Abstract
BackgroundCytoskeletal tension is an intracellular mechanism through which cells convert a mechanical signal into a biochemical response, including production of cytokines and activation of various signaling pathways.Methods/Principal FindingsAdipose-derived stromal cells (ASCs) were allowed to spread into large cells by seeding them at a low-density (1,250 cells/cm2), which was observed to induce osteogenesis. Conversely, ASCs seeded at a high-density (25,000 cells/cm2) featured small cells that promoted adipogenesis. RhoA and actin filaments were altered by changes in cell size. Blocking actin polymerization by Cytochalasin D influenced cytoskeletal tension and differentiation of ASCs. To understand the potential regulatory mechanisms leading to actin cytoskeletal tension, cDNA microarray was performed on large and small ASCs. Connective tissue growth factor (CTGF) was identified as a major regulator of osteogenesis associated with RhoA mediated cytoskeletal tension. Subsequently, knock-down of CTGF by siRNA in ASCs inhibited this osteogenesis.Conclusions/SignificanceWe conclude that CTGF is important in the regulation of cytoskeletal tension mediated ASC osteogenic differentiation.
Highlights
Connective tissue growth factor (CTGF, CCN2), a member of the CCN family of proteins, is a cysteine-rich pro-adhesive matricellular protein that plays an essential role in the formation of blood vessels, bone, and connective tissue [1]
Through the results of a gene array and siRNA knock-down experiments, we determined that connective tissue growth factor (CTGF) is highly induced in large Adipose-derived stromal cells (ASCs) and is a pro-osteogenic effector that plays an important role in RhoA mediated cytoskeletal tensionassociated osteogenesis
As demonstrated by crystal violet staining, ASCs spread into large cells when they were seeded at a lower density (1,250 cells/ cm2) and a medium density (2,500 cells/cm2); when ASCs were seeded at high density (25,000 cells/cm2), they were smaller in size microscopically (Figure 1A)
Summary
Connective tissue growth factor (CTGF, CCN2), a member of the CCN family of proteins, is a cysteine-rich pro-adhesive matricellular protein that plays an essential role in the formation of blood vessels, bone, and connective tissue [1]. Previous studies have shown that CTGF is highly expressed by osteoblasts, and CTGF null mice exhibit impaired chondrocytic cell proliferation and angiogenesis resulting in neonatal skeletal defects [6,7,8,9] These observations suggest that CTGF is important in bone and cartilage physiological events and repair. We manipulated the in vitro cell seeding densities of ASCs, resulting in large and small cells with distinguished microenvironments associated with actin cytoskeletal tension, and subsequently explored the influence on osteogenic and adipogenic differentiation of ASCs. Through the results of a gene array and siRNA knock-down experiments, we determined that CTGF is highly induced in large ASCs and is a pro-osteogenic effector that plays an important role in RhoA mediated cytoskeletal tensionassociated osteogenesis
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