Intestinal-enriched Krüppel-like Factor (Krüppel-like Factor 5) Is a Positive Regulator of Cellular Proliferation

Abstract

Intestinal-enriched Krüppel-like factor (IKLF or KLF5) belongs to the family of mammalian Krüppel-like transcription factors. Previous studies indicate that expression of IKLF is enriched in the proliferating crypt epithelial cells of the intestinal tract. However, the biological function of IKLF is unknown. In the current study, we have shown that the level of IKLF mRNA was nearly undetectable in serum-deprived NIH3T3 fibroblasts but became acutely and significantly increased upon the addition of fetal bovine serum or the phorbol ester, PMA. This induction required protein synthesis because it was prevented by cycloheximide. Transfection of IKLF into NIH3T3 cells resulted in the formation of foci in a manner similar to that caused by the activated Ha-ras oncogene. Constitutive expression of IKLF in transfected NIH3T3 cells significantly increased the rate of proliferation when compared with cells transfected with an empty vector. The growth ofIKLF-transfected cells was no longer inhibited by cell-cell contact or by low serum content. Moreover, these cells proliferated in an anchorage-independent fashion. We conclude thatIKLF encodes a delayed early response gene product that positively regulates cellular proliferation and may give rise to a transformed phenotype when overexpressed. Intestinal-enriched Krüppel-like factor (IKLF or KLF5) belongs to the family of mammalian Krüppel-like transcription factors. Previous studies indicate that expression of IKLF is enriched in the proliferating crypt epithelial cells of the intestinal tract. However, the biological function of IKLF is unknown. In the current study, we have shown that the level of IKLF mRNA was nearly undetectable in serum-deprived NIH3T3 fibroblasts but became acutely and significantly increased upon the addition of fetal bovine serum or the phorbol ester, PMA. This induction required protein synthesis because it was prevented by cycloheximide. Transfection of IKLF into NIH3T3 cells resulted in the formation of foci in a manner similar to that caused by the activated Ha-ras oncogene. Constitutive expression of IKLF in transfected NIH3T3 cells significantly increased the rate of proliferation when compared with cells transfected with an empty vector. The growth ofIKLF-transfected cells was no longer inhibited by cell-cell contact or by low serum content. Moreover, these cells proliferated in an anchorage-independent fashion. We conclude thatIKLF encodes a delayed early response gene product that positively regulates cellular proliferation and may give rise to a transformed phenotype when overexpressed. Krüppel-like factor gut-enriched KLF basic transcription element binding protein 2 cycloheximide Dulbecco's modified Eagle's medium early growth response gene-1 fetal bovine serum glyceraldehyde-3-phosphate dehydrogenase hemagglutinin intestinal-enriched Krüppel-like factor phorbol 12-myristate 13-acetate smooth muscle cell Krüppel is a zinc finger-containing transcription factor that is responsible for segmentation of the Drosophila melanogaster embryo (1Schuh R. Aicher W. Gual U. Cote S. Preiss A. Maier D. Seifert E. Nauber U. Shroder C. Kemler R. Jackle H. Cell. 1986; 47: 1025-1032Abstract Full Text PDF PubMed Scopus (329) Google Scholar). In vertebrates, a large number of proteins have been identified that exhibit homology to Krüppel (2Turner J. Crossley M. Trends Biochem. Sci. 1999; 24: 236-240Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). One prominent example is Sp1 (3Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1255) Google Scholar), a general transcription factor. Recently, a family of Krüppel-like factors (KLFs)1 that are highly related to the mammalian Krüppel protein erythroid Krüppel-like factor (EKLF) (4Miller I.J. Bieker J.J. Mol. Cell. Biol. 1993; 13: 2776-2786Crossref PubMed Scopus (658) Google Scholar) have been described (2Turner J. Crossley M. Trends Biochem. Sci. 1999; 24: 236-240Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 5Philipsen S. Suske G. Nucleic Acids Res. 1999; 27: 2991-3000Crossref PubMed Scopus (537) Google Scholar, 6Dang D.T. Pevsner J. Yang V.W. Int. J. Biochem. Cell Biol. 2000; 32: 1103-1121Crossref PubMed Scopus (372) Google Scholar). Many of these proteins were given a numerical designation by the Human Gene Nomenclature Committee (HGNC, Ref. 7White J.A. McAlpine P.J. Antonarakis S. Cann H. Eppig J.T. Frazer K. Frezal J. Lancet D. Nahmias J. Pearson P. Peters J. Scott A. Scott H. Spurr N. Talbot Jr., C. Povey S. Genomics. 1997; 45: 468-471Crossref PubMed Scopus (85) Google Scholar), with EKLF designated as KLF1. The genes encoding many KLFs are expressed in a tissue-specific or -selective manner. In addition, evidence suggests that KLFs collectively exert important regulatory functions in diverse biological processes such as growth, development, differentiation, and apoptosis. One tissue in which a number of KLFs appear to play an important regulatory role is the intestinal epithelium. This tissue is a dynamic system in which proliferation of stem cells located in the crypts is intimately coupled to their differentiation into mature daughter cells once they exit the crypts (8Stappenbeck T.S. Wong M.H. Saam J.R. Mysorekar I.U. Gordon J.I. Curr. Opin. Cell Biol. 1998; 10: 702-709Crossref PubMed Scopus (129) Google Scholar, 9Gordon J.I. Hermiston M.L. Curr. Opin. Cell Biol. 1994; 6: 795-803Crossref PubMed Scopus (215) Google Scholar). Expression of the genes encoding two KLFs, gut-enriched Krüppel-like factor (GKLF or KLF4, Ref. 10Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar,11Garrett-Sinha L.A. Eberspaecher H. Seldin M.F. de Crombrugghe B. J. Biol. Chem. 1996; 271: 31384-31390Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar) and intestinal-enriched Krüppel-like factor (IKLF or KLF5, Refs. 12Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar, 13Ohnishi S. Laub F. Matsumoto N. Asaka M. Ramirez F. Yoshida T. Terada M. Dev. Dyn. 2000; 217: 421-429Crossref PubMed Scopus (76) Google Scholar), is particularly active in the intestinal epithelium. However, their patterns of expression appear to be complementary rather than redundant. Whereas GKLF is primarily expressed in the differentiated epithelial cells, away from the proliferating zone,IKLF is found mainly in the proliferating crypt cell population. The in vivo pattern of GKLFexpression is mirrored by its in vitro pattern; it is found mostly in cells that are growth-arrested (10Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 14Shie J.L. Chen Z.Y. Fu M. Pestell R.G. Tseng C.C. Nucleic Acids Res. 2000; 28: 2969-2976Crossref PubMed Google Scholar). Moreover, constitutive expression of GKLF leads to the inhibition of DNA synthesis (10Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 15Shie J.L. Chen Z.Y. O'Brien M.J. Pestell R.G. Lee M.E. Tseng C.C. Am. J. Physiol. 2000; 279: G806-G814Crossref PubMed Google Scholar). In contrast, the physiological function of IKLF is less clear, although it has been proposed to have an opposing effect to GKLF in regulating epithelial cell differentiation (12Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar). The present study seeks to characterize the effect of IKLF on cellular proliferation. We demonstrate that expression ofIKLF in cultured cells responds transiently and acutely to growth stimuli. In addition, forced expression of IKLF in transfected cells results in an accelerated rate of proliferation and a transformed phenotype as evidenced by formation of foci, loss of contact inhibition, as well as acquisition of serum- and anchorage-independent growth. Our results indicate that IKLF has a proproliferative effect, which lends support to the previous hypothesis that it may counteract the function of GKLF. Cell culture media and fetal bovine serum (FBS) were purchased from Life Technologies (Gaithersburg, MD) and Hyclone Laboratories (Logan, UT), respectively. Radioisotopes were purchased from PerkinElmer Life Sciences. Phorbol 12-myristate 13-acetate (PMA) and cycloheximide (CHX) were purchased from Sigma. The monoclonal antibody directed against the hemagglutinin A (HA) epitope (F-7, sc-7392) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Expression constructs containing full-length IKLF and HA-tagged IKLF (pBK-CMV-IKLF and pBK-CMV-IKLF·HA, respectively) were kindly provided by Dr. Jerry Lingrel (12Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar). The expression construct containing activated Ha-Ras was a generous gift of Dr. Raul Urrutia (16Gebelein B. Fernandez-Zapico M. Imoto M. Urrutia R. J. Clin. Invest. 