Human neutrophil adherence to thrombospondin occurs through a CD11/CD18-independent mechanism.

Thrombospondin (TSP), a 450-kDa trimeric glycoprotein secreted by platelets and endothelial cells at sites of tissue injury or inflammation, may play an important role in polymorphonuclear leukocyte (PMN) adherence to blood vessel walls before diapedesis. We have examined the adherence of PMN to TSP and compared it to adherence to other extracellular matrix proteins. PMN adherence to TSP-coated plastic was complete by 60 min with spreading completed by 2 h. The kinetics of adhesion and spreading on TSP were similar to that of vitronectin (VN), laminin (LN), and fibronectin (FN). Activation of PMN with the calcium ionophore A23187 or the chemotactic peptide FMLP increased PMN adherence to LN and FN, but not to TSP or VN, suggesting that PMN activation may differentially regulate expression of TSP and VN receptors as compared to LN and FN receptors. The specificity of PMN adherence to TSP was confirmed by competition with saturating amounts of TSP and inhibition with anti-TSP antibodies. mAb A6.1, which binds to the protease-resistant core of TSP, was the most effective in blocking PMN adherence to TSP. Using TSP proteolytic fragments, we demonstrated that the primary interaction of PMN with TSP was mediated through the 140-kDa COOH-terminal domain. Inasmuch as the 140-kDa fragment of TSP contains an Arg-Gly-Asp sequence similar to the cell recognition site of FN and VN, we determined whether RGDS peptides would inhibit PMN adhesion. RGDS did not significantly inhibit PMN adhesion to TSP, VN, or LN, but reduced PMN adhesion to FN by 50%. To determine if PMN adhesion to TSP was mediated by a beta 2 integrin receptor such as LFA-1, MO-1, or p150,95, we performed adhesion assays using PMN isolated from patients with leukocyte adhesion deficiency that lack beta 2 receptors. Leukocyte adhesion deficiency PMN exhibited normal adherence to TSP. In contrast, adherence to VN, LN, and FN was reduced by 95%. Therefore, adherence to TSP is probably not mediated by a beta 2 integrin receptor. These data contribute to the accumulating evidence that PMN can interact with extracellular matrix proteins through a CD11/CD18-independent process.     

Enterocytic differentiation of the human Caco-2 cell line correlates with alterations in integrin signaling

We previously reported that the enterocytic differentiation of human colonic Caco-2 cells correlated with down-regulation of fibronectin (FN) and laminin (LN), two extracellular matrix components interacting with cell surface integrin receptors. We now investigated whether Caco-2 cell differentiation was associated with alterations in integrin signaling with special interest in the expression and activity of focal adhesion kinase (FAK) and mitogen-activated protein (MAP) kinase. The differentiation of Caco-2 cells was associated with: 1) down-regulation of beta1 integrin expression at the mRNA and protein levels; 2) increased FAK expression together with decreased FAK autophosphorylation; 3) decreased FAK's ability to associate with PI3-kinase and pp60c-src; and 4) increased MAP kinase expression along with decreased MAP activity. In addition, we show that FAK and MAP kinase belong to distinct integrin signaling pathways and that both pathways remain functional during Caco-2 cell differentiation since the coating of differentiating cells on FN and LN but not on polylysine increased the tyrosine phosphorylation of FAK and of its endogenous substrate paxillin, and stimulated MAP kinase activity. In conclusion, our results provide evidence that FAK and MAP kinase, two signaling molecules activated independently by beta1 integrins in Caco-2 cells, undergo alterations of both expression and activity during the enterocytic differentiation of this cell line.     

The basement membrane protein laminin-5 acts as a soluble cell motility factor

One of the basement membrane (BM) proteins, laminin-5 (LN5), is known to support efficient cell adhesion and migration through interaction with integrins on the basal plasma membrane. Here, we show that a soluble form of LN5 induced migration of human epithelial cells and carcinoma cells by interacting with integrins on the apical cell surface. Although both LN5 and laminin-10/11 (LN10/11) promoted cell migration when coated onto a plastic surface as insoluble substrata, only LN5 stimulated cell migration in its soluble form on other substrata such as fibronectin (FN), vitronectin (VN) and collagen. Soluble LN5 interacted with integrins alpha3beta1 and alpha6beta1 on the apical cell surface and stimulated cell migration, while the cell morphology was largely dependent on the underlying substratum. Thus, integrin signals from the apical surface and the basal surface synergistically regulated cytoskeletal organization and cell motility. Soluble and insoluble LN5 induced cell motility by activating signal pathways via protein kinase C (PKC), phosphoinositide 3-OH kinase (PI3-K) and MAP kinase. The PKC dependency was more prominent for soluble LN5 than insoluble LN5, and was absent in the stimulation by insoluble LN10/11. In vitro scratch assays with keratinocytes, self-produced soluble LN5 bound to the apical cell surface of migrating cells at the scratched edges, suggesting that soluble LN5 may contribute to cell migration in pathological conditions such as wound healing and tumor invasion.     

Involvement of caveolin‐1 in fibronectin‐induced mouse embryonic stem cell proliferation: Role of FAK,RhoA, PI3K/Akt,and ERK 1/2 pathways

Fibronectin (FN) is the foremost proliferation‐associated extracellular matrix component promoting cell adhesion, migration, and survival. We examined the effect of FN on cell proliferation and the related signaling pathways in mouse embryonic stem (ES) cells. FN increased integrin β1, Src, focal adhesion kinase (FAK), and caveolin‐1 phosphorylation levels in a time‐dependent manner. Phosphorylation of Src, FAK, and caveolin‐1 was attenuated by integrin β1 neutralizing antibody. Integrin β1, Src, and FAK coimmunoprecipitated with caveolin‐1 in the presence of FN. In addition, FN increased RhoA and Rho kinase activation, which were completely blocked by PP2, FAK small interfering RNA (siRNA), caveolin‐1 siRNA, or the caveolar disruptor methyl‐β‐cyclodextrin (MβCD). FN also increased phosphorylation of Akt and ERK 1/2, which were significantly blocked by either FAK siRNA, caveolin‐1 siRNA, MβCD, GGTI‐286 (RhoA inhibitor), or Y‐27632 (Rho kinase inhibitor). FN‐induced increase of protooncogenes (c‐fos, c‐myc, and c‐Jun) and cell‐cycle regulatory proteins (cyclin D1/CDK4 and cyclin E/CDK2) expression levels were attenuated by FAK siRNA or caveolin‐1 siRNA. Furthermore, inhibition of each pathway such as integrin β1, Src, FAK, caveolin‐1, RhoA, Akt, and ERK 1/2 blocked FN‐induced [³H]‐thymidine incorporation. We conclude that FN stimulates mouse ES cell proliferation via RhoA‐PI3K/Akt‐ERK 1/2 pathway through caveolin‐1 phosphorylation. J. Cell. Physiol. 226: 267–275, 2010. © 2010 Wiley‐Liss, Inc.     

Substrate modulation of osteoblast adhesion strength, focal adhesion kinase activation, and responsiveness to mechanical stimuli

Osteoblast interactions with extracellular matrix (ECM) proteins are known to influence many cell functions, which may ultimately affect osseointegration of implants with the host bone tissue. Some adhesion-mediated events include activation of focal adhesion kinase, and subsequent changes in the cytoskeleton and cell morphology, which may lead to changes in adhesion strength and cell responsiveness to mechanical stimuli. In this study we examined focal adhesion kinase activation (FAK), F-actin cytoskeleton reorganization, adhesion strength, and osteoblast responsiveness to fluid shear when adhered to type I collagen (ColI), glass, poly-L-lysine (PLL), fibronectin (FN), vitronectin (VN), and serum (FBS). In general, surfaces that bind cells through integrins (FN, VN, FBS) elicited the highest adhesion strength, FAK activation, and F-actin stress fiber formation after both 15 and 60 minutes of adhesion. In contrast, cells attached through non-integrin mediated means (PLL, glass) showed the lowest FAK activation, adhesion strength, and little F-actin stress fiber formation. When subjected to steady fluid shear using a parallel plate flow chamber, osteoblasts plated on FN released significantly more PGE2 compared to those on glass. In contrast, PGE2 release of osteoblasts attached to FN or glass was not different in the absence of fluid shear, suggesting that differences in binding alone are insufficient to alter PGE2 secretion. The increased adhesion strength as well as PGE2 secretion of osteoblasts adhered via integrins may be due to increased F-actin fiber formation, which leads to increased cell stiffness.     

FAK和ILK介导骨桥蛋白诱导的血管平滑肌细胞黏附和迁移

黏着斑激酶（FAK）和整合素偶联激酶（ILK）是整合素信号途径中的重要信号转导分子,为阐明两者在血管平滑肌细胞（VSMC）黏附和迁移中的作用,以骨桥蛋白（OPN）作为VSMC黏附和迁移的诱导剂,检测其对FAK和ILK磷酸化以及对两者之间结合的影响.在此基础上,用FAK磷酸化特异性抑制剂黏着斑相关非激酶（FRNK）或ILK反义RNA分别阻断FAK磷酸化或ILK表达,进一步探讨两者在VSMC黏附和迁移中所起的作用.结果显示,OPN诱导可促进FAK磷酸化,诱导10 min后FAK磷酸化水平升高到对照组的2.4倍;与此同时,ILK的磷酸化受到抑制,30 min降至对照细胞的44.6％.OPN诱导FAK磷酸化的同时使FAK与ILK的结合减少.外源性FRNK在VSMC中的过表达显著降低FAK的磷酸化水平,促进ILK磷酸化和FAK与ILK之间的结合,抑制VSMC的黏附和迁移.用ILK反义RNA抑制ILK表达使VSMC在OPN上的黏附增加1.8倍,迁移细胞数降低45.5％.结果提示,FAK和ILK介导OPN诱导的VSMC黏附和迁移过程,两者通过对同一刺激信号产生不同的磷酸化变化而对VSMC的黏附和迁移产生不同的影响.     

Osteopontin Promotes Mesenchymal Stem Cell Migration and Lessens Cell Stiffness via Integrin β1, FAK, and ERK Pathways

The use of mesenchymal stem cells (MSCs) for therapeutic applications has attracted great attention because MSCs home to and engraft to injured tissues after in vivo administration. The expression of osteopontin (OPN) is elevated in response to injury and inflammation, and its role on rat bone marrow-derived mesenchymal stem cells (rMSCs)-directed migration has been elucidated. However, the signaling pathways through the activation of which OPN promotes rMSCs migration and the involvement of cell mechanics during OPN-mediating rMSCs migration have not been well studied. In this study, we found that OPN activated focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways by the ligation of integrin β1 in rMSCs. Inhibitors of FAK and ERK pathways inhibited OPN-induced rMSCs migration, indicating the possible involvement of FAK and ERK activation in OPN-induced migration in rMSCs. In addition, atomic force microscopy analysis showed that OPN reduced cell stiffness in rMSCs via integrin β1, FAK, and ERK pathways, suggesting that the promotion of rMSCs migration might partially be contributing to the decrease in cell stiffness stimulated by OPN. To further examine the role of OPN on cell motility and stiffness, actin cytoskeleton of rMSCs was observed. The reduced well-defined F-actin filaments and the promoted formation of pseudopodia in rMSCs induced by OPN explained the reduction in cell stiffness and the increase in cell migration. The current study data have shown for the first time that OPN binding to integrin β1 promotes rMSCs migration through the activation of FAK and ERK pathways, which may be attributed to the change in cell stiffness caused by the reduction in the amount of organized actin cytoskeleton.     

Osteopontin increases migration and MMP‐9 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, αvβ3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)‐9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN‐induced increase of the migration and MMP‐9 up‐regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited OPN‐induced cell migration and MMP‐9 up‐regulation. Stimulation of JJ012 cells with OPN also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The OPN‐mediated increases in MMP‐9 and κB‐luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP‐9 expression through the αvβ3 integrin, FAK, MEK, ERK and NF‐κB signal transduction pathway. J. Cell. Physiol. 221: 98–108, 2009. © 2009 Wiley‐Liss, Inc     

A novel role of ERK5 in integrin‐mediated cell adhesion and motility in cancer cells via Fak signaling

In metastatic cancer, high expression levels of vitronectin (VN) receptors (integrins), FAK, and ERK5 are reported. We hypothesized that integrin‐mediated ERK5 activation via FAK may play a pivotal role in cell adhesion, motility, and metastasis. ERK5 and FAK phosphorylation when metastatic MDA‐MB‐231 and PC‐3 cells were plated on VN was enhanced. Further experiments showed co‐immunoprecipitation of integrins β1, αVβ3, or αVβ5 with ERK5 and FAK. To gain better insight into the mechanism of ERK5, FAK, and VN receptors in cell adhesion and motility, we performed loss‐of‐function experiments using integrin blocking antibodies, and specific mutants of FAK and ERK5. Ectopic expression of dominant negative ERK5/AEF decreased ERK5 and FAK (Y397) phosphorylation, cell adhesion, and haptotactic motility (micromotion) on VN. Additionally, DN FAK expression attenuated ERK5 phosphorylation, cell adhesion, and motility. This study documents the novel finding that in breast and prostate cancer cells, ERK5 is a critical target of FAK in cell adhesion signaling. Using different cancer cells, our experiments unveil a novel mechanism by which VN receptors and FAK could promote cancer metastasis via ERK5 activation. J. Cell. Physiol. 219: 152–161, 2009. © 2008 Wiley‐Liss, Inc.     

NG2, a novel proapoptotic receptor, opposes integrin alpha4 to mediate anoikis through PKCalpha-dependent suppression of FAK phosphorylation

Disruption of cell-matrix interactions can lead to anoikis - apoptosis due to loss of matrix contacts. Altered fibronectin (FN) induces anoikis of primary human fibroblasts by a novel signaling pathway characterized by reduced phosphorylation of focal adhesion kinase (FAK). However, the receptors involved are unknown. FAK phosphorylation is regulated by nerve/glial antigen 2 (NG2) receptor signaling through PKCalpha a point at which signals from integrins and proteoglycans may converge. We found that an altered FN matrix induced anoikis in fibroblasts by upregulating NG2 and downregulating integrin alpha4. Suppressing NG2 expression or overexpressing alpha4 rescued cells from anoikis. NG2 overexpression alone induced apoptosis and, by reducing FAK phosphorylation, increased anoikis induced by an altered matrix. NG2 overexpression or an altered matrix also suppressed PKCalpha expression, but overexpressing integrin alpha4 enhanced FAK phosphorylation independently of PKCalpha. Cotransfection with NG2 cDNA and integrin alpha4 siRNA did not lower PKCalpha and pFAK levels more than transfection with either alone. PKCalpha was upstream of FAK phosphorylation, as silencing PKCalpha decreased FAK phosphorylation. PKCalpha overexpression reversed this behavior and rescued cells from anoikis. Thus, NG2 is a novel proapoptotic receptor, and NG2 and integrin alpha4 oppositely regulate anoikis in fibroblasts. NG2 and integrin alpha4 regulate FAK phosphorylation by PKCalpha-dependent and -independent pathways, respectively.     

Modeled microgravity disrupts collagen I/integrin signaling during osteoblastic differentiation of human mesenchymal stem cells

Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following 7 days culture in modeled microgravity (MMG). One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen (Col I)-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix (ECM) proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of MMG on integrin expression and function in hMSC. We demonstrate that 7 days of culture in MMG leads to reduced expression of the ECM protein, Col I. Conversely, MMG consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin subunit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt protein kinase (Akt) is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-mitogen activated protein kinase (MAPK) pathway is evidenced by a reduction in Ras and extracellular signal-related protein kinase (ERK) activation. Taken together, our findings indicate that MMG decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.     

Effects of matrix proteins on the expression of matrix metalloproteinase-2, -9, and -14 and tissue inhibitors of metalloproteinases in human cytotrophoblast cells during the first trimester.

The activity of matrix metalloproteinases (MMPs) specifies the ability of the trophoblast cell to degrade extracellular matrix (ECM) substrates. Usually the process of normal human placentation involves a coordinated interaction between the fetal-derived trophoblast cells and their microenvironment in the uterus. In this study, the effects of ECM proteins on the expression of MMP-2, -9, and -14 (membrane-type MMP-1); and the production of tissue inhibitors of metalloproteinase (TIMP) types -1, -2, and -3 have been investigated. Cytotrophoblast cells at 9 or 10 wk of gestation were cultured on various ECM coated dishes under serum-free conditions. Gelatin zymography analysis showed that cells grown on fibronectin (FN), laminin (LN), and vitronectin (VN) secreted more MMP-9 (about 1.5- to 3-fold more) than cells cultured on collagen I (Col I), whereas the secretion of MMP-9 by cells cultured on collagen IV (Col IV) was only half that by the cells on Col I. Northern Blot analysis gave the same results as zymography, indicating that expression of the MMP-9 gene in cytotrophoblast cells can be affected by matrix proteins. There was no significant difference in the expression of MMP-2 either at protein or mRNA levels among the cells cultured on the different matrix substrates. The expression of MMP-14 was regulated in a manner similar to that of MMP-2. Using ELISA, we detected higher levels of TIMP-1 in the culture medium of cells grown on VN, LN, and FN compared with that grown on Col I. But the expression of TIMP-3 mRNA was remarkably inhibited by VN, and ECM proteins had no effect on TIMP-1 and TIMP-2 mRNA expression. It was also observed that cultured cytotrophoblast cells expressed the corresponding receptors for the tested matrix proteins, such as integrins alpha(1), alpha(5), alpha(6), beta(1), and beta(4). Furthermore, the adhesiveness of cytotrophoblast cells on Col I, Col IV, FN, and LN was increased by 62%, 45%, 21%, and 22%, respectively, when compared with adhesiveness on VN. Isolated cytotrophoblast cells remained stationary when cultured on dishes coated with Col I and Col IV, but they assumed a more motile morphology and aggregated into a network when cultured on LN and VN. These data indicate that human trophoblast cells interact with their microenvironment to control their behavior and function.     

Thrombospondin modulates alpha v beta 3 function through integrin- associated protein

Integrin-associated protein (IAP) is a receptor for the carboxyl- terminal "cell-binding domain" (CBD) of thrombospondin 1 (TS1). IAP associates with alpha v beta 3 integrin and mAbs against IAP inhibit certain integrin functions. Here we examine the effects of the TS1 CBD and 4N1K (KRFYVVMWKK), a cell-binding peptide derived from it, on the adhesion and spreading on vitronectin (VN) of C32 human melanoma cells which express IAP, alpha v beta 3, and alpha v beta 5. Cells adhere to VN at low surface densities via alpha v beta 5 and spread very slowly while adhesion to higher density VN involves both alpha v beta 5 and alpha v beta 3 and results in rapid spreading. Spreading of the cells, but not adhesion, on sparse VN coatings is markedly enhanced by the presence of soluble TS1, the recombinant CBD and 4N1K, but not the "mutant" peptide 4NGG, KRFYGGMWKK, which fails to bind IAP. This enhanced spreading is completely blocked by mAb LM609 against alpha v beta 3 and the anti-IAP mAb B6H12. Correlated with this enhanced spreading is increased tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and a protein of ca. 90 kD. The enhanced spreading induced by TS1 and 4N1K and the constitutive spreading on higher density VN are both blocked by calphostin C (100 nM), wortmannin (10 nM), and tyrosine kinase inhibitors. In contrast, pertussis toxin specifically blocks only the TS1 stimulated spreading on low density VN, indicating that IAP exerts its effects on signal transduction via a heterotrimeric Gi protein acting upstream of a common cell spreading pathway which includes PI-3 kinase, PKC, and tyrosine kinases.     

粘着斑相关非激酶对骨桥蛋白促血管平滑肌细胞迁移的抑制作用及分子机制

为阐明整合素 β3 粘着斑激酶 (FAK)信号途径在骨桥蛋白 (OPN)诱导血管平滑肌细胞(VSMC)迁移中的作用 ,用FAK磷酸化特异性抑制剂粘着斑相关非激酶 (FRNK)选择性阻断FAK磷酸化 ,观察对OPN 整合素 β3 相互作用所激活的FAK信号通路的影响及其与OPN诱导VSMC迁移之间的关系 .外源性FRNK在VSMC中的过表达可显著抑制OPN诱导的VSMC迁移 ,使跨膜迁移细胞数下降 5 0 5 8% (P <0 0 5 ) .OPN刺激不但明显诱导FAK表达 ,而且还促进其磷酸化 .外源性FRNK对OPN诱导的FAK磷酸化具有显著抑制作用 ,使磷酸化型FAK水平比相应对照细胞下降5 9 1% ,但其对FAK表达不产生明显的影响 .FRNK还具有下调整合素 β3 表达的作用 ,免疫荧光细胞化学分析结果显示 ,在转染FRNK的VSMC中 ,粘着斑蛋白的磷酸化水平降低 ,粘着斑数量明显减少 .结果提示 ,整合素 β3 FAK是介导VSMC迁移的重要信号途径 ,外源性FRNK通过下调 β3 表达、抑制FAK磷酸化和减少粘着斑蛋白磷酸化及粘着斑形成等机制 ,减弱OPN刺激信号的跨膜转导及沿胞内途径传递 ,发挥抑制OPN促VSMC迁移的效应 .     

Optimal intensity shock wave promotes the adhesion and migration of rat osteoblasts via integrin β1-mediated expression of phosphorylated focal adhesion kinase

To search for factors promoting bone fracture repair, we investigated the effects of extracorporeal shock wave (ESW) on the adhesion, spreading, and migration of osteoblasts and its specific underlying cellular mechanisms. After a single period of stimulation by 10 kV (500 impulses) of shock wave (SW), the adhesion rate was increased as compared with the vehicle control. The data from both wound healing and transwell tests confirmed an acceleration in the migration of osteoblasts by SW treatment. RT-PCR, flow cytometry, and Western blotting showed that SW rapidly increased the surface expression of α5 and β1 subunit integrins, indicating that integrin β1 acted as an early signal for ESW-induced osteoblast adhesion and migration. It has also been found that a significant elevation occurred in the expression of phosphorylated β-catenin and focal adhesion kinase (FAK) at the site of tyrosine 397 in response to SW stimulation after the increasing expression of the integrin β1 molecule. When siRNAs of integrin α5 and β1 subunit were added, the level of FAK phosphorylation elevated by SW declined. Interestingly, the adhesion and migration of osteoblasts were decreased when these siRNA reagents as well as the ERK1/2 signaling pathway inhibitors, U0126 and PD98059, were present. Further studies demonstrated that U0126 could inhibit the downstream integrin-dependent signaling pathways, such as the FAK signaling pathway, whereas it had no influence on the synthesis of integrin β1 molecule. In conclusion, these data suggest that ESW promotes the adhesion and migration of osteoblasts via integrin β1-mediated expression of phosphorylated FAK at the Tyr-397 site; in addition, ERK1/2 are also important for osteoblast adhesion, spreading, migration, and integrin expression.     

Temporal and spatial expression of integrins and their extracellular matrix ligands at the maternal-fetal interface in the rhesus monkey during pregnancy

The integrin and extracellular matrix protein (ECM)-mediated adhesion and invasion of the receptive maternal uterine endometrium by trophoblasts is a critical event in the complex physiological process of pregnancy. Although the process has been largely characterized in mice, the relevant mechanism in primates remains unclear. We investigated the expression patterns and dynamic alterations of integrin subunits (alpha1, alpha5, alpha6, beta1, and beta4) and their ECM ligands, such as laminin (LN), type IV collagen (Col IV), and fibronectin (FN), at the maternal-fetal interface during Gestational Days 15, 25, 50, and 100 and at full term in 20 pregnant rhesus monkeys. Immunohistochemistry and in situ hybridization revealed that a relatively high expression of integrins occurred in trophoblast cells at Gestational Day 15, with the peak level occurring at Day 25. The expression level decreased from Day 50 to term. Along the invasive pathway, expression levels of integrin alpha1, alpha5, and beta1 subunits were gradually elevated from the proximal to distal column, reaching peak level in the trophoblast shell, but were reduced in those invasive extravillous cytotrophoblast (EVCT) cells in contact with the decidua. Integrin alpha1, alpha5, beta1, and beta4 subunits were also highly expressed in decidual stromal cells and moderately expressed in the maternal epithelium and endothelium. Immunoreactive FN, LN, and Col IV were distributed in EVCT and decidual stromal cells and part of the uterine epithelial and endothelial cells. These data suggest that the correlated expression of integrins and their ECM ligands at the maternal-fetal interface might be involved in regulation of cell proliferation and differentiation and the counterbalanced invasion-accelerating and invasion-restraining processes in trophoblast cells during the early stage of pregnancy.     

Activation of RhoA and FAK induces ERK-mediated osteopontin expression in mechanical force-subjected periodontal ligament fibroblasts

The precise mechanism by which Rho kinase translates the mechanical signals into OPN up-regulation in force-exposed fibroblasts has not been elucidated. Human periodontal ligament fibroblasts (hPLFs) were exposed to mechanical force by centrifuging the culture plates at a magnitude of 50 g/cm² for 60 min. At various times of the force application, they were processed for analyzing cell viability, trypan blue exclusion, and OPN expression at protein and RNA levels. Cellular mechanism(s) of the force-induced OPN up-regulation was also examined using various kinase inhibitors or antisense oligonucleotides specific to mechanosensitive factors. Centrifugal force up-regulated OPN expression and induced a rapid and transient increase in the phosphorylation of focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and Elk1. Pharmacological blockade of RhoA/Rho-associated coiled coil-containing kinase (ROCK) signaling markedly reduced force-induced FAK and ERK1/2 phosphorylation. Transfecting hPLFs with FAK antisense oligonucleotide diminished ERK1/2 activation and force-induced OPN expression. Further, ERK inhibitor inhibited significantly OPN expression, Elk1 phosphorylation, and activator protein-1 (AP-1)-DNA binding activation, but not FAK phosphorylation, in the force-applied cells. These results demonstrate that FAK signaling plays critical roles in force-induced OPN expression in hPLFs through interaction with Rho/ROCK as upstream effectors and ERK-Elk1/ERK-c-Fos as downstream effectors.     

Beta 1 and beta 3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix.

Extracellular matrix receptors on ductus arteriosus smooth muscle cells (SMC) must enable the cells to migrate through both interstitial and basement membrane matrices to form intimal mounds during postnatal ductus closure. We examined the role of beta 1 and beta 3 integrin receptors on SMC adhesion and migration. Using a new assay to measure cell migration, we found that lamb ductus arteriosus SMC attach to and migrate over surfaces coated with fibronectin (FN), laminin (LN), vitronectin (VN), and collagens I (I) and IV (IV). Blocking antibodies, specific to different integrin complexes, showed that SMC adhesion to FN, LN, I, and IV depended exclusively on functioning beta 1 integrins with little, if any, contribution by the alpha V beta 3 integrin; on the other hand, cell migration over these substrates depended to a large extent on the alpha V beta 3 receptor. Immunofluorescent staining demonstrated that during the early phase of SMC migration, the beta 1 integrins organized rapidly into focal plaques that, with time, gradually covered the cell's basal surface; on the other hand, the beta 3 receptor remained concentrated at all times at the cell's margins. Ligand affinity chromatography and immunoprecipitation techniques identified a unique series of beta 1 integrins binding to each matrix component: FN (alpha 5 beta 1, alpha 3 beta 1, alpha V beta 1), LN (alpha 1 beta 1, alpha 7 beta 1), VN (alpha V beta 1), I (alpha 1 beta 1, alpha 2 beta 1), and IV (alpha 1 beta 1). In contrast, the beta 3 integrin, alpha V beta 3, bound to all the substrates tested: FN, LN, VN, I, and IV. The results indicate that beta 1 and beta 3 integrins may play different roles in attachment and migration as SMC move through the vascular extracellular matrix to produce obliteration of the ductus arteriosus lumen.