research article cytokeratin 18 is not required for...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 576472 8 pageshttpdxdoiorg1011552013576472

Research ArticleCytokeratin 18 Is Not Required for Morphogenesis ofDeveloping Prostates but Contributes to Adult ProstateRegeneration

Chenlu Zhang12 Yanjing Guo12 Jian Cui12 Helen He Zhu12 and Wei-Qiang Gao12

1 State Key Laboratory of Oncogenes and Related Genes Stem Cell Research Center Renji Hospital 160 Pu Jian RoadShanghai Jiao Tong University School of Medicine Shanghai 200127 China

2Med-X Research Institute Shanghai Jiao Tong University Shanghai 200030 China

Correspondence should be addressed toHelenHe Zhu zhuhecraneshsmueducn andWei-Qiang Gao gaoweiqiangsjtueducn

Received 30 April 2013 Accepted 17 October 2013

Academic Editor Paul Crispen

Copyright copy 2013 Chenlu Zhang 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

Cytokeratin 18 (CK18) is a key component of keratin-containing intermediate filaments and has long been used as a classic luminalcell marker in prostatic tissue However the in vivo function of CK18 in prostate is not known so far We reported in this studyunexpectedly that deletion of CK18 in a mouse model did not affect the morphological or the histological structures of adultprostate as the CK18 knockout prostate displayed a normal glandular ductal structure branching pattern and composition ofboth luminal and basal cells However CK18 loss compromised the regenerative tubular branching in dorsolateral prostate aftercastration and androgen replacement Therefore in contrast to its importance as luminal cell marker CK18 is dispensable for theprostate morphogenesis but contributes to adult prostate regeneration

1 Introduction

The glandular epithelium of prostate is composed of threetypes of differentiated cells luminal cells basal cells and rareneuroendocrine cells Different keratins are preferentiallyexpressed in specific prostatic epithelial cells and are routinelyused as markers for respective epithelial lineages The rect-angular lumen-lining luminal cells constitute the major partof the prostate epithelial and express cytokeratin 18 (CK18)cytokeratin 8 (CK8) and androgen receptor (AR) [1] whilethe small and oblong basal cells frequently found adjacent tothe basement membrane are positive for p63 CK5 and CK14but do not express CK8 CK18 or AR Deletion of basal cellmaker p63 in mouse model led to absence of basal cells inprostate explants [2ndash4]

CK18 is a type II cytokeratin and usually coexists withtype I cytokeratin CK8 to produce keratin-containing inter-mediate filaments [5] Keratin intermediate filaments formcage-like structures around the nucleus which play an essen-tial role in the maintenance of nuclear integrity Therefore

CK18 acts as an important scaffold protein in response toexternal stresses It is also involved in regulation of cellularprocesses including apoptosis mitosis and cell cycle [5]Although homozygous CK18 knockout mice are viable andfertile liver disorders with CK8-positive aggregates are foundin aged CK18minusminus animals [6] In contrast to the importance ofCK18 as a luminal cell marker its in vivo function in the pros-tate morphogenesis and regeneration remains unexplored sofar

The mouse prostate development initiates from the uro-genital sinus (UGS) [7] The prostatic epithelium buds outaround embryonic day 175 (E175) and elongates and branchesafter birth The branching morphogenesis almost completesat postnatal week two but fully matures by 60 to 90 days[7 8] The adult prostate is composed of three symmetricallobes the anterior prostate (AP) ventral prostate (VP)and dorsolateral prostate (DLP) [9] Prostate developmentbranching and maintaining rely on serial androgen releaseand response Deprivation of androgen induces intensiveatrophy of prostate lobes while androgen replacement

2 BioMed Research International

efficiently stimulates prostate regeneration [10 11] Thisatrophy-regeneration process can be repeated around thirtytimes in rats in support of the notion that stem-like cells existin castration-resistance prostate cells [10]

We performed in this study morphological and histolog-ical investigation of CK18minusminus prostate in both physiologicaland regenerative conditions We reported here against ouroriginal hypothesis that CK18 may be required for themorphogenesis and regeneration of prostate deletion ofCK18 in amousemodel did not affect the normalmorphologyand histology of prostate However loss of CK18 indeedcompromised the branching of regenerative DLP

2 Materials and Methods

21 Experimental Animals CK18minusminus mice were introducedfrom Jackson Laboratory originated deposited by Dr MBishr Omary Genotyping of CK18minusminus mice is performed aspreviously reported in [12] All mice were housed in exper-imental animal center at Renji Hospital Shanghai JiaotongUniversity in pathogen-free environment with controlledtemperature and humidity Experimental procedures followthe guidelines and recommendations from Institutional Lab-oratory Animal Use and Care Committee (IACUC) Forgenotyping 2mm tail samplewas obtained for genomicDNAextraction 150 uL 25mM NaOH02mM EDTA was addedto the tail sample and kept at 98∘C for 1 hour followedby neutralization with 150 uL of 40mM Tris-HCl (pH =55) After a brief centrifugation 2 120583L of supernatant wasused for PCR reaction The genotyping primers we usedare 51015840-AAGGAATCCAGGAAGGGAGA-31015840 51015840-AGCCC-CGGACTTACTTGACT-31015840 and 51015840-GCCAGAGGCCAC-TTGTGTAG-31015840 Thermal cycling parameters included (i) andenaturation at 95∘C for 5 min (ii) 30 cycles of denaturationat 95∘C for 30 s annealing at 60∘C for 1min and extension at72∘C for 1min and (iii) a final extension at 72∘C for 5minThe PCR products for mutant and wild-type allele are 187 bpand 312 bp respectively

22Hematoxylin andEosin (HampE) Staining Prostate sampleswere fixed in 4 paraformaldehyde dehydrated and paraffinembedded After that the paraffined prostates were cut at5 120583m thickness on a microtome Slides were stained withhematoxylin and eosin following standard protocols Mul-tiple fields were selected for each HampE-stained section tocompare the differences in prostate

23 Immunoblotting 100120583g protein samples extracted fromboth wild-type and CK18minusminus knockout prostates were usedfor electrophoresis in 10 sodium dodecyl sulfate polyacry-lamide gel Antibodies against CK18 (Abcam) were used asprimary antibody

24 Castration and Androgen Replacement Four 2-month-old male mice of each genotype are used in our experimentAfter anesthesia with avertin the testes and epididymideswere completely removed The distal end of the spermaticcord was ligated with suture 14 days after castration a 08 cm

sustained-release capsule of stanolone (30ndash40mg Sigma)was implanted subcutaneously The mice were sacrificed forprostate collection 14 days later

25 Prostate Microdissection The separated prostate wasmoderately digested in 10mgmL collagenase for 10minAfter digestion microdissection was performed to visualizethe individual ductal networks of prostate with fine for-ceps All specimens were observed and photographed withmicrodissectionmicroscope (Nikon SMZ800) for branch andtip quantification

26 Immunofluorescence Staining Immunofluorescencestaining was performed with 8 um cryosections After fixa-tion in 4 PFA at room temperature for 5min sections wereimmerged in sodium citrate solution (10mM pH = 60) andmicrowaved for antigen retrieval for 5min Sections weresubsequently treated with 05 triton for 10min followedby PBS wash and blocking with 10 normal goat serum(NGS) for 1 hour at room temperature Specimens werethen incubated with the primary antibodies overnight at4∘C Primary antibodies were washed at least three timeswith PBS containing 1 NGS After washing secondaryantibodies conjugated with Alexa-488 or 594 (JacksonLaboratory) were added to the specimens and incubatedfor 1 hour at room temperature Secondary antibody waswashed with PBS containing 1 NGS 3 times Slides werethen mounted with DAPI containing mounting mediumfrom Vector Laboratories We used the followed primaryantibodies in the immunofluorescence staining experimentsanti-p63 (Santa Cruz sc-8431) anti-CK5 (Epitomics 1988-1)anti-CK8 (Covance MMS-162P) anti-CK8 (Epitomics2032-1) and anti-CK18 (Epitomics 3258-1)

3 Results

31 HampE Staining Shows No Changes in Histology of CK18minusminusMouse Prostates To investigate the in vivo function of CK18in prostate morphogenesis we made use of CK18minusminus miceGenomic PCR detected the null alleles in CK18minusminus mice(Figure 1(a)) Immunoblotting further confirmed a completeCK18 deletion in prostates collected from knockout animals(Figure 2(a)) In order to assess the impact of CK18 deletionon the histology of developing prostate we performed HampEstaining of prostates from mutant and control mice atdifferent postnatal development stages Surprisingly we didnot find any difference in gross appearance and histologicalstructure between CK18minusminus and WT prostates As shownin Figure 2 size of prostate from 8-week-old CK18minusminus micewas comparable to WT HampE staining of mutant prostateAP VP and DLP did display a normal glandular structureand branching pattern (Figure 2) We examined carefully theprostate of 1-week-old and 4-week-old mutant mice as wellbut no histological changes were detected (data not shown)

32 Both Luminal and Basal Cell Compartments Are Detectedin the CK18minusminus Mouse Prostate CK18 is a frequently used

BioMed Research International 3

WT

WT alleleKnockout allele

CK18minusminus CK18+minus

(a)

CK18

120573-Actin

CK18minusminus WT(b)

Figure 1 CK18 deletion in prostate from CK18minusminus mouse is verified by PCR and immunoblotting (a) CK18 null allele and wild-type (WT)allele were detected by PCR analysis of tail samples (b) Immunoblotting verifies ablation of CK18 expression in the CK18minusminus mouse prostateTwo mice of each genotype are used

50120583m

AP

VP

DLP

WT

(a)

DLP

VP

AP

50120583m

CK18minusminus

(b)

Figure 2 HampE staining displays no histological difference between WT and CK18minusminus mouse prostates Representative HampE staining of APVP and DLP prostates from WT (a) and CK18minusminus (b) mice shows normal histology of CK18minusminus prostates Images were taken by Nikon lightmicroscope (Nikon Eclipse Ti) with 200x magnification Scale bars are 50 120583m for each related row

luminal cell marker To investigate if CK18 loss affects theluminal cell compartment we performed immunofluores-cence staining on mutant and WT prostates using well-characterized markers for luminal cells and basal cells Asexpected no expression of CK18 was detected from prostatesof mutant mice (Figure 3) P63 and CK5 are selectivelyexpressed in basal cells while CK8 CK18 and AR are prefer-entially expressed in luminal cells As shown in Figure 3 bothluminal and basal cells were detected in CK18minusminus prostates

with comparable numbers to WT controls implicating thatCK18 is not required for luminal cell differentiation in theprostate

33 CK18 Is Dispensable for Prostate Branching Tubularmor-phogenesis of the mouse prostate completes at two monthsafter birth The maturation of AP DP and DLP accomplishesby then (Figures 4(a) and 4(b)) We further examined thefunction of CK18 in prostate tubular branchingThe complex


DAPI AR

DAPI CK8 P63

DAPI CK5 CK8

DAPI CK18

50120583m

WT

(a)

DAPI AR CK8 P63

DAPI CK5 CK8

DAPI

DAPI CK18

50120583m

CK18minusminus

(b)

Figure 3 Normal basal and luminal cell compartments in CK18minusminus prostates Immunofluorescence staining of prostate frozen sectionsdisplays similar luminal and basal cell compartments in WT (a) and CK18minusminus (b) prostates Luminal cells are positive for AR CK8 andCK18 while the basal cells are CK5 and P63 positive At least 4 mice were sacrificed in each group for prostate collection sectioning andstaining Multiple fields were thoroughly examined under a microscope Representative images are shown Pictures were taken using a Nikonfluorescent microscope (Nikon Eclipse Ti) with 400x magnification Scale bars are 50 120583m for each related row


WT 2000 120583m 2000 120583mCK18minusminus

(a)

AP

DLP

VP

DLP

1000 120583m

2000 120583m

1000 120583m

1000 120583m

WT

(b)

AP

DLP DLP

VP

1000 120583m

1000 120583m1000 120583m

2000 120583m

CK18minusminus

(c)

300

200

100

0AP VP DLP

ns

nsns

Duc

tal t

ips

WTCK18minusminus

(d)

300

200

100

0AP VP DLP

ns

nsns

WTCK18minusminus

Bran

ch p

oint

s

(e)

Figure 4 CK18 is dispensable for prostate branching (a)The gross anatomic appearance ofWT and CK18minusminus prostates with AP VP and DLPScale bars are 2000120583m (b c) The microdissection images display the ductal networks of prostate in left or right lobe of AP VP and DLPfrom WT and CK18minusminus mice (a) Four mice of each group are used in the experiment Only representative pictures are shown Scale bar forAP image is 2000 120583m in VP and DLP images is 1000120583m (d e) Bar graph shows no significant differences of ductal tips and branch points inAP VP and DLP between WT and of CK18minusminus prostates (119899 = 4)

and extended prostatic ductal networks of all three lobes canbe readily seen under microscope after brief digestion andcareful microdissection (Figures 4(b) and 4(c)) The ductaltips and branching points were counted in each group toquantify the tubular formation Statistical analysis showedno significant difference of ductal tips and branching pointsbetween mutant and WT groups indicating normal tubularmorphogenesis in CK18minusminus prostates

34 CK18 Deletion Reduces the Branching of DLP dur-ing Prostate Regeneration Induced by Androgen Deprivationand Replacement Androgen deprivation induces atrophy ofprostate lobes while administration of androgen efficientlystimulates prostate regeneration in rodents To investigateif CK18 plays a role in a stress scenario such as prostate

regeneration we subjected both mutant and WT animalsto castration and androgen replacement Two-month-oldmice from each group were used for castration 14 dayslater the castrated mice were given androgen by subcuta-neous implants for another 14 days and then were sacri-ficed for prostate collection Regenerated CK18minusminus prostatesdisplayed similar gross anatomic appearances to control(Figure 5(a)) However when we carefully microdissectedeach regenerated prostatic lobes we did notice compromisedtubular restoration in DLP from CK18minusminus mice (Figures 5(b)and 5(c)) Consistently quantitative data showed significantreduction of both ductal tips and branch points in CK18minusminusDLPcompared toWTcontrol suggesting thatCK18 does playa role in the prostate in response to stress and contributes toprostate regeneration


CK18minusminusWT 2000 120583m 2000 120583m

(a)

AP VP

DLP DLP

2000 120583m

1000 120583m1000 120583m

1000 120583m

WT

(b)

CK18minusminus

VPAP

DLP DLP

1000 120583m1000 120583m

1000 120583m2000 120583m

(c)

300

200

100

0AP VP DLP

ns ns

Duc

tal t

ips

WT

400lowastlowast

CK18minusminus

(d)

