VAMP2 controls murine epidermal differentiation and carcinogenesis by regulation of nucleophagy.

Differentiation of murine epidermal stem/progenitor cells involves the permanent withdrawal from the cell cycle, the synthesis of various protein and lipid components for the cornified envelope, and the controlled dissolution of cellular organelles and nuclei. Deregulated epidermal differentiation contributes to the development of various skin diseases, including skin cancers. With a genome-wide shRNA screen, we identified vesicle-associated membrane protein 2 (VAMP2) as a critical factor involved in skin differentiation. Deletion of VAMP2 leads to aberrant skin stratification and enucleation in vivo. With quantitative proteomics, we further identified an autophagy protein, focal adhesion kinase family interacting protein of 200 kDa (FIP200), as a binding partner of VAMP2. Additionally, we showed that both VAMP2 and FIP200 are critical for murine keratinocyte enucleation and epidermal differentiation. Loss of VAMP2 or FIP200 enhances cutaneous carcinogenesis in vivo. Together, our findings identify important molecular mechanisms underlying epidermal differentiation and skin tumorigenesis.

BMP and activin receptor membrane bound inhibitor: BAMBI has multiple roles in gene expression and diseases (Review).

BMP and activin membrane-bound inhibitor (BAMBI) is a transmembrane glycoprotein, known as a pseudo-receptor for TGFß, as, while its extracellular domain is similar to that of type I TGFß receptors, its intracellular structure is shorter and lacks a serine/threonine phosphokinase signaling motif. BAMBI can regulate numerous biological phenomena, including glucose and lipid metabolism, inflammatory responses, and cell proliferation and differentiation. Furthermore, abnormal expression of BAMBI at the mRNA and protein levels contributes to various human pathologies, including obesity and cancer. In the present review, the structure of BAMBI is briefly introduced and its associated signaling pathways and physiological functions are described. Understanding of BAMBI structure and function may contribute to knowledge regarding the occurrence of diseases, including obesity and diabetes, among others. The present review provides a theoretical foundation for the development of BAMBI as a potential biomarker or therapeutic target.

MACF1 promotes osteoblastic cell migration by regulating MAP1B through the GSK3beta/TCF7 pathway.

RATIONALE: The migration of osteoblastic cells to bone formation surface is an essential step for bone development and growth. However, whether the migration capacity of osteoblastic cells is compromised during osteoporosis occurrence and how it contributes to bone formation reduction remain unexplored so far. In this work, we found, as a positive regulator of cell migration, microtubule actin crosslinking factor 1 (MACF1) enhanced osteoblastic cells migration. We also examined whether MACF1 could facilitate osteoblastic cells' migration to bone formation surface to promote bone formation through another cytoskeleton protein, microtubule associated protein 1 (MAP1B). METHODS: Preosteoblast cell line MC3T3-E1 with different MACF1 level was used for in vitro and in vivo cell migration assay; Primary cortical bone derived mesenchymal stem cells (C-MSCs) from bone tissue of MACF1 conditional knock out (cKO) mice was used for in vitro cell migration assay. Cell migration ability in vitro was evaluated by wound healing assay and transwell assay and in vivo by bone marrow cavity injection. Small interfering RNA (siRNA) was used for knocking down Map1b in MC3T3-E1 cell. Lithium chloride (LiCl) and Wortmannin (Wort) were used for inhibiting/activating GSK3ß pathway activity. Luciferase report assay was performed for detection of transcriptional activity of TCF7 for Map1b; Chromatin immunoprecipitation (ChIP) was engaged for the binding of TCF7 to Map1b promoter region. RESULTS: We found MACF1 enhanced MC3T3-E1 cell and C-MSCs migration in vitro through promoting microtubule (MT) stability and dynamics, and increased the injected MC3T3-E1 cell number on bone formation surface, which indicated a promoted bone formation. We further authenticated that MAP1B had a similar function to MACF1 and was regulated by MACF1 in osteogenic cell, and silencing map1b repressed MC3T3-E1 cell migration in vitro. Mechanistically, by adopting MC3T3-E1 cell with different MACF1 level or treated with LiCl/Wort, we discovered that MACF1 decreased the levels of 1265 threonine phosphorylated MAP1B (p[T1265] MAP1B) through inhibiting GSK3ß activity. Additionally, total MAP1B mRNA expression level was upregulated by MACF1 through strengthening the binding of TCF7 to the map1b promoter sequence. CONCLUSION: Our study uncovered a novel role of MACF1 in bone formation and MAP1B regulation, which suggested that MACF1 could be a potential therapeutic target for osteoporosis.

