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Endocrinology, doi:10.1210/en.2007-0024
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Endocrinology Vol. 148, No. 7 3507-3517
Copyright © 2007 by The Endocrine Society

Upstream Stimulatory Factor Regulates Constitutive Expression and Hormonal Suppression of the 90K (Mac-2BP) Protein

Antonino Grassadonia, Nicola Tinari, Bruno Fiorentino, Minoru Nakazato, Hyun-Kyung Chung, Cesidio Giuliani, Giorgio Napolitano, Stefano Iacobelli, T. Kevin Howcroft, Dinah S. Singer, Leonard D. Kohn on behalf of Consorzio Interuniversitario Nazionale per la Bioncologia

Cell Regulation Section (A.G., B.F., M.N., H.-K.C., C.G., G.N., L.D.K.), Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, and Experimental Immunology Branch (T.K.H., D.S.S.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; University "G. D’Annunzio" Foundation (A.G., N.T., B.F., C.G., G.N., S.I.), 66100 Chieti, Italy; and Edison Biotechnology Institute (L.D.K.), Department of Biomedical Sciences, Ohio University College of Osteopathic Medicine, Athens, Ohio 45701

Address all correspondence and requests for reprints to: Antonino Grassadonia, M.D., Ph.D., Dipartimento di Oncologia e Neuroscienze, Sezione di Oncologia Medica, Centro Servizi Biomedici (SEBI), via dei Vestini, 66100 Chieti, Italy. E-mail: grassadonia{at}unich.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously reported that hormones important for the normal growth and function of FRTL-5 rat thyroid cells, TSH, or its cAMP signal plus insulin or IGF-I, could transcriptionally suppress constitutive and {gamma}-interferon (IFN)-increased synthesis of the 90K protein (also known as Mac-2BP). Here we cloned the 5'-flanking region of the rat 90K gene and identified a minimal promoter containing an interferon response element and a consensus E-box or upstream stimulator factor (USF) binding site, which are highly conserved in both the human and murine genes. We show that suppression of constitutive and {gamma}-IFN-increased 90K gene expression by TSH/cAMP plus insulin/IGF-I depends on the ability of the hormones to decrease the binding of USF to the E-box, located upstream of the interferon response element. This site is required for the constitutive expression of the 90K gene. Transfection with USF1 and USF2 cDNAs increases constitutive promoter activity, attenuates the ability of TSH/cAMP plus insulin/IGF-I to decrease constitutive or {gamma}-IFN-increased 90K gene expression but does not abrogate the ability of {gamma}-IFN itself to increase 90K gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
90K (ALSO KNOWN as Mac-2BP) refers to a large oligomeric glycoprotein composed of 90-kDa subunits that has been identified in the culture fluid of human breast cancer cells (1). Supranormal serum levels of 90K have been found in patients with tumors or patients afflicted with HIV infection; in both cases elevated levels correlate negatively with prognosis (2, 3, 4, 5, 6, 7, 8).

The function of 90K is not known, although several lines of evidence support a role for the protein in the host immune defense. First, 90K is a member of a family of transmembrane or secreted glycoproteins containing a scavenger receptor, cysteine-rich domain (9, 10). Proteins containing a scavenger receptor, cysteine-rich domain are expressed by immunocompetent cells (B and T lymphocytes, macrophages) and are implicated in immune defense (9, 11, 12, 13). Second, in vitro generation of cytotoxic effector cells (natural killer and lymphokine-activated killer) has been observed after exposure of peripheral blood mononuclear cells (PBMCs) to 90K (9). Third, 90K increases cytokine production by PBMCs (9, 12) and accessory cells (12, 14). Finally, 90K increases major histocompatibility (MHC) class I antigen expression in human breast cancer cells (15).

A rat 90K was recently cloned (16) and found to be homologous to human 90K (9, 10) and to mouse adherent macrophage protein (also known as CyCap, Cyclophilin C associated protein) (17, 18). In rat thyroid FRTL-5 cells, 90K is increased by {gamma}-interferon (IFN), as in humans and mice, and acts in an autocrine or paracrine/exocrine manner to increase MHC class I (16). Hormones that regulate the growth and function of FRTL-5 cells, particularly TSH/cAMP plus insulin/IGF-I (19, 20, 21, 22, 23), coordinately decrease MHC class I and 90K expression (16).

The ability of TSH/cAMP plus insulin/IGF-I to decrease MHC class I (24) has been explained by a complex, interlocking array of trans factors and cis elements (25, 26, 27, 28, 29, 30). In contrast, the mechanisms involved in hormonal suppression of 90K are unknown.

Here we report the cloning of the 5' flanking region of the rat 90K gene and the identification of the critical regulatory element responsible for the suppression of 90K expression by TSH/cAMP plus insulin/IGF-I. We show that this element controls basal expression of 90K, is conserved in the human and mouse promoter, and corresponds to an E-box that is sensitive to regulation by the basic helix-loop-helix leucine zipper proteins, upstream stimulator factor (USF)-1 and -2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
Rat FRTL-5 thyroid cells (Interthyr Research Foundation, Baltimore, MD) were a fresh subclone (F1) with all properties described (31, 32). They were grown in 6H medium consisting of Coon’s modified F12 medium, 5% heat-treated, mycoplasma-free calf serum, 1 mM nonessential amino acids, and a six hormone mixture: bovine TSH (1 x 10–10 M), insulin (10 µg/ml), cortisol (0.4 ng/ml), transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml). Cells were fed every 2–3 d, were passaged every 7–10 d, were diploid, and were between their fifth and 25th passage. Their doubling time with TSH was 36 h; without TSH, they did not proliferate or exhibit most thyroid-specific functions. As noted, in some experiments cells have been maintained in 5H medium (no TSH). In other experiments forskolin (5 µM) and IGF-I (100 ng/ml) have been used instead of TSH and insulin, respectively.

