The molecular control of lens fiber cell lateral membrane structure

Duncan, Melinda K.For vision to occur, there needs to be coordination between tissues that detect light such as the retina, and those that control image quality, such as the iris, cornea, and lens. The lens contains two main cell types; epithelial cells, a monolayer of cells found on the anterior lens surface, and fiber cells, which make up the majority of the lens. For the lens to refract light, it must remain transparent which is regulated by a combination of inter-molecular interactions and its highly specialized cellular structure. Changes in the morphology of lens cells can result in improper light refraction and even cataract, a clouding of the ocular lens. The investigations in this dissertation contribute to the growing knowledge of the molecular control of lens fiber cell lateral membrane structure. ??1 integrin, a heterodimeric transmembrane cell adhesion molecule, is the most abundant ?? integrin in the lens and has multifaceted roles in lens biology. Conditional deletion of ??1 integrin from all lens cells during embryonic development results in profound lens defects; however, it is less clear whether this reflects functions in the lens epithelium alone or whether this protein plays a role in lens fibers. Thus, a conditional deletion approach was used to delete ??1 integrin solely from the lens fiber cells, while leaving lens epithelial expression intact. This deletion resulted in two distinct phenotypes, with some lenses exhibiting cataracts while others were clear, albeit with refractive defects. Analysis of "clear" conditional knockout lenses revealed that they had profound defects in fiber cell morphology associated with loss of the F-actin network. Physiological measurements found that the lens fiber cells had a two-fold increase in gap junctional coupling, perhaps due to the differential localization of connexins 46 and 50, as well as increased water permeability. This would presumably facilitate transport of ions and nutrients through the lens, and may partially explain how lenses with profound structural abnormalities can maintain transparency. In conclusion, I show that ??1-integrin has a large role in maintaining/specifying the structure of the lens fiber cell membrane during differentiation of the LFCs, presumably due to its function in maintaining the F-actin cytoskeleton underlying the lateral membrane, as opposed to functions mediated via its interactions with ECM. The lateral membranes contain a microstructure known as a membrane protrusion, which are the most commonly seen microstructure on lens fiber cells. However, little has been published on what the function of membrane protrusions are, although it could be speculated that they are needed to increase surface area and communication between cells in an avascular lens. Notably, elaborate membrane curvature is a key characteristic of these protrusions although little is known about the molecules that regulate these structures. However, in the past decade, a superfamily of proteins known as BAR domain proteins was discovered that are known to sense and induce membrane curvature. Bridging integrator protein 3 (Bin3), a ubiquitously expressed and evolutionarily conserved BAR domain protein, was first implicated in lens transparency since mice lacking this gene develop cataracts. However, at the beginning of my dissertation work, little was known about either the pathophysiology of the cataract or the possible underlying mechanisms by which Bin3 regulated lens transparency. The lack of cellular organization in the Bin3 null lenses implied that Bin3 may play a role in lens fiber cell differentiation as cells develop increased surface area along the lateral membranes. Bin3 null lenses displayed minor defects in lens packing and vacuole formation early in development (one to two weeks) followed by a complete loss of organization of fiber cell morphology by eight months, resulting in a loss of transparency and possibly disrupted ion transport. Further analysis of Bin3 null lenses revealed hypertrophy of interdigitations, a loss of spatial regulation in where these protrusions form, and disruption of the F-actin network. Bin3 null lenses were analyzed for Cdc42 since Bin3 is known to recruit Rho GTPases and Rho GEFs to allow for cytoskeletal remodeling. Cdc42, one of the most common RhoGTPases in the lens, was mislocalized to the broad sides of the membrane. The Rho GEF, Tuba, was downregulated in the Bin3 null lenses. Thus, the mislocalization of Cdc42, the down regulation of Tuba, and the disrupted F-actin network, may all lead to the disruption in cellular morphology and hypertrophied interdigitations. In conclusion, we proposed a mechanistic model for the formation of interdigitations during fiber differentiation. Bin3 may be needed to recruit Tuba (a Rho GEF) to the fiber cell membranes. Tuba in turn could then interact with Cdc42 (a Rho GTPase), and possibly WAVE2, activating an ARP2/3 complex to initiate nucleation and branching of cortical F-actin along the membrane pulling the membrane inward forming an invagination. (Abstract shortened by UMI.)University of Delaware, Department of Biological SciencesPh.D

Loss of Sip1 leads to migration defects and retention of ectodermal markers during lens development

SIP1 encodes a DNA-binding transcription factor that regulates multiple developmental processes, as highlighted by the pleiotropic defects observed in Mowat-Wilson syndrome, which results from mutations in this gene. Further, in adults, dysregulated SIP1 expression has been implicated in both cancer and fibrotic diseases, where it functionally links TGFβ signaling to the loss of epithelial cell characteristics and gene expression. In the ocular lens, an epithelial tissue important for vision, Sip1 is co-expressed with epithelial markers, such as E-cadherin, and is required for the complete separation of the lens vesicle from the head ectoderm during early ocular morphogenesis. However, the function of Sip1 after early lens morphogenesis is still unknown. Here, we conditionally deleted Sip1 from the developing mouse lens shortly after lens vesicle closure, leading to defects in coordinated fiber cell tip migration, defective suture formation, and cataract. Interestingly, RNA-Sequencing analysis on Sip1 knockout lenses identified 190 differentially expressed genes, all of which are distinct from previously described Sip1 target genes. Furthermore, 34% of the genes with increased expression in the Sip1 knockout lenses are normally downregulated as the lens transitions from the lens vesicle to early lens, while 49% of the genes with decreased expression in the Sip1 knockout lenses are normally upregulated during early lens development. Overall, these data imply that Sip1 plays a major role in reprogramming the lens vesicle away from a surface ectoderm cell fate towards that necessary for the development of a transparent lens and demonstrate that Sip1 regulates distinctly different sets of genes in different cellular contexts

90 Focus on a clear message: conserved RNA binding proteins function in mRNA control in eye lens development and their deficiency causes cataract

International audienceno abstrac

Coexpression of nos2 and cox2 accelerates tumor growth and reduces survival in estrogen receptor-negative breast cancer

Proinflammatory signaling pathways are commonly up-regulated in breast cancer. In estrogen receptor-negative (ER-) and triple-negative breast cancer (TNBC), nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) have been described as independent predictors of disease outcome. We further explore these findings by investigating the impact of their coexpression on breast cancer survival. Elevated coexpression of NOS2/COX2 proteins is a strong predictor of poor survival among ER-patients (hazard ratio: 21). Furthermore, we found that the key products of NOS2 and COX2, NO and prostaglandin E2 (PGE2), respectively, promote feed-forward NOS2/COX2 crosstalk in both MDA-MB-468 (basal-like) and MDA-MB-231 (mesenchymal-like) TNBC cell lines in which NO induced COX2 and PGE2 induced NOS2 proteins. COX2 induction by NO involved TRAF2 activation that occurred in a TNF alpha-dependent manner in MDA-MB-468 cells. In contrast, NO-mediated TRAF2 activation in the more aggressive MDA-MB-231 cells was TNF alpha independent but involved the endoplasmic reticulum stress response. Inhibition of NOS2 and COX2 using amino-guanidine and aspirin/indomethacin yielded an additive reduction in the growth of MDAMB-231 tumor xenografts. These findings support a role of NOS2/COX2 crosstalk during disease progression of aggressive cancer phenotypes and offer insight into therapeutic applications for better survival of patients with ER-and TNBC disease

Tumour irradiation combined with vascular-targeted photodynamic therapy enhances antitumour effects in pre-clinical prostate cancer

BACKGROUND: There is a need to improve the treatment of prostate cancer (PCa) and reduce treatment side effects. Vascular-targeted photodynamic therapy (VTP) is a focal therapy for low-risk low-volume localised PCa, which rapidly disrupts targeted tumour vessels. There is interest in expanding the use of VTP to higher-risk disease. Tumour vasculature is characterised by vessel immaturity, increased permeability, aberrant branching and inefficient flow. FRT alters the tumour microenvironment and promotes transient ‘vascular normalisation’. We hypothesised that multimodality therapy combining fractionated radiotherapy (FRT) and VTP could improve PCa tumour control compared against monotherapy with FRT or VTP. METHODS: We investigated whether sequential delivery of FRT followed by VTP 7 days later improves flank TRAMP-C1 PCa tumour allograft control compared to monotherapy with FRT or VTP. RESULTS: FRT induced ‘vascular normalisation’ changes in PCa flank tumour allografts, improving vascular function as demonstrated using dynamic contrast-enhanced magnetic resonance imaging. FRT followed by VTP significantly delayed tumour growth in flank PCa allograft pre-clinical models, compared with monotherapy with FRT or VTP, and improved overall survival. CONCLUSION: Combining FRT and VTP may be a promising multimodal approach in PCa therapy. This provides proof-of-concept for this multimodality treatment to inform early phase clinical trials

Systemic Nos2 Depletion and Cox inhibition limits TNBC disease progression and alters lymphoid cell spatial orientation and density

Antitumor immune polarization is a key predictor of clinical outcomes to cancer therapy. An emerging concept influencing clinical outcome involves the spatial location of CD8+ T cells, within the tumor. Our earlier work demonstrated immunosuppressive effects of NOS2 and COX2 tumor expression. Here, we show that NOS2/COX2 levels influence both the polarization and spatial location of lymphoid cells including CD8+ T cells. Importantly, elevated tumor NOS2/COX2 correlated with exclusion of CD8+ T cells from the tumor epithelium. In contrast, tumors expressing low NOS2/COX2 had increased CD8+ T cell penetration into the tumor epithelium. Consistent with a causative relationship between these observations, pharmacological inhibition of COX2 with indomethacin dramatically reduced tumor growth of the 4T1 model of TNBC in both WT and Nos2- mice. This regimen led to complete tumor regression in ∼20–25% of tumor-bearing Nos2- mice, and these animals were resistant to tumor rechallenge. Th1 cytokines were elevated in the blood of treated mice and intratumoral CD4+ and CD8+ T cells were higher in mice that received indomethacin when compared to control untreated mice. Multiplex immunofluorescence imaging confirmed our phenotyping results and demonstrated that targeted Nos2/Cox2 blockade improved CD8+ T cell penetration into the 4T1 tumor core. These findings are consistent with our observations in low NOS2/COX2 expressing breast tumors proving that COX2 activity is responsible for limiting the spatial distribution of effector T cells in TNBC. Together these results suggest that clinically available NSAID’s may provide a cost-effective, novel immunotherapeutic approach for treatment of aggressive tumors including triple negative breast cancer

Loss of Sip1 leads to migration defects and retention of ectodermal markers during lens development

SIP1 encodes a DNA-binding transcription factor that regulates multiple developmental processes, as highlighted by the pleiotropic defects observed in Mowat-Wilson Syndrome, which results from mutations in this gene. Further, in adults, dysregulated SIP1 expression has been implicated in both cancer and fibrotic diseases, where it functionally links TGFβ signaling to the loss of epithelial cell characteristics and gene expression. In the ocular lens, an epithelial tissue important for vision, Sip1 is co-expressed with epithelial markers, such as E-cadherin, and is required for the complete separation of the lens vesicle from the head ectoderm during early ocular morphogenesis. However, the function of Sip1 after early lens morphogenesis is still unknown. Here, we conditionally deleted Sip1 from the developing mouse lens shortly after lens vesicle closure, leading to defects in coordinated fiber cell tip migration, defective suture formation, and cataract. Interestingly, RNA-Sequencing analysis on Sip1 knockout lenses identified 190 differentially expressed genes, all of which are distinct from previously described Sip1 target genes. Furthermore, 34% of the genes with increased expression in the Sip1 knockout lenses are normally downregulated as the lens transitions from the lens vesicle to early lens, while 49% of the genes with decreased expression in the Sip1 knockout lenses are normally upregulated during early lens development. Overall, these data imply that Sip1 plays a major role in reprogramming the lens vesicle away from a surface ectoderm cell fate towards that necessary for the development of a transparent lens and demonstrate that Sip1 regulates distinctly different sets of genes in different cellular contexts