Long noncoding RNA AK039312 and AK079370 inhibits bone formation via miR-199b-5p.

Osteoporosis caused by aging and menopause had become an emerging threat to human health. The reduction of osteoblast differentiation has been considered to be an essential cause of osteoporosis. Osteoblast differentiation could be regulated by LncRNAs, and increasing evidences have proved that LncRNAs may be adopted as potential therapeutic targets for osteoporosis. However, reports on rescue effects of LncRNAs in vivo are relatively limited. In this study, two LncRNAs (AK039312 and AK079370) were screened as osteogenic related LncRNAs. Both AK039312 and AK079370 could inhibit osteoblast differentiation and bone formation through suppressing osteogenic transcription factors. This inhibitory effect was achieved via binding and sequestering miR-199b-5p, and enhanced GSK-3ß which further inhibited wnt/ß-catenin pathway. Moreover, the siRNAs of AK039312 and AK079370 significantly alleviated postmenopausal osteoporosis, and the combination of si-AK039312 and si-AK079370 was more efficient than applying one si-LncRNA alone. This study has provided new insights for the therapy of osteoporosis.

Silencing of miR-138-5p sensitizes bone anabolic action to mechanical stimuli.

Emerging evidence is revealing that microRNAs (miRNAs) play essential roles in mechanosensing for regulating osteogenesis. However, no mechanoresponsive miRNAs have been identified in human bone specimens. Methods: Bedridden and aged patients, hindlimb unloaded and aged mice, and Random Positioning Machine and primary aged osteoblasts were adopted to simulate mechanical unloading conditions at the human, animal and cellular levels, respectively. Treadmill exercise and Flexcell cyclic mechanical stretching were used to simulate mechanical loading in vivo and in vitro, respectively. Results: Here, we found increased miR-138-5p levels with a lower degree of bone formation in bone specimens from bedridden and aged patients. Loss- and gain-of-function studies showed that miR-138-5p directly targeted microtubule actin crosslinking factor 1 (MACF1) to inhibit osteoblast differentiation under different mechanical conditions. Regarding translational medicine, bone-targeted inhibition of miR-138-5p attenuated the decrease in the mechanical bone anabolic response in hindlimb unloaded mice. Moreover, bone-targeted inhibition of miR-138-5p sensitized the bone anabolic response to mechanical loading in both miR-138-5p transgenic mice and aged mice to promote bone formation. Conclusion: These data suggest that miR-138-5p as a mechanoresponsive miRNA accounts for the mechanosensitivity of the bone anabolic response and that inhibition of miR-138-5p in osteoblasts may be a novel bone anabolic sensitization strategy for ameliorating disuse or senile osteoporosis.

miR-129-5p Inhibits Bone Formation Through TCF4.

