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Endocrinology Vol. 143, No. 3 1008-1017
Copyright © 2002 by The Endocrine Society

INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS

High Glucose Levels Increase Major Histocompatibility Complex Class I Gene Expression in Thyroid Cells and Amplify Interferon-{gamma} Action

Giorgio Napolitano, Ines Bucci, Cesidio Giuliani, Cinzia Massafra, Cinzia Di Petta, Eleonora Devangelio, Dinah S. Singer, Fabrizio Monaco and Leonard D. Kohn

Chair of Endocrinology (G.N., I.B., C.G., C.M., C.D., E.D., F.M.), Department of Medicine and Sciences of Aging, University "G. D’Annunzio," Chieti 66100, Italy; Experimental Immunology Branch (D.S.S.), National Cancer Institute, National Institutes of Health, Bethesda Maryland 20892; and Edison Biotechnology Institute (L.D.K.), Ohio University, Athens Ohio 45701

Address all correspondence and requests for reprints to: Dr. Leonard D. Kohn, Edison Biotechnology Institute, Ohio University, The Ridges Building 25, Athens, Ohio 45701. E-mail: . kohnl{at}ohio.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increased major histocompatibility complex (MHC) class I gene expression in target tissues may be relevant to the pathogenesis of autoimmune diseases. In this study, we questioned whether high glucose levels might increase MHC class I levels and thereby contribute to autoimmune complications. We used thyrocytes in continuous culture, because there is an increased incidence of autoimmune thyroiditis in type 2 diabetics and because transcriptional regulation of MHC class I is well studied in these cells. Northern analysis and flow cytometry showed that 20 and 30 mM D-glucose up-regulated MHC class I expression and that the glucose effect was additive to and independent of interferon-{gamma}. The effect was specific, because L-glucose did not modify class I expression. The glucose acted transcriptionally, requiring both enhancer A and a cAMP-response element-like element located in the hormone-sensitive region of the MHC class I 5'flanking region. These elements are different from those activated by interferon-{gamma}. High glucose levels increase formation of the MOD-1 complex with enhancer A; MOD-1 is a heterodimer of fra-2 and the p50 subunit of NF-{kappa}B. Both TSH and insulin are required for full expression of the glucose activity in thyrocytes. The glucose effect is partially blocked by wortmannin, suggesting involvement of the PI3K signal system. The data support the possibility that high serum glucose levels in type 2 diabetic patients may increase MHC class I levels in target tissues and contribute to autoimmune complications of the disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HIGH GLUCOSE LEVELS may have diverse effects on the immune response system. A higher incidence of infectious diseases has been widely reported in diabetic patients, suggesting an immunosuppressive action of hyperglycemia (reviewed in Ref. 1). Deficits of both innate and the adaptive immunity (2, 3) have been described.

Hyperglycemia might, however, also contribute to an exaggerated immune response. Thus, it has been recently suggested (4, 5) that type 2 diabetes might be an immune-related disease. Increased blood concentrations of markers of the immune system’s acute-phase response to environmental stress are frequently detected in type 2 diabetic patients, e.g. C-reactive protein, sialic acid, serum amyloid A, and IL-6 (4, 5), suggesting an involvement of the innate immune system. IL-12 has also been shown to be elevated in type 2 (as well as type 1) diabetic patients, implicating the adaptive immune system (6, 7). Islet cell autoimmunity has been described in type 2 patients and is associated with impaired insulin secretion (8). Thus, glutamic acid decarboxylase 65 autoantibodies, which are the main marker of islet cell autoimmunity in type 2 diabetics, are present in about 10–30% of patients (8, 9) and are associated with an increased incidence of chronic autoimmune thyroiditis, as shown by positive TPO antibodies (9). Some autoimmune diseases, chronic autoimmune thyroiditis among them (9, 10, 11), are strongly associated with type 1 diabetes, but their incidence and prevalence are increased in type 2 diabetic patients as well.

For example, a 2- to 3-fold increase in annual incidence of thyroid autoimmune diseases has been reported in a Scottish type 2 diabetic population (12), by comparison with nondiabetics. These data are even more significant if only hypothyroidism (both overt and subclinical) is considered, with an annual incidence of 3.8% in males and 4.8% in type 2 diabetic females. Furthermore, the prevalence of hypothyroidism (both overt and subclinical) reported in the same study, 7.6% in type 2 diabetic patients (mean age, 53 yr), is increased by comparison with older patients in a study by Vanderpump et al. (13) in the survivors of the Wickham study. Indeed, the Vanderpump study had a slightly older population (mean age, 58 yr), from a geographical region situated nearby the area where the diabetic population lived, but had an overall prevalence of spontaneous hypothyroidism of only 4.9%.

It has been suggested (14) that the clinical association of chronic autoimmune thyroiditis and type 2 diabetes might be related to a common antigen shared by pancreatic ß-cells and thyroid follicular cells. Recent data (15, 16, 17, 18, 19) have separately supported the hypothesis that autoimmune diseases are correlated with an increased target tissue expression of major histocompatibility complex (MHC) class I surface antigens. Were this the case in type 2 diabetics, increased autoantigen presentation might provide an explanation for the onset of chronic autoimmune thyroiditis in addition to a common shared antigen. High glucose levels have already been shown to increase MHC class I antigen expression in pancreatic ß-cells (20). We therefore decided to test the hypothesis that high glucose levels might increase MHC class I expression not only in a strictly glucose-dependent cell-type where glucose has been previously shown to facilitate antigen expression (21) but also in a different cell-type, e.g. the thyroid follicular cells (FRTL-5).

To pursue this purpose, we choose to test the effect of glucose by increasing its levels by 10 and 20 mM. This range of increase of glucose concentration has already been shown to modulate the transcriptional activity of cultured cells (22, 23); moreover, keeping in mind that the normal medium for FRTL-5 already contains 10 mM glucose, we reached 20 and 30 mM glucose levels, which are comparable with high and very high serum glucose levels in type 2 diabetic patients.

We and others (24, 25, 26, 27, 28) have previously defined the 5' flanking region of the PD1 gene, a classical MHC class I gene whose properties are maintained when transfected into cells from different species. It has two main regions (24, 29, 30, 31, 32) that control the MHC level in a particular cell. The tissue-specific region is -771 to -689 bp from the start of transcription; it sets the constitutive level of class I expression in each tissue. The hormone-sensitive region, -203 to -56 bp, is responsible for the regulation of class I expression within each tissue and is modulated by drugs, cytokines, and hormones (26, 27, 28, 29, 30, 31, 32). In this report, we show that high glucose levels increase MHC class I levels in thyrocytes, show the glucose action is transcriptional, and link its action to the hormone-sensitive region of the MHC gene 5'-flanking region, which is also regulated by interferon (IFN)-{gamma}, TSH, and insulin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Purified bovine TSH was obtained from the hormone distribution program of the NIDDK, NIH (NIDDK-bTSH I-1; 30 U/mg). The wild-type and the mutated oligonucleotide spanning the enhancer A sequence were from Operon Technologies (Alameda, CA); all the other consensus oligonucleotides and antibodies for electrophoretic mobility shifts and for Western blotting were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Calf serum was a heat-treated, mycoplasma-free product from Life Technologies, Inc. (Grand Island, NY). The source of all other materials was Sigma (St. Louis, MO), unless otherwise specified.

Cells
The F1 subclone of FRTL-5 rat thyroid cells (Interthyr Research Foundation, Baltimore, MD) was grown in a six-hormone mixture (6H) medium consisting of Coon’s modified Ham’s F-12 supplemented with 5% calf serum, 2 mM glutamine, 1 mM nonessential amino acids, and 6H containing 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) (33, 34). Cells were diploid and had all the functional properties previously detailed (26, 27, 29, 30, 31, 32, 33, 34, 35, 36). Fresh medium was added every 2–3 d; cells were passaged every 7 d. In some experiments, cells were maintained without TSH (5H medium) for 5 d, after having achieved 60% confluency in the 6H medium with TSH. Where indicated, cells were shifted to a serum-deprived medium (0.2%), 24 h before D-glucose treatment, to avoid any possible confusing effect caused by variable glucose concentrations in sera.

Unless otherwise noted, glucose always refers to D-glucose, because L-glucose does not duplicate the effect of D-glucose. Coon’s modified Ham’s F-12 medium already contains 10 mM glucose; final glucose concentrations were therefore 10 mM for control experiments and 20 and 30 mM, respectively, for experiments where 10 and 20 mM glucose have been added.

Plasmids
Luciferase chimeras of the MHC class I swine (PD1) 5'-flanking region, p(-1100)Luc, p(-549)Luc, p(-203)Luc, and p(-127)Luc, have been obtained by linking previously described constructs (24, 25, 26, 27, 28, 29, 30, 31, 32) to the pGL-2 basic vector (Promega Corp., Madison, WI). Briefly, the 5'-flanking region of the swine class I (PD1) gene was isolated from pSV0-based construct by digestion with BamHI-HindIII for the 1,100- and 127-bp fragments, and XbaI-HindIII for the -549 and -203 fragments. They were inserted into the NheI-HindIII or BglII-HindIII site of the pGL-2 basic vector. They are numbered from the nucleotide at the 5' -end to +1 bp, the start of transcription. Luciferase constructs with mutated MHC class I sequences were created by two-step recombinant PCR methods (37); the PCR products were inserted into the multicloning site of the pGL2-based luciferase construct used as a control in all experiments. The sequences of all constructs were confirmed by a standard method (38); DNA was prepared and twice purified by CsCl gradient centrifugation (39).

Transfection and luciferase assay
Transient transfection used the class I promoter/Luc chimeras and a diethylaminoethyl (DEAE)-dextran procedure (40). Cells were grown to 60% confluency in 6H medium, shifted to 5H medium for 7 d, and then again to 6H for 20–24 h. Cells were washed twice with PBS, pH 7.4, and incubated 1 h with 5 ml serum-free medium, without hormones (0H), containing 20 µg class I-luciferase chimera plasmid DNA, 2 µg pRSV-GH (which was used to measure the efficiency of transfection), and 250 µg DEAE-dextran. Cells were then exposed to 10% dimethylsulfoxide in PBS for 3 min, washed twice in PBS, cultured in 6H medium for 16–20 h, and then treated with glucose for 40–48 h. Where appropriate, 24 h after transfection, cells were shifted to a serum-deprived (0.2%) medium. In some experiments, wortmannin (Sigma), 3 µM final concentration, was added after transfection and 6–12 h before exposure to glucose. All values were normalized for total cell protein; this correction did not change values more than 6%. Cells were cotransfected with 2 µg pRSV-GH, and medium was taken for RIA of human GH (Nichols Institute Diagnostics, San Juan Capistrano, CA) to measure transfection efficiency. In no experiment did this deviate by more than 2%; corrections were made for this, as appropriate. Cell viability was approximately 80% in all experiments.

In some experiments, we used FRTL-5 cells that had been stably transfected with the pGL2-based luciferase-PD1 chimeras. Briefly, near-confluent cells in 6H medium were cotransfected with 20 µg plasmid DNA and 2 µg pMAMneo (CLONTECH Laboratories, Inc., Palo Alto, CA) using LipofectAMINE Reagent (Life Technologies, Inc.). After 2 d, 400 µg/ml G418 (Life Technologies, Inc.) was added to the medium; and after 3 wk, the G418-resistant colonies were pooled and used for experiments herein. To test the effect of glucose, three individually isolated clones of each construct were grown to 60% confluency in 6H medium, maintained without TSH (5H medium) for 6 d, and exposed to glucose before luciferase activity was measured. Luciferase assays were performed as described (41).

RNA isolation and Northern analyses
FRTL-5 cells were maintained in medium without TSH (5H medium) for 5 d, then stimulated with 1 x 10-10 M TSH and/or 10 or 20 mM additional glucose for the indicated times. Because, as noted earlier, Coon’s modified Ham’s F-12 medium already contains 10 mM glucose, final glucose concentrations were 10 mM for control experiments and 20 or 30 mM in experiments where 10 or 20 mM glucose, respectively, was added. mRNA was prepared using a commercial kit (Amersham Pharmacia Biotech, Uppsala, Sweden); 10 µg of the different RNA samples were run on denatured agarose gels, capillary blotted on Nytran membranes (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH), UV cross-linked, and subjected to hybridization as described (29). A 1.0-Kb HpaI fragment of the MHC class I pH-7 clone, spanning the entire cDNA insert (29), and a ß-actin cDNA (kindly provided by Dr. B. Paterson, National Cancer Institute, NIH, Bethesda, MD) were used as probes. Quantitation was performed using a BAS 1500 Bioimaging Analyzer (Fuji Photo Film Co., Ltd. Medical Systems USA, Inc., Stanford, CA).

Cellular extracts
FRTL-5 cells were grown in the presence of complete 6H medium until 60% confluent, then maintained in 5H medium with 5% calf serum for 7 d. Finally, cells were cultured for 12 h in 5H or 6H medium with 5% calf serum, before being shifted in a serum-deprived (0.2%) medium, and exposed to glucose. Cellular extracts were prepared by a modification of described methods (31, 32, 42). In brief, cells were washed twice in cold PBS (pH 7.4), scraped, and centrifuged (500 x g). The cell pellet was resuspended in 2 vol Dignam buffer C [25% glycerol, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES-KOH), pH 7.9; 1.5 mM MgCl2; 0.42 M NaCl; 0.5 mM dithiothreitol (DTT); 1 µg/ml leupeptin; 1 µg/ml pepstatin; and 0.5 mM phenylmethylsulfonyl fluoride]. The final NaCl concentration was adjusted, on the basis of cell pellet volume, to 0.42 M. Cells were lysed by repeated cycles of freezing and thawing. The extracts were centrifuged (100,000 x g) at 4 C for 20 min. The supernatant was recovered, aliquoted, and stored at -70 C.

EMSAs
DNA probes were created by restriction enzyme treatment of the chimeric luciferase constructs described above and purified from 2% agarose gels using QIAEX (QIAGEN, Valencia, CA) (43). Oligonucleotides were from Santa Cruz Biotechnology, Inc. They were labeled with [{alpha}-32P]dCTP using Klenow or with [{gamma}-32P]ATP using T4 polynucleotide kinase, then purified on an 8% native polyacrylamide gel (26, 27, 29, 30, 31, 32).

EMSAs were performed as previously described (26, 27, 29, 30, 31, 32, 43). Binding reactions in low salts, and no detergent, included 1.5 fmol [32P]DNA, 3 µg cell extract and 1 µg poly(dI-dC) in 10 mM Tris-Cl (pH 7.9), 1 mM MgCl2, 1 mM DTT, 1 mM EDTA, and 5% glycerol in a 20-µl total vol. Binding reactions in high salts plus detergent included 1.5 fmol [32P]DNA, 2 µg extract, and 0.5 µg poly(dI-dC) in 10 mM Tris-Cl (pH 7.9), 5 mM MgCl2, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton X-100, and 12.5% glycerol (30). Incubations were at room temperature for 30 min. Where indicated, unlabeled oligonucleotide competitors, recombinant proteins, or antibodies were added to the binding reaction and incubated with the extract for 20 min before the addition of labeled DNA. After incubations, reaction mixtures were electrophoresed on 4–6% native polyacrylamide gels at 160 V in 0.5x Tris-borate-EDTA at room temperature. Gels were dried and autoradiographed.

Flow cytometry
Single-cell suspensions of FRTL-5 cells were prepared and stained as previously described (27, 32). For staining of FTRL-5 cells, a directly fluorescinated mAb against the rat MHC class I antigen, fluorescein isothiocyanate antirat FT1a (ox-18) (PharMingen, San Diego, CA) was used. An isotype-matched control was used for detection of background fluorescence. Events were gated on forward vs. side scatter, such that cell debris, doublets, and nonviable cells were excluded.

Protein assays and statistical significance
Protein concentration was determined using a BCA protein assays kit (Pierce Chemical Co., Rockford, IL); crystalline BSA was the standard. All experiments were repeated at least three times with different batches of cells. Significance between experimental values was determined by two-way ANOVA and P values < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
High glucose increases MHC class I mRNA levels and antigen presentation in FRTL-5 rat thyroid cells
An increase in MHC class I mRNA levels is evident in FRTL-5 cells cultured in medium containing TSH (6H) and high (20 or 30 mM) levels of D-glucose vs. control cells in TSH-containing medium with normal (10 mM) levels of D-glucose (Fig. 1AGo). After 24 h, mean ± SD values from four separate independent experiments, each performed in triplicate, were 195 ± 27% and 256 ± 23% at 20 or 30 mM D-glucose, respectively, vs. the control, which is set at 100% (Fig. 1AGo). The effect is not evident in medium without TSH (5H medium), values being 103 ± 11% and 94 ± 5% for 20 or 30 mM D-glucose, respectively, vs. the 10 mM control (Fig. 1AGo). After 48 h, the increase is even greater in 6H-treated cells, 208 ± 23 and 287 ± 18% for cells treated with 20 and 30 mM D-glucose, respectively, vs. control cells with only 10 mM glucose (100%). No increase was again detectable in 5H-treated cells, 108 ± 12 and 109 ± 18%, respectively, vs. control. No further increase was detected in cells treated with glucose for longer times; and higher glucose concentrations, 40 or 50 mM final concentration, seemed to be toxic, because there was a high proportion of detached cells.



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  		Figure 1. A, Effect of glucose on MHC class I mRNA. FRTL-5 cells were exposed to 20 or 30 mM glucose (G 20 or G 30), for 24 h, in serum-deprived (0.2%) medium with (+TSH) or without TSH (-TSH). Northern analysis was performed using class I and ß-actin probes. Results are presented as the ratio of class I/ß-actin. Black bars, Glucose 30 mM; gray bars, glucose 20 mM; white bars, controls. Controls are set at 100%. A representative blot is presented, as is the mean of four different experiments. *, Statistically significant increase, P < 0.05. B, Flow cytometry analysis of the expression of surface MHC class I molecules on FRTL-5 cells. Cells in the presence or absence of 30 mM glucose and IFN-{gamma} (100 U/ml) are stained with a fluorescinated monoclonal antibody that specifically reacts with epitopes of the rat MHC class I antigen (PharMingen) or with an isotype-matched control (PharMingen, negative control). Cells were grown to 60% confluency in a medium with TSH (6H) and then shifted to a serum-deprived (0.2%) medium with TSH for 24 h. Cells were then treated, as appropriate, for 48 h. Data shown are representative of four different experiments with similar results. FITC, Fluorescein isothiocyanate. C, Untreated cells; D–G, FRTL-5 cells exposed to 30 mM D-glucose; IFN-{gamma}, cells treated with 100 U/ml IFN-{gamma}; IFN-{gamma}/D–G, cells treated with combination of IFN-{gamma} and D-glucose; dotted line, the isotype-matched control. 6H medium consists of Coon’s modified Ham’s F-12 supplemented with 5% calf serum, 2 mM glutamine, 1 mM nonessential amino acids, and 6H containing 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) (33 34 ); 5H medium has the same composition of 6H medium except for the absence of TSH.

 
The increase in MHC class I mRNA was accompanied by an increase in MHC class I molecules on the surface of the cells (Fig. 1BGo). Thus, there was a shift in the fluorescence intensity of D-glucose-treated cells, by comparison with control cells, fluorescence intensity being measured after staining cells with an MHC class I-specific antibody (Fig. 1BGo). The observed phenomenon was specific and not an osmotic effect. Thus, L-glucose at the same 30-mM final concentration had no effect on fluorescence intensity, by comparison with D-glucose. The same negative result with L-glucose was obtained in studies of mRNA levels (data not shown). As a result of these data, glucose refers to D-glucose in all subsequent experiments.

In the flow cytometry experiments (Fig. 1BGo), and in the previous experiments studying mRNA levels (Fig. 1AGo), we used serum (0.2%)-deprived medium to avoid any possible confusion that might be caused by variable glucose concentrations in the sera. We evaluated the possibility that serum deprivation could alter our data; glucose was therefore added to 6H medium containing 5% serum. The up-regulation of MHC class I molecules was still evident, as measured by flow cytometry or mRNA levels (data not shown).

Finally, we compared the ability of glucose and IFN-{gamma} to up-regulate MHC class I antigen expression. As shown in Fig. 1BGo, the increase in MHC class I expression induced by glucose is only slightly lower than that induced by 100 U/ml IFN-{gamma}, a maximally effective concentration of the cytokine (data not shown). More interestingly, however, simultaneous treatment with glucose and IFN-{gamma} caused a much greater, nearly additive, up-regulation of MHC class I molecules, by comparison with either agent alone, whether measured by flow cytometry (Fig. 1BGo) or mRNA levels (data not shown).

Glucose acts transcriptionally to increase MHC class I levels
The ability of glucose to increase MHC gene expression was linked to the hormone-sensitive region of the 5'-flanking region. Thus, deletion of the tissue-specific region of the 5'-flanking region between -1100 and -203 bp (Fig. 2Go) did not alter the glucose-induced reporter gene activity of cells transiently transfected with luciferase chimeras of the MHC PD-1 gene. In contrast, deletion of the hormone-sensitive region between -203 and -56 bp eliminated the glucose-induced increase in promoter activity. Results of transiently transfected cells treated with 30 mM glucose are as follows: 191 ± 12%, 170 ± 5%, 185 ± 17%, and 104 ± 7% for the -1100-bp, -549-bp, -203-bp, and -56-bp constructs, respectively, with all the controls arbitrarily set at 100%. The absolute level of expression, calculated as the ratio of luciferase activity to protein concentration (µg/ml), was 69.0 ± 7.4 vs. 36.2 ± 4.5 for the -1100-bp construct, 31.4 ± 5.6 vs. 18.5 ± 2.8 for the -549-bp construct, 64.8 ± 11.3 vs. 35 ± 4.5 for the -203-bp construct, and 3.7 ± 0.6 vs. 3.6 ± 0.4 for the -56-bp construct. Thus, in all constructs tested, except for the -56-bp insert, luciferase activity was significantly (P < 0.05) higher in glucose-treated cells than in controls.



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  		Figure 2. Diagramatic representation of the PD1 5' flanking region with its regulatory elements.

 
When we cultured FRTL-5 cells transfected with the -203-bp class I/luciferase chimera for 12, 24, 48, and 72 h with 20 or 30 mM glucose, the maximum increase in class I promoter activity was measured at 48 h in cells exposed to 30 mM glucose (Table 1Go and Fig. 3AGo). The effect of different concentrations of IFN-{gamma} is shown as comparison in Fig. 3BGo. As in the case of the mRNA and flow cytometry studies, the glucose effect was measurable only in the presence of TSH (6H medium) and not in cells maintained in 5H medium with no TSH (Fig. 3AGo). Also, as in the case of the mRNA and flow cytometry studies, the D-glucose increase in promoter activity was not duplicated by L-glucose (Fig. 4AGo) and was measurable in medium with 5% serum as well as 0.2% serum (Fig. 4BGo). Finally, as in the case of the mRNA and flow cytometry studies, the glucose effect on p-203 class I promoter activity was quantitatively similar to that of 100 U/ml IFN-{gamma} (Fig. 5Go), was additive with IFN-{gamma} in increasing class I gene expression in studies of cells maintained in 6H medium with TSH (Table 2Go and Fig. 5Go), and (unlike IFN-{gamma}) was not evident in cells maintained in 5H medium with no TSH (Fig. 5Go). These data indicated that, both qualitatively and quantitatively, the glucose effect on class I mRNA levels and antigen presentation could be fully explained by a transcriptional action of glucose.


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  		Table 1. Time and concentration effect of glucose on MHC class I promoter activity in FRTL-5 cells stably transfected with the p (-203) bp class I/luciferase chimera

 


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  		Figure 3. Effect of time and different glucose concentrations on the promoter activity of the p-203 class I/luciferase chimera. FRTL-5 cells, stably transfected with the p-203 class I/luciferase chimera, were grown to 60% confluency in a medium with TSH (6H) and 5% serum and then shifted to a 5H medium containing no TSH for 7 d. Cells were then shifted to a serum-deprived (0.2%) medium with (6H, black bars) or without (5H, white bars) TSH. A, After 24 h, cells were cultured in the presence of different glucose concentrations (10, 20, or 30 mM) for 24 or 48 h; B, after 24 h, cells were treated with different amounts of IFN-{gamma}, for 48 h, in the presence (+TSH, black bars) or in the absence (-TSH, white bars) of TSH. Data are the mean of three different experiments, performed in duplicate, ± SD for three different stable transfectant clones. *, Statistically significant increase of P < 0.05, or better, vs. controls.

 


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  		Figure 4. Effect of D- vs. L-glucose on MHC class I gene expression. A, FRTL-5 cells were grown to 60% confluency in 6H medium with TSH and 5% serum, then shifted to 5H medium containing no TSH for 7 d. Cells were washed and then transiently transfected with the p-203/luciferase chimera, using a DEAE-dextran procedure (Materials and Methods). Cells were then cultured for 24 h in the 6H 5% serum medium and then shifted to a serum-deprived medium (0.2%) with TSH. After 24 h, cells were finally exposed to 20 or 30 mM D- or L-glucose or maintained in the basal medium with 10 mM D-glucose (control). Data are the mean of four different experiments, performed in duplicate, ± SD. The absolute level of expression, calculated as the ratio of luciferase activity to protein (µg/ml) concentration, was 29.8 ± 7.6 for control, 59.1 ± 15.9 and 80.6 ± 12.9 for D-glucose (20 and 30 mM, respectively), and 32.3 ± 10.2 and 30.5 ± 6.8 for L-glucose (20 and 30 mM, respectively). B, We used FRTL-5 cells, stably transfected with the p-203 class I/luciferase, as previously described. After growing the cells to 60% confluency in 6H medium plus 5% serum, cells were shifted to 5H medium containing 5% serum; TSH (1 x 10-10 M) was added, where indicated, for 24 h. Finally, cells were exposed to 20 or 30 mM glucose for 48 h; black bars, 6H (+TSH) 5% serum; white bars, 5H (-TSH) 5% serum. Data are the mean of two different experiments, performed in duplicate, ± SD for three separate stably transfected clones. *, Statistically significant increase, P < 0.05.

 


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  		Figure 5. Effect of D-glucose, IFN-{gamma}, or both on MHC class I promoter activity. FRTL-5 cells, stably transfected with the p-203 class I/luciferase chimera, were treated as described in Fig. 3Go, then exposed to 30 mM glucose, 100 U/ml IFN-{gamma}, or a combination of the above, for 48 h, in the presence (+TSH, black bars) or in the absence (-TSH, white bars) of TSH. Data are the mean of two different experiments, performed in duplicate, ± SD for three separate stably transfected clones. *, Statistically significant increase, P < 0.05.

 

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  		Table 2. Additive effect of glucose and IFN-{gamma} after 48 h exposure at different concentrations on MHC class I promoter activity in FRTL-5 cells stably transfected with the p (-203) bp class I/luciferase chimera

 
One possible explanation for the different results in the presence or absence of TSH might be related to the different glucose uptake of quiescent vs. activated cells, e.g. FRTL-5 cells maintained without TSH (5H medium) vs. those maintained with TSH (6H medium), respectively. We therefore measured the glucose concentration in the medium of cells exposed to 30 mM glucose in the absence and presence of TSH, both at the beginning of the treatment and after 48 h of exposure to glucose. The glucose concentration, at the end of the treatment, in 6H medium is significantly (P < 0.05) lower than that measured in 5H medium (4.4 ± 1.5 mM vs. 16.6 ± 3.2 mM).

Glucose uptake in FRTL-5 cells is regulated mainly by Glut-1 (44, 45), whose concentration and cellular distribution are TSH and insulin dependent (44, 46). As shown in Fig. 6AGo, cells maintained with TSH, but without insulin (4H plus TSH), also lose their response to glucose but not IFN-{gamma}. Furthermore, treatment with 3 µM wortmannin, which has been shown to prevent the translocation of Glut-1 to the plasma membrane (46), significantly, albeit partially, reduced the ability of glucose to increase p-203 MHC class I promoter activity (Fig. 6BGo). These data are not only consistent with the possibility that the glucose action requires active glucose porting by Glut-1, they also implicate the PI3K system as an important signal in the glucose-induced up-regulation of MHC class I gene expression. Only partial inhibition of Glut-1 translocation and Akt activation after wortmannin treatment has already been described (46, 47) and has been previously explained as a cooperation between TSH and insulin in activating Akt in a non-PI3K-dependent manner (47).



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  		Figure 6. Effect of insulin withdrawal or addition of wortmannin on the ability of glucose to increase class I promoter activity. A, FRTL-5 cells, stably transfected with the p-203 class I/luciferase chimera, were grown to 60% confluency in 6H 5% medium and then shifted to medium containing no TSH and no insulin (4H) for 7 d. Cells were then exposed to a serum-deprived medium (0.2%) in the presence of insulin plus TSH (6H) or in the absence of insulin plus TSH (4H + TSH) for 24 h. Cells were then exposed, for 48 h, to 20 or 30 mM glucose as indicated; black bars, 4H + TSH (-INS+TSH); white bars, 6H (+INS+TSH). Data are the mean of two different experiments, performed in duplicate, ± SD for each of three separate stably transfected clones. 4H medium consists of Coon’s modified Ham’s F-12 supplemented with calf serum, 2 mM glutamine, 1 mM nonessential amino acids, and a hormone mixture containing cortisol (0.4 ng/ml), transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml). Where appropriate, insulin (10 µg/ml) and/or bovine TSH (1 x 10-10 M) were added. *, The statistically significant increase induced by glucose vs. control is P < 0.05; **, the statistically significant increase induced by IFN-{gamma} + glucose vs. INF-{gamma} alone is P < 0.05. B, Cells, transiently transfected as described earlier, were cultured, where indicated, with 3 µM wortmannin for 6–12 h before exposure to glucose; black bars, 6H control cells; white dotted bars, 6H-wortmannin-treated cells. Data presented are the mean of four different experiments performed in duplicate ± SD. The absolute level of expression, calculated as the ratio of luciferase activity to protein concentration (µg/ml), was: 35.3 ± 9.4 and 97.8 ± 17.7 for control and glucose-treated cells, respectively, in the absence of wortmannin; and 40.3 ± 6.1 and 63.5 ± 11.8 for control and glucose-treated cells, in the presence of wortmannin. *, The statistically significant increase induced by glucose vs. control is P < 0.05; **, the statistically significant reduction in glucose effect by wortmannin is also P < 0.05.

 
Data on different deletion constructs would suggest that the transcriptional action of high glucose involves modulation of elements in the hormone-sensitive region of the 5'-flanking region of the class I promoter, -203 to -56 bp. This region contains three major elements: enhancer A, the IFN-response element (IRE), and the downstream regulatory element (DRE) whose activity depends on a cAMP-response element (CRE)-like element between -107 and -100 bp. To evaluate which of these, if any, might be involved in the transcriptional effects of high glucose, we mutated or deleted the CRE, the IRE, and the enhancer A elements. The mutations were those described (see Materials and Methods; Refs. 26 , 27 , 30, 31, 32 ; Fig. 7BGo) and are known to cause a loss of function of the particular element. Mutation of enhancer A and deletion of the CRE completely prevented the ability of glucose to increase class I promoter activity, whereas deletion of the IRE had no effect on glucose activity (Fig. 7Go, A and B). Aside from establishing that integrity of the CRE and enhancer A were needed for glucose activity, the data provide a rationale for the independent and additive actions of glucose and IFN-{gamma}, because their actions seem to involve different elements.



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  		Figure 7. Glucose effect on wild-type and mutated p-203 class I/luciferase chimeras. A, Individually isolated clones, stably transfected with the wild-type (p-203bp) class I/luciferase chimera or with mutated p-203 constructs containing either a mutation of the enhancer A (p-203Mut A), deletion of the IRE (p-203{Delta}IRE), or deletion of the CRE (p-203{Delta}CRE), were grown to 60% confluency in 6H medium with 5% calf serum and then maintained without TSH for 7 d. Cells were then cultured in a serum-deprived medium (0.2%) containing TSH (6H) for 24 h and finally exposed to glucose 30 mM for 48 h. Controls have been arbitrarily set at 100%. Data are the mean ± SD of four experiments, performed in duplicate, using three separate clones containing each construct. *, Statistically significant increase, P < 0.05. B depicts the mutations and a diagrammatic representation of the deletions.WT, Wild type; MA, mutation of enhancer A.

 
We performed an EMSA with a 74-bp probe encompassing the region between -203 and -130 bp that includes the IRE and enhancer A. The slowest migrating protein complex was more prominent in the extracts from glucose-treated cells (30 mM), by comparison with control cells (Fig. 8AGo, lane 2 vs. 1). This complex has been previously defined as MOD-1 and has been shown to be a complex involving enhancer A and a heterodimer of fra-2, a c-fos family member, and the p50 subunit of NF-{kappa}B. Competition with an oligonucleotide having the sequence of wild-type enhancer A (TGGGGAGTCCCCGTG), but not mutated enhancer A (TGCCGAGTCAAGGTG), prevented the formation of MOD-1 (Fig. 8AGo, lanes 4 and 3, respectively). Additionally, antibodies directed against fra-2 and the p50 subunit of NF-{kappa}B supershifted MOD-1 (Fig. 8BGo, lanes 6 and 4, respectively), whereas antibodies directed against ets-1 and the p65 subunit of NF-{kappa}B did not (Fig. 8BGo, lanes 5 and 3, respectively).



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  		Figure 8. Ability of glucose to increase formation of the MOD-1 complex with enhancer A. EMSA was performed, as detailed in Materials and Methods, using cellular extracts from FRTL-5 cells treated with or without glucose and using the -74-bp probe, which spans the region between -203 and -130 bp from the transcription start site. A, Lanes 1 and 2, incubations of radioactive probe with extracts from control and glucose-treated (30 mM) cells; lanes 3 and 4, incubations of radioactive probe with extracts from cells exposed to 30 mM glucose but preincubated with an excess amount (100x) of unlabeled oligonucleotide containing the wild-type (lane 4) or mutated (lane 3) sequence of the enhancer A (ENH-A) element; lane 5, incubation of radioactive probe with extracts from cells exposed to 30 mM glucose but preincubated with an excess amount (100x) of the unlabeled probe; lane 6, radioactive probe alone; arrows, MOD-1 complex. B, lane 1, radioactive probe alone; lane 2, incubation of radioactive probe with extracts of glucose-treated (30 mM) cells; lanes 3–6, incubations of extracts from the same glucose-treated cells, the radioactive probe, and rabbit serum containing monoclonal antibodies to the noted transcription factors. Arrow, Complex that is supershifted by antibodies against fra-2 and p50.

 
Hydrocortisone and high concentrations of iodide, phorbol esters, and TGF-ß1, all of which down-regulate MOD-1, decrease class I gene expression; whereas, thymosin-{alpha}1, which increases formation of this complex, up-regulates class I gene expression (26, 27, 30, 32). The presumption from these associations is that increased MOD-1 increases class I activity, whereas decreased MOD-1 decreases class I expression. This presumption has been directly supported by transfection data using fra-2 and the p50 subunit of NF-{kappa}B (27). In sum, these data are consistent with the conclusion that one action of high glucose is to increase MOD-1 and thereby increase MHC class I gene expression.


   Discussion
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 Abstract
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 Materials and Methods
 Results
 Discussion
References
 
The present study shows that high glucose levels act transcriptionally to increase MHC class I expression in thyroid cells. It also shows that TSH and insulin are both essential for the expression of this glucose effect, because deprivation of just one of the two hormones completely prevented the glucose action.

In FRTL-5 cells, the uptake of glucose has been shown to be mainly dependent on Glut-1, which is regulated by both TSH and insulin (44, 45, 46). TSH and insulin are required both for full expression of the Glut-1 protein and for translocation of the Glut-1 from the intracellular compartment to the plasma membranes (46). Consistent with the hypothesis that TSH/insulin-increased Glut-1 activity might be critical for the glucose effect, we show that wortmannin, which is able to block the translocation of Glut-1 to plasma membrane (46), is a significant inhibitor of the glucose action on class I and that TSH/insulin increase glucose utilization by the cells. Wortmannin has been shown to affect the transcriptional activation of Glut-1 gene and is effective in inhibiting the hormone-induced translocation of this glucose transporter. It seems, therefore, reasonable to explain our data by modulation of already existing and newly synthesized proteins. In this regard, evidence exists that AKT or PKB is an important kinase involved in signaling Glut-1 translocation (48) and, in FRTL-5 cells, is modulated by both insulin and TSH (49). The wortmannin data thus provide a clue that an important signal path involved in glucose regulation of MHC class I expression involves PI3K.

To ensure specificity of the glucose effect, we showed that L-glucose had no effect. Whereas L-glucose was shown to be completely ineffective, we did find that fructose was able to partially mimic the glucose effect (data not shown). Some of the transcriptional effects of high glucose levels, e.g. induction of plasminogen activator inhibitor-1 and TGF-ß1 expression, have been recently explained by the shunt of glucose into the hexosamine pathway, whose rate-limiting enzyme is glutamine:fructose-6-phosphate amidotransferase (22). Because fructose-6-phosphate is a metabolite in the fructose path, it is conceivable that fructose, which is usually transported by Glut-5 and secondarily by Glut-2, can partially duplicate the glucose effect. These possibilities are being directly investigated, as are the links of Glut-1 activation to the high glucose effects on MHC class I.

The transcriptional effect of glucose on MHC class I gene expression reflects its action on two different elements in the hormone-sensitive region of the 5'-flanking region of the class I gene, -203 to -56 bp enhancer A and the CRE. The interrelationship of these two elements in hormone and cytokine regulation of MHC class I expression has previously been reported (27, 30, 32); thus, TGF-ß1, iodide, and thymosin-{alpha}1 have been shown to require both enhancer A and the CRE to achieve their effects on MHC class I gene expression. High glucose therefore seems to act in the same manner. Indeed, we previously showed (27) that the enhancer A and the CRE-like element are functionally linked and mutually interacting; this interaction has been demonstrated by cross-competition experiments showing the ability of oligonucleotides with the sequence of one element to inhibit the binding properties of the other. This interaction can also explain the absence of effect with mutation of either element.

The CRE-like element, -107 to -100 bp from the transcription start site, lies within a wider element (-127 to -90 bp) termed the DRE (28). The DRE is regulated by TSH and forskolin (31), which increase cAMP levels and induce the formation of new regulatory complexes with the DRE, involving both thyroid-specific (thyroid transcription factor-1, Pax-8) as well as ubiquitous transcription factors (thyrotropin receptor suppressor element protein-1, activating transcription factor-1, CRE binding protein). Currently, we are investigating the effects of high glucose on these complexes with the DRE. It is noteworthy in this regard, however, that glucose has been shown to affect iodide uptake and hormone synthesis in thyroid cells (50) and that the thyroid-specific transcription factors involved in transcriptional regulation of thyroid-specific genes important for iodide uptake and thyroid hormone synthesis also regulate MHC class I expression (33, 35, 51, 52). It is therefore conceivable that glucose can modulate the thyroid-specific (as well as ubiquitous) trans factors involved in complexes with the DRE or CRE.

Interestingly, it has been shown (53) that glucose induces c-fos and its family members only in the presence of high cAMP levels. This may be relevant to this and our previous study (27, 28) that shows fra-2 is binding the p50 subunit of NF-{kappa}B, thus forming the MOD-1 complex with enhancer A. Also relevant are reports using different cell types that showed hyperglycemia activates the NF-{kappa}B transcription factor (23, 54), which is also induced by akt kinase (55). It seems, therefore, reasonable that activation of both c-fos or its family members (fra-2) and NF-{kappa}B, by high glucose levels, can facilitate the formation of MOD-1, which is associated to increased MHC class I regulation. Indeed, in a previous report (28), we have shown that overexpression of fra-2 and p50, by cotransfecting FRTL-5 cells with plasmids containing their cDNA, increases MHC class I gene regulation.

High glucose levels increase MHC class I surface molecules almost to the same degree of IFN-{gamma}. Moreover, treatment of cells with both glucose and IFN-{gamma} has an additive effect on MHC class I expression. A similar effect has already been described in pancreatic ß-cells (20); however, this is the first report, to our knowledge, that this phenomenon is observed in a cell that is not strictly glucose dependent. A 5- to 10-fold increase in MHC class I expression, as measured by mRNA levels, flow cytometry, and luciferase assays, might be relevant to the induction of autoimmune thyroiditis in type 2 diabetic patients, because MHC class I hyperexpression has been shown to be associated with increased autoimmune phenomena (17, 24, 35, 51). A relevant corollary observation may well be the high incidence of autoimmune thyroiditis in IFN-{alpha}-treated patients (56) who have no past evidence of thyroid abnormalities.

Type 2 diabetes is not usually considered an autoimmune disease; however, markers of cell autoimmunity can be detected in a significant percentage of patients. This subgroup of type 2 diabetic patients have been shown to be heterogenous in terms of age of diagnosis, requirement for insulin therapy, histocompatibility leucocyte antigen (HLA) class II genotyping, and cellular reactivity to islet proteins (8, 9, 57, 58). However, in this subgroup of patients, an increased incidence of a thyroid autoimmune phenomenon, as detected by TPO antibodies, has been reported (9). A genetic haplotype predisposing patients to autoimmune complications and a common antigen shared by thyroid follicular cells and pancreatic ß-cells have been proposed to be responsible for this association (9, 14). However, the HLA haplotype can only account for predisposition to the association of both type 2 diabetes and TPO antibodies; also, no increase in adrenal antibodies, which are associated in polyendocrine diseases, has been found. Moreover, reactivity to the 64-kDa protein proposed as the common antigen shared by thyroid and pancreatic cells has been detected in only one serum out of six tested in patients with chronic autoimmune thyroiditis (14).

Our data suggest an alternative or additional explanation for the autoimmune complications. High glucose levels might contribute to the onset of autoimmune complications, particularly chronic thyroiditis, by increasing MHC class I expression. Coordinate hyperexpression of both thyroid-specific autoantigens and MHC class I surface molecules might overcome self-tolerance and, in the presence of a cytokine stimulus, induce an autoimmune reaction.

To ensure the ability of high glucose levels to induce autoimmunity, studies of MHC class II regulation are necessary; preliminary data have shown that glucose can indeed up-regulate MHC class II genes. A manuscript in preparation (Montani, V., I. Bucci, G. Napolitano, C. Giuliani, C. Di Petta, L. D. Kohn, and F. Monaco) shows that hyperglycemia increases MHC class II gene regulation, especially in the presence of IFN-{gamma} (151 ± 7; 5,958 ± 82; and 15,950 ± 187 for glucose, IFN-{gamma}, and IFN-{gamma} plus glucose, respectively, vs. control arbitrarily set at 100% in stably class II/luciferase-transfected cells), thus further supporting the idea that high glucose levels might contribute to the induction of autoimmune diseases possibly in genetically susceptible individuals.

In summary, our data show that high glucose levels increase MHC class I gene regulation in FRTL-5 cells treated with TSH and insulin and that this increase is even more significant in the presence of IFN-{gamma}. We show that glucose acts, at least in part, by enhancing the binding of a heterodimeric complex of a c-fos family member, fra-2, and the p50 subunit of NF-{kappa}B to the enhancer A element of the class I 5'-flanking region. We speculate that this up-regulation of MHC class I gene may contribute to the increased incidence of autoimmune thyroiditis in type 2 diabetic patients.


   Footnotes
 
This work was supported by Juvenile Diabetes Foundation Innovative Grant 5-2000-750.

Abbreviations: CRE, cAMP-Response element; DEAE, diethylaminoethyl; DRE, downstream regulatory element; DTT, dithiothreitol; HLA, histocompatibility leucocyte antigen; IFN, interferon; IRE, IFN-response element; MHC, major histocompatibility complex; 6H, six-hormone mixture; TPO, thyroid peroxidase.

Received July 11, 2001.

Accepted for publication November 6, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C Giuliani, M Saji, I Bucci, G Fiore, M Liberatore, D S Singer, F Monaco, L D Kohn, and G Napolitano
Transcriptional regulation of major histocompatibility complex class I gene by insulin and IGF-I in FRTL-5 thyroid cells.
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