Bran

ch p

oint

s 300

200

100

0AP VP DLP

ns ns

WT

400

lowastlowast

CK18minusminus

(e)

Figure 5 CK18 is required for DLP branching in prostate regeneration (a) Prostates are carefully dissected after castration and androgenreplacement The gross anatomic appearance reveals no obvious difference between WT and CK18minusminus prostates Scale bars are 2000 120583m (bc)The images of microdissected prostates fromWT (a) and CK18minusminus (b) mice after castration and androgen replacement show compromisedbranching pattern in DLP but not in VP and AP of CK18minusminus prostate Four mice of each group are used in the experiment Only representativepictures are shownThe scale bar in AP image is 2000 120583m scale bars in VP and DLP images are 1000 120583m (d e) Bar graphs show significantlyreduced umbers of ductal tips (meanplusmn SEM) and branch points in CK18minusminus DLP (meanplusmn SEM lowastlowastdenotes 119875 lt 002)

4 Discussion

The morphogenesis of developing prostate is governed byextracellular signal molecules and intracellular genetic pro-grams [13ndash16] For example sonic hedgehog (Shh) pathwayinhibits epithelial ductal branching of the prostate as reducednumber of epithelial ducts was found in Shh-treated post-natal day 2 rat ventral prostate [17ndash19] In contrast cas-trated prostates could not regain normal size and structureafter androgen replacement when the hedgehog pathway isblocked [20] BMPs including BMP4 and BMP7 are addi-tionally shown to be negative regulators in prostate ductalbudding and branching [21 22] Basal-cell-specific transcrip-tional factor p63 is indispensable for prostate development

UGS of p63minusminus mice is only able to generate prostatic struc-tures with luminal and neuroendocrine cells but fails to giverise to any basal cells upon transplantation in renal capsule [423] CK18 is preferentially expressed in prostate luminal cellsand has long served as luminal cell marker We examined themorphology and histology of CK18minusminus prostates in this studyto investigate the in vivo function of CK18 in prostate Toour surprise CK18 deletion did not cause any morphologicalor histological change in homozygous ck18 knockout miceHowever when the prostate is under regenerative stress aftercastration and androgen replacement CK18 ablation leads topartially compromised branching of DLPs

CK18 and CK8 are the most abundant coexpressed ker-atins in mammals [24] In the liver CK18 and CK8 comprise


the hepatocellular keratin whileCK18 CK8 andCK19 are themajor keratins in hepatobiliary ductal cells [25] Mutations ofCK18 are associated with predisposition to subacute injuryapoptosis and fibrosis of liver possibly due to temperedCK18phosphorylation and instability of filament structure [25ndash27] In epidermis keratins are themajor proteins constitutingthe cytoskeleton and protecting cell from mechanistic stressCK5 and CK14 are often coexpressed as specific pairs in basalcell compartment Deletion of CK14 in mice dramaticallyincreases CK15 is that rarely expressed in basal cell [28]Moreover CK15 is able to reconstitute CK14 to form thekeratin networks with CK5 implicating a redundant functionof CK14 and CK15 in vivo CK1 and CK10 are keratin pairsin suprabasal layer of epidermis Similar phenomenon wasalso observed in CK10minusminus mice in which upregulation ofbasal keratins 5 and 14 permits the normal epidermal differ-entiation of CK10minusminus mice [29] Redundancy among CK6120572and CK61205734 CK4 and CK6 and CK19 and CK18 was furtheridentified in the epidermis [30ndash32] The result we show herethat CK18 deletion barely affects prostate morphogenesis isprobably also caused by upregulation of redundant cytoker-atin Increased CK19 expression is detected in CK18minusminus liversand double knockout of CK18 and CK19 led to much severephenotypes including trophoblast fragility and severe earlydevelopment defects [6 32ndash34] Therefore we propose thatCK19 which normally is not expressed in luminal cells maybe upregulated in CK18minusminus prostates However embryoniclethality in the double knockout animals prevents us from thein vivo study of CK18 and CK19 redundancy It is noteworthythat we did observe defects in CK18minusminus DLP branching mor-phogenesis when the prostate undergoes great stress duringregeneration Further analysis of expression level of otherkeratins in CK18minusminus prostate and experiment approaches likeadditional knocking down of relevant keratins will facilitatethe identification of CK18 redundant protein in prostaticmorphogenesis and regeneration

5 Conclusion

In summary our data suggest that although CK18 is animportant marker for luminal cells it has limited impact onthe histology and ductal formation of the prostatic epitheliumpossibly due to functional redundancy of other keratinsHowever in stress conditions such as regeneration processdeletion of CK18 partly compromises the branching and fullrecovery of DLP

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Chenlu Zhang YanjingGuo and JianCui contributed equallyto the paper

