John H. Kehrl

Activation of autophagy by inflammatory signals limits IL-1β production by targeting ubiquitinated inflammasomes for destruction

Autophagosomes delivers cytoplasmic constituents to lysosomes for degradation, whereas inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1β (IL-1β) and IL-18. Here we show that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered activation of the G protein RalB and autophagosome formation. The induction of autophagy did not depend on the adaptor ASC or capase-1 but was dependent on the presence of the inflammasome sensor. Blocking autophagy potentiated inflammasome activity, whereas stimulating autophagy limited it. Assembled inflammasomes underwent ubiquitination and recruited the autophagic adaptor p62, which assisted their delivery to autophagosomes. Our data indicate that autophagy accompanies inflammasome activation to temper inflammation by eliminating active inflammasomes.

Pancreas dorsal lobe agenesis and abnormal islets of Langerhans in Hlxb9-deficient mice

In most mammals the pancreas develops from the foregut endoderm as ventral and dorsal buds. These buds fuse and develop into a complex organ composed of endocrine, exocrine and ductal components. This developmental process depends upon an integrated network of transcription factors. Gene targeting experiments have revealed critical roles for Pdx1, Isl1, Pax4, Pax6 and Nkx2-2 (refs 3,4,5,6,7,8,9,10). The homeobox gene HLXB9 (encoding HB9) is prominently expressed in adult human pancreas, although its role in pancreas development and function is unknown. To facilitate its study, we isolated the mouse HLXB9 orthologue, Hlxb9. During mouse development, the dorsal and ventral pancreatic buds and mature β-cells in the islets of Langerhans express Hlxb9. In mice homologous for a null mutation of Hlxb9, the dorsal lobe of the pancreas fails to develop. The remnant Hlxb9–/– pancreas has small islets of Langerhans with reduced numbers of insulin-producing β-cells. Hlxb9–/– β-cells express low levels of the glucose transporter Glut2 and homeodomain factor Nkx 6-1. Thus, Hlxb9 is key to normal pancreas development and function.

Active Suppression of Interneuron Programs within Developing Motor Neurons Revealed by Analysis of Homeodomain Factor HB9

Sonic hedgehog (Shh) specifies the identity of both motor neurons (MNs) and interneurons with morphogen-like activity. Here, we present evidence that the homeodomain factor HB9 is critical for distinguishing MN and interneuron identity in the mouse. Presumptive MN progenitors and postmitotic MNs express HB9, whereas interneurons never express this factor. This pattern resembles a composite of the avian homologs MNR2 and HB9. In mice lacking Hb9, the genetic profile of MNs is significantly altered, particularly by upregulation of Chx10, a gene normally restricted to a class of ventral interneurons. This aberrant gene expression is accompanied by topological disorganization of motor columns, loss of the phrenic and abducens nerves, and intercostal nerve pathfinding defects. Thus, MNs actively suppress interneuron genetic programs to establish their identity.

MyD88 and Trif Target Beclin 1 to Trigger Autophagy in Macrophages

The Toll-like receptors (TLR) play an instructive role in innate and adaptive immunity by recognizing specific molecular patterns from pathogens. Autophagy removes intracellular pathogens and participates in antigen presentation. Here, we demonstrate that not only TLR4, but also other TLR family members induce autophagy in macrophages, which is inhibited by MyD88, Trif, or Beclin 1 shRNA expression. MyD88 and Trif co-immunoprecipitate with Beclin 1, a key factor in autophagosome formation. TLR signaling enhances the interaction of MyD88 and Trif with Beclin 1, and reduces the binding of Beclin 1 to Bcl-2. These findings indicate TLR signaling via its adaptor proteins reduces the binding of Beclin 1 to Bcl-2 by recruiting Beclin 1 into the TLR-signaling complex leading to autophagy.

TRAF6 and A20 regulate lysine 63-linked ubiquitination of Beclin-1 to control TLR4-induced autophagy.

Regulation of the ubiquitination of Beclin-1 may be a general mechanism that controls autophagy. Controlling the Inner Pac-Man Autophagy is a process by which cellular components can be recycled to provide much-needed raw materials to help cells survive conditions of acute stress. But if a cell has to rely on this process for too long, the cell will die. TLR4 is a receptor that responds to a bacterial component called LPS to activate the transcription factor NF-κB, which drives the expression of pro-inflammatory genes in a process that requires a modifying enzyme called TRAF6 to switch on the pathway and an opposing enzyme called A20 to switch it off. Shi and Kehrl now show that these two enzymes play an analogous role in regulating TLR4-induced autophagy: TRAF6 modifies (ubiquitinates) a protein called Beclin-1, which initiates autophagy, whereas A20 counters the effects of TRAF6 and shuts the process down. The authors’ examination of other pro-autophagy pathways suggests that regulation of the ubiquitination state of Beclin-1 may be a general mechanism for the induction of autophagy by pro-inflammatory stimuli. Autophagy delivers cytoplasmic constituents to autophagolysosomes and is linked to both innate and adaptive immunity. Toll-like receptor 4 (TLR4) signaling induces autophagy and recruits Beclin-1, the mammalian homolog of yeast Atg6, to the receptor complex. We found that tumor necrosis factor receptor (TNFR)–associated factor 6 (TRAF6)–mediated, Lys63 (K63)–linked ubiquitination of Beclin-1 is critical for TLR4-triggered autophagy in macrophages. Two TRAF6-binding motifs in Beclin-1 facilitated the binding of TRAF6 and the ubiquitination of Beclin-1. Lys117, which is strategically located in the Bcl-2 homology 3 (BH3) domain of Beclin-1, was a major site for K63-linked ubiquitination. The deubiquitinating enzyme A20 reduced the extent of K63-linked ubiquitination of Beclin-1 and limited the induction of autophagy in response to TLR signaling. Treatment of macrophages with either interferon-γ or interleukin-1 also triggered the K63-linked ubiquitination of Beclin-1 and the formation of autophagosomes. These results indicate that the status of K63-linked ubiquitination of Beclin-1 plays a key role in regulating autophagy during inflammatory responses.

Tumor Necrosis Factor Signaling to Stress-activated Protein Kinase (SAPK)/Jun NH2-terminal Kinase (JNK) and p38 GERMINAL CENTER KINASE COUPLES TRAF2 TO MITOGEN-ACTIVATED PROTEIN KINASE/ERK KINASE KINASE 1 AND SAPK WHILE RECEPTOR INTERACTING PROTEIN ASSOCIATES WITH A MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE UPSTREAM OF MKK6 AND p38

Tumor necrosis factor (TNF) elicits a diverse array of inflammatory responses through engagement of its type-1 receptor (TNFR1). Many of these responses require de novogene expression mediated by the activator protein-1 (AP-1) transcription factor. We investigated the mechanism by which TNFR1 recruits the stress-activated protein kinases (SAPKs) and the p38s, two mitogen-activated protein kinase (MAPK) families that together regulate AP-1. We show that the human SPS1 homologue germinal center kinase (GCK) can interact in vivo with the TNFR1 signal transducer TNFR-associated factor-2 (TRAF2) and with MAPK/ERK kinase kinase 1 (MEKK1), a MAPK kinase kinase (MAPKKK) upstream of the SAPKs, thereby coupling TRAF2 to the SAPKs. Receptor interacting protein (RIP) is a second TNFR signal transducer which can bind TRAF2. We show that RIP activates both p38 and SAPK; and that TRAF2 activation of p38 requires RIP. We also demonstrate that the RIP noncatalytic intermediate domain associates in vivo with an endogenous MAPKKK that can activate the p38 pathway in vitro. Thus, TRAF2 initiates SAPK and p38 activation by binding two proximal protein kinases: GCK and RIP. GCK and RIP, in turn, signal by binding MAPKKKs upstream of the SAPKs and p38s.

RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III

The heterotrimeric G-protein Gs couples cell-surface receptors to the activation of adenylyl cyclases and cyclic AMP production (reviewed in refs 1, 2). RGS proteins, which act as GTPase-activating proteins (GAPs) for the G-protein α-subunits αi and αq, lack such activity for αs (refs 3,4,5,6). But several RGS proteins inhibit cAMP production by Gs-linked receptors. Here we report that RGS2 reduces cAMP production by odorant-stimulated olfactory epithelium membranes, in which the αs family member αolf links odorant receptors to adenylyl cyclase activation. Unexpectedly, RGS2 reduces odorant-elicited cAMP production, not by acting on αolf but by inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons. Furthermore, whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous RGS2 negatively regulates odorant-evoked intracellular signalling. These results reveal a mechanism for controlling the activities of adenylyl cyclases, which probably contributes to the ability of olfactory neurons to discriminate odours.

Heterotrimeric G Protein Signaling: Roles in Immune Function and Fine-Tuning by RGS Proteins

The RGS proteins provide a mechanism by which cells can regulate both the duration and the magnitude of a signal generated through a heterotrimeric G protein. Such fine-tuning is undoubtedly essential for the finely orchestrated events that occur in response to the chemokines, hormones, and neuropeptides, which signal through GPCRs. Furthermore, certain key intracellular molecules such as Ras integrate output signals generated through heptahelical, antigen, and cytokine receptors. Discovering these key molecules and understanding how they discriminate between relevant signals and irrelevant background noise to coordinate the cellular responses that eventually lead to humoral and cellular immunity remains a sizable challenge for the future.*To whom correspondence should be addressed (e-mail: jkehrl@atlas.niaid.nih.gov).

Pericyte-specific expression of Rgs5: implications for PDGF and EDG receptor signaling during vascular maturation

RGS proteins finely tune heterotrimeric G‐protein signaling. Implying the need for such fine‐tuning in the developing vascular system, in situ hybridization revealed a striking and extensive expression pattern of Rgs5 in the arterial walls of E12.5–E17.5 mouse embryos. The distribution and location of the Rgs5‐positive cells typified that of pericytes and strikingly overlapped the known expression pattern of platelet‐derived growth factor receptor (PDGFR)‐β. Both E14.5 PDGFR‐β‐ and platelet‐derived growth factor (PDGF)‐B‐deficient mice exhibited markedly reduced levels of Rgs5 in their vascular plexa and small arteries. This likely reflects the loss of pericytes in the mutant mice. RGS5 acts as a potent GTPase activating protein for Giα and Gqα and it attenuated angiotensin II‐, endothelin‐1‐, sphingosine‐1‐phosphate‐, and PDGF‐induced ERK‐2 phosphorylation. Together these results indicate that RGS5 exerts control over PDGFR‐β and GPCR‐mediated signaling pathways active during fetal vascular maturation.

Transcription Profiling of Platelet-Derived Growth Factor-B-Deficient Mouse Embryos Identifies RGS5 as a Novel Marker for Pericytes and Vascular Smooth Muscle Cells

All blood capillaries consist of endothelial tubes surrounded by mural cells referred to as pericytes. The origin, recruitment, and function of the pericytes is poorly understood, but the importance of these cells is underscored by the severe cardiovascular defects in mice genetically devoid of factors regulating pericyte recruitment to embryonic vessels, and by the association between pericyte loss and microangiopathy in diabetes mellitus. A general problem in the study of pericytes is the shortage of markers for these cells. To identify new markers for pericytes, we have taken advantage of the platelet-derived growth factor (PDGF)-B knockout mouse model, in which developing blood vessels in the central nervous system are almost completely devoid of pericytes. Using cDNA microarrays, we analyzed the gene expression in PDGF-B null embryos in comparison with corresponding wild-type embryos and searched for down-regulated genes. The most down-regulated gene present on our microarray was RGS5, a member of the RGS family of GTPase-activating proteins for G proteins. In situ hybridization identified RGS5 expression in brain pericytes, and in pericytes and vascular smooth muscle cells in certain other, but not all, locations. Absence of RGS5 expression in PDGF-B and PDGFR beta-null embryos correlated with pericyte loss in these mice. Residual RGS5 expression in rare pericytes suggested that RGS5 is a pericyte marker expressed independently of PDGF-B/R beta signaling. With RGS5 as a proof-of-principle, our data demonstrate the usefulness of microarray analysis of mouse models for abnormal pericyte development in the identification of new pericyte-specific markers.