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Palo Alto, CA, United States

Litzenberger J.-B.,Bone and Joint Rehabilitation Center | Litzenberger J.-B.,Stanford University | Kim J.-B.,Stanford University | Kim J.-B.,Caliper Life Sciences Inc. | And 2 more authors.
Calcified Tissue International | Year: 2010

Integrins are cell-substrate adhesion proteins that initiate intracellular signaling and may serve as mechanosensors in bone. MLO-Y4 cells were stably transfected with a dominant negative form of the b1 integrin subunit (β1DN) containing the transmembrane domain and cytoplasmic tail of β1 integrin. Cells expressing β1DN had reduced vinculin localization to focal contacts but no change in intracellular actin organization. When exposed to oscillatory fluid flow, β1DN cells exhibited a significant reduction in the upregulation of cyclooxygenase-2 gene expression and prostaglandin E2 release. Similarly, the ratio of receptor activator of NF-κB ligand mRNA to osteoprotegerin mRNA decreased significantly after exposure to fluid flow in control cells but not in β1DN cells. Interfering with integrin signaling did not affect mechanically induced intracellular calcium mobilization. These data suggest that integrins may initiate the cellular response of osteocytes to dynamic fluid flow and may serve as mechanosensitive molecules in bone. Source

Tummala P.,Bone and Joint Rehabilitation Center | Arnsdorf E.J.,Bone and Joint Rehabilitation Center | Arnsdorf E.J.,Stanford University | Arnsdorf E.J.,Incube Labs | And 2 more authors.
Cellular and Molecular Bioengineering | Year: 2010

Primary cilia are sensory organelles that have been shown to play a critical role in lineage commitment. It was our hypothesis that the primary cilium is necessary for chemically induced differentiation of human mesenchymal stem cells (MSC). To investigate this, polaris siRNA was used to inhibit the primary cilia and the mRNA levels of transcription factors Runx2, PPARγ were measured by RT PCR as markers of osteogenic, adipogenic and chondrogenic differentiation, respectively. MSCs with inhibited primary cilia had significantly decreased basal mRNA expression levels of all three lineages specific transcription factors indicating that primary cilia are critical in multiple differentiation pathways. Furthermore, to determine if primary cilia play a role in the differentiation potential of MSCs, progenitor cells transfected with either scrambled or polaris siRNA were cultured in osteo-inductive, chondro-inductive, or adipo-inductive media and lineage commitment was ascertained. Interestingly, within 24 h of culture, cells transfected with polaris siRNA in both osteogenic and adipogenic media lost adhesion and released from the slides; however MSCs in chondrogenic media as well as cells transfected with scrambled siRNA did not. These results suggest that the primary cilium is necessary for the normal progression of chemically induced osteogenic and adipogenic differentiation. As a control, the experiment was repeated with NIH3T3 fibroblasts and none of the effects of inhibited primary cilia were observed indicating that the loss of adhesion may be specific to MSCs. Furthermore after biochemically inducing the cells to differentiate, polaris knockdown resulted in abrogation of both Runx2 and PPARγ mRNA while SOX9 mRNA expression was significantly lower. These results suggest that primary cilia play an essential role not only in the initiation of both osteogenic and adipogenic differentiation, but also in maintaining the phenotype of differentiated cells. Interestingly, chondrogenic differentiation appeared less dependent on a functional primary cilium. © 2010 Biomedical Engineering Society. Source

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