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ABSTRACT: KLF5 (Krüppel-like factor 5) is a multifunctional transcription factor involved in cell proliferation, differentiation and carcinogenesis. In addition to frequent inactivation in different types of human cancers, including breast cancer, KLF5 has been identified as an essential co-factor for the TGF-β (transforming growth factor β) tumour suppressor. In our previous study demonstrating a negative regulation of ER (oestrogen receptor α) function by KLF5 in breast cancer cells [Guo, Dong, Zhao, Sun, Li and Dong (2010) Int. J. Cancer 126, 81-89], we noticed that oestrogen reduced the protein level of KLF5. In the present study, we have tested whether and how oestrogen/ER signalling regulates KLF5 protein. We found that oestrogen caused the degradation of KLF5 protein, and the degradation was sensitive to proteasome inhibitors, but not other inhibitors. The oestrogen-inducible E3 ligase EFP (oestrogen-responsive finger protein) was identified as a key player in oestrogen-mediated degradation of KLF5, as knockdown and overexpression of EFP increased and decreased KLF5 protein levels respectively, and the decrease continued even when protein synthesis was blocked. EFP-mediated degradation impaired the function of KLF5 in gene transcription. Although only unubiquitinated EFP interacted with KLF5, overexpression of EFP appeared to prevent the ubiquitination of KLF5, while resulting in heavy ubiquitination of the E3 itself. Furthermore, ubiquitination of EFP interrupted its interaction with KLF5. Although the mechanism for how EFP degrades KLF5 remains to be determined, the results of the present study suggest that oestrogen causes the degradation of KLF5 protein by inducing the expression of EFP in ER-positive breast cancer cells.
Biochemical Journal 05/2011; 437(2):323-33. · 4.90 Impact Factor
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ABSTRACT: Kruppel-like factor 5 (KLF5) is implicated in human breast cancer by frequent genomic deletion and expressional deregulation, but the molecular mechanisms by which KLF5 affects breast tumorigenesis are still unknown. This study was conducted to examine whether and how KLF5 affects the function of estrogen receptor (ER) in breast cancer cells. Using different cell lines, we found that restored expression of KLF5 inhibited estrogen-promoted cell proliferation in ER-positive MCF-7 and T-47D cell lines but had no effect on ER-negative SK-BR-3 cells. Transcriptional activity of ER was also suppressed by KLF5, as detected by using estrogen-stimulated ER responsive element-mediated reporter assay and expression analysis of ER target genes including c-MYC and Cathepsin D (CSTD). Chromatin immunoprecipitation assays showed that KLF5 inhibited ERalpha binding to the promoter of c-myc and CSTD. Furthermore, estrogen induced an interaction between KLF5 and ERalpha. These results suggest that KLF5 inhibits the function of ERalpha in gene regulation and cell proliferation through protein interaction that interrupts the binding of ERalpha to target gene promoters to prevent target gene induction.
International Journal of Cancer 07/2009; 126(1):81-9. · 5.44 Impact Factor
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ABSTRACT: KLF5 plays important roles in a variety of cellular processes including proliferation and differentiation. Recently KLF5 was shown to reverse its function in proliferative and p15 regulation upon transforming growth factor-beta (TGFbeta)-mediated acetylation. To understand how KLF5 acetylation functions in TGFbeta-induced p15 transcription, we characterized the interactions of KLF5 with other transcription factors and promoter DNA elements in the context of TGFbeta. KLF5 interacted with Smad2-4 and Miz-1 in a TGFbeta-independent manner, but interacted with Myc only when TGFbeta was activated, and at least some of the interactions had an additive effect on TGFbeta-induced p15 transcription. Oligo pulldown assays showed that binding of Myc to the Inr element was KLF5-dependent, and TGFbeta could enhance the binding when more KLF5 was available. Furthermore, TGFbeta induced an interaction between KLF5 and the p300 acetylase, and acetylation of KLF5 was necessary for Smad4 to associate with p300. Failure in KLF5 acetylation not only prevented p300-assembled Smad4-KLF5 complex formation on p15 promoter but also affected the binding of Smad4 and FOXO3 on the p15 promoter in vivo. These findings suggest that without TGFbeta, some KLF5 associates with Smads in the nucleus and other KLF5 associates with Miz-1 on the p15 promoter to repress its transcription. Activation of TGFbeta recruits p300 to the KLF5-Smad complex to acetylate KLF5, and the complex with acetylated KLF5 binds to the Smad binding element and alters the binding of other factors to p15 promoter to induce its transcription.
Journal of Biological Chemistry 06/2009; 284(27):18184-93. · 4.77 Impact Factor
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ABSTRACT: KLF5 plays important roles in a variety of cellular processes including proliferation and differentiation. Recently KLF5 was
shown to reverse its function in proliferative and p15 regulation upon TGFβ-mediated acetylation. To understand how KLF5¡¦s
acetylation functions in TGFβ-induced p15 transcription, we characterized the interactions of KLF5 with other transcription
factors and promoter DNA elements in the context of TGFβ. KLF5 interacted with Smads2-4 and Miz-1 in a TGFβ-independent manner,
but interacted with Myc only when TGFβ was activated, and at least some of the interactions had an additive effect on TGFβ-induced
p15 transcription. Oligo pulldown assays showed that binding of Myc to the Inr element was KLF5-dependent, and TGFβ could
enhance the binding when more KLF5 was available. Furthermore, TGFβ induced an interaction between KLF5 and the p300 acetylase,
and acetylation of KLF5 was necessary for Smad4 to associate with p300. Failure in KLF5 acetylation not only prevented p300-assembled
Smad4-KLF5 complex formation on p15 promoter, but also affected the binding of Smad4 and FOXO3 on p15 promoter in vivo. These
findings suggest that without TGFβ, some KLF5 associates with Smads in the nucleus and other KLF5 associates with Miz-1 on
the p15 promoter to repress its transcription. Activation of TGFβ recruits p300 to the KLF5-Smads complex to acetylate KLF5,
and the complex with acetylated KLF5 binds to the SBE element and alters the binding of other factors to p15 promoter to induce
its transcription.
Journal of Biological Chemistry 05/2009; · 4.77 Impact Factor
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ABSTRACT: During epithelial homeostasis, stem cells divide to produce progenitor cells, which not only proliferate to generate the cell mass but also respond to cellular signaling to transition from a proliferative state to a differentiation state. Such a transition involves functional alterations of transcriptional factors, yet the underlying molecular mechanisms are poorly understood. Recent studies have implicated Kruppel-like factors (KLFs) including KLF5 in the renewal and maintenance of stem/progenitor cells. Here we demonstrate that the pro-proliferative factor KLF5 becomes anti-proliferative upon TGFbeta-mediated acetylation in an in vitro model of epithelial homeostasis. In the HaCaT epidermal cell line treated with or without TGFbeta, we found that KLF5 was not only essential for cell proliferation, it was also indispensable for TGFbeta-induced anti-proliferation in these cells. KLF5 inhibited the expression of p15 (CDKN2B), a cell cycle inhibitor, without TGFbeta, but became a coactivator in TGFbeta-induced p15 expression in the same cells. Mechanistically, TGFbeta recruited acetylase p300 to acetylate KLF5, and acetylation in turn altered the binding of KLF5 to p15 promoter, resulting in the reversal of KLF5 function. These studies not only demonstrate that a basic transcription factor can be both pro-proliferation and anti-proliferation in epithelial homeostasis, they also present a unique mechanism for how transcriptional regulation changes during the transition from proliferation to inhibition of proliferation. Furthermore, they establish KLF5 as an essential cofactor for TGFbeta signaling.
Journal of Biological Chemistry 01/2009; 284(10):6071-8. · 4.77 Impact Factor
