Beta1 Integrin Expression Research Articles

All-trans retinoic acid remodels extracellular matrix and suppresses laminin-enhanced contractility of cultured human retinal pigment epithelial cells

All-trans retinoic acid (atRA) has been reported to inhibit the proliferation of retinal pigment epithelial (RPE) cells and used in treatment of proliferative vitreoretinopathy (PVR) in animal model. This study aimed at examining the effectiveness of atRA in inhibiting the extracellular matrix (ECM) biosynthesis by RPE cells and the RPE cell-mediated collagen gel contraction. Cultured RPE cells were treated with atRA and the expression of four ECM proteins (collagen types I, III, IV and laminin beta1) was profiled. The results indicated that atRA treatment up-regulated de novo synthesis of collagen type I, but decreased that of laminin beta1 in a dose-dependent manner. Moreover, the effect of atRA on RPE cell contraction was evaluated by measuring the area of collagen gel where RPE cells populated. Treatment with atRA significantly inhibited RPE cell-mediated collagen gel contraction. Addition of exogenous laminin nonapeptide into gels promoted RPE cell contraction, while atRA reversed the laminin-enhanced contractility. atRA treatment significantly suppressed the gene expression of integrin beta3 but not alphaV subunit, and effectively inhibited the tyrosine phosphorylation of integrin beta3 at residue 747 in RPE cells grown on laminin-coated dish, suggesting that atRA may suppress the RPE contractility through either inhibiting integrin beta3 expression or abrogating the integrin beta3-mediated signaling. In conclusion, atRA pharmacologically possesses a tissue-remodeling capacity and inhibits contractility of RPE cells. Therefore, atRA might be potentially a therapeutic agent for certain ocular disorders such as PVR.

Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation

Aberrant activation of hedgehog (HH) pathway has been implicated in the development of human malignancies. This study aimed at investigating the role of HH molecules in human ovarian carcinogenesis. The expression profiles of HH molecules were examined in ovarian tumor samples and ovarian cancer cell lines and the in vitro effects of HH molecules on cell proliferation, apoptosis, migration, invasion and cell differentiation as well as related downstream target genes were assessed. Overexpression of Patched and Gli1 protein in ovarian cancers correlated with poor survival of the patients (P = 0.008; P = 0.004). Significantly elevated expression of Sonic hedgehog messenger RNA was observed in ovarian cancers compared with normal tissues and benign ovarian tumors and such differential expression was specific to histological types (P < 0.05). Ectopic Gli1 overexpression in ovarian cancer cells conferred increased cell proliferation, cell mobility, invasiveness and change in differentiation in association with increased expression of E-cadherin, vimentin, Bcl-2, caspases as well as β1 integrin, membrane type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF). Treatment with 3-keto-N-(aminoethyl-aminocaproyl-dihydrocinnamoyl)-cyclopamine induced cancer cell apoptosis, suppressed cell growth, mobility and invasiveness and induced cancer cell dedifferentiation with decreased expression of E-cadherin, cytokeratin 7, Snail, calretinin, vimentin, Bcl-2, caspases, β1 integrin, MT1-MMP and VEGF. Our data suggested that abnormal HH signaling activation plays important roles in the development and progression of ovarian cancers. Gli1 expression is an independent prognostic marker. Inhibition of the HH pathway molecules might be a valid therapeutic strategy for ovarian cancers.

C-Myc Is a Major Target of Rbm15 in the Regulation of Hematopoietic Stem Cell and Megakaryocyte Development

Acute megakaryoblastic leukemia (AMKL, FAB-M7) with the t(1;22)(p13;q13) chromosome translocation is a unique subtype of AMKL, characterized by its early onset, poor response to existing therapies, rapid progression, and poor prognosis. t(1;22) is often the sole abnormality found in infants with this AMKL subtype, suggesting that the RBM15-MKL1 (also known as OTT1-MAL) fusion gene created by the translocation plays a crucial role in the pathogenesis of this leukemia. Here we have explored the normal functions of one of the t(1;22) partner genes, Rbm15, by conditionally knocking it out in the mouse (Rbm15 CKO mice) and by crossing these mice with an Mx1-Cre transgenic line to delete Rbm15 in the hematopoietic system. We found both the percentage and absolute number of long-term hematopoietic stem cells (Lin-Sca1+ckit+/Flk2-, LT-HSCs) to be increased in Rbm15-deleted (Rbm15lx/lx;Mx1-Cre+) mice (0.23 ± 0.02% of total nucleated marrow cells [TNMC], 2.3-fold higher) compared to age- and gender-matched wild-type littermates (Rbm15lx/lx;Mx1-Cre-) (0.10 ± 0.01% TNMC) (P <0.0001, n = 18 mice per group). However, Rbm15-deleted bone marrow (BM) cells were markedly impaired in their ability to reconstitute normal hematopoiesis in competitive repopulation studies, with only 9.73 ± 2.32% donor-derived cells in the peripheral blood (PB) of recipient animals compared to the contribution by donor cells from littermate controls (47.52 ± 7.26%) (P = 0.00015; n = 10 mice per group) when transplanted at a 1:1 ratio of donor vs. wild-type competitor bone marrow. Additional analysis showed that Rbm15-deleted donor-derived cells were also significantly decreased in the recipient BM as a whole; importantly, however, the percentages of donor-derived HSC and LT-HSC populations were markedly increased (8.8-fold and 5.3-fold, respectively) in recipients transplanted with Rbm15 mutant cells compared to those transplanted with wild-type control marrow. This stem cell accumulation was more profound than that observed in the original Rbm15 mutant mice. Additional analysis that will be presented suggested an HSC activation/differentiation defect due to Rbm15 absence rather than abnormalities of HSC homing, survival or proliferation. Collectively, these alterations are highly reminiscent of those seen in c-Myc knockout (KO) mice, in which the expression levels of several adhesion molecules have been shown to be altered and an HSC-niche interaction defect posited to be the underlying mechanism of the HSC abnormalities observed. We found that similar to the c-Myc KO phenotype, the expression of both N-cadherin and beta1-integrin is increased on Rbm15-deficient HSCs and the N-cadherin expression level can be downregulated by ectopic expression of RBM15. Interestingly, in addition to the abnormalities of c-Myc-null HSCs that have been previously reported, we have identified a heretofore unrecognized significant increase of megakaryopoiesis in c-Myc knockout mice as well. We also determined that Rbm15 deficiency predisposes HSCs toward megakaryocyte lineage differentiation – the percentages of CD41-positive megakaryocytes were found to be increased both in vivo in the spleens of Rbm15 mutant mice and after the in vitro culture of Rbm15-deleted HSCs in a megakaryocyte liquid differentiation medium, as compared to wild-type littermate controls – thus uncovering another phenotypic similarity between mice lacking Rbm15 and c-Myc in their hematopoietic systems. Furthermore, c-Myc expression was decreased in Rbm15-deficient HSCs (nearly 2-fold lower compared to littermate control HSCs), as confirmed by TaqMan real-time PCR. Importantly, the megakaryocytic differentiation bias of Rbm15-deficient HSCs could be at least partially rescued by c-Myc overexpression in liquid culture conditions in vitro, suggesting a functional interaction between Rbm15 and c-Myc activity. Our data suggest that c-Myc may be a major target and effector of Rbm15 function in the regulation of HSC and megakaryocyte development. The putative oncoprotein RBM15-MKL1 has been hypothesized to be involved in the pathogenesis of AMKL in part through a dominant-negative mechanism that impairs normal RBM15 function; our data imply that such an inhibition of normal RBM15 function would in turn lead to diminished c-MYC activity, thus contributing to the aberrant megakaryocyte lineage involvement observed in t(1;22)-positive AMKL.

Homing of Mouse Spermatogonial Stem Cells to Germline Niche Depends on β1-Integrin

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis. In a manner comparable to hematopoietic stem cell transplantation, SSCs colonize the niche of recipient testes and reinitiate spermatogenesis following microinjection into the seminiferous tubules. However, little is known about the homing mechanism of SSCs. Here we examined the role of adhesion molecules in SSC homing. SSCs isolated from mice carrying loxP-tagged beta1-integrin alleles were ablated for beta1-integrin expression by in vitro adenoviral cre transduction. The beta1-integrin mutant SSCs showed significantly reduced ability to recolonize recipient testes in vivo and to attach to laminin molecules in vitro. In contrast, genetic ablation of E-cadherin did not impair homing, and E-cadherin mutant SSCs completed normal spermatogenesis. In addition, the deletion of beta1-integrin on Sertoli cells reduced SSC homing. These results identify beta1-integrin as an essential adhesion receptor for SSC homing and its association with laminin is critical in multiple steps of SSC homing.

Knockdown of integrin β4‐induced autophagic cell death associated with P53 in A549 lung adenocarcinoma cells

Integrin beta4 is a tissue-specific protein, but its role in autophagy of lung adenocarcinoma cells is not clear. In this study, we used microtubule-associated protein 1 light chain 3 processing and acridine orange staining to reveal that knockdown of integrin beta4 by its specific siRNA induced autophagic cell death in A549 lung cancer cells. Next, we investigated the effects of siRNA-mediated downregulation of integrin beta4 on cell death and the level of p53. The proportion of dead cells and level of p53 were significantly increased. Inhibition of autophagy by the inhibitor 3-methyladenine attenuated the cell death induced by integrin beta4 knockdown. To further understand the relationship between p53 and integrin beta4 in autophagic cell death, we inhibited the expression of integrin beta4 by its specific siRNA in p53-mutated H322 lung cancer cells. Knockdown of integrin beta4 could not induce autophagic cell death in H322 cells. The data suggest that integrin beta4 is implicated in and associated with p53 in autophagy of lung cancer cells.

Beta3 Integrin Expression within Uterine Endometrium and its Relationship with Unexplained Infertility

The aim of this study was to investigate whether the endometrium of women with unexplained infertility differs in the presence of the beta 3 (beta3) integrin molecule from the endometrium of normal fertile women. In a retrospective case-control study 30 endometrial biopsies from hysterectomies with nonendometrial pathology and 30 endometrial samples from women with a history of unexplained infertility were collected during the window of implantation. Immunohistochemically staining with a monoclonal antibody against beta3 integrin subunit in endometrial biopsies was performed and then assessed semiquantitively by microscope on different endometrial compartments including glandular epithelial cells, vessels, lymphocytes, macrophages and stromal cells. Chi-square test was used to compare the expression and defect of beta3 integrin subunit between two groups. The results showed that beta3 integrin molecules were present in fertile and infertile endometrial uterine tissues. The majority of glandular epithelial cells expressed beta3 integrin in fertile endometrium. However, the endometrial expression of beta3 integrin was reduced significantly in infertile endometrium during the window of implantation (p < 0.05). A few numbers of vessels and stromal cells expressed beta3 integrin molecule with no statistical significance between the two groups. In conclusion Abnormal endometrial integrin expression is a frequent finding in women with unexplained infertility. A therapeutic potential approach in improving uterine endometrium receptivity together with up-regulation of beta3 integrin in this population of women suggested.

N-Acetylglucosaminyltransferase V Regulates Extravillous Trophoblast Invasion through Glycosylation of α5β1 Integrin

For successful human placentation, invasion of trophoblast cells into the uterus and its associated vasculature is essential, and the regulation of this process is controlled by many factors at the fetal-maternal interface. N-acetylglucosaminyltransferase V (GnT-V) is a key enzyme that catalyzes beta1, 6-N-acetylglucosamine (beta1-6GlcNAc) branching on asparagine-linked oligosaccharides of cell proteins. GnT-V and its product, beta1-6GlcNAc, are known to regulate cellular transformation and correlate with the metastatic potential of various cancer cells. The aim of the present study was to determine whether extravillous trophoblast (EVT) expressed this molecule and examine the role of GnT-V in the regulation of human trophoblast invasion. Immunohistochemistry showed that GnT-V was strongly expressed within the cytoplasm of EVT in the anchoring villi; this expression was down-regulated in EVTs invading the decidua. Suppression of beta1-6GlcNAc glycosylation by swainsonine enhanced the migratory potential and invasive capability of both primary EVTs and the EVT cell line, HTR-8/SVneo. Down-regulation of GnT-V expression by small interfering RNA in the choriocarcinoma cell line Jar consistently enhanced the migration and invasive capacity of these cells and elevated cellular adhesion to extracellular matrix proteins, such as fibronectin and collagen type I/IV. The extent of beta1-6 branching of alpha5beta1 integrin was significantly reduced in small interfering GnT-V-transfected Jar cells compared with mock transfectants, although the expression of alpha1, alpha5, alpha6, and beta1 integrin on the cell surface was not changed. These results suggest that GnT-V is expressed in human EVT and is involved in regulating trophoblast invasion through modifications of the oligosaccharide chains of alpha5beta1 integrin.

Expression of integrin-beta1 in oral leukoplakia and early invasive carcinoma and its relationship with cell proliferation

To investigate the expression and significance of integrin-beta1 in oral leukoplakia and early invasive carcinoma. The expression of integrin-beta1 and Ki-67 was examined in 12 normal oral mucosa, 10 simple hyperplasia, 24 epithelial dysplasia and 14 early invasive carcinoma by immunohistochemistry. In normal and simple hyperplasia epithelium, integrin-beta1 was mostly expressed in the basal cell membrane, and Ki-67 in the nuclei of basal and the parabasal layers. In dysplasia epithelium and early invasive carcinoma, integrin-beta1 showed membrane staining and Ki-67 showed nuclear staining in dysplastic basal cells, sprinkle cells and SCC cells. Integrin-beta1 and Ki-67 were overexpressed in 7 and 10 of 24 dysplasia cases and in 8 of 14 early invasive carcinoma cases respectively. There was a significant difference in integrin-beta1 and Ki-67 expression among the four groups (chi2 = 10.651, P = 0.014; chi2 = 14.831, P = 0.002), in integrin-beta1 expression among normal oral mucosa, simple hyperplasia and early invasive carcinoma (P = 0.008, P = 0.013) and in Ki-67 expression among normal oral mucosa and dysplasia, early invasive carcinoma (P = 0.026, P = 0.001), and among simple hyperplasia and early invasive carcinoma (P = 0.005). A significant correlation between integrin-beta1 and Ki-67 (R = 0.442, P < 0.01) was found. Integrin-beta1 showed increased staining in dysplasia epithelium cells and SCC cells, and may correlate to the proliferation of the dysplasia cells.

The expression of integrinβ1 and FAK in pituitary adenomas and their correlation with invasiveness

The expression and possible role of integrin-focal adhesion kinase signal pathway in invasive pituitary adenomas were explored. Forty-nine human pituitary adenomas were detected for the expression of integrinbeta1 (INTbeta1) and focal adhesion kinase (FAK) by immunohistochemistry, and their correlation with the invasiveness of pituitary adenomas as well as between themselves was analyzed. The results showed that INTbeta1 was expressed in 46 cases (93.9%) and FAK in 36 cases (73.5%), respectively, and their expression levels were highly correlated with tumor invasiveness, but not with the tumor types. It was suggested that the integrin-focal adhesion kinase signal pathway plays a role in the invasiveness of pituitary adenomas.

Isoproterenol induces primary loss of dystrophin in rat hearts: correlation with myocardial injury

The mechanism of isoproterenol-induced myocardial damage is unknown, but a mismatch of oxygen supply vs. demand following coronary hypotension and myocardial hyperactivity is the best explanation for the complex morphological alterations observed. Severe alterations in the structural integrity of the sarcolemma of cardiomyocytes have been demonstrated to be caused by isoproterenol. Taking into account that the sarcolemmal integrity is stabilized by the dystrophin-glycoprotein complex (DGC) that connects actin and laminin in contractile machinery and extracellular matrix and by integrins, this study tests the hypothesis that isoproterenol affects sarcolemmal stability through changes in the DGC and integrins. We found different sensitivity of the DGC and integrin to isoproterenol subcutaneous administration. Immunofluorescent staining revealed that dystrophin is the most sensitive among the structures connecting the actin in the cardiomyocyte cytoskeleton and the extracellular matrix. The sarcomeric actin dissolution occurred after the reduction or loss of dystrophin. Subsequently, after lysis of myofilaments, gamma-sarcoglycan, beta-dystroglycan, beta1-integrin, and laminin alpha-2 expressions were reduced followed by their breakdown, as epiphenomena of the myocytolytic process. In conclusion, administration of isoproterenol to rats results in primary loss of dystrophin, the most sensitive among the structural proteins that form the DGC that connects the extracellular matrix and the cytoskeleton in cardiomyocyte. These changes, related to ischaemic injury, explain the severe alterations in the structural integrity of the sarcolemma of cardiomyocytes and hence severe and irreversible injury induced by isoproterenol.

