N-Acylation of Glucosamine Modulates Chondrocyte Growth, Proteoglycan Synthesis, and Gene Expression

DORIS E. TERRY, KAREN REES-MILTON, PATRICK SMITH, JOHN CARRAN, PARVIS PEZESHKI, CRAIG WOODS, PETER GREER, and TASSOS P. ANASTASSIADES

ABSTRACT.

Objective. To examine the effects of glucosamine (GlcN) and some N-acylated (GlcNAcyl) derivatives on the proliferation and proteoglycan (PG) synthesis of bovine articular chondrocyte (BAC); and to expand these results to human articular chondrocytes (HAC) and study the modulation of gene regulation by these compounds.

Methods. The compounds tested were: glucose (Glc), GlcN.HCl, N-acetyl GlcN (GlcNAc), and N-butyryl GlcN, (GlcNBu). GlcNBu was synthesized from GlcN and butyric anhydride. For the chondrocyte cultures, both anchorage-dependent (AD) and an anchorage-independent (AI) system (alginate beads) were evaluated. Following the various additions, BAC were assessed for total cell number, DNA, or total PG synthesis at different times. Utilizing similar conditions, human cDNA microarrays were performed for the HAC after harvesting total RNA.

Results. For AD cultures, the addition of GlcN.HCl (0.1–5.0 mM) to BAC or HAC cultures inhibited cell proliferation and total PG synthesis in a dose-dependent manner. For AI cultures, the inhibitory effects of GlcN.HCl on cell proliferation were less prominent, and PG synthesis increased slightly more for the GlcNAcyl than the GlcN additions. In the AD system, the addition of GlcNAc did not result in the inhibitory effect of GlcN.HCl, while GlcNBu addition resulted in an increase in BAC proliferation and PG synthesis that could not be explained by the Bu moiety alone. For the HAC, additions of 0.1 mM GlcNBu resulted in upregulation of a large number of genes, with only a few downregulated, while GlcN addition resulted in no upregulation and one downregulated gene, for preset stringency criteria.

Conclusion. Addition of GlcNBu to BAC or HAC to AD cultures generally stimulated cell proliferation and PG synthesis, while addition of GlcN resulted in inhibition of these indicators. The inhibitory effects of the GlcN molecule appear to be related to the unsubstituted amino group. Additions of GlcNBu, but not GlcN, to HAC resulted in upregulation in the expression of a large number of genes. (J Rheumatol 2005;32:1775-86)

Key Indexing Terms:

ARTICULAR CARTILAGE
CHONDROCYTE
GLUCOSAMINE
GLUCOSAMINE SULFATE
ACYLATION
GENE REGULATION

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From the Department of Medicine, Department of Pathology, and Department of Biochemistry, Queen's University, Kingston, Ontario, Canada; and Veterinary Products Laboratories, Farnam Companies, Phoenix, Arizona, USA.

Supported by the Canadian Institutes of Health Research (MOP 43833), the National Science and Engineering Research Council of Canada (STP 246 039-01), and Farnam Companies, Inc., Phoenix, Arizona.

D.E. Terry, PhD, Department of Medicine; K. Rees-Milton, PhD, Department of Medicine; P. Smith, MSc, Department of Pathology; J. Carran, PhD, Department of Medicine; P. Pezeshki, BSc, Department of Medicine; P. Greer, PhD, Department of Pathology; T.P. Anastassiades, MD, PhD, Departments of Medicine and Biochemistry, Queen's University; C. Woods, DVM, Veterinary Products Laboratories, a division of Farnam Companies, Phoenix, Arizona.

Address reprint requests to Dr. T. Anastassiades, Department of Medicine, Queen's University, Kingston, Ontario K7L 3N6, Canada. E-mail: anastass@post.queensu.ca

Accepted for publication May 17, 2005.

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