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First published online August 20, 2008
doi: 10.1242/10.1242/jcs.032417

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Accelerated re-epithelialization in Dpr2-deficient mice is associated with enhanced response to TGFβ signaling

¹ Protein Sciences Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China
² State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing 100071, People's Republic of China

View larger version (41K): [in this window] [in a new window]   Fig. 1. Generation of Dpr2-knockout mice by gene targeting. (A) Targeting strategy. The targeting vector pDpr2neo deleted a 2.7 kb genomic sequence containing exon 1 with the translation start codon (ATG) and a portion of intron 1 of Dpr2. The 3' external probe and Neo probe for Southern blot analyses are indicated. Arrows indicate positions of genotyping primers. Numbers indicate primers used for PCR. (B) Southern blot analysis of Dpr2+/Neo (+/N) ES clones. KpnI-digested genomic DNA of G418-resistant ES clones was hybridized to the 3' external probe, which produced a 15 kb band from the wild-type (WT) allele and a 6.2 kb band from the mutant allele. (C,D) Southern blot analysis of Dpr2–/– (–/–, Neo deleted) mice. Tail DNA digested with KpnI was hybridized with the Neo probe (C) or the 3' external probe (D). (E) PCR genotyping of Dpr2–/– mice. PCR of tail DNA was performed using primers 1, 2 and 4 (shown in panel A). The wild-type allele produces a 714 bp band, and the knockout allele a 284 bp band. (F) RT-PCR analysis of brain extracts of wild-type, Dpr2+/–, and Dpr2–/– mice. Dpr2 mRNA was not detected in Dpr2–/– mice. (G) Northern blot analysis of wild-type and Dpr2–/– mice. Br, brain; K, KpnI; E, exon; Kd, kidney; Neo, neomycin resistance gene; TK, thymidine kinase gene.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Expression of Dpr2 in adult mouse tissues. (A) Dpr2 expression in adult tissues and ES cells was detected by northern blot. (B-E) Dpr2 expression in keratinocytes. Colocalization of Dpr2 (B, in situ hybridization) and keratinocyte-specific marker K14 (C, red, immunofluorescence) was visualized on frozen brain sections of wild-type adult mice. The nuclei were stained blue with DAPI (D). E, epidermis; HF, hair follicle. Scale bar: 200 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 3. Wound closure is accelerated in skin of Dpr2–/– mice. (A) Appearance of wound areas of Dpr2–/– and wild-type (WT) mice at day 0, 3, 5 and 7 post wounding. (B) Wound areas were determined using image analysis and expressed as a percentage of the wound area at day 0 for Dpr2–/– and control mice at the time points indicated. Results represent the mean ± s.d., n=5 for each time point and genotype, *P<0.05. Scale bar: 4 mm.

View larger version (75K): [in this window] [in a new window]   Fig. 4. Re-epithelialization is accelerated in Dpr2–/– mice. (A) K14 staining (brown) of wounds of Dpr2–/– and wild-type (WT) control mice at various time points after injury. Arrows indicate migrated edges of keratinocytes; black lines on Day 3 and Day 5 mark the wound edge. Five individuals were analyzed for each time point and genotype. (B,C) Distance between the keratinocytes (B) and migration distance of the keratinocytes (C) in wounds of Dpr2–/– and control mice. (D) Immunofluorescence staining with BrdU (red) and K14 (green) antibody carried out on day 3 wild-type and Dpr2–/– wound sections. Arrows indicate examples of BrdU-positive keratinocyte nuclei. (E) Percentage of BrdU-positive keratinocytes. The total number of nuclei were counted based on DAPI staining. Five individuals for each genotype were analyzed. Results are mean ± s.d., *P<0.05, **P<0.01. D, dermis; ES, eschar; G, granulation tissue. Scale bar: 500 µm (A); 50 µm (D).

View larger version (23K): [in this window] [in a new window]   Fig. 5. Enhanced TGFβ signaling in Dpr2–/– keratinocytes. (A) Reporter gene assay. Dpr2–/– and wild-type (WT) keratinocytes were transfected with (CAGA)12-luciferase or cotransfected with constitutively active forms of ALK5 (caALK5) or mouse Dpr2 (mDpr2). Next day, the cells were treated with or without TGFβ1 (Tβ1, 5 ng/ml) for another 20 hours. Results are relative luciferase activity of transfected cells compared with the Renilla control. All values are expressed as mean ± s.d., n=3, *P<0.05, **P<0.01. (B,C) Western blot analyses of protein extracts from Dpr2–/– and wild-type keratinocytes for ALK5 (B) and phosphorylated Smad2 (Smad2-P) (C). Cells treated with or without TGFβ1 (Tβ1) for 48 hours. β-actin or total Smad2 served as loading controls. Bar charts show densitometry results. All values are mean ± s.d., n=3; **P<0.01.

View larger version (67K): [in this window] [in a new window]   Fig. 6. TGFβ1 induces acceleration of Dpr2–/– keratinocyte migration. (A) Confluent Dpr2–/– and wild-type (WT) keratinocyte monolayers were wounded by scraping and treated with or without TGFβ1 (Tβ1) or SB431542 (SB, 10 µM). Cell migration toward the wound surface was monitored from 0 to 72 hours. (B) Migration distance of the wound edge was quantified (n=5). (C) Transwell assay was performed with growth medium in the lower well of the chamber and 5x104 keratinocytes with or without Tβ1 seeded into the upper well. After 24 hours, the cells migrating to the lower surface of the membrane were examined. Migrated cells in five different areas were counted for each data point. All data represent means ± s.d.; *P<0.05, **P<0.01. Scale bar: 200 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Expression levels of integrins are upregulated in Dpr2–/– keratinocytes. (A-D) Dpr2–/– and wild-type (WT) keratinocytes were treated with TGFβ1 for the indicated time. Real-time RT-PCR was carried out using RNA isolated from each type of cells. Integrin (Itg) β6 (A), β1 (B), v (C) and 5 (D) gene expression was quantified and normalized to HPRT, and relative values expressed as mean ± s.d. of at least four independent experiments. (E) Adhesion of Dpr2–/– keratinocytes was stronger than that of wild-type keratinocytes. Wild-type and Dpr2–/– keratinocytes were treated with TGFβ1 (5 ng/ml) for 48 hours, and the harvested cells were regrown in wells coated with fibronectin (50 µg/ml). The optical density at 550 nm of retained cells stained with Crystal Violet is shown. Data are means ± s.d. of three independent experiments; *P<0.05, **P<0.01.

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