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First published online 19 August 2008
doi: 10.1242/jcs.029256

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HIF1 transcription factor regulates laminin-332 expression and keratinocyte migration

Giorgos Fitsialos^1,2,*, Isabelle Bourget^1,2,*, Séverine Augier^1,2, Amandine Ginouvès³, Roger Rezzonico^1,2, Teresa Odorisio⁴, Francesca Cianfarani⁴, Thierry Virolle^1,2, Jacques Pouysségur³, Guerrino Meneguzzi^1,2, Edurne Berra^3,5, Gilles Ponzio^1,2, and Roser Buscà^1,2

¹ INSERM U634, 28 avenue de Valombrose, F-06107 Nice, France
² Université de Nice Sophia Antipolis, Faculté de Médecine, F-06000 Nice, France
³ CNRS UMR 6543, Centre Antoine Lacassagne, 33, avenue de Valombrose, 06189 Nice, France
⁴ Laboratory of Molecular and Cellular Biology, IDI-IRCCS, via Monti di Creta, 104 00167 Roma, Italy
⁵ CICbioGUNE, Cell Biology and Stem Cells Unit, Technologic Park of Bizkaia, 48160 Derio, Spain

View larger version (35K): [in this window] [in a new window]   Fig. 1. Keratinocyte scratch wounding upregulates HIF1 target genes but not expression of HIF1 mRNA. (A) Wild-type human keratinocytes were scratch wounded using the `scarificator' device. Total mRNA was extracted from the cells at different times after injury and submitted to biochip screening. Histograms represent the mRNA increase of six different HIF1 target genes found induced in the DNA array screening. (B) HIF1 mRNA levels in wounded cells measured by DNA array (upper panel) and by real-time quantitative PCR (lower panel). NW, non-wounded cells. Data are mean ± s.e.m. of three independent experiments performed in triplicate.

View larger version (64K): [in this window] [in a new window]   Fig. 2. Wounding increases HIF1 protein concentration in keratinocyte cells. (A) Western blot detection of HIF1 from normal human keratinocytes (NHK) and HaCaT cell lysates at different times after scratch wounding. GAPDH was monitored as a gel-loading control. NW, not-wounded; W, wounded. (B) Immunofluorescence analysis of HIF1 in NHKs wounded using a micropipette tip (green labelling). Nuclei were stained with DAPI dye (blue). NW, not-wounded; W, 15 hours after wounding. In the upper panel, the dotted line corresponds to the wound incision and the arrows indicate the direction of migrating keratinocytes. The lower panel shows images depicting the activation of HIF1 in keratinocytes far from the wounded area of the cell monolayer. Scale bar: 20 µm. (C) HIF1 immunolabelling using peroxidase staining of paraffin slices obtained from mice epidermis harvested 1, 3, 5, 7 and 9 days (d) after injury. Arrows indicate HIF1-positive keratinocytes. Scale bar: 60 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 3. PI3K inhibition abrogates the injury-dependent increased levels of HIF1. HaCaT cells and normal human keratinocytes (NHK) were incubated for 1 hour in the presence of the PI3K inhibitor LY294002 (15 µM) and were then scratch wounded. Cells were collected 1 and 3 hours after injury and submitted to western blot analysis to detect HIF1 and phosphorylated AKT (as a reporter of the PI3K activity). NW, non-wounded cells. GAPDH was detected as a loading control.

View larger version (60K): [in this window] [in a new window]   Fig. 4. Depletion of HIF1 inhibits keratinocyte cell migration. (A) HaCaT keratinocytes transfected with a nonrelevant siRNA (siCtrl) and a siRNA targeting HIF1 (siHIF1) were scratch wounded with a micropipette tip and filmed for 24 hours using time-lapse videomicroscopy. The wound closure is illustrated by showing the wound size (arrow and a) immediately after injury and 12 and 24 hours after wounding. The right panel represents the quantification of the siHIF1 effect on the wound closure calculated by the measure of the diminution of the wound bed surface upon time by using Image J software. Results are represented as the percentage of the wound closure of monolayers obtained with cells transfected with the siHIF1 compared with that observed in the cultures of cells transfected with the nonrelevant siRNA (siCtrl) (taken as the 100% value). Each experiment was performed at least three times, and different areas of several scratches were randomly taken. (B) siRNA-transfected HaCaT keratinocytes were seeded in the upper chambers of Transwell inserts. After 24 hours, cells on the lower side of the filter were stained with DAPI and scored in five independent fields. Experiments were carried out three times and each condition was performed in triplicate. (C) Verification of HIF1 abrogation by the specific siRNA duplex. Transfected cells were scratch wounded, harvested 6 hours later then HIF1 mRNA and protein were analysed respectively by real time Q-PCR (left panel) and by western blotting (right panel).

View larger version (58K): [in this window] [in a new window]   Fig. 5. Expression of laminin-332 is upregulated upon scratch wounding and correlates with HIF1 induction. (A) Histograms representing the quantification obtained by microarray analysis of the LAMA3 transcripts at different time points after cell wounding (left panel). Results were confirmed by real time Q-PCR (right panel). Values are mean ± s.e.m. of three independent experiments performed in triplicate. NW, not-wounded; W, wounded. (B) Immunofluorescence detection of HIF1 (green) and laminin-332 (BM165 antibody; red), 15 hours after wounding (W), in NHKs (upper panel) and HaCaT (lower panel), compared with nonwounded counterparts (NW). Nuclei were detected using DAPI (blue). The dotted line in the upper panel highlights the original position of the scratch wound edge, and the arrows indicate the direction of cell migration. The lower panel shows HaCaT cells in a nonwounded area of the cell culture far from the wound site. Scale bar: 20 µm. (C) Left panel, HaCaT cells untreated (NS) or treated with Cobalt (Co2+) for 15 hours to increase HIF1 protein. HIF1 protein that was measured by western blot analysis. GAPDH was detected as a loading control. Right panel, real-time quantitative PCR showing normalised LAMA3 mRNA expression in HaCaT cells nonstimulated (NS) or treated with Co2+ for 15 hours. (D) HaCaT (lower panel) and NHK cells (upper panel) were either left untreated (NS) or treated with Co2+. After 24 hours, cells were fixed and immunofluorescence analysis was carried out to detect HIF1 protein (green) and laminin-332 (red). Nuclei were stained using the DAPI. Scale bar: 20 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 6. HIF1 depletion downregulates laminin-332. (A) Real-time quantitative PCR analysis of LAMA3 mRNA expression in HaCaT cells transfected with a nonrelevant siRNA (siCtrl) (grey) or a siRNA to HIF1 (siHIF1) (black). Q-PCR analysis was performed using transfected cells either not wounded (NW) or harvested 6 hours after wounding (W). Results were obtained from three independent experiments performed in triplicate and are expressed as normalised LAMA3 mRNA `fold increase' relative to the siCtrl-transfected non-wounded cells (siCtrl-NW). (B) Immunofluorescence detection of HIF1 (green) and laminin-332 (red) in HaCaT cells transfected with the nonrelevant siCtrl, a siRNA to HIF1 or a siRNA targeting PHD2 (that stabilised and upregulated HIF1 expression). Cells transfected with the siPHD2 siRNA were not wounded. Cells were fixed 15 hours after either scratch wounding (Wounded) or CoCl2 treatment (Co2+ treated). Nuclei were stained using DAPI. Scale bar: 40 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 7. HIF1 directly binds to the human LAMA3 promoter and stimulates its activity. (A) Luciferase reporter assays of the human LAMA3 promoter activity performed in HaCaT cells transfected with either an empty vector (pcDNA3) or constructs encoding either HIF1 or USF1. Results are expressed as the fold stimulation of the luciferase activity measured in cells transfected with the pcDNA3 empty vector. (B) Functional activity of the HIF1 construct was verified by cotransfecting HIF1 together with a reporter plasmid containing three HRE consensus sites cloned upstream a minimal thymidine kinase (TK) promoter and the luciferase reporter gene. (C) Partial DNA nucleotide sequence of the LAMA3 promoter. Nucleotide numbering is relative to the first nucleotide of the transcription initiation site (+1). AP1a, AP1b and AP1c indicate the position of the three reported AP1 sites. The HRE consensus sequence is represented by a box at position –108. Arrows indicate the transcription start site and the beginning of the DNA coding sequence. (D) Effect of HIF1 on the transcriptional activity of the human LAMA3 promoter mutated at the –108 HRE consensus site. The Mut hLAMA3 promoter driving the expression of the luciferase reporter gene was cotransfected in HaCaT cells with either an empty vector (pcDNA3) or a construct encoding HIF1. A control condition where the Mut hLAMA3-Luc promoter was cotransfected with an USF1-encoding plasmid was also performed. In A, B and D, the histograms show the mean ± s.e.m. of the results obtained in three independent experiments resulting from transfections performed using different plasmid preparations. (E) A ChIP assay was performed on extracts from 6 hour CoCl2-stimulated HaCaT keratinocytes. HIF1 immunoprecipitation was performed using a specific anti-HIF1 antibody. Primers spanning the LAMA3 (upper panel) or the VEGF (lower panel) promoter regions were used for PCR amplification. In (input) represents the control of the PCR amplification performed using HaCaT genomic DNA, which showed a 142 bp and a 104 bp band corresponding to the amplification of the LAMA3 and VEGF promoter regions, respectively. The IgG (M,R) and Pol2 lanes indicate the PCR amplification obtained when the extracts were immunoprecipitated with a non immune mouse immunoglobulin (M), a non relevant rabbit antibody (R) or an antibody raised against the RNA polymerase II (Pol2). The Pol2 antibody was included in the kit and represents a supplemental positive control, according to the manufacturer. PG, amplification after immunoprecipitation with protein-G-coupled beads only.

View larger version (20K): [in this window] [in a new window]   Fig. 8. Illustration of a hypothetical mechanism for the role of HIF1 in the control of keratinocyte migration during wound closure. The disruption of the integrity and cohesion of the keratinocyte monolayer consequent to scratch wounding activates PI3K signalling, which leads to an increased expression of HIF1 and results in the upregulation of the HIF1 target genes among which is LAMA3 that encodes the 3 subunit of laminin-332 and reflects the upregulated synthesis of laminin-332. Enhanced deposition of laminin-322 to the ECM, strongly influences the keratinocyte adhesion and their migration capacity.

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