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Originally published online as doi:10.1189/jlb.0408238 on December 3, 2008

Published online before print December 3, 2008
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(Journal of Leukocyte Biology. 2009;85:481-490.)
© 2009 by Society for Leukocyte Biology

Sequestration of PDLIM2 in the cytoplasm of monocytic/macrophage cells is associated with adhesion and increased nuclear activity of NF-{kappa}B

Nollaig C. Healy and Rosemary O'Connor1

Cell Biology Laboratory, Department of Biochemistry, BioSciences Institute, University College, Cork, Ireland

1 Correspondence: Cell Biology Laboratory, Department of Biochemistry, BioSciences Institute, University College, Cork, Ireland. E-mail: r.oconnor{at}ucc.ie


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ABSTRACT
 
PDLIM2 (Mystique/SLIM) is a postsynaptic density-95/discs large/zonula occludens-1-Lin-11, Isl-1, Mec-3 (PDZ-LIM) domain protein expressed in the nucleus of T lymphocytes, where it promotes degradation of the p65 subunit of NF-{kappa}B. It is also expressed at the cytoskeleton in epithelial cells, where it is essential for cell migration. It is not known whether PDLIM2 function at the nucleus and cytoskeleton is linked and whether PDLIM2 subcellular location is regulated in hematopoietic cells. To investigate this, we used the human monocytic leukemia cell line THP-1 that can differentiate into adherent macrophages and the adherent murine macrophage cell line RAW264.7. PMA-induced differentiation of THP-1 cells resulted in increased accumulation of PDLIM2. In differentiated cells, PDLIM2 exhibited retarded mobility indicative of serine phosphorylation, which could be reversed by phosphatases and by inhibition of protein kinase C or ERK kinases. In nondifferentiated THP-1 cells, PDLIM2 was located predominantly in the nucleus, whereas in differentiated cells, PDLIM2 was located predominantly in the cytoplasm. Suppression of PDLIM2 expression in THP-1 and RAW 264.7 cells resulted in decreased adhesion, increased NF-{kappa}B transcription reporter activity, and increased LPS-induced TNF-{alpha} production. Overexpression of PDLIM2 in THP-1 cells enhanced cell adhesion. Overall, these findings indicate that PDLIM2 sequestration in the cytoplasm is associated with cell adhesion and increased nuclear activity of NF-{kappa}B p65. The data suggest that sequestration of PDLIM2 at the cytoskeleton regulates its nuclear function.

Key Words: monocyte • PDZ-LIM • ERK


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INTRODUCTION
 
PDLIM2 (also known as Mystique and SLIM) is a postsynaptic density-95 (PSD-95)/discs large (DLG)/zonula occludens-1 (ZO-1)-Lin-11, Isl-1, Mec-3 (PDZ-LIM) domain protein that was identified in cells transformed by overexpression of the insulin-like growth factor-I receptor (IGF-IR) [1 ], in corneal epithelial cells [2 ], and in T lymphocytes [3 ]. It is a member of the actinin-associated LIM protein (ALP) family of proteins that contains a single N-terminal PDZ and C-terminal LIM domain. PDZ domains derive their name from the first three proteins in which these domains were identified; PSD-95 [4 ], Drosophila melanogaster DLG protein [5 ], and epithelial tight junction protein, ZO-1 [6 ]. The LIM domain, comprising a cysteine-rich sequence that forms two zinc fingers, was first discovered in the Caenorhabditis elegans cell-lineage protein LIN-11 [7 ], the rat insulin gene enhancer-binding protein Isl1 [8 ], and the C. elegans protein MEC-3 [9 ]. The PDZ and LIM domains mediate protein–protein interactions associated with cell differentiation. All members of the ALP family are known to interact with the actin cytoskeleton through their PDZ domain [1 , 10 , 11 ]. However, the LIM interactions are more diverse, ranging from nonreceptor and receptor kinases to other PDZ or LIM domains [12 13 14 ].

PDLIM2 expression can be enhanced by IGF-I and by cell adhesion, and it is highly expressed in motile epithelial cells including the androgen-independent prostate carcinoma cell lines, DU145 and PC3 [1 , 15 ]. Small interfering (si)RNA-mediated silencing of PDLIM2 results in loss of cell migration and adhesion [1 ].

PDLIM2 is located in the nucleus of most cells. It can interact with STAT proteins and has been proposed to function in the nucleus to promote degradation of phosphorylated STAT1 and STAT4 [3 ]. Cells from the PDLIM2 knockout mouse have increased expression of STAT proteins [3 ]. PDLIM2 was also reported to mediate the actions of the integrin ligand osteopontin in degradation of STAT1 in RAW264.7 murine macrophages and in mammary epithelial tumor cells [16 , 17 ]. More recently, PDLIM2 was shown to promote nuclear degradation of the p65 subunit of the NF-{kappa}B family of dimeric transcription factors [18 ]. NF-{kappa}B subunits are sequestered in the cytoplasm through an association with the inhibitory I{kappa}B proteins. Phosphorylation and subsequent ubiquitin-mediated degradation of I{kappa}B are prerequisites for the rapid translocation of NF-{kappa}B to the nucleus [19 , 20 ]. PDLIM2 has been shown to target nuclear p65 to promyelocytic leukemia protein (PML) nuclear bodies for ubiquitination and subsequent degradation [18 ]. PDLIM2-knockout mice produce more of the proinflammatory cytokines IL-6 and IL-12 in response to LPS. Thus, PDLIM2 is an important regulator of NF-{kappa}B-mediated immune and inflammatory responses.

Although the mechanism of action of PDLIM2 in regulating cytokine transcription in T lymphocytes has been elucidated, its mechanism of action in regulating adhesion and migration in adherent cells is not known. It is also not known whether the actions of PDLIM2 at these different cellular locations are linked by its activity toward NF-{kappa}B or STATs, both of which may regulate cell adhesion or migration. NF-{kappa}B activation is thought to act as a key link between inflammation and tumorigenesis [21 ], and its activity is enhanced in many tumors including estrogen receptor-negative breast cancer [22 ]. STAT3 has been implicated in the secretion of metalloprotease proteins [23 ] and IL-6 signaling [24 ]. For these reasons, we were interested to determine whether the functions of PDLIM2 at the cytoskeleton were linked with those in the nucleus and whether they converged on regulation of NF-{kappa}B. To do this, we used the THP-1 monocytic leukemia cell line that can be induced to differentiate into macrophage-like cells, which are accompanied by transition from a nonadherent to an adherent phenotype in culture. The function of PDLIM2 was also tested in the murine macrophage cell line RAW264.7.


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MATERIALS AND METHODS
 
Reagents, antibodies, and plasmids
PMA, MG132, LPS, fibronectin anti-actin, -{alpha}-actinin, and -tubulin mAb were from Sigma-Aldrich (Dublin, Ireland). Shrimp alkaline phosphatase (SAP) was from Roche (East Sussex, UK). VCAM-1 was from R&D Systems (Minneapolis, MN, USA). Anti-CD11b antibody was from BD PharMingen (Oxford, UK). Lamin B, heat shock protein (Hsp)90, and p65 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-poly-(ADP-ribose) polymerase (PARP) antibody was from Cell Signaling Technologies (Beverly, MA, USA). For Western blot analysis, IRDye® 680- and IRDye® 800CW-conjugated antibodies were from LI-COR Biosciences (Cambridge, UK). Cy2- and Cy3-conjugated secondary antibodies for immunofluorescence were purchased from Jackson ImmunoResearch Laboratories (Soham, Cambridgeshire, UK). 1,25-Dihydroxyvitamin D3 (VD3) and the pharmalogical inhibitors PD89059, LY294001, SB202190, bisindolymaleimide (BIM), Gö6796, and leptomycin B (LMB) were from Calbiochem (La Jolla, CA, USA). Expression plasmid encoding GFP-PDLIM2 has been described previously [1 ]. All other reagents were purchased from Sigma-Aldrich.

Cell culture, differentiation, and treatments
THP-1 (human monocyte-derived leukemia), HL-60 (human acute promyelocytic leukemia), U937 (human diffuse histiocytic lymphoma, displaying many monocytic characteristics), RAW264.7 (leukemic macrophage), and Jurkat (T cell leukemia) cell lines were from American Type Culture Collection (Manassas, VA, USA). THP-1 cells were cultured in RPMI 1640, supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 0.05 mM 2-ME. HL-60, U937, and Jurkat cells were maintained in RPMI 1640, supplemented with 10% FBS, 2 mM penicillin–streptomycin, and 2 mM L-glutamine. RAW264.7 cells were cultured in DMEM, supplemented with 10% FBS, 2 mM penicillin–streptomycin, and 2 mM L-glutamine. PBMC were isolated from whole blood using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient. Monocytes were separated from lymphocytes by adherence and cultured in RPMI 1640, supplemented with 10% FBS, 2 mM penicillin–streptomycin, and 2 mM L-glutamine.

To induce differentiation of THP-1 cells, the cells were incubated with 100 ng/ml PMA or 10–100 nM VD3 for 3 days. Cells were photographed under the 20x objective of a Nikon TE300 inverted microscope equipped with a SPOT digital camera to document the morphological changes. Differentiation of THP-1 cells into macrophages was confirmed by FACS analysis of the surface expression of CD11b, 24, 48, and 72 h after the addition of PMA. In brief, differentiated cells were harvested by scraping gently in PBS. Cells were centrifuged for 3 min, 500 g, washed in PBS containing 2% BSA, and resuspended in PBS containing 10% FBS and anti-CD11b. After 1 h incubation at 4°C, cells were washed twice in PBS containing 2% BSA. After the second wash, the pellet was resuspended and incubated in PBS containing 10% FBS and FITC-conjugated secondary antibody at 4°C for 30 min. Samples were washed twice prior to fixing in PBS containing 1% paraformaldehyde. Mean fluorescence of CD11b expression was quantified with a FACScan flow cytometer using CellQuest software (Becton Dickinson, San Jose, CA, USA).

Preparation of whole cell protein extracts and immunoprecipitations (IPs)
Cellular protein extracts were prepared by washing cells with PBS and then resuspending suspension cells or scraping adherent cells in ice-cold radio IP assay lysis buffer [50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40 (NP-40), 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM sodium fluoride (NaF), 1 mM sodium orthovanadate (Na3VO4), 1 mM PMSF, 1 µM pepstatin, and 1.5 µg/ml aprotinin]. Where indicated, cells were treated with 10 µM MG132 for 6 h prior to lysis. For examining PDLIM2 phosphorylation in the SAP assays, NaF and Na3VO4 were omitted from the lysis buffer. After incubation at 4°C for 20 min, nuclear and cellular debris was removed by microcentrifugation at 20,800 g for 15 min at 4°C. Protein concentration was estimated using the Bradford reagent (BioRad, Munich, Germany).

For IP of endogenous PDLIM2 or {alpha}-actinin, cells were lysed in IP lysis buffer: 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 50 mM NaF, 1% NP-40, 1 mM Na3VO4, 1 mM PMSF, 1 µM pepstatin, and 1.5 µg/ml aprotinin. Protein extracts were precleared using BSA-coated protein G agarose beads (Pierce, Rockford, IL, USA; 15 µl beads/400 µg total protein in 700 µl lysis buffer) by incubation at 4°C for 1 h with gentle rocking. The lysates were recovered from the beads by centrifugation at 1000 g for 3 min and transferred to fresh tubes for incubation with primary antibody overnight at 4°C with gentle rocking (~3 µg each antibody was used). Immune complexes were obtained by adding 20 µl protein G agarose beads for 3 h at 4°C. The beads were washed (three times) with ice-cold lysis buffer, and the immune complexes were then removed from the beads by boiling for 5 min in 20 µl 2x SDS-PAGE sample buffer for electrophoresis and Western blot analysis.

Western blot analysis
Cell lysates for Western blot analysis were subjected to electrophoresis on 10% SDS-polyacrylamide gels, transferred onto a nitrocellulose membrane (Protran, Dassel, Germany), and blocked at room temperature for 1 h with 5% BSA for PDLIM2 antibody incubation or 5% nonfat dried milk diluted in TBS/0.05% Tween 20 (Sigma Chemical Co., St. Louis, MO, USA) for all other antibody incubations. Primary antibodies were diluted in the relevant blocking buffer, and incubations were performed overnight at 4°C. The next day, membranes were incubated at room temperature for 1 h with IRDye® 680- or IRDye® 800CW-conjugated secondary antibodies, and immunoreactive bands were detected using a LI-COR Odyssey infrared imaging system (LI-COR Biosciences). Densitometric quantification was performed using the LI-COR Odyssey analysis program, according to the manufacturer’s instructions (LI-COR Biosciences).

Phosphatase and kinase inhibition assays
THP-1 cells were left untreated or treated with PMA (100 ng/ml) for 48–72 h. PDLIM2 was immunoprecipitated from cell lysates as described above. The immunoprecipitates were incubated for 1 h at 37°C in a final volume of 10 µl lysis buffer containing 5 µl SAP or no enzyme as a control. SDS sample buffer was added, and samples were boiled for 5 min and resolved on a 10% SDS-polyacrylamide gel.

To examine the effect of kinase inhibitors on PDLIM2 phosphorylation, inhibitors of MEK signaling (PD98059, 18 µM), PI-3K signaling (LY294001, 10 µM), p38MAPK signaling (SB202190, 3 µM), or protein kinase C (PKC) signaling (BIM, 2 µM) were added to THP-1 cells for 1 h prior to the addition of PMA (100 ng/ml). Following an overnight incubation, cell viability was confirmed by trypan blue exclusion and propidium iodide uptake. Whole cell lysates were analyzed by Western blot for PDLIM2 expression.

Immunofluorescence
For immunofluorescence analysis, undifferentiated THP-1 cells were captured by cytospinning, or cells were plated on glass coverslips prior to treatment with 100 ng/ml PMA for 48–72 h. Where indicated, cells were treated additionally with 0.1 µM LMB or methanol for 24 h. Cells were rinsed with 60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, pH 6.9 (PHEM), fixed in 3.7% formaldehyde in PHEM for 15 min, rinsed with PBS, and permeabilized with 0.1% Triton X (TX)-100 in PBS for 5 min. After preblocking with 5% normal goat serum (Sigma-Aldrich) in PBS for 30 min, cells were incubated with primary antibody, washed with PBS, and incubated with Cy2- or Cy3-conjugated secondary antibody and Hoechst (nuclear counterstain). F-actin was detected by incubating the cells with tetramethylrhodamine B isothiocyanate-phalloidin (Sigma-Aldrich). Fluorescence was monitored using a 100x Plan Fluor objective on a Nikon Eclipse E600 microscope. Images were acquired with a SPOT camera and adjusted using Adobe Photoshop software.

Subcellular fractionation
Cytoplasmic, soluble, and insoluble nuclear extracts were prepared as follows. An equal number of cells were lysed on ice for 10 min with 100 µl hypotonic buffer [20 mM HEPES, pH 8.0, 10 mM KCl, 1 mM MgCl2, 0.1% (v/v) TX-100, and 20% (v/v) glycerol]. After centrifugation of samples at 2300 g for 2 min, supernatants containing the cytoplasmic fractions were collected. Pellets were washed once in the hypotonic buffer and then lysed for 20 min on ice in 40 µl hypertonic buffer [20 mM HEPES, pH 8.0, 1 mM EDTA, 20% (v/v) glycerol, 0.1% (v/v) TX-100, and 400 mM NaCl] with brief vortexing. After centrifugation at 20,400 g for 5 min, supernatants containing soluble nuclear fractions were collected. Pellets were washed once in the hypertonic buffer and then lysed with 40 µl insoluble buffer [20 mM Tris, pH 8.0, 150 mM NaCl, 1% (w/v) SDS, 1% NP-40, and 10 mM iodoacetamide] by shaking for 50 min on a vortex genie at 4°C. After centrifugation at 20,400 g for 5 min, insoluble nuclear fractions were collected. All samples were boiled in 5x SDS-PAGE sample buffer, and equal volumes of each fraction were subjected to electrophoresis on 10% SDS-polyacrylamide gels. The purity of the fractions was confirmed by Western blotting with anti-Hsp90, -tubulin, or -actin (cytoplasm markers), anti-PARP (soluble nuclear fraction marker), and anti-lamin B (insoluble nuclear fraction marker).

Transient transfection of THP-1 and RAW264.7 cells with plasmid DNA or siRNA
Transfection of THP-1 cells with plasmid DNA or siRNA was performed using the Nucleofector Kit V (Amaxa Biosystems, Cologne, Germany), according to the manufacturer’s instructions. In brief, 1 x 106 cells/reaction were resuspended in 100 µl prewarmed Nucleofector Solution V. The cell solution was mixed with 0.5 µg GFP-PDLIM2 plasmid DNA or 2.5 µg siRNA. The siRNA targeted to human PDLIM2 (ON-TARGETplus SMART-pool, #L-010731-00-0020) was purchased from Dharmacon (Lafayette, CO, USA). The siRNA-negative control (#4611) used was purchased from Ambion (Austin, TX, USA). The Nucleofector Program U-001 was used, and protein expression was analyzed at 12–48 h postnucleofection. RAW264.7 cells were transfected with 50 nM mouse PDLIM2 (msPDLIM2) from Ambion (#4390818: 5'-caguugcccuuucuaaagatt-3') using Oligofectamine, according to the manufacturer’s instructions.

Measurement of TNF-{alpha} production and NF-{kappa}B luciferase activity
THP-1, before and after differentiation, or RAW264.7 cells transfected with control or msPDLIM2 siRNA were seeded at equal cell numbers and treated with 10 ng/ml LPS for 24 h. THP-1 cells were starved of serum and PMA for 4 h prior to addition of LPS. Supernatants of the culture media were collected, and levels of TNF-{alpha} were determined using a human or mouse TNF-{alpha} ELISA kit (eBioscience, San Diego, CA, USA), according to the manufacturer’s instructions.

The NF-{kappa}B dual-luciferase reporter plasmid was from SuperArray (Madison, WI, USA). RAW264.7 cells were first transfected with control or msPDLIM2 siRNA, and 24 h later, the cells were transfected with luciferase reporter plasmid and porcine CMV-p65 using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA), and equal cell numbers were seeded on half-area opaque 96-well plates (Corning, Corning, NY, USA). Cells were treated with 1 µg/ml LPS for 2 h prior to lysis with passive lysis buffer (Promega, Madison, USA). Lysates were analyzed using the dual luciferase reporter assay system kit (Promega). Luminescence was measured by programming a luminometer (Turner BioSystem, Sunnyvale, CA, USA) to perform a 2-s premeasurement delay, followed by a 10-s measurement period for each reporter assay. All experiments were performed at least in triplicate. The level of Firefly luciferase activity was normalized to that of the Renilla luciferase activity in each experiment, and the activity measured in cells transfected with the promoterless reporter construct was subtracted. Data are reported as mean-fold induction change in luciferase activity ± SD relative to the control siRNA samples.

Adhesion assays
THP-1 cell adhesion to VCAM-1 was examined by allowing cells to adhere to multiple wells of a 96-well tissue-culture plate, that was precoated with 3 µg/ml solution of VCAM-1 for 2 h at 37°C. RAW264.7 cells were examined adhering to fibronectin (25 µg/ml). Nonspecific binding sites were blocked with 2.5% BSA in PBS for 1 h at 37°C. THP-1 or RAW264.7 cells transfected with GFP-empty vector, GFP-PDLIM2, control siRNA, or PDLIM2 siRNA were plated at a density of 5 x 104 cells/well in multiple wells of the 96-well plate and allowed to attach for the indicated times. The medium was removed, and cells were washed three times with PBS. Attached cells were fixed with 100% methanol at –20°C for 5 min and stained by addition of freshly filtered 0.1% crystal violet for 15 min. Cells were destained by extensive washing in H2O and allowed to air dry. The crystal violet dye was solubilized by the addition of TX-100 (0.5%) overnight at room temperature. The absorbance for each well was measured at 590 nm on a spectrophotometer (SpectraMax 340, Molecular Devices, Sunnyvale, CA, USA). Data are represented as the mean and SD of absorbance in at least triplicate wells, and the graph is a representative of three separate experiments.


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RESULTS
 
PMA-induced differentiation of THP-1 cells results in increased PDLIM2 expression
We first investigated PDLIM2 expression levels in a series of leukemic cell lines. Western blot analysis shown in Figure 1A demonstrated that PDLIM2 was expressed in THP-1, HL-60, and U937 monocytic cell lines and in a Jurkat T cell line at levels that were comparable with MCF10A cells (a highly migratory, nontransformed breast myoepithelial cell line). In all of these cells, the expression of PDLIM2 was at least tenfold higher than in MCF-7 (an estrogen-dependent breast carcinoma cell line). PDLIM2 was also expressed in RAW264.7 macrophages and in human peripheral monocytes (Fig. 1A , right panel).


Figure 1
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  		Figure 1. PMA induces differentiation of THP-1 cells and an increase in PDLIM2 expression. (A) Leukemic cell lines were resolved on a 10% SDS-polyacrylamide gel, and PDLIM2 expression was determined by Western blot analysis. Actin levels were used as a loading control. Lysates from MCF10A cells were used as a positive control for PDLIM2 expression. (B) THP-1 cells were left untreated or treated with 100 ng/ml PMA for indicated times up until 72 h. Cell-surface expression of CD11b was detected by flow cytometry at indicated times. The shaded area represents staining with anti-CD11b at 0 h PMA, and the bold line represents staining with anti-CD11b at 24, 48, or 72 h plus PMA. The graph represents the mean fluorescence for CD11b expression at each time-point. (C) Following PMA stimulation, whole cell lysates were prepared at the indicated time-points, resolved by SDS-PAGE, and detected by Western blot using anti-PDLIM2 antibody. The slower-migrating band of PDLIM2 is indicated by {leftrightarrow}.[INSERT IMAGE] The right panel shows THP-1 and peripheral monocytes stimulated with 100 ng/ml for 72 h. (D) THP-1 cells were treated with 10 or 100 nM VD3 for 72 h, and PDLIM2 expression was analyzed by Western blot.

Previously, we demonstrated that PDLIM2 expression is increased by adhesion and decreased by de-adhesion in epithelial cells [1 ]. We hypothesized that PDLIM2 levels may be regulated during monocyte differentiation from nonadherent into adherent macrophage-like cells. To test this, we used the THP-1 cell line, which differentiates in response to the PMA [25 , 26 ]. PMA-induced differentiation of THP-1 cells over 72 h resulted in cell adhesion (not shown) as well as enhanced cell-surface expression of CD11b, which is a hallmark of macrophage differentiation (Fig. 1B) .

We then examined PDLIM2 expression in THP-1 cells at various times following induction of differentiation. As can be seen in Figure 1C , PDLIM2 levels increased greatly from 8 h up to 72 h following induction of differentiation. In differentiated cells, it was also apparent that the PDLIM2 protein exhibited retarded mobility in SDS-PAGE, which is indicative of post-translational modification such as phosphorylation. PMA-induced differentiation of human peripheral monocytes (Fig. 1C , right panel), HL-60, and U937 monocytic leukemic cells (data not shown) also resulted in increased cell adhesion and increased PDLIM2 expression. We also induced differentiation of THP-1 cells using VD3, which resulted in an adherent phenotype (data not shown) and an increase PDLIM2 expression accompanied by a retarded mobility in SDS-PAGE (Fig. 1D) . Taken together, these data indicate that PDLIM2 expression is enhanced upon monocyte-macrophage differentiation.

PDLIM2 expression is regulated by proteasomal degradation, and the protein is modified by serine phosphorylation
We next investigated whether the increased expression and retarded migration of PDLIM2 in differentiation THP-1 cells (shown at 72 h in Fig. 2A , left panel) were a result of increased protein accumulation and whether this was associated with protein phosphorylation. Pdlim2mRNA levels were not increased in differentiated THP-1 cells (not shown). This suggests that accumulation of PDLIM2 is a result of increased translation or increased stability of the protein in differentiated cells.


Figure 2
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  		Figure 2. PMA-induced differentiation of THP-1 cells results in retarded SDS-PAGE migration of PDLIM2, which can be reversed by phosphatases and PKC or ERK inhibitors. THP-1 cells were grown in complete medium and left untreated (–) or treated (+) with 100 ng/ml PMA for 72 h. Whole cell lysates were analyzed for PDLIM2 protein expression by Western blot (A and C). The slower-migrating band of PDLIM2 is indicated by {leftrightarrow}. (B) Following treatment with PMA for 72 h, cells were treated with 10 µM MG132, a proteasomal inhibitor, for 6 h prior to lysis. (C) PDLIM2 was immunoprecipitated from THP-1 cell lysates, and immunoprecipitates were incubated with 5 µl SAP for 1 h at 37°C and subjected to 10% SDS-PAGE, followed by Western blotting with anti-PDLIM2 antibody (right panel). Lysates prior to IP are shown in left panel. THP-1 cells were treated with 2 µM BIM, 2 µM Rottlerin, 18 µM PD98059 (PD), 3 µM SB202190 (SB), or 10 µM LY294001 (LY) for 1 h or DMSO as vehicle control and were then left untreated (–) or treated (+) with 100 ng/ml PMA for 14 h. Whole cell lysates were prepared, and PDLIM2 expression was assessed by Western blot. Tubulin levels were assessed as a loading control.

PDLIM2 stabilization could result from decreased ubiquitin-mediated degradation of the protein. To test this, nondifferentiated and differentiated THP-1 cells were treated with the proteasomal inhibitor MG132 prior to cell lysis. MG132 caused an increase in PDLIM2 levels in nondifferentiated and differentiated cells (Fig. 2B) . Interestingly, in nondifferentiated cells, MG132 caused a greater accumulation of the faster-migrating PDLIM2 species. This suggests that the faster-migrating species present in nondifferentiated cells is susceptible to proteasome-mediated degradation.

The retarded migration of PDLIM2 in SDS-PAGE suggests protein phosphorylation. Indeed, the PDLIM2 amino acid sequence contains 53 serine residues, 17 threonine, and seven tyrosine residues. To investigate PDLIM2 phosphorylation in differentiated THP-1 cells, a series of phosphatases and kinase inhibitors was tested for their ability to alter the migration of the protein in SDS-PAGE. Exposure of immunoprecipitated PDLIM2 to SAP resulted in loss of the slower-migrating form of PDLIM2 along with an accumulation of the faster-migrating form (Fig. 2C) . The serine/threonine phosphatase protein phosphatase 2A also prevented the accumulation of the slower-migrating form of PDLIM2 (data not shown). These results indicate that PDLIM2 becomes phosphorylated in differentiated THP-1 cells.

PMA can activate members of the PKC family of serine/threonine kinases [27 , 28 ] and has also been reported to increase activation of p42/44 ERK1/2 MAPK [29 ]. To test whether PKCs or ERKs mediate phosphorylation of PDLIM2, inhibitors of these kinases were used. THP-1 cells were incubated with the PKC inhibitor BIM or Rottlerin [30 ], the inhibitor of ERK activation PD98059 [31 ], or controls in the presence and absence of PMA prior to analysis of PDLIM2 mobility in SDS-PAGE. The PKC inhibitor BIM and ERK inhibitor reversed the appearance of the slower-migrating form of PDLIM2 (Fig. 2D) , whereas the PI-3K inhibitor LY294001 and p38MAPK inhibitor SB202190 did not affect the migration of PDLIM2.

Overall, these data indicate that increased PDLIM2 expression in differentiated cells is not a result of increased transcription but may be, in part, a result of increased stability of the protein. Furthermore, the data indicate that that PKC and ERK promote adhesion-mediated phosphorylation of PDLIM2 in differentiated cells and suggest that the phosphorylated form is resistant to proteasome-mediated degradation.

PDLIM2 accumulates in the cytoplasm of differentiated THP-1 cells
We next investigated the subcellular location of PDLIM2 in differentiated THP-1 cells compared with nondifferentiated cells by analyzing subcellular fractions for PDLIM2 expression. In undifferentiated THP-1 cells, the majority of PDLIM2 was localized in the nuclear fraction and was represented by the faster-migrating band, suggesting it is not phosphorylated in the nucleus (Fig. 3A ). However, in differentiated THP-1 cells, PDLIM2 was predominantly present in the cytoplasmic fractions (Fig. 3A) . In these cells, PDLIM2 was predominantly present as the slow-migrating, phosphorylated form, which is in agreement with our data in Figure 2 , showing that the phosphorylated species accumulates in response to differentiation. To exclude the possibility that the nuclear pool of PDLIM2 was degraded following differentiation, cells were incubated with the proteasomal inhibitor MG132 prior to fractionation. MG132 caused an increase in the levels of nuclear PDLIM2 in nondifferentiated cells (Fig. 3A ; –PMA samples). This is in agreement with the earlier observation that MG132 treatment caused an increase in the total cellular levels of the faster-migrating band of PDLIM2 in nondifferentiated cells (Fig. 2B) . Nuclear levels of PDLIM2 in differentiated cells did not increase following MG132 treatment (Fig. 3A ; +PMA). This suggests that in differentiated THP-1 cells, the decrease in nuclear PDLIM2 levels is not a result of degradation but could alternatively be a result of translocation of PDLIM2 out of the nucleus. Similar results were obtained when subcellular fractionation was performed on VD3-differentiated THP-1 cells (Fig. 3B) and RAW264.7 macrophages (data not shown).


Figure 3
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  		Figure 3. PDLIM2 accumulates in the cytoplasm PMA-differentiated monocytes. THP-1 cells cultured in complete medium were left untreated (–) or treated (+) with 100 ng/ml PMA for 72 h. Cells were treated with 10 µM MG132 or DMSO as a vehicle control for 6 h. Subcellular fractionation was performed to isolate cytoplasmic, nuclear-soluble, and nuclear-insoluble fractions, which were subjected to SDS-PAGE on a 10% polyacrylamide gel. PDLIM2 expression was assessed by Western blot. Equal loading and purity of the fractions were confirmed by probing the blot with antitubulin (cytoplasmic marker), anti-PARP (nuclear-soluble marker), or antilamin B (nuclear-insoluble marker) antibodies. (B) THP-1 cells were treated with VD3 for 72 h, and subcellular fractionation was performed. (C) THP-1 cells were cultured in complete medium in the absence or presence of PMA. After 48 h, cells were treated with 0.1 µM LMB or methanol (control) for a further 24 h. Cells were fixed, permeabilized, and stained for PDLIM2 using Cy2 secondary antibody.

Translocation of proteins between the nucleus and cytoplasm requires a nuclear localization signal (NLS) or nuclear export signal (NES; reviewed in refs. [32 , 33 ]). Although the amino acid sequence of PDLIM2 does not contain any predicted NLS [34 ], it contains a predicted NES at aa 71–79 in the PDZ domain (predicted using the NetNES server, www.expasy.com). LMB inhibits exportin1/chromosome region maintenance 1 (CRM1), which is the receptor for the leucine-rich NES [35 ]. This promotes nuclear accumulation of proteins with a functional NES, as the NES cannot be recognized by CRM1 and therefore, cannot be exported. We investigated whether LMB would affect translocation of PDLIM2 to the cytoplasm following differentiation of THP-1 cells. In nondifferentiated THP-1 cells, there was no obvious nuclear accumulation of PDLIM2 following exposure of cells to LMB (Fig. 3C ; –PMA). However, in differentiated cells, LMB caused a distinct accumulation of PDLIM2 in the nucleus (Fig. 3C ; +PMA). This result suggests that a NES signal in PDLIM2 becomes active when THP-1 cells are induced to differentiate.

Overall, these data indicate that PMA-induced differentiation results in increased PDLIM2 expression, phosphorylation, and cytoplasmic translocation, and suggest that PDLIM2 may have a nuclear function primarily in nondifferentiated cells and a cytoplasmic function in differentiated cells.

PMA-mediated translocation of PDLIM2 to the cytoplasm is associated with increased nuclear NF-{kappa}B p65
It has been demonstrated by Tanaka et al. [18 ] that nuclear PDLIM2 promotes degradation of the p65 subunit of NF-{kappa}B, and that Pdlim2–/– T cells display enhanced NF-{kappa}B signaling. Therefore, we predicted that PMA-induced accumulation of PDLIM2 in the cytoplasm may be accompanied by increased nuclear p65 expression. To test this, nondifferentiated and differentiated cells were treated with LPS to activate NF-{kappa}B signaling, and p65 nuclear levels were analyzed by subcellular fractionation. Western blot analysis demonstrated an increase in p65 expression in the soluble and insoluble nuclear fractions of differentiated cells, which correlated with the observed decrease in nuclear levels of PDLIM2 (Fig. 4 ). Similar results were observed in HL-60 and U937 cell lines (data not shown). TNF-{alpha} was measured by ELISA in the culture supernatants of differentiated and PMA-differentiated THP-1 cells that were stimulated with LPS. As expected, the differentiated cells showed increased TNF-{alpha} expression (Fig. 4B , open bar). Furthermore, RAW264.7 cells transfected with msPDLIM2 siRNA showed increased NF-{kappa}B luciferase activity and increased TNF-{alpha} levels in response to LPS compared with the control cells (Fig. 4C) . These data suggest that when PDLIM2 is sequestered in the cytoplasm of differentiated macrophages, there is enhanced accumulation of p65 protein and NF-{kappa}B target gene activation in the nucleus.


Figure 4
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  		Figure 4. Accumulation of PDLIM2 in the cytoplasm of PMA-differentiated THP-1 cells correlates with an increase in nuclear p65 levels. (A) THP-1 cells were incubated with 100 ng/ml LPS for the indicated times. Subcellular fractionation was performed to isolate cytoplasmic, nuclear-soluble, and nuclear-insoluble fractions, which were subjected to SDS-PAGE and Western blotting with anti-PDLIM2 and anti-p65 antibodies. Equal loading and purity of the fractions were confirmed by probing the blot with antitubulin (cytoplasmic), anti-PARP (nuclear-soluble), and antilamin B (nuclear-insoluble) antibodies. (B) ELISA assay of TNF-{alpha} in culture supernatants of differentiated and differentiated THP1 cells starved of serum and PMA for 4 h followed by stimulated with 10 ng/ml LPS for 24 h. (C) Effect of PDLIM2 suppression on p65-mediated gene activation of TNF-{alpha} production in RAW264.7 cells. Cells were transfected with control or msPDLIM2 siRNA. The left panel shows luciferase activity in cells transfected with p65 and a dual-luciferase plasmid containing a NF-{kappa}B promoter and treated with 1 µg/ml LPS for 2 h. Data are reported as mean-fold induction change in luciferase activity ± SE for triplicate samples. The right panel shows ELISA assay of TNF-{alpha} in culture supernatants of transfected cells stimulated for 24 h with 10 ng/ml LPS. The lower panel shows suppression of PDLIM2 in RAW264.7 cells. The graphs are representative of three separate experiments (*, P<0.001; **, P<0.05; Student’s t-test).

PDLIM2 promotes adhesion and associates with {alpha}-actinin in differentiated THP-1 cells
We next turned our attention to the function of PDLIM2 in the cytoplasm of differentiated THP-1 cells. As these cells are adherent, we hypothesized that the cytoplasmic pool of PDLIM2 may be important for regulating cytoskeletal function in adherent cells, as we have reported previously for epithelial cells [1 ]. Therefore, we investigated whether cells with overexpressed or suppressed PDLIM2 expression have altered adhesion to VCAM-1, which on the surface of activated endothelial cells, interacts with leukocyte integrins to promote transendothelial migration of leukocytes (reviewed in ref. [36 ]). Suppression of PDLIM2 using siRNA caused decreased adhesion to VCAM-1 compared with control siRNA-treated cells (Fig. 5A , left panel). In contrast, cells overexpressing GFP-PDLIM2 exhibited enhanced adhesion to VCAM-1 (Fig. 5A , right panel). Suppression of PDLIM2 in an adherent RAW264.7 macrophage cell line resulted in decreased adhesion to fibronectin (Fig. 5B) .


Figure 5
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  		Figure 5. PDLIM2 promotes adhesion to VCAM-1 and associates with {alpha}-actinin in PMA-differentiated monocytes. (A) THP-1 cells were transfected with control or PDLIM2 siRNA (left panel) or GFP or GFP-PDLIM2 (right panel) using the Amaxa Nucleofector Kit V, according to the manufacturer’s instructions. Thirty-six hours postnucleofection, cells were seeded onto VCAM-1-coated wells for 1 h, at which time, the cells were fixed and stained with crystal violet. Confirmation of transfection was measured by Western blot with anti-PDLIM2 antibody (left panel) or photographing the cells using the FITC channel of the Nikon TE300 inverted microscope equipped with a SPOT digital camera (right panel). (B) RAW264.7 cell adhesion to fibronectin was measured in cells transfected with control or msPDLIM2 siRNA. Western blot (right panel) shows suppression of PDLIM2. The data for adhesion assays are represented as the means and SD of absorbance (Abs.) at 590 nm in triplicate wells; a representative graph is shown for each assay (*, P< 0.01; **, P<0.001; Student’s t-test). (C) THP-1 cells, untreated or treated with 100 ng/ml PMA for 72 h, were subjected to IP with anti-PDLIM2 antibody followed by Western blotting with anti-{alpha}-actinin antibody or fixed and stained with anti-PDLIM2 (green) and anti-{alpha}-actinin (red) antibodies (D). Nuclei were stained with Hoechst stain. Fluorescence was monitored using a 100x Plan Fluor objective on a Nikon Eclipse E600 microscope.

As PDLIM2 is known to associate with the actin cytoskeleton in epithelial cells through interaction with {alpha}-actinin [1 , 2 ], we investigated whether the PMA-mediated change in subcellular location of PDLIM2 affected its association with {alpha}-actinin. Following IP of {alpha}-actinin from THP-1 cells, PDLIM2 was only detected in immunoprecipitates from PMA-differentiated cells (Fig. 5B) . The association between PDLIM2 and {alpha}-actinin in PMA-differentiated cells was confirmed further by immunofluorescence analysis. Colocalization of both proteins is evident from the yellow merge in the large cytoplasm of differentiated cells (Fig. 5C) .

Taken together, these data demonstrate that PDLIM2 promotes adhesion and associates with cytoskeletal proteins in differentiated macrophages.


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DISCUSSION
 
The objective of this study was to use monocyte-macrophage cell models to investigate regulation of expression and subcellular location of the PDLIM2 protein and to determine whether this is associated with its function in nonadherent and adherent cells. Our findings demonstrate that PMA-induced differentiation of THP-1 cells results in a dramatic increase in PDLIM2 protein expression. In contrast to a predominantly nuclear location in nondifferentiated THP-1 cells, PDLIM2 was located predominantly in the cytoplasm of adherent differentiated cells, where it was phosphorylated heavily on serine/threonine residues in an ERK- and PKC-dependent manner. Interestingly, in differentiated THP-1 cells, the nuclear expression levels of the NF-{kappa}B p65 subunit were increased, as was transcriptional activity and expression of target gene TNF-{alpha}. Decreased adhesion and increased NF-{kappa}B activity were also observed upon suppression of PDLIM2 with siRNA in THP-1 and RAW246.7 cells. The data suggest that PDLIM2 expression, phosphorylation, and cytoskeletal location are dependent on cell adhesion, which may sequester it from the nucleus and thereby, regulate its activity in promoting NF-{kappa}B degradation.

Increased expression of PDLIM2 in differentiated THP-1 cells was apparently, in part, a result of increased stability of the protein, which could be reversed by proteasome inhibition. Our data showing that LMB caused accumulation of PDLIM2 in the nucleus of differentiated cells also indicated that nuclear export could account for the accumulation of PDLIM2 in the cytoplasm. The increased expression of PDLIM2 was accompanied by greatly retarded mobility in SDS-PAGE, which could be reversed by serine/threonine phosphatase. It is likely that the phosphorylation of PDLIM2 is enhanced by the actions of PMA. Inhibition of PKCs or ERK was sufficient to reverse phosphorylation and also caused loss of adhesion of the cells. It is likely that PKC inhibitors reverse the induction of PKCs by PMA, but nonetheless these data indicate that PDLIM2 can be phosphorylated by PKCs [37 ]. This conclusion is supported by analysis of the putative phosphorylation sites in the protein (data not shown).

Different PKC isoforms can play antagonistic roles in the monocyte-differentiation process. For example, in the THP-1 monocytic cell line, PKC{alpha} has been shown to promote cellular differentiation, whereas PKCβ retards the differentiation process (reviewed in ref. [38 ]). In addition to increased PKC{alpha} expression, PMA-differentiated THP-1 cells have been shown to express constitutively active ERK [39 ]. PMA-induced activation of PKCs can also promote ERK activation in primary human macrophages [29 ]. The PMA-PKC-ERK signaling pathway has been implicated in the inhibition of THP-1 cell growth that occurs before differentiation of these cells [40 ].

It is also likely that the increase in PDLIM2 expression at the cytoplasm and its phosphorylation are, in part, a result of adhesion signaling. We have observed previously that adherent epithelial cells and fibroblasts have higher expression of PDLIM2 than de-adhered cells [1 ]. Cells treated with the PKC and ERK inhibitor displayed weak adherence and a rounded appearance following PMA treatment, which was in contrast to control cells treated that remained attached and displayed a spread phenotype following PMA stimulation (data not shown). The loss of adhesion in cells may contribute to the decrease in phosphorylation observed with PDLIM2.

Analysis of PDLIM2 expression in discrete, subcellular protein fractions demonstrated that PDLIM2 was located predominantly in the cytoplasm of differentiated cells. The increased accumulation of PDLIM2 in response to LM-mediated inhibition of nuclear export, in differentiated but not in nondifferentiated THP-1 cells, suggests that PDLIM2 accumulation in the cytoplasm is a result of activation of NES-mediated export. The retention of PDLIM2 in the nucleus of nondifferentiated cells suggests that its ability to undergo NES-mediated export may be suppressed. Activation of such a nuclear export mechanism has been shown in other proteins to require post-translational modification of the protein, which promotes its interaction with CRM1. For example, histone deacetylase 7 requires phosphorylation of three critical serines for CRM1-dependent nuclear export [41 ]. Therefore, the phosphorylation of PDLIM2 following PMA-mediated differentiation may be necessary for activation of NES-mediated export. However, as the predicted NES is located in the PDZ domain, a putative protein–protein interaction motif, we cannot rule out that an unidentified, interacting protein may interfere with the translocation of PDLIM2. This has been shown for inhibitor of apoptosis proteins (IAPs), whose expression and activity are regulated by modulation of their subcellular location. IAPs are sequestered in the nucleus of undifferentiated monocytic cell lines and migrate to the cytoplasm upon cell differentiation [42 ].

PMA-induced differentiation of monocytes into macrophages also correlates with increased nuclear translocation of NF-{kappa}B [43 ]. However, the molecular mechanisms regulating NF-{kappa}B translocation to the nucleus and the consequential increased NF-{kappa}B activity during macrophage differentiation remain unknown. It has been proposed that rather than driving monocyte differentiation, the persistent nuclear translocation of NF-{kappa}B confers a protection to cells undergoing the differentiation process [44 ]. However, as many NF-{kappa}B target genes encode potentially cytotoxic molecules, excessive activation and dysregulation of NF-{kappa}B can result in massive damage to host tissues, chronic inflammatory disorders [45 , 46 ], and cancers (reviewed in refs. [47 48 49 ]). Ubiquitin-mediated degradation of nuclear p65 is required for efficient termination of NF-{kappa}B-dependent transcription [50 ], and recently, PDLIM2 has been shown to mediate the translocation of p65 to PML bodies in the nucleus, where it undergoes proteasome-mediated degradation [18 ]. Here, we observed an increase of p65 expression and function in the nucleus of differentiated THP-1 cells, which correlates with the increased export, phosphorylation, and accumulation of PDLIM2 in the cytoplasm. We propose that the differentiation-induced nuclear export of PDLIM2 is required for the enhanced NF-{kappa}B activity, previously reported in differentiated cells.

As we had previously demonstrated a role for PDLIM2 in promoting epithelial cell adhesion and as a binding partner for {alpha}-actinin, we investigated whether PDLIM2 retained these functions in hematopoietic cells. As expected, PDLIM2 overexpression or suppression affected THP-1 cell adhesion to VCAM-1, which is thought to play a critical role in the firm adhesion of monocytes to the vascular endothelium and the subsequent transendothelial migration. A role for PDLIM2 in macrophage adhesion is supported by our observations in RAW246.7 cells, where suppression of PDLIM2 caused decreased adhesion to fibronectin. As a promoter of monocyte adhesion to VCAM-1 and epithelial cell migration [1 ], PDLIM2 may regulate transendothelial migration. Interestingly, PKC and ERK are required for phosphorylation of PDLIM2 and also for cell adhesion observed during differentiation. Our previous studies in epithelial cells provide a further connection between PDLIM2 phosphorylation and cell adhesion, as we demonstrated that the slower-migrating form of PDLIM2 in epithelial cells disappears when the cells are forced to grow in suspension on polyhema-coated dishes, but reappears when these cells are allowed to re-attach to a substratum [15 ]. These data suggest that phosphorylation of PDLIM2 is required for mediating its affect on cell adhesion and migration in leukemic and epithelial cells.

Altogether, the present study suggests that PDLIM2 function may be regulated by phosphorylation and subcellular location, which results in PDLIM2, predominantly localizing to the nucleus of undifferentiated cells where it regulates NF-{kappa}B activity, and upon differentiation, PDLIM2 is translocated to the cytoplasm where it regulates cell adhesion and possibly cell migration.


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ACKNOWLEDGEMENTS
 
The Science Foundation Ireland and the Health Research Board of Ireland funded this work. We are grateful to Kurt Tidmore for preparing the illustrations and to our colleagues in the Cell Biology Laboratory for helpful discussions.

Received April 14, 2008; revised October 23, 2008; accepted October 31, 2008.


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