Download Supplementary Materials (docx 48K)

Supplementary Information
p38 MAPK is required for inflammation-associated colon tumorigenesis
Ning Yin1, Xiaomei Qi1, Susan Tsai2, Yan Lu3, Zainab Basir4, Kiyok Oshima4, James P.
Thomas5, Charles R. Myers1, Gary Stoner5, Guan Chen1,6
Correspondence to [email protected]
1Department
5Medicine,
of Pharmacology and Toxicology,
and
6Zablocki
2Surgery,
3Physiology,
4Pathology,
Veterans Affairs Medical Center, Medical College of
Wisconsin, 8701 Watertown Plank Road, Milwaukee, MI 53226
This PDF file contains
Suppl. MATERIALS AND METHODS
SUPPL. REFERENCES
8 Supplementary Figures
3 Supplementary Tables
1
SUPPL. MATERIALS AND METHODS
DSS treatment, histopathology, and immunohistochemistry
The protocol for 5-day treatment with 2.0% DSS in drinking water was previously
published 1. Intestinal tissues and colon tumors were fixed in 10% neutral buffered
formalin overnight, and then paraffin-embedded and sectioned by our Histology Core.
Sections (4 μm thick) were stained with hematoxylin & eosin (H&E). Crypt damage was
blindly scored by a pathologist, and was independently confirmed. Based on the
percentage of colon tissue affected, a score was assigned as follows: 1 (1-25%), 2
(26%-50%), 3 (51%-75%) and 4 (76%-100%) as previously reported 1. PAS (periodic
acid Schiff) staining and scoring were performed as described 2. For IHC analysis,
slides were de-waxed, incubated in peroxidase blocking buffer (3% H2O2 in methanol)
and heated in citrate buffer (20 min, 95°C). After incubation with 3% bovine serum,
slides were incubated with primary antibody (Supplementary Table 3), washed, and
incubated with the peroxidase-conjugated second antibody as described 3.
Human colon cancer specimen analysis and DNA microarrays
Human colon cancer specimens were collected by the Department of Pathology,
Medical College of Wisconsin, with informed consent. IHC analysis was conducted in
accordance with Institutional Review Board (IRB) approval. The staining procedure and
score criteria were previously described 3. Staining results (intensity x percentage = 012) were examined independently by two observers including one board-certified
pathologist and a consensus score was assigned to each case 3. For DNA microarrays,
total RNA was prepared from dissected mouse colon tumor tissues and purified using
2
RNeasy Mini kit (74104, Qiagen) following the manufacturer’s protocol. Samples were
analyzed using Mouse Genome 430A 2.0 Array (Expression Analysis Inc). Gene set
enrichment analysis (GSEA) was performed to analyze the pattern of differential gene
expression between colon tumors from control p38flox/flox and p38 knockout mice. The
GSEA software is provided by the Broad Institute of MIT and Harvard University
(http://www.broad.mit.edu/gsea). Because of the small sample size, GSEA with the
gene permutation option was performed. Gene sets smaller than 15 or larger than 500
were excluded.
Isolation of intestinal epithelial cells
Intestinal epithelial cells were isolated using a rapid low-temperature method. Briefly,
the whole small intestine and colon were removed, cut longitudinally and washed with
ice-cold Hank’s balanced salt solution (HBSS). Tissues were then transferred into
chelating buffer (27 mmol/L trisodium citrate, 5 mmol/L Na 2PO4, 96 mmol/L NaCl, 8
mmol/L KH2PO4, 1.5 mmol/L KCl, 0.5 mmol/L dithiothreitol, 55 mmol/L D-sorbitol, 44
mmol/L sucrose, 6 mmol/L EDTA, 5 mmol/L Ethylene glycol bis (2-aminoethylether)N,N,N',N'-tetra acetic acid) for incubation for 1 hour at 4°C. Epithelial cells were
dissociated by vigorously shaking every 15 minutes, repeating 4 times. Tissue debris
was removed using a 100 μm cell strainer and epithelial cells were collected by
centrifugation at 150 x g for 10 min at 4°C.