Acknowledgments

The study is supported by funds to W-Q Gao from the Chi-nese Ministry of Science and Technology (2012CB966800)the National Natural Science Foundation of China (NSFC)(81130038 and 81372189) Science and Technology Commis-sion of Shanghai Municipality (Pujiang Program) ShanghaiHealth Bureau Key Disciplines and Specialties FoundationShanghai Education Committee Key Discipline and Special-ties Foundation (J50208) KC Wong foundation and KeyDiscipline and Specialty Foundation of Shanghai MunicipalCommission of Health and Family Planning and funds to HH Zhu from the NSFC (81270627) Science and TechnologyCommission of Shanghai Municipality (Pujiang Program12PJ1406100) and Shanghai Education Committee (12CG1613YZ030 and young investigator program)

References

[1] A Y Nikitin A Matoso and P Roy-Burman ldquoProstate stemcells and cancerrdquo Histology and Histopathology vol 22 no 9pp 1043ndash1049 2007

[2] A Yang R Schweitzer D Sun et al ldquop63 is essential forregenerative proliferation in limb craniofacial and epithelialdevelopmentrdquo Nature vol 398 no 6729 pp 714ndash718 1999

[3] A A Mills B Zheng X-J Wang H Vogel D R Roop andA Bradley ldquop63 is a p53 homologue required for limb andepidermal morphogenesisrdquo Nature vol 398 no 6729 pp 708ndash713 1999

[4] T Kurita R T Medina A A Mills and G R Cunha ldquoRole ofp63 and basal cells in the prostaterdquoDevelopment vol 131 no 20pp 4955ndash4964 2004

[5] Y R Weng Y Cui and J Y Fang ldquoBiological functions ofcytokeratin 18 in cancerrdquoMolecular Cancer Research vol 10 no4 pp 485ndash493 2012

[6] T M Magin R Schroder S Leitgeb et al ldquoLessons fromkeratin 18 knockout mice formation of novel keratin filamentssecondary loss of keratin 7 and accumulation of liver-specifickeratin 8-positive aggregatesrdquo The Journal of Cell Biology vol140 no 6 pp 1441ndash1451 1998

[7] P C Marker A A Donjacour R Dahiya and G R CunhaldquoHormonal cellular and molecular control of prostatic devel-opmentrdquo Developmental Biology vol 253 no 2 pp 165ndash1742003

[8] Y Sugimura G R Cunha and A A Donjacour ldquoMorpho-genesis of ductal networks in the mouse prostaterdquo Biology ofReproduction vol 34 no 5 pp 961ndash971 1986

[9] B G Timms T J Mohs and L J A Didio ldquoDuctal buddingand branching patterns in the developing prostaterdquoThe Journalof Urology vol 151 no 5 pp 1427ndash1432 1994

[10] A Tsujimura Y Koikawa S Salm et al ldquoProximal locationof mouse prostate epithelial stem cells a model of prostatichomeostasisrdquoThe Journal of Cell Biology vol 157 no 7 pp 1257ndash1265 2002

[11] Y Sugimura G R Cunha and A A Donjacour ldquoMorpholog-ical and histological study of castration-induced degenerationand androgen-induced regeneration in the mouse prostaterdquoBiology of Reproduction vol 34 no 5 pp 973ndash983 1986

[12] D M Toivola M I Nieminen M Hesse et al ldquoDisturbancesin hepatic cell-cycle regulation in mice with assembly-deficientkeratins 818rdquo Hepatology vol 34 no 6 pp 1174ndash1183 2001


[13] J J Meeks and E M Schaeffer ldquoGenetic regulation of prostatedevelopmentrdquo Journal of Andrology vol 32 no 3 pp 210ndash2172011

[14] G R Cunha P S Cooke and T Kurita ldquoRole of stromal-epithelial interactions in hormonal responsesrdquo Archives ofHistology and Cytology vol 67 no 5 pp 417ndash434 2004

[15] G S Prins and O Putz ldquoMolecular signaling pathways thatregulate prostate gland developmentrdquo Differentiation vol 76no 6 pp 641ndash659 2008

[16] C C Pritchard and P S Nelson ldquoGene expression profiling inthe developing prostaterdquo Differentiation vol 76 no 6 pp 624ndash640 2008

[17] B-E Wang J Shou S Ross H Koeppen F J de Sauvage andW-Q Gao ldquoInhibition of epithelial ductal branching in theprostate by sonic hedgehog is indirectly mediated by stromalcellsrdquo The Journal of Biological Chemistry vol 278 no 20 pp18506ndash18513 2003

[18] S H Freestone P Marker O C Grace et al ldquoSonic hedgehogregulates prostatic growth and epithelial differentiationrdquoDevel-opmental Biology vol 264 no 2 pp 352ndash362 2003

[19] D M Berman N Desai X Wang et al ldquoRoles for Hedgehogsignaling in androgen production and prostate ductal morpho-genesisrdquo Developmental Biology vol 267 no 2 pp 387ndash3982004

[20] S S Karhadkar G S Bova N Abdallah et al ldquoHedgehogsignalling in prostate regeneration neoplasia and metastasisrdquoNature vol 431 no 7009 pp 707ndash712 2004

[21] M L G Lamm C A Podlasek D H Barnett et al ldquoMesenchy-mal factor bone morphogenetic protein 4 restricts ductal bud-ding and branching morphogenesis in the developing prostaterdquoDevelopmental Biology vol 232 no 2 pp 301ndash314 2001

[22] I B Grishina S Y Kim C Ferrara H P Makarenkovaand P D Walden ldquoBMP7 inhibits branching morphogenesisin the prostate gland and interferes with Notch signalingrdquoDevelopmental Biology vol 288 no 2 pp 334ndash347 2005

[23] S Signoretti M M Pires M Lindauer et al ldquop63 regulatescommitment to the prostate cell lineagerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 32 pp 11355ndash11360 2005

[24] N-O Ku D M Toivola Q Zhou G-Z Tao B Zhong andM B Omary ldquoStudying simple epithelial keratins in cells andtissuesrdquoMethods in Cell Biology vol 78 pp 489ndash517 2004

[25] N-O Ku P Strnad B-H Zhong G-Z Tao and M B OmaryldquoKeratins let liver live mutations predispose to liver disease andcrosslinking generates Mallory-Denk bodiesrdquo Hepatology vol46 no 5 pp 1639ndash1649 2007

[26] P A Coulombe and M B Omary ldquolsquoHardrsquo and ldquosoftrdquo principlesdefining the structure function and regulation of keratinintermediate filamentsrdquo Current Opinion in Cell Biology vol 14no 1 pp 110ndash122 2002

[27] P StrnadQ Zhou SHanada et al ldquoKeratin variants predisposeto acute liver failure and adverse outcome race and ethnicassociationsrdquoGastroenterology vol 139 no 3 pp 828e3ndash835e32010

[28] C Lloyd Q-C Yu J Cheng et al ldquoThe basal keratin networkof stratified squamous epithelia defining K15 function in theabsence of K14rdquo The Journal of Cell Biology vol 129 no 5 pp1329ndash1344 1995

[29] J Reichelt H Bussow C Grund and T M Magin ldquoFormationof a normal epidermis supported by increased stability ofkeratins 5 and 14 in keratin 10 null micerdquo Molecular Biology ofthe Cell vol 12 no 6 pp 1557ndash1568 2001

[30] S M Wojcik M A Longley and D R Roop ldquoDiscovery of anovelmurine keratin 6 (K6) isoformexplains the absence of hairand nail defects in mice deficient for K6a and K6brdquoThe Journalof Cell Biology vol 154 no 3 pp 619ndash630 2001

[31] S L Ness W Edelmann T D Jenkins W Liedtke A K Rustgiand R Kucherlapati ldquoMouse keratin 4 is necessary for internalepithelial integrityrdquoThe Journal of Biological Chemistry vol 273no 37 pp 23904ndash23911 1998

[32] M Hesse T Franz Y Tamai M M Taketo and T M MaginldquoTargeted deletion of keratins 18 and 19 leads to trophoblastfragility and early embryonic lethalityrdquoThe EMBO Journal vol19 no 19 pp 5060ndash5070 2000

[33] R Moll M Divo and L Langbein ldquoThe human keratinsbiology and pathologyrdquo Histochemistry and Cell Biology vol129 no 6 pp 705ndash733 2008

[34] T M Magin P Vijayaraj and R E Leube ldquoStructural andregulatory functions of keratinsrdquo Experimental Cell Researchvol 313 no 10 pp 2021ndash2032 2007

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


efficiently stimulates prostate regeneration [10 11] Thisatrophy-regeneration process can be repeated around thirtytimes in rats in support of the notion that stem-like cells existin castration-resistance prostate cells [10]

We performed in this study morphological and histolog-ical investigation of CK18minusminus prostate in both physiologicaland regenerative conditions We reported here against ouroriginal hypothesis that CK18 may be required for themorphogenesis and regeneration of prostate deletion ofCK18 in amousemodel did not affect the normalmorphologyand histology of prostate However loss of CK18 indeedcompromised the branching of regenerative DLP

2 Materials and Methods

21 Experimental Animals CK18minusminus mice were introducedfrom Jackson Laboratory originated deposited by Dr MBishr Omary Genotyping of CK18minusminus mice is performed aspreviously reported in [12] All mice were housed in exper-imental animal center at Renji Hospital Shanghai JiaotongUniversity in pathogen-free environment with controlledtemperature and humidity Experimental procedures followthe guidelines and recommendations from Institutional Lab-oratory Animal Use and Care Committee (IACUC) Forgenotyping 2mm tail samplewas obtained for genomicDNAextraction 150 uL 25mM NaOH02mM EDTA was addedto the tail sample and kept at 98∘C for 1 hour followedby neutralization with 150 uL of 40mM Tris-HCl (pH =55) After a brief centrifugation 2 120583L of supernatant wasused for PCR reaction The genotyping primers we usedare 51015840-AAGGAATCCAGGAAGGGAGA-31015840 51015840-AGCCC-CGGACTTACTTGACT-31015840 and 51015840-GCCAGAGGCCAC-TTGTGTAG-31015840 Thermal cycling parameters included (i) andenaturation at 95∘C for 5 min (ii) 30 cycles of denaturationat 95∘C for 30 s annealing at 60∘C for 1min and extension at72∘C for 1min and (iii) a final extension at 72∘C for 5minThe PCR products for mutant and wild-type allele are 187 bpand 312 bp respectively

