IntroductionIn cancer cells, different pathways may be activated and/or different transcription factors may be expressed, resulting in the specific expression of a repertoire of genes in distinct cell types. A good example is the urokinase-type plasminogen activator (uPA) gene, which is expressed at high basal levels in PC3 cells but is not expressed in HeLa or LNCaP cells. [1][2][3] uPA is a serine protease involved in cell migration in nonpathological (eg, tissue remodeling) and pathological (eg, tumor invasion and metastases formation) events. 4,5 Many human cancers, in fact, overexpress uPA, and reduction of the invasive and metastatic phenotype has been obtained by down-regulating the expression of the gene or by inhibiting the enzymatic activity of this protease. 4,5 The minimal promoter (MP) of the human uPA gene extends approximately 86 bp upstream of the transcription start site 1,6 and contains 5 (high, 3 ϫ GGGCGG; low, 2 ϫ GGGAGG) affinity binding sites for the Sp1-family transcription factors immediately upstream of the TATA box. This cis-acting element binds the transcription factor Sp1 in vivo in PC3 cells, which constitutively express the uPA gene. In this cell line, a uPA minimal promoterdriven reporter construct is 10-fold transcriptionally more active than in HeLa cells. 1 This result correlates with the presence of the phosphorylated form of Sp1 in PC3 cells and with the absence of the phosphorylated transcription factor 1 and the lack of transcription of the endogenous uPA gene in HeLa cells. Thus, the uPA minimal promoter plays a very important role in uPA gene expression, invasion, and metastasis formation in cancer cells.The expression of the uPA gene is inducible in several cells by stimulating the enhancer, located about 2 kb upstream of the transcription start site, 6 which binds transcription factors belonging to different families. Despite its relevance in these cells, the uPA enhancer appears to have no role (or a very modest one) in uPA expression in PC3 cells, where, instead, transcriptional activity is dependent on the binding of the transcription factor Sp1 to the minimal promoter. 1 Phosphorylation of Sp1 seems to be an absolute requirement, because HeLa cells, which express but do not phosphorylate Sp1, are unable to activate the MP.Recently, it has been shown that in a CCL39 (Chinese hamster fibroblast)-derivative cell line, which expresses a chimeric, estrogeninducible Raf:ER chimera, vascular endothelial growth factor (VEGF) expression is under the control of the p42/p44 mitogenactivated protein (MAP) kinase pathway, 28 which targets the transcription factor Sp1 and promotes its phosphorylation at residues Thr453 and Thr739. 29 The phosphorylated form of Sp1, in turn, drives transcription from the VEGF minimal promoter, which contains 2 binding sites for this transcription factor, which bracket a binding site for the transcription factor AP-2. 28 The role of VEGF-MP in the overall expression of VEGF is not known. Interestingly, the constitutive levels of VEGF and uPA mRNA i...

Autogenous bone samples provided higher vital over comparable total bone levels than mHA-grafted sites. Osteoblast gene expression profiles from mHA grafts revealed higher expression of certain specific markers of osteoblast differentiation and bone formation, associated with a lower osteoclastogenic potential.

BackgroundDendritic cells (DCs) are highly specialized cells, which capture antigen in peripheral tissues and migrate to lymph nodes, where they dynamically interact with and activate T cells. Both migration and formation of DC-T cell contacts depend on cytoskeleton plasticity. However, the molecular bases governing these events have not been completely defined.Methodology/Principal FindingsUtilizing a T cell-dependent model of arthritis, we find that PLCγ2−/− mice are protected from local inflammation and bone erosion. PLCγ2 controls actin remodeling in dendritic cells, thereby affecting their capacity to prime T cells. DCs from PLCγ2−/− mice mature normally, however they lack podosomes, typical actin structures of motile cells. Absence of PLCγ2 impacts both DC trafficking to the lymph nodes and migration towards CCL21. The interaction with T cells is also affected by PLCγ2 deficiency. Mechanistically, PLCγ2 is activated by CCL21 and modulates Rac activation. Rac1/2−/− DCs also lack podosomes and do not respond to CCL21. Finally, antigen pulsed PLCγ2−/− DCs fail to promote T cell activation and induce inflammation in vivo when injected into WT mice. Conversely, injection of WT DCs into PLCγ2−/− mice rescues the inflammatory response but not focal osteolysis, confirming the importance of PLCγ2 both in immune and bone systems.Conclusions/SignificanceThis study demonstrates a critical role for PLCγ2 in eliciting inflammatory responses by regulating actin dynamics in DCs and positions the PLCγ2 pathway as a common orchestrator of bone and immune cell functions during arthritis.

The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional coactivator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte-osteoclast lineage. Both CIITA-overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro-computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone-forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA-overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased cfms and receptor activator of NF-kB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA-overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re-expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation, plays a novel, unexpected function in skeletal homeostasis, independent of MHC Class II expression and T cells, by exerting a selective and intrinsic control of osteoclast differentiation and bone resorption in vivo.