Role Of Microtubule Actin Crosslinking Factor 1 Research Articles

Comprehensive ceRNA network for MACF1 regulates osteoblast proliferation

BackgroundPrevious studies have shown that microtubule actin crosslinking factor 1 (MACF1) can regulate osteoblast proliferation and differentiation through non-coding RNA (ncRNA) in bone-forming osteoblasts. However, the role of MACF1 in targeting the competing endogenous RNA (ceRNA) network to regulate osteoblast differentiation remains poorly understood. Here, we profiled messenger RNA (mRNA), microRNA (miRNA), and long ncRNA (lncRNA) expression in MACF1 knockdown MC3TC‑E1 pre‑osteoblast cells.ResultsIn total, 547 lncRNAs, 107 miRNAs, and 376 mRNAs were differentially expressed. Significantly altered lncRNAs, miRNAs, and mRNAs were primarily found on chromosome 2. A lncRNA-miRNA-mRNA network was constructed using a bioinformatics computational approach. The network indicated that mir-7063 and mir-7646 were the most potent ncRNA regulators and mef2c was the most potent target gene. Pathway enrichment analysis showed that the fluid shear stress and atherosclerosis, p53 signaling, and focal adhesion pathways were highly enriched and contributed to osteoblast proliferation. Importantly, the fluid shear stress and atherosclerosis pathway was co-regulated by lncRNAs and miRNAs. In this pathway, Dusp1 was regulated by AK079370, while Arhgef2 was regulated by mir-5101. Furthermore, Map3k5 was regulated by AK154638 and mir-466q simultaneously. AK003142 and mir-3082-5p as well as Ak141402 and mir-446 m-3p were identified as interacting pairs that regulate target genes.ConclusionThis study revealed the global expression profile of ceRNAs involved in the differentiation of MC3TC‑E1 osteoblasts induced by MACF1 deletion. These results indicate that loss of MACF1 activates a comprehensive ceRNA network to regulate osteoblast proliferation.

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

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). 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 GSK3β 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. 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 GSK3β activity. Additionally, total MAP1B mRNA expression level was upregulated by MACF1 through strengthening the binding of TCF7 to the map1b promoter sequence. 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.

MiR-34a-5p suppresses colorectal cancer cell proliferation through silencing Microtubule Actin Crosslinking Factor 1 (MACF1) gene

BackgroundColorectal cancer (CRC) is the third most frequent cancer in both males and females. CRC results from multiple causes including genetic, epigenetic, and environmental insults. One of the newly identified oncogenes is the Microtubule Actin Crosslinking Factor 1 (MACF1) gene. MACF1 plays a critical role in colon cancer cell proliferation and progression through Wnt /β catenin pathway. Accordingly, we hypothesized that targeting MACF1 would affect CRC progression. Methodsbioinformatics analysis revealed that MACF1 is a target of miR-34a-5p. miR-34a-5p and MACF1 relative expression were measured in 40 colorectal cancers, 40 nonmalignant colon tissue samples using RT- qPCR. "CaCo2" cells line was transfected with miR-34a-5p mimic to assess the effect of MACF1 gene silencing on colorectal cell growth in vitro. ResultsmiR-34a-5p relative expression was significantly lowered in the colorectal cancer group than its expression in nonmalignant groups (P < 0.01). While MACF1 expression was significantly overexpressed in the colorectal cancer group as compared to the nonmalignant groups (P < 0.01). CaCo2 cells' proliferation significantly inhibited after transfection with miR-34a-5p mimic. This inhibited growth was associated with a significant decrease in MACF1 expression. ConclusionThese findings suggest that miR-34a-5p may inhibit colorectal cancer cell proliferation through downregulation of MACF1a member of Wnt/β catenin signaling. This study highlights the novel role of MACF1 as an indicative biomarker and therapeutic goal in colorectal cancer.

Alteration of scaffold: Possible role of MACF1 in Alzheimer’s disease pathogenesis

Alzheimer’s disease (AD) is a progressive neurodegenerative disease, with the sign of sensory or motor function loss, memory decline, and dementia. Histopathological study shows AD neuron has irregular cytoskeleton and aberrant synapse. Amyloid-β (Aβ) is believed as the trigger of AD, however, the detailed pathogenesis is not fully elucidated. Microtubule-actin crosslinking factor 1 (MACF1) is a unique giant molecule which can bind to all three types of cytoskeleton fibers, different linkers/adaptors, as well as various functional proteins. MACF1 is a critical scaffold for orchestrating the complex 3D structure, and is essential for correct synaptic function. MACF1’s binding ability to microtubule depends on Glycogen synthase kinase 3 Bate (GSK3β) mediated phosphorylation. While GSK3β can be regulated by the binding of Aβ and the receptor Paired immunoglobulin-like receptor B (PirB), possibly via Protein phosphatase 2A (PP2A). So based on literature search and logic analysis, we propose a hypothesis: Aβ binds to its receptor PirB, and triggers cytosol PP2A, which might activate GSK3β. GSK3β might further phosphorylates microtubule-binding domain (MTBD) of MACF1, causes the separation of microtubule and MACF1. Thus MACF1 might lose the control of the whole cytoskeleton system, synapse might change and AD might develop. That is Aβ-PirB-PP2A-GSK3β-MACF1 axis might give rise to AD. We hope our hypothesis might provide new clue and evidence to AD pathogenesis.

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.

Nervous-tissue-specific elimination of microtubule-actin crosslinking factor 1a results in multiple developmental defects in the mouse brain

The microtubule-actin crosslinking factor 1 (MACF1) is a ubiquitous cytoskeletal linker protein with multiple spliced isoforms expressed in different tissues. The MACF1a isoform contains microtubule and actin-binding regions and is expressed at high levels in the nervous system. Macf1−/− mice are early embryonic lethal and hence the role of MACF1 in the nervous system could not be determined. We have specifically knocked out MACF1a in the developing mouse nervous system using Cre/loxP technology. Mutant mice died within 24–36 h after birth of apparent respiratory distress. Their brains displayed a disorganized cerebral cortex with a mixed layer structure, heterotopia in the pyramidal layer of the hippocampus, disorganized thalamocortical and corticofugal fibers, and aplastic anterior and hippocampal commissures. Embryonic neurons showed a defect in traversing the cortical plate. Our data suggest a critical role for MACF1 in neuronal migration that is dependent on its ability to interact with both microfilaments and microtubules.

The role of microtubule actin cross-linking factor 1 (MACF1) in the Wnt signaling pathway

MACF1 (microtubule actin cross-linking factor 1) is a multidomain protein that can associate with microfilaments and microtubules. We found that MACF1 was highly expressed in neuronal tissues and the foregut of embryonic day 8.5 (E8.5) embryos and the head fold and primitive streak of E7.5 embryos. MACF1(-/-) mice died at the gastrulation stage and displayed developmental retardation at E7.5 with defects in the formation of the primitive streak, node, and mesoderm. This phenotype was similar to Wnt-3(-/-) and LRP5/6 double-knockout embryos. In the absence of Wnt, MACF1 associated with a complex that contained Axin, beta-catenin, GSK3beta, and APC. Upon Wnt stimulation, MACF1 appeared to be involved in the translocation and subsequent binding of the Axin complex to LRP6 at the cell membrane. Reduction of MACF1 with small interfering RNA decreased the amount of beta-catenin in the nucleus, and led to an inhibition of Wnt-induced TCF/beta-catenin-dependent transcriptional activation. Similar results were obtained with a dominant-negative MACF1 construct that contained the Axin-binding region. Reduction of MACF1 in Wnt-1-expressing P19 cells resulted in decreased T (Brachyury) gene expression, a DNA-binding transcription factor that is a direct target of Wnt/beta-catenin signaling and required for mesoderm formation. These results suggest a new role of MACF1 in the Wnt signaling pathway.