Lipopolysaccharide and Hypoxia Modulate Collagen Metabolism in Human Gingival Fibroblasts

Objectives: Collagens are key structural components of connective tissues. Biochemical alternation of collagen metabolism during periodontal bacterial infection leads to tissue breakdown. We examined collagen I metabolism in human gingival fibroblasts (HGF) and systemically studied the impact of hypoxia and lipopolysaccharides (LPS), which are both intimately involved in the pathogenesis of periodontal disease. Methods: Primary HGF were treated with Escherichia coli LPS, Toll-like receptor (TLR) 4-neutralizing antibody, and/or hypoxia-inducible factor α (HIF-α) inhibitor 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) under different oxygen concentrations. The transcript levels of 35 genes directly related to collagen I synthesis and degradation were examined using Real-Time PCR. The concentrations of collagen I and tissue inhibitor of metalloproteinase (TIMP)-3 from cultured cells were measured by ELISA. The activities of matrix metalloproteinase (MMP) -2 and -9 were examined using gelatin zymography. Results: Although the transcript and protein production of collagen I propeptide remained unchanged, hypoxia and/or LPS enhanced the transcription of several enzymes related to collagen assembly and crosslinking, including prolyl 4-hydroxylases, lysyl hydroxylases and lysyl oxidases. The mRNA levels of MMP-1, 2, 8, 9, 13 and 14 remained stable, as did the enzymatic activity of MMP-2 and -9. However, levels of TIMP-1 and -3 were increased by hypoxia. The observed cellular reactions under hypoxic conditions could be reversed by YC-1 treatment. Conclusions: Hypoxia and LPS appeared to alter cellular reactions in HGF by maintaining or upregulating signals involved in collagen I matrix homeostasis possibly mediated through HIF-α. Study supported by: HKUCRCG 10400006, 10400878; approved by IRB UW 06-376.