Abstract
Skeletal development and remodeling of adult bone are critically controlled by activated NOTCH signaling in genetically modified mice. It is yet unclear whether NOTCH signaling is activated by mechanical strain sensed by bone cells. We found that expression of specific NOTCH target genes is induced after in vivo tibial mechanical loading in wild-type mice. We further applied mechanical strain through cyclic stretching in human bone marrow-derived mesenchymal stromal cells (BMSCs) in vitro by using a bioreactor system and detected upregulation of NOTCH target gene expression. Inhibition of the NOTCH pathway in primary BMSCs as well as telomerase-immortalized human BMSCs (hMSC-TERT) through the gamma-secretase inhibitor GSI XII blocked mechanotransduction and modulated actin cytoskeleton organization. Short-hairpin RNA gene silencing identified NOTCH2 as the key receptor mediating NOTCH effects on hMSC-TERT cells. Our data indicate a functional link between NOTCH activation and mechanotransduction in human BMSCs. We suggest that NOTCH signaling is an important contributor to molecular mechanisms that mediate the bone formation response to mechanical strain.
Highlights
The NOTCH signaling pathway is evolutionarily highly conserved and regulates cell growth, cell death, and differentiation programs via cell-cell communication [1]
To evaluate NOTCH activation after tibial mechanical loading in mice, we performed quantitative PCR (qPCR) analysis of NOTCH target genes in osteocyte-enriched bone from left-loaded and right-nonloaded limbs [19]. 1 and 24 h after a single loading session, we detected up to 6-fold increases in mRNA expression of the NOTCH targets HES1, HEY1, and HEY2 dependent on the time point (Figures 1(a) and 1(b))
To clarify if NOTCH signaling is responsive for mechanical strain in osteogenic precursors, bone marrow-derived mesenchymal stromal cells (BMSCs) from five donors were seeded on PU dishes and mechanical strain (1 Hz, 1%) was applied
Summary
The NOTCH signaling pathway is evolutionarily highly conserved and regulates cell growth, cell death, and differentiation programs via cell-cell communication [1]. Conditional overexpression of NOTCH in the osteoblastic lineage at various differentiation stages confirms NOTCH’s role in maintaining the early differentiation stage of BMSCs. conflicting roles of NOTCH signaling in osteocyte development and function were reported: (1) Overexpression of NOTCH in mature osteocytes increases bone formation due to an induction of osteoprotegerin (OPG) production and a diminished secretion of the WNT inhibitors sclerostin (SOST) and dickkopf 1 (DKK1). Conflicting roles of NOTCH signaling in osteocyte development and function were reported: (1) Overexpression of NOTCH in mature osteocytes increases bone formation due to an induction of osteoprotegerin (OPG) production and a diminished secretion of the WNT inhibitors sclerostin (SOST) and dickkopf 1 (DKK1) This results in enhanced osteogenic canonical WNT signaling, which is coincident with suppressed bone resorption [3, 4]. Osteocyte-specific overexpression of β-catenin and subsequent activation of WNT signaling result in increased NOTCH signaling in the bone microenvironment of mice [6]
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