Genetic mutation and pharmacological inhibition of Bruton's tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopro-, in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fc receptor cross-linking-induced degranulation in mast cells (IC 50 ϭ 2.9 nM), Fc␥ receptor engagement-mediated tumor necrosis factor ␣ production in monocytes (IC 50 ϭ 7.0 nM), and B cell antigen receptor-induced expression of an activation marker, CD69, in B cells in whole blood (IC 50 ϭ 21.0 nM). RN486 displayed similar functional activities in rodent models, effectively preventing type I and type III hypersensitivity responses. More importantly, RN486 produced robust anti-inflammatory and boneprotective effects in mouse CIA and rat adjuvant-induced arthritis (AIA) models. In the AIA model, RN486 inhibited both joint and systemic inflammation either alone or in combination with methotrexate, reducing both paw swelling and inflammatory markers in the blood. Together, our findings not only demonstrate that Btk plays an essential and conserved role in regulating immunoreceptor-mediated immune responses in both humans and rodents, but also provide evidence and mechanistic insights to support the development of selective Btk inhibitors as small-molecule disease-modifying drugs for RA and potentially other autoimmune diseases.

Prostaglandin F 2␣ (PGF 2␣ ) exerts its biological effects by binding to and activating FP prostanoid receptors. These receptors, which include two isoforms, the FP A and FP B , have been cloned from a number of species and are members of the superfamily of G-protein-coupled receptors. Previous studies have shown that the activation of FP receptors leads to phosphatidylinositol hydrolysis, intracellular calcium release, and activation of protein kinase C. Here, we demonstrate that PGF 2␣ treatment of 293-EBNA (Epstein-Barr nuclear antigen) cells that have been stably transfected with either the FP A or FP B receptor isoforms leads to changes in cell morphology and in the cell cytoskeleton. Specifically, cells treated with PGF 2␣ show retraction of filopodia and become rounded, and actin stress fibers are formed. Pretreatment of the cells with bisindolylmaleimide I, a protein kinase C inhibitor, has no effect on the PGF 2␣ -induced changes in cell morphology, although it does block the effects of phorbol myristate acetate on cell morphology. On the other hand, the PGF 2␣ -induced changes in cell morphology and formation of actin stress fibers can be blocked by pretreatment of the cells with C3 exoenzyme, a specific inhibitor of the small G-protein, Rho. Consistent with FP receptor induced formation of actin stress fibers and focal adhesions, FP A receptor activation also leads to rapid (within two minutes) tyrosine phosphorylation of p125 focal adhesion kinase (FAK) which can be blocked by pretreating the cells with C3 exoenzyme. Taken together, these results suggest that the FP receptor isoforms are coupled to at least two second messenger pathways, one pathway associated with protein kinase C activation, and the other with activation of Rho. Prostaglandin F 2␣ (PGF 2␣ )1 is one of the biologically active prostanoids formed from the cyclooxygenase-catalyzed metabolism of arachidonic acid. Physiologically, PGF 2␣ is known to be important in regulating luteolysis or regression of the corpus luteum (1) and regulating intraocular pressure (2) and may also be involved in cardiac hypertrophy. Specifically, long term administration of the PGF 2␣ analog fluprostenol to rats led to an increase in both heart weight and ventricular weight relative to body weight (3). In vitro, PGF 2␣ treatment of isolated rat cardiomyocytes has been shown to lead to hypertrophy, to increased myofibrillar organization, and to increased atrial natriuretic factor expression (3-6). PGF 2␣ has been shown to exert its physiological effects by binding to its receptor, the FP prostanoid receptor. This receptor is a member of the superfamily of G-protein-coupled receptors and has been cloned from a number of species, including human (7), mouse (8), bovine (9), and ovine (10). In addition, we recently cloned a carboxyl-terminal isoform of the ovine FP receptor, the FP B isoform (11). This truncated isoform, which arises by alternative mRNA splicing, lacks 46 amino acids present in the original isoform, now called the FP A receptor isoform. In hetero...

1 Prostacyclin (PGI 2 ) possesses various physiological functions, including modulation of nociception, inflammation and cardiovascular activity. Elucidation of these functions has been hampered by the absence of selective IP receptor antagonists. 2 Two structurally distinct series of IP receptor antagonists have been developed: 4,5-dihydro-1H-imidazol-2-yl)-[4-(4-isopropoxy-benzyl)-phenyl]-amine (RO1138452) and R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO3244794). 3 RO1138452 and RO3244794 display high affinity for IP receptors. In human platelets, the receptor affinities (pK i ) were 9.370.1 and 7.770.03, respectively; in a recombinant IP receptor system, pK i values were 8.770.06 and 6.970.1, respectively. 4 Functional antagonism of RO1138452 and RO3244794 was studied by measuring inhibition of carbaprostacyclin-induced cAMP accumulation in CHO-K1 cells stably expressing the human IP receptor. The antagonist affinities (pK i ) of RO1138452 and RO3244794 were 9.070.06 and 8.570.11, respectively. 5 Selectivity profiles for RO1138452 and RO3244794 were determined via a panel of receptor binding and enzyme assays. RO1138452 displayed affinity at I 2 (8.3) and PAF (7.9) receptors, while RO3244794 was highly selective for the IP receptor: pK i values for EP 1 (o5), EP 3 (5.38), EP 4 (5.74) and TP (5.09). 6 RO1138452 (1-10 mg kg , p.o.) significantly reduced carrageenan-induced mechanical hyperalgesia and edema formation. RO3244794 (1 and 10 mg kg À1 , p.o.) also significantly reduced chronic joint discomfort induced by monoiodoacetate. 7 These data suggest that RO1138452 and RO3244794 are potent and selective antagonists for both human and rat IP receptors and that they possess analgesic and anti-inflammatory potential.

Diabetes mellitus (DM) is a common endocrine disease characterized by a state of hyperglycemia (higher level of glucose in the blood than usual). DM and its complications can lead to diabetic foot ulcer (DFU). DFU is associated with impaired wound healing, due to inappropriate cellular and cytokines response, infection, poor vascularization, and neuropathy. Effective therapeutic strategies for the management of impaired wound could be attained through a better insight of molecular mechanism and pathophysiology of diabetic wound healing. Nanotherapeutics-based agents engineered within 1–100 nm levels, which include nanoparticles and nanoscaffolds, are recent promising treatment strategies for accelerating diabetic wound healing. Nanoparticles are smaller in size and have high surface area to volume ratio that increases the likelihood of biological interaction and penetration at wound site. They are ideal for topical delivery of drugs in a sustained manner, eliciting cell-to-cell interactions, cell proliferation, vascularization, cell signaling, and elaboration of biomolecules necessary for effective wound healing. Furthermore, nanoparticles have the ability to deliver one or more therapeutic drug molecules, such as growth factors, nucleic acids, antibiotics, and antioxidants, which can be released in a sustained manner within the target tissue. This review focuses on recent approaches in the development of nanoparticle-based therapeutics for enhancing diabetic wound healing.

Highlights d Prdm16 is dispensable for cardiac development d Prdm16 cKO mice develop hypertrophy, adverse remodeling, and mitochondrial dysfunction d Prdm16 cKO mice are predisposed to develop heart failure in response to metabolic stress d Prdm16 and Ehmts act together to repress expression of hypertrophic genes

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G-protein coupled receptors (GPCRs) are regulated by numerous proteins including kinases, G-proteins, β-arrestins and accessory proteins. Several families of GPCR accessory proteins like Receptor Activity Modifying Proteins, Receptor Transporting Proteins and Melanocortin Receptor Accessory Proteins (MRAPs) have been identified as regulator of receptor trafficking, signaling and ligand specificity. The MRAP family contains two members, MRAP1 and MRAP2, responsible for the formation of a functional ACTH receptor and for the regulation of energy homeostasis respectively. Like all known GPCR accessory proteins, MRAPs are single transmembrane proteins, however, they form a unique structure since they assemble as an anti-parallel homodimer. Moreover, the accepted idea that MRAPs are specific regulators of melanocortin receptors was recently challenged by the discovery that MRAP2 inhibits the activity of prokineticin receptors. Recent studies are starting to explain the role of the unusual structure of MRAPs and to illustrate the importance of MRAP2 for the maintenance of both energy and glucose homeostasis. This article is part of a Special Issue entitled: Melanocortin Receptors - edited by Ya-Xiong Tao.

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