Modulation of expression and cellular distribution of p21 by macrophage migration inhibitory factor

MIF-/- mice were generated via homologous recombination in J1 embryonic stem cells as described previously [13]. They were maintained on a mixed background of 129/Sv C57BL/6. MIF+/+ wild-type (wt) mice of the same background were bred from MIF-/- littermates and used as controls. Mice were housed in a conventional housing facility. All animal experiments were performed in accordance with the regulations of Monash University Animal Ethics Committee.

Murine dermal fibroblasts (MDF) were obtained from MIF-/- and wt mice. Dermal fibroblasts were isolated by dissection of the dermal layer. A single cell suspension was obtained by digesting minced dermal tissue with 2.4 mg/ml Dispase (grade II, 5 U/mg; Boehringer Mannheim, Melbourne, Australia), 1 mg/ml collagenase (type II; Sigma, Melbourne, Australia) and DNase (type I; Boehringer Mannheim). MDF were propagated at a cell density of 1-5 × 10⁶ cells per 10 cm culture plates in RPMI (ICN Biomedicals, Cincinatti OH)/10% FCS (ICN Pty Ltd, Melbourne, Australia) at 37°C in a 5% CO₂ humidified incubator. Cells were used between passages 4 and 14. Comparisons between MIF-/- and wt cells were made using cells of the same passage.

MDF were seeded onto a 24 well plate at a density of 5 × 10⁴ cells/well (70% confluence) in RMPI (10%FCS) and incubated for 24 hours at 37°C. Cells were then washed twice with Hanks buffered saline solution (HBSS) before starving in 500 ul RPMI (0.1%BSA) at 37C for 24 hours and were then cultured for 30 hours in 10% FCS. Cells were pulsed with 1 uCi/ml ³H/Thymidine in 50 ul RPMI for 18 hours at 37°C. Radioactivity content was determined by scintillation counting using a Wallac 1409 Liquid Scintillation Counter (3H/Thy, 60 seconds/tube). DNA synthesis was estimated by measurement of ³H-Thy incorporation in fibroblasts. Results were expressed as the average CPM count with the standard error of the mean.

Wt and MIF-/- MDF were seeded onto a 24 well plate at a density of 5 × 10⁴ cells/well (70% confluence) in RMPI (10%FCS) and incubated for 72 hours at 37°C. At 24 hour time points, cells were washed with Hanks buffered saline solution (HBSS) and removed by trypsinisation. A single cell suspension was prepared 1:1 ratio with trypan blue (Sigma) and counted using a haemocytometer. Results were expressed as the total number of viable cells (cell count).

Murine Dermal Fibroblasts (MDF) seeded onto a 6 well plate at 2 × 10⁵ cells per well in RPMI (10% FCS) and allowed to adhere overnight. After 24 hours non adherent cells were washed off in HBSS and each well was replenished with RPMI (10% FCS) with or without SNP (0.50 mM)/(0.25 mM) and rMIF (100 ng/ml) treatment. 24 hours later apoptotic cells were differentiated by combined application of annexin V-FITC and PI. Briefly, cells were trypsinised, centrifuged and re-suspended in binding buffer (10 mM HEPES/NaOH, 0.14 M NaCl, 2.5 mM CaCl2, pH 7.5). Samples were incubated with annexin V-FITC and PI for 30 min at room temperature and analysed by flow cytometry. Annexin V-FITC and PI fluorescence was detected in the FL-1 (green) and FL-2 (red) channels respectively after correction to the spectral overlap between the two channels. Apoptotic and necrotic cells were distinguished on the basis of annexin V-FITC reactivity and PI exclusion. Live, non-apoptotic cells were not stained with any of the reagents. Apoptotic cells exhibited intense green (FITC) and low or intermediate red (PI) fluorescence (early and late stages of apoptosis, respectively). Permeability of late apoptotic cells for PI is the consequence of the compromised integrity of their plasma membrane. Results are expressed as % apoptosis, the percentage of annexin V/PI and annexin V positive cells relative to total number of cells gated.

Total RNA was extracted from MDF using TRIzol reagent (Gibco BRL, Grand Island, NY), according to the manufacturer's protocol. Two micrograms of total RNA was reverse transcribed using Superscript II reverse transcriptase and oligo (dT)18 (Gibco BRL). PCR amplification was performed on a LightCycler (Roche Diagnostics, Castle Hill, Australia) by using SYBR Green I as a double-stranded DNA-specific binding dye and continuous fluorescence monitoring. MIF, p21 and β-actin PCR products were purified on agarose gel electrophoresis using the QIAEX II gel extraction system (Qiagen, Clifton Hill, Australia). The level of expression of messenger RNA (mRNA) of MIF and or p21 was determined relative to the standard preparation using the LightCycler computer software.

For PCR, 5 μl each of the standard and the sample complementary DNA dilutions were added to individual capillary tubes. Amplification was carried out in a total volume of 10 μl containing primer concentrations of 3 pmoles and 1 μl of dNTP mix, Taq, reaction buffer, and SYBR Green I dye as supplied in the LightCycler DNA Master SYBR Green I kit (Roche). Forty cycles of PCR were programmed. Relative quantification of target mRNA expression was calculated and normalized to the expression of β-actin. The results, based on the ratio of target mRNA to β-actin amplification, are presented as the fold induction in mRNA expression relative to the amount present in control samples.

MDF were seeded on mini-slides placed within a 24 well plate at a semi confluent density (70%) of 2.5 × 10⁴ cells per slide for 24 hours. Adherent cells were then washed with HBSS, and fixed in 2% paraformaldehyde. Cells were again washed and then permeabilised in 0.1% Triton X (Sigma), or 0.1% Saponin (Sigma) for extranuclear staining, before being washed again in PBS/0.1% Na azide/0.1% BSA and incubated with specific monoclonal antibody to p21 (F-5) or isotype-matched negative control antibodies (Santa Cruz, CA) for 30 minutes. Unbound antibody was removed by washing in PBS/0.1% Na azide/0.1% BSA. Cells were then incubated for 30 minutes with FITC conjugated-secondary antibody (Silenus, Melbourne, Australia) before being washed and analysed under UV microscope.

MDF taken from semi confluent culture were washed with HBSS, and fixed in 2% paraformaldehyde. Cells were again washed and then permeabilised in 0.1% Triton X (Sigma) for 30 minutes before being washed again in PBS/0.1% Na azide/0.1% BSA and incubated with specific monoclonal antibody to p21 (F-5) or isotype-matched negative control antibodies (Santa Cruz, CA) for 30 minutes. Unbound antibody was removed by washing in PBS/0.1% Na azide/0.1% BSA. Cells were then incubated for 30 minutes with FITC conjugated-secondary antibody (Silenus, Melbourne, Australia) before being washed and analysed by flow cytometry. Results are expressed as delta mean fluorescence intensity (ΔMFI)

For intracellular analysis of p21 protein by flow cytometry 0.05% Saponin was used to permeabilise the cell wall leaving intracellular membranes intact. As above cells were incubated with p21 antibody or negative control antibody and analysed by flow cytometry. Nuclear p21 was estimated by subtracting ΔMFI of cytoplasmic p21 from ΔMFI of total p21.

RNA interference was used to knockdown MIF protein expression by introducing a homologous double-stranded RNA. The nucleotide sequences of dsRNA and complimentary dsRNA for mouse MIF mRNA were 5'-CCGCAACUACAGUAAGCUGdTdT-3' and 5'-CAGCUUACUGUAGUUGCGGdTdT-3', respectively. As a control RNA duplex 5'-GCGCGCUUUGUAGGAUUCGdTdT-3' and 5'-CGAAUCCUACAAAGCGCGCdTdT-3' were used. MDF grown in RPMI 1640 with FCS (10%) were transfected with either the MIF siRNA or the control RNA duplex using Amaxa nucleofection electroporation (Amaxa AG, Germany) according to the manufacturer's protocol [14]. After 24 hours, cells were transfected with either MIF siRNA or the control siRNA a second time. 24 hours after the second transfection the culture medium was removed and cells were examined for p21 immunofluorescence.

MDF taken from semi confluent culture via HBBS wash/trypsinisation were resuspended in PBS before being resuspended in ethanol and left on ice for 2 hours. The suspension was then centrifuged and ethanol thoroughly decanted. The pellet was resuspended in a staining solution of 0.1% triton-X, DNase free RNase and Propidium iodide (PI) for 30 minutes at room temperature. Samples were then analysed by flow cytometry.

ΔMFI was calculated by subtracting the mean fluorescence of the samples stained with an isotype matched negative control antibody from that of the samples stained with specific antibodies. Results are expressed as the mean +SEM. Statistical analysis was performed using the Students t test. P < 0.05 was considered statistically significant.

To determine the role of endogenous MIF in cell growth we examined proliferation in MIF-/- and wt MDF using a ³H-thymidine incorporation assay and cell count assay. After a 42 hour analysis of 6 independent experiments we found that cells lacking endogenous MIF displayed significantly reduced cell growth, (12437.9 +/- 1253.3), compared to wt MDF (20841.7 +/- 2040.6) (Figure 1a). This was confirmed by cell count assays which revealed significantly reduced numbers of viable MIF-/- cells compared to wt over 24, 48 and 72 hours (Figure 1b).

To further characterise the effects of endogenous MIF on cell cycle profile, we examined apoptotic events in MIF-/- and wt MDF by annexin V-FITC and PI dual labeling. Compared with wt MDF, MIF-/- MDF were more prone to undergo cell death exhibiting increased SNP induced apoptosis, (63.8 +/- 5.5), compared to wt MDF, (36.7 +/- 5.2) (Figure 1c).

To explore the potential for exogenous MIF to alter cell cycle events we examined both basal and induced apoptotic events in MIF-/- and wt MDF with and without rMIF treatment. Compared with control, treatment with rMIF protein was demonstrated to significantly increase basal survival and to significantly reduce SNP-induced apoptosis in MIF-/- MDF (Figure 2).

To examine the role of endogenous MIF in the regulation of p21 levels, MIF-/- and wt MDF expression of p21 was analysed using Western blotting and flow cytometry analysis. MIF-/- cells expressed lower levels of p21 protein compared to wt MDF (Figure 3a, 3b). Accordingly P21 mRNA was reduced in MIF-/- MDF as determined by RT-PCR (Figure 3c).

To further explore the potential for MIF to influence p21, we examined the subcellular localisation of p21 in MIF-/- and wt MDF. Interestingly, MIF-/- MDF exhibited diffuse, predominantly cytoplasmic p21 staining (Figure 4b) compared to wt MDF, which displayed strong nuclear staining for p21 (Figure 4a). Flow cytometric analysis of nuclear p21 protein revealed a significant reduction in MIF-/- compared to wt MDF (Figure 4c). Similar results were seen in wt MDF spiked with MIF siRNA which exhibited a significant reduction in nuclear p21 (figure 5b) compared to control siRNA (Figure 5a). The efficiency of MIF knockdown in wt MDF was quantified using RT-PCR. After 40 cycles of amplification MIF mRNA was not detectable in samples transfected with MIF siRNA (figure 5c).

To further confirm the involvement of MIF in p21 redistribution, MIF-/- and wt MDF were treated with rMIF and examined for p21 by immunofluorescence. Randomly cycling wt MDF from semi-confluent culture exhibited predominantly nuclear localisation of p21 and rMIF treatment of these cells did not yield any further increase in nuclear p21 expression. However compared to vehicle, rMIF induced a shift from predominantly cytoplasmic to nuclear localisation of p21 protein in MIF-/- MDF demonstrating MIF involvement in induction and nuclear translocation of p21 protein (Figure 6).

Cell cycle phase is well described to alter expression of key cell cycle proteins including p21. To address this issue, we examined the cell cycle phase profile of MIF-/- and wt MDF taken from semi-confluent culture using flow cytometric analysis of PI incorporation. No significant difference in the percentage of cells in each phase of the cell cycle was seen. Therefore the difference in p21 expression and subcellular localisation observed between MIF-/- and wt MDF could not be explained by cell cycle phase differences between the cells in these experiments (Figure 7).