Expression and activity of hyaluronidase in human periodontal ligament fibroblasts.
BACKGROUND
Hyaluronan is a major component of the extracellular matrix of periodontal ligament (PDL) contributing to the structural and functional integrity. Hyaluronans contribute to the buffering effect of the PDL during chewing, and they are also important in inflammation and wound healing. Hyaluronan is known to be synthesized and turned over by the resident PDL cells, although the mechanisms of hyaluronan metabolism still remain unclear. Hyaluronidase (HAase), an endoglycosidase, degrades hyaluronan into small fragments. Currently, 3 human HAases, HYAL1, HYAL2, and PH-20, have been identified and well characterized.


METHODS
This study was conducted to investigate the expression and activity of these HAases in cultured human PDL fibroblasts and to elucidate the mechanisms involved in hyaluronan metabolism under normal and inflammatory conditions. Human PDL fibroblasts derived from the periodontium of 3 premolars were cultured with or without interleukin (IL)-1beta (0.1 to 10 ng/ml) and tumor necrosis factor (TNF)-alpha (1 to 100 ng/ml) for 0 to 48 hours. The expression of HAase mRNA was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR, and the enzymatic activity was examined using hyaluronan zymography.


RESULTS
PDL fibroblasts expressed HYAL1 and HYAL2 mRNAs, but not PH-20 mRNA. The expression of HYAL1 mRNA was enhanced by about 3.5- and 3.7-fold at maximum after 1-hour stimulation with 1 ng/ml IL-1beta and after 3-hour stimulation with 10 ng/ml TNF-alpha, respectively. The expression of HYAL2 and PH-20 mRNAs was not affected by stimulation with cytokines. HAase activity was detected in conditioned medium from PDL fibroblast cultures, and the activity was enhanced by treatment with 10 ng/ml TNF-alpha.


CONCLUSIONS
These results suggest that PDL fibroblasts express HAases and generate HAase activity essential for extracellular hyaluronan metabolism under physiological and inflammatory conditions.
Ohno
Shigeru
S
Department of Orthodontics, Hiroshima University Faculty of Dentistry, Hiroshima, Japan. shigebon@hiroshima-u.ac.jp
Ijuin
Chise
C
Doi
Takeyoshi
T
Yoneno
Kiyoshi
K
Tanne
Kazuo
K
D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism*; D006821:Hyaluronoglucosaminidase / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology; D010513:Periodontal Ligament / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase