Distinct recognition of collagen subtypes by alpha(1)beta(1) and alpha(2)beta(1) integrins. Alpha(1)beta(1) mediates cell adhesion to type XIII collagen

Two integrin-type collagen receptors, alpha(1)beta(1) and alpha(2)beta(1), are structurally very similar. However, cells can concomitantly express the both receptors and they might have independent functions. Here, Chinese hamster ovary (CHO) cells, which lack endogenous collagen receptors, were transfected with either alpha(1) or alpha(2) integrin cDNA. Cells were allowed to adhere to various collagen types and their integrin function was tested by observing the progression of cell spreading. The cells expressing alpha(1)beta(1) integrin could spread on collagen types I, III, IV, and V but not on type II, while alpha(2)beta(1) integrin could mediate cell spreading on collagen types I-V. Type XIII is a transmembrane collagen and its interaction with the integrins has not been previously studied. CHO-alpha1beta1 cells could spread on human recombinant type XIII collagen, unlike CHO-alpha2beta1 cells. Integrins alpha(1)beta(1) and alpha(2)beta(1) recognize collagens with the specific alphaI domains. The alpha(1)I and alpha(2)I domains were produced as recombinant proteins, labeled with europium and used in a sensitive solid-phase binding assay based on time-resolved fluorescence. alpha(1)I domain, unlike the alpha(2)I domain, could attach to type XIII collagen. The results indicate, that alpha(1)beta(1) and alpha(2)beta(1) have different ligand binding specificity. Distinct recognition of different collagen subtypes by the alphaI domains can partially explain the differences seen in cell spreading. However, despite the fact that CHO-alpha1beta1 cells could not spread on type II collagen alpha(1)I domain could bind to this collagen type. Thus, the cell spreading on collagens may also be regulated by factors other than the integrins.     

Probing the Interaction Forces of Prostate Cancer Cells with Collagen I and Bone Marrow Derived Stem Cells on the Single Cell Level

Adhesion of metastasizing prostate carcinoma cells was quantified for two carcinoma model cell lines LNCaP (lymph node-specific) and PC3 (bone marrow-specific). By time-lapse microscopy and force spectroscopy we found PC3 cells to preferentially adhere to bone marrow-derived mesenchymal stem cells (SCP1 cell line). Using atomic force microscopy (AFM) based force spectroscopy, the mechanical pattern of the adhesion to SCP1 cells was characterized for both prostate cancer cell lines and compared to a substrate consisting of pure collagen type I. PC3 cells dissipated more energy (27.6 aJ) during the forced de-adhesion AFM experiments and showed significantly more adhesive and stronger bonds compared to LNCaP cells (20.1 aJ). The characteristic signatures of the detachment force traces revealed that, in contrast to the LNCaP cells, PC3 cells seem to utilize their filopodia in addition to establish adhesive bonds. Taken together, our study clearly demonstrates that PC3 cells have a superior adhesive affinity to bone marrow mesenchymal stem cells, compared to LNCaP. Semi-quantitative PCR on both prostate carcinoma cell lines revealed the expression of two Col-I binding integrin receptors, α1β1 and α2β1 in PC3 cells, suggesting their possible involvement in the specific interaction to the substrates. Further understanding of the exact mechanisms behind this phenomenon might lead to optimized therapeutic applications targeting the metastatic behavior of certain prostate cancer cells towards bone tissue.     

Inhibition of integrin-mediated crosstalk with epidermal growth factor receptor/Erk or Src signaling pathways in autophagic prostate epithelial cells induces caspase-independent death

In vivo in the prostate gland, basal epithelial cells adhere to laminin 5 (LM5) via alpha3beta1 and alpha6beta4 integrins. When placed in culture primary prostate basal epithelial cells secrete and adhere to their own LM5-rich matrix. Adhesion to LM5 is required for cell survival that is dependent on integrin-mediated, ligand-independent activation of the epidermal growth factor receptor (EGFR) and the cytoplasmic tyrosine kinase Src, but not PI-3K. Integrin-mediated adhesion via alpha3beta1, but not alpha6beta4 integrin, supports cell survival through EGFR by signaling downstream to Erk. PC3 cells, which do not activate EGFR or Erk on LM5-rich matrices, are not dependent on this pathway for survival. PC3 cells are dependent on PI-3K for survival and undergo caspase-dependent death when PI-3K is inhibited. The death induced by inhibition of EGFR or Src in normal primary prostate cells is not mediated through or dependent on caspase activation, but depends on the induction of reactive oxygen species. In addition the presence of an autophagic pathway, maintained by adhesion to matrix through alpha3beta1 and alpha6beta4, prevents the induction of caspases when EGFR or Src is inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in LM5-adherent primary prostate epithelial cells.     

Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell--collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I.

Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the alpha 1 beta 1 and alpha 2 beta 1 integrins. Different cyanogen bromide (CB) fragments of the alpha 1 (I) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed alpha 1 beta 1, primary rat cardiac fibroblasts alpha 1 beta 1 and alpha 2 beta 1, and Rat-1 cells only alpha 2 beta 1. All three cell types expressed alpha 3 beta 1 but this integrin did not bind to collagen--Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with alpha 1(I)CB3 and alpha 1(I)CB8; Rat-1 cells attached to alpha 1(I)CB3 but only poorly to alpha 1(I)CB8-coated substrata. Cardiac fibroblasts and Rat-1 cells spread and formed beta 1-integrin-containing focal adhesions when grown on substrata coated with native collagen or alpha 1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on alpha 1(I)CB8. The rat alpha 1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to alpha 1(I)CB3 and alpha 1(I)CB8, as well as the attachment of cardiac fibroblasts to alpha 1(I)CB8, but only partially inhibited the attachment of cardiac fibroblasts to alpha 1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat-1 cells to collagen type I or to alpha 1(I)CB3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parathyroid hormone-related protein upregulates integrin expression via an intracrine pathway in PC-3 prostate cancer cells

Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissue and prostate cancer cell lines, and enhances prostate tumor cell growth both in vivo and in vitro. PTHrP expression also plays a role in the development of bone metastasis, which is a frequent complication in patients with prostate carcinoma. Tumor cell adhesion to extracellular matrix (ECM) components is mediated via integrin subunits, and plays a major role in the invasion and metastasis of tumor cells. We previously showed that PTHrP overexpression increases adhesion of the human prostate cancer cell line PC-3 to the ECM molecules collagen type I, fibronectin, and laminin. Increased adhesion is accompanied by upregulation in the expression of alpha1, alpha5, alpha6, and beta4 integrin subunits. We used the same cell line to study the mechanism via which PTHrP upregulates integrin expression. Clonal PC-3 cells were established overexpressing wild-type PTHrP or PTHrP mutated in the nuclear localization sequence (NLS). Mutation of the NLS negated the effects of PTHrP on alpha1, alpha5, alpha6, and beta4 integrin expression, indicating that these effects are mediated via an intracrine pathway requiring nuclear localization. Expression of the alpha2, alpha3, alphav, and beta1 integrin subunits were comparable in wild-type and NLS-mutated PTHrP transfectants. These findings indicate that PTHrP may play a role in prostate tumor invasion and metastasis by upregulating the expression of specific integrin subunits via an intracrine pathway.     

Upregulation of endothelin 1 and its precursor by IL-1beta, TNF-alpha, and TGF-beta in the PC3 human prostate cancer cell line

Increasing evidence indicates that endothelin 1 (ET-1) is implicated in prostate tumour progression. However, data on ET-1 regulation in human prostate and prostate cancer cell lines are lacking. In this study, regulation of ET-1 and its precursor big ET-1, using PC3 cells, a human bone metastatic prostatic carcinoma cell line, was addressed. ET-1 and big ET-1 assays demonstrated greater secretion of both peptides in the presence of 10% fetal calf serum (FCS) as compared with 0.5% FCS. Incubation of PC3 cells in the absence and presence of various cytokines and growth factors known to be implicated in prostate stroma-epithelium interactions, revealed that IL-6, FGF7/KGF and FGF2/bFGF had no effect on ET-1 and big ET-1 secretion, whereas interleukin 1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) stimulated their secretion in a concentration-dependent manner. Binding experiments indicated the presence of specific ET-1 receptors in PC3 cells: Kdapp = 1.1 x 0.2 x 10(-10)M, Bmax = 2660 +/- 390 sites/cell. Data analysis demonstrated the presence of only the ETA receptor subtype in PC3 cells. In conclusion, our results indicate that the implication of ET-1 in prostate cancer is likely to be mediated via paracrine/autocrine control of cell factors.     

A neuronal cell line (PC12) expresses two beta 1-class integrins-alpha 1 beta 1 and alpha 3 beta 1-that recognize different neurite outgrowth-promoting domains in laminin

Integrins mediate neuronal process outgrowth on components of the ECM. Integrin alpha subunit-specific antibodies have been used to examine the roles of individual beta 1 integrins in attachment and neurite outgrowth by the neuronal cell line, PC12, in response to laminin and collagen. alpha 1 beta 1 and alpha 3 beta 1 were identified as the major beta 1 integrins expressed by PC12 cells. In functional assays, both alpha 1 beta 1 and alpha 3 beta 1 mediated PC12 cell interactions with laminin, whereas alpha 1 beta 1 alone mediated responses to collagen types I and IV. alpha 1 beta 1 and alpha 3 beta 1 were shown to recognize two different neurite-promoting sites in laminin: alpha 1 beta 1 interacted with the cross-region of laminin present in proteolytic fragments E1-4 and E1; alpha 3 beta 1 recognized a site in the long arm contained in laminin fragment E8. Thus, PC12 cells express two beta 1 integrins, which together function in attachment and neurite outgrowth on laminin and collagen. These integrins are candidates for mediating neurite outgrowth of sympathetic and other neurons in response to these ECM components.     

Substrate specificity of alpha(v)beta(3) integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT pathway activation

The alpha(v)beta(3) integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express alpha(v)beta(3) adhere and migrate on osteopontin and vitronectin in an alpha(v)beta(3)-dependent manner. However, exogenous expression of alpha(v)beta(3) in noninvasive prostate cancer LNCaP (beta(3)-LNCaP) cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of alpha(v)beta(3) by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. We show that alpha(v)beta(3)-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, alpha(v)beta(3) engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of beta(3)-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in beta(3)-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in beta(3)-LNCaP cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support alpha(v)beta(3)-mediated cell migration and PI 3-kinase/AKT pathway activation. We conclude therefore that alpha(v)beta(3) exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner.     

Hyaluronan synthase elevation in metastatic prostate carcinoma cells correlates with hyaluronan surface retention, a prerequisite for rapid adhesion to bone marrow endothelial cells

Bone marrow is the primary site of metastasis in patients with advanced stage prostate cancer. Prostate carcinoma cells metastasizing to bone must initially adhere to endothelial cells in the bone marrow sinusoids. In this report, we have modeled that interaction in vitro using two bone marrow endothelial cell (BMEC) lines and four prostate adenocarcinoma cell lines to investigate the adhesion mechanism. Highly metastatic PC3 and PC3M-LN4 cells were found to adhere rapidly and specifically (70-90%) to BMEC-1 and trHBMEC bone marrow endothelial cells, but not to human umbilical vein endothelial cells (15-25%). Specific adhesion to BMEC-1 and trHBMEC was dependent upon the presence of a hyaluronan (HA) pericellular matrix assembled on the prostate carcinoma cells. DU145 and LNCaP cells were only weakly adherent and retained no cell surface HA. Maximal BMEC adhesion and HA encapsulation were associated with high levels of HA synthesis by the prostate carcinoma cells. Up-regulation of HA synthase isoforms Has2 and Has3 relative to levels expressed by normal prostate corresponded to elevated HA synthesis and avid BMEC adhesion. These results support a model in which tumor cells with up-regulated HA synthase expression assemble a cell surface hyaluronan matrix that promotes adhesion to bone marrow endothelial cells. This interaction could contribute to preferential bone metastasis by prostate carcinoma cells.     

Bone matrix osteonectin limits prostate cancer cell growth and survival

There is considerable interest in understanding prostate cancer metastasis to bone and the interaction of these cells with the bone microenvironment. Osteonectin/SPARC/BM-40 is a collagen binding matricellular protein that is enriched in bone. Its expression is increased in prostate cancer metastases, and it stimulates the migration of prostate carcinoma cells. However, the presence of osteonectin in cancer cells and the stroma may limit prostate tumor development and progression. To determine how bone matrix osteonectin affects the behavior of prostate cancer cells, we modeled prostate cancer cell-bone interactions using the human prostate cancer cell line PC-3, and mineralized matrices synthesized by wild type and osteonectin-null osteoblasts in vitro. We developed this in vitro system because the structural complexity of collagen matrices in vivo is not mimicked by reconstituted collagen scaffolds or by more complex substrates, like basement membrane extracts. Second harmonic generation imaging demonstrated that the wild type matrices had thick collagen fibers organized into longitudinal bundles, whereas osteonectin-null matrices had thinner fibers in random networks. Importantly, a mouse model of prostate cancer metastases to bone showed a collagen fiber phenotype similar to the wild type matrix synthesized in vitro. When PC-3 cells were grown on the wild type matrices, they displayed decreased cell proliferation, increased cell spreading, and decreased resistance to radiation-induced cell death, compared to cells grown on osteonectin-null matrix. Our data support the idea that osteonectin can suppress prostate cancer pathogenesis, expanding this concept to the microenvironment of skeletal metastases.     

Hypoxia stabilizes GAS6/Axl signaling in metastatic prostate cancer

The receptor tyrosine kinase Axl is overexpressed in a variety of cancers and is known to play a role in proliferation and invasion. Previous data from our laboratory indicate that Axl and its ligand growth arrest-specific 6 (GAS6) may play a role in establishing metastatic dormancy in the bone marrow microenvironment. In the current study, we found that Axl is highly expressed in metastatic prostate cancer cell lines PC3 and DU145 and has negligible levels of expression in a nonmetastatic cancer cell line LNCaP. Knockdown of Axl in PC3 and DU145 cells resulted in decreased expression of several mesenchymal markers including Snail, Slug, and N-cadherin, and enhanced expression of the epithelial marker E-cadherin, suggesting that Axl is involved in the epithelial-mesenchymal transition in prostate cancer cells. The Axl-knockdown PC3 and DU145 cells also displayed decreased in vitro migration and invasion. Interestingly, when PC3 and DU145 cells were treated with GAS6, Axl protein levels were downregulated. Moreover, CoCl(2), a hypoxia mimicking agent, prevented GAS6-mediated downregulation of Axl in these cell lines. Immunochemical staining of human prostate cancer tissue microarrays showed that Axl, GAS6, and hypoxia-inducible factor-1α (Hif-1α; indicator of hypoxia) were all coexpressed in prostate cancer and in bone metastases compared with normal tissues. Together, our studies indicate that Axl plays a crucial role in prostate cancer metastasis and that GAS6 regulates the expression of Axl. Importantly, in a hypoxic tumor microenvironment Axl expression is maintained leading to enhanced signaling.     

Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins.

The alpha(1)beta(1) and alpha(2)beta(1) integrins are cell surface collagen receptors. Cells expressing the alpha(1)beta(1) integrin preferentially adhere to collagen IV, whereas cells expressing the alpha(2)beta(1) integrin preferentially adhere to collagen I. Recombinant alpha(1) and alpha(2) integrin I domains exhibit the same collagen type preferences as the intact integrins. In addition, the alpha(2) integrin I domain binds echovirus 1; the alpha(1) I domain does not. To identify the structural components of the I domains responsible for the varying ligand specificities, we have engineered several alpha(1)/alpha(2) integrin I domain chimeras and evaluated their virus and collagen binding activities. Initially, large secondary structural components of the alpha(2) I domain were replaced with corresponding regions of the alpha(1) I domain. Following analysis in echovirus 1 and collagen binding assays, chimeras with successively smaller regions of alpha(1) I were constructed and analyzed. The chimeras were analyzed by ELISA with several different alpha(2) integrin monoclonal antibodies to assess their proper folding. Three different regions of the alpha(1) I domain, when present in the alpha(2) I domain, conferred enhanced collagen IV binding activity upon the alpha(2) I domain. These include the alpha3 and alpha5 helices and a portion of the alpha6 helix. Echovirus 1 binding was lost in a chimera containing the alphaC-alpha6 loop; higher resolution mapping identified Asn(289) as playing a critical role in echovirus 1 binding. Asn(289) had not been implicated in previous echovirus 1 binding studies. Taken together, these data reveal the existence of multiple determinants of ligand binding specificities within the alpha(1) and alpha(2) integrin I domains.     

Purification and characterization of mammalian integrins expressed by a rat neuronal cell line (PC12): evidence that they function as alpha/beta heterodimeric receptors for laminin and type IV collagen

Cells of the rat neuronal line, PC12, adhere well to substrates coated with laminin and type IV collagen, but attach poorly to fibronectin. Adhesion and neurite extension in response to these extracellular matrix proteins are inhibited by Fab fragments of an antiserum (anti-ECMR) that recognizes PC12 cell surface integrin subunits of Mr 120,000, 140,000, and 180,000 (Tomaselli, K. J., C. H. Damsky, and L. F. Reichardt. 1987. J. Cell Biol. 105:2347-2358). Here we extend our study of integrin structure and function in PC12 cells using integrin subunit-specific antibodies prepared against synthetic peptides corresponding to the cytoplasmic domains of the human integrin beta 1 and the fibronectin receptor alpha (alpha FN) subunits. Anti-integrin beta 1 immunoprecipitated a 120-kD beta 1 subunit and two noncovalently associated integrin alpha subunits of 140 and 180 kD from detergent extracts of surface-labeled PC12 cells. Immunodepletion studies using anti-integrin beta 1 demonstrated that these two putative alpha/beta heterodimers are identical to those recognized by the adhesion-perturbing ECMR antiserum. Anti-alpha FN immunoprecipitated fibronectin receptor heterodimers in human and rat fibroblastic cells, but not in PC12 cells. Thus, low levels of expression of the integrin alpha FN subunit can explain the poor attachment of PC12 cells to FN. The PC12 cell integrins were purified using a combination of lectin and ECMR antibody affinity chromatography. The purified integrins: (a) completely neutralize the ability of the anti-ECMR serum to inhibit PC12 cell adhesion to laminin and collagen IV; (b) have hydrodynamic properties that are very similar to those of previously characterized integrin alpha/beta heterodimeric receptors for ECM proteins; and (c) can be incorporated into phosphatidylcholine vesicles that then bind specifically to substrates coated with laminin or collagen IV but not fibronectin. Thus, the ligand-binding specificity of the liposomes containing the purified PC12 integrins closely parallels the substrate-binding preference of intact PC12 cells. These results demonstrate that mammalian integrins purified from a neuronal cell line can, when incorporated into lipid vesicles, function as receptors for laminin and type IV collagen.     

Ligation of integrin alpha 3beta 1 by laminin 5 at the wound edge activates Rho-dependent adhesion of leading keratinocytes on collagen

Wounding of the epidermis signals the transition of keratinocytes from quiescent anchorage on endogenous basement membrane laminin 5 to migration on exposed dermal collagen. In this study, we attempt to characterize activation signals that transform quiescent keratinocytes into migratory leading cells at the wound edge. Previously, we reported that adhesion and spreading on collagen via integrin alpha(2)beta(1) by cultured human foreskin keratinocytes (HFKs) requires RhoGTP, a regulator of actin stress fibers. In contrast, adhesion and spreading on laminin 5 requires integrins alpha(3)beta(1) and alpha(6)beta(4) and is dependent on phosphoinositide 3-hydroxykinase (Nguyen, B. P., Gil, S. G., and Carter, W. G. (2000) J. Biol. Chem. 275, 31896-31907). Here, we report that quiescent HFKs do not adhere to collagen but adhere and spread on laminin 5. By using collagen adhesion as one criterion for conversion to a "leading wound cell," we found that activation of collagen adhesion requires elevation of RhoGTP. Adhesion of quiescent HFKs to laminin 5 via integrin alpha(3)beta(1) and alpha(6)beta(4) is sufficient to increase levels of RhoGTP required for adhesion and spreading on collagen. Consistently, adhesion of quiescent HFKs to laminin 5, but not collagen, also promotes expression of the precursor form of laminin 5, a characteristic of leading keratinocytes in the epidermal outgrowth. We suggest that wounding of quiescent epidermis initiates adhesion and spreading of keratinocytes at the wound edge on endogenous basement membrane laminin 5 via alpha(3)beta(1) and alpha(6)beta(4) in a Rho-independent mechanism. Spreading on endogenous laminin 5 via alpha(3)beta(1) is necessary but not sufficient to elevate expression of precursor laminin 5 and RhoGTP, allowing for subsequent collagen adhesion via alpha(2)beta(1), all characteristics of leading keratinocytes in the epidermal outgrowth.     

Prolyl hydroxylation of collagen type I is required for efficient binding to integrin alpha 1 beta 1 and platelet glycoprotein VI but not to alpha 2 beta 1

Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins alpha 1 beta 1 and alpha 2 beta 1. Collagen fibrils also bind to the integrin alpha 2 beta 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO)n, respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptor-binding properties of collagen. We show that alpha 2 beta 1 but not alpha 1 beta 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant alpha 1 beta 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use alpha 2 beta 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by alpha 1 beta 1. These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.     

Affinity of integrins for damaged extracellular matrix: alpha v beta 3 binds to denatured collagen type I through RGD sites.

Cellular adhesion receptors termed integrins play an important role in the interaction of cells with extracellular matrix (ECM) during wound healing, development and tumorigenesis. During such events, ECM may become modified or damaged which could alter the types of adhesive signals presented to cells. In this study, cell adhesion and affinity chromatography experiments were performed to determine whether different integrins interact with denatured versus native ECM molecules. Human melanoma cells were found to adhere to denatured versus native type I collagen through different integrins. The cells adhere to denatured collagen through the alpha v beta 3 integrin and this interaction is inhibited by an RGD containing peptide but not by a control peptide. In contrast, adhesion to native type I collagen appears to be mediated by several beta 1 integrins and thus, is not inhibited by either alpha v beta 3 antibodies or the RGD peptide. Affinity chromatography reveals a marked increase in the quantity of alpha v beta 3 isolated on denatured collagen versus native collagen-sepharose. These results suggest that RGD sites in type I collagen may be masked and that they become exposed upon denaturation of the molecule. Wounding of extracellular matrix may, thus, expose RGD sites in collagens that facilitate the interaction of cells with damaged extracellular matrix through RGD binding integrins.     

Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) is involved in the activation of endothelial integrins

We previously reported that mouse orthologue of puromycin insensitive leucyl-specific aminopeptidase (mPILSAP) played an important role in angiogenesis by regulating the proliferation and migration of endothelial cells (ECs) (Miyashita et al., 2002. Blood 99:3241-3249). Here, we examined the mechanism as to how mPILSAP regulates the migration of ECs. Cell adhesion through integrins plays a crucial role in cell migration, and ECs use at least type-1 collagen receptor integrin alpha2beta1, fibronectin receptor alpha5beta1, and vitronectin receptors alphavbeta3 and alphavbeta5. mPILSAP antisense oligodeoxynucleotide (AS-ODN) or leucinethiol (LT), a leucyl-aminopeptidase inhibitor, did not affect the attachment but did significantly inhibit the spreading of cells of the murine endothelial cell line MSS31 when they were plated on vitronectin-, fibronectin-, or type-1 collagen, although they did not affect the expression of integrin alpha2, alpha5, alphav, beta1, beta3, and beta5 subunits on the cell surface. AS-ODN and LT also inhibited the tyrosine phosphorylation of FAK when cells were plated on vitronectin, fibronectin, or type-1 collagen. This inhibition of cell spreading and of tyrosine phosphorylation of FAK could be negated by Mg(2+). These results suggest that mPILSAP is involved in the activation of endothelial integrins.     

Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins.

alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo.     

Effects of the Antiandrogen Flutamide on the Expression of Protein Kinase C Isoenzymes in LNCaP and PC3 Human Prostate Cancer Cells

Flutamide is a nonsteroidal antiandrogen that is frequently used for total androgen blockage in the treatment of advanced prostate cancer. We investigated the effect of this antiandrogen on the expression of protein kinase C (PKC) isoenzymes (alpha, beta1, epsilon, zeta) that are involved in cell growth, apoptosis and neoplastic transformation. Androgen-dependent (LNCaP) and independent (PC3) human prostate cancer cells were cultured in a medium that contained fetal bovine serum (FBS) or charcoal-stripped serum (CSS) and treated with 10 microM flutamide. The expression of PKC isoenzymes and the androgen receptor (AR) were analyzed by Western blot and RT-PCR, respectively. Serum steroids differentially regulate the expression of PKC isoenzymes in LNCaP and PC3 cells. Flutamide up-regulated the expression of alpha, beta1 and zeta, but not epsilon, PKC isoenzymes in CSS-LNCaP cells. These results were not homogeneously reproduced in the presence of androgens. We observed an opposite effect of flutamide, compared to CSS, on PKCbeta1 isoform expression in CSS-LNCaP suggesting that this antiandrogen exerts an agonistic effect. In PC3 cells flutamide potentiated the expression of the four PKC isoenzymes in almost all conditions tested (FBS- and CSS-cultured cells). Such effect of flutamide in PC3 cells is independent of AR since no expression of AR was detected. These results provide new evidence on antagonistic/agonistic responses of prostate cancer cells to antiandrogen drugs that are widely used in therapy and show that flutamide can elicit responses in prostate cancer cells that do not express AR.