In vitro evidence suggests that plasmacytoid dendritic cells (pDCs) are intimately involved in the pathogenesis of lupus. However, it remains to be determined whether these cells are required in vivo for disease development, and whether their contribution is restricted to hyperproduction of type I IFNs. To address these issues, we created lupus-predisposed mice lacking the IFN regulatory factor 8 (IRF8) or carrying a mutation that impairs the peptide/histidine transporter solute carrier family 15, member 4 (SLC15A4). IRF8-deficient NZB mice, lacking pDCs, showed almost complete absence of anti-nuclear, anti-chromatin, and anti-erythrocyte autoantibodies, along with reduced kidney disease. These effects were observed despite normal B-cell responses to Toll-like receptor (TLR) 7 and TLR9 stimuli and intact humoral responses to conventional T-dependent and -independent antigens. Moreover, Slc15a4 mutant C57BL/6-Fas lpr mice, in which pDCs are present but unable to produce type I IFNs in response to endosomal TLR ligands, also showed an absence of autoantibodies, reduced lymphadenopathy and splenomegaly, and extended survival. Taken together, our results demonstrate that pDCs and the production of type I IFNs by these cells are critical contributors to the pathogenesis of lupus-like autoimmunity in these models. Thus, IRF8 and SLC15A4 may provide important targets for therapeutic intervention in human lupus.E xtensive evidence suggests that type I IFNs are major pathogenic effectors in lupus-associated systemic autoimmunity. A well-documented pattern of expression of type I IFN-inducible genes occurs in peripheral blood mononuclear cells of patients with systemic lupus erythematosus (SLE) (1-3), and reduced disease is observed in some lupus-predisposed mice that either lack the common receptor (IFNAR) for these cytokines (4, 5) or have been treated with IFNAR-blocking antibody (6). Consequently, attention has focused on defining the cell subsets and signaling processes involved in type I IFN production, the mechanisms by which these mediators accelerate disease, and approaches to interfere with these pathogenic events.Early in vitro studies showed that type I IFN production can be induced in normal blood leukocytes by SLE autoantibodies complexed with nucleic acid-containing apoptotic/necrotic cell material, and further work demonstrated that this activity is sensitive to RNase and DNase digestion (7,8). These results were integrated in a more comprehensive scheme following the demonstration that type I IFN induction by these complexes is mediated by the engagement of endosomal Toll-like receptors (TLRs) (9-11). Similarly, antigenic cargo containing nucleic acids was found to promote B-cell proliferation in a TLR9-or TLR7-dependent manner, with this effect enhanced by type I IFN signaling (9, 12, 13). The contribution of nucleic acid-sensing TLRs to systemic autoimmunity was further corroborated by studies in lupus-predisposed mice lacking or overexpressing TLR7 and/or TLR9 (14-20), and in Unc93b1 (3d) mutant mice i...

Specific cleavage of the transmembrane molecule, CUB domain-containing protein-1 (CDCP1), by plasmin-like serine proteases induces outside–in signal transduction that facilitates early stages of spontaneous metastasis leading to tumor cell intravasation, namely cell escape from the primary tumor, stromal invasion and transendothelial migration. We identified active β1 integrin as a biochemical and functional partner of the membrane-retained 70-kDa CDCP1 fragment, newly generated from its full-length 135-kDa precursor though proteolytic cleavage by serine proteases. Both in cell cultures and in live animals, active β1 integrin complexed preferentially with functionally activated, phosphorylated 70-kDa CDCP1. Complexing of β1 integrin the 70-kDa with CDCP1 fragment induced intracellular phosphorylation signaling, involving focal adhesion kinase-1 (FAK) and PI3 kinase (PI3K)-dependent Akt activation. Thus, inhibition of FAK/PI3K activities by specific inhibitors as well as short-hairpin RNA downregulation of β1 integrin significantly reduced FAK/Akt phosphorylation under conditions where CDCP1 was processed by serine proteases, indicating that FAK/PI3K/Akt pathway operates downstream of cleaved CDCP1 complexed with β1 integrin. Furthermore, this complex-dependent signaling correlated positively with high levels of tumor cell intravasation and dissemination. Correspondingly, abrogation in vivo of CDCP1 cleavage either by unique cleavage-blocking monoclonal antibody 10-D7 or by inhibition of proteolytic activity of plasmin-like serine proteases with aprotinin prevented β1 integrin/CDCP1 complexing and downstream FAK/Akt signaling concomitant with significant reduction of stromal invasion and spontaneous metastasis. Therefore, β1 integrin appears to serve as a motility-regulating partner mediating cross-talk between proteolytically cleaved, membrane-retained CDCP1 and members of FAK/PI3K/Akt pathway. This CDCP1 cleavage-induced signaling cascade constitutes a unique mechanism, independent of extracellular matrix remodeling, whereby a proteolytically cleaved CDCP1 regulates in vivo locomotion and metastasis of tumor cells through β1 integrin partnering. Our findings indicate that CDCP1 cleavage, occurring at the apex of a β1 integrin/FAK/PI3K/Akt signaling cascade, may represent a therapeutic target for CDCP1-positive cancers.

Cell invasion is a tightly controlled process occurring during development and tumor progression. The nematode Caenorhabditis elegans serves as a genetic model to study cell invasion during normal development. In the third larval stage, the anchor cell in the somatic gonad first induces and then invades the adjacent epidermal vulval precursor cells. The homolog of the Evi-1 oncogene, egl-43, is necessary for basement membrane destruction and anchor cell invasion. egl-43 is part of a regulatory network mediating cell invasion downstream of the fos-1 proto-oncogene. In addition, EGL-43 is required to specify the cell fates of ventral uterus cells downstream of or in parallel with LIN-12 NOTCH. Comparison with mammalian Evi-1 suggests a conserved pathway controlling cell invasion and cell fate specification.

Intravasation, the active entry of primary tumor cells into the vasculature, remains the least studied step in the metastatic cascade. Protease-mediated escape and stromal invasion of tumor cells represent widely-accepted processes leading up to the intravasation step. However, molecular factors that contribute directly to tumor cell vascular penetration have not been identified. In this study, the in vivo role of the collagenolytic protease, MMP-1, in cancer cell intravasation and metastasis was analyzed by employing a highly-disseminating variant of human HEp3 epidermoid carcinoma, HEp3-hi/diss. Whereas naturally-acquired or experimentally-induced MMP-1 deficiency substantially suppressed HEp3-hi/diss intravasation, supplementation of recombinant MMP-1 to MMP-1-silenced primary tumors, restored their impaired vascular dissemination. Surprisingly, abrogation of MMP-1 production and activity did not affect significantly HEp3-hi/diss migration or matrix invasion, suggesting non-collagenolytic mechanisms underlying MMP-1-dependent cell intravasation. In support of such non-collagenolytic mechanisms, MMP-1 silencing in HEp3-hi/diss cells modulated the microarchitecture and integrity of the angiogenic vasculature in a novel microtumor model. Concomitantly, MMP-1 deficiency led to decreased levels of intratumoral vascular permeability, tumor cell intravasation and metastatic dissemination. Taking advantage of PAR1 deficiency of HEp3-hi/diss cells, we further demonstrate that endothelial PAR1 is a putative non-tumor-cell/non-matrix target, activation of which by carcinoma-produced MMP-1 regulates endothelial permeability and transendothelial migration. The inhibitory effects of specific PAR1 antagonists in live animals have also indicated that the mechanisms of MMP-1-dependent vascular permeability in tumors involve endothelial PAR1 activation. Together, our findings mechanistically underscore the contribution of a tumor MMP-1/endothelial PAR1 axis to actual intravasation events manifested by aggressive carcinoma cells.

Through an iterative process of computational modeling, prediction, and experimentation, a molecular synchronization mechanism is revealed by which the cell-cycle regulates Notch signaling to allow the formation of a stable cell fate pattern.

Morphogenesis is a developmental phase during which cell fates are executed. Mechanical forces shaping individual cells play a key role during tissue morphogenesis. By investigating morphogenesis of the Caenorhabditis elegans hermaphrodite vulva, we show that the force-generating actomyosin network is differentially regulated by NOTCH and EGFR/RAS/MAPK signaling to shape the vulval tube. NOTCH signaling activates expression of the RHO kinase LET-502 in the secondary cell lineage through the ETS-family transcription factor LIN-1. LET-502 induces actomyosin-mediated contraction of the apical lumen in the secondary toroids, thereby generating a dorsal pushing force. In contrast, MAPK signaling in the primary lineage downregulates LET-502 RHO kinase expression to prevent toroid contraction and allow the gonadal anchor cell to expand the dorsal lumen of the primary toroids. The antagonistic action of the MAPK and NOTCH pathways thus controls vulval tube morphogenesis linking cell fate specification to morphogenesis.

Significance A large body of evidence has indicated that recognition of self-nucleic acids by endosomal toll-like receptors (TLRs) is central to the pathogenesis of lupus-like systemic autoimmunity in spontaneous mouse models, and the solute carrier SLC15A4 is required for this recognition. Here we describe a mechanism in which SLC15A4 is a major contributor to the proper trafficking of TLRs and their ligands to endolysosomes, wherein recognition and signaling is initiated. This finding supports ongoing efforts to identify pharmacologic inhibitors for this carrier as a means to treat lupus and other inflammatory disorders.

Mutations in the human Mid1 gene cause Opitz G/BBB syndrome, which is characterized by various midline closure defects. The Caenorhabditis elegans homolog of Mid1, madd-2, positively regulates signaling by the unc-40 Netrin receptor during the extension of muscle arms to the midline and in axon guidance and branching. During uterine development, a specialized cell called anchor cell (AC) breaches the basal laminae separating the uterus from the epidermis and invades the underlying vulval tissue. AC invasion is guided by an UNC-6 Netrin signal from the ventral nerve cord and an unknown guidance signal from the vulval cells. Using genetic epistasis analysis, we show that madd-2 regulates AC invasion downstream of or in parallel with the Netrin signaling pathway. Measurements of AC shape, polarity and dynamics indicate that MADD-2 prevents the formation of ectopic AC protrusions in the absence of guidance signals. We propose that MADD-2 represses the intrinsic invasive capacity of the AC, while the Netrin and vulval guidance cues locally overcome this inhibitory activity of MADD-2 to guide the AC ventrally into the vulval tissue. Therefore, developmental cell invasion depends on a precise balance between pro- and anti-invasive factors.