pBD-GAL4/Jun was carried out with YRG-2 competent cells at an efficiency of 104 colony forming units per 1 mg DNA. By repeated screenings of a total of 2×105 transformants, four clones (porcine anterior pituitary clones; pAP1-4) were obtained on SD-Trp-Leu-Hisagar plates. Phagemid DNAs of these four clones were amplified in E. coli cells and re-transformed into yeast cells together with pBD-GAL4/Jun. Co-transformants were obtained by incubation on SD-agar plates with or without essential amino acids. They showed no requirement of tryptophane, leucine or histidine (Fig. 1a). On the other hand, the transformation of yeast cells with pAP1-4 DNAs alone did show amino acid requirement of tryptophane and histidine (data not

154

Y. Kato et al. / Molecular and Cellular Endocrinology 154 (1999) 151–159

shown). This indicates that prototrophs of the transformed yeast cells had arisen by protein – protein interaction between the products of the pAP-cDNA clone and pBD-GAL4/Jun, and the resulting activation of the HIS3 reporter gene.

3.2. b-Galactosidase acti6ity To confirm the activation of the b-galactosidase reporter gene, four yeast transformants that harbored pBD-GAL4/Jun and/or pAP1-4 were grown on filter papers on SD-Trp-Leu-agar plates and then incubated with X-Gal. Those colonies turned blue in color due to the activation of the b-galactosidase gene (Fig. 1b). Transformants harboring pAP1-4 or pBD-GAL4/Jun alone did not show b-galactosidase activity (data not shown). These results indicated that pAP1-4 clones encoded molecules capable of interacting with the leucine zipper of porcine cJun.

3.3. Nucleotide sequence of cDNAs pAP1-4 cDNAs were amplified in E. coli cells and the sizes of the cDNA inserts were analyzed (data not shown). The lengths of the cDNA inserts were each less than 900 base pairs (bp). The nucleotide sequence of the four clones indicated that they contained the same sequence, and that the 5% region of the pAP1 clone was 100 bp longer than the others. A homology search with the nucleotide sequence revealed that the pAP clones had high homology to human ATF4 (Hai et al., 1989), including its cognates human T-cell leukemia TAXREB67 (Tsujimoto et al., 1991), human Jurkat CREB2 (Karpinski et al., 1992) and 3T3-L1 mouse adipocyte C/ATF (Vallejo et al., 1993).

Since none of the pAP clones contained the initiation codon, the full length sequence was obtained by PCR from a pBluescript porcine pituitary cDNA library, using a T3 primer and a primer containing the stop codon (nucleotide number (n.n.) 1285–1266). The amplified DNA was ligated to Bluescript vector and the nucleotide sequence was determined. Fig. 2 shows the composite sequence of 1399 bp encoding 348 amino acids determined for pAP1-4 and the PCR product. The deduced amino acid sequence showed the presence of a typical leucine zipper consisting of five leucine repeats every seven amino acid residues and an adjacent basic region at the carboxyl terminus. When the deduced amino acid sequence was compared to those of human and mouse ATF4 (Fig. 3), there was definite homology among the three sequences, although some deletions/insertions were present. The amino acid sequence had 88.8 and 85.6% identity to human and mouse ATF4, respectively. The bZIP region showed a high level of conservation.

3.4. Western blot analysis Western blot analysis was performed on proteins prepared from yeast transformed with pBD-GAL4/Jun and pAP-1 to identify fusion proteins. The monoclonal antibodies raised against the GAL4 DNA binding domain or the GAL4 activation domain each reacted with a single protein of 30 and 60 kDa (Fig. 4), respectively. These sizes coincided with the expected sizes of the fusion proteins of the GAL4 DNA binding domain and cJun leucine zipper, and of the GAL4 activation domain and a coding length of the pAP-1 insert. Western blot analysis using anti-ATF4 gave a positive band of about 60 kDa, the same size as that identified by the anti-GAL4 activation domain antibody.

3.5. Interaction of porcine ATF4 with porcine cJun and cFos

Fig. 1. Two-hybrid assay results of amino acid requirements (a) and b-galactosidase activity (b). The amino acid requirements, which are indicated in the figure, and b-galactosidase activity of transformants grown on SD-Trp-Leu-agar plates were examined. The transformants contained pAP1 and pBD-GAL4/Jun (1), pAP2 and pBD-GAL4/Jun (2), pAP3 and pBD-GAL4/Jun (3), pAP4 and pBD-GAL4/Jun (4), pBD-GAL4/p53 and pAD-GAL4/pSV40 used as positive control (5), pAD-GAL4 and pBD-GAL4 (6), pBD-GAL4 (7), and pAD-GAL4 (8).

To investigate the interactions of porcine ATF4 with porcine cJun and cFos, DNA sequences coding for the leucine zipper region of each protein were synthesized by PCR and constructed in yeast expression vectors to obtain pBD-GAL4/ATF4, pADGAL4/Jun and pAD-GAL4/Fos. Then the pBDGAL4/ATF4, together with pAD-GAL4/Jun or pADGAL4/Fos, were expressed in yeast cells and selected on SD-Trp-Leu-His-agar plates. Both transformations gave activation of the HIS3 gene as well as the bgalactosidase gene (data not shown), indicating that porcine ATF4 can interact with not only porcine cJun, but also porcine cFos.

Y. Kato et al. / Molecular and Cellular Endocrinology 154 (1999) 151–159

155

Fig. 2. Nucleotide sequence of porcine ATF4. The nucleotide sequence was determined by the analysis of clones obtained by two-hybrid cloning and PCR products. The deduced amino acid sequence is indicated below the nucleotide sequence. The 5% ends of the pAP1-4 clones corresponded to nucleotide numbers 408, 478, 478 and 520, respectively.

3.6. Localization and expression of ATF4 RT-PCR on 11 porcine tissues was performed to examine the tissue distribution of ATF4. Oligonucleotide primers, corresponding to n.n. 678-702 and n.n. 1089-1065 in Fig. 2, were designed and PCR was car-

ried out on cDNAs prepared from the same amount of poly(A) enriched RNAs from cerebrum, cerebellum, hypothalamus, anterior pituitary, posterior lobe, heart, liver, pancreas, kidney, spleen and testis (Fig. 5). The results demonstrated that porcine ATF4 is present in all tissues examined.

156

Y. Kato et al. / Molecular and Cellular Endocrinology 154 (1999) 151–159

Fig. 3. Comparison of the amino acid sequences of ATF4s. The amino acid sequence of porcine ATF4 was compared with those of human (Tsujimoto et al., 1991; Karpinski et al., 1992) and mouse (Vallejo et al., 1993). Each amino acid identical to that of porcine ATF4 is indicated by a hyphen (-) and the open spaces represent gaps to optimize homology. Repeats of leucine residues are indicated above the sequence (*).

3.7. Immunohistochemistry of ATF4 Immunostaining with anti-ATF4 antibody showed that positive cells were present and scattered throughout the whole anterior pituitary (data not shown). Immunoreactivity with anti-ATF4 was located in the nuclei of pituitary cells (closed arrowheads in Fig. 6a). Interestingly, some of the cells (open arrowhead in Fig. 6a) did not react with the antibody. Double immunostaining using antibodies for anti-ATF4 (Fig. 6b) and anti-LHb, as an example of a typical pituitary hormone, (Fig. 6c) was performed to look for expression of ATF4 in pituitary hormone producing cells. The results showed immunoreactive cells with both antibodies. When individual immunoreactive cells were examined, some of the cells reactive to the anti-LHb antibody were also reactive to the anti-ATF4 antibody (indicated by an arrow), suggesting that ATF4 and LH can be present in the same cells. In addition, some cells reacted

only to anti-ATF4 antibody (arrowheads in Fig. 6b and c).

Fig. 4. Western blot analysis of fusion proteins. Proteins prepared from yeast transformants were separated and transferred to nitrocellulose membranes and enzyme-linked immunoreaction was performed using the antibodies indicated. Lanes 1 and 2: protein samples from yeast YRG-2 cells harboring both pBD-GAL4/Jun and pAP1 and untransformed yeast YRG-2 cells, respectively.

Y. Kato et al. / Molecular and Cellular Endocrinology 154 (1999) 151–159

Fig. 5. The presence of porcine ATF4 in porcine tissues. Poly(A)-enriched fractions from porcine tissues were subjected to RT-PCR using specific primers and the products were analyzed. Molecular mass markers (M) are indicated on the left. Lanes: 1, cerebrum; 2, cerebellum; 3, hypothalamus; 4, anterior pituitary; 5, posterior lobe; 6, heart; 7, liver; 8, pancreas; 9, kidney; 10, spleen; and 11, testis.

Fig. 6. Immunohistochemistry of ATF4 in the porcine anterior pituitary. (a) Nuclei immunoreactive and non-reactive for anti-ATF4 antibody are indicated by closed and open arrowheads, respectively. Double immunostaining was carried out with anti-ATF4 antibody (b) and anti-LHb antibody (c). The cells reactive to both antibodies are indicated by arrows. The arrowheads indicate the cells reactive to anti-ATF4 antibody, but not to anti-LHb antibody.

4. Discussion This study has addressed to identify novel transcription factors interacting with cJun in the porcine anterior pituitary using the yeast two-hybrid system. The result is, to the best of our knowledge, the first to demonstrate the presence of the transcription factor ATF4 in this tissue. As cJun, a dimerization partner of ATF4, is also present in the porcine anterior pituitary (Chung et al., 1996), the present data suggests that heterodimerization between dissimilar transcription fac-

157

tors may be a novel mechanism by which gene expression in the anterior pituitary is regulated. In a previous study, we demonstrated that several molecular species of transcription factor bind to CRE and/or AP1 protein binding sites by forming unique heterodimers with different partners, and that these are present in the porcine anterior pituitary (Ezashi et al., 1992). Furthermore, heterodimerization among the transcription factors that recognize pituitary hormone genes has been postulated to occur by several investigators (Hoeffler et al., 1989; Habener, 1990). It is noteworthy that such multifarious dimerizations result in a novel diversity of recognition sequences, and thus an increase in the number of target genes (Habener, 1990; Lamb and McKnight, 1991; Miner and Yamamoto, 1991). It will, therefore, be important to obtain further information about the transcription factors in the anterior pituitary in order to understand the molecular basis underlying the mechanisms that regulate hormone genes. ATF4 was originally cloned from HeLa and human osteosarcoma MG63 cells (Hai et al., 1989). It binds preferentially to the CRE, in contrast to members of the cJun/cFos family and another bZIP protein subfamily which bind preferentially to the AP1 protein binding site. The cDNAs of TAXREB67 from T-cell leukemia cells (Tsujimoto et al., 1991), CREB-2 from Jurkat T-cells (Karpinski et al., 1992), C/ATF from mouse 3T3-L1 cells (Vallejo et al., 1993) and porcine pituitary ATF4 have been cloned and can be regarded as the same protein or homologue. The common transcription factors, cJun and cFos, which were identified in the anterior pituitary in a previous study (Chung et al., 1996), form heterodimers known as AP1 proteins that bind to AP1 protein binding sites. Both cJun and cFos are also known to form heterodimers with other transcription factors belonging to the ATF/CREB family, generating diverse recognition sequences (Habener, 1990; Hai and Curran, 1991; Lamb and McKnight, 1991; Miner and Yamamoto, 1991). We previously observed that hypothalamic factors modulate the pituitary transcription factors cJun and cFos (Chung et al., 1996, 1998). It is noteworthy that pituitary genes expressed in the anterior pituitary (Kato et al., 1991; Peers et al., 1991; Steinfelder et al., 1992; Shepard et al., 1994; Kraus and Hollt, 1995) as well as the gene for the pituitary specific transcription factor Pit-1 (Chen et al., 1990), are modulated through 5% upstream CRE and/or AP1 protein binding sites. Double immunostaining (Fig. 6b and c) demonstrated that ATF4 is clearly present in gonadotropin producing cells as well as other pituitary cells, suggesting that ATF4 may regulate several genes in the pituitary cells. We previously observed that pituitary nuclear factors, which bind to CRE and/or AP1 protein binding sites, form heterodimers in the anterior

158

Y. Kato et al. / Molecular and Cellular Endocrinology 154 (1999) 151–159

pituitary (Ezashi et al., 1992; Kato et al., 1993). The evidence that ATF4 is present in the porcine pituitary and forms heterodimers with cJun and cFos suggests another possible mechanism by which ATF4 may regulate gonadotropin subunit genes, through the formation of a homodimer or heterodimers with cJun, cFos and/ or other members of the bZIP family. Although the exact function of ATF4 in the anterior pituitary is not yet clear, there are several interesting reports of ATF4 function in other cells and tissues. ATF4 (CREB2) was reported to be involved in the negative regulation of transcription through binding to CRE in monkey CV-1 cells (Karpinski et al., 1992). In contrast, heterodimerization of ATF4 (C/ATF) with C/EBPb, which itself binds to the CAAT box and related enhancer motifs (Williams et al., 1991), resulted in a marked increase in transcriptional activity through binding to CREs (Vallejo et al., 1993). Furthermore, ApCREB1 and ApCREB2, which were cloned from an Aplysia central nervous system cDNA library, are homologous to mammalian CREB and ATF4, respectively (Bartsch et al., 1995). It is noteworthy that ApCREB2 alone activates CRE-mediated transcription, but represses it in the presence of ApCREB1, showing that the function of ApCREB2 changes depending on its dimerization partner. Therefore, it is possible that ATF4 and its cognates participate in a versatile mechanism that modulates transcription. More recently, several interesting reports supporting these versatile functions have been accumulating. ATF4 mediated activation of human T-cell leukemia virus by interacting with Tax transactivator (Reddy et al., 1997). However, ATF4 also suppressed the elevated promoter activity of the rat cyclin gene with ATF2-JunD dimer (Shimizu et al., 1998), and similarly suppressed both transcription of the cHa-ras oncogene and cellular transformation in NIH3T3 fibroblasts (Mielnicki et al., 1996). A new type of heterodimerization was recently described, in which ZIP kinase, a novel serine/threonine kinase which mediates apoptosis, requires ATF4 for the activation of kinase activity (Kawai et al., 1998). Thus, heterodimerization of ATF4 provides versatile and important biological functions in gene regulation. RT-PCR analysis of the tissue distribution of ATF4 (Fig. 5) suggested that ATF4 is expressed ubiquitously in porcine tissues. This result is consistent, in part, with those from mouse (Karpinski et al., 1992) and rat (Vallejo et al., 1993) tissues. The present immunohistochemistry demonstrated (Fig. 6) that some of the pituitary cells lack ATF4, indicating that ATF4 is not a prerequisite for these cells. An alternative explanation is that the appearance of ATF4 in some pituitary cells may depend on the cell function. However, this remains to be clarified in the future. The many studies of ATF4 suggest that it participates in versatile biological functions through interac-

tion with other cognates, with such important functions as gene activation, co-activation, and kinasion activation. The finding that ATF4 is present in the anterior pituitary provides new insight into gene regulation of pituitary hormones, although further elucidation of the function of ATF4 in the pituitary is clearly required.

Acknowledgements We wish to thank Drs K. Shimotono and A. Tsujimoto for providing the anti-TAXREB67 antibody. This study was supported, in part, by grant-in-aid from the Ministry of Education, Science, Sports and Culture (Nos. 02640578 and 06454019) to YK.

References Bartsch, D., Ghirardi, M., Skehel, P.A., et al., 1995. Aplysia CREB2 represses long-term facilitation: Relief of repression converts transient facilitation into long-term functional and structural change. Cell 83, 979 – 992. Becker, D.M., 1994. Manipulation of yeast genes. In: Current Protocols in Molecular Biology, vol. 2. Wiley, Chichester, pp. 13.17.11 – 13.17.10. Benbrook, D.M., Jones, N.C., 1990. Heterodimer formation between CREB and JUN proteins. Oncogene 5, 295 – 302. Benbrook, D.M., Jones, N.C., 1994. Different binding specificities and transactivation of variant CRE’s by CREB complexes. Nucleic Acids Res. 22, 1463 – 1469. Bertherat, J., Chanson, P., Montminy, M., 1995. The cyclic adenosine 3%,5%-monophosphate-responsive factor CREB is constitutively activated in human somatotroph adenomas. Mol. Endocrinol. 9, 777 – 783. Brindle, P., Linke, S., Montminy, M., 1993. Protein-kinase-A-dependent activator in transcription factor CREB reveals new role for CREM repressors. Nature 364, 821 – 824. Chen, R., Ingraham, H.A., Treacy, M.N., Alert, V.R., Wilson, L., Rosenfeld, M.G., 1990. Autoregulation of pit-1 gene expression mediated by two cis-active promoter elements. Nature 346, 583– 586. Chien, C.-T., Bartel, P.L., Sternglanz, R., Fields, S., 1991. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA 88, 9578 – 9582. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by guanidium thiocyanate – phenol – chloroform extraction. Anal. Biochem. 162, 156 – 159. Chung, H.-O., Kato, T., Kato, Y., 1996. Molecular cloning of c-jun and c-fos cDNAs from porcine anterior pituitary and their involvement in gonadotropin-releasing hormone stimulation. Mol. Cell. Endocrinol. 119, 75 – 82 Errata 120, 113 – 114. Chung, H.-O., Kato, T., Tomizawa, K., Kato, Y., 1998. Molecular cloning of pit-1 cDNA from porcine anterior pituitary and its involvement in pituitary stimulation by growth hormone-releasing factor. Exp. Clin. Endocrinol. Diabetes 106, 203 – 210. Ezashi, T., Kato, T., Wakabayashi, K., Kato, Y., 1992. Presence of nuclear factors bound to both cAMP-responsive element and AP1 factor binding site in the porcine anterior pituitary. Biochem. Biophys. Res. Commun. 188, 170 – 176. Fields, S., Song, O.-K., 1989. A novel genetic system to detect protein – protein interactions. Nature 340, 245 – 246.

Y. Kato et al. / Molecular and Cellular Endocrinology 154 (1999) 151–159 Habener, J.F., 1990. Cyclic AMP response element binding proteins: a cornucopia of transcription factors. Mol. Endocrinol. 4, 1087 – 1094. Hai, T., Curran, T., 1991. Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc. Natl. Acad. Sci. USA 88, 3720–3724. Hai, T., Liu, F., Allegretto, E.A., Karin, M., Green, M.R., 1988. A family of immunologically related transcription factors that includes multiple forms of ATF and AP-1. Genes Dev. 2, 1216 – 1226. Hai, T., Liu, F., Coukos, W.J., Green, M.R., 1989. Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes Dev. 3, 2083 – 2090. Hoeffler, J.P., Deutsch, P.J., Lin, J.C., Habener, J.F., 1989. Distinct adenosine 3%,5%-monophosphate and phorbol ester-responsive signal transduction pathways converge at the level of transcriptional activation by the interactions of DNA-binding proteins. Mol. Endocrinol. 3, 868 – 880. Karpinski, B.A., Morle, G.D., Huggenvik, J., Uhler, M.D., Leiden, J.M., 1992. Molecular cloning of human CREB-2: an ATF/CREB transcription factor that can negatively regulate transcription from the cAMP response element. Proc. Natl. Acad. Sci. USA 89, 4820 – 4824. Kato, Y., Ezashi, T., Kato, T., 1991. The gene for the common a subunit of porcine glycoprotein hormone. J. Mol. Endocrinol. 7, 27– 34. Kato, Y., Kato, T., Ezashi, T., Inoue, K., 1993. In situ binding for detection of proteins bound to regulatory elements. Biochem. Biophys. Res. Commun. 195, 963–968. Kawai, T., Matsumoto, M., Takeda, K., Sanjo, H., Akira, S., 1998. ZIP kinase, a novel serine/threonine kinase which mediates apoptosis. Mol. Cell. Biol. 18, 1642–1651. Kraus, J., Hollt, V., 1995. Identification of a cAMP-response element on the human proopiomelanocortin gene upstream promoter. DNA Cell Biol. 14, 103–110. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lamb, P., McKnight, S.L., 1991. Diversity and specificity in transcriptional regulation: the benefits of heterotypic dimerization. Trend. Biochem. Sci. 16, 417–422. Maniatis, T., Goodbourn, S., Fischer, J., 1987. Regulation of inducible and tissue-specific gene expression. Science 236, 1237 – 1245. Mielnicki, L.M., Hughes, R.G., Chevray, P.M., Pruitt, S.C., 1996. Mutated Atf4 suppresses c-Ha-ras oncogene transcript levels and cellular t ransformation in NIH3T3 fibroblasts. Biochem. Biophys. Res. Commun. 228, 586–595. Miner, J.N., Yamamoto, K.R., 1991. Regulatory crosstalk at composite response elements. Trend. Biochem. Sci. 16, 423–426.

