Integrin-mediated adhesion as self-sustained waves of enzymatic activation.

Integrin receptors mediate interaction between the cellular actin-cytoskeleton and extracellular matrix. Based on their activation properties, we propose a reaction-diffusion model where the kinetics of the two-state receptors is modulated by their lipidic environment. This environment serves as an activator variable, while a second variable plays the role of a scaffold protein and controls the self-sustained activation of the receptors. Due to receptor diffusion which couples dynamically the activator and the inhibitor, our model connects major classes of reaction diffusion systems for excitable media. Spot and rosette solutions, characterized by receptor clustering into localized static or dynamic structures, are organized into a phase diagram. It is shown that diffusion and kinetics of receptors determines the dynamics and the stability of these structures. We discuss this model as a precursor model for cell signaling in the context of podosomes forming actoadhesive metastructures, and we study how generic signaling defects influence their organization.

Cooperativity between integrin activation and mechanical stress leads to integrin clustering.

Integrins are transmembrane receptors involved in crucial cellular biological functions such as migration, adhesion, and spreading. Upon the modulation of integrin affinity toward their extracellular ligands by cytoplasmic proteins (inside-out signaling) these receptors bind to their ligands and cluster into nascent adhesions. This clustering results in the increase in the mechanical linkage among the cell and substratum, cytoskeleton rearrangements, and further outside-in signaling. Based on experimental observations of the distribution of focal adhesions in cells attached to micropatterned surfaces, we introduce a physical model relying on experimental numerical constants determined in the literature. In this model, allosteric integrin activation works in synergy with the stress build by adhesion and the membrane rigidity to allow the clustering to nascent adhesions independently of actin but dependent on the integrin diffusion onto adhesive surfaces. The initial clustering could provide a template to the mature adhesive structures. Predictions of our model for the organization of focal adhesions are discussed in comparison with experiments using adhesive protein microarrays.

Excitable waves at the margin of the contact area between a cell and a substrate.

In this paper, we study a new physical mechanism to generate an activator field which signals the extreme margin of the contact area between an adherent cell and the substrate. This mechanism is based on the coupling between the adhesive bridges connecting the substrate to the cytoskeleton and a cytosolic activator. Once activated by adhesion on the adhesive bridges, this activator is free to diffuse on the membrane. We propose that this activator is part of the mecano-transduction pathway which links adhesion to actin polymerization and, thus, to cellular motility. The consequences of our model are as follows: (a) the activator is localized at the rim of the contact area, (b) the adhesion is reinforced at the margin of the contact area between the cell and the substrate, (c) excitable waves of the activator can propagate along the adhesion rim.

Early enterocytic differentiation of HT-29 cells: biochemical changes and strength increases of adherens junctions.

We have characterized the modulation of cell-cell adhesion and the structure of adherens junctions in the human colon adenocarcinoma HT-29 cell line that differentiates into enterocytes after glucose substitution for galactose in the medium. We demonstrate that differentiated cells (HT-29 Gal) rapidly established E-cadherin-mediated interactions in aggregation assays. This effect is not due to an increase in E-cadherin expression during this early stage of cell differentiation, but rather results from the maturation of preexisting adherens junctions. These junctions are characterized by the redistribution of E-cadherin to the basolateral membrane and its co-localization with the actin cytoskeleton. Subcellular fractionation studies indicate that actin-associated E-cadherins bind beta-catenin and p120ctn. Furthermore, the p120ctn/E-cadherin association is upregulated. These data reveal a cooperative interaction between p120ctn and E-cadherin that corresponds to mature functional adherens junctions able to initiate tight cell-cell adhesion required for epithelium architecture and further affirm the gatekeeper role of p120ctn.

RhoA-dependent switch between alpha2beta1 and alpha3beta1 integrins is induced by laminin-5 during early stage of HT-29 cell differentiation.

Integrin-mediated interactions between the basement membrane and epithelial cells control the differentiation of epithelia. We characterized the modulation of adhesive behaviors to basement membrane proteins and of integrin function in the human colon adenocarcinoma HT-29 cell line, which differentiates into enterocytes after the substitution of galactose for glucose in the medium. We demonstrate an increased capability of these cells to adhere to collagen type IV during the early stage of differentiation. This effect occurs without any changes in integrin cell surface expression but rather results from an alpha2beta1/alpha3beta1 integrin switch, alpha3beta1 integrin becoming the major collagen receptor. The increase in laminin-5 secretion and deposit on the matrix is a key factor in the mechanism regulating cell adhesion, because it is responsible for the activation of alpha3beta1 integrin. Furthermore, down-regulation of RhoA GTPase activity occurs during HT-29 cell differentiation and correlates with the activation of the integrin alpha3beta1. Indeed, C3 transferase, a RhoA GTPase inhibitor, induces a similar alpha2beta1/alpha3beta1 switch in undifferentiated HT-29 cells. These results indicate that the decrease in RhoA activation is the biochemical mechanism underlying this integrin switch observed during cell differentiation. The physiological relevance of such modulation of integrin activity in the functioning of the crypt-villus axis is discussed.

