two-methods-for-establishing-primary-human-endometrial-stromal-cells

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

University of Virginia

Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca(2+)-induced differentiation, stress-induced apoptosis, and modulating TGF-beta signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin alpha and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor alpha-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity. Inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor alpha-induced nitrosylation and association with importin alpha and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution.

S-nitrosylation regulates nuclear translocation of chloride intracellular channel protein CLIC4.

Chloride intracellular channel (CLIC) 4 is a member of a redox-regulated, metamorphic multifunctional protein family, first characterized as intracellular chloride channels. Current knowledge indicates that CLICs participate in signaling, cytoskeleton integrity and differentiation functions of multiple tissues. In metabolically stressed skin keratinocytes, cytoplasmic CLIC4 is S-nitrosylated and translocates to the nucleus where it enhances transforming growth factor-β (TGF-β) signaling by protecting phospho-Smad 2 and 3 from dephosphorylation. CLIC4 expression is diminished in multiple human epithelial cancers, and the protein is excluded from the nucleus. We now show that CLIC4 expression is reduced in chemically induced mouse skin papillomas, mouse and human squamous carcinomas and squamous cancer cell lines, and the protein is excluded from the nucleus. The extent of reduction in CLIC4 coincides with progression of squamous tumors from benign to malignant. Inhibiting antioxidant defense in tumor cells increases S-nitrosylation and nuclear translocation of CLIC4. Adenoviral-mediated reconstitution of nuclear CLIC4 in squamous cancer cells enhances TGF-β-dependent transcriptional activity and inhibits growth. Adenoviral targeting of CLIC4 to the nucleus of tumor cells in orthografts inhibits tumor growth, whereas elevation of CLIC4 in transgenic epidermis reduces de novo chemically induced skin tumor formation. In parallel, overexpression of exogenous CLIC4 in squamous tumor orthografts suppresses tumor growth and enhances TGF-β signaling. These results indicate that CLIC4 suppresses the growth of squamous cancers, that reduced CLIC4 expression and nuclear residence detected in cancer cells is associated with the altered redox state of tumor cells and the absence of detectable nuclear CLIC4 in cancers contributes to TGF-β resistance and enhances tumor development.

CLIC4 is a tumor suppressor for cutaneous squamous cell cancer.

Nuclear translocation of cytosolic CLIC4 is an essential feature of its proapoptotic and prodifferentiation functions. Here we demonstrate that CLIC4 is induced concurrently with inducible nitric oxide synthase (iNOS) and S-nitrosylated in proinflammatory peritoneal macrophages. Chemical inhibition or genetic ablation of iNOS inhibits S-nitrosylation and nuclear translocation of CLIC4. In macrophages, iNOS-induced nuclear CLIC4 coincides with the pro- to anti-inflammatory transition of the cells because IL-1β and CXCL1 mRNA remain elevated in CLIC4 and iNOS knockout macrophages at late time points, whereas TNFα mRNA is elevated only in the iNOS knockout macrophages. Active IL-1β remains elevated in CLIC4 knockout macrophages and in macrophages in which CLIC4 nuclear translocation is prevented by the NOS inhibitor l-NAME. Moreover, overexpression of nuclear-targeted CLIC4 down-regulates IL-1β in stimulated macrophages. In mice, genetically null for CLIC4, the number of phagocytosing macrophages stimulated by LPS is reduced. Thus, iNOS-induced nuclear CLIC4 is an essential part of the macrophage deactivation program.

Kasey Jividen

Department of Pathology

University of Virginia

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Kasey Jividen

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pathology

University of Virginia

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Department of Pathology