Biomedical subjects

Tamsin Thomas

Publications and source records attributed to Tamsin Thomas.

6 recordsLinked to original sources

PubMed2006

Cx26 inhibits breast MDA-MB-435 cell tumorigenic properties by a gap junctional intercellular communication-independent mechanism.

It has been well established that the restoration of connexin expression in tumor cells often leads to a partial reversion of tumor cell phenotypes and increased growth control. In this study, a less-aggressive variant of MDA-MB-435 cells obtained from the MDA-MB-435 cell line was engineered to express gap junctional intercellular communication (GJIC)-competent Cx26, a GJIC-incompetent cell surface transported GFP-Cx26 chimera, or a Golgi apparatus-localized, disease-linked Cx26 mutant (D66H). Collectively, these cell lines were designed to establish whether Cx26 regulates tumor properties, such as migration, invasion and growth, by (i) a GJIC-dependent pathway; (ii) a mechanism requiring Cx26 transport to the cell surface; or (iii) a mechanism where Cx26 expression alone was sufficient. The expression of Cx26 and green fluorescent protein (GFP)-Cx26 decreased cell proliferation while all three Cx26 variants inhibited anchorage-independent cell growth. All three Cx26 variants also altered the distribution of filamentous actin and significantly reduced cell migration, while only the D66H mutant failed to inhibit cell invasion through matrigel. Furthermore, expression of all the Cx26 variants reduced the levels of total beta1 integrin, and decreased the activity of matrix metalloproteinase-9 (MMP-9) while increasing tissue inhibitors of MMP-1 (TIMP-1) activity. Interestingly, the expression of Cx43 regulated the same gene products without significantly affecting the tumorigenic properties of the MDA-MB-435 cells. Together, these results suggest that Cx26 expression, independent of the necessity for gap junctional intercellular communication, partially reverted MDA-MB-435 cell properties associated with tumorigenesis, and regulated the expression of genes important in cell migration and invasion.

Animals↗

PubMed2005

Mechanisms of Cx43 and Cx26 transport to the plasma membrane and gap junction regeneration.

Previous reports have suggested that Cx26 exhibits unique intracellular transport pathways en route to the cell surface compared with other members of the connexin family. To directly examine and compare nascent and steady-state delivery of Cx43 and Cx26 to the plasma membrane and gap junction biogenesis we expressed fluorescent-protein-tagged Cx43 and Cx26 in BICR-M1Rk and NRK cells. Static and time-lapse imaging revealed that both connexins were routed through the Golgi apparatus prior to being transported to the cell surface, a process inhibited in the presence of brefeldin A (BFA) or the expression of a dominant-negative form of Sar1 GTPase. During recovery from BFA, time-lapse imaging of nascent connexin Golgi-to-plasma membrane delivery revealed many dynamic post-Golgi carriers (PGCs) originating from the distal side of the Golgi apparatus consisting of heterogeneous vesicles and long, tubular-like extensions. Vesicles and tubular extensions were also observed in HBL-100 cells expressing a human, disease-linked, Golgi-localized Cx26 mutant, D66H-GFP. A diffuse cell surface rim of fluorescent-protein-tagged wild-type connexins was observed prior to the appearance of punctate gap junctions, which suggests that random fusion of PGCs occurred with the plasma membrane followed by lateral diffusion of connexins into clusters. Fluorescence recovery after photobleaching studies revealed that Cx26-YFP was more mobile within gap junction plaques compared with Cx43-GFP. Intriguingly, Cx43-GFP delivery and gap junction regeneration was inhibited by BFA and nocodazole, whereas Cx26-GFP delivery was prevented by BFA but not nocodazole. Collectively, these studies suggest that during gap junction biogenesis two phylogenetically distinct members of the connexin family, Cx43 and Cx26, share common secretory pathways, types of transport intermediates and turnover dynamics but differ in their microtubule-dependence and mobility within the plasma membrane, which might reflect differences in binding to protein scaffolds.

Animals↗

PubMed2004

Functional domain mapping and selective trans-dominant effects exhibited by Cx26 disease-causing mutations.

Mutations in Cx26 are a major cause of autosomal dominant and recessive forms of sensorineural deafness. Some mutations in Cx26 are associated not only with deafness but also with skin disease. We examined the subcellular localization and function of two green fluorescent protein (GFP)-tagged Cx26 point mutants that exhibit both phenotypes, G59A-GFP and D66H-GFP. D66H-GFP was retained within the brefeldin A-insensitive trans-Golgi network, whereas a population of G59A-GFP was transported to the cell surface. Neither G59A nor D66H formed gap junctions that were permeable to small fluorescent dyes, suggesting they are loss-of-function mutations. When co-expressed with wild-type Cx26, both G59A and D66H exerted dominant-negative effects on Cx26 function. G59A also exerted a trans-dominant negative effect on co-expressed wild type Cx32 and Cx43, whereas D66H exerted a trans-dominant negative effect on Cx43 but not Cx32. We propose that the severity of the skin disease is dependent on the specific nature of the Cx26 mutation and the trans-dominant selectivity of the Cx26 mutants on co-expressed connexins. Additional systematic mutations at residue D66, in which the overall charge of this motif was altered, suggested that the first extracellular loop is critical for Cx26 transport to the cell surface as well as function of the resulting gap junction channels.

Amino Acid Substitution↗

PubMedDate not supplied

Rat epidermal keratinocytes as an organotypic model for examining the role of Cx43 and Cx26 in skin differentiation.

In order to characterize connexin expression and regulation in the epidermis, we have characterized a rat epidermal keratinocyte (REK) cell line that is phenotypically similar to basal keratinocytes in that they have the ability to differentiate into organotypic epidermis consisting of a basal cell layer, 2-3 suprabasal cell layers, and a cornified layer. RT-PCR revealed that REK cells express mRNA for Cx26, Cx31, Cx31.1, Cx37, and Cx43, which mimics the reported connexin profile for rat tissue. In addition, we report the expression of Cx30, Cx30.3, Cx40, and Cx45 in rat keratinocytes, highlighting the complexity of the connexin complement in rat epidermis. Furthermore, 3-dimensional analysis of organotypic skin revealed that Cx26 and Cx43 are exquisitely regulated during the differentiation process. The life-cycle of these connexins including their expression, transport, assembly into gap junctions, internalization, and degradation are elegantly depicted in organotypic epidermis as keratinocytes proceed from differentiation to programmed cell death.

Animals↗

PubMedDate not supplied

Transport and function of cx26 mutants involved in skin and deafness disorders.

We examined the subcellular localization and function of several Cx26 mutants that exhibit both sensorineural deafness and various skin disease phenotypes. To facilitate these aims, all Cx26 mutants were tagged at the carboxyl-terminal with green fluorescent protein (GFP), which has previously been shown not to affect Cx26 transport, assembly or function. In this article we focus on two point mutations (R75W and DeltaE42) that occur in the first extracellular loop region of Cx26, a region hypothesized to be critical for correct hemichannel docking between contacting cells. In gap junctional intercellular communication (GJIC)-deficient HeLa cells, both R75W-GFP and DeltaE42-GFP were transported to the cell surface and assembled into gap junction-like structures. Neither R75W-GFP nor DeltaE42-GFP formed gap junctions that were permeable to Lucifer Yellow suggesting they are loss-of-function mutations. We also examined the phenotype of these two mutations in a rat epidermal keratinocyte (REK) cell line that is capable of undergoing differentiation. Using antibodies against several members of the connexin family reportedly expressed by epidermal keratinocytes, we found these cells endogenously expressed Cx43 and Cx26 but not Cx30, Cx32, or Cx37. When expressed in REK cells, similar to in HeLa cells, R75W-GFP and DeltaE42-GFP were assembled at the cell surface into structures that resembled gap junctions. Future experiments will examine the effect of the Cx26 mutants on the function and differentiation of these epidermal keratinocytes.

Animals↗

PubMedDate not supplied

Connexin26 regulates the expression of angiogenesis-related genes in human breast tumor cells by both GJIC-dependent and -independent mechanisms.

We previously reported that over-expression of connexins in mammary tumor cells retarded tumor growth in vivo in the absence of appreciable gap junction formation, highlighting a possible connexin-linked, but gap junctional intercellular communication (GJIC)-independent mechanism. In the current study, we engineered GJIC-deficient MDA-MB-435 human breast tumor cells to express a chimeric Cx26 where the green fluorescent protein was fused to the amino-terminal of Cx26 (GFP-Cx26). Characterization of this chimeric protein revealed that GFP-Cx26 assembled into non-functional gap junction-like clusters that were impermeable to Lucifer Yellow. In contrast, expression of wild-type Cx26 or Cx26 tagged at the carboxy terminal with yellow fluorescent protein, efficiently rescued GJIC in these tumor cells. Interestingly, by screening 96 tumor-related genes, we observed that the expression of Cx26 or GFP-Cx26 in the tumor cells up-regulated both the transcription and the translation of thrombospondin-1 (TSP-1), an anti-angiogenic molecule. Affymetrix array analysis extended the list of Cx26 or GFP-Cx26 regulated genes by ten candidates including connective tissue growth factor (CTGF), another angiogenesis-related gene. CTGF mRNA and protein levels were found to be down-regulated by both Cx26 and GFP-Cx26. Thus, our data indicates that Cx26 regulates angiogenesis-related molecules by mechanisms that are both GJIC-dependent and -independent.

Connective Tissue Growth Factor↗