22Hematoxylin andEosin (HampE) Staining Prostate sampleswere fixed in 4 paraformaldehyde dehydrated and paraffinembedded After that the paraffined prostates were cut at5 120583m thickness on a microtome Slides were stained withhematoxylin and eosin following standard protocols Mul-tiple fields were selected for each HampE-stained section tocompare the differences in prostate

23 Immunoblotting 100120583g protein samples extracted fromboth wild-type and CK18minusminus knockout prostates were usedfor electrophoresis in 10 sodium dodecyl sulfate polyacry-lamide gel Antibodies against CK18 (Abcam) were used asprimary antibody

24 Castration and Androgen Replacement Four 2-month-old male mice of each genotype are used in our experimentAfter anesthesia with avertin the testes and epididymideswere completely removed The distal end of the spermaticcord was ligated with suture 14 days after castration a 08 cm

sustained-release capsule of stanolone (30ndash40mg Sigma)was implanted subcutaneously The mice were sacrificed forprostate collection 14 days later

25 Prostate Microdissection The separated prostate wasmoderately digested in 10mgmL collagenase for 10minAfter digestion microdissection was performed to visualizethe individual ductal networks of prostate with fine for-ceps All specimens were observed and photographed withmicrodissectionmicroscope (Nikon SMZ800) for branch andtip quantification

26 Immunofluorescence Staining Immunofluorescencestaining was performed with 8 um cryosections After fixa-tion in 4 PFA at room temperature for 5min sections wereimmerged in sodium citrate solution (10mM pH = 60) andmicrowaved for antigen retrieval for 5min Sections weresubsequently treated with 05 triton for 10min followedby PBS wash and blocking with 10 normal goat serum(NGS) for 1 hour at room temperature Specimens werethen incubated with the primary antibodies overnight at4∘C Primary antibodies were washed at least three timeswith PBS containing 1 NGS After washing secondaryantibodies conjugated with Alexa-488 or 594 (JacksonLaboratory) were added to the specimens and incubatedfor 1 hour at room temperature Secondary antibody waswashed with PBS containing 1 NGS 3 times Slides werethen mounted with DAPI containing mounting mediumfrom Vector Laboratories We used the followed primaryantibodies in the immunofluorescence staining experimentsanti-p63 (Santa Cruz sc-8431) anti-CK5 (Epitomics 1988-1)anti-CK8 (Covance MMS-162P) anti-CK8 (Epitomics2032-1) and anti-CK18 (Epitomics 3258-1)

3 Results

31 HampE Staining Shows No Changes in Histology of CK18minusminusMouse Prostates To investigate the in vivo function of CK18in prostate morphogenesis we made use of CK18minusminus miceGenomic PCR detected the null alleles in CK18minusminus mice(Figure 1(a)) Immunoblotting further confirmed a completeCK18 deletion in prostates collected from knockout animals(Figure 2(a)) In order to assess the impact of CK18 deletionon the histology of developing prostate we performed HampEstaining of prostates from mutant and control mice atdifferent postnatal development stages Surprisingly we didnot find any difference in gross appearance and histologicalstructure between CK18minusminus and WT prostates As shownin Figure 2 size of prostate from 8-week-old CK18minusminus micewas comparable to WT HampE staining of mutant prostateAP VP and DLP did display a normal glandular structureand branching pattern (Figure 2) We examined carefully theprostate of 1-week-old and 4-week-old mutant mice as wellbut no histological changes were detected (data not shown)

32 Both Luminal and Basal Cell Compartments Are Detectedin the CK18minusminus Mouse Prostate CK18 is a frequently used


WT



(a)

CK18

120573-Actin



50120583m

AP

VP

DLP

WT

(a)

DLP

VP

AP

50120583m

CK18minusminus

(b)






DAPI AR

DAPI CK8 P63

DAPI CK5 CK8

DAPI CK18

50120583m

WT

(a)

DAPI AR CK8 P63

DAPI CK5 CK8

DAPI

DAPI CK18

50120583m

CK18minusminus

(b)




(a)

AP

DLP

VP

DLP

1000 120583m

2000 120583m

1000 120583m

1000 120583m

WT

(b)

AP

DLP DLP

VP

1000 120583m

1000 120583m1000 120583m

2000 120583m

CK18minusminus

(c)

300

200

100

0AP VP DLP

ns

nsns

Duc

tal t

ips

WTCK18minusminus

(d)

300

200

100

0AP VP DLP

ns

nsns

WTCK18minusminus

Bran

ch p

oint

s

(e)







(a)

AP VP

DLP DLP

2000 120583m

1000 120583m1000 120583m

1000 120583m

WT

(b)

CK18minusminus

VPAP

DLP DLP

1000 120583m1000 120583m

1000 120583m2000 120583m

(c)

300

200

100

0AP VP DLP

ns ns

Duc

tal t

ips

WT

400lowastlowast

CK18minusminus

(d)

Bran

ch p

oint

s 300

200

100

0AP VP DLP

ns ns

WT

400

lowastlowast

CK18minusminus

(e)


4 Discussion






5 Conclusion






Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















WT



(a)

CK18

120573-Actin



50120583m

AP

VP

DLP

WT

(a)

DLP

VP

AP

50120583m

CK18minusminus

(b)






DAPI AR

DAPI CK8 P63

DAPI CK5 CK8

DAPI CK18

50120583m

WT

(a)

DAPI AR CK8 P63

DAPI CK5 CK8

DAPI

DAPI CK18

50120583m

CK18minusminus

(b)




(a)

AP

DLP

VP

DLP

1000 120583m

2000 120583m

1000 120583m

1000 120583m

WT

(b)

AP

DLP DLP

VP

1000 120583m

1000 120583m1000 120583m

2000 120583m

CK18minusminus

(c)

300

200

100

0AP VP DLP

ns

nsns

Duc

tal t

ips

WTCK18minusminus

(d)

300

200

100

0AP VP DLP

ns

nsns

WTCK18minusminus

Bran

ch p

oint

s

(e)







(a)

AP VP

DLP DLP

2000 120583m

1000 120583m1000 120583m

1000 120583m

WT

(b)

CK18minusminus

VPAP

DLP DLP

1000 120583m1000 120583m

1000 120583m2000 120583m

(c)

300

200

100

0AP VP DLP

ns ns

Duc

tal t

ips

WT

400lowastlowast

CK18minusminus

(d)

Bran

ch p

oint

s 300

200

100

0AP VP DLP

ns ns

WT

400

lowastlowast

CK18minusminus

(e)


4 Discussion






5 Conclusion






Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DAPI AR

DAPI CK8 P63

DAPI CK5 CK8

DAPI CK18

50120583m

WT

(a)

DAPI AR CK8 P63

DAPI CK5 CK8

DAPI

DAPI CK18

50120583m

CK18minusminus

(b)




(a)

AP

DLP

VP

DLP

1000 120583m

2000 120583m

1000 120583m

1000 120583m

WT

(b)

AP

DLP DLP

VP

1000 120583m

1000 120583m1000 120583m

2000 120583m

CK18minusminus

(c)

300

200

100

0AP VP DLP

ns

nsns

Duc

tal t

ips

WTCK18minusminus

(d)

300

200

100

0AP VP DLP

ns

nsns

WTCK18minusminus

Bran

ch p

oint

s

(e)







(a)

AP VP

DLP DLP

2000 120583m

1000 120583m1000 120583m

1000 120583m

WT

(b)

CK18minusminus

VPAP

DLP DLP

1000 120583m1000 120583m

1000 120583m2000 120583m

(c)

300

200

100

0AP VP DLP

ns ns

Duc

tal t

ips

WT

400lowastlowast

CK18minusminus

(d)

Bran

ch p

oint

s 300

200

100

0AP VP DLP

ns ns

WT

400

lowastlowast

CK18minusminus

(e)


4 Discussion






5 Conclusion






Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

AP

DLP

VP

DLP

1000 120583m

2000 120583m

1000 120583m

1000 120583m

WT

(b)

AP

DLP DLP

VP

1000 120583m

1000 120583m1000 120583m

2000 120583m

CK18minusminus

(c)

300

200

100

0AP VP DLP

ns

nsns

Duc

tal t

ips

WTCK18minusminus

(d)

300

200

100

0AP VP DLP

ns

nsns

WTCK18minusminus

Bran

ch p

oint

s

(e)







(a)

AP VP

DLP DLP

2000 120583m

1000 120583m1000 120583m

1000 120583m

WT

(b)

CK18minusminus

VPAP

DLP DLP

1000 120583m1000 120583m

1000 120583m2000 120583m

(c)

300

200

100

0AP VP DLP

ns ns

Duc

tal t

ips

WT

400lowastlowast

CK18minusminus

(d)

Bran

ch p

oint

s 300

200

100

0AP VP DLP

ns ns

WT

400

lowastlowast

CK18minusminus

(e)


4 Discussion






5 Conclusion






Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

AP VP

DLP DLP

2000 120583m

1000 120583m1000 120583m

1000 120583m

WT

(b)

CK18minusminus

VPAP

DLP DLP

1000 120583m1000 120583m

1000 120583m2000 120583m

(c)

300

200

100

0AP VP DLP

ns ns

Duc

tal t

ips

WT

400lowastlowast

CK18minusminus

(d)

Bran

ch p

oint

s 300

200

100

0AP VP DLP

ns ns

WT

400

lowastlowast

CK18minusminus

(e)


4 Discussion






5 Conclusion






Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion






Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article cytokeratin 18 is not required for...

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