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1.
Int J Mol Sci ; 25(18)2024 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-39337350

RESUMEN

The basal cell maintains the airway's respiratory epithelium as the putative resident stem cell. Basal cells are known to self-renew and differentiate into airway ciliated and secretory cells. However, it is not clear if every basal cell functions as a stem cell. To address functional heterogeneity amongst the basal cell population, we developed a novel monoclonal antibody, HLO1-6H5, that identifies a subset of KRT5+ (cytokeratin 5) basal cells. We used HLO1-6H5 and other known basal cell-reactive reagents to isolate viable airway subsets from primary human airway epithelium by Fluorescence Activated Cell Sorting. Isolated primary cell subsets were assessed for the stem cell capabilities of self-renewal and differentiation in the bronchosphere assay, which revealed that bipotent stem cells were, at minimum 3-fold enriched in the HLO1-6H5+ cell subset. Crosslinking-mass spectrometry identified the HLO1-6H5 target as a glycosylated TFRC/CD71 (transferrin receptor) proteoform. The HLO1-6H5 antibody provides a valuable new tool for identifying and isolating a subset of primary human airway basal cells that are substantially enriched for bipotent stem/progenitor cells and reveals TFRC as a defining surface marker for this novel cell subset.


Asunto(s)
Diferenciación Celular , Células Epiteliales , Queratina-5 , Mucosa Respiratoria , Células Madre , Humanos , Células Madre/citología , Células Madre/metabolismo , Queratina-5/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Receptores de Transferrina/metabolismo , Anticuerpos Monoclonales , Antígenos CD/metabolismo , Células Cultivadas , Citometría de Flujo/métodos , Biomarcadores/metabolismo , Separación Celular/métodos
2.
J Cell Sci ; 131(5)2018 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-29361528

RESUMEN

Defects in assembly of gap junction-forming proteins, called connexins (Cxs), are observed in a variety of cancers. Connexin32 (Cx32; also known as GJB1) is expressed by the polarized cells in epithelia. We discovered two dileucine-based motifs, which govern the intracellular sorting and endocytosis of transmembrane proteins, in the C-terminal tail of Cx32 and explored their role in regulating its endocytosis and gap junction-forming abilities in pancreatic and prostate cancer cells. One motif, designated as LI, was located near the juxtamembrane domain, whereas the other, designated as LL, was located distally. We also discovered a non-canonical motif, designated as LR, in the C-terminal tail. Our results showed that rendering these motifs non-functional had no effect on the intracellular sorting of Cx32. However, rendering the LL or LR motif nonfunctional enhanced the formation of gap junctions by inhibiting Cx32 endocytosis by the clathrin-mediated pathway. Rendering the LI motif nonfunctional inhibited gap junction formation by augmenting the endocytosis of Cx32 via the LL and LR motifs. Our studies have defined distinct roles of these motifs in regulating the endocytosis of Cx32 and its gap junction-forming ability.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Clatrina/metabolismo , Conexinas/genética , Endocitosis/genética , Uniones Comunicantes/genética , Secuencias de Aminoácidos/genética , Línea Celular Tumoral , Polaridad Celular/genética , Clatrina/genética , Células Epiteliales/metabolismo , Humanos , Leucina/genética , Masculino , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteína beta1 de Unión Comunicante
3.
J Biol Chem ; 290(8): 4647-4662, 2015 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-25548281

RESUMEN

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size.


Asunto(s)
Conexinas/biosíntesis , Uniones Comunicantes/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neoplasias Pancreáticas/metabolismo , Neoplasias de la Próstata/metabolismo , Línea Celular Tumoral , Conexinas/genética , Uniones Comunicantes/genética , Uniones Comunicantes/patología , Humanos , Masculino , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Permeabilidad , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Estructura Terciaria de Proteína , Proteína beta1 de Unión Comunicante
4.
PLoS One ; 9(9): e106437, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25188420

RESUMEN

1α-25(OH)2 vitamin D3 (1-25D), an active hormonal form of Vitamin D3, is a well-known chemopreventive and pro-differentiating agent. It has been shown to inhibit the growth of several prostate cancer cell lines. Gap junctions, formed of proteins called connexins (Cx), are ensembles of cell-cell channels, which permit the exchange of small growth regulatory molecules between adjoining cells. Cell-cell communication mediated by gap junctional channels is an important homeostatic control mechanism for regulating cell growth and differentiation. We have investigated the effect of 1-25D on the formation and degradation of gap junctions in an androgen-responsive prostate cancer cell line, LNCaP, which expresses retrovirally-introduced Cx32. Connexin32 is expressed by the luminal and well-differentiated cells of normal prostate and prostate tumors. Our results document that 1-25D enhances the expression of Cx32 and its subsequent assembly into gap junctions. Our results further show that 1-25D prevents androgen-regulated degradation of Cx32, post-translationally, independent of androgen receptor (AR)-mediated signaling. Finally, our findings document that formation of gap junctions sensitizes Cx32-expressing LNCaP cells to the growth inhibitory effects of 1-25D and alters their morphology. These findings suggest that the growth-inhibitory effects of 1-25D in LNCaP cells may be related to its ability to modulate the assembly of Cx32 into gap junctions.


Asunto(s)
Andrógenos/metabolismo , Colecalciferol/farmacología , Uniones Comunicantes/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Conexinas/metabolismo , Humanos , Masculino , Retinoides/farmacología , Proteína beta1 de Unión Comunicante
5.
Mol Biol Cell ; 24(6): 715-33, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23363606

RESUMEN

The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell-cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif-deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43's endocytosis and assembly into gap junctions.


Asunto(s)
Conexina 43/metabolismo , Endocitosis , Uniones Comunicantes/metabolismo , Neoplasias Pancreáticas/metabolismo , Comunicación Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Clatrina/metabolismo , Técnicas de Cocultivo , Conexina 26 , Conexina 43/genética , Conexinas , Humanos , Mutación , Fosforilación , Fosfoserina/metabolismo , Transporte de Proteínas , Interferencia de ARN , ARN Interferente Pequeño
6.
PLoS One ; 7(4): e32846, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22514600

RESUMEN

The retinoids, the natural or synthetic derivatives of Vitamin A (retinol), are essential for the normal development of prostate and have been shown to modulate prostate cancer progression in vivo as well as to modulate growth of several prostate cancer cell lines. 9-cis-retinoic acid and all-trans-retinoic acid are the two most important metabolites of retinol. Gap junctions, formed of proteins called connexins, are ensembles of intercellular channels that permit the exchange of small growth regulatory molecules between adjoining cells. Gap junctional communication is instrumental in the control of cell growth. We examined the effect of 9-cis-retinoic acid and all-trans retinoic acid on the formation and degradation of gap junctions as well as on junctional communication in an androgen-responsive prostate cancer cell line, LNCaP, which expressed retrovirally introduced connexin32, a connexin expressed by the luminal cells and well-differentiated cells of prostate tumors. Our results showed that 9-cis-retinoic acid and all-trans retinoic acid enhanced the assembly of connexin32 into gap junctions. Our results further showed that 9-cis-retinoic acid and all-trans-retinoic acid prevented androgen-regulated degradation of gap junctions, post-translationally, independent of androgen receptor mediated signaling. Finally, our findings showed that formation of gap junctions sensitized connexin32-expressing LNCaP cells to the growth modifying effects of 9-cis-retinoic acid, all-trans-retinoic acid and androgens. Thus, the effects of retinoids and androgens on growth and the formation and degradation of gap junctions and their function might be related to their ability to modulate prostate growth and cancer.


Asunto(s)
Andrógenos/farmacología , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Retinoides/farmacología , Alitretinoína , Línea Celular Tumoral , Conexinas/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Tretinoina/farmacología , Proteína beta1 de Unión Comunicante
7.
Int J Syst Evol Microbiol ; 59(Pt 9): 2291-6, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19620380

RESUMEN

The taxonomic position of a yellow-pigmented bacterial strain, designated DS20T, isolated from a hexachlorocyclohexane dump site at Lucknow, India was determined based on a polyphasic taxonomic characterization. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain DS20T occupied a distinct phylogenetic position in the Sphingobium cluster, showing highest similarity with 'Pseudomonas abikonensis' IAM 12404 (98.8%), followed by Sphingobium rhizovicinum CC-FH12-1T (97.4%) and Sphingobium olei IMMIB HF-1T (97.2%). Therefore, the taxonomic characterization of 'P. abikonensis' NBRC 16140 was also undertaken. Phylogenetic, chemotaxonomic and morphological analyses, based on signature sequences, DNA-DNA hybridizations, fatty acid profiles, physiological characterizations and polar lipid profiles confirmed that both strains DS20T and 'P. abikonensis' NBRC 16140 represent two distinct species of the genus Sphingobium. Therefore, two novel Sphingobium species are proposed, Sphingobium lactosutens sp. nov. (type strain, DS20T=CCM 7540T=MTCC 9471T) and Sphingobium abikonense sp. nov. (type strain, NBRC 16140T=IAM 12404T=KCTC 2864T).


Asunto(s)
Hexaclorociclohexano/metabolismo , Aceites/metabolismo , Microbiología del Suelo , Contaminantes del Suelo/metabolismo , Sphingomonadaceae/clasificación , Sphingomonadaceae/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Ácidos Grasos/análisis , India , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fosfolípidos/análisis , Filogenia , Pigmentos Biológicos/biosíntesis , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Sphingomonadaceae/química , Sphingomonadaceae/genética
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