1998; 102: 1911-1919Crossref PubMed Scopus (43) Google Scholar). The expression construct containing full-length GKLF, PMT3-GKLF, was previously described (10Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C in a 5% CO2 atmosphere. For experiments involving mitogenic stimulation, cells were first rendered quiescent by removal of serum from the medium for 24 h. Cells were then stimulated with fresh medium containing 15% FBS or 100 ng/ml PMA for various duration. In experiments in which CHX was included, quiescent cells were pretreated with 10 μg/ml CHX for 1 h before the addition of FBS or PMA. Treatments were then continued for another 1 h with the respective mitogen and CHX. RNA was isolated using the Trizol method (Life Technologies) and resolved by denaturing agarose gel electrophoresis followed by transfer to nylon membranes. Complementary DNA probes encoding IKLF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were labeled using [α-32P]dATP and the random-primed DNA labeling kit (Roche Molecular Biochemicals). Conditions of hybridization and washing were previously described (10Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). Western blot analysis was performed according to a previous protocol (10Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar) using proteins extracted from stably transfected cells. The blots were probed with a monoclonal antibody directed against the HA epitope at a concentration of 200 ng/ml. Blots were then incubated with a peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) at a dilution of 1:2,000. Signals were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech). Focus formation assays were performed according to a previously published protocol (17Cox A.D. Der C.J. Methods Enzymol. 1994; 238: 277-294Crossref PubMed Scopus (87) Google Scholar). Briefly, 5 μg of plasmid DNA containing the various effectors was transfected into NIH3T3 cells using LipofectAMINE (Life Technologies, Inc.). Cells were then maintained in DMEM and 10% FBS for 2–3 weeks at which time they were stained with methylene blue to demonstrate the foci. The number of foci in each dish was then manually counted. NIH3T3 cells were transfected with pBK-CMV-IKLF·HA or the pBK-CMV empty vector using LipofectAMINE. Two mg/ml G418 was added to the medium beginning 24 h after transfection to select for resistant clones, which were isolated 2 weeks later and expanded. The presence of HA-tagged IKLF was detected by Western blot analysis using the anti-HA monoclonal antibody. Cell proliferation assays were performed by seeding theIKLF·HA- or empty vector-transfected cells at a density of 2 × 105 cells per well in 6-well plates. Cells were fed DMEM with 10% FBS every other day, and the number of cells in the wells were manually counted with a hemocytometer daily for up to 5 days following seeding. For the serum dependence assay, cells were seeded at a density 2 × 105 cells per well in 6-well plates and maintained in DMEM with 1% FBS. Cells were counted every other day up to 6 days after seeding. Anchorage dependence assay was performed according to a published protocol (17Cox A.D. Der C.J. Methods Enzymol. 1994; 238: 277-294Crossref PubMed Scopus (87) Google Scholar).Ha-ras-, IKLF·HA-, or empty vector-transfected cells were seeded at a density of 5 × 105 cells per 10-cm dish in a 0.33% top agar suspension, which was overlaid onto a 0.5% agar bottom layer. Cells were fed DMEM, 10% FBS, and 2 mg/ml G418 every other day. Colonies developed in the agar suspension were examined 3 weeks following seeding, and the number was tabulated under an inverted phase-contrast microscope. Photodocumentation was accomplished with a Nikon digital camera. To investigate the responsiveness of IKLFexpression to growth stimulation, we conducted Northern blot analyses in cultured fibroblasts. NIH3T3 cells were first rendered quiescent by removing the serum from the medium and then stimulated by 15% FBS or the phorbol ester, PMA. As seen in Fig.1, A and B, the level of IKLF transcripts was barely detectable in serum-starved, quiescent cells (time 0). Upon addition of FBS (Fig. 1 A) or PMA (Fig. 1 B), the levels ofIKLF transcripts increased acutely and transiently, reaching a maximum after 2–3 h of treatment before returning to baseline levels. The enhancing effect of both stimuli was prevented in cells pretreated with the protein synthesis inhibitor, cycloheximide (Fig.1 C, CHX). These results indicate that IKLF is an early response gene to growth stimulation although this responsiveness requires protein synthesis. IKLF would therefore fall into the category of delayed early response genes (18Winkles J.A. Prog. Nucleic Acid Res. Mol. Biol. 1998; 58: 41-78Crossref PubMed Scopus (80) Google Scholar, 19Lanahan A. Williams J.B. Sanders L.K. Nathans D. Mol. Cell. Biol. 1992; 12: 3919-3929Crossref PubMed Scopus (299) Google Scholar). As a means to measure the effect of IKLF on cell proliferation, we conducted the focus formation assay previously developed for testing the transforming activity of oncogenes such as ras (17Cox A.D. Der C.J. Methods Enzymol. 1994; 238: 277-294Crossref PubMed Scopus (87) Google Scholar, 20Clark G.J. Cox A.D. Graham S.M. Der C.J. Methods Enzymol. 1995; 255: 395-412Crossref PubMed Scopus (182) Google Scholar). NIH3T3 fibroblasts were transfected with the various effector constructs and foci scored 2–3 weeks after transfection. As shown in Fig.2, an expression plasmid containing the HA-tagged IKLF produced ∼30% of the number of foci caused by activated Ha-Ras. A second construct containing IKLF without the HA-tag gave rise to similar results (not shown). In contrast, an expression plasmid containing GKLF failed to produce any foci, as were mock-transfected cells (Fig. 2). These results suggest that forced expression of IKLF causes focus formation in a manner similar to activated Ha-Ras, an activity that was not paralleled by GKLF. To further investigate the effect of IKLF on cell proliferation, we established several clonally derived NIH3T3 cell lines that had been transfected with pBK-CMV-IKLF·HA or the pBK-CMV empty vector and selected with the antibiotic, G418. Two independent clones from each construct were chosen and examined. Fig.3 A shows that the two pBK-CMV-IKLF·HA- but not the two pBK-CMV-transfected clones (lanes 1 and 2 versus lanes 3 and 4, respectively) produced a full-length HA-tagged IKLF as detected by Western blot using a monoclonal antibody against the HA epitope. When the growth characteristics of these cells were examined and compared over a course of 5 days following seeding at a low density, it became apparent that the IKLF·HA-expressing cells proliferated at a much faster rate compared with the control, empty vector-transfected cells (Fig. 3 B). Moreover, whereas the control cells ceased to proliferate after reaching confluency, theIKLF·HA-transfected cells continued to grow to several layers (Fig. 3 C). These findings suggest that the growth of IKLF·HA-transfected cells was no longer subject to contact inhibition, providing further evidence for a proproliferative effect of IKLF. The stably transfected NIH3T3 cells were subjected to additional and more stringent tests of cellular proliferation and transformation. In one, cells were seeded and maintained in medium containing only 1% FBS. Under these conditions, empty vector-transfected cells failed to proliferate. In fact, many perished because of the low-serum content. In contrast, IKLF·HA-transfected cells continued to proliferate at a relatively brisk rate albeit slightly slower than that when maintained in 10% FBS (compare Figs.4 A and 3 B). A second test involved growth in soft agar (17Cox A.D. Der C.J. Methods Enzymol. 1994; 238: 277-294Crossref PubMed Scopus (87) Google Scholar). As seen in Fig.4 B, IKLF·HA-transfected cells formed colonies in an agar suspension as were Ha-ras-transfected cells at a ∼1:3 ratio. In contrast, empty vector-transfected cells remained as single cells in the agar suspension without ever forming any colonies (Fig. 4 C). The morphology and size of the colonies produced by IKLF·HA- andHa-ras-transfected cells were very similar to each other (Fig. 4 C). Complementary DNA clones encoding IKLF were identified because of homology to (12Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar). A was isolated on its binding to a in the of the gene H. S. Nucleic Acids Res. 1999; 27: PubMed Scopus Google Scholar). In hybridization studies of both (12Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar) and fetal intestinal S. Laub F. Matsumoto N. Asaka M. Ramirez F. Yoshida T. Terada M. Dev. Dyn. 2000; 217: 421-429Crossref PubMed Scopus (76) Google Scholar) that expression ofIKLF is in the of intestinal vivo pattern of IKLF expression in the intestinal therefore with a although a effect of IKLF on cellular proliferation was not established by these be that IKLF is to the previously isolated K. H. H. H. Nucleic Acids Res. 1993; PubMed Scopus Google Scholar), because of a has a than IKLF S. Laub F. Matsumoto N. Asaka M. Ramirez F. Yoshida T. Terada M. Dev. Dyn. 2000; 217: 421-429Crossref PubMed Scopus (76) Google Scholar). In a expression of is in activated smooth muscle cells in the of N. M. K. M. M. M. T. R. Res. 1999; PubMed Scopus Google Scholar). increased has been in proliferating at T. M. S. R. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and this increased expression is a factor for in M. T. A. H. K. N. T. A. R. 2000; 102: PubMed Scopus Google Scholar). these studies suggest that expression is with a The results of the current study indicate that expression ofIKLF in quiescent NIH3T3 cells is acutely and transiently upon mitogenic stimulation by factors such as serum and phorbol This induction is on protein synthesis as it is in the presence of cycloheximide. A similar response of was in cultured with PMA or basic growth factor K. M. R. Res. 1999; PubMed Scopus Google Scholar). The study that expression of the early response is highly by PMA and that to and the of gene K. M. R. Res. 1999; PubMed Scopus Google Scholar). is therefore that is an of induction of IKLF evidence from in vitro and in a IKLF expression and proliferation, it is not IKLF regulates cellular proliferation. The results of the current study are the first to that constitutive expression of IKLF proliferation. We that IKLF is to a transformed phenotype as by focus formation (Fig. loss of contact inhibition (Fig. 3 and of serum- A) and anchorage-independent growth (Fig. 4, C). These therefore suggest that IKLF is a of cellular proliferation in the vivo and in vitro described IKLF in regulating the cell or expression of IKLF is increased in such as to be on the opposing patterns of expression of GKLF in the Lingrel and (12Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar) proposed that their gene may have opposing in regulating proliferation and differentiation of the intestinal epithelium. the effect of GKLF by previous studies (10Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google J.L. Chen Z.Y. Fu M. Pestell R.G. Tseng C.C. Nucleic Acids Res. 2000; 28: 2969-2976Crossref PubMed Google Scholar) and the proproliferative effect of IKLF by this study support their evidence suggests that the two proteins may have opposing For the of the differentiation is by GKLF but activated by IKLF P.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). studies from indicate that GKLF and IKLF in an opposing manner. and W. In of the highly in the zinc of the two proteins and the similar DNA to which they (10Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, L.A. Eberspaecher H. Seldin M.F. de Crombrugghe B. J. Biol. Chem. 1996; 271: 31384-31390Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 12Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar, 14Shie J.L. Chen Z.Y. Fu M. Pestell R.G. Tseng C.C. Nucleic Acids Res. 2000; 28: 2969-2976Crossref PubMed Google Scholar, H. S. Nucleic Acids Res. 1999; 27: PubMed Scopus Google Scholar), it would not be that the two KLFs may the expression of a of genes similar not studies demonstrate the by which GKLF and IKLF each other in the of regulating biological processes such as cellular proliferation and We M. and Dr. J. Lingrel for providing pBK-CMV-IKLF·HA and (12Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar) and Dr. R. Urrutia for providing the activated Ha-Ras (16Gebelein B. Fernandez-Zapico M. Imoto M. Urrutia R. J. Clin. Invest. 1998; 102: 1911-1919Crossref PubMed Scopus (43) Google Scholar) expression