Conformation, localization, and integrin binding of talin depend on its interaction with phosphoinositides.

Talin is a structural component of focal adhesion sites and is thought to be engaged in multiple protein interactions at the cytoplasmic face of cell/matrix contacts. Talin is a major link between integrin and the actin cytoskeleton and was shown to play an important role in focal adhesion assembly. Consistent with the view that talin must be activated at these sites, we found that phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) bound to talin in cells in suspension or at early stages of adhesion, respectively. When phosphoinositides were associated with phospholipid bilayer, talin/phosphoinositide association was restricted to PI4,5P(2). This association led to a conformational change of the protein. Moreover, the interaction between integrin and talin was greatly enhanced by PI4,5P(2)-induced talin activation. Finally, sequestration of PI4,5P(2) by a specific pleckstrin homology domain confirms that PI4,5P(2) is necessary for proper membrane localization of talin and that this localization is essential for the maintenance of focal adhesions. Our results support a model in which PI4,5P(2) exposes the integrin-binding site on talin. We propose that PI4,5P(2)-dependent signaling modulates assembly of focal adhesions by regulating integrin-talin complexes. These results demonstrate that activation of the integrin-binding activity of talin requires not only integrin engagement to the extracellular matrix but also the binding of PI4,5P(2) to talin, suggesting a possible role of lipid metabolism in organizing the sequential assembly of focal adhesion components.

Talin controls the exit of the integrin alpha 5 beta 1 from an early compartment of the secretory pathway.

Talin is a major cytosolic protein that links the intracellular domains of beta1 and beta3 integrins to the cytoskeleton. It is required for focal adhesion assembly. However, its downregulation not only slows down cell spreading and organization of focal adhesions but also impairs the maturation of some beta1 integrins, including the fibronectin receptor alpha5beta1. To investigate this, we characterized the beta1 integrin synthesized in cells expressing talin anti-sense RNA (AT22 cells). We identified a large intracellular pool of beta1 integrins that is abnormally accumulated in an earlier compartment of the secretory pathway. In this report, we show that in talin-deficient AT22 cells, the aberrant glycosylation of integrin receptors is accompanied by a delay in the export of the integrin alpha5beta1. In normal cells, talin was found associated with beta1 integrins in an enriched membrane fraction containing Golgi and endoplasmic reticulum. Finally, microinjection of anti-talin antibodies resulted in accumulation of the integrins within the cells. These data strongly suggest that talin plays a specific role in the export of newly synthesized integrins. We propose that talin binding to the integrin may disclose a diphenylalanine export signal, which is present in the membrane-proximal GFFKR motif conserved in all integrin alpha chains.

Adherence of human erythroleukemia cells inhibits proliferation without inducing differentiation.

To investigate the effect of extracellular matrix molecules in the megakaryocytic lineage, we studied the role of integrin engagement in the proliferation and differentiation of human erythroleukemia (HEL) cells. HEL cells grew in suspension, but their adherence depended upon the presence of matrix proteins or protein kinase C signaling. Adherence by itself did not trigger commitment of these cells but accelerated phorbol 12-myristate 13-acetate-induced differentiation. HEL cells adhered to fibronectin mainly through alpha5beta1, and this receptor acted synergetically with alpha4beta1. Integrin engagement induced cell growth arrest through mitogen-activated protein kinase inactivation. Such down-regulation of the mitogen-activated protein kinase pathway by integrin engagement was suggested as a megakaryocytic-platelet lineage specificity. This signaling was not restricted to a peculiar integrin but was proposed as a general mechanism in these cells.

Grafting an RGD motif onto an epidermal growth factor-like module: chemical synthesis and functional characterization of the chimeric molecule.

A novel protein was engineered by inserting the GRGDS motif of fibronectin within the 14-residue loop of the EGF-like module from human complement protease C1r. The resulting chimeric EGF-RGD module (52 residues, three disulfide bridges) was assembled by automated solid-phase synthesis using the t-Boc strategy. Using reduced/oxidized glutathione, the EGF-RGD module was folded as efficiently as the natural C1r-EGF module, resulting in formation of the appropriate disulfide bridge pattern as shown by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. Circular dichroism and NMR measurements provided further indication that introduction of the GRGDS motif had no significant effect on the folding. Using Chinese Hamster Ovary (CHO) cells bearing the integrin receptors specific for fibronectin and vitronectin, EGF-RGD was shown to induce cell adhesion via the introduced GRGDS motif. Cell binding was inhibited specifically and efficiently by the synthetic peptide GRGDSP and by fibronectin, and to a much lesser extent by vitronectin, whereas the monoclonal antibody PB1 directed to the alpha5 subunit of alpha5beta1 integrin had no effect. The ability of EGF-RGD to trigger significant cell spreading and intracellular signaling was also demonstrated using immunofluorescence and confocal microscopy.

Calcium/calmodulin-dependent protein kinase II controls integrin alpha5beta1-mediated cell adhesion through the integrin cytoplasmic domain associated protein-1alpha.

This paper provided evidence that the regulation of CHO cell adhesion on fibronectin by calcium/calmodulin-dependent protein kinase II (CaMKII) is mediated through the recently described integrin cytoplasmic domain associated protein-1alpha (ICAP-1alpha). The point mutation T38D localized within the optimal CaMKII recognition motif of ICAP-1alpha results in a strong defect in cell spreading which cannot be overcome by the inhibition of the endogenous CaMKII. This fact strongly suggests that the phosphorylation of Threonine 38 by CaMKII modulates the alpha5beta1 integrin function. Conversely, the mutation T38A produces an analog of ICAP-1alpha that cannot be phosphorylated and that stimulates cell spreading on fibronectin to a similar extent when CaMKII is inhibited.

Adhesion of mature polyploid megakaryocytes to fibronectin is mediated by beta 1 integrins and leads to cell damage.

Human CD34+ bone marrow cells were committed to the megakaryocytic lineage in serum-free liquid cultures by the following cytokines: thrombopoietin, erythropoietin, and IL-6. Megakaryocyte maturation has been described as being regulated by the extracellular matrix. These cells express receptors for laminin, collagen, and vitronectin, but they selectively adhere to and spread on fibronectin, a major component of the bone marrow environment. Function-perturbing antibodies against beta 1 integrins totally abolished the adhesion of megakaryocytes on fibronectin, whereas antibodies to beta 3 did not, suggesting that beta 1 integrins were responsible for the adhesive phenotype of these polyploid cells. beta 1-positive clusters were visualized in close contact with the extremities of stress fibers at the cell surface. In the course of cell spreading, we observed morphological modifications such as the disorganization of the compact nuclei structure and the appearance of holes in the cytoplasm leading to the release of alpha IIb beta 3-positive cellular fragments. This process appeared to be a specific feature of megakaryocytes and is correlated neither to apoptosis nor to integrin signaling.

Calcium/calmodulin-dependent protein kinase II controls alpha5beta1 integrin-mediated inside-out signaling.

Fibronectin binding on alpha5beta1 integrin is strictly dependent on intracellular calcium. Using an in vitro assay, we previously found that either calcineurin inhibitors or a blocking calcineurin monoclonal antibody added to cell lysates completely abolished the fibronectin/integrin interaction, which suggested that the activity of calcineurin, a calcium/calmodulin-dependent phosphatase, was required to counteract some kinase activity and maintain the high affinity state of alpha5beta1. In this paper, we show that blocking of the calcium/calmodulin kinase II (CaMKII) activity with the specific inhibitor KN-62 or with its pseudosubtrate Autocamtide-2 preserved the high affinity state of the integrin even under experimental conditions that inhibit calcineurin. Conversely, the addition of purified CaMKII to the cell lysate inhibited alpha5beta1 binding to fibronectin in vitro. Consistent with these results, cell adhesion on fibronectin was stimulated by KN-62. Moreover, Scatchard analysis of fibronectin binding on CHO cells revealed that KN-62 decreased the Kd value from 0.3 to 0.05 microM. Finally the expression of exogenous constitutively active CaMKII resulted in a dramatic defect in cell adhesion with no significant modification in alpha5beta1 cell surface expression. In summary our results demonstrate that CaMKII controls the affinity state of the integrin alpha5beta1 in vitro and in living cells.

The 'usual care' of major depression in primary care practice.

OBJECTIVE: To determine how primary care physicians treat patients with major depression in the course of routine practice and the degree to which such practice produces outcomes anticipated with interventions recommended by the Agency for Health Care Policy and Research Depression Guideline Panel. DESIGNS: Prospective cohort study. SETTINGS: Academically affiliated ambulatory family practice centers and internal medicine clinics in urban neighborhoods of Pittsburgh, Pa. PATIENTS: Ninety-two patients who were seen in primary care practices and who met criteria for a current major depression as determined by the Diagnostic Interview Schedule and a psychiatrist's assessment. INTERVENTION: Physicians were informed of the patient's psychiatric diagnosis, and were urged to treat it in whatever manner and for whatever duration they deemed appropriate (ie, with "usual care"). MAIN OUTCOME MEASURES: The treatments that were provided, the patients' clinical course, and the relationship between the type of treatment and clinical course. RESULTS: Health center records indicated that 67 patients (73%) received a depression-specific treatment in the 8 months following study entry. A majority of the total cohort were prescribed an antidepressant drug. Of the 92 patients, 18 (20%) were asymptomatic at 8 months (Hamilton Rating Scale for Depression score, < or = 7). The treatment pattern was not clearly related to the clinical course. CONCLUSIONS: The recovery rates for the patients with major depression who were treated with usual care in routine primary care practices were lower than those anticipated from treatments consistent with the Agency for Health Care Policy and Research guidelines. Further studies of the caregiving elements that influence the effectiveness of depression-specific treatments of patients in primary care settings are needed.

NPXY motifs control the recruitment of the alpha5beta1 integrin in focal adhesions independently of the association of talin with the beta1 chain.

With the exception of the divergent beta4 and beta8 chains, the integrin beta subunit cytoplasmic domains are short and highly conserved sequences. Consensus motifs are found among the different cytoplasmic beta chains. Experiments using chimeric receptors demonstrated that the 47 amino acids of the beta1 subunit cytoplasmic domain contain sufficient information to target integrins to adhesion plaques. Three clusters of amino acids, named cyto-1, cyto-2 and cyto-3, seem to contribute to this localization. Cyto-2 and cyto-3 exhibit NPXY motifs. At present, the exact function of these motifs remains unknown but it is likely that these sequences are involved in protein-protein interactions. Although NPXY motifs often act as internalization signals at the cytoplasmic tail of membrane receptors, our previous results showed that the two NPXY motifs are not responsible for the alpha5beta1 integrin endocytosis. Herein, we address the question of the role of the two highly conserved NPXY motifs found in the beta1 cytoplasmic domain, and which correspond to the conserved domains cyto-2 and cyto-3. We demonstrate that, within the integrin beta1 cytoplasmic tail, the two NPXY motifs are required for the recruitment of the integrin in focal adhesions. In addition, our results indicate that these two motifs control but do not belong to the talin-binding sites. Finally, the analysis of the phenotypes of NPXY mutants reveals that the interaction of talin with the beta1 cytosolic domain is not sufficient to target the integrins to focal adhesions.

Symptoms of major depression and tricyclic side effects in primary care patients.

OBJECTIVE: To examine the prevalence and course of symptoms resembling side effects of tricyclic antidepressants among primary care patients experiencing major depression and receiving nortriptyline pharmacotherapy. DESIGN: Prospective cohort study. PATIENTS: Seventy-five patients meeting DSM-III-R criteria for a current major depression. SETTING: Four Pittsburgh (Pa.) ambulatory health centers affiliated with residency programs. MEASUREMENTS AND MAIN RESULTS: Symptoms resembling tricyclic side effects were assessed at baseline and at monthly intervals using the Somatic Symptoms Checklist. The Hamilton Rating Scale for Depression and Diagnostic Interview Schedule were used to assess depressive severity and history of generalized anxiety or panic disorder, respectively. Symptoms resembling tricyclic side effects, including thirst (54%), palpitations (51%), and dry mouth (48%), were commonly experienced before commencing pharmacotherapy. Patients with severe depressive episodes and those with a history of an anxiety or panic disorder had significantly more physical symptoms than those with milder episodes of depression and were more likely to drop out of care (n = 25) before completing the acute phase of pharmacotherapy. Patients who completed the acute phase of pharmacotherapy and those who entered its continuation phase (n = 43) experienced significant reduction in many depressive and physical symptoms (p < .001). CONCLUSIONS: Symptoms resembling tricyclic side effects are common among depressed primary care patients before beginning pharmacotherapy and generally remit with the depressive episode. Better awareness of major depression's somatic effects and the consequences of therapy could result in better management of both physicians' and patients' expectations regarding antidepressant pharmacotherapy.

Treating depressed primary care patients improves their physical, mental, and social functioning.

BACKGROUND: This study describes the functioning of primary care patients with major depressive disorder, the relationship of medical comorbidity to functional status, and the effects of depression-specific treatment on functional status after 8 months. METHODS: Patients were randomized to a protocol intervention (nortriptyline hydrochloride or interpersonal psychotherapy) or to usual care with the patient's physician in a clinical trial of primary care treatments of depression. Their functional status was evaluated using the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36) and the Global Assessment Scale. Medical comorbidity was assessed with the Duke Severity of Illness Checklist. The Hamilton Rating Scale for Depression and Beck Depression Inventory were used to measure depressive severity. Assessments were conducted at baseline and at 1, 2, 4, and 8 months after randomization. RESULTS: At baseline, patients reported substantial impairments in the functional domains as assessed by the SF-36 and Global Assessment Scale. Severity of general medical illness and depression were not correlated. Greater medical comorbidity was associated with diminished physical, but not psychological, functioning. Mean scores on SF-36 scales and the Global Assessment Scale improved significantly during the 8 months of follow-up. Patients assigned to protocol treatments showed greater improvement, compared with those assigned to usual care, on the SF-36 mental summary scale and most individual scales but not on the SF-36 physical summary scale. However, patients who completed protocol treatment also experienced significant improvement on the physical summary scale. Medical comorbidity was only a weak predictor of outcome. CONCLUSIONS: Primary care patients with major depressive disorder report substantial impairments in physical, psychological, and social functioning on initial assessment. Severity of baseline medical comorbidity did not correlate with severity of depression and only weakly correlated with functional status at 8 months. Functional impairments improve with time, but standardized depression-specific treatment is associated with greater improvement in more domains of functioning than is a physician's usual care.

Treating major depression in primary care practice. Eight-month clinical outcomes.

BACKGROUND: We studied whether standardized treatments of major depression whose efficacy was established with psychiatric patients are equally effective when provided to primary care patients, and whether standardized treatments are more effective than a primary care physician's usual care. METHODS: A randomized controlled trial was conducted, in which primary care patients meeting DSM-III-R criteria for a current major depression were assigned to nortriptyline (n = 91) or interpersonal psychotherapy (n = 93) provided within well-structured parameters, or a physician's usual care (n = 92). The main outcome measures were degree and rate of improvement in severity of depressive symptoms and proportion of patients recovered at 8 months. RESULTS: Severity of depressive symptoms was reduced more rapidly and more effectively among patients randomized to pharmacotherapy or psychotherapy than among patients assigned to a physician's usual care. Among treatment completers, approximately 70% of patients participating in the full pharmacotherapy or psychotherapy protocol but only 20% of usual care patients were judged as recovered at 8 months. CONCLUSIONS: Pharmacotherapy and psychotherapy effectively treat major depression among primary care patients when provided within specific parameters and for the full acute and continuation phases. Treatment principles recommended by the Depression Guideline Panel of the Agency for Health Care Policy and Research are supported.

Cotranscription of two RNA coding for the cell adhesion regulator and its variant in Reh leukemia cells.

Human genomic DNA analysis reveals the existence of polymorphisms at the cell molecular adhesion regulator (CMAR) locus. In order to choose between the two possible open frames deduced from the variant sequence, we have sequence both the human 5' non-coding region and the mouse CMAR variant DNA. We found that both mRNA species coexist in human cells.

Down regulation of talin alters cell adhesion and the processing of the alpha 5 beta 1 integrin.

The role of talin was addressed by down regulating its expression using an antisense RNA strategy. HeLa cells were transfected with a talin 5' cDNA fragment under the control of the inducible human metallothionein promotor. Isolated clones displayed a decrease in talin level down to 10% of control. The reduction in talin expression dramatically slowed down the kinetics of cell spreading. Mock-transfected cells, spread out onto fibronectin, exhibited large peripheral adhesion plaques. In contrast, cells with reduced talin expression showed smaller focal contacts localized all over the ventral face, and displayed a marked decrease in the number of stress fibers. Immunoprecipitation experiments carried out with a polyclonal antibody on surface-labeled receptor indicated a shift in the mobility for both alpha 5 and beta 1 subunits. Surprisingly, beta 1 integrin chains could not be detected by indirect immunofluorescence using monoclonal antibodies in talin deficient clones. Western blot analysis indicated the presence of two forms of beta 1. We analyzed the processing of beta 1 in normal and talin deficient cells using pulse chase experiments. Normal cells required a minimum of 5 hours for the processing of mature beta 1, while the talin deficient AT22 clone showed that the beta 1 precursor was slowly converted into a very low molecular mass product. Our data demonstrate that talin plays a central role in the establishment of cell-matrix contacts. In addition, down regulation of talin impairs the folding and processing of beta 1 integrins.