Characterization of TIMP-3 in human articular talar cartilage.

Tissue inhibitor of matrix metalloproteinase 3 (TIMP-3) is an inhibitor of matrix degradation; however, little else is known about the role(s) of this protein in articular cartilage. In this study we compared levels of TIMP-3 in human knee and ankle cartilages and in normal and degraded cartilages. In addition, our studies focused on the compartmentalization of TIMP-3 in human adult articular cartilage matrix, identification of its potential binding partners, and determining the effects of cytokines on its matrix compartment deposition. We extracted TIMP-3 from cartilage and found that while TIMP-3 was localized throughout the matrix, it was predominately associated with the chondrocyte. We also found that more TIMP-3 was extracted from normal compared to degraded cartilage and more in ankle than knee cartilage suggesting the potential of this inhibitor as a protective agent. Our data suggest that TIMP-3 interacts with heparan sulfate and heparan sulfate proteoglycans and to a lesser extent with heparin and chondroitin sulfate. Stimulation with Interleukin-1ß and osteogenic protein-1 decreased while tumor necrosis factor alpha and transforming growth factor beta increased TIMP-3 protein levels; however, TIMP-3 mRNA was not significantly affected by any of these treatments. These characteristics indicate the chondroprotective nature of TIMP-3 and its potential as a therapeutic agent for osteoarthritis.

RNA interference-mediated inhibition of hepatocyte nuclear factor 1alpha identifies target genes.

Hepatocyte nuclear factor 1alpha (HNF1alpha) is a homeodomain transcription factor that is central to co-ordinated differentiation of a number of cell lineages, including hepatocytes in the liver and islet cells in the pancreas. HNF1alpha interacts directly with other transcription factors and co-factors and is involved in chromatin modification to alter gene expression. To further investigate the pivotal role of HNF1alpha in transcriptional control pathways we utilized RNA interference. An siRNA oligonucleotide specific for HNF1alpha reduced HNF1alpha protein levels by up to 70% in transient transfections of Caco2 cells. The same sequence incorporated into an shRNAi reduced protein levels by up to 90% in stable transfections. Microarray analysis of RNA from cell lines with stable RNAi-mediated down-regulation of HNF1alpha, identified genes known to be regulated by this transcription factor and also novel genes.

Collagen XV inhibits epithelial to mesenchymal transition in pancreatic adenocarcinoma cells.

Collagen XV (COLXV) is a secreted non-fibrillar collagen found within basement membrane (BM) zones of the extracellular matrix (ECM). Its ability to alter cellular growth in vitro and to reduce tumor burden and increase survival in vivo support a role as a tumor suppressor. Loss of COLXV during the progression of several aggressive cancers precedes basement membrane invasion and metastasis. The resultant lack of COLXV subjacent to the basement membrane and subsequent loss of its interactions with other proteins in this zone may directly impact tumor progression. Here we show that COLXV significantly reduces invasion of pancreatic adenocarcinoma cells through a collagen I (COLI) matrix. Moreover, we demonstrate that epithelial to mesenchymal transition (EMT) in these cells, which is recapitulated in vitro by cell scattering on a COLI substrate, is inhibited by over-expression of COLXV. We identify critical collagen-binding surface receptors on the tumor cells, including the discoidin domain receptor 1 (DDR1) and E-Cadherin (E-Cad), which interact with COLXV and appear to mediate its function. In the presence of COLXV, the intracellular redistribution of E-Cad from the cell periphery, which is associated with COLI-activated EMT, is inhibited and concurrently, DDR1 signaling is suppressed. Furthermore, continuous exposure of the pancreatic adenocarcinoma cells to high levels of COLXV suppresses endogenous levels of N-Cadherin (N-Cad). These data reveal a novel mechanism whereby COLXV can function as a tumor suppressor in the basement membrane zone.

Tumor suppression by collagen XV is independent of the restin domain.

Non-fibrillar collagen XV is a chondroitin sulfate modified glycoprotein that is associated with the basement membrane zone in many tissues. Its precise functions remain to be fully elucidated though it clearly plays a critical role in the structural integrity of the extracellular matrix. Loss of collagen XV from the basement membrane zone precedes invasion of a number of tumor types and we previously showed that collagen XV functions as a dose-dependent suppressor of tumorigenicity in cervical carcinoma cells. The carboxyl terminus of another non-fibrillar collagen (XVIII) is cleaved to produce endostatin, which has anti-angiogenic effects and thus may act as a tumor suppressor in vivo. Since collagen XV has structural similarity with collagen XVIII, its C-terminal restin domain could confer tumor suppressive functions on the molecule, though our previous data did not support this. We now show that expression of collagen XV enhances the adhesion of cervical carcinoma cells to collagen I in vitro as does the N-terminus and collagenous regions of collagen XV, but not the restin domain. Destruction of a cysteine residue in the collagenous region that is critical for intermolecular interactions of collagen XV abolished the enhanced adhesion to collagen I. Finally, we demonstrate that unlike full length collagen XV, expression of the restin domain alone does not suppress tumorigenicity of cervical carcinoma cells in vivo; hence, this process is dependent on functions and interactions of other parts of the protein.