Myeloid dermal dendritic cells (DCs) accumulate in chronically inflamed tissues such as psoriasis. The importance of these cells for psoriasis pathogenesis is suggested by comparative T cell and DC cell counts, where DCs outnumber T cells. We have previously identified CD11c+BDCA-1+ cells as the main resident dermal DC population found in normal skin. We now show that psoriatic lesional skin has two populations of dermal DCs: 1) CD11c+BDCA-1+ cells which are phenotypically similar to those contained in normal skin, and 2) CD11c+BDCA-1− cells which are phenotypically immature and produce inflammatory cytokines. While BDCA-1+ DCs are not increased in number in psoriatic lesional skin compared to normal skin, BDCA-1− DCs are increased 30-fold. For functional studies, we FACS-sorted psoriatic dermal single cell suspensions to isolate these two cutaneous DC populations, and cultured them as stimulators in an allo-MLR. Both BDCA-1+ and BDCA-1− myeloid dermal DC populations induced T cell proliferation, and polarized T cells to become Th1 and Th17 cells. In addition, psoriatic dermal DCs induced a population of activated T cells that simultaneously produced IL-17 and IFN-γ, which was not induced by normal skin dermal DCs. As psoriasis is believed to be a mixed Th17/Th1 disease, it is possible that induction of these IL-17+IFNγ+ cells is pathogenic. These cytokines, the T cells that produce them, and the inducing inflammatory DCs may all be important new therapeutic targets in psoriasis.

We developed a model of 545 components (nodes) and 1259 interactions representing signaling pathways and cellular machines in the hippocampal CA1 neuron. Using graph theory methods, we analyzed ligand-induced signal flow through the system. Specification of input and output nodes allowed us to identify functional modules. Networking resulted in the emergence of regulatory motifs, such as positive and negative feedback and feedforward loops, that process information. Key regulators of plasticity were highly connected nodes required for the formation of regulatory motifs, indicating the potential importance of such motifs in determining cellular choices between homeostasis and plasticity.

The intracellular movement of the bacterial pathogen Listeria monocytogenes has helped identify key molecular constituents of actin-based motility (recent reviews ). However, biophysical as well as biochemical data are required to understand how these molecules generate the forces that extrude eukaryotic membranes. For molecular motors and for muscle, force-velocity curves have provided key biophysical data to distinguish between mechanistic theories. Here we manipulate and measure the viscoelastic properties of tissue extracts to provide the first force-velocity curve for Listeria monocytogenes. We find that the force-velocity relationship is highly curved, almost biphasic, suggesting a high cooperativity between biochemical catalysis and force generation. Using high-resolution motion tracking in low-noise extracts, we find long trajectories composed exclusively of molecular-sized steps. Robust statistics from these trajectories show a correlation between the duration of steps and macroscopic Listeria speed, but not between average step size and speed. Collectively, our data indicate how the molecular properties of the Listeria polymerization engine regulate speed, and that regulation occurs during molecular-scale pauses.

The G(alpha)o/i-coupled CB1 cannabionoid receptor induces neurite outgrowth in Neuro-2A cells. The mechanisms of signaling through G(alpha)o/i to induce neurite outgrowth were studied. The expression of G(alpha)o/i reduces the stability of its direct interactor protein, Rap1GAPII, by targeting it for ubiquitination and proteasomal degradation. This results in the activation of Rap1. G(alpha)o/i-induced activation of endogenous Rap1 in Neuro-2A cells is blocked by the proteasomal inhibitor lactacystin. G(alpha)o/i stimulates neurite outgrowth that is blocked by the expression of dominant negative Rap1. Expression of Rap1GAPII also blocks the G(alpha)o/i-induced neurite outgrowth and treatment with proteasomal inhibitors potentiates this inhibition. The endogenous G(alpha)o/i-coupled cannabinoid (CB1) receptor in Neuro-2A cells stimulates the degradation of Rap1GAPII; activation of Rap1 and treatment with pertussis toxin or lactacystin blocks these effects. The CB1 receptor-stimulated neurite outgrowth is blocked by treatment with pertussis toxin, small interfering RNA for Rap, lactacystin, and expression of Rap1GAPII. Thus, the G(alpha)o/i-coupled cannabinoid receptor, by regulating the proteasomal degradation of Rap1GAPII, activates Rap1 to induce neurite outgrowth.

Background Previous work has identified CD11c+CD1c- dendritic cells (DCs) as the major “inflammatory” dermal DC population in psoriasis vulgaris and CD1c+ DCs as the “resident” cutaneous DC population. Objective To further define molecular differences between these two myeloid dermal DC populations. Methods Inflammatory and resident DCs were single-cell sorted from psoriasis lesional skin biopsies, and the transcriptome of CD11c+CD1c- versus CD1c+ DCs was determined. Results were confirmed with RT-PCR, flow cytometry, immunohistochemistry, and double label immunofluorescence. Human keratinocytes were cultured for functional studies. Results TNF-related apoptosis-inducing ligand (TRAIL), Toll-like receptors (TLRs) 1 and 2, S100A12/EN-RAGE, CD32, and many other inflammatory products were differentially expressed in inflammatory DCs compared to resident DCs. Flow cytometry and immunofluorescence confirmed higher protein expression on CD1c- versus CD1c+ DCs. TRAIL receptors, death receptor 4 (DR4), and decoy receptor 2 (DcR2) were expressed in keratinocytes and dermal cells. In vitro culture of keratinocytes with TRAIL induced CCL20 chemokine. Conclusions CD11c+CD1c- inflammatory DCs in psoriatic lesional skin express a wide range of inflammatory molecules compared to skin resident CD1c+ DCs. Some molecules made by inflammatory DCs, including TRAIL, could have direct effects on keratinocytes or other skin cell types to promote disease pathogenesis.

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3′,5′-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element–binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor–driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease.

Cell signaling pathways interact with one another to form networks in mammalian systems. Such networks are complex in their organization and exhibit emergent properties such as bistability and ultrasensitivity. Analysis of signaling networks requires a combination of experimental and theoretical approaches including the development and analysis of models. This review focuses on theoretical approaches to understanding cell signaling networks. Using heterotrimeric G protein pathways an example, we demonstrate how interactions between two pathways can result in a network that contains a positive feedback loop and function as a switch. Different mathematical approaches that are currently used to model signaling networks are described, and future challenges including the need for databases as well as enhanced computing environments are discussed.

Our group recently described a population of antigen presenting cells that appear to be critical in psoriasis pathogenesis, termed inflammatory myeloid dendritic cells (CD11c+ BDCA1−). Triggering receptor expressed on myeloid cells type-1 (TREM-1) Signaling was a major canonical pathway in the published transcriptome of these cells. TREM-1 is a member of the immunoglobulin superfamily, active through the DAP12 signaling pathway, with an unknown ligand. Activation through TREM-1 induces inflammatory cytokines including IL-8, MCP/CCL2 and TNF. We now show that TREM-1 was expressed in the skin of healthy and psoriatic patients, and there was increased soluble TREM-1 in the circulation of psoriasis patients. In psoriasis lesions, TREM-1 was co-localized with dendritic cells as well as CD31+ endothelial cells. TREM-1 expression was reduced with successful NB-UVB, etanercept and anti-IL-17 treatments. An in vitro model of PGN-activated monocytes as inflammatory myeloid DCs was developed to study TREM-1 blockade, and treatment with a TREM-1 blocking chimera decreased allogeneic Th17 activation as well as IL-17 production. Furthermore, TREM-1 blockade of ex vivo psoriatic dendritic cells in an alloMLR also showed a decrease in IL-17. Together, these data suggest that the TREM-1 signaling pathway may be a previously unidentified therapeutic target to prevent the effects of inflammatory myeloid DCs in psoriasis.