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First published online June 25, 2007
doi: 10.1242/10.1242/jcs.03467

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Impaired tight junction sealing and precocious involution in mammary glands of PKN1 transgenic mice

Andreas Fischer¹, Heiko Stuckas¹, Markus Gluth¹, Tanya D. Russell², Michael C. Rudolph², Neal E. Beeman², Sebastian Bachmann³, Shinobu Umemura⁴, Yasuhiro Ohashi^5,, Margaret C. Neville^2,*, and Franz Theuring^1,*,

¹ Institute of Pharmacology, Center for Cardiovascular Research, Charité University Medicine, 10115 Berlin, Germany
² Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Aurora, CO 80045, USA
³ Department of Anatomy, Center for Cardiovascular Research, Charité University Medicine, Berlin, Germany
⁴ Department of Pathology, Tokai University School of Medicine, Isehara, Kanagawa, Japan
⁵ Laboratory of Molecular Biology, Nihon Schering K.K., Osaka 532-0004, Japan

View larger version (48K): [in this window] [in a new window]   Fig. 1. Generation of PKN1 transgenic mice. (A) Structure of the construct used to generate transgenic mice. Binding sites for the primers Flag.for and PKN.rev used to identify transgenic animals are indicated. (B) Transgene expression in animals of line 933 and 995. RNA was isolated from mammary glands of lactating animals of the indicated lines and subjected to RT-PCR using the primers Flag.for and PKN.rev (PKN-KD). Bands for the murine -actin transcript are shown as control (lower panel). (C) Expression of the Flag-tagged PKN1 protein in mammary glands of lines 933 and 995. Protein extracts from wild-type (WT) and transgenic animals of the indicated lines were immunoprecipitated with an antibody against the FLAG epitope tag and western blotting was performed with an antibody against the C-terminus of human PKN1. (D) Temporal expression pattern of the transgene in line 933. RNA was prepared from virgin animals (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 was conducted using a transgene specific probe covering the C-terminus of the human PKN1. The blot was subsequently hybridized with a probe for the S26 antigen as a loading control. Note that, compared with lactation, more RNA was necessary to obtain a hybridization signal in virgin and pregnant animals.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Impaired lactational competence in PKN1 transgenic mice. Body weights of pups suckled by mothers of the indicated genotype were recorded after 10 (L10) and 21 (L21) days of suckling. ***P<0.0001 significantly different from the wild type using Student's t-test. Error bars depict the s.e.m.

View larger version (69K): [in this window] [in a new window]   Fig. 3. Mammary gland morphology in PKN1 transgenic mice of line 933. Whole mount preparations (A,C,G) and tissue 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, Lactation day two; L10, Lactation day ten). (A) Regular side branching and alveolar budding in late pregnancy in PKN1 transgenic mice. (B) Accumulation of intracellular lipid droplets (arrow) in the wild type as well as in PKN1 transgenic mammary epithelial cells indicative of proper differentiation at the end of pregnancy. (C) Sparse alveoli with significant intervening adipose tissue in PKN1 transgenic mice at the onset of lactation. (D) Failure of transgenic alveoli to expand at secretory activation. (E) Condensed alveoli in PKN1 transgenic mice on the second day of lactation. Note that alveolar lumina are filled with thickened secretion product frequently containing apoptotic bodies (arrow). (F) In situ hybridization to detect transgene expression at the second day of lactation. Tissue sections from transgenic animals of line 933 were hybridized to a probe comprising the transcribed portion of the SV40 polyadenylation sequence in either the sense or antisense direction. Note the abundance of transgene-expressing cells in condensed alveoli (arrow). (G) Mammary gland regression in PKN1 transgenic mice after ten days of lactation. Note the segmental distribution of the epithelial decline.

View larger version (99K): [in this window] [in a new window]   Fig. 4. Milk protein gene expression at secretory activation in PKN1 transgenic mice. (A) Expression of the milk proteins WAP, -casein, -lactalbumin and WDNM on lactation day 1 in animals of the indicated genotype. Data were derived from microarray analyses of triplicate samples for each group. There were no significant differences by Student's t-test. Error bars represent the s.e.m. (B) Immunohistochemical detection of -casein in mammary glands of mice of the indicated genotype on the first day of lactation using a secondary antibody coupled to Cy3. Nuclei were stained with DAPI and Oregon-Green-488-coupled WGA was used to label the alveolar lumina. In contrast to wild-type animals, -casein staining is not restricted to the apical part of the cells in PKN1 transgenic mice (lower right panel). (C) Transmission electron micrographs of wild-type and transgenic mammary epithelial cells at the second day of lactation. Cytoplasm is reduced in transgenic animals and highly vacuolarized with a big lipid droplet and casein (arrow) being detained in the cytoplasm.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Precocious involution in PKN1 transgenic mice. (A,B) Tissue sections were subjected to TUNEL assays at pregnancy day 18 (P18), lactation day one (L1), lactation day 2 (L2), and lactation day ten (L10). TUNEL-positive cells were identified by the green fluorescence and nuclei were stained with DAPI. Representative TUNEL-stained mammary gland sections from wild-type and transgenic mice on the second day of lactation are shown. Fraction of apoptotic cells at the analyzed developmental stages calculated from the ratio of TUNEL-positive epithelial cells to the total number of DAPI stained epithelial nuclei. Significance was determined using the Student's t-test (*P<0.05); error bars depict the s.e.m. (C) Raw expression levels of the involution associated genes Tgfb3 and Igfbp5 on the first day of lactation in animals of the indicated genotype. Data were derived from microarray experiments involving triplicate samples for each group and tested for significance using the Student's t-test (*P<0.05). Error bars represent the s.e.m. (D) Expression of Tgfb3 (arrow) on the second day of lactation in wild-type (wt) and transgenic (tg) animals as assessed by northern blot hybridization. The samples shown are representative of four animals analyzed in each group. Ethidium-bromide-stained ribosomal RNA is shown as a loading control in the lower panel.

View larger version (82K): [in this window] [in a new window]   Fig. 6. Impaired tight junction closure at secretory activation in PKN1 transgenic mice. (A) Increased permeability of mammary tight junctions in PKN1 transgenic mice. [14C]sucrose was injected into the milk duct of animals of the indicated genotype at the first postpartum day, blood samples were taken 5 minutes after the injection and counted for 14C. The displayed results depict averages and s.e.m. from four wild-type and five transgenic animals (*P<0.05, Student's t-test). (B) Immunohistochemical detection of the tight-junction-associated molecule ZO-1 in wild-type and transgenic mice on the first day of lactation. ZO-1 was visualized with a secondary antibody coupled to Cy3; nuclei were stained with DAPI. To avoid focus artifacts, z-stacks were taken and projected into a two-dimensional image. Broadened ZO-1 staining is marked with an arrow.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Effect of hydrocortisone on tight junction function in EpH4 cells grown on Transwell filters. Hydrocortisone (5 µmol/l; HC) was added to the culture medium where indicated. (A) The TER was measured daily in treated and untreated cultures. (B) At the indicated times [3H]mannitol was added to the top well; 5 hours later a sample was taken from the top and bottom well, counted, and the percentage of isotope passing from the top to the bottom chamber was calculated.

View larger version (83K): [in this window] [in a new window]   Fig. 8. The distribution of occludin and ZO-1 in EpH4 cells at various times after plating determined by dual label immunohistochemistry. Occludin and ZO-1 staining of the same region are shown separately in black and white images. Images were taken in cultures fixed after plating (subconfluent) and 24 and 72 hours later in the presence and absence of hydrocortisone (+ or –HC, respectively).

View larger version (46K): [in this window] [in a new window]   Fig. 9. Impaired tight junction sealing in response to hydrocortisone in EpH4 cells expressing constitutively activated PKN1. (A) Expression of constitutively activated PKN1 in EpH4 cells. RNA was prepared from cells stably transfected either with constitutively activated PKN1 (PKN) or vector alone (con) and subjected to RT-PCR using transgene specific primers (left panel). Bands for -actin are shown as a control (right panel). (B) TER in PKN1 (PKN) and mock-transfected (con) cells either grown in the absence (–HC) or presence (+HC) of hydrocortisone. Data were derived from 12-15 filters in each group and experiments were performed on three independent occasions. Error bars depict the s.e.m. Hydrocortisone-stimulated PKN1 transfected cells were compared to stimulated mock-transfected cells using the Student's t-test (***P<0.0005; **P<0.005). (C) Immunohistochemical detection of ZO-1 and occludin in PKN1 (PKN) and mock-transfected (con) cells. ZO-1 was visualized using a secondary antibody coupled to Cy3 whereas occludin was detected by a FITC-labeled secondary antibody. Nuclei were stained with DAPI.

View larger version (36K): [in this window] [in a new window]   Fig. 10. Stimulation of tight junction sealing in EpH4 cells expressing a dominant-negative PKN1 mutant. (A) Expression of the dominant-negative PKN1 mutant PKN1 T774A (PKNT774A) in EpH4 cells. RNA prepared from cells stably transfected either with a construct encoding the dominant-negative PKN1 mutant PKN1 T774A or vector alone was subjected to RT-PCR using primers for the house keeping gene Gapdh (left panel) or the dominant-negative PKN1 mutant (right panel). In lanes marked with –RT, reverse transcriptase was omitted during RT-PCR as a control for DNA contamination; + 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 in each group and experiments were performed on three independent occasions. Error bars depict the s.e.m. and cells expressing PKN1 T774A were compared with control cells growing under the same conditions using the Student's t-test. Significant differences with P<0.05 are marked with # for cells grown in the absence of hydrocortisone and * for cells stimulated with hydrocortisone.