A Sue Menko - US grants

? DESCRIPTION (provided by applicant): Salivary gland dysfunctions that accompany disease states pose a substantial health and economic burden in the US and worldwide. The debilitating consequences of radiation treatment for head and neck cancer, and of the autoimmune disorder Sjögren's Syndrome (SS), could be ameliorated with effective strategies to regenerate functional salivary epithelia and to prevent the development of fibrosis. The goal of this exploratory R21 proposal is to generate new knowledge about salivary gland repair and prevention of disease-associated fibrosis. Our preliminary studies identified a novel subpopulation of vimentin-rich cells within the SMG epithelia. These cells underwent expansion in response to injury and functioned in wound repair. They closely resembled vimentin-rich repair cell progenitors of mesodermal lineage within the lens epithelia that mediate injury repair, and whose ablation results in ineffective repair of the epithelium. Importantly, when these repair cells encounter a rigid extracellular matrix environment characteristic of injured tissues, they ca differentiate into fibrotic disease-causing myofibroblasts. Since diseases of the salivary glands result in structural defects, they are likely to trigger the repair process that involves activatio of these vinmentin-rich repair cells. In this proposal, we aim to characterize the SMG repair cells and to elucidate their role in SMG repair, including their ability to acquire myofibroblast phenotypes when the healing process is complete. Our hypothesis is that repair cell progenitors of mesodermal lineage in the SMG function as immediate responders to injury, that they mediate effective wound repair and that their fates include elimination by apoptosis and differentiation into myofibroblasts. We will test this hypothesis using genetic lineage tracing in transgenic mice that express a tamoxifen-inducible CreER under control of the endogenous vimentin promoter, coupled with ex vivo and in vivo SMG injury models. Two aims are proposed: 1) investigate the lineage of SMG repair cells, their response to injury, and the cell signaling and cytoskeletal functions essential to their reparative function; and 2) determine the fate of the repair cells following wound healing, including their ability to cause fibrotic disease Our proposed studies are significant and innovative because they will determine the identity of the SMG repair cells and explore whether these mesenchymal cells can potentially serve in salivary tissue regeneration. In addition, we will gain knowledge into how to manipulate these repair cells to prevent the development of salivary gland fibrotic disease. Our findings have the potential to be translated into effective therapeutic approaches for the regeneration of salivary gland structure and function, and to the fields of tissue injury repair and regeneration, in genera.

DESCRIPTION (provided by applicant): Fibrosis reduces the quality of life for millions and negatively impacts vision in the cornea by causing haze and scarring, in the lens by causing Posterior Capsular Opacification (PCO), and in the retina by causing fibrovascular membrane contraction leading to macular holes. During the previous funding period we showed that in the mouse and chick lens, as in the cornea, there is an innate population of mesodermal cells that are CD45+ and that these cells go to the leading edge of an injured lens epithelium to regulate migration of the epithelium to repair the wound in a mock cataract surgery model. This same population can be induced to express ?-SMA, acquiring a myofibroblast phenotype associated with causing PCO. The fact that these innate repair cells express CD45 suggests they are leukocytes. Because the lens was believed to consist exclusively of ectodermally derived cells, these data change our fundamental understanding of the lens and how it is formed and maintained. In this proposal, we propose to: 1) Establish that the lens contains a diverse resident population of mesodermally derived leukocytes with tissue specific properties, by identifying the leukocyte type(s) present in the lens and cornea that modulate the repair process following injury to ocular epithelia, examining how leukocytes impact the rate of epithelial sheet movement and the reestablishment of a normal epithelium following wounding of the lens and cornea, assessing the ability of injury-induced cytokines to mediate lens leukocyte activation, determining whether immune surveillance is induced in the lens following injury to other ocular tissues, and investigating the hypothesis that lens leukocyte activation in response to injury can recruit leukocytes from the outside the lens. 2) Establish that integrin-matrix signaling converts resident immune cells in the lens and cornea to myofibroblasts by investigating the role played by tenascin-C in the provisional matrix needed for FN(EDA+) expression and assembly, examining the mechanism by which FN(EDA+) signals myofibroblast differentiation, determining the mechanism by which ?9 integrin mediates myofibroblast differentiation, investigating whether collagen assembly and stiffening modulate persistence of the myofibroblast phenotype in the lens. Leukocyte integrins are known to mediate immune cell migration after injury and leukocytes can convert into ?-SMA expressing myofibroblasts. The proposed studies use well-characterized lens and cornea models to study myofibroblast formation and persistence from innate leukocytes with the goal of developing new treatments that induce myofibroblasts to revert into non-pathologic cells and or to undergo apoptosis to reduce the burden of scarring diseases in vision.

? DESCRIPTION (provided by applicant): This application will establish new pathways required for initiation of lens cell differentiation and will reveal a long-sought after mechanism for lens organelle elimination upon lens cell maturation to achieve lens transparency. We have recently discovered that low-level caspase-3 activation is required for initiation of lens cell differentiaton and that a new macroautophagy pathway initiated by JNK down-regulation directs organelle elimination during lens cell maturation. AIM 1 will establish a novel function for ?-crystallin in regulating mitochondrial cytochrome c release to ensure low-level caspase-3 activation required for lens cell differentiation. AIM 2 will establish how low-level caspase-3 activity transmits its differentiation initiation signal through activation of caspase 3-dependent DNAase (CAD) and Mst1 (and its downstream effector ?H2AX) to direct chromatin remodeling events that we propose are required for lens cell differentiation. AIM 3 will functionally define the macroautophagy proteins and pathways that direct the temporal and spatial removal of organelles during lens cell maturation. The logic, feasibility, and potential success of these AIMs is supported by our strong preliminary data demonstrating that: ?-crystallin translocates to the mitochondria where it complexes with cytochrome c to modulate its release and likely ensure low-level caspase-3 activation required for initiation of lens cell differentiation; CAD is activatd and H2AX phosphorylated and bound to DNA in a caspase-3-dependent manner during initiation of lens cell differentiation likely to initiate DNA strand breaks and ?H2AX DNA binding leading to nucleosome positioning changes required for initiation of lens differentiation- specific gene expression; and key macroautophagy regulatory proteins exhibit expression patterns consistent with their functioning in the JNK-regulated MTORC1 pathway that leads to removal of lens cell organelles upon lens cell maturation. The AIMs proposed are significant because their testing will establish new mechanisms crucial to regulation of the decision of lens cells to begin their differentiation program, as well as developmental processes critical for lens transparency. The work will be applicable towards understanding the differentiation, development and disease states of other tissues since the regulatory molecules examined are common to many other tissues. The proposed AIMs are conceptually innovative since they establish new roles and requirements for classic cell death regulators in normal signaling roles in the cell that regulate differentiation and development. The AIMs are technically innovative since they employ sophisticated lens organ culture methods, gene expression and deletion strategies, signaling assays and chromatin monitoring techniques developed and established in our laboratories.

Using a combined ATACseq and RNAseq approach our studies have provided the first evidence that changes in chromatin accessibility are crucial to the differentiation state-specific expression of a wide variety genes essential to the transition from lens epithelial to fiber cells. These studies also identified key DNA regulatory regions and transcription factor binding sites likely to regulate a wide-range of lens genes, most importantly FOXO4. Genes with requisite roles in lens cell differentiation that contain a consensus DNA binding sequence for FOXO4 include EPHA2, NrCAM, ?-crystallin, Notch1 and FYCO1. The FOXO family, including FOXO4, is regulated by the PI3K/Akt signaling pathway. Phosphorylation of FOXO4 by PI3K/Akt sequesters it in the cytoplasm and suppression of PI3K/Akt signaling is required for FOXO4 import to the nucleus for its role in regulating gene expression. The function of FOXO4 has never been examined in the lens. We propose to establish the role of PI3K/AKT regulation of FOXO4-dependent gene expression in lens fiber cell differentiation. This will be accomplished by 1) identifying the requirement for PI3K/Akt inhibition for the nuclear translocation of FOXO4 in the transition of lens epithelial to fiber cells; 2) establishing the link between PI3K/Akt inhibition and the expression of lens differentiation-specific genes containing FOXO4 binding sequences; 3) demonstrating the binding of FOXO4 to distinct chromatin accessible DNA regulatory regions in genes crucial to lens differentiation using targeted-CHIP assays; and 4) establishing the spectrum, range and spatial expression patterns of genes regulated by FOXO4 during lens cell differentiation. We also discovered that the PI3K/Akt signaling axis plays an essential role in regulating the timing and mechanism that removes mitochondria, ER and Golgi from the central light path and that multiple PI3K-downstream signaling pathways are required for the process of eliminating nuclei to form the lens Organelle Free Zone (OFZ). This process is required to create a mature lens capable of focusing light images on the retina. We will explore how PI3K signaling pathways regulate the elimination of nuclei and other organelles to form the lens Organelle Free Zone in studies aimed at 1) identifying the functions of individual PI3K p110 catalytic subunits in regulating formation of the OFZ; 2) confirming that the induction of autophagy following inhibition of the PI3K/Akt signaling axis is responsible for the removal of mitochondria, ER and Golgi from the developing lens; 3) determining the link between inactivation of different PI3K signaling pathways and activation of the mechanistic targets required to eliminate nuclei to form the OFZ; and 4) investigating the potential link between the ring of Akt activity at the border of cortical and nuclear fiber cells and the regulation of the outer boundary of the OFZ.