Epithelial Cell Entry Research Articles

Crystal structure of the Epstein-Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) complex.

The Epstein-Barr virus (EBV) is a γ-herpesvirus that infects B cells and epithelial cells and that has been linked to malignancies in both cell types in vivo. EBV, like other herpesviruses, has three glycoproteins, glycoprotein B (gB), gH, and gL, that form the core membrane fusion machinery mediating viral penetration into the cell. The gH and gL proteins associate to form a heterodimeric complex, which is necessary for efficient membrane fusion and also implicated in direct binding to epithelial cell receptors required for viral entry. To gain insight into the mechanistic role of gH/gL, we determined the crystal structure of the EBV gH/gL complex. The structure is comprised of four domains organized along the longest axis of the molecule. Comparisons with homologous HSV-2 gH/gL and partial pseudorabies virus gH structures support the domain boundaries determined for the EBV gH/gL structure and illustrate significant differences in interdomain packing angles. The gL subunit and N-terminal residues of gH form a globular domain at one end of the structure, implicated in interactions with gB and activation of membrane fusion. The C-terminal domain of gH, proximal to the viral membrane, is also implicated in membrane fusion. The gH/gL structure locates an integrin binding motif, implicated in epithelial cell entry, on a prominent loop in the central region of the structure. Multiple regions of gH/gL, including its two extreme ends, are functionally important, consistent with the multiple roles of gH/gL in EBV entry.

Update on the current status of cytomegalovirus vaccines

Human cytomegalovirus (HCMV) is ubiquitous in all populations, and is the most commonly recognized cause of congenital viral infection in developed countries. On the basis of the economic costs saved and the improvement in quality of life that could potentially be conferred by a successful vaccine for prevention of congenital HCMV infection, the Institute of Medicine has identified HCMV vaccine development as a major public health priority. An effective vaccine could potentially also be beneficial in preventing or ameliorating HCMV disease in immunocompromised individuals. Although there are no licensed HCMV vaccines currently available, enormous progress has been made in the last decade, as evidenced by the recently reported results of a Phase II trial of a glycoprotein B vaccine for the prevention of HCMV infection in seronegative women of childbearing age. HCMV vaccines currently in clinical trials include: glycoprotein B subunit vaccines; alphavirus replicon particle vaccines; DNA vaccines; and live-attenuated vaccines. A variety of vaccine strategies are also being examined in preclinical systems and animal models of infection. These include: recombinant vesicular stomatitis virus vaccines; recombinant modified vaccinia virus Ankara; replication-deficient adenovirus-vectored vaccines; and recombinant live-attenuated virus vaccines generated by mutagenesis of cloned rodent CMV genomes maintained as bacterial artificial chromosomes in Escherichia coli. In this article, we provide an overview of the current state of clinical trials and preclinical development of vaccines against HCMV, with an emphasis on studies that have been conducted in the past 5 years. We also summarize a number of recent advances in the study of the biology of HCMV, particularly with respect to epithelial and endothelial cell entry of the virus, which have implications for future vaccine design.

CFTR is required for cellular entry and internalization of Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular Gram-negative pathogen affecting over 600 million people worldwide with 92 million new cases occurring globally each year. C. trachomatis enter the cells and replicate to infect different tissues/organs, giving rise to a spectrum of pathological conditions; however, the exact mechanism or receptor(s) for their entry is not well understood. Here we report that CFTR (cystic fibrosis transmembrane conductance regulator), an apical epithelial anion channel, is required for cellular entry and internalization of C. trachomatis. Human epithelial cell lines expressing functional CFTR internalized more C. trachomatis than the cells expressing mutant Delta508 CFTR. The in vitro cellular uptake of C. trachomatis can be blocked by CFTR inhibitors or antibody, and the in vivo cellular uptake of C. trachomatis in CFTR mutant (CFTR(-/-)) mice was significantly less compared with that in the wild-type. Direct interaction between CFTR and C. trachomatis LPS (lipopolysaccharide) is demonstrated by their immune-co-localization and co-immunoprecipitation. Despite an increase in CFTR expression observed upon C. trachomatis LPS challenge, a reduction in its ion channel activity is observed, consistent with the notion that CFTR functions as a receptor for cellular entry and internationization of C. trachomatis, with compromised ion-channel function. These findings, for the first time, demonstrate that CFTR functions as a cell-surface receptor for epithelial cell entry, and internalization of C. trachomatis and these findings may lead to the development of new treatment strategies to curtail the spread of chlamydial infections.

Polychlorinated biphenyl induced ROS signaling delays the entry of quiescent human breast epithelial cells into the proliferative cycle

Polychlorinated biphenyls (PCBs) are environmental chemical contaminants that can produce reactive oxygen species (ROS) by autoxidation of dihydroxy-PCBs and redox-cycling. We investigate the hypothesis that PCB induced perturbations in ROS signaling regulate the entry of quiescent cells into the proliferative cycle. Quiescent MCF-10A human breast epithelial cells were incubated with 0-3 micromolar of 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), 2, 2′, 4, 4′, 5, 5′-hexachlorobiphenyl (PCB 153), and Aroclor 1254 for 4 days. Cells were replated at a lower density and analyzed for cell cycle phase distributions, ROS levels, MnSOD expression, and cyclin D1 protein levels. Quiescent cells incubated with 4-Cl-BQ showed the maximal delay in entering S phase. This delay was associated with a decrease in MnSOD activity, protein and mRNA levels, and an increase in cellular ROS levels. Results from the mRNA turnover assay showed that the 4-Cl-BQ treatment selectively enhanced the degradation of the 4.2 kb MnSOD transcript, while the half-life of the 1.5 kb transcript did not change. Accumulation of cyclin D1 protein levels in replated cells was suppressed in cells treated with 4-Cl-BQ. Pretreatment of quiescent cells with polyethylene glycol-conjugated superoxide dismutase and catalase suppressed 4-Cl-BQ induced increase in ROS levels, which was consistent with an increase in cyclin D1 accumulation, and entry into S phase. These results showed 4-Cl-BQ induced perturbations in ROS signaling inhibit the entry of quiescent cells into S phase.

Palm domains of the epithelial sodium channel and sodium self‐inhibition

Epithelial Na+ channels (ENaC) play an essential role in body fluid volume homeostasis by mediating apical Na+ entry in epithelial cells. The structure of chicken acid sensing ion channel 1 (cASIC1) homologous to ENaCs reveals that the palm domain offers the sole link between the extracellular domain and the channel pore within a subunit. We investigated the functional roles of the two charged residues within a palm domain β‐sheet (β12) in the mechanism of Na+ self‐inhibition by site‐directed mutagenesis. The Na+ self‐inhibition responses for wild type and mutant channels expressed in Xenopus oocytes were examined by two electrode voltage clamp. Charge reversal mutations at a homologous site (αK561E and γR520E) of β12 significantly reduced the magnitude and speed of Na+ self‐inhibition response. The Na+ inhibition was absent in oocytes expressing the double mutant channel (αK561E βγR520E). Mutation of the other charged residue in γ subunit (γE524R) also significantly suppressed Na+ self‐inhibition whereas the homologous mutation in α(α E565R) did not alter the inhibition. Our results suggest that certain charged residues in αand γpalm domains are critical determinants for Na+ self‐inhibition. Based on our observations and the insights from the cASIC1 structure, we propose that the palm domains play a pivotal role in Na+ self‐inhibition of ENaC. (Supported by NIH ES14701, DK54354 and DK079307)

Regulation of calcium entry in exocrine gland cells and other epithelial cells

Regulation of calcium entry in exocrine gland cells and other epithelial cells

Joint Requirement for Rac and ERK Activities Underlies the Mid-G1 Phase Induction of Cyclin D1 and S Phase Entry in Both Epithelial and Mesenchymal Cells

Cyclin D1 gene induction is a key event in G1 phase progression. Our previous studies indicated that signaling to cyclin D1 is cell type-dependent because the timing of cyclin D1 gene expression in MCF10A mammary epithelial cells and mesenchymal cells such as fibroblasts and vascular smooth muscle cells is very different, with epithelial cells first expressing cyclin D1 in early rather than mid-G1 phase. In this report, we induced a mesenchymal phenotype in MCF10A cells by long-term exposure to TGF-beta and used the control and transitioned cells to examine cell type specificity of the signaling pathways that regulate cyclin D1 gene expression. We show that early-G1 phase cyclin D1 gene expression in MCF10A cells is under the control of Rac, whereas mid-G1 phase cyclin D1 induction requires parallel signaling from Rac and ERK, both in the control and transitioned cells. This combined requirement for Rac and ERK signaling is associated with an increased requirement for intracellular tension, Rb phosphorylation, and S phase entry. A similar co-regulation of cyclin D1 mRNA by Rac and ERK is seen in primary mesenchymal cells. Overall, our results reveal two mechanistically distinct phases of Rac-dependent cyclin D1 expression and emphasize that the acquisition of Rac/ERK co-dependence is required for the mid-G1 phase induction of cyclin D1 associated with S phase entry.

