Qing Luo

Osteopontin Promotes Mesenchymal Stem Cell Migration and Lessens Cell Stiffness via Integrin β1, FAK, and ERK Pathways

The use of mesenchymal stem cells (MSCs) for therapeutic applications has attracted great attention because MSCs home to and engraft to injured tissues after in vivo administration. The expression of osteopontin (OPN) is elevated in response to injury and inflammation, and its role on rat bone marrow-derived mesenchymal stem cells (rMSCs)-directed migration has been elucidated. However, the signaling pathways through the activation of which OPN promotes rMSCs migration and the involvement of cell mechanics during OPN-mediating rMSCs migration have not been well studied. In this study, we found that OPN activated focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways by the ligation of integrin β1 in rMSCs. Inhibitors of FAK and ERK pathways inhibited OPN-induced rMSCs migration, indicating the possible involvement of FAK and ERK activation in OPN-induced migration in rMSCs. In addition, atomic force microscopy analysis showed that OPN reduced cell stiffness in rMSCs via integrin β1, FAK, and ERK pathways, suggesting that the promotion of rMSCs migration might partially be contributing to the decrease in cell stiffness stimulated by OPN. To further examine the role of OPN on cell motility and stiffness, actin cytoskeleton of rMSCs was observed. The reduced well-defined F-actin filaments and the promoted formation of pseudopodia in rMSCs induced by OPN explained the reduction in cell stiffness and the increase in cell migration. The current study data have shown for the first time that OPN binding to integrin β1 promotes rMSCs migration through the activation of FAK and ERK pathways, which may be attributed to the change in cell stiffness caused by the reduction in the amount of organized actin cytoskeleton.

Liver cancer stem cell markers: Progression and therapeutic implications

Cancer stem cells (CSCs) are a small subpopulation in cancer, have been proposed to be cancer-initiating cells, and have been shown to be responsible for chemotherapy resistance and cancer recurrence. The identification of CSC subpopulations inside a tumor presents a new understanding of cancer development because it implies that tumors can only be eradicated by targeting CSCs. Although advances in liver cancer detection and treatment have increased the possibility of curing the disease at early stages, unfortunately, most patients will relapse and succumb to their disease. Strategies aimed at efficiently targeting liver CSCs are becoming important for monitoring the progress of liver cancer therapy and for evaluating new therapeutic approaches. Herein, we provide a critical discussion of biological markers described in the literature regarding liver cancer stem cells and the potential of these markers to serve as therapeutic targets.

A synthetic mechano-growth factor E peptide promotes rat tenocyte migration by lessening cell stiffness and increasing F-actin formation via the FAK-ERK1/2 signaling pathway.

Tendon injuries are common in sports and are frequent reasons for orthopedic consultations. The management of damaged tendons is one of the most challenging problems in orthopedics. Mechano-growth factor (MGF), a recently discovered growth repair factor, plays positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury-induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. We used a scratch wound assay in this study to demonstrate that MGF-C25E (a synthetic mechano-growth factor E peptide) promotes the migration of rat tenocytes and that this promotion is accompanied by an elevation in the expression of the following signaling molecules: focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2). Inhibitors of the FAK and ERK1/2 pathways inhibited the MGF-C25E-induced tenocyte migration, indicating that MGF-C25E promotes tenocyte migration through the FAK-ERK1/2 signaling pathway. The analysis of the mechanical properties showed that the Youngs modulus of tenocytes was decreased through treatment of MGF-C25E, and an obvious formation of pseudopodia and F-actin was observed in MGF-C25E-treated tenocytes. The inhibition of the FAK or ERK1/2 signals restored the decrease in Youngs modulus and inhibited the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25E promotes rat tenocyte migration by lessening cell stiffness and increasing pseudopodia formation via the FAK-ERK1/2 signaling pathway.

Mechano-growth factor induces migration of rat mesenchymal stem cells by altering its mechanical properties and activating ERK pathway

Mechano-growth factor (MGF) generated by cells in response to mechanical stimulation has been identified as a mechano effector molecule, playing a key role in regulating mesenchymal stem cell (MSC) function, including proliferation and migration. However, the mechanism(s) underlying how MGF-induced MSC migration occurs is still unclear. In the present study, MGF motivated migration of rat MSCs (rMSCs) in a concentration-dependent manner and optimal concentration of MGF at 50 ng/mL (defined as MGF treatment in this paper) was demonstrated. Notably, enhancement of mechanical properties that is pertinent to cell migration, such as cell traction force and cell stiffness were found to respond to MGF treatment. Furthermore, MGF increased phosphorylation of extracellular signal-regulated kinase (ERK), ERK inhibitor (i.e., PD98059) suppressed ERK phosphorylation, and abolished MGF-induced rMSC migration were found, demonstrating that ERK is involved molecule for MGF-induced rMSC migration. These in vitro evidences of MGF-induced rMSC migration and its direct link to altering rMSC mechanics and activating the ERK pathway, uncover the underlying biomechanical and biological mechanisms of MGF-induced rMSC migration, which may help find MGF-based application of MSC in clinical therapeutics.

Mesenchymal stem cells require integrin β1 for directed migration induced by osteopontin in vitro

Mesenchymal stem cells (MSCs) are characterized by their ability of self-renewal paired with the capacity to differentiate into multiple mesenchymal cell lineages. Numerous studies have reported beneficial effects of MSCs in tissue repair and regeneration. After in vivo administration, MSCs home to and engraft to injured tissues. However, the molecular mechanisms are not clear. Osteopontin (OPN) has been found to be elevated in response to injury and inflammation and its role on cell mobilization has been studied. Therefore, the facts imply that OPN may contribute to the recruitment of MSCs to the sites of injury. In this study, using transwell assay, we found that rat bone marrow-derived mesenchymal stem cells (rMSCs) migrated towards OPN in a concentration-dependent manner. To further examine the involved molecular mechanisms for OPN-induced rMSCs migration, RT-PCR, and Western blot were used to detect the expressions of integrin β1 and CD44v6, the two receptors of OPN. OPN promoted integrin β1 mRNA and protein expression while CD44v6 mRNA level was not altered. Blockade of integrin β1 also inhibited OPN-induced rMSCs migration, indicating the possible involvement of integrin β1 in OPN-induced migration in rMSCs. Our data have shown for the first time that OPN increases integrin β1 expression in rMSCs and promotes rMSCs migration through the ligation to integrin β1.

Cell stiffness determined by atomic force microscopy and its correlation with cell motility

BACKGROUND Cell stiffness is a crucial mechanical property that is closely related to cell motility. AFM is the most prevalent method used to determine cell stiffness by the quantitative parameter designated as Youngs modulus. Youngs modulus is regarded as a biomarker of cell motility, especially in estimating the metastasis of cancer cells, because in recent years, it has been repeatedly shown that cancerous cells are softer than their benign counterparts. However, some conflicting evidence has shown that cells with higher motility are sometimes stiffer than their counterparts. Thus, the correlation between cell stiffness and motility remains a matter of debate. SCOPE OF REVIEW In this review, we first summarize the reports on correlations between cell motility and stiffness determined by AFM and then discuss the major determinants of AFM-determined cell stiffness with a focus on the cytoskeleton, nuclear stiffness and methodological issues. Last, we propose a possible correlation between cell stiffness and motility and the possible explanations for the conflicting evidence. MAJOR CONCLUSIONS The AFM-determined Youngs modulus is greatly affected by the characteristics of the cytoskeleton, as well as the procedures and parameters used in detection. Youngs modulus is a reliable biomarker for the characterization of metastasis; however, reliability is questioned in the evaluation of pharmacologically or genetically modified motility. GENERAL SIGNIFICANCE This review provides an overview of the current understanding of the correlation between AFM-determined cell stiffness and motility, the determinants of this detecting method, as well as clues to optimize detecting parameters.

Biomechanical profile of cancer stem-like cells derived from MHCC97H cell lines

Biomechanical properties and cytoskeletal organization of cancer cells are known to be closely related with their aggressive phenotype. In this study, based on atomic force microscopy (AFM), we aimed to evaluate the mechanical property of liver cancer stem-like cells (LCSCs) and compare it with human hepatoma cells (HHCs). LCSCs were enriched from human hepatoma cell line MHCC97H through a sphere culture system. AFM nanoindentation was investigated as a method for measuring the cell stiffness, and reflecting by Young׳s modulus. Microfilament bundles of F-actin were observed with immunofluorescence staining by confocal microscopy. We found that LCSCs show lower Young׳s modulus and higher migration ability compared to MHCC97H cells. Moreover, the decrease in Young׳s modulus is accompanied with a dramatic decline in F-actin content. These results demonstrated a close relationship between the cell Young׳s modulus and metastatic potential of HHCs, which suggest that Young׳s modulus detected by AFM can be used to evaluate metastatic potential of cancer cells.

Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells via depolymerizing F-actin to impede TAZ nuclear translocation

Microgravity induces observed bone loss in space flight, and reduced osteogenesis of bone mesenchymal stem cells (BMSCs) partly contributes to this phenomenon. Abnormal regulation or functioning of the actin cytoskeleton induced by microgravity may cause the inhibited osteogenesis of BMSCs, but the underlying mechanism remains obscure. In this study, we demonstrated that actin cytoskeletal changes regulate nuclear aggregation of the transcriptional coactivator with PDZ-binding motif (TAZ), which is indispensable for osteogenesis of bone mesenchymal stem cells (BMSCs). Moreover, we utilized a clinostat to model simulated microgravity (SMG) and demonstrated that SMG obviously depolymerized F-actin and hindered TAZ nuclear translocation. Interestingly, stabilizing the actin cytoskeleton induced by Jasplakinolide (Jasp) significantly rescued TAZ nuclear translocation and recovered the osteogenic differentiation of BMSCs in SMG, independently of large tumor suppressor 1(LATS1, an upstream kinase of TAZ). Furthermore, lysophosphatidic acid (LPA) also significantly recovered the osteogenic differentiation of BMSCs in SMG through the F-actin-TAZ pathway. Taken together, we propose that the depolymerized actin cytoskeleton inhibits osteogenic differentiation of BMSCs through impeding nuclear aggregation of TAZ, which provides a novel connection between F-actin cytoskeleton and osteogenesis of BMSCs and has important implications in bone loss caused by microgravity.

Cyclic mechanical stretching promotes migration but inhibits invasion of rat bone marrow stromal cells.

Bone marrow stromal cells (BMSCs, also broadly known as bone marrow-derived mesenchymal stem cells) are multipotent stem cells that have a self-renewal capacity and multilineage differentiation potential. Mechanical stretching plays a vital role in regulating the proliferation and differentiation of BMSCs. However, little is known about the effects of cyclic stretching on BMSC migration and invasion. In this study, using a custom-made cell-stretching device, we studied the effects of cyclic mechanical stretching on rat BMSC migration and invasion using a Transwell Boyden Chamber. The protein secretion of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) was detected by gelatin zymography, and the activation of focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2) was measured by western blot. We found that cyclic mechanical stretching with 10% amplitude at 1Hz frequency for 8h promotes BMSC migration, but reduces BMSC invasion. FAK and ERK1/2 signals were activated in BMSCs after exposure to cyclic stretching. In the presence of the FAK phosphorylation blocker PF573228 or the ERK1/2 phosphorylation blocker PD98059, the cyclic-stretch-promoted migration of BMSCs was completely suppressed. On the other hand, cyclic mechanical stretching reduced the secretion of MMP-2 and MMP-9 in BMSCs, and PF573228 suppressed the cyclic-stretch-reduced secretion of MMP-2 and MMP-9. The decrease of BMSC invasion induced by mechanical stretching is partially restored by PF573228 but remained unaffected by PD98059. Taken together, these data show that cyclic mechanical stretching promotes BMSC migration via the FAK-ERK1/2 signalling pathway, but reduces BMSC invasion by decreasing secretion of MMP-2 and MMP-9 via FAK, independent of the ERK1/2 signal.

Nucleus and nucleus-cytoskeleton connections in 3D cell migration.

Cell migration plays an important role in many physiological and pathological settings, ranging from embryonic development to cancer metastasis. Currently, accumulating data suggest that cells migrating in three-dimensional (3D) environments show well-defined differences compared to their well-established two-dimensional (2D) counterparts. During 3D migration, the cell body and nucleus must deform to allow cellular passage through the available spaces, and the deformability of the relatively rigid nucleus may constitute a limiting step. Here, we highlight the key evidence regarding the role of the nuclear mechanics in 3D migration, including the molecular components that govern the stiffness of the nucleus and review how the nuclear dynamics are connected to and controlled by cytoskeleton-based migration machinery. Intriguingly, nuclear movement must be coordinated with the cytoskeletal dynamics at the leading and trailing edges, which in turn impact the cytoplasmic dynamics that affect the migration efficiency. Thus, we suggest that alterations in the nuclear structure may facilitate cellular reorganizations that are necessary for efficient migration.