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1.
J Comp Neurol ; 530(9): 1494-1506, 2022 06.
Article in English | MEDLINE | ID: mdl-34958682

ABSTRACT

Glaucoma is a group of eye diseases characterized by retinal ganglion cell (RGC) loss and optic nerve damage. Studies, including this study, support that RGCs degenerate and die in a type-specific manner following the disease insult. Here we specifically examined one RGC type, the intrinsically photosensitive retinal ganglion cell (ipRGC), and its associated functional deficits in a mouse model of experimental glaucoma. We induced chronic ocular hypertension (OHT) by laser photocoagulation and then characterized the survival of ipRGC subtypes. We found that ipRGCs suffer significant loss, similar to the general RGC population, but ipRGC subtypes are differentially affected following chronic OHT. M4 ipRGCs, which are involved in pattern vision, are susceptible to chronic OHT. Correspondingly, mice with chronic OHT experience reduced contrast sensitivity and visual acuity. By comparison, M1 ipRGCs, which project to the suprachiasmatic nuclei to regulate circadian rhythmicity, exhibit almost no cell loss following chronic OHT. Accordingly, we observed that circadian re-entrainment and circadian rhythmicity are largely not disrupted in OHT mice. Our study demonstrates the link between subtype-specific ipRGC survival and behavioral deficits in glaucomatous mice. These findings provide insight into glaucoma-induced visual behavioral deficits and their underlying mechanisms.


Subject(s)
Glaucoma , Retinal Ganglion Cells , Animals , Glaucoma/metabolism , Mice , Retinal Ganglion Cells/physiology , Rod Opsins/metabolism , Suprachiasmatic Nucleus , Vision, Ocular
2.
Mol Cancer Res ; 13(3): 483-92, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25516960

ABSTRACT

UNLABELLED: RhoGDI2 (ARHGDIB) suppresses metastasis in a variety of cancers but the mechanism is unclear, thus hampering development of human therapeutics. RhoGDI2 is a guanine nucleotide dissociation inhibitor (GDI) for the Rho family of GTPases thought to primarily bind to Rac1; however, Rac1 activation was not decreased by RhoGDI2 expression in bladder cancer cells. To better understand the GTPase-binding partners for RhoGDI2, a mass spectrometry-based proteomic approach was used in bladder cancer cells. As expected, endogenous RhoGDI2 coimmunoprecipitates with Rac1 and unexpectedly also with RhoC. Further analysis demonstrated that RhoGDI2 negatively regulates RhoC, as knockdown of RhoGDI2 increased RhoC activation in response to serum stimulation. Conversely, overexpression of RhoGDI2 decreased RhoC activation. RhoC promoted bladder cancer cell growth and invasion, as knockdown increased cell doubling time, decreased invasion through Matrigel, and decreased colony formation in soft agar. Importantly, RhoC knockdown reduced in vivo lung colonization by bladder cancer cells following tail vein injection in immunocompromised mice. Finally, unbiased transcriptome analysis revealed a set of genes regulated by RhoGDI2 overexpression and RhoC knockdown in bladder cancer cells. IMPLICATIONS: RhoGDI2 suppresses bladder cancer metastatic colonization via negative regulation of RhoC activity, providing a rationale for the development of therapeutics that target RhoC signaling.


Subject(s)
Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Urinary Bladder Neoplasms/metabolism , rho GTP-Binding Proteins/metabolism , rho Guanine Nucleotide Dissociation Inhibitor beta/metabolism , Animals , Cell Movement , Cell Proliferation , Female , Gene Expression Regulation, Neoplastic , Humans , Mice , Neoplasm Transplantation , Proteomics , Urinary Bladder Neoplasms/pathology , rac1 GTP-Binding Protein/metabolism , rhoC GTP-Binding Protein
3.
BMC Cell Biol ; 14: 42, 2013 Sep 25.
Article in English | MEDLINE | ID: mdl-24063632

ABSTRACT

BACKGROUND: Epithelial tissues depend on intercellular homodimerization of E-cadherin and loss of E-cadherin is central to the epithelial to mesenchymal transition seen in multiple human diseases. Signaling pathways regulate E-cadherin function and cellular distribution via phosphorylation of the cytoplasmic region by kinases such as casein kinases but the protein phosphatases involved have not been identified. RESULTS: This study shows protein Ser/Thr phosphatase-6 catalytic subunit (PP6c) is expressed in epithelial tissue and its mRNA and protein are robustly up-regulated in epithelial cell lines at high vs. low density. PP6c accumulates at adherens junctions, not tight junctions, co-immunoprecipitates with E-cadherin-catenin complexes without a canonical SAPS subunit, and associates directly with the E-cadherin cytoplasmic tail. Inducible shRNA knockdown of PP6c dispersed E-cadherin from the cell surface and this response was reversed by chemical inhibition of casein kinase-1 and prevented by alanine substitution of Ser846 in murine E-cadherin. CONCLUSIONS: PP6c associates with E-cadherin in adherens junctions and is required to oppose casein kinase-1 to maintain cell surface localization of E-cadherin. There is feedback signaling to enhance PP6c transcription and boost protein levels in high density epithelial cells.


Subject(s)
Adherens Junctions/metabolism , Cadherins/genetics , Casein Kinase I/genetics , Phosphoprotein Phosphatases/genetics , RNA, Messenger/genetics , Adherens Junctions/genetics , Adherens Junctions/ultrastructure , Amino Acid Substitution , Caco-2 Cells , Cadherins/metabolism , Casein Kinase I/metabolism , Catenins/genetics , Catenins/metabolism , Cell Adhesion , Epithelial-Mesenchymal Transition/genetics , Feedback, Physiological , Gene Expression Regulation , Humans , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/metabolism , Protein Binding , Protein Structure, Tertiary , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Transcription, Genetic
4.
Cell ; 149(7): 1594-606, 2012 Jun 22.
Article in English | MEDLINE | ID: mdl-22726444

ABSTRACT

Axon pruning and synapse elimination promote neural connectivity and synaptic plasticity. Stereotyped pruning of axons that originate in the hippocampal dentate gyrus (DG) and extend along the infrapyramidal tract (IPT) occurs during postnatal murine development by neurite retraction and resembles axon repulsion. The chemorepellent Sema3F is required for IPT axon pruning, dendritic spine remodeling, and repulsion of DG axons. The signaling events that regulate IPT axon pruning are not known. We find that inhibition of the small G protein Rac1 by the Rac GTPase-activating protein (GAP) ß2-Chimaerin (ß2Chn) mediates Sema3F-dependent pruning. The Sema3F receptor neuropilin-2 selectively binds ß2Chn, and ligand engagement activates this GAP to ultimately restrain Rac1-dependent effects on cytoskeletal reorganization. ß2Chn is necessary for axon pruning both in vitro and in vivo, but it is dispensable for axon repulsion and spine remodeling. Therefore, a Npn2/ß2Chn/Rac1 signaling axis distinguishes DG axon pruning from the effects of Sema3F on repulsion and dendritic spine remodeling.


Subject(s)
Axons/metabolism , Hippocampus/cytology , Hippocampus/metabolism , Neoplasm Proteins/metabolism , Neuropeptides/metabolism , Signal Transduction , rac GTP-Binding Proteins/metabolism , Animals , Dentate Gyrus/metabolism , Humans , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Synapses , rac1 GTP-Binding Protein
5.
Cancer Metastasis Rev ; 31(3-4): 519-28, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22718398

ABSTRACT

RhoGDI2 is a guanine nucleotide dissociation inhibitor (GDI) specific for the Rho family of small GTPases that plays dual opposite roles in tumor progression, being both a promoter in tissues such as breast and a metastasis suppressor in tissues such as the bladder. Despite a clear role for this protein in modulating the invasive and metastatic process, the mechanisms through which RhoGDI2 executes these functions remain unclear. This review will highlight the current state of our knowledge regarding how RhoGDI2 functions in metastasis with a focus on bladder cancer and will also seek to highlight other potential underappreciated avenues through which this protein may affect cancer cell behavior.


Subject(s)
Neoplasm Metastasis , rho Guanine Nucleotide Dissociation Inhibitor beta/physiology , Animals , Apoptosis , Endothelin-1/physiology , GTP Phosphohydrolases/metabolism , Humans , Neoplasm Metastasis/prevention & control , Protein Kinase C-alpha/physiology , Urinary Bladder Neoplasms/pathology
6.
J Biol Chem ; 285(22): 16931-41, 2010 May 28.
Article in English | MEDLINE | ID: mdl-20335173

ABSTRACT

Although the family of chimaerin Rac-GAPs has recently gained significant attention for their involvement in development, cancer, and neuritogenesis, little is known about their molecular regulation. Chimaerins are activated by the lipid second messenger diacylglycerol via their C1 domain upon activation of tyrosine kinase receptors, thereby restricting the magnitude of Rac signaling in a receptor-regulated manner. Here we identified a novel regulatory mechanism for beta2-chimaerin via phosphorylation. Epidermal growth factor or the phorbol ester phorbol 12-myristate 13-acetate caused rapid phosphorylation of beta2-chimaerin on Ser(169) located in the SH2-C1 domain linker region via protein kinase Cdelta, which retained beta2-chimaerin in the cytosol and prevented its C1 domain-mediated translocation to membranes. Furthermore, despite the fact that Ser(169) phosphorylation did not alter intrinsic Rac-GAP activity in vitro, a non-phosphorylatable beta2-chimaerin mutant was highly sensitive to translocation, and displayed enhanced association with activated Rac, enhanced Rac-GAP activity, and anti-migratory properties when expressed in cells. Our results not only revealed a novel regulatory mechanism that facilitates Rac activation, but also identified a novel mechanism of cross-talk between diacylglycerol receptors that restricts beta2-chimaerin relocalization and activation.


