Similarity and difference between systemic lupus erythematosus and NZB/W F1 mice by multi-omics analysis.

OBJECTIVES: Several animal disease models have been used to understand the mechanisms of systemic lupus erythematosus (SLE); however, the translation of findings from animals to humans has not been sufficiently examined in drug development. To confirm the validity of New Zealand black x New Zealand white (NZB/W) F1 mice as an SLE model, we extensively characterized SLE patients and NZB/W F1 mice by omics analysis. METHODS: Peripheral blood from patients and mice and spleen and lymph node tissue from mice were analysed using cell subset analysis, cytokine panel assays, and transcriptome analysis. RESULTS: CD4+ effector memory T cells, plasmablasts, and plasma cells were increased in both SLE patients and NZB/W F1 mice. Levels of tumor necrosis factor-α, interferon gamma induced protein-10, and B cell activating factor in plasma were significantly higher in SLE patients and NZB/W F1 mice than in their corresponding controls. Transcriptome analysis revealed an upregulation of genes involved in the interferon signalling pathway and T-cell exhaustion signalling pathway in both SLE patients and the mouse model. In contrast, death receptor signalling genes showed changes in the opposite direction between patients and mice. CONCLUSION: NZB/W F1 mice are a generally suitable model of SLE for analysing the pathophysiology and treatment response of T/B cells and monocytes/macrophages and their secreted cytokines.

Conversion of antigen-specific effector/memory T cells into Foxp3-expressing Treg cells by inhibition of CDK8/19.

A promising way to restrain hazardous immune responses, such as autoimmune disease and allergy, is to convert disease-mediating T cells into immunosuppressive regulatory T (Treg) cells. Here, we show that chemical inhibition of the cyclin-dependent kinase 8 (CDK8) and CDK19, or knockdown/knockout of the CDK8 or CDK19 gene, is able to induce Foxp3, a key transcription factor controlling Treg cell function, in antigen-stimulated effector/memory as well as naïve CD4+ and CD8+ T cells. The induction was associated with STAT5 activation, independent of TGF-ß action, and not affected by inflammatory cytokines. Furthermore, in vivo administration of a newly developed CDK8/19 inhibitor along with antigen immunization generated functionally stable antigen-specific Foxp3+ Treg cells, which effectively suppressed skin contact hypersensitivity and autoimmune disease in animal models. The results indicate that CDK8/19 is physiologically repressing Foxp3 expression in activated conventional T cells and that its pharmacological inhibition enables conversion of antigen-specific effector/memory T cells into Foxp3+ Treg cells for the treatment of various immunological diseases.

Metabolomic approach to the exploration of biomarkers associated with disease activity in rheumatoid arthritis.

We aimed to investigate metabolites associated with the 28-joint disease activity score based on erythrocyte sedimentation rate (DAS28-ESR) in patients with rheumatoid arthritis (RA) using capillary electrophoresis quadrupole time-of-flight mass spectrometry. Plasma and urine samples were collected from 32 patients with active RA (DAS28-ESR≥3.2) and 17 with inactive RA (DAS28-ESR<3.2). We found 15 metabolites in plasma and 20 metabolites in urine which showed a significant but weak positive or negative correlation with DAS28-ESR. When metabolites between active and inactive patients were compared, 9 metabolites in plasma and 15 in urine were found to be significantly different. Consequently, we selected 11 metabolites in plasma and urine as biomarker candidates which significantly correlated positively or negatively with DAS28-ESR, and significantly differed between active and inactive patients. When a multiple logistic regression model was built to discriminate active and inactive cohorts, three variables-histidine and guanidoacetic acid from plasma and hypotaurine from urine-generated a high area under the receiver operating characteristic (ROC) curve value (AUC = 0.8934). Thus, this metabolomics approach appeared to be useful for investigating biomarkers of RA. Combination of plasma and urine analysis may lead to more precise and reliable understanding of the disease condition. We also considered the pathophysiological significance of the found biomarker candidates.

Constitutive Activation of Integrin α9 Augments Self-Directed Hyperplastic and Proinflammatory Properties of Fibroblast-like Synoviocytes of Rheumatoid Arthritis.

Despite advances in the treatment of rheumatoid arthritis (RA), currently approved medications can have significant side effects due to their direct immunosuppressive activities. Additionally, current therapies do not address residual synovial inflammation. In this study, we evaluated the role of integrin α9 and its ligand, tenascin-C (Tn-C), on the proliferative and inflammatory response of fibroblast-like synoviocytes (FLSs) from RA patients grown in three-dimensional (3D)-micromass culture. FLSs from osteoarthritis patients, when grown in the 3D-culture system, formed self-directed lining-like structures, whereas FLSs from RA tissues (RA-FLSs) developed an abnormal structure of condensed cellular accumulation reflective of the pathogenic features of RA synovial tissues. Additionally, RA-FLSs grown in 3D culture showed autonomous production of proinflammatory mediators. Predominant expression of α9 and Tn-C was observed in the condensed lining, and knockdown of these molecules abrogated the abnormal lining-like structure formation and suppressed the spontaneous expression of matrix metalloproteinases, IL-6, TNFSF11/RANKL, and cadherin-11. Disruption of α9 also inhibited expression of Tn-C, suggesting existence of a positive feedback loop in which the engagement of α9 with Tn-C self-amplifies its own signaling and promotes progression of synovial hyperplasia. Depletion of α9 also suppressed the platelet-derived growth factor-induced hyperplastic response of RA-FLSs and blunted the TNF-α-induced expression of matrix metalloproteinases and IL-6. Finally, α9-blocking Ab also suppressed the formation of the condensed cellular lining by RA-FLSs in 3D cultures in a concentration-related manner. This study demonstrates the central role of α9 in pathogenic behaviors of RA-FLSs and highlights the potential of α9-blocking agents as a nonimmunosuppressive treatment for RA-associated synovitis.

A sphingosine-1-phosphate receptor type 1 agonist, ASP4058, suppresses intracranial aneurysm through promoting endothelial integrity and blocking macrophage transmigration.

BACKGROUND AND PURPOSE: Intracranial aneurysm (IA), common in the general public, causes lethal subarachnoid haemorrhage on rupture. It is, therefore, of utmost importance to prevent the IA from rupturing. However, there is currently no medical treatment. Recent studies suggest that IA is the result of chronic inflammation in the arterial wall caused by endothelial dysfunction and infiltrating macrophages. The sphingosine-1-phosphate receptor type 1 (S1P1 receptor) is present on the endothelium and promotes its barrier function. Here we have tested the potential of an S1P1 agonist, ASP4058, to prevent IA in an animal model. EXPERIMENTAL APPROACH: The effects of a selective S1P1 agonist, ASP4058, on endothelial permeability and migration of macrophages across an endothelial cell monolayer were tested in vitro using a Transwell system, and its effects on the size of IAs were evaluated in a rat model of IA. KEY RESULTS: S1P1 receptor was expressed in endothelial cells of human IA lesions and control arterial walls. ASP4058 significantly reduced FITC-dextran leakage through an endothelial monolayer and suppressed the migration of macrophages across the monolayer in vitro. Oral administration of ASP4058 reduced the vascular permeability, macrophage infiltration and size of the IAs by acting as an S1P1 agonist in the rat model. This effect was mimicked by another two structurally-unrelated S1P1 agonists. CONCLUSION AND IMPLICATIONS: A selective S1P1 agonist is a strong drug candidate for IA treatment as it promotes the endothelial cell barrier and suppresses the trans-endothelial migration of macrophages in IA lesions.

Glucose regulates RMI1 expression through the E2F pathways in adipose cells.

RecQ-mediated genome instability 1 (RMI1) has been identified as a novel energy homeostasis-related molecule. While recent studies have suggested that change in RMI1 expression levels in adipose tissue may affect the body's energy balance, no reports have identified the mechanism behind this expression regulation. In the present study, we found that RMI1 expression increased on differentiation of 3T3-L1 fibroblasts to adipocytes. In addition, glucose stimulation induced RMI1 expression to approximately eight times the baseline level. Further, knockdown of either E2F5 or E2F8 mRNA using siRNA suppressed this glucose-induced up-regulation of RMI1 expression. These results suggest that RMI1 expression may be regulated by glucose, at least in part, via E2F expression.

Glucose metabolism activation by SHIP2 inhibitors via up-regulation of GLUT1 gene in L6 myotubes.

Lipid phosphatase SH2 domain-containing inositol 5'-phosphatase 2 (SHIP2) plays an important role in the regulation of insulin signaling. In this report, we identified AS1938909, a novel small-molecule SHIP2 inhibitor. AS1938909 showed potent inhibition of SHIP2 (Ki=0.44 microuM) and significant selectivity over other related phosphatases. Further, AS1938909 increased Akt phosphorylation, glucose consumption, and glucose uptake in L6 myotubes. Treatment of L6 myotubes with SHIP2 inhibitors for 48 h significantly induced expression of GLUT1 mRNA, but not that of GLUT4. These results suggest that pharmacological inhibition of SHIP2 activates glucose metabolism due, at least in part, to up-regulation of GLUT1 gene expression.

RMI1 deficiency in mice protects from diet and genetic-induced obesity.

The aim of this study is to discover and characterize novel energy homeostasis-related molecules. We screened stock mouse embryonic stem cells established using the exchangeable gene trap method, and examined the effects of deficiency of the target gene on diet and genetic-induced obesity. The mutant strain 0283, which has an insertion at the recQ-mediated genome instability 1 (RMI1) locus, possesses a number of striking features that allow it to resist metabolic abnormalities. Reduced RMI1 expression, lower fasting-blood glucose and a reduced body weight (normal diet) were observed in the mutant mice. When fed a high-fat diet, the mutant mice were resistant to obesity, and also showed improved glucose intolerance and reduced abdominal fat tissue mass and food intake. In addition, the mutants were also resistant to obesity induced by the lethal yellow agouti (A(y)) gene. Endogenous RMI1 genes were found to be up-regulated in the liver and adipose tissue of KK-A(y) mice. RMI1 is a component of the Bloom's syndrome gene helicase complex that maintains genome integrity and activates cell-cycle checkpoint machinery. Interestingly, diet-induced expression of E2F8 mRNA, which is an important cell cycle-related molecule, was suppressed in the mutant mice. These results suggest that the regulation of energy balance by RMI1 is attributable to the regulation of food intake and E2F8 expression in adipose tissue. Taken together, these findings demonstrate that RMI1 is a novel molecule that regulates energy homeostasis.

Novel method for selecting immunosuppressive histone deacetylase (HDAC) inhibitors with minimal thrombocytopenia.

Histone deacetylase (HDAC) inhibitors repress interleukin-2 (IL-2) gene expression in T cells and possess immunosuppressive activity in vivo. In addition to its immunosuppressive activity, HDAC inhibitors block GATA binding protein-1 (GATA-1) gene expression in megakaryocytes and elicit thrombocytopenia. In this report we state that for a given immunosuppressive dose of HDAC inhibitor, the ratio of GATA-1 reporter gene activity relative to IL-2 reporter gene assay (G/I ratio of measured IC(50)) can be predictive of a HDAC inhibitor's thrombocytopenic effect. This study utilized nine HDAC inhibitors at a minimal effective dose in a rat heterotopic cardiac transplantation model and the resultant G/I ratios and platelet depletion rates were highly correlated (r=0.933). These results indicate that calculation of G/I ratio can be a novel method for selecting immunosuppressive HDAC inhibitor having minimal thrombocytopenic effect which will benefit the search for new immunosuppressants of greater safety and efficacy.

Mechanism of HDAC inhibitor FR235222-mediated IL-2 transcriptional repression in Jurkat cells.

Interleukin (IL)-2 is an essential cytokine in T cell proliferation and homeostasis. The importance of IL-2 down-regulation in preventing acute rejection in organ transplantation and the development of autoimmune diseases has been demonstrated by the therapeutic usefulness of the widely used immunosuppressants cyclosporine A and FK506. Recently, a histone deacetylase (HDAC) inhibitor, FR235222, has been shown to inhibit IL-2 gene expression and to possess immunosuppressive activity in vivo. To elucidate the inhibitory mechanism of FR235222 in IL-2 gene expression, we performed Affymetrix GeneChip analysis of activated Jurkat cells treated with or without FR235222. Here, we show that many NF-kappaB-regulated genes are transcriptionally down-regulated by FR235222 in activated Jurkat cells. Further, luciferase reporter assays revealed that FR235222 selectively inhibits NF-kappaB activity without impairing NF-AT or AP-1 at the concentrations at which it potently inhibits IL-2 promoter activation. These results indicate that FR235222 inhibits IL-2 gene expression via a different mechanism to CsA and FK506, and that FR235222 has the ability to inhibit NF-kappaB activity, which may be partly related to the potent inhibition of IL-2 gene expression by FR235222. Our findings may help our understanding of the molecular mechanism of the inhibition of IL-2 gene expression by HDAC inhibitors and provide insight into the development of more effective and safer new immunosuppressants.

Mechanisms of HDAC inhibitor-induced thrombocytopenia.

Histone deacetylase inhibitors (HDAC inhibitors) are an emerging class of anticancer agents. To elucidate the mechanism of HDAC inhibitor-induced thrombocytopenia, we focused on the effects of HDAC inhibitors on megakaryocyte differentiation and performed Affymetrix GeneChip analysis of human megakaryocytic HEL cells treated with or without HDAC inhibitors. Here, we report that GATA-1 and 10 haematopoietic factors (SCL, NF-E2, EKLF, Pleckstrin, Thrombin-R, LMO2, PU.1, Fli-1, AML1, and TCF11) are transcriptionally repressed by HDAC inhibitors in a similar pattern (R>0.98), and putative GATA-1-binding sites are found in almost all promoters of these genes. In addition, luciferase reporter assays reveal that mutations of GATA-1-binding sites in the GATA-1 promoter abolish its sensitivity to HDAC inhibitor-mediated down-regulation in HEL cells. Further, this report also asserts that HDAC inhibitor increases megakaryocyte counts and inhibits GATA-1 gene expression in rat spleen. Together, these results suggest that HDAC inhibitors inhibit GATA-1 gene expression by decreasing the transactivation function of GATA-1 itself, and that this may in turn lead to a delay in megakaryocyte maturation and finally cause thrombocytopenia. Our findings may help our understanding of the molecular mechanism of HDAC inhibitor-mediated GATA-1 transcriptional repression and to reduce the risk of HDAC inhibitor-induced thrombocytopenia.

Disruption of HDAC4/N-CoR complex by histone deacetylase inhibitors leads to inhibition of IL-2 gene expression.

Previous studies have shown that HDAC inhibitors selectively inhibit IL-2 gene expression, but the mechanism of this inhibition remains to be elucidated. It was recently reported that HDAC4, a component of the nuclear hormone receptor corepressor (N-CoR) complex, associates with the IL-2 promoter via the transcription factor myocyte enhancer factor 2 (MEF2). We therefore focused on the role of HDAC4/N-CoR complex in the transcriptional regulation of IL-2. Four approaches were used to characterize this role and to investigate the relation between the regulatory function of HDAC4/N-CoR complex and HDAC4-enzymatic activity: (i) HDAC4 silencing by RNA interference, (ii) overexpression of N-CoR repression domain 3 (RD3), (iii) overexpression of HDAC4 point mutants, and (iv) treatment with HDAC inhibitors. Here, we report that HDAC4 plays an essential role in IL-2 promoter activation, and that the formation of the HDAC4/N-CoR complex, which is closely related to HDAC4-enzymatic activity, might be involved in HDAC inhibitor-mediated inhibition of IL-2 gene expression. These observations indicate that the selective inhibition of HDAC4 or the interaction of HDAC4 with N-CoR is likely a potential target for the development of novel immunosuppressants.

Tacrolimus (FK506) limits accumulation of granulocytes and platelets and protects against brain damage after transient focal cerebral ischemia in rat.

We investigated the neuroprotective effect of tacrolimus (FK506) on the ischemia-reperfusion injury caused by transient focal brain ischemia induced by middle cerebral artery (MCA) occlusion for 60 min in rats. Neuronal damage visualized as a decrease of MAP2 immunoreactivity was observed in the cerebral cortex at 9 h after MCA occlusion and further expanded at 24 h. Hypoxic areas visualized with an immunohistochemical reaction for 2-nitroimidazole, a hypoxia marker (hypoxyprobe-1), and accumulation of granulocytes and platelets were also observed at 9 h and 24 h after MCA occlusion. Tacrolimus (1 mg/kg, i.v.), administered immediately after MCA occlusion, attenuated cortical damage and decreased the hypoxyprobe-1 positive area, as well as the number of granulocytes and platelets at 24 h after MCA occlusion. Immunohistochemical analysis showed that tacrolimus reduced the number of blood vessels positively stained for ICAM-1, E-selectin and P-selection. These results suggested that tacrolimus limited attachment of granulocytes and platelets to blood vessels by inhibiting the expression of adhesion molecules and protected neuronal tissue from hypoxic insults.

Temporal and topographic profiles of tissue hypoxia following transient focal cerebral ischemia in rats.

Intravascular accumulation of blood cells after brain ischemia-reperfusion can cause obstruction of cerebral blood flow and tissue hypoxia/ischemia as a consequence. In the present study, we examined temporal and topographic changes of tissue hypoxia/ischemia after occlusion of the middle cerebral artery (MCA) for 60 min in rats with immunohistochemical staining for hypoxia (2-nitroimidazole hypoxia marker: hypoxyprobe-1 adducts). Our results showed that tissue hypoxia expressed as positive staining for hypoxyprobe-1 adducts preceded neuronal degeneration. Platelets and granulocytes were detected close to the hypoxyprobe-1 adducts positive area. These results suggested that the hypoxic environment could persist even after reperfusion of MCA, because of vascular obstruction with accumulation of platelets and granulocytes.

VIP and PACAP receptor pharmacology: a comparison of intracellular signaling pathways.

VIP/PACAP receptor activation stimulates the production of [cAMP]i and [Ca2+]i by coupling to independent G-protein subunits, although agonist potencies for the different transduction pathways appear to differ. Using CHO-K1 cells stably expressing the human VIP/PACAP receptors (hVPAC1R, hVPAC2R, and hPAC1R), functional assays ([cAMP]i and [Ca2+]i) were established and the receptor pharmacology was characterized with five peptide agonists (VIP, PACAP-27, PACAP-38, [Ala(11,22,28)]VIP, and R3P65). The rank order of potency (ROP) was consistent between assays for the individual receptor subtypes, however, higher agonist concentrations (approximately 100-fold) were required for stimulating [Ca2+]i when compared to [cAMP]i.

A systematic comparison of intracellular cyclic AMP and calcium signalling highlights complexities in human VPAC/PAC receptor pharmacology.

VPAC/PAC receptor activation classically results in cyclic-AMP production, with limited reports evaluating calcium signalling. These studies systematically characterise intracellular cyclic-AMP ([cAMP](i)) and calcium ([Ca(2+)](i)) responses in CHO-cells expressing recombinant human (h) VPAC/PAC receptors (hVPAC(1)R, hVPAC(2)R, hPAC(1)R), using two simple, non-radioactive, HT-amenable assays. The rank order of potency (ROP) of the agonists VIP, PACAP-27 and PACAP-38 was similar in both assays for each individual receptor subtype, although potencies (EC(50)) in the [Ca(2+)](i) assay were approximately 100-fold lower. Importantly, this shift was also evident in SHSY-5Y cells endogenously expressing hPAC(1)R. Furthermore, [Ala(11,22,28)]VIP and maxadilan were selective hVPAC(1)R and hPAC(1)R agonists, respectively, and although R3P65 had no demonstrable hVPAC(2)R selectivity, these compounds exhibited comparable reductions in [Ca(2+)](i) EC(50) values. In contrast, PG97-269 and PG99-465, putatively selective hVPAC(1)R and hVPAC(2)R antagonists, respectively, were marginally less potent in [cAMP](i) studies, whereas M65 was equipotent at hPAC(1)R. Moreover, PG99-465 alone increased [cAMP](i) at all three hVPAC/PAC receptor subtypes, with full hVPAC(1)R and hPAC(1)R agonism. With equivalent agonist ROPs generated in both assays, [Ca(2+)](i) signalling provides an alternative approach to examine hVPAC/PAC receptor pharmacology. However, these studies underscore the paucity of receptor selective compounds, complexities in comparing drug potencies across assays, and the pleiotropic nature of VPAC/PAC-receptor signalling.

A selective peroxisome proliferator-activated receptor gamma modulator with distinct fat cell regulation properties.

Adipogenesis is an important process for the improvement of insulin resistance by peroxisome proliferator-activated receptor (PPAR) gamma agonists, such as rosiglitazone and pioglitazone. FK614 [3-(2,4-dichlorobenzyl)-2-methyl-N-(pentylsulfonyl)-3-Hbenzimidazole-5-carboxamide] is a structurally novel class of PPARgamma agonist that improves insulin sensitivity in animal models of type 2 diabetes. Herein, we characterize FK614, a selective PPARgamma modulator (SPPARM) with differential properties affecting the regulation of fat cell function. FK614 behaves as a partial agonist in inducing the interaction of PPARgamma with both transcriptional coactivators, cAMP response element-binding protein-binding protein and steroid receptor coactivator-1, but as a full agonist with both PPAR-binding protein and PPAR-interacting protein, which are required for PPARgamma-mediated adipogenesis. In the differentiating 3T3-L1 adipocytes, the levels of adipose fatty acid-binding protein (aP2) mRNA expression and triglyceride accumulation induced by FK614 were as efficacious as those of rosiglitazone and pioglitazone. In contrast, the effect of FK614 on aP2 gene expression in mature adipocytes was less than that of the other PPARgamma agonists. Furthermore, the long-term treatment of mature adipocytes with rosiglitazone and pioglitazone reduced the expression of phosphodiesterase 3B, the down-regulation of which has an important role in the development of insulin resistance; however, FK614 had no such effect in mature adipocytes. Thus, FK614 behaves as an SPPARM with differential effects on the activation of PPARgamma at each stage of adipocyte differentiation. The stage-dependent selectivity of FK614 may contribute to its enhanced insulin sensitization in differentiating adipocytes and to reduced insulin resistance at the stage of adipocyte hypertrophy.

Unique properties of coactivator recruitment caused by differential binding of FK614, an anti-diabetic agent, to peroxisome proliferator-activated receptor gamma.

FK614 is a structurally novel class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, with the mechanism of its insulin-sensitizing action most likely due to activation of PPARgamma. In this study, properties of FK614 for PPARgamma binding, ability to induce conformational change, and coactivator recruitment were investigated. FK614, rosiglitazone, and pioglitazone competed specific binding of [3H]rosiglitazone to PPARgamma with Ki values of 11 nM, 47 nM, and 1.3 microM, respectively. Limited trypsin digestion of PPARgamma with FK614 or rosiglitazone produced distinct patterns of digested polypeptides, suggesting that FK614 directly binds to PPARgamma but induces specific alterations in receptor conformation. FK614 induced interaction of PPARgamma with nuclear receptor coactivator CBP but of lower magnitude than rosiglitazone and pioglitazone. The estimated Kd values of FK614-, rosiglitazone-, and pioglitazone-PPARgamma complex to CBP peptide were 1.8, 0.64, and 0.72 microM, respectively, indicating FK614-PPARgamma complex exhibits a lower affinity for CBP peptide compared to other agonist-PPARgamma complexes. When tested the effect of FK614 on CBP recruitment induced by 9(S)-hydroxyoctadecadienoic acid, an endogenous ligand, FK614 negatively modulated PPARgamma activation. The unique properties of FK614 may underlie the molecular basis of ligand-dependent transcriptional modulation mediated by PPARgamma.

FK614, a novel peroxisome proliferator-activated receptor gamma modulator, induces differential transactivation through a unique ligand-specific interaction with transcriptional coactivators.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcriptional factor implicated in regulating adipogenesis, glucose homeostasis, and in mediating the action of the insulin sensitizing anti-diabetic thiazolidinedione (TZD) compounds. [3-(2,4-Dichlorobenzyl)-2-methyl-N-(pentylsulfonyl)-3-H-benzimidazole-5-carboxamide] (FK614) is a structurally novel PPARgamma agonist that demonstrates potent anti-diabetic activity in vivo. Herein, we describe that FK614 is a selective PPARgamma ligand with specific transactivation properties that are dependent upon the context of coactivators. FK614 dissociates the corepressors NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptors) from PPARgamma as effectively as rosiglitazone and pioglitazone, but can also differentially induce a ligand specific interaction of PPARgamma with coactivators. The amount of CBP (CREB-binding protein) and SRC-1 (steroid receptor coactivator-1) recruited by FK614 was less than that induced by rosiglitazone and pioglitazone, but FK614 caused similar PGC-1alpha (PPARgamma coactivator-1alpha) recruitment as these compounds. As a consequence of these ligand-specific differences in the strength of ligand-type specific interactions of PPARgamma and coactivators, FK614 functions as a partial or full agonist for transcriptional activation depending upon the amount of specific coactivators in cells following overexpression. In conclusion, FK614 is a novel, non-TZD type, and selective PPARgamma modulator whose pharmacological properties are distinct from rosiglitazone and pioglitazone.

FK1706, a novel non-immunosuppressive immunophilin: neurotrophic activity and mechanism of action.

Immunophilin ligands are neuroregenerative agents, characterized by binding to FK506 binding proteins (FKBPs), which stimulate recovery of neurons in a variety of injury paradigms. Here we report the discovery of a novel, non-immunosuppressive immunophilin ligand, FK1706. FK1706, a derivative of FK506, showed similarly high affinity for two FKBP subtypes, FKBP-12 and FKBP-52, but inhibited T-cell proliferation and interleukin-2 cytokine production with much lower potency and efficacy than FK506. FK1706 (0.1 to 10 nM) significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in SH-SY5Y cells, as did FK506. This neurite potentiation could be blocked by an anti-FKBP-52 antibody, as well as by specific pharmacological inhibitors of phospholipase C (PLC), phosphatidylinositol 3-kinase (PI3K), and the Ras/Raf/Mitogen-Activated Protein Kinase (MAPK) signaling pathway. FK1706 also potentiated NGF-induced MAPK activation, with a similar dose-dependency to that necessary for potentiating neurite outgrowth. Taken together, these data suggest that FK1706 is a non-immunosuppressive immunophilin ligand with significant neurotrophic effects, putatively mediated via FKBP-52 and the Ras/Raf/MAPK signaling pathway, and therefore that FK1706 may have therapeutic potential in a variety of neurological disorders.