Identification and functional analysis of SKA2 interaction with the glucocorticoid receptor.

Glucocorticoid (GC) receptors (GRs) have profound anti-survival effects on human small cell lung cancer (SCLC). To explore the basis of these effects, protein partners for GRs were sought using a yeast two-hybrid screen. We discovered a novel gene, FAM33A, subsequently identified as a SKA1 partner and involved in mitosis, and so renamed Ska2. We produced an anti-peptide antibody that specifically recognized full-length human SKA2 to measure expression in human cell lines and tissues. There was a wide variation in expression across multiple cell lines, but none was detected in the liver cell line HepG2. A xenograft model of human SCLC had intense staining and archival tissue revealed SKA2 in several human lung and breast tumours. SKA2 was found in the cytoplasm, where it co-localized with GR, but nuclear expression of SKA2 was seen in breast tumours. SKA2 overexpression increased GC transactivation in HepG2 cells while SKA2 knockdown in A549 human lung epithelial cells decreased transactivation and prevented dexamethasone inhibition of proliferation. GC treatment decreased SKA2 protein levels in A549 cells, as did Staurosporine, phorbol ester and trichostatin A; all agents that inhibit cell proliferation. Overexpression of SKA2 potentiated the proliferative response to IGF-I exposure, and knockdown with shRNA caused cells to arrest in mitosis. SKA2 has recently been identified in HeLa S3 cells as part of a complex, which is critical for spindle checkpoint silencing and exit from mitosis. Our new data show involvement in cell proliferation and GC signalling, with implications for understanding how GCs impact on cell fate.

Signaling pathways regulating intercellular adhesion molecule 1 expression by endothelin 1: comparison with interleukin-1beta in normal and scleroderma dermal fibroblasts.

OBJECTIVE: Endothelin 1 (ET-1) has been implicated in the pathogenesis of fibrotic and inflammatory diseases, including scleroderma. In addition to modulating vascular tone and extracellular matrix turnover, ET-1 up-regulates cell surface adhesion molecules including intercellular adhesion molecule 1 (ICAM-1), which is key to cell-cell and cell-matrix adhesion and leukocyte infiltration. This study was undertaken to delineate the signal transduction pathways utilized by ET-1 and compare them with those adopted by proinflammatory cytokine interleukin-1beta (IL-1beta) in normal and scleroderma dermal fibroblasts. METHODS: Protein expression induced by ET-1 and IL-1beta on normal dermal fibroblasts, with or without signaling inhibitors, was detected by enzyme-linked immunosorbent assay, while messenger RNA (mRNA) levels were analyzed by LightCycler polymerase chain reaction. Expression of protein kinase Cdelta (PKCdelta) and PKCepsilon protein in normal dermal fibroblasts and scleroderma dermal fibroblasts was determined by Western blotting, and PKCepsilon involvement in ET-1 signaling was confirmed through transfection of an ICAM-1 promoter construct into murine PKCepsilon-/- fibroblasts. NF-kappaB activation was confirmed via electrophoretic mobility supershift assay, and analysis of the ICAM-1 promoter region was achieved via transfection of deletion constructs into human dermal fibroblasts. RESULTS: In normal dermal fibroblasts, ET-1 induced ICAM-1 mRNA and surface protein expression in a dose- and time-dependent manner via both receptor subtypes, ET(A) and ET(B); antagonism of both abolished the ET-1 response. MEK was involved in the signaling cascade, but phosphatidylinositol 3-kinase and p38 MAPK were not. Key to the cascade was activation of NF-kappaB, achieved by ligation of either receptor subtype. PKCepsilon activation led to downstream activation of MEK and, in part, NF-kappaB. IL-1beta signaling required NF-kappaB and MEK activation, along with activation of PKCdelta. ET-1 and IL-1beta each utilized the same ICAM-1 promoter region and the same NF-kappaB site at -157 bp. Responses to ET-1 and IL-1beta differed in scleroderma dermal fibroblasts, with ET-1 sensitivity decreasing and IL-1beta responses remaining intact. Expression of PKCepsilon and PKCdelta in scleroderma dermal fibroblasts was also altered. CONCLUSION: The findings of this study indicate that differences in sensitivity to ET-1 and IL-1beta in scleroderma dermal fibroblasts may be explained by altered expression of the PKC isoforms and cytokine receptors.