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First published online 21 November 2006
doi: 10.1242/jcs.03298

Journal of Cell Science 119, 5067-5076 (2006)
Published by The Company of Biologists 2006
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An unexpected role for keratin 10 end domains in susceptibility to skin cancer

Jiangli Chen1, Xing Cheng1, Maria Merched-Sauvage1, Carlos Caulin1, Dennis R. Roop1,2 and Peter J. Koch1,2,*

1 Department of Dermatology, Baylor College of Medicine, Houston, TX 77030, USA
2 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA


Figure 1
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  		Fig. 1. Generation of K1014chim mice. (a) The mouse K14 gene consists of 8 exons (labeled I-VIII). (b) We generated a gene-targeting vector in which the sequence encoding the K14 N-terminal head domain in exon 1 was replaced with the sequence for the K10 head domain (EcoRV/BspEI fragment). Next, we deleted exons 7 and 8 and fused a cDNA encoding the K10 tail domain (BamHI/XhoI fragment) with K14 exon 6. A neomycin selection cassette (PGK-neo), flanked by LoxP sites, was inserted into a unique PmlI site in intron 1. (c) The targeting construct was introduced into mouse ES cells and recombinant clones were identified by Southern blot analysis using an array of internal and external probes (data not shown). Recombinant ES cell clones were injected into blastocysts and several independent mouse lines were established. (d) Next, we crossed a mouse line carrying the recombinant K1014chim locus with CMV-Cre mice to remove the floxed neomycin selection cassette from K14 intron 1, thereby generating the founder for the K1014chim line used in the present study. Using RT-PCR and DNA sequence analysis, we demonstrated that the K1014chim locus generated an mRNA that encodes the predicted K1014chim fusion protein. (e) Schematic representation of the K1014chim protein and the keratin sub-domains (1A, 1B, 2A, 2B, L1, L12, L2) (Kirfel et al., 2003Go and references therein).

 

Figure 2
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  		Fig. 2. K1014chim expression in the epidermis of newborn homozygous mutant mice. Sections of mutant (a,b) and wild-type (c,d) skin were stained with the antibodies indicated. (a,b) K5 and K1014chim are co-expressed in the basal layer of the epidermis and the ORS of hair follicles in mutant mice. Note that BrdU-positive nuclei (white arrows in b) are present in K1014chim-positive cell layers, demonstrating that the presence of the K10 end domains is compatible with keratinocyte proliferation. (c,d) In wild-type epidermis, the K10 antibody only stains suprabasal keratinocytes. Note the lack of K10 staining along the ORS of wild-type hair follicles.

 

Figure 3
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  		Fig. 3. K1014chim expression in cultured keratinocytes from newborn mutant mice. The antibodies used [K5, K10, Desmoplakin (Dp)] are indicated. Both keratin antibodies (K5, K10) used in this experiment were raised against the C-terminal tail domains of their targets. The K10 antibody therefore recognizes both K1014chim and K10. However, the keratinocytes shown were cultured for 8 days in low calcium medium to eliminate differentiating cells. Under these culture conditions, K10 is not expressed in wild-type control cells (see Fig. 4). The homogenous staining of mutant cultures with the K10 antibody therefore indicates expression of the K1014chim protein. (a-c) Staining of mutant cells (maintained in low calcium medium) with the keratin antibodies demonstrates co-distribution of K5 and K1014chim, suggesting that the chimeric keratin can form a functional intermediate filament cytoskeleton with its partner K5. (d-f) Keratinocytes cultured in high calcium medium for 1 hour to induce cell junction formation. Dp is a general marker for desmosomes (reviewed in Cheng et al., 2005Go; Cheng and Koch, 2004Go). Dp antibodies stain cell-cell borders in a punctuated fashion. Each dot represents a single desmosome. The double staining with Dp and K10 antibodies indicates that the K1014chim filaments terminate in desmosomes at the plasma membrane.

 

Figure 4
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  		Fig. 4. Keratin expression in mutant (Mut) and wild-type (WT) keratinocytes as judged by western blotting. The antibodies used are indicated on the left (K5, K14, K10, ß-actin). Total protein extracts were prepared from K1014chim and wild-type keratinocytes cultured in low calcium medium for 8 days. Expression of the differentiation marker K10 is not detectable in wild-type cells under these culture conditions. The signal obtained with the K10 antibody in K1014chim extracts therefore represents expression of the chimeric protein. Note that both the K10 and K14 antibody were raised against the C-termini of the keratins. Consequently, the K14 antibody does not detect its antigen in K1014chim extracts. ß-actin was used as a loading control to normalize keratin expression. K5 expression was similar in mutant and wild-type cells. As expected, the molar ratio of K5/K14 in wild-type cells was ~1:1. The ratio of K5/K1014chim in mutant cells was also calculated as ~1:1, indicating that the steady state levels of K14 (wild-type) and K1014chim (mutant) are similar.

 

Figure 5
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  		Fig. 5. Migration and proliferation of mutant cells. (a) Primary mouse keratinocytes from newborn K1014chim (Mut) and wild-type (WT) littermates were tested for their ability to migrate in vitro (see Materials and Methods for details). The number of migrating wild-type cells was set to 100%. Note that we did not observe a statistically significant difference in the migratory behavior of the two genotypes. The graphic summarizes the results obtained with three mutant and three wild-type isolates. (b) Keratinocyte proliferation as measured by BrdU incorporation by cells grown in low calcium medium at low cell density. Four mutant and four wild-type keratinocyte isolates were used in this experiment. BrdU incorporation of wild-type cells was defined as 1. Note that both genotypes showed essentially identical proliferation rates. Error bars show standard deviation.

 

Figure 6
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  		Fig. 6. Effects of K1014chim expression on tumor development in mice subjected to a skin carcinogenesis protocol (DMBA/TPA protocol). 20 mutants and 20 wild-type mice (gender and age matched) were subjected to the protocol. We used mutants that were congenic with the C57Bl/6 background for this experiment. All mice were treated once with DMBA (50 µg/mouse), followed by TPA treatment (20 µg/mouse) once a week for 25 weeks. (a) K1014 mutants developed papillomas earlier than control mice and on average carried 2-3 times as many tumors per mouse. (b) The percentage of tumor-free mutant mice declined more rapidly throughout the 25-week period than the percentage of tumor-free wild-type controls. For example, 10 weeks into the TPA treatment about 50% of the mutants were tumor free, whereas 95% of the wild-type controls had not developed tumors (P<0.05).

 

Figure 7
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  		Fig. 7. K1014 transgene expression in an SCC of a K1014chim mouse subjected to the DMBA/TPA protocol. (a) SCC tumor cells from the skin stained with antibodies against K10 (green) and BrdU (red). Note that the K1014chim keratin is homogenously expressed throughout the tumor and apparently does not interfere with proliferation as indicated by the K10/BrdU double-positive cells. (b) Histological section of a lung SCC and (c) staining with K10 (green) and BrdU (red) antibodies. Note that the tumor tissue stains for the K1014chim protein. (d) As expected, the SCC was negative for TTF-1 (thyroid transcription factor-1), a marker for normal airway epithelial cells. Note the nuclear staining (white arrows) of normal lung tissue, suggesting that the SCC represents a metastasis of the primary tumor shown in panel a. The dashed line demarcates the border between the tumor and normal lung tissue. The asterisk indicates the bronchus.

 

Figure 8
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  		Fig. 8 Impaired apoptotic response of K1014chim skin in response to UV-B irradiation. The back skin of newborn K1014chim mutants (Mut; n=5) and wild-type littermates (WT; n=4) was irradiated with 100 mJ/cm2 UV-B. 24 hours later, the skin was isolated and processed for (a) TUNEL and (b) BrdU staining. The number of TUNEL- and BrdU-positive keratinocytes in the interfollicular epidermis was determined (the average value for the wild-type samples was defined as 100%). Note that K1014 skin showed a 27% decrease in TUNEL-positive cells compared with wild-type control skin (Mut: 57±4, WT: 78±11; P<0.05). The proliferation indices of mutant and wild-type samples were essentially the same (Mut: 26±5; WT: 25±5). Error bars show standard deviation.

 

Figure 9
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  		Fig. 9. Impaired apoptotic response of mutant keratinocytes exposed to TNF{alpha}. Primary keratinocytes from newborn mutants (Mut) and wild-type (WT) littermates were exposed to various concentrations of TNF{alpha} in the presence of cycloheximide (CHX), an inhibitor of protein translation (Caulin et al., 2000Go). (a) Western blot detection of pro-caspase 3 and active caspase 3. ß-Actin was used as a loading control. (b) Quantitative evaluation of the western blot signals shown in panel a. The relative amount of active caspase 3 (normalized against ß-actin) in the two genotypes is shown as a function of the TNF{alpha} concentration. Note the reduced apoptotic response of mutant cells compared with wild-type controls at a TNF{alpha} concentration >=10 ng/ml. This experiment was repeated twice with independent isolates of newborn keratinocytes from mutant and wild-type littermates. Similar results were obtained in these experiments (data not shown) (P=0.04).

 

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© The Company of Biologists Ltd 2006