Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells

Abstract

Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes, but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study, we demonstrated that stabilizing actin filaments by inhibiting gene expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs, enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro, as well as heterotopic bone formation in vivo. Similarly, treating hMSC with Phalloidin, which is known to stabilize polymerized actin filaments, increased hMSCs viability and OB differentiation. Conversely, Cytocholasin D, an inhibitor of actin polymerization, reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level, preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1 (LIMK1) decreased cell viability and impaired OB differentiation of hMSCs. Moreover, depolymerizing actin reduced FAK, p38 and JNK activation during OB differentiation of hMSCs, while polymerizing actin enhanced these signaling pathways. Our results demonstrate that the actin dynamic reassembly and Cofilin phosphorylation loop is involved in the control of hMSC proliferation and osteoblasts differentiation.

Transgelin is a TGFβ-inducible gene that regulates osteoblastic and adipogenic differentiation of human skeletal stem cells through actin cytoskeleston organization

“…It has been reported that significant changes took place in the cytoskeleton during lineage commitment and differentiation of hMSC. 33, 34 During hMSCs' lineage commitment, the cells undergo significant modifications in morphology and actin cytoskeletal organization, and this participates in cell-fate determination. 33 In addition, during osteoblast differentiation, hMSCs morphologically change from fibroblast-like cells to cuboidal morphology, which is associated with changes in the cytoskeleton.…”

“…33, 34 During hMSCs' lineage commitment, the cells undergo significant modifications in morphology and actin cytoskeletal organization, and this participates in cell-fate determination. 33 In addition, during osteoblast differentiation, hMSCs morphologically change from fibroblast-like cells to cuboidal morphology, which is associated with changes in the cytoskeleton. 35 Similarly, during differentiation of hMSCs to adipocytes, significant changes in cell morphology takes place.…”

“…40, 41, 42 A recent study from our group has demonstrated that, during osteoblast differentiation of hMSCs, alteration in ADF expression leads to changes in the ratio of G-actin (monomer) and F-actin (polymerized), and inhibit actin depolymerization. 33 …”

Single-cell high-content imaging parameters predict functional phenotype of cultured human bone marrow stromal stem cells

“…The association of cellular shape and functional outcome may be explained by changes in actin dynamics. We have previously demonstrated that changes in cell shape induced by alternations in the actin cytoskeleton structure determine differentiation outcome of hBM‐MSCs with cellular changes associated with actin de‐polymerization led to enhanced adipocytic differentiation, whereas inhibition of actin de‐polymerization increased osteoblast formation of hBM‐MSCs . Furthermore, one study also shows that stimulation of a immunomodulating pathway can lead to a morphological response in BM‐MSCs .…”

“…The relationship between cell morphology and biological functions have been studied in respect to changes in cytoskeletal fibers (actin and tubulin) that mediate cellular adaptation to microenvironmental stimuli and facilitate intracellular signal transduction. For example, the actin cytoskeleton plays a role in hBM‐MSC lineage commitment, and microtubules are key players in cell proliferation and have been reported to contribute to osteoblast differentiation . Alterations in cell shape affects actin stress fibers, which through intracellular signaling pathways initiate cell lineage commitment .…”