Extracellular Matrix Ligands Research Articles

Extracellular matrix rigidity modulates neuroblastoma cell differentiation and N-myc expression

Neuroblastoma is a pediatric malignancy characterized by tremendous clinical heterogeneity, in which some tumors are extremely aggressive while others spontaneously differentiate into benign forms. Because the degree of differentiation correlates with prognosis, and because differentiating agents such as retinoic acid (RA) have proven to decrease mortality, much effort has been devoted to identifying critical regulators of neuroblastoma differentiation in the cellular microenvironment, including cues encoded in the extracellular matrix (ECM). While signaling between tumor cells and the ECM is classically regarded to be based purely on biochemical recognition of ECM ligands by specific cellular receptors, a number of recent studies have made it increasingly clear that the biophysical properties of the ECM may also play an important role in this cross-talk. Given that RA-mediated neuroblastoma differentiation is accompanied by profound changes in cell morphology and neurite extension, both of which presumably rely upon mechanotransductive signaling systems, it occurred to us that mechanical cues from the ECM might also influence RA-mediated differentiation, which in turn might regulate clinically-relevant aspects of neuroblastoma biology. In this study, we tested this hypothesis by subjecting a series of neuroblastoma culture models to ECM microenvironments of varying mechanical stiffness and examined the regulatory role of ECM stiffness in proliferation, differentiation, and expression of tumor markers. We find that increasing ECM stiffness enhances neuritogenesis and suppresses cell proliferation. Remarkably, increasing ECM stiffness also reduces expression of N-Myc, a transcription factor involved in multiple aspects of oncogenic proliferation that is used for evaluating prognosis and clinical grading of neuroblastoma. Furthermore, the addition of RA enhances all of these effects for all ECM stiffnesses tested. Together, our data strongly support the notion that the mechanical signals from the cellular microenvironment influence neuroblastoma differentiation and do so synergistically with RA. These observations support further investigation of the role of microenvironmental mechanical signals in neuroblastoma proliferation and differentiation and suggest that pharmacological agents that modulate the underlying mechanotransductive signaling pathways may have a role in neuroblastoma therapy.

CRT-1/Calreticulin and the E3 Ligase EEL-1/HUWE1 Control Hemidesmosome Maturation in C. elegans Development

Hemidesmosomes connect the extracellular matrix (ECM) to intermediate filaments through ECM receptors and plakins (plectin and BPAG1e). They affect tissue integrity, wound healing, and carcinoma invasion. Although biochemical and time-lapse studies indicate that alpha6beta4-integrin (ECM receptor) and plectin play a central role in modulating hemidesmosome disassembly, the mechanisms controlling hemidesmosome biogenesis in vivo remain poorly understood. The nematode C. elegans provides a powerful genetic model to address this issue. We performed a genome-wide RNA interference screen in C. elegans, searching for genes that decrease the viability of a weak VAB-10A/plakin mutant. We identified 14 genes that have human homologs with predicted roles in different cellular processes. We further characterized two genes encoding the chaperone CRT-1/calreticulin and the HECT domain E3 ubiquitin ligase EEL-1/HUWE1. CRT-1 controls by as little as 2-fold the abundance of UNC-52/perlecan, an essential hemidesmosome ECM ligand. Likewise, EEL-1 fine tunes by 2-fold the abundance of myotactin, the putative hemidesmosome ECM receptor. CRT-1 and EEL-1 activities, and by extension other genes identified in our screen, are essential during embryonic development to enable hemidesmosomes exposed to mechanical tension to mature into a tension-resistant form. Our findings should help understand how hemidesmosome dynamics are regulated in vertebrate systems.

Slowdown promotes muscle integrity by modulating integrin-mediated adhesion at the myotendinous junction

The correct assembly of the myotendinous junction (MTJ) is crucial for proper muscle function. In Drosophila, this junction comprises hemi-adherens junctions that are formed upon arrival of muscles at their corresponding tendon cells. The MTJ mainly comprises muscle-specific alphaPS2betaPS integrin receptors and their tendon-derived extracellular matrix ligand Thrombospondin (Tsp). We report the identification and functional analysis of a novel tendon-derived secreted protein named Slowdown (Slow). Homozygous slow mutant larvae exhibit muscle or tendon rupture, sluggish larval movement, partial lethality, and the surviving adult flies are unable to fly. These defects result from improper assembly of the embryonic MTJ. In slow mutants, Tsp prematurely accumulates at muscle ends, the morphology of the muscle leading edge changes and the MTJ architecture is aberrant. Slow was found to form a protein complex with Tsp. This complex is biologically active and capable of altering the morphology and directionality of muscle ends. Our analysis implicates Slow as an essential component of the MTJ, crucial for ensuring muscle and tendon integrity during larval locomotion.

Vascular endothelial growth factor enhances cancer cell adhesion to microvascular endothelium in vivo

To investigate whether vascular endothelial growth factor (VEGF) enhances cancer cell adhesion to normal microvessels, we used in vivo video microscopy to measure adhesion rates of MDA‐MB‐435s human breast cancer cells and ErbB2‐transformed mouse mammary carcinomas in the postcapillary venules of rat mesentery. An individual postcapillary venule in the mesentery was injected via a glass micropipette with cancer cells either in a perfusate of mammalian Ringer solution containing 1% bovine serum albumin as a control, or with the addition of 1 nm VEGF for test measurements. Cell adhesion was measured as either the number of adherent cells or the fluorescence intensity of adherent cells in a vessel segment for ∼60 min. Our results showed that during both control and VEGF treatments, the number of adherent cells increased almost linearly with time over 60 min. The VEGF treatment increased the adhesion rates of human tumour cells and mouse carcinomas 1.9‐fold and 1.8‐fold, respectively, over those in control conditions. We also measured cancer cell adhesion after pretreatment of cells with an antibody blocking VEGF or an antibody blocking α6 integrin, and pretreatment of the microvessel with VEGF receptor (KDR/Flk‐1) inhibitor, SU1498, or anti‐integrin extracellular matrix ligand antibody, anti‐laminin‐5. All antibodies and inhibitor significantly reduced adhesion, with anti‐VEGF and SU1498 reducing it the most. Our results indicate that VEGF enhances cancer cell adhesion to the normal microvessel wall, and further suggest that VEGF and its receptor, KDR/Flk‐1, as well as integrins of tumour cells and their ligands at the endothelium, contribute to mammary cancer cell adhesion to vascular endothelium in vivo.

Changes in cell migration‐related molecules expressed by thymic microenvironment during experimental Plasmodium berghei infection: consequences on thymocyte development

We previously showed alterations in the thymus during experimental infection with Plasmodium berghei. Such alterations comprised histological changes, with loss of cortical-medullary limits, and the intrathymic presence of parasites. As the combination of chemokines, adhesion molecules and extracellular matrix (ECM) is critical to appropriate thymocyte development, we analysed the thymic expression of ECM ligands and receptors, as well as chemokines and their respective receptors during the experimental P. berghei infection. Increased expression of ECM components was observed in thymi from infected mice. In contrast, down-regulated surface expression of fibronectin and laminin receptors was observed in thymocytes from these animals. Moreover, in thymi from infected mice there was increased CXCL12 and CXCR4, and a decreased expression of CCL25 and CCR9. An altered thymocyte migration towards ECM elements and chemokines was seen when the thymi from infected mice were analysed. Evaluation of ex vivo migration patterns of CD4/CD8-defined thymocyte subpopulations revealed that double-negative (DN), and CD4(+) and CD8(+) single-positive (SP) cells from P. berghei-infected mice have higher migratory responses compared with controls. Interestingly, increased numbers of DN and SP subpopulations were found in the spleens of infected mice. Overall, we show that the thymic atrophy observed in P. berghei-infected mice is accompanied by thymic microenvironmental changes that comprise altered expression of thymocyte migration-related molecules of the ECM and chemokine protein families, which in turn can alter the thymocyte migration pattern. These thymic disturbances may have consequences for the control of the immune response against this protozoan.

Dynamic regulation of ROCK in tumor cells controls CXCR4-driven adhesion events

CXCR4 is a chemokine receptor often found aberrantly expressed on metastatic tumor cells. To investigate CXCR4 signaling in tumor cell adhesion, we stably overexpressed CXCR4 in MCF7 breast tumor cells. Cell attachment assays demonstrate that stimulation of the receptor with its ligand, CXCL12, promotes adhesion of MCF7-CXCR4 cells to both extracellular matrix and endothelial ligands. To more closely mimic the conditions experienced by a circulating tumor cell, we performed the attachment assays under shear stress conditions. We found that CXCL12-induced tumor cell attachment is much more pronounced under flow. ROCK is a serine/threonine kinase associated with adhesion and metastasis, which is regulated by CXCR4 signaling. Thus, we investigated the contribution of ROCK activity during CXC12-induced adhesion events. Our results demonstrate a biphasic regulation of ROCK in response to adhesion. During the initial attachment, inhibition of ROCK activity is required. Subsequently, re-activation of ROCK activity is required for maturation of adhesion complexes and enhanced tumor cell migration. Interestingly, CXCL12 partially reduces the level of ROCK activity generated by attachment, which supports a model in which stimulation with CXCL12 regulates tumor cell adhesion events by providing an optimal level of ROCK activity for effective migration.

Finite Element Modeling of Cell-Matrix Adhesive Interaction

Finite Element Modeling of Cell-Matrix Adhesive InteractionWhen cells adhere to extracellular matrix (ECM) or other bioactive surfaces (substrates), the bond formation is mediated by the bindings of cell receptors, which can diffuse along the cell membrane surface, to immobilized ligands in ECM. Cells spread and the adhesion zone grows as bond formation at the adhesion front increases to a critical level. This process consists of multiple physical and chemical mechanisms and involves the coupling of reaction-diffusion and mechanical contact between cells and ECM. In this study, we have developed a finite element code to incorporate the kinetics of receptor-ligand interaction into the mass diffusion of cell receptor. For the mechanical interactions attributed to the cell adhesion development and spreading, this code is implemented in a commercial finite element program, through features of user subroutine provided, using its coupled diffusion-displacement solver. This can take into account the fully coupling of reaction-diffusion with mechanics of cell/substrate contact and deformation. In the finite element model, interaction forces between cell and substrate include the specific attraction due to the receptor-ligand binding and the nonspecific repulsion due to glycocalyx proteins associated with cell surface. Parametric studies are also performed to investigate effect of various system parameters, such as ligand density and mechanical properties of the receptor-ligand interaction, on the kinetics and mechanics of cell adhesion process. More importantly, this study provides a computational framework, with multi-scales and multi-physics, that can be extended for better controlling of cell interactions at the cell-biomaterial interface and for modeling the cell motility.

Integrin αIIbβ3 Activation in Chinese Hamster Ovary Cells and Platelets Increases Clustering Rather than Affinity

Integrin alphaIIbbeta3 affinity regulation by talin binding to the cytoplasmic tail of beta3 is a generally accepted model for explaining activation of this integrin in Chinese hamster ovary cells and human platelets. Most of the evidence for this model comes from the use of multivalent ligands. This raises the possibility that the activation being measured is that of increased clustering of the integrin rather than affinity. Using a newly developed assay that probes integrins on the surface of cells with only monovalent ligands prior to fixation, I do not find increases in affinity of alphaIIbbeta3 integrins by talin head fragments in Chinese hamster ovary cells, nor do I observe affinity increases in human platelets stimulated with thrombin. Binding to a multivalent ligand does increase in both of these cases. This assay does report affinity increases induced by either Mn(2+), a cytoplasmic domain mutant (D723R) in the cytoplasmic domain of beta3, or preincubation with a peptide ligand. These results reconcile the previously observed differences between talin effects on integrin activation in Drosophila and vertebrate systems and suggest new models for talin regulation of integrin activity in human platelets.

Matrix mechanics and receptor–ligand interactions in cell adhesion

Cell adhesions to both soluble and insoluble extracellular matrix ligands are critical in inter and intra-cellular signaling that mediates numerous physiological processes. These adhesions are complex structures composed of many scaffolding and signaling proteins. There are four distinct types of cell-matrix adhesions: focal complexes, focal adhesions, fibrillar adhesions, and 3D cell-matrix adhesions, which vary in composition, organization and function. The primary mediators of cell-matrix adhesions are integrins, which are mechanosensitive transmembrane receptor proteins that directly bind to matrix ligands to initiate adhesion formation. The development of cell-matrix adhesions is affected by a number of factors including matrix properties such as dimensionality and rigidity, and forces, both internally and externally generated, exerted on the adhesion sites. In this article, we discuss how matrix mechanics and forces affect the assembly and maturation of cell-matrix adhesions.

Black Tea Polyphenol (Theaflavin) Downregulates MMP-2 in Human Melanoma Cell Line A375 by Involving Multiple Regulatory Molecules

The tumor-inhibiting property of black tea polyphenol, theaflavin, is well documented. Matrix metalloproteinases (MMPs) play a pivotal role in tumor invasion through degradation of extracellular matrix (ECM). In the present study, we observed the effect of theaflavin on MMP-2, which is upregulated in most tumor types, and its regulatory molecules, in human melanoma cell line, A375. The treatment of theaflavin downregulated the gelatinolytic activity, mRNA and protein expression of MMP-2. It reduced the mRNA and protein expression of membrane type-1 MMP (MT1-MMP) and induced mRNA and protein expression of tissue inhibitor of MMP-2 (TIMP-2), suggesting theaflavin's inhibitory effect on MMP-2 activation. Theaflavin reduced the binding of A375 cell to ECM ligands demonstrating that theaflavin treatment hinders cell-ECM adhesion, cell motility, and integrin-mediated MMP-2 activation. Theaflavin treatment inhibited the protein expression FAK EGFR and ERK, suggesting that, theaflavin treatment downregulates the molecules participating in MMP-2 secretion and regulation. The downregulation of NFchiB suggests downregulation of MMP-2 transactivation. Theaflavin also reduced the tumor volume in syngenic black mice. Thus, we report that theaflavin causes an inhibition of the expression and activity of pro-MMP-2 by a process involving multiple regulatory molecules in human melanoma cells, A375.

Analysis of Single Integrin Behavior in Living Cells

Integrins are trans-plasma membrane receptors that mediate linkages between the extracellular matrix (ECM) and the actin cytoskeleton. In migrating cells, integrins cluster into focal adhesions (FAs). Integrin binding to ECM involves induced conformational changes that increase the affinity of the extracellular domains to ECM ligands, while indirect protein-protein interactions in FAs mediate cytoskeletal linkages. How the motion of individual integrin molecules in the plasma membrane relates to integrin activation, ECM or cytoskeletal binding, and FA formation is unknown. To address these questions, we analyzed the dynamics of single integrin molecules in migrating U2OS osteosarcoma cells. Cells expressing integrin αv or a farnesylated peptide (CAAX) fused to the photoconvertible fluorescent protein, tdEos, were analyzed by single particle tracking photoactivation localization microscopy (SPT-PALM). We imaged a high density of single photoconverted tdEos molecules by total internal reflection fluorescence microscopy (TIRFM) and tracked their trajectories using a globally-optimal SPT algorithm. Analysis of CAAX trajectories revealed that a majority of molecules (84%) exhibited Brownian diffusive behavior with 7% showing confined diffusion (D = 2.5-3×10−13 m2/s). In contrast, a majority of integrin αv trajectories exhibited confined diffusive behavior (74% confined, 21% Brownian; D = 2-5×10−14 m2/s). Image segmentation allowed classification of trajectories inside and outside FAs. Surprisingly, this showed that the proportion of confined or Brownian diffusive behavior was independent of whether molecules were inside or outside FAs. Analysis of integrin αv molecules specifically within FA showed that treatment with Mn2+ to induce integrin activation reduced the confinement radius of diffusion, whereas disruption of the actin cytoskeleton with latrunculin-A significantly increased the confinement radius. These results indicate that integrins can be immobilized without clustering, but can also remain mobile while clustered within FAs. In addition, both activation and cytoskeletal connection contribute to reduced integrin mobility within FAs.

The use of surface modified poly(glycerol-co-sebacic acid) in retinal transplantation

Retinal transplantation experiments have advanced considerably during recent years, but remaining diseased photoreceptor cells in the host retina and inner retinal cells in the transplant physically obstruct the development of graft-host neuronal contacts which are required for vision. Recently, we developed methods for the isolation of donor photoreceptor layers in vitro, and the selective removal of host photoreceptors in vivo using biodegradable elastomeric membranes composed of poly(glycerol-co-sebacic acid) (PGS). Here, we report the surface modification of PGS membranes to promote the attachment of photoreceptor layers, allowing the resulting composite to be handled surgically as a single entity. PGS membranes were chemically modified with peptides containing an arginine-glycine-aspartic acid (RGD) extracellular matrix ligand sequence. PGS membranes were also coated with electrospun nanofiber meshes, containing laminin and poly(epsilon-caprolactone) (PCL). Following in vitro co-culture of biomaterial membranes with isolated embryonic retinal tissue, composites were tested for surgical handling and examined with hematoxylin and eosin staining and immunohistochemical markers. Electrospun nanofibers composed of laminin and PCL promoted sufficient cell adhesion for simultaneous transplantation of isolated photoreceptor layers and PGS membranes. Composites developed large populations of recoverin and rhodopsin labeled photoreceptors. Furthermore, ganglion cells, rod bipolar cells and AII amacrine cells were absent in co-cultured retinas as observed by neurofilament, PKC and parvalbumin labeling respectively. These results facilitate retinal transplantation experiments in which a composite graft composed of a biodegradable membrane adhered to an immature retina dominated by photoreceptor cells may be delivered in a single surgery, with the possibility of improving graft-host neuronal connections.

The integrin adhesion complex changes its composition and function during morphogenesis of an epithelium

Cell adhesion to the extracellular matrix (ECM) is mediated by the integrin family of transmembrane receptors. Integrins link ECM ligands to the cytoskeleton, providing strong attachment to enable cell-shape change and tissue integrity. This connection is made possible by an intracellular complex of proteins, which links to actin filaments and controls signalling cascades that regulate cytoskeletal rearrangements. We have identified stress-fibre-associated focal adhesions that change their composition during tissue morphogenesis. Early expression of alphaPS1betaPS integrin decreases the levels of the actin-nucleating factors Enabled, Diaphanous and profilin, as well as downregulating the amount of F-actin incorporated into the stress fibres. As follicle cells mature in their developmental pathway and become squamous, the integrin in the focal adhesions changes from alphaPS1betaPS to alphaPS2betaPS. During the switch, stress fibres increase their length and change orientation, first changing by 90 degrees and then reorienting back. The normal rapid reorientation requires new expression of alphaPS2betaPS, which also permits recruitment of the adaptor protein tensin. Unexpectedly, it is the extracellular portion of the alphaPS2 subunit that provides the specificity for intracellular recruitment of tensin. Molecular variation of the integrin complex is thus a key component of developmentally programmed morphogenesis.

Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.

Up‐regulation of oligodendrocyte precursor cell αV integrin and its extracellular ligands during central nervous system remyelination

To determine the role of extracellular matrix molecules and their integrin ligands in CNS remyelination, we have examined in experimentally induced focal demyelinated lesions the expression of the two classes of integrins implicated in oligodendrocyte development and myelination: alpha6 laminin-binding integrins and alphaV integrins that bind a range of extracellular matrix proteins containing the -Arg-Gly-Asp- (RGD) recognition sequence. Only alphaV integrins were up-regulated during remyelination, being expressed on oligodendrocyte precursor cells during their recruitment into the lesion. Next, therefore, we examined the expression of extracellular matrix ligands for alphaV integrins and documented increased expression of tenascin-C, tenascin-R, fibronectin, and vitronectin. Taken together with our previous discovery of high levels of expression of another alphaV ligand, osteopontin, during remyelination in these lesions, our findings suggest that alphaV integrins make an important contribution to successful repair in the CNS.

The Direct Binding of Insulin-like Growth Factor-1 (IGF-1) to Integrin αvβ3 Is Involved in IGF-1 Signaling

It has been proposed that ligand occupancy of integrin alphavbeta3 with extracellular matrix ligands (e.g. vitronectin) plays a critical role in insulin-like growth factor-1 (IGF-1) signaling. We found that expression of alphavbeta3 enhanced IGF-1-induced proliferation of Chinese hamster ovary cells in serum-free conditions (in the absence of vitronectin). We hypothesized that the direct integrin binding to IGF-1 may play a role in IGF-1 signaling. We demonstrated that alphavbeta3 specifically and directly bound to IGF-1 in cell adhesion, enzyme-linked immunosorbent assay-type binding, and surface plasmon resonance studies. We localized the amino acid residues of IGF-1 that are critical for integrin binding by docking simulation and mutagenesis. We found that mutating two Arg residues at positions 36 and 37 in the C-domain of IGF-1 to Glu (the R36E/R37E mutation) effectively reduced integrin binding. Interestingly, although the mutant still bound to IGF1R, it was defective in inducing IGF1R phosphorylation, AKT and ERK1/2 activation, and cell proliferation. Furthermore wild type IGF-1 mediated co-precipitation of alphavbeta3 and IGF1R, whereas the R36E/R37E mutant did not, suggesting that IGF-1 mediates the interaction between alphavbeta3 and IGF1R. These results suggest that the direct binding to IGF-1 to integrin alphavbeta3 plays a role in IGF-1 signaling through ternary complex formation (alphavbeta3-IGF-IGF1R), and integrin-IGF-1 interaction is a novel target for drug discovery.

Fibronectin is a good catch for integrin

Kong et al. find a touch of romance in the engagement between integrin adhesion receptors and their extracellular matrix ligands. The more life tries to pull them apart, the more they want to stay together. As a cell crawls along, integrin heterodimers bind extracellular matrix molecules such as

Agonizing Integrin Antagonists?

A recent study published in Nature Medicine reports that low-dose treatment with RGD-mimetic integrin inhibitors may paradoxically enhance angiogenesis and tumor growth. This work implies that delivery of these agents should be redesigned in order to avoid nanomolar plasma concentrations and to improve their efficacy to treat human cancers.

Loss of α-Dystroglycan Laminin Binding in Epithelium-derived Cancers Is Caused by Silencing of LARGE

The interaction between epithelial cells and the extracellular matrix is crucial for tissue architecture and function and is compromised during cancer progression. Dystroglycan is a membrane receptor that mediates interactions between cells and basement membranes in various epithelia. In many epithelium-derived cancers, β-dystroglycan is expressed, but α-dystroglycan is not detected. Here we report that α-dystroglycan is correctly expressed and trafficked to the cell membrane but lacks laminin binding as a result of the silencing of the like-acetylglucosaminyltransferase (LARGE) gene in a cohort of highly metastatic epithelial cell lines derived from breast, cervical, and lung cancers. Exogenous expression of LARGE in these cancer cells restores the normal glycosylation and laminin binding of α-dystroglycan, leading to enhanced cell adhesion and reduced cell migration in vitro. Our findings demonstrate that LARGE repression is responsible for the defects in dystroglycan-mediated cell adhesion that are observed in epithelium-derived cancer cells and point to a defect of dystroglycan glycosylation as a factor in cancer progression.

Diphenylhydantoin plays a role in gene expression related to cytoskeleton and protein adhesion in human normal palate fibroblasts

Morphogenetic processes during palate development are related to extracellular matrix composition. The cell-extracellular matrix relation plays a role in cell activity and in gene expression. We studied the effect of diphenylhydantoin, a teratogen known to induce cleft palate in human newborns, on extracellular matrix production. We investigated whether diphenylhydantoin treatment caused any differences in glycosaminoglycans, collagen synthesis and gene expression in human normal palate fibroblasts. Human palate fibroblasts were maintained for 24 hours in serum-free 199 medium containing 5 microg/mL (3)H-glucosamine or (3)H proline hydrochloride. Collagen and glycosaminoglycan classes were then measured using biochemical methods, gene expression with microarray analysis and cytoskeleton components with immunofluorescent antibodies and computer analysis. In normal fibroblasts diphenylhydantoin reduced collagen and glycosaminoglycan synthesis with a marked effect on sulphated glycosaminoglycans. There were also substantial decreases in tubulin, vimentin and alpha-actin staining and an increase of vinculin compared to controls. Diphenylhydantoin acted on several genes related to the synthesis of cytoskeleton and adhesion membrane proteins. It inhibited caderin, caveolin, RTK and alpha-actin, and increased nectin, cytoplasmatic FRG vinculin, ITGA, ITGB extracellular matrix ligand and EDG2 gene expression. DNA binding gene expression, which plays a role in cell growth and senescence, was activated. Since cell activity is dependent on the cell morphology and extracellular matrix composition, these findings indicate that in human normal palate fibroblasts diphenylhydantoin can modify cytoskeletal components and extracellular matrix-cell adhesion, with consequent effects on gene expression. These changes might be related to anomalous palate development.