OBJECTIVES: To investigate CEACAM6 expression in nasopharyngeal carcinoma (NPC) and its regulatory effects on tumor cell proliferation, migration, and epithelial-mesenchymal transition (EMT). METHODS: CEACAM6 expression in NPC was analyzed using GEO datasets and validated by immunohistochemistry in NPC tissues and by Western blotting and RT-qPCR in NPC cell lines (HNE1, C666-1, HK1, 5-8F and CNE2Z) and normal nasopharyngeal epithelial NP69 cells. In the NPC cell lines, the effects of lentivirus-mediated CEACAM6 overexpression and knockdown on cell proliferation, migration, invasion and cytoskeletal structures were evaluated using CCK-8 assay, Edu staining, wound healing assay, Transwell assay, and phalloidin staining. Western blotting was performed to determine the expressions of EMT-related proteins (FN1, ITGA5, ITGB1, E-cadherin, N-cadherin and vimentin) in the NPC cells and the effect of FN1 overexpression on ITGA5 and ITGB1 protein expressions. RESULTS: Analysis of the data from the GEO datasets suggested that CEACAM6 was significantly downregulated in NPC, which was associated with poor patient prognosis. Immunohistochemistry also showed low expressions of CEACAM6 in clinical NPC tissues (P<0.05). In NPC cells, CEACAM6 overexpression significantly suppressed cell proliferation, migration and invasion and reduced the fluorescence intensity of actin. CEACAM6 overexpression also resulted in significant downregulation of FN1, ITGA5, ITGB1, N-cadherin and vimentin expressions and upregulation of E-cadherin expression, and FN1 overexpression obviously attenuated the inhibitory effect of CEACAM6 overexpression on ITGA5 and ITGB1 expressions. CONCLUSIONS CEACAM6 inhibits NPC cell migration and invasion by inhibiting EMT via regulating FN1, ITGA5 and ITGB1 expressions.
Humans ; Epithelial-Mesenchymal Transition ; Cell Movement ; Cell Proliferation ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms/metabolism* ; Cell Line, Tumor ; Cell Adhesion Molecules/genetics* ; Antigens, CD/metabolism* ; GPI-Linked Proteins ; Integrin alpha5/metabolism* ; Integrin beta1/metabolism* ; Cadherins/metabolism* ; Fibronectins ; Integrins

Patients suffering from nerve injury often experience exacerbated pain responses and complain of memory deficits. The dorsal hippocampus (dHPC), a well-defined region responsible for learning and memory, displays maladaptive plasticity upon injury, which is assumed to underlie pain hypersensitivity and cognitive deficits. However, much attention has thus far been paid to intracellular mechanisms of plasticity rather than extracellular alterations that might trigger and facilitate intracellular changes. Emerging evidence has shown that nerve injury alters the microarchitecture of the extracellular matrix (ECM) and decreases ECM rigidity in the dHPC. Despite this, it remains elusive which element of the ECM in the dHPC is affected and how it contributes to neuropathic pain and comorbid cognitive deficits. Laminin, a key element of the ECM, consists of α-, β-, and γ-chains and has been implicated in several pathophysiological processes. Here, we showed that peripheral nerve injury downregulates laminin β1 (LAMB1) in the dHPC. Silencing of hippocampal LAMB1 exacerbates pain sensitivity and induces cognitive dysfunction. Further mechanistic analysis revealed that loss of hippocampal LAMB1 causes dysregulated Src/NR2A signaling cascades via interaction with integrin β1, leading to decreased Ca²⁺ levels in pyramidal neurons, which in turn orchestrates structural and functional plasticity and eventually results in exaggerated pain responses and cognitive deficits. In this study, we shed new light on the functional capability of hippocampal ECM LAMB1 in the modulation of neuropathic pain and comorbid cognitive deficits, and reveal a mechanism that conveys extracellular alterations to intracellular plasticity. Moreover, we identified hippocampal LAMB1/integrin β1 signaling as a potential therapeutic target for the treatment of neuropathic pain and related memory loss.
Animals ; Laminin/genetics* ; Hippocampus/metabolism* ; Neuralgia/metabolism* ; Cognitive Dysfunction/etiology* ; Male ; Peripheral Nerve Injuries/metabolism* ; Extracellular Matrix/metabolism* ; Integrin beta1/metabolism* ; Pyramidal Cells/metabolism* ; Signal Transduction

Endothelial-mesenchymal transition (EndMT) disrupts vascular endothelial integrity and induces atherosclerosis. Active integrin β1 plays a pivotal role in promoting EndMT by facilitating TGFβ/Smad signaling in endothelial cells. Here, we report a novel anthraquinone compound, Kanglexin (KLX), which prevented EndMT and atherosclerosis by activating MAP4K4 and suppressing integrin β1/TGFβ signaling. First, KLX effectively counteracted the EndMT phenotype and mitigated the dysregulation of endothelial and mesenchymal markers induced by TGFβ1. Second, KLX suppressed TGFβ/Smad signaling by inactivating integrin β1 and inhibiting the polymerization of TGFβR1/2. The underlying mechanism involved the activation of FGFR1 by KLX, resulting in the phosphorylation of MAP4K4 and Moesin, which led to integrin β1 inactivation by displacing Talin from its β-tail. Oral administration of KLX effectively stimulated endothelial FGFR1 and inhibited integrin β1, thereby preventing vascular EndMT and attenuating plaque formation and progression in the aorta of atherosclerotic Apoe^-/- mice. Notably, KLX (20 mg/kg) exhibited superior efficacy compared with atorvastatin, a clinically approved lipid-regulating drug. In conclusion, KLX exhibited potential in ameliorating EndMT and retarding the formation and progression of atherosclerosis through direct activation of FGFR1. Therefore, KLX is a promising candidate for the treatment of atherosclerosis to mitigate vascular endothelial injury.
Animals ; Atherosclerosis/prevention & control* ; Mice ; Receptor, Fibroblast Growth Factor, Type 1/metabolism* ; Signal Transduction/drug effects* ; Anthraquinones/pharmacology* ; Humans ; Integrin beta1/metabolism* ; Epithelial-Mesenchymal Transition/drug effects* ; Male ; Transforming Growth Factor beta/metabolism* ; Disease Models, Animal ; Mice, Inbred C57BL ; Human Umbilical Vein Endothelial Cells/drug effects*

Objective To study the characteristics of positive expression of integrin β1 in the rat brain tissue of two kinds of traumatic brain injury models and to explore the feasibility of inferring the mode of traumatic brain injury using the positive expression of integrin β1. Methods The occipital region of rats was hit by hydraulic impact method and pendulum striking method to produce two closed brain injury models of linear and rotation acceleration respectively, then 120 SD rats were randomly divided into linear acceleration injury group, rotation acceleration injury group, sham operation group and normal control group. Immunohistochemistry staining and Western blotting method were used to detect the positive expression of integrin β1 in different parts of the brain tissue at 30 min, 3 h, 6 h, 12 h, 3 d and 7 d after rat injury. The data was processed statistically by SPSS 18.0 software. Results The positive expression of integrin β1 was detected 30 min after brain injury and reached the peak 6 h after brain injury. With the extension of injury time, the expression tended to enhance. At the same time points after injury, the differences in the positive expression of integrin β1 between the linear acceleration injury group and the rotation acceleration injury group in the occipital strike point and thalamus had no statistical significance （ P>0.05）, but the differences in the expression of integrin β1 in the frontal lobe and brain stem had statistical significance （P<0.05）. Conclusion The characteristics of positive expression of integrin β1 in brain tissue can be used to infer the strike point and the manner of injury and has application value for the reconstruction of craniocerebral injury process.
Animals ; Brain/metabolism* ; Brain Injuries ; Brain Injuries, Traumatic ; Integrin beta1/metabolism* ; Rats ; Rats, Sprague-Dawley

BACKGROUNDThe initial osteoblastic adhesion to materials characterizes the first phase of cell-material interactions and influences all the events leading to the formation of new bone. In a previous work, we developed a novel amorphous calcium phosphate (ACP)/poly(L-lactic acid) (PLLA) material that demonstrated morphologic variations in its microstructure. The aim of this study was to investigate the initial interaction between this material and osteoblastic cells. Cellular attachment and the corresponding signal transduction pathways were investigated.

METHODSA porous ACP/PLLA composite and PLLA scaffold (as a control) were incubated in fetal bovine serum (FBS) containing phosphate-buffered saline (PBS), and the protein adsorption was determined. Osteoblastic MG63 cells were seeded on the materials and cultured for 1, 4, 8, or 24 hours. Cell attachment was evaluated using the MTS method. Cell morphology was examined using scanning electron microscopy (SEM). The expression levels of the genes encoding integrin subunits α1, α5, αv, β1, focal adhesion kinase (FAK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using real-time reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThe ACP/PLLA material significantly increased the protein adsorption by 6.4-fold at 1 hour and 2.4-fold at 24 hours, compared with the pure PLLA scaffold. The attachment of osteoblastic cells to the ACP/PLLA was significantly higher than that on the PLLA scaffold. The SEM observation revealed a polygonal spread shape of cells on the ACP/ PLLA, with the filopodia adhered to the scaffold surface. In contrast, the cells on the PLLA scaffold exhibited a spherical or polygonal morphology. Additionally, real-time RT-PCR showed that the genes encoding the integrin subunits α1, αv, β1, and FAK were expressed at higher levels on the ACP/PLLA composite.

CONCLUSIONSThe ACP/PLLA composite promoted protein adsorption and osteoblastic adhesion. The enhanced cell adhesion may be mediated by the binding of integrin subunits α1, αv, and β1, and subsequently may be regulated through the FAK signal transduction pathways.

Biocompatible Materials ; chemistry ; Calcium Phosphates ; chemistry ; Cell Adhesion ; physiology ; Cells, Cultured ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Humans ; Integrin alpha1 ; metabolism ; Integrin alpha5 ; metabolism ; Integrin alphaV ; metabolism ; Integrin beta1 ; metabolism ; Integrins ; genetics ; metabolism ; Lactic Acid ; chemistry ; Osteoblasts ; cytology ; Porosity ; Tissue Engineering ; methods

OBJECTIVETo investigate the effect of Ganfukang (GFK) on connective tissue growth factor (CTGF) and focal adhesion kinase (FAK)/protein kinase B (PKB or Akt) signal pathway in a hepatic fibrosis rat model and to explore the underlying therapeutic molecular mechanisms of GFK.

METHODSFifty SD rats were randomly divided into five groups as follows: the control group, the model group (repeated subcutaneous injection of CCl4), and the three GFK treatment groups (31.25, 312.5, and 3125 mg/kg, intragastric administration). Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry were used to examine the expression of CTGF, integrin α5, integrin β1, FAK/Akt signal pathway, cyclinD1, and collagen in the different-treated rats.

RESULTSGFK attenuated the up-regulation of CTGF, integrin α5, and integrin β1 in hepatic fibrosis rats and suppressed both the phosphorylation of FAK and the phosphorylation of Akt simultaneously (P<0.01). At the same time, the expression of cyclinD1, collagen I, and collagen III was decreased by GFK significantly (P<0.01).

CONCLUSIONSCTGF and FAK/Akt signal pathway were activated in the CCl4-induced hepatic fibrosis rats, which contribute to increased expression of cyclinD1 and collagen genes. The mechanisms of the anti-fibrosis activity of GFK may be due to its effects against CTGF and FAk/Akt signal pathway.

Animals ; Collagen ; genetics ; metabolism ; Connective Tissue Growth Factor ; genetics ; metabolism ; Cyclin D1 ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Gene Expression Regulation ; drug effects ; Integrin alpha5 ; genetics ; metabolism ; Integrin beta1 ; genetics ; metabolism ; Liver ; drug effects ; enzymology ; pathology ; Liver Cirrhosis ; drug therapy ; enzymology ; genetics ; pathology ; Male ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects

BACKGROUNDRecent studies have indicated that human nucleus pulposus contain mesenchymal stem cells (NP-MSCs). However, the immunophenotypic variation of NP-MSCs in vitro was unclear. The present study was conducted to address the immunophenotypic variation of mesenchymal stem cells in nucleus pulposus under continuous proliferation in vitro and show the difference between mesenchymal stem cells and nucleus pulposus cell.

METHODSTissue samples were obtained from thoracolumbar burst fracture patients and degenerative disc disease patients who underwent discectomy and fusion procedures. Flow cytometric and laser scanning confocal microscope (LSCM) were used to detect the variation of mesenchymal stem cells in nucleus pulposus which were expressing CD105 and CD24 in condition with or without transforming growth factor β1 (TGF-β1).

RESULTSMore than 90% of the analyzed primary cells of mesenchymal stem cells in nucleus pulposus fulfilled the general immunophenotyping criteria for MSCs, such as CD44, CD105 and CD29, but the marker of mature NP cells characterized as CD24 was negative. In continuous cultures, the proportion of mesenchymal stem cells which were expressing CD44, CD105 and CD29 in nucleus pulposus gradually decreased. The mesenchymal stem cells in nucleus pulposus cells were positive for CD105 and CD29, with slight positivity for CD44. The CD24 expression gradually increased in proliferation. Biparametric flow cytometry and laser scanning confocal microscopy confirmed the presence of cells which were expressing CD105 and CD24 independently, and only a small part of cells expressed both CD105 and CD24 simultaneously. TGF-β1 could stimulate mesenchymal stem cells in nucleus pulposus to express CD24.

CONCLUSIONSNon-degenerative and degenerative NP contains mesechymal stem cells. The variation of CD24 can be used as a marker to identify the NP-MSCs differentiation into NP-like cells.

Adult ; Antigens, CD ; metabolism ; CD24 Antigen ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Endoglin ; Female ; Flow Cytometry ; Humans ; Hyaluronan Receptors ; metabolism ; Integrin beta1 ; metabolism ; Intervertebral Disc ; cytology ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Receptors, Cell Surface ; metabolism ; Young Adult

OBJECTIVETo observe the changes in expression of microRNA-203 and P63 in human epidermal stem cells and KCs, and to investigate their effects and significance in the epidermal proliferation and differentiation.

METHODS(1) Five normal foreskin tissue specimens were collected from 5 patients by circumcision in Department of Urinary Surgery of the First Affiliated Hospital of Nanchang University from March to June in 2013. Then single cell suspension was obtained by separating epidermis with trypsin digestion method. The cells were divided into quick adherent cells and non-quick adherent cells by type IV collagen differential adherent method. The biological characteristics of cells were observed by inverted phase contrast microscope immediately after isolation and on post culture day (PCD) 3. The expression of CD29, keratin 19, keratin 1, and keratin 10 was identified by immunocytochemical staining. The expression of microRNA-203 and mRNA of P63 was determined by real-time fluorescent quantitative RT-PCR. The protein expression of P63 was determined by Western blotting. Data were processed with t test and Pearson correlation analysis.

RESULTS(1) Immediately after isolation, quick adherent cells were small, round, and dispersed uniformly. On PCD 3, the cells adhered firmly, and they grew in clones. Immediately after isolation, non-quick adherent cells appeared in different shapes and sizes, and dispersed unevenly. On PCD 3, the cells adhered precariously and did not show clonal growth. Quick adherent cells showed positive expression of CD29 and keratin 19, while non-quick adherent cells showed positive expression of keratin 1 and keratin 10. Quick adherent cells were identified as epidermal stem cells, and non-quick adherent cells were identified as KCs. (2)The expression level of microRNA-203 in epidermal stem cells (0.74 ± 0.20) was lower than that in KCs (3.66 ± 0.34, t =16.582, P <0.001). The mRNA expression level of P63 in epidermal stem cells (4. 16 ± 0.28) was higher than that in KCs (2.90 ± 0.39, t =5. 850, P =0.001). The protein expression level of P63 in epidermal stem cells (1.42 ± 0.05) was higher than that in KCs (0.73 ± 0.03, t =26.460, P <0. 001). (3) The expression level of microRNA-203 was in significantly negative correlation with the expression levels of mRNA and protein of P63 (with r values respectively - 0. 94 and -0.98 , P values below 0.05).

CONCLUSIONSThe expression levels of microRNA-203 and P63 in human epidermal stem cells and KCs were significantly different, which might be related to the different characteristics of proliferation and differentiation of the cells.

Cell Differentiation ; Cells, Cultured ; Epidermis ; cytology ; growth & development ; Epithelial Cells ; cytology ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Humans ; Integrin beta1 ; Keratin-10 ; genetics ; metabolism ; Keratin-19 ; genetics ; metabolism ; Keratinocytes ; Male ; Membrane Proteins ; genetics ; metabolism ; MicroRNAs ; genetics ; metabolism ; Stem Cells ; cytology ; metabolism

Liver fibrosis is a pathological process of the excessive accumulation of extracellular matrix, especially collagen al (I) in liver. Ultimately, hepatic fibrosis leads to cirrhosis or hepatic failure. Liver fibrosis and early cirrhosis can be reversed, thus control of the development of liver fibrosis is very important for preventive treatment of cirrhosis and hepatic failure. This is a review of potential targets for anti-hepatic fibrosis based on plenty of publications, including TGF-β1 and integrin α(v) and so on, aimed at providing novel therapeutic targets in liver fibrosis.
Collagen ; metabolism ; Humans ; Integrin alphaV ; metabolism ; Liver ; pathology ; Liver Cirrhosis ; drug therapy ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo study the effect of β8 expression on transforming growth factor β1(TGF-β1) activation in astrocytes with oxygen glucose deprivation (OGD).

METHODSAstrocytes were cultured and then subjected to OGD to generate hypoxia-ischemia (HI) model in vitro. Immunocytochemistry was used to detect the expression and distribution of β8 in nomoxia cultured cells. β8 protein expression was quantified by Western blot at 12 hours, 1 day and 2 days after OGD. Astrocytes and luciferase reporter cells (TMLC) were co-cultured. β8 RNA interference system was established to specifically inhibit β8 expression in cultured astrocytes. TGF-β1 activation was then detected in the co-culture system.

RESULTSβ8 was mainly located in the cytoplasm and neurites of astrocytes. OGD resulted in increase of β8 protein expression at 12 hours after reoxygenation in astrocytes, which was peaked at 1 day after reoxygenation. TGF-β1 activation was in accordance with β8 expression in astrocyte-TMLC co-culture system after reoxygenation. After the inhibition of β8, TGF-β1 activation was significantly reduced in all time points.

CONCLUSIONSThe highly expressed β8 plays important roles in the regulation of TGF-β1 activation in neonatal rats with hypoxic-ischemic brain damage.

Animals ; Astrocytes ; metabolism ; Female ; Glucose ; metabolism ; Hypoxia-Ischemia, Brain ; metabolism ; Integrin beta Chains ; physiology ; Male ; Oxygen ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism