Aryl Hydrocarbon Receptor Activation by TCDD Modulates ... · using the free software application, FiberFit, which uses image processing techniques to analyze two-dimensional imagesoffibernetworks[35].ResultsindicatethatTCDD

Research ArticleAryl Hydrocarbon Receptor Activation by TCDD ModulatesExpression of Extracellular Matrix Remodeling Genes duringExperimental Liver Fibrosis

Cheri L Lamb1 Giovan N Cholico1 Daniel E Perkins2 Michael T Fewkes2

Julia Thom Oxford23 Trevor J Lujan4 Erica E Morrill4 and Kristen A Mitchell12

1Biomolecular Sciences PhD Program Boise State University Boise ID 83725 USA2Department of Biological Sciences Boise State University Boise ID 83725 USA3Biomolecular Research Center Boise State University Boise ID 83725 USA4Department of Mechanical and Biomedical Engineering Boise State University Boise ID 83725 USA

Correspondence should be addressed to Kristen A Mitchell kristenmitchellboisestateedu

Received 7 May 2016 Revised 26 July 2016 Accepted 4 August 2016

Academic Editor Kim Bridle

Copyright copy 2016 Cheri L Lamb et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aryl hydrocarbon receptor (AhR) is a soluble ligand-activated transcription factor that mediates the toxicity of 2378-tetrachlorodibenzo-p-dioxin (TCDD) Increasing evidence implicates the AhR in regulating extracellular matrix (ECM)homeostasis We recently reported that TCDD increased necroinflammation and myofibroblast activation during liver injuryelicited by carbon tetrachloride (CCl

4) However TCDD did not increase collagen deposition or exacerbate fibrosis in CCl

4-treated

mice which raises the possibility that TCDD may enhance ECM turnover The goal of this study was to determine how TCDDimpacts ECMremodeling gene expression in the liverMale C57BL6micewere treated for 8weekswith 05mLkgCCl

4 andTCDD

(20120583gkg) was administered during the last two weeks Results indicate that TCDD increased mRNA levels of procollagen types IIII IV and VI and the collagen processing molecules HSP47 and lysyl oxidase TCDD also increased gelatinase activity andmRNAlevels of matrix metalloproteinase- (MMP-) 3 MMP-8 MMP-9 andMMP-13 Furthermore TCDDmodulated expression of genesin the plasminogen activatorplasmin system which regulates MMP activation and it also increased TIMP1 gene expressionThesefindings support the notion that AhR activation by TCDD dysregulates ECM remodeling gene expression and may facilitate ECMmetabolism despite increased liver injury

1 Introduction

The aryl hydrocarbon receptor (AhR) is a soluble pro-tein in the basic helix-loop-helix PerARNTSim family oftranscriptional regulators that contribute to developmentalprocesses adaptation to environmental stress and xenobioticmetabolism [1ndash3] The AhR mediates the toxicity associ-ated with exposure to 2378-tetrachlorodibenzo-p-dioxin(TCDD) which is an environmental contaminant and high-affinity ligand for this receptor [4] After ligand binding theAhR translocates from the cytoplasm to the nucleus whereit forms a heterodimer with the AhR nuclear translocatorprotein (ARNT) The AhRARNT complex binds to DNAat xenobiotic response elements (XREs) and modulates gene

transcription A growing body of evidence indicates thatthe AhR also interacts with other coregulatory proteins inaddition to ARNT and can modulate the expression ofgenes that do not contain XREs [5] which underscoresthe increasing complexity of AhR-mediated gene regulationSuchAhR-dependent changes in gene expression are believedto underlie most of the toxic responses to TCDD In theabsence of TCDD endogenous AhR activation is implicatedin regulating the expression of genes important for a numberof developmental and physiological processes [6 7]

Emerging evidence implicates a role for AhR signalingin the deposition and metabolism of extracellular matrix(ECM) components The ECM is comprised of a network ofproteins such as collagens which are deposited in interstitial

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 5309328 14 pageshttpdxdoiorg10115520165309328

2 BioMed Research International

spaces and providemechanical and structural support to cells[8] The ECM also regulates various cellular processes suchas survival migration proliferation and differentiation bymodulating tissue stiffness communicating with the intra-cellular cytoskeleton and sequestering and releasing growthfactors [9] AhR activation by TCDD has been shown tomodulate the expression of ECM proteins such as collagenand fibronectin [10ndash14] Expression of matrix metallopro-teinases (MMPs) which are responsible for the degradationof ECM components also appears to be targeted by TCDDFor example in vitro TCDD treatment was found to increaseMMP expression in human keratinocytes prostate cancercells and melanoma cells [15ndash17] Insight into the effect ofTCDD on ECM maintenance and remodeling also stemsfrom studies in a zebrafish regeneration model in whichamputation of the caudal (tail) fin initiates epimorphic regen-eration accompanied by a wound healing response Usingthis model Andreasen et al reported that TCDD treatmentincreased the expression of MMP-9 and MMP-13 [18] Inaddition exposure to TCDD induced a localized fibrosisin the regenerating fin where collagen accumulated as anunorganized fibrotic deposit at the basement membraneIn a separate study gene expression analysis revealed thatthe largest numbers of genes impacted by TCDD duringfin regeneration were those involved in ECM remodelingand structure [10] Collectively these reports support thenotion that TCDD dysregulates ECM homeostasis and thismost likely occurs through a mechanism that includes AhR-mediated changes in gene expression

Disruptions of ECM metabolism and deposition areknown to impact the development of liver disease [19 20]Liver fibrosis is a pathological condition characterized bythe deposition of excessive or abnormal ECM componentsincluding collagen type I [19] In the liver collagen issynthesized by myofibroblast precursors namely hepaticstellate cells (HSCs) Upon liver injury HSCs transition fromquiescent vitamin A-rich cells into activated myofibroblastscharacterized by increased proliferation contractility andsynthesis of collagen type I [21] One well-established modelsystem to investigate HSC activation and ECM modulationis experimental liver fibrosis induced by chronic carbontetrachloride (CCl

4) administration In the liver CCl

4is

metabolized by cytochrome P4502E1 to a trichloromethylradical that elicits membrane damage through lipid perox-idation [22] Chronic treatment of mice with CCl

4causes

widespread centrilobular necrosis and inflammation whichdrive HSC activation and the development of fibrosis [23]

We recently found that exposure to TCDD increased liverdamage and HSC activation in mice treated with CCl

4for 8

weeks [24] However TCDD did not increase the depositionof collagen or the severity of liver fibrosis in CCl

4-treated

mice despite increased expression of genes encoding collagentype I and the potent profibrogenic mediator transforminggrowth factor-1205731 (TGF-1205731) Results further indicated thatTCDD increased collagenase activity in the liver of CCl

4-

treated mice Increased breakdown of ECM in CCl4TCDD-

treated mice could explain why collagen deposition andfibrosis development were not exacerbated despite increasesin other endpoints of fibrogenesis

Collagen biosynthesis begins with the transcription ofprocollagen genes and is facilitated by various intercellularand extracellularmolecules [25] For example heat shock pro-tein-47 (HSP47) is required for proper triple helical fold-ing and trafficking of procollagen within the endoplasmicreticulum [26]Anothermolecule decorin regulates collagenfibrillogenesis [27ndash29] Lysyl oxidase (LOX) catalyzes cross-linking of collagen fibers which marks the last step incollagen biosynthesis [30]

Collagen breakdown is achieved through the activityof numerous MMPs MMP expression is regulated at thetranscriptional level and these proteins are synthesizedas inactive zymogens called proMMPs [31] MMP activityis regulated by enzymatic inhibition and activation Forexample endogenous tissue inhibitors of metalloproteinases(TIMPs) inhibit MMP activity Numerous mechanisms acti-vate MMPs including the plasminogen activatorplasminsystem [20] Plasmin is produced through the cleavageof plasminogen by tissue plasminogen activator (tPA) andurokinase plasminogen activator (uPA) and this pathwayis suppressed by plasminogen activator inhibitor-1 (PAI-1)Plasmin can directly convert proMMPs into enzymaticallyactive MMPs and some of these active MMPs can furtheractivate other proMMPs [32] MMP activity is central tothe resolution of fibrosis and scar-associated macrophageshave been identified as an abundant cellular source of theseenzymes in the fibrotic liver [33 34]

The goal of the present study was to determine howTCDD treatment impacts the expression of genes related toECM synthesis deposition and breakdown during chronicliver injury induced by CCl

4administration We measured

gene expression related to collagen synthesis processingand cross-linking and assessed the impact of TCDD onthe organization and dispersion of fibrillar collagens in theinjured liver Expression of MMPs and the molecules thatactivate or inhibit themwere alsomeasured to determine howTCDDmodulates ECM turnover

2 Materials and Methods

21 Animals Male C57BL6 mice (8ndash10 weeks old CharlesRiver Wilmington MA) were injected ip with 05mLkgCCl4(Sigma-Aldrich St Louis MO) diluted in corn oil or

with corn oil alone (Ctrl) twice a week for 8 weeks Duringthe last two weeks of the experiment mice were treated byoral gavage once weekly with 20120583gkg TCDD (CambridgeIsotope Laboratories Andover MA) diluted in peanut oilor with peanut oil vehicle alone (Veh) At the end of theexperiment animals were euthanized and liver was eitherflash-frozen in liquid nitrogen or fixed in UltraLight ZincFormalin Fixative (PSL Equipment Vista CA) All animalexperiments were approved by the Institutional Animal Careand Use Committee at Boise State University and conductedaccording to the established policies and guidelines of thiscommittee

22 Quantitative Real-Time RT-PCR Total RNA was ex-tracted using the Omega Bio-Tek EZNA Total RNA Kit

BioMed Research International 3

(Norcross GA) from 20mg of frozen liver tissue GenomicDNA was eliminated using the Omega RNase Free DNaseSet (Norcross GA) RNA concentration and purity weremeasured by ultraviolet (UV) absorbance RNA quality andelimination of genomic DNA were assessed using an aga-rose bleach gel [36] RNA was reverse-transcribed using theApplied Biosystems High Capacity cDNA reverse transcrip-tion kit (Thermo Fisher Scientific Waltham MA) Gene-specific primers (Table 1) were used for quantitative real-timeRT-PCR (qRT-PCR)whichwas performedusing a LightCycler 96 Thermocycler and FastStart Essential DNAGreen Master Reaction Mix (Roche Indianapolis IN) Allsamples were analyzed in duplicate from three mice pertreatment group Relative quantification was estimated usingΔΔ119862119902method normalized to GAPDH [37]

23 Measurement of Collagen Fibril Organization Fixed livertissue was paraffin-embedded cut into 2 120583m sections andstained with Sirius Red as described elsewhere [38] Bire-fringence of stained liver tissues was visualized using anOlympus BX53F polarizing microscope Photographs weretaken at 600x magnification to focus on septa formation inthe damaged liver of CCl

4-treated mice Images were then

converted to 8 bit grayscale and analyzed with FiberFit soft-ware to calculate fiber dispersion (120581) [35] Ten images wereanalyzed from each mouse liver four mice were evaluatedper treatment group Septa formation was not detected in thelivers of vehicle- or TCDD-treated mice that did not receiveCCl4 and these samples were excluded from the FiberFit

analysis

24 In Situ Zymography Gelatinase activity was examinedusing in situ zymography of zinc-formalin-fixed liver tissueas described elsewhere [39 40] Briefly tissue sections (8 120583m)were heated at 58∘C for 12 hours then deparaffinized andrehydrated DQ-gelatin (Thermo Fisher Scientific) was dis-solved in reagent-grade water and diluted at 1 50 in a 50mMTris-HCl buffer containing 150mM NaCl and 5mM CaCl

2

(pH 76) Tissue sections were incubated with the DQ-gelatin solution for 12 hours at 37∘C Nuclei were stained with410158406-diamidino-2-phenylindole (DAPI) and cover slips weremounted with Permount (Fisher Scientific Pittsburgh PA)Fluorescent images were taken with an EVOS fluorescencemicroscope (Thermo Fisher Scientific) with 20x objectiveFluorescence was quantified using ImageJ software (USNational Institutes of Health) and expressed as a percentageof the area in the microscope field of view

25Western Blotting Frozen liver tissue was homogenized in50mM HEPES 150mM NaCl 10 glycerol 01 Tween 2075mM EDTA and 75mM MgCl

2lowast 6H2O Protein content

was determined using a DC Protein Assay kit (Bio-RadLaboratories Inc Hercules CA) and homogenates werediluted to 5mgmL and resuspended in SDS loading buffer(100mM Tris-Cl pH 68 4 SDS 02 bromophenol blueand 20 glycerol) containing 400mM 120573-mercaptoethanol

Table 1 qRT-PCR primers and annealing temperatures used in thisstudy

Gene Primer sequence Annealingtemp (∘C)

Col1a1 FWD GTCCCTGAAGTCAGCTGCATA 60REV TGGGACAGTCCAGTTCTTCAT

Col3a1 FWD CCTGGTGGAAAGGGTGAAAT 62REV CGTGTTCCGGGTATACCATTAG

Col4a3 FWD TCCTGGGGAAATGGGAAAGC 64REV CTGCCTACGGATGGTTCTCC

Col4a5 FWD TGCTCCTGAGAGATCGGCTT 58REV GTTATGCTGGTGCACTTGGG

Col6a1 FWD TCCCACCCACACAGAACAAC 58REV CACTGAGAGGTGTCGTGTCC

Col6a2 FWD TGACGCTGTTCTCTGACCTG 58REV TTGTGGAAGTTCTGCTCGCC

Col6a3 FWD CTGATGGCACCTCTCAGGAC 58REV GTCACTTCCAACATCGAGGC

Dcn FWD AAGGGGGCCGATAAAGTTTC 58REV CTGGGTTGAAAACCTCCTGC

Lox FWD CTGCACACACACAGGGATTG 56REV AGCTGGGGTTTACACTGACC

Mmp2 FWD ACCCAGATGTGGCCAACTAC 63REV TACTTTTAAGGCCCGAGCAA

Mmp3 FWD GTCCTCCACAGACTTGTCCC 65REV GGGAGTTCCATAGAGGGACTGA

Mmp8 FWD TACAGGGAACCCAGCACCTA 64REV GGGGTTGTCTGAAGGTCCATAG

Mmp9 FWD AAGGCAGCGTTAGCCAGAAG 63REV GCGGTACAAGTATGCCTCTGC

Mmp13FWD

GCCCTGGGAAGGAGAGACTCCAGG 55REV GGATTCCCGCAAGAGTCGCAGG

Mmp14 FWD GCCCTCTGTCCCAGATAAGC 58REV ACCATCGCTCCTTGAAGACA

Plat FWD CAGAGATGAGCCAACGCAGA 58REV TTCGCTGCAACTTCGGACAG

Plau FWD CATCCAGTCCTTGCGTGTCT 62REV CCAAGTACACTGCCACCTTCA

Plg FWD ACTCAAGGGACTTTCGGTGC 58REV TCAGATACTCGACGCGGTTG

Serpine1 FWD TTCAGCCCTTGCTTGCCTC 60REV ACACTTTACTCCGAAGTCGGT

Serpinh1 FWD GGGAACGGATCGCTCCAAA 67REV GGACCTGTGAGGGTTTACCAG

Timp1 FWD CACGGGCCGCCTAAGGAACG 60REV GGTCATCGGGCCCCAAGGGA

Timp2 FWD GCCAAAGCAGTGAGCGAGAAG 56REV CACACTGCTGAAGAGGGGGC

Timp3 FWD AAGAAAAGAGCGGCAGTCCC 60REV TTTGGCCCGGATCACGATG

Timp4 FWD TATGGTAGGTGGGCTGACTGT 64REV AGTTGAGACAGTGGGAGTAGGA

Samples (25 120583g proteinlane) were resolved on a 10 SDS-polyacrylamide gel transferred to nitrocellulose and incu-bated with the following antibodies purchased from SantaCruz Biotech (Dallas TX) anti-actin (sc-1615) anti-uPa (sc-59727) or anti-tPA (sc-5239) Blots were then incubatedwith HRP-conjugated secondary antibodies and bands were


visualized with Pierce ECL Western Blotting Substrate(Thermo Scientific)

26 Immunohistochemistry Liver tissue was fixed in Ultra-Light Zinc Formalin Fixative (PSL Equipment Vista CA)paraffin-embedded and cut into 2 120583m sections Tissue sec-tions were incubated overnight at 4∘C with an anti-F480antibody (MCA497R AbD Serotec Raleigh NC) and thenstained with 33-diaminobenzidine (DAB) using a commer-cially available kit (RampD SystemsMinneapolis MN) Tissueswere counterstained with hematoxylin Images were takenwith anOlympus BX53 compoundmicroscope at 10x and 20xmagnifications

27 Statistical Analysis Statistical analyses were performedusing Prism (version 6 GraphPad Software La Jolla Ca)With the exception of data in Figure 3 all data were evaluatedby two-way analysis of variance followed by Bonferronirsquospost hoc testing to evaluate significance among all possiblepairwise comparisons in the four treatment groups Statisticalsignificance between pairwise comparisons is indicated withletters above the bar for each treatment group If two groupsshare the same letter then the difference between the meansis not statistically significant at 119901 lt 005 If two meanshave different letters then they are significantly different fromeach other at 119901 lt 005 For the analysis of collagen fiberorganization in Figure 3 an unpaired two-tailed Studentrsquos119905-test was used and data were also considered significantlydifferent at 119901 lt 005

3 Results

31 Consequences of TCDD Treatment on Procollagen mRNALevels in CCl

4-Treated Mice To determine how TCDD treat-

ment impacts procollagen synthesis during chronic liverinjury we measured the mRNA levels of genes that encodeprocollagen type I and III (fibrillar collagens) and typesIV and VI (nonfibrillar collagens) Chronic CCl

4treatment

significantly increased Col1a1 Col3a1 and Col4a5 in themouse liver (Figure 1) Administration of TCDD to CCl

4-

treatedmice further increased expression ofCol1a1 comparedto mice that received CCl

4alone The combination of TCDD

and CCl4markedly increased transcript levels of Col6a1

Col6a2 and Col6a3 compared to mice that did not receiveCCl4 Finally TCDD treatment elevatedCol4a3mRNA levels

in mice that were not treated with CCl4 but this increase was

not observed in mice that received both TCDD and CCl4

Collectively these findings support a general trend in whichexposure to TCDD increases procollagen gene expression inthe liver of CCl

4-treated mice Moreover TCDD impacts the

expression of procollagen isoforms that encode both fibrillarand nonfibrillar types of collagens

32 TCDD Modulates mRNA Levels of Collagen ProcessingMolecules in CCl

4-Treated Mice Collagen synthesis requires

not only expression of procollagen genes but also processingof procollagen assembly of fibrils and cross-linking of fibers

Col1a1 Col3a1 Col4a3 Col4a5 Col6a1 Col6a2 Col6a3

0

2

4

6

8

CtrlVehCtrlTCDD

A

B

B

C

AAB

B

B B

A AA

ABB B

AAB

ABA

B

ABABA

B

B

AA

A

CCl4TCDDCCl4Veh

mRN

AΔΔCq

Figure 1 Consequences of TCDD treatment on collagen mRNAlevels in the liver of CCl

4-treated mice Collagen mRNA expression

was measured by qRT-PCR and normalized to GAPDH Datarepresent mean (plusmnSEM) of three mice per treatment group Withinthe data set for each gene all possible pairwise comparisons weremeasured Means that do not share a letter are significantly differentfrom each other (119901 lt 005) whereas means that share a letter arenot

Serpinh1 Dcn Lox

minus2

0

2

4

6

8

A

AA

B

BABAA

B

AA

A

CtrlVehCtrlTCDD CCl4TCDD

CCl4Veh

mRN

AΔΔCq

Figure 2 TCDD treatment alters expression of collagen processingmolecules in the liver of CCl

4-treated mice Transcript levels of

Serpinh1 (HSP47) Dcn (decorin) and Lox (lysyl oxidase) weremeasured by qRT-PCR and normalized to GAPDH Data representmean (plusmnSEM) of three mice per treatment group Within the dataset for each gene all possible pairwise comparisons were measuredMeans that do not share a letter are significantly different from eachother (119901 lt 005) whereas means that share a letter are not

To identify how TCDD impacts these events during CCl4-

induced liver injury wemeasured transcript levels of Serpinh1(HSP47) Lox (LOX) and Dcn (decorin) HSP47 is requiredfor proper folding and trafficking of procollagen whereasdecorin and lysyl oxidase contribute to fibril assembly andfiber cross-linking in the ECM [26 41] When TCDD wasadministered to CCl

4-treated mice Serpinh1 and LoxmRNA

levels increased 4- to 6-fold compared to mice treatedwith CCl

4alone (Figure 2) In contrast Dcn mRNA levels


CCl4TCDDCCl4Veh

(a)

CCl4 + TCDDCCl4 + Veh

4

3

2

1

0

Mea

n fib

er d

isper

sion

(k)

(b)

Figure 3 Exposure to TCDD does not impact collagen fiber organization in the liver of CCl4-treated mice (a) Polarized microscopy

facilitates the visualization of collagen fiber birefringence in liver tissue stained with Sirius Red (600x magnification) Photomicrographsdepict representative fibers in septa of liver from a mouse treated with CCl

4and peanut oil vehicle (left) or with CCl

4and TCDD (right)

Scale bars represent 10120583m (b) Collagen network organization was evaluated by analyzing Sirius Red-stained liver tissues with the FiberFitsoftware application [35] Ten photomicrographs were evaluated per mouse four mice were analyzed in each treatment group Data representmean (plusmnSEM) fiber dispersion 119896 (greater 119896 values = increase in fiber alignment) No statistically significant changes were found betweentreatment groups (119901 = 036 based on unpaired two-tailed Studentrsquos 119905-test)

were significantly decreased in CCl4TCDD-treated mice

These results support the notion that TCDD modulates theexpression of genes encoding collagen-processing moleculesduring chronic liver injury

33 TCDD Treatment Does Not Affect Collagen Fiber Organi-zation in the Liver of CCl

4-TreatedMice Theobservation that

TCDD altered the expression of ECM processing moleculesin CCl

4-treated mice led us to speculate that it would subse-

quently impact the fibrillar collagen network To test this col-lagen fibers were visualized in liver tissue stained with SiriusRed which aligns with fibrillar collagens and enhances theirbirefringence under cross-polarized light [42 43] Polarizedmicroscopy of stained tissue revealed the presence of thickstrongly birefringent yellow fibers in the septa of livers from

CCl4-treated mice (Figure 3(a)) Based on visual inspection

TCDDhadno overt impact on fiber appearanceThe effects ofTCDD on collagen fiber organization were further evaluatedusing the free software application FiberFit which usesimage processing techniques to analyze two-dimensionalimages of fiber networks [35] Results indicate that TCDDhad no effect on fiber dispersion which is a measure offiber network disorder (Figure 3(b)) Hence despite theTCDD-mediated increase in expression of genes encodingprocollagen and collagen-processing molecules TCDD didnot appear to impact collagen organization in the ECM ofCCl4-treated mice No collagen fibers were detected in either

vehicle- or TCDD-treated mice that did not receive CCl4

(data not shown) and these samples were excluded from theFiberFit analysis


Mmp8 Mmp13 Mmp2 Mmp9 Mmp3 Mmp14minus2

02468

10 Collagenases

Gelatinases

Stromelysin

Membrane-type

A

B

A

C BB

B

A AA

AA

AAB B

AB

AB

AA

B

AB ABBA


CCl4Veh

mRN

AΔΔCq

Figure 4 Effects of TCDD treatment on mRNA levels of MMPsin the liver of CCl

4-treated mice MMP mRNA expression was

measured by qRT-PCR and normalized to GAPDH Data representmean (plusmnSEM) of three mice per treatment group Within the dataset for each gene all possible pairwise comparisons were measuredMeans that do not share a letter are significantly different from eachother (119901 lt 005) whereas means that share a letter are not

34 Expression of ECM Remodeling Enzymes Is Elevated inthe Presence of TCDD ECM maintenance requires not onlythe synthesis and deposition of matrix molecules but alsotheir degradation and turnover which is regulated by theproteolytic activity of MMPs MMP expression is largelyregulated at the transcriptional level [44] To determinehow TCDD treatment impacts MMP gene expression in theliver of CCl

4-treated mice we measured transcript levels of

mouse MMPs known to be important in chronic liver injuryMmp8 and Mmp13 encode enzymes that function primarilyas collagenases and expression of these genes was markedlyincreased by TCDD regardless of CCl

4treatment (Figure 4)

Mmp2 and Mmp9 are referred to as gelatinases and theydegrade not only gelatin but also collagen type IV lamininelastin and fibronectin [44] While TCDD had no effecton Mmp2 transcript levels it increased Mmp9 expression inCCl4-treated mice Likewise the combination of TCDD and

CCl4increased Mmp14 (membrane-type MMP) expression

compared to mice treated with TCDD alone although thisincrease was modestMmp3 (stromelysin) mRNA levels weresignificantly higher in TCDD-treated mice regardless ofCCl4treatment Generally speaking these results support

the conclusion that TCDD treatment increases MMP geneexpression during CCl

4-induced liver injury

35 TCDD Increases Gelatinase Activity in the Liver of CCl4-

Treated Mice MMP activity is central to ECM remodelingand is implicated in both the promotion and attenuation ofliver injury [20] We recently found that TCDD treatmentincreased collagenase activity in the liver of CCl

4-treated

mice [24] During fibrotic liver injury collagenases cleave thenative helix of fibrillar collagens to produce gelatin whichcan be degraded by MMPs namely MMP-2 and MMP-9 [45] We used in situ zymography to measure gelatinaseactivity in the liver Whereas gelatinase activity was barelydetectable in mice treated with CCl

4Veh (Figure 5(a)) it

was significantly induced when TCDD was administered toCCl4-treated mice (Figure 5(b)) When administered alone

TCDD did not increase gelatinase activity In fact there wasno detectable gelatinase activity in either vehicle- or TCDD-treated mice in the absence of CCl

4(data not shown)

36 Consequences of TCDD Treatment on TIMP mRNAExpression in CCl

4-Treated Mice MMP activity is controlled

by enzymatic activation and inhibition [31] The activity ofMMPs can be inhibited by four homologous members of theTIMP family TIMP1 is a strong inhibitor of many MMPsbut the gelatinases MMP-2 and MMP-9 are also inhibitedby other TIMPs For example TIMP2 TIMP3 and TIMP4can inhibit MMP-2 activity and TIMP3 inhibits MMP-9[31] Analysis of TIMP gene expression revealed that TCDDtreatment increased Timp1 but had no impact on Timp2Timp3 or Timp4 regardless of CCl


Hence modulation of TIMP gene expression by TCDDappears to be limited to Timp1

37 TCDD Treatment Modulates Expression of Molecules inthe Plasminogen ActivatorPlasmin System MMP activationis regulated through numerous mechanisms including theplasminogen activatorplasmin system in which tPA anduPA mediate the conversion of plasminogen to plasminwhich directly activates numerous proMMPs [20] PAI-1 sup-presses MMP proteolytic activity by inhibiting tPAuPA andPAI-1 gene is known to be regulated by AhR activity [46ndash48]To determine how TCDD impacted this pathway of MMPactivation we measured expression of Plg (plasminogen)Plat (tPA) Plau (uPA) and Serpine1 (PAI-1) TCDD induceda modest yet statistically significant decrease in Plg mRNAlevels in CCl

4-treated mice (Figure 7(a)) Levels of Plat and

Plau expression were markedly elevated in CCl4TCDD-

treated mice A corresponding increase in the Plat-encodedprotein tPa was measured in CCl

4TCDD-treated mice

whereas no changes were detected in the expression of uPawhich is encoded by Plau (Figure 7(b)) Finally exposure toTCDD increased PAI-1 (Serpine1) gene expression regardlessof CCl

4-treatment (Figure 7(a)) Hence these observations

indicate that TCDD treatment modulated the expression ofthe plasminogen activatorplasmin system

38 TCDD Treatment Increases the Localization of HepaticMacrophages around Fibrotic Scars in the Liver of CCl

4-Treated

Mice Macrophages contribute to ECM remodeling duringboth the injury and recovery phase of CCl

4-induced liver

fibrosis [49] and are an abundant source of MMP-9 andMMP-13 [33 50] Scar-associated macrophages populate thefibrotic scar during injury and repair produce MMPs andsecrete cytokines that induce MMP production in other cells[51] Given that TCDD treatment increasedMMP expressionand activity we investigated the possibility that TCDDincreased the prevalence of hepatic macrophages aroundscar areas in the fibrotic liver As shown in Figure 8 CCl

4

administration elicited the infiltration of inflammatory cellsto the fibrotic scar and the prevalence of F480+ hepaticmacrophageswasmarkedly increased inCCl

4TCDD-treated

mice compared to CCl4Veh-treated mice


CCl4TCDDCCl4Veh

(a)

Ctrl0

1

2

3

4

5

o

f are

a with

gel

atin

ase a

ctiv

ity

VehTCDD

A

B

AA

CCl4

(b)

Figure 5 TCDD treatment increases gelatinase activity in the liver of CCl4-treated mice (a) In situ zymography of zinc-buffered formalin-

fixed liver tissue usingDQ-gelatin Green fluorescence indicates gelatinase activity nuclei were stained withDAPI Photomicrographs (100xmagnification) are representative of three mice per treatment group Scale bars represent 400 120583m (b) Quantification of gelatinase activitybased on the percentage of green fluorescence coverage per field of liver tissue Ten fields were analyzed per mouse three mice were evaluatedper treatment group Data represent mean (plusmnSEM) of three mice per treatment group All possible pairwise comparisons were measured forstatistical significance Means that do not share a letter are significantly different from each other (119901 lt 005) whereas means that share a letterare not

4 Discussion

The present study investigated the consequences of TCDDtreatment on expression of molecules involved in colla-gen biosynthesis and ECM metabolism during chronicliver injury We recently reported that exposure to TCDDincreased HSC activation and mRNA levels of TGF-1205731and collagen type I in the injured liver without increasinghepatic collagen content or exacerbating fibrosis [24] Thisled us to speculate that TCDD treatment may dysregulateECM remodeling activities including collagen synthesis orturnover

During fibrosis the collagen content in the liver canincrease up to tenfold [52] Our results indicate that TCDDtreatment alone increased Col1a1 and Col4a3 This obser-vation corroborates other reports in which exposure toTCDD increased collagen types I and IV [11ndash14 53] InCCl4TCDD-treated mice there was a marked increase in

expression of Col3a1 Col4a5 Col6a1 Col6a2 and Col6a3compared to CtrlVeh-treated mice Collagen type III isstructurally similar to collagen type I and is the first collagento increase during chronic liver disease [54] Collagen typeIV is the primary component of basement membranes andits expression increases during fibrosis [55] Collagen type VI


Timp1 Timp2 Timp3 Timp4

0

1

2

3

AA

AA

A

A

AAA

AA

A

A

B

AB

A

minus1


CCl4Veh

mRN

AΔΔCq

Figure 6 Consequences of TCDD treatment onTIMPmRNA levelsin the liver of CCl

4-treated mice TIMP mRNA expression was

measured by qRT-PCR and normalized to GAPDH Data representmean (plusmnSEM) of three mice per treatment group Within the dataset for each gene all possible pairwise comparisons were measuredfor statistical significance Means that do not share a letter aresignificantly different from each other (119901 lt 005) whereas meansthat share a letter are not

is also upregulated in liver fibrosis and has been shown tostimulate DNA synthesis and inhibit apoptotic cell death inHSCs in vitro [56] This is intriguing because we previouslyreported that exposure to TCDD increases HSC proliferationin vitro [57] and increases HSC activation markers in theliver of CCl

4-treated mice [24] It is possible that increased

expression of collagen type VI as well as other types ofcollagen contributes to the effects of TCDD in the CCl

4

model systemThe finding that certain collagen genes were upregulated

by TCDD treatment only while others were increased bythe combination of TCDD and CCl

4 implies that the AhR

may differentially regulate gene expression in the healthyand injured liver It is well established that most if notall of the biochemical and toxic effects of TCDD occurthrough the AhR [2 58] Increasing evidence supports arole for endogenous AhR signaling in regulating collagendeposition including the discovery that AhR knockout micedevelop liver fibrosis and have elevated TGF-1205731 and collagenexpression [59ndash61] In addition it was recently reported thatAhR knockdown increased Col1a1 and Col4a4 mRNA levelsin retinal pigment epithelial cells and choroidal endothelialcells [62] Collectively these findings implicate a role forAhR activity in regulating collagen gene expression Futurestudies that investigate how AhR knockdown impacts geneexpression during chronic liver injury will expand our under-standing of how the AhR regulates ECM remodeling duringstates of health and disease Furthermore the use of mice inwhich the AhR is conditionally depleted from discrete livercell populations could help identify which cells are directlytargeted by TCDD to produce ECM dysregulation

Not only did TCDD increase the expression of collagengenes but it also modulated gene expression for severalkey proteins involved in collagen synthesis For exampleadministration of TCDD to CCl

4-treated mice increased

gene expression of HSP47 which resides in the endoplasmicreticulum and is involved in the folding and shuttling ofcollagen molecules to the Golgi [63] Increased HSP47 levelsreportedly contribute to fibrosis by facilitating the exces-sive assembly and intracellular processing of procollagenmolecules leading to fibrotic lesions [64] Furthermoresuppression of HSP47 expression was reported to reducecollagen accumulation and delay fibrotic progression [65]Both procollagen and HSP47 gene expression are regulatedby TGF-1205731 [66] We previously found that TGF-1205731 geneexpression was increased in CCl

4TCDD-treated mice and

speculate that this could drive HSP47 and Col1a1 expressionin our model system However TCDD treatment was shownto suppress bothCol1a1 andHSP47 gene expression during finregeneration in zebrafish despite increased TGF-1205731 expres-sion [10 18]

Decorin is a secreted proteoglycan that has a dual rolein liver fibrosis First it functions as a naturally occurringTGF-1205731 antagonist and its genetic ablation has been shownto increase ECM deposition impair matrix degradation andincrease HSC activation [67] Second decorin facilitates thedevelopment of normal collagen morphology by binding tothe collagen triple helix and preventing the lateral fusion offibrils [28] We found that TCDD suppressed decorin geneexpression in CCl

4-treated mice Other studies demonstrate

a possible role for AhR signaling in decorin expression Forinstance decorin expression was increased in fibroblasts andvascular smooth muscle cells from AhR knockout mice [6869]

LOX mediates the cross-linking of collagen fibers whichis important for collagen organization and perhaps also forconferring resistance to proteolytic degradation by MMPs[70] Consistent with this role of LOX administration ofthe irreversible LOX inhibitor 120573-aminopropionitrile (BAPN)to CCl

4-treated mice was recently found to reduce collagen

cross-linking and produced fibrotic septa with less organizedcollagen fibers [30] Our finding that TCDD increased LOXexpression in CCl

4-treated mice could possibly be explained

as a compensatory response to increased collagen synthesisas could the TCDD-induced increase in HSP47 It is worthnoting that Andreasen et al reported that TCDD treatmentsuppressed not only LOX2 and HSP47 expression duringzebrafish fin regeneration but also prolyl-4-hydroxylase 1205721and 2 which help stabilize collagen cross-links [10] Basedon the role of these molecules in collagen processing andorganization their reduced expression may underlie theaccumulation of disorganized collagen observed in the regen-erating fin tissue [18] In contrast we found no evidence thatTCDD impacted collagen fiber organization in the liver ofCCl4-treated mice Increased expression of LOX and HSP47

as well as decreased expression of decorin could be onepossible explanation for this observation

One of the most consistently reported consequences ofTCDD treatment on ECM remodeling is increased MMPexpression [71] TCDD treatment increases the expressionand activity of MMPs in numerous and diverse cell typesincluding keratinocytes macrophages and endometrial cells[72ndash74] In the zebrafish model of fin regeneration TCDD


Plg Plat Plau Serpine1

0

5

10

AA

B

AA

A B

B

AA

B

A A

BB

A

A


CCl4Veh

mRN

AΔΔCq

minus5

(a)

0

1

2

3

uPa d

ensit

omet

ry (f

old

chan

ge)

A

A

A

A

0

1

2

3

tPa d

ensit

omet

ry (f

old

chan

ge)

B

AA

A

CtrlVeh CtrlTCDD CCl4TCDD

uPa

tPa

Actin

CCl4Veh

CtrlVeh CtrlTCDD CCl4TCDDCCl4Veh CtrlVeh CtrlTCDD CCl4TCDDCCl4Veh

(b)

Figure 7 Exposure to TCDD modulates expression of genes in the plasminogen activatorplasmin system (a) Transcript levels of Plg(plasminogen) Plat (tPA) Plau (uPA) and Serpine1 (PAI-1) were measured by qRT-PCR and normalized to GAPDH Data represent mean(plusmnSEM) of three mice per treatment group Within the data set for each gene all possible pairwise comparisons were measured for statisticalsignificance Means that do not share a letter are significantly different from each other (119901 lt 005) whereas means that share a letter arenot (b) uPa and tPa protein levels were measured byWestern blot Band densitometry was normalized to actin and expressed as fold changerelative to the CtrlVeh treatment group Means that do not share a letter are significantly different from each other (119901 lt 005)

upregulatedMMP-13 [10] Similarly TCDD increased expres-sion of MMP-13 as well as other MMPs in the fetalmouse heart [53]These reports support our observation thatTCDD increasedMmp3Mmp8Mmp9Mmp13 andMmp14genes in the mouse liver MMP-8 and MMP-13 function

primarily as collagenases and these were robustly increasedby TCDD regardless of CCl

4treatment which corroborates

our previous finding that TCDD increases collagenase activ-ity in the liver of CCl

4-treated mice [24] During ECM

breakdown MMPs with collagenase activity will partially


50120583m 50120583m


Figure 8 TCDD treatment increases localization of F480+ macrophages around fibrotic scar Immunohistochemistry was performed toidentify hepatic macrophages (F480+ cells) localized around the fibrotic scar in CCl

4-treated mice (20x magnification) Scale bars represent

50 120583m

denature collagen resulting in the production of gelatinwhich is metabolized primarily by the gelatinases MMP-2and MMP-9 [20] Decreased gelatinase activity particularlyMMP-2 is associated with increased liver fibrosis develop-ment [75] The increase in gelatinase activity we observedin CCl

4TCDD-treated mice could reflect a compensatory

response to increased collagenase activity FurthermoreTCDD also increased expression of MMP-3 (stromelysin)and MMP-14 (membrane-type) both of which reportedlyexhibit some collagenase and gelatinase activity

MMP activity is inhibited through interactions withTIMP proteins as well as other endogenous inhibitors [76]TIMP1 in particular is associated with ECM proteolysisduring fibrosis and Timp1minusminus mice display increased liverinjury inflammation and fibrosis following CCl

4treatment

[77] TIMP1 is a strong inhibitor of most MMPs exceptsome of the membrane-type MMPs However the gelatinaseMMPs are inhibited by other TIMPs as well SpecificallyTIMP1 and TIMP3 inhibit MMP-9 and TIMPs 2 3 and4 inhibit MMP-2 [78] In the CCl

4model system TCDD

treatment increased TIMP1 but had no effect on expressionof TIMPs 2 3 or 4 Thus it is possible that the expressionof TIMPs in CCl

4TCDD-treated mice was not sufficient to

counteract MMP activity Other studies have reported thatTIMP expression is modulated by in vitro and in vivo TCDDexposure as well [10 79ndash81]

Our results demonstrate that TCDD treatment producedchanges in the plasminogen activatorplasmin system thatmodulates MMP activation TCDD was found to modestlybut significantly decrease plasminogen expression in CCl

4-

treated mice Because MMPs are activated by plasmin whichis produced from plasminogen this would presumably leadto decreased MMP activation Given that TCDD increasedboth collagenase and gelatinase activity in the CCl

4model

system it is possible that the observed decrease in plas-minogen gene expression was not physiologically relevantIt is also possible that increased expression of tPA and uPAcompensated for any decrease in plasminogen expressionThe TCDD-mediated increase in uPA gene expression cor-roborates another report showing that TCDD upregulated

uPA protein in a human keratinocyte cell line [82] It isinteresting to note that this TCDD-induced increase in uPAappeared to occur through a posttranscriptional mechanismthat included changes in mRNA stability [82 83] We foundno significant increase in uPa protein expression among allfour treatment groups in our study although protein levels oftPa were markedly increased in CCl

4TCDD-treated mice

The Serpine1 gene that encodes PAI-1 is recognized as anAhR-regulated target gene It is transcriptionally induced byTCDD through a mechanism that involves heterodimeriza-tion of the AhR with the transcription factor KLF-6 and therecruitment of this complex to a nonconsensus XRE [46ndash48] We found that TCDD treatment increased PAI-1 geneexpression regardless ofCCl

4treatment andpresume that this

reflects a direct effect of TCDD through AhR-regulated geneexpression However it is also possible that increased PAI-1expression by TCDD occurs as a consequences of activationof the TGF-1205731 pathway as PAI-1 is an early TGF-1205731 activatedgene [84] and other studies have described crosstalk betweenthe AhR and TGF-1205731 signaling axes [69 85] Based on ourfinding that TCDD did not suppress collagenase or gelatinaseactivity in CCl

4-treated mice it is possible that increased

PAI-1 expression in CCl4TCDD-treated mice failed to offset

increased tPAuPA activity However MMPs can also be acti-vated through nonplasmin pathways which leaves open thepossibility that MMP activation is increased in CCl

4TCDD-

treated mice despite inhibition of the plasminogen activa-torplasmin system by PAI-1

The observation that TCDD treatment may enhancethe prevalence of scar-associated macrophages is intriguingbecause these cells are a rich source of MMPs and maycontribute to both the injury and regression phase of fibrosisinduced by CCl

4[49] We have previously found that TCDD

treatment increases liver damage and inflammation in CCl4-

treated mice [24] yet the direct cellular targets of TCDDin this model system have not been determined It is pos-sible that increased hepatocellular necrosis in CCl

4TCDD-

treated mice evokes a heightened inflammatory responseresulting in increased numbers of infiltrating neutrophils andmonocytesmacrophages However it is also conceivable that


ECM synthesis

Collagen synthesis

Collagen processing molecules

ECM metabolism

MMP expression

MMP activityuarr gelatinase activity

MMP inhibitors

Increased scar-associated macrophagesas source of MMPs

Plasminogen activatorplasmin system

uarr uPa protein

uarr Col1a1 uarr Col3a1 uarr Col6a3 mRNA levels

uarr Lox uarr Hsp47 darr Dcn mRNA levels

uarr Mmp3 uarr Mmp8 uarr Mmp9 mRNA levels

uarr Timp1 mRNA levels

darr Plg (plasminogen) uarr Plat (tpa) mRNA levels

uarr Serpine1 (PAI-1) mRNA levels

Figure 9 Summary of the consequences of TCDD treatment onECM remodeling activities and regulatory processes during CCl

4-

induced liver injury

TCDD treatment directly modulates hepatic macrophagesin CCl

4-treated mice Further investigation is warranted to

identify the cellular source of increased MMP activity anddetermine how TCDD treatment impacts the localizationof resident macrophages and infiltrating monocytes to thefibrotic scar

In conclusion results from this study demonstrate thatAhR activation by TCDD modulates ECM remodeling dur-ing chronic liver injury although the precise mechanismremains unclear At least three interrelated components ofECM homeostasis could be targeted by TCDD collagen syn-thesis ECM metabolism and regulation of enzyme activityby the plasminogen activatorplasmin system As summa-rized in Figure 9 TCDD treatment increased expressionof procollagen genes and altered expression of moleculesinvolved in collagen processing and maturation Further-more TCDD enhanced gelatinase activity increased mRNAlevels of several MMPs and increased the localization ofhepatic macrophages to the fibrotic scar Finally TCDDtreatment had multiple effects on the plasminogen activa-torplasmin system Future studies will be needed to distin-guish between TCDD-induced changes that directly impactECM remodeling and changes that occur as secondarycompensatory responses to system perturbations

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

This work was supported by Institutional DevelopmentAwards (IDeA) from the National Institute of GeneralMedical Sciences of the National Institutes of Health[P20GM103408 P20GM109095] The authors also acknowl-edge support from the Biomolecular ResearchCenter at BoiseState University with funding from the National ScienceFoundation [0619793 0923535] the MJ Murdock CharitableTrust and the Idaho State Board of Education

References

[1] T V Beischlag J L Morales B D Hollingshead and GH Perdew ldquoThe aryl hydrocarbon receptor complex and thecontrol of gene expressionrdquo Critical Reviews in Eukaryotic GeneExpression vol 18 no 3 pp 207ndash250 2008

[2] O Hankinson ldquoThe aryl hydrocarbon receptor complexrdquoAnnual Review of Pharmacology andToxicology vol 35 pp 307ndash340 1995

[3] Y-Z Gu J B Hogenesch and C A Bradfield ldquoThe PAS super-family sensors of environmental and developmental signalsrdquoAnnual Review of Pharmacology andToxicology vol 40 pp 519ndash561 2000

[4] P M Fernandez-Salguero D M Hllbert S Rudikoff JM Ward and F J Gonzalez ldquoAryl-hydrocarbon receptor-deficient mice are resistant to 2378-tetrachlorodibenzo-p-dioxin-induced toxicityrdquo Toxicology and Applied Pharmacologyvol 140 no 1 pp 173ndash179 1996

[5] D P Jackson A D Joshi and C J Elferink ldquoAh receptorpathway intricacies signaling through diverse protein partnersand DNA-motifsrdquo Toxicology Research vol 4 no 5 pp 1143ndash1158 2015

[6] K A Mitchell and C J Elferink ldquoTiming is everything con-sequences of transient and sustained AhR activityrdquo BiochemicalPharmacology vol 77 no 6 pp 947ndash956 2009

[7] R Barouki X Coumoul and P M Fernandez-Salguero ldquoThearyl hydrocarbon receptor more than a xenobiotic-interactingproteinrdquo FEBS Letters vol 581 no 19 pp 3608ndash3615 2007

[8] M A Karsdal T Manon-Jensen F Genovese et al ldquoNovelinsights into the function and dynamics of extracellularmatrix in liver fibrosisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 308 no 10 pp G807ndashG830 2015

[9] S-H Kim J Turnbull and S Guimond ldquoExtracellular matrixand cell signalling the dynamic cooperation of integrin pro-teoglycan and growth factor receptorrdquo Journal of Endocrinologyvol 209 no 2 pp 139ndash151 2011

[10] E A Andreasen L K Mathew and R L Tanguay ldquoRegener-ative growth is impacted by TCDD gene expression analysisreveals extracellular matrix modulationrdquo Toxicological Sciencesvol 92 no 1 pp 254ndash269 2006

[11] A C Aragon P G Kopf M J Campen J K Huwe and M KWalker ldquoIn utero and lactational 2378-tetrachlorodibenzo-p-dioxin exposure effects on fetal and adult cardiac gene expres-sion and adult cardiac and renal morphologyrdquo ToxicologicalSciences vol 101 no 2 pp 321ndash330 2008

[12] C Nottebrock K Riecke M Kruse M Shakibaei and RStahlmann ldquoEffects of 2378-tetrachloro-dibenzo-p-dioxin onthe extracellular matrix of the thymus in juvenile marmosets(Callithrix jacchus)rdquo Toxicology vol 226 no 2-3 pp 197ndash2072006


[13] K Riecke D Grimm M Shakibaei et al ldquoLow doses of 2378-tetrachlorodibenzo-p-dioxin increase transforming growth fac-tor 120573 and cause myocardial fibrosis in marmosets (Callithrixjacchus)rdquo Archives of Toxicology vol 76 no 5-6 pp 360ndash3662002

[14] S Pierre A Chevallier F Teixeira-Clerc et al ldquoAryl hydrocar-bon receptor-dependent induction of liver fibrosis by dioxinrdquoToxicological Sciences vol 137 no 1 pp 114ndash124 2014

[15] M Haque J Francis and I Sehgal ldquoAryl hydrocarbon exposureinduces expression of MMP-9 in human prostate cancer celllinesrdquo Cancer Letters vol 225 no 1 pp 159ndash166 2005

[16] K A Murphy C M Villano R Dorn and L A White ldquoInter-action between the aryl hydrocarbon receptor and retinoic acidpathways increases matrix metalloproteinase-1 expression inkeratinocytesrdquo Journal of Biological Chemistry vol 279 no 24pp 25284ndash25293 2004

[17] C M Villano K A Murphy A Akintobi and L A Whiteldquo2378-Tetrachlorodibenzo-p-dioxin (TCDD) induces matrixmetalloproteinase (MMP) expression and invasion in A2058melanoma cellsrdquoToxicology andApplied Pharmacology vol 210no 3 pp 212ndash224 2006

[18] E A Andreasen L K Mathew C V Lohr R Hasson and R LTanguay ldquoAryl hydrocarbon receptor activation impairs extra-cellular matrix remodeling during zebra fish fin regenerationrdquoToxicological Sciences vol 95 no 1 pp 215ndash226 2007

[19] S L Friedman ldquoMolecular regulation of hepatic fibrosis anintegrated cellular response to tissue injuryrdquo Journal of Biologi-cal Chemistry vol 275 no 4 pp 2247ndash2250 2000

[20] S Duarte J Baber T Fujii and A J Coito ldquoMatrix metallopro-teinases in liver injury repair and fibrosisrdquoMatrix Biology vol44ndash46 pp 147ndash156 2015

[21] S L Friedman ldquoHepatic stellate cells protean multifunctionaland enigmatic cells of the liverrdquo Physiological Reviews vol 88no 1 pp 125ndash172 2008

[22] F W-Y Wong W-Y Chan and S S-T Lee ldquoResistance tocarbon tetrachloride-induced hepatotoxicity inmicewhich lackCYP2E1 expressionrdquo Toxicology and Applied Pharmacology vol153 no 1 pp 109ndash118 1998

[23] I Mederacke C C Hsu J S Troeger et al ldquoFate tracing revealshepatic stellate cells as dominant contributors to liver fibrosisindependent of its aetiologyrdquo Nature Communications vol 4article 2823 2013

[24] C L Lamb G N Cholico X Pu G D Hagler K A Cor-nell and K A Mitchell 2378-Tetrachlorodibenzo-p-dioxin(TCDD) increases necroinflammation and hepatic stellate cellactivation but does not exacerbate experimental liver fibrosis inmice

[25] C Frantz K M Stewart and V M Weaver ldquoThe extracellularmatrix at a glancerdquo Journal of Cell Science vol 123 no 24 pp4195ndash4200 2010

[26] C Widmer J M Gebauer E Brunstein et al ldquoMolecu-lar basis for the action of the collagen-specific chaperoneHsp47SERPINH1 and its structure-specific client recognitionrdquoProceedings of the National Academy of Sciences vol 109 no 33pp 13243ndash13247 2012

[27] K G Danielson H Baribault D F Holmes H Graham K EKadler and R V Iozzo ldquoTargeted disruption of decorin leads toabnormal collagen fibril morphology and skin fragilityrdquo Journalof Cell Biology vol 136 no 3 pp 729ndash743 1997

[28] I T Weber R W Harrison and R V Iozzo ldquoModel structureof decorin and implications for collagen fibrillogenesisrdquo The

Journal of Biological Chemistry vol 271 no 50 pp 31767ndash317701996

[29] G Zhang Y Ezura I Chervoneva et al ldquoDecorin regulatesassembly of collagen fibrils and acquisition of biomechanicalproperties during tendon developmentrdquo Journal of CellularBiochemistry vol 98 no 6 pp 1436ndash1449 2006

[30] S B Liu N Ikenaga Z Peng et al ldquoLysyl oxidase activitycontributes to collagen stabilization during liver fibrosis pro-gression and limits spontaneous fibrosis reversal in micerdquo TheFASEB Journal vol 30 no 4 pp 1599ndash1609 2016

[31] M P Caley V L Martins and E A OrsquoToole ldquoMetallopro-teinases and wound healingrdquo Advances in Wound Care vol 4no 4 pp 225ndash234 2015

[32] H Nagase R Visse and G Murphy ldquoStructure and func-tion of matrix metalloproteinases and TIMPsrdquo CardiovascularResearch vol 69 no 3 pp 562ndash573 2006

[33] J A Fallowfield M Mizuno T J Kendall et al ldquoScar-associated macrophages are a major source of hepatic matrixmetalloproteinase-13 and facilitate the resolution of murinehepatic fibrosisrdquoThe Journal of Immunology vol 178 no 8 pp5288ndash5295 2007

[34] P Ramachandran A PellicoroMA Vernon et al ldquoDifferentialLy-6C expression identifies the recruited macrophage pheno-type which orchestrates the regression of murine liver fibrosisrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 109 no 46 pp E3186ndashE3195 2012

[35] E EMorrill A N Tulepbergenov C J Stender R LamichhaneR J Brown and T J Lujan ldquoA validated software applicationto measure fiber organization in soft tissuerdquo Biomechanics andModeling in Mechanobiology 2016

[36] P S Aranda DM Lajoie and C L Jorcyk ldquoBleach gel a simpleagarose gel for analyzing RNA qualityrdquo Electrophoresis vol 33no 2 pp 366ndash369 2012

[37] T D Schmittgen and K J Livak ldquoAnalyzing real-time PCR databy the comparative CT methodrdquo Nature Protocols vol 3 no 6pp 1101ndash1108 2008

[38] L C U Junqueira G Bignolas and R R Brentani ldquoPicrosiriusstaining plus polarization microscopy a specific method forcollagen detection in tissue sectionsrdquoTheHistochemical Journalvol 11 no 4 pp 447ndash455 1979

[39] E Hadler-Olsen P Kanapathippillai E Berg G Svineng J-OWinberg and L Uhlin-Hansen ldquoGelatin in situ zymographyon fixed paraffin-embedded tissue zinc and ethanol fixationpreserve enzyme activityrdquo Journal of Histochemistry and Cyto-chemistry vol 58 no 1 pp 29ndash39 2010

[40] P Kumar T Smith K Rahman N E Thorn and F A AnanialdquoAdiponectin agonist ADP355 attenuates CCL

4-induced liver

fibrosis in micerdquo PLoS ONE vol 9 no 10 Article ID e1104052014

[41] E G Canty and K E Kadler ldquoProcollagen trafficking process-ing and fibrillogenesisrdquo Journal of Cell Science vol 118 no 7 pp1341ndash1353 2005


[43] L Rich and PWhittaker ldquoCollagen and picrosirius red staininga polarized light assessment of fibrillar hue and spatial distribu-tionrdquo Journal of Morphological Science vol 22 no 2 pp 97ndash1042005


[44] D Rodrıguez C J Morrison and C M Overall ldquoMatrixmetalloproteinases what do they not do New substrates andbiological roles identified by murine models and proteomicsrdquoBiochimica et Biophysica ActamdashMolecular Cell Research vol1803 no 1 pp 39ndash54 2010

[45] J P Iredale AThompson and N C Henderson ldquoExtracellularmatrix degradation in liver fibrosis biochemistry and regula-tionrdquoBiochimica et BiophysicaActamdashMolecular Basis of Diseasevol 1832 no 7 pp 876ndash883 2013

[46] G M Huang and C J Elferink ldquoA novel nonconsensus xeno-biotic response element capable of mediating aryl hydrocarbonreceptor-dependent gene expressionrdquoMolecular Pharmacologyvol 81 no 3 pp 338ndash347 2012

[47] D-S Son and K K Rozman ldquo2378-tetrachlorodibenzo-p-dioxin (TCDD) induces plasminogen activator inhibitor-1through an aryl hydrocarbon receptor-mediated pathway inmouse hepatoma cell linesrdquo Archives of Toxicology vol 76 no7 pp 404ndash413 2002

[48] S R Wilson A D Joshi and C J Elferink ldquoThe tumorsuppressor kruppel-like factor 6 is a novel aryl hydrocarbonreceptor DNA binding partnerrdquo Journal of Pharmacology andExperimental Therapeutics vol 345 no 3 pp 419ndash429 2013

[49] J S Duffield S J Forbes C M Constandinou et al ldquoSelectivedepletion of macrophages reveals distinct opposing roles dur-ing liver injury and repairrdquoThe Journal of Clinical Investigationvol 115 no 1 pp 56ndash65 2005

[50] T Knittel M Mehde D Kobold B Saile C Dinter and GRamadori ldquoExpression patterns of matrix metalloproteinasesand their inhibitors in parenchymal and non-parenchymal cellsof rat liver regulation by TNF-120572 and TGF-1205731rdquo Journal ofHepatology vol 30 no 1 pp 48ndash60 1999

[51] A Pellicoro P Ramachandran and J P Iredale ldquoReversibilityof liver fibrosisrdquo Fibrogenesis amp Tissue Repair vol 5 no 1 p 12012

[52] MRojkindMAGiambrone and L Biempica ldquoCollagen typesin normal and cirrhotic liverrdquo Gastroenterology vol 76 no 4pp 710ndash719 1979

[53] E A Thackaberry Z Jiang C D Johnson K S Ramos and MK Walker ldquoToxicogenomic profile of 2378-tetrachlorodi-benzo-p-dioxin in the murine fetal heart modulation of cellcycle and extracellular matrix genesrdquo Toxicological Sciences vol88 no 1 pp 231ndash241 2005

[54] R G Wells ldquoCellular sources of extracellular matrix in hepaticfibrosisrdquo Clinics in Liver Disease vol 12 no 4 pp 759ndash7682008

[55] L Ala-Kokko T Pihlajaniemi J C Myers K I Kivirikko andE R Savolainen ldquoGene expression of type I III and IV collagensin hepatic fibrosis induced by dimethylnitrosamine in the ratrdquoBiochemical Journal vol 244 no 1 pp 75ndash79 1987

[56] K M Mak P Sehgal and C K Harris ldquoType VI collagen itsbiology and value as a biomarker of hepatic fibrosisrdquo AustinBiomarkers amp Diagnosis vol 1 no 2 article 9 2014

[57] W A Harvey K Jurgensen X Pu et al ldquoExposure to 2378-tetrachlorodibenzo-p-dioxin (TCDD) increases human hepaticstellate cell activationrdquo Toxicology vol 344ndash346 pp 26ndash332016

[58] J AWalisser E Glover K Pande A L Liss andCA BradfieldldquoAryl hydrocarbon receptor-dependent liver development andhepatotoxicity are mediated by different cell typesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 102 no 49 pp 17858ndash17863 2005

[59] JM Carvajal-Gonzalez A C RomanM I Cerezo-Guisado EM Rico-Leo GMartin-Partido and PM Fernandez-SalgueroldquoLoss of dioxin-receptor expression accelerates wound healingin vivo by amechanism involving TGF120573rdquo Journal of Cell Sciencevol 122 no 11 pp 1823ndash1833 2009

[60] P Fernandez-Salguero T Pineau D M Hilbert et al ldquoImmunesystem impairment and hepatic fibrosis in mice lacking thedioxin-binding Ah receptorrdquo Science vol 268 no 5211 pp 722ndash726 1995

[61] H Zaher P M Fernandez-Salguero J Letterio et al ldquoTheinvolvement of aryl hydrocarbon receptor in the activationof transforming growth factor-beta and apoptosisrdquo MolecularPharmacology vol 54 no 2 pp 313ndash321 1998

[62] M Choudhary D Kazmin P Hu R S Thomas D P McDon-nell andGMalek ldquoAryl hydrocarbon receptor knock-out exac-erbates choroidal neovascularization via multiple pathogenicpathwaysrdquo Journal of Pathology vol 235 no 1 pp 101ndash112 2015

[63] K Kawasaki R Ushioda S Ito K Ikeda Y Masago and KNagata ldquoDeletion of the collagen-specific molecular chaperoneHsp47 causes endoplasmic reticulum stress-mediated apoptosisof hepatic stellate cellsrdquoThe Journal of Biological Chemistry vol290 no 6 pp 3639ndash3646 2015

[64] T Taguchi and M S Razzaque ldquoThe collagen-specific molec-ular chaperone HSP47 is there a role in fibrosisrdquo Trends inMolecular Medicine vol 13 no 2 pp 45ndash53 2007

[65] M Sunamoto K Kuze H Tsuji et al ldquoAntisense oligonu-cleotides against collagen-binding stress protein HSP47 sup-press collagen accumulation in experimental glomerulonephri-tisrdquo Laboratory Investigation vol 78 no 8 pp 967ndash972 1998

[66] H Pan and J Halper ldquoRegulation of heat shock protein 47 andtype I procollagen expression in avian tendon cellsrdquo Cell andTissue Research vol 311 no 3 pp 373ndash382 2003

[67] K Baghy R V Iozzo and I Kovalszky ldquoDecorin-TGF120573 axisin hepatic fibrosis and cirrhosisrdquo Journal of Histochemistry andCytochemistry vol 60 no 4 pp 262ndash268 2012

[68] J Guo M Sartor S Karyala et al ldquoExpression of genes in theTGF-120573 signaling pathway is significantly deregulated in smoothmuscle cells from aorta of aryl hydrocarbon receptor knockoutmicerdquo Toxicology and Applied Pharmacology vol 194 no 1 pp79ndash89 2004

[69] X Chang Y Fan S Karyala et al ldquoLigand-independentregulation of transforming growth factor 1205731 expression and cellcycle progression by the aryl hydrocarbon receptorrdquoMolecularand Cellular Biology vol 27 no 17 pp 6127ndash6139 2007

[70] H M Kagan and W Li ldquoLysyl oxidase properties specificityand biological roles inside and outside of the cellrdquo Journal ofCellular Biochemistry vol 88 no 4 pp 660ndash672 2003

[71] J M Hillegass K A Murphy C M Villano and L AWhite ldquoThe impact of aryl hydrocarbon receptor signaling onmatrix metabolism implications for development and diseaserdquoBiological Chemistry vol 387 no 9 pp 1159ndash1173 2006

[72] K A Murphy C M Villano R Dorn and L A White ldquoInter-action between the aryl hydrocarbon receptor and retinoic acidpathways increases matrix metalloproteinase-1 expression inkeratinocytesrdquoThe Journal of Biological Chemistry vol 279 no24 pp 25284ndash25293 2004

[73] C F A Vogel E Sciullo and F Matsumura ldquoActivation ofinflammatory mediators and potential role of Ah-receptorligands in foam cell formationrdquo Cardiovascular Toxicology vol4 no 4 pp 363ndash373 2004

[74] T M Igarashi K L Bruner-Tran G R Yeaman et al ldquoReducedexpression of progesterone receptor-B in the endometrium of


women with endometriosis and in cocultures of endometrialcells exposed to 2378-tetrachlorodibenzo-p-dioxinrdquo Fertilityand Sterility vol 84 no 1 pp 67ndash74 2005

[75] A-M Preaux A Mallat J T Van Nhieu M-P DrsquoOrthoR M Hembry and P Mavier ldquoMatrix metalloproteinase-2activation in human hepatic fibrosis regulation by cell-matrixinteractionsrdquo Hepatology vol 30 no 4 pp 944ndash950 1999

[76] V Arpino M Brock and S E Gill ldquoThe role of TIMPs inregulation of extracellular matrix proteolysisrdquo Matrix Biologyvol 44ndash46 pp 247ndash254 2015

[77] HWang F Lafdil LWang S Yin D Feng and B Gao ldquoTissueinhibitor of metalloproteinase 1 (TIMP-1) deficiency exacer-bates carbon tetrachloride-induced liver injury and fibrosis inmice Involvement of hepatocyte STAT3 inTIMP-1 productionrdquoCell and Bioscience vol 1 no 1 article 140 2011

[78] A H Baker D R Edwards and GMurphy ldquoMetalloproteinaseinhibitors biological actions and therapeutic opportunitiesrdquoJournal of Cell Science vol 115 no 19 pp 3719ndash3727 2002

[79] T Mizutani M Yoshino T Satake et al ldquoIdentification of2378-tetrachlorodibenzo-p-dioxin (TCDD)-inducible and -suppressive genes in the rat placenta induction of interferon-regulated genes with possible inhibitory roles for angiogenesisin the placentardquo Endocrine Journal vol 51 no 6 pp 569ndash5772004

[80] P R Hanlon M A Cimafranca X Liu Y C Cho andC R Jefcoate ldquoMicroarray analysis of early adipogenesis inC3H10T12 cells cooperative inhibitory effects of growth fac-tors and 2378-tetrachlorodibenzo-p-dioxinrdquo Toxicology andApplied Pharmacology vol 207 no 1 pp 39ndash58 2005

[81] J M Martinez C A Afshari P R Bushel A MasudaT Takahashi and N J Walker ldquoDifferential toxicogenomicresponses to 2378-tetrachlorodibenzo-p-dioxin in malignantand nonmalignant human airway epithelial cellsrdquo ToxicologicalSciences vol 69 no 2 pp 409ndash423 2002

[82] K W Gaido and S C Maness ldquoPost-transcriptional stabi-lization of urokinase plasminogen activator mRNA by 2378-tetrachlorodibenzo-p-dioxin in a human keratinocyte cell linerdquoToxicology and Applied Pharmacology vol 133 no 1 pp 34ndash421995

[83] S Shimba M Hayashi H Sone J Yonemoto and M Tezukaldquo2378-Tetrachlorodibenzo-p-dioxin (TCDD) induces bindingof a 50mdashkDaprotein on the 31015840 untranslated region of urokinase-type plasminogen activator mRNArdquo Biochemical and Biophysi-cal Research Communications vol 272 no 2 pp 441ndash448 2000

[84] J R Boehm S M Kutz E H Sage L Staiano-Coico and PJ Higgins ldquoGrowth state-dependent regulation of plasminogenactivator inhibitor type-1 gene expression during epithelialcell stimulation by serum and transforming growth factor-1205731rdquoJournal of Cellular Physiology vol 181 no 1 pp 96ndash106 1999

[85] A Starsıchova E Hruba E Slabakova et al ldquoTGF-1205731 signalingplays a dominant role in the crosstalk between TGF-1205731 and thearyl hydrocarbon receptor ligand in prostate epithelial cellsrdquoCellular Signalling vol 24 no 8 pp 1665ndash1676 2012


spaces and providemechanical and structural support to cells[8] The ECM also regulates various cellular processes suchas survival migration proliferation and differentiation bymodulating tissue stiffness communicating with the intra-cellular cytoskeleton and sequestering and releasing growthfactors [9] AhR activation by TCDD has been shown tomodulate the expression of ECM proteins such as collagenand fibronectin [10ndash14] Expression of matrix metallopro-teinases (MMPs) which are responsible for the degradationof ECM components also appears to be targeted by TCDDFor example in vitro TCDD treatment was found to increaseMMP expression in human keratinocytes prostate cancercells and melanoma cells [15ndash17] Insight into the effect ofTCDD on ECM maintenance and remodeling also stemsfrom studies in a zebrafish regeneration model in whichamputation of the caudal (tail) fin initiates epimorphic regen-eration accompanied by a wound healing response Usingthis model Andreasen et al reported that TCDD treatmentincreased the expression of MMP-9 and MMP-13 [18] Inaddition exposure to TCDD induced a localized fibrosisin the regenerating fin where collagen accumulated as anunorganized fibrotic deposit at the basement membraneIn a separate study gene expression analysis revealed thatthe largest numbers of genes impacted by TCDD duringfin regeneration were those involved in ECM remodelingand structure [10] Collectively these reports support thenotion that TCDD dysregulates ECM homeostasis and thismost likely occurs through a mechanism that includes AhR-mediated changes in gene expression

Disruptions of ECM metabolism and deposition areknown to impact the development of liver disease [19 20]Liver fibrosis is a pathological condition characterized bythe deposition of excessive or abnormal ECM componentsincluding collagen type I [19] In the liver collagen issynthesized by myofibroblast precursors namely hepaticstellate cells (HSCs) Upon liver injury HSCs transition fromquiescent vitamin A-rich cells into activated myofibroblastscharacterized by increased proliferation contractility andsynthesis of collagen type I [21] One well-established modelsystem to investigate HSC activation and ECM modulationis experimental liver fibrosis induced by chronic carbontetrachloride (CCl

4) administration In the liver CCl

4is

metabolized by cytochrome P4502E1 to a trichloromethylradical that elicits membrane damage through lipid perox-idation [22] Chronic treatment of mice with CCl

4causes

widespread centrilobular necrosis and inflammation whichdrive HSC activation and the development of fibrosis [23]

We recently found that exposure to TCDD increased liverdamage and HSC activation in mice treated with CCl

4for 8

weeks [24] However TCDD did not increase the depositionof collagen or the severity of liver fibrosis in CCl

4-treated

mice despite increased expression of genes encoding collagentype I and the potent profibrogenic mediator transforminggrowth factor-1205731 (TGF-1205731) Results further indicated thatTCDD increased collagenase activity in the liver of CCl

4-

treated mice Increased breakdown of ECM in CCl4TCDD-

treated mice could explain why collagen deposition andfibrosis development were not exacerbated despite increasesin other endpoints of fibrogenesis

Collagen biosynthesis begins with the transcription ofprocollagen genes and is facilitated by various intercellularand extracellularmolecules [25] For example heat shock pro-tein-47 (HSP47) is required for proper triple helical fold-ing and trafficking of procollagen within the endoplasmicreticulum [26]Anothermolecule decorin regulates collagenfibrillogenesis [27ndash29] Lysyl oxidase (LOX) catalyzes cross-linking of collagen fibers which marks the last step incollagen biosynthesis [30]

Collagen breakdown is achieved through the activityof numerous MMPs MMP expression is regulated at thetranscriptional level and these proteins are synthesizedas inactive zymogens called proMMPs [31] MMP activityis regulated by enzymatic inhibition and activation Forexample endogenous tissue inhibitors of metalloproteinases(TIMPs) inhibit MMP activity Numerous mechanisms acti-vate MMPs including the plasminogen activatorplasminsystem [20] Plasmin is produced through the cleavageof plasminogen by tissue plasminogen activator (tPA) andurokinase plasminogen activator (uPA) and this pathwayis suppressed by plasminogen activator inhibitor-1 (PAI-1)Plasmin can directly convert proMMPs into enzymaticallyactive MMPs and some of these active MMPs can furtheractivate other proMMPs [32] MMP activity is central tothe resolution of fibrosis and scar-associated macrophageshave been identified as an abundant cellular source of theseenzymes in the fibrotic liver [33 34]

The goal of the present study was to determine howTCDD treatment impacts the expression of genes related toECM synthesis deposition and breakdown during chronicliver injury induced by CCl

4administration We measured

gene expression related to collagen synthesis processingand cross-linking and assessed the impact of TCDD onthe organization and dispersion of fibrillar collagens in theinjured liver Expression of MMPs and the molecules thatactivate or inhibit themwere alsomeasured to determine howTCDDmodulates ECM turnover

2 Materials and Methods

21 Animals Male C57BL6 mice (8ndash10 weeks old CharlesRiver Wilmington MA) were injected ip with 05mLkgCCl4(Sigma-Aldrich St Louis MO) diluted in corn oil or

with corn oil alone (Ctrl) twice a week for 8 weeks Duringthe last two weeks of the experiment mice were treated byoral gavage once weekly with 20120583gkg TCDD (CambridgeIsotope Laboratories Andover MA) diluted in peanut oilor with peanut oil vehicle alone (Veh) At the end of theexperiment animals were euthanized and liver was eitherflash-frozen in liquid nitrogen or fixed in UltraLight ZincFormalin Fixative (PSL Equipment Vista CA) All animalexperiments were approved by the Institutional Animal Careand Use Committee at Boise State University and conductedaccording to the established policies and guidelines of thiscommittee

22 Quantitative Real-Time RT-PCR Total RNA was ex-tracted using the Omega Bio-Tek EZNA Total RNA Kit






analysis


2




















Mmp13FWD

















3 Results




4treatment


4-














0

2

4

6

8

CtrlVehCtrlTCDD

A

B

B

C

AAB

B

B B

A AA

ABB B

AAB

ABA

B

ABABA

B

B

AA

A

CCl4TCDDCCl4Veh

mRN

AΔΔCq




Serpinh1 Dcn Lox

minus2

0

2

4

6

8

A

AA

B

BABAA

B

AA

A


CCl4Veh

mRN

AΔΔCq










CCl4TCDDCCl4Veh

(a)


4

3

2

1

0

Mea

n fib

er d

isper

sion

(k)

(b)




4and TCDD (right)















02468

10 Collagenases

Gelatinases

Stromelysin

Membrane-type

A

B

A

C BB

B

A AA

AA

AAB B

AB

AB

AA

B

AB ABBA


CCl4Veh

mRN

AΔΔCq















4-treated





4(data not shown)










4TCDD-treated mice





4-Treated


4-induced liver


4


4TCDD-treated



CCl4TCDDCCl4Veh

(a)

Ctrl0

1

2

3

4

5

o

f are

a with

gel

atin

ase a

ctiv

ity

VehTCDD

A

B

AA

CCl4

(b)



4 Discussion






0

1

2

3

AA

AA

A

A

AAA

AA

A

A

B

AB

A

minus1


CCl4Veh

mRN

AΔΔCq







4






















0

5

10

AA

B

AA

A B

B

AA

B

A A

BB

A

A


CCl4Veh

mRN

AΔΔCq

minus5

(a)

0

1

2

3

uPa d

ensit

omet

ry (f

old

chan

ge)

A

A

A

A

0

1

2

3

tPa d

ensit

omet

ry (f

old

chan

ge)

B

AA

A


uPa

tPa

Actin

CCl4Veh


(b)









50120583m 50120583m




50 120583m





4treatment


4model system TCDD





4-


4model



4TCDD-treated mice







4TCDD-






4TCDD-



ECM synthesis

Collagen synthesis


ECM metabolism

MMP expression


MMP inhibitors



uarr uPa protein








4-






Competing Interests


Acknowledgments


References











































4-induced liver























































analysis


2




















Mmp13FWD

















3 Results




4treatment


4-














0

2

4

6

8

CtrlVehCtrlTCDD

A

B

B

C

AAB

B

B B

A AA

ABB B

AAB

ABA

B

ABABA

B

B

AA

A

CCl4TCDDCCl4Veh

mRN

AΔΔCq




Serpinh1 Dcn Lox

minus2

0

2

4

6

8

A

AA

B

BABAA

B

AA

A


CCl4Veh

mRN

AΔΔCq










CCl4TCDDCCl4Veh

(a)


4

3

2

1

0

Mea

n fib

er d

isper

sion

(k)

(b)




4and TCDD (right)















02468

10 Collagenases

Gelatinases

Stromelysin

Membrane-type

A

B

A

C BB

B

A AA

AA

AAB B

AB

AB

AA

B

AB ABBA


CCl4Veh

mRN

AΔΔCq















4-treated





4(data not shown)










4TCDD-treated mice





4-Treated


4-induced liver


4


4TCDD-treated



CCl4TCDDCCl4Veh

(a)

Ctrl0

1

2

3

4

5

o

f are

a with

gel

atin

ase a

ctiv

ity

VehTCDD

A

B

AA

CCl4

(b)



4 Discussion






0

1

2

3

AA

AA

A

A

AAA

AA

A

A

B

AB

A

minus1


CCl4Veh

mRN

AΔΔCq







4






















0

5

10

AA

B

AA

A B

B

AA

B

A A

BB

A

A


CCl4Veh

mRN

AΔΔCq

minus5

(a)

0

1

2

3

uPa d

ensit

omet

ry (f

old

chan

ge)

A

A

A

A

0

1

2

3

tPa d

ensit

omet

ry (f

old

chan

ge)

B

AA

A


uPa

tPa

Actin

CCl4Veh


(b)









50120583m 50120583m




50 120583m





4treatment


4model system TCDD





4-


4model



4TCDD-treated mice







4TCDD-






4TCDD-



ECM synthesis

Collagen synthesis


ECM metabolism

MMP expression


MMP inhibitors



uarr uPa protein








4-






Competing Interests


Acknowledgments


References











































4-induced liver






















































3 Results




4treatment


4-














0

2

4

6

8

CtrlVehCtrlTCDD

A

B

B

C

AAB

B

B B

A AA

ABB B

AAB

ABA

B

ABABA

B

B

AA

A

CCl4TCDDCCl4Veh

mRN

AΔΔCq




Serpinh1 Dcn Lox

minus2

0

2

4

6

8

A

AA

B

BABAA

B

AA

A


CCl4Veh

mRN

AΔΔCq










CCl4TCDDCCl4Veh

(a)


4

3

2

1

0

Mea

n fib

er d

isper

sion

(k)

(b)




4and TCDD (right)















02468

10 Collagenases

Gelatinases

Stromelysin

Membrane-type

A

B

A

C BB

B

A AA

AA

AAB B

AB

AB

AA

B

AB ABBA


CCl4Veh

mRN

AΔΔCq















4-treated





4(data not shown)










4TCDD-treated mice





4-Treated


4-induced liver


4


4TCDD-treated



CCl4TCDDCCl4Veh

(a)

Ctrl0

1

2

3

4

5

o

f are

a with

gel

atin

ase a

ctiv

ity

VehTCDD

A

B

AA

CCl4

(b)



4 Discussion






0

1

2

3

AA

AA

A

A

AAA

AA

A

A

B

AB

A

minus1


CCl4Veh

mRN

AΔΔCq







4






















0

5

10

AA

B

AA

A B

B

AA

B

A A

BB

A

A


CCl4Veh

mRN

AΔΔCq

minus5

(a)

0

1

2

3

uPa d

ensit

omet

ry (f

old

chan

ge)

A

A

A

A

0

1

2

3

tPa d

ensit

omet

ry (f

old

chan

ge)

B

AA

A


uPa

tPa

Actin

CCl4Veh


(b)









50120583m 50120583m




50 120583m





4treatment


4model system TCDD





4-


4model



4TCDD-treated mice







4TCDD-






4TCDD-



ECM synthesis

Collagen synthesis


ECM metabolism

MMP expression


MMP inhibitors



uarr uPa protein








4-






Competing Interests


Acknowledgments


References











































4-induced liver



















































CCl4TCDDCCl4Veh

(a)


4

3

2

1

0

Mea

n fib

er d

isper

sion

(k)

(b)




4and TCDD (right)















02468

10 Collagenases

Gelatinases

Stromelysin

Membrane-type

A

B

A

C BB

B

A AA

AA

AAB B

AB

AB

AA

B

AB ABBA


CCl4Veh

mRN

AΔΔCq















4-treated





4(data not shown)










4TCDD-treated mice





4-Treated


4-induced liver


4


4TCDD-treated



CCl4TCDDCCl4Veh

(a)

Ctrl0

1

2

3

4

5

o

f are

a with

gel

atin

ase a

ctiv

ity

VehTCDD

A

B

AA

CCl4

(b)



4 Discussion






0

1

2

3

AA

AA

A

A

AAA

AA

A

A

B

AB

A

minus1


CCl4Veh

mRN

AΔΔCq







4






















0

5

10

AA

B

AA

A B

B

AA

B

A A

BB

A

A


CCl4Veh

mRN

AΔΔCq

minus5

(a)

0

1

2

3

uPa d

ensit

omet

ry (f

old

chan

ge)

A

A

A

A

0

1

2

3

tPa d

ensit

omet

ry (f

old

chan

ge)

B

AA

A


uPa

tPa

Actin

CCl4Veh


(b)









50120583m 50120583m




50 120583m





4treatment


4model system TCDD





4-


4model



4TCDD-treated mice







4TCDD-






4TCDD-



ECM synthesis

Collagen synthesis


ECM metabolism

MMP expression


MMP inhibitors



uarr uPa protein








4-






Competing Interests


Acknowledgments


References











































4-induced liver




















































02468

10 Collagenases

Gelatinases

Stromelysin

Membrane-type

A

B

A

C BB

B

A AA

AA

AAB B

AB

AB

AA

B

AB ABBA


CCl4Veh

mRN

AΔΔCq















4-treated





4(data not shown)










4TCDD-treated mice





4-Treated


4-induced liver


4


4TCDD-treated



CCl4TCDDCCl4Veh

(a)

Ctrl0

1

2

3

4

5

o

f are

a with

gel

atin

ase a

ctiv

ity

VehTCDD

A

B

AA

CCl4

(b)



4 Discussion






0

1

2

3

AA

AA

A

A

AAA

AA

A

A

B

AB

A

minus1


CCl4Veh

mRN

AΔΔCq







4






















0

5

10

AA

B

AA

A B

B

AA

B

A A

BB

A

A


CCl4Veh

mRN

AΔΔCq

minus5

(a)

0

1

2

3

uPa d

ensit

omet

ry (f

old

chan

ge)

A

A

A

A

0

1

2

3

tPa d

ensit

omet

ry (f

old

chan

ge)

B

AA

A


uPa

tPa

Actin

CCl4Veh


(b)









50120583m 50120583m




50 120583m





4treatment


4model system TCDD





4-


4model



4TCDD-treated mice







4TCDD-






4TCDD-



ECM synthesis

Collagen synthesis


ECM metabolism

MMP expression


MMP inhibitors



uarr uPa protein








4-






Competing Interests


Acknowledgments


References











































4-induced liver



















































CCl4TCDDCCl4Veh

(a)

Ctrl0

1

2

3

4

5

o

f are

a with

gel

atin

ase a

ctiv

ity

VehTCDD

A

B

AA

CCl4

(b)



4 Discussion






0

1

2

3

AA

AA

A

A

AAA

AA

A

A

B

AB

A

minus1


CCl4Veh

mRN

AΔΔCq







4






















0

5

10

AA

B

AA

A B

B

AA

B

A A

BB

A

A


CCl4Veh

mRN

AΔΔCq

minus5

(a)

0

1

2

3

uPa d

ensit

omet

ry (f

old

chan

ge)

A

A

A

A

0

1

2

3

tPa d

ensit

omet

ry (f

old

chan

ge)

B

AA

A


uPa

tPa

Actin

CCl4Veh


(b)









50120583m 50120583m




50 120583m





4treatment


4model system TCDD





4-


4model



4TCDD-treated mice







4TCDD-






4TCDD-



ECM synthesis

Collagen synthesis


ECM metabolism

MMP expression


MMP inhibitors



uarr uPa protein








4-






Competing Interests


Acknowledgments


References











































4-induced liver




















































0

1

2

3

AA

AA

A

A

AAA

AA

A

A

B

AB

A

minus1


CCl4Veh

mRN

AΔΔCq







4






















0

5

10

AA

B

AA

A B

B

AA

B

A A

BB

A

A


CCl4Veh

mRN

AΔΔCq

minus5

(a)

0

1

2

3

uPa d

ensit

omet

ry (f

old

chan

ge)

A

A

A

A

0

1

2

3

tPa d

ensit

omet

ry (f

old

chan

ge)

B

AA

A


uPa

tPa

Actin

CCl4Veh


(b)









50120583m 50120583m




50 120583m





4treatment


4model system TCDD





4-


4model



4TCDD-treated mice







4TCDD-






4TCDD-



ECM synthesis

Collagen synthesis


ECM metabolism

MMP expression


MMP inhibitors



uarr uPa protein








4-






Competing Interests


Acknowledgments


References











































4-induced liver




















































0

5

10

AA

B

AA

A B

B

AA

B

A A

BB

A

A


CCl4Veh

mRN

AΔΔCq

minus5

(a)

0

1

2

3

uPa d

ensit

omet

ry (f

old

chan

ge)

A

A

A

A

0

1

2

3

tPa d

ensit

omet

ry (f

old

chan

ge)

B

AA

A


uPa

tPa

Actin

CCl4Veh


(b)









50120583m 50120583m




50 120583m





4treatment


4model system TCDD





4-


4model



4TCDD-treated mice







4TCDD-






4TCDD-



ECM synthesis

Collagen synthesis


ECM metabolism

MMP expression


MMP inhibitors



uarr uPa protein








4-






Competing Interests


Acknowledgments


References











































4-induced liver



















































50120583m 50120583m




50 120583m





4treatment


4model system TCDD





4-


4model



4TCDD-treated mice







4TCDD-






4TCDD-



ECM synthesis

Collagen synthesis


ECM metabolism

MMP expression


MMP inhibitors



uarr uPa protein








4-






Competing Interests


Acknowledgments


References











































4-induced liver



















































ECM synthesis

Collagen synthesis


ECM metabolism

MMP expression


MMP inhibitors



uarr uPa protein








4-






Competing Interests


Acknowledgments


References











































4-induced liver
















































































4-induced liver












































































































Documents

Aryl Hydrocarbon Receptor Activation by TCDD Modulates ... · using the free software application, FiberFit, which uses image processing techniques to analyze two-dimensional imagesoffibernetworks[35].ResultsindicatethatTCDD