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The osteoblast-secreted molecule osteocalcin favors insulin secretion, but how this function is regulated in vivo by extracellular signals is for now unknown. In this study, we show that leptin, which instead inhibits insulin secretion, partly uses the sympathetic nervous system to fulfill this function. Remarkably, for our purpose, an osteoblast-specific ablation of sympathetic signaling results in a leptin-dependent hyperinsulinemia. In osteoblasts, sympathetic tone stimulates expression of Esp, a gene inhibiting the activity of osteocalcin, which is an insulin secretagogue. Accordingly, Esp inactivation doubles hyperinsulinemia and delays glucose intolerance in ob/ob mice, whereas Osteocalcin inactivation halves their hyperinsulinemia. By showing that leptin inhibits insulin secretion by decreasing osteocalcin bioactivity, this study illustrates the importance of the relationship existing between fat and skeleton for the regulation of glucose homeostasis.

Chemoattractant signaling induces the polarization and directed movement of cells secondary to the activation of multiple effector pathways. In addition, chemotactic signals can be amplified and relayed to proximal cells via the synthesis and secretion of additional chemoattractant. The mechanisms underlying such remarkable features remain ill defined. We show that the asymmetrical distribution of adenylyl cyclase (ACA) at the back of Dictyostelium discoideum cells, an essential determinant of their ability to migrate in a head-to-tail fashion, requires vesicular trafficking. This trafficking results in a local accumulation of ACA-containing intracellular vesicles and involves intact actin, microtubule networks, and de novo protein synthesis. We also show that migrating cells leave behind ACA-containing vesicles, likely secreted as multivesicular bodies and presumably involved in the formation of head-to-tail arrays of migrating cells. We propose that similar compartmentalization and shedding mechanisms exist in mammalian cells during embryogenesis, wound healing, neuron growth, and metastasis.

Mechanosensing followed by mechanoresponses by cells is well established, but the mechanisms by which mechanical force is converted into biochemical events are poorly understood. Vascular endothelial cells (ECs) exhibit flow- and stretch-dependent responses and are widely used as a model for studying mechanotransduction in mammalian cells. Platelet EC adhesion molecule 1 (PECAM-1) is tyrosine phosphorylated when ECs are exposed to flow or when PECAM-1 is directly pulled, suggesting that it is a mechanochemical converter. We show that PECAM-1 phosphorylation occurs when detergent-extracted EC monolayers are stretched, indicating that this phosphorylation is mechanically triggered and does not require the intact plasma membrane and soluble cytoplasmic components. Using kinase inhibitors and small interfering RNAs, we identify Fyn as the PECAM-1 kinase associated with the model. We further show that stretch- and flow-induced PECAM-1 phosphorylation in intact ECs is abolished when Fyn expression is down-regulated. We suggest that PECAM-1 and Fyn are essential components of a PECAM-1-based mechanosensory complex in ECs.

During migration, cells must couple direction sensing to signal transduction and actin remodeling. We previously identified GIV/Girdin as a G[alpha]i3 binding partner. We demonstrate that in mammalian cells G[alpha]i3 controls the functions of GIV during cell migration. We find that G[alpha]i3 preferentially localizes to the leading edge and that cells lacking G[alpha]i3 fail to polarize or migrate. A conformational change induced by association of GIV with G[alpha]i3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane. Activation of G[alpha]i3 serves as a molecular switch that triggers dissociation of G[beta][gamma] and GIV from the Gi3-GIV complex, thereby promoting cell migration by enhancing Akt signaling and actin remodeling. G[alpha]i3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the G[alpha]i3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

Atherosclerotic plaques develop in regions of the vasculature associated with chronic inflammation due to disturbed flow patterns. Endothelial phenotype modulation by flow requires the integration of numerous mechanotransduction pathways, but how this is achieved is not well understood. We show here that, in response to flow, the adaptor protein Shc is activated and associates with cell--cell and cell--matrix adhesions. Shc activation requires the tyrosine kinases vascular endothelial growth factor receptor 2 and Src. Shc activation and its vascular endothelial cadherin (VE-cadherin) association are matrix independent. In contrast, Shc binding to integrins requires VE-cadherin but occurs only on specific matrices. Silencing Shc results in reduction in both matrix-independent and matrix-dependent signals. Furthermore, Shc regulates flow-induced inflammatory signaling by activating nuclear factor [kappa]B-dependent signals that lead to atherogenesis. In vivo, Shc is activated in atherosclerosis-prone regions of arteries, and its activation correlates with areas of atherosclerosis. Our results support a model in which Shc orchestrates signals from cell--cell and cell--matrix adhesions to elicit flow-induced inflammatory signaling.

Reactive oxygen species (ROS) function as intracellular signaling molecules in a diverse range of biological processes. However, it is unclear how freely diffusible ROS dictate specific cellular responses. In this study, we demonstrate that nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (Nox4), a major Nox isoform expressed in nonphagocytic cells, including vascular endothelium, is localized to the endoplasmic reticulum (ER). ER localization of Nox4 is critical for the regulation of protein tyrosine phosphatase (PTP) 1B, also an ER resident, through redox-mediated signaling. Nox4-mediated oxidation and inactivation of PTP1B in the ER serves as a regulatory switch for epidermal growth factor (EGF) receptor trafficking and specifically acts to terminate EGF signaling. Consistent with this notion, PTP1B oxidation could also be modulated by ER targeting of antioxidant enzymes but not their untargeted counterparts. These data indicate that the specificity of intracellular ROS-mediated signal transduction may be modulated by the localization of Nox isoforms within specific subcellular compartments.

Many studies of [Ca.sup.2+] signaling use PC12 cells, yet the balance of [Ca.sup.2+] clearance mechanisms ill these cells is unknown. We used pharmacological inhibition of [Ca.sup.2+] transporters to characterize [Ca.sup.2+] clearance after depolarizations in both undifferentiated and nerve growth factor-differentiated PC12 cells. Sarco-endoplasmic reticulum [Ca.sup.2+] ATPase (SERCA), plasma membrane [Ca.sup.2+] ATPase (PMCA), and [Na.sup.+]/[Ca.sup.2+] exchanger (NCX) account for almost all [Ca.sup.2+] clearance in both cell states, with NCX and PMCA making the greatest contributions. Any contribution of mitochondrial uniporters is small. The ATP pool in differentiated cells was much more labile than that of undifferentiated cells in the presence of agents that dissipated mitochondrial proton gradients. Differentiated PC12 cells have a small component of [Ca.sup.2+] clearance possessing pharmacological characteristics consistent with secretory pathway [Ca.sup.2+] ATPase (SPCA), potentially residing on Golgi and/or secretory granules. Undifferentiated and differentiated cells are similar in overall [Ca.sup.2+] transport and in the small transport due to SERCA, but they differ in the fraction of transport by PMCA and NCX. Transport in neurites of differentiated PC 12 cells was qualitatively similar to that in the somata, except that the ER stores in neurites sometimes released [Ca.sup.2+] instead of clearing it after depolarization. We formulated a mathematical model to simulate the observed [Ca.sup.2+] clearance and to describe the differences between these undifferentiated and NGF-differentiated states quantitatively. The model required a value for the endogenous [Ca.sup.2+] binding ratio of PC 12 cell cytoplasm, which we measured to be 268 [+ or -] 85. Our results indicate that [Ca.sup.2+] transport in undifferentiated PC12 cells is quite unlike transport ill adrenal chromaffin cells, for which they often are considered models. Transport in both cell states more closely resembles that of sympathetic neurons, for which differentiated PC12 cells often are considered models. Comparison with other cell types shows that different cells emphasize different [Ca.sup.2+] transport mechanisms.

Receptor-operated [Ca.sup.2+] entry (ROCE) and store-operated [Ca.sup.2+] entry (SOCE) into cells are functions performed by all higher eukaryotic cells, and their impairment is life-threatening. The main molecular components of this pathway appear to be known. However, the molecular make-up of channels mediating ROCE and SOCE is largely unknown. One hypothesis proposes SOCE channels to be formed solely by Oral proteins. Another proposes SOCE channels to be composed of both Oral and C-type transient receptor potential (TRPC) proteins. Both hypotheses propose that the channels are activated by STIM1, a sensor of the filling state of the [Ca.sup.2+] stores that activates [Ca.sup.2+] entry when stores are depleted. The role of Oral in SOCE has been proven. Here we show the TRPC-dependent reconstitution of Icrac, the electrophysiological correlate to SOCE, by expression of Orai1; we also show that R91W-Orai1 can inhibit SOCE and ROCE and that Orai1 and STIM1 expression leads to functional expression of Gd-resistant ROCE. Because channels that mediate ROCE are accepted to be formed with the participation of TRPCs, our data show functional interaction between ROCE and SOCE components. We propose that SOCE/Icrac channels are composed of heteromeric complexes that include TRPCs and Oral proteins. capacitative calcium entry | signal transduction | store depletion

The complexity of biochemical intracellular signal transduction networks has led to speculation that the high degree of interconnectivity that exists in these networks transforms them into an information processing network. To test this hypothesis directly, a large scale model was created with the logical mechanism of each node described completely to allow simulation and dynamical analysis. Exposing the network to tens of thousands of random combinations of inputs and analyzing the combined dynamics of multiple outputs revealed a robust system capable of clustering widely varying input combinations into equivalence classes of biologically relevant cellular responses. This capability was nontrivial in that the network performed sharp, nonfuzzy classifications even in the face of added noise, a hallmark of real-world decision-making. information processing | systems biology

A question preoccupying many researchers is how signal transduction pathways control metabolic processes and energy production. A study by Jang et al. (Jang, C., G. Lee, and J. Chung. 2008. J. Cell Biol. 183:11-17) provides evidence that in Drosophila melanogaster a signaling network controlled by the LKB1 tumor suppressor regulates trafficking of an SIn/dMCT1 monocarboxylate transporter to the plasma membrane. This enables cells to import additional energy sources such as lactate and butyrate, enhancing the repertoire of fuels they can use to power vital activities.