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J. Biol. Chem., Vol. 282, Issue 17, 12475-12483, April 27, 2007

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A Rapid Transient Increase in Hyaluronan Synthase-2 mRNA Initiates Secretion of Hyaluronan by Corneal Keratocytes in Response to Transforming Growth Factor ^*

From the UPMC Eye Center, Ophthalmology and Visual Sciences Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

Keratocytes of the corneal stroma produce transparent extracellular matrix devoid of hyaluronan (HA); however, in corneal pathologies and wounds, HA is abundant. We previously showed primary keratocytes cultured under serum-free conditions to secrete matrix similar to that of normal stroma, but serum and transforming growth factor (TGF) induced secretion of fibrotic matrix components, including HA. This study found HA secretion by primary bovine keratocytes to increase rapidly in response to TGF, reaching a maximum in 12 h and then decreasing to <5% of the maximum by 48 h. Cell-free biosynthesis of HA by cell extracts also exhibited a transient peak at 12 h after TGF treatment. mRNA for hyaluronan synthase enzymes HAS1 and HAS2 increased >10- and >50-fold, respectively, in 4–6 h, decreasing to near original levels after 24–48 h. Small interfering RNA against HAS2 inhibited the transient increase of HAS2 mRNA and completely blocked HA induction, but small interfering RNA to HAS1 had no effect on HA secretion. HAS2 mRNA was induced by a variety of mitogens, and TGF acted synergistically to induce HAS2 by as much as 150-fold. In addition to HA synthesis, treatment with TGF induced degradation of fluorescein-HA added to culture medium. These results show HA secretion by keratocytes to be initiated by a rapid transient increase in the HAS2 mRNA pool. The very rapid induction of HA expression in keratocytes suggests a functional role of this molecule in the fibrotic response of keratocytes to wound healing.

Received for publication, October 2, 2006 , and in revised form, January 19, 2007.

^* This work was supported by National Institutes of Health Grants EY09368 and P30-EY08098, Research to Prevent Blindness, and the Eye and Ear Foundation of Pittsburgh. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Jules and Doris Stein Research to Prevent Blindness Professor. To whom correspondence should be addressed: Dept. of Ophthalmology, University of Pittsburgh, 1009 Eye and Ear Institute, 203 Lothrop St., Pittsburgh, PA 15213-2588. Tel.: 412-647-3853; Fax: 412-647-5880; E-mail: jlfunder{at}pitt.edu.

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