Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization.

We have previously demonstrated that Stat3 regulates lysosomal-mediated programmed cell death (LM-PCD) during mouse mammary gland involution in vivo. However, the mechanism that controls the release of lysosomal cathepsins to initiate cell death in this context has not been elucidated. We show here that Stat3 regulates the formation of large lysosomal vacuoles that contain triglyceride. Furthermore, we demonstrate that milk fat globules (MFGs) are toxic to epithelial cells and that, when applied to purified lysosomes, the MFG hydrolysate oleic acid potently induces lysosomal leakiness. Additionally, uptake of secreted MFGs coated in butyrophilin 1A1 is diminished in Stat3-ablated mammary glands and loss of the phagocytosis bridging molecule MFG-E8 results in reduced leakage of cathepsins in vivo. We propose that Stat3 regulates LM-PCD in mouse mammary gland by switching cellular function from secretion to uptake of MFGs. Thereafter, perturbation of lysosomal vesicle membranes by high levels of free fatty acids results in controlled leakage of cathepsins culminating in cell death.This work was supported by a grant from the Medical Research Council programme grant no. MR/J001023/1 (T.J.S. and B. L-L.) and a Cancer Research UK Cambridge Cancer Centre PhD studentship (H.K.R.).This is the accepted manuscript. The final version is available from Nature Publishing at http://www.nature.com/ncb/journal/vaop/ncurrent/full/ncb3043.html

Effects of glucosamine sulfate on intracellular UDP-hexosamine and UDP-glucuronic acid levels in bovine primary chondrocytes

SummaryObjectiveTo analyze the effects of exogenously added glucose (Glc), glucosamine (GlcN) and glucosamine sulfate (GS) on the intracellular UDP-hexoses (UDP-Hex), UDP-N-acetylhexosamines (UDP-HexN) and UDP-glucuronic acid (UDP-GlcA) levels in bovine primary chondrocytes.MethodsChondrocytes were incubated with different concentrations of Glc, GlcN and GS either in high- or low-glucose DMEM for up to 120min to analyze the intracellular levels of UDP-Hex, UDP-GlcA and UDP-HexN by a reversed-phase high-performance liquid chromatography–electrospray ionization mass spectrometry analysis. Glycosaminoglycan (GAG) synthesis rate and aggrecan mRNA expression levels were quantified using 35S-sulfate incorporation assay and quantitative real-time RT-PCR, respectively. The cells were cultivated for 2 days or 8 days before UDP-sugar analysis.ResultsLevels of UDP-HexN and UDP-GlcA were unchanged at 10μM concentration of GS in low-glucose DMEM, while addition of 1mM GlcN or GS in low-glucose DMEM for 10min increased UDP-HexN level. The highest intracellular level of UDP-HexN was reached at 30min after addition of 1mM GS to the cells. The intracellular contents of UDP-HexN and UDP-GlcA related to UDP-Hex were higher after prolonged cultivation of chondrocytes for 8 days compared with 2-day-old cultures. Aggrecan mRNA expression and GAG synthesis remained at control level after the cells were treated with 10, 100μM or 1mM of GS for 24h.ConclusionPhysiologically relevant level of GS could not increase the intracellular UDP-HexN and UDP-GlcA levels in bovine primary chondrocyte, while longer-time culture itself appeared to increase the intracellular UDP-HexN and UDP-GlcA levels

Bioengineering Cartilage Growth, Maturation, and Form

Cartilage of articular joints grows and matures to achieve characteristic sizes, forms, and functional properties. Through these processes, the tissue not only serves as a template for bone growth but also yields mature articular cartilage providing joints with a low-friction, wear-resistant bearing material. The study of cartilage growth and maturation is a focus of both cartilage biologists and bioengineers with one goal of trying to create biologic tissue substitutes for the repair of damaged joints. Experimental approaches both in vivo and in vitro are being used to better understand the mechanisms and regulation of growth and maturation processes. This knowledge may facilitate the controlled manipulation of cartilage size, shape, and maturity to meet the criteria needed for successful clinical applications. Mathematical models are also useful tools for quantitatively describing the dynamically changing composition, structure and function of cartilage during growth and maturation and may aid the development of tissue engineering solutions. Recent advances in methods of cartilage formation and culture which control the size, shape, and maturity of these tissues are numerous and provide contrast to the physiologic development of cartilage