Volume 123, Issue 3, Pages 764-779 (September 2002) JunD stabilization results in inhibition of normal intestinal epithelial cell growth through P21 after polyamine depletion  Li Li, *,‡, Lan Liu, *,‡, Jaladanki N. Rao, *,‡, Ali Esmaili, *, Eric D. Strauch, *, Barbara L. Bass, *,‡, Jian–ying Wang, *,‡,§  Gastroenterology  Volume 123, Issue 3, Pages 764-779 (September 2002) DOI: 10.1053/gast.2002.35386 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Changes in junD gene expression and growth of IEC-6 cells in control cultures, cultures in which ornithine decarboxylase activity was inhibited with 5 mmol/L DFMO, and cultures inhibited with DFMO and supplemented with 5 μmol/L spermidine (SPD) for 6 days. (A) Levels of junD mRNA and protein. (A [a]) Representative autoradiograms of Northern blots. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum, and medium was changed every second day. Total cellular RNA was isolated and examined by Northern blotting analysis using a junD cDNA probe. Hybridization to the labeled GAPDH probe served as a marker for equal loading of the lanes. (A [b]) Representative autoradiograms of Western blots from cells described in A [a]. Whole cell lysates were harvested, applied to each lane (20 μg), and subjected to electrophoresis on 7.5% acrylamide gel and Western immunoblotting. The observed JunD protein molecular mass is ~40 kilodaltons, and the position is indicated by JunD. (A [c]) Quantitative analysis of Northern or Western blots by densitometry from A [a] and A [b]. Values are means ± SE of data from 3 separate experiments; relative levels of mRNA and protein for JunD were corrected for RNA and protein loading as measured by densitometry of GAPDH and actin, respectively. *P < 0.05 compared with controls. (B) Growth of cells described in A [a]. (B[a]) Cell numbers. Values are means ± SE of data from 6 dishes. *P < 0.05 compared with controls. (B [b]) Percentages of cells arrested in G1 phase. Cells were harvested and stained with propidium iodide and analyzed by flow cytometry. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with controls. Gastroenterology 2002 123, 764-779DOI: (10.1053/gast.2002.35386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Rate of junD gene transcription in response to polyamine depletion in IEC-6 cells. (A) Representative autoradiograms of nuclear run-on transcription assays. Cells were grown in control cultures and in cultures containing either DFMO (5 mmol/L) or DFMO plus 5 μmol/L spermidine (SPD) for 4 and 6 days. Nuclei were isolated, and the rate of transcription of the junD gene was measured by nuclear run-on transcription analysis. Conditions for nuclear run-on assay are described in Materials and Methods. Equal amounts of [α-32P] uridine triphosphate–labeled RNA (5 × 106 counts/min) were hybridized to filter containing immobilized plasmids of junD, GAPDH, and control pCRII. (B) Quantitative analysis derived from densitometric scans of nuclear run-on transcription analysis of junD from cells described in A. Relative transcription rates were normalized as measured by densitometry of GAPDH, and values are means ± SE of data from 3 separate experiments. Gastroenterology 2002 123, 764-779DOI: (10.1053/gast.2002.35386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Cytoplasmic half-life studies of JunD mRNA from IEC-6 cells described in Figure 1. (A) Representative autoradiograms of Northern blots. Cells were (a) untreated or treated (b) with DFMO alone or (c) DFMO plus spermidine (SPD) for 6 days and incubated further with 5 μmol/L actinomycin D per milliliter for indicated times. Total cellular RNA was isolated, and JunD mRNA levels were assayed by Northern blotting analysis using a junD cDNA probe. Loading of RNA was monitored by hybridization to labeled GAPDH probe. (B) Percent of JunD mRNA remaining in IEC-6 cells described in A. Values are means ± SE of data from 3 separate experiments, and relative levels of JunD mRNA were corrected for RNA loading as measured by densitometry of GAPDH. Gastroenterology 2002 123, 764-779DOI: (10.1053/gast.2002.35386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Rapid effect of exogenous spermidine on JunD mRNA expression in normal and polyamine-deficient IEC-6 cells. (A) Representative autoradiograms of Northern blots. Cells were initially grown in the (a) absence or (b) presence of 5 mmol/L DFMO for 6 days and then exposed to 5 μmol/L spermidine for indicated times. Total cellular RNA was isolated, and JunD mRNA levels were measured by Northern blot analysis by using junD cDNA probe. Loading of RNA was monitored by hybridization to labeled GAPDH probe. (B) Quantitative analysis of autoradiograms by densitometry from cells described in A. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with cells treated without spermidine (0 hours). Gastroenterology 2002 123, 764-779DOI: (10.1053/gast.2002.35386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of antisense JunD oligodeoxyribonucleotides on cell growth and JunD protein expression in polyamine-deficient cells. (A) Cell numbers. Cells were initially treated with 5 mmol/L DFMO for 4 days and then exposed to different concentrations of JunD antisense oligomers in the presence of DFMO and 0.5 μmol/L putrescine. JunD sense oligomers served as a control. Assays were performed 48 hours after treatment with JunD antisense or sense oligomers. Values are means ± SE of data from 6 dishes. +P < 0.05 compared with cells exposed to control sense. (B) Changes in JunD and JunB proteins in cells described in A. (B [a]) Representative autoradiograms of Western blots. After cells were treated with DFMO for 4 days, they were exposed to 4 μmol/L sense or antisense oligomers for 48 hours. Levels of JunD and JunB were measured by Western immunoblotting analysis by using a specific antibody against JunD or JunB. (B [b]) Quantitative analysis derived from densitometric scans of Western blots of JunD protein from cells described in B [a]. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with controls; +P < 0.05 compared with cells treated with DFMO plus sense oligomers. Gastroenterology 2002 123, 764-779DOI: (10.1053/gast.2002.35386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 JunD protein distribution as assessed by immunohistochemical staining in cells described in Figure 5. (A) Control; (B) cells treated with DFMO alone; (C) cells treated with DFMO for 4 days and then exposed to 4 μmol/L JunD sense oligomers for 48 hours; (D) cells treated with DFMO for 4 days and then exposed to 4 μmol/L JunD antisense oligomers for 48 hours. Cells were fixed, permeabilized, and incubated with anti-JunD antibody used for Western blot analysis. Bound antibody was visualized with biotinylated second antibody and avidin-biotin complexes. (Original magnification 600×.) Gastroenterology 2002 123, 764-779DOI: (10.1053/gast.2002.35386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of polyamine depletion on expression of p21, CDK2, CDK4, cyclin D1, cyclin D2, cyclin E, cyclin A, and E2F4 in IEC-6 cells. Cells were grown in control cultures and in cultures containing either DFMO (5 mmol/L) or DFMO plus 5 μmol/L spermidine (SPD) for 4 and 6 days. Whole cell lysates from each group were subjected to electrophoresis on a 7.5% acrylamide gel and immunoblotting analysis. p21 (~21 kilodaltons), CDK2 (~34 kilodaltons), CKD4 (~34 kilodaltons), cyclin D1 (~33 kilodaltons), cyclin D2 (~34 kilodaltons), cyclin E (~50 kilodaltons), cyclin A (~40 kilodaltons), and E2F4 (~55 kilodaltons) proteins were identified by probing nitrocellulose with the specific antibodies. After the blot was stripped, actin (~45 kilodaltons) immunoblotting was performed as an internal control for equal loading. Three separate experiments were performed that showed similar results. Gastroenterology 2002 123, 764-779DOI: (10.1053/gast.2002.35386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Transcriptional activation of the p21 gene in IEC-6 cells described in Figure 7. (A) Northern analysis of p21 mRNA levels in cells treated with either DFMO (5 mmol/L) alone or DFMO plus spermidine (SPD) (5 μmol/L) for 4 and 6 days. Total cellular RNA was isolated, and p21 mRNA levels were assayed by using a p21 cDNA probe. Loading of RNA was monitored by hybridization to labeled GAPDH probe. Three separate experiments were performed that showed similar results. (B) p21-promoter activity. (B [a]) Structure of the p21-promoter luciferase (Luc). The 3' boundary is 16 base pairs downstream of the p21 transcription initiation site, fused to the Luc reporter gene. S1 and S2 indicate the relative locations of the 2 p53 DNA-binding sites within the 2.3-kilobase regulatory region upstream of the p21 gene. (B [b]) Luciferase activity. Cells were grown in the media containing either DFMO alone or DFMO plus SPD for 4 days and then transfected using the p21-promoter luciferase reporters by lipofectamine technique. After 48 hours of incubation, transfected cells were harvested and assayed for luciferase activity. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with controls. Gastroenterology 2002 123, 764-779DOI: (10.1053/gast.2002.35386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 Effect of inhibition of JunD expression by using antisense JunD oligodeoxyribonucleotides on p21 expression and promoter activity in polyamine-deficient IEC-6 cells. (A) JunD and p21 protein levels. (A [a]) Representative autoradiograms of Western blots. Cells were initially treated with 5 mmol/L DFMO for 4 days and then exposed to 4 μmol/L JunD antisense oligomers in the presence of DFMO and 0.5 μmol/L putrescine. JunD sense oligomers served as a control. JunD and p21 protein levels were measured 48 hours after treatment with JunD antisense or sense oligomers by Western immunoblotting analysis. (A [b]) Quantitative analysis derived from densitometric scans of Western blots of JunD and p21 proteins from cells described in A [a]. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with controls; +P < 0.05 compared with cells treated with JunD sense oligomers. (B) p21-promoter activity from cells described in A [a]. After cells are grown in the presence of DFMO for 4 days, they were transfected using the p21-promoter luciferase reporters. JunD antisense or sense oligomers (4 μmol/L) with 0.5 μmol/L putrescine were given immediately after the transfection. The activity of p21-promoter luciferase reporter was measured 48 hours later. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with controls; a+P < 0.05 compared with cells treated with sense oligomers. Gastroenterology 2002 123, 764-779DOI: (10.1053/gast.2002.35386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 10 Effect of overexpression of the junD gene on p21-promoter activity and cell growth in Caco-2 cells. (A [a]) Structure of expression vector. The complete open reading frame of human junD cDNA was amplified by PCR, sequenced, and then cloned to an expression vector pcDNA3.1(+). PMCV, human cytomegalovirus immediate-early promoter. (A [b]) Representative autoradiograms of Western blot for JunD protein. Caco-2 cells were grown in minimum essential medium containing 5% dialyzed fetal bovine serum and transfected using the expression vector containing human junD cDNA (JunD) by lipofectamine technique. JunD protein levels were measured 24 and 48 hours after the transfection. (A [c]) Quantitative analysis of Western blots by densitometry from A [b]. Values are means ± SE of data from 3 separate experiments, and relative levels of JunD protein were corrected for protein loading as measured by densitometry of actin. *P < 0.05 compared with cells transfected with the vector lacking junD cDNA (null). (B [a]) p21-promoter activity. Cells were cotransfected with the JunD expression vector and the p21-promoter luciferase reporter, and the activity of p21 promoter was measured 48 hours after transfection. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with cells transfected with the null. (B [b]) Cell numbers. After cells were grown in standard minimum essential medium for 24 hours, they were transfected with either the expression vector or null. Assays were performed at various times after the transfection. Values are means ± SE of data from 6 dishes. *P < 0.05 compared with cells transfected with the null. Gastroenterology 2002 123, 764-779DOI: (10.1053/gast.2002.35386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 11 Schematic diagram showing the involvement of induced JunD/AP-1 transcription factor in the process of inhibition of normal intestinal epithelial cell proliferation following polyamine depletion. Decreased cellular polyamine levels, by either inhibition of their synthesis, stimulation of the catabolism, or suppression of polyamine uptake, stabilize JunD mRNA and induce JunD protein expression, leading to an increase in nuclear JunD-JunD homodimers. This polyamine depletion-induced JunD/AP-1 activity transactivates p21-promoter activity and enhances expression of the p21 gene. The resultant increase in p21 suppresses cyclin E (CycE) dependent-kinase 2 (CDK2) and cyclin D (CycD) dependent-kinase 4 (CDK4) activities, blocks the G1 to S phase transition during the cell cycle, and inhibits proliferation of normal intestinal epithelial cells. Gastroenterology 2002 123, 764-779DOI: (10.1053/gast.2002.35386) Copyright © 2002 American Gastroenterological Association Terms and Conditions