Impaired tight junction sealing and precocious involution ...Fig. 1. Generation of PKN1 transgenic...

2272 Research Article

IntroductionThe mammary gland undergoes most of its morphologicaldevelopment and functional differentiation in the adult animalunder the control of reproductive hormones. For this reason, itis a valuable model for studies of development and hormoneregulation as well as for the in vivo analysis of signalingpathways (Daniel and Smith, 1999). During the transition frompregnancy to lactation, the mammary gland undergoes aprogrammed set of complex changes during which manymetabolic and secretory pathways are coordinated to establishcopious milk secretion in a process termed secretory activationor lactogenesis 2 (McManaman and Neville, 2003). Thesealing (or closure) of the tight junctions between neighboringmammary epithelial cells has been identified as a keycomponent of this transition, and a fall of systemicprogesterone levels as well as the presence of prolactin andglucocorticoids is required for this sealing to occur (Nguyen etal., 2001b; Nguyen and Neville, 1998).

The precise mechanisms governing tight junction closure atsecretory activation have not yet been elucidated. A potentialsignaling pathway, the Rho family of small GTPases is linkedby some tantalizing evidence to the regulation of tight junctions

in the mammary gland and other epithelial and endothelial cells(Matter and Balda, 2003). In con8 mammary epithelial tumorcells, it has been demonstrated that glucocorticoiddownregulation of RhoA is required for the organization of theapical junctional complex and tight junction formation(Rubenstein et al., 2003). Furthermore, Jou et al. studyingMDCK cells, found that both constitutively active anddominant-negative RhoA and Rac1 impaired tight junctionfunctionality in a dose-dependent and reversible manner (Jouet al., 1998). Notably, constitutive activation of RhoA resultedin a disorganization of the tight junction complexes in thesecells reminiscent of freeze-fracture studies of the tight junctioncomplexes in the pregnant mammary gland (Pitelka et al.,1973). On the basis of these studies we hypothesize that adecrease in Rho signaling may be involved in secretoryactivation, and particularly in the tight junction closure thatoccurs at that time.

The small GTPases of the Rho family act as molecularswitches that cycle between an inactive GDP-bound and anactive GTP-bound state to regulate a plethora of cellularfunctions including transcriptional activation, the regulation ofmembrane trafficking, cell adhesion, migration and cell cycle

The mammary gland undergoes a complex set of changesto establish copious milk secretion at parturition. To testthe hypothesis that signaling through the Rho pathwayplays a role in secretory activation, transgenic miceexpressing a constitutively activated form of the Rhoeffector protein PKN1 in the mammary epithelium weregenerated. PKN1 activation had no effect in late pregnancybut inhibited milk secretion after parturition, diminishingthe ability of transgenic dams to support a litter. Mammarygland morphology as well as increased apoptosis andexpression of IFGBP5 and TGF�3 suggest precociousinvolution in these animals. Furthermore, tight junctionsealing at parturition was impaired in transgenicmammary glands as demonstrated by intraductal injection

of [14C]sucrose. Consistent with this finding, tight junctionsealing in response to glucocorticoid stimulation was highlyimpaired in EpH4 mammary epithelial cells expressingconstitutively activated PKN1, whereas expression of adominant-negative PKN1 mutant resulted in acceleratedtight junction sealing in vitro. Tight junction formation wasnot impaired as demonstrated by the correct localization ofoccludin and ZO1 at the apical cell borders. Our resultsprovide evidence that PKN1 participates in the regulationof tight junction sealing in the mammary gland byinterfering with glucocorticoid signaling.

Key words: PKN1, Tight junction sealing, Mammary glanddevelopment, Secretory activation

Summary

Impaired tight junction sealing and precociousinvolution in mammary glands of PKN1 transgenicmiceAndreas Fischer1, Heiko Stuckas1, Markus Gluth1, Tanya D. Russell2, Michael C. Rudolph2, Neal E. Beeman2,Sebastian Bachmann3, Shinobu Umemura4, Yasuhiro Ohashi5,‡, Margaret C. Neville2,*,§ andFranz Theuring1,*,§

1Institute of Pharmacology, Center for Cardiovascular Research, Charité University Medicine, 10115 Berlin, Germany2Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Aurora, CO 80045, USA3Department of Anatomy, Center for Cardiovascular Research, Charité University Medicine, Berlin, Germany4Department of Pathology, Tokai University School of Medicine, Isehara, Kanagawa, Japan5Laboratory of Molecular Biology, Nihon Schering K.K., Osaka 532-0004, Japan*These authors contributed equally to this work‡Present address: Shin-Kyowa Hospital, Osaka 538-0043, Japan§Authors for correspondence (e-mail: [email protected]; [email protected])

Accepted 26 April 2007Journal of Cell Science 120, 2272-2283 Published by The Company of Biologists 2007doi:10.1242/jcs.03467

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2273Mammary development in PKN1 transgenic mice

progression (Etienne-Manneville and Hall, 2002). Thesefunctions are coordinated by the differential activation ofeffector molecules including kinases of the ROCK/Rho-kinaseand the PKN/PRK family as well as a number of other proteins(Bishop and Hall, 2000). Among these targets, protein kinaseN1 (PKN1) is a conserved, ubiquitously expressed Ser/Thrkinase having a catalytic domain highly homologous to PKC(Mukai, 2003). In addition to RhoA and Rac1 (Owen et al.,2003), PKN1 can be activated by unsaturated fatty acids,limited proteolysis and PDK1 (Mukai, 2003). A considerablenumber of molecules interacting with PKN1 in vitro have beenidentified, and there is accumulating evidence that some of thefunctions of this kinase are independent of the cytoskeletaleffects of RhoA. PKN1 has been established as part of asignaling cascade that links Rho to the Jun promoter(Marinissen et al., 2001) and results from various experimentalsystems support a role for this kinase in the regulation oftranscription (Kitagawa et al., 1998; Morissette et al., 2000).The fact that PKN1 is able to activate the androgen receptorimplicates it in the progression of prostate cancer and recentdata suggest that it may be involved in the action of othersteroid hormones (Metzger et al., 2003). Despite a growingnumber of binding partners for PKN1, its physiological roleremains elusive, in part because of a lack of selective inhibitorsand suitable in vivo models to study its function.

To begin to dissect the role of the Rho pathway, andspecifically PKN1 in secretory activation, we generatedtransgenic mice that express a constitutively active form ofPKN1 within the mammary epithelium. PKN1 transgenic micefailed to lactate and displayed a phenotype of precociousinvolution, accompanied by a perturbation of tight junctionsealing at parturition. We also provide evidence that PKN1 alsointerferes with glucocorticoid-mediated tight junction sealingin vitro.

ResultsGeneration of MMTV-PKN1 transgenic miceTo direct expression to the mammary gland, a constitutivelyactive fragment of PKN1 with the Flag tag at the N-terminuswas placed under the control of the MMTV-LTR promoter andthe polyadenylation sequence of the SV40 virus was used tofacilitate transcription (Fig. 1A). Founder animals wereidentified by Southern blot as well as a PCR screen usingtransgene-specific primers and mated with wild-type animalsto establish six transgenic lines. To assess transgeneexpression, RNA isolated from mammary glands of each linewas subjected to RT-PCR analysis. The transgenic transcriptwas detected in animals of lines 933 and 995 (Fig. 1B).Northern blot analysis with a probe consisting of the humanPKN1 kinase domain confirmed these results (data not shown).

We next evaluated transgene expression at the protein levelin the positive lines by immunoprecipitation using an antibodyagainst the Flag epitope. We were able to demonstrate thepresence of a protein of ~53 kDa that reacted with an antibodydirected against the C-terminus of the human PKN1 in animalsof both these lines. The mass of the protein is consistent withthe reported molecular mass of the PKN1 kinase domain(Ueyama et al., 2001). By contrast, no signal was obtained inother transgenic lines or wild-type animals (Fig. 1C).

The temporal expression pattern of the transgene withrespect to distinct phases of mammary gland development was

analyzed by northern blot and a band of the expected size couldbe detected at all developmental stages. Fig. 1D demonstratesthat compared with lactation, expression of the transgene waslow in virgin animals and during the first half of pregnancy andmore RNA was needed to obtain a hybridization signal. Strongsignals were obtained from lactating tissues, whereas duringinvolution, the expression level of the transgene decreasedagain. This temporal expression pattern is consistent with thepreviously reported activity of the MMTV-LTR promoter(Wagner et al., 2001). Transgenic animals of both lines showedno gross phenotypic abnormalities and developed normally tothe adult stage. They were fertile and transgenic females gavebirth to normal sized litters.

Impaired lactational competence in PKN1 transgenicfemalesTo determine whether the expression of constitutively activePKN1 interferes with the functionality of the mammary gland,mortality and growth rate of the pups were monitored. With littersizes not significantly differing between transgenic (11.4 pups)and wild-type animals (12.9 pups, P=0.29), the perinatalmortality determined as the fraction of pups dying within thesuckling period was significantly higher in transgenic animals(19.3%) compared with wild-type animals (0.35%, P=0.003). Of

Fig. 1. Generation of PKN1 transgenic mice. (A) Structure of theconstruct used to generate transgenic mice. Binding sites for theprimers Flag.for and PKN.rev used to identify transgenic animals areindicated. (B) Transgene expression in animals of line 933 and 995.RNA was isolated from mammary glands of lactating animals of theindicated lines and subjected to RT-PCR using the primers Flag.forand PKN.rev (PKN-KD). Bands for the murine �-actin transcript areshown as control (lower panel). (C) Expression of the Flag-taggedPKN1 protein in mammary glands of lines 933 and 995. Proteinextracts from wild-type (WT) and transgenic animals of the indicatedlines were immunoprecipitated with an antibody against the FLAGepitope tag and western blotting was performed with an antibodyagainst the C-terminus of human PKN1. (D) Temporal expressionpattern of the transgene in line 933. RNA was prepared from virginanimals (virg) and on pregnancy day ten (P10); lactation day 2 (L2);lactation, day ten (L10); involution, day five (I5); involution day ten(I10), involution day 60 (I60). Northern blot hybridization wasconducted using a transgene specific probe covering the C-terminusof the human PKN1. The blot was subsequently hybridized with aprobe for the S26 antigen as a loading control. Note that, comparedwith lactation, more RNA was necessary to obtain a hybridizationsignal in virgin and pregnant animals.

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the remaining pups, offspring suckled by transgenic mothers ofeither line gained significantly less weight than pups suckled bywild-type dams (Fig. 2). To determine whether this phenotypewas primarily due to lactational failure in transgenic mothers orrelated to the inability of their offspring to suckle, foster motherexperiments were conducted. Pups of transgenic mothersfostered to wild-type dams had completely normal growth ratesand mortality. By contrast, wild-type pups fostered to transgenicmothers displayed decreased weight gain and increasedmortality (data not shown) showing that this phenotype dependssolely on the genotype of the mother. We therefore analyzedmammary gland morphology in transgenic animals to determinewhether structural changes in the mammary gland accompaniedthe observed functional deficiency.

Morphological abnormalities in the mammary gland ofPKN1 transgenic miceNeither whole mount preparations nor conventional histologydemonstrated morphological abnormalities in transgenic miceof line 933 in either the virgin state or the first half ofpregnancy (data not shown). Toward the end of pregnancy,transgenic mammary glands were indistinguishable from wild-type organs with respect to side branching and alveolarbudding (Fig. 3A). As in their wild-type counterparts,epithelial cells in transgenic animals were organized in alveoliand displayed large cytoplasmic lipid droplets indicative of aproper differentiation in late pregnancy (Fig. 3B, arrow)(Richert et al., 2000). However, with the onset of lactation,striking differences were observed between transgenic andwild-type animals. Glands from transgenic mice appearedsparse in whole mounts, in contrast to wild-type glands, whichnearly filled the fat pad (Fig. 3C). Histological sectionsrevealed that transgenic alveoli had failed to expand at theonset of lactation as indicated by the small size of their lumina.In addition, the contents of the alveolar lumina appearedcondensed and stained intensely with eosin (Fig. 3D). Thisfinding became more pronounced at the second day oflactation, when a dense amorphous eosinophilic substanceresembling thickened secretion product could frequently be

Journal of Cell Science 120 (13)

observed in condensed transgenic alveoli. In addition, pyknoticnuclei were seen in transgenic mammary glands at this stageand frequently, apoptotic bodies appeared in the lumen ofmammary milk ducts and alveoli (Fig. 3E, arrows). Consistentmorphological findings were obtained in both transgenic linesat this stage. In addition to alveoli exhibiting this alteredphenotype, alveoli with a normal appearance were also presentin the mammary glands of transgenic animals in early lactation.

Because transgenes driven by the MMTV promoter havebeen shown previously to be expressed in patches (Wagner etal., 2001), we attempted to determine whether transgeneexpression was correlated with the altered morphologyobserved in parts of the transgenic mammary glands employingin situ hybridization. As Fig. 3F shows, constitutively activatedPKN1 was expressed in a patchy manner throughout the gland.Notably, small alveoli that had failed to expand at secretoryactivation were characterized by a higher rate of transgeneexpressing cells compared with alveoli that had a more normalmorphology, where only a few cells expressed the transgene.Thus, the morphological abnormalities observed in mammaryglands of transgenic animals correlated with the expression ofthe transgene.

As lactation progressed, wild-type mammary glandsmaintained a fully differentiated state and stromal adipocyteswere largely regressed until lactation ceased after 21 days. Bycontrast, a progressive loss of epithelial tissue was observed intransgenic mammary glands over the course of lactation. After5 days, focal loosening of the epithelium was seen resulting inwidely regressed glands after 10 days of lactation (Fig. 3G).Taken together, the appearance of apoptotic bodies and themorphological findings over the course of lactation areconsistent with precocious involution in transgenic mice. Nodifferences were observed in fully involuted glands betweenwild-type and transgenic animals (data not shown).

In addition to the these morphological findings in lactation,common to both transgenic lines, transgenic mice of line 995frequently exhibited a defect in ductal elongation and sidebranching during their pubertal development, resulting in avery inhomogeneous morphology in pregnancy and lactation.As these additional defects were only observed in one of thetransgenic lines, the contribution of an effect related to theintegration site of the transgene can not be investigated usingthe existing transgenic lines. However, because themorphological findings in lactation were common to bothindependent lines, they probably reflect specific effects relatedto the expression of the transgene. For this reason we focusedon line 933 for further analysis,

Milk protein synthesis in PKN1 transgenic animalsThe ability to synthesize and secrete milk proteins is one of thedefining characteristics of the mammary epithelial cell duringlactation. To further characterize the developmental defect inPKN1 transgenic mice, we therefore assessed the expressionlevel of the major milk proteins WAP, �-casein, �-lactalbuminand WDNM in transgenic mice at the onset of lactation usingmicroarray technology and, as shown in Fig. 4A, none of thesegenes was significantly altered in its expression. Northern blothybridizations using a probe specific for the murine WAPtranscript confirmed these results (data not shown) suggestingthat milk protein gene transcription and turnover are normal intransgenic mice on a global level. To determine whether a

Fig. 2. Impaired lactational competence in PKN1 transgenic mice.Body weights of pups suckled by mothers of the indicated genotypewere recorded after 10 (L10) and 21 (L21) days of suckling.***P<0.0001 significantly different from the wild type usingStudent’s t-test. Error bars depict the s.e.m.

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secretory defect was present and whether individualcells in alveoli that had failed to expand at parturitionwere capable of milk protein synthesis, we examinedthe localization of �-casein by immunohistochemistry.As Fig. 4B demonstrates, a strong signal for �-caseincould be detected in the lumen of morphologicallyabnormal alveoli by immunohistochemistry (upperpanel). Remarkably, whereas at high power (lowerpanels) immunoreactivity for �-casein was localizedmainly to the alveolar lumina and the very apicalportions of the cell in wild-type glands, caseinlocalization appeared to be altered in transgenicanimals (Fig. 4B, lower panel). In addition to densestaining in the alveolar lumen, large pools of �-caseinwere present throughout the epithelial cell and notrestricted to the apical part of the cells as in the wild-type animals. At the ultrastructural level, large vesicleswith retained casein micelles within the cells wereobserved (Fig. 4C). These observations suggest asecretory defect that prevents efficient movement ofsecretory vesicles and possibly lipid droplets to theapical membrane. As such defects are oftenaccompanied by premature involution (Schwertfeger etal., 2003), we next sought evidence of precociousinvolution in transgenic mammary glands.

Precocious involution in the mammary glands ofPKN1 transgenic miceDuring the first phase of mammary gland involution,mammary epithelial cells undergo apoptosis followedby the clearance of these cells by neighboringepithelial cells or shedding into the ductal lumen(Furth et al., 1997). The TUNEL method was used todetect apoptotic cells in various developmental stages

Fig. 3. Mammary gland morphology in PKN1 transgenicmice of line 933. Whole mount preparations (A,C,G) andtissue sections stained with hematoxylin and eosin (B,D,E)of wild-type and transgenic mammary glands are shown.The respective developmental stage is indicated (P18,Pregnancy day 18; L1, Lactation day one; L2, Lactationday two; L10, Lactation day ten). (A) Regular sidebranching and alveolar budding in late pregnancy in PKN1transgenic mice. (B) Accumulation of intracellular lipiddroplets (arrow) in the wild type as well as in PKN1transgenic mammary epithelial cells indicative of properdifferentiation at the end of pregnancy. (C) Sparse alveoliwith significant intervening adipose tissue in PKN1transgenic mice at the onset of lactation. (D) Failure oftransgenic alveoli to expand at secretory activation.(E) Condensed alveoli in PKN1 transgenic mice on thesecond day of lactation. Note that alveolar lumina are filledwith thickened secretion product frequently containingapoptotic bodies (arrow). (F) In situ hybridization to detecttransgene expression at the second day of lactation. Tissuesections from transgenic animals of line 933 werehybridized to a probe comprising the transcribed portion ofthe SV40 polyadenylation sequence in either the sense orantisense direction. Note the abundance of transgene-expressing cells in condensed alveoli (arrow).(G) Mammary gland regression in PKN1 transgenic miceafter ten days of lactation. Note the segmental distributionof the epithelial decline.

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and, as shown in Fig. 5A,B, the fraction of apoptotic cells waslow in late pregnancy in the wild type as well as in transgenicanimals. However, an increase in the percentage of apoptoticcells was observed in transgenic mice at the onset of lactationreaching statistical significance at the second day postpartum(Fig. 5B). At later stages of lactation, the number of apoptoticcells in transgenic mammary glands returned to the low levelof their wild-type counterparts.

In addition to abundant apoptosis, the early phase ofmammary gland involution is characterized by the induction ofnumerous genes, of which Tgfb3 and Igfbp5, in particular, havebeen shown to be induced solely by local factors independentlyof systemic hormone levels (Nguyen and Pollard, 2000; Tonneret al., 1997). We therefore analyzed the expression of thesegenes at the onset of lactation using microarray technologyand, as depicted in Fig. 5C, both Igfbp5 and Tgfb3 weresignificantly upregulated on the first day of lactation intransgenic mice. Furthermore Tgfb3 expression was virtuallyabsent in wild-type animals at the second day of lactation when


analyzed by northern blot, whereas clear signals were obtainedin transgenic animals (Fig. 5D). Taken together, these dataindicate that precocious involution occurs in PKN1 transgenicmice.

Impaired tight junction closure at secretory activation inPKN1 transgenic miceOne important change that occurs during secretory activationis the sealing of the tight junctions between mammaryepithelial cells (Nguyen and Neville, 1998). Injection ofradiolabeled sucrose into the mammary milk duct has provedto be a valid tool to assess the functionality of tight junctionsin the mouse mammary gland (Nguyen et al., 2001b). Wetherefore used this technique to determine whether tightjunction closure at secretory activation is impaired in PKN1transgenic mice.

When injected into the milk duct of wild-type mice at thefirst day of lactation, only a small fraction of the injected tracercould be recovered in blood samples 5 minutes after injection.

Fig. 4. Milk protein gene expression atsecretory activation in PKN1 transgenic mice.(A) Expression of the milk proteins WAP, �-casein, �-lactalbumin and WDNM on lactationday 1 in animals of the indicated genotype.Data were derived from microarray analyses oftriplicate samples for each group. There wereno significant differences by Student’s t-test.Error bars represent the s.e.m.(B) Immunohistochemical detection of �-caseinin mammary glands of mice of the indicatedgenotype on the first day of lactation using asecondary antibody coupled to Cy3. Nucleiwere stained with DAPI and Oregon-Green-488-coupled WGA was used to label thealveolar lumina. In contrast to wild-typeanimals, �-casein staining is not restricted tothe apical part of the cells in PKN1 transgenicmice (lower right panel). (C) Transmissionelectron micrographs of wild-type andtransgenic mammary epithelial cells at thesecond day of lactation. Cytoplasm is reducedin transgenic animals and highly vacuolarizedwith a big lipid droplet and casein (arrow) beingdetained in the cytoplasm.

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By contrast, transgenic animals of line 933 injected at the samedevelopmental stage displayed a significantly higherconcentration of [14C]sucrose in their circulation indicative ofan increased permeability of their tight junctions at the onsetof lactation (Fig. 6A).

We next investigated whether this functional impairment wasaccompanied by structural changes of the tight junctions inPKN1 transgenic mice. Using transmission electron microscopy,we were not able to detect any differences in the ultrastructuralappearance of the tight junction complexes between wild-typeanimals and PKN1 transgenic mice (data not shown). We thenanalyzed the localization of the tight junction associated proteinZO-1 by immunohistochemistry (Fig. 6B). In wild-type animals,ZO-1 staining was restricted to a narrow band at the cell borderin the plane of the tight junction. In PKN1 transgenic mice, ZO-1 localized to the apical borders of the cells as well, althoughfocally broadened ZO-1 staining was observed in these animalssimilar to that seen in wild-type mice during pregnancy (arrowin Fig. 6B). When assessed by immunohistochemistry, thedistribution of occludin was not altered in transgenic animals.Similarly, no difference was observed with respect to thelocalization of the adherens-junction-associated molecules E-cadherin and �-catenin (data not shown). In sum, tight junctionformation, as indicated by localization of tight junction proteinsto the junctional complexes of the alveolar cells, was not alteredby expression of PKN1, but tight junction sealing, indicated bysucrose permeability, was at least partially inhibited in thetransgenic mice.

To determine whether similar effects could be seen in vitro,we examined the effect of expression of PKN1 and dominant-negative PKN1 in a mammary cell culture system.

Glucorticoid-mediated tight junction sealing in EpH4cellsThe EPH4 cell line was subcloned from the Ip-1 cell line(Fialka et al., 1996), in turn a derivative of the lactogenic-hormone-sensitive parental line IM-2 originally cultured fromthe fourth mammary gland of mid-pregnant BALB/c mice(Reichmann et al., 1992). The line produces a monolayer ofuniform cuboidal cells with a transepithelial resistance (TER)of up to 7000 ohm cm2 when grown on a Transwell filter(Vietor et al., 2001). In our hands these high resistances wereachieved much more rapidly in the presence of glucocorticoidsas can be seen in Fig. 7A. In the absence of hydrocortisone,the cells achieved a TER of about 1000 ohm cm2 after 4 daysin culture. Addition of hydrocortisone initiated a rapid increasein resistance and as depicted in Fig. 7B, a reduction in theparacellular permeability of radiolabeled mannitol paralleledthe observed increase in the TER. Immunohistochemistry forthe tight junction proteins occludin and ZO-1 showed that evenat subconfluent density these proteins were present in thesecultures at the appropriate apical localization (Fig. 8). Withtime in culture, the cell borders became more regular but therewas no difference in the localization of the tight junctionproteins. Thus hydrocortisone is not necessary for tightjunction formation in this cell line, but does promote tightjunction sealing, similarly to its action on tight junctions duringthe transition from pregnancy to lactation (Nguyen et al.,2001a). Therefore, the EpH4 system represents a valid modelsystem to investigate the sealing of the mammary tightjunctions at secretory activation.

Constitutively activated PKN1 interferes with tightjunction sealing in EpH4 cellsTo investigate the effect of PKN1 activation on mammary tightjunction function in vitro, EpH4 cells were stably transfected

Fig. 5. Precocious involution in PKN1 transgenic mice. (A,B) Tissuesections were subjected to TUNEL assays at pregnancy day 18(P18), lactation day one (L1), lactation day 2 (L2), and lactation dayten (L10). TUNEL-positive cells were identified by the greenfluorescence and nuclei were stained with DAPI. RepresentativeTUNEL-stained mammary gland sections from wild-type andtransgenic mice on the second day of lactation are shown. Fraction ofapoptotic cells at the analyzed developmental stages calculated fromthe ratio of TUNEL-positive epithelial cells to the total number ofDAPI stained epithelial nuclei. Significance was determined usingthe Student’s t-test (*P<0.05); error bars depict the s.e.m. (C) Rawexpression levels of the involution associated genes Tgfb3 and Igfbp5on the first day of lactation in animals of the indicated genotype.Data were derived from microarray experiments involving triplicatesamples for each group and tested for significance using theStudent’s t-test (*P<0.05). Error bars represent the s.e.m.(D) Expression of Tgfb3 (arrow) on the second day of lactation inwild-type (wt) and transgenic (tg) animals as assessed by northernblot hybridization. The samples shown are representative of fouranimals analyzed in each group. Ethidium-bromide-stainedribosomal RNA is shown as a loading control in the lower panel.

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to express constitutively activated PKN1 under the control ofthe RSV promoter (Fig. 9A). Transfected cells grew withkinetics similar to those of untransfected cells and showed nomorphological abnormalities. Tight junction permeability wasmonitored by measuring the TER and as demonstrated in Fig.9B, both PKN1 and mock-transfected cells exhibited a gradualincrease in the TER when grown for 5 days in the absence ofglucocorticoids. In response to hydrocortisone, monolayers ofmock-transfected cells displayed a rapid increase in TER asdemonstrated before; this response was dramatically delayedin PKN1-transfected cells.

Tight junction formation did not appear to be altered inPKN1-transfected cells because ZO-1 and occludin localizedcorrectly to the apical cell borders in both transfected andcontrol cells after hydrocortisone treatment for 5 days (Fig.9C). Taken together, PKN1 activation highly diminished thehydrocortisone effect on transepithelial resistance withoutaffecting ZO-1 and occludin targeting to the tight junction.

Expression of a dominant-negative PKN1 mutantstimulates tight junction sealing in EpH4 cellsAs the data obtained so far suggest a negative role for PKN1in the regulation of mammary tight junction sealing, we nexttested the effect of a dominant-negative PKN1 mutant on TERdevelopment in EpH4 cells. Stable transfection of these cellswith PKN1 T774A, a point mutant rendered catalyticallyinactive by an amino acid exchange within its activation loop(Mukai, 2003), again did not significantly affect growthkinetics or cell morphology. However, when grown onTranswell filters, tight junction sealing was greatly acceleratedin these cells as reflected by significantly higher TERscompared with control cells either in the absence or presenceof hydrocortisone stimulation (Fig. 10). Notably, cellsexpressing the dominant-negative form of PKN1, when grownwithout hydrocortisone, displayed a TER pattern very similar


to control cells stimulated with steroids, suggesting thatinhibition of PKN1 signaling is sufficient to mimic thestimulatory effects of hydrocortisone on tight junction sealingin this model system. Stimulation of EpH4 cells expressingPKN1 T774A with hydrocortisone resulted in an additionalincrease of the TER, furthermore supporting the notion thatPKN1 negatively regulates glucocorticoid-mediated tightjunction sealing in vitro.

DiscussionTo test the hypothesis that signaling through the Rho pathwayplays a role in secretory activation, transgenic mice expressinga constitutively activated form of the Rho effector kinase PKN1in the mammary epithelium were used. We found that activatedPKN1 had no effect on differentiation in late pregnancy, butthat it inhibited both secretion of milk components and tightjunction closure on day 1 of lactation, leading to a phenotypeof precocious involution that diminished the ability of the damsto support a litter.

Although the developmental cycle of the mouse mammarygland is highly complex (Richert et al., 2000), consistentmorphological alterations in two independent transgenic lineswere observed only at the onset of lactation. Although thefinding of a defect largely confined to this stage could reflectthe hormone dependence of the MMTV promoter, numerousmodels using the same promoter display defects in either thevirgin state or during pregnancy (Jager et al., 2003; Jiang andZacksenhaus, 2002). It therefore seems reasonable to argue thatPKN1 activation selectively interferes with cellular processesoccurring at secretory activation. Our observation that cells inmorphologically abnormal alveoli were capable of milk proteinsynthesis furthermore suggests that PKN1 does not blockdifferentiation per se but rather interferes with a more definedset of processes that lead to a delayed switch between thepregnant and lactating state upon parturition.

Fig. 6. Impaired tight junction closure at secretoryactivation in PKN1 transgenic mice. (A) Increasedpermeability of mammary tight junctions in PKN1transgenic mice. [14C]sucrose was injected into the milkduct of animals of the indicated genotype at the firstpostpartum day, blood samples were taken 5 minutes afterthe injection and counted for 14C. The displayed resultsdepict averages and s.e.m. from four wild-type and fivetransgenic animals (*P<0.05, Student’s t-test).(B) Immunohistochemical detection of the tight-junction-associated molecule ZO-1 in wild-type and transgenic miceon the first day of lactation. ZO-1 was visualized with asecondary antibody coupled to Cy3; nuclei were stainedwith DAPI. To avoid focus artifacts, z-stacks were takenand projected into a two-dimensional image. BroadenedZO-1 staining is marked with an arrow.

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Among the processes occurring at secretory activation, tightjunction sealing has been postulated to be a prerequisite forsuccessful lactation (Itoh and Bissell, 2003). Using intraductalinjection of a radiolabeled tracer as well as determining theTER in cultivated mammary epithelial cells, we demonstratedthat PKN1 activation results in impaired tight junction sealingin vitro as well as in vivo. This is of particular interest, becauseRho activation has previously been linked to a perturbation oftight junction function in various experimental systems(Hopkins et al., 2003; Jou et al., 1998; Wojciak-Stothard et al.,2001). In Con8 cells, expression of constitutively activatedRhoA prevented tight junction formation upon glucocorticoidtreatment (Rubenstein et al., 2003) and expression ofconstitutively activated RhoA increased tight junctionpermeability in MDCK cells (Jou et al., 1998). However, Rhoinhibition has also been demonstrated to perturb tight junctionfunction, suggesting that the activity of these molecules iscarefully balanced and highly dependent on cellular context(Matter and Balda, 2003). One reason for this complexity maybe that a plethora of molecules is involved in the downstreamtransduction of Rho signals within the cell. A number ofpreviously published reports support a role for PKN1 in theregulation of tight junctions, although there has been no directinvestigation of its involvement. For example, an increase inparacellular permeability in response to Rho activation wasreversed by the kinase inhibitor Y-27632 in a number of

experimental systems (Stamatovic et al., 2003; Wojciak-Stothard et al., 2001). This compound has been widely used asan inhibitor for Rho kinase, but a detailed analysis revealed thatit equally inhibits the PKN1 isoform PKN2 (Davies et al.,2000) as well as PKN1 itself (Y.O., unpublished results). InMDCK II cells, RhoA activation increased the TER whichcould be reversed by expression of either of two Rho mutantsRhoAV14/L40 and RhoAV14/C42 (Fujita et al., 2000), both ofwhich have been shown to be defective in their binding toPKN1 (Sahai et al., 1998). These findings are consistent withour notion that PKN1 might be involved in the regulation oftight junction permeability downstream of Rho GTPases.

It is important to note that two distinct processes areinvolved in tight junction regulation: assembly or formation of

Fig. 7. Effect of hydrocortisone on tight junction function in EpH4cells grown on Transwell filters. Hydrocortisone (5 �mol/l; HC) wasadded to the culture medium where indicated. (A) The TER wasmeasured daily in treated and untreated cultures. (B) At the indicatedtimes [3H]mannitol was added to the top well; 5 hours later a samplewas taken from the top and bottom well, counted, and the percentageof isotope passing from the top to the bottom chamber wascalculated.

Fig. 8. The distribution of occludin and ZO-1 in EpH4 cells atvarious times after plating determined by dual labelimmunohistochemistry. Occludin and ZO-1 staining of the sameregion are shown separately in black and white images. Images weretaken in cultures fixed after plating (subconfluent) and 24 and 72hours later in the presence and absence of hydrocortisone (+ or –HC,respectively).

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tight junctions and their sealing or closure, both of which havebeen shown to be independently regulated (Walsh et al., 2001;Woo et al., 1999). Assembly involves the movement of tightjunction components to the apical borders of the cells. In Con8


cells, it is this process that is primarily regulated byglucocorticoids (Woo et al., 1999). However, in the in vivomammary gland and 31EG4 cells (Zettl et al., 1992), proteinssuch as ZO1 and occludin are normally localized at the tightjunction in the absence of glucocorticoids. We show here thatthe same is true for EpH4 cells and that glucocorticoids bringabout tight junction sealing. We observed that this process wasdramatically diminished in EpH4 cells transfected with activePKN1, whereas no changes could be detected in the absenceof glucocorticoids, arguing against the assumption that a non-specific effect of activated PKN1 on cell monolayercharacteristics is responsible for the observed perturbation ofparacellular permeability. This finding was further confirmedby our observation that expression of a dominant-negativePKN1 mutant resulted in accelerated tight junction sealing inEpH4 cells. The fact that the distribution of ZO-1 and occludin

Fig. 9. Impaired tight junction sealing in response to hydrocortisonein EpH4 cells expressing constitutively activated PKN1.(A) Expression of constitutively activated PKN1 in EpH4 cells. RNAwas prepared from cells stably transfected either with constitutivelyactivated PKN1 (PKN) or vector alone (con) and subjected to RT-PCR using transgene specific primers (left panel). Bands for �-actinare shown as a control (right panel). (B) TER in PKN1 (PKN) andmock-transfected (con) cells either grown in the absence (–HC) orpresence (+HC) of hydrocortisone. Data were derived from 12-15filters in each group and experiments were performed on threeindependent occasions. Error bars depict the s.e.m. Hydrocortisone-stimulated PKN1 transfected cells were compared to stimulatedmock-transfected cells using the Student’s t-test (***P<0.0005;**P<0.005). (C) Immunohistochemical detection of ZO-1 andoccludin in PKN1 (PKN) and mock-transfected (con) cells. ZO-1was visualized using a secondary antibody coupled to Cy3 whereasoccludin was detected by a FITC-labeled secondary antibody. Nucleiwere stained with DAPI.

Fig. 10. Stimulation of tight junction sealing in EpH4 cellsexpressing a dominant-negative PKN1 mutant. (A) Expression of thedominant-negative PKN1 mutant PKN1 T774A (PKNT774A) inEpH4 cells. RNA prepared from cells stably transfected either with aconstruct encoding the dominant-negative PKN1 mutant PKN1T774A or vector alone was subjected to RT-PCR using primers forthe house keeping gene Gapdh (left panel) or the dominant-negativePKN1 mutant (right panel). In lanes marked with –RT, reversetranscriptase was omitted during RT-PCR as a control for DNAcontamination; + indicates plasmid DNA used as positive control.(B) TER in EpH4 cells transfected with PKN1 T774A (PKN1 d.n.)or vector alone and either grown in the absence (–HC) or presence(+HC) of hydrocortisone. Data were derived from eight filters ineach group and experiments were performed on three independentoccasions. Error bars depict the s.e.m. and cells expressing PKN1T774A were compared with control cells growing under the sameconditions using the Student’s t-test. Significant differences withP<0.05 are marked with # for cells grown in the absence ofhydrocortisone and * for cells stimulated with hydrocortisone.

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remained unchanged suggests that PKN1 rather regulatessealing but not formation of tight junctions. Taken together,both our in vivo and in vitro experiments support thehypothesis that activated PKN1 inhibits tight junction closurein the mammary gland, suggesting a role for this molecule inmaintaining the open tight junctions of pregnancy. In addition,it has been recently reported that the balance between the twoisoforms of the Ser/Thr kinase ROCK participates in theglucocorticoid regulation of tight junction permeabilitydownstream of RhoA in con8 cells (Rubenstein et al., 2007).Our observations point to an additional pathway, by which Rhosignaling can modulate the regulation of paracellularpermeability by glucocorticoids.

As PKN1 has been implicated in transcriptional activationin various systems (Mukai, 2003), its action on tight junctionpermeability potentially might involve changes in thetranscription of tight-junction-associated proteins, possibly byinterfering with glucocorticoid-regulated transcriptionalactivation. In this regard, it seems noteworthy that microarrayanalyses revealed a significant downregulation of theglucocorticoid receptor in PKN1 transgenic mice. Furthermore,significant alterations in the expression of claudin 3 andclaudin 8 were found by microarray analysis in these animals(unpublished data). Claudins represent a large class ofmolecules implicated in the regulation of tight junctionfunction (Schneeberger and Lynch, 2004), yet theirsignificance in the mammary gland is presently unclear.Determining the localization of the various claudin familymembers in the mammary gland could significantly advanceour understanding of the permeability switch occurring atsecretory activation and experiments addressing this questionare currently underway in our laboratory.

Accompanying impaired tight junction sealing, mammaryglands of transgenic animals displayed numerousmorphological abnormalities in early lactation and transgeneexpression correlated with these abnormalities as shown by insitu hybridization. Over the course of lactation, wedemonstrated a phenotype of precocious involution in PKN1transgenic mice reflected in increased apoptosis in earlylactation followed by a progressive loss of epithelium. Whereasthe precise mechanism underlying this observation remainsunclear, the fact that the relative expression level of thetransgene remained unchanged during lactation suggests thatprogrammed cell death does not selectively affect transgene-expressing cells and is therefore unlikely to be a directconsequence of PKN1 activation, consistent with previousreports (Ueyama et al., 2001). As the tight junction has beenidentified as a central signaling interface within the cell (Matterand Balda, 2003), it is possible that a signaling pathway fromthe mammary tight junction initiates or maintains secretion andcell survival. Indeed, recent results from our laboratory suggestthat disruption of the mammary tight junctions results inincreased apoptosis under various conditions in vitro (N. E.Beeman and M.C.N., unpublished results), thereby furthersupporting the idea of a causal connection between impairedtight junction sealing and the induction of involution in themammary gland.

Taken together, our observations suggest that the Rhosignaling pathway and PKN1 are part of the program thatmaintains the state of pregnancy including open tight junctionsin the mammary gland and that prolonging this program by

activation of PKN1 results in precocious involution. Thus,whereas the precise molecular mechanism by which PKN1elicits this effect remains to be elucidated, our data demonstratefor the first time a role for the Rho signaling pathway in theregulation of secretory activation.

Materials and MethodsAnimal proceduresNMRI outbred mice were obtained from Harlan-Winkelmann (Paderborn,Germany). Pregnancies were staged by observing a vaginal plug after mating andthis day was subsequently considered the first day of pregnancy (P1). Intraductalinjection of tracer substances into the murine mammary gland has been describedin detail before (Nguyen et al., 2001b; Nguyen et al., 2000). The InstitutionalAnimal Care and Use Committee of the University of Colorado Health SciencesCenter and German authorities approved all procedures, which follow the guidelinesof the United States Department of Agriculture and the European CommunitiesCouncil Directive for care of laboratory animals.

Generation of PKN1 transgenic miceA 2.3 kb fragment encoding the MMTV-LTR promoter (kindly provided by DiegoWalther, Max Planck Institute for Molecular Genetics, Berlin, Germany) and a900 bp fragment encoding the SV40 small intron and polyadenylation site (kindlyprovided by Yvan de Launoit, Department of Molecular Virology, University ofBrussels, Belgium) were ligated into the pBluescript II SK+ vector (Stratagene, LaJolla, CA). The C-terminus of human PKN1 (a.a. 511-942), acting as constitutivelyactive form of this molecule (Yoshinaga et al., 1999), was tagged with the Flagepitope and cloned between these fragments (Fig. 1A). Transgenic mice weregenerated as previously described (Theuring et al., 1990) and identified by PCR(annealing temperature 60°C, 30 cycles) using the transgene-specific primersPKN.Flag (5�-TGGACTACAAGGACGACGACG-3�) and PKN.rev (5�-GCCGC -CAATATCCGCTTCTCAC-3�).

RNA analysisTotal RNA was extracted from snap-frozen tissue using Trizol Reagent (Invitrogen,Life Technologies, Karlsruhe, Germany) whereas the RNeasy kit (Qiagen, Valencia,CA) was used to isolate RNA from EpH4 cells. Standard northern blot hybridizationwas performed using the radiolabelled human PKN1 kinase domain to detecttransgene expression. Probes for Tgfb3 and the murine whey acidic protein (WAP)were kindly provided by Jeffrey W. Pollard (Department of Developmental andMolecular Biology, Albert Einstein College of Medicine, New York, NY) andAkihiko Yoshimura (Department of Immunobiology and Neuroscience, KyushuUniversity, Fukuoda, Japan), respectively.

Blots were analyzed using a STORM PhosphorImager and ImageQuant TLsoftware (Amersham Biosciences, Freiburg, Germany). Transgene expression wasassessed by standard RT-PCR using the primers PKN.Flag and PKN.rev. As acontrol, samples in which the reverse transcriptase was omitted were analyzed inparallel.

In situ hybridization using digoxigenin-labeled probes of the SV40polyadenylation sequence was performed as previously described (Ansorge et al.,2004). The procedures for microarray hybridizations to Mu74AV2 chips(Affymetrix, Santa Clara, CA) have been described in detail elsewhere (Rudolph etal., 2003).

Protein analysisFor protein isolation, snap-frozen tissue was pulverized and extracted in TSA (10mM Tris-HCl, pH 8.0, 140 mM NaCl, 0.025% NaN3) containing 0.5% Triton X-100 and proteinase inhibitors (Complete proteinase inhibitor cocktail, RocheDiagnostics, Mannheim, Germany) on ice for 60 minutes. Immunoprecipitation wascarried out by incubation of protein extracts with 2.5% anti FLAG M2 affinity gel(Sigma, Taufkirchen, Germany) and western blots were performed using the rabbitpolyclonal antibody PKN-H234 (Santa Cruz Biotechnology, Santa Cruz, CA) at aconcentration of 1 �g/ml.

HistologyFormalin-fixed tissue was paraffin embedded and 5 �m sections were stained withhematoxylin and eosin according to standard procedures. Mammary whole mountpreparations were obtained as previously described (Binas et al., 1995). For electronmicroscopy, mammary tissue was dissected into cubes of approximately 1 mm3 sizeand fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodiumcacodylate buffer overnight. Standard electron microscopy was then performedusing EPON 812 as embedding medium and an EM 900 transmission electronmicroscope (LEO, Oberkochen, Germany).

ImmunohistochemistryImmunohistochemistry was performed as previously described (Russell et al.,2003). Primary antibodies against ZO-1 (Chemicon, Temecula, CA), occludin

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(Zymed, San Francisco, CA) and �-casein (Ab 7781, MCN) were diluted 1:100 andsecondary antibodies coupled to Cy3 or fluorescein isothiocyanate (FITC) (JacksonImmunoResearch Laboratories, West Grove, PA) were applied at a dilution of 1:150.Oregon-Green-488-conjugated wheat germ agglutinin (Molecular Probes, Eugene,OR) was used to visualize the alveolar lumen. All slides were mounted using theProLong antifade kit (Molecular Probes). Images were collected using SlideBooksoftware (Intelligent Imaging Innovations, Denver, CO) on a Nikon Diaphot TMDmicroscope equipped for fluorescence with a Xenon lamp and filter wheels (SutterInstruments, Novato, CA), fluorescent filters (Chroma, Brattleboro, VT), cooledCCD camera (Cooke, Tonawanda, NY), and stepper motor (Intelligent ImagingInnovations). Images were digitally deconvolved using the No Neighbors algorithm(Slidebook software, Intelligent Imaging Innovations).

TUNEL assaysApoptotic rates were determined from 10 �m paraffin sections of formalin-fixedtissue using the Fluorescein in situ cell death detection kit (Roche Diagnostics,Indianapolis, IN) according to the manufacturer’s instructions. Nuclei werecounterstained with DAPI (Sigma) and images were taken as described above. Atleast four animals were analyzed for each time point and genotype and 1000-2500nuclei per animal were counted to determine the apoptotic rate calculated as thepercentage of TUNEL-positive epithelial nuclei of all epithelial nuclei.

Cell cultureEpH4 mammary epithelial cells (kindly provided by Irene Fialka, Research Instituteof Molecular Pathology, Vienna, Austria) were grown in DMEM (GibcoBRL, GrandIsland, NY) supplemented with 5% FCS (Gemini Bioproducts, Calabasas, CA), 1%penicillin-streptomycin (GibcoBRL, Grand Island, NY) and 10 mM HEPES(Sigma) and subcultured every 3-4 days. For measurement of the TER, cells wereseeded at confluent density on Transwell inserts with a pore size of 0.4 �m (CorningCostar Corporation, Cambridge, MA). For the induction of tight junction sealing,hydrocortisone (Sigma) was added to the growth medium at a concentration of 5�mol/l. For transfection of EpH cells, the Flag-tagged contitutively activated PKN1fragment described above was cloned into the pRC/RSV vector (Invitrogen,Carlsbad, CA), whereas a construct encoding the Flag-tagged catalytically inactivepoint mutant PKN1 T774A was the kind gift of Hideyuki Mukai (BiosignalResearch Center, Kobe University, Japan). The Lipofectamine Plus system(Invitrogen) was used according to the manufacturer’s recommendations and controlcells were obtained by transfection of the respective empty vectors alone.Expression was validated by standard RT-PCR using the primers PKN.FLAG andPKN.rev for the constitutively active fragment and PKN.for (5�-GTCT -TCGACAGCATCGTCAA-3�) and Flag.rev (5�-CTTATCGTCGTCGTCCTTGT-3�) for the catalytically inactive mutant, respectively. For immunocytochemistry,Transwell inserts were fixed in 2% paraformaldehyde for 10 minutes at 4°C andprocessed as described above. The TER was measured with an EVOM-Gvoltohmmeter (World Precision Instruments, New Haven, CT). To monitor[3H]mannitol permeability (Toddywalla et al., 1997), approximately 5�105 d.p.m.[3H]mannitol were added to the top of the Transwell. The filters were maintainedat 37°C on a rocker at low speed and 50 �l and 150 �l samples were taken fromthe top and bottom wells, respectively, 5 hours later. Radioactivity was determinedwith �-scintillation counting using Budget Solve (Research Products International,Mount Pleasant, IL).

This work contains experimental data that are part of the M.D.thesis of A.F., which has been submitted to the Faculty of Medicineof the Charité University Medicine (Berlin, Germany). The authorswould like to thank Petra Schrade and Julia Foo for technicalassistance, B. J. Davies and J. L. McManaman for helpful discussion,and J. W. Pollard, A. Yoshimura, I. Fialka, H. Mukai, D. Walther, Y.de Launoit and E. Hara for providing materials. This work wassupported by a fellowship from Charité University Medicine, Berlin,and from the Biomedical Sciences Exchange Program between NorthAmerica and Europe Inc to A.F. and NIH grants PO1-HD38129 andR37 HD19547 to M.C.N.

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