Role Of Krüppel-like Factor 4 Research Articles

Abstract 284: Down-regulated Kruppel-like factor 4 induces the migration and epithelial-mesenchymal transition in human hepatoma cells.

Abstract The transcriptional factor Kruppel-like factor 4 (KLF4) plays important roles in development, stemness, and tumorigenesis. Several studies have suggested that KLF4 is important in various tumors; however limited information is available on the detailed function of KLF4 in hepatocellular carcinoma. The objective of this study was to examine the functional roles of KLF4 in metastasis of hepatoma cells. In order to investigate the role of KLF4 in the progression of hepatocellular carcinoma, we used lentiviral transduction method to down-regulate KLF4 expression in Hep3B and HA22T/VGH hepatoma cell lines. The effects of KLF4 on cell migration, invasion and the expression of epithelial-mesenchymal transition (EMT) related proteins were evaluated in KLF4-knockdown hepatoma cells. Transwell migration assay and matrigel invasion assay revealed the migration and invasion abilities were significantly increased in KLF4-knockdown Hep3B or HA22T/VGH cells. Knockdown KLF4 in hepatoma cells resulted in decreased mRNA and protein expression of E-cadherin, and a concomitant increase of vimentin, MMP2 and MMP9. These results corresponded well with our finding that overexpression of KLF4 in Mahlavu hepatoma cells led to significant inhibition of cell migration and invasion through up-regulating E-cadherin and down-regulating vimentin, MMP2 and MMP9. Snail and Bmi1, repressors of E-cadherin gene expression, were also markedly decreased in KLF4-overexpressing Mahlavu cells. Therefore, these results together suggested that KLF4 is tightly involved in the epithelial-mesenchymal transition and metastasis in the tumor progression of hepatocellular carcinoma. Citation Format: Ming-Ta Sung, Hsin-Chen Lee, Cheng-Yuan Hsia, Chin-Wen Chi. Down-regulated Kruppel-like factor 4 induces the migration and epithelial-mesenchymal transition in human hepatoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 284. doi:10.1158/1538-7445.AM2013-284

Krüppel-Like Factor 4, a Tumor Suppressor in Hepatocellular Carcinoma Cells Reverts Epithelial Mesenchymal Transition by Suppressing Slug Expression

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that plays an important role in differentiation and pathogenesis. KLF4 has been suggested to act as an oncogene or tumor suppressor in different tumor types. However, the role of KLF4 in hepatocellular carcinoma (HCC) remains unclear. Here, we demonstrate that forced expression of Klf4 in murine HCC cell lines reduced anchorage-independent growth in soft agar as well as cell migration and invasion activities in vitro. Ectopic Klf4 expression impaired subcutaneous tumor growth and lung colonization in vivo. By contrast, Klf4 knockdown enhanced HCC cell migration. Interestingly, ectopic expression of Klf4 changed the morphology of murine HCC cells to a more epithelial phenotype. Associated with this, we found that expression of Slug, a critical epithelial mesenchymal transition (EMT)-related transcription factor, was significantly down-regulated in Klf4-expressing cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays showed that Klf4 is able to bind and repress the activity of the Slug promoter. Furthermore, ectopic Slug expression partially reverts the Klf4-mediated phenotypes. Consistent with a role as a tumor suppressor in HCC, analysis of the public microarray databases from Oncomine revealed reduced KLF4 expression in human HCC tissues in comparison with normal liver tissues in 3 out of 4 data sets. By quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we found reduced KLF4 mRNA in 50% of HCC tissues. Importantly, an inverse correlation between the expression of KLF4 and SLUG was found in HCC tissues. Our data suggest that KLF4 acts as a tumor suppressor in HCC cells, in part by suppressing SLUG transcription.

Abstract 3442: Overexpression of Kruppel-like factor 4 inhibits the growth and migration in human hepatoma cells

Abstract The zinc-finger transcriptional factor Kruppel-like factor 4 (KLF4), can inhibit cell proliferation and maintain the differentiation of epithelial cells by regulating the expression of genes with GC-rich promoters, plays important roles in development and tumorigenesis. Recently, reduction of KLF4 expression has been reported in multiple gastrointestinal cancers such as gastric cancer, colorectal cancer and hepatocellular carcinoma (HCC), and this observation was associated well with tumor progression and poor clinical outcome. However, the exact role of KLF4 in the progression of HCC is still unclear. In this study, we used lentiviral transduction method to overexpress KLF4 in a highly metastatic HA22T/VGH hepatoma cell line. Using MTT and sulforhodamine B assay, we found that cell growth was decreased in KLF4-overexpressing HA22T/VGH cells as compared to parental cells. Moreover, wound healing assay revealed the migration ability was significantly suppressed in KLF4-overexpressing HA22T/VGH cells. Consistent with these findings, KLF4 overexpression also resulted in the up-regulation of E-cadherin expression and concomitant down-regulation of vimentin expression in HA22T/VGH cells. Moreover, our preliminary finding showed that KLF4 knockdown by transfection of KLF4 shRNA caused the decrease in E-cadherin expression in HEK293T cells. E-cadherin, a key adhesion molecule in the process of epithelial-mesenchymal transition (EMT) and tumor metastasis, was regulated by many transcription factors such as Snail. Interestingly, in this study we have found that KLF4 overexpression led to an obvious reduction in the Snail expression in HA22T/VGH cells. Therefore, these results together suggest that KLF4-induced E-cadherin expression may result from regulation at E-cadherin transcription level or inhibition of the expression of Snail. Furthermore, our findings also suggested that the role of KLF4 may be associated with the cell growth, EMT process and metastasis in the tumor progression of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3442. doi:1538-7445.AM2012-3442

Abstract 4582: Regulation of Krüppel-Like Factor 4 (KLF4) expressions via Yin Yang 1 (YY1) in B Non-Hodgkin's Lymphomas (B-NHLs): KLF4 upregulation is associated with unfavorable overall survival in pediatric B-NHL

Abstract Krüppel-like factor 4 (KLF4) is a transcription factor expressed in a variety of tissues in humans and has been implicated in several physiologic processes including development, differentiation, and tissue homeostasis. KLF4 is a bi-functional and can either activate or repress transcription depending on the target gene. For instance, KLF4 acts as a tumor suppressor gene (colon, gastric, esophageal, bladder, and NSCLC) or as an oncogene (laryngeal carcinoma, squamous cell carcinoma, ductal carcinoma of the breast). However, the role of KLF4 in hematological malignancies is still poorly understood. Studies in leukemia suggest that KLF4 may be a tumor suppressor. The goal of this study was to investigate the expression and the clinical significance of KLF4 in B cell non-Hodgkin's lymphomas (B-NHLs). Both B-NHL cell lines and patient-derived tumor tissues (TMA) were examined by western blot and immunohistochemistry (IHC), respectively. Using IHC, the expression of KLF4 was calculated based on the intensity and percentage of the area stained, and scoring was corroborated by two pathologists. The complete absence of KLF4 expression was considered as negative. A significant overexpression of KLF4 in Ramos and Raji (Burkitt's lymphoma) and 2F7 (AIDS lymphoma) B-NHL cell lines. However, the DHL4 (DBLCL) cell line showed a level of similar to that seen in normal cells. Among the 73 childhood lymphomas studied, 13/23 (57%) of lymphoblastic lymphoma, 7/20 (35%) of large B-cell lymphoma, 4/4 (100%) of anaplastic large cell lymphoma and 5/6 (83%) NHL not specified were KLF4 positive. Notably, 20/20 (100%) Burkitt's lymphoma were KLF4 positive. Nuclear expression of KLF4 was significantly higher in Burkitt's lymphoma (90%) compared to the remaining subtypes. The 3-year event-free survival rate (EFS) for the whole cohort was 67% (43% to 79%) compared to 23% (13% to 38%) in those who has tumors that were KLF4 positive, (p< 0.05). Multivariate analyses confirmed the association of KLF4 expression with unfavorable overall survival (OS; P<.005). Previous findings demonstrated overexpression of the transcription factor YY1 in B-NHL. In silico analysis of the KLF4 promoter identified the presence of four putative binding sites for YY1. We confirmed that –126 and –298 sites were binding sites for YY1 by ChIP analyses. The transcriptional regulation of KLF4 by YY1 was demonstrated following transfection with YY1 siRNA. We also found a positive correlation between the expression of YY1 and KLF4 in the NHL tissues, suggesting that YY1 regulates KLF4 in vivo. The present findings suggest that KLF4 may be considered as an oncogene in Burkitt's lymphoma, and in certain subsets of other types of lymphoma, and that KLF4 may be a potential prognostic factor. We propose that KLF4 may be a therapeutic target in patients with B-NHL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4582. doi:1538-7445.AM2012-4582

Krüppel-like Factor 4 Regulates Intestinal Epithelial Cell Morphology and Polarity

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mice demonstrated a decrease in number of goblet cells in the colon, and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. However, the role of KLF4 in differentiated intestinal cells and colon cancer cells, as well as the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine is not well understood. In our study, KLF4 was partially depleted in the differentiated intestinal epithelial cells by a tamoxifen-inducible Cre recombinase. We found a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers through its function in inhibiting cell proliferation. The number and position of Paneth cells also changed. This is consistent with the KLF4 knockout study using villin-Cre [1]. Through immunohistochemistry (IHC) staining and statistical analysis, we found that a stem cell and/or tuft cell marker, DCAMKL1, and a proliferation marker, Ki67, are affected by KLF4 depletion, while an enteroendocrine cell marker, neurotensin (NT), was not affected. In addition, we found KLF4 depletion altered the morphology and polarity of the intestinal epithelial cells. Using a three-dimensional (3D) intestinal epithelial cyst formation assay, we found that KLF4 is essential for cell polarity and crypt-cyst formation in human colon cancer cells. These findings suggest that, as a tumor suppressor in colorectal cancer, KLF4 affects intestinal epithelial cell morphology by regulating proliferation, differentiation and polarity of the cells.

Krüppel‐like factor 4 functions as a tumor suppressor in cervical carcinoma

The Krüppel-like factor 4 (KLF4) protein, a zinc finger transcription factor that is highly expressed in epithelial tissues such as the gut and skin, has been implicated in both tumor suppression and progression. However, the role of KLF4 in human cervical carcinoma is still unclear. The expression of KLF4 in cervical carcinoma tissues and cervical cancer cell lines was examined with immunohistochemistry and Western blot assay. The effects of KLF4 silencing and overexpression on the cell proliferation, cell viability, and tumor formation of cervical cancer cells were investigated. KLF4 protein expression showed a pattern of gradual decrease from normal cervix to cervical carcinoma in situ and then to invasive cervical carcinomas (P < .05). Overexpression of KLF4 in SiHa and C33A cells resulted in significantly inhibited cell growth and significantly attenuated tumor formation. Consistently, KLF4 silencing in HeLa cells significantly promoted cell growth and tumor formation. Furthermore, KLF4 overexpression caused cell cycle arrest at the G1/S transition, along with the up-regulated expression of p27(Kip1) protein. Promoter analysis revealed that KLF4 transactivated the expression of p27(Kip1) through the specific motif that is between the nucleotides of -435 and -60 in its promoter. The results from chromatin immunoprecipitation assays demonstrated the physical interaction between KLF4 protein and this specific motif in p27(Kip1) promoter. KLF4 may function as a tumor suppressor in cervical carcinoma by inhibiting cell growth and tumor formation.

Abstract B26: Expression of the KLF4 and SOX10 predicting for survival in colorectal cancer.

Abstract The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is involved in many important cellular processes such as growth, development, differentiation, proliferation, and apoptosis. A loss of KLF4 expression has been observed in human tumors, particularly in the gastrointestinal tract. Sry box 10 (SOX10) is a transcription factor expressed in nerve cell and melanocytes. SOX10 is believed to be essential for neural crest fate determination and to maintain the multipotency of neural crest cells. Here, to investigate the roles of KLF4 and SOX10 in colorectal cancers (CRC), we examined the expression of KLF4 and SOX10 in tumor tissues and paired normal tissues in 125 CRC patients by real-time reverse transcription-polymerase chain (real-time RT-PCR). CRC had significantly lower KLF4 and SOX10 levels compared with matched normal tissues (KLF4: 2007±1531 Copies/ul in CRC, 6585±2833 Copies/ul in normal tissues, SOX10: 60±100 Copies/ul in CRC, 399±215 Copies/ul in normal tissues) (P&amp;lt;0.0001). Statistically, KLF4 and SOX10 expression was not associated with age, sex, stage, lymph node metastasis, differentiation, tumor location. However, in univariate analyses, KLF4 and SOX10 were significant predictors of survival (P=0.002, P=0.039) and in multivariate analyses, KLF4 was significantly predictor of survival (P=0.045). Therefore, these findings suggest that KLF4 and SOX10 might have a potent oncogenic role in CRC tumorigenesis and prognostic value in predicting survival of CRC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B26.

Loss of Heterozygosity Status of D9S105 Marker Is Associated with Downregulation of Krüppel-Like Factor 4 Expression in Pancreatic Ductal Adenocarcinoma and Pancreatic Intraepithelial Lesions

The transcription factor Krüppel-like factor 4 (KLF4) may act both as an oncogene and a tumor suppressor in a tissue-dependent manner, and further studies on its role in pancreatic ductal adenocarcinoma (PDAC) progression and clinical outcome are warranted. Therefore, we investigated the loss of heterozygosity (LOH) in the 9q22.3–32 region and loss of KFL4 gene expression in epithelial cells from 35 PDAC, 6 pancreatic intraductal neoplasias (PanINs) and 6 normal ducts, isolated by laser microdissection, as well as their correlation with overall survival (OS) in patients treated with gemcitabine in the adjuvant setting. LOH was evaluated with 4 microsatellite markers and in situ hybridization, while KLF4 expression was studied by reverse transcription-PCR and immunohistochemistry. LOH in at least 1 locus was observed in 25 of 35 PDAC cases and in 5 of 6 PanINs, respectively. In particular, the loss of the D9S105 marker was present in 46.9% of PDAC and 83.3% of PanlNs, becoming the most deleted marker, while no LOH in D9S105 was observed in normal Wir-sung pancreatic duct. Lack of KLF4 mRNA expression was significantly associated with: (1) genomic deletion flanking KLF4 in PDAC and in PanINs (with LOH of D9S105), (2) low-grade PDAC-associated PanIN, (3) lack of KLF4 protein expression, and (4) shorter OS. These results strongly suggest a relationship between D9S105 deletion and downregulation of KLF4 gene expression as an early event in PDAC progression, as well as a possible role of KLF4 as a prognostic biomarker in gemcitabine-treated patients.

Persistent Krüppel‐like factor 4 expression predicts progression and poor prognosis of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is one prevalent human cancer worldwide. No molecular markers are presently used for predicting prognosis in HNSCC. Krüppel-like factor 4 (KLF4) is a transcription factor with diverse physiological functions, and possesses opposing roles in different human cancers. The expression and roles of KLF4 in HNSCC remain to be elucidated. In this study, immunohistochemical (IHC) analysis of KLF4 in 62 HNSCC was firstly performed. IHC results demonstrated that 42 (67.7%) had decreased KLF4 expression compared with surrounding normal epithelium, while persistent KLF4 expression was demonstrated in 20 (32.3%). The IHC results were further verified by Western blot and real-time PCR analyses to confirm the robustness of staining and interpretation. Interestingly, persistent KLF4 expression independently correlated with a worse disease-specific survival (P = 0.005), especially in patients with advanced disease. In consistent with clinical observation, all five HNSCC cell lines tested revealed a low level of baseline KLF4 expression. Moreover, enforced KLF4 expression in cell line SAS significantly increased in vitro migration/invasion abilities, multi-drug resistance, and in vivo tumorigenicity. These results clearly illustrate that persistent KLF4 expression predicts poor prognosis and confers aggressiveness in HNSCC. Our data therefore provides valuable information that HNSCC with persistent KLF4 expression might require intensified combination treatment in future practice.

Krüppel-like Factor 4 Promotes Differentiation by Transforming Growth Factor-β Receptor-mediated Smad and p38 MAPK Signaling in Vascular Smooth Muscle Cells

KLF4 (Krüppel-like factor 4) has been implicated in vascular smooth muscle cell (VSMC) differentiation induced by transforming growth factor beta (TGF-beta). However, the role of KLF4 and mechanism of KLF4 actions in regulating TGF-beta signaling in VSMCs remain unclear. In this study, we showed that TGF-beta1 inhibited cell cycle progression and induced differentiation in cultured rat VSMCs. This activity of TGF-beta1 was accompanied by up-regulation of KLF4, with concomitant increase in TbetaRI (TGF-beta type I receptor) expression. KLF4 was found to transduce TGF-beta1 signals via phosphorylation-mediated activation of Smad2, Smad3, and p38 MAPK. The activation of both pathways, in turn, increased the phosphorylation of KLF4, which enabled the formation of KLF4-Smad2 complex in response to TGF-beta1. Chromatin immunoprecipitation studies and oligonucleotide pull-down assays showed the direct binding of KLF4 to the KLF4-binding sites 2 and 3 of the TbetaRI promoter and the recruitment of Smad2 to the Smad-responsive region. Formation of a stable KLF4-Smad2 complex in the promoter's Smad-responsive region mediated cooperative TbetaRI promoter transcription in response to TGF-beta1. These results suggest that KLF4-dependent regulation of Smad and p38 MAPK signaling via TbetaRI requires prior phosphorylation of KLF4 through Smad and p38 MAPK pathways. This study demonstrates a novel mechanism by which TGF-beta1 regulates VSMC differentiation.

Krüppel-like Factor 4 Inhibits Epithelial-to-Mesenchymal Transition through Regulation of E-cadherin Gene Expression

The Krüppel-like factor 4 (KLF4) is a transcriptional regulator of proliferation and differentiation in epithelial cells, both during development and tumorigenesis. Although KLF4 functions as a tumor suppressor in several tissues, including the colon, the role of KLF4 in breast cancer is less clear. Here, we show that KLF4 is necessary for maintenance of the epithelial phenotype in non-transformed MCF-10A mammary epithelial cells. KLF4 silencing led to alterations in epithelial cell morphology and migration, indicative of an epithelial-to-mesenchymal transition. Consistent with these changes, decreased levels of KLF4 also resulted in the loss of E-cadherin protein and mRNA. Promoter/reporter analyses revealed decreased E-cadherin promoter activity with KLF4 silencing, while chromatin immunoprecipitation identified endogenous KLF4 binding to the GC-rich/E-box region of this promoter. Furthermore, forced expression of KLF4 in the highly metastatic MDA-MB-231 breast tumor cell line was sufficient to restore E-cadherin expression and suppress migration and invasion. These findings identify E-cadherin as a novel transcriptional target of KLF4. The clear requirement for KLF4 to maintain E-cadherin expression and prevent epithelial-to-mesenchymal transition in mammary epithelial cells supports a metastasis suppressive role for KLF4 in breast cancer.

KLF 4 is involved in ATRA‐induced mitofusin 2 expression in vascular smooth muscle cells

The expression of mitofusin 2 (mfn‐2) and Krüppel‐like factor 4 (KLF4) was increased by all‐trans retinoic acid (ATRA) in vascular smooth muscle cell (VSMC) differentiation. However, the role of KLF4 and mechanism of KLF4 actions in regulating mfn‐2 expression remain unknown. In this report, Western blotting and RT‐PCR showed that overexpression of KLF4 in VSMCs increases mfn‐2 expression at both the protein and mRNA levels. Silencing of KLF4 expression by KLF4‐specific small interfering RNA (siRNA) abrogated the inducing effects of ATRA on mfn‐2 expression, indicating that KLF4 mediates ATRA‐induced mfn‐2 expression. Chromatin immunoprecipitation (ChIP) and reporter gene assays revealed that KLF4 binds directly to the mfn‐2 promoter and activates its transcription. Promoter deletion from 5' region and point mutation analysis indicated that the KLF4‐binding site 3 of mfn‐2 promoter is required for the induction of the mfn‐2 promoter activity by KLF4. Furthermore, co‐immunoprecipitation assays showed that ATRA increases the interaction of KLF4 with p300 through inducing KLF4 phosphorylation mediated by the JNK and p38 MAPK signaling, subsequently leading to the increase in KLF4 acetylation and KLF4‐mediated transcriptional activation of the mfn‐2 promoter. Our results provide a novel mechanism by which KLF4 activates mfn‐2 expression and further expand the net work of KLF4 actions in VSMCs.

