ABSTRACT
Small molecule inhibitors of Bruton's tyrosine kinase (BTK) have been approved for the treatment of multiple B-cell malignancies and are being evaluated for autoimmune and inflammatory diseases. Various BTK inhibitors (BTKi) have distinct potencies, selectivity profiles, and binding modes within the ATP-binding site. On the basis of the latter feature, BTKis can be classified into those that occupy the back-pocket, H3 pocket, and the hinge region only. Hypothesizing that differing binding modes may have differential impact on the B-cell receptor (BCR) signaling pathway, we evaluated the activities of multiple BTKis in B-cell lymphoma models in vitro and in vivo. We demonstrated that, although all three types of BTKis potently inhibited BTK-Y223 autophosphorylation and phospholipase C gamma 2 (PLCγ2)-Y1217 transphosphorylation, hinge-only binders were defective in inhibiting BTK-mediated calcium mobilization upon BCR activation. In addition, PLCγ2 activation was effectively blocked by back-pocket and H3 pocket binders but not by hinge-only binders. Further investigation using TMD8 cells deficient in Rac family small GTPase 2 (RAC2) revealed that RAC2 functioned as a bypass mechanism, allowing for residual BCR signaling and PLCγ2 activation when BTK kinase activity was fully inhibited by the hinge-only binders. These data reveal a kinase activity-independent function of BTK, involving RAC2 in transducing BCR signaling events, and provide mechanistic rationale for the selection of clinical candidates for B-cell lymphoma indications.
Subject(s)
Lymphoma, B-Cell , Protein-Tyrosine Kinases , Humans , Phospholipase C gamma/metabolism , Signal Transduction , Agammaglobulinaemia Tyrosine Kinase , Lymphoma, B-Cell/drug therapy , Receptors, Antigen, B-Cell/metabolism , Protein Kinase Inhibitors/pharmacologyABSTRACT
Inosine is an endogenous purine nucleoside that is produced by catabolism of adenosine. Adenosine has a short half-life (approximately 10s) and is rapidly deaminated to inosine, a stable metabolite with a half-life of approximately 15h. Resembling adenosine, inosine acting through adenosine receptors (ARs) exerts a wide range of anti-inflammatory and immunomodulatory effects in vivo. The immunomodulatory effects of inosine in vivo, at least in part, are mediated via the adenosine A2A receptor (A2AR), an observation that cannot be explained fully by in vitro pharmacological characterization of inosine at the A2AR. It is unclear whether the in vivo effects of inosine are due to inosine or a metabolite of inosine engaging the A2AR. Here, utilizing a combination of label-free, cell-based, and membrane-based functional assays in conjunction with an equilibrium agonist-binding assay we provide evidence for inosine engagement at the A2AR and subsequent activation of downstream signaling events. Inosine-mediated A2AR activation leads to cAMP production with an EC50 of 300.7µM and to extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation with an EC50 of 89.38µM. Our data demonstrate that inosine produces ERK1/2-biased signaling whereas adenosine produces cAMP-biased signaling at the A2AR, highlighting pharmacological differences between these two agonists. Given the in vivo stability of inosine, our data suggest an additional, previously unrecognized, mechanism that utilizes inosine to functionally amplify and prolong A2AR activation in vivo.
Subject(s)
Adenosine A2 Receptor Agonists/pharmacology , Inosine/pharmacology , Receptor, Adenosine A2A/metabolism , Signal Transduction , Animals , CHO Cells , Cricetinae , Cricetulus , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Drug Inverse Agonism , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , MiceSubject(s)
Epidermis/drug effects , Hesperidin/administration & dosage , Animals , Cell Proliferation , Epidermis/metabolism , Hesperidin/chemistry , Homeostasis , Hydrogen-Ion Concentration , Inflammation , Lipids/chemistry , Mice , NF-E2-Related Factor 2/metabolism , Permeability , Proliferating Cell Nuclear Antigen/metabolism , Skin Aging , Skin Physiological Phenomena , Wound HealingABSTRACT
Systemic and topical glucocorticoids (GC) can cause significant adverse effects not only on the dermis, but also on epidermal structure and function. In epidermis, a striking GC-induced alteration in permeability barrier function occurs that can be attributed to an inhibition of epidermal mitogenesis, differentiation and lipid production. As prior studies in normal hairless mice demonstrated that topical applications of a flavonoid ingredient found in citrus, hesperidin, improve epidermal barrier function by stimulating epidermal proliferation and differentiation, we assessed here whether its topical applications could prevent GC-induced changes in epidermal function in murine skin and the basis for such effects. When hairless mice were co-treated topically with GC and 2% hesperidin twice-daily for 9 days, hesperidin co-applications prevented the expected GC-induced impairments of epidermal permeability barrier homoeostasis and stratum corneum (SC) acidification. These preventive effects could be attributed to a significant increase in filaggrin expression, enhanced epidermal ß-glucocerebrosidase activity and accelerated lamellar bilayer maturation, the last two likely attributable to a hesperidin-induced reduction in stratum corneum pH. Furthermore, co-applications of hesperidin with GC largely prevented the expected GC-induced inhibition of epidermal proliferation. Finally, topical hesperidin increased epidermal glutathione reductase mRNA expression, which could counteract multiple functional negative effects of GC on epidermis. Together, these results show that topical hesperidin prevents GC-induced epidermal side effects by divergent mechanisms.
Subject(s)
Clobetasol/adverse effects , Clobetasol/antagonists & inhibitors , Epidermis/drug effects , Glucocorticoids/adverse effects , Glucocorticoids/antagonists & inhibitors , Hesperidin/administration & dosage , Administration, Topical , Animals , Cell Proliferation/drug effects , Clobetasol/administration & dosage , Epidermis/pathology , Epidermis/physiopathology , Female , Filaggrin Proteins , Glucocorticoids/administration & dosage , Glutathione Reductase/genetics , Intermediate Filament Proteins/genetics , Lipid Metabolism/drug effects , Mice , Mice, Hairless , Mice, Inbred C57BL , RNA, Messenger/genetics , RNA, Messenger/metabolism , Up-Regulation/drug effectsABSTRACT
Acute psychological stress (PS) mobilizes metabolic responses that are of immediate benefit to the host, but the current medical paradigm holds that PS exacerbates systemic and cutaneous inflammatory disorders. Although the adverse consequences of PS are usually attributed to neuroimmune mechanisms, PS also stimulates an increase in endogenous glucocorticoids (GCs) that compromises permeability barrier homeostasis, stratum corneum cohesion, wound healing, and epidermal innate immunity in normal skin. Yet, if such PS-induced increases in GC were uniformly harmful, natural selection should have eliminated this component of the stress response. Hence, we hypothesized here instead that stress-induced elevations in endogenous GC could benefit, rather than aggravate, cutaneous function and reduce inflammation in three immunologically diverse mouse models of inflammatory diseases. Indeed, superimposed exogenous (motion-restricted) stress reduced, rather than aggravated inflammation and improved epidermal function in all three models, even normalizing serum IgE levels in the atopic dermatitis model. Elevations in endogenous GC accounted for these apparent benefits, because coadministration of mifepristone prevented stress-induced disease amelioration. Thus, exogenous stress can benefit rather than aggravate cutaneous inflammatory dermatoses through the anti-inflammatory activity of increased endogenous GC.
Subject(s)
Glucocorticoids/metabolism , Skin Diseases/metabolism , Skin Diseases/psychology , Skin/metabolism , Stress, Psychological/metabolism , Animals , Cell Membrane Permeability/physiology , Disease Models, Animal , Female , Homeostasis/physiology , Immunity, Innate/physiology , Immunoglobulin E/blood , Male , Mice , Mice, Hairless , Mice, Inbred C57BL , Skin/physiopathology , Skin Diseases/physiopathology , Stress, Psychological/physiopathology , Wound Healing/physiologyABSTRACT
In terrestrial animals, the epidermal barrier transitions from covering an organism suspended in a liquid environment in utero, to protecting a terrestrial animal postnatally from air and environmental exposure. Tight junctions (TJ) are essential for establishing the epidermal permeability barrier during embryonic development and modulate normal epidermal development and barrier functions postnatally. We now report that TJ function, as well as claudin-1 and occludin expression, change in parallel during late epidermal development. Specifically, TJ block the paracellular movement of Lanthanum (La(3+)) early in rat in vivo prenatal epidermal development, at gestational days 18-19, with concurrent upregulation of claudin-1 and occludin. TJ then become more permeable to ions and water as the fetus approaches parturition, concomitant with development of the lipid epidermal permeability barrier, at days 20-21. This sequence is recapitulated in cultured human epidermal equivalents (HEE), as assessed both by ultrastructural studies comparing permeation of large and small molecules and by the standard electrophysiologic parameter of resistance (R), suggesting further that this pattern of development is intrinsic to mammalian epidermal development. These findings demonstrate that the role of TJ changes during epidermal development, and further suggest that the TJ-based and lipid-based epidermal permeability barriers are interdependent.
Subject(s)
Epidermis/embryology , Epidermis/metabolism , Tight Junctions/metabolism , Animals , Claudin-1/metabolism , Gestational Age , Humans , Occludin/metabolism , Permeability , Rats , Tissue Culture TechniquesABSTRACT
Darier's disease (DD), caused by mutations in the endoplasmic reticulum (ER) Ca(2+) ATPase ATP2A2 (SERCA2b), is a skin disease that exhibits impaired epidermal cell-to-cell adhesion and altered differentiation. Although previous studies have shown that keratinocyte Ca(2+) sequestration and fluxes are controlled by sphingolipid signaling, the role of this signaling pathway in DD previously has not been investigated. We show here that sphingosine levels increase and sphingosine kinase (SPHK1) expression decreases after inactivating SERCA2b with the specific SERCA2 inhibitors thapsigargin (TG) or small interfering RNA to SERCA2b. Conversely, inhibiting sphingosine lyase rescues the defects in keratinocyte differentiation, E-cadherin localization, desmoplakin (DP) translocation, and ER Ca(2+) sequestration seen in TG-treated keratinocytes. Here, we report early evidence that the keratinocyte sphingolipid and Ca(2+) signaling pathways intersect in ATP2A2-controlled ER Ca(2+) sequestration, E-cadherin and DP localization, and Ca(2+)-controlled differentiation, and thus may be important mediators in DD.
Subject(s)
Calcium/physiology , Cell Differentiation/physiology , Darier Disease/physiopathology , Keratinocytes/pathology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/physiology , Signal Transduction/physiology , Sphingolipids/physiology , Cadherins/metabolism , Calcium Signaling/drug effects , Calcium Signaling/physiology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Differentiation/drug effects , Cells, Cultured , Darier Disease/drug therapy , Desmoplakins/metabolism , Enzyme Inhibitors/pharmacology , Humans , Keratinocytes/drug effects , Keratinocytes/metabolism , Mutation/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , RNA, Small Interfering/pharmacology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/antagonists & inhibitors , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Thapsigargin/pharmacologyABSTRACT
BACKGROUND: In Aedes and Anopheles mosquitoes, ribosomal protein RPS6 has an unusual C-terminal extension that resembles histone H1 proteins. To explore homology between a mosquito H1 histone and the RPS6 tail, we took advantage of the Anopheles gambiae genome database to clone a histone H1 gene from an Anopheles stephensi mosquito cell line. RESULTS: We designed specific primers based on RPS6 and histone H1 alignments to recover an Anopheles stephensi histone H1 corresponding to a conceptual An. gambiae protein, with 92% identity. Southern blots suggested that Anopheles stephensi histone H1 gene has multiple variants, as is also the case for histone H1 proteins in Chironomid flies. CONCLUSIONS: Histone H1 proteins from Anopheles stephensi and Anopheles gambiae mosquitoes share 92% identity to each other, but only 50% identity to a Drosophila homolog. In a phylogenetic analysis, Anopheles, Chironomus and Drosophila histone H1 proteins cluster separately from the histone H1-like, C-terminal tails on RPS6 in Aedes and Anopheles mosquitoes. These observations suggest that the resemblance between histone H1 and the C-terminal extensions on mosquito RPS6 has been maintained by convergent evolution.