MACF1 overexpression in BMSCs alleviates senile osteoporosis in mice through TCF4/miR-335-5p signaling pathway.

Background: The decreased osteogenic differentiation ability of mesenchymal stem cells (MSCs) is one of the important reasons for SOP. Inhibition of Wnt signaling in MSCs is closely related to SOP. Microtubule actin crosslinking factor 1 (MACF1) is an important regulator in Wnt/ß-catenin signal transduction. However, whether the specific expression of MACF1 in MSC regulates SOP and its mechanism remains unclear. Methods: We established MSC-specific Prrx1 (Prx1) promoter-driven MACF1 conditional knock-in (MACF-KI) mice, naturally aged male mice, and ovariectomized female mice models. Micro-CT, H&E staining, double calcein labeling, and the three-point bending test were used to explore the effects of MACF1 on bone formation and bone microstructure in the SOP mice model. Bioinformatics analysis, ChIP-PCR, qPCR, and ALP staining were used to explore the effects and mechanisms of MACF1 on MSCs' osteogenic differentiation. Results: Microarray analysis revealed that the expression of MACF1 and positive regulators of the Wnt pathway (such as TCF4, ß-catenin, Dvl) was decreased in human MSCs (hMSCs) isolated from aged osteoporotic than non-osteoporotic patients. The ALP activity and osteogenesis marker genes (Alp, Runx2, and Bglap) expression in mouse MSCs was downregulated during aging. Furthermore, Micro-CT analysis of the femur from 2-month-old MSC-specific Prrx1 (Prx1) promoter-driven MACF1 conditional knock-in (MACF-cKI) mice showed no significant trabecular bone changes compared to wild-type littermate controls, whereas 18- and 21-month-old MACF1 c-KI animals displayed increased bone mineral densities (BMD), improved bone microstructure, and increased maximum compression stress. In addition, the ovariectomy (OVX)-induced osteoporosis model of MACF1 c-KI mice had significantly higher trabecular volume and number, and increased bone formation rate than that in control mice. Mechanistically, ChIP-PCR showed that TCF4 could bind to the promoter region of the host gene miR-335-5p. Moreover, MACF1 could regulate the expression of miR-335-5p by TCF4 during the osteogenic differentiation of MSCs. Conclusion: These data indicate that MACF1 positively regulates MSCs osteogenesis and bone formation through the TCF4/miR-335-5p signaling pathway in SOP, suggesting that targeting MACF1 may be a novel therapeutic approach against SOP. The translational potential of this article: MACF1, an important switch in the Wnt signaling pathway, can alleviate SOP through the TCF4/miR-335-5p signaling pathway in mice model. It might act as a therapeutic target for the treatment of SOP to improve bone function.

Calycosin prevents bone loss induced by hindlimb unloading.

Bone loss induced by microgravity exposure seriously endangers the astronauts' health, but its countermeasures still have certain limitations. The study aims to find potential protective drugs for the prevention of the microgravity-induced bone loss. Here, we utilized the network pharmacology approach to discover a natural compound calycosin by constructing the compound-target interaction network and analyzing the topological characteristics of the network. Furthermore, the hind limb unloading (HLU) rats' model was conducted to investigate the potential effects of calycosin in the prevention of bone loss induced by microgravity. The results indicated that calycosin treatment group significantly increased the bone mineral density (BMD), ameliorated the microstructure of femoral trabecular bone, the thickness of cortical bone and the biomechanical properties of the bone in rats, compared that in the HLU group. The analysis of bone turnover markers in serum showed that both the bone formation markers and bone resorption markers decreased after calycosin treatment. Moreover, we found that bone remodeling-related cytokines in serum including IFN-γ, IL-6, IL-8, IL-12, IL-4, IL-10 and TNF-α were partly recovered after calycosin treatment compared with HLU group. In conclusion, calycosin partly recovered hind limb unloading-induced bone loss through the regulation of bone remodeling. These results provided the evidence that calycosin might play an important role in maintaining bone mass in HLU rats, indicating its promising application in the treatment of bone loss induced by microgravity.

Acacetin Prevents Bone Loss by Disrupting Osteoclast Formation and Promoting Type H Vessel Formation in Ovariectomy-Induced Osteoporosis.

