Elevated PIN1 expression by C/EBPα-p30 blocks C/EBPα induced granulocytic differentiation via c-Jun in AML

Cells, transfections and reagents

Kasumi-6 cells were obtained from ATCC (Manassas, United States). Blast cells from AML patients were obtained from the Laboratory for Leukemia Diagnostics at the University of Munich. All samples were karyotyped and molecular genetics analysis was performed for C/EBPα mutations. Prior to therapy, all patients gave their informed consent for participation in the Acute Myeloid Leukemia Cooperative Group (AMLCG) studies. Details of the study protocol have been published ²⁰.

K562-C/EBPα-p42-ER and K562-C/EBPα-p30-ER cells ²¹ were maintained in RPMI 1640 without phenol red supplemented with 10% charcoal treated fetal bovine serum, 1% penicillin-streptomycin and 2 μg/ml puromycin; Kasumi-6 cells ²² were cultured in RPMI 1640 supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin and 2 ng/ml GM-CSF; AML blast cells were cultured in IMDM supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 20 mM HEPES; U937 cells and NB4 cells were cultured in RPMI supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin. K562-C/EBPα-p30-BRM2-ER cells were established as reported before ²¹. Briefly, C/EBPα-p30 was amplified by PCR using rat C/EBPα having BRM2 mutation (kind gift from Bruno Calabretta) with BamHI flanking primers (Forward primer 5’ GGG GAT CCG CCA CCA TGT CCG CGG GGG CGC AC 3’ and reverse primer 5’ATG GAT CCG GCG CGC AGT TG 3’) and subsequently cloned into pBabe-ER digested with BamHI with the 30-KDa C/EBPα peptide in frame with the C-terminal ER domain. K562 cells (10⁶ cells) were electroporated with AMAXA transfection method with 3 μg of ScaI linearized plasmid and plated on 6 well plate in phenol red free RPMI/10 % charcoal treated FBS. Selection with 1 μg/ml puromycin began 48 hours after transfection.

293T cells (2 × 10⁴ cells) were transfected with the LipofectAMINE Plus reagent (Invitrogen, Germany) according to manufacturer’s instruction. Transfection of U937 and Kasumi-6 cells was performed with the Nucleofector kit (AMAXA, Germany) as described by the manufacturer. 2 μg DNA plasmid were used for each transfection and the transfection efficiency was analyzed using a plasmid with eGFP marker. U937 and Kasumi-6 cells were transfected with nucleofection programmes V-01 and T-03, respectively. Transfection efficiencies of around 55-70% and 66-75% were observed in these cell lines, respectively.

Peptidylprolyl isomerase-parvulin inhibitor PiB (Calbiochem, United States) was prepared in ethanol and used at 5 μM. C/EBPα siRNA was purchased from Invitrogen.

Proteomics screening

To induce C/EBPα-p30, K562-C/EBPα-p30-ER cells were treated with 5 μM of β-estradiol for 6 hours followed by lysis. The protein identification by mass spectrometry was done essentially as reported before ⁶.

Promoter assay

293T cells were transiently transfected using LipofectAMINE (Invitrogen, Germany) as described by the manufacturer. Firefly luciferase activities from the PIN1 promoter constructs and Renilla luciferase activity from the internal control plasmid pRL-null were determined 24 h after transfection using the Dual-Luciferase Reporter Assay System (Promega, Germany). Firefly luciferase activities were normalized to the Renilla luciferase values of pRL-null.

Immunoblot analysis

Immunoblot analysis was performed with 50 μg total protein lysates using anti-C/EBPα (sc-61), anti-PIN1 (sc-15340), anti-c-Jun (sc-1694), anti-β-Tubulin (sc-9104) and anti-E2F1 (sc-251) antibodies from Santa Cruz Biotechnology, Germany. The immunodetection was performed with an ECL reagent (Amersham Biosciences, Germany). The band intensities were quantified using ImageJ software.

Chromatin Immunoprecipitation

The crosslinking of proteins to DNA was accomplished by the addition of 1% formaldehyde for 10 min to cultured cells (6×10⁶) at 37°C. After sonication, the chromatin was immunoprecipitated with 5 μg of the antibodies anti-E2F1 (sc-193X) or anti-IgG (SC-2027, Santacruz) at 4°C overnight.

Flow cytometry analysis

For flow cytometry analysis, 10⁶ cells were washed twice with PBS and resuspended in 50 μl of PBS with 2 μl of the respective antibody. Incubation was performed for 20 minutes in ice. After the incubation, cells were washed with PBS, resuspended in PBS and analyzed by flow cytometry on a FACScan (Becton Dickinson).

mRNA expression analysis

Total RNA was isolated from leukemic patient samples, processed and analyzed on the Affymetrix HG-U133A and HG-U133B chips as described before ²³. The data from Affymetrix analysis were normalized according to the procedure described before ²⁴. Normalized expression data were then analyzed with the R software package and the “boxplot” function (www.r-project.org). Expression signal intensities are expressed on a logarithmic scale.

Real-time RT-PCR

Total RNA was isolated from cells with Trizol reagent (Invitrogen, Germany). 750 ng of RNA was used to synthesize cDNA by Reverse Transcription. Equal amounts of cDNA were taken for a subsequent quantitative real-time PCR (Q-RT-PCR) using the SYBR Green PCR kit (Qiagen, Germany) in a Rotor-Gene RG-3000 cycler (Corbett Research, Australia). The fold change was calculated using delta C_T method reported before ²⁵.

Protein stability Assay

293T cells were transfected with 6.6 μg of PIN1 or non silencing siRNA (Qiagen, Germany) using lipofectamine. After 24 hours, cells were treated with cyclohexamide (100 μg/ml) for various periods and lysed. The amount of c-Jun protein was analysed by Western blot and quantified by densitometric scanning.

Ubiquitination Assay

293T cells were transiently transfected with different constructs as described for promoter assay, 24 hours after transfection cells were lysed in RIPA buffer followed by c-Jun immunoprecipitation from 500 μg total protein. The protein samples after immunoprecipitation were analysed in a 10% SDS-PAGE gel and probed with an HA antibody (Roche Applied Science, Germany).

Statistical analysis

We used Student’s t-tests to determine the statistical significance of experimental results. A P value of 0.05 or less was considered significant.

C/EBPα-p30 induces PIN1 expression in leukemia

The K562-ER cell line is an early multipotential cell line derived from the K562 cell line, which was originally obtained from a patient with chronic myeloid leukemia. To derive the K562-ER cell line, K562 cells were stably transfected with a plasmid encoding an estrogen inducible C/EBPα-p30 estrogen receptor fusion protein (C/EBPα-p30-ER)²¹. K562-ER cells have been shown to be a good model system to study granulopoiesis in the context of C/EBPα proteins (Supplementary figures 1 and 2). We have previously reported a proteomic approach for the identification of C/EBPα-p30 target proteins in C/EBPα-p30-ER cell line ⁶. In this screen, PIN1 was identified as one of the targets of C/EBPα-p30 (Figure 1a).

As a first step to elucidate the importance and role of PIN1 as a target of C/EBPα-p30, we investigated the PIN1 mRNA levels in leukemic patient samples. Affymetrix analysis showed that PIN1 mRNA expression is increased in different subtypes of leukemia compared to normal bone marrow controls (Figure 1b). We also observed elevated PIN1 levels in AML samples with C/EBPα mutation in comparison to AML samples without C/EBPα mutation (Figure 1c). The increased expression of PIN1 in different leukemic subtypes suggests that mechanisms other than C/EBPα mutations can increase PIN1 levels (See Discussion). Next we examined the ability of C/EBPα-p30 to induce PIN1 protein expression in the K562- C/EBPα-p30-ER cell line. Our data revealed that PIN1 protein expression was upregulated by C/EBPα-p30 during ß-estradiol induction (Figure 1d left, supplementary figure 3). These data confirm our proteomic findings and reveal the relevance of PIN1 as a target in leukemia. We also investigated regulation of PIN1 during C/EBPα-p30 overexpression in two AML cell lines - Kasumi-6 (C/EBPα mutation positive) and NB4 (C/EBPα mutation negative),). We observed that C/EBPα-p30 is able to up-regulate PIN1 (Supplementary figure 4). We observed slightly lower expression of PIN1 in Kasumi-6 cells in comparison to NB4 cells (Supplementary figure 4). Similar to the upregulation of PIN1 protein levels, we could observe an upregulation of PIN1 mRNA levels upon C/EBPα-p30 induction (Supplementary figure 5).

C/EBPα-p42 and C/EBPα-p30 have been shown to have different biological properties ³. Based on our finding that C/EBPα-p30 is able to induce PIN1 expression, we asked whether C/EBPα-p42 is able to regulate PIN1 protein levels. Our data show that in contrast to the C/EBPα-p30 form, C/EBPα-p42 repressed PIN1 protein levels (Figure 1d, right). This demonstrates that C/EBPα-p42 and C/EBPα-p30 have opposing effects on PIN1 protein levels. We could not find any regulation of PIN1 during C/EBPα-p42 silencing by siRNA (supplementary Figure 4). So we assume that silencing of C/EBPα-p42 alone is not sufficient for the regulation of PIN1.

We also analysed the role of C/EBPα with mutations in the E2F1 protein binding domain (C/EBPα-BRM2) ^{21, 26}, in regulating PIN1 protein expression. Our data suggests C/EBPα- BRM2 fails to downregulate PIN1 protein levels (data not shown). This denotes E2F1 interaction of C/EBPα-p42 is necessary for its inhibitory action over PIN1.

C/EBPα-p30 induces PIN1 promoter activity in association with E2F1

It has been shown previously that there are three E2F1 binding sites in the PIN1 promoter and E2F1 can transactivate PIN1 promoter activity ²⁷. Since we could observe C/EBPα-p30 induces PIN1 expression, we next investigated how C/EBPα proteins regulate PIN1 transcription. To address this, we performed promoter assays in the human embryonic kidney cell line 293T with different PIN1 promoter constructs ²⁷. Our data revealed that overexpression of E2F1 could transactivate PIN1 promoter as published before (Figure 2a, compare bars 1 and 4). In comparison with E2F1 alone, co-transfection of C/EBPα-p30 enhanced the transactivation of the PIN1 promoter activity to around 2 fold (Figure 2a, compare bars 4 and 6). This indicates that C/EBPα-p30 co-operates with E2F1 to regulate PIN1 mRNA levels. Interestingly, co-transfection of C/EBPα-p42 downregulated PIN1 promoter activity (Figure 2a, compare bars 4 and 5).

To determine the importance of the three E2F1 binding sites for the C/EBPα-p30 co-operation with E2F1 in regulating PIN1 promoter activity, we performed promoter assays with different deletion mutants of PIN1 promoter (Figure 2b). Compared to the wild type PIN1 promoter vector (-2300 Luc), deletion of the E2F1 binding sites (sites located at -557 to -550, -312 to -305 and at -288 to -281) exhibited decrease in the PIN1 promoter activity in a site dependent manner (Figure 2c). These data suggest that E2F1 binding sites are important for the association of C/EBPα-p30 with E2F1.