Role of KLF2 in endocardial cushion development in mice

Krüppel‐like factor 2 (KLF2) belongs to a family of transcription factors that have 3 C2/H2 zinc finger domains. KLF2 is expressed in endothelial cells and is induced by shear stress. KLF2−/− embryos in FVB/N genetic background die by embryonic day 11.5 (E11.5) (chi‐square p = 0.0044), whereas in mixed genetic background, they die by E14.5. Our studies show that unlike WT hearts, in E9.5 KLF2 KO hearts (n = 3) the cells lining the atrioventricular (AV) cushion, the area that will become the AV valves, form multiple, disorganized layers and the number of cells is 2‐fold higher. Electron microscopy revealed that compared to WT, the E9.5 KLF2−/− AV canal is narrower in some locations but patent and lined by endothelial cells having numerous cytoplasmic processes extending toward the lumen. In E10.5 KLF2−/− embryos (n = 3), the AV and outflow tract cushions are hypoplastic and the cells lining the AV canal form multiple disorganized layers. Alcian blue staining of E10.5 KLF2−/− hearts showed an absence of glycosaminoglycans in the cardiac jelly. These findings suggest that KLF2, directly or indirectly, regulates endothelial to mesenchymal transformation in AV cushion development. The AV phenotype is observed in FVB/N KLF2−/− embryos but not in embryos of mixed background (n = 2). Therefore, KLF2 is required for normal endocardial cushion development in FVB/N genetic background mice. (Supported by NIH R01DK074694)

Kruppel‐like factor 4 is involved in TGF‐beta1‐induced VSMC differentiation through trans‐activating TbetaRI expression

Krüppel‐like factor 4 (KLF4) is involved in phenotypic switching of vascular smooth muscle cells (VSMCs) induced by transforming growth factor beta (TGF‐β). However, as a non‐Smad protein, the role of KLF4 in TGF‐β‐regulated biological processes remains unclear. In this study, we found that TGF‐β1 inhibited cell cycle progression and induced expression of differentiation marker genes in cultured VSMCs, which was associated with the up‐regulation KLF4 and TGF‐β type I receptor (TβRI) expression induced by TGF‐β1. TGF‐β1 induced KLF4 phosphorylation in VSMCs, Which was blocked by pretreating cells with either SB431542, the TGF‐β inhibitor, or SB203580, the p38 MAP kinase inhibitor. We further demonstrated that treating VSMCs stimulated KLF4 interaction with Smad2, indicating that the phosphorylation of KLF4 is required for the interaction of KLF4 with Smad2 and KLF4 mediated TβRI expression via activation Smad and p38 MAP kinase signaling. In addition, TGF‐β1 increased DNA binding activity of KLF4 and Smad2 to TβRI promoter. We demonstrated that two potential KLF4 binding sites could activate TβRI promoter, and meanwhile, KLF4 formed complex with Smad2 to bind to the Smad‐binding region and activate the TβRI transcription cooperatively. These results indicate that KLF4 plays a positive regulatory effect on cell differentiation through trans‐activating TβRI expression via synergistic interaction with Smad2 in TGF‐β‐induced VSMCs.

KLF4 inhibition of lung cancer cell invasion by suppression of SPARC expression

Krüppel-Like Factor 4 (KLF4) functions as a tumor suppressor in some cancers, but its molecular mechanism is not clear. Our recent study also showed that the expression of KLF4 is dramatically reduced in primary lung cancer tissues. To investigate the possible role of KLF4 in lung cancer, we stably transfected KLF4 into cells from lung cancer cell lines H322 and A549 to determine the cells' invasion ability. Our results showed that ectopic expression of KLF4 extensively suppressed lung cancer cell invasion in Matrigel. This effect was independent of KLF4-mediated p21 up-regulation because ectopic expression of p21 had minimal effect on cell invasion. Our analysis of the expression of 12 genes associated with cell invasion in parental, vector-transfected, and KLF4-transfected cells showed that ectopic expression of KLF4 resulted in extensively repressed expression of secreted protein acidic and rich in cysteine (SPARC), an extracellular matrix protein that plays a role in tumor development and metastasis. Knockdown of SPARC expression in H322 and A549 cells led to suppression of cell invasion, comparable to that observed in KLF4-transfected cells. Moreover, retrovirus-mediated restoration of SPARC expression in KLF4-transfected cells abrogated KLF4-induced anti-invasion activity. Together, our results indicate that KLF4 inhibits lung cancer cell invasion by suppressing SPARC gene expression.

Putative Tumor-Suppressive Function of Krüppel-Like Factor 4 in Primary Lung Carcinoma

Krüppel-like factor 4 (KLF4) is a zinc-finger protein that plays important roles in stem cells and the development of gastric cancers. However, the role of KLF4 in primary lung cancer is unknown. The purpose of this study is to determine possible roles of KLF4 in lung cancer. The KLF4 expression in primary lung cancer tissues and case-matched normal lung tissues were determined by protein and mRNA analyses. The effects of KLF4 on cell proliferation, clonogenic formation, and cell cycle progression were determined in cultured lung cancer cells or bronchial epithelial cells after enforced KLF4 overexpression or small interfering RNA knockdown. The in vivo antitumor activity of KLF4 was evaluated by using stably transfected lung cancer cells and by adenovector-mediated gene delivery. The effect of KLF4 in regulating p21 and cyclin D1 was also evaluated. KLF4 protein and mRNA levels were dramatically decreased in most primary lung tumors compared with in case-matched normal lung tissues. Enforced expression of KLF4 resulted in marked inhibition of cell growth and clonogenic formation. The tumor-suppressive effect of KLF4 was associated with its role in up-regulating p21 and down-regulating cyclin D1, leading to cell cycle arrest at the G(1)-S checkpoint. Knockdown of KLF4 promoted cell growth in immortalized human bronchial epithelial cells. The enforced expression of KLF4 gene to lung cancer cells by ex vivo transfection or adenovector-mediated gene transfer suppressed tumor growth in vivo. Our results suggest that KLF4 plays an important role in suppressing the growth of lung carcinoma.

Kruppel-like Factor 4 is Required for the Expression of Vascular Smooth Muscle Cell Differentiation Marker Genes Induced by All-Trans Retinoic Acid

Krüppel-like factor 4 (KLF4) is involved in phenotypic modulation of vascular smooth muscle cells (VSMCs). All-trans retinoic acid (ATRA) inhibits VSMC proliferation and induces VSMC differentiation. However, the role of KLF4 in ATRA-elicited VSMC phenotypic modulation remains unclear. Here, we show that treatment of VSMCs with ATRA resulted in significant inhibition of proliferation and migration of VSMCs, as well as up-regulation of KLF4 and the VSMC differentiation marker genes SM22alpha and SM alpha-actin (alpha-SMA). At the same time, the KLF4 target gene p53 was up-regulated, while the VSMC dedifferentiation marker gene nonmuscle myosin heavy chain-B (SMemb) was down-regulated. We also show that overexpression of KLF4 in VSMCs increased the expression of p53, SM22alpha and alpha-SMA, but decreased the expression of SMemb and VSMC proliferation and migration. Silencing of KLF4 expression by KLF4-specific small interfering RNA (siRNA) abrogated the inducing effects of ATRA on p53, SM22alpha and alpha-SMA expression and neutralized the inhibitory effects of ATRA on SMemb expression and VSMC proliferation and migration. Thus, our data suggest that KLF4 is required for the expression of VSMC differentiation marker genes induced by ATRA and that this transcription factor is one of the key mediators of retinoid actions in VSMCs.

Induction of KLF4 in response to heat stress

Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor with diverse regulatory functions in cell growth, proliferation, differentiation, and embryogenesis. However, little is known about the response of KLF4 to heat stress. In this study, Western blot and reverse transcriptase-polymerase chain reaction were performed to determine the changes in KLF4 expression in response to heat stress. The results showed that heat stress up-regulated KLF4 messenger RNA and protein levels in a time-dependent manner in vivo and in 4 cell lines. Moreover, a study with heat shock transcription factor 1 (Hsf1) gene knockout mice indicated that the induction of KLF4 in response to heat stress was mediated by Hsf1. This process occurred rapidly, indicating that KLF4 is an immediate early response gene of heat stress. Next, the roles of KLF4 under heat stress conditions were analyzed for cells overexpressing or deficient in KLF4. The results showed overexpression of KLF4 increased the death rate of C2C12 cells, whereas KLF4 deficiency decreased the injury of C2C12 cells from heat stress conditions, suggesting that KLF4 might play an important role in cell injury induced by heat stress. KLF4 might be an immediate early response gene and could play an important role in cell injury induced by heat stress.