TGF-beta regulates the expression of transcription factor KLF6 and its splice variants and promotes co-operative transactivation of common target genes through a Smad3-Sp1-KLF6 interaction

Abstract

KLF6 (Krüppel-like factor 6) is a transcription factor and tumour suppressor with a growing range of biological activities and transcriptional targets. Among these, KLF6 suppresses growth through transactivation of TGF-beta1 (transforming growth factor-beta1). KLF6 can be alternatively spliced, generating lower-molecular-mass isoforms that antagonize the full-length WT (wild-type) protein and promote growth. A key target gene of full-length KLF6 is endoglin, which is induced in vascular injury. Endoglin, a homodimeric cell membrane glycoprotein and TGF-beta auxiliary receptor, has a pro-angiogenic role in endothelial cells and is also involved in malignant progression. The aim of the present work was to explore the effect of TGF-beta on KLF6 expression and splicing, and to define the contribution of TGF-beta on promoters regulated by co-operation between KLF6 and Sp1 (specificity protein 1). Using co-transfection, co-immunoprecipitation and fluorescence resonance energy transfer, our data demonstrate that KLF6 co-operates with Sp1 in transcriptionally regulating KLF6-responsive genes and that this co-operation is further enhanced by TGF-beta1 through at least two mechanisms. First, in specific cell types, TGF-beta1 may decrease KLF6 alternative splicing, resulting in a net increase in full-length, growth-suppressive KLF6 activity. Secondly, KLF6-Sp1 co-operation is further enhanced by the TGF-beta-Smad (similar to mothers against decapentaplegic) pathway via the likely formation of a tripartite KLF6-Sp1-Smad3 complex in which KLF6 interacts indirectly with Smad3 through Sp1, which may serve as a bridging molecule to co-ordinate this interaction. These findings unveil a finely tuned network of interactions between KLF6, Sp1 and TGF-beta to regulate target genes.

Figure 1. Expression of KLF6 after TGF-β treatment in different cells

After different types of cells had been treated with 10 ng/ml TGF-β1 for various times up to 24 h, cells were fixed and the KLF6 protein content of each cell type was assayed by flow cytometry and are shown as the Expression Index (see the Materials and methods section for a definition of this term). Representative results from three different experiments with similar results are shown. *Statistical significance at least P < 0.05.

Figure 2. Changes in KLF6 alternative splicing after TGF-β1 treatment in different cells

(A and B) HEK-293T cells were co-transfected with pCD105 (− 50/+ 350)-pXP2 (pEndoglin, A) or pGL-Col1 (pCollagen, B) and 1 μg of either WT KLF6 (KLF6wt) (black bar 2), KLF6 Sv1 (Sv1) (black bar 3), KLF6 Sv2 (Sv2) (black bar 4) or a mixture of 0.5 μg of WT and 0.5 μg of Sv1 (black bar 5)/Sv2 (black bar 6). For the first two bars in (A), endogenous KLF6 was knocked down with KLF6 siRNA (white bar). In addition, the effect of TGF-β treatment (10 ng/ml) was assessed in hatched bars 1–4 in (A). The results are given in arbitrary units of LUC activity. Representative results obtained from three different experiments with replicable results are shown. *Statistical significance at least P < 0.05 between control (pEndoglin, pCollagen) and KLF6 co-transfected. In case of the white bar (siRNA), the asterisk (*) means that the value was statistically significant (P<0.05) compared with the corresponding black bar 2. (C–E) After THP-1 (C), HUVEC (D) and HepG2 (E), cells were treated with 10 ng/ml TGF-β1 for the various times indicated and cell lysates were prepared. Total RNA was extracted from each cell lysate, and levels of WT KLF6 and its Svs in each sample were measured using real-time PCR. To calculate the fold change in mRNA levels of KLF6 alternative splicing, the fold change in mRNA levels of total KLF6 (WT KLF6 plus alternatively spliced KLF6 transcripts) was divided by the fold change in wild-type KLF6 alone. *Statistical significance at least P < 0.05.

Figure 3. Effect of Sp1, KLF6 and Smad3 on transactivation of ENG and COL promoters in mammalian and insect cells

(i) Panels A and B: the transactivation effect of different expression vectors for KLF6, Smad3, and a combination of both, on the endoglin (ENG, A) and collagen (COL, B) promoter constructs, respectively in HeLa cells. In each transfection, 1 μg of reporters, 0.5 μg of KLF6 and 250 ng of Smad3 expression vectors were used. At 24 h after transfection, cell lysates were prepared and the LUC activity in each lysate was determined as described in the Materials and methods section. The results are expressed as fold induction, comparing with values obtained with vacant vector 1. Lane 1, reporter (endoglin or collagen-1); lane 2, reporter + KLF-6; lane 3, reporter + Smad3; lane 4, reporter + KLF6 + Smad3. Black bars represent the untreated samples, whereas hatched bars represent samples that were additionally treated with TGF-β (10 ng/ml). Panel C: the same experiment was carried out using HEK-293T cells. The transactivation effect of different expression vectors for KLF6 and Smad3 and in combination with the endoglin promoter construct. In each transfection, 1 μg of reporters, 0.5 μg of KLF6 and 250 ng of Smad3 expression vectors were used. At 24 h after transfection, cell lysates were prepared and the LUC activity in each lysate was determined as described in the Materials and methods section. The results are expressed as fold induction, comparing with values obtained with vacant vector (sample 1). Lane 1, reporter (endoglin); lane 2, reporter + KLF6; lane 3, reporter + Smad3; lane 4, reporter + KLF6 + Smad3. Moreover, the co-operative effect between KLF6 and Smad3 was also assessed by silencing Sp1 using transfection with 0.5 μg of siRNA (siSp1; white bar) and using as a control a random siRNA sequence (si Random, hatched bar). Panels D and E: the same type of experiment as in panels A–C was performed, but instead Schneider Drosophila cells were used, which express neither Sp1 nor KLF6. The transactivation effect of different expression vectors for Sp1, KLF6 and Smad3, and a combination of them, on both on the endoglin (ENG, D) and collagen (Col, E) promoter constructs respectively was assayed. In each transfection, 1 μg of reporters, 0.5 μg of KLF6 and Sp1, and 250 ng of Smad3 expression vectors, were used. At 48 h after transfection, cell lysates were prepared and the LUC activity in each lysate was determined as described in the Materials and methods section. The results are expressed as fold induction, comparing with values obtained with vacant vector (sample 1). In the eight samples shown, ‘R’ means the reporter. The experiments were repeated five times, and each experiment had two or three replicate points. The experiments shown are representative of the results obtained. (ii) Panel A: RNA from KLF6 was quantitated after 0, 3, 12 and 25 h of WH in HUVECs by real-time PCR. The increase in RNA is expressed as fold induction. Panels B and C: RNA from endoglin and type I collagen was quantified after 0, 3,12 and 25 h in HUVECs either, untreated or subjected to TGF-β treatment (10 ng/ml), WH or the combination of both treatments by real-time PCR. The increase in RNA is expressed as fold induction. Asterisks show that the differences are significant with P at least <0.05. Experiments were repeated at least twice, using triplicates, and referred to GAPDH RNA as endogenous control.