Other Assays
3
Quantitative RT-PCR (qRT-PCR)-Total RNA was extracted with Trizol Reagent
(Invitrogen), and qRT-PCR was performed using the iScriptTM One-Step RT-PCR Kit
with SYBR Green (170-8893, Bio-Rad). Samples were analyzed by ΔΔCt method for
fold changes in expression and for the ratio of target genes over Glyceraldehyde 3phosphate dehydrogenase (GAPDH) as previously described 4. All experiments were
repeated at least three times. Wnt reporter assay (TOP/FOPflash)-The TOPflash and
FOPflash luciferase vectors were gifts from Dr. H. P. Howe 5. To measure the luciferase
activity, cells were co-transfected with TOPflash (1 μg/well) or FOPflash (1μg/well)
together with different plasmids using Lipofectamine 2000 (Invitrogen). Luciferase
activity was determined 24 or 48 hours later using a dual-luciferase assay kit (Promega)
6.
In vitro kinase assay-Kinase assay for p38 activity was performed as previously
described 6. Briefly, the kinase and substrate proteins were added together in the
reaction buffer. The mixture was incubated at 30°C for 30 minutes and phosphorylated
proteins were detected with phospho-specific antibodies as indicated. Protein analysis
and stability assay-Colon tissues and tumors were immediately rinsed in cold PBS and
then frozen in liquid nitrogen. Frozen tissues were prepared for protein analysis as
described 3. Immuno-precipitation (IP) and Western Blot (WB) analysis were conducted
as previously described 3. For protein stability analysis, cycloheximide (CHX) (Sigma)
was added into cultured cells at 100g/ml. Thereafter, cells were harvested at the
indicated times for WB analysis. For WB, the band intensity was measured using
ImageQuant 5.0 software
7.
Protein ubiquitination and degradation assays were
performed in 293T cells as previously described 8.
4
REFERENCES
1
Dupaul-Chicoine J, Yeretssian G, Doiron K, Bergstrom KSB, Mcintire CR,
LeBlanc PM et al. Control of intestinal homeostasis, colitis, and colitis-associated
colorectal cancer by the inflammatory caspases Immunity 2010; 32: 367-378.
2
Otsuka M, Kang YJ, Ren J, Jiang H, Wang Y, Omata M et al. Distinct effects of
p38 deletion in myeloid lineage and gut epithelia in mouse models of
inflammatory bowel disease. Gastroenterol 2010; 138: 1255-1265.
3
Hou SW, Zhi H, Pohl N, Loesch M, Qi X, Li R et al. PTPH1 dephosphorylates
and cooperates with p38 MAPK to increases Ras oncogenesis through PDZmediated interaction. Cancer Res 2010; 70: 2901-2910.
4
Loesch M, Zhi H, Hou S, Qi X, Li R, Basir Z et al. p38 MAPK cooperates with cJun in trans-activating matrix metalloproteinase 9. J Biol Chem 2010; 285:
15149-15158.
5
Jiang Y, He X, Howe PH. Disabled-2 (Dab2) inhibits Wnt/-catenin signaling by
binding LRP6 and promoting its internalization through clathrin. EMBO J 2012;
31: 2336-2349.
6
Qi X, Zhi H, Lepp A, Wang P, Huang J, Basir Z et al. p38 mitogen-activated
protein kinase (MAPK) confers breast cancer hormone sensitivity by switching
estrogen receptor (ER) signaling from classical to nonclassical pathway via
stimulating ER phosphorylation and c-Jun transcription. J Biol Chem 2012; 287:
14681-14691.
7
Hou S, Padmanaban S, Qi X, Leep A, Mirza S, Chen G. p38 MAPK signals
through phosphorylating its phosphatase PTPH1 in regulating Ras oncogenesis
and stress response. J Biol Chem 2012; 287: 27895-27905.
8
Qi X, Pohl NM, Loesch M, Hou S, Li R, Qin JZ et al. p38  antagonizes p38
activity through c-Jun-dependent ubiquitin-proteasome pathways in regulating
Ras transformation and stress response J Biol Chem 2007; 282: 31398-31408.
5
Supplementary Figure S1. Effects of IEC-specific p38 knockout on PAS staining and
colonic epithelial cell proliferation and effects of 1-day DSS treatment on p38 MAPK
activities. (a) Specific deletion of p38 in p38fl/fl + villin-Cre {p38 knockout (KO)} mice.
Isolated intestinal epithelial cells from p38fl/fl and p38KO mice were analyzed by
Western blot (WB). (b) PCR of genomic DNA with appropriate primers for the loxPflanked p38 alleles in the heterozygous, homozygous and wild-type mice with loxPflanked p38 alleles. (c) p38 protein expression in different tissues of p38 KO mice
was analyzed by WB. (d) IHC for p38 and H&E staining of small intestine and colon
sections from p38fl/fl and p38 KO mice. (e-g) IHC for Ki-67 and PAS staining of colon
tissues from the control p38fl/fl and p38KO mice. Representative images are shown in
(e) and the numbers of PAS-positive cells (f) and Ki-67-positive cells (g) per gland in the
p38fl/fl and p38 KO mice are shown (mean ± SD, n = 4 mice, * p < 0.05, ** p < 0.005
for f and g). (h) The levels of phosphorylated and total p38 and p38 in colon epithelial
cells from indicated mice in response to 1-day treatment with DSS (6 mice each). Colon
epithelial cells were isolated and lysates were prepared and examined for p38/
expression by direct WB (input) and for p38/ phosphorylation by p-p38 IP and
Western Blotting with an isoform-specific antibody. The bar graph (mean + SD, n = 6
mice per group) shows fold-differences of each p38 (from input) and p-p38 (from IP) in
DSS animals relative to water controls {measured with Image Quant 5.0 software).
Scale bars for (d) and (e), 100 μm.
Supplementary Figure S2. p38 is required for pro-inflammatory cytokine expression
and protects colon tissue from damage in response to 5-day treatment with DSS. (a).
6
IHC staining for TNF and IL-6 was performed on colon sections derived from p38fl/fl and
p38 KO mice on day 5 after treatment with 2% DSS water or water (H2O). (b) H&E
staining of colon sections on day 5 after treatment with 2% DSS or H2O. (c)
Histopathology scores indicate more crypt damage in colon tissues of p38fl/fl mice than
in p38 KO mice on day 5 after 2% DSS (mean + SD, n = 3 mice, * p < 0.05). Scale bar
= 100 M.
Supplementary Figure S3. p38 knockout attenuates colitis symptoms in mice. (a-c)
Mice were i.p. injected with AOM and then subjected to 3 cycles of DSS (Figure 2a) one
week later. Stools were monitored for nonvisible bleeding using the ColoScreen kit
(Helena Laboratories, a, right: 0, negative, 1 positive). Visible bleeding was scored as 2
(moderate, a, middle) or 3 (severe, a, left). Typical images for scoring bleeding are
shown in (a), and the combined scores at the indicated times for each group are
presented in (b) (mean + SD, for p38fl/fl mice, n = 13; for p38 KO mice, n=12, * p <
0.05; *** P < 0.001). Percent change (relative to week 1) in body weight during the colon
cancer induction is shown in c (mean ± SD, * p < 0.05, n = 10 mice per group). (d)
Levels of phosphorylated and total p38 and p38 in mouse colon tumors from
indicated mice (5 mice each). Mouse colon tumors were isolated and lysates were
prepared and examined for p38/ expression by direct WB (input) and for p38/
phosphorylation by p-p38 IP and Western Blotting with an isoform-specific antibody. (e,
f) MEF cells were plated for colony formation in soft agar. The data are relative colony
numbers (mean + SD, n = 3, * P < 0.05, see Figure 3f for p38 protein expression).
7
Supplementary Figure S4. Pirfenidone inhibits p38 activities and -catenin expression
in AOM/DSS-induced colon tumors from p38fl/fl but not from p38 KO mice. (a) PTPH1
and p-PTPH1 protein levels were measured by WB using protein lysates from a group
of mouse colon tumors of p38fl/fl mice. (b) The same amount of protein lysates (1 mg)
from each colon tumor of p38fl/fl mice (4 separate mice per group as indicated) were
subjected to Hsp90 IP and precipitates were examined for HSP90 phosphorylation
using a p-S/TP antibody with a portion of lysates analyzed by direct WB (input). (c)
Tumor lysates from a group of p38 KO mice (5 mice per group, from Supplementary
Figure 5) were analyzed for protein expression and phosphorylation, with the sample
from p38fl/fl mouse as a positive control for p38. (d) Levels of β-catenin protein
expression in colon tumors from indicated mice treated with PFD or water (mean of 4
p38γfl/fl mice and 5 p38γ KO mice, + SD, * P < 0.05). The bar graph shows folddifferences of β-catenin (normalized with β-actin) relative to water controls.
Supplementary Figure S5. Treatment with PFD failed to show substantial effects on
AOM/DSS-induced tumorigenesis and pro-inflammatory cytokine expression in IECspecific p38knockout mice. (a) Photograph of colon tumors of p38 KO mice treated
with or without PFD (with the same protocol in Figure 4a) at the end of experiment (Day
63), Scale bar = 1 cm. (b) Representative photomicrographs of colonic tumors showing
IHC staining for TNF, IL-6, p-PTPH1, and -catenin. Scale bar = 100 M. (c) Tumor
number and tumor size in p38 KO mice treated with AOM/DSS + PFD or water (mean +
SD, n = 6 per group). (d, e) Expression levels of TNF (d) and IL-6 (e) in indicated
colonic tumors as determined by qRT-PCR. Data are relative RNA levels in control p38
8
KO mice treated with PFD versus water (mean ± SD, n = 3 mice).
Supplementary Figure S6. p38promotes Wnt transcription in mouse colon tumors
and human cells. (a) Relative RNA levels of representative Wnt target genes in mouse
colonic tumors (the level in p38 KO mice was assigned a value of 1.0) as determined
by qRT-PCR (mean + SD, n = 3 mice, * P < 0.05). (b) Transient transfection with
p38but not p38 significantly increases the Wnt reporter activity. TOPflash/FOPflash
ratio was determined and calculated from luciferase activities in 293T and SW480 cells
expressed with the indicated constructs (mean + SD, n = 3, * P < 0.05)
Supplementary Figure 7. S605 phosphorylation suppresses -catenin ubiquitination
and degradation. Flag-tagged -catenin and its mutant S605A were co-transfected with
CA-p38 or pcDNA3 into 293T cells for 24 hours. Cells were incubated for another 24 h
with and without the last 6 h-treatment with MG132 (10 M) before analyzed by Flag IP
and WB.
Supplementary Figure 8. -catenin/S605 is important for p38-stimulation of Wnt
transcription and for colon cancer growth. (a) TOPflash or FOPflash luciferase reporter
was co-transfected with the indicated constructs in 293T cells for 24 h. Luciferase
activity was determined and their ratio was calculated (mean + SD, n = 3, * P < 0.05)
(b) SW480 cells were stably transfected with -catenin and its S605A mutant by
antibiotic selection and resistant cells were analyzed for colony formation and protein
expression (the bar graph, mean + SD, n = 3, * p < 0.05), with WB results and
9
representative images of colonies shown at right. 
Supplemental Table 1. Pathways down-regulated by IEC-specific p38 knockout in
mouse colon tumors as compared to tumors from the control (only those that were
statistically significantly down-regulated were shown).
Supplemental Table 2. Wnt target genes down-regulated in IEC-specific p38 knockout
mouse colon tumors as compared to tumors from the control mouse (only those that
were statistically significantly down-regulated were shown).
Supplemental Table 3. Information about antibodies and primers used.
10