α11ß1 integrin-mediated MMP-13-dependent collagen lattice contraction by fibroblasts: evidence for integrin-coordinated collagen proteolysis.

We have previously determined that integrin α11ß1 is required on mouse periodontal ligament (PDL) fibroblasts to generate the force needed for incisor eruption. As part of the phenotype of α11(-/-) mice, the incisor PDL (iPDL) is thickened, due to disturbed matrix remodeling. To determine the molecular mechanism behind the disturbed matrix dynamics in the PDL we crossed α11(-/-) mice with the Immortomouse and isolated immortalized iPDL cells. Microarray analysis of iPDL cells cultured inside a 3D collagen gel demonstrated downregulated expression of a number of genes in α11-deficient iPDL cells, including matrix metalloproteinase-13 (MMP-13) and cathepsin K. α11(-/-) iPDL cells in vitro displayed disturbed interactions with collagen I during contraction of attached and floating collagen lattices and furthermore displayed reduced MMP-13 protein expression levels. The MMP-13 specific inhibitor WAY 170523 and the Cathepsin K Inhibitor II both blocked part of the α11 integrin-mediated collagen remodeling. In summary, our data demonstrate that in iPDL fibroblasts the mechanical strain generated by α11ß1 integrin regulates molecules involved in collagen matrix dynamics. The positive regulation of α11ß1-dependent matrix remodeling, involving MMP-13 and cathepsin K, might also occur in other types of fibroblasts and be an important regulatory mechanism for coordinated extracellular and intracellular collagen turnover in tissue homeostasis.

Use of siRNA in dental tissue-derived cell cultures: integrin knockdown in fibroblasts.

Short (or small) interfering RNAs (siRNAs) are double-stranded RNA molecules about 21-25 nucleotides long that have the capacity to disrupt the activity of genes on a posttranscriptional level. This sequence homology-driven gene silencing capacity has been utilized by researchers to selectively block the translation of mRNA to proteins in order to study specific gene functions and identify target molecules. Importantly, siRNAs have the potential to be used in treatment of disease. Here, we describe how the siRNA technology can be used to knock down genes in dental tissue-derived cells using integrin α11 knockdown as an example.

Mutation in the heparan sulfate biosynthesis enzyme EXT1 influences growth factor signaling and fibroblast interactions with the extracellular matrix.

Heparan sulfate (HS) chains bind and modulate the signaling efficiency of many ligands, including members of the fibroblast growth factor (FGF) and platelet-derived growth factor families. We previously reported the structure of HS synthesized by embryonic fibroblasts from mice with a gene trap mutation of Ext1 that encodes a glycosyltransferase involved in HS chain elongation. The gene trap mutation results in low expression of Ext1, and, as a consequence, HS chain length is substantially reduced. In the present study, Ext1 mutant and wild-type mouse embryonic fibroblasts were analyzed for the functional consequences of the Ext1 mutation for growth factor signaling and interaction with the extracellular matrix. Here, we show that the phosphorylation of ERK1/2 in response to FGF2 stimulation was markedly decreased in the Ext1 mutant fibroblasts, whereas neither PDGF-BB nor FGF10 signaling was significantly affected. Furthermore, Ext1 mutants displayed reduced ability to attach to collagen I and to contract collagen lattices, even though no differences in the expression of collagen-binding integrins were observed. Reintroduction of Ext1in the Ext1 mutant fibroblasts rescued HS chain length, FGF2 signaling, and the ability of the fibroblasts to contract collagen. These data suggest that the length of the HS chains is a critical determinant of HS-protein interactions and emphasize the essential role of EXT1 in providing specific binding sites for growth factors and extracellular matrix proteins.