Library screening
To clone the 5'-flanking region of the rat 90K, we used the Promoter Finder DNA Walking kit (CLONTECH, Palo Alto, CA). Briefly, the method uses a rat library of uncloned, adaptor-ligated genomic DNA fragments digested with five different restriction endonucleases. Long-distance PCR (33) was performed to synthesize the promoter region using a forward primer designed to match the adaptor sequence and a reverse primer to match the 90K-cDNA sequence spanning base +4 through +30.

DNA sequencing and sequence analysis
The longest amplified fragment was cloned into pCR 2.1 (Invitrogen, Portland, OR) and sequenced using the dideoxynucleotide chain termination method (34) and T7, M13 reverse, or site-specific synthetic oligonucleotide primers. Sequence alignments and comparisons with the human and mouse 5'-flanking regions (35, 36) were performed using Gene Works (IntelliGenetics, Inc., Mountain View, CA).

Identification of the transcription start sites in the 5'-flanking region
An uncloned library of adaptor-ligated double-strand cDNA from FRTL-5 was created using the Marathon cDNA amplification kit (CLONTECH). Briefly, total FRTL-5 cell RNA was used to synthesize the first-strand cDNA with Moloney murine leukemia virus reverse transcriptase; second-strand synthesis was performed as described by Gubler and Hoffman (37). The double-strand cDNA was then ligated to the Marathon cDNA adaptor and 5'-rapid amplification of cDNA ends (RACE) was performed using 10 pmol of the adaptor primer (5'-ACT CAC TAT AGG GCT CGA GCG GC-3') and 10 pmol of the gene-specific primer (5'-CGA GAA GAC ACC GAG GAG AGA CAC AAG-3'), which is complementary to nucleotides +4 to +30 of the rat 90K cDNA. The reaction mixture was sequentially incubated at 94 C (1 min), 60 C (2 min), and 72 C (2 min) for 35 cycles and the PCR products extended at 72 C for 7 min. The 5'-RACE products were analyzed on 1.8% agarose gels, ligated to pCR2.1, and sequenced as described above. Sequence alignments and comparisons were again performed using Gene Works (IntellliGenetics).

Oligonucleotides and plasmids
USF expression plasmids were those previously described (38, 39). To construct the rat 90K promoter-luciferase chimeric plasmids, 11 different fragments of the 90K 5'-flanking region, termed 3B (–2,605 to +30 bp), L2 (–2605 to –1834 bp), M2 (–2527 to –1810 bp), N7 (–2335 to –1810 bp), O2 (–2072 to –1772 bp), OP (–2013 to –1820 bp), P2 (–1963 to 1772 bp), R4 (–1874 to –1772 bp), 4B (–1769 to +30 bp), U3 (–951 to +30 bp), and L3 (–1769 to –952 bp) were generated by either PCR or digestion with restriction endonucleases from the cloned full-length 5' region and were subcloned into the pGL3 basic luciferase reporter vector (Promega, Madison, WI). Sequence numbers are based on defining the start of translation as +1. In the case of PCR, MluI and BglII were the 5' and 3' insertion sites, respectively.

Oligo I, Oligo II, and Oligo I-II define the region spanning from –1963 to –1912 bp in the minimal promoter; Oligo II is from –1963 to –1932 bp: 5'-AGC CTT GTC TGC AGC CAA CCC AGA GGC AGC C-3'; Oligo I is from –1937 to –1912 bp: 5'-GCA GCC TCC GTC ATG TGT TTT CTG GA-3'. CM3, CM2, CM1, TM1, TM2, and EM are two base substitutions of Oligo I: CM3, 5'-GCA aCa TCC GTC ATG TGT TTT CTG GA-3'; CM2, 5'-GCA GCa TCa GTC ATG TGT TTT CTG GA-3'; CM1, 5'-GCA GCC TCa GgC ATG TGT TTT CTG GA-3'; TM1, 5'-GCA GCC TCC GTC ATc TGT TTc CTG GA-3'; TM2, 5'-GCA GCC TCC GTC ATG TGT TTc aTG GA-3'; and EM, 5'-GCA GCC TCC GTC ATG gaT TTT CTG GA-3'. Lowercase, bold letters denote the substituted nucleotides.

The double-strand oligonucleotides used for EMSA were obtained by annealing each oligonucleotide with its complementary antisense strand.

The mutated oligonucleotides described above were used as primers to create P2 mutants (P2CM3, P2CM2, P2CM1, P2TM1, and P2TM2, respectively) according to a two-step PCR procedure (40). In the first step, two PCR products that overlap the mutated sequence were separately created by using the mutated single-strand oligonucleotides and their respective antisense counterparts as inside primers and the primers used to generate P2 as outside primers. Products were separated from excess primer, mixed, and reannealed to obtain recombinant molecules with the inside primer sequence overlapped. These products were 3' extended and used as template for a second round of PCR using the same primers used to generate P2. The mutated P2 fragments were subcloned into the pGL3 basic luciferase reporter vector (Promega); MluI and BglII were the 5' and 3' insertion sites, respectively.

Using the same site-specific PCR mutagenesis procedure (40), another P2 mutant with four base substitutions in the interferon response element (IRE) (–1912 to –1902 bp) was created. This construct, indicated as P2IREM, has the IRE sequence changed to tcAgCtGAAGCT. Lowercase, bold letters are the substituted nucleotides.

To verify that the region from –1963 to –1912 bp contained an important element for the activity of the 5'-flanking region of the rat 90K and to better match functional data with EMSA, Oligo I-II, Oligo II, and Oligo I, with and without mutations, were also subcloned into a pGL3 promoter luciferase reporter vector containing the Simian virus 40 (SV40) minimal promoter. Also in this case, MluI and BglII were the 5' and 3' insertion sites, respectively.

All the cloned inserts were analyzed in their entirety to check direction and sequence.

Transient expression
Transient transfections in FRTL-5 cells used a diethylaminoethyl procedure (41, 42). In brief, cells were grown in 6-well plates in 6H medium until 50–70% confluent. They were washed twice with 6H medium without serum and then incubated at 37 C for 1 h in 1 ml of the same medium containing 125 µg diethylaminoethyl-dextran and 2 µg of the pGL3 basic luciferase reporter gene or equivalent molar amounts of pGL3 containing the different rat 90K constructs with or without mutation. pSVGH (5 µg) was added to measure transfection efficiency. Cells were shocked for 3 min with 10% dimethyl sulfoxide in PBS at room temperature and then maintained 3–6 h in 6H medium containing 5% serum before being shifted to 5H medium with 5% serum but no TSH. After 24 h in 5H medium, cells were treated with TSH (1 x 10–10 M) and/or {gamma}-IFN (100 U/ml). Luciferase activity was assayed after 24 h.

In some experiments, cotransfections were performed adding 3 µg USF1, USF2, or the control vector, pSG5. In other experiments, the same procedure was used to transfect cells with 2 µg of the pGL3 promoter luciferase reporter vector or equivalent molar amounts of pGL3 promoter containing Oligo I-II or Oligo I with or without the different mutations, plus pSVGH. Cells were treated as above and luciferase activity assayed 24 h after TSH or {gamma}-IFN treatments.

Values were normalized for total cell protein and GH activity; these corrections did not change values more than 10%. The activity of the empty vector was considered as the control value unless otherwise noted.

Nuclear extracts
To prepare nuclear extracts, FRTL-5 cells were grown in complete 6H medium until 50–70% confluent. Cells were shifted to 5H medium with no TSH for 5 d and then stimulated with {gamma}-IFN (100 U/ml), TSH (1 x 10–10 M), or both for the times noted, usually 24 or 48 h. Nuclear extracts were prepared as described (43, 44) with minor modifications. In brief, cells were washed twice in PBS, scraped, centrifuged at 500 x g and resuspended in 5 pellet volumes of 0.3 M sucrose, 2% Tween 40 in buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride, 2 mg/ml leupeptin, and 2 mg/ml pepstatin A]. After vigorous pipetting, the cell lysate was overlaid on 1 ml 1.5 M sucrose in buffer A and centrifuged at 16,000 x g for 10 min at 4 C. The white nuclear pellet was then resuspended in 50 µl buffer B [10 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 2 mg/ml leupeptin, and 2 mg/ml pepstatin A] per 100 µl starting cell pellet, incubated for 20 min in ice with occasionally mixing, and centrifuged at 16,000 x g for 20 min at 4 C. The supernatant was collected and protein concentration determined by Bradford’s method (Bio-Rad Laboratories, Hercules, CA) with crystalline BSA as standard. The extracts were aliquoted and stored at –70 C.

EMSA
DNA probes were the oligonucletides described above. They were 5' labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) (45). Binding reactions, 20 µl in volume, included 5 µg nuclear extract, 2% glycerol, 50 ng/ml polydI-dC, and 2 mM DTT in binding buffer: 20 mM HEPES, 0.1% Nonidet P-40, 5 mM MgCl2, and 50 mM KCl. Where indicated, the noted concentrations (20 or 50 times) of unlabeled oligonucleotide competitors were added for 15 min at room temperature, before 50,000 cpm-labeled DNA probe was added to the reaction mixture. After a 15-min incubation at room temperature, samples were electrophoresed at 160 V on 5% native polyacrylamide gels in 1x Tris-borate EDTA at room temperature. Gels were dried and autoradiography performed.

Statistical significance
All experiment were repeated at least three times with different batches of cells. Values are the mean ± SD of these experiments where noted. Significance between experimental values was determined by two-way ANOVA.

Materials
Highly purified bovine TSH was obtained from the hormone distribution program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIDDK-bTSH I-1; 30 U/mg) or was a previously described preparation 26 ± 3 U/mg, homogeneous in the ultracentrifuge, about 27,500 in molecular weight, with the amino acid and carbohydrate composition of TSH (44). Rat {gamma}IFN was from Amgen (Thousand Oaks, CA); recombinant IGF-I was from the Fujisawa Pharmaceutical Co. (Osaka, Japan). Antibodies against USF1 and USF2 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and are highly specific for these transcription factors with no cross-reactivity (46). The source of other materials was the Sigma Chemical Co. (St. Louis, MO) unless otherwise noted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the 5'-flanking region of the rat 90K gene
A genomic DNA fragment containing 2605 bp of the 5' flanking region of the rat 90K gene was cloned and sequenced. Analysis of the nucleotide sequence reveals the presence of 11 leader binding protein-1 consensus motifs. A putative IRE is identified between –1913 and –1902 bp, assuming the ATG start codon as +1 (see Figs. 1Go and 2Go and text below). The sequence has been deposited in GenBank (DQ062745).


Figure 1
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  		FIG. 1. Transcriptional start sites identified by 5'-RACE. Using the procedure described in Materials and Methods, 5'-RACE identified the same terminating sequence region between –1874 and –1864 bp in six separate clones using an amplification primer, which is complementary to nucleotides 4–30 of the rat 90K cDNA: 5'-GCT CTT CTG TGG CTC CTC TCT GTG TTC-3'. In each case sequence, identity of the full-length 5'-flanking region was lost 5' to nucleotide –1864 and sequence terminated at nucleotide –1874. The intron that the 5'-RACE sequence bypassed is noted by the heavy bars between nucleotides –1729 and –21. Numbering of the full-length clone is relative to the translational start site, which is defined as +1. The putative IRE is noted between –1913 and –1902 bp.

 

Figure 2
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  		FIG. 2. Diagrammatic representation of the 5'-flanking regions of the rat, mouse, and human 90K genes (A) and a sequence comparison of a putative minimal promoter region flanking the IRE common to the three genes (B). Data from the mouse and human gene are derived published elsewhere (35 36 , respectively). Sequence numbers for the mouse and human gene are based on defining the major transcriptional start site as +1. Sequence numbers for the rat gene are based on defining the start of translation as +1; we used this convention because there was no single nucleotide as transcriptional start site and because it is common to define the translation start site as +1 in housekeeping-type genes without TATA or CAAT core promoter elements. In A the schematic representation shows the full-length 5'-flanking region of the three genes with the IRE (black box) close to the transcription start site and the putative minimal promoters (white box) upstream of the IRE. In contrast to the human gene, the rat and mouse genes have a large intron between the transcription start site and translation start site (ATG) of 2200 and 1707 bp, respectively. In B the minimal promoter sequences are compared. The IRE sequence is enclosed in a black box. Alignment analysis comparing the sequences of the three species showed that this region is the most conserved. The framed nucleotides are common to all three genes; an identity of the rat and mouse sequence is denoted by a dot; an identity between the rat and human sequence is noted by a star.

 
To identify the transcription start site of the rat 90K gene, eight different 5'-RACE products, subcloned into pCR2.1 vector, were analyzed for sequence. Six of them had nearly the same size and sequence; two smaller products were considered to result from premature termination of the reverse transcriptase. The 5' ends of the first six products clustered between –1874 and –1864 bp (Fig. 1Go), indicating the presence of a major transcriptional initiation site in this region. Additionally, a large intron of 1707 bp was identified between the transcriptional and translational start sites. The transcriptional start site has no typical TATA or CCAAT boxes, and it is not in a GC-rich region (Fig. 1Go). RNase protection assays confirmed these results (data not shown).

When the putative IREs of the rat, mouse, and human 90K 5'-flanking region are aligned (Fig. 2Go, A and B), the rat transcription start sites lie between those of mouse and human genes (Fig. 2BGo). A best-fit alignment of all three genes indicates a high degree of homology in a region of 110 bp extending from the start sites through –1952 (rat), –66 (mouse), and –99 bp (human) of the 5'-flanking region (Fig. 2Go). This region has been reported to be the minimal promoter of the mouse and human 90K genes (35, 36) and, as will be evident below, corresponds to the minimal functional promoter of the rat 90K as well.

Identification of a minimal promoter responsive to TSH/cAMP plus insulin/IGF-I and to {gamma}-IFN
Promoter activity exhibited by the full-length 2605-bp genomic fragment or 5' or 3' deletions thereof were measured by linking each fragment to a luciferase reporter gene (Fig. 3Go). After transfection with the different constructs, FRTL-5 cells in 5H medium containing insulin were treated with {gamma}-IFN and/or TSH. Although high levels of promoter activity were measured in the full-length clone with or without the intron (3B and L2, respectively, in Fig. 3Go) and in 5'-deletion mutants through –2072 bp (M2, N7, and O2 in Fig. 3Go), no effect was seen after exposure of the cells to either {gamma}-IFN or TSH. Conversely, deletion between –2072 and –2013 bp significantly decreased basal activity of the promoter in 5H medium but uncovered significant stimulation by {gamma}-IFN as well as suppression of constitutive or {gamma}-IFN-increased activity by TSH (OP and P2 in Fig. 3Go). As expected, deletions close to or 3' to the transcriptional start sites (R4, 4B, U3, L3 in Fig. 3Go), including those within the intron, resulted in a total loss of promoter activity. Similar results were obtained when forskolin or IGF-I was substituted for TSH and insulin, respectively (data not shown).


Figure 3
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  		FIG. 3. Promoter activity of 5'- or 3'-deletion constructs of the full-length rat 90K 5'-flanking region measured by transient transfection in FRTL-5 cells after 24-h stimulation with 1 x 10–10 M TSH or 100 U/ml {gamma}-IFN. FRTL-5 cells were grown in 6H medium to approximately 70% confluency in six-well plates and then transfected with 2 µg of the pGL3 basic luciferase reporter vector or the pGL3 vector containing the various 90K gene 5'-flanking regions diagrammatically presented on the left side of the figure; pSVGH was added to measure transfection efficiency. After transfection, cells were maintained in 6H medium for 3–6 h at which time 5H medium with insulin but no TSH was added to duplicate sets of cells. After 24 h, cells were stimulated with TSH (1 x 10–10 M), i.e. 6H medium, {gamma}-IFN (100 U/ml), or both. Cells were harvested 24 h later to measure GH and luciferase activity. Cell viability was 87.5% in all transfections. Luciferase activity was corrected for cell protein and GH activity; these corrections did not change values more than 10%. The luciferase activity of cells transfected with the pGL3 vector alone was considered as background and was similar to the R4, 4B, U3, and L3 construct activities. The data in the inset are expressed relative to the 5H control with insulin but no TSH or {gamma}-IFN. A single asterisk (*) denotes a significant increase in {gamma}-IFN-increased promoter activity over the 5H control (P < 0.01); two asterisks (**) denote a significant TSH-induced decrease in constitutive or {gamma}-IFN-increased promoter activity (P < 0.01). Data in both panels represent the mean ± SD of duplicate values determined in three separate experiments performed on different batches of cells.

 
Based on the activity of the P2 construct, a minimal promoter with {gamma}-IFN and TSH responsiveness could be defined as being located between –1963 and –1820 bp (Fig. 3Go, P2). This region includes the IRE (Figs. 2Go and 3Go) and corresponds to the minimal promoter defined by sequence homology among rat, human, and mouse genes (Fig. 2Go). Further analyses were focused on this region. We did not further study the region between –2072 and –2013 bp, despite our realization that this region contains an enhancer that negates responsiveness to {gamma}-IFN and TSH/insulin.

Different elements of the minimal rat 90K promoter control {gamma}-IFN increased and TSH/insulin decreased activities
In 5H medium, the constitutive activity of the P2-luciferase chimera was not affected by a 4-bp mutation in the IRE (Fig. 4Go, top, black bars, P2IREM vs. P2), but the ability of {gamma}-IFN to increase promoter activity was lost (Fig. 4Go, top, P2IREM vs. P2, gray bars). The ability of TSH to suppress constitutive promoter activity in the presence of insulin was unaffected by the IRE mutation (Fig. 4Go, top, P2IREM vs. P2, open bars), suggesting that a cis element important for this suppression was distinct from the IRE.


Figure 4
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  		FIG. 4. The {gamma}-IFN and TSH-responsive sites in the minimal rat 90K promoter are distinct. The P2 construct (in pGL3 basic) is depicted diagrammatically to include the IRE, the transcription start sites and luciferase reporter gene 3' to the IRE, and the region 5' to the IRE termed Oligo I-II, –1963 to –1912 bp. In the top, the IRE sequence is boxed to compare the wild-type construct (P2) with P2 construct with four-point mutations in the IRE created by PCR mutagenesis (P2 IREM). The Oligo I-II sequence inserted into a pGL3 luciferase vector with an SV40 promoter (pGL3 Promoter) is also diagrammatically presented. In the top panel, the luciferase activity of P2 and P2 IREM in 5H medium (with insulin) or in 5H medium plus 1 x 10–10 M TSH or 100 U/ml {gamma}-IFN is shown. Transfection conditions are the same as in Fig. 3Go. The ability of {gamma}-IFN to increase the activity of P2 is completely abolished in P2 IREM, but the effect of TSH to decrease the promoter activity is conserved. The basal promoter activity (5H) is also conserved. Data are normalized for protein and GH activity and are expressed in arbitrary units per milligram protein. In the bottom panel, the Oligo I-II sequence of the P2 construct, which does not include the IRE, was inserted in the pGL3 vector with the SV40 promoter and transfected into FRTL-5 cells exactly as was the P2 promoter above or in Fig. 3Go. Cells were maintained with insulin (5H medium), plus TSH or {gamma}-IFN, or both. Measurements of relative luciferase activity of the construct with or without the Oligo I-II insert are presented after protein and GH corrections. Data show that the ability of TSH plus insulin to significantly decrease constitutive promoter activity is retained but not the {gamma}-IFN-increased response. A single asterisk (*) denotes a significant increase in promoter activity induced by {gamma}-IFN. Two asterisks (**) denote a significant decrease in promoter activity induced by TSH in the presence of insulin (P < 0.01). Data in both panels represent the mean ± SD of values determined in duplicate from three separate experiments performed on different batches of cells.

 
Oligo I-II, corresponding to the P2 sequence upstream of the IRE, was inserted into a pGL3 vector with a SV40 promoter (Fig. 4Go, bottom). In 5H medium, the Oligo I-II construct displayed a significant constitutive activity relative to the control vector (Fig. 4Go, bottom). As expected, the construct was ineffective in the presence of {gamma}-IFN, whereas the addition of TSH was able to suppress constitutive promoter activity (Fig. 4Go, bottom), suggesting that the cis element important for this suppression was located upstream of the IRE. Experiments wherein 5 µM forskolin were used instead of TSH or 100 ng/ml IGF-I were used instead of insulin did not alter the data (data not shown).

To further define the site responsible for the suppressive effect of TSH on constitutive or {gamma}-IFN-increased 90K promoter activity, the Oligo I-II sequence was split into two parts: Oligo II (–1963 to –1932 bp) and Oligo I (–1937 to –1912 bp). Each was assayed for TSH suppression after insertion into the pGL3 promoter vector (Fig. 5Go). The Oligo I fragment retained significant constitutive promoter activity and responsiveness to TSH (Fig. 5AGo), whereas the Oligo II fragment was ineffective (data not shown). Mutations of Oligo I in the region between –1929 and –1917 bp (CM1 and TM1) resulted in the loss of constitutive promoter activity and TSH-responsiveness (Fig. 5BGo). Mutations upstream of –1929 bp had no effect (see below); mutations downstream of –1917 bp recovered from the losses observed in CM1 and TM1. Again, experiments wherein 5 µM forskolin were used for TSH or 100 ng/ml IGF-I substituted for insulin did not alter the data (data not shown).


Figure 5
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  		FIG. 5. The region spanning –1937 to –1912 bp of the rat 90K minimal promoter (Oligo I) contains the important element for TSH responsiveness. The regions between –1963 and –1912 bp (Oligo I-II) and –1937 and –1912 bp (Oligo I) were subcloned into the pGL3 promoter luciferase reporter vector containing an SV40 minimal promoter as diagrammatically depicted. Each was transfected into FRTL-5 cells using the same protocol as in Fig. 3Go and cells maintained in 5H medium with or without 1 x 10–10 M TSH or 100 U/ml {gamma}-IFN as described in Figs. 3Go or 4. In A the data show that Oligo I has a significant ability to increase promoter activity by comparison with transfectants with Oligo I-II and is responsive to TSH. In B the activity of the pGL3 promoter luciferase reporter vector containing an SV40 minimal promoter and Oligo I was compared with the same vector containing Oligo I with two mutated inserts: CM1 and TM1. The mutations are noted in the diagrammatic representation. The data are normalized for the pGL3 promoter activity without the insert (relative luciferase activity ± insert) when assayed in the same stimulatory conditions. Data in both panels represent the mean ± SD of values determined in duplicate from three separate experiments performed on three different batches of cells. The region of the IRE, downstream of –1913 bp, is noted.

 
To validate these data, the same mutations were included in the P2 construct. CM1 and TM1 mutations markedly decreased constitutive activity and eliminated both the TSH and {gamma}-IFN response (Fig. 6Go). Other mutations upstream of –1929 bp (CM3, CM2) had no effect on either activity. The mutation at –1917/–1916 bp, termed TM2 and located between the TM1 mutation and the IRE, partially restored constitutive activity and both {gamma}-IFN- and TSH/insulin-responsiveness. Interestingly, CM1 mutations were also able to decrease the promoter activity of the full-length construct (3B) (data not shown).


Figure 6
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  		FIG. 6. The region between –1929 and –1917 bp in the minimal 90K promoter is essential for constitutive as well as TSH plus insulin suppressive activity. Using PCR mutagenesis, two base substitutions were made in the P2 fragment: P2CM3, P2CM2, P2CM1, P2TM1, and P2TM2. These are diagrammatically presented in the top of the figure. Each was transfected into FRTL-5 cells as in Fig. 3Go and the cells maintained in 5H medium with insulin or 5H medium with 100 U/ml {gamma}-IFN or 1 x 10–10 M TSH as described. The luciferase activity was measured 24 h later and data expressed as arbitrary luciferase unit per milligram protein after normalization for GH activity. A single asterisk (*) denotes a significant increase in promoter activity over the control induced by {gamma}-IFN (P < 0.01); two asterisks (**) denote a significant decrease in promoter activity induced by TSH in the presence of insulin (P < 0.05). Three asterisks (***) denote a significant decrease in constitutive promoter activity (P < 0.01). Data represent the mean ± SD of duplicate values determined in three separate experiments performed on different batches of cells.

 
The results suggested the existence of an element important for the constitutive activity of the rat 90K minimal promoter in the region between –1929 and –1917 bp. This same element was also important for TSH suppression of constitutive or {gamma}-IFN-increased 90K activity and was distinct from the IRE.

An E-box cis element and USF transcription factors regulate 90K gene expression
We performed EMSA to evaluate the effects of the CM1, CM2, CM3, TM1, and TM2 mutations on trans factor binding to the region between –1929 and –1917 bp (Fig. 7AGo). A prominent protein/DNA complex was noted in incubations of radiolabeled Oligo I with nuclear extracts of cells maintained in 5H medium (Fig. 7AGo, lane 2). This complex was unaltered using oligonucleotides with the CM3 and CM2 mutations (Fig. 7AGo, lanes 3 and 4) but was nearly eliminated in incubations with CM1 and TM1 mutations (Fig. 7AGo, lanes 5 and 6) and only partially present in those with TM2 (Fig. 7AGo, lane 7).


Figure 7
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  		FIG. 7. Identification of a specific binding complex to the region between –1937 and –1912 bp of the rat 90K minimal promoter that seems to be important for constitutive expression of the 90K gene (A) and the ability of TSH/forskolin to decrease the formation of the complex (B). In A double-strand olignucleotides with the sequences presented on the bottom of the figure were radiolabeled and incubated with nuclear extracts from cells maintained in 5H medium containing insulin exactly as described in the protocols for measuring functional expression of the gene. Incubations were evaluated by EMSA, as described in Materials and Methods. In B a double-strand olignucleotide with the sequence between –1937 and –1912 bp (Oligo I, on the top of the figure) was radiolabeled and incubated with nuclear extracts from cells maintained in 5H medium containing insulin, exactly as described in the protocols for measuring functional expression of the gene (Fig. 3Go), or with TSH, 5 µM forskolin, or 100 ng/ml IGF-I substituted for the insulin. Incubations were evaluated by EMSA, as described in Materials and Methods. Noted incubations also contained a 50x concentration of the same unlabeled oligonucleotide (self) as a specific competitor or a 50x concentration of an oligonucleotide with an unrelated sequence (nonself): TCA GCC GCA ACA TTG TTG TTT TAT CA, –2052 to –2027 bp.

 
The formation of this complex was specific, being inhibited by unlabeled self but not by unrelated oligonucleotide (Fig. 7BGo, lane 3 vs. lane 4). More importantly, its formation was decreased in extracts from cells treated with TSH in the presence of insulin (Fig. 7BGo, lane 1 vs. lanes 2, 4, and 5). This result was duplicated when forskolin was used for TSH or when IGF-I was used for insulin (Fig. 7BGo, lane 8 vs. lanes 7 and 9 vs. lane 10, respectively).

The sum of these data suggested that this protein/DNA complex was important for constitutive 90K gene expression and was decreased by TSH/cAMP.

In reviewing the sequence of Oligo I, we observed a potential cAMP-responsive element, but the formation of the protein/DNA complex was not affected by the presence of an oligonucleotide with a cAMP-responsive element consensus (data not shown). We also noted a potential binding site for the foxO family of forkhead transcription factors (TGTTTTC), but mutations affecting the foxO-binding site in the TM2 construct ruled out any regulatory role of this element.

Additionally, we noted an E Box, CANNTG, at –1927 to –1922 bp. The formation of the complex was inhibited by an oligonucleotide with an E box consensus and no other sequence homology to Oligo I (Fig. 8AGo, EWT). The complex was not inhibited by an oligonucleotide with a mutation of two critical nucleotides within the E box (Fig. 8AGo, EM). P2 with the EM mutation had no promoter activity, as observed in the case of CM1 and TM1 mutations (data not shown).


Figure 8
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  		FIG. 8. Inhibition of the specific complex binding to the region between –1937 and –1912 bp of the rat 90K minimal promoter by an oligonucleotide with a consensus E box sequence (A); shift of the complex using specific antibodies against USF1 or USF2 (B); and basal promoter activity of the P2 or P2CM1 90K-luciferase chimera in the presence or absence of USF1 or USF2 cotransfections; and ability of USF1/USF2 expression vectors to increase the binding of the specific complex (C). In A a double-strand olignucleotide with the sequence of Oligo I, –1937 to –1912 bp, was radiolabeled and incubated with nuclear extracts from cells maintained in 5H medium containing insulin exactly as described in the protocols for measuring functional expression of the gene. Incubations included 20x concentrations (over labeled probe) of unlabeled oligonucleotides with a consensus E box (EWT) or mutated E box (EM). The E box in Oligo I and the other oligonucleotides is boxed. Gels were evaluated by EMSA, as described in Materials and Methods. In B EMSA was performed as in A after incubation with specific antibodies for USF1 or USF2. Normal serum was used as control. In C FRTL-5 cells were grown in 6H medium to approximately 70% confluency in six-well plates and then transfected with 2 µg of the pGL3 vector containing the luciferase chimera with the P2 (E-box wild type) or the P2CM1construct (E-box mutated) of the 90K gene, –1963 to –1772 bp (see Fig. 3Go or 6) along with plasmid containing full-length cDNAs encoding USF1 or USF2 as well as their control vector, pSG5. pSVGH was added to measure transfection efficiency. Cells were harvested 24 h later to measure GH and luciferase activity. Cell viability was 89.7% in all transfections. Luciferase activity was corrected for cell protein and GH activity; these corrections did not change values more than 10%. The luciferase activity of cells transfected with the pGL3 vector alone was considered as background and was the same as the R4, 4B, U3, and L3 constructs in Fig. 3Go. The asterisk (*) denotes a significant increase in promoter activity over the control in absence of pUSFs (P < 0.01); in D EMSA was performed incubating radiolabeled Oligo 1 with nuclear extract from cells transfected as in C with plasmid containing full-length cDNAs encoding USF1 or USF2 as well as their control vector, pSG5.

 
To determine the factors that specifically bind the 90K E-box, EMSAs were performed with antibodies to known E-box binding proteins added to the DNA binding reaction. Antibodies against basic helix-loop-helix zipper proteins, such as Myc, Max, and sterol regulatory element-binding protein-1 and antibodies against unrelated factors, such as activating transcription factor 2, c-jun, or p50 subunit of nuclear factor-{kappa}B, had no effect on either complex formation or complex mobility (data not shown). In contrast, when antibodies specific for USF1 and USF2 were included in EMSA, the native complex was eliminated and supershifted complexes appeared, whereas control nonimmune serum did not alter the mobility of the complex (Fig. 8BGo, lanes 2 and 3 vs. lane 1).

Cotransfecting FRTL-5 cells with the P2 construct, containing the wild-type E-box, or the P2CM1 construct, containing the mutated E-box, along with USF1 or USF2 expression vectors, the luciferase activity of the wild-type promoter, but not that of the mutated promoter was significantly increased by USF overexpression (Fig. 8CGo). Moreover, EMSA performed after incubation of Oligo I with nuclear extracts from FRTL5 cells transfected with USF1 or USF2 vectors showed a marked increase of the specific protein-DNA complex (Fig. 8CGo, lanes 3 and 4 vs. lanes 1 and 2).

The functional involvement of USF1 and USF2 in 90K gene regulation was further investigated evaluating the hormone effects on the P2 luciferase vector in FRTL-5 cells cotransfected with USF1/USF2 expression vectors. The addition of USF1 or USF2 to transfections enhanced constitutive expression of P2 eliminated its suppression by TSH/cAMP and attenuated the ability of TSH/cAMP to suppress {gamma}-IFN-increased P2 expression (Fig. 9Go).


Figure 9
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  		FIG. 9. Promoter activity of the P2 90K-luciferase chimera in the presence or absence of USF1 or USF2 cotransfections. FRTL-5 cells were cotransfected as in Fig. 8CGo and then maintained in 6H medium for 3–6 h at which time 5H medium with insulin but no TSH was added to duplicate sets of cells. After 24 h, cells were stimulated with TSH (1 x 10–10 M), i.e. 6H medium, {gamma}-IFN (100 U/ml), or both. Cells were harvested 24 h later to measure GH and luciferase activity. A single asterisk (*) denotes a significant increase in {gamma}-IFN-increased promoter activity over the 5H control (P < 0.01); two asterisks (**) denote a significant TSH-induced decrease in constitutive or {gamma}-IFN-increased promoter activity (P < 0.01). Three asterisks (***) denote a loss of TSH suppressed constitutive or {gamma}-IFN-increased promoter activity (P < 0.01). A filled circle represents a significant increase in constitutive promoter activity in medium with insulin (P < 0.01). Data in both panels represent the mean ± SD of duplicate values determined in three separate experiments performed on different batches of cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In a previous report we showed that in FRTL-5 cells expression of endogenous 90K is regulated by {gamma}-IFN and TSH/cAMP plus insulin or IGF-I (16). To investigate the molecular basis of transcriptional regulation of rat 90K by TSH, we cloned the 5'-flanking region of the rat 90K gene and analyzed its function and structure. Surprisingly, promoter activity exhibited by the full-length 2605-bp genomic fragment or 5' deletions through –2072 was very high, and no effect was seen after exposure of the cells to either {gamma}-IFN or TSH. Conversely, deletion between –2072 and –2013 bp significantly decreased basal activity of the promoter but uncovered significant stimulation by {gamma}-IFN as well as suppression by TSH. The hormonal regulation of the shorter fragment was thereby consistent with the reported regulation of the endogenous 90K both at mRNA and protein level (16). We focused our further investigation on this region, which functionally defines the minimal promoter, although we realized that the region between –2072 and –2013 might contain an enhancer that confers to the full-length construct and its 5' deletion fragments through –2072, an unusually high basal luciferase activity that covers responsiveness to {gamma}-IFN and TSH/insulin. The functional meaning of this enhancer will be investigated in a future work.

Sequence analysis showed that the identified rat 90K minimal promoter is homologous to that of the human and mouse 90K gene. As reported for the human and mouse (35, 36), the promoter from rat has no typical TATA or CCAAT boxes, and it is not in a GC-rich region. Additionally, all three genes show identical loci for the IRE in the minimal promoter, which is IFN responsive. Major differences are represented by a large intron separating transcriptional from translational start sites in the rat and the mouse genes but not the human one.

The rat minimal promoter contains a cis element upstream of the IRE, which displays the sequence of an E box, CANNTG, and binds USF1/USF2 transcription factors. These factors are important for constitutive expression of 90K. In fact, mutations within the E-box preventing binding of USFs completely abolished promoter activity, even in the presence of {gamma}-IFN, whereas transfection of FRTL-5 cells with USF1/USF2 increased both constitutive and {gamma}-IFN-induced activity of the 90K gene. Our data indicate that these cis/trans regulatory elements are implicated in the suppression of the 90K gene expression by TSH/cAMP in presence of insulin. In fact, this suppressive effect was associated with a reduced binding of USF1/USF2 to the E box, whereas overexpression of these trans factors counteracted the effect of TSH/cAMP on both constitutive and {gamma}-IFN-induced 90K expression. To the best of our knowledge, this is the first evidence of an implication of USFs in gene regulation by TSH/cAMP. Because the E-box is present in the human and mouse promoter as well, it is reasonable to presume that all three genes have similar control of constitutive 90K gene expression via this element.

USFs are ubiquitous transcription factors expressed in different tissues (47, 48), including thyroid gland (UniGene database Hs.414880). Consideration of the roles of USFs in regulating the immune response may provide a conceptual basis to interpret our findings. USFs are key regulators of genes implicated in both the humoral-antibody response mediated by B cell production of immunoglobulins and cell-mediated immune response involving cytotoxic and helper T lymphocytes (49). For example, USFs are known to promote transcription of {lambda}-Ig light chain (50); Ig J chain (51), which links IgM monomers; and pIgR (52), the polymeric Ig receptor that mediates transport of IgA and IgM across epithelia. Moreover, USFs are known to induce ß-2-microglobulin (53); Class II transactivator (54), which is required for MHC class II gene induction by interferon-{gamma}; and MHC class I genes (46). Given the important role of USFs in the immune response, it is perhaps not surprising that the 90K gene is regulated by these factors. In fact, evidence has been presented to show that 90K displays positive effects on host defense as well. For example, enhanced generation of cytotoxic effector cells (natural killer and lymphokine-activated killer) (9, 12) and T cell costimulation by secretion of IL-1, IL-6, and TNF-{alpha} by accessory cells have been observed after in vitro exposure of PBMCs to purified 90K (12).

Finally, 90K induces MHC class I expression (15, 16), and the concomitant suppression of both 90K and MHC class I by TSH has been suggested as a mechanism to preserve self-tolerance and prevent autoimmunity (16, 25, 26, 28). On the basis of these considerations, we suggest that induction of 90K by USFs may be another mechanism through which these transcription factors activate immune responses.

The role of 90K as an immune stimulator has been apparently challenged by the finding that the protein is overexpressed during infection by viruses. In particular, 90K serum levels have been found to be elevated in HIV-infected individuals, to correlate with plasma viral loads, and ultimately to be an independent predictor of evolution to full-blown AIDS (6, 7, 8). In our opinion, the induction of 90K expression by USF may represent a way to mechanistically justify these findings. In fact, the activation of USF by the kind of stress triggered by viral infection, which stimulates transcription of HIV long-terminal repeat, may also be responsible for the increase of 90K expression.

In sum, the results of the present study on the effect of TSH/cAMP on 90K transcription have uncovered a novel and important interaction of USF1 and USF2 with a conserved E box in the minimal promoter of the 90K gene. This observation not only is of relevance to the regulation of 90K gene expression but also may contribute to better understanding the biological function of the 90K glycoprotein.


   Acknowledgments
 
The sequence of the rat 90K promoter has been deposited in the GenBank database under accession no. DQ062745.


   Footnotes
 
This work was in part supported by grants from the Associazione Italiana per la Ricerca sul Cancro and the Consiglio Nazionale delle Ricerche.

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 19, 2007

Abbreviations: DTT, Dithiothreitol; IFN, interferon; IRE, interferon response element; MHC, major histocompatibility; PBMC, peripheral blood mononuclear cell; RACE, rapid amplification of cDNA ends; SV40, Simian virus 40; USF, upstream stimulator factor.

Received January 10, 2007.

Accepted for publication April 11, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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