Lysophosphatidic Acid Facilitates Proliferation of Colon Cancer Cells via Induction of Krüppel-like Factor 5
2007; Elsevier BV; Volume: 282; Issue: 21 Linguagem: Inglês
10.1074/jbc.m700702200
ISSN1083-351X
AutoresHuanchun Zhang, Agnieszka B. Bialkowska, Raluca Rusovici, Sengthong Chanchevalap, Hyunsuk Shim, Jonathan P. Katz, Vincent W. Yang, C. Chris Yun,
Tópico(s)Cancer-related gene regulation
ResumoAmong the multiple cellular effects mediated by lysophosphatidic acid (LPA), the effect on cell proliferation has extensively been investigated. A recent study showed that LPA-mediated proliferation of colon cancer cells requires activation of β-catenin. However, the majority of colon cancer cells have deregulation of the Wnt/β-catenin pathway. This prompted us to hypothesize the presence of additional pathway(s) activated by LPA resulting in an increase in the proliferation of colon cancer cells. Krüppel-like factor 5 (KLF5) is a transcriptional factor highly expressed in the crypt compartment of the intestinal epithelium. In this work, we investigated a role of KLF5 in LPA-mediated proliferation. We show that LPA stimulated the expression levels of KLF5 mRNA and protein in colon cancer cells and this stimulation was mediated by LPA2 and LPA3. Silencing of KLF5 expression by small interfering RNA significantly attenuated LPA-mediated proliferation of SW480 and HCT116 cells. LPA-mediated KLF5 induction was partially blocked by inhibition of the mitogen-activated protein kinase kinase and protein kinase C-δ. Moreover, we observed that LPA regulates KLF5 expression via eukaryotic elongation factor 2 kinase (eEF2k). Inhibition of calmodulin or silencing of eEF2k blocked the stimulation in KLF5 expression. Knockdown of eEF2k specifically inhibited KLF5 induction by LPA but not by fetal bovine serum or phorbol 12-myristate 13-acetate. These results identify KLF5 as a target of LPA-mediated signaling and suggest a role of KLF5 in promoting proliferation of intestinal epithelia in response to LPA. Among the multiple cellular effects mediated by lysophosphatidic acid (LPA), the effect on cell proliferation has extensively been investigated. A recent study showed that LPA-mediated proliferation of colon cancer cells requires activation of β-catenin. However, the majority of colon cancer cells have deregulation of the Wnt/β-catenin pathway. This prompted us to hypothesize the presence of additional pathway(s) activated by LPA resulting in an increase in the proliferation of colon cancer cells. Krüppel-like factor 5 (KLF5) is a transcriptional factor highly expressed in the crypt compartment of the intestinal epithelium. In this work, we investigated a role of KLF5 in LPA-mediated proliferation. We show that LPA stimulated the expression levels of KLF5 mRNA and protein in colon cancer cells and this stimulation was mediated by LPA2 and LPA3. Silencing of KLF5 expression by small interfering RNA significantly attenuated LPA-mediated proliferation of SW480 and HCT116 cells. LPA-mediated KLF5 induction was partially blocked by inhibition of the mitogen-activated protein kinase kinase and protein kinase C-δ. Moreover, we observed that LPA regulates KLF5 expression via eukaryotic elongation factor 2 kinase (eEF2k). Inhibition of calmodulin or silencing of eEF2k blocked the stimulation in KLF5 expression. Knockdown of eEF2k specifically inhibited KLF5 induction by LPA but not by fetal bovine serum or phorbol 12-myristate 13-acetate. These results identify KLF5 as a target of LPA-mediated signaling and suggest a role of KLF5 in promoting proliferation of intestinal epithelia in response to LPA. Lysophosphatidic acid (LPA) 4The abbreviations used are: LPA, lysophosphatidic acid; KLF, Krüppel-like factor; S1P, sphingosine 1-phosphate; eEF2k, eukaryotic elongation factor 2 kinase; PKC, protein kinase C; MAPK, mitogen-activate protein kinase; MEK, mitogen-activate protein kinase kinase; ERK, extracellular signal-regulated kinase; PTX, pertussis toxin; siRNA, small interfering RNA; APC, adenomatous polyposis coli; PMA, phorbol 12-myristate 13-acetate; GSK-3β, glycogen synthase kinase 3β; CaM, calmodulin; FBS, fetal bovine serum; PBS, phosphate-buffered saline; mTOR, mammalian target of rapamycin; EGF, epidermal growth factor. is a biologically active lysophospholipid that mediates a plethora of cellular effects, including cell survival, proliferation, migration, and induction of cytokines and growth factors. Signaling by LPA is mediated through LPA1, LPA2, and LPA3, which are members of a family of G protein-coupled receptors (1An S. Dickens M.A. Bleu T. Hallmark O.G. Goetzl E.J. 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In the absence of Wnt, the cytoplasmic level of β-catenin is kept low via a multiprotein complex consisting of APC, axin, and the glycogen synthase kinase 3β (GSK-3β). GSK-3β phosphorylates β-catenin, directing it to degradation by proteosome (17Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3517) Google Scholar). When activated, β-catenin accumulates in the nucleus where it binds to the transcription factors, T-cell factor, and lymphoid enhancer-binding protein, leading to transcriptional activation of multiple target genes, such as c-myc and cyclin D (17Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3517) Google Scholar, 18Moon R.T. Kohn A.D. De Ferrari G.V. Kaykas A. Nat. Rev. Genet. 2004; 5: 691-701Crossref PubMed Scopus (1599) Google Scholar). Recently, work by Yang et al. (12Yang M. Zhong W.W. Srivastava N. Slavin A. Yang J. Hoey T. An S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6027-6032Crossref PubMed Scopus (164) Google Scholar) showed that LPA can activate the β-catenin pathway leading to nuclear translocation of β-catenin. This work defines another entry point to the Wnt/β-catenin pathway and adds a new dimension to the biological effects by LPA. However, the majority of colon cancer cells have mutations that result in inactivation of APC or activation of β-catenin and, hence, it is expected that β-catenin is constitutively activated in these cells. Therefore, it remains to be determined whether LPA can further activate the β-catenin pathway in these cells. Krüppel is a zinc finger-containing transcription factor that is responsible for segmentation of the Drosophila melanogaster embryo (19Schuh R. Aicher W. Gaul U. Cote S. Preiss A. Maier D. Seifert E. Nauber U. Schroder C. Kemler R. Jäckle H. 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KLF4 expression is enriched in differentiated enterocytes found in the upper villus region, whereas KLF5 is found mainly in the proliferating crypt cell population where it positively regulates cell proliferation (24Conkright M. Wani M. Anderson K. Lingrel J. Nucleic. Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar, 25Dang D.T. Bachman K.E. Mahatan C.S. Dang L.H. Giardiello F.M. Yang V.W. FEBS Lett. 2000; 476: 203-207Crossref PubMed Scopus (88) Google Scholar, 26Sun R. Chen X. Yang V.W. J. Biol. Chem. 2001; 276: 6897-6900Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Despite the effect of LPA on proliferation of many types of cells, the mechanism of LPA-mediated proliferation is not fully elucidated and led us to hypothesize that KLF5 might be required for LPA-mediated signaling. In this work, we report that LPA stimulates the expression level of KLF5 in colon cancer cells. LPA-mediated induction of KLF5 is observed in intestinal cells regardless of the mutational status of APC and KLF5 plays a crucial role in LPA-induced proliferation of colon cancer cells. Furthermore, we found that LPA induces KLF5 via pathways dependent on MEK1/2, PKC, and eukaryotic elongation factor 2 kinase (eEF2k). Materials—1-Oleoyl LPA (18:1 LPA) and sphingosine 1-phosphate (S1P) were obtained from Avanti Polar Lipids. All antibodies were from Cell Signaling. LY290042, U0126, GF109203X, rotterlin, trifluoperazine, and rapamycin were from Calbiochem. All kinase inhibitors were added to the culture medium 10 min before the addition of LPA. PTX was added 14 h before LPA treatment. The concentrations used are as follow: GF109203X at 5 μm, rottlerin at 10 μm, LY294002 at 50 μm, SB203580 at 5 μm, U0126 at 10 μm, rapamycin at 100 nm, U73122 at 5 μm, AG1478 at 250 nm, PTX at 50 ng/ml, trifluoperazine at 3 μm, and calmidazolium at 6 μm. Stock solutions were prepared in dimethyl sulfoxide at a 1,000× concentration with respect to the working concentrations indicated above and dimethyl sulfoxide alone was added to control cells. All other chemicals were obtained from Sigma. Cell Culture—SW480 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C in 95% air, 5% CO2 atmosphere as previously described (27Yun C.C. Chen Y. Lang F. J. Biol. Chem. 2002; 277: 7676-7683Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). For Caco-2 cells, RPMI 1640 was replaced with Dulbecco's modified Eagle's medium supplemented with 0.5% non-essential amino acids. HCT116 cells were maintained in McCoy's 5A medium supplemented with 10% FBS, 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C in 95% air, 5% CO2. IEC6 cells were obtained from the Emory Digestive Disease Research Development Center and cultured at 37 °C in 95% air, 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 5% FBS, 0.1 unit/ml insulin, 50 μg/ml streptomycin, and 50 units/ml penicillin. All the cells were serum starved 24 h before LPA treatment in their appropriate media without FBS. Northern Blot Analysis—Total RNA from intestinal cells was isolated using TRIzol (Invitrogen). Thirty μg of total RNA for each sample was hybridized with [α-32P]dATP-labeled full-length mouse KLF5 cDNA probe (28Chanchevalap S. Nandan M.O. McConnell B.B. Charrier L. Merlin D. Katz J.P. Yang V.W. Nucleic Acids Res. 2006; 34: 1216-1223Crossref PubMed Scopus (81) Google Scholar) using ExpressHyb hybridization solution (BD Biosciences) at 68 °C with continuous shaking for 1 h. Washing was performed at 50 °C with 3 changes of washing solution. Phosphoprotein 36B4 (29Laborda J. Nucleic Acids Res. 1991; 193998Crossref PubMed Scopus (434) Google Scholar) was used as a loading control to normalize the expression levels of KLF5 mRNA. siRNA Transfection—Double-stranded siRNA oligonucleotides targeting LPA2, LPA3, PKCα, and eEF2k were from Dharmacon. siRNA targeting KLF5 or PKCδ was purchased from Invitrogen and Upstate, respectively. As a control, a scrambled 21-nucleotide RNA duplex was used. Cells seeded at 60% confluence on 60-mm culture plates were transfected with 40 nm siRNA using Lipofectamine 2000 (Invitrogen). Twenty-four h after transfection, cells were serum deprived for 16–24 h and then treated with LPA or carrier. The efficacy of gene silencing of LPA2 and LPA3 was determined by reverse transcriptase-PCR using a primer set specific for LPA2 and LPA3, respectively (30Yun C.C. Sun H. Wang D. Rusovici R. Castleberry A. Hall R.A. Shim H. Am. J. Physiol. 2005; 289: C2-C11Crossref PubMed Scopus (100) Google Scholar). Expression of KLF5, PKC, and eEF2k was determined by Western blot. Luciferase Assay—The promoter of KLF5 was previously described (31Du J.X. Yun C.C. Bialkowska A. Yang V.W. J. Biol. Chem. 2007; 282: 4782-4793Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). HCT116 cells were transfected with pGL3 harboring KLF5 promoter (32Ziemer L.T. Pennica D. Levine A.J. Mol. Cell. Biol. 2001; 21: 562-574Crossref PubMed Scopus (58) Google Scholar). A Renilla luciferase control vector was co-transfected to normalize the transfection efficiency. Cell lysis and reporter assay were performed 2 days after the transfection with the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Fluorescence-activated Cell Sorter Analysis—Cell cycle analysis was performed as previously described (33Yoon H.S. Chen X. Yang V.W. J. Biol. Chem. 2003; 278: 2101-2105Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Cells were rinsed twice in phosphate-buffered saline (PBS), treated with trypsin, and resuspended in their corresponding medium containing 10% FBS. Cells were then collected by centrifugation, washed with PBS, collected again by centrifugation, resuspended in 70% ethanol, and fixed at -20 °C overnight. Cells were pelleted once again by centrifugation and resuspended in a solution containing 50 μg/ml propidium iodide, 50 μg/ml RNase A, 0.1% Triton X-100, and 0.1 mm EDTA at room temperature for 30 min. Flow cytometry was performed on a FACSCalibur cytometer (BD Biosciences). Cell Proliferation—Cells were seeded at 20,000 cells/well in a 24-well plate. Cells were serum starved 24 h and maintained in Dulbecco's modified Eagle's medium, 0.1% FBS supplemented with either 0.1% bovine serum albumin/PBS or 0.1–10 μm LPA in 0.1% bovine serum albumin/PBS for up 3 days. On the day of cell counting, cells were trypsinized and the number of cells was counted by using a hemacytometer. Nuclear Extraction—SW480 and HCT cells were serum starved for 24 h followed by exposure to LPA. Nuclear proteins were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce). Western Immunoblot—Cells were rinsed three times with ice-cold PBS buffer, and lysed in lysis buffer composed of 10 mm Tris-Cl, pH 7.4, 100 mm NaCl, 1 mm EDTA, 1 mm EGTA, 0.1 mm phenylmethylsulfonyl fluoride, 10% glycerol, 2 mm sodium orthovanadate, 10 mm sodium fluoride, 20 mm sodium pyrophosphate, 25 mm β-glycerophosphate, 1% Triton X-100, and protease inhibitors. The lysates were cleared by centrifugation at 14,000 × g at 4 °C for 10 min. Protein concentration was determined by the bicinchoninic acid assay (Sigma). The equal amount of lysate in 2× Laemmli sample buffer was resolved by 10% SDS-PAGE, and Western immunoblot analysis was performed as previously described (30Yun C.C. Sun H. Wang D. Rusovici R. Castleberry A. Hall R.A. Shim H. Am. J. Physiol. 2005; 289: C2-C11Crossref PubMed Scopus (100) Google Scholar). The amount of KLF5 protein was determined using polyclonal anti-KLF5 antibody generated against amino acids 106–122 of the mouse KLF5 (24Conkright M. Wani M. Anderson K. Lingrel J. Nucleic. Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar). The expression level of KLF5 was normalized against the expression levels of β-actin. Densitometric analyses were performed using ImageJ (Scion Corp). Statistical significance was assessed by one-way analysis of variance using Origin software (OriginLab). Data are presented as the mean ± S.E. LPA Induces KLF5 Expression—Previous studies have shown that LPA promotes proliferation of colon cancer cells, such as DLD1 and HCT116 (12Yang M. Zhong W.W. Srivastava N. Slavin A. Yang J. Hoey T. An S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6027-6032Crossref PubMed Scopus (164) Google Scholar, 34Shida D. Kitayama J. Yamaguchi H. Okaji Y. Tsuno N.H. Watanabe T. Takuwa Y. Nagawa H. Cancer Res. 2003; 63: 1706-1711PubMed Google Scholar). Similarly, we found that LPA stimulated the rate of proliferation of human colon cancer Caco-2 and SW480 cells in a concentration-dependent manner (Fig. 1). It has recently been reported that LPA-mediated signaling activates β-catenin in HCT116 and LS174T cells, which express wild type APC gene, and a decrease in β-catenin expression attenuates LPA-mediated proliferation of HCT116 cells (12Yang M. Zhong W.W. Srivastava N. Slavin A. Yang J. Hoey T. An S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6027-6032Crossref PubMed Scopus (164) Google Scholar). The APC gene is the most commonly mutated gene in colorectal cancer with the mutations resulting in deregulation of β-catenin activity (35Ilyas M. Tomlinson I.P.M. Rowan A. Pignatelli M. Bodmer W.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10330-10334Crossref PubMed Scopus (442) Google Scholar). To determine whether LPA can also activate β-catenin in colon cancer cells with the mutated APC gene, we examined nuclear translocation of β-catenin in SW480 cells. As shown in Fig. 2, LPA treatment of serum-starved SW480 cells did not induce the nuclear translocation of β-catenin. In contrast, LPA treatment resulted in an increase in β-catenin expression in nuclear fractions in HCT116 cells, which have the wild type APC gene, consistent with a previous report (12Yang M. Zhong W.W. Srivastava N. Slavin A. Yang J. Hoey T. An S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6027-6032Crossref PubMed Scopus (164) Google Scholar). These data suggest that an alternative pathway(s) is responsible for the proliferation of SW480 and others, in which the APC gene is mutated. KLF5 is a transcription factor highly expressed in epithelial cells in the proliferating compartment of the gastrointestinal tract (24Conkright M. Wani M. Anderson K. Lingrel J. Nucleic. Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar), but whether KLF5 plays a role in LPA-mediated proliferation has not been studied. To test this, we first determined whether LPA induces KLF5 gene expression. Fig. 3A shows that LPA treatment led to an increase in the KLF5 mRNA level in Caco-2 and SW480 cells within 1–2 h of treatment, which was sustained for at least 24 h. The induction of KLF5 mRNA by LPA was not limited to cancer cells but also observed in the nontransformed rat intestinal cell line IEC-6 as well. We next determined the levels of the KLF5 protein. In SW480, Caco-2, and IEC6 cells LPA treatment resulted in a sustained increase in KLF5 protein level in these cells following 2–4 h treatment (Fig. 3B). It is noteworthy that the increase at 2 h was not always observed but the increase much more consistently occurred after 4 h of treatment. Interestingly, LPA also induced the expression level of KLF5 protein in HCT116 cells, in which LPA induced nuclear translocation of β-catenin (Fig. 2). The induction of KLF5 was maximal at 10 μm LPA, whereas submaximal activation was observed at 0.1–1.0 μm LPA (data not shown). To further substantiate the increase in KLF5 expression by LPA, a luciferase reporter driven by a KLF5 promoter was transiently transfected in SW480 or HCT116 cells for the reporter gene assay (31Du J.X. Yun C.C. Bialkowska A. Yang V.W. J. Biol. Chem. 2007; 282: 4782-4793Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Exposure of SW480 cells to LPA for 2 h had a minimal effect on promoter activity (Fig. 3C), as we observed that a similar treatment resulted in an inconsistent increase in KLF5 protein level (Fig. 3B). In contrast, there was a significant increase at 4 h, which further increased after 7 h of treatment. Similarly, LPA treatment of HCT116 stimulated the luciferase activity by 85% at 3 h and 152% at 5 h (Fig. 3D). As a control, 4 h incubation with 100 nm phorbol 12-myristate 13-acetate (PMA), a known agonist of KLF5 expression (26Sun R. Chen X. Yang V.W. J. Biol. Chem. 2001; 276: 6897-6900Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), resulted in an increase in the KLF5 promoter activity in both cell lines (Fig. 3, D–E). A previous report showed that another lysophospholipid S1P induces KLF5 expression (36Usui S. Sugimoto N. Takuwa N. Sakagami S. Takata S. Kaneko S. Takuwa Y. J. Biol. Chem. 2004; 279: 12300-12311Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Hence, we next compared the induction of KLF5 expression by LPA and S1P. As shown in Fig. 3E, S1P did not show a significant effect on the expression level of KLF5 in SW480 cells (upper panel). On the other hand, we observed an increase in KLF5 expression in HCT116 cells (lower panel) treated with S1P, but the effect of S1P was relatively smaller and was short lived compared with LPA. Induction of KLF5 Is Important for LPA-mediated Proliferation—Previous findings that KLF5 positively regulates cell proliferation led us to hypothesize that KLF5 may be a mediator of LPA-mediated cell proliferation (26Sun R. Chen X. Yang V.W. J. Biol. Chem. 2001; 276: 6897-6900Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 37Chanchevalap S. Nandan M.O. Merlin D. Yang V.W. FEBS Lett. 2004; 578: 99-105Crossref PubMed Scopus (72) Google Scholar, 38Nandan M.O. Yoon H.S. Zhao W. Ouko L.A. Chanchevalap S. Yang V.W. Oncogene. 2004; 23: 3404-3413Crossref PubMed Scopus (120) Google Scholar, 39Nandan M.O. Chanchevalap S. Dalton W.B. Yang V.W. FEBS Lett. 2005; 579: 4757-4762Crossref PubMed Scopus (71) Google Scholar). Because many colon cancer cells such as Caco-2 and SW480 have high basal levels of KLF5 expression, we examined the role of KLF5 via silencing of the KLF5 gene using siRNA. Fig. 4A shows that KLF5 siRNA decreased KLF5 expression by ∼90% compared with control siRNA-transfected SW480 cells (Fig. 4A). Despite the knockdown, LPA treatment led to an increase in KLF5 expression in siKLF5-treated cells. Silencing of the KLF5 gene resulted in a significant decrease in LPA-induced proliferation of SW480 cells (Fig. 4B). Similarly, knockdown of KLF5 decreased the rate of LPA-mediated proliferation of HCT116 cells (Fig. 4C). However, LPA treatment resulted in a significant increase in cell numbers in siKLF5-treated cells compared with untreated cells, which may be attributed to the small induction of KLF5 observed in these cells (Fig. 4A). To further understand the role of KLF5, we performed cell-cycle analysis on cells treated with siKLF5 or control siRNA. Following LPA treatment for 72 h, the population of cells in S phase is almost 2-fold greater in control cells compared with siKLF5-treated cells (15.55 versus 8.29%). These data indicate that the induction of KLF5 is important for LPA-mediated proliferation of colon cancer cells and it stimulates cell proliferation by promoting G1/S transition. LPA2 and LPA3 Receptor Mediate LPA-induced KLF5 Induction—We previously reported that colon cancer cells, such as SW480 and HCT116, overexpress LPA2 (30Yun C.C. Sun H. Wang D. Rusovici R. Castleberry A. Hall R.A. Shim H. Am. J. Physiol. 2005; 289: C2-C11Crossref PubMed Scopus (100) Google Scholar). At the same time, LPA1 expression was significantly decreased in these cells compared with normal colon epithelial cells (30Yun C.C. Sun H. Wang D. Rusovici R. Castleberry A. Hall R.A. Shim H. Am. J. Physiol. 2005; 289: C2-C11Crossref PubMed Scopus (100) Google Scholar). We next sought to determine which LPA receptors, LPA2 or LPA3, are responsible for the induction of KLF5. We omitted LPA1 based on the previous finding that the level of LPA1 in these cells is low (30Yun C.C. Sun H. Wang D. Rusovici R. Castleberry A. Hall R.A. Shim H. Am. J. Physiol. 2005; 289: C2-C11Crossref PubMed Scopus (100) Google Scholar). LPA2 and LPA3 expression in HCT116 cells was blocked using siRNA specific for LPA2 or LPA3. Silencing of either LPA2 or LPA3 markedly decreased activation of ERK 1 and 2 in these cells. Similarly, the induction of KLF5 by LPA was abrogated by knockdown of either LPA2 or LPA3 and when combined a greater inhibition was achieved (Fig. 5). Induction of KLF5 Is Dependent on PKCδ and MEK1/2—A myriad of signaling pathways regulates LPA-mediated cell proliferation. Earlier studies have indicated involvement of ERK1/2, the phosphoinositide 3-kinase-Akt cascade, and in some cases the p38 group of protein kinases. To determine the signal transduction pathway involved in the induction of KLF5, we used pharmaceutical inhibitors specific against these protein kinases. The specific Gαi/o inhibitor PTX, the phosphoinositide 3-kinase inhibitor LY290042, the p38 inhibitor SB203580, the phospholipase Cβ inhibitor U37122, and the epidermal growth factor (EGF) receptor inhibitor AG1478 did not block the induction of KLF5 in SW480 and HCT116 cells (Fig. 6, A and B). On the other hand, the MEK1/2 inhibitor U0126 was able to partially block the effect of LPA on KLF5 protein expression in both SW480 and HCT116 cells (Fig. 6, C and D). Previous studies have shown that LPA regulates PKC (12Yang M. Zhong W.W. Srivastava N. Slavin A. Yang J. Hoey T. An S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6027-6032Crossref PubMed Scopus (164) Google Scholar, 40Fang X. Yu S. Tanyi J.L. Lu Y. Woodgett J.R. Mills G.B. Mol. Cell. Biol. 2002; 22: 2099-2110Crossref PubMed Scopus (153) Google Scholar). Moreover, LPA induces interleukin-8 secretion via a PKCδ-dependent mechanism (41Cummings R. Zhao Y. Jacoby D. Spannhake E.W. Ohba M. Garcia J.G.N. Watkins T. He D. Saatian B. Natarajan V. J. Biol. Chem. 2004; 279: 41085-41094Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 42Zachos N.C. Tse M. Donowitz M. Ann. Rev. Physiol. 2005; 67: 411-443Crossref PubMed Scopus (307) Google Scholar). We, therefore, examined whether PKC is involved in the activation of KLF5. SW480 cells were treated with LPA in the presence or absence of the broad-spectrum PKC inhibitor GF109203X or the PKCδ-specific inhibitor rottlerin. GF109203X showed a modest inhibitory effect in both SW480 and HCT116 cells (Fig. 6, C and D). Interestingly, when the cells were pretreated with both U0126 and GF109203X, the inhibitory effect on KLF5 was substantially greater than with each inhibitor in both cell lines. The effects of the inhibitors were confirmed by the KLF5 promoter reporter assay, which shows a significant decrease in the luciferase activity in the presence of U0126, GF109203X, or both (Fig. 6E). This indicates that the effects of these inhibitors are additive. In addition to GF109203X, 10 μm rottlerin drastically blocked the inducti
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