Transcript Slide 1

TEMPORAL AND DOSE-DEPENDENT AHR-MEDIATED GENE EXPRESSION
ANALYSIS IN HUMAN LIVER ADULT STEM CELLS
1,2,3
Kim ,
1,2
Dere ,
1,2,3
Burgoon ,
2,4
Chang
1,2,3
Zacharewski .
S
E
LD
CC
& TR
1Department of Biochemistry & Molecular Biology, 2National Food Safety & Toxicology Center,
3Center for Integrative Toxicology, 4Department of Pediatrics & Human Development,
Michigan State University, East Lansing, MI, 48824
ABSTRACT
FUNCTIONAL CATEGORIZATION OF PUTATIVE PRIMARY
RESPONSE GENES
TCDD INDUCES CYP1A1 mRNA IN HL1-1 CELLS
Time course and dose response studies with the human liver cell line, HL1-1, which possesses stem cell
characteristics, were conducted to assess gene expression responses elicited by 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD). For the dose response study, cells were treated for with 0.001, 0.01, 0.1, 1, 10 or 100 nM
TCDD or DMSO vehicle control for 12 hrs. The time course study involved treating cells with 10 nM TCDD or
vehicle for 1, 2, 4, 8, 12, 24 or 48 hrs. TCDD-elicited changes in gene expression were monitored using
human cDNA microarrays consisting of 7,844 genes. Empirical Bayes analysis identified 251 genes that were
differentially expressed at one or more time points. Most of these genes also exhibited dose-dependent
responses. Dose- and time- dependent induction of CYP1B1, ALDH1A3 and SLC7A5 gene expressions were
confirmed by QRT-PCR. Comparisons of the HL1-1 temporal responses with human HepG2 and mouse
Hepa1c1c7 hepatoma cell lines identified 75 genes and 18 orthologs common to HL1-1 cells, respectively.
Further comparison of temporal gene expression in HL1-1 with hepatic tissue from immature ovariectomized
C57BL/6 mice treated with 30 mg/kg TCDD identified 32 commonly regulated orthologous genes, primarily
associated with signal transduction and transcriptional regulation. Metabolism and transport related genes
were also commonly regulated between all comparisons. This comparative analysis further demonstrates the
species- and model-specific AhR-mediated gene expression elicited by TCDD.
A
B
4 hr CHX Study (79 active genes)
TCDD is the prototypical ligand of the aryl hydrocarbon receptor (AhR) and an ubiquitous and
bioaccumulative environmental contaminant that causes various adverse and biological effects in animals
and humans including endocrine, immuno- and hepato- toxicity, teratogenesis, and multi-site tumor
promotion. Various in vivo and in vitro model systems have been utilized to investigate the molecular
networks and mechanisms responsible for the diverse toxic responses.
An in vitro model system derived from human target tissue is generally expected to be more predictive of
human toxicity. Human stem cells provide an attractive in vitro alternative, which may more closely mimic
human responses, and be a potentially unlimited source of human cells.
Toxicogenomic analysis provides extensive global differential gene expression profile information, that
can be used to more accurately predict a compound's toxicity and support quantitative risk assessments. In
addition, 'omics' technologies may facilitate the development of predictive toxicity screening assays as well
as more comprehensive computational modeling. Application of toxicogenomics to human liver stem cells
will provide expression profiles that can be used to investigate species-specific responses and the
identification putative biomarkers.
Primary
response
Secondary
response
Unclassifi
able
Primary
response
Secondary
response
Unclassifi
able
48
20
11
53
95
55
48
53
26
79 putative primary
response genes
31
22
4hr
12hr
Table 1: Functional categorization of putative primary response genes elicited by TCDD (Part of whole table)
Functional
Category a
MICROARRAY DATA: TIME COURSE
Figure 4
Number of genes exhibiting significant
expression changes in the TCDD time course study.
The number of active genes showed steady increase
between 1 and 8 hr, followed by a decrease at 12 hr but
further increases at 24 and 48 hr.
Entrez
GeneId
Metabolizing
Enzymes
1543 CYP1A1d
1545 CYP1B1
224 ALDH3A2
220 ALDH1A3
BC023019
AA448157
AA633569
AA465614
cytochrome P450, family 1, subfamily A, polypeptide 1
cytochrome P450, family 1, subfamily B, polypeptide 1
aldehyde dehydrogenase 3 family, member A2
aldehyde dehydrogenase 1 family, member A3
13
7
6
3
Cell growth/
Differentiation
23189 KANK1
5069 PAPPA
113130 CDCA5
5270 SERPINE2
1490 CTGF
AA464605
R02529
W00895
N57754
AA598794
KN motif and ankyrin repeat domains 1
pregnancy-associated plasma protein A, pappalysin 1
cell division cycle associated 5
serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2
connective tissue growth factor
6
3
2
0
2
2.2
2.5
8.6
2.9
-2.3
2.1
2.4
14.8
2.6
-2.2
1.0
1.6
1.4
1.6
1.0
1.6
1.7
2.8
2.0
-1.7
1.7
5.8
33.3
7.1
-1.9
-1.3
1.7
2.1
1.9
1.7
crystallin, lambda 1
phospholipase D family, member 3
7
6
2.4
1.6
2.7
2.9
1.0
1.6
2.2
1.1
10.1
2.7
1.0
-1.1
Gene Name
 Comparatively evaluate the model-specific and model-conserved gene expression responses elicited by
TCDD
51084 CRYL1
23646 PLD3
H99932
H15746
Apoptosis
79370 BCL2L14
317 APAF1
AA620708 BCL2-like 14 (apoptosis facilitator)
N51014
apoptotic peptidase activating factor
9
1
2.1
-1.2
2.5 1.2
-1.2 -1.1
1.8
-1.7
5.0
-2.2
1.3
-1.7
Regulation of
transcription
83463 MXD3
10522 DEAF1
22938 SNW1
10062 NR1H3
2002 ELK1
22936 ELL2
9792 SERTAD2
10370 CITED2
H95253
AA425806
H17512
H61726
AA844141
AA284232
AA489839
AA115076
MAX dimerization protein 3
deformed epidermal autoregulatory factor 1 (Drosophila)
SNW domain containing 1
nuclear receptor subfamily 1, group H, member 3
ELK1, member of ETS oncogene family
elongation factor, RNA polymerase II, 2
SERTA domain containing 2
Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2
13
13
8
8
7
4
1
3
2.4
1.2
2.0
2.4
2.5
1.4
1.6
-1.9
2.8 -1.1
1.1 1.0
1.6 1.1
1.8 1.2
4.9 2.0
-1.1 -1.4
6.7 2.8
-1.7 -1.1
2.1
1.7
2.6
3.3
2.4
2.1
2.0
1.4
9.2
1.9
2.8
3.7
9.5
1.5
8.7
2.3
1.0
1.3
1.4
1.8
2.8
-1.3
4.4
1.0
Transport
29066
6583
6566
6513
55937
23788
23788
AA885491
N39791
AA043133
H58872
N74679
AA463946
AA463946
zinc finger CCCH-type containing 7A
solute carrier family 22 (organic cation transporter), member 4
solute carrier family 16 (monocarboxylic acid transporters), member 1
solute carrier family 2 (facilitated glucose transporter), member 1
apolipoprotein M
mitochondrial carrier homolog 2 (C. elegans)
mitochondrial carrier homolog 2 (C. elegans)
13
13
11
9
8
4
4
1.6
2.2
2.4
3.3
1.2
2.9
-1.6
1.6
4.1
2.2
6.5
1.2
5.0
-1.6
1.0
1.9
1.1
1.4
1.0
1.0
1.1
1.3
2.2
2.0
1.7
2.0
1.6
-1.4
1.7
7.5
4.5
8.2
1.1
5.8
-1.7
-1.1
3.2
1.5
1.0
-1.2
-1.1
1.2
5
9
•
•
1.6
-1.6
•
•
3.3
-1.3
•
•
2.6
1.0
•
•
1.8
-1.8
•
•
9.4
-2.0
•
•
5.3
-1.4
•
•
CLCN3
SLC22A4
SLC16A1
SLC2A1
APOM
MTCH2
MTCH2
1965 EIF2S1
1983 EIF5
•
•
•
•
W60015
eukaryotic translation initiation factor 2, subunit 1 alpha, 35kDa
AA669443 eukaryotic translation initiation factor 5
•
•
•
•
1hr
2hr
4hr
8hr
12hr
HL1-1 vs. HepG2
HL1-1 HepG2
1057
• 55 genes were regulated in the same direction 251
- 13 putative primary responsive genes
were included
177 74 983
• 19 genes were divergently regulated
- No putative primary responsive gene
- Signal transduction, development related genes
24hr 48hr
Figure 5 Hierarchical clustering of 273 active features expression profiles by gene and time in the TCDD time course study. Three
time-dependent clusters were formed; early (2 and 4hrs), middle (8 and 12hrs), and late (24 and 48hrs). The early and middle time
points show significant differences in their expression pattern, suggesting the characteristic differences in expression profiles
between primary and secondary response genes.
EXPERIMENTAL DESIGN
A. TIME COURSE DESIGN
4V
8V
CHX*
-1
12T 24T 48T
V6
V1
V5
V2
V4
V3
1nM
0.1nM
T :TCDD
V :DMSO vehicle
CHX
+T
24
Figure 8: Comparative analysis of temporal gene
expression profiles among human liver stem cell (HL11), human hepatoma cell line (HepG2), mouse
hepatoma cell line (Hepa1c1c7) and hepatic tissue of
C57BL/6 mice (Mm liver) studies. 251 active genes
were identified from HL1-1 time course study, and
1,057, 770 and 1,465 active genes were identified from
HepG2, Hepa1c1c7 and mouse liver, respectively. 75,
18 and 32 genes were identified as conserved between
model systems compared with HL1-1 cell study.
: Dose response study
* : CHX co-treatment study
10nM
0.01nM
CHX
8
48 (hr)
†
100nM
12V 24V 48V
T
4*
12†*
Cell Harvest
C. CHX STUDY DESIGN
V
0 1 2
CHX :cycloheximide
Cy3
Cy5
• Cell cycle progression and arrest
- CDCA5↑, CDKN1C↓, MCM3AP↓
• Matrix metalloproteinase
- MMP13↑
QRT-PCR CONFIRMATION OF MICROARRAY DATA
Dosed with TCDD or
DMSO vehicle
B. DOSE RESPONSE DESIGN Figure 2 Microarray experimental designs for
0.001nM
2V
HL1-1 vs. Mm liver
HL1-1 vs. Hepa1c1c7
Figure 1 HL1-1 TCDD time course, dose response and cycloheximide
(CHX) co-treatment study designs. Time course study: HL1-1 cells were
treated with either 10 nM TCDD or 0.1% DMSO and harvested at 1, 2, 4,
8, 12, 24, or 48 hrs post-treatment. Dose response study: HL1-1 cells
were treated with 0.001, 0.01, 0.1, 1, 10, 100 nM TCDD or 0.1 % DMSO
vehicle and harvested 12 hrs post-treatment (as indicated †). CHX cotreatment study: 10 mg/mL CHX was treated 1 hr precedent to 10 nM
TCDD or 0.1% DMSO treatment. Each combination of treatment group
was harvested at 4 and 12 hrs post-treatment (as indicated *). N=3 for
each study.
