Human Arrest Defective 1 Acetylates and Activates B-Catenin, Promoting Lung Cancer Cell Proliferation

Arrest defective 1 (ARD1), an acetyltransferase, is essential for the yeast life cycle. Although its human homologue (hARD1) has been identified, its biological functions in human cells remain unclear. In the present study, we examined the biological function of hARD1. In H1299 and A549 lung cancer cells, hARD1-silencing RNA inhibited cell proliferation and induced G1 arrest. Cyclin D1 was also found to be downregulated in these growth-arrested cells, and the ectopic expression of cyclin D1 rescued cell growth. hARD1 knockdown repressed the promoter activity of the cyclin D1 gene, which inhibited the transcription of cyclin D1. Moreover, hARD1 knockdown reduced the binding of B-catenin/TCF4 transcription factor to cyclin D1 promoter and repressed its transcriptional activity. Inversely, hARD1 expression increased the transcriptional activity of B-catenin. Both endogenous and ectopically expressed hARD1 was coimmunoprecipitated with B-catenin. hARD1 knockdown did not affect B-catenin expression or degradation but noticeably reduced acetylated B-catenin. The B-catenin binding and acetylation by hARD1 were observed in vitro . Therefore, it is suggested that hARD1 participates in proliferation of lung cancer cells via the activation of B-catenin. (Cancer Res 2006; 66(22): 10677-82)


Introduction
Acetylation is a common means of protein modification in eukaryotes, and protein NH 2 -terminal a-acetylation is the most frequent of these protein modifications (1).q-Amino lysine acetylation, unlike NH 2 -terminal acetylation, reversibly regulates the function and/or stabilities of many proteins (2).Of mammalian protein acetyltransferases, homologues of yeast arrest defective 1 protein (Ard1p) show both a-protein and q-protein acetylation activities.Ard1p was originally described in Saccharomyces cerevisiae (3), where it functions as a catalytic subunit of the NH 2 -terminal acetyltransferase complex with N-acetyltransferase 1 protein (Nat1p; ref. 4).Similarly, both mouse and human Ard1p homologues (mARD1 and hARD1), in complexes with homologues of yeast Nat1p, express a-protein acetylation activity (5,6).In addition, ARD1 has been reported to show q-protein acetylation activity in mammalian cells; for example, it acetylates Lys 532 in hypoxia-inducible factor 1a (HIF-1a) and thereby degrades HIF-1a via the ubiquitin-proteasome system (7).However, two research groups recently posed questions about the involvement of ARD1 in HIF-1a regulation (8,9).
In terms of its biological functions, Ard1p has been linked with G 0 entry, growth, and sporulation in yeast cells (3,10,11).However, its function in mammalian cells has not been defined.Recently, Fisher et al. (12) showed that hARD1 knockdown is correlated with the down-regulation of proliferative genes and up-regulation of antiproliferative genes.Because the expressions of HIF-1-regulated genes were not found to be affected by the small interfering RNA (siRNA), hARD1 was suggested to be directly linked to cell proliferation, regardless of HIF-1.However, the molecular entities that mediate gene expressions by hARD1 remain unidentified.
In the present study, we evaluated the biological functions of hARD1 and identified the transcription factor regulated by hARD1.In H1299 and A549 lung cancer cells, hARD1-silencing RNA inhibited cell proliferation and induced G 1 arrest, and this was attributed to the transcriptional repression of the cyclin D1 gene.hARD1 knockdown inhibited the activity of h-catenin/TCF, which is a transcription factor for cyclin D1.We also showed that hARD1 associated with and acetylated h-catenin, which in turn induced the binding of TCF4 to cyclin D1 promoter.We conclude that hARD1 may participate in cell proliferation as an activator of h-catenin.

Materials and Methods
Cell culture and proliferation analysis.Two non-small cell lung carcinoma cell lines, H1299 and A549, were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 supplemented with 10% FCS in a 5% CO 2 humidified atmosphere at 37jC.To analyze cell proliferation, bromodeoxyuridine (BrdUrd) incorporation assays were done using a FITC BrdUrd Flow kit purchased from BD PharMingen (San Diego, CA).Total DNAs were stained with 7-aminoactinomycin D (7-AAD).To analyze cell cycle, cells were fixed in 75% ethanol and labeled with propidium iodide (0.05 mg/mL) for 30 minutes.FITC, 7-AAD, and propidium iodide were detected using a FACStar flow cytometer (BD Biosciences, San Jose, CA).
Reporter assays.Cells were cotransfected with reporter plasmids and cytomegalovirus-h-gal plasmid or siRNAs using LipofectAMINE (Invitrogen).The transfected cells were then allowed to stabilize for 48 hours before being used in experiments.The cells were lysed and assayed for luciferase activity, and h-gal assays were done for normalization of transfection efficiency.
Immunoblotting and immunoprecipitation.Proteins were separated on SDS/polyacrylamide gels and transferred to Immobilon-P membranes.Membranes were blocked with 5% nonfat milk, incubated overnight at 4jC with primary antibodies (1:1,000), incubated with a HRP-conjugated secondary antibodies (1:5,000) for 2 hours, and visualized using an enhanced chemiluminescence kit (GE Healthcare Bio-Sciences, Piscataway, NJ).For immunoprecipitation, cell lysates (1 mg protein) were incubated with 10 AL of antiserum and further incubated with 10 AL of protein A/G-Sepharose beads (GE Healthcare Bio-Sciences) for 4 hours.Immunocomplexes were eluted in a sample buffer containing 2% SDS and 10 mmol/ L DTT and then subjected to immunoblotting.
In vitro acetylation assays.To assay the activity of hARD1 expressed from HEK293 cells, cells were transfected separately with pARD1 or pHAcatenin.The HA-catenin lysates were incubated with the hARD1 lysates.Acetylated h-catenin was identified using immunoprecipitation with antih-catenin and immunoblotting with anti-acetyl-lysine.To assay the activity of bacterially expressed hARD1, 500 ng GST-h-catenin and 400 ng His-hARD1 were incubated in the reaction mixture [50 mmol/L Tris-HCl (pH 8.0), 0.1 mmol/L EDTA, 1 mmol/L DTT, 10 mmol/L sodium butyrate, 20 Amol/L acetyl-CoA, 10% glycerol] for 1.5 hours at 37jC or 4jC.Acetylated GST-h-catenin was identified by immunoblotting with anti-acetyl-lysine, and protein loading was verified by Coomassie staining.
Statistical analysis.Results are expressed as mean and SD using Microsoft Excel 2002 software.The Mann-Whitney U test (SPSS, Chicago, IL) was used to compare BrdUrd incorporations, cell cycle populations, and reporter activities.All statistical tests were two sided, and Ps < 0.05 were considered to be significant.

Results and Discussion
hARD1 promotes cell proliferation by regulating cyclin D1.In hARD1 knockdown cells, BrdUrd incorporation to DNA was significantly reduced by 62% in H1299 or by 33% in A549 (Fig. 1A).In addition, the G 0 -G 1 population significantly increased by 21.5% in H1299 or by 13.8% in A549 at the expense of the S population (Fig. 1B).Of cyclins, cyclin D1 was markedly reduced by si-ARD1 at the protein and mRNA levels (Fig. 1C).Moreover, cyclin D1 expression rescued growth arrest induced by si-ARD1 (Fig. 1D).These results suggest that hARD1 is required for the proliferation of non-small cell lung cancer cells by expressing cyclin D1. with anti-ARD1 antiserum or nonimmunized rat serum (Pre-IgG ).Left, coprecipitation of h-catenin with hARD1 was analyzed by immunoblotting (IB ) using anti-catenin antibody; right, coprecipitation of hARD1 with h-catenin was also analyzed.B, association of expressed hARD1 and h-catenin.HEK293 cells were cotransfected with 1 Ag of pGFP-hARD1 and 1.5 Ag of pHA-catenin.Left, GFP-hARD1 was immunoprecipitated with anti-GFP antibody, and HA-catenin coprecipitates were analyzed using anti-HA antibody; right, HA-catenin was also immunoprecipitated, and the coprecipitation of GFP-hARD1 was identified.C, in vitro binding of hARD1 to h-catenin.Purified recombinant His-hARD1 and GST-h-catenin (or GST) peptides were coincubated, and then His-hARD1 was pulled down using glutathione beads.Input and pull-down proteins were analyzed by SDS-PAGE and Coomassie staining.D, hARD1 was required for h-catenin acetylation.After transfected with siRNAs (20 nmol/L), H1299 cells were treated with 10 mmol/L sodium butyrate to prevent protein deacetylation.Top, after cell lysates were immunoprecipitated with h-catenin antibody, acetylated and total (acetylated plus unacetylated) h-catenins were identified using acetyl-lysine (Ac-Lys ) and h-catenin antibodies, respectively; bottom, this immunoprecipitation was cross-checked by changing the sequence of antibody treatment.The heavy chain of IgG (H.Chain ) was used as a loading control.
hARD1 is required for the activation of B-catenin/TCF.In the cycD1-Luc reporter system, the cyclin D1 promoter activity was significantly repressed by si-ARD1 (Fig. 2A, left).We next examined whether hARD1 activates h-catenin/TCF, which is known to regulate cyclin D1 (15).A chromatin immunoprecipitation analysis showed that si-ARD1 inhibited the recruitment of h-catenin/TCF on the cyclin D1 promoter (Fig. 2A, right).Interestingly, the h-catenin/TCF complex was shown to bind the cyclin D1 promoter (Fig. 2A) and h-catenin was translocated into the nucleus (Supplementary Fig. S2) without additional Wnt signaling stimulants, which suggest that H1299 and A549 cells have the activated h-catenin.Indeed, many non-small cell lung cancer cells, including H1299 and A549, have been reported to show basal activation of Wnt signaling by overexpressing Wnt ligands (16,17).The activity of a TCF reporter plasmid, TOP-FLASH, was significantly reduced by si-ARD1 in H1299 cells but that of FOP-FLASH lacking TCF-binding site was not (Fig. 2B).In HEK293 cells stimulated by LiCl and Wnt3a, TOP-FLASH activity was also inhibited by si-ARD1 (Fig. 2C).Moreover, hARD1 expression augmented the TOP-FLASH activation by h-catenin (Fig. 2D).These results suggest that hARD1 induces cyclin D1 by activating h-catenin/TCF.hARD1 binds B-catenin and is required for B-catenin acetylation.h-Catenin was coimmunoprecipitated with hARD1 and vice versa (Fig. 3A), and expressed HA-catenin and GFP-hARD1 were also coimmunoprecipitated (Fig. 3B).In an in vitro GST pulldown assay using recombinant proteins, His-hARD1 directly interacted with GST-h-catenin (Fig. 3C).To examine the mechanism by which hARD1 activates h-catenin, we tested the possibility that h-catenin is activated by hARD1-mediated acetylation and found that the h-catenin acetylation was attenuated by si-ARD1 (Fig. 3D).However, hARD1 did not affect the cellular level or the ubiquitination of h-catenin (Supplementary Fig. S3).
hARD1 directly acetylates B-catenin.hARD1 acetylated hcatenin in HEK293 lysates (Fig. 4A).Moreover, recombinant hcatenin was acetylated by 37jC incubation with recombinant hARD1 but not by 4jC incubation (Fig. 4B), suggesting that hARD1 directly acetylates lysine residues of h-catenin by enzymatic reaction without other cellular components (e.g., p300).Because p300 is also known to activate h-catenin via acetylation (18)(19)(20), we test the possibility that p300 is linked with the h-catenin acetylation by hARD1.p300 knockdown attenuated p300 binding to h-catenin but did not affect the association of hARD1 and h-catenin (Fig. 4C), suggesting that p300 is not necessary for hARD1-h-catenin interaction.However, h-catenin acetylation was inhibited noticeably (>60%) by si-ARD1 or si-p300 each and further suppressed by si-ARD1 and si-p300 combination (Fig. 4D).Although hARD1 and p300 bind h-catenin independently, it cannot be ruled out that they cooperatively acetylate h-catenin in vivo.containing HA-catenin were incubated with lysates (500, 250, or 100 ng protein) containing expressed hARD1 at 30jC for 2 hours and then precipitated with h-catenin antibody.Total and acetylated h-catenins were identified by immunoblotting with h-catenin and acetyl-lysine antibodies, respectively.Bottom, the amounts of hARD1 in the reaction mixture were analyzed by immunoblotting with hARD1 antibody.B, hARD1 directly acetylated h-catenin.Top, GST-h-catenin was incubated with His-hARD1 and acetyl-CoA at 37jC or 4jC (as a negative control to verify enzymatic reaction), and then the lysine acetylation was identified using anti-acetyl-lysine antibody; bottom, Coomassie staining showed proteins present in reaction mixtures.C, p300 was not required for hARD1 binding to h-catenin.HEK293 cells were transfected with 40 nmol/L of si-p300 or si-con.hARD1 or p300 was immunoprecipitated with anti-ARD1 or anti-p300 antiserum, and coprecipitation of h-catenin was analyzed using anti-catenin antibody.D, hARD1 and p300 both were responsible for h-catenin acetylation.After transfected with hARD1 or/and p300 siRNAs (20 nmol/L each), H1299 cells were treated with 10 mmol/L sodium butyrate.Cell lysates were immunoprecipitated with anti-catenin antibody and immunoblotted with anti-acetyl-lysine or anti-catenin antibody.Protein band intensities were quantified using ImageJ 1.36b image analysis software (NIH, Bethesda, MD).Bottom, ratios of acetylated form to total h-catenin and the percentages of h-catenin acetylation inhibited by siRNAs.
Implication and speculation.Wnt signaling plays crucial roles in normal lung development and in lung cancer promotion, similar to its roles in other organs and tumors.In lung cancers, Wnt pathway is found to be aberrantly activated at multiple steps: overexpression of Wnt ligands, up-regulation of h-catenin, and down-regulation of Wnt inhibitors, including Wnt inhibitory factor-1, secreted frizzled-related proteins, and Dickkopf proteins (17).Therefore, each component of the Wnt pathway is reviewed as a potential target for development of new cancer therapy.In the present study, it is suggested that hARD1, as a component of the Wnt pathway to activate h-catenin, is involved in the Wnt signaling-dependent proliferation of non-small cell lung cancer cells.Indeed, hARD1 siRNA showed antiproliferative activities in lung cancer cells.We thus propose that hARD1 be a novel target for cancer therapy.

Figure 2 .
Figure 2. hARD1 inhibition represses h-catenin/TCF activity.A, left, si-ARD1 repressed the cyclin D1 promoter activity.After transfected with 40 nmol/L of si-ARD1 or si-con, H1299 cells were cotransfected with 0.25 Ag of cycD1-Luc and 0.25 Ag of h-gal plasmids.Luciferase activities were measured using a luminometer, and h-gal assays were done to normalize transfection efficiencies.Right, hARD1 inhibition prevented the binding of h-catenin/TCF to cyclin D1 promoter.Soluble chromatin was prepared from cells transfected with 40 nmol/L siRNA and immunoprecipitated with anti-catenin, anti-TCF4, or preimmune serum (Pre-IgG ).Final DNA extractions were amplified by primers spanning cyclin D1 promoter.B, si-ARD1 reduced the transcriptional activity of h-catenin/TCF.H1299 cells were cotransfected with 0.25 Ag of TCF reporter plasmid, 0.25 Ag of h-gal plasmid, and 20 nmol/L of siRNAs.C, si-ARD1 prevented h-catenin/TCF activation by LiCl or Wnt3a.HEK293 cells were cotransfected with TCF reporter, h-gal plasmid, and siRNAs and then treated for 16 hours with 30 mmol/L LiCl or 10 AL of Wnt3a extract.D, hARD1 expression enhanced h-catenin activity.HEK293 cells were cotransfected with 0.25 Ag of TCF reporter plasmid, 0.25 Ag of h-gal plasmid, 0.1 Ag of h-catenin plasmid, and 0.2/0.6Ag of hARD1 plasmid.Columns, mean of eight experiments; bars, SD.

