Inositol-requiring enzyme 1 (IRE1) is the most highly conserved signaling node of the unfolded protein response (UPR) and represents a potential therapeutic target for a number of diseases associated with endoplasmic reticulum stress. IRE1 activates the XBP-1 transcription factor by site-specific cleavage of two hairpin loops within its mRNA to facilitate its nonconventional splicing and alternative translation. We screened for inhibitors using a construct containing the unique cytosolic kinase and endoribonuclease domains of human IRE1α (hIRE1α-cyto) and a mini-XBP-1 stem-loop RNA as the substrate. One class compounds was salicylaldehyde analogs from the hydrolyzed product of salicylaldimines in the library. Salicylaldehyde analogs were active in inhibiting the site-specific cleavage of several mini-XBP-1 stem-loop RNAs in a dose-dependent manner. Salicyaldehyde analogs were also active in inhibiting yeast Ire1 but had little activity inhibiting RNase L or the unrelated RNases A and T1. Kinetic analysis revealed that one potent salicylaldehyde analog, 3-ethoxy-5,6-dibromosalicylaldehyde, is a non-competitive inhibitor with respect to the XBP-1 RNA substrate. Surface plasmon resonance studies confirmed this compound bound to IRE1 in a specific, reversible and dose-dependent manner. Salicylaldehydes inhibited XBP-1 splicing induced pharmacologically in human cells. These compounds also blocked transcriptional up-regulation of known XBP-1 targets as well as mRNAs targeted for degradation by IRE1. Finally, the salicylaldehyde analog 3-methoxy-6-bromosalicylaldehyde strongly inhibited XBP-1 splicing in an in vivo model of acute endoplasmic reticulum stress. To our knowledge, salicylaldehyde analogs are the first reported specific IRE1 endoribonuclease inhibitors.

Sunlight (UVB) triggers cutaneous (CLE) and systemic lupus through an unknown mechanism. We tested the hypothesis that UVB triggers CLE through a CSF-1-dependent, macrophage (Mø) -mediated mechanism in MRL-Faslpr mice. By constructing mutant MRL-Faslpr strains expressing varying levels of CSF-1 (high, intermediate, none), and use of an ex-vivo gene transfer to deliver CSF-1 intra-dermally, we determined that CSF-1 induces CLE in lupus-susceptible, MRL-Faslpr mice, but not in lupus-resistant, BALB/c mice. Notably, UVB incites an increase in Mø, apoptosis in the skin and CLE in MRL-Faslpr, but not in CSF-1-deficient MRL-Faslpr mice. Furthermore, UVB did not induce CLE in BALB/c mice. Probing further, UVB stimulates CSF-1 expression by keratinocytes leading to recruitment and activation of Mø that, in turn, release mediators, which induce apoptosis in keratinocytes. Thus, sunlight triggers a CSF-1-dependent, Mø-mediated destructive inflammation in the skin leading to CLE in lupus-susceptible MRL-Faslpr, but not lupus-resistant BALB/c mice. Taken together, we envision CSF-1 as the “match” and lupus-susceptibility as the “tinder” leading to CLE.

MRL/MpJ-Faslpr (MRL-Faslpr) mice develop a spontaneous T cell and macrophage (Mø) dependent autoimmune disease that shares features with human lupus. Interactions via the PD-1/PD-L1 pathway down-regulate immune responses and provide a negative regulatory checkpoint in mediating tolerance and autoimmune disease. Therefore, we tested the hypothesis that the PD-1/PD-L1 pathway suppresses lupus nephritis and the systemic illness in MRL-Faslpr mice. For this purpose, we compared kidney and systemic illness (lymph nodes, spleen, skin, lung, glands) in PD-L1 null (−/−) and PD-L1 intact (wild-type, WT) MRL-Faslpr mice. Unexpectedly, PD-L1−/−;MRL-Faslpr mice died as a result of autoimmune myocarditis and pneumonitis prior to developing renal disease or the systemic illness. Dense infiltrates, consisting of Mø and T cells (CD8+ > CD4+), were prominent throughout the heart (atria and ventricles) and localized specifically around vessels in the lung. In addition, once disease was evident, we detected heart specific autoantibodies (autoAbs) in PD-L1−/−;MRL-Faslpr mice. This unique phenotype is dependent on MRL-specific background genes as PD-L1−/−;MRL-+/+ mice lacking the Fas-lpr mutation developed autoimmune myocarditis and pneumonitis. Notably, the transfer of PD-L1−/−;MRL-+/+ bone marrow (BM) cells induced myocarditis and pneumonitis in WT;MRL-+/+ mice, in spite of a dramatic up-regulation of PD-L1 expression on endothelial cells in the heart and lung of WT;MRL-+/+ mice. Taken together, we suggest that PD-L1 expression is central to autoimmune heart and lung disease in lupus-susceptible (MRL) mice.

The gene encoding the human muscarinic receptor, type 1 (CHRM1), was genotyped from 245 samples of the Coriell Collection (Coriell Institute for Medical Research, Camden, NJ). Fifteen single nucleotide polymorphisms (SNPs) were discovered, 9 of which are located in the coding region of the receptor. Of these, 8 represent synonymous SNPs, indicating that CHRM1 is highly conserved in humans. Only a single allele was found to contain a nonsynonymous SNP, which encodes an amino acid change of Cys to Arg at position 417. This may have functional consequences because a C417S point mutation in rat M1 was previously shown to affect receptor binding and coupling. Furthermore, 0 of 4 SNPs within CHRM1 previously deduced from sequencing of the human genome were found in this study despite a prediction that a majority of such inferred SNPs are accurate. The consensus sequence of CHRM1 obtained in our study differs from the deposited reference sequence (AC NM_000738) in 2 adjacent nucleotides, leading to a V173M change, suggesting a sequencing error in the reference sequence. The extraordinary sequence conservation of the CHRM1 gene-coding region was unexpected as M1-knockout mice show only minimal functional impairments.

Numerous genes encode G protein-coupled receptors (GPCRs)-a main molecular target for drug therapy. Estimates indicate that the human genome contains approximately 600 GPCR genes. This article addresses therapeutic implications of sequence variations in GPCR genes. A number of inactivating and activating receptor mutations have been shown to cause a variety of (mostly rare) genetic disorders. However, pharmacogenetic and pharmacogenomic studies on GPCRs are scarce, and therapeutic relevance of variant receptor alleles often remains unclear. Confounding factors in assessing the therapeutic relevance of variant GPCR alleles include 1) interaction of a single drug with multiple closely related receptors, 2) poorly defined binding pockets that can accommodate drug ligands in different orientations or at alternative receptor domains, 3) possibility of multiple receptor conformations with distinct functions, and 4) multiple signaling pathways engaged by a single receptor. For example, antischizophrenic drugs bind to numerous receptors, several of which might be relevant to therapeutic outcome. Without knowing accurately what role a given receptor subtype plays in clinical outcome and how a sequence variation affects drug-induced signal transduction, we cannot predict the therapeutic relevance of a receptor variant. Genome-wide association studies with single nucleotide polymorphisms could identify critical target receptors for disease susceptibility and drug efficacy or toxicity.