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First published online 2 December 2003
doi: 10.1242/jcs.00831

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Hyaluronan-oligosaccharide-induced transcription of metalloproteases

Christina Fieber^1,*, Petra Baumann¹, Rüdiger Vallon^1,, Christian Termeer², Jan C. Simon^2,**, Martin Hofmann^1,, Peter Angel^1,¶, Peter Herrlich¹ and Jonathan P. Sleeman^1,

¹ Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, and University of Karlsruhe, Institute of Genetics, PO Box 3640, 76021 Karlsruhe, Germany
² Freiburg University, Department of Dermatology, Hauptstr. 7, 79104 Freiburg, Germany

View larger version (49K): [in a new window]   Fig. 1. HA induces proteinase activity in 3LL cells. (A) HA induces gelatinolytic activity. 3LL cells were incubated with rooster comb HA or chondroitin sulfate A (CS-A) for 48 hours. Proteins in the conditioned medium were then TCA precipitated and used in a zymogram analysis to examine gelatinolytic activties in the conditioned medium. A major gelatinolytic band migrated close to 100 kDa and a minor band at 55 kDa. As a control, culture medium alone was also TCA precipitated and subjected to zymogram analysis (M). (B) 3LL cells were incubated with or without rooster comb HA for 24 hours. The cells were lysed in nonreducing SDS-PAGE sample buffer (lysate). Proteins in the conditioned medium were then TCA precipitated (CM). As a control, culture medium alone containing 0.5 mg ml-1 HA was also TCA precipitated (M+). (C) HA enhances mRNA levels of several proteases. After treatment of 3LL cells with HA, cells were harvested at different times and poly(A)+ RNA was isolated. RNA (4 µg per sample) was analysed by northern blot for expression of the indicated genes.

View larger version (42K): [in a new window]   Fig. 2. Low-molecular-weight HA fragments induce MMP gene expression in MEFs and 3LL cells. (A) MEFs were treated or not with rooster comb HA of mixed molecular weight (R.C. HA), chondroitin sulphate A (CS-A), HealonTM high-molecular-weight HA (High HA), sonificated healon of intermediate molecular weight (Int HA) or with low-molecular-weight fragments of four to six residues (Low HA). Numbers refer to the final concentration of HA in the culture medium (mg ml-1). RT-PCR was performed to analyse the expression levels of Gapdh, Mmp-9 and Mmp-13. (B) 3LL cells were treated or not with 0.1 mg ml-1 Low HA. RT-PCR was performed to analyse the expression of Gapdh, Mmp-9 and Mmp-13. In one lane, cDNA was omitted (H2O control) as a negative control.

View larger version (50K): [in a new window]   Fig. 3. HA mediates the expression of Mmp-9 through interaction with a membrane receptor. MEFs were either treated or not with Suramin before stimulation with rooster comb HA. As a control, MEFs were treated with TNF. Total RNA was prepared and analysed for Mmp-9 and Mmp-13 transcripts by RT-PCR. RT-PCR of GAPDH mRNA was carried out to ensure equal cDNA amounts in the different samples.

View larger version (39K): [in a new window]   Fig. 4. CD44 and RHAMM/IHABP are not the HA receptors that mediate the synthesis of MMP-9 and MMP-13 induced by HA. (A) For a HA-binding assay, soluble FITC-labelled HA (HA-FITC) was added to 3LL cells. To determine whether HA binding is CD44-dependent, cells were preincubated with CD44-specific antibodies KM81 (left) or IM7 8.1 (right) before HA-FITC was added. As a negative control, cells were incubated with unlabelled HA (HA). (B) HA binding to CD44 was blocked in 3LL cells by incubation with KM81 antibody (IgG or F(ab')2) for 45 minutes on ice. The cells were then seeded with or without 0.5 mg ml-1 rooster comb HA and together with KM81 antibody IgG or F(ab')2 (as appropriate). Mmp-9 expression was then analysed by RT-PCR. (C) Expression of Mmp-9 and Mmp-13 was determined by RT-PCR analysis in MEFs deficient for either CD44 or RHAMM/IHABP treated with or without rooster comb HA (1 mg ml-1).

View larger version (49K): [in a new window]   Fig. 5. Activation of Mmp-9 by HA is not abolished in TLR4-deficient embryonic fibroblasts. Wild-type (WT) or TLR4-negative (TLR4-/-) fibroblasts were treated with or without rooster comb HA (1 mg ml-1). Detection of Mmp-9 transcripts was then performed by RT-PCR analysis.

View larger version (33K): [in a new window]   Fig. 6. The HA-mediated induction of MMP-9 and MMP-13 is sensitive to actinomycin D, TPCK and emetine. MEFs were pretreated with either actinomycin D (A), TPCK (B) or left untreated before stimulation with rooster comb HA or TPA. For the detection of MMP-9 and MMP-13 mRNA equal amounts of total RNA from the cells were reverse transcribed and subjected to PCR amplification. As a control for equal amounts of cDNA, RT-PCR analysis of Gapdh was also performed. (C) 3LL cells were pretreated with 50 µM emetine for 45 min, then incubuated for 10 hours with 0.5 mg ml-1 HA and 80 ng ml-1 TPA as indicated. Poly(A)+ RNA was then prepared from the cells and analysed in a northern blot. Filters were hybridised with probes for Mmp-9, Mmp-13 and Gapdh.

View larger version (57K): [in a new window]   Fig. 7. HA causes activation of NF-B (p65/p50) in embryonic fibroblasts. (A) MEFs were treated with or without rooster comb HA. Total cell extracts were prepared at the indicated time points and equal amounts of proteins were analysed for NF-B activity by EMSA. Pretreatment with TPCK blocked the appearance of p65/p50. (B) To determine the specificity of binding, a ten- and 100-fold excess of the unlabelled NF-B-binding oligonucleotide or of the AP-1-binding oligonucleotide were used as competitors. The position of the shift produced by the p65/p50 heterodimer is indicated.

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