May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Keratan Sulfate Biosynthesis Is Selectively Regulated by Cell-Cell Junctions in Keratocytes
Author Affiliations & Notes
  • M. L. Funderburgh
    Ophthalmology and Visual Science Res Ctr, Univ of Pittsburgh Sch of Med, Pittsburgh, Pennsylvania
  • M. M. Mann
    Ophthalmology and Visual Science Res Ctr, Univ of Pittsburgh Sch of Med, Pittsburgh, Pennsylvania
  • J. L. Funderburgh
    Ophthalmology and Visual Science Res Ctr, Univ of Pittsburgh Sch of Med, Pittsburgh, Pennsylvania
  • Footnotes
    Commercial Relationships  M.L. Funderburgh, None; M.M. Mann, None; J.L. Funderburgh, None.
  • Footnotes
    Support  NIH Grants EY09368, EY16415 (JLF), P30-EY08098, Eye Ear Foundation of Pittsburgh, Research To Prevent Blindness
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4827. doi:https://doi.org/
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    • Get Citation

      M. L. Funderburgh, M. M. Mann, J. L. Funderburgh; Keratan Sulfate Biosynthesis Is Selectively Regulated by Cell-Cell Junctions in Keratocytes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4827. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Keratan sulfate (KS) has unique abundance in the corneal stroma and is important for maintenance of corneal transparency. KS secretion by stromal keratocytes is tightly regulated and is reduced in a variety of stromal pathologies. Similarly, KS biosynthesis by keratocytes in vitro responds to stimuli and is rapidly downregulated in response to serum and other mitogens. The molecular mechanisms controlling KS synthesis are still unknown. This study explores our observation that cellular density has an effect on KS biosynthesis by cultured keratocytes.

Methods: : Primary bovine keratocytes were cultured in serum-free media on tissue culture plastic or on non-adhesive surfaces coated with polyhema. Cell-cell association was prevented by methylcellulose in culture media. Glycosaminoglycans in media were detected by western blotting, labeled with 35S-sulfate, or analyzed by FACE (fluorophore-assisted carbohydrate electrophoresis). Abundance of cell-cell junctions was estimated from the ratio of connexin 43 (Cx43) sedimenting in high-speed pellets (cytoskeletal fraction) to soluble Cx43 in Triton-X-100 cell lysates.

Results: : We previously reported (ARVO2006) that keratocytes plated on polyhema aggregated into viable spheroids not attached to substratum. Such aggregates secrete up to 5-fold more KS than attached cells. In spheroids cytoskeletal-associated Cx43 was about 4-fold more abundant than in monolayer cells, demonstrating increased cell-cell junctions. In attached cultures plated at different cell densities, KS synthesis and the proportion of junctional Cx43 increased in direct relation with cell density as did mRNA for CHST6, a corneal KS-specific sulfotransferase. Dermatan sulfate (DS), conversely, was downregulated in the more dense cultures. Prevention of cell-cell association with methylcellulose eliminated KS biosynthesis but stimulated DS. Calcium-free medium similarly disrupted cell-cell junctions, eliminated KS synthesis, and stimulated DS.

Conclusions: : KS biosynthesis is dependent on the presence of keratocyte cell-cell junctions whereas DS, a closely related glycosaminoglycan, is not. Selective dependence of KS synthesis on cell-cell junctions is consistent with its downregulation during mitogenic activation, migration, and cell division in vitro and wound healing in vivo. Signaling pathways controlled by cell-cell junctions such as the Wnt pathway may be involved in regulation of KS biosynthesis.

Keywords: cornea: stroma and keratocytes • extracellular matrix • cell adhesions/cell junctions 
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