Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1beta [IL-1beta], TNFalpha, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA(+) cytotoxic T lymphocytes (CTLs) induce IL-1beta from primary mouse macrophages, and GzmA(-/-) mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator.

Purpose To understand the role of HER2-associated signaling network in breast cancer stem cells (BCSCs); using radiation-resistant breast cancer cells and clinical recurrent breast cancers to evaluate HER2-targeted therapy as a tumor eliminating strategy for recurrent HER2−/low breast cancers. Experimental Design HER2-expressing BCSCs (HER2+/CD44+/CD24−/low) were isolated from radiation-treated breast cancer MCF7 cells and in vivo irradiated MCF7 xenograft tumors. Tumor aggressiveness and radiation resistance were analyzed by gap filling, Matrigel invasion, tumor-sphere formation, and clonogenic survival assays. The HER2/CD44 feature was analyzed in 40 primary and recurrent breast cancer specimens. Protein expression profiling in HER2+/CD44+/CD24−/low versus HER2−/CD44+/CD24−/low BCSCs was conducted with 2-D DIGE and HPLC-MS/MS analysis and HER2-mediated signaling network was generated by MetaCore™ program. Results Compared to HER2-negative BCSCs, HER2+/CD44+/CD24−/low cells showed elevated aldehyde dehydrogenase (ALDH) activity and aggressiveness tested by matrigel invasion, tumor sphere formation and in vivo tumorigenesis. The enhanced aggressive phenotype and radioresistance of the HER2+/CD44+/CD24−/low cells were markedly reduced by inhibition of HER2 via siRNA or Herceptin treatments. Clinical breast cancer specimens revealed that cells co-expressing HER2 and CD44 were more frequently detected in recurrent (84.6%) than primary tumors (57.1%). In addition, 2-D DIGE and HPLC-MS/MS of HER2+/CD44+/CD24−/low versus HER2−/CD44+/CD24−/low BCSCs reported a unique HER2-associated protein profile including effectors involved in tumor metastasis, apoptosis, mitochondrial function and DNA repair. A specific feature of HER2-STAT3 network was identified. Conclusion This study provides the evidence that HER2-mediated pro-survival signaling network is responsible for the aggressive phenotype of breast cancer stem cells that could be targeted to control the therapy-resistant HER2−/low breast cancer.

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We screened a human osteoclast (OCL) cDNA expression library for OCL inhibitory factors and identified a clone that blocked both human and murine OCL formation and bone resorption by more than 60%. This clone was identical to human legumain, a cysteine endopeptidase. Legumain significantly inhibited OCL-like multinucleated cell formation induced by 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) and parathyroid hormone-related protein (PTHrP) in mouse and human bone marrow cultures, and bone resorption in the fetal rat long bone assay in a dose-dependent manner. Legumain was detected in freshly isolated marrow plasma from normal donors and conditioned media from human marrow cultures. Furthermore, treatment of human marrow cultures with an antibody to legumain induced OCL formation to levels that were as high as those induced by 1,25-(OH) 2 D 3 . Implantation in nude mice of 293 cells transfected with the legumain cDNA and constitutively expressing high levels of the protein significantly reduced hypercalcemia induced by PTHrP by about 50%, and significantly inhibited the increase in OCL surface and in OCL number expressed per mm 2 bone area and per mm bone surface induced by PTHrP. These results suggest that legumain may be a physiologic local regulator of OCL activity that can negatively modulate OCL formation and activity.The osteoclast (OCL), 1 the primary bone-resorbing cell, is derived from cells in the monocyte-macrophage lineage (1). Osteoclast activity and formation are regulated by both systemic hormones and locally produced factors. We have used an expression cloning approach with a cDNA expression library prepared from highly purified human OCL-like cells formed in vitro to identify local factors that modulate OCL formation. We have recently identified two novel stimulators of OCL formation, annexin II (2) and osteoclast stimulatory factor (3), and an inhibitor of OCL formation, human ScaI (4). We have screened this library for additional factors that enhance or inhibit OCL formation, and report here the identification and characterization of a previously unknown inhibitor of the OCL, legumain. Legumain inhibited OCL formation in both long term human and murine marrow cultures and blocked 45 calcium ( 45 Ca) release from fetal rat long bones stimulated by 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ). Interestingly, legumain was detected in normal human marrow plasma, and addition of an antibody to legumain induced high levels of OCL formation in human marrow cultures in the absence of any added stimulator of OCL formation. In addition, legumain was active in vivo and inhibited PTHrP-stimulated OCL bone resorption in nude mice implanted with human kidney fibroblast 293 cells genetically engineered to constitutively express human legumain.

The osteoclast (OCL), the primary cell responsible for bone resorption, is under control of several factors that regulate its formation and/or activation. These factors can be systemic, such as 1,25-(OH) 2 D 3 , calcitonin, and parathyroid hormone (PTH); or can be produced locally, such as IL-6 and RANK ligand (1), by cells in the bone microenvironment such as osteoblasts, stromal cells, and immune cells (1). Recently, we and others have demonstrated that the OCL secretes factors that enhance or inhibit OCL formation and/or activation. IL-6 was one of the first cytokines identified among the autocrine/paracrine factors produced by OCLs that stimulated OCL formation (2). TGF-β is also produced by OCLs and can inhibit OCL activity (3). We have also identified and cloned a novel factor secreted by OCLs (OIP-1) that blocks OCL formation and bone resorption (4). Recently, Takahashi et al. (5), using an expression cloning approach, identified annexin II (AXII) as an OCL-secreted product that stimulates human and murine OCL formation and can also enhance the stimulatory effects of suboptimal concentrations of 1,25-(OH) 2 D 3 on OCL formation in marrow cultures and on bone resorption in long bones of fetal rats. Others have shown that rabbit OCL activity is inhibited by neutralizing antibodies to AXII (6). However, the mechanisms responsible for the stimulatory effects of AXII on OCL activity are unclear.Until recently, AXII was considered an intracellular or a membrane protein that played a role in cell-cell adhesion and plasminogen activation and acted as a cell surface receptor (7). Nesbitt andHorton (6) proposed that intracellular AXII in the OCL was involved in the clearance of matrix protein degraded by OCLs by acting as a binding protein. However, this mechanism does not explain the actions of secreted AXII on OCL formation.OCLs are multinucleated cells that share a common ancestor with monocytes/macrophages and CFU-GM, the granulocyte-macrophage colony-forming cell. CFU-GM proliferate and differentiate under the influence of osteoclastogenic factors and fuse to form multinucleated OCLs that finally are activated to resorb bone (1). Osteotrophic factors enhance OCL formation by expanding the OCL precursor pool through cell proliferation (e.g., GM-CSF or IL-6), fusion of precursors [e.g., PTH and 1,25-(OH) 2 D 3 ], or both (e.g., IL-1) (8). These effects could be direct, such as GM-CSF, or through induction of a second secreted factor, as with PTH, Annexin II (AXII), a calcium-dependent phospholipid-binding protein, has been recently found to be an osteoclast (OCL) stimulatory factor that is also secreted by OCLs. In vitro studies showed that AXII induced OCL formation and bone resorption. However, the mechanism of action by which AXII acts as a soluble extracellular protein to induce OCL formation is unknown. In this paper, we demonstrate that AXII gene expression is upregulated by 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ] and that addition of AXII significantly increased OCL-like multinucleated cell formation...

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Cultured microglial cells were examined for their ability to metabolize 25-hydroxyvitamin D3 (25-(OH) D3). Upon exposure to lipopolysaccharide, microglial cells produced a vitamin D metabolite which comigrated with synthetic 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in two different systems of high performance liquid chromatography. This metabolite had the same affinity as synthetic 1,25-(OH)2D3 for the chick intestinal 1,25-(OH)2D3 receptor. Lipopolysaccharide-stimulated microglial cells incubated with 3 nM of 25-(OH) D3 synthesized up to 5.76 fmol 1,25-(OH)2D3/8 x 10(5) cells/2 hr. Microglial cells stimulated for 48 hr with interferon-gamma also produced a significant amount of 1,25-(OH)2D3 (4.17 fmol/8 x 10(5) cells/2 hr). In contrast, levels of 1,25-(OH)2D3 produced by resting microglial cells were barely detectable. It is concluded that activated brain macrophages may be committed to synthesize 1,25-(OH)2D3 in vitro. This raises the possibility that activation of microglial cells in vivo may be followed by an increase in the level of 1,25-(OH)2D3 in the central nervous system (CNS). These results support the emerging concept that the brain constitutes a target tissue for vitamin D metabolites.