1. Volume 124, Issue 7, Pages 1855-1865 (June 2003)
IL-1α-induced COX-2 expression in human intestinal myofibroblasts is dependent on a PKCζ-ROS pathway1  
John F Di Mari, Randy C Mifflin, Patrick A Adegboyega, Jamal I Saada, Don W Powell 
Gastroenterology 
Volume 124, Issue 7, Pages (June 2003)
DOI: /S (03)

2. Figure 1
BIS, but not CC, inhibits IL-1α-mediated COX-2 expression. Western blot analysis was performed using a COX-2-specific antibody on protein (10 μg/lane) 24 hours after treatment in the absence (−) or presence (+) of 500 pg/mL IL-1α. (A) 18Co, a neonatal colonic primary isolate. (B) Adult primary colonic IMF isolates, CMF-3 and CMF-5. The effect of PKC inhibition was examined by pretreating cells with 10 μmol/L BIS or 0.5 μmol/L CC for 1 hour before IL-1α addition. Blots were reprobed with an α smooth muscle actin antibody to ensure equal loading. BIS was found to inhibit IL-1-induced COX-2 expression, whereas CC did not.
Gastroenterology  , DOI: ( /S (03) )

3. Figure 2
IL-1α activates all classes of PKC whereas BIS and CC inhibit with different specificities. (A) IPK analysis was performed on 50 μg of protein isolated from 18Co cells 5 minutes after IL-1α treatment (IL-1) and from control cells (C) using antibodies specific for PKCα, δ, or ζ, and kinase activity was measured by the level of histone H1 phosphorylation. All of these classes were activated by IL-1α. (B) The effect of a 1-hour pretreatment with 10 μmol/L BIS and 0.5 μmol/L CC on IL-1α-induced PKC activation was examined by IPK analysis 5 minutes after the addition of IL-1α as described. Relative amounts of protein compared with control for each kinase assay was performed as described in the Materials and Methods section and all sample protein concentrations were within ± 20% of control. BIS inhibited all 3 classes, whereas CC inhibited α and δ, but not ζ.
Gastroenterology  , DOI: ( /S (03) )

4. Figure 3
PKCζ inhibition prevents IL-1α-induced COX-2 expression. (A) IPK analysis showing the time course of IL-1α-mediated PKCζ kinase activation. Immunoprecipitation was performed using a PKCζ-specific antibody and kinase activity was measured as described in the Materials and Methods section. Protein was isolated from control cells (c) and at 5, 10, 20, and 30 minutes after IL-1α addition. (B) Western blot analysis was performed using the PKCζ antibody on protein isolated from control and IL-1α-treated cells 5 minutes after IL-1α addition. Molecular size markers are indicated on the left in kilodaltons. The 76-kilodalton PKCζ band was present, whereas the 69-kilodalton PKCι band was not detected. (C) IPK analysis of protein isolated 5 minutes after treatment in the presence or absence of IL-1α and the PKCζ inhibitor (PKCζI). Kinase activity was measured as in A. The myristoylated PKCζ inhibitor markedly inhibited PKCζ activity. (D) Western blot analysis was performed as described for COX-2 expression on 10 μg/lane of protein isolated from 18Co cells 24 hours after treatment in the presence or absence of IL-1α and 25 μmol/L of the myristoylated PKCζ inhibitor as indicated. Blots were reprobed with an αsmooth muscle antibody to ensure equal loading. The PKCζ inhibitor completely blocked IL-1-induced COX-2 expression.
Gastroenterology  , DOI: ( /S (03) )

5. Figure 4
PKCζ gene ablation by siRNA inhibits IL-1α-mediated COX-2 expression. 18Co were transfected with 10, 50, or 100 nmol/L of PKCζ300 or PKCζ412 siRNA and then treated with 500 pg/mL of IL-1α as described. Western blot analysis was then performed and the blots probed for COX-2 and PKCζ expression. The amount of siRNA used for transfection is indicated. Sham transfections were performed using no siRNA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) controls (GAP) were performed using 10 nmol/L of a control GAPDH siRNA. The PKCζ siRNA inhibited COX-2 expression in a dose-dependent manner.
Gastroenterology  , DOI: ( /S (03) )

6. Figure 5
IL-1α treatment results in PKCζ intranuclear translocation. (A) Control and IL-1α-treated 18Co were subjected to immunofluorescence analysis using confocal microscopy as described. Nuclei were stained with propidium iodide (PI) (red) and PKCζ was detected using a polyclonal anti PKCζ primary and an Alexa Fluor 647 labeled secondary antibody (green). The time of treatment (0, 5, and 20 minutes) after IL-1 addition is indicated on the left and the fluorescence pattern for PI, PKCζ, or both are shown as indicated. Note the translocation of PKCζ from the cytosol to the nucleus after IL-1α addition. (B) Orthogonal cut of 18Co showing nuclear localization of PKCζ. A z-series was performed on cells 20 minutes after IL-1 addition, 3-dimensional reconstruction was performed, and orthogonal plane cuts were made showing PKCζ intranuclear localization after IL-1 treatment (colocalization of red and green in both planes).
Gastroenterology  , DOI: ( /S (03) )

7. Figure 6
Antioxidant treatment inhibits IL-1α-induced COX-2 expression. (A) 18Co cells were pretreated with 400 μmol/L BHA, 20 μmol/L DPI, or 200 μmol/L PDTC for 1 hour as described in the Materials and Methods section. Protein was then isolated 24 hours after IL-1α addition where indicated and subjected to Western blot analysis. Blots were probed with a COX-2-specific antibody and reprobed with an α smooth muscle actin antibody. All of these antioxidants inhibited COX-2 expression. (B) Northern blot analysis was performed on RNA isolated from control and IL-1α-treated cells in the presence and absence of 20 μmol/L DPI. RNA was isolated 4 hours after IL-1α addition and the blot was probed using a probe specific for COX-2 mRNA. DPI inhibited IL-1α-induced COX-2 mRNA expression. (C) DPI was chosen as a representative antioxidant to determine if ROS inhibition prevented PKCζ activation by IL-1α. IPK analysis was performed and PKCζ activity was measured as described from 50 μg of protein isolated 5 minutes after IL-1α addition after a 1-hour pretreatment in the presence or absence of 20 μmol/L DPI. No effect of DPI on PKCζ activity was noted.
Gastroenterology  , DOI: ( /S (03) )

8. Figure 7
Measurement of ROS generation by single-cell fluorescence. 18Co cells were loaded with 50 μmol/L DCF for 5 minutes, washed, and intracellular ROS levels were measured by single-cell fluorescence for 30 minutes. Cells were treated as follows: (A) IL-1α alone; (B) 10 μmol/L BIS ± IL-1α; (C) 0.5 μmol/L CC ± IL-1α; (D) 25 μmol/L of the PKCζ inhibitor (PKCζI) ± IL-1α; (E) 400 μmol/L BHA ± IL-1α. Relative fluorescence is shown over time as compared with fluorescence before IL-1α addition.
Gastroenterology  , DOI: ( /S (03) )

9. Figure 8
Absolute cellular ROS levels and distribution are PKCζ dependent. (A) Total ROS levels from control cells and cells treated for 15 minutes with IL-1α in the presence or absence of CC, BIS, and the PKCζ inhibitor (PKCζI). The average single-cell fluorescence is given in arbitrary units. (B) Fluorescence micrographs of representative cells from the experiments described earlier. An analysis of variance was performed on samples taken from 3 separate experiments with a minimum of 4 cells measured per field. The difference between means was evaluated by using a Duncan multiple comparison test that showed a statistical difference between the IL-1α and control groups and between the CC + IL-1α and IL-1α groups (P < 0.05). No significant difference was seen between the BIS + IL-1α, PKCζI + IL-1α, and the control cells. Note that absolute ROS levels exceeded basal level with IL-1α alone and IL-1α in the presence of CC. The myristoylated PKCζ inhibitor not only inhibited absolute increases in ROS, but also caused a different staining pattern within the nucleus.
Gastroenterology  , DOI: ( /S (03) )

10. Figure 9
Antioxidants and PKCζ inhibitors have no effect on NF-κB transcriptional activity. 18Co were infected with an adenoviral luciferase reporter vector for NF-κB transcriptional activity. Cells were pretreated with the indicated inhibitors for 1 hour and then treated with IL-1 (500 pg/mL) for 6 hours. Infected cells treated with the specific NF-κB inhibitor triptolide (TRIP) were included as controls. Relative luciferase activity is shown in arbitrary units. Although statistical differences were seen within each group with the addition of IL-1 (P < 0.01), no statistically significant difference was seen between the IL-1-treated groups. Neither antioxidant (BHA, DPI, PDTC) nor PKC inhibition (BIS and CC) inhibited NF-κB translocation or activity, but a specific NF-κB inhibitor (TRIP) did.
Gastroenterology  , DOI: ( /S (03) )

11. Figure 10
Possible mechanism by which PKCζ-ROS regulates IL-1α-induced COX-2 expression. Binding of IL-1α to its receptor leads to the activation of PKCζ and the MAP kinase kinase kinases (MAPKKKs). The MAPKKKs result in both activation of the MAPKs (JNK, ERK, and p38) as well as NF-κB through phosphorylation of IKB. PKCζ activation results in ROS generation. Once activated, all of these kinases migrate into the nucleus and can act on the COX-2 promoter that contains NF-κB, and other cis-acting elements (i.e., Jun/ATF). Although the PKCζ-ROS pathway does not affect NF-κB translocation or activity, it is possible that PKCζ-ROS can affect accessibility of one or both COX-2 NF-κB sites via modulation of histone acetyl transferase (HAT) activity or histone phosphorylation (p-H3). Alternatively, PKCζ-ROS may modulate the transcriptional activation of factors bound to other cis-acting elements in the COX-2 promoter.
Gastroenterology  , DOI: ( /S (03) )