Signaling by RANKL is essential for terminal differentiation of monocytes/macrophages into osteoclasts. The TRAF6 and c-Fos signaling pathways both play important roles downstream of RANKL. We show here that RANKL selectively induces NFATc1 expression via these two pathways. RANKL also evokes Ca(2+) oscillations that lead to calcineurin-mediated activation of NFATc1, and therefore triggers a sustained NFATc1-dependent transcriptional program during osteoclast differentiation. We also show that NFATc1-deficient embryonic stem cells fail to differentiate into osteoclasts in response to RANKL stimulation, and that ectopic expression of NFATc1 causes precursor cells to undergo efficient differentiation without RANKL signaling. Thus, NFATc1 may represent a master switch for regulating terminal differentiation of osteoclasts, functioning downstream of RANKL.

Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men. Similar gender disparity is seen in mice given a chemical carcinogen, diethylnitrosamine (DEN). DEN administration caused greater increases in serum interleukin-6 (IL-6) concentration in males than it did in females. Furthermore, ablation of IL-6 abolished the gender differences in hepatocarcinogenesis in mice. DEN exposure promoted production of IL-6 in Kupffer cells (KCs) in a manner dependent on the Toll-like receptor adaptor protein MyD88, ablation of which also protected male mice from DEN-induced hepatocarcinogenesis. Estrogen inhibited secretion of IL-6 from KCs exposed to necrotic hepatocytes and reduced circulating concentrations of IL-6 in DEN-treated male mice. We propose that estrogen-mediated inhibition of IL-6 production by KCs reduces liver cancer risk in females, and these findings may be used to prevent HCC in males.H epatocellular carcinoma (HCC), the most common primary liver cancer, is a dreaded complication of chronic liver disease that occurs in the setting of risk factors such as hepatitis B (HBV) and hepatitis C (HCV) viral infections, alcoholic liver disease, hemochromatosis, and nonalcoholic steatohepatitis (1). Most HCC appears in cirrhotic livers after years of chronic inflammation. The 5-year survival rate for patients with HCC, the increasing incidence of which is likely due to the spread of HCV (2), is only about 7%. Notably, men are about three to five times more likely to develop HCC than women (3). A similar or even more pronounced gender disparity is seen in rodent HCC models (4, 5). Furthermore, administration of estrogens to male mice inhibits development of chemically (DEN)-induced HCC (6). Nonetheless, the mechanisms that account for this gender disparity and the anticarcinogenic activity of estrogens are unknown.Inflammation is a major contributing factor to carcinogenesis (7). HCC represents a classic case of inflammation-linked cancer (8), and chemically or genetically induced HCC depends on inflammatory signaling (5, 9, 10). To understand the mechanisms underlying gender disparity in HCC, we used the chemical carcinogen diethylnitrosamine (DEN), which causes HCC in 100% of male mice but only in 10 to 30% of female littermates (5, 6). The pathogenesis of HCC in this mouse model differs from that in humans and thus may not be directly comparable to human HCC. Nevertheless, the mouse model of DENinduced HCC has a histology and genetic signature similar to that of human HCCs with poor prognosis (11) and recapitulates a dependence on inflammation and gender disparity seen in human HCC.Interleukin-6 (IL-6) is a multifunctional cytokine largely responsible for the hepatic re- (B) IL-6 mRNA levels in livers of male, female, or ovariectomized (OVX; ovariectomy was done 2 weeks before DEN administration) female mice 4 hours after DEN injection. E2 (50 mg/kg) in corn oil was injected intraperitoneally 2 hours before DEN was administered. (C) Male B6 mice (n = 3) were injected with ERa-specific ago...

Metastatic progression depends on genetic alterations intrinsic to cancer cells as well as the inflammatory microenvironment of advanced tumors 1,2 . To understand how cancer cells affect the inflammatory microenvironment, we conducted a biochemical screen for macrophage activating factors secreted by metastatic carcinomas. Amongst the cell lines screened, Lewis lung carcinoma (LLC) 3 were the most potent macrophage activators leading to production of IL-6 and TNF-α through activation of the Toll-like receptor family members 4 TLR2 and TLR6. Both TNF-α and TLR2 were found to be required for LLC metastasis. Biochemical purification of LLC conditional medium (LCM) led to identification of the extracellular matrix proteoglycan versican, which is upregulated in many human tumors including lung cancer 5,6 , as a macrophage activator that acts via TLR2 and its co-receptors TLR6 and CD14. By activating TLR2:TLR6 complexes and inducing TNF-α secretion by myeloid cells, versican strongly enhances LLC metastatic growth. These results explain how advanced cancer cells usurp components of the host innate immune system, including bone marrow-derived myeloid progenitors 7 , to generate an inflammatory microenvironment hospitable for metastatic growth.Distant site metastases are the leading cause of cancer-associated mortality and depend on genetic and/or epigenetic alterations that are intrinsic to cancer cells or extrinsic factors provided by the tumor microenvironment 1 . For instance, cytokines produced by inflammatory cells can enhance metastatogenesis by repressing the metastasis suppressor maspin within primary prostate carcinoma cells 8 . Furthermore, tumor progression and metastasis positively correlate with presence of infiltrates containing myeloid and lymphoid cells 2,9 . It was shown that certain carcinomas secrete factors that upregulate fibronectin and recruit vascular endothelial growth factor receptor 1 (VEGFR1)-positive hematopoetic progenitors to sites of Correspondence and requests for materials should be addressed to M.K. future metastatic growth, termed the pre-metastatic niche 7 . To examine whether cancer cells secrete factors that directly activate myeloid cells to produce tumor promoting cytokines 10 , we collected serum free conditioned medium (CM) from different cancer cell lines, derived mainly from C57BL6 mice, and applied it to bone marrow (BM)-derived macrophages (BMDM), which were assayed for production of IL-1β, IL-6 and TNF-α. The screen included 1C1C7 and TrampC1, which are liver and prostate cancer cell lines, respectively, with little or no metastatic activity, and two metastatic breast and lung carcinomas, 4T1 and LLC, respectively. CM from metastatic cells, especially LLC, induced higher amounts of IL-6 and TNF-α secretion than CM from non-metastatic cells (Fig. 1A). IL-1β secretion was undetectable and the CM did not contain IL-6 or TNF-α (data not shown). LLC-CM (LCM) also induced expression of Il1β, Il6 and Tnfα mRNAs, whereas serum free medium (SFM) and NIH3T3 CM were inacti...

Costimulatory signals are required for activation of immune cells, but it is not known whether they contribute to other biological systems. The development and homeostasis of the skeletal system depend on the balance between bone formation and resorption. Receptor activator of NF-kappaB ligand (RANKL) regulates the differentiation of bone-resorbing cells, osteoclasts, in the presence of macrophage-colony stimulating factor (M-CSF). But it remains unclear how RANKL activates the calcium signals that lead to induction of nuclear factor of activated T cells c1, a key transcription factor for osteoclastogenesis. Here we show that mice lacking immunoreceptor tyrosine-based activation motif (ITAM)-harbouring adaptors, Fc receptor common gamma subunit (FcRgamma) and DNAX-activating protein (DAP)12, exhibit severe osteopetrosis owing to impaired osteoclast differentiation. In osteoclast precursor cells, FcRgamma and DAP12 associate with multiple immunoreceptors and activate calcium signalling through phospholipase Cgamma. Thus, ITAM-dependent costimulatory signals activated by multiple immunoreceptors are essential for the maintenance of bone homeostasis. These results reveal that RANKL and M-CSF are not sufficient to activate the signals required for osteoclastogenesis.

Osteoclasts are cells of monocyte/macrophage origin that erode bone matrix: regulation of their differentiation is central to the understanding of the pathogenesis and treatment of bone diseases such as osteoporosis. Signalling by RANKL (receptor activator of NF-kappaB ligand), also known as Tnfsf11, is essential for the induction of osteoclast differentiation, and it must be strictly regulated to maintain bone homeostasis. But it is not known whether RANKL signalling to the cell interior is linked to any regulatory mechanisms. Here we show that RANKL induces the interferon-beta (IFN-beta) gene in osteoclast precursor cells, and that IFN-beta inhibits the differentiation by interfering with the RANKL-induced expression of c-Fos, an essential transcription factor for the formation of osteoclasts. This IFN-beta gene induction mechanism is distinct from that induced by virus, and is dependent on c-Fos itself. Thus an autoregulatory mechanism operates-the RANKL-induced c-Fos induces its own inhibitor. The importance of this regulatory mechanism for bone homeostasis is emphasized by the observation that mice deficient in IFN-beta signalling exhibit severe osteopenia (loss of bone mass) accompanied by enhanced osteoclastogenesis. Our study places the IFN-beta system in a new context, and may offer a molecular basis for the treatment of bone diseases.

Bone remodeling is central to maintaining the integrity of the skeletal system, wherein the developed bone is constantly renewed by the balanced action of osteoblastic bone formation and osteoclastic bone resorption. In the present study, we demonstrate a novel function of the Stat1 transcription factor in the regulation of bone remodeling. In the bone of the Stat1-deficient mice, excessive osteoclastogenesis is observed, presumably caused by a loss of negative regulation of osteoclast differentiation by interferon (IFN)-␤. However, the bone mass is unexpectedly increased in these mice. This increase is caused by excessive osteoblast differentiation, wherein Stat1 function is independent of IFN signaling. Actually, Stat1 interacts with Runx2 in its latent form in the cytoplasm, thereby inhibiting the nuclear localization of Runx2, an essential transcription factor for osteoblast differentiation. The new function of Stat1 does not require the Tyr 701 that is phosphorylated when Stat1 becomes a transcriptional activator. Our study provides a unique example in which a latent transcription factor attenuates the activity of another transcription factor in the cytoplasm, and reveals a new regulatory mechanism in bone remodeling.[Keywords: Stat1; Runx2; osteoblast; bone remodeling] Supplemental material is available at http://www.genesdev.org.

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BackgroundStudies have shown that long‐term exposure to air pollution such as fine particulate matter (≤2.5 μm in aerodynamic diameter [PM 2.5]) increases the risk of all‐cause and cardiovascular mortality. To date, however, there are limited data on the impact of air pollution on specific cardiovascular diseases. This study aimed to evaluate cardiovascular effects of long‐term exposure to air pollution among residents of Seoul, Korea.Methods and ResultsHealthy participants with no previous history of cardiovascular disease were evaluated between 2007 and 2013. Exposure to air pollutants was estimated by linking the location of outdoor monitors to the ZIP code of each participant's residence. Crude and adjusted analyses were performed using Cox regression models to evaluate the risk for composite cardiovascular events including cardiovascular mortality, acute myocardial infarction, congestive heart failure, and stroke. A total of 136 094 participants were followed for a median of 7.0 years (900 845 person‐years). The risk of major cardiovascular events increased with higher mean concentrations of PM 2.5 in a linear relationship, with a hazard ratio of 1.36 (95% confidence interval, 1.29–1.43) per 1 μg/m3 PM 2.5. Other pollutants including PM 2.5–10 of CO, SO 2, and NO 2, but not O3, were significantly associated with increased risk of cardiovascular events. The burden from air pollution was comparable to that from hypertension and diabetes mellitus.ConclusionsThis large‐scale population‐based study demonstrated that long‐term exposure to air pollution including PM 2.5 increases the risk of major cardiovascular disease and mortality. Air pollution should be considered an important modifiable environmental cardiovascular risk factor.