No abstract

Transforming growth factor-β (TGF-β) is implicated in numerous pathological disorders, including cancer and mediates a broad range of biological responses by signaling through the type I and II TGF-β receptors. Internalization of these receptors via the clathrin-coated pits pathway facilitates SMAD-mediated signaling, whereas internalization via the caveolae pathway is associated with receptor degradation. Thus, molecules that modulate receptor endocytosis are likely to play a critical role in regulating TGF-β action. We previously identified CD109, a GPI-anchored protein, as a TGF-β co-receptor and a negative regulator of TGF-β signaling. Here, we demonstrate that CD109 associates with caveolin-1, a major component of the caveolae. Moreover, CD109 increases binding of TGF-β to its receptors and enhances their internalization via the caveolae. In addition, CD109 promotes localization of the TGF-β receptors into the caveolar compartment in the presence of ligand and facilitates TGF-β-receptor degradation. Thus, CD109 regulates TGF-β receptor endocytosis and degradation to inhibit TGF-β signaling. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Chitosan (CS) shows in vitro and in vivo efficacy for siRNA delivery but with contradictory findings for incompletely characterized systems. For understanding which parameters produce effective delivery, a library of precisely characterized chitosans was produced at different degrees of deacetylation (DDAs) and average molecular weights (M). Encapsulation and transfection efficiencies were characterized in vitro. Formulations were selected to examine the influence of M and N:P ratio on nanoparticle uptake, metabolic activity, genotoxicity, and in vitro transfection. Hemocompatibility and in vivo biodistribution were then investigated for different M, N:P ratios, and doses. Nanoparticle uptake and gene silencing correlated with increased surface charge, which was obtained at high DDA and high M. A minimum polymer length of ∼60-70 monomers (∼10 kDa) was required for stability and knockdown. In vitro knockdown was equivalent to lipid control with no metabolic or genotoxicity. An inhibitory effect of serum on biological performance was dependent on DDA, M, and N:P. In vivo biodistribution in mice show accumulation of nanoparticles in kidney with 40-50% functional knockdown.

This PLM method offers a novel means for systematically evaluating collagen organization in repair cartilage. We propose that it be used to supplement current gold standard histological scoring systems for a more complete assessment of repair tissue quality.

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone.