Cooperation of p300 and PCAF in the Control of MicroRNA 200c/141 Transcription and Epithelial Characteristics

Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype

Replicative Senescence of Biliary Epithelial Cells Precedes Bile Duct Loss in Chronic Liver Allograft Rejection : Increased Expression of p21WAF1/Cip1 as a Disease Marker and the Influence of Immunosuppressive Drugs

Early chronic liver allograft rejection (CR) is characterized by distinctive cytological changes in biliary epithelial cells (BECs) that resemble cellular senescence, in vitro, and precede bile duct loss. If patients suffering from early CR are treated aggressively, the clinical and histopathological manifestations of CR can be completely reversed and bile duct loss can be prevented. We first tested whether the senescence-related p21WAF1/Cip1 protein is increased in BECs during early CR, and whether treatment reversed the expression. The percentage of p21+ BECs and the number of p21+ BECs per portal tract is significantly increased in early CR (26 ± 17% and 3.6 ± 3.1) compared to BECs in normal liver allograft biopsies or those with nonspecific changes (1 ± 1% and 0.1 ± 0.3; P < 0.0001 and P < 0.02), chronic hepatitis C (2 ± 3% and 0.7 ± 1; P < 0.0001 and P < 0.04) or obstructive cholangiopathy (7 ± 7% and 0.7 ± 0.6; P < 0.006 and P = 0.04). Successful treatment of early CR is associated with a decrease in the percentage of p21+ BECs and the number of p21+ BECs per portal tract. In vitro, nuclear p21WAF1/Cip1 expression is increased in large and multinucleated BECs, and is induced by transforming growth factor (TGF)-β. TGF-β1 also increases expression of TGF-β receptor II, causes phosphorylation of SMAD-2 and nuclear translocation of p21WAF1/Cip1, which inhibits BEC growth. Because conversion from cyclosporine to tacrolimus is an effective treatment for early CR, we next tested whether these two immunosuppressive drugs directly influenced BEC growth in vitro. The results show that cyclosporine, but not tacrolimus, stimulates BEC TGF-β1 production, which in turn, causes BEC mito-inhibition and up-regulation of nuclear p21WAF1/Cip1. In conclusion, expression of the senescence-related p21WAF1/Cip1 protein is increased in BECs during early CR and decreases with successful recovery. Replicative senescence accounts for the characteristic BEC cytological alterations used for the diagnosis of early CR and lack of a proliferative response to injury. The ability of cyclosporine to inhibit the growth of damaged BECs likely accounts for the relative duct sparing properties of tacrolimus

A Putative model illustrating the interactions of P300/PCAF/ZEB1 in modulating cell phenotype through control of <i>miR-200c/141</i> transcription.

<p>(AC = acetylation).</p

EMT induced by SPRR2a in HuCCT-1 involves loss of E-cadherin, increased vimentin, and reduction of miR-200 family transcription as compared to vector transfected controls.

<p>Examples of the morphological changes and changes in E-cadherin and vimentin expression in stable SPRR2a clones (NC = negative control) (<b>A</b>). Transcriptional loss of the miR200 family in SPRR2a expressing cells does not involve SH3 domain containing tyrosine kinases. Real-time PCR analysis of miR-200 family after 72 hrs treatment with ABL1 siRNA (<b>B</b>) and PP2 treatment (<b>C</b>) did not alter miR-200 expression. All clones used in this paper stably express SPRR2a (<b>D</b>). Real time PCR analysis: comparative 2-ΔΔCT method (miRNA: U6 internal control; ABL1: GAPDH internal control). (n = 2 independent experiments).</p

Changes in miR-200c/141 and P300 result in expected EMT/MET responses.

<p>Representative western blot and corresponding real time PCR analysis shows inhibition of miR200c/141 causes partial EMT shifts in vector cells as evidenced by increased Vimentin, increased ZEB1 and phenotypic changes. No change is seen in E-cadherin expression. (84 hr post transfection; cells grown on glass coverslips; n≥2 independent experiments) (<b>A</b>). Representative western blot (48 hr) showing partial MET in SPRR2A cells (increased E-cadherin; phenotypic changes on tissues culture plastic) following transfection with pre-miR-200c and pre-miR-141. Western blot results were verified by real time PCR analysis and immunofluorescence staining (n = 2 independent experiments) (<b>B</b>). Representative western blot (48 hr) and real time PCR showing changes in EMT markers following knock down of P300. Phenotypic changes were not observed, but EP300siRNA significantly reduced vector cell miR200c expression. (n≥2 independent experiments) (<b>C</b>) Real time PCR analysis: comparative 2−ΔΔCT method (U6 or GAPDH internal control); *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001; Student's <i>t</i>-test).</p

P300/PCAF complexes with ZEB1 on the <i>miR-200c/141</i> promoter and requires the CH3 domain of P300 for transcription.

<p>Immunoprecipitation of P300 following transfection with the indicated protein expression vectors verifies P300/ZEB1 and P300/PCAF interactions, which were unaffected by TGF-β1 treatments (5 ng/mL; 24 hrs) (<b>A</b>). Immunoprecipitation experiments show PCAF acetylates ZEB1 following transfection with ZEB1 ± PCAF expression vectors (24 hrs) (<b>B</b>). DNA pull-down assay using a wild type (wt) or mutational E-box/Z-box sequence for the <i>miR-200c/141</i> promoter after co-transfection of HuCCT-1 shows binding of ZEB1 and PCAF to the wt promoter sequence (<b>C</b>), and p300/PCAF/ZEB1 binding to the wt promoter, which is unaffected by TGF-β1 treatments (5 ng/mL; 24 hrs) (<b>D</b>). Luciferase assay for <i>miR-200c/141</i> promoter activity following transfection with wild type or CH3 deleted P300 expression vector in HuCCT-1 parent cells shows the CH3 domain is required for miR transcription. (n = 3 independent experiments; ***, <i>P</i><0.001; Student's <i>t</i> -test) (<b>E</b>).</p