Overlap of cargo binding sites on myosin V coordinates the inheritance of diverse cargoes.

During cell division, organelles are distributed to distinct locations at specific times. For the yeast vacuole, the myosin V motor, Myo2, and its vacuole-specific cargo adaptor, Vac17, regulate where the vacuole is deposited and the timing of vacuole movement. In this paper, we show that Mmr1 functions as a mitochondria-specific cargo adaptor early in the cell cycle and that Mmr1 binds Myo2 at the site that binds Vac17. We demonstrate that Vac17 and Mmr1 compete for binding at this site. Unexpectedly, this competition regulates the volume of vacuoles and mitochondria inherited by the daughter cell. Furthermore, eight of the nine known Myo2 cargo adaptors overlap at one of two sites. Vac17 and Mmr1 overlap at one site, whereas Ypt11 and Kar9 bind subsets of residues that also bind Ypt31/Ypt32, Sec4, and Inp2. These observations predict that competition for access to Myo2 may be a common mechanism to coordinate the inheritance of diverse cargoes.

PTC1 is required for vacuole inheritance and promotes the association of the myosin-V vacuole-specific receptor complex.

Organelle inheritance occurs during cell division. In Saccharomyces cerevisiae, inheritance of the vacuole, and the distribution of mitochondria and cortical endoplasmic reticulum are regulated by Ptc1p, a type 2C protein phosphatase. Here we show that PTC1/VAC10 controls the distribution of additional cargoes moved by a myosin-V motor. These include peroxisomes, secretory vesicles, cargoes of Myo2p, and ASH1 mRNA, a cargo of Myo4p. We find that Ptc1p is required for the proper distribution of both Myo2p and Myo4p. Surprisingly, PTC1 is also required to maintain the steady-state levels of organelle-specific receptors, including Vac17p, Inp2p, and Mmr1p, which attach Myo2p to the vacuole, peroxisomes, and mitochondria, respectively. Furthermore, Vac17p fused to the cargo-binding domain of Myo2p suppressed the vacuole inheritance defect in ptc1Delta cells. These findings suggest that PTC1 promotes the association of myosin-V with its organelle-specific adaptor proteins. Moreover, these observations suggest that despite the existence of organelle-specific receptors, there is a higher order regulation that coordinates the movement of diverse cellular components.

Increased glucocorticoid receptor beta alters steroid response in glucocorticoid-insensitive asthma.

RATIONALE: Glucocorticoids (GCs) are highly effective in the treatment of asthma. However, some individuals have GC-insensitive asthma. OBJECTIVES: To evaluate the functional response to steroids of bronchoalveolar lavage (BAL) cells from sites of airway inflammation from patients with GC-insensitive versus GC-sensitive asthma. As well, to attempt to define the functional role of glucocorticoid receptor (GCR)beta (a splicing variant, and dominant negative inhibitor of, the classic GCRalpha) in controlling GCRalpha nuclear translocation and transactivation at a molecular level. METHODS AND MEASUREMENTS: Fiberoptic bronchoscopy with collection of BAL fluid was performed on seven patients with GC-sensitive asthma and eight patients with GC-insensitive asthma. GCRalpha cellular shuttling in response to 10(-6) M dexamethasone treatment and GCRbeta expression were analyzed in BAL cells by immunofluorescence staining. The effects of overexpression and silencing of GCRbeta mRNA on GCRalpha function were assessed. MAIN RESULTS: Significantly reduced nuclear translocation of GCRalpha in response to steroids was found in BAL cells from patients with GC-insensitive asthma. BAL macrophages from patients with GC-insensitive asthma had significantly increased levels of cytoplasmic and nuclear GCRbeta. It was demonstrated that GCRalpha nuclear translocation and its transactivation properties were proportionately reduced by level of viral transduction of the GCRbeta gene into the DO-11.10 cell line. RNA silencing of GCRbeta mRNA in human BAL macrophages from patients with GC-insensitive asthma resulted in enhanced dexamethasone-induced GCRalpha transactivation. CONCLUSIONS: GC insensitivity is associated with loss of GCRalpha nuclear translocation in BAL cells and elevated GCRbeta, which may inhibit GCRalpha transactivation in response to steroids.

A comparative study of the effect of pigment on drug toxicity in human choroidal melanocytes and retinal pigment epithelial cells.

The aim of this study was to investigate whether the presence of pigment affects the sensitivity of pigmented cells of the eye, retinal pigment epithelium (RPE) and choroidal melanocytes (CMs) to the cytotoxic effects of xenobiotic drugs. Two approaches were used to compare pigmented versus unpigmented cells: RPE cells were repigmented by phagocytosis of synthetic melanin; UVB irradiation was used to induce an increase in pigment in both RPE and CMs. Three drugs known to induce toxicity in the eye, tamoxifen, chloroquine and thioridazine, were used to assess the sensitivity of cells to xenobiotic drugs. RPE cells were more resistant than CMs to the cytotoxic effects of all three drugs by a factor of 5-fold for tamoxifen, 7-fold for thioridazine and 30-fold for chloroquine. When RPE cells were repigmented using synthetic melanin, their sensitivity to tamoxifen was unchanged, they showed a slightly improved response to thioridazine (after 3 days of incubation with this drug), but they showed greatly increased toxicity to chloroquine (after 1 and 3 days of exposure to the drug), suggesting accumulation of this latter drug on the synthetic melanin. UVB irradiation was used to achieve an increase in the pigment content of both RPE and CMs. CMs were much more sensitive to UVB than RPE cells. CMs appeared to synthesise pigment via DOPA oxidase activity; RPE cells showed an increase in fluorescent material independent of any detectable DOPA oxidase activity. Irrespective of the nature of the pigment that UVB induced in melanocytes and RPE cells, their subsequent response to thioridazine and chloroquine was unchanged by the presence of this pigment.

Characterization of an in vitro model of human melanoma invasion based on reconstructed human skin.

The purpose of this study was to compare the invasive properties of normal human cutaneous melanocytes and of a cutaneous melanoma cell line (HBL) in a three-dimensional model of reconstructed human skin. Specifically, we asked to what extent the pigmentary and invasive behaviour of both cells is influenced by their interaction with adjacent skin cells (keratinocytes and fibroblasts) and the basement membrane (BM). In the presence of a BM, normal human melanocytes within this model remained within the basal layer of keratinocytes and did not pigment spontaneously. When the BM was removed, melanocytes were found suprabasally and pigmented extensively. No significant invasion of melanocytes into the dermis was detected in the presence or absence of the BM. HBL melanoma cells showed no significant ability to invade into the dermis in the absence of other cells, irrespective of the presence or absence of the BM. However, when added to keratinocytes and fibroblasts, HBL cells showed a capacity to invade into the dermis, both in the presence and absence of the BM. Associated with HBL invasion into the dermis, we noted significant keratinocyte entry into the dermis. On their own, keratinocytes entered the dermis in the absence of the BM but showed no significant penetration into the dermis when the BM was present. In summary, this model demonstrates clear differences between melanocytes and a melanoma cell line with respect to their invasive properties. It also allows demonstration of interactions between cells, and between cells and the BM. The study also provides evidence for a synergistic interaction between this melanoma cell line and keratinocytes in penetrating the BM.

Melanoma cell migration is upregulated by tumour necrosis factor-alpha and suppressed by alpha-melanocyte-stimulating hormone.

We reported recently that the inflammatory cytokine tumour necrosis factor alpha (TNF-alpha) can upregulate integrin expression, cell attachment and invasion of cells through fibronectin in a human melanoma cell line (HBL). Furthermore, the actions of TNF-alpha were suppressed by the addition of an anti-inflammatory peptide alpha-melanocyte-stimulating hormone (alpha-MSH). In the current study, we extend this work investigating to what extent TNF-alpha might stimulate melanoma invasion by promoting cell migration and whether alpha-MSH is also inhibitory. Two human melanoma cell lines were examined in vitro (HBL and C8161) using a scratch migration assay. Analysis using either time-lapse video microscopy or imaging software analysis of migrating 'fronts' of cells revealed that C8161 cells migrated more rapidly than HBL cells. However, when cells were stimulated with TNF-alpha both cell types responded with a significant increase in migration distance over a 16-26 h incubation time. alpha-Melanocyte-stimulating hormone had an inhibitory effect on TNF-alpha-stimulated migration for HBL cells, completely blocking migration at 10(-9) M. In contrast, C8161 cells did not respond to alpha-MSH (as these cells have a loss-of-function melanocortin-1 receptor). However, stable transfection of C8161 cells with the wild-type melanocortin-1 receptor produced cells whose migration was significantly inhibited by alpha-MSH. In addition, the use of a neutralising antibody to the beta(1)-integrin subunit significantly reduced migration in both cell types. This data therefore supports an inflammatory environment promoting melanoma cell migration, and in addition shows that alpha-MSH can inhibit inflammatory stimulated migration. The data also support a fundamental role of the beta(1)-integrin receptor in melanoma cell migration.

Oestrogenic steroids and melanoma cell interaction with adjacent skin cells influence invasion of melanoma cells in vitro.

The invasion of melanoma is complex and multi-staged and involves changes in both cell/extracellular matrix (ECM) and cell/cell interactions. Female steroids and alpha-MSH have also been reported to influence metastatic melanoma progression, but their mechanisms of action are unknown. Accordingly, our aim was to establish in vitro models to examine (a) the influence of sex steroids and alpha-melanocyte-stimulating hormone (alpha-MSH) on tumour invasion and the influence of (b) ECM proteins and (c) adjacent cells on melanoma invasion. In the first model, melanoma cell invasion through fibronectin over 20 hr under serum-free conditions was used to investigate the effects of 17beta-oestradiol and oestrone on the invasion of human melanoma cell lines, A375-SM and HBL. A375-SM, but not HBL cells, proved very susceptible to inhibition by female steroids. However, invasion of the HBL line was inhibited by alpha-MSH. Using the second model of reconstructed human skin based on de-epidermised acellular dermis, we found that the HBL cells on their own failed to invade into the dermis (irrespective of the presence or absence of the basement membrane). However, there was a significant synergistic interaction between keratinocytes, fibroblasts and HBL cells, such that a modest invasion of HBLs into the dermis was seen within 2 weeks when other skin cells were present. In contrast, A375-SM cells showed a significant ability to invade the dermis in the absence of other cells, with less invasion when other skin cells were present. In summary, these models have provided new information on the extent to which melanoma cell invasion is sensitive to oestrogenic steroids and to alpha-MSH and to interaction, not only with adjacent skin cells but also to the presence of basement membrane antigens.

Anti-inflammatory and anti-invasive effects of alpha-melanocyte-stimulating hormone in human melanoma cells.

Alpha-melanocyte stimulating hormone (alpha-MSH) is known to have pleiotrophic functions including pigmentary, anti-inflammatory, antipyretic and immunoregulatory roles in the mammalian body. It is also reported to influence melanoma invasion with levels of alpha-, beta- and gamma-MSH correlated clinically with malignant melanoma development, but other studies suggest alpha-MSH acts to retard invasion. In the present study, we investigated the action of alpha-MSH on three human melanoma cell lines (HBL, A375-SM and C8161) differing in metastatic potential. alpha-melanocyte-simulating hormone reduced invasion through fibronectin and also through a human reconstructed skin composite model for the HBL line, and inhibited proinflammatory cytokine-stimulated activation of the NF-kappaB transcription factor. However, A375-SM and C8161 cells did not respond to alpha-MSH. Immunofluorescent microscopy and Western blotting identified melanocortin-1 receptor (MC-1R) expression for all three lines and MC-2R on HBL and A375-SM lines. Receptor binding identified a similar affinity for alpha-MSH for all three lines with the highest number of binding sites on HBL cells. Only the HBL melanoma line demonstrated a detectable cyclic adenosine monophosphate (cAMP) response to alpha-MSH, although all three lines responded to acute alpha-MSH addition (+(-)-N(6)-(2-phenylisopropyl)-adenosine (PIA)) with an elevation in intracellular calcium. The nonresponsive lines displayed MC-1R polymorphisms (C8161, Arg (wt) 151/Cys 151; A375-SM, homozygous Cys 151), whereas the HBL line was wild type. Stable transfection of the C8161 line with wild-type MC-1R produced cells whose invasion was significantly inhibited by alpha-MSH. From this data, we conclude that alpha-MSH can reduce melanoma cell invasion and protect cells against proinflammatory cytokine attack in cells with the wild-type receptor (HBL).

Evidence for the existence of a novel pregnancy-associated soluble variant of the vascular endothelial growth factor receptor, Flt-1.

Angiogenesis is essential in physiological processes including ovulation, implantation and pregnancy. One of the most potent regulators is the cytokine vascular endothelial growth factor (VEGF). We provide evidence for a novel pregnancy-associated soluble variant of the VEGF receptor Flt-1. VEGF ranged from undetectable to 157.3 pg/ml (mean 49.9 pg/ml, SD 48.4 pg/ml) in plasma samples from normal volunteers (n = 10), but was undetectable in plasma from pregnant women (n = 12) and amniotic fluid (n = 10). Recoveries of spiked VEGF were poor in pregnancy-related samples, indicating the presence of VEGF-binding activity which was confirmed using biosensor and chromatographic techniques. Partial purification and protein sequencing indicated a novel soluble form of Flt-1 with a subunit size of 150 kDa. Normally present as a multimeric structure of approximately 400-550 kDa, complexes of 600-700 kDa were formed following binding of multiple VEGF molecules. Reverse transcriptase polymerase chain reaction of Flt-1 in placenta, amnion, chorion, human umbilical vein endothelial cells and cord blood samples produced bands of the predicted sizes but failed to identify any additional RNA species, and possible reasons for this are discussed. Soluble Flt-1 may be important in regulating the actions of VEGF in angiogenesis and trophoblast invasion and may have therapeutic implications in diseases with inappropriate angiogenesis such as proliferative retinopathies and cancer.