1. Upregulation of CD44 and Hyaluronate Synthases by Topical Retinoids in Mouse Skin 
Gürkan Kaya, Denise Grand, Raymonde Hotz, Eric Augsburger, Pierre Carraux, Liliane Didierjean, Jean-Hilaire Saurat 
Journal of Investigative Dermatology 
Volume 124, Issue 1, Pages (January 2005)
DOI: /j X x
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Topical application of RAL increases the protein expression of CD44 in mouse skin. Histological sections of vehicle- (A) or retinaldehyde (RAL)- (B) treated SKH1 hairless mouse back skin. Note the hyperplasia and increase in CD44 expression in the epidermis. Groups of five adult (>3 mo old) SKH1 hairless mice (The Jackson Laboratory, Bar Harbor, Maine) were used. RAL was used at the concentration of 0.05% compounded in an oil-in-water cream (Saurat et al, 1994). Retinoid cream samples of 0.5 g or the vehicle were applied daily for 7 d on the back skin of SKH1 hairless mice. The daily amount of retinoid delivered corresponded to 250 μg. Twenty hours after the last application, the animals were killed by decapitation, back skin was fixed in 10% phosphate-buffered formaldehyde, embedded in paraffin, and processed for histological analysis. Sections were cut at 5 μm, mounted onto slides, dewaxed in xylene, and rehydrated in a graded ethanol series. Endogenous peroxidase activity was blocked by incubating the sections with 0.03% H2O2 in anhydrous methanol for 45 min at room temperature. Antigen demasking was performed by subjecting the sections to microwave treatment in citrate buffer (pH 6.0) for 20 min at 1200 W. Then the sections were incubated with rat IgG2b isotype control (PharMingen, San Diego, California) for 30 min at room temperature and biotinylated rabbit anti-rat antibodies (DakoCytomation, Glostrup, Denmark) for 30 min at room temperature, or only with biotinylated rat anti-mouse CD44 antibodies (IgG2b; PharMingen, San Diego, California; 2.5 μg per mL) for 30 min at room temperature, washed thrice as above, and treated with avidin–biotin–peroxidase for 30 min at room temperature. The sections were then washed with the buffer and incubated in 0.05% DAB (3,3′-diaminobenzidine; Sigma, St. Louis, Missouri) and 0.03% H2O2 in the phosphate buffer at room temperature. All sections were examined under a Zeiss axiophot microscope (Zeiss, Oberkochen, Germany) using appropriate filters.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Topically applied RAL increases the HA content of mouse skin. (a) Histological sections of vehicle- (A) or retinaldehyde (RAL)- (B) treated SKH1 hairless mouse back skin (three animals per group) stained with hyaluronate (HA) binding protein (HABP). Strong epidermal and dermal HABP reactivity was observed in RAL-treated skin (B). Histochemistry was performed by using biotinylated HABP (Seikagaku Kogyo, Tokyo, Japan; 25 ng per mL) instead of biotinylated anti-CD44 antibodies as described in the legend of Figure 1. (b) Epidermal HA content in untreated and vehicle- or RAL-treated SKH1 hairless mouse back skin quantified by an enzyme-linked binding protein assay. Quantification of HA by this assay was performed using Corgenix Hyaluronic Acid Quantitative Test Kit (Endotell, Allschwil, Switzerland) on mouse epidermal extracts according to the manufacturer's instructions. Post mortem immediately frozen (-80°C) mouse skin samples were incubated with NaBr (2 M) for 90 min at 37.5°C to separate the epidermis from the dermis. Contamination of the epidermis with the dermis was excluded by standard histological analysis. The epidermal samples were then put in extraction buffer containing 20 mM Tris/HCl (pH 7.5), 100 mM NaCl, 10 mM EDTA, 1% SDS, 10% glycerol, and protease inhibitor cocktail (complete Boehringer, Biberach, Germany), minced, polytron-homogenized, and sonicated on ice. Cell culture extracts were treated with the same buffer. Homogenates were spun in a microfuge for 20 min, and the soluble fraction was extracted and subjected to the enzyme-linked binding protein assay. The results were represented as the mean HA concentration±SD of three animals per group. ***p<0.01 versus RAL; **p<0.05 versus RAL (student's t test).
Journal of Investigative Dermatology  , DOI: ( /j X x)
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4. Figure 3
Topical RAL increases the RNA expression of CD44 and hyaluronate synthases in mouse skin. (a) Northern blot analysis of CD44 RNA expression in vehicle- (cont) or retinaldehyde (RAL)-treated SKH1 hairless mouse back skin (three animals per group). Note the increase of the three CD44 transcripts (5.2, 4.0, and 3.2 kb) in RAL-treated skin. Total RNA from the whole back skin of SKH1 hairless mice topically treated with RAL was prepared using a TRIzol reagent kit (Invitrogen, Carlsbad, California) and digested with RNase-free DNase 1 (Ambion, Austin, Texas). Eight micrograms of isolated RNAs were separated electrophoretically on a 1% agarose gel containing glyoxal and transferred onto a nitrocellulose membrane. The membrane was UV-cross-linked and probed with [32P]UTP-labeled CD44 cDNA (1 × 106 cpm per mL). Hybridization with the CD44 probe prepared using random primers and a 1.1 kb CD44 fragment purified from HindIII-EcoRI-digested mCD44H-pBS plasmid template (Kaya et al, 1997) was performed overnight at 60°C in 50% formamide, 1 × sodium chloride-sodium citrate, 5 × Denhardt's reagent, and 0.2% tRNA. The membrane was washed twice at 68°C for 30 min in 0.1 × sodium chloride-sodium citrate and 0.1% sodium dodecyl sulfate. Kodak (Rochester, New York) X-Omat AR film was exposed overnight at -70°C. The integrity of total RNA is good, and the ratio of 28S and 18S ribosomal RNAs is ∼2:1 in all samples. (b) Northern blot analysis of HAS2 and HAS3 RNA expression in vehicle- (cont) or RAL-treated SKH1 hairless mouse back skin (three animals per group). Note the increase of the 3.2 and 4.8 kb HAS2, and 4.0 and 6.0 kb HAS3 transcripts in RAL-treated skin. Hybridization was performed with mouse HAS2 (1656 bp) and HAS3 (1662 bp) cDNA probes (kindly provided by Professor I. Stamenkovic, University Hospital of Lausanne, Switzerland) as described above.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions