TGF-ß1- and CCN2-Stimulated Sirius Red Assay for Collagen Accumulation in Cultured Cells.

A simple method for the determination of relative levels of insoluble collagen accumulation in fibroblast cultures is presented. Confluent cell cultures are provided with sodium ascorbate which is then permissive for collagen deposition. At intervals, cultures are fixed and stained successively with sirius red and then crystal Violet to, respectively, assess for relative changes in collagen accumulation in response to factors such as TGF-ß1 or matricellular CCN2 and changes in DNA content as an index of changes in cell density.

A novel function for lysyl oxidase in pluripotent mesenchymal cell proliferation and relevance to inflammation-associated osteopenia.

Lysyl oxidase is a multifunctional enzyme required for collagen biosynthesis. Various growth factors regulate lysyl oxidase during osteoblast differentiation, subject to modulation by cytokines such as TNF-α in inflammatory osteopenic disorders including diabetic bone disease. Canonical Wnt signaling promotes osteoblast development. Here we investigated the effect of Wnt3a and TNF-α on lysyl oxidase expression in pluripotent C3H10T1/2 cells, bone marrow stromal cells, and committed osteoblasts. Lysyl oxidase was up-regulated by a transcriptional mechanism 3-fold in C3H10T1/2 cells, and 2.5-fold in bone marrow stromal cells. A putative functional TCF/LEF element was identified in the lysyl oxidase promoter. Interestingly, lysyl oxidase was not up-regulated in committed primary rat calvarial- or MC3T3-E1 osteoblasts. TNF-α down-regulated lysyl oxidase both in Wnt3a-treated and in non-treated C3H10T1/2 cells by a post-transcriptional mechanism mediated by miR203. Non-differentiated cells do not produce a collagen matrix; thus, a novel biological role for lysyl oxidase in pluripotent cells was investigated. Lysyl oxidase shRNAs effectively silenced lysyl oxidase expression, and suppressed the growth of C3H10T1/2 cells by 50%, and blocked osteoblast differentiation. We propose that interference with lysyl oxidase expression under excess inflammatory conditions such as those that occur in diabetes, osteoporosis, or rheumatoid arthritis can result in a diminished pool of pluripotent cells which ultimately contributes to osteopenia.