Expression regulation of hyaluronan synthase in corneal endothelial cells.
OBJECTIVE
Our previous study showed that hyaluronan synthase (HAS), the enzyme protein of hyaluronan (HA) biosynthesis, is expressed in ocular tissues including the corneal endothelium. In the current study, the mechanism that regulates HAS expression in bovine corneal endothelial cells (BCECs) was investigated.


METHODS
Cultured BCECs were used. HAS expression in BCECs at the mRNA level was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis. The effects of transforming growth factor (TGF)-beta and platelet-derived growth factor (PDGF)-BB on HAS expression were examined by quantitative RT-PCR. The involvement of the Smad family (intracellular signal transducer of TGF-beta) was also investigated. The expression of HAS in BCECs at the protein level was confirmed by immunocytochemistry and Western blot analysis.


RESULTS
Three HAS isoforms in BCECs were expressed at the mRNA level. The transcriptional sizes of each HAS in BCECs were 4.9 kb for HAS1, 2.8 kb for HAS2, and 1.6 kb for HAS3. The expression of HAS2 at the mRNA level was stimulated by TGF-beta1 and/or PDGF-BB treatment. In contrast, HAS1 and HAS3 expression was not affected by these growth factors. The additive effects of TGF-beta1 and PDGF-BB were observed in the stimulation of the expression levels of HAS2. HAS2 upregulation by these growth factors was also detected by Western blot analysis. The stimulation of the expression of HAS2 at the mRNA level by TGF-beta was accelerated by the overexpression of Smad2, Smad3, and Smad4 and inhibited by that of Smad7, all of which were confirmed to be involved in the signal transduction from TGF-beta through HAS expression.


CONCLUSIONS
Although three HAS isoforms were expressed in the corneal endothelial cells, the expression of HAS2 was upregulated by TGF-beta1 and/or PDGF-BB. HAS2 expression was regulated by TGF-beta through Smad family members.
Usui
T
T
Department of Ophthalmology, Faculty of Medicine, University of Tokyo.
Amano
S
S
Oshika
T
T
Suzuki
K
K
Miyata
K
K
Araie
M
M
Heldin
P
P
Yamashita
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000077214:Becaplermin; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D014453:Glucuronosyltransferase / Q000096:biosynthesis / Q000235:genetics*; D016695:Glycosyltransferases; D000076002:Hyaluronan Synthases; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014166:Transferases; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D015854:Up-Regulation; D029867:Xenopus Proteins
D017931:DNA Primers; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D029867:Xenopus Proteins; D000077214:Becaplermin; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases