- Open Access
LIM kinase1 modulates function of membrane type matrix metalloproteinase 1: implication in invasion of prostate cancer cells
© Tapia et al; licensee BioMed Central Ltd. 2011
Received: 28 July 2010
Accepted: 10 January 2011
Published: 10 January 2011
LIM kinase 1 (LIMK1) is an actin and microtubule cytoskeleton modulatory protein that is overexpressed in a number of cancerous tissues and cells and also promotes invasion and metastasis of prostate and breast cancer cells. Membrane type matrix metalloproteinase 1 (MT1-MMP) is a critical modulator of extracellular matrix (ECM) turnover through pericellular proteolysis and thus plays crucial roles in neoplastic cell invasion and metastasis. MT1-MMP and its substrates pro-MMP-2 and pro-MMP-9 are often overexpressed in a variety of cancers including prostate cancer and the expression levels correlate with the grade of malignancy in prostate cancer cells. The purpose of this study is to determine any functional relation between LIMK1 and MT1-MMP and its implication in cell invasion.
Our results showed that treatment with the hydroxamate inhibitor of MT1-MMP, MMP-2 and MMP-9 ilomastat inhibited LIMK1-induced invasion of benign prostate epithelial cells. Over expression of LIMK1 resulted in increased collagenolytic activity of MMP-2, and secretion of pro-MMP2 and pro-MMP-9. Cells over expressing LIMK1 also exhibited increased expression of MT1-MMP, transcriptional activation and its localization to the plasma membrane. LIMK1 physically associates with MT1-MMP and is colocalized with it to the Golgi vesicles. We also noted increased expression of both MT1-MMP and LIMK1 in prostate tumor tissues.
Our results provide new information on regulation of MT1-MMP function by LIMK1 and showed for the first time, involvement of MMPs in LIMK1 induced cell invasion.
LIM kinase 1 (LIMK1) is a downstream effector of Rho signaling pathway, which modulates actin dynamics. LIMK1, a unique serine/threonine kinase containing two N-terminal LIM domains in tandem and a PDZ domain  is a newly identified candidate that promotes prostate and breast cancer metastasis [2–4]. High levels of LIMK1 have been observed in highly invasive prostate cancer cell lines and in human prostate tumors [2, 3, 5]. LIMK1 expression increased invasiveness of non-invasive prostate and breast cancer cells and expression of antisense RNA or dominant negative kinase-dead LIMK1 greatly reduced invasion of prostate and breast cancer cells [2–4]. LIMK1 regulates actin cytoskeleton remodeling through inactivating phosphorylation of cofilin on Ser3 residue  resulting in accumulation of actin polymer. The catalytic activity of LIMK1 requires activating phosphorylation at the T508 residue in its kinase domain, which changes conformation of the kinase domain and favors dissociation of the autoinhibitory N-terminal LIM domains from the C-terminal kinase domain making the kinase domain accessible to its substrate . Activating phosphorylation of LIMK1 is mediated by p21 kinase (PAK1 & PAK4) and Rho kinase (ROCK), which in turn are activated by the members of Rho subfamily of small GTPases (Rho, Rac and Cdc42) . LIMK1 is also involved in Rac-mediated lamellipodia formation .
Membrane type matrix metalloproteinase 1 (MT1-MMP) belongs to a family of zinc binding collagenase that is involved in extracellular matrix (ECM) turnover . The ability of MT1-MMP to degrade ECM has established its role in physiological and pathological tissue remodeling such as angiogenesis and tumor development. Expression of MT1-MMP is documented in various tumor cells and strongly implicated in tumor progression and metastasis . MT1-MMP shares conserved structural features with other MMPs, such as an N-terminal signal peptide, a propeptide and a catalytic domain . In its active form MT1-MMP is a membrane-tethered metalloproteinase, which anchors to the plasma membrane with its transmembrane domain so that the catalytic domain is exposed on the surface of the cells .
Activation of MT1-MMP requires removal of the propeptide by furin convertase, resulting in a 57 kDa active enzyme  and its targeting into the plasma membrane. Tissue inhibitor of matrix metalloproteinase 2 (TIMP-2) interacts with the membrane-tethered MT1-MMP with its catalytic domain and inhibits its proteolytic activity . MT1-MMP bound with TIMP-2 acts as a receptor for binding of soluble pro-MMP-2 with its hemopexin domain. The trimolecular complex of MT1-MMP/TIMP-2/pro-MMP-2 then present pro-MMP-2 to a neighboring TIMP-2 free MT1-MMP, which cleaves pro-MMP2 to its active form . To position another molecule of MT1-MMP next to the ternary complex, MT1-MMP forms a homo-oligomeric complex through its hemopexin and or transmembrane/cytoplasmic domain [17, 18]. Recent studies linked the function of MT1-MMP and MMP-2 on ECM degradation and metastasis by showing the processing , membrane targeting , autocatalysis  and internalization  of MMPs. These studies showed that MT1-MMP and MMP-2 function through balanced activation and inactivation process and any alteration in the activation and processing of MMPs influence the overall maintenance of ECM homeostasis, which may trigger excessive ECM degradation leading to cancer metastasis. MT1-MMP/TIMP-2/MMP-2 activation complex also processes proMMP-9 to its active form, which is mediated by TIMP-2-regulated cascade of zymogen activation initiated by MT1-MMP . Recent studies also showed activation of MMP-9 by an MT1-MMP associated protein through RhoA activation and actin remodeling . Because MT1-MMP, MMP-2 and MMP-9 are all overexpressed in invasive prostate cancers, it is likely that increased activation of MT1-MMP/MMP-2 complex also activates proMMP-9 and acts as a major mediator of pericellular proteolysis [13, 25].
Earlier studies showed the involvement of activated Rac1 and RhoA in induction of metastasis in animals suggesting that the signaling pathway regulated by these proteins may play a role in acquisition of the metastatic phenotype . Rac1 is essential for growth factor-induced cell invasion and lamellipodia formation through modulation of actin cytoskeleton . Later on, the role of Rac1 in tumor cell invasion mediated through expression, processing and activation of MMPs was established . These observations indicate a possible link between activation of MMP and LIMK1 function. In this study, we examined the involvement of MMPs in cell invasion induced by LIMK1 and the role of LIMK1 in regulation of expression and activation of MT1-MMP in prostate epithelial cells.
Materials and methods
Cell lines and antibodies
The parental BPH-1 cells (a gift from P Narayana, University of Florida) , and its transfected sub-lines, BPHLCA and BPHV, were maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich). PC3 cells (ATCC) were maintained in HAM 12 medium (Sigma-Aldrich). All media were supplemented with 10% fetal bovine serum, 2 mM glutamine and 1× antibiotic and antimycotic solution (Invitrogen). BPHLCA and BPHV cells were developed by stable transfection of constitutively active (phosphomimic mutant) LIMK1 gene containing a Flag tag at the 3' end cloned in pRevTRE (Clontech, Mountain View, CA), and an empty vector, respectively. A number of hygromycin resistant clones were isolated and mixed for subsequent experiments to avoid clonal bias. The phosphomimic mutant of human LIMK1 (LIMKT508EE) was generated by site directed mutagenesis of T508 to EE. Transfected cells were routinely maintained in antibiotic (hygromycin) containing media. All cells were grown in appropriate growth media in a humidified atmosphere containing 5% CO2, at 37°C. Monoclonal or polyclonal antibodies specific for LIMK1 (BD Biosciences and Santa Cruz Biotechnology), Trans-Golgi Network Golgi marker TGN46 (Novous Biologicals, Littleton CO). MT1-MMP (Neomarker, Fremont, CA, Thermo Fisher, Rockford, IL, and Chemicon, Millipore, Billerica, MA) and Flag (Sigma) were used for various experiments.
Human multiple prostate cancer tissue microarrays (TMA) (PR483) from US Biomax, Inc. (Rockville, MD) were used to detect expressions of MT1-MMP and LIMK1. PR483 contained 40 cases of prostate cancer tissues with 8 cases of normal tissues from autopsy. TMA containing formalin fixed and paraffin embedded tissue samples were cut at 5 μm thickness and mounted on positively charged SuperFrost Plus glass slides. Individual cores in TMA sections were 1.5 mm in diameter. All tumors were malignant in nature with Gleason Scores ranged between 2(1+1) to 10(5+5) and with no detectable local invasion or metastasis. TNM classification showed that tumors in the TMA were either T2NxM0 or T3NxM0 or T4NxM0. The composition of tumor tissues included 20 tumors of grades between 1 and 2 (low grade LG) and 20 tumors of grades between 3-4 (high grade HG). Tumor tissues were obtained from patients with ages ranged from 20-87 years.
BPHLCA and BPHV cells were maintained in phenol red-free medium and were seeded at a density of 1.25 × 105 in serum-free medium in the upper chamber containing matrigel coated inserts (8 μM pore) of the in vitro invasion chamber (ECM 554, Chemicon). EGF was added to the cell suspension at a final concentration of 10 ng/ml. Serum free media containing EGF (100 nM) was added to the bottom chamber as the chemoattractant and chambers were incubated at 37°C in a CO2 incubator for 48 hrs. Parallel experiments were performed in the presence or absence of GM6001 (Ilomastat, Chemicon) at a concentration of 25 μM in serum free media. Next, cells migrated to the inner side of the inserts were detached, stained with fluorescent dye solution and lysed. Fluorescence was measured in a Wallac Victor 2 spectroflurimeter. Cells that traversed through the matrigel and accumulated in the bottom chamber were also counted. Invasion was confirmed by staining the underside of the membrane with 0.1% Crystal violet solution. Data was calculated as fold changes in the averaged values obtained from relative fluorescence unit (RFU) and cell enumeration.
Transfected cells (2.5 × 105) cells were seeded in equal volumes of culture media into six well dishes and incubated for 24 hrs. Next day, media was replaced with phenol red free DMEM supplemented with 10% charcoal stripped FBS and cells were incubated at 37°C in a CO2 incubator for 48 hrs. Cells were then serum starved for 24 hrs; conditioned media were collected and centrifuged at 500 × g for 5 mins at 4°C. The supernatants were separated and used for zymography. Equal volume of each sample with or without concentration was incubated with non-reducing loading buffer at room temperature for 15-20 mins. Samples were then separated on a 10% SDS gel co-polymerized with gelatin (1 mg/mL). Next, gels were incubated in renaturing buffer (2.5% Triton X-100 in distilled water) for 30 mins, washed and incubated in developing buffer (50 mM Tris pH 7.8, 0.2 M NaCl, 5 mM CaCl2, 0.02% NP-40) at 37°C for 24 hrs. Gelatinoic bands were visualized by staining with Coomassie blue followed by destaining of the gels and quantified by densitometric analysis of the dried gels using Gene Snap software.
Quantitative Real time PCR
Total RNA from BPHLCA and BPHV cells was extracted using a total RNA extraction kit (Promega, Madison, WI) and used for quantitative real time PCR. For cDNA synthesis, BioRad iScript kit was used according to manufacturer's protocol. Briefly, RNase free water and 5× reaction mix (provided with kit) were added to total RNA samples (1 μg). Next, samples were denatured at 65°C for 15 mins and cooled to 37°C for 3 mins. Reverse transcription was carried out at 42°C for 1.5 hrs. Real-time PCR was performed using a SYBR green based PCR kit (Biorad) and cDNAs. A RT-PCR reaction without reverse transcriptase was used as a control. Specific primers for MMP-2 (F: 5'GTCTCCTGCTCCCCCT3', R: 5' CGAACATTGGCCTTGATCTCA3') and GAPDH (F: 5'GCAAGTTTCCGTTCCGCTTCC3', R: 5'CAGTACCAGTGTCAGTATCAGC3') were used for QPCR (40 cycles) of MMP-2 and GAPDH transcripts. Reactions were carried out in BioRad iCycler thermocycler. Quantification of the relative expression of MMP-2 gene was performed using 2-ΔΔCt method and GAPDH as a reference gene. To calculate relative expression, MMP-2 expression was normalized for each sample using GAPDH expression. Fold change expression was calculated as a ratio of normalized expression of MMP-2 in BPHLCA cells and in BPHV cells.
Immunoblot and immunoprecipitation
Total cell extracts from BPH-1, BPHLCA, BPHV and PC3 cells were prepared using the lysis buffer (50 mM Tris pH 8.0, 120 mM NaCl, 2.5 mM EDTA, 1 mM PMSF, 1%NP-40, 10 μg/mL leupeptin/aprotinin) and freeze-thaw cycles. Total proteins (50 μg) were separated in SDS-PAGE and subjected to immunoblot analysis using primary antibodies against the Flag tag, LIMK1 or MT1-MMP to monitor expression of specific proteins. A chemiluminescence detection kit (Thermo Scientific, Rockford, IL) was used to detect target proteins using corresponding secondary antibodies. For immunoprecipitation, crude PC3 cell extracts were diluted in RIPA buffer containing proteinase inhibitor mixture set III (Calbiochem EMD, Gibbstown, NJ) and treated with antibodies against LIMK1 or MT1-MMP using the standard protocol. Antigen-antibody complexes were immunoprecipitated using protein A/G PLUS sepharose beads (Santa Cruz Biotechnology) and detected by immunoblot analysis using specific antibodies.
Gene silencing using small interfering RNA
Inhibition of LIMK1 expression in PC3 cells was conducted by transfection of HuSH shRNA constructs against LIMK1 (AAGGACAAGA GGCTCAACTTCATCACTGA) in pGFP-V-RS vector (Origene Technologies). Initially four different shRNAs of LIMK1 were screened to identify the shRNA that caused maximum inhibition of LIMK1 expression for subsequent experiments. An shRNA construct for scrambled RNA was used to evaluate the off target effect of the shRNA. Cells were transiently transfected using Lipofectamine LTX (Invitrogen, Carlsbad, CA) reagent or FuGENE HD and shRNA constructs, and incubated for 55-72 hrs for optimum knockdown of LIMK1.
The TMA sections were deparaffinized in xylene, hydrated with a graded series of alcohol (100%, 95%, and 80% ethanol [vol/vol] in deionized H2O), and re-hydrated in de-ionized water. Sections were incubated for 5 mins in 3% H2O2 in water to block endogenous peroxidase and washed. Antigen retrieval was achieved by placing slides in 1× antigen retrieval solution (Target Retrieval solution, S-1699, DakoCytomation) for 30 mins in microwave oven with simmering conditions then cooled down for 15 mins at room temperature. Slides were then washed with PBS that contained 0.1% triton and 0.1% BSA. Nonspecific binding was blocked with (2.5%) normal horse blocking serum and 2% BSA in PBS. The slides were then incubated for 1 hr at room temperature with one of the following: 1) monoclonal mouse anti-LIMK1 (1:600 dilution) or 2) rabbit anti-MMP-14/MT1-MMP antibodies (Millipore Ab-1) (1:800 dilution). Slides were then washed with PBS that contained 0.1% triton and 0.1% BSA. Slides were then incubated with ImmPRESS™ Reagent anti-Rabbit or anti-Mouse Ig (peroxidase)(Vector Laboratories) for 30 mins at room temperature. Slides were washed next and incubated in peroxidase substrate DAB solution (DAKO Cytomation). Finally, sections were washed in tap water and counterstained with Hematoxylin QS (Vector Labs). Slides were mounted with permanent mounting medium (C0487, Sigma). IHC staining was evaluated by an independent pathologist from US Biomax, Inc. Manual scoring of intensity, negative (0), weak (1+), moderate (2+), or strong (3+), location and cell types of staining were performed by the pathologist and the scores were then converted to number from 0 to 3 scales. Images of the stained sections were scanned and the total positive cell numbers and intensity of anti-LIMK1 and anti-MT1-MMP staining were computed and measured by ImageScope from Aperio Scanning System (US Biomax, Inc).
Dual and triple label Immunofluorescence analysis
Because each pixel is subtracted by the average pixel intensity, the value for Correlation R can range from -1 to 1. A value of 1 would mean that the patterns are perfectly similar (colocalized), while a value of -1 would mean that the patterns are perfectly opposite.
Surface staining of MT1-MMP and cell surface biotinylation
BPHLCA or BPHV cells were seeded in complete growth medium to 80% confluence and harvested by incubating in cell stripper (Cell Gro, Manassas, VA) at room temperature. Phosphate buffered saline (PBS) was added to the dish and cells (5 × 105) were collected by centrifugation. Cells were suspended in PBS containing 3%FBS and MT1-MMP antibodies against the extracellular catalytic domain (Chemicon) (5 μg/1 × 106 cells) and incubated at 4°C with rocking for 3 hrs. Cells were washed with PBS containing 3% FBS and incubated with secondary antibodies conjugated with Alexa 488 (Molecular Probes, Carlsbad, CA) (1:800) for 30 mins with rocking at 4°C. Cells were washed and fixed with sterile 2% paraformaldehyde in PBS. Cells were analyzed in a flow cytometer (FACS Calibur/BD Biosciences, San Jose, CA). For biotin labeling of cell surface proteins, PC3 cells (3 × 105 cells/well) were seeded on 6-well dishes and after 24 hrs transfected with cDNAs for LIMK1 shRNA or control shRNA. After 68 hrs of incubation, cells were incubated with cell impermeable Ez-Link Sulfo-NHS-LC-Biotin (Pierce) (0.5 mg/ml) at 4°C with rocking for 30 mins and quenched with 100 mM glycine to remove excess Biotin according to the method described in [30, 31]. Next, cells were harvested in RIPA Buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 0.1% SDS) with proteinase inhibitors (1 μg/ml aprotinin, 1 μM pepstatin, and 10 μM leupeptin) by scraping. Cells were lysed and clarified by centrifugation. Biotin-labeled surface proteins were separated from equal amounts of cell lysate proteins, by incubating with washed UltraLink Streptavidin sepharose beads (Pierce) at 4°C with mixing for 14 hrs. Bead-bound proteins were separated on SDS-PAGE and immunoblotted for MT1-MMP (1:500) on the plasma membrane using antibodies against the hinge region (Millipore).
Dual Luciferase reporter assay
The MT1-MMP promoter-luciferase construct containing firefly luciferase driven by a 7.2 KB promoter fragment of MT1-MMP (kindly provided by Jorma Keski-Oja, University of Helsinki) was used for transient transfection using Lipofectamine LTX according to our published protocol . A construct containing Renilla Luciferase driven by thymidine kinase promoter was used for cotransfection as the transcription control. Transfected BPH-1 sublines and PC3 cells with or without co-transfection of cDNAs for LIMK1 shRNA or control shRNA were used for luciferase reporter assays. Cells were harvested at 62 hrs post transfection and luciferase expression was determined using a Dual luciferase assay kit (Promega) according to supplier's protocol.
Quantitative results are presented as meam ± SD of the number of independent experiments performed. Statistical differences were calculated using Student's t-test in GraphPad/Prism 4.0a. A p value of < 0.05 was considered significant. IHC scoring data were analyzed using GraphPad/Prism 4.0a.
LIMK1-induced invasion is mediated by MMPs
LIMK1 expression was associated with increased secretion of pro-MMP-2 and pro-MMP-9 in the conditioned media
LIMK1 expression positively correlated with expression of MT1-MMP in prostate cancer cells
LIMK1 and MT1-MMP are overexpressed in cancerous prostate
LIMK1 colocalizes and physically associates with MT1-MMP
LIMK1 facilitates transport of MT1-MMP to the plasma membrane through Golgi vesicles
Expression of LIMK1 increased surface localization of MT1-MMP
Expression of LIMK1 was associated with increased transcriptional activation of MT1-MMP
LIMK1 being an actin and microtubule modulatory protein is likely to be involved in acquisition of an invasive phenotype commonly noted in tumors exhibiting advanced stages of malignancy. Accordingly, earlier studies including ours clearly demonstrated an important role of LIMK1 in induction of invasion and metastasis of prostate and breast cancer cells and tumors [2–4]. More recently, role of LIMK1 in induction of metastasis in pancreatic cancer in zebrafish xenograft assays ; and mesenchymal and ameboid modes of invasion of fibrosarcoma cells in 3D matrices [43, 44] were shown. These studies further strengthened the importance of LIMK1 and Rho/Rock signaling pathway in generation of protrusive forces of tumor cells through collagen matrices.
Our studies presented here demonstrated that LIMK1 is involved in regulating MT1-MMP functions at various levels. The role of MT1-MMP in invasion and metastasis through direct and indirect collagenolytic activities is well documented therefore it is likely that the critical role of LIMK1 in facilitation of cell motility and invasion is at least partly mediated through modulation of MMP functions. We confirmed the association of MMPs such as MT1-MMP, MMP-2 and MMP-9 in LIMK1-induced invasion of BPHLCA cells in invasion assays. Expression of phosphomimic LIMK1 in BPH-1 cells changed their noninvasive phenotype to invasive ones, but use of the hydoxamate inhibitor (GM6001) of MMPs, specifically MT1-MMP, MMP-2 and MMP-9 completely abrogated the invasive property of these cells. To our knowledge, this is the first report showing a functional link between LIMK1 and MT1-MMP.
The question that we asked next is that if LIMK1 expression also increased the proteolytic function of MMPs. Indeed, our results showed that processing of pro-MMP-2 to its active form was increased in BPHLCA cells (Figure 2). In addition, there was an overall increase in secreted MMP-2 (latent) as commonly noted in invasive prostate cancer cells. We investigated whether LIMK1 expressing cells also have a higher expression of mRNAs of pro-MMP-2, which was also the case and a significant increase in MMP-2 mRNA was noted in these cells. Increased expression of LIMK1, either ectopically or endogenously, was associated with a significant increase in expressions of the latent and active forms of MT1-MMPs, which were substantially diminished upon knockdown of LIMK1. It is speculated that increased expression of the latent MT1-MMP resulted in increased levels of active MT1-MMP as seen in BPHLCA and PC3 cells. The correlative expression profile of LIMK1 and MT1-MMP was also detected in clinical samples pathological reports of which showed higher tumor grades. In addition, most of tumor cells of prostate adenocarcinoma had higher cytoplasmic and nuclear expression of LIMK1 and MT1-MMP, compared to normal prostate tissues. A number of studies showed increased expression of MT1-MMP in advanced cancers including prostate cancer. Increased expression of LIMK1 in luminal cells has been reported also in advanced prostate tumors. Our study shows increased expression of both proteins in the same tumor samples, which suggests a clinical relevance of overexpression of both LIMK1 and MT1-MMP.
Our studies further provide evidence that LIMK1 physically interacts with MT1-MMP and regulates its vesicular transport to the plasma membrane. Colocalization of MT1-MMP and LIMK1 was in abundance in the Golgi areas at the perinuclear region in both PC3 and BPHLCA cells. In addition, distinct vesicles at various distances between perinuclear region to the plasma membrane with colocalized LIMK1, MT1-MMP and TGN46 were seen in PC3 cells. This observation indicating a role of LIMK1 in vesicular trafficking is in support of earlier reports showing that LIMK1 regulates endocytic or exocytic vesicular transport in endothelial cells for transport of SREBP cleavage activating protein (SCAP) . Earlier studies also showed that LIMK1 modulates Golgi dynamics through protein-protein interaction through its LIM domain and trafficking of Golgi transport vesicles between ER and Golgi in primary neuronal cells . Recent studies by Nishimura et al  indicated a role of LIMK1 in regulating endocytic trafficking of EGFR wherein LIMK1 delays internalization of EGFR bound EGF, thereby maintaining sustained activation of EGF/EGFR axis in invasive tumor cells. Nonetheless, it is not clear if LIMK1 also regulates internalization of MT1-MMP and requires further study. Importantly, inhibition of LIMK1 dramatically reduced plasma membrane targeting of MT1-MMP (Figure 6), which confirms a distinct regulatory role of LIMK1 in vesicular transport of MT1-MMP for its surface localization.
Surface localization of MT1-MMP is another essential event for MT1-MMP to be functionally active for its collagenolytic activities either directly or through increased activation of soluble pro-MMP-2 through the ternary complex formation (MT1-MMP/TIMP-2/pro-MMP-2), and pro-MMP-9 through activation of MMP-2/TIMP2 axis [14, 23]. In support of our immunoblot results, increased surface localization of MT1-MMP was noted in BPHLCA cells expressing LIMKT508EE as shown by flow cytometry. Surface biotinylation assays in PC3 cells following knockdown of LIMK1 further confirmed the role of LIMK1 in MT1-MMP surface localization. We speculate that LIMK1 regulates surface localization of MT1-MMP through its physical interaction with MT1-MMP, as our immunofluorescence analysis and coimmunoprecipitation studies confirmed such interaction. It is possible that interaction between LIMK1 and latent MT1-MMP helps the proteolytic processing of MT1-MMP and its targeting to the plasma membrane.
Our studies using luciferase reporter assays showed that LIMK1 expression increased activation of MT1-MMP promoter in BPHLCA cells and knockdown of LIMK1 significantly reduced luciferase expression in PC3 cells. This result indicates that LIMK1 has a regulatory role in transcription of MT1-MMP and thereby increases pro-MT1-MMP levels when overexpressed in cells. Role of LIMK1 in promoter activation has been reported earlier, which showed that LIMK1 expression increased activation of uPA promoter in breast cancer cells . However, how LIMK1 induces transcriptional activation of MT1-MMP is not clear and studies are underway to determine the mechanism of LIMK1-induced increased transcription of MT1-MMP. To this end, the importance of this study lies in the realm of a possibly better therapeutic approach for metastatic cancer by inhibition of LIMK1 instead of MMP inhibitors which showed higher toxicity.
We thank Dr. Debopam Chakrabarti for critical review and helpful discussion with the manuscript. This work was supported in part by the Prostate Cancer Research Program of the Department of Defense (PC041048-RC) and from National Cancer Institute (R15CA125681-RC).
- Okano I, Hiraoka J, Otera H, Nunoue K, Ohashi K, Iwashita S, Hirai M, Mizuno K: Identification and characterization of a novel family of serine/threonine kinases containing two N-terminal LIM motifs. J Biol Chem. 1995, 270: 31321-31330. 10.1074/jbc.270.52.31321View ArticlePubMedGoogle Scholar
- Davila M, Frost AR, Grizzle WE, Chakrabarti R: LIM kinase 1 is essential for the invasive growth of prostate epithelial cells: implications in prostate cancer. J Biol Chem. 2003, 278: 36868-36875. 10.1074/jbc.M306196200View ArticlePubMedGoogle Scholar
- Yoshioka K, Foletta V, Bernard O, Itoh K: A role for LIM kinase in cancer invasion. Proc Natl Acad Sci USA. 2003, 100: 7247-7252. 10.1073/pnas.1232344100PubMed CentralView ArticlePubMedGoogle Scholar
- Bagheri-Yarmand R, Mazumdar A, Sahin AA, Kumar R: LIM kinase 1 increases tumor metastasis of human breast cancer cells via regulation of the urokinase-type plasminogen activator system. Int J Cancer. 2006, 118: 2703-2710. 10.1002/ijc.21650View ArticlePubMedGoogle Scholar
- Davila M, Jhala D, Ghosh D, Grizzle WE, Chakrabarti R: Expression of LIM kinase 1 is associated with reversible G1/S phase arrest, chromosomal instability and prostate cancer. Mol Cancer. 2007, 6: 40- 10.1186/1476-4598-6-40PubMed CentralView ArticlePubMedGoogle Scholar
- Arber S, Barbayannis FA, Hanser H, Schneider C, Stanyon CA, Bernard O, Caroni P: Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature. 1998, 393: 805-809. 10.1038/31729View ArticlePubMedGoogle Scholar
- Edwards DC, Gill GN: Structural features of LIM kinase that control effects on the actin cytoskeleton. J Biol Chem. 1999, 274: 11352-11361. 10.1074/jbc.274.16.11352View ArticlePubMedGoogle Scholar
- Edwards DC, Sanders LC, Bokoch GM, Gill GN: Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat Cell Biol. 1999, 1: 253-259. 10.1038/12963View ArticlePubMedGoogle Scholar
- Yang N, Higuchi O, Ohashi K, Nagata K, Wada A, Kangawa K, Nishida E, Mizuno K: Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature. 1998, 393: 809-812. 10.1038/31735View ArticlePubMedGoogle Scholar
- Itoh Y, Seiki M: MT1-MMP: a potent modifier of pericellular microenvironment. J Cell Physiol. 2006, 206: 1-8. 10.1002/jcp.20431View ArticlePubMedGoogle Scholar
- Deryugina EI, Quigley JP: Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev. 2006, 25: 9-34. 10.1007/s10555-006-7886-9View ArticlePubMedGoogle Scholar
- Li XY, Ota I, Yana I, Sabeh F, Weiss SJ: Molecular dissection of the structural machinery underlying the tissue-invasive activity of membrane type-1 matrix metalloproteinase. Mol Biol Cell. 2008, 19: 3221-3233. 10.1091/mbc.E08-01-0016PubMed CentralView ArticlePubMedGoogle Scholar
- Nagakawa O, Murakami K, Yamaura T, Fujiuchi Y, Murata J, Fuse H, Saiki I: Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) on prostate cancer cell lines. Cancer Lett. 2000, 155: 173-179. 10.1016/S0304-3835(00)00425-0View ArticlePubMedGoogle Scholar
- Yana I, Weiss SJ: Regulation of membrane type-1 matrix metalloproteinase activation by proprotein convertases. Mol Biol Cell. 2000, 11: 2387-2401.PubMed CentralView ArticlePubMedGoogle Scholar
- Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA, Goldberg GI: Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J Biol Chem. 1995, 270: 5331-5338. 10.1074/jbc.270.10.5331View ArticlePubMedGoogle Scholar
- Wang Z, Juttermann R, Soloway PD: TIMP-2 is required for efficient activation of proMMP-2 in vivo. J Biol Chem. 2000, 275: 26411-26415. 10.1074/jbc.M001270200PubMed CentralView ArticlePubMedGoogle Scholar
- Itoh Y, Takamura A, Ito N, Maru Y, Sato H, Suenaga N, Aoki T, Seiki M: Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion. EMBO J. 2001, 20: 4782-4793. 10.1093/emboj/20.17.4782PubMed CentralView ArticlePubMedGoogle Scholar
- Lehti K, Lohi J, Juntunen MM, Pei D, Keski-Oja J: Oligomerization through hemopexin and cytoplasmic domains regulates the activity and turnover of membrane-type 1 matrix metalloproteinase. J Biol Chem. 2002, 277: 8440-8448. 10.1074/jbc.M109128200View ArticlePubMedGoogle Scholar
- Golubkov VS, Chekanov AV, Shiryaev SA, Aleshin AE, Ratnikov BI, Gawlik K, Radichev I, Motamedchaboki K, Smith JW, Strongin AY: Proteolysis of the membrane type-1 matrix metalloproteinase prodomain: implications for a two-step proteolytic processing and activation. J Biol Chem. 2007, 282: 36283-36291. 10.1074/jbc.M706290200View ArticlePubMedGoogle Scholar
- Nie J, Pei J, Blumenthal M, Pei D: Complete restoration of cell surface activity of transmembrane-truncated MT1-MMP by a glycosylphosphatidylinositol anchor. Implications for MT1-MMP-mediated prommp2 activation and collagenolysis in three-dimensions. J Biol Chem. 2007, 282: 6438-6443. 10.1074/jbc.M607337200View ArticlePubMedGoogle Scholar
- Remacle AG, Chekanov AV, Golubkov VS, Savinov AY, Rozanov DV, Strongin AY: O-glycosylation regulates autolysis of cellular membrane type-1 matrix metalloproteinase (MT1-MMP). J Biol Chem. 2006, 281: 16897-16905. 10.1074/jbc.M600295200View ArticlePubMedGoogle Scholar
- Lafleur MA, Mercuri FA, Ruangpanit N, Seiki M, Sato H, Thompson EW: Type I collagen abrogates the clathrin-mediated internalization of membrane type 1 matrix metalloproteinase (MT1-MMP) via the MT1-MMP hemopexin domain. J Biol Chem. 2006, 281: 6826-6840. 10.1074/jbc.M513084200View ArticlePubMedGoogle Scholar
- Toth M, Chvyrkova I, Bernardo MM, Hernandez-Barrantes S, Fridman R: Pro-MMP-9 activation by the MT1-MMP/MMP-2 axis and MMP-3: role of TIMP-2 and plasma membranes. Biochem Biophys Res Commun. 2003, 308: 386-395. 10.1016/S0006-291X(03)01405-0View ArticlePubMedGoogle Scholar
- Hoshino D, Tomari T, Nagano M, Koshikawa N, Seiki M: A novel protein associated with membrane-type 1 matrix metalloproteinase binds p27(kip1) and regulates RhoA activation, actin remodeling, and matrigel invasion. J Biol Chem. 2009, 284: 27315-27326. 10.1074/jbc.M109.041400PubMed CentralView ArticlePubMedGoogle Scholar
- Wilson SR, Gallagher S, Warpeha K, Hawthorne SJ: Amplification of MMP-2 and MMP-9 production by prostate cancer cell lines via activation of protease-activated receptors. Prostate. 2004, 60: 168-174. 10.1002/pros.20047View ArticlePubMedGoogle Scholar
- del Peso L, Hernandez-Alcoceba R, Embade N, Carnero A, Esteve P, Paje C, Lacal JC: Rho proteins induce metastatic properties in vivo. Oncogene. 1997, 15: 3047-3057. 10.1038/sj.onc.1201499View ArticlePubMedGoogle Scholar
- Malliri A, Symons M, Hennigan RF, Hurlstone AF, Lamb RF, Wheeler T, Ozanne BW: The transcription factor AP-1 is required for EGF-induced activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells. J Cell Biol. 1998, 143: 1087-1099. 10.1083/jcb.143.4.1087PubMed CentralView ArticlePubMedGoogle Scholar
- Zhuge Y, Xu J: Rac1 mediates type I collagen-dependent MMP-2 activation. role in cell invasion across collagen barrier. J Biol Chem. 2001, 276: 16248-16256.View ArticlePubMedGoogle Scholar
- Hayward SW, Dahiya R, Cunha GR, Bartek J, Deshpande N, Narayan P: Establishment and characterization of an immortalized but non-transformed human prostate epithelial cell line: BPH-1. In Vitro Cell Dev Biol Anim. 1995, 31: 14-24. 10.1007/BF02631333View ArticlePubMedGoogle Scholar
- Munshi HG, Wu YI, Mukhopadhyay S, Ottaviano AJ, Sassano A, Koblinski JE, Platanias LC, Stack MS: Differential regulation of membrane type 1-matrix metalloproteinase activity by ERK 1/2- and p38 MAPK-modulated tissue inhibitor of metalloproteinases 2 expression controls transforming growth factor-beta1-induced pericellular collagenolysis. J Biol Chem. 2004, 279: 39042-39050. 10.1074/jbc.M404958200View ArticlePubMedGoogle Scholar
- Munshi HG, Wu YI, Ariztia EV, Stack MS: Calcium regulation of matrix metalloproteinase-mediated migration in oral squamous cell carcinoma cells. J Biol Chem. 2002, 277: 41480-41488. 10.1074/jbc.M207695200View ArticlePubMedGoogle Scholar
- Mallik I, Davila M, Tapia T, Schanen B, Chakrabarti R: Androgen regulates Cdc6 transcription through interactions between androgen receptor and E2F transcription factor in prostate cancer cells. Biochim Biophys Acta. 2008, 1783: 1737-1744. 10.1016/j.bbamcr.2008.05.006View ArticlePubMedGoogle Scholar
- Zhang L, Shi J, Feng J, Klocker H, Lee C, Zhang J: Type IV collagenase (matrix metalloproteinase-2 and -9) in prostate cancer. Prostate Cancer Prostatic Dis. 2004, 7: 327-332. 10.1038/sj.pcan.4500750View ArticlePubMedGoogle Scholar
- Bonfil RD, Dong Z, Trindade Filho JC, Sabbota A, Osenkowski P, Nabha S, Yamamoto H, Chinni SR, Zhao H, Mobashery S, Vessella RL, Fridman R, Cher ML: Prostate Cancer-Associated Membrane Type 1-Matrix Metalloproteinase. A Pivotal Role in Bone Response and Intraosseous Tumor Growth. Am J Pathol. 2007Google Scholar
- Nelson AR, Fingleton B, Rothenberg ML, Matrisian LM: Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol. 2000, 18: 1135-1149.PubMedGoogle Scholar
- Boghaert ER, Chan SK, Zimmer C, Grobelny D, Galardy RE, Vanaman TC, Zimmer SG: Inhibition of collagenolytic activity relates to quantitative reduction of invasion in vitro in a c-Ha-ras transfected glial cell line. J Neurooncol. 1994, 21: 141-150. 10.1007/BF01052898View ArticlePubMedGoogle Scholar
- Golubkov VS, Boyd S, Savinov AY, Chekanov AV, Osterman AL, Remacle A, Rozanov DV, Doxsey SJ, Strongin AY: Membrane type-1 matrix metalloproteinase (MT1-MMP) exhibits an important intracellular cleavage function and causes chromosome instability. J Biol Chem. 2005, 280: 25079-25086. 10.1074/jbc.M502779200View ArticlePubMedGoogle Scholar
- Cao J, Chiarelli C, Kozarekar P, Adler HL: Membrane type 1-matrix metalloproteinase promotes human prostate cancer invasion and metastasis. Thromb Haemost. 2005, 93: 770-778.PubMedGoogle Scholar
- Daja MM, Niu X, Zhao Z, Brown JM, Russell PJ: Characterization of expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in prostate cancer cell lines. Prostate Cancer Prostatic Dis. 2003, 6: 15-26. 10.1038/sj.pcan.4500609View ArticlePubMedGoogle Scholar
- Sroka IC, Nagle RB, Bowden GT: Membrane-type 1 matrix metalloproteinase is regulated by sp1 through the differential activation of AKT, JNK, and ERK pathways in human prostate tumor cells. Neoplasia. 2007, 9: 406-417. 10.1593/neo.07193PubMed CentralView ArticlePubMedGoogle Scholar
- Gamell C, Osses N, Bartrons R, Ruckle T, Camps M, Rosa JL, Ventura F: BMP2 induction of actin cytoskeleton reorganization and cell migration requires PI3-kinase and Cdc42 activity. J Cell Sci. 2008, 121: 3960-3970. 10.1242/jcs.031286View ArticlePubMedGoogle Scholar
- Vlecken DH, Bagowski CP: LIMK1 and LIMK2 are important for metastatic behavior and tumor cell-induced angiogenesis of pancreatic cancer cells. Zebrafish. 2009, 6: 433-439. 10.1089/zeb.2009.0602View ArticlePubMedGoogle Scholar
- Rosel D, Brabek J, Tolde O, Mierke CT, Zitterbart DP, Raupach C, Bicanova K, Kollmannsberger P, Pankova D, Vesely P, Folk P, Fabry B: Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces. Mol Cancer Res. 2008, 6: 1410-1420. 10.1158/1541-7786.MCR-07-2174View ArticlePubMedGoogle Scholar
- Mishima T, Naotsuka M, Horita Y, Sato M, Ohashi K, Mizuno K: LIM-kinase is critical for the mesenchymal-to-amoeboid cell morphological transition in 3D matrices. Biochem Biophys Res Commun. 392: 577-581.Google Scholar
- Lin T, Zeng L, Liu Y, DeFea K, Schwartz MA, Chien S, Shyy JY: Rho-ROCK-LIMK-cofilin pathway regulates shear stress activation of sterol regulatory element binding proteins. Circ Res. 2003, 92: 1296-1304. 10.1161/01.RES.0000078780.65824.8BView ArticlePubMedGoogle Scholar
- Rosso S, Bollati F, Bisbal M, Peretti D, Sumi T, Nakamura T, Quiroga S, Ferreira A, Caceres A: LIMK1 regulates Golgi dynamics, traffic of Golgi-derived vesicles, and process extension in primary cultured neurons. Mol Biol Cell. 2004, 15: 3433-3449. 10.1091/mbc.E03-05-0328PubMed CentralView ArticlePubMedGoogle Scholar
- Nishimura Y, Yoshioka K, Bernard O, Bereczky B, Itoh K: A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells. Histochem Cell Biol. 2006, 126: 627-638. 10.1007/s00418-006-0198-xView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.