Figure 4. Interactions of Sp1, KLF6 and Smad3 based on co-immunoprecipitation

(A) Cos-7 cells were transfected with expression vectors for Smad3 and Smad4 in the presence or absence of constitutively active TβRI (TβRI C. A.) (Cos cells have very low amounts of this receptor and of Smad proteins). GST–KLF6 fusion pull-down was generated from the protein extracts of transfected cells. As a control for specificity of the method, when Smad3 was not transfected (lane 3), only Sp1 was recovered by GST–KLF6 pull-down. (B) Co-immunoprecipitation (IP) experiments in HUVECs, either untreated (C) or after treatment with TGF-β (10ng/ml) and recovery from WH for 24 h (T). IgG is a control lane following immunoprecipitation using human IgG. (C) Co-immunoprecipitation experiments were carried out in Schneider cells, which do not express Sp1, or KLF6. Schneider cells were co-transfected with pPACSp1 and Flag Smad3 expression vectors on the left side of (B), or with pPACKLF6 and Flag Smad3 on the right side. When the immunoprecipitation was made using the Flag tag to recover Smad3, Sp1 could be revealed by Western Blot (WB) in the immunoprecipitate (lane 1, left side). However, when transfecting with pPACKLF6 and Smad3, in the Flag Smad3 immunoprecipitate we could not detect KLF6. In the upper part, Western blots (WBs) show the expression of the transfected proteins in all the cases of transfected proteins. Lane 3C (293T) is a control of the migration for KLF6. The co-immunoprecipitation experiments were repeated three times and the results are representative. (D) Sequential co-immunoprecipitation in HEK-293T cells. HEK-293T cells were doubly or triply transfected with KLF6 and Smad3, or KLF6, Smad 3 and Sp1 expression vectors respectively. Lane 1 shows the results obtained with double transfection of KLF6 and Smad3, whereas lane 2 displays the triple-transfection results. On the left-hand side, Western blots of total extracts are shown. On the right-hand side the results for the first immunoprecipitation using KLF6 antibody and the second immunoprecpipation with the anti-Flag tag are shown. There is a substantial enrichment of Smad3 in the ternary complex after the second immunoprecipitation, which is dependent on the transfection of Sp1.

Figure 5. Physical interaction between Sp1 and the different domains of KLF6

COS-7 cells were transiently transfected with expression vectors for the DNA of the KLF6 DBD, (pcDNA3.DBDKLF6-Flag) or for the KLF6 transactivation domain (ZAD) (pcDNA3. DBDKLF6-Flag), both tagged with the Flag domain. (A) The left-hand side of the Western blot (WB) shows that Sp1 is present in all the cell extracts. (B) On the left-hand side the proteins corresponding to the transfected vectors could be detected in total extracts. When these extracts were subjected to immunoprecipitation (IP) using anti-Flag as an antibody, we could detect Sp1 by Western blot only in the immunoprecipitate of cells transfected with the expression vector of KLF6 transactivation domain (ZAD). (C) COS-7 cells were transfected with the different pcDNA3 expression vectors of KLF6, including full-length (WT), Sv1 and Sv2 and the DBD and ZAD KLF6 domains. As all the expression vectors were Flag-tagged, the immunoprecipitates after Flag incubation were separated and subjected to Western blot developed by Sp1 antibody. The experiments were repeated three times.

Figure 6. Model of basal and injury-associated endoglin-gene regulation

This model defines the sequence of events involving KLF6, Sp1 and Smad3. (A) As TGF-β increases, the alternative splicing of KLF6 is inhibited, leading to more net unopposed KLF6-mediated growth suppression. Binding of Sv1 and Sv2 to Sp1 suggests a potential mechanism whereby these splice forms may antagonize endoglin-gene expression through cytoplasmic sequestration of this bridging factor, which is critical to maximal endoglin gene transactivation. (B) Basal transcription of the endoglin gene. On TGF-β1 stimulation associated with injury and/or inflammation, this cytokine signals through its cognate receptors to Smad3/4 (C), which translocate to the nucleus where they physically interact with Sp1. Smad3/4 also interact with the GTF machinery, thereby synergizing transcription. Endothelial injury or inflammatory events stimulates de novo synthesis of KLF6 and its translocation to the nucleus (as an early event). In the nucleus, KLF6 interacts with Sp1, the binding domain (BD) or C-terminal part of Sp1 with the transactivating domain (AD) of KLF6, enhancing endoglin transcription (D) and other KLF6-responsive promoters involved in the TGF-β system, thereby increasing expression and activation of TGF-β. This may eventually culminate in the formation of the Smad3–Sp1–KLF6 multimeric complex (E) (as a late event), where Sp1 would be the bridge between the KLF6 transactivating domain and the MH1 domain of Smad3.