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

Journal of Cell Science 122, 1495-1498 (2009)
Published by The Company of Biologists 2009
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Participation of the lipoprotein receptor LRP1 in hypoxia-HSP90{alpha} autocrine signaling to promote keratinocyte migration

David T. Woodley1, Jianhua Fan1, Chieh-Fang Cheng1, Yong Li1,*, Mei Chen1, Guojun Bu2 and Wei Li1,{ddagger}

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

{ddagger} Author for correspondence (e-mail: wli{at}usc.edu)

Accepted 23 January 2009

Summary

Hypoxia is a microenvironmental stress in many pathological conditions, including wound healing and tumor invasion. Under hypoxia, the cells are forced to adapt alternative and self-supporting mechanisms. Understanding these mechanisms may lead to new insights into human disorders. We report here a novel autocrine signaling mechanism by which hypoxia promotes human keratinocyte (HK) migration. First, hypoxia triggers HKs to secrete heat shock protein 90-alpha (HSP90{alpha}) via a HIF1-dependent pathway. The secreted HSP90{alpha} in turn promotes migration, but not proliferation, of the cells. Disruption of the secretion or extracellular function of HSP90{alpha} blocked hypoxia-stimulated HK migration. The ubiquitously expressed surface receptor, LRP1 (LDL-receptor-related protein 1), mediates the HSP90{alpha} signaling. Inhibition of LRP1 binding to extracellular HSP90{alpha} by neutralizing antibodies or genetic silencing of the LRP1 receptor by RNAi completely nullified hypoxia-driven HK migration. Finally, re-introducing a RNAi-resistant LRP1 cDNA into LRP1-downregulated HKs rescued the motogenic response of the cells to hypoxia. We propose that the hypoxia-HSP90{alpha}-LRP1 autocrine loop provides previously unrecognized therapeutic targets for human disorders such as chronic wounds and cancer invasion.

Key words: Keratinocytes, Hypoxia, HSP90{alpha}, LRP1, Cell motility

Introduction

The microenvironment of wounded skin is hypoxic because of vascular disruption and high oxygen consumption by cells in the wound (Hunt et al., 1972Go). To adapt to the hypoxic environment, the cells activate a number of novel signaling pathways to induce synthesis and secretion of a wide variety of gene products such as growth factors and extracellular matrices (ECMs). The new gene expression presumably achieves a temporary self-support status for continued cell survival in the absence of an adequate blood supply. One of the most-studied signaling pathways in cells under hypoxia is the hypoxia inducible factor 1 (HIF1)-dependent pathway (Semenza, 2003Go). HIF1 is a ubiquitously expressed heterodimeric transcription factor that consists of {alpha}- and β-subunits and a key regulator of cellular oxygen homeostasis (Semenza, 2000Go).

Hypoxia promotes migration of human keratinocytes (HKs) (O'Toole et al., 1997Go; Xia et al., 2001Go) and dermal fibroblasts (Mogford et al., 2002Go; Lerman et al., 2003Go; Li et al., 2007Go). Acute hypoxia is probably a driving force during skin wound healing (Tandara and Mustoe, 2004Go). We demonstrated that hypoxia triggers human dermal fibroblasts to secrete heat shock protein 90-alpha (HSP90{alpha}), which in turn stimulates cell migration (Li et al., 2007Go). In the current study, we report a novel autocrine loop that hypoxia uses to promote HK migration.

Results and Discussion

HIF1 is critical for hypoxia's pro-motility signaling in HKs
Using an established HK model to study hypoxia-induced cell motility (O'Tool et al., 1997), we wished to identify the key pathway for hypoxia-driven HK migration. Using the single-cell-based colloidal gold migration assay as shown in Fig. 1A, we observed that hypoxia stimulated HK migration (compare panels b and c). Similar results were obtained using the cell-population-based in vitro wound-healing assay, namely hypoxia significantly enhanced HK migration (Fig. 1B, panels b,c).


Figure 1
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  		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., 2004Go). 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{alpha} (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{alpha} (WT), HIF1{alpha}CA (CA) and HIF1{alpha}DN (DN). 48 hours following infection, the lysates of the cells were immunoblotted with anti-HIF1{alpha} 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 HIF1{alpha}WT or HIF1{alpha}CA or HIF1{alpha}DN 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.

 
We then studied whether induction of HIF1{alpha} is necessary and/or sufficient to mediate the effect of hypoxia on motility. First, in hypoxic HKs, we detected a dramatic accumulation of HIF1{alpha} protein in a time-dependent fashion (Fig. 1C, panel a). The maximum accumulation of HIF1{alpha} appeared to occur after 3 hours under 1% O2 (lane 3). By contrast, duplicate HK cultures under normoxia (20% O2) showed no detectable HIF1{alpha} (Fig. 1C, panel c).

Second, we constructed the following cDNAs: (1) wt HIF1, (2) a constitutively activated (non-degradable) HIF1 (HIF1{alpha}CA5) and (3) a dominant negative HIF1 (HIF1{alpha}DN) (Jiang et al., 1996Go; Kelly et al., 2003Go) into the lentiviral vector, pRRLsin.MCS-Deco. This system offers more than 90% gene transduction efficiency in primary HKs (Cheng et al., 2008Go). Following infection, expression of these exogenous HIF1 genes in HKs was confirmed by anti-HIF1 antibody immunoblot analyses. As shown in Fig. 1D, both the wt HIF1 and HIF1{alpha}CA5 proteins were detected even under normoxia (lanes 2 and 3). By contrast, endogenous HIF1 was undetectable (panel a, lane 1). Since the HIF1{alpha}DN mutant lacks the region amino acids 391-826 that contains the epitopes for anti-HIF1{alpha} antibodies (lane 4), we used anti-HA-tag antibodies to confirm the expression of the HA-tagged HIF1{alpha}DN. As shown in Fig. 1E, an expected 36 kDa protein species was detected in HIF1{alpha}DN-infected HKs (lane 2), but not in vector-infected cells (lane 1). Infection with an HA-tagged Nck cDNA served as a control for the anti-HA antibodies (lane 3).

The cells expressing vector alone, HIF1{alpha}WT, HIF1{alpha}CA or HIF1{alpha}DN were then subjected to colloidal gold migration assays under either normoxia or hypoxia in the absence of any exogenously added growth factors. As shown in Fig. 1F, HIF1{alpha}WT- or HIF1{alpha}CA-overexpressing HKs migrated further than the vector control cells, even under normoxia (bars 3 and 5 versus bar 1), similar to the effect of hypoxia on the parental cells (bar 2). Hypoxia increased migration of control cells as expected (bar 2), but not the maximally stimulated HKs expressing HIF1{alpha}WT or HIF1{alpha}CA gene (bars 4 and 6). By contrast, expression of the HIF1{alpha}DN blocked hypoxia-stimulated migration (bar 8 versus lane 7). These data demonstrated that HIF1 is necessary and sufficient to mediate the entire hypoxia pro-motility signaling.

Hypoxia promotes HK migration via secretion and extracellular function of HSP90{alpha}
Hypoxia triggers cells to secrete HSP90{alpha} into the extracellular environment, which in turn promotes cell migration (Li et al., 2007Go). An important question is whether disruption of (1) the secretion of HSP90{alpha} or (2) the extracellular function of HSP90{alpha} would block hypoxia-induced cell migration. As shown in Fig. 2A, hypoxia and HIF1 expression caused HKs to secrete HSP90{alpha} into the culture medium. Equal quantities (see figure legend for details) of HK-conditioned media were immunoblotted with anti-HSP90{alpha} antibodies, HSP90{alpha} proteins were clearly detected in the preparations from hypoxic (lane 2), but very little from those of normoxic (lane 1) HKs. Expression of the HIF1{alpha}CA mutant showed a slightly increased secretion of HSP90{alpha}, even under normoxia (lanes 5). By contrast, the dominant-negative HIF1 mutant, HIF1{alpha}DN, blocked HSP90{alpha} secretion (lanes 3 and 4).


Figure 2
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  		Fig. 2. Hypoxia-triggered and HIF1-mediated secretion of HSP90{alpha} is essential for hypoxia-induce HK migration. (A) The HIF1{alpha}-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{alpha} 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{alpha} (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{alpha} 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).

 
We used two independent approaches to study the role of secreted HSP90{alpha} in hypoxia signaling. First, since HSP90{alpha} secretion is mediated by the unconventional exosomal trafficking pathway (Hegmans et al., 2004Go; Clayton et al., 2005Go; Yu et al., 2006Go), we investigated whether inhibition of the exosome-mediated protein trafficking affects hypoxia-stimulated HK migration. Dimethyl amiloride (DMA), a specific inhibitor of the exosomal trafficking pathway, inhibited hypoxia-stimulated HK migration in a dose-dependent manner (Fig. 2B, bars 4-7 versus bar 2). By contrast, brefeldin A (BFA), a specific inhibitor of the classical endoplasmic reticulum/Golgi trafficking pathway, showed little inhibition of hypoxia-driven HK migration (Fig. 2B, bar 3). We also used an anti-HSP90{alpha} neutralizing antibody to specifically block the function of extracellular HSP90{alpha}, since antibodies do not have access to intracellular HSP90{alpha}. As shown in Fig. 2C, the presence of the antibody, but not an IgG control, inhibited hypoxia-stimulated HK migration in a dose-dependent fashion (Fig. 2C bars 4-7 versus bars 1 and 3). These results demonstrate that hypoxia-triggered secretion and extracellular function of HSP90{alpha} are required for hypoxia-driven HK migration.

LRP1 is essential for both hypoxia- and HSP90{alpha}-driven HK migration
LRP1 (LDL receptor-related protein 1) is known as a common receptor for a variety of heat shock proteins, including extracellular HSP90{alpha} (Basu et al., 2001Go). Therefore, we speculated that hypoxia creates an `HSP90{alpha}-LRP1 autocrine loop' to support cell migration. LRP1 consists of a 515 kDa extracellular subunit and a membrane-anchoring 85 kDa subunit, two proteolytic products from a common 600 kDa precursor (Strickland et al., 1990Go). We undertook three independent approaches to assess the importance of LRP1: (1) we used a neutralizing antibody against the extracellular ligand binding domain of LRP1 to block HSP90{alpha} binding to LRP1; (2) we used the lentiviral FG12 system to express two independent siRNAs to downregulate the endogenous LRP1 to an undetectable level; and (3) we re-introduced a siRNA-resistant LRP1 receptor cDNA into LRP1-downregulated HKs to rescue the responsiveness of the cells to hypoxia. Hypoxia and HSP90{alpha} strongly stimulated HK migration (Fig. 3A, bars 2 and 3 versus bar 1) as expected. Control IgG showed little effect (Fig. 3A, bars 2 and 3). However, the addition of anti-LRP1 antibodies completely blocked both hypoxia-induced (Fig. 3A, bar 5 versus bar 2) and HSP90{alpha}-stimulated (Fig. 3A, bar 6 versus bar 3), HK migration. Second, two designed RNAi sequences, RNAi-1 and RNAi-2, completely downregulated endogenous LRP1, as shown in Fig. 3B (insert, lane 2 and 3 versus lane 1). When these LRP1-null HKs were subjected to migration assays, either under normoxia plus HSP90{alpha} or hypoxia, they failed to exhibit increased migration in response to either hypoxia (Fig. 3B, bars 5 and 8) or HSP90{alpha} (Fig. 3B, bars 6 and 9). Third, to ensure the specificity of LRP1 function, we re-introduced a siRNA-resistant functional fragment of the LRP1 receptor, mLRP1 (Obermoeller-McCormick et al., 2001Go), into the LPR1-downregulated HKs. As shown in Fig. 3C, an expected major 150 kDa m-LRP1 receptor and a partially processed 85 kDa subunit were detected by anti-LRP1 antibody immunoblotting (against the p85 subunit of LRP1). The HKs expressing mLRP1 regained migratory responsiveness to both HSP90{alpha} and hypoxia (bars 8 and 9 versus bars 5 and 6). Taken together, these data provided evidence of a novel autocrine mechanism by which hypoxia promotes HK migration: hypoxia -> HIF1 -> HSP90{alpha} secretion -> LRP1 receptor -> cell migration. A schematic representation of these findings is shown in Fig. 3D.


Figure 3
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  		Fig. 3. LRP1 mediates autocrine HSP90{alpha} to promote HK migration. (A) The effect of anti-LRP1 neutralizing antibody on HK migration in response to hypoxia or HSP90{alpha}. 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{alpha} (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{alpha} secretion. Secreted HSP90{alpha} 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{alpha}, remains to be determined.

 

LRP1 belongs to a family of seven members related to the LDL receptor (Lillis et al., 2008Go). Deletion of the LRP1 gene leads to embryonic lethality in mice (Herz et al., 1992Go). It has been reported to bind a wide variety of extracellular molecules, including lipoproteins, proteases and their inhibitors, ECMs and growth factors (Lillis et al., 2008Go). Whereas LRP1 is widely expressed in different cell types, its expression is altered in breast cancer cells. In these cells, this alteration correlates with the invasiveness of the cancer (Lillis et al., 2008Go). How LRP1 mediates HSP90{alpha} signaling to promote HK migration remains largely unclear. A number of reports linked LRP1 signaling to the PDGF receptor. Activation of the PDGF receptor led to LRP1 tyrosine phosphorylation (Broucher et al., 2002; Loukinova et al., 2002Go). However, we observed that downregulation of LRP1 had little effect on TGF{alpha}-stimulated HK migration (Cheng et al., 2008Go) or PDGF-BB-stimulated dermal fibroblast migration (C.-F.C. and W.L., unpublished). Nonetheless, the main discovery of this work, that the HSP90{alpha}-LRP1 autocrine loop plays an essential role in hypoxia-driven cell migration, may have broad implications.

Materials and Methods

Primary human neonatal keratinocytes (HKs) were purchased from Clonetics (San Diego, CA) and cultured in EpiLife medium with added human keratinocyte growth supplements (HKGS), according to the manufacturer's instructions. HKs at passages four to five were used in all the experiments throughout this study. Native rat-tail type I collagen was from BD Biosciences (Bedford, MA). Colloidal gold (gold chloride, G4022) was purchased from Sigma (St Louis, MI). The cDNAs that encode HIF1{alpha} (WT), HIF1{alpha}CA5 (constitutively active) and HIF1{alpha}{Delta}NB{Delta}AB (dominant negative) were gifts from Gregg Semenza (Johns Hopkins University, Baltimore, MA). Anti-HIF1{alpha} antibody (#610958) and anti-HIF1β antibody (#611078) were from BD Transduction Laboratories (Lexington, KY). Anti-HSP90{alpha} antibody (SPA-840) for western analysis and anti-HSP90{alpha} neutralizing antibody (SPS-771) were from Stressgen (Victoria, BC, Canada). Recombinant HSP90{alpha} was prepared in our laboratory as previously described (Cheng et al., 2008Go). Anti-LRP1/CD91 neutralizing antibody was purchased from Progen Biotechnik (Heidelberg, Germany).

Hypoxia
Multi-chamber OxyCycler C42 from BioSpherix (Redfield, NY) was used as an oxygen content controller in this study. This equipment allows creation of a full-range oxygen content regulation from 0.1% to 99.9%, as well CO2 control from 0.1% to 20.0%. All media used in hypoxia experiments were pre-incubated in the chambers with the designated oxygen content overnight.

Cell migration assays
An updated protocol for the colloidal gold cell motility assay, including data and statistical analysis, and a recently modified in vitro wound-healing assay (including procedures for pre-coating with ECMs, cell plating, scratching and quantification of migration data) were recently described by us (Li et al., 2004Go; Fan et al., 2006Go).

Sub-cloning, production of lentivirus stocks and infection
Wild-type or mutant HIF1{alpha} cDNAs were inserted into the lentivirus-derived vector pRRLsinhCMV at BamHI-EcoRI sites, tested, and confirmed as previously described (Li et al., 2007Go).

FG-12 RNAi delivery system
Four potential sites were selected and synthesized using the RNAi Selection Program (Yuan et al., 2004Go). The effectiveness of synthetic double strand siRNA in downregulation of LRP1/CD91 was measured in 293 cells by transfection and the cell lysates blotted with a corresponding anti-LPR1 antibody. The two most effective RNAi sequences were cloned into the lentiviral RNAi delivery vector, FG-12, as previously described (Qin et al., 2003Go). The two selected RNAi sequences (sense) against human LRP1 were used for FG-12 cloning, as previously described (Cheng et al., 2008Go).

Footnotes

We wish to thank Greg Semenza for the cDNAs of HIF1 and HIF1 mutants. This study was supported by NIH grant GM/AR67100-01 (to W.L.) and AR46538 (to D.T.W.). Deposited in PMC for release after 12 months.

* Present address: Department of Plastic Surgery, Xijing Hospital, FMMU, China Back

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