QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 11 November 2008
doi: 10.1242/jcs.037549

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by D'Alessio, S. Articles by Blasi, F. Search for Related Content PubMed PubMed Citation Articles by D'Alessio, S. Articles by Blasi, F. Social Bookmarking                         What's this?

uPAR-deficient mouse keratinocytes fail to produce EGFR-dependent laminin-5, affecting migration in vivo and in vitro

¹ Università Vita Salute San Raffaele and Istituto Scientifico H San Raffaele, via Olgettina 60, 20132 Milano, Italy
² IFOM (Fondazione Istituto FIRC di Oncologia Molecolare), via Adamello 16, 20139 Milano, Italy

View larger version (26K): [in this window] [in a new window]   Fig. 1. Skin wound healing is delayed in uPAR-KO mice. uPAR-wt and -KO skins were wounded, and cross-sections of skin samples were collected 3, 5 and 11 days later. (A) Representative wt and uPAR-KO cross-sections of 5-day-old wounds stained for keratin 5 (K5) are shown. Arrows indicate the position of the advancing epithelial edges and the wound diameter. Below the arrows are reported the mean wound diameters 5 days after injury. (B) Average wound diameters (± s.d.) from 10 wt and 13 KO mice for each time point (Student's t-test).

View larger version (41K): [in this window] [in a new window]   Fig. 2. The absence of uPAR delays keratinocyte migration and proliferation in vivo. (A) uPAR-wt and -KO paraffin-embedded cross-sections of 3-day-old wounds stained with hematoxylin and eosin. The length of the epidermal tongue was measured blindly at x10 magnification in eight sections per wound in eight mice per genotype by computer-assisted morphometry from the tip of the epidermal tongue (black arrowhead) to the start of the proliferating keratinocyte zone (blue arrowhead). (B) uPAR-wt and -KO paraffin-embedded cross-sections of 5-day-old wounds were immunostained using an anti-PCNA antibody and counterstained with hematoxylin. Measurements of PCNA-positive nuclei are reported. The numbers (± s.d.) refer to the average of eight wounds per genotype. (C) wt and uPAR-KO frozen cross-sections of 15-day-old wounds stained for keratin 5 (K5) are shown. Arrows indicate the thickness of the epidermis.

View larger version (43K): [in this window] [in a new window]   Fig. 3. uPAR promotes keratinocyte proliferation in vitro and is required for EGFR and ERK1/2 activation in cultured cells. (A,B) Primary keratinocytes from 2-day-old newborn wt and uPAR-KO littermates were grown in S-MEM containing 1% (A) or 8% (B) chelexed FCS, and their growth rate measured every day over a 7-day period. Values are expressed as absorbance at 650 nm after staining with 0.1% crystal violet in 200 mM MES, pH 6.0. The mean (± s.d.) of triplicate samples is reported. (C,D) Semi-confluent primary wt (C) and uPAR-KO (D) keratinocytes were grown in S-MEM 1% chelexed FCS in the presence or absence of EGF (10 ng/ml) and/or AG1478 (10 µM). The growth rate was measured every day over a 7-day period as described above. (E) The EGFR levels were evaluated by western blotting of wt and uPAR-KO cell lysates using an anti-EGFR antibody. (F) Semi-confluent primary wt and uPAR-KO keratinocytes were serum-starved for 18 hours and then stimulated with EGF (10 ng/ml) for 15 minutes. Cell lysates were immunoprecipitated with an antibody against EGFR and immunoblotted with anti-phospho-Tyr (pTyr), anti-EGFR and anti-uPAR antibodies. (G) Wt and uPAR-KO cell lysates were immunoblotted using antibodies against phospho-ERK1/2 (pERK1/2) and ERK2 (totERK). (H) Semi-confluent wt and uPAR-KO primary keratinocytes were serum-starved for 18 hours and stimulated with EGF (10 ng/ml) for 15 minutes. Cells were pre-treated for 20 minutes with AG1478 (10 µM). Protein extracts were then immunoblotted for phospho-ERK1/2 and ERK2 or immunoprecipitated with an anti-EGFR antibody and immunoblotted for phospho-Tyr, uPAR and EGFR.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Murine uPAR rescues the EGF response in uPAR-KO keratinocytes. (A) uPAR-KO keratinocytes were infected with either a murine or human uPAR (muPAR or huPAR, respectively) retroviral expression vector and the expression analyzed by western blot using an anti-uPAR antibody (kindly provided by Steven Rosenberg) that recognizes both the human and mouse receptor. An empty vector was used as a negative control. (B,C) The growth rate of infected uPAR-KO keratinocytes was monitored in the presence (C) or absence (B) of exogenous EGF (10 ng/ml). (D) Semi-confluent puromycin-selected cells were serum-starved for 18 hours and then stimulated with EGF (10 ng/ml) for 15 minutes. After protein extraction, the levels of huPAR, muPAR, active ERK (pERK1/2) and total ERK (totERK2) were determined by western blot analysis on total cell lysates.

View larger version (42K): [in this window] [in a new window]   Fig. 5. uPAR is required for keratinocyte adhesion and migration in vitro. (A) Wt and uPAR-KO primary keratinocytes were seeded on ColIV-, FN-, VN- or LN5-coated 24-well plates, or in the absence of any exogenous substrate. After 8 hours, the number of adherent cells was quantitated by staining with 0.1% crystal violet in water. Values are expressed as absorbance at 650 nm and the mean ± s.d. of triplicate samples is reported. (B) Wt and uPAR-KO primary keratinocytes were seeded for 12 hours in the absence of an exogenous substrate. Representative phase-contrast images (x20) of deposited cells are shown. (C) Wt and uPAR-KO keratinocytes were seeded on LN5- or ColIV-coated dishes and grown to confluency. Cell surfaces were scraped with a pipette tip in either a single stripe or a grid pattern. The wounds were incubated in S-MEM containing 1% chelexed FCS and photographed at various times, as indicated, with the indentation marks aligned. The lines indicate the wound edge at the start of the experiment (t=0). (D) Haptotaxis of wt and uPAR-KO keratinocytes was analyzed by 48-well Boyden chamber assay in S-MEM 1% chelexed serum through FN-, ColIV-, VN- or LN5-coated 8-µm-pore-size polycarbonate filters at 37°C for 6 hours. Results, expressed as the mean number of migrated cells ± s.d. from triplicate samples, are representative of at least three experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Production and secretion of LN5 by uPAR-wt and -KO keratinocytes. (A) Suspended (susp.) wt or uPAR-KO keratinocytes, or uPAR-KO keratinocytes infected with a murine uPAR (muPAR) retrovirus, were seeded on a ColIV-coated surface. After 45 minutes, attached cells were detached with 10 mM EDTA and secreted LN5 was detached from the surface with SDS-sample buffer. As a control, suspended cells were lysed. The samples were immunoblotted with anti-LN5 (2 chain) antibody. (Right) The amounts of secreted LN5 (2 chain) levels in the western blot were quantitated relative to levels in the suspended wt sample (=100%) ± s.d. The values are expressed as the average of at least three experiments. (B) Suspended wt or uPAR-KO keratinocytes, or uPAR-KO keratinocytes infected with a muPAR retrovirus, were seeded on a ColIV-coated surface. After 45 minutes, attached cells were detached for mRNA isolation and the amount of LN5 (2 chain) mRNA and actin (control) measured by semi-quantitative RT-PCR. The histogram represents the increase in LN5 mRNA levels relative to the suspended wt sample (=100%). The values are expressed as the average of at least three independent experiments. Error bars represent the s.d.

View larger version (35K): [in this window] [in a new window]   Fig. 7. EGFR regulates the migration of uPAR-wt keratinocytes by controlling LN5 deposition. (A) Migration of keratinocytes was analyzed by 48-well Boyden chamber assay in S-MEM 1% chelexed serum with or without AG1478 (10 µM) through FN-, ColIV-, VN- or LN5-coated 8-µm-pore-size polycarbonate filters at 37°C for 6 hours. The mean number of migrated cells ± s.d. from triplicate samples is representative of at least three experiments. (B,C) Suspended wt and uPAR-KO keratinocytes were seeded on a ColIV-coated surface with or without AG1478 (10 µM). After 45 minutes, attached cells were detached with 10 mM EDTA, used for mRNA isolation, and subjected to RT-PCR using LN5 (2 chain) primers (B), whereas secreted LN5 was detached from the surface with SDS-sample buffer and immunoblotted (IB) with anti-LN5 (2 chain) antibodies (C). Actin was used as a control. The amounts of secreted LN5 (2 chain) (protein and mRNA) were quantitated and expressed relative to levels in wt samples (=100%) ± s.d., and are representative of at least three experiments.

View larger version (55K): [in this window] [in a new window]   Fig. 8. uPAR upregulates LN5 (2 chain) in vivo during wound healing. (A) Representative wt and uPAR-KO cross-sections of 3-day-old wounds stained for LN5 (2 chain) and counterstained with hematoxylin and eosin (HE). (B) Wounded skins of wt and uPAR-KO mice were collected at different times after incision, lysed and immunoblotted with an anti-LN5 (2 chain) antibody. The immunoblot shows one representative experiment, whereas the histogram shows the average values obtained with eight wt and seven uPAR-KO mice. The amounts of LN5 (2 chain) are expressed as fold increase relative to the uninjured uPAR-KO mice (left-most bar, arbitrary value of 1) ± s.d.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter