OBJECTIVETo investigate the expression of integrin alpha5 and actin in the cells of intervertebral disc under cyclic hydrostatic pressure in vitro.

METHODSThe porcine lumbar intervertebral disc cells were isolated and cultured in vitro, and the cells underwent cyclic hydrostatic loading. After that, the expression of integrin alpha5 and actin in intervertebral disc cells were studied by means of morphology observing, Western blot and immunohistochemistry staining.

RESULTSThe morphology of intervertebral disc cells were changed into smaller and flatten shape, and the expression of integrin alpha5 and actin were decreased after loading.

CONCLUSIONSThe expression of integrin alpha5 decreases under cyclic hydrostatic pressure, and the actin is affected at the same time when signals are transferred into the cells by integrin alpha5. That may be one of the important mechanisms of the mechanotransduction in the cells of intervertebral disc.

Actins ; metabolism ; Animals ; Cells, Cultured ; Hydrostatic Pressure ; adverse effects ; Integrin alpha5 ; metabolism ; Intervertebral Disc ; cytology ; Mechanotransduction, Cellular ; Swine

OBJECTIVETo explore the correlation between the expression of integrin subunits alpha5 and beta1 in prostate cancer (PCa) and its clinicopathological data including tumor grade and clinical stage.

METHODSExpressions of integrin subunits alpha5 and beta1 were examined in 30 cases of PCa and 30 cases of normal prostatic tissues by immunohistochemical assay.

RESULTSExpressions of integrin subunits alpha5 and beta1 in PCa were lower than those in normal prostatic tissues (P <0.05) respectively.

CONCLUSIONCompared with normal prostatic tissues, expressions of integrin subunits alpha5 and beta1 in PCa were rather weaker or even faded. Expressions of integrin subunits alpha5 and beta1 revealed an positive correlation with tumor's Gleason grade and negative with clinical TNM stage. The results indicate that integrin subunits alpha5 and beta1 have potential values in the diagnosis and are predictable indices in the proliferation of PCa.

Aged ; Aged, 80 and over ; Animals ; Humans ; Integrin alpha5 ; biosynthesis ; Integrin alpha5beta1 ; biosynthesis ; Male ; Mice ; Middle Aged ; Neoplasm Staging ; Prostatic Hyperplasia ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology

OBJECTIVETo observe the expressions of α-SMA, integrin α5 and fibronectin (Fn) in acute paraquat poisoned rats and the effect of PDTC. To investigate the mechanism of paraquat-induced pulmonary fibrosis.

METHODSSprague-Dawley rats were randomly divided into three experimental groups: Control group (6 rats), PQ group (36 rats) and PQ+PDTC group (36 rats). On the 1st, 3rd, the 7th, the 14th, the 28th and the 56th day after exposure, the protein expression of α smooth muscle actin (α-SMA) was evaluated by western blot. The mRNA levels of integrin α5 and fibronectin (Fn) were analyzed with real-time quantitative PCR (RT-PCR). Meanwhile, the lung pathological changes were observed and semi-quantified.

RESULTST With the time passing, the expression of α-SMA in PQ group increased gradually compared with control group (P < 0.05 or P < 0.01). The increasing extent was gently on the 3 rd, the 7 th day. While increasing extent was rapidly from the 28 th to the 56 th day. RT-PCR showed PQ significantly increased Fn mRNA level on all time points and increased integrin α5 mRNA level from the 7 rd to 56 th day compared with control group (P < 0.05 or P < 0.01). PDTC treatment significantly deceased α-SMA, Fn, and integrin α5 levels compared with PQ group in corresponding time points (P < 0.05 or P < 0.01) Noteworthy, in PQ+PDTC group, the occurrence of pathological changes were drastically attenuated and pathologic score significantly decreased (P < 0.05 or P < 0.01).

CONCLUSIONSα-SMA, integrin α5 and fibronectin could play an important role in the development of pulmonary fibrosis caused by paraquat poisoning. PDTC, asa strong NF-κB inhibitor, may inhibit NF-κB activity and further significantly decreased expressions of α-SMA, integrin α5 and fibronectin which were important part of ECM, leading to drastically attenuated pulmonary fibrosis. However, the mechanisms of PDTC intervention still remains to be explored.

Actins ; metabolism ; Animals ; Disease Models, Animal ; Fibronectins ; metabolism ; Integrin alpha5 ; metabolism ; Male ; Paraquat ; poisoning ; Pulmonary Fibrosis ; chemically induced ; metabolism ; Pyrrolidines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Thiocarbamates ; pharmacology

OBJECTIVETo investigate the differentiation induction effect of 2-methoxyestradiol (2ME2), an estrogen derivative on myeloma cell line CZ-1.

METHODSThe changes of CZ-1 cells in morphology, expression of surface CD49e and quantity of light chain secretion in the supernatant were observed when treated with 0.1 approximately 0.5 micromol/L 2ME2 for 48 h.

RESULTS2ME2 could induce differentiation of CZ-1 cells. The cells appeared decreased in size of nucleus, increased in cytoplasma, decreased in the ratio of nucleus to plasma, decreased in number or disappearance of nucleolus, and thickness and pyknosis of chromatin. The expression of CD49e was increased from (12.20 +/- 1.57)% to (24.80 +/- 1.26)% (P < 0.05). Light chain secretion in the supernatant was increased from (35.97 +/- 2.60) microg/ml to (79.67 +/- 1.88) microg/ml (P < 0.05).

CONCLUSIONLow concentrations of 2ME2 could induce differentiation of myeloma cell line CZ-1.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Estradiol ; analogs & derivatives ; pharmacology ; Flow Cytometry ; Humans ; Integrin alpha5 ; analysis ; Multiple Myeloma ; metabolism ; pathology ; Tubulin Modulators ; pharmacology

The aim of this study was to evaluate the homing capabilities of hematopoietic stem/progenitor cells (HSPCs) derived from human placenta tissues (PT). Single cell suspension of human PT was prepared by mechanical method. The expression levels of homing-related adhesion molecules (HRAM) including CD11a, CD49d, CD44, CD49e, CD62L and CD54 on CD34(+) cells and the percentages of CD34(+) cells and their subpopulations in nucleated cells (NC) from fresh human PT, umbilical cord arterial blood (UCAB) and umbilical cord venous blood (UCVB) were detected by using flow cytometry. The results showed that the percentage of CD34(+) cells and CD34(+)CD38(-) cells in placenta were higher than those in UCAB and UCVB. There were no significant difference in percentage of HSPC between UCAB and UCVB. Placenta-derived CD34(+) cells strongly expressed CD11a, CD49d, CD44, CD49e and CD54, among which expression levels of CD49e and CD54 on placenta-derived CD34(+) cells were significantly higher than those on UCAB and UCVB-derived CD34(+) cells. While the percentage of CD34(+)CD62L(+) cells in placenta was only lower than that in UCVB. It is concluded that human placenta is rich in HSPC. Moreover, the expression levels of most HRAM in CD34(+) cells from PT are higher than those from UCAB and UCVB or are close to them. It suggested that HSPCs derived from PT might have stronger homing capabilities than those from UCB.
Antigens, CD34 ; biosynthesis ; Cell Adhesion Molecules ; biosynthesis ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; metabolism ; Humans ; Hyaluronan Receptors ; biosynthesis ; Integrin alpha5 ; biosynthesis ; Intercellular Adhesion Molecule-1 ; biosynthesis ; Placenta ; cytology

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

In postmenopausal osteoporosis, estrogen deficiency leads to unbalance of bone metabolism, decreased bone formation and increased bone resorption, and the result is reduced bone mineral density (BMD) and bone stiffness. The processes of bone formation and resorption involves the expression of integrins in anchoragedependent cells, such as osteoblast and osteoclast. The osteoporosis-induced rats frequently demonstrated the loss of trabecular bone volume in the tibia, vertebra and mandible due to estrogen depletion. However, in maxilla, study has been rare because of its anatomical limits. So the objective of this study was to investigate bony change and property of integrin expression in maxilla of osteoporosis-induced rats. 12-week-old female Sprague-Dawley rats were bilaterally ovariectomized (OVX). At 1, 2, 3, 4, 8 and 12 weeks, control and OVX group rats were sacrificed respectively. BMD of maxilla of the rats was measured using dual-energy X-ray absorptiometry (DEXA). And then the histopathologic observation, histomorphometric analysis and immunohistochemistry with CD44, alpha2 integrin, alpha5 integrin, alpha6 integrin, alphav integrin and beta3 integrin were done. BMD of alveolar bone in maxilla was decreased with significance statistically after OVX 4 weeks and was decreased 18.15% at OVX 12 weeks group compared to control group. From OVX 4 to 12 weeks, the thickness of periodontal ligament space was decreased, the number of osteoclast and the size of marrow stroma were increased than control group. By histomorphometric analysis, the size of marrow stroma of alveolar bone in maxilla was increased 86.42% at OVX 12 weeks group compared to control group. CD44 was widely expressed throughout the odontoblast, cementoblast, dental pulp, preiodontal ligament, osteocyte, osteoclast and perivascular tissue at control group, and CD44 immunoreactivity was increased the odontoblast, osteoblast and osteoclast at OVX groups. alpha2 integrin was expressed the odontoblast and osteoblast at control group, but alpha2 integrin immunoreactivity was decreased the osteoblast at OVX 12 weeks group. alpha5 integrin was expressed the cementoblast, osteoblast and osteoclast at control group, and alpha5 integrin immunoreactivity was decreased the osteoblast and was increased the osteoclast from OVX 4 weeks group. alpha6 integrin was weakly expressed the odontoblast, cementoblast, osteoblast and osteoclast at control group, and alpha6 integrin immunoreactivity was weakly increased the osteoclast from OVX 4 weeks. alphav integrin was expressed the odontoblast and osteoclast at control group, and alphav integrin immunoreactivity was strongly increased the osteoclast from OVX 4 weeks. beta3 integrin was expressed the osteocyte and osteoclast at control group, and beta3 integrin immunoreactivity was strongly increased the osteoclast from OVX 4 weeks. From these results, alveolar bone in maxilla of OVX rats was decreased BMD gradually. Moreover, alpha2 and alpha5 integrin expression of osteoblast was decreased, and alpha5, alphav and beta3 integrin expression of osteoclast was increased in OVX rats. Thus, this study indicates that consideration of reduced BMD is necessary in dental procedure of postmenopausal women.
Absorptiometry, Photon ; Animals ; Bone Density* ; Bone Marrow ; Bone Resorption ; Dental Cementum ; Dental Pulp ; Estrogens ; Female ; Humans ; Immunohistochemistry ; Integrin alpha2 ; Integrin alpha5 ; Integrin alpha6 ; Integrin alphaV ; Integrin beta3 ; Integrins ; Ligaments ; Mandible ; Maxilla* ; Metabolism ; Odontoblasts ; Osteoblasts ; Osteoclasts ; Osteocytes ; Osteogenesis ; Osteoporosis, Postmenopausal ; Ovariectomy* ; Periodontal Ligament ; Rats* ; Rats, Sprague-Dawley ; Spine ; Tibia

OBJECTIVETo investigate the biological behavior of stromal cell-derived factor-1 (SDF-1) on multiple myeloma (MM) cell migration and adhesion and it related signaling pathways.

METHODSExpression of adhesion molecules on MM cells of RPMI8226, XG-1 and XG-7 cells was analysed by flow cytometry, the influence of SDF-1 on CD29 and CD49e distribution by immunofluorescence, the effect of SDF-1 on chemotaxis of MM cells by transwell assay. Activation of phosphoinositide-3 kinase (PI3K) in MM cells treated with SDF-1 and by immunoblotting.

RESULTS3 strains of MM cell line expressed many adhesion molecule. RPMI8226, XG-7 cells were all high level of expression of CD29 (> 70%). XG-1, XG-7 cells were all high level of expression of CD44 (> 80%), and XG-7 cells was of CD49d (> 90%). In all of 3 strains, the levels of expression of CD49e were low (< 30%). SDF-1 could not upregulate their expression, but could trigger the establishment of polarized morphology of MM cells and the redistribution of CD29 and CD49e. SDF-1 promoted MM cells adhesion to endothelial cells, stimulated phosphorylation of P85 subunit of PI3K in MM cells and induced MM cells migration, which were inhibited by G protein inhibitor PTX and PI3K inhibitor wortmannin.

CONCLUSIONSDF-1 can promote MM cell adhesion to endothelial cells, trigger establishment of a polarized morphology of MM cells and redistribution of adhesion molecules and induce MM cells migration via PI3K signaling pathway.

Blotting, Western ; Cell Adhesion ; drug effects ; physiology ; Cell Adhesion Molecules ; metabolism ; Cell Line, Tumor ; Cell Movement ; drug effects ; physiology ; Chemokine CXCL12 ; pharmacology ; physiology ; Enzyme Activation ; drug effects ; Flow Cytometry ; Fluorescent Antibody Technique ; Humans ; Integrin alpha4 ; metabolism ; Integrin alpha5 ; metabolism ; Integrin beta1 ; metabolism ; Multiple Myeloma ; metabolism ; pathology ; physiopathology ; Phosphatidylinositol 3-Kinases ; metabolism ; Signal Transduction ; drug effects ; physiology

OBJECTIVETo investigate the regulatory mechanisms of integrin α5 and β1 in osteoblast in the process of gingipains-induced apoptosis.

METHODSGingipains were isolated and purified from supernatants of Porphyromonas gingivalis W83 which was cultured under standard anaerobic conditions. MC3T3-E1 was challenged with or without 8.3480 U/L gingipains for 48 h and apoptosis was examined by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (TUNEL-DAPI) staining. The expression of integrin α5 and β1 was analyzed by Western blotting after MC3T3-E1 was treated under different conditions.

RESULTSArginine-specific proteinases(Rgp) activity was (41.74 ± 2.11) U/L and lysine-specific proteinase(Kgp) was (1.02 ± 0.25) U/L.Gingipains induced MC3T3-E1 cells apoptosis after 48 h. Compared with control group, expression of integrin α5 and β1 was down-regulated by gingipains in a time-dependent manner within short periods ( ≤ 72 h), integrin α5 and β1 relative expression was (0.485 ± 0.039),(0.504 ± 0.002) at 48 h,(0.398 ± 0.058),(0.179 ± 0.001) at 72 h respectively (P < 0.05). After 72 h, integrin α5 expression in MC3T3-E1 cells was stable compared with control group while integrin β1 was still lower(control group:1.000 ± 0.000, 96 h:0.604 ± 0.003, 120 h: 0.357 ± 0.002) (P < 0.05). Proteinase inhibitor tosyl- L- lysine-chloromethyl-ketone(TLCK) effectively blocked the activity of gingipain and inhibited down-regulation of integrin α5 and β1 induced by gingipains from (0.398 ± 0.058,0.179 ± 0.001 ) to (0.781 ± 0.012, 0.857 ± 0.060) (P < 0.05). TLCK alone did not have any effect on integrin α5 and β1(P > 0.05). Gingipains also decreased integrin α5 and β1 in a dose-dependent manner.When cells were treated with 20.8700 U/L gingipains, integrin α5 and β1 relative expression reached to the lowest(0.105 ± 0.004,0.020 ± 0.000) (P < 0.05).

CONCLUSIONSGingipains inhibited the expression of integrin α5 and β1 in a time- and dose- dependent manner in osteoblasts in the process of apoptosis, which may not be mediated by direct proteolytic effect.

Adhesins, Bacterial ; administration & dosage ; isolation & purification ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cysteine Endopeptidases ; administration & dosage ; isolation & purification ; pharmacology ; Dose-Response Relationship, Drug ; Down-Regulation ; Integrin alpha5 ; metabolism ; Integrin beta1 ; metabolism ; Mice ; Osteoblasts ; cytology ; metabolism ; Porphyromonas gingivalis ; chemistry ; Serine Proteinase Inhibitors ; pharmacology ; Time Factors ; Tosyllysine Chloromethyl Ketone ; pharmacology

Melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from malignant melanoma cells and from chondrocytes. Recently, we revealed that MIA/CD-RAP can modulate bone morphogenetic protein (BMP)2-induced osteogenic differentiation into a chondrogenic direction. In the current study we aimed to find the molecular details of this MIA/CD-RAP function. Direct influence of MIA on BMP2 by protein-protein-interaction or modulating SMAD signaling was ruled out experimentally. Instead, we revealed inhibition of ERK signaling by MIA/CD-RAP. This inhibition is regulated via binding of MIA/CD-RAP to integrin alpha5 and abolishing its activity. Active ERK signaling is known to block chondrogenic differentiation and we revealed induction of aggrecan expression in chondrocytes by treatment with MIA/CD-RAP or PD098059, an ERK inhibitor. In in vivo models we could support the role of MIA/CD-RAP in influencing osteogenic differentiation negatively. Further, MIA/CD-RAP-deficient mice revealed an enhanced calcified cartilage layer of the articular cartilage of the knee joint and disordered arrangement of chondrocytes. Taken together, our data indicate that MIA/CD-RAP stabilizes cartilage differentiation and inhibits differentiation into bone potentially by regulating signaling processes during differentiation.
Animals ; Bone Morphogenetic Proteins/metabolism ; Cartilage/*cytology/metabolism ; *Cell Differentiation ; Chondrocytes/cytology/enzymology ; Extracellular Matrix Proteins/deficiency/*metabolism ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Integrin alpha5/metabolism ; Mesenchymal Stem Cells/cytology/metabolism ; Mice ; Neoplasm Proteins/deficiency/*metabolism ; Osteogenesis ; Protein Binding ; Signal Transduction ; Smad Proteins/metabolism