Functional Live-Cell Imaging Demonstrates that β1-Integrin Promotes Type IV Collagen Degradation by Breast and Prostate Cancer Cells

The ability of tumor cells to adhere to, migrate on, and remodel extracellular matrices is mediated by cell surface receptors such as beta1-integrins. Here we conducted functional live-cell imaging in real time to investigate the effects of modulating beta1-integrin expression and function on proteolytic remodeling of the extracellular matrix. Human breast and prostate cancer cells were grown on reconstituted basement membrane containing a quenched fluorescent form of collagen IV. Generation of cleavage products and the resulting increases in fluorescence were imaged and quantified. Decreases in the expression and activity of beta1-integrin reduced digestion of quenched fluorescent-collagen IV by the breast and prostate cancer cells and correspondingly their invasion through and migration on reconstituted basement membrane. Decreased extracellular matrix degradation also was associated with changes in the constituents of proteolytic pathways: decreases in secretion of the cysteine protease cathepsin B, the matrix metalloproteinase (MMP)-13, and tissue inhibitors of metalloproteinases (TIMP)-1 and 2; a decrease in expression of MMP-14 or membrane type 1 MMP; and an increase in secretion of TIMP-3. This is the first study to demonstrate through functional live-cell imaging that downregulation of beta1-integrin expression and function reduces proteolysis of collagen IV by breast and prostate cancer cells.

Modulation of β1-integrins on hemopoietic progenitor cells after allergen challenge in asthmatic subjects

Mobilization of hemopoietic progenitor cells from the bone marrow (BM) is a feature of inflammatory asthmatic responses. Understanding the mechanisms regulating progenitor cell mobilization and trafficking to the peripheral circulation might be important for the development of effective asthma therapies. We investigated the role of adhesion molecules in the mobilization of hemopoietic progenitor cells from the BM during an allergen-induced asthmatic response. BM and peripheral blood samples were obtained from dual-responders with mild asthma before and at several time points after allergen challenge. Fluctuations in expression and adhesive properties of beta1- and beta2-integrins on CD34(+)CD45(+) progenitor cells were assessed by using flow cytometry and adhesion to protein-coated wells, respectively. On BM-derived CD34(+)CD45(+) cells, expression of very late antigen (VLA) 4, but not VLA-5 or Mac-1, decreased significantly 24 hours after allergen challenge and had begun to recover by 48 hours after challenge. In peripheral blood allergen challenge induced a significant decrease in VLA-4 levels after 6 hours, which had not recovered by 96 hours after challenge. Similarly, VLA-5 expression decreased, most prominently at 72 to 96 hours after allergen challenge. In contrast, Mac-1 levels did not change. Chemokine-stimulated adhesion of BM-derived CD34(+)CD45(+) cells to fibronectin was significantly attenuated 24 hours after challenge. Furthermore, adhesion to fibronectin and vascular cell adhesion molecule 1 was greatly reduced by anti-VLA-4 or anti-VLA-5 antibodies. Preferential downregulation of beta1-integrin expression on progenitor cells can reduce the tethering forces to BM components, thus facilitating their egress to the peripheral circulation during an allergic inflammatory response.

