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Endocrinology Vol. 145, No. 10 4728-4736
Copyright © 2004 by The Endocrine Society

The 90K Protein Increases Major Histocompatibility Complex Class I Expression and Is Regulated by Hormones, {gamma}-Interferon, and Double-Strand Polynucleotides

Antonino Grassadonia, Nicola Tinari, Bruno Fiorentino, Koichi Suzuki, Minoru Nakazato, Michele De Tursi, Cesidio Giuliani, Giorgio Napolitano, Dinah S. Singer, Stefano Iacobelli and Leonard D. Kohn

Cell Regulation Section (A.G., B.F., K.S., M.N., C.G., G.N., L.D.K.), Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; Section of Medical Oncology (A.G., N.T., B.F., M.D.T., S.I.), Department of Oncology and Neurosciences and Section of Endocrinology (C.G., G.N.), Department of Medicine and Science of Aging, Università degli Studi G. D’Annunzio, Faculty of Medicine and Surgery, 66100 Chieti, Italy; Experimental Immunology Branch (D.S.S.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; 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: Stefano Iacobelli, M.D., Dipartimento di Oncologia e Neuroscienze, Sezione di Oncologia Medica, SEBI, via dei Vestini, 66100 Chieti, Italy. E-mail: iacobell{at}unich.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Here we report the cloning of the rat 90K, a homolog of the mouse cyclophilin C-associated protein/mouse adherent macrophage and human 90K. The protein is constitutively expressed by FRTL-5 thyrocytes, and its levels are modulated by TSH, insulin/IGF-I, and {gamma}-interferon. Transfection of the cells with 90K cDNA or exposure to purified 90K resulted in a significant increase of the expression of major histocompatibility complex class I but not class II antigens. An increased expression of 90K was obtained after viral infection or introduction into the cells of fragments of viral, bacterial, or mammalian double-strand polynucleotides. The increase was sequence independent, not CpG mediated, and associated with the expression of molecules characterizing antigen-presenting-cell phenotype. The present data along with results from previous studies suggest that 90K plays an important role in the maintenance of an appropriate level of immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A LARGE OLIGOMERIC human protein composed of approximately 90-kDa subunits, designated 90K/Mac-2BP, has been originally identified as a tumor-associated antigen (1, 2, 3) and as a ligand of galectin-3 (formerly Mac-2) (4, 5). Characterization of 90K by cDNA cloning and sequencing revealed a multidomain organization of 567 amino acid residues in the mature protein. Notably, a region in the N-terminal portion of the protein shows a high degree of homology with members of the macrophage scavenger receptor cysteine-rich (SRCR) domain family (4, 5). A mouse homolog of human 90K with a 69% sequence identity has been independently cloned as cyclophilin C-associated protein (CyCAP) (6) and mouse adherent macrophage (MAMA) (7).

The function of 90K is not well defined yet. Like other members of the SRCR family (8), the protein might be involved in host defense. In vitro, tumor-derived 90K induces production of IL-1, IL-6, and other cytokines by blood monocytes and stimulates natural killer cell and lymphokine-activated killer cell activity (5, 9). Elevated expression levels of the protein have been observed in tissues and serum of patients with different types of cancer (3, 10, 11, 12, 13) or infected by viruses (14, 15, 16) and proven to be of prognostic value. Expression of 90K in normal tissues has been less well established, and there is little knowledge of its biological role in normal cells or how its synthesis or secretion might be regulated.

The FRTL-5 rat thyroid cells are a continuously cultured line that have no attributes of tumor cells and exhibit normal TSH/insulin/IGF-I-regulated growth and function (17, 18). They represent, therefore, a reasonable model to evaluate the role of 90K in normally functioning cells with a differentiated phenotype.

Here, we report the cloning of rat 90K and demonstrate that its expression is modulated by {gamma}-interferon ({gamma}-IFN) and hormones controlling growth and function of FRTL-5 cells. Additionally, we show that rat 90K increases major histocompatibility complex (MHC) class I and that both 90K and MHC class I appear to be coordinately increased by introducing double-strand (ds) polynucleotides into the cells. The data support a role of 90K in immune self defense mechanisms of nonimmune cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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; 30 U/mg). Rat {gamma}-IFN was from Amgen (Thousand Oaks, CA); recombinant IGF-I was from the Fujisawa Pharmaceutical Co. (Osaka, Japan). [{alpha}-32P]dCTP (3000 Ci/mmol), [32P]UTP (3000 Ci/mmol), and [35S]methionine were from DuPont/NEN Life Science Products (Boston, MA). Synthetic polynucleotides were from Pharmacia Biotech (Piscataway, NJ), salmon sperm DNA from Stratagene (La Jolla, CA), calf thymus DNA from Sigma Chemical Co. (St. Louis, MO), and pcDNA3 and pRc/RSV plasmids from Invitrogen (San Diego, CA). Genomic DNA was purified using Wizard Genomic DNA purification Kit (Promega, Madison, WI). The source of other materials was Sigma unless otherwise noted.

Cells
Rat FRTL-5 thyroid cells (Interthyr Research Foundation, Baltimore, MD) were a fresh subclone (F1) with all properties previously described (19, 20, 21). Cells were grown in Coon’s modified F12 medium supplemented with 5% heat-treated, mycoplasma-free calf serum (Life Technologies, Inc., Grand Island, NY), 1 mM nonessential amino acids (Life Technologies), and a six-hormone mixture (6H medium) including bovine TSH (1 x 10–10 M), insulin (10 µg/ml), hydrocortisone (0.4 ng/ml), transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml) (27, 28). Complete medium containing no TSH is referred to as 5H medium. In some experiments, cells were maintained in medium with no TSH, insulin, or hydrocortisone (3H medium) and then stimulated with the three hormones, separately or in combination. Unless otherwise noted, cells were stimulated with 100 U/ml rat {gamma}-IFN, 1 x 10–10 M TSH (or 5 µM forskolin), or 10 µg/ml insulin (or 100 ng/ml IGF-I).

Library screening, DNA sequencing, and sequence analysis
To isolate rat 90K cDNA, a previously described {lambda}gt11 rat cDNA library, constructed using FRTL-5 cell poly(A+) RNA (22), was screened by plaque hybridization with 32P-labeled human 90K cDNA. Hybridization was performed at 68 C; washes were done at room temperature and at 37 C. DNA fragments from the screening were subcloned into pGEM7zf(+) (Promega) and sequenced, using the dideoxynucleotide chain termination method (23). Sequence alignments and comparisons were performed by using Gene Works (IntelliGenetics, Inc., Mountain View, CA).

Protein production in Escherichia coli and FRTL-5 cells
Recombinant protein was produced by using the pET system (Novagen, Madison, WI). The rat 90K cDNA was subcloned into the EcoRI site of the pET-30(+) expression vector. The protein was produced in E. coli BL21 (DE3) after a 4-h stimulation with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) and purified according to the manufacturer’s procedures under reducing conditions.

Rat 90K protein was also purified from the culture medium of FRTL-5 cells. To increase the concentration of the protein in the medium, cells were transfected with a pcDNA3 expression vector (Invitrogen) containing the 90K cDNA insert in its EcoRI site (pcDNA3/90K). The vector was subjected to restriction enzyme analysis to identify and isolate plasmids with 90K incorporated in a sense or antisense direction. The protein was purified by high-pressure gel permeation chromatography using a 7.5 x 300-mm TSK-4000 SW column (Tosohaas, Montgomeryville, PA). A Gilson HPLC system (Gilson, Inc., Middletown, WI) pumping prefiltered, degassed PBS (pH 7.5) at 0.5 ml/min, was used to resolve 0.2-ml samples; detection was by dual-wavelength 280/256 monitoring.

Peptide synthesis and antibody production
A 17-amino-acid peptide (amino acids 530–546), identified as potentially immunogenic with the aid of the GeneWorks software, was synthesized (Genemed Biotechnologies, San Francisco, CA) and used to immunize rabbits after conjugation with keyhole limpet hemocyanin (24). The antiserum was purified by affinity chomatography using CNBr-activated Sepharose (Sigma) coupled with the immunizing peptide.

Western blotting
Transformed bacteria or FRTL-5 cells were directly lysed in Laemmli buffer (25) and protein content determined with the BCA protein assay (Pierce Chemical Co., Rockford, IL). Proteins (30 µg) were separated by SDS-gel electrophoresis under reducing conditions on precast 8% Tris-glycine gels (Novex, San Diego, CA). Proteins were transferred to nitrocellulose membranes according to standard procedures (26) and processed for immunoblotting using the polyclonal anti-90K followed by a horseradish peroxidase-conjugated donkey antirabbit IgG (Amersham Life Science, Cleveland, OH). The nitrocellulose membrane was developed using an enhanced chemiluminescence kit (Amersham).

Immunofluorescence
FRTL-5 cells were plated at a density of 1 x 105 cells per well on Permanox chamber slides (Nalge Nunc International, Rochester, NY) and grown in 6H medium for 2–3 d before shifting to 5H medium for 6 d. Cells were rinsed in PBS, fixed in 3.5% formaldehyde in PBS for 1 h, and permeabilized with methanol at –20 C for 5 min. After a 1-h incubation in blocking solution (5% BSA/0.05% Tween 20 in PBS), cells were incubated overnight at 4 C with polyclonal anti-90K antibody in 1% BSA/0.05% Tween 20 in PBS. After washing with PBS/0.05% Tween 20, cells were incubated for 1 h with an ALEXA-594-conjugated donkey antirabbit antibody (Molecular Probes Inc., Eugene, OR). Images were taken using a confocal laser-scanning microscope (LSM-510 META, Zeiss) equipped with a x100 objective.

In vivo labeling and immunoprecipitation
FRTL-5 cells were grown overnight in methionine-free Coon’s modified F-12 medium (0.5 ml/well) containing 1% dialyzed calf serum (Life Technologies) in the presence of [35S]methionine (50 µCi/ml). Spent medium was precleared with rabbit preimmune serum and incubated with the polyclonal anti-90K antibody followed by protein A-Sepharose (Sigma) The immunoprecipitate was separated by SDS-PAGE and proteins detected by autoradiography.

Northern blot analysis
Total cellular RNA was isolated using RNeasy Mini Kits (QIAGEN, Valencia, CA). Northern blot analysis was performed as described (19, 27) using nitrocellulose membranes (Nytran Plus, Schleicher & Schuell, Keene, NH). Filters were sequentially hybridized with probes for rat 90K, MHC class I, MHC class II, rat class II transactivator (CIITA), proteasomal subunit low-molecular-mass polypeptide 2 (LMP2), transporters of antigen peptide (TAP)-1, invariant chain, human leukocyte antigen (HLA)-DMB, B7.1, and glyceraldehyde-3 phosphate dehydrogenase (GAPDH). Labeling of probes and hybridization (1.0 x 106 cpm/ml) were performed as described (19, 27). The rat 90K cDNA was the full-length clone described herein; all other probes have been described elsewhere (19).

Nucleic acid transfections
Plasmid DNAs were purified using EndoFree Plasmid Maxi Kits (QIAGEN). Methylation was by treatment with SssI methylase (New England BioLabs, Beverly, MA) at 37 C for 2 h; methylation of CpG motifs was confirmed by resistance to BstUI (New England BioLabs). DNase I (Promega) digestion was performed at 37 C for 30 min and was followed by phenol-chloroform extraction and ethanol precipitation. Digestion was confirmed by agarose gel electrophoresis. For transfection, 5 µg of DNA was mixed with 30 µl of Lipofectamine Plus reagent (GIBCO BRL, Gaithersburg, MD) and 750 µl of serum-free medium, then incubated at room temperature for 15 min. Cells were washed with serum-free medium before the addition of DNA. After 3 h, medium was replaced with serum-containing medium.

Flow cytometry
One-hundred microliters of single-cell suspensions (106 cells) were placed in a 96-well flat-bottomed plate and incubated on ice for 30 min with 100 µl of fluorescein isothiocyanate-labeled antirat MHC class I monoclonal antibody (Serotec, Raleigh, NC). FACS analysis was performed using a FACSort instrument and CellQuest software (Becton Dickinson, San Jose, CA).

Statistical analysis
All experiments were done in triplicate. Differences among mean expression levels of three or more groups were assessed by one-way ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of rat 90K
Five positive clones were isolated by screening the rat {lambda}gt11 FRTL-5 expression library with human 90K cDNA. The longest one was sequenced and found to extend 2016 nucleotides (GenBank accession no. AY552591). The open reading frame starts with an ATG initiation codon at nucleotide 18 and ends at a TAG termination codon at position 1740, encoding a protein of 574 amino acids with a calculated molecular mass of 67,490 Da. There are seven potential glycosylation sites and 16 cysteine residues. The first 18 amino acids have the characteristics of a signal peptide (28).

The amino acid sequence of rat 90K has a high degree of homology with CyCAP/MAMA and the human 90K proteins (Fig. 1Go). The three proteins diverge in a region spanning residues 431–449 of human 90K; however, all cysteine residues are conserved, as well as the region coding for the SRCR domain (amino acids 24–128). This domain is also found in CD5, CD6, M130, complement factor 1, Workshop cluster 1 (WC1), and the speract receptor, which are either secreted or membrane glycoproteins expressed by immune cells (8).



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  		FIG. 1. Sequence comparison of rat 90K with human and murine homologs. Amino acid identities in all three homologs are boxed; an identity of the rat 90K sequence with only one of the homologs is denoted by a dot. Nonidentical but similar residues are in white in the black boxes.