Osteoporosis is a frequently occurring bone disease in middle-aged and aged men and women. However, current therapies on this disease are still not ideal. MicroRNAs (miRNAs) are a class of endogenous non-protein-coding RNA with a length of 18-25 nucleotides. miRNAs have been identified as important regulators for development, metabolism, carcinogenesis, and bone formation. miR-129-5p has been reported as a regulator of cancer and neuroscience, whereas studies about its function on bone formation is still limited. In this study, we investigated the function and mechanism of miR-129-5p on osteoblast differentiation and bone formation. We have assessed the expression of miRNAs in bone mesenchymal stem cells from aging and menopause osteoporosis C57BL6 mice. The expression of miR-129-5p was altered in all osteoporosis models. Besides, the expression of miR-129-5p was negatively correlated with osteoblastic differentiation markers in the femur tissues of C57BL/6 mice of different ages. We further demonstrated that overexpression of miR-129-5p inhibited osteoblast differentiation in MC3T3-E1 cell line, as well as bone formation of C57BL/6 mice. On the other hand, down-regulation of miR-129-5p enhanced osteoblast differentiation and bone formation. We also found that miR-129-5p inhibited Wnt/ß-catenin pathway in osteoblast. The target gene of miR-129-5p has been forecasted and proved as Tcf4. We further found that plasmid containing Tcf4-3' UTR sequence enhanced osteoblast differentiation, as well as Wnt/ß-catenin pathway in MC3T3-E1 cells. To further investigate the rescue effect of miR-129-5p inhibitor, we manufactured bioengineered novel recombinant miR-129-5p inhibitor through Escherichia coli system and then tested its function. The results showed that the novel recombinant miR-129-5p inhibitor promoted osteoblast differentiation and greatly ameliorated menopause osteoporosis in C57BL6 mice. In conclusion, we have discovered miR-129-5p as an inhibitor of bone formation. miR-129-5p inhibited downstream transcription factors of Wnt/ß-catenin pathway through targeting Tcf4. Moreover, novel recombinant miR-129-5p inhibitor showed rescue effect on osteoporosis. This study has revealed a new mechanism of osteogenic differentiation and provided novel therapeutic strategies for treatment of skeletal disorders.

Bone Microenvironment and Osteosarcoma Metastasis.

The bone microenvironment is an ideal fertile soil for both primary and secondary tumors to seed. The occurrence and development of osteosarcoma, as a primary bone tumor, is closely related to the bone microenvironment. Especially, the metastasis of osteosarcoma is the remaining challenge of therapy and poor prognosis. Increasing evidence focuses on the relationship between the bone microenvironment and osteosarcoma metastasis. Many elements exist in the bone microenvironment, such as acids, hypoxia, and chemokines, which have been verified to affect the progression and malignance of osteosarcoma through various signaling pathways. We thoroughly summarized all these regulators in the bone microenvironment and the transmission cascades, accordingly, attempting to furnish hints for inhibiting osteosarcoma metastasis via the amelioration of the bone microenvironment. In addition, analysis of the cross-talk between the bone microenvironment and osteosarcoma will help us to deeply understand the development of osteosarcoma. The cellular and molecular protagonists presented in the bone microenvironment promoting osteosarcoma metastasis will accelerate the exploration of novel therapeutic strategies towards osteosarcoma.

Mesenchymal MACF1 Facilitates SMAD7 Nuclear Translocation to Drive Bone Formation.

Microtubule actin crosslinking factor 1 (MACF1) is a large crosslinker that contributes to cell integrity and cell differentiation. Recent studies show that MACF1 is involved in multiple cellular functions such as neuron development and epidermal migration, and is the molecular basis for many degenerative diseases. MACF1 is highly abundant in bones, especially in mesenchymal stem cells; however, its regulatory role is still less understood in bone formation and degenerative bone diseases. In this study, we found MACF1 expression in mesenchymal stem cells (MSCs) of osteoporotic bone specimens was significantly lower. By conditional gene targeting to delete the mesenchymal Macf1 gene in mice, we observed in MSCs decreased osteogenic differentiation capability. During early stage bone development, the MACF1 conditional knockout (cKO) mice exhibit significant ossification retardation in skull and hindlimb, and by adulthood, mesenchymal loss of MACF1 attenuated bone mass, bone microarchitecture, and bone formation capability significantly. Further, we showed that MACF1 interacts directly with SMAD family member 7 (SMAD7) and facilitates SMAD7 nuclear translocation to initiate downstream osteogenic pathways. Hopefully these findings will expand the biological scope of the MACF1 gene, and provide an experimental basis for targeting MACF1 in degenerative bone diseases such as osteoporosis.

MACF1 promotes preosteoblast migration by mediating focal adhesion turnover through EB1.

Microtubule actin crosslinking factor 1 (MACF1) is a widely expressed cytoskeletal linker and plays an essential role in various cells' functions by mediating cytoskeleton organization and dynamics. However, the role of MACF1 on preosteoblast migration is not clear. Here, by using MACF1 knockdown and overexpressed MC3T3-E1 cells, we found MACF1 positively regulated preosteoblast migration induced by cell polarization. Furthermore, immunofluorescent staining showed that MACF1 increased end-binding protein (EB1) distribution on microtubule (MT), and decreased EB1 distribution on focal adhesion (FA) complex. Moreover, upregulation of MACF1 activated Src level and enhanced the colocalization of EB1 with activated Src. In addition, MACF1 diminished colocalization of EB1 with adenomatous polyposis coli (APC), which induced EB1 release from FA and promoted FA turnover. These results indicated an important role and mechanism of MACF1 in regulating preosteoblast migration through promoting FA turnover by mediating EB1 colocalization with Src and APC, which inferred that MACF1 might be a potential target for preventing and treating bone disorders.

The Roles of FoxO Transcription Factors in Regulation of Bone Cells Function.

Forkhead box class O family member proteins (FoxOs) are evolutionarily conserved transcription factors for their highly conserved DNA-binding domain. In mammalian species, all the four FoxO members, FoxO1, FoxO3, FoxO4, and FoxO6, are expressed in different organs. In bone, the first three members are extensively expressed and more studied. Bone development, remodeling, and homeostasis are all regulated by multiple cell lineages, including osteoprogenitor cells, chondrocytes, osteoblasts, osteocytes, osteoclast progenitors, osteoclasts, and the intercellular signaling among these bone cells. The disordered FoxOs function in these bone cells contribute to osteoarthritis, osteoporosis, or other bone diseases. Here, we review the current literature of FoxOs for their roles in bone cells, focusing on helping researchers to develop new therapeutic approaches and prevent or treat the related bone diseases.

Silencing of lncRNA AK045490 Promotes Osteoblast Differentiation and Bone Formation via ß-Catenin/TCF1/Runx2 Signaling Axis.

Osteoporosis, a disease characterized by both loss of bone mass and structural deterioration of bone, is the most common reason for a broken bone among the elderly. It is known that the attenuated differentiation ability of osteogenic cells has been regarded as one of the greatest contributors to age-related bone formation reduction. However, the effects of current therapies are still unsatisfactory. In this study we identify a novel long noncoding RNA AK045490 which is correlated with osteogenic differentiation and enriched in skeletal tissues of mice. In vitro analysis of bone-derived mesenchymal stem cells (BMSCs) showed that AK045490 inhibited osteoblast differentiation. In vivo inhibition of AK045490 by its small interfering RNA rescued bone formation in ovariectomized osteoporosis mice model. Mechanistically, AK045490 inhibited the nuclear translocation of ß-catenin and downregulated the expression of TCF1, LEF1, and Runx2. The results suggest that Lnc-AK045490 suppresses ß-catenin/TCF1/Runx2 signaling and inhibits osteoblast differentiation and bone formation, providing a novel mechanism of osteogenic differentiation and a potential drug target for osteoporosis.

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.

MiR-21-5p, miR-34a, and human telomerase RNA component as surrogate markers for cervical cancer progression.

OBJECTIVE: This study aimed to demonstrate the predictive value of miR-21-5p, miR-34a, and human telomerase RNA component (hTERC) in cervical cancer (CC) development and evaluated their potential possibility for future clinical applications. METHODS: Specimens were collected from the normal cervix, cervical intraepithelial neoplasia (CIN) I, CIN II/III, cervical squamous cell carcinoma. Cytological evaluations and histopathologic examinations were conducted in all subjects, along with the assessment of human papillomavirus (HPV) DNA. The expression levels of the miR-21-5p and miR-34a were detected by RT-PCR. hTERC amplification was detected by dual-color interphase fluorescence in situ hybridization (FISH). Then miRNA, hTERC expressions were compared with the cytological and histologic examination. RESULTS: Compared to that in the benign samples, the expression of miR-21-5p and miR-34a in abnormal samples was significantly upregulated and downregulated, gradually corresponding to the severity of cervical lesions (P < 0.05). There was a trend toward an increasing amplification of hTERC with the increasing severity of cervical lesions. miR-21-5p and miR-34a expression, and hTERC amplification were more specific than HPV positivity in differentiating low-grade cervical disorders from high-grade ones (P < 0.05). CONCLUSIONS: MiR-21-5p upregulation, miR-34a downregulation, and hTERC amplification were associated with the aggressive progression of CC, which suggests that miR-21-5p, miR-34a and hTERC might serve as surrogate markers for CC progression and potential molecular targets for blockage of the development of CC.

MACF1 Overexpression by Transfecting the 21 kbp Large Plasmid PEGFP-C1A-ACF7 Promotes Osteoblast Differentiation and Bone Formation.

Microtubule actin crosslinking factor 1 (MACF1) is a large spectraplakin protein known to have crucial roles in regulating cytoskeletal dynamics, cell migration, growth, and differentiation. However, its role and action mechanism in bone remain unclear. The present study investigated optimal conditions for effective transfection of the large plasmid PEGFP-C1A-ACF7 (∼21 kbp) containing full-length human MACF1 cDNA, as well as the potential role of MACF1 in bone formation. To enhance MACF1 expression, the plasmid was transfected into osteogenic cells by electroporation in vitro and into mouse calvaria with nanoparticles. Then, transfection efficiency, osteogenic marker expression, calvarial thickness, and bone formation were analyzed. Notably, MACF1 overexpression triggered a drastic increase in osteogenic gene expression, alkaline phosphatase activity, and matrix mineralization in vitro. Mouse calvarial thickness, mineral apposition rate, and osteogenic marker protein expression were significantly enhanced by local transfection. In addition, MACF1 overexpression promoted ß-catenin expression and signaling. In conclusion, MACF1 overexpression by transfecting the large plasmid containing full-length MACF1 cDNA promotes osteoblast differentiation and bone formation via ß-catenin signaling. Current data will provide useful experimental parameters for the transfection of large plasmids and a novel strategy based on promoting bone formation for prevention and therapy of bone disorders.

Mechanical unloading reduces microtubule actin crosslinking factor 1 expression to inhibit ß-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/ß-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 ß-catenin expression and activity, and mechanical unloading decreased ß-catenin expression through MACF1. Moreover, ß-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 ß-catenin signaling. This study has thus provided a mechanism for mechanical unloading-induced inhibited osteoblast proliferation.

Microtubule actin crosslinking factor 1 promotes osteoblast differentiation by promoting ß-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 ß-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 ß-catenin/TCF1. In addition, MACF1-KD increased the active level of glycogen synthase kinase-3ß (GSK-3ß), which is a key regulator for ß-catenin signal transduction. Moreover, the reduction of nuclear ß-catenin amount and decreased expression of TCF1 and Runx2 were significantly reversed in MACF1-KD cells when treated with lithium chloride, an agonist for ß-catenin by inhibiting GSK-3ß activity. Taken together, these findings suggest that knockdown of MACF1 in osteoblastic cells inhibits osteoblast differentiation through suppressing the ß-catenin/TCF1-Runx2 axis. Thus, a novel role of MACF1 in and a new mechanistic insight of osteoblast differentiation are uncovered.

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.

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, Parkinson's disease, cancer and osteoporosis. This demonstrates the crucial roles of MACF1 in physiology and pathology. Here, we review the research advances of MACF1's roles in specific tissue and in human diseases, providing the perspectives of MACF1 for future studies.

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.

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.