Biomedical subjects

C A McCulloch

Publications and source records attributed to C A McCulloch.

At least 19 recordsLinked to original sources

PubMed2007

Beyond the epithelium: cadherin function in fibrous connective tissues.

In fibrous connective tissues, fibroblasts are organized into syncytia, cellular networks that enable matrix remodeling and that are interconnected by intercellular adherens junctions (AJs). The AJs of fibroblasts are mediated by N-cadherin, a broadly expressed classical cadherin that is critically involved in developmental processes, wound healing and several diseases of mesenchymal tissues. In contrast to E-cadherin-dependent junctions of epithelia, the formation of AJs in fibrous connective tissues is relatively uncharacterized. Work over the last several years has documented an expanding list of molecules which function to regulate N-cadherin mediated junctions such as: Fer, PTP1B, cortactin, calcium, gelsolin, PIP5KIgamma, PIP2, and the Rho family of GTPases. We present an overview on the regulation of N-cadherin-mediated junction formation that highlights recent molecular advances in the field and rationalizes the roles of N-cadherin in connective tissue function.

Adherens Junctions↗

PubMed2006

Role of p38 in stress activation of Sp1.

Cell stressors such as physical forces can activate Sp1-dependent genes but the regulatory mechanisms are not defined. We determined if the stress-induced MAP kinase, p38, can phosphorylate Sp1 and thereby regulate the Sp1 target gene FLNA. We used Rat-2 cells and human gingival fibroblasts to examine stress-induced activation of an Sp1-dependent gene and SL2 cells, an Sp1-deficient model system, to facilitate interaction studies of transfected Sp1 with regulatory factors. Mechanical stress applied to Rat-2 cells increased promoter activity of the Sp1 target gene filamin A by >5-fold; activation was blocked by mutations to Sp1 binding sites in the filamin A promoter. Transfection experiments in SL2 cells with Sp1 expression vectors showed that when co-transfected with constitutively active p38, wild-type Sp1 but not an Sp1 binding mutant, increased promoter activity of the Sp1 target gene, filamin A, and enhanced binding of nuclear extracts to a filamin A promoter oligonucleotide. Filamin A promoter activity was blocked by dominant negative p38. Sp1 that was phosphorylated at Thr453 and Thr739 by constitutively active p38 bound to the filamin A promoter more effectively than un-phosphorylated Sp1. Recombinant active p38 phosphorylated wild-type Sp1 in vitro while the Sp1 Thr453Thr739 double mutant protein showed >3-fold reduction of phosphorylation. We conclude that stress activation of p38 phosphorylates Sp1 at specific threonine residues, modifications which in turn enhance the expression of Sp1-dependent genes.

Animals↗

PubMed2005

Separate functions of gelsolin mediate sequential steps of collagen phagocytosis.

Collagen phagocytosis is a critical mediator of extracellular matrix remodeling. Whereas the binding step of collagen phagocytosis is facilitated by Ca2+-dependent, gelsolin-mediated severing of actin filaments, the regulation of the collagen internalization step is not defined. We determined here whether phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] regulation of gelsolin is required for collagen internalization. In gelsolin null fibroblasts transfected with gelsolin severing mutants, actin severing and collagen binding were strongly impaired but internalization and actin monomer addition at collagen bead sites were much less affected. PI(4,5)P2 accumulated around collagen during internalization and was associated with gelsolin. Cell-permeable peptides mimicking the PI(4,5)P2 binding site of gelsolin blocked actin monomer addition, the association of gelsolin with actin at phagosomes, and collagen internalization but did not affect collagen binding. Collagen beads induced recruitment of type 1 gamma phosphatidylinositol phosphate kinase (PIPK1gamma661) to internalization sites. Dominant negative constructs and RNA interference demonstrated a requirement for catalytically active PIPK1gamma661 for collagen internalization. We conclude that separate functions of gelsolin mediate sequential stages of collagen phagocytosis: Ca2+-dependent actin severing facilitates collagen binding, whereas PI(4,5)P2-dependent regulation of gelsolin promotes the actin assembly required for internalization of collagen fibrils.

Actins↗

PubMed2005

Mechanical force activates eIF-2alpha phospho-kinases in fibroblast.

Mechanical forces can induce differentiation of fibroblasts into myofibroblasts, a process which requires activation of the MAP kinase p38. Currently, the identification of other phospho-kinases involved in myofibroblast differentiation has not been explored. We applied static tensile forces to rat cardiac fibroblasts via collagen-coated magnetite beads and examined activation of protein phospho-kinases by the Kinexus phospho-antibody screening system. Of 75 candidate protein kinases screened, 39 were detected and, of these, 31 phospho-kinases were analyzed. Following force application, 12 out of 31 phospho-kinases exhibited increases of phosphorylation including PKR (>4-fold), MKK3 (3-fold), MKK6 ( approximately 2-fold), and p38 ( approximately 2-fold). In several types of mechanically sensitive, contractile fibroblasts including rat cardiac, human gingival, and Rat-2 fibroblasts, tensile forces increased eIF-2alpha phosphorylation, a downstream effector of PKR. We conclude that phospho-antibody screening is an efficient method for discovery of novel mechanical force-induced phospho-kinases and force can activate eIF-2alpha phospho-kinases in fibroblasts.

Animals↗

PubMed2004

Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway.

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN(-/-)) mice were treated in vitro with H(2)O(2) to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN(-/-) cells but was increased to only 20% in WT cells. In contrast, after 1-8 h of treatment with H(2)O(2), the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN(-/-) cells. Electron microscopy of WT cells treated with H(2)O(2) showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H(2)O(2)-treated OPN(-/-) cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN(-/-) and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN(-/-) cells by approximately 30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN(-/-) cells was not altered. Restoration of OPN expression in OPN(-/-) fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H(2)O(2) treatment. Thus H(2)O(2)-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.

Animals↗

PubMed2003

Mechanical force regulation of myofibroblast differentiation in cardiac fibroblasts.

The myocardium responds to chronic pressure or volume overload by activation and proliferation of cardiac fibroblasts and their differentiation into myofibroblasts. Because alpha-smooth muscle actin (SMA) expression is the classical marker for myofibroblast differentiation, we examined force-induced SMA expression and regulation by specific MAPK pathways. Rat cardiac fibroblasts were separated from myocytes and smooth muscle cells, cultured, and phenotyped by using the presence of SMA, vimentin, and ED-A fibronectin and the absence of desmin as myofibroblast markers. Static tensile forces (0.65 pN/microm2) were applied to fibroblasts via collagen-coated magnetite beads. In neonatal cardiac fibroblasts cultured for 1 day, immunostaining and Western and Northern blotting showed very low basal levels of SMA. After the application of force, there were 1.5- to 2-fold increases of SMA protein and mRNA within 4 h. Force-induced SMA expression was dependent on ERK phosphorylation and on intact actin filaments. In contrast to cells cultured for 1 day, cells grown for 3 days on rigid substrates showed prominent stress fibers and high basal levels of SMA, which were reduced by 32% within 4 h after force application. ERK was not activated by force, but p38 phosphorylation was required for force-induced inhibition of SMA expression. These results indicate that mechanical force-induced regulation of SMA content is dependent on myofibroblast differentiation and by selective activation of MAPKs.

Actins↗

PubMed2001

Intercellular mechanotransduction: cellular circuits that coordinate tissue responses to mechanical loading.

Physical forces play an important role in modulating cell function and shaping tissue structure. Mechanotransduction, the process by which cells transduce physical force-induced signals into biochemical responses, is critical for mediating adaptations to mechanical loading in connective tissues. While much is known about mechanotransduction in cells involving forces delivered through extracellular matrix proteins and integrins, there is limited understanding of how mechanical signals are propagated through the interconnected cellular networks found in tissues and organs. We propose that intercellular mechanotransduction is a critical component for achieving coordinated remodeling responses to force application in connective tissues. We examine here recent evidence on different pathways of intercellular mechanotransduction and suggest a general model for how multicellular structures respond to mechanical loading as an integrated unit.

Adaptation, Physiological↗

PubMed2001

Cadherins mediate intercellular mechanical signaling in fibroblasts by activation of stretch-sensitive calcium-permeable channels.

Cells in mechanically active environments form extensive, cadherin-mediated intercellular junctions that are important in tissue remodeling and differentiation. Currently, it is unknown whether adherens junctions in connective tissue fibroblasts transmit mechanical signals and coordinate multicellular adaptations to physical forces. We hypothesized that cadherins mediate intercellular mechanotransduction by activating calcium-permeable, stretch-sensitive channels. Human gingival fibroblasts in suspension were plated on established homotypic monolayer cultures. The cells formed intercellular adherens junctions. Controlled mechanical forces were applied to intercellular junctions by electromagnets acting on cells containing internalized magnetite beads. At early but not later stages of intercellular attachment, force application visibly displaced magnetite bead-loaded cells and induced robust Ca(2+) transients (65 +/- 9.4 nm above base line). Similar Ca(2+) transients were induced by force application to anti-N-cadherin antibody-coated magnetite beads. Ca(2+) responses depended on influx of extracellular Ca(2+) through mechanosensitive channels because both Ca(2+) chelation and gadolinium chloride abolished the response and MnCl(2) quenched fura-2 fluorescence after force application. Force application induced accumulation of microinjected rhodamine-actin at intercellular contacts; actin assembly was inhibited by buffering intracellular calcium fluxes. Our results indicate that mechanical forces applied to adherens junctions activate stretch-sensitive calcium-permeable channels and increase actin polymerization. We suggest that N-cadherins in fibroblasts are intercellular mechanotransducers.

Cadherins↗

PubMed2001

Cytoprotection against mechanical forces delivered through beta 1 integrins requires induction of filamin A.

Cells in mechanically active environments can activate cytoprotective mechanisms to maintain membrane integrity in the face of potentially lethal applied forces. Cytoprotection may be mediated by expression of membrane-associated cytoskeletal proteins including filamin A, an actin-binding protein that increases the rigidity of the subcortical actin cytoskeleton. In this study, we tested the hypotheses that applied forces induce the expression of filamin A specifically and that this putative protective response inhibits cell death. Magnetically generated forces were applied to protein-coated magnetite beads bound to human gingival fibroblasts, cells with constitutively low basal levels of filamin A mRNA and protein. Forces applied through collagen or fibronectin, but not bovine serum albumin or poly-l-lysine-coated beads, increased mRNA and protein content of filamin A by 3-7-fold. Forces had no effect on the expression of other filamin isotypes or other cytoskeletal proteins. This effect was dependent on the duration of force and was blocked by anti-beta(1) integrin antibodies. Force also stimulated a 60% increase in expression of luciferase under the control of a filamin A promoter in transiently transfected Rat2 fibroblasts and was dependent on Sp1 transcription factor binding sites located immediately upstream of the transcription start site. Experiments with actinomycin D-treated cells showed that the increased filamin A expression after force application was due in part to prolongation of mRNA half-life. Antisense filamin oligonucleotides blocked force-induced filamin A expression and increased cell death by >2-fold above controls. The force-induced regulation of filamin A was dependent on intact actin filaments. We conclude that cells from mechanically active environments can couple diverse signals from forces applied through beta-integrins to up-regulate the production of cytoprotective cytoskeletal proteins, typified by filamin A.

Animals↗

PubMed2001

A spirochete surface protein uncouples store-operated calcium channels in fibroblasts: a novel cytotoxic mechanism.

The cytotoxicity of infectious agents can be mediated by disruption of calcium signaling in target cells. Outer membrane proteins of the spirochete Treponema denticola, a periodontal pathogen, inhibit agonist-induced Ca(2+) release from internal stores in gingival fibroblasts, but the mechanism is not defined. We determined here that the major surface protein (Msp) of T. denticola perturbs calcium signaling in human fibroblasts by uncoupling store-operated channels. Msp localized in complexes on the cell surface. Ratio fluorimetry showed that in cells loaded with fura-2 or fura-C18, Msp induced cytoplasmic and near-plasma membrane Ca(2+) transients, respectively. Increased conductance was confirmed by fluorescence quenching of fura-2-loaded cells with Mn(2+) after Msp treatment. Calcium entry was blocked with anti-Msp antibodies and inhibited by chelating external Ca(2+) with EGTA. Msp pretreatment reduced the amplitude of [Ca(2+)](i) transients upon challenge with ATP or thapsigargin. In experiments using cells loaded with mag-fura-2 to report endoplasmic reticulum Ca(2+), Msp reduced Ca(2+) efflux from endoplasmic reticulum stores when ATP was used as an agonist. Msp alone did not induce Ca(2+) release from these stores. Msp inhibited store-operated influx of extracellular calcium following intracellular Ca(2+) depletion by thapsigargin and also promoted the assembly of subcortical actin filaments. This actin assembly was blocked by chelating intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester. The reduced amplitude of agonist-induced transients and inhibition of store-operated Ca(2+) entry due to Msp were reversed by latrunculin B, an inhibitor of actin filament assembly. Thus, Msp retards Ca(2+) release from endoplasmic reticulum stores, and it inhibits subsequent Ca(2+) influx by uncoupling store-operated channels. Actin filament rearrangement coincident with conformational uncoupling of store-operated calcium fluxes is a novel mechanism by which surface proteins and toxins of pathogenic microorganisms may damage host cells.

Actins↗

PubMed2001

Transplantation of labeled periodontal ligament cells promotes regeneration of alveolar bone.

Regeneration of damaged periodontal tissues is mediated by periodontal cells, but a major sub-population comprises highly differentiated cells that do not renew. To overcome the loss of specialized cell types caused by disease, various therapeutic approaches including cell transplants have been developed to promote cell re-population in periodontal tissues. As previous transplantation studies used unlabeled cells, that are indistinguishable from host cells, it has been difficult to assess the contributions of transplanted cells to the healing processes. To track the fate and differentiation of rat periodontal cells transplanted into periodontal wounds, we used collagen-coated fluorescent beads as a permanent endocytosed marker, or cells constitutively expressing beta-galactosidase. We assessed osteogenic cell differentiation with immunohistochemical staining for osteopontin and bone sialoprotein. Cells were transplanted into periodontal wounds created in Sprague--Dawley male rats that are null for beta-galactosidase. Defects were allowed to heal spontaneously (controls), or were closed with collagen implants mixed with beta-galactosidase-positive (Lac-Z) periodontal cells, or closed with collagen implants mixed with periodontal cells loaded with fluorescent beads. Animals were killed at 1 and 2 weeks after surgery and tissues were prepared for morphometric assessment and immunostaining for osteopontin (OPN) and bone sialoprotein (BSP). Transplanted cells were easily distinguished by fluorescent beads or by beta-galactosidase-positive expression and were distributed throughout the regenerating periodontal ligament (PL) and alveolar bone. At 1 week after wounding, animals treated with beta-galactosidase-positive cells exhibited a slightly higher percentage of labeled cells in the PL compared with the fluorescent bead-labeled cell implant group (2% vs. 1% respectively; P > 0.2). At Week 2 percentages of labeled cells were slightly increased in the regenerating PL (approximately 3% for both groups, P > 0.2). In regenerating alveolar bone at 1 week, animals that were treated with beta-galactosidase-positive cells and fluorescent bead-loaded cells exhibited approximately 30% and 25% of labeled cells respectively. At 2 weeks after wounding there was an increase in the percentage of transplanted beta-galactosidase-positive cells (approximately 39% at week 2; P < 0.05), but not of transplanted cells with fluorescent beads (approximately 25% at week 2). In sites with transplanted cells there were higher percentages of OPN positive and BSP positive cells in nascent bone and more newly formed bone than in controls (>40%; P < 0.05). Transplantation of beta-galactosidase-positive cells or cells loaded with fluorescent beads is a useful method for assessing the fate and differentiation of periodontal cells in vivo. Fluorescent beads, however, are diluted at mitosis and this method underestimates the percentage of transplanted cells. As transplanted periodontal cells in both groups promoted regeneration of alveolar bone, cell transplantation could improve the restoration of periodontium destroyed by periodontitis.

Alveolar Bone Loss↗

PubMed2001

Mechanism of cyclosporin-induced inhibition of intracellular collagen degradation.

The immunosuppressant cyclosporin A (CsA) markedly inhibits collagen degradation by an intracellular phagocytic pathway in fibroblasts, an effect that can lead to massive gingival overgrowth. We used a collagen bead model of collagen phagocytosis to determine whether CsA inhibits internalization by blocking efflux of calcium from endoplasmic reticulum (ER) and mitochondrial calcium stores. CsA caused dose-dependent inhibition of phagocytosis of collagen-coated (but not bovine serum albumin-coated) beads. Chelation of intracellular Ca(2+) with BAPTA/AM or inhibition of Ca(2+)-ATPase of ER stores with thapsigargin reduced collagen bead phagocytosis. Measurement of intracellular calcium by ratio fluorometry showed increases in response to collagen-coated beads. Preincubation with CsA or thapsigargin caused a >3-fold decrease in intracellular calcium elevations in response to stimulation with collagen beads. Direct measurements of Ca(2+) in mitochondrial and ER stores showed that CsA only slightly inhibited collagen bead-induced discharge of calcium from mitochondria, but almost completely blocked discharge from ER stores. We reduced the numbers of mitochondria with chronic ethidium bromide treatment to test for the importance of ER/mitochondrial interactions. In these cells, CsA delayed collagen bead-induced calcium discharge from mitochondria. Collectively, these data indicate that CsA inhibits collagen phagocytosis by blocking calcium release from ER stores and may perturb functional interactions between the ER and mitochondria that regulate calcium stores.

Animals↗

PubMed2001

Tissue specificity of lymphocyte migration into sheep gingival tissue.

T cells show a bias in their migration pathways: some migrate preferentially to peripheral lymph nodes, some to mucosal tissues and some to peripheral tissues such as skin. The aim here was to determine the types of T cells that migrate preferentially into inflamed gingival tissue and compare this migration to that found in inflamed subcutaneous and mucosal tissues. The experiments were designed so that the simultaneous 3 h localization of two, differentially radiolabelled, lymphocyte populations (subcutaneously and mucosally derived) into sites of purified protein derivative/bacillus Calmette-Guerin-induced, delayed-type hypersensitivity, inflammatory lesions in skin, bowel and gingiva in the sheep model could be compared. The relative migration of two populations in each of the tissues was expressed as a ratio of the radioactivity of intestinal/subcutaneous lymphocytes recovered from that tissue. From nine experiments, the ratios [mean+/-S.E.M. (n)] for skin, bowel and gingiva were 0.53+/-0.02 (84), 1.98+/-0.11 (85), and 0.73+/-0.05 (29), respectively. These findings suggest that inflammation in skin and gingiva favoured the localization of subcutaneously derived lymphocytes (ratio significantly <1, P<0.025), while in bowel, the localization of intestinally derived lymphocytes was favoured (ratio significantly >1, P<0.025). Statistical analysis demonstrated that the relative localization of the two lymphocyte populations to the gingival lesions differed significantly from that for inflamed skin and bowel lesions (P<0.05). When tumour necrosis factor-alpha was used as a non-antigenic inflammatory agent to induce lymphocyte migration into skin and gingiva, a similarly greater increase in the localization of subcutaneously derived lymphocytes was detected, but the relative localization of lymphocytes was not significantly different between the two tissues. Therefore, it appears that there is tissue specificity in the migration of lymphocytes into the inflamed gingival tissues and that antigen is required for distinct tissue-specific lymphocyte traffic to occur.

Analysis of Variance↗

PubMed2001

Phenotypic comparison of periodontal ligament cells in vivo and in vitro.

The mammalian periodontal ligament contains heterogeneous populations of connective tissue cells, the precise function of which is poorly understood. Despite close proximity to bone and the application of high amplitude physical forces, cells in the periodontal ligament (PL) are capable of expressing regulatory factors that maintain PL width during adult life. The study of PL homeostasis and PL cell differentiation requires culture and phenotypic methods for precise characterization of PL cell populations, in particular those cells with an inherently osteogenic program. Currently it is unknown if cells cultured from the PL are phenotypically similar to the parental cells that are present in the tissues. We have compared the phenotype of cells in vivo with cells derived from the PL and expanded in vitro to assess the general validity of in vitro models for the study of phenotypic regulation in vivo. Rat PL cells were isolated by either scraping the root of the extracted first mandibular molars (Group A), or by scraping the alveolar socket following extraction of first mandibular molars (Group B), or by obtaining a mixture of cells after disaggregating a block of tissue consisting of first mandibular molar, PL and the surrounding alveolar bone (Group C). Cultured cells at confluence were fixed and immunostained for alpha-smooth muscle actin (alpha-SMA), osteopontin (OPN), alkaline phosphatase (AP), or bone sialoprotein (BSP). For in vivo assessments, frontal sections of rat first mandibular molar were immunostained for alpha-SMA, OPN, AP and BSP. We examined osteogenic differentiation of cultured PL cell cultures by bone nodule-forming assays. In vivo and at all examined sites, > 68% of PL cells were immunostained for AP; approximately 50% and approximately 51% for OPN and alpha-SMA (p = 0.3), respectively, while only approximately 8% were positively stained for BSP (p < 0.01). Analysis of cultured PL cells in Groups A, B and C showed 54%, 53%, and 56% positive staining for alpha-SMA respectively; 51%, 56%, 54% for OPN; 66%, 70%, 69% for AP and 2.2%, 1.4% and 2.8% for BSP. The mean percentage of PL cells in situ stained for the different markers was similar to that of cultured PL cells (Group A approximately Group B approximately Group C in situ for p > 0.2) except for BSP which was 3 to 4 fold higher in vitro (p < 0.01). PL cell cultures treated with dexamethasone showed mineralized tissue formation for all groups (A, B, C), but no mineralized tissue formation was detected in the absence of dexamethasone. As PL cells express quantitatively similar phenotypes in vitro and in vivo, we conclude that the in vitro models used here for assessment of PL cell differentiation appear to be appropriate and are independent of the cell sampling method. Further, dexamethasone-dependent progenitors are present both on the root and bone-related sides of the PL.

Actins↗

PubMed2001

Use of conditionally immortalized mouse cardiac fibroblasts to examine the effect of mechanical stretch on alpha-smooth muscle actin.

Mechanical stretch regulates alpha-smooth muscle actin (SMA) expression in myofibroblasts but limited replication and cellular heterogeneity have hampered definitive studies in vitro. We examined the role of applied force in regulating SMA expression in conditionally immortalized cardiac fibroblast lines derived from H-2Kb-tsA58 transgenic mice. When plated in differentiating conditions (37 degrees C without interferon-gamma), transgenic myofibroblasts exhibited vimentin staining, no desmin staining and abundant SMA in well-developed stress fibers that were indistinguishable from controls. Magnetically-generated tensile forces (approximately 500 pN/cell) applied through collagen-coated magnetite beads selectively reduced SMA but not beta-actin mRNA and protein content in both cell types. The early loss of SMA was due in part to selective leakage into the cell culture medium. Depolymerization of actin filaments with cytochalasin D blocked the force-induced reduction of SMA. Cardiac fibroblast lines established from H-2Kb-tsA58 transgenic mice provide a phenotypically stable source of cells for studying the role of physical forces in regulating SMA.

Actins↗

PubMed2001

Neutrophil-mediated epithelial injury during transmigration: role of elastase.

Neutrophil-mediated injury to gut epithelium may lead to disruption of the epithelial barrier function with consequent organ dysfunction, but the mechanisms of this are incompletely characterized. Because the epithelial apical junctional complex, comprised of tight and adherens junctions, is responsible in part for this barrier function, we investigated the effects of neutrophil transmigration on these structures. Using a colonic epithelial cell line, we observed that neutrophils migrating across cell monolayers formed clusters that were associated with focal epithelial cell loss and the creation of circular defects within the monolayer. The loss of epithelial cells was partly attributable to neutrophil-derived proteases, likely elastase, because it was prevented by elastase inhibitors. Spatially delimited disruption of epithelial junctional complexes with focal loss of E-cadherin, beta-catenin, and zonula occludens 1 was observed adjacent to clusters of transmigrating neutrophils. During neutrophil transmigration, fragments of E-cadherin were released into the apical supernatant, and inhibitors of neutrophil elastase prevented this proteolytic degradation. Addition of purified leukocyte elastase also resulted in release of E-cadherin fragments, but only after opening of tight junctions. Taken together, these data demonstrate that neutrophil-derived proteases can mediate spatially delimited disruption of epithelial apical junctions during transmigration. These processes may contribute to epithelial loss and disruption of epithelial barrier function in inflammatory diseases.

Adherens Junctions↗

PubMed2001

Involvement of actin filaments and integrins in the binding step in collagen phagocytosis by human fibroblasts.

In physiological conditions, collagen degradation by fibroblasts occurs primarily via phagocytosis, an intracellular pathway that is thought to require collagen receptors and actin assembly for fibril internalization and degradation. Currently it is unclear which specific steps of collagen phagocytosis in fibroblasts involve actin filament assembly. As studies of phagocytosis in fibroblasts are complicated by the relatively slow rate of particle internalization compared to professional phagocytes, we have examined the role of collagen receptors and actin only in the initial collagen binding step. Prior to the binding of collagen-coated fluorescent beads by human gingival fibroblasts, a cell type that is avidly phagocytic in vitro, cells were treated with cytochalasin D (actin filament barbed-end capping) or swinholide A (actin dimer sequestering and severing) or latrunculin B (actin monomer sequestering). Bead binding and immunostaining of (alpha)(2)(beta)(1) and (alpha)(3)(beta)(1) integrin collagen receptors were measured by flow cytometry. After 1-3 hours of coincubation with beads, cytochalasin D or swinholide A eliminated actin filaments stained by rhodamine-phalloidin and inhibited collagen bead binding (reductions of 25% and 50%, respectively), possibly because of cell rounding and restricted interactions with beads. In contrast, latrunculin enhanced binding dose-dependently over controls (twofold at 1 microM) and induced the formation of brightly staining aggregates of actin and the retention of long cytoplasmic extensions. Latrunculin also reduced surface (beta)(1), (alpha)(2) and (alpha)(3) integrin staining up to 40% in bead-free and bead-loaded cells, indicating that latrunculin enhanced collagen receptor internalization. As determined by fluorescence recovery after photobleaching, latrunculin increased the mobility of surface-bound (beta)(1) integrin. The stimulatory effect of latrunculin on collagen bead binding was reduced to control levels by treatment with a (beta)(1) integrin inactivating antibody while a (beta)(1) integrin blocking antibody abrogated both bead binding and the latrunculin-induced stimulation. Immunoblotting of bead-associated proteins showed that latrunculin completely eliminated binding of (beta)-actin to collagen beads but did not affect (beta)(1) integrin binding. These data indicate that latrunculin-induced sequestration of actin monomers facilitates the disengagement of actin from (beta)(1) integrin receptors, increases collagen bead binding and enhances collagen receptor mobility. We suggest that these alterations increase the probability of adhesive bead-to-cell interactions.

Actins↗

PubMed2001

Cell-cell adhesion in human fibroblasts requires calcium signaling.

In connective tissues, intercellular adhesion is essential for tissue morphogenesis, development and wound healing. However, the signaling mechanisms initiated by cell-cell adhesion in fibroblasts and that regulate it are not known. In this study we tested the hypothesis that intracellular calcium signaling is required to mediate intercellular adhesion between fibroblasts. Fura-2 or fluo-3 labeled human fibroblasts were used to investigate calcium homeostasis during intercellular adhesion. After contact with suspended fibroblasts there was a rise in cytosolic free calcium ([Ca2+]i) and multiple calcium oscillations in substrate-attached cells. Antibodies against the extracellular but not the cytoplasmic domain of cadherin induced a similar calcium response, indicating that these responses were initiated by cadherin binding. As shown by the near-plasma membrane Ca2+ indicator (Fura-C18) and by confocal microscopy of fluo-3-loaded cells, [Ca2+]i transients probably originated at sites of cell-cell contact. Cell-cell adhesion was dependent on both calcium influx through membrane channels and release of Ca2+ from internal calcium stores, because the calcium channel inhibitor LaCl3 or pretreatment of cells with thapsigargin significantly inhibited (>35%) cell-cell attachment. The [Ca2+]i changes induced by cell-cell adhesion were temporally correlated with increased recruitment of intercellular junctional proteins into the cytoskeleton and movement of GFP-actin to sites of cell-cell contact. [Ca2+]i responses induced by intercellular adhesion were essential for both junctional protein recruitment and the establishment of strong cell-cell contacts, as loading cells with BAPTA/AM significantly inhibited cell-cell adhesion and recruitment of cadherins and beta-catenin to the actin cytoskeleton. Actin depolymerization by cytochalasin D dramatically reduced cell-cell adhesion and recruitment of cadherins and catenin to the actin cytoskeleton. These results demonstrate that cadherin-cadherin interaction induces [Ca2+]i transients during cell-cell adhesion in fibroblasts, and these calcium signals regulate cell-cell adhesion through remodeling of cortical actin and recruitment of cadherins and beta-catenin into intercellular junctions.

Actin Cytoskeleton↗