 
The rat 90K cDNA, subcloned into a pET expression vector and expressed in IPTG-stimulated BL-21 E. coli, produced a protein migrating at approximately 65 kDa on Western blots, consistent with its predicted amino acid sequence (Fig. 2AGo, lanes 2 and 3 vs. lane 1). Analysis of FRTL-5 whole-cell lysates by Western blot revealed a major band migrating at approximately 83 kDa, with a minor component at approximately 90 kDa (Fig. 2AGo, lane 4). The higher molecular mass of the rat 90K produced by FRTL-5 is consistent with the known glycosylation state of the mammalian protein (4, 5, 6, 7). Autoradiography of [35S]methionine-labeled proteins released into the medium by FRTL-5 cells and immunoprecipitated with antirat 90K antibody revealed the presence of three different molecular species of approximately 200, 90, and 57 kDa, respectively (Fig. 2AGo, lane 5). The 200- and 57-kDa bands are likely to correspond to 90K dimers and glycosylation intermediates, respectively (4). When FRTL-5 cells were transfected with the expression vector pcDNA3/90K sense, a marked increase in the intensity of all three bands of the secreted 90K was observed (Fig. 2AGo, lane 6). Immunofluorescence staining localized the protein predominantly in the perinuclear region (Fig. 2BGo).



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  		FIG. 2. Rat 90K expression in transformed BL-21 bacteria and in FRTL-5 cells. A, 30 µg of BL-21 bacteria lysates (lanes 1 and 2), recombinant purified protein (lane 3), or FRTL-5 cell lysates (lane 4) were separated by SDS-PAGE and Western blots performed with the rabbit antibody raised against a synthetic peptide encompassing residues 530–546 of the deduced amino acid sequence of rat 90K. Proteins were detected by enhanced chemiluminescence. Recombinant protein was present in bacteria lysates after, but not before, induction with IPTG (lane 2 vs. lane 1). Lane 3 depicts the recombinant product of approximately 65 kDa after purification on a His-tagged column, and lane 4 shows the 83- and 90-kDa components in the whole-cell lysates of FRTL-5 maintained in 5H medium. To verify whether rat 90K was secreted, FRTL-5 cells were metabolically labeled overnight with [35S]methionine and medium incubated with the anti-90K antibody followed by protein-A Sepharose. The immunoprecipitate was separated by SDS-PAGE, and autoradiography revealed three bands at 55, 90, and 200 kDa, respectively (lane 5). These bands were clearly increased when FRTL-5 cells were transfected with the 90K expression vector pcDNA3/90K sense (lane 6). B, FRTL-5 cells, grown in chamber slides and maintained in 5H medium, were fixed, permeabilized, and incubated with the antirat 90K antibody, followed by an ALEXA-594-conjugated donkey antirabbit antibody. The red fluorescence shows a perinuclear localization. Control staining with preimmune serum showed no fluorescence signal (data not shown).

 
Regulation of 90K by hormones and by {gamma}-IFN
FRTL-5 cells maintained in minimal medium (3H) constitutively express 90K mRNA (Fig. 3BGo, lane 1). To assess whether the expression of 90K was regulated by hormones, FRTL-5 cells were exposed to hydrocortisone, insulin, and TSH, separately or in combination. Hydrocortisone significantly decreased 90K mRNA levels (Fig. 3AGo, bottom; 3B, lane 2), with a maximal effect after 24 h. Insulin or TSH increased 90K mRNA levels (Fig. 3AGo, top; 3B, lanes 3 and 4), although their effect differed as a function of time; insulin caused a progressive increase that maximized after 24 h, whereas the TSH-induced increase was maximal at 6 h and plateaued or slightly decreased thereafter. Time-course experiments were then performed on 3H cells using TSH and insulin in combination. Surprisingly, a decrease in 90K RNA/GAPDH ratios was observed, with a half-maximal decrease at 12 h and maximal decrease at 24 h lasting up to 48 h (data not shown). Therefore, 24- or 48-h time points were chosen to evaluate the action of the combined hormones in subsequent experiments. The presence of hydrocortisone attenuated the effect of insulin (Fig. 3BGo, lane 5 vs. 3) but not that of TSH (Fig. 3BGo, lane 6 vs. 4). The simultaneous presence of insulin and TSH significantly decreased 90K mRNA levels, independently from the presence of hydrocortisone (Fig. 3BGo, lanes 7 and 8). Similar results were obtained by substituting TSH with forskolin (Fig. 3BGo, lane 9) and insulin with IGF-I (data not shown).



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  		FIG. 3. Hormonal modulation of rat 90K mRNA levels. A, FRTL-5 cells were maintained in 3H medium and then stimulated with TSH or insulin (top), or hydrocortisone (bottom). At the time noted, total RNA was isolated and subjected (10 µg/lane) to Northern analysis using 32P-labeled rat 90K cDNA and GAPDH probes. The 90K data were normalized to GAPDH mRNA levels on the same blots. The 90K/GAPDH ratio was compared with the 3H control; results are the mean ± SD of three different experiments on three batches of cells. B, FRTL-5 cells were maintained in 3H medium and then stimulated with TSH, insulin (INS), or hydrocortisone (CORT) individually or in combination. On lane 9, forskolin (FSK, 5 µM) was used instead of TSH. After 24 h, total RNA was isolated and subjected to Northern analysis. Representative Northern data are presented at the top. Quantitative results from three different experiments on three batches of cells are presented on the bottom, expressed as the mean ± SD after 90K/GAPDH ratios were calculated. Data in 3H medium (lane 1) were set at unity and other results expressed relative to these. *, Significant decrease in 90K mRNA levels (P < 0.05); **, significant increase (P < 0.01).

 
We previously showed that IFN is able to increase human 90K both in vitro and in vivo (29, 30, 31). Exposure of FRTL-5 cells cultured in 3H medium to {gamma}-IFN resulted in a significant increase of 90K mRNA level (P < 0.01) (Fig. 4AGo, lane 2 vs. 1). The {gamma}-IFN effect was not influenced by TSH, insulin, hydrocortisone, hydrocortisone plus insulin, or hydrocortisone plus TSH (Fig. 4AGo, lanes 3–7); however, it was significantly decreased by the combination of TSH and insulin (P < 0.01) (Fig. 4AGo, lanes 8 and 9 vs. 2–7). This last result was reproduced in run-on experiments (Fig. 4BGo), suggesting an effect at the transcriptional level.



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  		FIG. 4. Effect of hormones on the ability of {gamma}-IFN to increase 90K mRNA levels. A, 32P-labeled rat 90K cDNA and GAPDH probes were used in Northern analyses to characterize mRNA levels in FRTL-5 cells maintained in 3H medium and stimulated with {gamma}-IFN, TSH, hydrocortisone (CORT), or insulin (INS) for 24 h. Total RNA was isolated and subjected (10 µg/lane) to Northern analysis. Representative Northern data are presented at the top of the figure. Results from three different experiments on three batches of cells are presented on the bottom as the mean ± SD after 90K/GAPDH ratios were calculated and the value of 3H set at unity. *, Significant increase (P < 0.01) in 90K mRNA levels; **, significant decrease in the effect of {gamma}-IFN (P < 0.01). B, FRTL-5 cells maintained in 3H medium were treated with insulin (10 µg/ml) plus {gamma}-IFN, {gamma}-IFN plus TSH, or {gamma}-IFN plus 5 µM forskolin (FSK) for 24 h. Nuclei were isolated and incubated with [32P]UTP before mRNA was purified, denatured, and hybridized to an excess of the noted unlabeled cDNA probes.

 
Effect of rat 90K on MHC class I antigens
Human 90K has been found to increase the expression of MHC class I antigens in human breast cancer cells (32). In pcDNA3/90K-transfected FRTL-5 cells, Northern blot analysis revealed a clear increase of MHC class I (Fig. 5AGo). A similar increase of MHC class I, but not of MHC class II, antigen mRNA was obtained when parental FRTL-5 cells were exposed to rat 90K purified from the culture fluid of the same cells (P < 0.01) (Fig. 5BGo). The effect was evident at the protein level as well (Fig. 5CGo).



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  		FIG. 5. 90K increases MHC class I mRNA expression in FRTL-5 cells. A, FRTL-5 cells were grown in 6H medium to 80% confluency and transfected with pcDNA3 containing the 90K cDNA in a sense (pcDNA3/90K sense) or antisense (pcDNA3/90K antisense) direction. After 24 h, the levels of 90K, MHC class I, and GAPDH mRNAs were measured by Northern analysis as described earlier. B, Fresh FRTL-5 cells maintained in 5H medium were incubated with 10 µg/ml purified 90K for 24 h, and MHC class I and II levels were measured by Northern analysis. A representative Northern blot is presented on the top; results from three different experiments on three batches of cells are presented on the bottom as the mean ± SD after 90K/GAPDH ratios were calculated and the value with no 90K set at unity. Densitometry indicated that MHC class I increase by 90K was statistically significant (P < 0.01). C, FACS analysis for measuring MHC class I expression on FRTL-5 cells treated as in B.

 
Effect of viral infection and ds polynucleotides on rat 90K
TSH/cAMP and insulin/IGF-I are known to down-regulate MHC class I antigen in FRTL-5 cells (33, 34, 35) (Fig. 6AGo). As these same hormones decrease rat 90K, it was worthwhile to assess whether other experimental conditions known to modulate MHC class I antigens (viral infection, transfection with ds bacterial DNA, cell genomic DNA, etc.) (19) were effective in influencing rat 90K level as well. Infection of FRTL-5 cells with herpes simplex coordinately increased 90K and class I mRNA levels within 16 h (Fig. 6BGo). Similar results were observed when ds bacterial DNA, salmon sperm DNA, calf thymus DNA, or FRTL-5 cell genomic DNA was introduced into the cytoplasm of the cells (Fig. 6CGo, lanes 2–5). Additionally, transfection of the cells with herpes or foamy virus dsDNA, pcDNA3, or pRc/RSV (Fig. 6CGo, lanes 7–10), but not with single-strand (ss) herpesvirus DNA (Fig. 6CGo, lane 6) increased 90K and MHC class I mRNA. Similar results were obtained using different transfection procedures, i.e. electroporation or diethylaminoethyl dextran (data not shown). The increase in rat 90K mRNA was DNase but not methylase sensitive (Fig. 7AGo, lanes 5 and 8) as for MHC class I antigens (19). The ss oligodeoxynucleotides (ODNs) having one or more CpG motifs (CpG-1; CpG-2) or non-CpG control counterparts had no effect on 90K mRNA levels (Fig. 7AGo, lanes 9–12), whereas the expression was increased by ds-phosphorothioate (s)ODNs, but not by ss sODNs (Fig. 7AGo, lanes 13–16). Furthermore, as seen for MHC class I (19), dsDNA copolymers (Fig. 7BGo, lanes 9–12) or duplexes (Fig. 7BGo, lanes 6–8) increased 90K expression, whereas ss polymers did not (Fig. 7BGo, lanes 3–5). The ds oligonucleotides as short as 35 bp were effective in increasing 90K mRNA levels (Fig. 7CGo). The effect of dsDNA on 90K mRNA appeared to be as good or better then that of {gamma}-IFN at maximal concentration of each (Fig. 7DGo). 90K gene expression was also increased by dsRNA but not by ssRNA (data not shown). Finally, the increase of 90K expression after transfection with dsDNA was additive to that of {gamma}-IFN (Fig. 7EGo), but it was mechanistically different because it was not paralleled by the increase of CIITA (Fig. 7FGo) nor attenuated by TSH plus insulin (Fig. 8Go).



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  		FIG. 6. Coordinate regulation of MHC class I and 90K mRNAs by TSH plus insulin, by herpes simplex infection, or by dsDNA transfected into FRTL-5 cells. A, FRTL-5 cells were maintained in 5H medium, then maintained 48 h in the same medium with TSH. RNA was isolated and Northern analyses performed using 10 µg RNA and radiolabeled 90K, MHC class I, and GAPDH probes. B, FRTL-5 cells that had been grown to 80% confluency in 6H medium were infected with herpes simplex virus (HSV-2). RNA was isolated at the times noted and Northern analyses performed using radiolabeled 90K, MHC class I, and GAPDH probes. C, FRTL-5 cells, grown to 80% confluency in 6H medium, were transfected with 5 µg each of the noted dsDNAs (lanes 2–5), with ss or ds herpes simplex virus DNA fragments (lanes 6 and 7, respectively), with a 54-bp ds oligonucleotide from Foamy virus (lane 8), or with two plasmid DNAs, pcDNA3, and pRc/RSV (lanes 9 and 10). Total RNA was prepared and Northern analysis performed 48 h after transfection using MHC class I, 90K, or GAPDH probes, as described above. Lipofectamine treatment alone served as a control of the transfection procedure (Mock).

 


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  		FIG. 7. Properties of the nucleic acid needed to induce 90K expression in FRTL-5 cells. Transfection and Northern analysis using rat 90K and GAPDH probes were performed 48 h after treatment, exactly as described in Fig. 6CGo. A, FRTL-5 cells were transfected with 10 µg intact, methylated or DNase-treated plasmid, pcDNA3 or pRc/RSV (lanes 3–8) or 10 µg each of the ss-CpG oligodeoxynucleotides (ODNs) or control ODNs (CpG oligos; lanes 9–12) or ss- or ds-phosphorothioate ODNs (s-oligos; lanes 13–16). Lane 1 contains RNA from nontreated cells and lane 2 from cells subjected to the transfection procedure only (Mock). B, Ten micrograms of the noted synthetic polymer nucleotides and their duplexes were used to transfect cells (lanes 3–12). C, Cells were transfected with 5 µg of dsDNA fragments from 1004 to 24 bp in length (lanes 2–10) or with indicated amount of a 35-bp dsODNs (lanes 12–15) as described above. RNA from cells subjected to a mock transfection with lipofectamine alone is in lane 1; RNA from cells treated with 100 U/ml {gamma}-IFN is in lane 11. These serve as negative and positive controls, respectively. D, The 90K mRNA levels were compared after either transfecting cells with different amounts of dsDNA or incubating them with different amounts of {gamma}-IFN as noted. RNA was isolated 48 h after transfection or treatment and Northern analyses performed as above. E, 90K mRNA levels were compared 48 h after transfecting cells with 10 µg dsDNA (pcDNA3), incubating them with 1000 U/ml {gamma}-IFN, or subjecting them to both treatments. A representative Northern analysis is presented at the top; the mean ± SD of the calculated 90K/GAPDH ratios from four separate experiments using different batches of cells is presented at the bottom of the panel. The control value is set at unity. *, Significant increase (P < 0.01) in 90K RNA levels induced by {gamma}-IFN or dsDNA; **, significant increase in the individual effect of {gamma}-IFN or dsDNA (P < 0.05). F, 90K and CIITA mRNA levels were compared 48 h after transfecting cells with 10 µg dsDNA (pcDNA3) or incubating them with 100 U/ml {gamma}-IFN.

 


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  		FIG. 8. TSH plus insulin decreases 90K mRNA levels induced by {gamma}-IFN, but not by dsDNA. 32P-labeled rat 90K cDNA and GAPDH probes were used in Northern analyses to characterize mRNA levels in FRTL-5 cells maintained in 5H with 5% serum, then shifted to fresh medium with {gamma}-IFN (100 U/ml) and with or without TSH (1 x 10–10 M) for 48 h. Alternatively, cells were transfected with 10 µg/ml dsDNA and then placed in medium with or without TSH for 48 h. Total RNA was isolated and subjected (10 µg/lane) to Northern analysis as described above. Results from three different experiments on three batches of cells are presented on the bottom as the mean ± SD after 90K/GAPDH ratios were calculated and the value of 5H set at unity. *, Significant increase (P < 0.01) in 90K RNA levels induced by {gamma}-IFN or dsDNA; **, significant decrease in the effect of {gamma}-IFN (P < 0.01).

 
The ds-polynucleotide effect on rat 90K was associated with that on gene products characterizing the antigen-presenting-cell phenotype, such as proteasomal subunit LMP2, TAP molecules, MHC class II, invariant chain, HLA-DMB proteins, and the costimulatory molecule B7.1 (Fig. 9Go).



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  		FIG. 9. The dsDNA increased 90K and MHC class I in FRTL-5 cells is associated with conversion of the cells to a potential antigen-presenting cell. Changes in 90K, MHC class I, and GAPDH mRNA levels in cells transfected with dsDNA are compared with changes in genes important for the antigen-presenting-cell activity of a cell: the proteasome-processing protein (LMP2); the transporter of antigen peptide (TAP1); MHC class II; invariant chain (Ii); HLA-DMB, and the costimulatory molecule B7.1. Total RNA was isolated and subjected to Northern blot analysis as described above.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Most of the available information on the function of the mammalian 90K proteins is derived from studies on cancer cells, whereas little is known about the role and the regulation of these proteins in normal cells. To address these issues, we used rat FRTL-5 thyrocytes, a model of normal, hormonally responsive cells. Several novel observations emerged.

First, rat 90K expression was modulated by TSH and insulin/IGF-I, which are both required for the growth and function of normal thyrocytes (36, 37), and by {gamma}-IFN. Interestingly, when TSH and insulin were used in combination, a marked decrease of the constitutive and {gamma}-IFN-induced 90K expression levels were seen, whereas when these same hormones were used individually an increased expression of the protein was noticed. The reason for this differential behavior of 90K when hormones are used individually or in combination is not clear, although it very likely results from the activation of different pathways. The mechanisms responsible for 90K hormonal regulation are currently being investigated at the promoter level. Moreover, studies on the expression of 90K in thyrocytes of normal vs. hypothyroid mice are in progress to evaluate the physiological relevance of this phenomenon.

Second, the expression levels of MHC class I antigens increased when FRTL-5 cells were engineered to overexpress rat 90K or exposed to the protein purified from the culture fluid of the same cells, even in the presence of TSH and insulin, which are known to down-regulate MHC class I antigens. These data, which duplicate those obtained with human 90K (32), suggest that these proteins may behave as an autocrine or paracrine/exocrine factor able to up-regulate MHC class I antigens. Studies are in progress to clarify the mechanisms underlying the 90K effect on MHC class I and to verify whether this phenomenon is specific for thyrocytes or can apply also to other normal endocrine cells, e.g. ß-cells.

The functional meaning of these findings could be explained in light of the mechanism regulating immune surveillance in the thyroid gland. In this tissue, TSH and insulin stimulate cell proliferation and function, increasing de novo synthesis of proteins (17, 18, 36, 37). Peptides derived from these self-proteins would complex with MHC class I antigens, increasing the cell surface density of these complexes, possibly leading to a break of self-tolerance (38). To avoid this, down-regulation of MHC class I antigens by TSH and insulin is required (38, 39, 40, 41). As these same hormones decrease the expression of 90K, which is able to induce MHC class I antigens, it can be argued that the decrease of this molecule is aimed at down-regulating the immune response, thus preserving self-tolerance. The need for a coordinate expression of 90K and MHC class I is further supported by the finding that both these immune-related proteins are up-regulated by {gamma}-IFN and that this induction is counteracted by TSH/cAMP (33, 34).

Last, and perhaps most intriguing, the level of rat 90K expression is increased after virus infection or the introduction of DNAs into the cells. In all respects, this phenomenon reproduced what is seen for MHC class I (19). In fact, 90K expression does not seem to be mediated by CIITA or suppressed by TSH/insulin/IGF-I, as observed in cells exposed to {gamma}-IFN, supporting the hypothesis that ds polynucleotides and {gamma}-IFN act through additive although independent mechanisms, in contrast with previous suggestions (42). The present findings add new insights to the role of 90K in the immune defense against viruses and possibly cancer. The introduction of DNA into the cells experimentally reproduces the migration of self genomic or mitochondrial DNA into the cytoplasm, phenomena frequently observed in injured tissues (43) and in tumor cells (44, 45). These same procedures have been found to induce MHC class I antigens (19). This induction is highly desirable because the presentation of peptides by MHC antigens is required for the development of an effective immune response against cells infected by viruses or altered by malignant transformation (46). Interestingly, these pathogenic events are associated with an increased expression of 90K, which in turn promotes an additional MHC class I response. Therefore, it can be argued that the enhanced expression of 90K may be part of a mechanism aimed at triggering immune effectors against cells infected by viruses or altered after an oncogenic insult. This hypothesis is further supported by the finding that the increase of 90K and MHC class I is accompanied by enhanced expression of MHC class II, the costimulatory molecule B7–1, and other genes or gene products known to be important for antigen presentation, including LMP2, TAP, invariant chain, and HLA-DMB proteins (47, 48).

Some of the features of 90K, including the increased levels found in the tumors and after virus infection or interferon treatment of the cell, may resemble those of heat shock proteins. However, unlike these proteins, heating the cells up to 42 C does not result in an increased production or release of 90K (data not shown).

In sum, we have shown that 90K, constitutively expressed by FRTL-5 thyrocytes, increases MHC class I antigens and that the expression of these proteins is coordinately regulated by hormones. We suggest that 90K may participate in mechanisms ensuring an appropriate level of immune response in all instances. The decrease of this protein in cells proliferating under physiological conditions may contribute to the maintenance of the immune response within the limits of self-tolerance. On the other hand, the increase of 90K that follows viral infection or conditions resembling self DNA leakage into the cytoplasm could be part of the host defense mechanisms facilitating the elimination of infected or tumor-transformed cells.


   Footnotes
 
This work was supported in part by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC) and Ministero dell’Istruzione, Università e Ricerca (MIUR).

The sequence of rat 90K has been submitted to the GenBank database under accession no. AY552591.

Abbreviations: CIITA, Class II transactovator; CyCAP, cyclophilin C-associated protein; ds, double-strand; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; HLA, human leukocyte antigen; IFN, interferon; IPTG, isopropyl-ß-D-thiogalactopyranoside; LMP, low-molecular-mass polypeptide; MAMA, mouse adherent macrophage; MHC, major histocompatibility complex; ODN, oligodeoxynucleotide; SRCR, scavenger receptor cysteine-rich; s, phosphorothioate; ss, single-strand; TAP, transporter of antigen peptide.

Received April 19, 2004.

Accepted for publication June 23, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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