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

First published online 21 April 2009
doi: 10.1242/jcs.047894

This Article Summary Full Text Full Text (PDF) 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 Woodley, D. T. Articles by Li, W. Search for Related Content PubMed PubMed Citation Articles by Woodley, D. T. Articles by Li, W. Social Bookmarking                         What's this?

Participation of the lipoprotein receptor LRP1 in hypoxia-HSP90 autocrine signaling to promote keratinocyte migration

David T. Woodley¹, Jianhua Fan¹, Chieh-Fang Cheng¹, Yong Li^1,*, Mei Chen¹, Guojun Bu² and Wei Li^1,

¹ Department of Dermatology and the USC-Norris Comprehensive Cancer Center, the University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA
² The Department of Pediatrics, Washington University School of Medicine, St Louis, MO 63130, USA

View larger version (57K): [in this window] [in a new window]   Fig. 1. Hypoxia promotes HK migration through the action of HIF1. HKs were serum-starved and subjected to two cell migration assays (both 15 hours). (A) Colloidal gold migration assay. Representative images of cell migration tracks are shown together with the migration index (MI). An average migration track under each experimental condition is highlighted with a dotted circle for visual purpose only. (B) The in vitro wound healing assay. Cell migrations were photographed and the remaining cell-free space was quantified as the average gap (AG; double-headed arrows) (Li et al., 2004). Values are the means ± s.e.m. of three independent experiments. (C) Lysates of the cells, which were subjected to hypoxia (1% O2) or normoxia (20% O2) for the indicated time, were analyzed by western immunoblotting analysis using antibodies specifically against HIF1 (panels a and c). Anti-GAPDH antibody blotting of duplicate membranes was used as a sample loading control (panels b and d). (D,E) HKs were infected with lentivirus-carrying vector (Vec.), wild-type HIF1 (WT), HIF1CA (CA) and HIF1DN (DN). 48 hours following infection, the lysates of the cells were immunoblotted with anti-HIF1 antibodies (D, panel a) or anti-HA tag antibody (E, panel a). Anti-GAPDH antibody was used as a sample loading and densitometry scan control (panel b). (F) The HKs carrying vector or HIF1WT or HIF1CA or HIF1DN were subjected to colloidal gold migration assays under either hypoxia (1% O2) or normoxia (20% O2). The computer-assistant quantification of the migration is shown as a migration index, as previously described. *Statistically significantly different (P<0.01) from normoxia (20% oxygen). The experiment was carried out four times.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Hypoxia-triggered and HIF1-mediated secretion of HSP90 is essential for hypoxia-induce HK migration. (A) The HIF1-engineered HKs were cultured in serum-free medium under either hypoxia (1% O2) or normoxia (20% O2) for 15 hours. Cell-free conditioned medium (CM) was collected from each culture condition, concentrated and analyzed by western blot analysis with an anti-HSP90 antibody. Equal loading of these samples was based on two parameters: (1) equal volumes of medium were added to each cell culture plate and (2) the volumes of loaded samples were further calibrated according to the cell counts. Recombinant HSP90 (100 ng) was included as the positive control (lane 7). (B) HKs were subjected to colloidal gold migration assays either under normoxia (20% O2) or hypoxia (1% O2) in the absence or presence of the indicated concentrations of dimethyl amiloride (DMA) or brefeldin A (BFA) for 15 hours. Only MIs are shown, as previously described. Values are means ± s.e.m. of the MIs from four independent experiments. *Statistically significantly different (P<0.03) from the controls under the same experimental condition (i.e. bar 1). (C) HKs were subjected to colloidal gold migration assays under hypoxia or normoxia in the absence (bars 1 and 2) or presence of 5 µg of control IgG (bar 3) or increasing amounts of anti-HSP90 neutralizing antibodies (SPS-771; bars 4-7). Values are means ± s.e.m. of the MIs from four independent experiments. *Statistically significantly different (P<0.0%) from their controls under the same experimental condition (bar 1).

View larger version (38K): [in this window] [in a new window]   Fig. 3. LRP1 mediates autocrine HSP90 to promote HK migration. (A) The effect of anti-LRP1 neutralizing antibody on HK migration in response to hypoxia or HSP90. HKs were subjected to colloidal gold migration assays in the presence of either a control IgG or increasing concentrations of an anti-LRP1 neutralizing antibody under hypoxia. (B) Lysates of HKs infected with lentivirus carrying either a control siRNA (lacZ-siRNA) or two siRNAs against LRP1 (RNAi-1 and RNAi-2) were analyzed by western blot with an anti-LRP1 antibody (inset). Colloidal gold migration assays were used to assess the response of the cells to hypoxia or HSP90 (10 µg/ml). (C) Lysates of HKs expressing vector alone, siRNA-1 or siRNA-1 plus a rescuing LRP1 mutant (mlRLP1) were analyzed by western blot with an anti-LRP1 antibody, which detected the p85 subunit of LRP1 and the p150 mLRP1 mutant (Inset). The HKs were then subjected to colloidal gold migration assays. *Statistically significant (P<0.03-0.05) over serum-free controls, n=3 or 4. (D) Hypoxia drives HSP90 secretion. Secreted HSP90 binds LRP1 and promotes migration of HKs (and probably the surrounding LRP1-positive dermal cells, as well, during wound healing). The mechanism, by which hypoxia causes HKs to secrete intracellular HSP90, remains to be determined.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter