Unique and non-redundant function of csf1r paralogues in regulation and evolution of post-embryonic development of the zebrafish.

Evolution is replete with reuse of genes in different contexts, leading to multifunctional roles of signaling factors during development. Here, we explore osteoclast regulation during skeletal development through analysis of colony-stimulating factor 1 receptor (csf1r) function in the zebrafish. A primary role of Csf1r signaling is to regulate the proliferation, differentiation and function of myelomonocytic cells, including osteoclasts. We demonstrate the retention of two functional paralogues of csf1r in zebrafish. Mutant analysis indicates that the paralogues have shared, non-redundant roles in regulating osteoclast activity during the formation of the adult skeleton. csf1ra, however, has adopted unique roles in pigment cell patterning not seen in the second paralogue. We identify a unique noncoding element within csf1ra of fishes that is sufficient for controlling gene expression in pigment cells during development. As a role for Csf1r signaling in pigmentation is not observed in mammals or birds, it is likely that the overlapping roles of the two paralogues released functional constraints on csf1ra, allowing the signaling capacity of Csf1r to serve a novel function in the evolution of pigment pattern in fishes.

MAZ induces MYB expression during the exit from quiescence via the E2F site in the MYB promoter.

Most E2F-binding sites repress transcription through the recruitment of Retinoblastoma (RB) family members until the end of the G1 cell-cycle phase. Although the MYB promoter contains an E2F-binding site, its transcription is activated shortly after the exit from quiescence, before RB family members inactivation, by unknown mechanisms. We had previously uncovered a nuclear factor distinct from E2F, Myb-sp, whose DNA-binding site overlapped the E2F element and had hypothesized that this factor might overcome the transcriptional repression of MYB by E2F-RB family members. We have purified Myb-sp and discovered that Myc-associated zinc finger proteins (MAZ) are major components. We show that various MAZ isoforms are present in Myb-sp and activate transcription via the MYB-E2F element. Moreover, while forced RB or p130 expression repressed the activity of a luciferase reporter driven by the MYB-E2F element, co-expression of MAZ proteins not only reverted repression, but also activated transcription. Finally, we show that MAZ binds the MYB promoter in vivo, that its binding site is critical for MYB transactivation, and that MAZ knockdown inhibits MYB expression during the exit from quiescence. Together, these data indicate that MAZ is essential to bypass MYB promoter repression by RB family members and to induce MYB expression.

Specific calcineurin targeting in macrophages confers resistance to inflammation via MKP-1 and p38.

Macrophages contribute to tissue homeostasis and influence inflammatory responses by modulating their phenotype in response to the local environment. Understanding the molecular mechanisms governing this plasticity would open new avenues for the treatment for inflammatory disorders. We show that deletion of calcineurin (CN) or its inhibition with LxVP peptide in macrophages induces an anti-inflammatory population that confers resistance to arthritis and contact hypersensitivity. Transfer of CN-targeted macrophages or direct injection of LxVP-encoding lentivirus has anti-inflammatory effects in these models. Specific CN targeting in macrophages induces p38 MAPK activity by downregulating MKP-1 expression. However, pharmacological CN inhibition with cyclosporin A (CsA) or FK506 did not reproduce these effects and failed to induce p38 activity. The CN-inhibitory peptide VIVIT also failed to reproduce the effects of LxVP. p38 inhibition prevented the anti-inflammatory phenotype of CN-targeted macrophages, and mice with defective p38-activation were resistant to the anti-inflammatory effect of LxVP. Our results identify a key role for CN and p38 in the modulation of macrophage phenotype and suggest an alternative treatment for inflammation based on redirecting macrophages toward an anti-inflammatory status.

NFATc3 promotes Ca(2+) -dependent MMP3 expression in astroglial cells.

Increase in intracellular calcium ([Ca(2+) ]i ) is a key mediator of astrocyte signaling, important for activation of the calcineurin (CN)/nuclear factor of activated T cells (NFAT) pathway, a central mediator of inflammatory events. We analyzed the expression of matrix metalloproteinase 3 (Mmp3) in response to increases in [Ca(2+) ]i and the role of the CN/NFAT pathway in this regulation. Astrocyte Mmp3 expression was induced by overexpression of a constitutively active form of NFATc3, whereas other MMPs and tissue inhibitor of metalloproteinases (TIMP) were unaffected. Mmp3 mRNA and protein expression was also induced by calcium ionophore (Io) and 2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (Bz-ATP) and Mmp3 upregulation was prevented by the CN inhibitor cyclosporin A (CsA). Ca(2+) -dependent astrocyte Mmp3 expression was also inhibited by actinomycin D, and a Mmp3 promoter luciferase reporter was efficiently activated by increased [Ca(2+) ]i , indicating regulation at the transcriptional level. Furthermore, Ca(2+) /CN/NFAT dependent Mmp3 expression was confirmed in pure astrocyte cultures derived from neural stem cells (Ast-NSC), demonstrating that the induced Mmp3 expression occurs in astrocytes, and not microglial cells. In an in vivo stab-wound model of brain injury, MMP3 expression was detected in NFATc3-positive scar-forming astrocytes. Because [Ca(2+) ]i increase is an early event in most brain injuries, these data support an important role for Ca(2+) /CN/NFAT-induced astrocyte MMP3 expression in the early neuroinflammatory response. Understanding the molecular pathways involved in this regulation could provide novel therapeutic targets and approaches to promoting recovery of the injured brain.

NFATc3 regulates the transcription of genes involved in T-cell activation and angiogenesis.

The nuclear factor of activated T cells (NFAT) family of transcription factors plays important roles in many biologic processes, including the development and function of the immune and vascular systems. Cells usually express more than one NFAT member, raising the question of whether NFATs play overlapping roles or if each member has selective functions. Using mRNA knock-down, we show that NFATc3 is specifically required for IL2 and cyclooxygenase-2 (COX2) gene expression in transformed and primary T cells and for T-cell proliferation. We also show that NFATc3 regulates COX2 in endothelial cells, where it is required for COX2, dependent migration and angiogenesis in vivo. These results indicate that individual NFAT members mediate specific functions through the differential regulation of the transcription of target genes. These effects, observed on short-term suppression by mRNA knock-down, are likely to have been masked by compensatory effects in gene-knockout studies.

Bioavailability of Cariban® Capsules: A Modified-Release Fixed-Dose Combination of Doxylamine and Pyridoxine to Relieve Nausea and Vomiting During Pregnancy.

BACKGROUND: Nausea and vomiting is a very prevalent condition during pregnancy. Combination of doxylamine and pyridoxine is placed as first-line pharmacological option for its treatment in most clinical guidelines. Among different release forms available, Cariban® is a fixed-dose combination of doxylamine/pyridoxine 10/10 mg, formulated as modified-release capsules. OBJECTIVES: In the present study, we aimed to characterize the bioavailability performance of Cariban® in vitro and in vivo. METHODS: An in vitro dissolution test was performed to evaluate the release profile of Cariban®, together with immediate- and delayed-release formulations available on the market. A single-center, single-dose, open-label bioavailability study following Cariban® administration in 12 healthy adult female patients was carried out to explore the drug behavior in vivo (protocol NBR-002-13; EUDRA-CT 2013-005422-35). These data were additionally used to perform a computational pharmacokinetic simulation of the posology approved for this drug. RESULTS: Cariban® capsules demonstrate a prolonged-release performance, with an early, gradual, and progressive release of both actives until reaching a complete dissolution after 4-5 h in solution. The pharmacokinetic features of these capsules show that doxylamine and pyridoxine metabolites are early absorbed, being all detectable in plasma within 1 h following oral administration. Computational pharmacokinetic simulation predicts that different posology provides distinct profiles of metabolites in plasma, with 1-1-2 (morning-midafternoon-night) being the one that concentrates higher plasma levels but lower dose dumping for 24 h. CONCLUSION: Cariban® behaves as a prolonged-release formulation, which correlates with rapid absorption and arising of the actives in the plasma, but also long-lasting and sustained bioavailability, especially when administered following the complete posology. These results would underlie its demonstrated efficacy to relieve nausea and vomiting of pregnancy (NVP) under clinical settings.

Iron Protein Succinylate in the Management of Iron Deficiency Anemia: A Comparative Study with Ferrous Sulphate at Low and High Therapeutic Doses.

Oral iron supplementation constitutes the first line treatment for iron deficiency anemia (IDA), with daily doses between 80 mg and 200 mg of elemental iron. Ferrous salts, such as ferrous sulphate (FeSO4), while efficacious, frequently give rise to gastrointestinal side effects. In the present paper we attempted to directly compare the efficacy of an alternative to the FeSO4 formulation, which presents a better tolerability profile, iron protein succinylate (Ferplex®). In a diet-induced anemia model, rats were treated by oral gavage with vehicle, FeSO4, or Ferplex® at a human-dose equivalent of 80 mg and 200 mg of elemental iron. We evaluated the change in anemia-related hematological and biochemical parameters, conducting a histological examination of the intestine at sacrifice. Results indicate that both types of iron supplementation are equally effective in the treatment of IDA, restoring hemoglobin, hematocrit, erythrocytes, free iron and transferrin levels in 15 days, with no statistical differences between treated groups and control. The impact of anemia on body weight was also attenuated following treatment with both iron supplements. Thrombocyte and reticulocyte levels, altered by the anemic condition, returned to homeostasis after 15 days of either FeSO4 or Ferplex® treatment. Importantly, the lower and higher doses of iron were equally effective, thus supporting the current school of thought which states that lower therapeutic doses are sufficient for management of IDA. In addition, the study shows for the first time that oral treatment with Ferplex® does not increase serum hepcidin. Finally, Ferplex® induced minimal iron depositions in the intestinal tissue compared to FeSO4.

NFATc3 controls tumour growth by regulating proliferation and migration of human astroglioma cells.

Calcium/Calcineurin/Nuclear Factor of Activated T cells (Ca/CN/NFAT) signalling pathway is the main calcium (Ca2+) dependent signalling pathway involved in the homeostasis of brain tissue. Here, we study the presence of NFATc members in human glioma by using U251 cells and a collection of primary human glioblastoma (hGB) cell lines. We show that NFATc3 member is the predominant member. Furthermore, by using constitutive active NFATc3 mutant and shRNA lentiviral vectors to achieve specific silencing of this NFATc member, we describe cytokines and molecules regulated by this pathway which are required for the normal biology of cancer cells. Implanting U251 in an orthotopic intracranial assay, we show that specific NFATc3 silencing has a role in tumour growth. In addition NFATc3 knock-down affects both the proliferation and migration capacities of glioma cells in vitro. Our data open the possibility of NFATc3 as a target for the treatment of glioma.

Conditional deletion of Rcan1 predisposes to hypertension-mediated intramural hematoma and subsequent aneurysm and aortic rupture.

Aortic intramural hematoma (IMH) can evolve toward reabsorption, dissection or aneurysm. Hypertension is the most common predisposing factor in IMH and aneurysm patients, and the hypertensive mediator angiotensin-II induces both in mice. We have previously shown that constitutive deletion of Rcan1 isoforms prevents Angiotensin II-induced aneurysm in mice. Here we generate mice conditionally lacking each isoform or all isoforms in vascular smooth muscle cells, endothelial cells, or ubiquitously, to determine the contribution to aneurysm development of Rcan1 isoforms in vascular cells. Surprisingly, conditional Rcan1 deletion in either vascular cell-type induces a hypercontractile phenotype and aortic medial layer disorganization, predisposing to hypertension-mediated aortic rupture, IMH, and aneurysm. These processes are blocked by ROCK inhibition. We find that Rcan1 associates with GSK-3ß, whose inhibition decreases myosin activation. Our results identify potential therapeutic targets for intervention in IMH and aneurysm and call for caution when interpreting phenotypes of constitutively and inducibly deficient mice.

Stretching Reduces Skin Thickness and Improves Subcutaneous Tissue Mobility in a Murine Model of Systemic Sclerosis.

OBJECTIVE: Although physical therapy can help preserve mobility in patients with systemic sclerosis (SSc), stretching has not been used systematically as a treatment to prevent or reverse the disease process. We previously showed in rodent models that stretching promotes the resolution of connective tissue inflammation and reduces new collagen formation after injury. Here, we tested the hypothesis that stretching would impact scleroderma development using a mouse sclerodermatous graft-versus-host disease (sclGvHD) model. METHODS: The model consists in the adoptive transfer (allogeneic) of splenocytes from B10.D2 mice (graft) into Rag2-/- BALB/c hosts (sclGvHD), resulting in skin inflammation followed by fibrosis over 4 weeks. SclGvHD mice and controls were randomized to stretching in vivo for 10 min daily versus no stretching. RESULTS: Weekly ultrasound measurements of skin thickness and subcutaneous tissue mobility in the back (relative tissue displacement during passive trunk motion) successfully captured the different phases of the sclGvHD model. Stretching reduced skin thickness and increased subcutaneous tissue mobility compared to no stretching at week 3. Stretching also reduced the expression of CCL2 and ADAM8 in the skin at week 4, which are two genes known to be upregulated in both murine sclGvHD and the inflammatory subset of human SSc. However, there was no evidence that stretching attenuated inflammation at week 2. CONCLUSION: Daily stretching for 10 min can improve skin thickness and mobility in the absence of any other treatment in the sclGvHD murine model. These pre-clinical results suggest that a systematic investigation of stretching as a therapeutic modality is warranted in patients with SSc.

Utility of quantitative micro-computed tomographic analysis in zebrafish to define gene function during skeletogenesis.

The zebrafish is a powerful experimental model to investigate the genetic and morphologic basis of vertebrate development. Analysis of skeletogenesis in this fish is challenging as a result of the small size of the developing and adult zebrafish. Many of the bones of small fishes such as the zebrafish and medaka are quite thin, precluding many standard assays of bone quality and morphometrics commonly used on bones of larger animals. Microcomputed tomography (microCT) is a common imaging technique used for detailed analysis of the skeleton of the zebrafish and determination of mutant phenotypes. However, the utility of this modality for analysis of the zebrafish skeleton, and the effect of inherent variation among individual zebrafish, including variables such as sex, age and strain, is not well understood. Given the increased use and accessibility of microCT, we set out to define the sensitivity of microCT methods in developing and adult zebrafish. We assessed skeletal shape and density measures in the developing vertebrae and parasphenoid of the skull base. We found most skeletal variables are tightly correlated to standard length, but that at later growth stages (>3months) there are age dependent effects on some skeletal measures. Further we find modest strain but not sex differences in skeletal measures. These data suggest that the appropriate control for assessing mutant phenotypes should be age and strain matched, ideally a wild-type sibling. By analyzing two mutants exhibiting skeletal dysplasia, we show that microCT imaging can be a sensitive method to quantify distinct skeletal parameters of adults. Finally, as developing zebrafish skeletons remain difficult to resolve by radiographic means, we define a contrast agent specific for bone that enhances resolution at early stages, permitting detailed morphometric analysis of the forming skeleton. This increased capability for detection extends the use of this imaging modality to leverage the zebrafish model to understand the development causes of skeletal dysplasias.

Anion Exchanger 2 Regulates Dectin-1-Dependent Phagocytosis and Killing of Candida albicans.

Anion exchanger 2 (Ae2; gene symbol, Slc4a2) is a plasma membrane Cl-/HCO3- exchanger expressed in the gastrointestinal tract, kidney and bone. We have previously shown that Ae2 is required for the function of osteoclasts, bone resorbing cells of the macrophage lineage, to maintain homeostatic cytoplasmic pH and electroneutrality during acid secretion. Macrophages require endosomal acidification for pathogen killing during the process known as phagocytosis. Chloride is thought to be the principal ion responsible for maintaining electroneutrality during organelle acidification, but whether Cl-/HCO3- exchangers such as Ae2 contribute to macrophage function is not known. In this study we investigated the role of Ae2 in primary macrophages during phagocytosis. We find that Ae2 is expressed in macrophages where it regulates intracellular pH and the binding of Zymosan, a fungal cell wall derivative. Surprisingly, the transcription and surface expression of Dectin-1, the major phagocytic receptor for Candida albicans (C. albicans) and Zymosan, is reduced in the absence of Ae2. As a consequence, Zymosan-induced Tnfα expression is also impaired in Ae2-deficient macrophages. Similar to Ae2 deficiency, pharmacological alkalinization of lysosomal pH with bafilomycin A decreases both Dectin-1 mRNA and cell surface expression. Finally, Ae2-deficient macrophages demonstrate defective phagocytosis and killing of the human pathogenic fungus C. albicans. Our results strongly suggest that Ae2 is a critical factor in the innate response to C. albicans. This study represents an important contribution to a better understanding of how Dectin-1 expression and fungal clearance is regulated.

IL4RA on lymphatic endothelial cells promotes T cell egress during sclerodermatous graft versus host disease.

Systemic sclerosis (SSc) is a potentially fatal autoimmune disorder with limited therapeutic options. Sclerodermatous graft versus host disease (sclGvHD), induced by transfer of B10.D2 splenocytes into BALB/c Rag2-/- mice, models an inflammatory subset of SSc characterized by a prominent IL13-induced gene expression signature in the skin. Host mice deficient in IL4RA, a subunit of the type II IL4/IL13 receptor, are protected from sclGvHD. While IL4RA has a well-established role in Th2 differentiation and alternative macrophage activation, we report here a previously unappreciated function for IL4RA in lymphatic endothelial cells (LECs): regulation of activated T cell egress. Seven days after splenocyte transfer, Il4ra-/- hosts had increased numbers of activated graft CD4+ T cells in skin draining lymph nodes (dLNs) but fewer T cells in efferent lymph, blood, and skin. Sphingosine-1 phosphate (S1P), master regulator of lymphocyte egress from LNs, was lower in dLNs of Il4ra-/- hosts with a corresponding decrease of S1P kinase 1 (Sphk1) expression in LECs. Bypassing the efferent lymphatics via i.v. injection of CD4+ T cells from dLNs of Il4ra-/- sclGvHD mice restored clinical GvHD in secondary Il4ra-/- recipients. These results identify a role for IL4RA and suggest that modulation of lymphocyte egress from LNs may be effective in SSc and GvHD.

A major role for RCAN1 in atherosclerosis progression.

Atherosclerosis is a complex inflammatory disease involving extensive vascular vessel remodelling and migration of vascular cells. As RCAN1 is implicated in cell migration, we investigated its contribution to atherosclerosis. We show RCAN1 induction in atherosclerotic human and mouse tissues. Rcan1 was expressed in lesional macrophages, endothelial cells and vascular smooth muscle cells and was induced by treatment of these cells with oxidized LDLs (oxLDLs). Rcan1 regulates CD36 expression and its genetic inactivation reduced atherosclerosis extension and severity in Apoe(-/-) mice. This effect was mechanistically linked to diminished oxLDL uptake, resistance to oxLDL-mediated inhibition of macrophage migration and increased lesional IL-10 and mannose receptor expression. Moreover, Apoe(-/-) Rcan1(-/-) macrophages expressed higher-than-Apoe(-/-) levels of anti-inflammatory markers. We previously showed that Rcan1 mediates aneurysm development and that its expression is not required in haematopoietic cells for this process. However, transplantation of Apoe(-/-) Rcan1(-/-) bone-marrow (BM) cells into Apoe(-/-) recipients confers atherosclerosis resistance. Our data define a major role for haematopoietic Rcan1 in atherosclerosis and suggest that therapies aimed at inhibiting RCAN1 expression or function might significantly reduce atherosclerosis burden.