RESUMEN
Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.
Asunto(s)
Antiportadores/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mutación/genética , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Antiportadores/química , Proteínas Portadoras , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas de la Matriz de Golgi , Células HEK293 , Humanos , Inmunoprecipitación , Proteínas de la Membrana , Proteínas de Transporte de Membrana , Modelos Biológicos , Dominios PDZ , Péptidos/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Transportadores de SulfatoRESUMEN
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 ComunicanteRESUMEN
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 ComunicanteRESUMEN
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 PequeñoRESUMEN
Uroplakin (UP)3a is critical for urinary tract development and function; however, its role in these processes is unknown. We examined the function of the UP3a-like protein Upk3l, which was expressed at the apical surfaces of the epithelial cells that line the pronephric tubules (PTs) of the zebrafish pronephros. Embryos treated with upk3l-targeted morpholinos showed decreased pronephros function, which was attributed to defects in PT epithelial cell morphogenesis and polarization including: loss of an apical brush border and associated phospho-ERM proteins, apical redistribution of the basolateral Na(+)/K(+)-ATPase, and altered or diminished expression of the apical polarity complex proteins Prkcz (atypical protein kinase C zeta) and Pard3 (Par3). Upk3l missing its C-terminal cytoplasmic domain or containing mutations in conserved tyrosine or proline residues did not rescue, or only partially rescued the effects of Upk3l depletion. Our studies indicate that Upk3l promotes epithelial polarization and morphogenesis, likely by forming or stimulating interactions with cytoplasmic signaling or polarity proteins, and that defects in this process may underlie the pathology observed in UP3a knockout mice or patients with renal abnormalities that result from altered UP3a expression.
Asunto(s)
Polaridad Celular , Células Epiteliales/citología , Túbulos Renales/citología , Túbulos Renales/crecimiento & desarrollo , Morfogénesis , Uroplaquina III/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Perros , Edema Cardíaco/genética , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Riñón/anomalías , Túbulos Renales/fisiología , Túbulos Renales/fisiopatología , Ratones , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Ratas , Anomalías Urogenitales/genética , Uroplaquina III/química , Uroplaquina III/deficiencia , Uroplaquina III/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
It is as yet unknown how the assembly of connexins (Cx) into gap junctions (GJ) is initiated upon cell-cell contact. We investigated whether the trafficking and assembly of Cx43 and Cx32 into GJs were contingent upon cell-cell adhesion mediated by E-cadherin. We also examined the role of the carboxyl termini of these Cxs in initiating the formation of GJs. Using cadherin and Cx-null cells, and by introducing Cx43 and Cx32, either alone or in combination with E-cadherin, our studies demonstrated that E-cadherin-mediated cell-cell adhesion was neither essential nor sufficient to initiate GJ assembly de novo in A431D human squamous carcinoma cells. However, E-cadherin facilitated the growth and assembly of preformed GJs composed of Cx43, although the growth of cells on Transwell filters was required to initiate the assembly of Cx32. Our results also documented that the carboxyl termini of both Cxs were required in this cell type to initiate the formation of GJs de novo. Our findings also showed that GJ puncta composed of Cx43 co-localized extensively with ZO-1 and actin fibers at cell peripheries and that ZO-1 knockdown attenuated Cx43 assembly. These findings suggest that the assembly of Cx43 and Cx32 into GJs is differentially modulated by E-cadherin-mediated cell-cell adhesion and that direct or indirect cross-talk between carboxyl tails of Cxs and actin cytoskeleton via ZO-1 may regulate GJ assembly and growth.
Asunto(s)
Cadherinas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Adhesión Celular , Conexina 43/metabolismo , Conexinas/metabolismo , Uniones Comunicantes/fisiología , Actinas/metabolismo , Biotinilación , Western Blotting , Cadherinas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Comunicación Celular , Permeabilidad de la Membrana Celular , Conexina 43/genética , Conexinas/genética , Humanos , Técnicas para Inmunoenzimas , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Fracciones Subcelulares , Células Tumorales Cultivadas , Proteína de la Zonula Occludens-1 , Proteína beta1 de Unión ComunicanteRESUMEN
The constituent proteins of gap junctions, called connexins (Cxs), have a short half-life. Despite this, the physiological stimuli that control the assembly of Cxs into gap junctions and their degradation have remained poorly understood. We show here that in androgen-responsive human prostate cancer cells, androgens control the expression level of Cx32-and hence the extent of gap junction formation-post-translationally. In the absence of androgens, a major fraction of Cx32 is degraded presumably by endoplasmic reticulum-associated degradation, whereas in their presence, this fraction is rescued from degradation. We also show that Cx32 and Cx43 degrade by a similar mechanism. Thus, androgens regulate the formation and degradation of gap junctions by rerouting the pool of Cxs, which normally would have been degraded from the early secretory compartment, to the cell surface, and enhancing assembly into gap junctions. Androgens had no significant effect on the formation and degradation of adherens and tight junction-associated proteins. The findings that in a cell culture model that mimics the progression of human prostate cancer, degradation of Cxs, as well as formation of gap junctions, are androgen-dependent strongly implicate an important role of junctional communication in the prostate morphogenesis and oncogenesis.