Osteoporosis, characterized by the destruction of bone resorption and bone formation, is a serious disease that endangers human health. Osteoporosis prevention and treatment has become one of the important research contents in the field of medicine. Acacetin, a natural flavonoid compound, could promote osteoblast differentiation, and inhibit osteoclast formation in vitro. However, the mechanisms of acacetin on osteoclast differentiation and type H vessel formation, as well as the effect of preventing bone loss, remain unclear. Here, we firstly used primary bone marrow derived macrophages (BMMs), endothelial progenitor cells (EPCs), and ovariectomized (OVX) mice to explore the function of acacetin on bone remodeling and H type vessel formation. In this study, we found that acacetin inhibits osteoclast formation and bone resorption of BMMs induced by the macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) in a concentration of 20 µM without exerting cytotoxic effects. It was accompanied by downregulation of osteoclast differentiation marker genes (Ctsk, Acp5, and Mmp9) and cell fusion genes (CD9, CD47, Atp6v0d2, Dc-stamp, and Oc-stamp). Moreover, acacetin disrupted actin ring formation and extracellular acidification in osteoclasts. Mechanistic analysis revealed that acacetin not only inhibits the expression of the major transcription factor NFATc1 and NF-κB during RANKL-induced osteoclast formation, but also suppresses RANKL-induced the phosphorylation of Akt, GSK3ß, IκBα, and p65. Additionally, acacetin enhanced the ability of M-CSF and RANKL-stimulated BMMs to promote angiogenesis and migration of EPCs. We further established that, in vivo, acacetin increased trabecular bone mass, decreased the number of osteoclasts, and showed more type H vessels in OVX mice. These data demonstrate that acacetin prevents OVX-induced bone loss in mice through inhibition of osteoclast function and promotion of type H vessel formation via Akt/GSK3ß and NF-κB signalling pathway, suggesting that acacetin may be a novel therapeutic agent for the treatment of osteoporosis.

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.

Deficiency of Macf1 in osterix expressing cells decreases bone formation by Bmp2/Smad/Runx2 pathway.

Microtubule actin cross-linking factor 1 (Macf1) is a spectraplakin family member known to regulate cytoskeletal dynamics, cell migration, neuronal growth and cell signal transduction. We previously demonstrated that knockdown of Macf1 inhibited the differentiation of MC3T3-E1 cell line. However, whether Macf1 could regulate bone formation in vivo is unclear. To study the function and mechanism of Macf1 in bone formation and osteogenic differentiation, we established osteoblast-specific Osterix (Osx) promoter-driven Macf1 conditional knockout mice (Macf1f/f Osx-Cre). The Macf1f/f Osx-Cre mice displayed delayed ossification and decreased bone mass. Morphological and mechanical studies showed deteriorated trabecular microarchitecture and impaired biomechanical strength of femur in Macf1f/f Osx-Cre mice. In addition, the differentiation of primary osteoblasts isolated from calvaria was inhibited in Macf1f/f Osx-Cre mice. Deficiency of Macf1 in primary osteoblasts inhibited the expression of osteogenic marker genes (Col1, Runx2 and Alp) and the number of mineralized nodules. Furthermore, deficiency of Macf1 attenuated Bmp2/Smad/Runx2 signalling in primary osteoblasts of Macf1f/f Osx-Cre mice. Together, these results indicated that Macf1 plays a significant role in bone formation and osteoblast differentiation by regulating Bmp2/Smad/Runx2 pathway, suggesting that Macf1 might be a therapeutic target for bone disease.

Circulating miR-181c-5p and miR-497-5p Are Potential Biomarkers for Prognosis and Diagnosis of Osteoporosis.

CONTEXT: Osteoporosis is a degenerative bone disease in aging men and women. MiRNAs associated with progressive bone loss in osteoporosis had not been clearly demonstrated. OBJECTIVE: The evaluation of the differentially expressed miRNAs in the bone tissue and serum of osteoporotic women with aging. METHODS: MiRNAs GeneChip and real-time PCR were used to screen differently expressed miRNAs in bone tissues of 21 osteoporotic women ages 60-69 years and 80-89 years. Identified miRNAs were detected in the serum of the validation cohort, which consisted of 14 healthy premenopausal women and 86 postmenopausal women with osteopenia or osteoporosis. MiR-181c-5p and miR-497-5p expression were validated in aging and OVX mice models, and osteoblasts. Their role in osteogenesis was validated in vitro. RESULTS: Twenty-four miRNAs showed the highest differential expression in bone tissues of osteoporotic women in initial screening. Among them, four miRNAs were identified both in the bone tissue and serum in the validation cohort. The levels of miR-181c-5p and miR-497-5p were decreased in the serum of postmenopausal women with osteopenia or osteoporosis, but increased in subjects treated with bisphosphonate plus calcitriol. MiR-181c-5p and miR-497-5p were significantly downregulated in the bone tissue of aging and OVX mice models, and upregulated during the osteogenic differentiation of hFOB1.19 and MC3T3-E1 cells. Overexpression of miR-181c-5p and miR-497-5p promoted the differentiation and mineralization of osteoblasts. CONCLUSIONS: MiR-181c-5p and miR-497-5p are involved in bone metabolism and associated with progressive bone loss of due to osteoporosis, suggesting that circulating miR-181c-5p and miR-497-5p might act as potential biomarkers for monitoring the effects of antiosteoporotic therapies or the diagnostic approach.

Phosphate glass fibers facilitate proliferation and osteogenesis through Runx2 transcription in murine osteoblastic cells.

Cell-material interactions and compatibility are important aspects of bioactive materials for bone tissue engineering. Phosphate glass fiber (PGF) is an attractive inorganic filler with fibrous structure and tunable composition, which has been widely investigated as a bioactive filler for bone repair applications. However, the interaction of osteoblasts with PGFs has not been widely investigated to elucidate the osteogenic mechanism of PGFs. In this study, different concentrations of short PGFs with interlaced oriented topography were cocultured with MC3T3-E1 cells for different periods, and the synergistic effects of fiber topography and ionic product of PGFs on osteoblast responses including cell adhesion, spreading, proliferation, and osteogenic differentiation were investigated. It was found that osteoblasts were more prone to adhere on PGFs through Vinculin protein, leading to enhanced cell proliferation with polygonal cell shape and spreading cellular actin filaments. In addition, osteoblasts incubated on PGF meshes showed enhanced alkaline phosphatase activity, extracellular matrix mineralization, and increased expression of osteogenesis-related marker genes, which could be attributed to the Wnt/ß-catenin/Runx2 signaling pathway. This study elucidated the possible mechanism of PGF on triggering specific osteoblast behavior, which would be highly beneficial for designing PGF-based bone graft substitutes with excellent osteogenic functions.

Microtubule actin crosslinking factor 1 (MACF1) knockdown inhibits RANKL-induced osteoclastogenesis via Akt/GSK3ß/NFATc1 signalling pathway.

Osteoclasts are responsible for bone resorption and play essential roles in causing bone diseases such as osteoporosis. Microtubule actin crosslinking factor 1 (MACF1) is a large spectraplakin protein that has been implicated in regulating cytoskeletal distribution, cell migration, cell survival and cell differentiation. However, whether MACF1 regulates the differentiation of osteoclasts has not been elucidated. In this study, we found that the expression of MACF1 was increased in primary bone marrow-derived monocytes (BMMs) of osteoporotic mice and was downregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis of pre-osteoclast cell lines RAW264.7 cells. RAW264.7 cells were transfected with shMACF1 using a lentiviral vector to study the role of MACF1 in osteoclastogenic differentiation. Knockdown of MACF1 in RAW264.7 cells inhibited the formation of multinucleated osteoclasts and decreased the expression of osteoclast-marker genes (Ctsk, Acp5, Mmp9 and Oscar) during RANKL-induced osteoclastogenesis. Additionally, knockdown of MACF1 disrupted actin ring formation in osteoclasts and further blocked the bone resorption activity of osteoclasts by reducing the area and depth of pits. Knockdown of MACF1 had no effect on the survival of pre-osteoclasts and mature osteoclasts. We further established that knockdown of MACF1 attenuated the phosphorylation of Akt and GSK3ß and inhibited the expression of its downstream target NFATc1. Akt activator rescued the inhibition of osteoclast differentiation by MACF1 knockdown. These data demonstrate that MACF1 positively regulates osteoclast differentiation via the Akt/GSK3ß/NFATc1 signalling pathway, suggesting that targeting MACF1 may be a novel therapeutic approach against osteoporosis.

MiR-199b-5p Suppresses Tumor Angiogenesis Mediated by Vascular Endothelial Cells in Breast Cancer by Targeting ALK1.

Angiogenesis is a crucial event during cancer progression that regulates tumor growth and metastasis. Activin receptor-like kinase 1 (ALK1), predominantly expressed in endothelial cells, plays a key role in the organization of neo-angiogenic vessels. Therapeutic targeting of ALK1 has been proposed as a promising strategy for cancer treatment, and microRNAs (miRNAs) are increasingly being explored as modulators of angiogenesis. However, the regulation of ALK1 by miRNAs is unclear. In this study, we identified that ALK1 is directly targeted by miR-199b-5p, which was able to inhibit angiogenesis in vitro and in vivo. Moreover, it was found that miR-199b-5p was repressed in breast cancer cells and its expression was decreased during the VEGF-induced angiogenesis process of human umbilical vein endothelial cells (HUVECs). Overexpression of miR-199b-5p inhibited the formation of capillary-like tubular structures and migration of HUVECs. Furthermore, overexpression of miR-199b-5p inhibited the mRNA and protein expression of ALK1 in HUVECs by directly binding to its 3'UTR. Additionally, overexpression of miR-199b-5p attenuated the induction of ALK1/Smad/Id1 pathway by BMP9 in HUVECs. Finally, overexpression of miR-199b-5p reduced tumor growth and angiogenesis in in vivo. Taken together, these findings demonstrate the anti-angiogenic role of miR-199b-5p, which directly targets ALK1, suggesting that miR-199b-5p might be a potential anti-angiogenic target for cancer therapy.

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.

BPAG1, a distinctive role in skin and neurological diseases.

Spectraplakins are multifunctional cytoskeletal linker proteins that act as important communicators, connecting cytoskeletal components with each other and to cellular junctions. Bullous pemphigoid antigen 1 (BPAG1)/dystonin is a member of spectraplakin family and expressed in various tissues. Alternative splicing of BPAG1 gene produces various isoforms with unique structure and domains. BPAG1 plays crucial roles in numerous biological processes, such as cytoskeleton organization, cell polarization, cell adhesion, and cell migration as well as signaling transduction. Genetic mutation of BPAG1 isoforms is the miscreant of epidermolysis bullosa and multifarious, destructive neurological diseases. In this review, we summarize the recent advances of BPAG1's role in various biological processes and in skin and neurological diseases.