Peihong Su

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells.

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function. [BMB Reports 2015; 48(10): 583-588]

Isoforms, structures, and functions of versatile spectraplakin MACF1

Spectraplakins are crucially important communicators, linking cytoskeletal components to each other and cellular junctions. Microtubule actin crosslinking factor 1 (MACF1), also known as actin crosslinking family 7 (ACF7), is a member of the spectraplakin family. It is expressed in numerous tissues and cells as one extensively studied spectraplakin. MACF1 has several isoforms with unique structures and well-known function to be able to crosslink F-actin and microtubules. MACF1 is one versatile spectraplakin with various functions in cell processes, embryo development, tissue-specific functions, and human diseases. The importance of MACF1 has become more apparent in recent years. Here, we summarize the current knowledge on the presence and function of MACF1 and provide perspectives on future research of MACF1 based on our studies and others. [BMB Reports 2016; 49(1): 37-44]

The Impact of Oxidative Stress on the Bone System in Response to the Space Special Environment

The space special environment mainly includes microgravity, radiation, vacuum and extreme temperature, which seriously threatens an astronaut’s health. Bone loss is one of the most significant alterations in mammalians after long-duration habitation in space. In this review, we summarize the crucial roles of major factors—namely radiation and microgravity—in space in oxidative stress generation in living organisms, and the inhibitory effect of oxidative stress on bone formation. We discussed the possible mechanisms of oxidative stress-induced skeletal involution, and listed some countermeasures that have therapeutic potentials for bone loss via oxidative stress antagonism. Future research for better understanding the oxidative stress caused by space environment and the development of countermeasures against oxidative damage accordingly may facilitate human beings to live more safely in space and explore deeper into the universe.

Microtubule actin crosslinking factor 1 promotes osteoblast differentiation by promoting β-catenin/TCF1/Runx2 signaling axis

Osteoblast differentiation is a multistep process delicately regulated by many factors, including cytoskeletal dynamics and signaling pathways. Microtubule actin crosslinking factor 1 (MACF1), a key cytoskeletal linker, has been shown to play key roles in signal transduction and in diverse cellular processes; however, its role in regulating osteoblast differentiation is still needed to be elucidated. To further uncover the functions and mechanisms of action of MACF1 in osteoblast differentiation, we examined effects of MACF1 knockdown (MACF1‐KD) in MC3T3‐E1 osteoblastic cells on their osteoblast differentiation and associated molecular mechanisms. The results showed that knockdown of MACF1 significantly suppressed mineralization of MC3T3‐E1 cells, down‐regulated the expression of key osteogenic genes alkaline phosphatase (ALP), runt‐related transcription factor 2 (Runx2) and type I collagen α1 (Col Iα1). Knockdown of MACF1 dramatically reduced the nuclear translocation of β‐catenin, decreased the transcriptional activation of T cell factor 1 (TCF1), and down‐regulated the expression of TCF1, lymphoid enhancer‐binding factor 1 (LEF1), and Runx2, a target gene of β‐catenin/TCF1. In addition, MACF1‐KD increased the active level of glycogen synthase kinase‐3β (GSK‐3β), which is a key regulator for β‐catenin signal transduction. Moreover, the reduction of nuclear β‐catenin amount and decreased expression of TCF1 and Runx2 were significantly reversed in MACF1‐KD cells when treated with lithium chloride, an agonist for β‐catenin by inhibiting GSK‐3β activity. Taken together, these findings suggest that knockdown of MACF1 in osteoblastic cells inhibits osteoblast differentiation through suppressing the β‐catenin/TCF1‐Runx2 axis. Thus, a novel role of MACF1 in and a new mechanistic insight of osteoblast differentiation are uncovered.

MACF1, versatility in tissue-specific function and in human disease

Spectraplakins are a family of evolutionarily conserved gigantic proteins and play critical roles in many cytoskeleton-related processes. Microtubule actin crosslinking factor 1 (MACF1) is one of the most versatile spectraplakin with multiple isoforms. As a broadly expressed mammalian spectraplakin, MACF1 is important in maintaining normal functions of many tissues. The loss-of-function studies using knockout mouse models reveal the pivotal roles of MACF1 in embryo development, skin integrity maintenance, neural development, bone formation, and colonic paracellular permeability. Mutation in the human MACF1 gene causes a novel myopathy genetic disease. In addition, abnormal expression of MACF1 is associated with schizophrenia, Parkinsons disease, cancer and osteoporosis. This demonstrates the crucial roles of MACF1 in physiology and pathology. Here, we review the research advances of MACF1s roles in specific tissue and in human diseases, providing the perspectives of MACF1 for future studies.

Mechanical unloading reduces microtubule actin crosslinking factor 1 expression to inhibit β-catenin signaling and osteoblast proliferation

Mechanical unloading was considered a major threat to bone homeostasis, and has been shown to decrease osteoblast proliferation although the underlying mechanism is unclear. Microtubule actin crosslinking factor 1 (MACF1) is a cytoskeletal protein that regulates cellular processes and Wnt/β‐catenin pathway, an essential signaling pathway for osteoblasts. However, the relationship between MACF1 expression and mechanical unloading, and the function and the associated mechanisms of MACF1 in regulating osteoblast proliferation are unclear. This study investigated effects of mechanical unloading on MACF1 expression levels in cultured MC3T3‐E1 osteoblastic cells and in femurs of mice with hind limb unloading; and it also examined the role and potential action mechanisms of MACF1 in osteoblast proliferation in MACF1‐knockdown, overexpressed or control MC3T3‐E1 cells treated with or without the mechanical unloading condition. Results showed that the mechanical unloading condition inhibited osteoblast proliferation and MACF1 expression in MC3T3‐E1 osteoblastic cells and mouse femurs. MACF1 knockdown decreased osteoblast proliferation, while MACF1 overexpression increased it. The inhibitory effect of mechanical unloading on osteoblast proliferation also changed with MACF1 expression levels. Furthermore, MACF1 was found to enhance β‐catenin expression and activity, and mechanical unloading decreased β‐catenin expression through MACF1. Moreover, β‐catenin was found an important regulator of osteoblast proliferation, as its preservation by treatment with its agonist lithium attenuated the inhibitory effects of MACF1‐knockdown or mechanical unloading on osteoblast proliferation. Taken together, mechanical unloading decreases MACF1 expression, and MACF1 up‐regulates osteoblast proliferation through enhancing β‐catenin signaling. This study has thus provided a mechanism for mechanical unloading‐induced inhibited osteoblast proliferation.

Mesenchymal Stem Cell Migration during Bone Formation and Bone Diseases Therapy

During bone modeling, remodeling, and bone fracture repair, mesenchymal stem cells (MSCs) differentiate into chondrocyte or osteoblast to comply bone formation and regeneration. As multipotent stem cells, MSCs were used to treat bone diseases during the past several decades. However, most of these implications just focused on promoting MSC differentiation. Furthermore, cell migration is also a key issue for bone formation and bone diseases treatment. Abnormal MSC migration could cause different kinds of bone diseases, including osteoporosis. Additionally, for bone disease treatment, the migration of endogenous or exogenous MSCs to bone injury sites is required. Recently, researchers have paid more and more attention to two critical points. One is how to apply MSC migration to bone disease therapy. The other is how to enhance MSC migration to improve the therapeutic efficacy of bone diseases. Some considerable outcomes showed that enhancing MSC migration might be a novel trick for reversing bone loss and other bone diseases, such as osteoporosis, fracture, and osteoarthritis (OA). Although plenty of challenges need to be conquered, application of endogenous and exogenous MSC migration and developing different strategies to improve therapeutic efficacy through enhancing MSC migration to target tissue might be the trend in the future for bone disease treatment.

Methods of studying mammalian cell migration and invasion in vitro

Cell migration is a critical event for many physiological processes, such as embryonic development, renewal of skin and intestine, tissue repair, immune surveillance and so on. The abnormality of cell migration affects normal physiological processes and causes diseases, such as wound-healing delay of skin. Cell culture in vitro is essential for studying the migration process and the underlying mechanism. For cell migration detection in vitro, there are a number of methods and each has its own advantages and limitations. Transwell and wound healing assay are the most common methods used to study migration of different cells under different conditions. Besides, other assays are developed in the process of studying cell migration. In recently years, 3D cell migration attracts more and more attention for its accurate mimic of in vivo environment and it may be a trend of detecting cell migration and invasion in vitro in future. This paper summarizes current methods of studying cell migration in vitro and provides reference to researchers.