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A role for the primary cilium in paracrine signaling between mechanically stimulated osteocytes and mesenchymal stem cells.

ABSTRACT: Bone turnover is a mechanically regulated process, coordinated in part by the network of mechanosensitive osteocytes residing within the tissue. The recruitment and bone forming activity of the mesenchymal derived osteoblast is determined by numerous factors including mechanical loading. It is therefore somewhat surprising that although mechanically regulated signaling between the coordinating osteocytes and mesenchymal stem cells (MSCs) should exist, to date it has not been directly demonstrated. In this study, conditioned media from mechanically stimulated osteocytes (MLO-Y4 cell line) was collected and added to MSCs (C3H10T1/2 cell line). The addition of mechanically stimulated osteocyte conditioned media resulted in a significant upregulation of the osteogenic genes OPN and COX-2 in MSCs compared to statically cultured conditioned media, demonstrating a novel paracrine signaling mechanism between the two cell types. The same mechanically conditioned media did not alter gene expression in osteoblasts (MC3T3 cell line), and mechanically stimulated osteoblast conditioned media did not alter gene expression in MSCs demonstrating that this signaling is unique to osteocytes and MSCs. Finally, the upregulation in osteogenic genes in MSCs was not observed if primary cilia formation was inhibited prior to mechanical stimulation of the osteocyte. In summary, the results of this study indicate that soluble factors secreted by osteocytes in response to mechanical stimulation can enhance osteogenic gene expression in MSCs demonstrating a novel, unique signaling mechanism and introduces a role for the primary cilium in flow mediated paracrine signaling in bone thereby highlighting the cilium as a potential target for therapeutics aimed at enhancing bone formation.

SUBMITTER: Hoey DA

PROVIDER: S-EPMC3160132 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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A role for the primary cilium in paracrine signaling between mechanically stimulated osteocytes and mesenchymal stem cells.

Hoey David A DA Kelly Daniel J DJ Jacobs Christopher R CR

Biochemical and biophysical research communications 20110723 1

Bone turnover is a mechanically regulated process, coordinated in part by the network of mechanosensitive osteocytes residing within the tissue. The recruitment and bone forming activity of the mesenchymal derived osteoblast is determined by numerous factors including mechanical loading. It is therefore somewhat surprising that although mechanically regulated signaling between the coordinating osteocytes and mesenchymal stem cells (MSCs) should exist, to date it has not been directly demonstrate ...[more]

PMID: 21810408

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Project description:BackgroundMechanical loading is an essential factor for bone formation. A previous study indicated that mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz for 8 h promoted osteogenesis and corresponding mechanoresponsive microRNAs (miRs) were identified in osteoblasts. However, in osteocytes, it has not been identified which miRs respond to the mechanical strain, and it is not fully understood how the mechanoresponsive miRs regulate osteoblast differentiation.MethodsMouse MLO-Y4 osteocytes were applied to the same mechanical tensile strain in vitro. Using molecular and biochemical methods, IGF-1 (insulin-like growth factor-1), PGE2 (prostaglandin E2), OPG (osteoprotegerin) and NOS (nitric oxide synthase) activities of the cells were assayed. MiR microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays were applied to select and validate differentially expressed miRs, and the target genes of these miRs were then predicted. MC3T3-E1 osteoblasts were stimulated by the mechanical tensile strain, and the miR-29b-3p expression was detected with miR microarray and RT-qPCR. Additionally, the effect of miR-29b-3p on IFG-1 secretion of osteocytes and the influence of conditioned medium of osteocytes transfected with miR-29b-3p on osteoblast differentiation were investigated.ResultsThe mechanical strain increased secretions of IGF-1 and PGE2, elevated OPG expression and NOS activities, and resulted in altered expression of the ten miRs, and possible target genes for these differentially expressed miRs were revealed through bioinformatics. Among the ten miRs, miR-29b-3p were down-regulated, and miR-29b-3p overexpression decreased the IGF-1 secretion of osteocytes. The mechanical strain did not change expression of osteoblasts' miR-29b-3p. In addition, the conditioned medium of mechanically strained osteocytes promoted osteoblast differentiation, and the conditioned medium of osteocytes transfected with miR-29b-3p mimic inhibited osteoblast differentiation.ConclusionsIn osteocytes (but not osteoblasts), miR-29b-3p was responsive to the mechanical tensile strain and regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically strained osteocytes.

Project description:BackgroundSecretome profiles of mesenchymal stem cells (MSCs) are reflective of their local microenvironments. These biologically active factors exert an impact on the surrounding cells, eliciting regenerative responses that create an opportunity for exploiting MSCs towards a cell-free therapy for cartilage regeneration. The conventional method of culturing MSCs on a tissue culture plate (TCP) does not provide the physiological microenvironment for optimum secretome production. In this study, we explored the potential of electrospun fiber sheets with specific orientation in influencing the MSC secretome production and its therapeutic value in repairing cartilage.MethodsConditioned media (CM) were generated from MSCs cultured either on TCP or electrospun fiber sheets of distinct aligned or random fiber orientation. The paracrine potential of CM in affecting chondrogenic differentiation, migration, proliferation, inflammatory modulation, and survival of MSCs and chondrocytes was assessed. The involvement of FAK and ERK mechanotransduction pathways in modulating MSC secretome were also investigated.ResultsWe showed that conditioned media of MSCs cultured on electrospun fiber sheets compared to that generated from TCP have improved secretome yield and profile, which enhanced the migration and proliferation of MSCs and chondrocytes, promoted MSC chondrogenesis, mitigated inflammation in both MSCs and chondrocytes, as well as protected chondrocytes from apoptosis. Amongst the fiber sheet-generated CM, aligned fiber-generated CM (ACM) was better at promoting cell proliferation and augmenting MSC chondrogenesis, while randomly oriented fiber-generated CM (RCM) was more efficient in mitigating the inflammation assault. FAK and ERK signalings were shown to participate in the modulation of MSC morphology and its secretome production.ConclusionsThis study demonstrates topographical-dependent MSC paracrine activities and the potential of employing electrospun fiber sheets to improve the MSC secretome for cartilage regeneration.

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A role for the primary cilium in paracrine signaling between mechanically stimulated osteocytes and mesenchymal stem cells.

Publications

A role for the primary cilium in paracrine signaling between mechanically stimulated osteocytes and mesenchymal stem cells.

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