Conceived and designed the experiments: JB MN. Performed the experiments: JB MN. Analyzed the data: JB MN. Wrote the paper: JB MN.
The authors have declared that no competing interests exist.
Posttransplantation diabetes mellitus (PTDM) is a frequent complication in immunosuppressive therapy. To better understand the molecular events associated with PTDM we investigated the effect of cyclosporine on expression and activity of hepatic nuclear factor (HNF)1alpha and 4alpha and on genes coding for glucose metabolism in cultures of the rat insulinoma cell line INS-1E, the human epithelial cell line Caco-2 and with Zucker diabetic fatty (ZDF) rats. In the pancreas of untreated but diabetic animals expression of HNF4alpha, insulin1, insulin2 and of phosphoenolpyruvate carboxykinase was significantly repressed. Furthermore, cyclosporine treatment of the insulinoma-1E cell line resulted in remarkable reduction in HNF4alpha protein and INS1 as well as INS2 gene expression, while transcript expression of HNF4alpha, apolipoprotein C2, glycerolkinase, pyruvatekinase and aldolase B was repressed in treated Caco-2 cells. Furthermore, with nuclear extracts of cyclosporine treated cell lines protein expression and DNA binding activity of hepatic nuclear factors was significantly repressed. As cyclosporine inhibits the calcineurin dependent dephosphorylation of nuclear factor of activated T-cells (NFAT) we also searched for binding sites for NFAT in the pancreas specific P2 promoter of HNF4alpha. Notably, we observed repressed NFAT binding to a novel DNA binding site in the P2 promoter of HNF4alpha. Thus, cyclosporine caused inhibition of DNA binding of two important regulators for insulin signaling, i.e. NFAT and HNF4alpha. We further investigated HNF4alpha transcript expression and observed >200-fold differences in abundance in n = 14 patients. Such variability in expression might help to identify individuals at risk for developing PTDM. We propose cyclosporine to repress HNF4alpha gene and protein expression, DNA-binding to targeted promoters and subsequent regulation of genes coding for glucose metabolism and of pancreatic beta-cell function.
In organ transplantation there is a need to suppress an immune response against the
grafted organ. Immunosuppressive therapies with calcineurin inhibitors result,
however, in unwanted secondary effects. This includes risk of infections of all
types, lymphomas and other malignancies
Taken collectively, HNF1α and HNF4α regulate various members of the
glucose-dependent insulin secretory pathways
Initially, we investigated expression of
(A) HNF4α western blotting of 20 µg Caco-2 cell nuclear extracts [control or cyclosporine treatment, 25 µM (30 µg/ml) for 72 h]. (B) HNF1α western blotting of 30 µg Caco-2 cell nuclear extracts [control or cyclosporine treatment, 25 µM (30 µg/ml)for 72 h] (C) Actin western blotting of 15 µg Caco-2 cell nuclear extracts [control or cyclosporine treatment, 25 µM (30 µg/ml) for 72 h]. The lower panels represent the quantification of protein amounts for HNF4α (A) and HNF1α (B) relative to the actin expression. (D) Electrophoretic mobility shift assays with 2,5 µg Caco-2 cell nuclear extract [control or cyclosporine treatment, 25 µM (30 µg/ml) for 72 h] and 32P labeled oligonucleotides to probe for DNA binding to HNF4α binding-sites within promoters of HNF1α (HNF1α), apolipoprotein C2 (ApoC2), glycerol kinase (GK), pyruvate kinase (PKLR), aldolase B (ALDOB), and insulin2 (INS2). In EMSA supershift assays an antibody directed against HNF4α (+) was added. Shifted (HNF4α) and supershifted bands (HNF4α ss) were marked. (E) Dried EMSA gels were analyzed with a Molecular Imager (BioRad, Muenchen, Germany) using the Quantity One software (BioRad, Muenchen, Germany). HNF4α binding of control extracts to the respective binding sites was set to 100% and inhibition of binding to the respective binding sites after treatment with cyclosporine [25 µM (30 µg/ml) for 72 h] was quantified.
Gene | Organ | Treatment | Mean±SD | % of the control | p-value |
Pancreas | Control | 0.013±0.002 | |||
ZDF 9 months | 0.008±0.004 | 61.5 |
|
||
|
Pancreas | Control | 0.857±0.849 | ||
ZDF 9 months | 0.365±0.541 | 42.6 |
|
||
|
Pancreas | Control | 0.146±0.076 | ||
ZDF 9 months | 0.109±0.217 | 74.5 |
|
||
|
Pancreas | Control | 0.960±0.487 | ||
ZDF 9 months | 0.456±0.871 | 47.5 |
|
||
Liver | Control | 1.379±0.611 | |||
ZDF 14 weeks | 0.835±0.365 | 60.6 |
|
||
|
Liver | Control | 1.180±0.330 | ||
ZDF 14 weeks | 0.694±0.228 | 58.8 |
|
Gene expression was measured by real-time qRT-PCR in 14 weeks and 9
months old ZDF rats and lean controls
(n = 10 animals, respectively) and was
determined relative to expression of cyclophilin, which served as a
housekeeping gene. Gene expression in control rats was set to 100 and
values for ZDF rats represent transcript abundance relative to control.
Non-parametric Mann-Whitney-U-Test was used to compare ZDF and control
groups. Results are considered significant at p<0.05 (gene names
and p-values in bold). Gene expression of HNF4α in the liver of
this cohort of ZDF rats has been previously reported
To further probe for HNF4α and HNF1α function we cultured the human
intestinal cell line Caco-2. This cell line enables mechanistic studies with
HNF4α protein expression being comparable to its expression levels in the
liver
(A) Electrophoretic mobility shift assays with 2,5 µg Caco-2 cell nuclear extract [control or cyclosporine treatment, 25 µM (30 µg/ml) for 72 h] and 32P labeled oligonucleotides to probe for DNA binding to the HNF1α binding-site within the HNF4α P2 promoter (HNF1-site in HNF4α P2). In EMSA supershift assays an antibody directed against HNF1α was added. Control and treated probes were run on same gels. (B) Dried EMSA gels were analyzed with a Molecular Imager (BioRad) using the Quantity One software (BioRad). HNF1α binding of control extracts was set to 100% and inhibition of binding after treatment with cyclosporine [25 µM (30 µg/ml) for 72 h] was quantified.
Gene | Treatment | Mean±SD | % of the control | p-value |
|
Control | 0.698±0.060 | ||
Cyclosporine | 0.267±0.008 | 38.3 |
|
|
HNF1α | Control | 0.910±0.094 | ||
Cyclosporine | 0.968±0.069 | 106.4 | 0.5127 | |
|
Control | 1.105±0.066 | ||
Cyclosporine | 0.601±0.251 | 54.4 |
|
|
ALDH2 | Control | 0.503±0.167 | ||
Cyclosporine | 0.539±0.063 | 107.2 | 0.8273 | |
PCK1 | Control | 1.056±0.136 | ||
Cyclosporine | 0.840±0.266 | 79.5 | 0.2753 | |
|
Control | 0.647±0.231 | ||
Cyclosporine | 0.251±0.098 | 38.8 |
|
|
|
Control | 0.784±0.229 | ||
Cyclosporine | 0.290±0.126 | 37.0 |
|
|
|
Control | 0.204±0.067 | ||
Cyclosporine | 0.035±0.025 | 17.2 |
|
|
NFATc1 | Control | 0.449±0.236 | ||
Cyclosporine | 0.498±0.065 | 110.9 | 0.5127 | |
NFATc2 | Control | 0.655±0.193 | ||
Cyclosporine | 0.495±0.196 | 75.6 | 0.5127 | |
NFATc3 | Control | 1.154±0.260 | ||
Cyclosporine | 0.938±0.134 | 81.3 | 0.2752 | |
NFATc4 | Control | 0.974±0.251 | ||
Cyclosporine | 0.793±0.151 | 81.4 | 0.2752 | |
Calcineurin | Control | 1.234±0.222 | ||
Cyclosporine | 0.906±0.533 | 73.3 | 0.5127 |
Gene expression was measured by RT-PCR in Caco-2 cells 72 h after treatment with 25 µM (30 µg/ml) cyclosporine (n = 3, respectively) and was determined relative to expression of mitATPase6, which served as a housekeeping gene. Gene expression in untreated Caco-2 cells was set to 100 and values for cyclosporine treatment represent transcript abundance relative to control. Non-parametric Mann-Whitney-U-Test was used to compare cyclosporine treated and control groups. Results are considered significant at p<0.05 (gene names and p-values in bold).
Gene | Treatment | Mean±SD | p-value |
HNF4α | Control | 1.373±0.347 | |
Tacrolimus | 1.166±0.127 | 0.5127 |
Gene expression was measured by real time qRT-PCR in Caco-2 cells 72 h after treatment with 25 µM (20 µg/ml) tacrolimus (Astellas Pharma GmbH, Munich, Germany) (n = 3, respectively) and was determined relative to expression of mitATPase6, which served as a housekeeping gene. Non-parametric Mann-Whitney-U-Test was used to compare tacrolimus treated and control groups. Results are considered significant at p<0.05.
To further confirm cyclosporine mediated dysregulation of HNF4α we analyzed
different rat and mouse beta cell lines, i.e. INS-1E, RINm5F and MIN6 cells, for its
(A) Cell viability of INS-1E cells after multiple treatments with cyclosporine for 72 h. (B) Actin western blotting of 10 µg INS-1E cell nuclear extracts [control or cyclopsorin treatment, 10 µg/mL (8.3 µM) for 72 h]. (C) Electrophoretic mobility shift assays with 20 µg INS-1E cell nuclear extract [control or cyclosporine treatment, 8.3 µM (10 µg/ml) for 72 h] and 32P labeled oligonucleotide to probe for DNA binding to the HNF4α binding-site within the promoter of HNF1α (HNF1α). In EMSA supershift assays an antibody directed against HNF4α (+) was added. Shifted (HNF4α) and supershifted bands (HNF4α ss) were marked. (D) Dried EMSA gels were analyzed with a Molecular Imager (BioRad) using the Quantity One software (BioRad). HNF4α binding of control extracts was set to 100% and inhibition of binding after treatment with cyclosporine [8.3 µM (10 µg/ml) for 72 h] was quantified.
Beta cell line | Species | Gene | % Expression |
INS-1E | Rat | HNF4α | 25.039±7.968 |
RIN-m5F | Rat | HNF4α | 1.289±0.071 |
MIN6 | Mouse | HNF4α | 0.094±0.026 |
HNF4α gene expression was measured by real time qRT-PCR in INS-1E, Rin-m5F or MIN6 cells after 6 days in culture (n = 3, respectively). Gene expression was determined relative to expression of mitATPase6, which served as a housekeeping gene. Gene expression in untreated liver was set to 100% and values for gene expression in beta cells were calculated respectively.
Gene | Treatment | Mean±SD | % of the control | p-value |
HNF4α | Control | 0.849±0.308 | ||
Cyclosporine | 0.984±0.066 | 0.5127 | ||
|
Control | 0.128±0.003 | ||
Cyclosporine | 0.087±0.004 | 68.0 |
|
|
|
Control | 1.076±0.237 | ||
Cyclosporine | 0.335±0.039 | 31.3 |
|
Gene expression was measured by real-time qRT-PCR in INS-1E cells 72 h after treatment with 8.3 µM (10 µg/ml) cyclosporine (n = 3, respectively) and was determined relative to expression of mitATPase6, which served as a housekeeping gene. Gene expression in untreated INS-1E cells was set to 100 and values for cyclosporine treatment represent transcript abundance relative to control. Non-parametric Mann-Whitney-U-Test was used to compare cyclosporine treated and control groups. Results are considered significant at p<0.05 (gene names and p-values in bold).
Taken collectively, HNF4α and HNF1α expression and DNA-binding
activity was repressed after cyclosporine treatment as was transcription of genes in
the glucose and insulin signaling pathways targeted by HNF4α and
HNF1α. Our study is the first report to determine a direct connection
between cyclosporine treatment and activity of hepatic nuclear factors and our
findings provide a molecular rational for PTDM observed in transplant patients. We
suggest individual differences in the HNF4α gene and protein expression
amongst patients to be of critical importance for the diabetogenic potential of
cyclosporine. Indeed, on average 1/10 of cyclosporine treated patients develop PTDM.
Consequently, repression of HNF4α by cyclosporine depends on the abundance
of HNF4α protein. In
Gene expression was determined by real-time qPCR in
n = 14 patients. Characteristics of
patients are given in
(A) Electrophoretic mobility shift assays with 2,5 µg Caco-2 cell nuclear extract [control or cyclosporine treatment, 25 µM (30 µg/ml) for 72 h] and 32P labeled oligonucleotides to probe for DNA binding to the NFAT binding site within the HNF4α P2 promoter (NFAT-site in HNF4α P2). In EMSA supershift assays an antibody directed against NFAT was added. Control and treated probes were run on same gels. (B) Dried EMSA gels were analyzed with a Molecular Imager (BioRad) using the Quantity One software (BioRad). NFAT binding of control extracts was set to 100% and inhibition of binding after treatment with cyclosporine [25 µM (30 µg/ml) for 72 h] was quantified.
HNF4α isoform | Mean±SD |
HNF4αP1 | 0 |
HNF4αP2 | 418.18±225.99 |
HNF4α isoform expression was measured by real time qRT-PCR in INS-1E cells after 6 days in culture (n = 3, respectively). Gene expression was determined relative to expression of mitATPase6, which served as a housekeeping gene. Gene expression in rat liver served as positive control for HNF4αP1 expression, gene expression in rat pancreas served as positive control for HNF4αP2 expression.
Patient Identification | Sex | Age | Tissue | Information |
P1 | F | 40 | Healthy tissue from liver resection | Colorectal liver metastasis |
P2 | M | 42 | Colorectal liver metastasis | |
P3 | F | 48 | Colorectal liver metastasis | |
P4 | F | 61 | Colorectal liver metastasis | |
P5 | F | 61 | Colorectal liver metastasis | |
P6 | M | 67 | Hepatocellular carcinoma | |
P7 | F | 70 | Hepatocellular carcinoma | |
P8 | F | 57 | Hepatocellular carcinoma | |
P9 | M | 67 | Hepatocellular carcinoma | |
P10 | M | 67 | Liver metastasis, stomach cancer | |
P11 | M | 72 | Liver metastasis, gastrointestinal stromal tumor | |
P12 | M | 69 | Colorectal liver metastasis | |
P13 | M | 76 | Hepatocellular carcinoma | |
P14 | F | 57 | Epitheloidal angiolipoma |
Patient material was used with a permission from the ethics committee of the Medical School Hannover, Germany.
In conclusion, cyclosporine repressed HNF4α/HNF1α expression, DNA-binding to targeted promoters and subsequent expression of genes involved in glucose metabolism and pancreatic β-cell function. We propose a molecular mechanism for PTDM based on dysregulation of HNF4α/HNF1α and of NFAT insulin signaling pathway targeted by cyclosporine.
Caco-2 cells, a human intestinal cell line derived from a colon adeno-carcinoma,
were obtained from and cultivated as recommended by DSMZ (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). INS-1E cells (rat
beta cells derived from insulinomas) were kindly provided by C. Wollheim
(University Medical Center, Geneva, Switzerland)
Pancreas (animals aged 9 months) and liver (animals aged 14 weeks) of fa/fa obese
Zucker diabetic fatty (ZDF) rats and of +/fa lean nondiabetic control
rats were kindly provided by W. Linz and H. Ruetten (Sanofi-Aventis, Frankfurt,
Germany)
Nuclear extracts were isolated by the method of Dignam et al
Total RNA was isolated using the nucleospin RNA Isolation Kit (Macherey-Nagel)
according to the manufacturers recommendations. 4 µg total RNA from
each sample was used for reverse transcription (Omniscript Reverse
Transcriptase, Qiagen, Hilden, Germany). PCR was done in a mixture containing a
cDNA equivalent to 25 ng of total RNA, 1 µM of each primer, 0.25 mM
dNTP mixture, 0.625 U Thermostart-Taq (Abgene, Hamburg, Germany) and
1× PCR-buffer (Abgene, with 1.5 mM MgCl2) in a total volume
of 20 µl. PCR-reactions were carried out with a thermocycler (T3,
Biometra, Göttingen, Germany) with the following conditions: initial
denaturation at 95°C for 15 min (Thermostart activation), denaturation
at 94°C for 30 sec, annealing at different temperatures for 45 sec (see
below), extension at 72°C for 45 sec, final extension at 74°C
for 10 min. The following primer pairs were used: HNF4α (human,
NM_000457), fwd:
Real-time RT-PCR measurement was done with the Lightcycler (Roche Diagnostics,
Mannheim, Germany) with the following conditions: denaturation at 94°C
for 120 sec, annealing at different temperatures for 8 sec (see below),
extension at 72°C for different times (see below), fluorescence at
different temperatures (see below). The PCR reaction was stopped after a total
of 40–45 cycles and at the end of each extension phase, fluorescence
was observed and used for quantification within the linear range of
amplification. Exact quantification was achieved by serial dilution with cDNA
produced from total RNA extracts using 1∶5 dilution steps. Gene
expression levels were normalized to cyclophilin, which was found to be stably
expressed. The following primer pairs were used: HNF4α (rat, NM_022180),
fwd:
All values are expressed as mean±standard deviation. To determine significance between two groups, comparison was made using the non-parametric two-tailed Mann-Whitney-U-Test. Therefore, Statistica software, version 7.1 (StatSoft) was used. The results are considered significant when the p value was less than 0.05.
We thank S. Marschke, A. Pfanne and A. Schulmeyer for valuable technical assistance, S. Reymann for assistance in bioinformatics and advice on design of PCR primers, Dres. W. Linz and H. Ruetten for providing pancreas of ZDF rats, Dr. C. Wollheim for providing INS-1E cells, Dr. J. Miyazaki for providing MIN6 cells and Dr. S. Lenzen for providing RIN-m5F cells.