Rho‐kinase inhibitor Y‐27632 facilitates the proliferation, migration and pluripotency of human periodontal ligament stem cells

Abstract

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell‐based regenerative therapies, while technical challenges have limited cell yield and thus affected the success of these potential treatments. The Rho GTPases and downstream Rho kinases are central regulators of cytoskeletal dynamics during cell cycle and determine the balance between stem cells self‐renewal, lineage commitment and apoptosis. Trans‐4‐[(1R)‐aminoethyl]‐N‐(4‐pyridinyl)cylohexanecarboxamidedihydrochloride (Y‐27632), Rho‐associated kinase (ROCK) inhibitor, involves various cellular functions that include actin cytoskeleton organization, cell adhesion, cell motility and anti‐apoptosis. Here, human periodontal ligament stem cells (PDLSCs) were isolated by limiting dilution method. Cell counting kit‐8 (CCK8), 5‐ethynyl‐2′‐deoxyuridine (EdU) labelling assay, cell apoptosis assay, cell migration assay, wound‐healing assay, alkaline phosphatase (ALP) activity assay, Alizarin Red S staining, Oil Red O staining, quantitative real‐time polymerase chain reaction (qRT‐PCR) were used to determine the effects of Y‐27632 on the proliferation, apoptosis, migration, stemness, osteogenic and adipogenic differentiation of PDLSCs. Afterwards, Western blot analysis was performed to elucidate the mechanism of cell proliferation. The results indicated that Y‐27632 significantly promoted cell proliferation, chemotaxis, wound healing, fat droplets formation and pluripotency, while inhibited ALP activity and mineral deposition. Furthermore, Y‐27632 induced PDLSCs proliferation through extracellular‐signal‐regulated kinase (ERK) signalling cascade. Therefore, control of Rho‐kinase activity may enhance the efficiency of stem cell‐based treatments for periodontal diseases and the strategy may have the potential to promote periodontal tissue regeneration by facilitating the chemotaxis of PDLSCs to the injured site, and then enhancing the proliferation of these cells and maintaining their pluripotency.

Modulation of microenvironment for controlling the fate of periodontal ligament cells: the role of Rho/ROCK signaling and cytoskeletal dynamics

“…The mineralization process was significantly inhibited by Y-27632 in a dose-dependent manner as shown by decreased ALP activity, calcified nodule formation, and expression of osteogenic genes such as ALP and Runx2 while Y-27632 induced adipogenesis in PDL cells. These reports indicate that ROCK dominantly regulates osteogenic differentiation of PDL cells Ugawa et al 2017;Wang et al 2017). Therefore, ROCK activity governs osteo-adipogenic transdifferentiation by modifying the actin cytoskeleton.…”

“…The ROCKdependent actomyosin hyperactivation can cause apoptosis, which is triggered by the loss of E-cadherin-dependent intercellular contact and involves Rho activation and Rac inhibition (Ohgushi et al 2010). In PDL cells, the effect of Y-27632 on apoptosis is not significant (Wang et al 2017). Because constitutively active expression of Rho mutants (Rho-V14; a generous gift from Dr. Christopher S. Chen, Boston University) in PDL cells ceased proliferation in prolonged culture (our unpublished observation), there might be some effects of Rho/ROCK signaling on apoptosis.…”

“…Y-27632-treated hES cells also retain autonomous proliferation ability and enhanced pluripotency (Watanabe et al 2007). In PDL cells, Y-27632 remarkably reduce the rate of appearance of ALP-positive colonies; however, the total number of colonies is not affected (Ugawa et al 2017) and the size of Y-27632-treated colonies is larger than that of untreated colonies (Wang et al 2017). Moreover, Y-27632 significantly increase the rate of cell proliferation and the expression of pluripotent markers, MYC protooncogene (c-Myc), Nanog homeobox (Nanog), Kruppel-like factor 4 (Klf4), and Oct3/4 (Wang et al 2017).…”

“…In contrast, Y-27632 decreased stromal cell-derived factor 1 alpha (SDF-1α)-induced migration distance but maintained directionality in MSCs (Park et al 2017), suggesting an interactive behavior with other components of microenvironment. In PDL cells, Y-27632 is capable of enhancing migration (Wang et al 2017), probably by activating Rac signaling (our unpublished observation). Integrin-mediated cell adhesion to ECM provides essential signals for cell migration.…”

“…In PDL cells, Y-27632 remarkably reduce the rate of appearance of ALP-positive colonies; however, the total number of colonies is not affected (Ugawa et al 2017) and the size of Y-27632-treated colonies is larger than that of untreated colonies (Wang et al 2017). Moreover, Y-27632 significantly increase the rate of cell proliferation and the expression of pluripotent markers, MYC protooncogene (c-Myc), Nanog homeobox (Nanog), Kruppel-like factor 4 (Klf4), and Oct3/4 (Wang et al 2017). These findings indicate that ROCK inhibitor is capable of increasing cell proliferation and pluripotency of PDL-derived somatic stem cells.…”