1V
506.7 4135.9 28.0 222.2 26794.4 512.9
9.0
9.0 1.3 11.1
14.9
2.9
10.0
8.3 -1.2
5.5
15.1 -1.4
8.6
7.5 -1.1
5.3
12.0 -1.3
Lipid metabolism
 Determine and verify primary response genes via cycloheximide (CHX) co-treatment studies
8T
12 hr Fold
Changec
T/V T+C/V C/V
MODEL COMPARISON OF CONSERVED EXPRESSION RESPONSES
 Comprehensively assess TCDD induced temporal and dose dependent changes in gene expression
4T
4 hr Fold
Changec
T/V T+C/V C/V
GenBank
Accession
a. Funtional categories was performed using an in-house Gene Ontology tool
b. DRE identified in -10kb to transcriptional start site (TSS) and 5' UTR
c. Expression fold changes determined by microarray analysis and numbers in colored font indicate |Fold change| > 1.5
d. Gene expressiion data was measured by QRT-PCR
 Assess TCDD responsiveness of human adult liver stem cells with CYP1A1 quantitative real-time PCR
(QRT-PCR)
2T
DRE
Count b
Gene
Symbol
Regulation of
translation
•
•
OBJECTIVES
1T
Figure 7: Identification of putative primary TCDD responsive
genes from CHX co-treatment study. Microarray analysis
identified 79 and 203 TCDD-responsive genes at 4 and 12 hr,
respectively. CHX co-treatment analysis resulted 48 and 53
genes classified as putative primary responsive genes, 20 and
95 genes were classified to putative secondary responsive
genes, and 11 and 55 genes were unclassifiable at 4 and 12 hr,
respectively. The Venn diagram illustrates that 79 of the putative
primary responsive genes were identified in 4 and 12 hour time
points CHX co-treatment studies.
Primary response
Figure 3 QRT-PCR verification of CYP1A1 gene expression levels from the dose response (A) and time course (B) studies
in HL1-1 cells treated with TCDD. Based on CYP1A1 induction levels, the EC50 for CYP1A1 expression was 8.30 nM. Error
bars represent the SEM for the average fold change. * p < 0.05.
INTRODUCTION
12 hr CHX Study (203 active genes)
(A) time course, (B) dose response and (C)
cycloheximide
co-treatment
study.
(A)
Temporal gene expression changes were
analyzed using an independent reference
design that results in two independent
labeling of each sample. Numbers indicate
time of cell harvesting (hours), T indicates
TCDD treatment and V indicates DMSO
vehicle treatment. (B) Dose response gene
expression changes were analyzed using a
spoke design. Each dose treatment sample
was compared with independent vehicle
control. (C) Cycloheximide co-treatment gene
expression changes were analyzed using a
2x2 factorial design. This array design allows
for multiple comparisons to identify significant
changes in gene expression between
treatments. Each arrow represents a single
microarray where arrow heads represent Cy5labeled samples and tails represent Cy3labeled samples and double headed arrows
indicate dye swap labeled on different arrays.
HL1-1
251
233
Hepa1c1c7
770
18
• Transcription factors
• Signal transductions
• Lipid metabolism
HL1-1
251
752
219
Mm Liver
1465
32
1433
Divergent responses
• ID3 : Mm liver↑ / HL1-1 & Hepa1c1c7↓
• SLC12A7 : HL1-1↑ / Hepa1c1c7 & Mm liver↓
CONCLUSIONS
 TCDD responsiveness of HL1-1 cell was confirmed with CYP1A1 mRNA expression induction by QRTPCR
 TCDD-elicited temporal and dose response microarray analysis has identified the conserved classical
responses in accordance with literature data
 Gene expression changes verified by QRT-PCR correlate well with the microarray data
 Comparative studies across models and species confirmed that some toxicity-related, conserved and
model- specific responses were observed in HL1-1 cells
Figure 6 Quantitative real-time PCR (QRT-PCR) verification of microarray results in time course and dose response studies. Three
putative primary TCDD-responsive genes (CYP1B1, ALDH1A3 and SLC7A5) were examined for microarray result verification. Fold
changes were calculated relative to time-matched vehicle controls. Bar (left axis) and lines (right axis) represent QRT-PCR and cDNA
microarray data, respectively. Results are represented as the average of three biological replicates. Error bars for QRT-PCR data
represent the SEM for the average fold change. *p < 0.05 for QRT-PCR.
 The limited differential gene expression conservation between human and mouse model systems
provides further evidence for species-specific AhR regulons.
Email: [email protected]
Supported by NIGMS R21 GM075838
Web: www.bch.msu.edu/~zacharet/