Steven H. Young

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism.

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

Expression of bitter taste receptors of the T2R family in the gastrointestinal tract and enteroendocrine STC-1 cells

Although a role for the gastric and intestinal mucosa in molecular sensing has been known for decades, the initial molecular recognition events that sense the chemical composition of the luminal contents has remained elusive. Here we identified putative taste receptor gene transcripts in the gastrointestinal tract. Our results, using reverse transcriptase–PCR, demonstrate the presence of transcripts corresponding to multiple members of the T2R family of bitter taste receptors in the antral and fundic gastric mucosa as well as in the lining of the duodenum. In addition, cDNA clones of T2R receptors were detected in a rat gastric endocrine cell cDNA library, suggesting that these receptors are expressed, at least partly, in enteroendocrine cells. Accordingly, expression of multiple T2R receptors also was found in STC-1 cells, an enteroendocrine cell line. The expression of α subunits of G proteins implicated in intracellular taste signal transduction, namely Gαgust, and Gαt-2, also was demonstrated in the gastrointestinal mucosa as well as in STC-1 cells, as revealed by reverse transcriptase–PCR and DNA sequencing, immunohistochemistry, and Western blotting. Furthermore, addition of compounds widely used in bitter taste signaling (e.g., denatonium, phenylthiocarbamide, 6-n-propil-2-thiouracil, and cycloheximide) to STC-1 cells promoted a rapid increase in intracellular Ca2+ concentration. These results demonstrate the expression of bitter taste receptors of the T2R family in the mouse and rat gastrointestinal tract.

Agonists of proteinase‐activated receptor 1 induce plasma extravasation by a neurogenic mechanism

Thrombin, generated in the circulation during injury, cleaves proteinase‐activated receptor 1 (PAR1) to stimulate plasma extravasation and granulocyte infiltration. However, the mechanism of thrombin‐induced inflammation in intact tissues is unknown. We hypothesized that thrombin cleaves PAR1 on sensory nerves to release substance P (SP), which interacts with the neurokinin 1 receptor (NK1R) on endothelial cells to cause plasma extravasation. PAR1 was detected in small diameter neurons known to contain SP in rat dorsal root ganglia by immunohistochemistry and in situ hybridization. Thrombin and the PAR1 agonist TFLLR‐NH2 (TF‐NH2) increased [Ca2+]i >50% of cultured neurons (EC50s 24 mu ml−1 and 1.9 μM, respectively), assessed using Fura‐2 AM. The PAR1 agonist completely desensitized responses to thrombin, indicating that thrombin stimulates neurons through PAR1. Injection of TF‐NH2 into the rat paw stimulated a marked and sustained oedema. An NK1R antagonist and ablation of sensory nerves with capsaicin inhibited oedema by 44% at 1 h and completely by 5 h. In wild‐type but not PAR1−/− mice, TF‐NH2 stimulated Evans blue extravasation in the bladder, oesophagus, stomach, intestine and pancreas by 2–8 fold. Extravasation in the bladder, oesophagus and stomach was abolished by an NK1R antagonist. Thus, thrombin cleaves PAR1 on primary spinal afferent neurons to release SP, which activates the NK1R on endothelial cells to stimulate gap formation, extravasation of plasma proteins, and oedema. In intact tissues, neurogenic mechanisms are predominantly responsible for PAR1‐induced oedema.

Amino Acid-stimulated Ca2+ Oscillations Produced by the Ca2+-sensing Receptor Are Mediated by a Phospholipase C/Inositol 1,4,5-Trisphosphate-independent Pathway That Requires G12, Rho, Filamin-A, and the Actin Cytoskeleton

The G protein-coupled Ca2+-sensing receptor (CaR) is an allosteric protein that responds to two different agonists, Ca2+ and aromatic amino acids, with the production of sinusoidal or transient oscillations in intracellular Ca2+ concentration ([Ca2+]i). Here, we examined whether these differing patterns of [Ca2+]i oscillations produced by the CaR are mediated by separate signal transduction pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in single cells, we found that stimulation of CaR by an increase in the extracellular Ca2+ concentration ([Ca2+]o) leads to periodic synthesis of inositol 1,4,5-trisphosphate, whereas l-phenylalanine stimulation of the CaR does not induce any detectable change in the level this second messenger. Furthermore, we identified a novel pathway that mediates transient [Ca2+]i oscillations produced by the CaR in response to l-phenylalanine, which requires the organization of the actin cytoskeleton and involves the small GTPase Rho, heterotrimeric proteins of the G12 subfamily, the C-terminal region of the CaR, and the scaffolding protein filamin-A. Our model envisages that Ca2+ or amino acids stabilize unique CaR conformations that favor coupling to different G proteins and subsequent activation of distinct downstream signaling pathways.

Protein Kinase Cν/Protein Kinase D3 Nuclear Localization, Catalytic Activation, and Intracellular Redistribution in Response to G Protein-coupled Receptor Agonists

The protein kinase D (PKD) family consists of three serine/threonine kinases: PKCμ/PKD, PKD2, and PKCν/PKD3. Whereas PKD has been the focus of most studies, virtually nothing is known about the effect of G protein-coupled receptor agonists (GPCR) on the regulatory properties and intracellular distribution of PKD3. Consequently, we examined the mechanism that mediates its activation and intracellular distribution. GPCR agonists induced a rapid activation of PKD3 by a protein kinase C (PKC)-dependent pathway that leads to the phosphorylation of the activation loop of PKD3. Comparison of the steady-state distribution of endogenous or tagged PKD3 versus PKD and PKD2 in unstimulated cells indicated that whereas PKD and PKD2 are predominantly cytoplasmic, PKD3 is present both in the nucleus and cytoplasm. This distribution of PKD3 results from its continuous shuttling between both compartments by a mechanism that requires a nuclear import receptor and a competent CRM1-nuclear export pathway. Cell stimulation with the GPCR agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD3 that is PKC-dependent. Interestingly, the nuclear accumulation of PKD3 can be dramatically enhanced in response to its activation. Thus, this study demonstrates that the intracellular distribution of PKD isoenzymes are distinct, and suggests that their signaling properties are regulated by differential localization.

Protein Kinase D Mediates Mitogenic Signaling by Gq-coupled Receptors through Protein Kinase C-independent Regulation of Activation Loop Ser744 and Ser748 Phosphorylation

Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to Gq-coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3–10 nm) or with vasopressin, a different Gq-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser744 and Ser748. Transphosphorylation targeted Ser744, whereas autophosphorylation was the predominant mechanism for Ser748 in cells stimulated with Gq-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to Gq-coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of Gq-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.

Negative cross-talk between calcium-sensing receptor and β-catenin signaling systems in colonic epithelium.

Here, we examined the role of the extracellular Ca2+-sensing receptor (CaSR) in the control of colonic epithelial cell proliferation in vivo and changes in β-catenin triggered by CaSR stimulation in human colonic epithelial cells in vitro. The in vivo studies, using a novel Casr intestinal-specific knock-out mouse, indicate that the genetic ablation of the Casr leads to hyperproliferation of colonic epithelial cells, expansion of the proliferative zone, changes in crypt structure, and enhanced β-catenin nuclear localization. The in vitro results indicate that stimulation of the CaSR, by Ca2+ or by the calcimimetic R-568, produced a striking and time-dependent decrease in the phosphorylation of β-catenin at Ser-552 and Ser-675, two amino acid residues that promote β-catenin transcriptional activity. The reduced phosphorylation of β-catenin coincided with a decline in its nuclear localization and a marked redistribution to the plasma membrane. Furthermore, CaSR stimulation promoted a down-regulation of β-catenin-mediated transcriptional activation. These studies demonstrate that signaling pathways emanating from the CaSR control colonic epithelial cell proliferation in vivo and suggest that the mechanism involves regulation of β-catenin phosphorylation.

Extracellular calcium sensing receptor stimulation in human colonic epithelial cells induces intracellular calcium oscillations and proliferation inhibition.

The extracellular Ca2+‐sensing receptor (CaR) is increasingly implicated in the regulation of multiple cellular functions in the gastrointestinal tract, including secretion, proliferation and differentiation of intestinal epithelial cells. However, the signaling mechanisms involved remain poorly defined. Here we examined signaling pathways activated by the CaR, including Ca2+ oscillations, in individual human colon epithelial cells. Single cell imaging of colon‐derived cells expressing the CaR, including SW‐480, HT‐29, and NCM‐460 cells, shows that stimulation of this receptor by addition of aromatic amino acids or by an elevation of the extracellular Ca2+ concentration promoted striking intracellular Ca2+ oscillations. The intracellular calcium oscillations in response to extracellular Ca2+ were of sinusoidal pattern and mediated by the phospholipase C/diacylglycerol/inositol 1,4,5‐trisphosphate pathway as revealed by a biosensor that detects the accumulation of diacylglycerol in the plasma membrane. The intracellular calcium oscillations in response to aromatic amino acids were of transient type, that is, Ca2+ spikes that returned to baseline levels, and required an intact actin cytoskeleton, a functional Rho, Filamin A and the ion channel TRPC1. Further analysis showed that re‐expression and stimulation of the CaR in human epithelial cells derived from normal colon and from colorectal adenocarcinoma inhibits their proliferation. This inhibition was associated with the activation of the signaling pathway that mediates the generation of sinusoidal, but not transient, intracellular Ca2+ oscillations. Thus, these results indicate that the CaR can function in two signaling modes in human colonic epithelial cells offering a potential link between gastrointestinal responses and food/nutrients uptake and metabolism. J. Cell. Physiol. 225: 73–83, 2010.

Blockade of P-Selectin Is Sufficient to Reduce MHC I Antibody-Elicited Monocyte Recruitment In Vitro and In Vivo

Donor‐specific HLA antibodies significantly lower allograft survival, but as yet there are no satisfactory therapies for prevention of antibody‐mediated rejection. Intracapillary macrophage infiltration is a hallmark of antibody‐mediated rejection, and macrophages are important in both acute and chronic rejection. The purpose of this study was to investigate the Fc‐independent effect of HLA I antibodies on endothelial cell activation, leading to monocyte recruitment. We used an in vitro model to assess monocyte binding to endothelial cells in response to HLA I antibodies. We confirmed our results in a mouse model of antibody‐mediated rejection, in which B6.RAG1−/− recipients of BALB/c cardiac allografts were passively transferred with donor‐specific MHC I antibodies. Our findings demonstrate that HLA I antibodies rapidly increase intracellular calcium and endothelial presentation of P‐selectin, which supports monocyte binding. In the experimental model, donor‐specific MHC I antibodies significantly increased macrophage accumulation in the allograft. Concurrent administration of rPSGL‐1‐Ig abolished antibody‐induced monocyte infiltration in the allograft, but had little effect on antibody‐induced endothelial injury. Our data suggest that antagonism of P‐selectin may ameliorate accumulation of macrophages in the allograft during antibody‐mediated rejection.

Requirement of the TRPC1 Cation Channel in the Generation of Transient Ca2+ Oscillations by the Calcium-sensing Receptor

The calcium-sensing receptor (CaR) is an allosteric protein that responds to extracellular Ca2+ ([Ca2+]o) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca2+ concentration ([Ca2+]i). An increase in [Ca2+]o stimulates phospholipase C-mediated production of inositol 1,4,5-trisphosphate and causes sinusoidal oscillations in [Ca2+]i. Conversely, aromatic amino acid-induced CaR activation does not stimulate phospholipase C but engages an unidentified signaling mechanism that promotes transient oscillations in [Ca2+]i. We show here that the [Ca2+]i oscillations stimulated by aromatic amino acids were selectively abolished by TRPC1 down-regulation using either a pool of small inhibitory RNAs (siRNAs) or two different individual siRNAs that targeted different coding regions of TRPC1. Furthermore, [Ca2+]i oscillations stimulated by aromatic amino acids were also abolished by inhibition of TRPC1 function with an antibody that binds the pore region of the channel. We also show that aromatic amino acid-stimulated [Ca2+]i oscillations can be prevented by protein kinase C (PKC) inhibitors or siRNA-mediated PKCα down-regulation and impaired by either calmodulin antagonists or by the expression of a dominant-negative calmodulin mutant. We propose a model for the generation of CaR-mediated transient [Ca2+]i oscillations that integrates its stimulation by aromatic amino acids with TRPC1 regulation by PKC and calmodulin.