To gain further insights into the mechanism of association of C/EBPα-p30 with E2F1 in regulating PIN1 expression, we performed chromatin immunoprecipitation assays in K562-C/EBPα-p30-ER cells. Cells were treated with β-estradiol to induce C/EBPα-p30 and chromatin fragments were immunoprecipitated with an anti-E2F1 antibody. DNA from the immunoprecipitates was PCR amplified using primers flanking the two E2F1 binding sites PIN1 promoter region shown in figure 2d. We observed increased binding of E2F1 during C/EBPα-p30 induction in a time dependent manner (Figure 2e). We analysed whether increased binding of E2F1 in the PIN1 promoter is associated with induction of E2F1. Our data shows that C/EBPα-p30 does not induce E2F1 protein (Supplementary figure 6). It has been previously shown that C/EBPα-p30 is able to interact with E2F protein ²¹. To evaluate interaction of C/EBPα-p30 with E2F1 plays a role in the induction of PIN1 by C/EBPα-p30, we analysed induction of PIN1 in a cell line which has mutation for E2F1 interacting domain of C/EBPα-p30. Our data shows that that E2F1 interaction is not needed for PIN1 induction (Supplementary figure 7). Taken together, our data demonstrate that induction of C/EBPα-p30 leads to a recruitment of E2F1 to the PIN1 promoter and this is independent of interaction of C/EBPα-p30 with E2F1. These data suggest that PIN1 might represent an important target of E2F1 activation in the C/EBPα-p30 induced differentiation block. We also analysed binding of C/EBPα-p30 to the PIN1 promoter in a similar experimental setting. We didn’t observe any binding of C/EBPα-p30 to the PIN1 promoter (data not shown).

To find the possibility of C/EBPα-p30 co-operation with E2F1 in regulating other E2F1 target genes, we performed chromatin immunoprecipitation in K562-C/EBPα-p30-ER cells for c-Myc promoter (Supplementary Figure 8). Even though we could observe a slight increase in E2F1 binding in c-Myc promoter during C/EBPα-p30 induction, the effects were minor. This suggests co-operation of C/EBPα-p30 with E2F1 is more effective in the regulation of PIN1 expression.

PIN1 inhibition by PiB can overcome the differentiation block observed in human myeloid cells

To further understand how silencing of the PIN1 activity is biologically significant in the context of C/EBPα mutation, we carried out myeloid differentiation experiments in AML blast cells with C/EBPα mutation and in Kasumi-6 cells. Kasumi-6 is a myeloid leukemia cell line established from the bone marrow cells of an individual with acute myeloid leukemia, subtype M2 having C/EBPα mutation and expressing both the p42 and the p30 isoforms of the C/EBPα protein endogenously ²². For silencing PIN1, we used the PIN1 inhibitor, PiB ²⁸. PiB has been shown to inhibit the PIN1 activity by binding to the PPIase domain of PIN1. The myeloid cell differentiation was assessed by CD11b and CD15 expression by flow cytometry analysis as well as by the level of G-CSFR expression by Real Time RT-PCR. Treatment of AML blast cells and the Kasumi-6 cells with PiB induced myeloid differentiation (Figure 3). We could not observe any significant change in granulocyte morphology during PIN1 inhibition by PiB (data not shown). So we assume PIN1 inhibition by PiB is not sufficient to induce terminal stages of differentiation. We investigated the role of PIN1 silencing in NB4 cells, which do not have any CEBPA mutation. We could observe that PIN1 inhibition in NB4 cells leads to granulocytic differentiation (Supplementary figure 9). How ever, compared to Kasumi-6, the differentiation effects were modest during PIN1 silencing in NB4 cells. We also analysed the effect of PIN1 silencing in normal bone marrow cells. We could not observe any change in granulocytic differentiation (data not shown). These data suggest that CEBPA mutation status (presence of C/EBPa-p30) is significant in granulocytic differentiation during PIN1 silencing. The granulocytic differentiation during PIN1 inhibition by PiB was found independent of apoptosis (Supplementary figure 10) and cell proliferation (data not shown).

PIN1 blocks granulocytic differentiation

C/EBPα-p30 has been shown to block granulocytic differentiation mediated by C/EBPα ³ as well as to be able to induce AML ⁵. Based on our data that C/EBPα-p30 induces PIN1 in AML (Figure 1) and silencing PIN1 leads to granulocytic differentiation (Figure 3), we assessed the ability of PIN1 to block granulocytic differentiation. We overexpressed PIN1 in myeloid U937 cells and treated the cells with retinoic acid to induce granulocytic differentiation. Overexpression of PIN1 blocked granulocytic differentiation as shown by around 50% reduction in CD15 expression as well as reduction in G-CSFR expression (Figure 4). Taken together, these data demonstrates that PIN1 could have significant role in granulocytic differentiation block observed in AML.

PIN1 controls the stability of the c-Jun protein

We have previously reported that c-Jun expression is high in AML patient samples with C/EBPα mutation ¹⁷. Upon JNK activation, PIN1 binds to c-Jun which is phosphorylated on Ser63/73-Pro motifs ¹⁰. Due to the fact that C/EBPα-p30 could induce PIN1 and PIN1 regulates the protein stability of many targets with which it interacts ⁸, we hypothesized that PIN1 could have a significant role in regulating c-Jun protein stability. To test this hypothesis, PIN1 expression was silenced in 293T cells by RNA interference and c-Jun protein stability was then assessed by blocking protein synthesis with cyclohexamide (CHX) and measuring the c-Jun protein remaining at various time points. Our data shows that the stability of c-Jun was reduced when PIN1 expression was knocked down by a specific siRNA (Figure 5a and supplementary figure 11). These results indicate that PIN1 is an important factor in regulating the stability of c-Jun protein.

PIN1 protects c-Jun from ubiquitination mediated protein degradation

c-Jun has been shown to be subjected to ubiquitin-mediated degradation in a JNK dependent manner ^{29, 30}. Based on our finding that PIN1 regulated c-Jun protein stability (Figure 5A), we tested the role of PIN1 in the degradation of c-Jun. We performed ubiquitination assays in 293T cells by overexpressing c-Jun, PIN1 and HA-tagged ubiquitin. When c-Jun was co-transfected with HA-tagged ubiquitin, higher molecular weight bands were detected in the immunoprecipitation with the HA antibody which reflects c-Jun ubiquitination (Figure 5b, lane 4). Interestingly, when PIN1 was co-transfected, the ubiquitination of c-Jun was prevented suggesting a role for PIN1 in the regulation of c-Jun degradation (Figure 5b, lane 5).

Our data suggest that PIN1 prevents the degradation of c-Jun protein (Figure 5a, b). The Jun proto-oncogene is positively autoregulated by its product, Jun/AP-1 ³¹. Based on these findings, we hypothesized that PIN1 could have a major role in regulating c-Jun mRNA levels. We overexpressed PIN1 in Kasumi-6 cells and analysed c-Jun mRNA levels by quantitative Real-Time RT-PCR. We observed that overexpression of PIN1 leads to an increase in c-Jun mRNA levels (Supplementary figure 12a). We next investigated the effect of silencing PIN1 on the c-Jun mRNA levels. We found that silencing PIN1 with the inhibitor PiB downregulated the c-Jun mRNA level (Supplementary figure 12b). These data demonstrate that PIN1 stabilizes c-Jun protein, which in turn induces c-Jun mRNA by auto regulation.

c-Jun blocks granulocytic differentiation

We have previously shown that downregulation of the proto-oncogene c-Jun by C/EBPα is important for granulocytic lineage commitment ¹⁶. We have also reported that c-Jun expression is high in different subtypes of AML including AML samples with C/EBPα mutation and that c-Jun is able to block the DNA binding of C/EBPα ¹⁷. Based on these findings, we hypothesized that c-Jun is able to block the granulocytic differentiation induced by C/EBPα. To assess this, we overexpressed c-Jun in human myeloid U937 cells and treated the cells with retinoic acid. The granulocyte differentiation was assessed by FACS analysis for CD15 expression and quantitative Real-Time RT-PCR for G-CSFR. The expression of CD15 during granulocytic differentiation was downregulated to around 3 fold in the presence of c-Jun (Figure 5c). The myeloid markers such as G-CSFR was also found downregulated during overexpression of c-Jun (Figure 5c). These data suggests that c-Jun is able to block granulocytic differentiation induced by C/EBPα.