Role for nectin‐1 in herpes simplex virus 1 entry and spread in human retinal pigment epithelial cells

Herpes simplex virus 1 (HSV-1) demonstrates a unique ability to infect a variety of host cell types. Retinal pigment epithelial (RPE) cells form the outermost layer of the retina and provide a potential target for viral invasion and permanent vision impairment. Here we examine the initial cellular and molecular mechanisms that facilitate HSV-1 invasion of human RPE cells. High-resolution confocal microscopy demonstrated initial interaction of green fluorescent protein (GFP)-tagged virions with filopodia-like structures present on cell surfaces. Unidirectional movement of the virions on filopodia to the cell body was detected by live cell imaging of RPE cells, which demonstrated susceptibility to pH-dependent HSV-1 entry and replication. Use of RT-PCR indicated expression of nectin-1, herpes virus entry mediator (HVEM) and 3-O-sulfotransferase-3 (as a surrogate marker for 3-O-sulfated heparan sulfate). HVEM and nectin-1 expression was subsequently verified by flow cytometry. Nectin-1 expression in murine retinal tissue was also demonstrated by immunohistochemistry. Antibodies against nectin-1, but not HVEM, were able to block HSV-1 infection. Similar blocking effects were seen with a small interfering RNA construct specifically directed against nectin-1, which also blocked RPE cell fusion with HSV-1 glycoprotein-expressing Chinese hamster ovary (CHO-K1) cells. Anti-nectin-1 antibodies and F-actin depolymerizers were also successful in blocking the cytoskeletal changes that occur upon HSV-1 entry into cells. Our findings shed new light on the cellular and molecular mechanisms that help the virus to enter the cells of the inner eye.

TRPV4 channel participates in receptor-operated calcium entry and ciliary beat frequency regulation in mouse airway epithelial cells

The rate of mucociliary clearance in the airways is a function of ciliary beat frequency (CBF), and this, in turn, is increased by increases in intracellular calcium. The TRPV4 cation channel mediates Ca(2+) influx in response to mechanical and osmotic stimuli in ciliated epithelia. With the use of a TRPV4-deficient mouse, we now show that TRPV4 is involved in the airways' response to physiologically relevant physical and chemical stimuli. Ciliary TRPV4 expression in tracheal epithelial cells was confirmed with immunofluorescence in TRPV4(+/+) mice. Ciliated tracheal cells from TRPV4(-/-) mice showed no increases in intracellular Ca(2+) and CBF in response to the synthetic activator 4alpha-phorbol 12,13-didecanoate (4alphaPDD) and reduced responses to mild temperature, another TRPV4-activating stimulus. Autoregulation of CBF in response to high viscosity solutions is preserved in TRPV4(-/-) despite a reduced Ca(2+) signal. More interestingly, TRPV4 contributed to an ATP-induced increase in CBF, providing a pathway for receptor-operated Ca(2+) entry but not store-operated Ca(2+) entry as the former mechanism is lost in TRPV4(-/-) cells. Collectively, these results suggest that TRPV4 is predominantly located in the cilia of tracheal epithelial cells and plays a key role in the transduction of physical and chemical stimuli into a Ca(2+) signal that regulates CBF and mucociliary transport. Moreover, these studies implicate the participation of TRPV4 in receptor-operated Ca(2+) entry.

Cytomegalovirus vaccines fail to induce epithelial entry neutralizing antibodies comparable to natural infection

Antibodies that neutralize cytomegalovirus (CMV) entry into fibroblasts are predominantly directed against epitopes within virion glycoproteins that are required for attachment and entry. However, the mechanism of CMV entry into epithelial and endothelial cells differs from fibroblast entry. Using assays that simultaneously measured neutralizing activities against CMV entry into fibroblasts and epithelial cells, we found that human immune sera and CMV-hyperimmuneglobulins have on on average 48-fold higher neutralizing activities against epithelial cell entry compared to fibroblast entry, suggesting that natural CMV infections elicit neutralizing antibodies that are epithelial entry-specific. This activity could not be adsorbed with recombinant gB. The Towne vaccine and the gB/MF59 subunit vaccine induced epithelial entry-specific neutralizing activities that were on on average 28-fold (Towne) or 15-fold (gB/MF59) lower than those observed following natural infection. These results suggest that CMV vaccine efficacy may be enhanced by the induction of epithelial entry-specific neutralizing antibodies.

Peptides derived from the Mycobacterium tuberculosis Rv1490 surface protein implicated in inhibition of epithelial cell entry: Potential vaccine candidates?

This study reports the Rv1490 gene presence and transcription in members of the Mycobacterium tuberculosis complex, and characterises the encoded Rv1490 putative membrane protein in M. tuberculosis H37Rv. Rv1490 derived peptides were synthesised and their A549 and U937 cell binding ability was tested, finding five high activity binding peptides (HABPs) for A549 and five for U937. Only two HABPs (11060 and 11073) were shared by both cell lines, both of which affected M. tuberculosis’ invading ability to target cells, thus indicating an important role for these sequences in M. tuberculosis entry to A549 alveolar epithelial cells and supporting their inclusion in further studies on the development of a subunit-based multi-epitopic, chemically synthesised anti-tuberculosis vaccine.

Role of the Clp System in Stress Tolerance, Biofilm Formation, and Intracellular Invasion inPorphyromonas gingivalis

Clp proteases and chaperones are ubiquitous among prokaryotes and eukaryotes, and in many pathogenic bacteria the Clp stress response system is also involved in regulation of virulence properties. In this study, the roles of ClpB, ClpC, and ClpXP in stress resistance, homotypic and heterotypic biofilm formation, and intracellular invasion in the oral opportunistic pathogen Porphyromonas gingivalis were investigated. Absence of ClpC and ClpXP, but not ClpB, resulted in diminished tolerance to high temperatures. Response to oxidative stress was not affected by the loss of any of the Clp proteins. The clpC and clpXP mutants demonstrated elevated monospecies biofilm formation, and the absence of ClpXP also enhanced heterotypic P. gingivalis-Streptococcus gordonii biofilm formation. All clp mutants adhered to gingival epithelial cells to the same level as the wild type; however, ClpC and ClpXP were found to be necessary for entry into host epithelial cells. ClpB did not play a role in entry but was required for intracellular replication and survival. ClpXP negatively regulated the surface exposure of the minor fimbrial (Mfa) protein subunit of P. gingivalis, which stimulates biofilm formation but interferes with epithelial cell entry. Collectively, these results show that the Clp protease complex and chaperones control several processes that are important for the colonization and survival of P. gingivalis in the oral cavity.

Basal Calcium Entry in Retinal Pigment Epithelial Cells Is Mediated by TRPC Channels

Ca(2+) is a major regulator of cell function. In the retinal pigment epithelium (RPE), intracellular free Ca(2+) concentration ([Ca(2+)](i)) is essential for the maintenance of normal retinal function. Therefore, accurate control of [Ca(2+)](i) is vital in these cells. Because Ca(2+) is permanently extruded from the cytosol, RPE cells need a basal Ca(2+) entry pathway that counteracts this Ca(2+) efflux. The purpose of this study was to identify the molecular basis of basal Ca(2+) entry into the RPE. [Ca(2+)](i) was measured using Fura-2-loaded ARPE-19 cells. The expression pattern of TRPC channels was investigated by RT-PCR with RNA extracted from ARPE-19 cells and freshly isolated RPE cells from human donor eyes. In most cells, basal [Ca(2+)](i) is highly controlled by cell membranes that are only slightly permeable to Ca(2+) and by the activity of Ca(2+) pumps and transporters. The authors show here that RPE cells have a basal Ca(2+) conductance that is dose dependently blocked by La(3+). Basal [Ca(2+)](i) was also strongly reduced by the TRP channel blockers Gd(3+), Ni(2+), 2-APB, and SKF96365 and was insensitive to blockers of other Ca(2+) channels. In confirmation of this pharmacologic profile, RPE cells expressed TRPC1 and TRPC4 channels, as shown by RT-PCR experiments. Ca(2+) is needed for several permanently occurring regulatory processes in RPE cells. The Ca(2+) influx pathway identified in this study is essential to define a resting basal [Ca(2+)](i). This resting [Ca(2+)](i) may contribute, for example, to basal cytokine secretion essential for the maintenance of normal retinal function.

584: Ex-vivo assessment of the effect of airway epithelial barrier disruption on transduction efficiency of a lentiviral vector in fetal rat tracheas

Gene therapy is contemplated as a causative treatment of cystic fibrosis. The advantages of in utero gene therapy include small target volume, presence of progenitor and stem cells, long contact time with the vector and reduced immune response. Even during fetal life conducting airways are protected by anatomical and physiological barriers comparable to postnatal life. Effective receptor-mediated entry of epithelial cells is only possible if the (viral) vector reaches their basolateral side hence after disruption of tight junctions.

Human Papillomavirus Type 31 Uses a Caveolin 1- and Dynamin 2-Mediated Entry Pathway for Infection of Human Keratinocytes

Papillomaviruses are species-specific and epitheliotropic DNA viruses that cause tumors in their natural hosts. Certain infections with genital human papillomavirus (HPV) types are causally related to cervical cancer development. Most papillomaviruses are thought to infect cells via a clathrin-dependent pathway, yet no studies have determined the entry route in permissive host epithelial cells. Employing fluorescently labeled and native virions, we tested the effects of dominant-negative and biochemical inhibitors of cellular endocytosis pathways. Infections of human keratinocytes, a natural host cell type for HPVs, were assessed visually and by infectious entry assays. We found that HPV type 31 (HPV31) entry and initiation of early infection events require both caveolin 1 and dynamin 2 and occur independently of clathrin-mediated endocytosis. Treatment with chlorpromazine and filipin had opposing effects on HPV31 and HPV16 infection. HPV31 entry was remarkably slow, with a half-time of approximately 14 h, whereas the entry half-time of HPV16 was 4 h. Consistent with a caveola-mediated entry pathway for HPV31, the virions associated with detergent-resistant lipid rafts. During a 16-h microscopic tracking of HPV31 and HPV16 virions, no colocalization of the two viral types was observed. These data suggest that HPV31 and HPV16 virions use distinct routes for host epithelial cell entry.

Humoral Immune Response to Tissue Transglutaminase Is Related to Epithelial Cell Proliferation in Celiac Disease

Tissue transglutaminase (tTG) autoantibodies are markers of celiac disease, and the enzyme is required for several crucial biological processes. The aim of this study was to determine whether these autoantibodies are involved in the pathogenesis of the mucosal lesion typical of celiac disease. Using rhodamine-conjugated phalloidin staining, we evaluated whether tTG antibodies, both commercially available and cloned from patients with celiac disease, cause cytoskeletal changes in Caco-2, MCF7, and NIH 3T3 cells. We monitored cell levels of bromodeoxyuridine incorporation to determine whether tTG autoantibodies are able to induce NIH 3T3 fibroblasts and epithelial mucosal cells into S phase. Treatment with tTG antibodies caused a dose-dependent increase of membrane ruffling in Caco-2, MCF7, and NIH 3T3 cells. It also dose-dependently induced G(0)-synchronized NIH 3T3 fibroblasts into S phase but did not affect the rate of apoptosis. Similarly, tTG antibodies induced S-phase entry of epithelial cells in cultured intestinal biopsy specimens from patients with celiac disease. They did not affect biopsy specimens from patients without celiac disease. Our results suggest that tTG autoantibodies per se, by interacting with the extracellular membrane-bound transglutaminase, may play an important role in epithelial cell proliferation in celiac disease.

Molecular links between obesity and breast cancer

For over 40 years there has been a statistical linkage between obesity and cancer, yet little is known about the molecular mechanisms underlying this long standing deleterious association. Recent work has shown that adipocyte-derived peptides can affect cell cycle entry in mammary epithelial cells. Thus we examined the paracrine role of the adipokines leptin and adiponectin on mammary epithelial cell cycle regulation. G0 synchronized MCF7 cells were treated with leptin and adiponectin, separately and in concert, in time and dose dependent experiments. Immunoblotting demonstrated that p27 protein levels decreased with leptin treatment which suggested cell cycle entry. In contrast adiponectin increased p27 levels suggesting cell cycle arrest. To complement these experiments MCF7 cells were co-cultured with subcutaneous adipocytes isolated from lean rats to mimic in vivo paracrine conditions. Moderate increases in adipocyte concentrations increased p27 levels up to a peak, mimicking the effects of adiponectin. At high adipocyte concentrations p27 levels decreased mirroring the effects of leptin. The relevance of these findings is that obese individuals exhibit high leptin levels and low adiponectin levels suggesting a stoichiometric imbalance may underlie cancer development and progression. This may also provide the basis for the development of novel cancer therapeutic and prevention strategies.

Differential Role of Transient Receptor Potential Channels in Ca2+ Entry and Proliferation of Prostate Cancer Epithelial Cells

One major clinical problem with prostate cancer is the cells' ability to survive and proliferate upon androgen withdrawal. Because Ca2+ is central to growth control, understanding the mechanisms of Ca2+ homeostasis involved in prostate cancer cell proliferation is imperative for new therapeutic strategies. Here, we show that agonist-mediated stimulation of alpha1-adrenergic receptors (alpha1-AR) promotes proliferation of the primary human prostate cancer epithelial (hPCE) cells by inducing store-independent Ca2+ entry and subsequent activation of nuclear factor of activated T cells (NFAT) transcription factor. Such an agonist-induced Ca2+ entry (ACE) relied mostly on transient receptor potential canonical 6 (TRPC6) channels, whose silencing by antisense hybrid depletion decreased both hPCE cell proliferation and ACE. In contrast, ACE and related growth arrest associated with purinergic receptors (P2Y-R) stimulation involved neither TRPC6 nor NFAT. Our findings show that alpha1-AR signaling requires the coupled activation of TRPC6 channels and NFAT to promote proliferation of hPCE cells and thereby suggest TRPC6 as a novel potential therapeutic target.

Pseudomonas aeruginosa Acquires Biofilm-Like Properties within Airway Epithelial Cells

Pseudomonas aeruginosa can notably cause both acute and chronic infection. While several virulence factors are implicated in the acute phase of infection, advances in understanding bacterial pathogenesis suggest that chronic P. aeruginosa infection is related to biofilm formation. However, the relationship between these two forms of disease is not well understood. Accumulating evidence indicates that, during acute infection, P. aeruginosa enters epithelial cells, a process viewed as either a host-mediated defense response or a pathogenic mechanism to avoid host-mediated killing. We investigated the possibility that epithelial cell entry during early P. aeruginosa-epithelial cell contact favors bacterial survival and is linked to chronic infection. Using electron microscopy and confocal microscopy to analyze primary culture airway epithelial cells infected with P. aeruginosa, we found that epithelial cells developed pod-like clusters of intracellular bacteria with regional variation in protein expression. Extracellular gentamicin added to the medium after acute infection led to the persistence of intracellular P. aeruginosa for at least 3 days. Importantly, compared to bacterial culture under planktonic conditions, the intracellular bacteria were insensitive to growth inhibition or killing by antibiotics that were capable of intraepithelial cell penetration. These findings suggest that P. aeruginosa can use airway epithelial cells as a sanctuary for persistence and develop a reversible antibiotic resistance phenotype characteristic of biofilm physiology that can contribute to development of chronic infection.

Epidermal growth factor receptor-dependent regulation of integrin-mediated signaling and cell cycle entry in epithelial cells.

Integrin-mediated adhesion of epithelial cells to extracellular matrix (ECM) proteins induces prolonged tyrosine phosphorylation and partial activation of epidermal growth factor receptor (EGFR) in an integrin-dependent and EGFR ligand-independent manner. Integrin-mediated activation of EGFR in epithelial cells is required for multiple signal transduction events previously shown to be induced by cell adhesion to matrix proteins, including tyrosine phosphorylation of Shc, Cbl, and phospholipase Cgamma, and activation of the Ras/Erk and phosphatidylinositol 3'-kinase/Akt signaling pathways. In contrast, activation of focal adhesion kinase, Src, and protein kinase C, adhesion to matrix proteins, cell spreading, migration, and actin cytoskeletal rearrangements are induced independently of EGFR kinase activity. The ability of integrins to induce the activation of EGFR and its subsequent regulation of Erk and Akt activation permitted adhesion-dependent induction of cyclin D1 and p21, Rb phosphorylation, and activation of cdk4 in epithelial cells in the absence of exogenous growth factors. Adhesion of epithelial cells to the ECM failed to efficiently induce degradation of p27, to induce cdk2 activity, or to induce Myc and cyclin A synthesis; subsequently, cells did not progress into S phase. Treatment of ECM-adherent cells with EGF, or overexpression of EGFR or Myc, resulted in restoration of late-G(1) cell cycle events and progression into S phase. These results indicate that partial activation of EGFR by integrin receptors plays an important role in mediating events triggered by epithelial cell attachment to ECM; EGFR is necessary for activation of multiple integrin-induced signaling enzymes and sufficient for early events in G(1) cell cycle progression. Furthermore, these findings suggest that EGFR or Myc overexpression may provoke ligand-independent proliferation in matrix-attached cells in vivo and could contribute to carcinoma development.