Subject(s)
Diglycerides/metabolism , GTPase-Activating Proteins/metabolism , Neoplasm Proteins/chemistry , Protein Kinase C-delta/metabolism , rac GTP-Binding Proteins/metabolism , Animals , COS Cells , Chlorocebus aethiops , Cytosol/metabolism , Diglycerides/chemistry , HeLa Cells , Humans , Mice , Mutation , Neurons/metabolism , Phorbol Esters/chemistry , Phosphorylation , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/chemistry , Signal Transduction
7.
Nat Rev Cancer ; 7(4): 281-94, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17384583

ABSTRACT

Almost three decades after the discovery of protein kinase C (PKC), we still have only a partial understanding of how this family of serine/threonine kinases is involved in tumour promotion. PKC isozymes - effectors of diacylglycerol (DAG) and the main targets of phorbol-ester tumour promoters - have important roles in cell-cycle regulation, cellular survival, malignant transformation and apoptosis. How do PKC isozymes regulate these diverse cellular processes and what are their contributions to carcinogenesis? Moreover, what is the contribution of all phorbol-ester effectors, which include PKCs and small G-protein regulators? We now face the challenge of dissecting the relative contribution of each DAG signal to cancer progression.


Subject(s)
Diglycerides/physiology , Neoplasms/etiology , Protein Kinase C/physiology , Apoptosis , Cell Cycle , Cell Proliferation , Diglycerides/chemistry , Enzyme Activation , GTP Phosphohydrolases/physiology , Humans , Isoenzymes , Neovascularization, Pathologic , Peptide Hydrolases/metabolism , Phorbol Esters/pharmacology
8.
Growth Factors ; 23(4): 245-52, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16338787

ABSTRACT

Growth factors exert their cellular effects through signal transduction pathways that are initiated by the ligation of growth factors to their cell surface receptors. One of the well-established effectors of growth factor receptors is protein kinase C (PKC), a family of serine-threonine kinases that have been known for years as the main target of the phorbol ester tumor promoters. While there is abundant information regarding downstream PKC effectors and partners, how individual PKC isozymes become activated by growth factors and the regulation of receptor function by PKCs is only partially understood. Moreover, the identification of novel "non-kinase" DAG-binding proteins has added a new level of complexity to the field of DAG signaling.


Subject(s)
Diglycerides/physiology , Protein Kinase C/physiology , Receptors, Growth Factor/physiology , Animals , ErbB Receptors/physiology , Growth Substances/physiology , Humans , Isoenzymes/physiology , Neoplasm Proteins/physiology , Phosphorylation , Protein Structure, Tertiary , Proto-Oncogene Proteins c-met/physiology , Signal Transduction
9.
Appl Environ Microbiol ; 69(2): 1159-71, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12571043

ABSTRACT

The analysis of functional diversity and its dynamics in the environment is essential for understanding the microbial ecology and biogeochemistry of aquatic systems. Here we describe the development and optimization of a DNA microarray method for the detection and quantification of functional genes in the environment and report on their preliminary application to the study of the denitrification gene nirS in the Choptank River-Chesapeake Bay system. Intergenic and intragenic resolution constraints were determined by an oligonucleotide (70-mer) microarray approach. Complete signal separation was achieved when comparing unrelated genes within the nitrogen cycle (amoA, nifH, nirK, and nirS) and detecting different variants of the same gene, nirK, corresponding to organisms with two different physiological modes, ammonia oxidizers and denitrifying halobenzoate degraders. The limits of intragenic resolution were investigated with a microarray containing 64 nirS sequences comprising 14 cultured organisms and 50 clones obtained from the Choptank River in Maryland. The nirS oligonucleotides covered a range of sequence identities from approximately 40 to 100%. The threshold values for specificity were determined to be 87% sequence identity and a target-to-probe perfect match-to-mismatch binding free-energy ratio of 0.56. The lower detection limit was 10 pg of DNA (equivalent to approximately 10(7) copies) per target per microarray. Hybridization patterns on the microarray differed between sediment samples from two stations in the Choptank River, implying important differences in the composition of the denitirifer community along an environmental gradient of salinity, inorganic nitrogen, and dissolved organic carbon. This work establishes a useful set of design constraints (independent of the target gene) for the implementation of functional gene microarrays for environmental applications.


Subject(s)
Bacteria/metabolism , Fresh Water/microbiology , Gene Expression Profiling , Nitrogen/metabolism , Oligonucleotide Array Sequence Analysis , Seawater/microbiology , Bacteria/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA, Bacterial/analysis , Ecosystem , Nitrite Reductases/genetics , Nitrite Reductases/metabolism , Oligonucleotide Probes
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