PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells.

Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases.

Green tea polyphenol epigallocatechin-3-gallate suppresses melanoma growth by inhibiting inflammasome and IL-1ß secretion.

Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, has been demonstrated to possess anti-inflammatory, antioxidant, anti-mutagenic and anti-carcinogenic properties. The anti-melanoma effect of EGCG has been previously suggested, but no clear mechanism of action has been established. In this study, we demonstrated that EGCG inhibits melanoma cell growth at physiological doses (0.1-1 µM). In the search for mechanisms of EGCG-mediated melanoma cell suppression, we found that NF-κB was inhibited, and that reduced NF-κB activity was associated with decreased IL-1ß secretion from melanoma cells. Since inflammasomes are involved in IL-1ß secretion, we investigated whether IL-1ß suppression was mediated by inflammasomes, and found that EGCG treatment led to downregulation of the inflammasome component, NLRP1, and reduced caspase-1 activation. Furthermore, silencing the expression of NLRP1 abolished EGCG-induced inhibition of tumor cell proliferation both in vitro and in vivo, suggesting a key role of inflammasomes in EGCG efficacy. This paper provides a novel mechanism for EGCG-induced melanoma inhibition: inflammasome downregulation→decreased IL-1ß secretion→decreased NF-κB activities→decreased cell growth. In addition, it suggests inflammasomes and IL-1ß could be potential targets for future melanoma therapeutics.

Constitutively active inflammasome in human melanoma cells mediating autoinflammation via caspase-1 processing and secretion of interleukin-1beta.

Interleukin-1beta (IL-1beta) is a pleiotropic cytokine promoting inflammation, angiogenesis, and tissue remodeling as well as regulation of immune responses. Although IL-1beta contributes to growth and metastatic spread in experimental and human cancers, the molecular mechanisms regulating the conversion of the inactive IL-1beta precursor to a secreted and active cytokine remains unclear. Here we demonstrate that NALP3 inflammasome is constitutively assembled and activated with cleavage of caspase-1 in human melanoma cells. Late stage human melanoma cells spontaneously secrete active IL-1beta via constitutive activation of the NALP3 inflammasome and IL-1 receptor signaling, exhibiting a feature of autoinflammatory diseases. Unlike human blood monocytes, these melanoma cells require no exogenous stimulation. In contrast, NALP3 functionality in intermediate stage melanoma cells requires activation of the IL-1 receptor to secrete active IL-1beta; cells from an early stage of melanoma require stimulation of the IL-1 receptor plus the co-stimulant muramyl dipeptide. The spontaneous secretion of IL-1beta from melanoma cells was reduced by inhibition of caspase-1 or the use of small interfering RNA directed against ASC. Supernatants from melanoma cell cultures enhanced macrophage chemotaxis and promoted in vitro angiogenesis, both prevented by pretreating melanoma cells with inhibitors of caspases-1 and -5 or IL-1 receptor blockade. These findings implicate IL-1-mediated autoinflammation as contributing to the development and progression of human melanoma and suggest that inhibiting the inflammasome pathway or reducing IL-1 activity can be a therapeutic option for melanoma patients.

LEDGF/DFS70, a major autoantigen of atopic dermatitis, is a component of keratohyalin granules.

Lens epithelium-derived growth factor/dense fine speckles 70 kDa protein (LEDGF/DFS70) is a transcriptional cofactor, a transcriptional activator, survival factor, and HIV-1 transporter. It is also a major autoantigen in patients with atopic dermatitis (AD), because autoantibodies to this protein are found in approximately 30% of AD patients. To better understand the role of autoantibodies and autoantigens in the pathogenesis of AD, we examined the distribution of LEDGF/DFS70 in the epidermis of normal human skin by light and electron microscopic immunocytochemistry. Increased amounts of LEDGF/DFS70 were located in the nuclei of cells in the basal layer, whereas the cytoplasm of cells in the granular layer stained for LEDGF/DFS70 by light microscopy. Using immunoelectron microscopy, we observed the accumulation of LEDGF/DFS70 in keratohyalin granules (KGs) in the cytoplasm of cells in the granular layer. In addition, Ig heavy chain-binding protein/glucose-regulated protein, 78-kDa (Bip/GRP78), a stress sensing protein in the endoplasmic reticulum, colocalized with LEDGF/DFS70 in the KGs. These results suggest that LEDGF/DFS70 is predominantly located in the nucleus of the basal epidermal cells and translocates into the cytoplasm during differentiation. Once in the cytoplasm, LEDGF/DFS70 accumulates in the KGs in the granular layer. Finally, LEDGF/DFS70, a "nuclear" autoantigen in AD, may play a functional role in the KGs.

A case of refractory bullous pemphigoid with plasmapheresis-associated thrombopenia: efficacy of pulsed intravenous cyclophosphamide therapy.

We present a 69-year-old woman with refractory bullous pemphigoid successfully treated with pulsed intravenous cyclophosphamide therapy. Because various other early treatments including 4,4'-diaminodiphenylsulphone (DDS), minocycline/nicotinamide, cyclosporin, azathioprine, high-dose oral prednisolone, and methylprednisolone pulse therapy were either ineffective or intolerable, she was treated with double filtration plasmapheresis, but she responded poorly with the rare complication of severe transient thrombopenia. Finally, she gradually recovered with pulsed intravenous cyclophosphamide therapy.

Autoantibodies to DFS70/LEDGF are increased in alopecia areata patients.

Alopecia areata (AA) has been suspected to be an autoimmune disease, although there is no distinct evidence, we investigated the relationship between AA and autoantibodies against dense fine speckles 70 kDa (DFS70) in 111 patients with alopecia and 105 healthy controls. The sera from 59 out of 111 (53%) Japanese alopecia patients were positive for anti-nuclear antibody (ANA), as compared to the sera of 16 out of 105 (15%) healthy controls (p < 0.001). Twenty percent (22/111) of the alopecia patients were shown to be positive for the prevalence of anti-DFS70 antibodies, as compared to 8% (8/105) of the healthy controls (p < 0.01). IgG subclass analysis by ELISA showed that IgG1 and IgG2-anti-DFS70 antibodies were dominant in alopecia patients. The DFS70 gene expression in the hair structures was clearly detected in both those with and those without the anti-DFS70 antibody by RT-PCR. Immunohistochemical techniques showed that the DFS70 was localized predominantly in the outer root sheath (ORS) cells. The elevated anti-DFS70 antibodies in alopecia patients and the localization of the DFS70 in the ORS suggest that autoantibodies against the DFS70 are related to the etiology in a certain population of AA.