C10orf99/GPR15L Regulates Proinflammatory Response of Keratinocytes and Barrier Formation of the Skin.

The epidermis, outermost layer of the skin, forms a barrier and is involved in innate and adaptive immunity in an organism. Keratinocytes participate in all these three protective processes. However, a regulator of keratinocyte protective responses against external dangers and stresses remains elusive. We found that upregulation of the orphan gene 2610528A11Rik was a common factor in the skin of mice with several types of inflammation. In the human epidermis, peptide expression of G protein-coupled receptor 15 ligand (GPR15L), encoded by the human ortholog C10orf99, was highly induced in the lesional skin of patients with atopic dermatitis or psoriasis. C10orf99 gene transfection into normal human epidermal keratinocytes (NHEKs) induced the expression of inflammatory mediators and reduced the expression of barrier-related genes. Gene ontology analyses showed its association with translation, mitogen-activated protein kinase (MAPK), mitochondria, and lipid metabolism. Treatment with GPR15L reduced the expression levels of filaggrin and loricrin in human keratinocyte 3D cultures. Instead, their expression levels in mouse primary cultured keratinocytes did not show significant differences between the wild-type and 2610528A11Rik deficient keratinocytes. Lipopolysaccharide-induced expression of Il1b and Il6 was less in 2610528A11Rik deficient mouse keratinocytes than in wild-type, and imiquimod-induced psoriatic dermatitis was blunted in 2610528A11Rik deficient mice. Furthermore, repetitive subcutaneous injection of GPR15L in mouse ears induced skin inflammation in a dose-dependent manner. These results suggest that C10orf99/GPR15L is a primary inducible regulator that reduces the barrier formation and induces the inflammatory response of keratinocytes.

Glycyl-tRNA synthetase from Nanoarchaeum equitans: The first crystal structure of archaeal GlyRS and analysis of its tRNA glycylation.

This study reports the X-ray crystallographic structure of the glycyl-tRNA synthetase (GlyRS) of Nanoarchaeum equitans - a hyperthermophilic archaeal species. This is the first archaeal GlyRS crystal structure elucidated. The GlyRS comprises an N-terminal catalytic domain and a C-terminal anticodon-binding domain with a long ß-sheet inserted between these domains. An unmodified transcript of the wild-type N. equitans tRNAGly was successfully glycylated using GlyRS. Substitution of the discriminator base A73 of tRNAGly with any other nucleotide caused a significant decrease in glycylation activity. Mutational analysis of the second base-pair C2G71 of the acceptor stem of tRNAGly elucidated the importance of the base-pair, especially G71, as an identity element for recognition by GlyRS. Glycylation assays using tRNAGly G71 substitution mutants and a GlyRS mutant where Arg223 is mutated to alanine strengthen the possibility that the carbonyl oxygen at position 6 of G71 would hydrogen-bond with the guanidine nitrogen of Arg223 in N. equitans GlyRS.

Possible new therapeutic strategy to regulate atopic dermatitis through upregulating filaggrin expression.

BACKGROUND: Nonsense mutations in filaggrin (FLG) represent a significant genetic factor in the cause of atopic dermatitis (AD). OBJECTIVE: It is of great importance to find drug candidates that upregulate FLG expression and to determine whether increased FLG expression controls the development of AD. METHODS: We screened a library of bioactives by using an FLG reporter assay to find candidates that promoted FLG mRNA expression using a human immortalized keratinocyte cell line (HaCaT). We studied the effect of the compound on keratinocytes using the human skin equivalent model. We examined the effect of the compound on AD-like skin inflammation in NC/Nga mice. RESULTS: JTC801 promoted FLG mRNA and protein expression in both HaCaT and normal human epidermal keratinocytes. Intriguingly, JTC801 promoted the mRNA and protein expression levels of FLG but not the mRNA levels of other makers for keratinocyte differentiation, including loricrin, keratin 10, and transglutaminase 1, in a human skin equivalent model. In addition, oral administration of JTC801 promoted the protein level of Flg and suppressed the development of AD-like skin inflammation in NC/Nga mice. CONCLUSION: This is the first observation that the compound, which increased FLG expression in human and murine keratinocytes, attenuated the development of AD-like skin inflammation in mice. Our findings provide evidence that modulation of FLG expression can be a novel therapeutic target for AD.

Intravital analysis of vascular permeability in mice using two-photon microscopy.

Blood vessel endothelium forms a semi-permeable barrier and its permeability controls the traffics of plasma contents. Here we report an intravital evaluation system for vascular permeability in mice using two-photon microscopy. We used various sizes of fluorescein-conjugated dextran as a tracer and its efflux was quantified by measuring the changes of fluorescent intensity both on the blood vessel area and the interstitial space. Using this system, we demonstrated that skin blood vessels limited the passage of dextran larger than 70 kDa under homeostatic conditions. We evaluated the kinetics of vascular permeability in histamine- or IgE-induced type I allergic models and a hapten-induced type IV allergic model. In such inflammatory conditions, the hyperpermeability was selectively induced in the postcapillary venules and dextran as large as 2000-kDa leaked from the bloods. Taken together, our study provides a convenient method to characterize the skin blood vessels as a traffic barrier in physiological conditions.

Discovery of CS-2100, a potent, orally active and S1P3-sparing S1P1 agonist.

S1P(3)-sparing S1P(1) agonists have attracted attention as a suppressant of autoimmunity with reduced side effects. Our synthetic efforts and extensive SAR studies led to the discovery of 10b named CS-2100 with the EC(50) value of 4.0 nM for human S1P(1) and over 5000-fold selectivity against S1P(3). The in vivo immunosuppressive efficacy was evaluated in rats on host versus graft reaction and the ID(50) value was determined at 0.407mg/kg. The docking studies of CS-2100 with the homology model of S1P(1) and S1P(3) showed that the ethyl group on the thiophene ring of CS-2100 was sterically hindered by Phe263 in S1P(3), not in the case of Leu276 in S1P(1). This observation gives an explanation for the excellent S1P(3)-sparing characteristic of CS-2100.

PGD2 induces eotaxin-3 via PPARγ from sebocytes: a possible pathogenesis of eosinophilic pustular folliculitis.

BACKGROUND: Eosinophilic pustular folliculitis (EPF) is a chronic intractable pruritic dermatosis characterized by massive eosinophil infiltrates involving the pilosebaceous units. Recently, EPF has been regarded as an important clinical marker of HIV infection, and its prevalence is increasing in number. The precise mechanism by which eosinophils infiltrate into the pilosebaceous units remains largely unknown. Given that indomethacin, a COX inhibitor, can be successfully used to treat patients with EPF, we can assume that COX metabolites such as prostaglandins (PGs) are involved in the etiology of EPF. OBJECTIVE: To determine the involvement of PGs in the pathogenesis of EPF. METHODS: We performed immunostaining for PG synthases in EPF skin lesions. We examined the effect of PGD(2) on induction of eotaxin, a chemoattractant for eosinophils, in human keratinocytes, fibroblasts, and sebocytes and sought to identify its responsible receptor. RESULTS: Hematopoietic PGD synthase was detected mainly in infiltrating inflammatory cells in EPF lesions, implying that PGD(2) was produced in the lesions. In addition, PGD(2) and its immediate metabolite 15-deoxy-Δ 12,14-PGJ(2) (15d-PGJ(2)) induced sebocytes to produce eotaxin-3 via peroxisome proliferator-activated receptor gamma. Consistent with the above findings, eotaxin-3 expression was immunohistochemically intensified in sebaceous glands of the EPF lesions. CONCLUSION: The PGD(2)/PGJ(2)-peroxisome proliferator-activated receptor gamma pathway induces eotaxin production from sebocytes, which may explain the massive eosinophil infiltrates observed around pilosebaceous units in EPF.

Discovery of CS-0777: A Potent, Selective, and Orally Active S1P1 Agonist.

CS-0777 (3) is phosphorylated in vivo, and the phosphate of CS-0777 (CS-0777-P) (4) acts as a selective S1P receptor-1 (S1P1) modulator. We report herein the synthesis of CS-0777 and CS-0777-P, pharmacological effects such as S1P1 and S1P3 agonist activity in vitro, peripheral blood lymphocyte lowering effects and the suppressive effect on experimental autoimmune encephalomyelitis (EAE), and also the pharmacokinetics in rats. CS-0777-P had ∼320-fold greater agonist activity for human S1P1 (EC50; 1.1 nM) relative to S1P3 (EC50; 350 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of CS-0777 in rats, lymphocyte counts decreased significantly, with a nadir at 12 h postdose and recovery to vehicle control levels by 5 days postdose. In the EAE model compared to the vehicle-treated group, significant decreases in the cumulative EAE scores were observed for the 0.1 and 1 mg/kg CS-0777 groups in rats. CS-0777 is currently in clinical trials for the treatment of multiple sclerosis (MS).

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: part 3.

In order to develop a new class of anti-rheumatic drug which inhibits production of proinflammatory cytokines such as TNFalpha, IL-1beta, IL-6, and IL-8, a series of 3-pyridylpyrrole derivatives possessing a bicyclic tetrahydropyridine moiety at the 4-position of the pyrrole ring were synthesized and their pharmacological activities were evaluated. The derivatives were found to have potent inhibitory activities on the production of the cytokines both in vitro and in vivo. Among them, compound 4a, (S)-2-(4-fluorophenyl)-4-(1,2,3,5,6,8a-hexahydroindolizin-7-yl)-3-(pyridin-4-yl)-1H-pyrrole (R-132811), achieved the most promising results in various in vitro and in vivo tests including several rheumatoid arthritis models ((i) inhibition of p38alpha, p38beta, p38gamma, and p38delta MAP kinases: IC(50)=0.034, 0.572, >10, and >10 microM, respectively; (ii) inhibition of TNFalpha, IL-1beta, IL-6, and IL-8 production in human whole blood: IC(50)=0.026, 0.020, 0.88, and 0.016 microM, respectively; (iii) inhibition of LPS induced TNFalpha, IL-1beta and IL-6 production in mice: ID(50)=0.93, 8.63, and 0.11 mg/kg, p.o., respectively; (iv) inhibition of anti-collagen antibody-induced arthritis in mice: ID(50)=2.22 mg/kg, p.o.; (v) inhibition of collagen-induced arthritis in mice: ID(50)=2.38 mg/kg, p.o.; (vi) prophylactic effect on adjuvant-induced arthritis in rats: ID(50)=3.1 mg/kg, p.o.; (vii) therapeutic effect on adjuvant-induced arthritis in rats: ID(50)=4.9 mg/kg, p.o.; (viii) analgesic effect on adjuvant-induced arthritic pain in rats: ID(50)=2.9 mg/kg, p.o.). As a result, compound 4a was chosen as a candidate for further pre-clinical studies.

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: part 2.

We previously reported a novel pyrrole derivative 1 which possesses a tetrahydropyridine group at the beta-position with a proinflammatory cytokine TNFalpha production inhibitor. Herein, we report the synthesis and biological activity of N- and alpha-position substituted tetrahydropyridine derivatives. In this series, we found that compound 3o showed good inhibitory activity in vitro (inhibition of lipopolysaccharide (LPS)-induced TNFalpha production in human whole blood, IC(50)=0.44 microM) and compound 3i demonstrated potent inhibitory activity in vivo (inhibition of LPS-induced TNFalpha production in mice, ID(50)=1.42 mg/kg).