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1.
FIGURE 1.

FIGURE 1. From: p38γ MAPK Cooperates with c-Jun in trans-Activating Matrix Metalloproteinase 9.

p38γ requires both phosphorylation and the C terminus to stimulate invasion. A–C, p38γ but not its AGF mutant, stimulates invasion and/or migration in IEC-6 cells. Cells were incubated with and without Tet for 24 h and collected for protein expression by Western blotting (A) (p-p38 antibody reacts both with p-p38α and p-38γ) and assessed for Matrigel invasion (B) and migration (C). Results of B and C are shown as relative over those without Tet from at least three separate experiments (*, p < 0.05; error bars, S.D.). D and E, p38γ increases invasion in IEC-6/K-Ras cells. Cells were transfected and selected, followed by retroviral infection and a second antibiotic selection, and were then analyzed by Western blotting (D) and Matrigel invasion (E) (*, p < 0.05 versus vector control; ** p < 0.05 versus p38γ). GADPH, glyceraldehyde-3-phosphate dehydrogenase.

Mathew Loesch, et al. J Biol Chem. 2010 May 14;285(20):15149-15158.
2.
FIGURE 4.

FIGURE 4. From: p38γ MAPK Cooperates with c-Jun in trans-Activating Matrix Metalloproteinase 9.

p38γ stimulates c-Jun expression through a complex formation with c-Jun and plays an important role in c-Jun/MMP9 expression. A and B, p38γ increases c-Jun promoter activity and RNA expression by phosphorylation and C terminus-dependent mechanisms. IEC-6 cells were transiently transfected with indicated constructs, and luciferase activity was analyzed 48 h later (A). To measure c-Jun RNA expression, p38γ stably expressed IEC-6/K-Ras cells were subjected to qRT-PCR analysis, and the obtained signals were normalized to β-actin (B) (*, p < 0.05 for p38γ versus vector for both A and B; error bars, S.D.). C–E, p38γ plays a role in endogenous c-Jun and MMP9 expression. Immortalized p38γ+/+ and p38γ−/− cells were analyzed for protein expression by Western blotting and for c-Jun/MMP9 RNA expression by qRT-PCR (C). To reexpress p38γ and its mutants, p38γ−/− cells were infected with retroviruses and selected with antibiotics, and resistant cells were then subjected to Western blotting (D) and qRT-PCR analyses (E) (*, p < 0.05 versus respective control for C and E).

Mathew Loesch, et al. J Biol Chem. 2010 May 14;285(20):15149-15158.
3.
FIGURE 5.

FIGURE 5. From: p38γ MAPK Cooperates with c-Jun in trans-Activating Matrix Metalloproteinase 9.

The p38γ/c-Jun/MMP9 pathway is functionally active in stimulating invasion. A and B, inhibition of MMP9 activity blocks p38γ-induced invasion. Tet-p38γ expressed cells were incubated with MMP9 inhibitors for 24 h and subjected to Matrigel invasion (A) and zymography (B) (A, *, p < 0.05 versus p38γ; **, p < 0.05 versus no Tet). Error bars, S.D. C and D, increased p38γ protein expression couples with elevated c-Jun/MMP9 expression in human colon cancer cells. Human colon cancer cells were analyzed by Western blotting for p38γ/c-Jun protein expression and by qRT-PCR for MMP9 RNA expression. Normalized MMP9 RNA levels are significantly higher in Ras-activated cells (**) than those without (*). E–H, p38γ controls endogenous c-Jun/MMP9 expression, and both p38γ and MMP9 are required for human colon cancer invasion. Human colon cancer cells were infected with lentiviral shLuc or shp38γ and subjected to Western blotting/zymography (E) and qRT-PCR (F) at 72 h and Matrigel invasion at 48 h (G). Effects of MMP9 inhibitors on invasion (H) were analyzed as in A (*, p < 0.05 versus shLuc or solvent control, F–H).

Mathew Loesch, et al. J Biol Chem. 2010 May 14;285(20):15149-15158.
4.
FIGURE 2.

FIGURE 2. From: p38γ MAPK Cooperates with c-Jun in trans-Activating Matrix Metalloproteinase 9.

p38γ stimulates MMP9 via AP-1. A–C, p38γ increases MMP9 transcription. Cells were transiently expressed with the indicated plasmids, and luciferase activities were determined 48 h later (*, p < 0.05 versus vector or no Tet control where Tet was present for total 72 h in the Tet group, 24 h before the transfection, and 48 h thereafter). D and E, p38γ activates MMP9. Cells were incubated with and without Tet for 48 h, RNAs were prepared for qRT-PCR, and levels of MMP9 and MMP2 RNAs were normalized to the β-actin and expressed as relative over no Tet control (D, *, p < 0.05 versus no Tet). To assess the MMP9 activity, Tet-on cells were changed to a serum-free medium for the last 24 h, and concentrated medium was assayed for MMP9 activity by zymography and MMP9 protein expression by WB (E, top) in which cell lysates were also analyzed for protein expression (E, bottom). F, stable transfected p38γ increases MMP9 RNA expression. RNA was prepared and subjected to qRT-PCR, and the ratio of MMP9/β-actin was expressed as a fold change over the vector alone (*, p < 0.05 versus vector; **, p < 0.05 versus p38γ).

Mathew Loesch, et al. J Biol Chem. 2010 May 14;285(20):15149-15158.
5.
FIGURE 3.

FIGURE 3. From: p38γ MAPK Cooperates with c-Jun in trans-Activating Matrix Metalloproteinase 9.

p38γ is recruited into the MMP9 promoter through interaction with c-Jun. A, p38γ depends on its phosphorylation and C terminus to bind c-Jun protein and MMP9 promoter. Stably transfected p38γs from IEC-6/K-Ras cells were isolated and precipitates subjected to PCR analyses for their activity in binding MMP9 promoter (top) with a set of cells in parallel analyzed by immunoprecipitation (IP)/Western blotting for p38γ interacting with c-Jun proteins (bottom). B and C, p38γ and c-Jun bind the MMP9 promoter through a complex formation. Tet-on p38γ IEC-6 cells were incubated with and without Tet and p38γ/c-Jun proteins isolated by their specific antibodies, and precipitates subjected to PCR (B, top), Western blotting (B, bottom) or a second IP/PCR for ChIP-re-ChIP (C). D, p38γ does not induce c-Jun/ATF2 phosphorylation or vitamin D receptor (VDR) expression in IEC-6 cells. Cells were incubated with and without a p38 inhibitor SB203580 (SB) for 24 h and then pulse-treated with a p38 activator arsenite (ARS) for 2 h, and analyzed by Western blotting (see supplemental Fig. S2A for the relationship between c-Jun expression and c-Jun phosphorylation in response to Tet addition). E, endogenous p38γ forms a complex with c-Jun on the MMP9 promoter in K-Ras-transformed IEC-6 cells. Cells were treated with SB or solvent control (Co) as previously described in these cells (), and p38γ and c-Jun proteins were isolated and assessed for their MMP9 promoter binding activity by ChIP and their complex formation by Western blotting.

Mathew Loesch, et al. J Biol Chem. 2010 May 14;285(20):15149-15158.
6.
FIGURE 6.

FIGURE 6. From: p38γ MAPK Cooperates with c-Jun in trans-Activating Matrix Metalloproteinase 9.

Role of the p38γ-MMP9 pathway in human colon cancer. A, p38γ protein expression is increased in primary human colon cancer tissues, whereas MMP9 is expressed in both benign and malignant tissues, but its luminal expression is only detectable in the malignant tissues. Please note that the brown MMP9 staining signals are detectable in the matrix of normal as well as colon cancer tissues, but significant portions of the MMP9 immunoreactivity are present inside lumens of the malignant (filled arrow) but not normal (open arrow) tissues (bottom two panels, from a separate set of specimens). H&E, hematoxylin and eosin. B, increased p38γ protein expression significantly correlates with higher levels of MMP9 expression/secretion in primary colon cancer tissues. p38γ signals in a group of invasive colon adenocarcinomas were subtracted from those of the matched normal tissues, which were compared with MMP9 intensity within the malignant lumens from the same group by the χ2 test. C, an experimental model shows that p38γ both acts as a c-Jun activator and a c-Jun cofactor in trans-activating MMP9 to stimulate colon cancer invasion. p38γ was shown to depend on its phosphorylation and C terminus to activate c-Jun by increasing its synthesis. Activated c-Jun then recruits p38γ as a cofactor into the AP-1 site of the MMP9 promoter through a complex formation, thereby stimulating MMP9 transcription and colon cancer invasion. Our model suggests that c-Jun alone, in the absence of p38γ, is not active in the MMP9 promoter binding, and therefore, p38γ acts as a critical activator and an essential cofactor for c-Jun trans-activating MMP9.

Mathew Loesch, et al. J Biol Chem. 2010 May 14;285(20):15149-15158.

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