ABSTRACT
Hypertrophic terminally differentiated cardiac myocytes show increased sarcomeric organization and altered gene expression. Previously, we established a role for the nonreceptor tyrosine kinase Src in signaling cardiac myocyte hypertrophy. Here we report evidence that p130Cas (Cas) and focal adhesion kinase (FAK) regulate this process. In neonatal cardiac myocytes, tyrosine phosphorylation of Cas and FAK increased upon endothelin (ET) stimulation. FAK, Cas, and paxillin were localized in sarcomeric Z-lines, suggesting that the Z-line is an important signaling locus in these cells. Cas, alone or in cooperation with Src, modulated basal and ET-stimulated atrial natriuretic peptide (ANP) gene promoter activity, a marker of cardiac hypertrophy. Expression of the C-terminal focal adhesion-targeting domain of FAK interfered with localization of endogenous FAK to Z-lines. Expression of the Cas-binding proline-rich region 1 of FAK hindered association of Cas with FAK and impaired the structural stability of sarcomeres. Collectively, these results suggest that interaction of Cas with FAK, together with their localization to Z-lines, is critical to assembly of sarcomeric units in cardiac myocytes in culture. Moreover, expression of the focal adhesion-targeting and/or the Cas-binding proline-rich regions of FAK inhibited ANP promoter activity and suppressed ET-induced ANP and brain natriuretic peptide gene expression. In summary, assembly of signaling complexes that include the focal adhesion proteins Cas, FAK, and paxillin at Z-lines in the cardiac myocyte may regulate, either directly or indirectly, both cytoskeletal organization and gene expression associated with cardiac myocyte hypertrophy.
Subject(s)
Cardiomegaly/metabolism , Gene Expression Regulation , Myocardium/cytology , Phosphoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Proteins , Sarcomeres/physiology , Animals , Atrial Natriuretic Factor/genetics , Cardiomegaly/genetics , Cell Fractionation , Cells, Cultured , Crk-Associated Substrate Protein , Culture Media, Serum-Free , Cytoskeletal Proteins/metabolism , Endothelins/metabolism , Endothelins/pharmacology , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Genes, Reporter , Immunoblotting , Immunohistochemistry , Microfilament Proteins/metabolism , Myocardium/metabolism , Myosin Heavy Chains/metabolism , Paxillin , Phosphoproteins/genetics , Phosphorylation , Promoter Regions, Genetic , Protein Structure, Tertiary , Protein-Tyrosine Kinases/genetics , Rats , Recombinant Fusion Proteins/metabolism , Retinoblastoma-Like Protein p130 , Sarcomeres/drug effects , Signal Transduction , Tensins , src-Family Kinases/metabolismABSTRACT
During human pregnancy specialized placental cells of fetal origin, termed cytotrophoblasts, invade the uterus and its blood vessels. This tumor-like process anchors the conceptus to the mother and diverts the flow of uterine blood to the placenta. Previously, we showed that the expression of molecules with important functional roles, including a number of extracellular matrix integrin receptors, is precisely modulated during cytotrophoblast invasion in situ. Here we exploited this observation to study the role of the focal adhesion kinase (FAK), which transduces signals from the extracellular matrix and recruits additional signaling proteins to focal adhesions. Immunolocalization studies on tissue sections showed that FAK is expressed by cytotrophoblasts in all stages of differentiation. Because extracellular matrix-induced integrin clustering results in FAK (auto)phosphorylation on tyrosine 397 (Y397FAK), we also localized this form of the molecule. Immunolocalization experiments detected Y397FAK in a subset of cytotrophoblasts near the surface of the uterine wall. To assess the functional relevance of this observation, we used an adenovirus strategy to inhibit cytotrophoblast expression of FAK as the cells differentiated along the invasive pathway in vitro. Compared to control cells transduced with a wild-type virus, cytotrophoblasts that expressed antisense FAK exhibited a striking reduction in their ability to invade an extracellular matrix substrate. When cytotrophoblast differentiation was compromised (hypoxia in vitro, preeclampsia in vivo), Y397FAK levels associated with the plasma membrane were strikingly lower, although total FAK levels did not change. Together our results suggest that (auto)phosphorylation of Y397 on FAK is a critical component of the signaling pathway that mediates cytotrophoblast migration/invasion.
Subject(s)
Protein-Tyrosine Kinases/metabolism , Trophoblasts/physiology , Biomarkers , Cell Membrane/metabolism , Cell Movement/drug effects , Cell Movement/physiology , Chorionic Villi/drug effects , Chorionic Villi/physiology , Enzyme Activation , Female , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Hypoxia/enzymology , Hypoxia/physiopathology , Immunologic Techniques , In Vitro Techniques , Oligonucleotides, Antisense/pharmacology , Oxygen/pharmacology , Phosphorylation , Placenta/enzymology , Pre-Eclampsia/enzymology , Pre-Eclampsia/physiopathology , Pregnancy , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Trophoblasts/drug effects , Trophoblasts/enzymology , Uterus/cytology , Uterus/physiologyABSTRACT
Most transformed cells have lost anchorage and serum dependence for growth and survival. Previously, we established that when serum is absent, fibronectin survival signals transduced by focal adhesion kinase (FAK), suppress p53-regulated apoptosis in primary fibroblasts and endothelial cells (Ilic et al. 1998. J. Cell Biol. 143:547-560). The present goals are to identify survival sequences in FAK and signaling molecules downstream of FAK required for anchorage-dependent survival of primary fibroblasts. We report that binding of the SH3 domain of p130Cas to proline-rich region 1 of FAK is required to support survival of fibroblasts on fibronectin when serum is withdrawn. The FAK-p130Cas complex activates c-Jun NH2-terminal kinase (JNK) via a Ras/Rac1/Pak1/MAPK kinase 4 (MKK4) pathway. Activated (phospho-) JNK colocalizes with FAK in focal adhesions of fibroblasts cultured on fibronectin, which supports their survival, but not in fibroblasts cultured on collagen, which does not. Cells often survive in the absence of extracellular matrix if serum factors are provided. In that case, we confirm work of others that survival signals are transduced by FAK, phosphatidylinositol 3'-kinase (PI3-kinase), and Akt/protein kinase B (PKB). However, when serum is absent, PI3-kinase and Akt/PKB are not involved in the fibronectin-FAK-JNK survival pathway documented herein. Thus, survival signals from extracellular matrix and serum are transduced by FAK via two distinct pathways.
Subject(s)
Cell Adhesion , Extracellular Matrix/metabolism , Fibronectins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases/metabolism , Proteins , Animals , Cell Survival , Culture Media, Serum-Free , Fibroblasts , Focal Adhesion Protein-Tyrosine Kinases , Immunohistochemistry , JNK Mitogen-Activated Protein Kinases , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/metabolism , Protein Binding , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rabbits , Retinoblastoma-Like Protein p130 , Signal Transduction , Transfection , src Homology DomainsABSTRACT
Here we show that cells lacking focal adhesion kinase (FAK) are refractory to motility signals from platelet-derived and epidermal growth factors (PDGF and EGF respectively), and that stable re-expression of FAK rescues these defects. FAK associates with activated PDGF- and EGF-receptor (PDGFR and EGFR) signalling complexes, and expression of the band-4.1-like domain at the FAK amino terminus is sufficient to mediate an interaction with activated EGFR. However, efficient EGF-stimulated cell migration also requires FAK to be targeted, by its carboxy-terminal domain, to sites of integrin-receptor clustering. Although the kinase activity of FAK is not needed to promote PDGF- or EGF-stimulated cell motility, kinase-inactive FAK is transphosphorylated at the indispensable Src-kinase-binding site, FAK Y397, after EGF stimulation of cells. Our results establish that FAK is an important receptor-proximal link between growth-factor-receptor and integrin signalling pathways.
Subject(s)
Cell Movement/physiology , Integrins/metabolism , MAP Kinase Signaling System/physiology , Platelet-Derived Growth Factor/pharmacology , Protein-Tyrosine Kinases/metabolism , Cell Movement/drug effects , Cells, Cultured , Epidermal Growth Factor/pharmacology , ErbB Receptors/physiology , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/enzymology , Focal Adhesion Kinase 1 , Focal Adhesion Kinase 2 , Focal Adhesion Protein-Tyrosine Kinases , Humans , MAP Kinase Signaling System/drug effects , Mutagenesis/physiology , Protein Structure, Tertiary , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/genetics , Receptors, Platelet-Derived Growth Factor/physiology , src-Family Kinases/metabolismABSTRACT
Integrin-mediated adhesion of monocytes and macrophages initiates a signal transduction pathway that leads to actin cytoskeletal reorganization, cell migration and immunologic activation. This signaling pathway is critically dependent on tyrosine kinases. To investigate the role of the Src-family of tyrosine kinases in integrin signal transduction, we have examined the adhesive properties of macrophages isolated from hck-/-fgr-/- double knockout mice which lack two of the three predominant Src-family kinases expressed in myeloid cells. Previous examination of polymorphonuclear leukocytes from these animals indicated that these kinases were critical in initiating the actin cytoskeletal rearrangements that lead to respiratory burst and granule secretion following integrin ligation. Double mutant peritoneal exudate macrophages demonstrated markedly reduced tyrosine phosphorylation responses compared to wild-type cells following plating on fibronectin, collagen or vitronectin-coated surfaces. Tyrosine phosphorylation of several actin-associated proteins (cortactin, paxillin, and tensin), as well as the Syk and Pyk2 tyrosine kinases, were all significantly reduced in double mutant cells. The subcellular localization of focal-adhesion associated proteins was also dramatically altered in mutant macrophages cultured on fibronectin-coated surfaces. In wild-type cells, filamentous actin, paxillin, and talin were concentrated along leading edges of the plasma membrane, suggesting that these proteins contribute to cellular polarization during migration in culture. Double mutant cells failed to show the polarized subcellular localization of these proteins. Likewise, double mutant macrophages failed to form normal filopodia under standard culture conditions. Together, these signaling and cytoskeletal defects may contribute to the reduced motility observed in in vitro assays. These data provide biochemical and morphological evidence that the Src-family kinases Hck and Fgr are required for normal integrin-mediated signal transduction in murine macrophages.
Subject(s)
Cell Movement/physiology , Cytoskeleton/metabolism , Integrins/physiology , Macrophages/metabolism , Protein-Tyrosine Kinases/deficiency , Proto-Oncogene Proteins/deficiency , Actins/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antigens, CD/chemistry , Antigens, CD/immunology , Bone Marrow Cells/cytology , Caseins/pharmacology , Cell Adhesion/drug effects , Cell Adhesion Molecules/metabolism , Cells, Cultured , Cross-Linking Reagents/pharmacology , Enzyme Activation , Enzyme Precursors/drug effects , Enzyme Precursors/metabolism , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/pharmacology , Fibronectins/pharmacology , Focal Adhesion Kinase 2 , Integrin alpha5 , Integrin beta1/chemistry , Integrin beta1/immunology , Integrins/metabolism , Intracellular Signaling Peptides and Proteins , Macrophages/cytology , Macrophages/drug effects , Macrophages, Peritoneal/cytology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/metabolism , Mice , Mice, Knockout , Phosphorylation/drug effects , Protein-Tyrosine Kinases/drug effects , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-hck , Pseudopodia , Signal Transduction , Syk Kinase , Tyrosine/metabolism , src-Family KinasesABSTRACT
Previous work with cultured primary cells, from our group and from other laboratories, has shown that signals from extracellular matrix, transduced by integrins, play critical roles in regulating gene expression, tissue-specific differentiation, and survival of primary osteoblasts and fibroblasts. This summary will focus on our most recent work, dealing with the role of cell-extracellular matrix interactions and focal adhesion kinase in regulating cell survival in osteoblasts and fibroblasts, and the role of beta1 integrins in tissue organization and remodeling in bone.
Subject(s)
Bone Remodeling/physiology , Extracellular Matrix/metabolism , Integrins/metabolism , Osteoblasts/physiology , Cell Adhesion Molecules/metabolism , Cell Survival/physiology , Fibroblasts/physiology , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Genes, p53 , Humans , Protein-Tyrosine Kinases/metabolismABSTRACT
Studies in several cell types indicate that the actions of integrin receptors for extracellular matrix and receptors for growth factors are synergistic in regulating cellular differentiation and function. We studied the roles of the alpha1beta1 and alpha2beta1 integrin collagen receptors in regulating the differentiation of 2T3 osteoblastic cells in response to bone morphogenetic protein (BMP)-2. The immortalized 2T3 cell line was established from the calvaria of mice transgenic for a BMP-2 promoter driving SV40 T-antigen. These cells require exogenous BMP-2, as well as ascorbic acid and beta-glycerolphosphate, for expression of a mature osteoblast phenotype and formation of a mineralized matrix. To determine how integrin receptors for collagen-I affect BMP-2 signaling, function-perturbing anti-rat alpha1 and/or alpha2 integrin subunit, or anti-type I collagen (Col-I), antibodies were added to human recombinant (hr)BMP-2-treated 2T3 cultures at confluence (C0) or at 4 or 8 days postconfluence (C4, C8). After 4 days of exposure to the antibodies, cultures were assayed for alkaline phosphatase (ALP) mRNA levels and enzyme activity and for cAMP production in response to parathyroid hormone. Addition of anti-collagen-I or both anti-integrin-alpha1 and -alpha2 antibodies to C0 cultures blocked expression of these early osteoblast markers by more than 90%, and also blocked mineralization (0.5-1.8% control) of these cells. In all cases, adding anti-alpha1 or anti-alpha2 antibodies separately produced partial effects, while their combined effect approached that of anti-collagen-I. When antibodies were added to more differentiated 2T3 cells, the inhibitory effects decreased. 2T3 cells carrying constitutively active BMP receptor (caBMPR-IB) showed elevated ALP activity without hrBMP-2; this constitutive activity was also suppressed by alpha1 and alpha2 integrin antibodies and by anti-Col-I antibody. Together, our data suggest that a signal(s) from collagen integrin receptors regulates the response to BMP downstream of BMPR-IB and upstream of the regulation of ALP mRNA and other early markers of osteoblast differentiation.
Subject(s)
Bone Morphogenetic Proteins/pharmacology , Integrins/physiology , Osteoblasts/cytology , Transforming Growth Factor beta , Alkaline Phosphatase/metabolism , Animals , Antibodies, Blocking/pharmacology , Blotting, Northern , Bone Morphogenetic Protein 2 , Bone Morphogenetic Protein Receptors, Type I , Bone Morphogenetic Proteins/antagonists & inhibitors , Calcification, Physiologic/drug effects , Cell Differentiation/drug effects , Cell Line , Extracellular Matrix/metabolism , Humans , Integrin alpha1beta1 , Integrins/metabolism , Mice , Osteoblasts/drug effects , Osteoblasts/metabolism , Parathyroid Hormone/pharmacology , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/metabolism , Rats , Receptors, Collagen , Receptors, Growth Factor/genetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/pharmacology , TransfectionABSTRACT
Activation of the atrial natriuretic peptide (ANP) gene is regarded as one of the earliest and most reliable markers of hypertrophy in the ventricular cardiac myocyte. We have examined the role of the nonreceptor tyrosine kinases in the signaling mechanism(s) leading to hypertrophy using human ANP gene promoter activity as a marker. Endothelin (ET), a well known hypertrophic agonist, increased activity of c-Src, c-Yes, and Fyn within minutes and promoted a selective redistribution of each of these kinases within the cell. Overexpression of c-Src effected a significant increase in activity of a cotransfected human ANP promoter-driven chloramphenicol acetyl transferase reporter, while expression of either c-Yes or Fyn was considerably less effective in this regard. ET-dependent stimulation of the human ANP gene promoter was partially inhibited by co-transfection with dominant negative Ras or dominant negative Src or Csk or by treatment with the potent Src family-selective tyrosine kinase inhibitor PP1, suggesting that the Src family kinases are involved in signaling ET-dependent activation of this promoter. Both ET- and Src-dependent activation of the ANP promoter required the presence of a CArG motif in a serum response element-like structure between -422 and -413 but did not appear to require assembly of a ternary complex for full activity. These findings support a role for Src in the activation of ANP gene expression and suggest that this kinase may contribute in an important way to the signaling mechanisms that activate hypertrophy in the cardiac myocyte.
Subject(s)
Atrial Natriuretic Factor/biosynthesis , Cardiomegaly/metabolism , Endothelins/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Receptors, Endothelin/metabolism , Animals , Animals, Newborn , Atrial Natriuretic Factor/genetics , Biological Transport , CSK Tyrosine-Protein Kinase , Cell Compartmentation , Cells, Cultured , Humans , Myocardium/cytology , Phosphoprotein Phosphatases/metabolism , Promoter Regions, Genetic , Protein-Tyrosine Kinases , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-fyn , Proto-Oncogene Proteins c-yes , Rats , Signal Transduction , ras Proteins/genetics , ras Proteins/metabolism , src-Family Kinases/metabolismABSTRACT
During early development, a subset of fetal (placental) cytotrophoblasts exhibits tumor-like behavior and invades the uterus. To access a supply of maternal blood, they invade arterioles and form heterotypic interactions with, and replace, resident maternal endothelium, creating a hybrid uterine vasculature. Recently, it has become clear that invading cytotrophoblasts transform their adhesion receptor phenotype to resemble the endothelial cells they replace. Furthermore, they express vasculogenic factors and receptors. Is this a form of vasculogenesis?
Subject(s)
Cell Adhesion Molecules/biosynthesis , Endothelium, Vascular/cytology , Neovascularization, Physiologic , Trophoblasts/physiology , Uterus/blood supply , Cell Differentiation , Epithelial Cells/cytology , Female , Humans , PregnancyABSTRACT
In many malignant cells, both the anchorage requirement for survival and the function of the p53 tumor suppressor gene are subverted. These effects are consistent with the hypothesis that survival signals from extracellular matrix (ECM) suppress a p53-regulated cell death pathway. We report that survival signals from fibronectin are transduced by the focal adhesion kinase (FAK). If FAK or the correct ECM is absent, cells enter apoptosis through a p53-dependent pathway activated by protein kinase C lambda/iota and cytosolic phospholipase A2. This pathway is suppressible by dominant-negative p53 and Bcl2 but not CrmA. Upon inactivation of p53, cells survive even if they lack matrix signals or FAK. This is the first report that p53 monitors survival signals from ECM/FAK in anchorage- dependent cells.
Subject(s)
Apoptosis/physiology , Cell Adhesion Molecules/metabolism , Extracellular Matrix/enzymology , Protein-Tyrosine Kinases/metabolism , Signal Transduction/physiology , Tumor Suppressor Protein p53/metabolism , Animals , Annexins/metabolism , Blood Proteins/pharmacology , Caspases/chemistry , Caspases/metabolism , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Culture Media, Serum-Free/pharmacology , Endothelium/cytology , Extracellular Matrix/chemistry , Fibroblasts/chemistry , Fibroblasts/cytology , Fibroblasts/enzymology , Fibronectins/metabolism , Focal Adhesion Protein-Tyrosine Kinases , Green Fluorescent Proteins , In Situ Nick-End Labeling , Indicators and Reagents , Isoenzymes/metabolism , Luminescent Proteins , Phospholipases A/metabolism , Phospholipases A2 , Protein Kinase C/metabolism , Protein Structure, Tertiary , Rabbits , Signal Transduction/drug effects , Synovial Membrane/cytologyABSTRACT
The focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways. We show that expression of the FAK-related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak-/- embryos (FAK-) compared with cells from fak+/+ embryos (FAK+). Pyk2 was localized to perinuclear regions in both FAK+ and FAK- cells. Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK- but not FAK+ cells. Increased Pyk2 tyrosine phosphorylation paralleled the time-course of Grb2 binding to Shc and activation of ERK2 in FAK- cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK- cells. However, Pyk2 associated with active Src-family PTKs after FN but not poly-L-lysine replating of the FAK- cells. Overexpression of both wild-type (WT) and kinase-inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN-stimulated c-Src in vitro kinase activity in FAK- cells, but only WT Pyk2 overexpression enhanced FN-stimulated activation of co-transfected ERK2. Interestingly, Pyk2 overexpression only weakly augmented FAK- cell migration to FN whereas transient FAK expression promoted FAK- cell migration to FN efficiently compared with FAK+ cells. Significantly, repression of endogenous Src-family PTK activity by p50(csk) overexpression inhibited FN-stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK- but not in FAK+ cells. These studies show that Pyk2 and Src-family PTKs combine to promote FN-stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK- cell migration defects.
Subject(s)
Cell Adhesion Molecules/analysis , Cell Adhesion Molecules/physiology , Cell Movement/physiology , Fibronectins/metabolism , Protein-Tyrosine Kinases/analysis , Protein-Tyrosine Kinases/deficiency , Protein-Tyrosine Kinases/physiology , Signal Transduction/physiology , src-Family Kinases/physiology , Amino Acid Sequence , Animals , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Cells, Cultured , Fibroblasts , Focal Adhesion Kinase 1 , Focal Adhesion Kinase 2 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Mice , Mitogen-Activated Protein Kinase 1 , Molecular Sequence Data , Protein-Tyrosine Kinases/biosynthesis , Rabbits , Vinculin/analysisABSTRACT
This review article focuses on the unique process by which the human placenta normally forms and how changes in this process can lead to serious pregnancy complications such as pre-eclampsia. One way to compare normal and pathologic pregnancies is to examine biopsy specimens of the placenta and placental bed for disease-associated morphological changes in cellular architecture. Our recent work has verified the decades-old observation that pre-eclampsia is associated with abnormally shallow placentation. We also discuss how these morphological observations prompted us to use a combination of in vitro modeling and in situ immunolocalization techniques to gain insights into the molecular bases of normal placentation and how these mechanisms go awry in pre-eclampsia.
Subject(s)
Endothelium, Vascular/cytology , Oxygen/pharmacology , Pre-Eclampsia/pathology , Trophoblasts/physiology , Cadherins/physiology , Cell Differentiation/drug effects , Female , Humans , Integrins/physiology , Placenta/pathology , Pregnancy , Vascular Cell Adhesion Molecule-1/physiologyABSTRACT
The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin, which regulates the adhesion, differentiation and function of various adherent cells. Interactions with fibronectin are required for osteoblast differentiation in vitro, since fibronectin antagonists added to cultures of immature fetal calvarial osteoblasts inhibit their progressive differentiation. To determine if fibronectin plays a unique role in fully differentiated osteoblasts, cultures that had already formed mineralized nodules in vitro were treated with fibronectin antagonists. Fibronectin antibodies caused >95% of the cells in the mature cultures to display characteristic features of apoptosis (nuclear condensation, apoptotic body formation, DNA laddering) within 24 hours. Cells appeared to acquire sensitivity to fibronectin antibody-induced apoptosis as a consequence of differentiation, since antibodies failed to kill immature cells and the first cells killed were those associated with mature nodules. Intact plasma fibronectin, as well as fragments corresponding to the amino-terminal, cell-binding, and carboxy-terminal domains of fibronectin, independently induced apoptosis of mature (day-13), but not immature (day-4), osteoblasts. Finally, transforming growth factor-beta1 partially protected cells from the apoptotic effects of fibronectin antagonists. Thus, in the course of maturation cultured osteoblasts switch from depending on fibronectin for differentiation to depending on fibronectin for survival. These data suggest that fibronectin, together with transforming growth factor-beta1, may affect bone formation, in part by regulating the survival of osteoblasts.
Subject(s)
Apoptosis/physiology , Fibronectins/metabolism , Osteoblasts/cytology , Animals , Antibodies/isolation & purification , Antibodies/pharmacology , Apoptosis/drug effects , Cell Differentiation/physiology , Cells, Cultured , Cellular Senescence/physiology , Fibronectins/immunology , Immunoglobulin G/isolation & purification , Immunoglobulin G/pharmacology , Microscopy, Electron , Osteoblasts/chemistry , Osteoblasts/ultrastructure , Rats , Skull/cytology , Staphylococcal Protein A , Transforming Growth Factor beta/pharmacologyABSTRACT
We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes alpha5ss1, alpha3ss1 and alpha8ss1. We further conclude that the specific alpha5ss1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.
Subject(s)
Fibronectins/metabolism , Osteoblasts/chemistry , Osteoblasts/cytology , Receptors, Fibronectin/metabolism , Alkaline Phosphatase/genetics , Animals , Antibodies/pharmacology , Binding, Competitive/immunology , Calcification, Physiologic/physiology , Cell Differentiation/physiology , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Fetus/cytology , Gene Expression Regulation, Enzymologic/physiology , Integrin alpha3beta1 , Integrins/immunology , Integrins/metabolism , Morphogenesis/physiology , Osteoblasts/enzymology , Osteocalcin/genetics , RNA, Messenger/analysis , Rats , Receptors, Fibronectin/immunology , Receptors, Laminin/immunology , Receptors, Laminin/metabolism , Receptors, Vitronectin/immunology , Receptors, Vitronectin/metabolism , Skull/cytology , Time FactorsABSTRACT
Establishment of the human placenta requires that fetal cytotrophoblast stem cells in anchoring chorionic villi become invasive. These cytotrophoblasts aggregate into cell columns and invade both the uterine interstitium and vasculature, anchoring the fetus to the mother and establishing blood flow to the placenta. Cytotrophoblasts colonizing spiral arterioles replace maternal endothelium as far as the first third of the myometrium. We show here that differentiating cytotrophoblasts transform their adhesion receptor phenotype so as to resemble the endothelial cells they replace. Cytotrophoblasts in cell columns show reduced E-cadherin staining and express VE-(endothelial) cadherin, platelet-endothelial adhesion molecule-1, vascular endothelial adhesion molecule-1, and alpha-4-integrins. Cytotrophoblasts in the uterine interstitium and maternal vasculature continue to express these receptors, and, like endothelial cells during angiogenesis, also stain for alphaVbeta3. In functional studies, alphaVbeta3 and VE-cadherin enhance, while E-cadherin restrains, cytotrophoblast invasiveness. Cytotrophoblasts expressing alpha4 integrins bound immobilized VCAM-1 in vitro, suggesting that this receptor-pair could mediate cytotrophoblast-endothelium or cytotrophoblast-cytotrophoblast interactions in vivo, during endovascular invasion. In the pregnancy disorder preeclampsia, in which endovascular invasion remains superficial, cytotrophoblasts fail to express most of these endothelial markers (Zhou et al., 1997. J. Clin. Invest. 99:2152-2164.), suggesting that this adhesion phenotype switch is required for successful endovascular invasion and normal placentation.
Subject(s)
Chorion/blood supply , Chorion/metabolism , Neovascularization, Physiologic , Trophoblasts/cytology , Antigens, CD , Cadherins/immunology , Cadherins/metabolism , Cell Adhesion , Cell Differentiation , Cells, Cultured , Chorion/cytology , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Female , Humans , Immunohistochemistry , Integrins/immunology , Integrins/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/immunology , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Pregnancy , Stem Cells/physiology , Uterus/blood supply , Uterus/metabolism , Vascular Cell Adhesion Molecule-1/immunology , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
In human pregnancy, placental cytotrophoblasts that invade the uterus downregulate the expression of adhesion receptors that are characteristic of their epithelial origin, and upregulate the expression of adhesion receptors that are expressed by vascular cells. We suggest that this transformation could be critical to endovascular invasion, the process whereby cytotrophoblasts invade the uterine spiral arterioles and line their walls (Zhou et al. J. Clin. Invest. 1997. 99: 2139-2151.). To better understand the in vivo significance of these findings, we tested the hypothesis that in preeclampsia, an important disease of pregnancy in which endovascular invasion is abrogated, cytotrophoblasts fail to adopt a vascular adhesion phenotype. In experiments described here we stained placental bed biopsy specimens from age-matched control pregnancies and from those complicated by preeclampsia with antibodies that recognize adhesion molecules that are normally modulated during this transformation. In preeclampsia, differentiating/invading cytotrophoblasts fail to express properly many of these molecules, including integrin, cadherin, and Ig superfamily members. These results suggest that preeclampsia is associated with failure of cytotrophoblasts to mimic a vascular adhesion phenotype. The functional consequences of this abnormality are unknown, but are likely to affect negatively cytotrophoblast endovascular invasion and uterine arteriole remodeling, thereby compromising blood flow to the maternal-fetal interface.
Subject(s)
Neovascularization, Physiologic , Placenta/blood supply , Placenta/metabolism , Pre-Eclampsia/physiopathology , Antigens, CD , Cadherins/biosynthesis , Cadherins/immunology , Cell Differentiation , Endothelium, Vascular/metabolism , Female , Humans , Immunohistochemistry , Integrins/biosynthesis , Integrins/immunology , Placenta/cytology , Platelet Endothelial Cell Adhesion Molecule-1/biosynthesis , Platelet Endothelial Cell Adhesion Molecule-1/immunology , Pre-Eclampsia/etiology , Pregnancy , Receptors, IgG/biosynthesis , Receptors, IgG/immunology , Uterus/blood supply , Uterus/metabolism , Vascular Cell Adhesion Molecule-1/biosynthesis , Vascular Cell Adhesion Molecule-1/immunologyABSTRACT
Cell interactions with extracellular matrix and with other cells play critical roles in morphogenesis during development and in tissue homeostasis and remodeling throughout life. Extracellular matrix is information-rich, not only because it is comprised of multifunctional structural ligands for cell surface adhesion receptors, but also because it contains peptide signaling factors, and proteinases and their inhibitors. The functions of these groups of molecules are extensively interrelated. In this review, three primary cell culture models are described that focus on adhesion receptors and their roles in complex aspects of morphogenesis and remodeling: the regulation of proteinase expression by fibronectin and integrins in synovial fibroblasts; the regulation of osteoblast differentiation and survival by fibronectin, and the regulation of trophoblast differentiation and invasion by integrins, cadherins and immunoglobulin family adhesion receptors.
Subject(s)
Embryonic and Fetal Development , Extracellular Matrix/physiology , Integrins/physiology , Animals , Cell Differentiation , Female , Fibronectins/physiology , Homeostasis , Humans , Osteoblasts/physiology , Pre-Eclampsia/etiology , PregnancySubject(s)
Cell Movement/physiology , Embryonic and Fetal Development , Extracellular Matrix/physiology , Mesoderm/physiology , Signal Transduction/physiology , Animals , Cardiovascular System/embryology , Epithelium/embryology , Germ Cells/growth & development , Humans , Neural Crest/embryology , Proteoglycans/physiology , ResearchABSTRACT
Morphogenetic processes during development, including cell migration, depend on signals from both the extracellular matrix (ECM) and soluble signaling factors. Extensive evidence has shown that the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is activated in response to both kind of signal. The most definitive evidence that FAK is directly downstream of signals initiated by the ECM comes from comparing the phenotypes of mice deficient for FAK and the ECM molecule, fibronectin: in both cases embryos die at about E8.5 and display almost identical severe vascular and other mesodermal defects. It is now clear that there are additional FAK-like proteins, indicating the existence of a FAK family. Furthermore, FAK is not located at adhesive sites in all cells where it is expressed. This, plus extensive data indicating that FAK becomes activated in response to several soluble signaling factors, suggests that the FAK family may be at the crossroads of multiple signaling pathways that affect cell and developmental processes.
Subject(s)
Cell Adhesion Molecules/metabolism , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Animals , Cell Adhesion Molecules/chemistry , Cell Movement , Enzyme Activation , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Molecular Structure , Morphogenesis , Phosphorylation , Protein-Tyrosine Kinases/chemistryABSTRACT
Vitronectin, a principal cell adhesion molecule in plasma and extracellular matrix, mediates cell adhesion and spreading via the alpha V family of integrins. In this study we demonstrate that decorin, a small dermatan sulfate proteoglycan, regulates extracellular matrix remodeling in rabbit synovial fibroblasts adhering to vitronectin. Decorin induced the expression of the matrix metalloproteinase collagenase (MMP-1) when present on the substrate with vitronectin, or with the 120-kDa cell-binding domain of fibronectin, but not when present with intact fibronectin or Type I collagen. Secreted collagenase was detected within 8 h of adhesion, there was no associated alteration in cell shape or focal contact formation in cells adhering to decorin plus vitronectin, whereas cell rounding was observed in cells adhering to decorin plus the 120-kDa fragment of fibronectin. The core protein of decorin, but not the glycosaminoglycan moiety, was sufficient to induce collagenase expression on both substrates; however, the glycosaminoglycan moiety of decorin as well as the core were required for cell rounding observed in cells adhering to the 120-kDa domain of fibronectin. The collagenase-inducing effect of decorin seems to be independent of its effects on transforming growth factor-beta, as function-blocking antibodies against transforming growth factor-beta did not interfere with the collagenase-inducing effects of decorin. These data indicate that decorin has specific gene regulatory effects in cells when present in the matrix with vitronectin or the 120-kDa fragment of fibronectin, polypeptides that are present in actively remodeling tissues. Thus, in combination, these adhesion regulatory molecules transduce novel signals that may contribute to the tissue remodeling process in morphogenesis, wound healing and disease states.