Regulation of Insulin Gene Transcription by a Ca2+-Responsive Pathway Involving Calcineurin and Nuclear Factor of Activated T Cells

Michael C. Lawrence, Harshika S. Bhatt, Jeannette M. Watterson and Richard A. Easom

Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas 76107-2699

Address all correspondence and requests for reprints to: Dr. Richard A. Easom, Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107-2699. E-mail: reasom{at}hsc.unt.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Immunosuppressants such as FK506 (tacrolimus), the primary cellular target of which is calcineurin, decrease ß-cell insulin content and preproinsulin mRNA expression. This study offers an explanation for this effect by establishing that calcineurin is an important regulator of insulin gene expression through the activation of a transcription factor, nuclear factor of activated T cells. Three putative nuclear factor of activated T cells binding sites were located within the proximal region of the rat insulin I gene promoter (-410 to +1 bp). Expression of nuclear factor of activated T cells in both clonal (INS-1) and primary (islet) ß-cells was confirmed by immunoblot and immunocytochemical analyses. Moreover, nuclear factor of activated T cells DNA-binding activity was detected in INS-1 and islet nuclear extracts by EMSAs. Activation of the insulin gene promoter by glucose or elevated extracellular K+ (to depolarize the ß-cell) was totally prevented by FK506 (5–10 µM). K+-induced promoter activation was suppressed (>65%) by a 2-bp mutation of a single nuclear factor of activated T cells binding site in -410 rInsI. Both stimulants also activated a minimal promoter-reporter construct containing tandem nuclear factor of activated T cells consensus sequences. The effects of FK506 on K+-induced nuclear factor of activated T cells reporter or insulin gene promoter activity were not mimicked by rapamycin, indicating specificity toward calcineurin. These findings suggest that the activation of calcineurin by ß-cell secretagogues that elevate cytosolic Ca2+ plays a fundamental role in maintenance of insulin gene expression via the activation of nuclear factor of activated T cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
POSTTRANSPLANT DIABETES MELLITUS is among the most serious adverse effects of immunosuppressive therapy using FK506 (tacrolimus) and is manifested by hyperglycemia, insulin resistance, and the appearance of islet cell antibodies (1). The incidence of diabetes in recipients of kidney transplants has been reported to be as high as 20% (2). The onset of diabetes may be even more widespread since tacrolimus is not only established for primary immunosuppression in liver and kidney transplantation, but has also been considered for therapies after solid organ transplantation of heart, lung, and pancreas (1).

The diabetogenic action of FK506 is not understood, but a direct effect on ß-cell function is invoked. The chronic administration of FK506 in vivo results in a marked but reversible reduction in insulin content of endocrine islets (3). This precedes morphological changes that are partially characterized by a loss of dense core secretory granules (4). Similar results are observed with the use of another immunosuppressant, cyclosporin A (CsA), although often to a lesser extent. FK506, or the structural analog L-683,590, also reduces insulin content and ultimately insulin secretion in isolated islets or cultured ß-cells in vitro (5, 6). Since FK506 does not acutely affect insulin secretion (7), these effects are presumed to be explained by a reduced capacity to synthesize insulin. At the molecular level, FK506 has been shown to reduce ß-cell preproinsulin mRNA expression (3, 5, 6) and dampen glucose activation of the insulin promoter (6). It is reasoned, therefore, that a primary effect of FK-506 is to interfere with the transcriptional activation of the insulin gene.

This mechanistic scenario is similar to the activated T cell in which the immunosuppressant properties of FK506 and CsA are accounted for by their common action to inhibit the Ca2+/calmodulin-dependent phosphatase 2B, calcineurin (8, 9, 10). Both compounds target calcineurin via their interaction with immunophilins, FK506 binding proteins (FKBPs) and cyclophilin for FK506 and CsA, respectively. Calcineurin is critically required for the induced expression of cytokine genes necessary for the initiation and coordination of an immune response (10). Under normal conditions, the action of calcineurin in the cell cytosol results in the dephosphorylation (on multiple serines) of NFAT (nuclear factor of activated T cells) (11, 12). The resultant exposure of a nuclear localization sequence promotes the rapid translocation of NFAT to the nucleus (13) where it binds, generally in cooperation with other trans-acting factors such as fos/jun components of activator protein-1 (14, 15), to cis-elements located in the promoters of several cytokine genes (9, 16). To date, most of the known therapeutic and toxic effects of FK506 and cyclosporin A are attributable to the inhibition of calcineurin (10).

Calcineurin is widely distributed among tissues (17), and several reports have documented its expression in islet cells of the endocrine pancreas (3, 6, 7, 18). It is now apparent that NFAT expression is also diverse in that it is detected in nonimmune tissues and cell types such as skeletal muscle, heart, neurons, adipocytes, and the pancreas (12, 19). The current study was therefore initiated to assess the involvement of NFAT in ß-cell gene expression. The evidence generated suggests that calcineurin, via NFAT, is an important regulator of insulin gene transcription and that the disruption of this pathway may contribute to the diabetogenic effects of FK506.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of NFAT Consensus Sites within the Insulin Gene Promoter
Examination of the first 410 bp of the rat I insulin gene promoter (-410 rInsI), a region that controls >90% of the transcriptional regulation of preproinsulin gene (23), revealed the presence of three putative NFAT binding sequences [consensus (T/A)GGAAA(A/N)(A/T/C), where N = any base] (Fig. 1Go). These sequences are located at positions -139 to -131 (1NFAT), -299 to -291 (2NFAT), and -308 to -316 (3NFAT) on -410 rInsI relative to the transcription start site (+1). Two of these binding sequences (1NFAT and 3NFAT) are positionally conserved in other mammalian insulin gene promoters, such as in human, mouse, and dog. The insulin gene promoter resembles other known NFAT-dependent promoters in that it displays, in common, a multiplicity of NFAT-binding sites (12), implying that higher-order interactions among NFAT-containing complexes are required for effective transcription.



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Figure 1. Multiple NFAT Consensus Sequences Are Present within the Insulin Gene Promoter

Shown is the promoter region (-410 bp to +1) for the rat insulin 1 (-410 rInsI) gene. The boxes represent select sequence elements previously identified as regulatory sites. The three conceptualized NFAT sites (1–3NFAT) are indicated above the promoter along with the DNA sequence conforming to the NFAT consensus motif.

 
NFAT Is Expressed in Pancreatic ß-Cells
Three independent pieces of evidence were acquired to support a role of NFAT in ß-cell function. First, the expression of NFAT in ß-cells was ascertained based on immunochemical analyses in rat pancreatic slices. Using an antibody raised against a peptide common to all known NFAT family members, NFAT immunoreactivity was primarily associated with islets of Langerhans with minimal reactivity in surrounding exocrine tissue (Fig. 2AGo). Within the islet, NFAT labeling was detected in cells also expressing insulin, supporting its association with the ß-cell. By this analysis, it was apparent that NFAT was also expressed in peripheral cells of the islet, but no further attempt was made to ascertain their identity as {alpha}, {delta}, or pp-cells. In addition, by immunoblot, NFAT was identified in isolated rat islets, as well as in the cultured clonal ß-cell line (INS-1) (Fig. 2BGo) and calculated to have an approximate Mr of 70,000. Furthermore, by EMSA, nuclear extracts of islets and INS-1 cells displayed specific binding activity toward DNA probes harboring NFAT consensus sequences from -410 rInsI. The NFAT DNA-binding complex supershifts in the presence of anti-NFAT antibody raised against a peptide sequence common to all known NFAT family members (Fig. 2CGo). The figure shown (2NFAT) is representative of what is exhibited from EMSAs of all three identified -410 rInsI promoter NFAT sites. Although two complexes were routinely resolved, only the upper band was found to represent a specific binding event under these experimental conditions based on its competition by excess NFAT DNA probe. These observations confirm a functional expression of NFAT in the ß-cell.



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Figure 2. NFAT Is Expressed in Pancreatic Cells

A, Immunocytochemistry: Cryosections of a rat pancreas double-stained (same section) with antiinsulin (left, Cy-2) and anti-NFAT796 (right, Texas-red) antibodies. B, Western analysis: Whole cell extracts from INS-1, pancreatic islets, and Jurkat cells were probed with an anti-NFATp antibody. C, EMSA: An NFAT probe (2NFAT) from the rat insulin I promoter was used to detect NFAT-DNA binding activity in INS-1 and pancreatic islet cells. The NFAT-DNA binding complex was competed with excess nonlabeled probe (2X, 20X, or 200X) incubated before (pre) or after (post) the addition of the radiolabeled NFAT probe of the insulin gene promoter. The lower band was not competed by the unlabeled probe, indicative of nonspecific binding. The NFAT-DNA binding complex was supershifted (complex indicated by arrow) in the presence of anti-NFAT796 antibody; lane1: no nuclear extract; lanes 2–4: INS-1 nuclear extract; lanes 5–7: islet nuclear extract; lanes 3 and 6: anti-NFAT796 Ab (Ab+); lanes 4 and 7: nonimmunized rabbit serum (Ab-).

 
Glucose and Potassium-Induced Insulin Gene Transcription
To permit an assessment of the involvement of calcineurin/NFAT signaling in insulin gene transcription, INS-1 ß-cells were transfected with an insulin promoter-reporter construct (pGL2-rInsI). The activity of this promoter was initially monitored in cells stimulated by glucose (11 mM) or by high extracellular concentrations (30 mM) of K+, conditions reasoned to induce the elevation of intracellular Ca2+ ([Ca2+]i) and the activation of calcineurin (24). Stimulatory concentrations of either glucose (11 mM) or K+ (30 mM) induced a similar (~7-fold) elevation in reporter enzyme activity (luciferase) within 6 h of stimulation relative to basal conditions (2 mM glucose, 5 mM K+) (Fig. 3AGo). Since high K+ induces cell depolarization without the influence of glucose metabolism, these observations support the conclusion that an elevation in [Ca2+]i is capable of enhancing insulin gene promoter activity.



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Figure 3. Glucose (Glc)- and K+-Induced Insulin Gene Transcription Is Inhibited by FK506

INS-1 cells were transfected with pGL2-rInsI and then incubated in basal (2 mM glucose) or stimulatory (11 mM glucose or 30 mM K+) media for 6 h. Inhibitors were added 2 h before cell stimulation. Luciferase activity in cell lysates was normalized with respect to CAT activity and expressed as fold over basal. A, FK506 (5 µM) or control vehicle (DMSO) was added. B, Increasing concentrations of FK506 (1–10 µM) were added. C, Increasing concentrations of rapamycin (1–10 µM) were added. Data are expressed as a fold increase in luciferase activity (normalized to CAT activity) over controls in the presence of 2 mM glucose. Data are means ± SE for three or more independent determinations.

 
Calcineurin Is Required for Up-Regulating Insulin Gene Promoter Activity
Significantly, the effect of glucose or K+ to enhance insulin gene promoter activity was antagonized by the presence of FK506, a selective inhibitor of calcineurin (Fig. 3Go). FK506 dose-dependently inhibited insulin promoter activity; complete inhibition was observed at a concentration of 5–10 µM FK506 (Fig. 3BGo). This effect appeared to be specific since it had no effect on the expression of a control vector (pSV-CAT) cotransfected with pGL2-rInsI. Furthermore, K+-induced insulin promoter activity was not inhibited by rapamycin (Fig. 3CGo), an analog of FK506 that binds the same intracellular receptor as FK506 (FKBP-12) but does not affect calcineurin activity (25, 26). In contrast, rapamycin inhibited insulin promoter activity driven by glucose nearly as efficiently as FK506. This latter observation suggests other signaling mechanisms independent of calcineurin are also necessary for glucose activation of insulin gene transcription.

The involvement of changes in [Ca2+]I in the activation of the insulin promoter by K+ was investigated using two different Ca2+ inhibitors. First, BAPTA, an intracellular Ca2+ chelator, completely blocked the effect of K+ to activate the insulin promoter (Fig. 4AGo). Second, verapamil, a selective inhibitor of L-type Ca2+ channels, also profoundly decreased this response to K+ (Fig. 4AGo), providing evidence that the activation of the insulin promoter was, at least in part, the result of Ca2+ influx. A direct effect of calcineurin to modulate insulin gene transcription was further demonstrated in INS-1 cells overexpressing a constitutively active form of calcineurin A (CaN{Delta}CaM-AI) lacking the autoinhibitory domain and a functional calmodulin binding domain. In cells cotransfected with pGL2-rInsI and constitutive calcineurin (CaN{Delta}CaM-AI) in basal glucose concentrations, reporter activity was increased 6-fold relative to cells transfected with the control vector (no calcineurin). This stimulation approximated reporter activation achieved in the presence of 30 mM K+ (Fig. 4BGo, cf. Fig. 3AGo). This effect was further heightened by the addition of 11 mM glucose, which enhanced reporter activities 14-fold over those observed under basal conditions (Fig. 4AGo). These observations demonstrate that calcineurin can directly up-regulate insulin gene transcription and enhance the ability of glucose to modulate the activity of the insulin promoter.



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Figure 4. Calcium and Calcineurin Can Modulate Insulin Gene Transcription

A, INS-1 cells were transfected with pGL2-rInsI and then incubated in basal conditions (2 mM glucose, 3 mM K+) or stimulatory K+ (30 mM) for 6 h. The cells were treated with calcium inhibitors verapamil or BAPTA for 2 h and then stimulated with 30 mM K+ for 6 h. B, INS-1 cells were cotransfected with pGL2-rInsI and constitutively active calcineurin A (pSR{alpha}–CaN{Delta}CaM-AI) or empty vector. Data are expressed as a fold increase in luciferase activity (normalized to CAT activity) over controls in the presence of 2 mM glucose. Data are means ± SE for three or more independent determinations.

 
Activation of NFAT in ß-Cells
To evaluate whether glucose and cell depolarization by K+ activate NFAT in ß-cells, INS-1 cells were transfected with an NFAT-reporter construct (NFAT-Luc) in which multiple NFAT-consensus sites were inserted upstream of a minimal promoter (IL-2) (8). Stimulatory concentrations of glucose (11 mM) and K+ (30 mM) increased NFAT-Luc reporter activity by 6-fold and 8-fold, respectively, over basal conditions (Fig. 5Go). In both cases, reporter activity was completely blocked by 5 µM FK506 as observed in cells transfected with pGL2-rInsI (cf. Fig. 3CGo). In contrast, rapamycin up to a concentration of 10 µM, had no significant effect on NFAT-mediated transcription induced by either glucose or K+. Thus, insulin secretagogues activate NFAT in pancreatic ß-cells by a calcineurin-dependent mechanism.



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Figure 5. Glucose and K+ Induce NFAT Activation in ß-Cells

INS-1 cells were transfected with a NFAT-Luc reporter construct and incubated with basal (2 mM glucose) or stimulatory (11 mM glucose, Glc, or 30 mM K+) conditions for 6 h. A, Cells were incubated in the presence of 10 µM FK506 (gray bar) or 10 µM rapamycin (striped bar). Control cells were supplemented with vehicle alone (white and black bars). B, Cells were incubated in increasing concentrations of FK506 (solid symbols) or rapamycin (open symbols). Data are expressed as a fold increase in luciferase activity (normalized to CAT activity) over controls in the presence of 2 mM glucose. Data are means ± SE for three or more independent determinations.

 
Effect of an NFAT-Mutated Insulin Gene Promoter on Transcription
To confirm the importance of calcineurin/NFAT in the activation of insulin gene transcription, site-directed mutagenesis was employed to eliminate an NFAT element from the insulin gene promoter. In light of the poorly defined binding of PDX-1 to A-box and the potential overlap with NFAT binding sequences, an NFAT site was chosen (2NFAT) that did not exist within either of the A and E element enhancers (Far-FLAT and Nir-P1) (Fig. 1Go). The double-point-mutated 2NFAT site does not disrupt any known elemental binding sites, such as that for pancreas duodenal homeobox-1 (PDX-1) or insulin enhancer factor-1, which are essential transcription factors for insulin gene promoter activity. There was little difference between luciferase reporter activities in cells transfected with wild-type (pGL2-rInsI) or mutant (pGL2–2NFATm) promoter constructs in the presence of stimulatory concentrations of glucose (Fig. 6AGo). In contrast, the mutation of 2NFAT resulted in a marked suppression (~68%) of luciferase reporter expression induced by depolarizing concentrations of extracellular K+. This mutation also resulted in the dramatic loss (~60%) of the insulin promoter to stimulation by the over-expression of constitutively active calcineurin (Fig. 6BGo).



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Figure 6. Elimination of 2NFAT Binding Element Reduces Insulin Promoter Activity in Response to K+

INS-1 cells were transfected with either rInsI-Luc (WT) or INS-2NFATm (2NFATm). A, Cells were incubated with 11 mM glucose or 30 mM K+ in the absence and presence of FK506 or rapamycin (5 µM each). B, rInsI-Luc or INS-2NFATm was cotransfected with a plasmid expressing constitutively active calcineurin (CaN; pSR{alpha}CaN{Delta}CaM-AI). Data are expressed as fold increase in luciferase activity (normalized to CAT activity) over controls in the presence of 2 mM glucose. Data are means ± SE for three or more independent determinations. *, P < 0.05 vs. rInsI-Luc, 2 mM glc).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In addition to its central role in the coordination of cytokine expression in the activated T cell, calcineurin is now known to influence transcriptional regulation in a variety of nonimmune cells (27). Most dramatic perhaps is its role in the transcriptional regulation of genes associated with hypertrophic growth in cardiac and skeletal muscles (28). Calcineurin has also been implicated in the transcriptional regulation of a noninsulin gene (i.e. glucagon) in the ß-cell (24). This study has now established that calcineurin has the capacity to directly modulate the insulin gene promoter. This is exhibited directly by the effect of overexpression of constitutively active calcineurin (CaN A) to up-regulate rInsI and is further supported by the attenuation of promoter activity by FK506. These data suggest that calcineurin may be required for physiological regulation of insulin gene expression by ß-cell stimuli.

Most significantly, the modulation of insulin gene transcription by calcineurin was found to be mediated via NFAT and thus is similar to that of the activated T cell. The observation that both primary (islet) and clonal ß-cells express NFAT was not unexpected based on the widened scope of detection of this transcription factor in nonimmune system cells. However, it is not yet determined which of the isoforms of this large multigene family of proteins (16) are represented in ß-cells. Nevertheless, the application of an NFAT promoter-reporter system demonstrates that the insulin secretagogues, glucose and K+, both activate NFAT in the ß-cell. Despite the fact that NFAT can be dephosphorylated by a number of phosphatases, the specific involvement of calcineurin is supported by the complete inhibition achieved in the presence of FK506, but not rapamycin. The same discriminatory sensitivity was observed with the rInsI insulin gene promoter, at least in response to K+, arguing that NFAT activation by calcineurin is also required for insulin gene expression under these conditions. This link is strengthened by the observation that the influence of calcineurin and K+ on insulin gene promoter activity were both significantly dampened by the 2-bp mutation of a single NFAT site (2NFAT) in this promoter. Collectively, these data establish a functional pathway by which calcineurin can modulate insulin promoter activity through the interaction of NFAT with specific sites and argue that NFAT should be added to the already large repertoire of transcription factors capable of influencing insulin gene transcription.

The lack of effect of rapamycin on K+-induced insulin promoter activity is in contrast to a previous study, which emphasized an autocrine effect of insulin secreted, in response to cell depolarization, on insulin gene transcription (29). This autocrine effect may be an important contributor to the regulation of insulin biosynthesis (30), but the identification of a calcineurin/NFAT pathway in the ß-cell forwards a direct mechanism by which Ca2+-responsiveness may be conferred on the insulin promoter (31, 32). Numerous other studies have established that cell depolarization-induced regulation of gene expression in the ß-cell is dependent on Ca2+ influx (33, 34). In the case of glucagon gene promoter, activation in HIT cells (ß-cells) is mediated by calcineurin modulation of cAMP response element binding protein interaction with a cAMP response element in this promoter (34, 35). Despite the presence of a cAMP response element within the -410 rInsI promoter, the context of this site does not appear to permit it to be responsive to FK506 (36). Consideration of these studies suggests that the prevention of NFAT activation is a primary mechanism by which FK506 perturbs insulin gene transcription.

The involvement of calcineurin in glucose regulation of the insulin promoter is less clear and confused by the observation that rapamycin mimics the effects of FK506 on glucose activation of the insulin promoter. This action of rapamycin has been observed previously (29). However, in the current study, glucose-induced activation of NFAT-reporter construct was refractory to rapamycin treatment, suggesting that the early cellular events of calcineurin and NFAT activation are not disrupted. Rather than inhibiting calcineurin, the rapamycin/FKBP12 complex targets mammalian target of rapamycin (also named FRAP, RAFT1, and RAPT1) (37) and then PHAS1 (38) and p70S6K (39), which are involved in the regulation of protein translation. The effect of rapamycin is unlikely to be a nonspecific, global inhibition of transcription because it has no effect on the insulin promoter activation induced by K+ or on the constitutive chloramphenicol transferase (CAT) expression from the control vector, pSV-CAT (data not shown). A potential suggestion, therefore, is that rapamycin affects the biosynthesis of insulin promoter-specific factors required to sustain insulin gene transcription induced by glucose. Alternatively, the inhibition of p70S6K, an integral component of the insulin signaling pathway, may interfere with an autocrine effect of secreted insulin to regulate its own transcription in the ß-cell (29), but this suggestion is minimized by the lack of effect of rapamycin on K+-induced activation of -410 rInsI (see above). Considering the increased complexity of glucose signaling relative to cell depolarization (23), it is more likely that rapamycin interferes indirectly with some aspect of transcriptional regulation by glucose. In any case, it is evident that there are at least two distinct pathways arising from glucose metabolism that effect insulin gene transcription: a rapamycin-sensitive pathway and a calcium-dependent (FK506-sensitive) pathway (Fig. 7Go). The rapamycin-sensitive pathway, which provides factors that are responsible for determining the full effect of glucose-stimulated insulin gene transcription, requires the glucose-induced calcium-dependent pathway.



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Figure 7. Schematic of the Activation of the Insulin Gene Promoter by Glucose

High glucose (11 mM) activates the insulin gene transcription by at least two distinct pathways. The calcium-dependent pathway involves calcineurin and NFAT, whereas the rapamycin-sensitive pathway involves factors derived or activated by glucose metabolism which target the insulin gene promoter.

 
A more extensive interaction of trans-acting factors on -410 rInsI, relative to K+, may account for the lack of effect of 2NFATm mutation on glucose-induced activation of the insulin promoter. This site may have greater significance in the context of the action of incretins, such as GLP-1, which can heighten intracellular calcium and up-regulate insulin gene transcription to a higher level than induced by glucose alone (40). In fact, our preliminary data (not shown) suggest that 2NFAT is important for maximal promoter activity in the combined presence of glucose and GLP-1. Divergent heterologous partnering between transcription factors interacting among the distinctly arranged NFAT sites may account for differences in response to a complex combination of integrated signals to which the ß-cell is exposed. Furthermore, preliminary experiments, in which we tested the effect of mutations of the other NFAT sites, suggest that 1NFAT may be most important to glucose signaling, and may therefore form the primary target of FK506 in experimental conditions involving glucose alone (data not shown).

Curiously, of the two NFAT sites that are conserved among mammalian insulin promoters, the site most proximal to the transcriptional start site is in close proximity to the A2 binding for the homeodomain protein PDX1 that is acutely activated by glucose (41, 42). This site also overlaps with a CAAT/enhancer box binding site for CAAT/enhancer-binding protein ß, a known repressor of insulin gene transcription but only in conditions of persisting hyperglycemia (45). Although deciphering the significance of this NFAT site may prove challenging, trans-acting factors to these sites may represent intricate mechanisms by which the ß-cell fine tunes the activity of the insulin gene promoter in response to various signals. Extensive studies in immune-system cells have shown that NFAT commonly, but not always, binds to DNA in concert with a partner, e.g. AP-1 activator protein, from the bZIP family of transcription factors (12, 43). A full understanding of how NFAT regulates the -410 rInsI promoter in the ß-cell thus hinges on the identification of other factors with which it cooperates and the DNA sequences with which they interact.

In summary, this study has demonstrated that calcineurin regulates insulin gene transcription via a mechanism involving NFAT interaction with specific elements within the insulin promoter. It is suggested that the disruption of this pathway in vivo under chronic FK506 treatment contributes to the diabetogenic effect of immunosuppressant therapy involving FK506. It is worth noting that immunosuppressant therapies involving low-dose FK506 treatment result in long-term survival of islet transplants (44). The further study of this mechanism is necessary to permit the development of new pharmacological approaches that clinically prevent tissue rejection with reduced risk of posttransplant diabetes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
INS-1 and Jurkat cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, streptomycin (100 µg/ml), and penicillin (100 U/ml) at 37 C under an atmosphere of 95% air/5% CO2.

Plasmids and Mutagenesis
A vector construct (pSYNT) harboring the promoter region (-410 to +1) of the rat insulin 1 gene (-410 rInsI) (20) was kindly provided by Dr. M. German (San Francisco, CA). The -410 rInsI fragment was amplified by PCR with primers incorporating a 5'-XhoI linker and directionally cloned into the pGL2-Basic luciferase promoter-reporter mammalian expression vector (Promega Corp., Madison, WI). The resultant construct is designated pGL2-rInsI. Mutagenesis of the second NFAT site within the insulin gene promoter (2NFAT) (Fig. 1Go) was achieved by a four-primer mutagenesis method designed to create two-point mutations and thus the disruption of the core NFAT sequence (5'-GGAAA to 5'-TCAAA). The PCR fragment was cloned into the pCR2.1 TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced for verification of the site-directed point mutations. The mutated fragment was then amplified to incorporate 5'-XhoI linker and cloned into pGL2-Basic as described above. The expression vector, pSR{alpha}CaN{Delta}CaM-AI, harboring constitutively active calcineurin A (CaN-A) in which the calmodulin binding and autoinhibitory domains were deleted (21), was a generous gift from Dr. Stephen O’Keefe (Merck Research Laboratories, Whitehouse Station, NJ). For calcineurin overexpression experiments, a control vector was generated by religation of pSR{alpha} after restriction enzyme digestion to eliminate the CaN-A insert. The NFAT-luciferase (NFAT-Luc) reporter plasmid was a generous gift of Dr. Gerald Crabtree (Stanford, CA).

Isolation of Islets
Pancreati were isolated from male Wistar rats by collagenase P (Roche Molecular Biochemicals, Indianapolis, IN) digestion followed by centrifugation on a discontinuous Ficoll gradient. Islets were cultured in CMRL-1066 containing 5.5 mM glucose and supplemented with 2 mM L-glutamine, 10% heat-inactivated FBS, 100 µg/ml streptomycin, and 100 U/ml penicillin overnight at 24 C under an atmosphere of 95% air/5% CO2. Immediately before experimentation, the islets were incubated at 37 C for a minimum of 60 min.

Western Blot Analysis
Cell extracts from INS-1, pancreatic islets, and Jurkat cells were prepared by lysis in Laemmli buffer. Samples were boiled for 5 min and loaded (30 µg protein per lane) on an SDS-6% polyacrylamide gel. The proteins were electrotransferred to a nitrocellulose membrane (Osmonics, Westborough, MA) and blotted with affinity-purified NFATp antibody (a gift from Dr. Karen L. Leach, Pharmacia & Upjohn, Inc., Kalamazoo, MI). Washes were done in PBS with 0.1% polyoxyethylene sorbitan monolaurate (Tween-20). The enhanced chemiluminescence system was used as the method of detection by a secondary goat antirabbit IgG antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, Piscataway, NJ).

Immunocytochemistry
Rat pancreati were excised from Wistar rats and fixed on ice for 4–6 h by immersion in PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 100 mM Na2HPO4, pH 7.2) supplemented with 4% paraformaldehyde. After overnight equilibration at 4 C in PBS containing 30% sucrose, the pancreati were embedded in tissue freezing medium (OCT compound) and cryosectioned (~70 nm) as previously described (22). On the day of immunocytochemistry, frozen pancreatic sections were rehydrated and permeabilized with PBS containing 0.2% Triton X-100 and blocked with PBS containing 4% BSA and 5% serum from the host animal species in which the secondary antibody was raised. Incubations with primary antibodies anti-NFAT796 (a generous gift of Dr. Nancy Rice, ABL-Basic Research Program, Frederick, MD) or antiinsulin (1:200 dilution) (Linco Research, Inc., St. Charles, MO) were continued overnight at 4 C and followed by incubation with fluorochrome-conjugated secondary antibodies (1:200) for 1 h at 37 C. All washes were done in PBS containing 0.1% Triton X-100. Visualization of slides was conducted on a Nikon Microphot FXA microscope.

EMSAs
Complementary oligonucleotides (5'-ATGAGGTGGAAAATGCTCAG) containing a -410 rInsI NFAT consensus site (2NFAT) were synthesized (Genosys, MO), hybridized, and end-labeled by T4 polynucleotide kinase (Amersham Pharmacia Biotech) in the presence of [{gamma}-32P]-ATP. INS-1 cells (~4 x 106 cells) were lysed in 400 µl of buffer A [10 mM Tris (pH 8.0), 1.5 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 10 mM NaF, 0.6% NP-40, 1 mM dithiothreitol (DTT), 0.5 phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin]. Nuclear pellets were spun down and resuspended in 50 µl buffer B [10 mM Tris (pH 8.0), 1.5 mM MgCl2, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin] to harvest extracts. Equal amounts of nuclear extract (20 µg) were incubated for 30 min with double-stranded 32P-labeled NFAT probe (20,000 cpm) in reaction buffer (10 mM Tris, pH 8.0, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 6% glycerol). Increasing amounts (2-, 20-, or 200-fold after; 2- or 20-fold before) excess of cold probe were added to competition reactions either 15 min before or after the labeled probe. Anti-NFAT antibody was added 15 min after labeled probe in supershift experiments. The reactions were subjected to electrophoresis on 6% polyacrylamide gels, and bands were detected using a Packard Instant Imager Electronic Autoradiography System (Packard, CT).

Cell Transfections and Reporter Assays
INS-1 cells were cultured in 12-well plates in RPMI medium as described, and then brought to 2 mM glucose 6 h before transfection. INS-1 cell transfection was achieved using FuGene-6 (Roche Molecular Biochemicals) according to the manufacturer’s directions. All cells were cotransfected with a control vector (pSV-CAT) for the normalization of transfection efficiency. Eighteen hours after transfection, the cells were stimulated with either 11 mM glucose or 30 mM KCl. In the latter case, the cell incubations (post 18 h) were performed using a modified Krebs Ringer bicarbonate medium (25 mM HEPES, pH 7.4, 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2) with 0.1% BSA; a 30 mM KCl isotonic Krebs Ringer bicarbonate solution was generated by adjusting the relative concentrations of KCl and NaCl to 30 mM and 90 mM, respectively. For inhibitor studies, FK506 (1, 5, 10 µM) or rapamycin (1, 5, 10 µM) was added to the media 2 h before cell stimulation. The cells were harvested 24 h after transfection by lysis in Reporter Lysis Buffer, (Promega Corp., Madison, WI). After brief centrifugation (~16,000 x g, 5 min) to remove cell debris, the supernatant was assayed for luciferase activity based on Luciferase Assay System (Promega Corp.) using a TD-20/20 bioluminometer (Turner Designs) or CAT activity by the CAT-ELISA method (Roche Molecular Biochemicals).

Statistical Analysis
Statistical significance was calculated by one-tailed t test.


    ACKNOWLEDGMENTS
 
The authors wish to thank Anne Marie Brun for generating pancreas cryosections.


    FOOTNOTES
 
This work was supported by a grant (009768-022 to R.A.E.) from the Advanced Research Program of the Texas Higher Education Coordinating Board.

Abbreviations: [Ca2+]i, Intracellular Ca2+; CAT, chloramphenicol acetyltransferase; CsA, cyclosporin A; DTT, dithiothreitol; NFAT, nuclear factor of activated T cells; PDX-1, pancreas duodenal homeobox-1; rInsI, rat I insulin gene promoter.

Received for publication November 10, 2000. Accepted for publication June 8, 2001.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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