Figure 3 .
Figure3.hARD1 binds h-catenin and is required for h-catenin acetylation.A, association of hARD1 and h-catenin.hARD1 of H1299 cells was immunoprecipitated (IP ) with anti-ARD1 antiserum or nonimmunized rat serum (Pre-IgG ).Left, coprecipitation of h-catenin with hARD1 was analyzed by immunoblotting (IB ) using anti-catenin antibody; right, coprecipitation of hARD1 with h-catenin was also analyzed.B, association of expressed hARD1 and h-catenin.HEK293 cells were cotransfected with 1 Ag of pGFP-hARD1 and 1.5 Ag of pHA-catenin.Left, GFP-hARD1 was immunoprecipitated with anti-GFP antibody, and HA-catenin coprecipitates were analyzed using anti-HA antibody; right, HA-catenin was also immunoprecipitated, and the coprecipitation of GFP-hARD1 was identified.C, in vitro binding of hARD1 to h-catenin.Purified recombinant His-hARD1 and GST-h-catenin (or GST) peptides were coincubated, and then His-hARD1 was pulled down using glutathione beads.Input and pull-down proteins were analyzed by SDS-PAGE and Coomassie staining.D, hARD1 was required for h-catenin acetylation.After transfected with siRNAs (20 nmol/L), H1299 cells were treated with 10 mmol/L sodium butyrate to prevent protein deacetylation.Top, after cell lysates were immunoprecipitated with h-catenin antibody, acetylated and total (acetylated plus unacetylated) h-catenins were identified using acetyl-lysine (Ac-Lys ) and h-catenin antibodies, respectively; bottom, this immunoprecipitation was cross-checked by changing the sequence of antibody treatment.The heavy chain of IgG (H.Chain ) was used as a loading control.

Figure 4 .
Figure 4. hARD1 acetylates h-catenin independently of p300.A, hARD1 acetylated h-catenin in cell extracts.HEK293 cells were transfected separately with 1 Ag pARD1 or 1 Ag pHA-catenin.Lysates (200 ng protein) containing HA-catenin were incubated with lysates (500, 250, or 100 ng protein) containing expressed hARD1 at 30jC for 2 hours and then precipitated with h-catenin antibody.Total and acetylated h-catenins were identified by immunoblotting with h-catenin and acetyl-lysine antibodies, respectively.Bottom, the amounts of hARD1 in the reaction mixture were analyzed by immunoblotting with hARD1 antibody.B, hARD1 directly acetylated h-catenin.Top, GST-h-catenin was incubated with His-hARD1 and acetyl-CoA at 37jC or 4jC (as a negative control to verify enzymatic reaction), and then the lysine acetylation was identified using anti-acetyl-lysine antibody; bottom, Coomassie staining showed proteins present in reaction mixtures.C, p300 was not required for hARD1 binding to h-catenin.HEK293 cells were transfected with 40 nmol/L of si-p300 or si-con.hARD1 or p300 was immunoprecipitated with anti-ARD1 or anti-p300 antiserum, and coprecipitation of h-catenin was analyzed using anti-catenin antibody.D, hARD1 and p300 both were responsible for h-catenin acetylation.After transfected with hARD1 or/and p300 siRNAs (20 nmol/L each), H1299 cells were treated with 10 mmol/L sodium butyrate.Cell lysates were immunoprecipitated with anti-catenin antibody and immunoblotted with anti-acetyl-lysine or anti-catenin antibody.Protein band intensities were quantified using ImageJ 1.36b image analysis software (NIH, Bethesda, MD).Bottom, ratios of acetylated form to total h-catenin and the percentages of h-catenin acetylation inhibited by siRNAs.