Immunoregulatory Properties of Immune Cells that Associate with the Lens Capsule Surface during Acute and Resolution Phases of Experimental Autoimmune Uveitis.

Inflammation in the eye is tightly regulated to prevent vision impairment and irreversible blindness. Emerging evidence shows that immune cells are specifically recruited to the lens capsule in response to autoimmune uveitis, yet the potential that they have a role in regulating this inflammatory disease remained unexplored. Here, using an immunolocalization approach combined with high-resolution confocal microscopy we investigated whether the immune cells that become stably associated with the lens capsule in the eyes of C57BL/6J mice with experimental autoimmune uveitis (EAU) have an immunoregulatory phenotype. These studies revealed that during the acute phase of uveitis, at day 18 after disease induction, the immune cells specifically recruited to the lens capsule included those with putative anti-inflammatory, pro-resolution roles such as regulatory T cells (FoxP3+CD4+) and M2 macrophages (CD68+Arg1+IL10+). The frequency of these lens capsule associated immunomodulatory phenotypes increased at day 35 after induction, during the resolution phase of EAU inflammation. At this later stage of resolution most of the macrophages expressed CD206, a mannose receptor responsible for removing inflammatory molecules, in addition to Arg1 and IL10. Our results suggest a previously unknown role for the lens as a site for recruitment of immune cells whose role is to suppress inflammation, promote resolution, and maintain remission of EAU.

Multiomic analysis implicates FOXO4 in genetic regulation of chick lens fiber cell differentiation.

A classic model for identification of novel differentiation mechanisms and pathways is the eye lens that consists of a monolayer of quiescent epithelial cells that are the progenitors of a core of mature fully differentiated fiber cells. The differentiation of lens epithelial cells into fiber cells follows a coordinated program involving cell cycle exit, expression of key structural proteins and the hallmark elimination of organelles to achieve transparency. Although multiple mechanisms and pathways have been identified to play key roles in lens differentiation, the entirety of mechanisms governing lens differentiation remain to be discovered. A previous study established that specific chromatin accessibility changes were directly associated with the expression of essential lens fiber cell genes, suggesting that the activity of transcription factors needed for expression of these genes could be regulated through binding access to the identified chromatin regions. Sequence analysis of the identified chromatin accessible regions revealed enhanced representation of the binding sequence for the transcription factor FOXO4 suggesting a direct role for FOXO4 in expression of these genes. FOXO4 is known to regulate a variety of cellular processes including cellular response to metabolic and oxidative stress, cell cycle withdrawal, and homeostasis, suggesting a previously unidentified role for FOXO4 in the regulation of lens cell differentiation. To further evaluate the role of FOXO4 we employed a multiomics approach to analyze the relationship between genome-wide FOXO4 binding, the differentiation-specific expression of key genes, and chromatin accessibility. To better identify active promoters and enhancers we also examined histone modification through analysis of H3K27ac. Specific methods included CUT&RUN (FOXO4 binding and H3K27ac modification), RNA-seq (differentiation state specific gene expression), and ATAC-seq (chromatin accessibility). CUT&RUN identified 20,966 FOXO4 binding sites and 33,921 H3K27ac marked regions across the lens fiber cell genome. RNA-seq identified 956 genes with significantly greater expression levels in fiber cells compared to epithelial cells (log2FC > 0.7, q < 0.05) and 2548 genes with significantly lower expression levels (log2FC < -0.7, q < 0.05). Integrated analysis identified 1727 differentiation-state specific genes that were nearest neighbors to at least one FOXO4 binding site, including genes encoding lens gap junctions (GJA1, GJA3), lens structural proteins (BFSP1, CRYBB1, ASL1), and genes required for lens transparency (HSF4, NRCAM). Multiomics analysis comparing the identified FOXO4 binding sites in published ATAC-seq data revealed that chromatin accessibility was associated with FOXO4-dependent gene expression during lens differentiation. The results provide evidence for an important requirement for FOXO4 in the regulated expression of key genes required for lens differentiation and link epigenetic regulation of chromatin accessibility and H3K27ac histone modification with the function of FOXO4 in controlling lens gene expression during lens fiber cell differentiation.

Uveitis-mediated immune cell invasion through the extracellular matrix of the lens capsule.

While the eye is considered an immune privileged site, its privilege is abrogated when immune cells are recruited from the surrounding vasculature in response to trauma, infection, aging, and autoimmune diseases like uveitis. Here, we investigate whether in uveitis immune cells become associated with the lens capsule and compromise its privilege in studies of C57BL/6J mice with experimental autoimmune uveitis. These studies show that at D14, the peak of uveitis in these mice, T cells, macrophages, and Ly6G/Ly6C+ immune cells associate with the lens basement membrane capsule, burrow into the capsule matrix, and remain integrated with the capsule as immune resolution is occurring at D26. 3D surface rendering image analytics of confocal z-stacks and scanning electron microscopy imaging of the lens surface show the degradation of the lens capsule as these lens-associated immune cells integrate with and invade the lens capsule, with a subset infiltrating both epithelial and fiber cell regions of lens tissue, abrogating its immune privilege. Those immune cells that remain on the surface often become entwined with a fibrillar net-like structure. Immune cell invasion of the lens capsule in uveitis has not been described previously and may play a role in induction of lens and other eye pathologies associated with autoimmunity.

Suppression of PI3K signaling is linked to autophagy activation and the spatiotemporal induction of the lens organelle free zone.

The terminal steps of lens cell differentiation require elimination of all organelles to create a central Organelle Free Zone (OFZ) that is required for lens function of focusing images on the retina. Previous studies show that the spatiotemporal elimination of these organelles during development is autophagy-dependent. We now show that the inhibition of PI3K signaling in lens organ culture results in the premature induction of autophagy within 24 h, including a significant increase in LAMP1+ lysosomes, and the removal of lens organelles from the center of the lens. Specific inhibition of just the PI3K/Akt signaling axis was directly linked to the elimination of mitochondria and ER, while pan-PI3K inhibitors that block all PI3K downstream signaling removed all organelles, including nuclei. Therefore, blocking the PI3K/Akt pathway was alone insufficient to remove nuclei. RNAseq analysis revealed increased mRNA levels of the endogenous inhibitor of PI3K activation, PIK3IP1, in differentiating lens fiber cells preceding the induction of OFZ formation. Co-immunoprecipitation confirmed that PIK3IP1 associates with multiple PI3K p110 isoforms just prior to formation of the OFZ, providing a likely endogenous mechanism for blocking all PI3K signaling and activating the autophagy pathway required to form the OFZ during lens development.

Resident immune cells of the avascular lens: Mediators of the injury and fibrotic response of the lens.

Tissues typically harbor subpopulations of resident immune cells that function as rapid responders to injury and whose activation leads to induction of an adaptive immune response, playing important roles in repair and protection. Since the lens is an avascular tissue, it was presumed that it was absent of resident immune cells. Our studies now show that resident immune cells are a shared feature of the human, mouse, and chicken lens epithelium. These resident immune cells function as immediate responders to injury and rapidly populate the wound edge following mock cataract surgery to function as leader cells. Many of these resident immune cells also express MHCII providing them with antigen presenting ability to engage an adaptive immune response. We provide evidence that during development immune cells migrate on the ciliary zonules and localize among the equatorial epithelial cells of the lens adjacent to where the ciliary zonules associate with the lens capsule. These findings suggest that the vasculature-rich ciliary body is a source of lens resident immune cells. We identified a major role for these cells as rapid responders to wounding, quickly populating each wound were they can function as leaders of lens tissue repair. Our findings also show that lens resident immune cells are progenitors of myofibroblasts, which characteristically appear in response to lens cataract surgery injury, and therefore, are likely agents of lens pathologies to impair vision like fibrosis.

Immune cells in lens injury repair and fibrosis.

Immune cells, both tissue resident immune cells and those immune cells recruited in response to wounding or degenerative conditions, are essential to both the maintenance and restoration of homeostasis in most tissues. These cells are typically provided to tissues by their closely associated vasculatures. However, the lens, like many of the tissues in the eye, are considered immune privileged sites because they have no associated vasculature. Such absence of immune cells was thought to protect the lens from inflammatory responses that would bring with them the danger of causing vision impairing opacities. However, it has now been shown, as occurs in other immune privileged sites in the eye, that novel pathways exist by which immune cells come to associate with the lens to protect it, maintain its homeostasis, and function in its regenerative repair. Here we review the discoveries that have revealed there are both innate and adaptive immune system responses to lens, and that, like most other tissues, the lens harbors a population of resident immune cells, which are the sentinels of danger or injury to a tissue. While resident and recruited immune cells are essential elements of lens homeostasis and repair, they also become the agents of disease, particularly as progenitors of pro-fibrogenic myofibroblasts. There still remains much to learn about the function of lens-associated immune cells in protection, repair and disease, the knowledge of which will provide new tools for maintaining the core functions of the lens in the visual system.

Descemet's membrane injury and regeneration, and posterior corneal fibrosis, in rabbits.

The purpose of this investigation was to study Descemet's membrane and corneal endothelial regeneration, myofibroblast generation and disappearance, and TGF beta-1 localization after Descemet's membrane-endothelial excision (Descemetorhexis) in rabbits. Thirty-six rabbits had 8 mm Descemetorhexis and standardized slit lamp photos at 1, 2 and 4 days, 1, 2 and 4 weeks, and 2, 4 and 6 months, as well as multiplex IHC for stromal cell markers keratocan, vimentin, and alpha-smooth muscle actin (SMA); basement membrane (BM) components perlecan, nidogen-1, laminin alpha-5, and collagen type IV; and corneal endothelial marker Na,K-ATPase ß1, and TGF beta-1, with ImageJ quantitation. Stromal transparency increased from the periphery beginning at two months after injury and progressed into the central cornea by six months. At six months, central transparency was primarily limited by persistent mid-stromal neovascularization. Stromal myofibroblast zone thickness in the posterior stroma peaked at one month after injury, and then progressively decreased until to six months when few myofibroblasts remained. The regeneration of a laminin alpha-5 and nidogen-1 Descemet's membrane "railroad track" structure was accompanied by corneal endothelial closure and stromal cell production of BM components in corneas from four to six months after injury. TGF beta-1 deposition at the posterior corneal surface from the aqueous humor peaked at one day after Descemetorhexis and diminished even before regeneration of the endothelium and Descemet's membrane. This decrease was associated with collagen type IV protein production by corneal fibroblasts, and possibly myofibroblasts, in the posterior stroma. Descemet's membrane and the corneal endothelium regenerated in the rabbit cornea by six months after eight mm Descemetorhexis. Real-time quantitative RT-PCR experiments in vitro with marker-verified rabbit corneal cells found that 5 ng/ml or 10 ng/ml TGF beta-1 upregulated col4a1 or col4a2 mRNA expression after 6 h or 12 h of exposure in corneal fibroblasts, but not in myofibroblasts. Stromal cells produced large amounts of collagen type IV that likely decreased TGF beta-1 penetration into the stroma and facilitated the resolution of myofibroblast-generated fibrosis.

TGFß1 and TGFß2 proteins in corneas with and without stromal fibrosis: Delayed regeneration of apical epithelial growth factor barrier and the epithelial basement membrane in corneas with stromal fibrosis.

The purpose of this study was to investigate the expression and localization of transforming growth factor (TGF) ß1 and TGFß2 in rabbit corneas that healed with and without stromal fibrosis, and to further study defective perlecan incorporation in the epithelial basement membrane (EBM) in corneas with scarring fibrosis. A total of 120 female rabbits had no surgery, -4.5D PRK, or -9D PRK. Immunohistochemistry (IHC) was performed at time points from unwounded to eight weeks after surgery, with four corneas at each time point in each group. Multiplex IHC was performed for TGFß1 or TGFß2, with Image-J quantitation, and keratocan, vimentin, alpha-smooth muscle actin (SMA), perlecan, laminin-alpha 5, nidogen-1 or CD11b. Corneas at the four-week peak for myofibroblast and fibrosis development were evaluated using Imaris 3D analysis. Delayed regeneration of both an apical epithelial growth factor barrier and EBM barrier function, including defective EBM perlecan incorporation, was greater in high injury -9D PRK corneas compared to -4.5D PRK corneas without fibrosis. Defective apical epithelial growth factor barrier and EBM allowed epithelial and tear TGFß1 and tear TGFß2 to enter the corneal stroma to drive myofibroblast generation in the anterior stroma from vimentin-positive corneal fibroblasts, and likely fibrocytes. Vimentin-positive cells and unidentified vimentin-negative, CD11b-negative cells also produce TGFß1 and/or TGFß2 in the stroma in some corneas. TGFß1 and TGFß2 were at higher levels in the anterior stroma in the weeks preceding myofibroblast development in the -9D group. All -9D corneas (beginning two to three weeks after surgery), and four -4.5D PRK corneas developed significant SMA + myofibroblasts and stromal fibrosis. Both the apical epithelial growth factor barrier and/or EBM barrier functions tended to regenerate weeks earlier in -4.5D PRK corneas without fibrosis, compared to -4.5D or -9D PRK corneas with fibrosis. SMA-positive myofibroblasts were markedly reduced in most corneas by eight weeks after surgery. The apical epithelial growth factor barrier and EBM barrier limit TGFß1 and TGFß2 entry into the corneal stroma to modulate corneal fibroblast and myofibroblast development associated with scarring stromal fibrosis. Delayed regeneration of these barriers in corneas with more severe injuries promotes myofibroblast development, prolongs myofibroblast viability and triggers stromal scarring fibrosis.

An immune response to the avascular lens following wounding of the cornea involves ciliary zonule fibrils.

The lens and central cornea are avascular. It was assumed that the adult lens had no source of immune cells and that the basement membrane capsule surrounding the lens was a barrier to immune cell migration. Yet, microfibril-associated protein-1 (MAGP1)-rich ciliary zonules that originate from the vasculature-rich ciliary body and extend along the surface of the lens capsule, form a potential conduit for immune cells to the lens. In response to cornea debridement wounding, we find increased expression of MAGP1 throughout the central corneal stroma. The immune cells that populate this typically avascular region after wounding closely associate with this MAGP1-rich matrix. These results suggest that MAGP1-rich microfibrils support immune cell migration post-injury. Using this cornea wound model, we investigated whether there is an immune response to the lens following cornea injury involving the lens-associated MAGP1-rich ciliary zonules. Our results provide the first evidence that following corneal wounding immune cells are activated to travel along zonule fibers that extend anteriorly along the equatorial surface of the lens, from where they migrate across the anterior lens capsule. These results demonstrate that lens-associated ciliary zonules are directly involved in the lens immune response and suggest the ciliary body as a source of immune cells to the avascular lens.

Lens differentiation is characterized by stage-specific changes in chromatin accessibility correlating with differentiation state-specific gene expression.

Changes in chromatin accessibility regulate the expression of multiple genes by controlling transcription factor access to key gene regulatory sequences. Here, we sought to establish a potential function for altered chromatin accessibility in control of key gene expression events during lens cell differentiation by establishing genome-wide chromatin accessibility maps specific for four distinct stages of lens cell differentiation and correlating specific changes in chromatin accessibility with genome-wide changes in gene expression. ATAC sequencing was employed to generate chromatin accessibility profiles that were correlated with the expression profiles of over 10,000 lens genes obtained by high-throughput RNA sequencing at the same stages of lens cell differentiation. Approximately 90,000 regions of the lens genome exhibited distinct changes in chromatin accessibility at one or more stages of lens differentiation. Over 1000 genes exhibited high Pearson correlation coefficients (r â€‹> â€‹0.7) between altered expression levels at specific stages of lens cell differentiation and changes in chromatin accessibility in potential promoter (-7.5kbp/+2.5kbp of the transcriptional start site) and/or other potential cis-regulatory regions ( ±10 â€‹kb of the gene body). Analysis of these regions identified consensus binding sequences for multiple transcription factors including members of the TEAD, FOX, and NFAT families of transcription factors as well as HIF1a, RBPJ and IRF1. Functional mapping of genes with high correlations between altered chromatin accessibility and differentiation state-specific gene expression changes identified multiple families of proteins whose expression could be regulated through changes in chromatin accessibility including those governing lens structure (BFSP1,BFSP2), gene expression (Pax-6, Sox 2), translation (TDRD7), cell-cell communication (GJA1), autophagy (FYCO1), signal transduction (SMAD3, EPHA2), and lens transparency (CRYBB1, CRYBA4). These data provide a novel relationship between altered chromatin accessibility and lens differentiation and they identify a wide-variety of lens genes and functions that could be regulated through altered chromatin accessibility. The data also point to a large number of potential DNA regulatory sequences and transcription factors whose functional analysis is likely to provide insight into novel regulatory mechanisms governing the lens differentiation program.

Functional role for stable microtubules in lens fiber cell elongation.

The process of tissue morphogenesis, especially for tissues reliant on the establishment of a specific cytoarchitecture for their functionality, depends a balanced interplay between cytoskeletal elements and their interactions with cell adhesion molecules. The microtubule cytoskeleton, which has many roles in the cell, is a determinant of directional cell migration, a process that underlies many aspects of development. We investigated the role of microtubules in development of the lens, a tissue where cell elongation underlies morphogenesis. Our studies with the microtubule depolymerizing agent nocodazole revealed an essential function for the acetylated population of stable microtubules in the elongation of lens fiber cells, which was linked to their regulation of the activation state of myosin. Suppressing myosin activation with the inhibitor blebbistatin could attenuate the loss of acetylated microtubules by nocodazole and rescue the effect of this microtubule depolymerization agent on both fiber cell elongation and lens integrity. Our results also suggest that acetylated microtubules impact lens morphogenesis through their interaction with N-cadherin junctions, with which they specifically associate in the region where lens fiber cell elongate. Disruption of the stable microtubule network increased N-cadherin junctional organization along lateral borders of differentiating lens fiber cells, which was prevented by suppression of myosin activity. These results reveal a role for the stable microtubule population in lens fiber cell elongation, acting in tandem with N-cadherin cell-cell junctions and the actomyosin network, giving insight into the cooperative role these systems play in tissue morphogenesis.

N-cadherin regulates signaling mechanisms required for lens fiber cell elongation and lens morphogenesis.

Tissue development and regeneration involve high-ordered morphogenetic processes that are governed by elements of the cytoskeleton in conjunction with cell adhesion molecules. Such processes are particularly important in the lens whose structure dictates its function. Studies of our lens-specific N-cadherin conditional knockout mouse (N-cadcKO) revealed an essential role for N-cadherin in the migration of the apical tips of differentiating lens fiber cells along the apical surfaces of the epithelium, a region termed the Epithelial Fiber Interface (EFI), that is necessary for normal fiber cell elongation and the morphogenesis. Studies of the N-cadcKO lens suggest that N-cadherin function in fiber cell morphogenesis is linked to the activation of Rac1 and myosin II, both signaling pathways central to the regulation of cell motility including determining the directionality of cellular movement. The absence of N-cadherin did not disrupt lateral contacts between fiber cells during development, and the maintenance of Aquaporin-0 and increased expression of EphA2 at cell-cell interfaces suggests that these molecules may function in this role. E-cadherin was maintained in newly differentiating fiber cells without interfering with expression of lens-specific differentiation proteins but was not able to replace N-cadherin function in these cells. The dependence of migration of the fiber cell apical domains along the EFI for lens morphogenesis on N-cadherin provides new insight into the process of tissue development.

BNIP3L/NIX is required for elimination of mitochondria, endoplasmic reticulum and Golgi apparatus during eye lens organelle-free zone formation.

The formation and life-long growth of the ocular lens depends on the continuous differentiation of lens epithelial cells into lens fiber cells. To achieve their mature structure and transparent function, newly formed lens fiber cells undergo a series of cellular remodeling events including the complete elimination of cellular organelles to form the lens organelle-free zone (OFZ). To date, the mechanisms and requirements for organelle elimination by lens fiber cells remain to be fully elucidated. In previous studies, we detected the presence of mitochondria contained within autophagolysosomes throughout human and chick lenses suggesting that proteins targeting mitochondria for degradation by mitophagy could be required for the elimination of mitochondria during OFZ formation. Consistently, high-throughput RNA sequencing of microdissected embryonic chick lenses revealed that expression of a protein that targets mitochondria for elimination during erythrocyte formation, called BCL2 interacting protein 3-like (BNIP3L/NIX), peaks in the region of lens where organelle elimination occurs. To examine the potential role for BNIP3L in the elimination of mitochondria during lens fiber cell remodeling, we analyzed the expression pattern of BNIP3L in newborn mouse lenses, the effect of its deletion on organelle elimination and its co-localization with lens organelles. We demonstrate that the expression pattern of BNIP3L in the mouse lens is consistent with it playing an important role in the elimination of mitochondria during lens fiber cell organelle elimination. Importantly, we demonstrate that deletion of BNIP3L results in retention of mitochondria during lens fiber cell remodeling, and, surprisingly, that deletion of BNIP3L also results in the retention of endoplasmic reticulum and Golgi apparatus but not nuclei. Finally, we show that BNIP3L localizes to the endoplasmic reticulum and Golgi apparatus of wild-type newborn mouse lenses and is contained within mitochondria, endoplasmic reticulum and Golgi apparatus isolated from adult mouse liver. These data identify BNIP3L as a novel requirement for the elimination of mitochondria, endoplasmic reticulum and Golgi apparatus during lens fiber cell remodeling and they suggest a novel function for BNIP3L in the regulation of endoplasmic reticulum and Golgi apparatus populations in the lens and non-lens tissues.

α6 integrin transactivates insulin-like growth factor receptor-1 (IGF-1R) to regulate caspase-3-mediated lens epithelial cell differentiation initiation.

The canonical mitochondrial death pathway was first discovered for its role in signaling apoptosis. It has since been found to have a requisite function in differentiation initiation in many cell types including the lens through low level activation of the caspase-3 protease. The ability of this pathway to function as a molecular switch in lens differentiation depends on the concurrent induction of survival molecules in the Bcl-2 and IAP families, induced downstream of an IGF-1R/NFκB coordinate survival signal, to regulate caspase-3 activity. Here we investigated whether α6 integrin signals upstream to this IGF-1R-mediated survival-linked differentiation signal. Our findings show that IGF-1R is recruited to and activated specifically in α6 integrin receptor signaling complexes in the lens equatorial region, where lens epithelial cells initiate their differentiation program. In studies with both α6 integrin knock-out mice lenses and primary lens cell cultures following α6 integrin siRNA knockdown, we show that IGF-1R activation is dependent on α6 integrin and that this transactivation requires Src kinase activity. In addition, without α6 integrin, activation and expression of NFκB was diminished, and expression of Bcl-2 and IAP family members were down-regulated, resulting in high levels of caspase-3 activation. As a result, a number of hallmarks of lens differentiation failed to be induced; including nuclear translocation of Prox1 in the differentiation initiation zone and apoptosis was promoted. We conclude that α6 integrin is an essential upstream regulator of the IGF-1R survival pathway that regulates the activity level of caspase-3 for it to signal differentiation initiation of lens epithelial cells.

Endogenous hydrogen peroxide production in the epithelium of the developing embryonic lens.

PURPOSE: Hydrogen peroxide (H2O2) is an endogenously produced reactive oxygen species (ROS) present in a variety of mammalian systems. This particular ROS can play dichotomous roles, being beneficial in some cases and deleterious in others, which reflects the level and location of H2O2 production. While much is known about the redox regulation of ROS by antioxidant and repair systems in the lens, little is known about the endogenous production of H2O2 in embryonic lens tissue or the physiologic relevance of endogenous H2O2 to lens development. This gap in knowledge exists primarily from a lack of reagents that can specifically detect endogenous H2O2 in the intact lens. Here, using a recently developed chemoselective fluorescent boronate probe, peroxyfluor-6 acetoxymethyl ester (PF6-AM), which selectively detects H2O2 over related ROS, we examined the endogenous H2O2 signals in the embryonic lens. METHODS: Embryonic day 10 chick whole lenses in ex vivo organ culture and lens epithelial cells in primary culture were loaded with the H2O2 probe PF6-AM. To determine the relationship between localization of mitochondria with active membrane potential and the region of H2O2 production in the lens, cells were exposed to the mitochondrial probe MitoTracker Red CMXRos together with PF6-AM. Diphenyleneiodonium (DPI), a flavin inhibitor that blocks generation of intracellular ROS production, was used to confirm that the signal from PF6-AM was due to endogenous ROS production. All imaging was performed by live confocal microscopy. RESULTS: PF6-AM detected endogenous H2O2 in lens epithelial cells in whole lenses in ex vivo culture and in lens epithelial cells grown in primary culture. No endogenous H2O2 signal could be detected in differentiating lens fiber cells with this probe. Treatment with DPI markedly attenuated the fluorescence signal from the peroxide-specific probe PF6-AM in the lens epithelium, suggesting that basal generation of ROS occurs in this region. The lens epithelial cells producing an endogenous H2O2 signal were also rich in actively respiring mitochondria. CONCLUSIONS: PF6-AM can be used as an effective reagent to detect the presence and localization of endogenous H2O2 in live lens cells.

Distinct roles for N-Cadherin linked c-Src and fyn kinases in lens development.

BACKGROUND: Src family tyrosine kinases (SFKs) are often coincidently expressed but few studies have dissected their individual functions in the same cell during development. Using the classical embryonic lens as our model, we investigated SFK signaling in the regulation of both differentiation initiation and morphogenesis, and the distinct functions of c-Src and Fyn in these processes. RESULTS: Blocking SFK activity with the highly specific inhibitor PP1 induced initiation of the lens differentiation program but blocked lens fiber cell elongation and organization into mini lens-like structures called lentoids. These dichotomous roles for SFK signaling were discovered to reflect distinct functions of c-Src and Fyn and their differentiation-state-specific recruitment to and action at N-cadherin junctions. c-Src was highly associated with the nascent N-cadherin junctions of undifferentiated lens epithelial cells. Its siRNA knockdown promoted N-cadherin junctional maturation, blocked proliferation, and induced lens cell differentiation. In contrast, Fyn was recruited to mature N-cadherin junctions of differentiating lens cells and siRNA knockdown suppressed differentiation-specific gene expression and blocked morphogenesis. CONCLUSIONS: Through inhibition of N-cadherin junction maturation, c-Src promotes lens epithelial cell proliferation and the maintenance of the lens epithelial cell undifferentiated state, while Fyn, signaling downstream of mature N-cadherin junctions, promotes lens fiber cell morphogenesis.

Insulin-like growth factor receptor-1 and nuclear factor κB are crucial survival signals that regulate caspase-3-mediated lens epithelial cell differentiation initiation.

It is now known that the function of the caspase family of proteases is not restricted to effectors of programmed cell death. For example, there is a significant non-apoptotic role for caspase-3 in cell differentiation. Our own studies in the developing lens show that caspase-3 is activated downstream of the canonical mitochondrial death pathway to act as a molecular switch in signaling lens cell differentiation. Importantly, for this function, caspase-3 is activated at levels far below those that induce apoptosis. We now have provided evidence that regulation of caspase-3 for its role in differentiation induction is dependent on the insulin-like growth factor-1 receptor (IGF-1R) survival-signaling pathway. IGF-1R executed this regulation of caspase-3 by controlling the expression of molecules in the Bcl-2 and inhibitor of apoptosis protein (IAP) families. This effect of IGF-1R was mediated through NFκB, demonstrated here to function as a crucial downstream effector of IGF-1R. Inhibition of expression or activation of NFκB blocked expression of survival proteins in the Bcl-2 and IAP families and removed controls on the activation state of caspase-3. The high level of caspase-3 activation that resulted from inhibiting this IGF-1R/NFκB signaling pathway redirected cell fate from differentiation toward apoptosis. These results provided the first evidence that the IGF-1R/NFκB cell survival signal is a crucial regulator of the level of caspase-3 activation for its non-apoptotic function in signaling cell differentiation.

The Hippo signaling pathway is required for salivary gland development and its dysregulation is associated with Sjogren's syndrome.

Sjogren's syndrome (SS) is a complex autoimmune disease that primarily affects salivary and lacrimal glands and is associated with high morbidity. Although the prevailing dogma is that immune system pathology drives SS, increasing evidence points to structural defects, including defective E-cadherin adhesion, to be involved in its etiology. We have shown that E-cadherin has pivotal roles in the development of the mouse salivary submandibular gland (SMG) by organizing apical-basal polarity in acinar and ductal progenitors and by signaling survival for differentiating duct cells. Recently, E-cadherin junctions have been shown to interact with effectors of the Hippo signaling pathway, a core pathway regulating the organ size, cell proliferation, and differentiation. We now show that Hippo signaling is required for SMG-branching morphogenesis and is involved in the pathophysiology of SS. During SMG development, a Hippo pathway effector, TAZ, becomes increasingly phosphorylated and associated with E-cadherin and α-catenin, consistent with the activation of Hippo signaling. Inhibition of Lats2, an upstream kinase that promotes TAZ phosphorylation, results in dysmorphogenesis of the SMG and impaired duct formation. SMGs from non-obese diabetic mice, a mouse model for SS, phenocopy the Lats2-inhibited SMGs and exhibit a reduction in E-cadherin junctional components, including TAZ. Importantly, labial specimens from human SS patients display mislocalization of TAZ from junctional regions to the nucleus, coincident with accumulation of extracellular matrix components, fibronectin and connective tissue growth factor, known downstream targets of TAZ. Our studies show that Hippo signaling has a crucial role in SMG-branching morphogenesis and provide evidence that defects in this pathway are associated with SS in humans.

Unique precursors for the mesenchymal cells involved in injury response and fibrosis.

We investigated an alternative pathway for emergence of the mesenchymal cells involved in epithelial sheet wound healing and a source of myofibroblasts that cause fibrosis. Using a mock cataract surgery model, we discovered a unique subpopulation of polyploid mesenchymal progenitors nestled in small niches among lens epithelial cells that expressed the surface antigen G8 and mRNA for the myogenic transcription factor MyoD. These cells rapidly responded to wounding of the lens epithelium with population expansion, acquisition of a mesenchymal phenotype, and migration to the wound edges where they regulate the wound response of the epithelium. These mesenchymal cells also were a principal source of myofibroblasts that emerged following lens injury and were responsible for fibrotic disease of the lens that occurs following cataract surgery. These studies provide insight into the mechanisms of wound-healing and fibrosis.

Examining PI3K-signaling-dependent regulation of lens organelle free zone formation via immunolocalization and immunoblotting in chick embryos.

The elimination of lens organelles during development, required for mature lens function, is an autophagy-dependent mechanism induced through suppression of PI3K signaling. Here, we present a protocol for investigating the signaling pathways responsible for induction of the formation of this lens organelle free zone. We describe steps for preparation of lens organ culture and use of signaling pathway inhibitors. We then detail procedures for analyzing their impact using both confocal microscopy imaging of immunolabeled lens cryosections and immunoblot approaches. For complete details on the use and execution of this protocol, please refer to Gheyas et al. (2022).1.