.

159

Mitchell, P.J., Tjian, R., 1989. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245, 371 – 378. Passegue, E., Laverriere, J.N., Gourdji, D., 1994. Thyrotropin-releasing hormone stimulates in parallel jun B and c-fos messenger ribonucleic acids in GH3B6 pituitary cells: comparison with PRL secretion. Mol. Cell. Neurosci. 5, 109 – 118. Peers, B., Monget, P., Nalda, M.A., et al., 1991. Transcriptional induction of the human prolactin gene by cAMP requires two cis-acting elements and at least the pituitary-specific factor Pit-1. J. Biol. Chem. 266, 18127 – 18134. Ptashne, M., Gann, A., 1990. Activators and targets. Nature 346, 329 – 331. Reddy, T.R., Tang, H., Li, X., Wong-Staal, F., 1997. Functional interaction of the HTLV-1 transactivator Tax with activating transcription factor-4 (ATF4). Oncogene 14, 2785 – 2792. Shepard, A.R., Zhang, W., Eberhardt, N.L., 1994. Two CGTCA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells. J. Biol. Chem. 269, 1804 – 18014. Sherman, F., Fink, G.R., Lawrence, C.W., 1979. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Shimizu, M., Nomura, Y., Suzuki, H.E.I., et al., 1998. Activation of the rat cyclin A promoter by ATF2 and Jun family members and its suppression by ATF4. Exp. Cell Res. 239, 93 – 103. Shin, R.-Y., Iwaki, T., Kitamoto, T., Tateishi, J., 1991. Hydrated autoclave pretreatment enhances TAU immunoreactivity in formalin-fixed normal and Alzheimer’s disease brain tissues. Lab. Invest. 64, 693 – 702. Steinfelder, H.J., Radovick, S., Mroczynski, M., et al., 1992. Role of a pituitary-specific transcription factor (pit-1/GHF-1) or a closely related protein in cAMP regulation of human thyrotropin-beta subunit gene expression. J. Clin. Invest. 89, 409 – 419. Tsujimoto, A., Nyunoya, H., Morita, T., Sato, T., Shimotono, K., 1991. Isolation of cDNAs for DNA-binding proteins which specifically bind to a tax-responsive enhancer element in the long terminal repeat of human T-cell leukemia virus type I. J. Virol. 65, 1420 – 1426. Turner, R., Tjian, R., 1989. Leucine repeats and an adjacent DNA binding domain mediate the formation of functional cFos-cJun heterodimers. Science 243, 1689 – 1694. Vallejo, M., Ron, D., Miller, C.P., Habener, J.F., 1993. C/ATF, a member of the activating transcription factor family of DNAbinding proteins, dimerizes with CAAT/enhancer-binding proteins and directs their binding to cAMP response elements. Proc. Natl. Acad. Sci. USA 90, 4679 – 4683. Williams, S.C., Cantwell, C.A., Johnson, P.F., 1991. A family of C/EBP-related proteins capable of forming covalently linked leucine zipper dimers in vitro. Genes Dev. 5, 1553 – 1567.