BMP‐2 increases migration of human chondrosarcoma cells via PI3K/Akt pathway

Bone morphogenetic protein-2 (BMP-2), a member of transforming growth factor-beta superfamily, plays a crucial role in migration and metastasis of human cancer cells. Integrins are the major adhesive molecules in mammalian cells. Here we found that BMP-2 directed the migration and increased cell surface and mRNA expression of beta1 integrin in human chondrosarcoma cancer cells (JJ012). Pretreated of JJ012 cells with phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002) or Akt inhibitor inhibited the BMP-2-mediated migration and integrin expression. BMP-2 increased the phosphorylation of p85 subunit of PI3K and serine 473 of Akt. In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited BMP-2-mediated cells migration and integrin upregulation. Stimulation of JJ012 cells with BMP-2 induced IkappaB kinase (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the BMP-2-mediated increasing of IKKalpha/beta phosphorylation, IkappaB phosphorylation, and p65 Ser(536) phosphorylation were inhibited by Ly294002 and Akt inhibitor. Co-transfection with p85 and Akt mutants also reduced the BMP-2-induced kappaB-luciferase activity. Taken together, these results suggest that the BMP-2 acts through PI3K/Akt, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of beta1 integrin and contributing the migration of human chondrosarcoma cells.

TGF-β–dependent suppressive function of Tregs requires wild-type levels of CD18 in a mouse model of psoriasis

Dysfunctional Tregs have been identified in individuals with psoriasis. However, their role in the pathogenesis of the disease remains unclear. Here we explored the effect of diminished CD18 (beta2 integrin) expression on the function of CD4+CD25+CD127(-) Tregs using the Cd18 hypomorphic (Cd18hypo) PL/J mouse model of psoriasis that closely resembles the human disease. We found that reduced CD18 expression impaired cell-cell contact between Tregs and DCs. This led to dysfunctional Tregs, which both failed to suppress the pathogenic T cells and promoted the onset and severity of the disease. This failure was TGF-beta-dependent, as Tregs derived from Cd18hypo PL/J mice had diminished TGF-beta1 expression. Adoptive transfer of Tregs expressing wild-type levels of CD18 into affected Cd18hypo PL/J mice resulted in a substantial improvement of the psoriasiform skin disease, which did not occur upon coinjection of the cells with TGF-beta-specific neutralizing antibody. Our data indicate a primary dysfunction of Cd18hypo Tregs, allowing subsequent hyperproliferation of pathogenic T cells in the Cd18hypo PL/J mouse model of psoriasis. This study may provide a step forward in our understanding of the unique role of CD18 expression levels in avoiding autoimmunity.

SILAC Mouse for Quantitative Proteomics Uncovers Kindlin-3 as an Essential Factor for Red Blood Cell Function

Stable isotope labeling by amino acids in cell culture (SILAC) has become a versatile tool for quantitative, mass spectrometry (MS)-based proteomics. Here, we completely label mice with a diet containing either the natural or the (13)C(6)-substituted version of lysine. Mice were labeled over four generations with the heavy diet, and development, growth, and behavior were not affected. MS analysis of incorporation levels allowed for the determination of incorporation rates of proteins from blood cells and organs. The F2 generation was completely labeled in all organs tested. SILAC analysis from various organs lacking expression of beta1 integrin, beta-Parvin, or the integrin tail-binding protein Kindlin-3 confirmed their absence and disclosed a structural defect of the red blood cell membrane skeleton in Kindlin-3-deficient erythrocytes. The SILAC-mouse approach is a versatile tool by which to quantitatively compare proteomes from knockout mice and thereby determine protein functions under complex in vivo conditions.

044 Up-Regulated p38, PCNA, and Decreased Smooth Muscle Actin in Cyclophilin C-Associated Protein Null Mice During Skin Wound Healing

Introduction: Cyclophilin C-associated protein (CyC-AP) expression is up-regulated during wound healing. CyC-AP deletion causes greater expression of TNFα, MMP-3, MMP-13, and MMP-14 during cutaneous repair. Furthermore, CyC-AP is a mediator for fibronectin-fragment induced MMP-13 expression. In order to understand the effects of the deletion of CyC-AP at the cellular level, we investigated the intracellular signal transduction pathways of CyC-AP null and wild-type mice during skin wound healing. Methods: Two-month-old CyC-AP null or wild-type mice were anesthetized and two 0.6 cm diameter excisional skin wounds were made on the dorsum of each mouse. Wounds were collected up to 10 days after injury. Total protein was purified from each wound and the protein expression of TNFα converting enzyme (TACE), integrin beta 1, ERK 1, ERK 2, p38, PCNA, and smooth muscle actin (sm-actin) was analyzed by immunoblot. Results: TNFα expression was higher in CyC-AP null mice in unwounded skin. However, the elevated TNFα in CyC-AP null wounds was lower compared to the wild-type mice. To further understand the relationship of CyC-AP and TNFα, TACE expression was examined. TACE expression was less in the skin of CyC-AP null mice compared to wild-type mice. Expression of p38 and PCNA increased while sm-actin decreased in both unwounded and wounded CyC-AP null skin compared to wild type. PCNA expression in CyC-AP null fibroblasts was also higher compared to wild type, supporting our in vivo observations. In addition, the expression of integrin beta 1 was lower during wound healing. Conclusion: The increased PCNA and decreased sm-actin expression suggests that CyC-AP null fibroblasts have higher proliferation and lower differentiation rates compared to wild types. These effects might be regulated through the p38 signaling pathway, which was increased in CyC-AP null mice. The decreased TACE expression in CyC-AP null skin suggests CyC-AP may also regulate TACE via the altered expression of integrin beta 1. Our data suggest CyC-AP is involved in TNFα activation.

Myocardial ischaemia–reperfusion injury in haematopoietic cell‐restricted β1 integrin knockout mice

Evidence indicates that the intercellular adhesion molecule-1 and its counter-receptor beta2 integrin are cardioprotective proteins during myocardial ischaemia-reperfusion, but no data are available concerning the role of blood cell beta1 integrins in this process. We studied the effects of temporary myocardial ischaemia and reperfusion in blood cell-restricted beta1 integrin knockout mice (beta1(-/-)). The left descending coronary artery in conditional beta1(-/-) integrin (beta1(-/-)), beta1 integrin +/+ (beta1(+/+)) and beta1 integrin -/- bone marrow chimeric (beta1(-/-) BM) mice was ligated for 30 min, followed by reperfusion of either 3 h or 3 weeks. Plasma levels of troponin T were evaluated as an index of cardiac cellular damage. The histological evaluation of tissue damage was performed with Haematoxylin and Eosin stained sections. Cell infiltrations in the ischaemic area were investigated by immunofluorescence studies. It was found that plasma troponin T was at a similar level in beta1(-/-), beta1(+/+) and beta1(-/-) BM mice treated with 30 min ischaemia and 3 h reperfusion. Histological analysis showed that ischaemia-reperfusion resulted in marked myocardial injury in all groups of animals, but the damage score of the hearts was not significantly different between beta1(-/-), beta1(+/+) and beta1(-/-) BM mice after 3 h of reperfusion following 30 min of ischaemia (2.8 +/- 0.5, 2.6 +/- 0.5 and 2.8 +/- 0.6, respectively, n.s.). Furthermore, no difference in scar sizes in ischaemia-injured hearts was found 3 weeks after ischaemia. Semi-quantification of cells demonstrated that, compared with beta1(+/+) mice, the number of infiltrating neutrophils was significantly reduced in beta1(-/-) and beta1(-/-) BM mice, whereas MAC-1(CD11b/CD18)-positive cells in the ischaemic regions were similar in myocardial tissues of all groups. We conclude that absence of beta1 integrin expression in haematopoietic cells results in reduced neutrophil infiltration in the ischaemic regions, but does not influence myocardial damage of ischaemic hearts.

Down-Regulation of Osteopontin Suppresses Growth and Metastasis of Hepatocellular Carcinoma Via Induction of Apoptosis

Expression of osteopontin correlates with tumor progression and metastasis. The mechanisms by which osteopontin promotes tumor cell survival remain unclear. Here we used short-hairpin RNA-mediated gene silencing to investigate the antitumor effects by osteopontin depletion in hepatocellular carcinoma (HCC). We applied polyethylenimine nanoparticles to deliver a short-hairpin RNA for depletion of osteopontin expression in HCC cells. Tumorigenicity and metastatic potentials of HCC cells were studied in vitro and in nude mice. Nuclear factor-kappaB (NF-kappaB) activation was analyzed by gel shift assay and luciferase analysis. The expressions of integrins were examined by real-time reverse-transcription polymerase chain reaction. Apoptosis was examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and mitochondrial membrane potential analysis. Down-regulation of osteopontin inhibited HCC cell growth, anchorage-independent growth, adhesion with fibronectin and invasion through extracellular matrix in vitro, and suppressed tumorigenicity and lung metastasis in nude mice. Osteopontin silencing resulted in suppression of alphav, beta1, and beta3 integrin expressions, blockade of NF-kappaB activation, inhibition of Bcl-2/Bcl-xL and XIAP expressions, increase of Bax expression, and induction of a mitochondria-mediated apoptosis. Furthermore, down-regulation of osteopontin inhibited drug-induced NF-kappaB activation and sensitized HCC cells to chemotherapeutic agents in vitro, which led to complete regression of HCC xenografts in nude mice. Osteopontin may facilitate tumorigenesis and metastasis through prevention of tumor cells from apoptosis. RNA interference-mediated depletion of osteopontin may be a promising strategy for the treatment of HCC by sensitizing the chemotherapeutic drugs.

Migration of renal carcinoma cells is dependent on protein kinase Cδ via β1 integrin and focal adhesion kinase

Migration and adhesion of tumor cells are essential prerequisites for the formation of metastases in malignant diseases. Protein kinase C (PKC) has been shown to regulate cell migration, adhesion and proliferation. In order to identify a connection between PKC isoforms and tumor progression in renal cell carcinoma (RCC), the influence of PKC isoforms on cell migration, adhesion and proliferation and possible influences of the activity of integrins and focal adhesion kinase (FAK) were analyzed in RCC cells. The experiments were performed in the RCC cell line CCF-RC1 after pre-incubation of the cells with the PKC inhibitors GF109203X, GO6976, RO31-8220 and rottlerin. Cell migration and adhesion were assessed through chemotaxis analysis and adhesion to an endothelial monolayer, respectively. Cell proliferation was analysed by a BrdU incorporation assay. The expression and activity of beta1 integrins and FAK were analysed by Western blot analysis. GF109203X reduced cell migration to 69%, the activity of beta1 integrins to 63% and FAK expression to 82% compared to untreated cells. Rottlerin reduced cell migration in a concentration-dependent manner to 36%, cell proliferation to 81%, expression and activity of beta1 integrins to 72 and 79%, and expression and activity of FAK to 56 and 76% of untreated cells, respectively. RO31-8220 also reduced the expression and activity of beta1 integrins as well as the expression of FAK to 84, 66 and 66% of untreated cells, respectively. GO6976 reduced the expression of FAK to 60% of untreated cells. Cell migration was only slightly reduced by GO6976 to 84% of untreated cells, and cell adhesion remained uninfluenced. These findings show a critical role of PKCdelta in the regulation of tumor cell migration, which seems to be caused by affecting the expression and activity of beta1 integrins and FAK. These results can provide a basis for new strategies in preventing metastases of renal cell carcinoma.

Interaktion zwischen humanen Chondrozyten und extrazellulärer Matrix in vitro

Autologous chondrocyte transplantation (ACT) has had reasonable success for repairing small articular cartilage defects. A limiting factor for ACT is, however, the in vitro cultivation of chondrocytes because it leads to dedifferentiation. Therefore, the goal of this work was to optimize the monolayer culture of chondrocytes in vitro.Human articular chondrocytes were plated on either collagen type II or untreated surfaces. The cells were evaluated morphologically and with immunoblotting.On collagen type II surfaces, a stable chondrogenic phenotype, expression of beta1-integrin, and a significant activation of phosphorylated intracellular proteins and the adaptor protein Shc could be observed up to day 20 in culture. Treatment with beta1 integrin antibody led to a loss of cell adhesion (82%). The results indicate that on collagen type II, beta1-integrin receptors are activated. Through the activation of Shc, these stimulate the Ras-MAPK pathway, which stabilizes the chondrogenic phenotype.Our results provide a practical and low-cost solution for improved long-term chondrocyte cultivation, thus providing a new perspective for using ACT on larger or arthrotic cartilage defects.