Janice L. Walker

Integrin-dependent signal transduction regulating cyclin D1 expression and G1 phase cell cycle progression.

Integrins and growth factor receptors coordinately regulate proliferation in nontransformed cells. Coordinate signaling from these receptors controls the activation of the G1 phase cyclin-dependent kinases, largely by regulating levels of cyclin D1 and p27kip1. Induction of cyclin D1 is one of the best understood examples of an integrin/growth factor receptor-regulated G1 phase target. This review focuses on the integrin-dependent signal transduction events that regulate the expression of cyclin D1 during G1 phase.

γ-Secretase Activation of Notch Signaling Regulates the Balance of Proximal and Distal Fates in Progenitor Cells of the Developing Lung

Little is known about the mechanisms by which the lung epithelial progenitors are initially patterned and how proximal-distal boundaries are established and maintained when the lung primordium forms and starts to branch. Here we identified a number of Notch pathway components in respiratory progenitors of the early lung, and we investigated the role of Notch in lung pattern formation. By preventing γ-secretase cleavage of Notch receptors, we have disrupted global Notch signaling in the foregut and in the lung during the initial stages of murine lung morphogenesis. We demonstrate that Notch signaling is not necessary for lung bud initiation; however, Notch is required to maintain a balance of proximal-distal cell fates at these early stages. Disruption of Notch signaling dramatically expands the population of distal progenitors, altering morphogenetic boundaries and preventing formation of proximal structures. Our data suggest a novel mechanism in which Notch and fibroblast growth factor signaling interact to control the proximal-distal pattern of forming airways in the mammalian lung.

Diverse Roles of E-Cadherin in the Morphogenesis of the Submandibular Gland: Insights Into the Formation of Acinar and Ductal Structures

The formation of acinar and ductal structures during epithelial tissue branching morphogenesis is not well understood. We report that in the mouse submandibular gland (SMG), acinar and ductal cell fates are determined early in embryonic morphogenesis with E‐cadherin playing pivotal roles in development. We identified two morphologically distinct cell populations at the single bud stage, destined for different functions. The outer layer of columnar cells with organized E‐cadherin junctions expressed the neonatal acinar marker B1 by E13.5, demonstrating their acinar fate. The interior cells initially lacked distinct E‐cadherin junctions, but with morphogenesis formed cytokeratin 7 (K7) ‐positive ductal structures with organized E‐cadherin junctions and F‐actin filaments. Inhibition of E‐cadherin function with either siRNA or function blocking antibody caused extensive apoptosis of ductal cells and aberrantly dilated lumens, providing the first evidence that E‐cadherin regulates ductal lumen formation during branching morphogenesis of the salivary gland. Developmental Dynamics 237:3128–3141, 2008.

Role for α6 integrin during lens development: Evidence for signaling through IGF‐1R and ERK

We show that α6 integrin function was required for normal lens cell differentiation by using an antisense construct to suppress α6 integrin expression. To elucidate the mechanism by which this integrin functions in the regulation of the lens cell differentiation process, we determined the molecular composition of α6 integrin signaling complexes at distinct stages of differentiation in vivo. Because both α6 integrin and insulin‐like growth factor‐1 (IGF‐1) have been implicated in signaling lens cell differentiation, we examined the possibility that they formed a signaling complex in the embryonic lens. Coprecipitation analysis revealed that α6 integrin/IGF‐1 receptor complexes were present and that their association was greatest in the equatorial zone, the region of the embryonic lens in which lens cells proliferate and then initiate their differentiation. These results provide in vivo support for the formation of integrin/growth factor receptor signaling complexes. We also found that extracellular signal‐regulated kinase (ERK), a downstream effector of both integrin and growth factor receptor signaling pathways, was associated with the α6 integrin signaling complexes in the embryonic lens. This result was supported by our findings that activated ERK, in addition to its nuclear location, localized to lens cell membranes in specific regions of cell‐matrix and cell–cell contact. A connection between integrin ligand engagement and ERK activation was shown in vitro after lens cell attachment to laminin. These results demonstrate that α6 integrin function is required for the early stages of lens cell differentiation most likely through its association with the IGF‐1 receptor and the activation of ERK.

MNL1 Regulates Weak Acid-induced Stress Responses of the Fungal Pathogen Candida albicans

MNL1, the Candida albicans homologue of an orphan Msn2-like gene (YER130c in Saccharomyces cerevisiae) has no known function. Here we report that MNL1 regulates weak acid stress responses. Deletion of MNL1 prevents the long-term adaptation of C. albicans cells to weak acid stresses and compromises their global transcriptional response under these conditions. The promoters of Mnl1-dependent genes contain a novel STRE-like element (SLE) that imposes Mnl1-dependent, weak acid stress-induced transcription upon a lacZ reporter in C. albicans. The SLE (HHYYCCCCTTYTY) is related to the Nrg1 response element (NRE) element recognized by the transcriptional repressor Nrg1. Deletion of NRG1 partially restores the ability of C. albicans mnl1 cells to adapt to weak acid stress, indicating that Mnl1 and Nrg1 act antagonistically to regulate this response. Molecular, microarray, and proteomic analyses revealed that Mnl1-dependent adaptation does not occur in cells exposed to proapoptotic or pronecrotic doses of weak acid, suggesting that Ras-pathway activation might suppress the Mnl1-dependent weak acid response in dying cells. Our work defines a role for this YER130c orthologue in stress adaptation and cell death.

Integrins in lens development and disease.

Integrins are the major cell surface receptors for proteins in the extracellular matrix. These receptors form major cell signaling centers that are bidirectional, communicating messages between the cell and its environment. They are a large receptor family, with members well-known to regulate cellular processes essential to both development and disease. In this review we examine the literature regarding integrins in the lens. Here we cover integrin function in lens cell differentiation, in the development of the lens and in protection of the lens epithelial cell phenotype. In addition, we analyze the role of integrins in the progression of lens fibrotic diseases, focusing particularly on integrin regulation of TGFbeta signaling pathways in posterior capsule opacification (PCO) and anterior subcapsular cataract (ASC).

A central role for vimentin in regulating repair function during healing of the lens epithelium

A unique ex vivo mock cataract surgery model is used to study the role of vimentin in repair cell function during wound healing within a clinically relevant setting. Vimentin is found to be critical for the function of repair cells in directing the collective migration of the epithelium during wound healing.

Overexpression of DPAGT1 leads to aberrant N-glycosylation of E-cadherin and cellular discohesion in oral cancer

Cancer cells are frequently characterized by aberrant increases in protein N-glycosylation and by disruption of E-cadherin-mediated adherens junctions. The relationship between altered N-glycosylation and loss of E-cadherin adhesion in cancer, however, remains unclear. Previously, we reported that complex N-glycans on the extracellular domains of E-cadherin inhibited the formation of mature adherens junctions. Here, we examined whether dysregulated N-glycosylation was one of the underlying causes for cellular discohesion in oral cancer. We show that dense cultures of human salivary epidermoid carcinoma A253 cells exhibited elevated expression of DPAGT1, the gene that initiates protein N-glycosylation. Overexpression of DPAGT1 correlated with the production of E-cadherin-bearing complex N-glycans in nascent adherens junctions. Partial inhibition of DPAGT1 with small interfering RNA reduced the complex N-glycans of E-cadherin and increased the abundance of alpha-catenin and stabilizing proteins in adherens junctions. This was associated with the assembly of functional tight junctions. The inverse relationship between DPAGT1 expression and intercellular adhesion was a feature of oral squamous cell carcinoma. Oral squamous cell carcinomas displayed overexpression of DPAGT1 that correlated with diminished localization of E-cadherin and alpha-catenin at the sites of adherens junctions. Our studies show for the first time that DPAGT1 is an upstream regulator of E-cadherin N-glycosylation status and adherens junction composition and suggest that dysregulation of DPAGT1 causes disturbances in intercellular adhesion in oral cancer.

A Skp2 autoinduction loop and restriction point control

We describe a self-amplifying feedback loop that autoinduces Skp2 during G1 phase progression. This loop, which contains Skp2 itself, p27kip1 (p27), cyclin E–cyclin dependent kinase 2, and the retinoblastoma protein, is closed through a newly identified, conserved E2F site in the Skp2 promoter. Interference with the loop, by knockin of a Skp2-resistant p27 mutant (p27T187A), delays passage through the restriction point but does not interfere with S phase entry under continuous serum stimulation. Skp2 knock down inhibits S phase entry in nontransformed mouse embryonic fibroblasts but not in human papilloma virus–E7 expressing fibroblasts. We propose that the essential role for Skp2-dependent degradation of p27 is in the formation of an autoinduction loop that selectively controls the transition to mitogen-independence, and that Skp2-dependent proteolysis may be dispensable when pocket proteins are constitutively inactivated.

Transition between proliferation and differentiation for lens epithelial cells is regulated by Src family kinases

As in many cell types, lens cells must withdraw from the cell cycle before they initiate their differentiation. The involvement of Src family kinases (SFKs) in this key initiating event in cell differentiation was examined in lens epithelial cell cultures. SFK activity was suppressed with the specific inhibitor PP1. This induced expression of the cyclin‐dependent kinase (CDK) inhibitors p27 and p57 and suppressed lens epithelial cell proliferation. Therefore, inhibition of SFK activity created conditions permissive for undifferentiated lens epithelial cells to withdraw from the cell cycle. Growth of the lens epithelial cell cultures in the presence of PP1 induced expression of filensin and CP49, lens differentiation‐specific intermediate filament proteins, providing evidence that suppression of SFK activity also promoted the initiation of lens cell differentiation. The mechanism by which PP1 signaled cell cycle withdrawal and commitment to differentiation was shown to involve induction of N‐cadherin cell–cell junction assembly and reorganization of the actin cytoskeleton from stress fibers to cortical filaments. This result was supported by the compaction of the epithelial monolayer in response to PP1, a morphogenetic change that we have previously shown to be dependent on N‐cadherin function and a hallmark of the commencement of the lens differentiation program in culture. The results presented in this study suggest that the decision of lens epithelial cells to withdraw from the cell cycle and initiate differentiation requires inhibition of SFKs and the formation of N‐cadherin cell–cell junctions.