A T-cell-related signature for prognostic stratification and immunotherapy response in hepatocellular carcinoma based on transcriptomics and single-cell sequencing.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most frequently diagnosed malignancy and the third leading cause of cancer death globally. T cells are significantly correlated with the progression, therapy and prognosis of cancer. Limited systematic studies regarding the role of T-cell-related markers in HCC have been performed. METHODS: T-cell markers were identified with single-cell RNA sequencing (scRNA-seq) data from the GEO database. A prognostic signature was developed with the LASSO algorithm in the TCGA cohort and verified in the GSE14520 cohort. Another three eligible immunotherapy datasets, GSE91061, PRJEB25780 and IMigor210, were used to verify the role of the risk score in the immunotherapy response. RESULTS: With 181 T-cell markers identified by scRNA-seq analysis, a 13 T-cell-related gene-based prognostic signature (TRPS) was developed for prognostic prediction, which divided HCC patients into high-risk and low-risk groups according to overall survival, with AUCs of 1 year, 3 years, and 5 years of 0.807, 0.752, and 0.708, respectively. TRPS had the highest C-index compared with the other 10 established prognostic signatures, suggesting a better performance of TRPS in predicting the prognosis of HCC. More importantly, the TRPS risk score was closely correlated with the TIDE score and immunophenoscore. The high-risk score patients had a higher percentage of SD/PD, and CR/PR occurred more frequently in patients with low TRPS-related risk scores in the IMigor210, PRJEB25780 and GSE91061 cohorts. We also constructed a nomogram based on the TRPS, which had high potential for clinical application. CONCLUSION: Our study proposed a novel TRPS for HCC patients, and the TRPS could effectively indicate the prognosis of HCC. It also served as a predictor for immunotherapy.

AFAP1L1 promotes gastric cancer progression by interacting with VAV2 to facilitate CDC42-mediated activation of ITGA5 signaling pathway.

BACKGROUND: The actin filament-associated protein (AFAP) family genes include AFAP1/AFAP-110, AFAP1L1 and AFAP1L2/XB130. Increasing evidence indicates these three AFAP family members participate in tumor progression, but their clinical significance and molecular mechanisms in gastric cancer (GC) remain unclear. METHODS: We first analyzed expression of AFAP family genes using public datasets and verified the results. The clinical significance of AFAP family genes in GC patients was also analyzed. In vitro and in vivo experiments were applied to explore the function of AFAP1L1. Enrichment analysis was used to explore potential molecular mechanisms. We then performed additional experiments, such as cell adhesion assay, co-immunoprecipitation and so on to confirm the downstream molecular mechanisms of AFAP1L1. RESULTS: Public data analyses and our verification both showed AFAP1L1 was the only AFAP family members that was significantly upregulated in GC compared with normal gastric tissues. Besides, only AFAP1L1 could predict poor prognosis and act as an independent risk factor for GC patients. In addition, AFAP1L1 promotes GC cells proliferation, migration, invasion in vitro and tumor growth, metastasis in vivo by inducing epithelial-to-mesenchymal transition (EMT). In terms of mechanism, AFAP1L1 interacts with VAV guanine nucleotide exchange factor 2 (VAV2) to activate Rho family GTPases CDC42, which finally promotes expression of integrin subunit alpha 5 (ITGA5) and activation of integrin signaling pathway. CONCLUSION: AFAP1L1 promotes GC progression by inducing EMT through VAV2-mediated activation of CDC42 and ITGA5 signaling pathway, indicating AFAP1L1 may be a promising prognostic biomarker and therapeutic target for GC patients.

TNF-α/TNFR1 regulates the polarization of Kupffer cells to mediate trichloroethylene-induced liver injury.

We have previously shown trichloroethylene (TCE) induced immune liver injury, and TNF-α/TNFR1 pathway as a probably mechanism underlying the immune damage, but the pathogenic mechanism is still unclear. The study aims to investigate whether TNF-α and its receptors regulate Kupffer cell polarization and downstream inflammation signaling pathways during TCE sensitization, to clarify the mechanism of TCE-mediated immune liver injury. 6-8 weeks old SPF BALB/c female mice were used to establish a TCE sensitization model. We found that in the TCE sensitization positive group, liver injury was aggravated, Kupffer cells activated and polarized to M1 type. The expression of M1 Kupffer cell marker proteins CD11c and CD16/32 increased in the TCE positive group, so did TNF-α and TNFR1 in liver. The expression of P-IKK protein, PP65 protein and P-STAT3 protein increased in the TCE sensitization positive group, and the downstream inflammatory factors IL-1ß and IL-6 also increased in the TCE sensitization positive group. After using the TNFR1 inhibitor R7050, we found that M1 Kupffer cell polarization, TNF-α expression, signal pathway expression and inflammatory factors IL-1ß and IL-6 expression declined, and the liver damage relieved. Briefly, the use of R7050 to inhibit TNF-α/TNFR1 changing the polarization of liver M1 Kupffer cell, thereby inhibiting the activation of related downstream signaling pathways and reducing the secretion of inflammatory factors. TNF-α/TNFR1 regulates the polarization of M1 Kupffer cells inflammatory play an important role in liver immune damage.

Effect of PLC-ß1/CaM signaling pathway mediated by AT1R on the occurrence and development of hepatocellular carcinoma.

OBJECTIVE: To study the roles of AT1R, PLC-ß1, CaM and other related signal molecules in the formation and development of hepatocellular carcinoma (HCC) and their correlation. METHODS: ELISA and immunohistochemistry were used to analyze the expressions of target proteins in serum and liver tissue of HCC patients, and the correlation between AT1R, PLC-ß1 and CaM and postoperative survival status of patients was followed up and determined. CCK-8 method was used to screen the doses of Ang II and candesartan sensitive to HepG2 and HCCLM3 cells. Transwell experiment was used to observe the effects of different drugs on the migration and invasion activity of HCC cells. Meanwhile, flow cytometry and Western blot were used to detect the expression levels of AT1R, PLC-ß1 and CaM in the cells. Then PLC-ß1 siRNA was selected to transfect HCC cells, so as to further clarify the mechanism of the above signal proteins. HepG2 cells were inoculated under the hepatic capsule of mice to induce the formation of HCC in situ. Ang II and candesartan were used to stimulate HCC mice to observe the difference in liver appearance and measure the liver index. Finally, ELISA and immunofluorescence experiments were selected to analyze the levels of target proteins in mouse serum and liver tissue. RESULTS: The expression levels of target proteins in serum and liver tissue of HCC patients were significantly increased, and the postoperative survival time of patients with high expression of AT1R, PLC-ß1 or CaM was obviously shortened. Ang II and candesartan could significantly promote and inhibit the motility of HCC cells, and had different effects on the levels of AT1R, PLC-ß1 and CaM in cells. However, in hepatocellular carcinoma cells transfected with PLC-ß1 siRNA, the intervention ability of drugs was obviously weakened. Ang II could significantly promote the formation and progression of mouse HCC, while candesartan had the opposite effect. Meanwhile, medications could affect the expressions of target proteins in mouse serum and liver tissue. CONCLUSION: AT1R, PLC-ß1 and CaM may be risk factors affecting the formation and prognosis of HCC, and the PLC-ß1/CaM signaling pathway mediated by AT1R is an important way to regulate the migration and invasion activity of HCC cells.

Mechanisms underlying the changes in acetaldehyde dehydrogenase 1 in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is the most recurrent malignant tumor found in the biliary system. It originates from the bile duct epithelial cells characterized by easy metastasis, high intermittent rate, and poor prognosis. Acetaldehyde dehydrogenase 1 (ALDH1), a marker of cancer stem cells, the levels of which are particularly elevated in various of malignant tumors. Additionally, the increased ALDH1 levels are closely related to the degree and prognosis of malignant tumors. This study reviewed the mechanisms underlying the changes in ALDH1 levels in CCA.

Application of symptom-based mind mapping combined with PBL teaching method in emergency trauma standardized resident training in MDT model.

Explore the feasibility and effectiveness of accepting mind mapping combined with problem-based learning (PBL) teaching method in the standardized training of emergency surgery residents in the multi-disciplinary team (MDT) model of emergency trauma. Eighty-nine doctors under training who rotated in the Department of Emergency Surgery of the First Affiliated Hospital of Anhui Medical University from January 2021 to January 2022 were selected as the study subjects, and randomly divided into a group receiving mind mapping combined with PBL teaching and a group receiving traditional lecture-based learning teaching. Mini-clinical evaluation exercise (Mini-CEX), direct observation of procedural skills (DOPS), teaching adherence, and satisfaction assessments were completed at the time of discharge from the department. There were no significant differences between the observation and control group trainees in terms of gender, age, education, and entry grades. Both groups of doctors were better able to participate in their respective teaching modes and made significant progress. The participants in the observation group had significantly higher Mini-CEX, DOPS, and teaching satisfaction scores than the control group (P < .05). Under the MDT model of emergency trauma, the combination of mind mapping and PBL teaching can improve the comprehensive clinical ability of the trainees more than participating in the traditional lecture-based learning teaching, which is worth promoting and implementing in the clinical standardized training.

Anti-TNF and immunosuppressive combination therapy is preferential to inducing clinical remission in patients with active inflammatory bowel disease: A systemic review and meta-analysis.

OBJECTIVE: To compare the efficacy and safety of a combination therapy of biologics and immunosuppressants with biological monotherapy in inflammatory bowel disease (IBD) in a systematic review and meta-analysis. METHODS: Eligible randomized controlled trials (RCTs) on the comparison of the efficacy and safety of biologics and immunomodulators with biological monotherapy were identified from the EMBASE, PubMed and the Cochrane Library databases published up to 1 May 2020. Raw data were extracted, pooled relative risk (RR) and 95% confidence interval (CI) was calculated, the fixed-effect and inverse variance models were used. Funnel plots were performed to analyze publication bias. RESULTS: Twelve RCTs were eligible for analysis. Overall, there was statistically a benefit for combination treatment over biologic monotherapy (IFX/ADA) in inducing clinical remission and preventing relapse in patients with IBD (RR 0.89, 95% CI 0.80-0.98). Moreover, the combination therapy was superior to biological monotherapy for active CD (RR 0.83, 95% CI 0.73-0.94). Also, there were significant benefits for combination therapy in the subgroup treated with infliximab (IFX) (RR 0.83, 95% CI 0.70-0.97). CONCLUSIONS: Combination therapy has slight benefits in inducing clinical remission in active CD compared with biological monotherapy. Patients with IBD who receive therapy with IFX and immunomodulator also have a mild advantage in comparison with those treated with IFX monotherapy.

Undercarboxylated osteocalcin inhibits the early differentiation of osteoclast mediated by Gprc6a.

Osteocalcin (OCN) was the most abundant noncollagen protein and considered as an endocrine factor. However, the functions of Undercarboxylated osteocalcin (ucOCN) on osteoclast and bone resorption are not well understood. In the present study, preosteoclast RAW264.7 cells and bone marrow mononuclear cells (BMMs) were treated with ucOCN purified from prokaryotic bacteria. Our results showed that ucOCN attenuated the proliferation of RAW264.7 cells with a concentration dependant manner by MTS assay. Scrape wounding assay revealed the decreased motility of RAW264.7 cells after ucOCN treatment. RT-qPCR results manifested the inhibitory effects of ucOCN on the expression of osteoclastic marker genes in RAW264.7 cells during inducing differentiation of RANKL. It was also observed that ucOCN inhibited the formation of multinucleated cells from RAW264.7 cells and BMMs detected by TRAP staining. The number and area of bone resorb pits were also decreased after treatment with ucOCN during their osteoclast induction by toluidine blue staining. The formation and integrity of the osteoclast actin ring were impaired by ucOCN by immunofluorescent staining. Time dependant treatment of ucOCN during osteoclastic induction demonstrated the inhibitory effects mainly occurred at the early stage of osteoclastogenesis. Signaling analysis of luciferase activity of the CRE or SRE reporter and ERK1/2 phosphorylation showed the selective inhibitor or siRNA of Gprc6a (a presumptive ucOCN receptor) could attenuate the promotion of ucOCN on CRE-luciferase activity. Taken together, we provided the first evidence that ucOCN had negative effects on the early differentiation and bone resorption of osteoclasts via Gprc6a.

Folic acid-modified nonionic surfactant vesicles for gambogenic acid targeting: Preparation, characterization, and in vitro and in vivo evaluation.

The aim of present study was to develop folic acid (FA)-modified nonionic surfactant vesicles (NISVs, niosomes) as carrier systems for targeted delivery of gambogenic acid (GNA). The FA-GNA-NISVs exhibited a mean particle size of 180.77 ± 2.41 nm with a narrow poly dispersion index of 0.147 ± 0.08 determined by dynamic light scattering. Transmission electron microscopy also revealed that the FA-GNA-NISVs were spherical with double-layer structure. Entrapment efficiency (EE%) and zeta potential of the optimal FA-GNA-NISVs were 87.84 ± 1.06% and -37.33 ± 0.33 mV, respectively. Differential scanning calorimetry demonstrated that the GNA was in a molecular or amorphous state inside the FA-NISVs in vitro release profiles suggested that FA-GNA-NISVs could release GNA at a sustained manner, and less than 60% of GNA was released from the FA-NISVs within 12 hours of dialysis. in vivo pharmacokinetic results illustrated that FA-GNA-NISVs had considerably higher Cmax , area under curve (AUC0 - t ) and accumulation in lung. The cell proliferation study shown that the FA-GNA-NISVs significantly enhanced the in vitro cytotoxicity against A549 cells. Flow cytometry and fluorescence microscopy further demonstrated that the FA-GNA-NISVs increased apoptosis compared with nonmodified GNA-NISVs and free GNA. Moreover, FA-GNA-NISVs induced A549 cell apoptosis in a dose-dependent manner. In addition, cellular uptake assays showed a higher uptake of FA-GNA-NISVs than GNA-NISVs as well as free GNA. Taken together, it could be concluded that FA-GNA-NISVs were proposed as a novel targeting carriers for efficient delivering of GNA to cancers cells.

Serum Metabolomics Associating With Circulating MicroRNA Profiles Reveal the Role of miR-383-5p in Rat Hippocampus Under Simulated Microgravity.

Microgravity impacts various aspects of human health. Yet the mechanisms of spaceflight-induced health problems are not elucidated. Here, we mapped the fusion systemic analysis of the serum metabolome and the circulating microRNAome in a hindlimb unloading rat model to simulate microgravity. The response of serum metabolites and microRNAs to simulated microgravity was striking. Integrated pathway analysis of altered serum metabolites and target genes of the significantly altered circulating miRNAs with Integrated Molecular Pathway-Level Analysis (IMPaLA) software was mainly suggestive of modulation of neurofunctional signaling pathways. Particularly, we revealed significantly increased miR-383-5p and decreased aquaporin 4 (AQP4) in the hippocampus. Using rabies virus glycoprotein-modified exosomes, delivery of miR-383-5p inhibited the expression of AQP4 not only in rat C6 glioma cells in vitro but also in the hippocampus in vivo. Using bioinformatics to map the crosstalk between the circulating metabolome and miRNAome could offer opportunities to understand complex biological systems under microgravity. Our present results suggested that the change of miR-383-5p level and its regulation of target gene AQP4 was one of the potential molecular mechanisms of microgravity-induced cognitive impairment in the hippocampus.

A novel drug delivery system of mixed micelles based on poly(ethylene glycol)-poly(lactide) and poly(ethylene glycol)-poly(ɛ-caprolactone) for gambogenic acid.

In this study, a novel mixed polymeric micelles formed from biocompatible polymers, poly(ethylene glycol)-poly(lactide) (mPEG-PLA) and poly(ethylene glycol)-poly(ɛ-caprolactone) (mPEG-PCL), used as a novel nanocarrier to encapsulate gambogenic acid (GNA). GNA-loaded mixed polymeric micelles (GNA-MMs) was prepared by cosolvent evaporation method. The mean average size of GNA-MMs was (83.23 ± 1.06) nm (n = 3) and entrapment efficiency (EE%) of GNA-MMs was (90.18 ± 2.59) % (n = 3) as well as (12.36 ± 0.64) % (n = 3) for drug loading (DL%). Transmission electron microscopy revealed that the GNA-MMs were spherical with "core-shell" structures. Compared with free GNA solution, in vitro release of GNA from GNA-MMs showed a two-phase sustained release profile: an initial relatively fast phase and followed by a slower release phase. Pharmacokinetic results also indicated that the GNA-MMs have longer systemic circulation time and slower plasma elimination rate than free GNA solution. Moreover, the in vitro cytotoxicity assay showed that the IC50 values on HepG2 cells for GNA-MMs and free GNA were (5.67 ± 0.02) µM and (9.02 ± 0.03) µM, respectively. In addition, GNA-MMs significantly increased the HepG2 cellular apoptosis in a concentration-dependent manner. In conclusion, the results showed that mPEG-PLA/mPEG-PCL mixed micelles may serve as an ideal drug delivery system for GNA to prolong drug circulation time in body, enhance bioavailability and retained its potent antitumor effect.

Actin cytoskeleton mediates BMP2-Smad signaling via calponin 1 in preosteoblast under simulated microgravity.

Microgravity influences the activity of osteoblast, induces actin microfilament disruption and leads to bone loss during spaceflight. Mechanical stress such as gravity, regulates cell function, response and differentiation through dynamic cytoskeleton changes, but the mechanotransduction mechanism remains to be fully elucidated. Previous, we demonstrated actin microfilament mediated osteoblast Cbfa1 responsiveness to BMP2 under simulated microgravity (SMG). Here, we explored a potential molecular and its detailed mechanism of actin cytoskeleton functioning on BMP2-Smad signaling in MC3T3-E1 under SMG. Results showed that the actin microfilament-disrupting agent, cytochalasin B (CB), reduced BMP2-induced activation, translocation of Smad1/5/8 and Runx2 expression. SMG also inhibited BMP2-Smad signaling, which was rescued by actin cytoskeleton stabilizing agent, Jasplakinolide (JAS). Furthermore, we found that siRNA mediated knockdown of calponin 1 (CNN1), an actin binding protein, markedly promoted BMP2-Smad signaling and abolished both inhibition of CB, SMG on BMP2-Smad signaling and the rescue action of JAS. Overexpression of CNN1 inhibited the p-Smad induced by BMP2. Bidirectional Co-IP experiments demonstrated CNN1 could interacted with Smad or p-Smad protein. Furthermore, CB or SMG decreased the phosphorylated CNN1 and increased its interaction with Smad or p-Smad. Combined with the phosphorylation of CNN1 inhibites its actin binding activity, these results indicate that actin cytoskeleton depolymerization inhibites BMP2 signaling via blocking of Smad by dephosphorylated CNN1 in osteoblast cells. Thus, we provide new important insights into the mechanism of mechanotransduction under SMG condition, which probably contribute to bone formation decrease induced by SMG.

[Disruption of microfilament cytoskeleton induced by simulated microgravity increases the activity of COL1A1 promoter].

It is well known that cytoskeleton system is the sensor of gravity in cells. Under microgravity condition, cytoskeleton is associated with the changes of cell shape, function, signaling and so on; but the relationship between cytoskeleton and gene expression is not fully understood. In present study, we discussed the effects of cell microfilament on the activity of collagen type I alpha 1 chain gene (COL1A1) promoter under microgravity simulated by clinostat and/or cytochalasin B as microfilament depolymerizer in the established EGFP-ROS cell line using the method of fluorescence semi-quantitative analysis and the fluorescent stain of microfilament. Compared with the normal control, the microfilament of ROS17/2.8 cell tended to disassemble, marginal distribution of fiber stress, and showed reducing stress fibers after spaceflight in Photon-M1 or clinorotation simulated microgravity, which suggested that microgravity destroyed the well-order cell cytoskeleton and induced a rearrangement. Treatment with suitable concentration of cytochalasin B in normal gravity induced disruption of microfilament, increased the activity of COL1A1 promoter and resulted in a dose-dependent increase of EGFP fluorescence. Therefore, a certain extent disruption of the microfilament system was associated with increased activity of the COL1A1 promoter. All above demonstrate that microfilament cytoskeleton system takes part in the regulation of COL1A1 promoter activity and plays an important role in the signaling of microgravity.

[Functions of human periodontal myofibroblast in vitro].

OBJECTIVE: To investigate the functions of human periodontal myofibroblast (MFB) in vitro. METHODS: Human periodontal fibroblast (hPDLFs) was cultured and induced to MFB by transforming growth factor-ß1 (TGF-ß1). MFB was denoted as the experimental group, whereas the hPDLFs was the control group. The groups were continuously cultured and harvested at 0, 12, 24, 48, and 72 h. The MFB marker α-smooth muscle actin (α-SMA) was examined by immunocytochemistry. The expression of fibronectin (FN) between MFB was examined by immunocytochemistry to detect the MFB contact relationship. The mRNA expression levels of α-SMA, collagen (Col) I, and Col III were measured by reverse transcription-polymerase chain reaction (RT-PCT) to analyze extracellular matrix secretion. The protein expression levels of α-SMA and Col I were also assessed by Western blot. RESULTS: The experimental group had significantly higher α-SMA expression than the control group at 0 h (P < 0.001). A positive expression of FN was found between MFB. The experimental group had significantly higher expression levels of Col I and Col III than the control group at 24 h (P < 0.001). CONCLUSION: Human periodontal MFB presents a continuous, high expression of α-SMA. MFB could interact through FN. MFB is significantly capable of extracellular matrix secretion.

Effects of clinorotation on COL1A1- EGFP gene expression.

Bone-formation related gene plays a critical role in bone loss induced by space microgravity, however the exact mechanism is unclear. In this study, we aim to investigate the effect of microgravity on the activity of alpha 1(I) collagen (COL1A1) gene promoter and the expression of osteoblast-related genes. COL1A1 promoter was digested by restriction enzymes resulting in three DNA fragments. The fragments were ligated with the enhanced green fluorescent protein report gene, and subcloned into expression vectors. ROS17/2.8 cells transfected by these vectors were screened by G418, and enhanced green fluorescent protein (EGFP) positive colonies were isolated and cultured under clinostat condition. EGFP and Collagen type I expression level were detected by fluorescence intensity analysis and immunocytochemistry methods respectively. The results showed that the expression of EGFP and collagen type I was increased 24 h, 48 h after the cells were cultured under stimulated microgravity, illustrating that the activity of COL1A1 promoter might be increased. In conclusion, osteoblasts can compensatively increase the expression of type I collagen by enhancing the activity of COL1A1 promoter under short-term simulated microgravity conditions.

[Establishment of COL1A1-EGFP stably transfected ROS17/2.8 cell lines].

OBJECTIVE: To obtain ROS17/2.8 cell lines which were stably expressing EGFP reporter gene drived by COL1A1 promoter. METHOD: A 3.6 Kb COL1A1 promoter from rat was cloned into pMD-18-T vector by PCR. This amplified promoter vector was digested to get several different length fragments which were then fused with EGFP reporter gene to construct eukaryotic expression vectors. ROS17/2.8 cell was stably transfected with these vectors by LipofectAMINE(TM) and selected by G418. RESULT: The COL1A1-EGFP stably transfected cell lines were established. CONCLUSION: The cell lines will be useful for studying the effects of microgravity on the activity of COL1A1 promoter and expression of gene related with bone form.

Differential diagnosis of intestinal tuberculosis from Crohn's disease and primary intestinal lymphoma in China.

BACKGROUND/AIMS: There are many similarities and overlaps in clinical, radiological, endoscopic, and histological features among intestinal tuberculosis (ITB), Crohn's disease (CD), and primary intestinal lymphoma (PIL), and the differential diagnosis of ITB can be very challenging for clinicians. PATIENTS AND METHODS: The clinical, radiologic, endoscopic, and pathological data of 213 patients were analyzed retrospectively. According to the diagnostic criteria and exclusive criteria of ITB, CD, and PIL, 83 patients were recruited and divided into three groups, including 30 cases in the ITB group, 38 cases in the CD group, and 15 cases in the PIL group, and the medical data and statistical analysis were recorded. RESULTS: Rural patients with abdominal pain as the first symptom and with transverse ulcer and caseating granulomas were more common in the ITB group than the CD group, whereas urban patients with stool change as the first symptom, moderate or severe anemia, thickening of intestinal wall, rectal involvement, skipping distribution, prominent lymphoid aggregates, and irregular glands were more common in CD group than ITB group (P < 0.05). Young patients (age < 30 years) with fever, weakness, fatigue, abdominal mass, intestinal perforation, and emergent operation were more common in ITB group than PIL group, whereas thickening of intestinal wall, malignant lymphocytes, limited distribution, and involvement of small intestine occurred more in PIL group than ITB group (P < 0.05). CONCLUSION: The differential diagnosis of ITB from CD and PIL can be made by a combination of clinical manifestation, endoscopy, and pathological examinations.

Actin microfilament mediates osteoblast Cbfa1 responsiveness to BMP2 under simulated microgravity.

Microgravity decreases osteoblastic activity, induces actin microfilament disruption and inhibits the responsiveness of osteoblast to cytokines, but the mechanisms remains enigmatic. The F-actin cytoskeleton has previously been implicated in manifold changes of cell shape, function and signaling observed under microgravity. Here we investigate the involvement of microfilament in mediating the effects of microgravity and BMP2 induction on Cbfa1 activity. For this purpose we constructed a fluorescent reporter cell line (OSE-MG63) of Cbfa1 activity by stably transfecting MG63 cells with a reporter consisting of six tandem copies of OSE2 and a minimal mOG2 promoter upstream of enhanced green fluorescent protein (EGFP). The fluorescence intensity of OSE-MG63 showed responsiveness to bone-related cytokines (IGF-I, vitamin D3 and BMP2) and presented an accordant tendency with alkaline phosphatase (ALP) activity. Using OSE-MG63 reporter fluorescence, we performed a semi-quantitative analysis of Cbfa1 activity after treatment with simulated microgravity, microfilament-disrupting agent (cytochalasin B, CB), microfilament-stabilizing agent (Jasplakinolide, JAS) or any combination thereof. In parallel, ALP activity, DNA binding activity of Cbfa1 to OSE2 (ChIP), F-actin structure (immunofluorescence) and EGFP mRNA expression (RT-qPCR) were analyzed. Simulated microgravity inhibited Cbfa1 activity, affected the responsiveness of Cbfa1 to cytokine BMP2, and caused a thinning and dispersed distribution of microfilament. Under normal gravity, CB significantly attenuated BMP2 induction to Cbfa1 activity as well as DNA binding activity of Cbfa1 to OSE2. The addition of JAS reversed the inhibitory effects of microgravity on the responsiveness of Cbfa1 to BMP2. Our study demonstrates that disrupting the microfilament organization by CB or simulated microgravity attenuates the responsiveness of Cbfa1 to BMP2. A stabilization of the microfilament organization by JAS reverses this inhibition. Taken together, these results suggest that actin microfilament participates in BMP2's induction to Cbfa1 activity and that their disruption might be an important contributor to microgravity's inhibition on BMP2's osteogenic induction.