The activity of S.pombe DSC-1-like factor is cell cycle regulated and dependent on the activity of p34cdc2.

In the eukaryotic cell cycle, there are major control points in late G2 to determine the timing of the initiation of mitosis, and in late G1, regulating entry into S phase. In yeasts, this latter control is called start. Traverse of the start control and progression to S phase is accompanied by an increase in the expression of some of the genes whose products are required for DNA synthesis. In Saccharomyces cerevisiae, the coordinate expression of these genes in late G1 is dependent on a cis-acting sequence element called the MluI cell cycle box (MCB). A transcription factor called DSC-1 binds these elements and mediates cell cycle regulated transcription, though it is unclear whether this is by cell cycle-dependent changes in its activity. A DSC-1-like factor has also been identified in the fission yeast S.pombe. This is composed of at least the products of the cdc10 and sct1/res1 genes, and binds to the promoters of genes whose expression increases prior to S phase. We demonstrate that p85cdc10 is a nuclear protein and that the activity of the S.pombe DSC-1 factor varies through the cell cycle; it is high in cells that have passed start, decreases at the time of anaphase, remains low during the pre-start phase of G1 and increases at the time of the next S phase. We also show that the reactivation in late G1 is dependent on the G1 form of p34cdc2.

Effect of different G protein-coupled receptor kinases on phosphorylation and desensitization of the alpha1B-adrenergic receptor.

The alpha1B-adrenergic receptor (alpha1BAR), its truncated mutant T368, different G protein-coupled receptor kinases (GRK) and arrestin proteins were transiently expressed in COS-7 or HEK293 cells alone and/or in various combinations. Coexpression of beta-adrenergic receptor kinase (betaARK) 1 (GRK2) or 2 (GRK3) could increase epinephrine-induced phosphorylation of the wild type alpha1BAR above basal as compared to that of the receptor expressed alone. On the other hand, overexpression of the dominant negative betaARK (K220R) mutant impaired agonist-induced phosphorylation of the receptor. Overexpression of GRK6 could also increase epinephrine-induced phosphorylation of the receptor, whereas GRK5 enhanced basal but not agonist-induced phosphorylation of the alpha1BAR. Increasing coexpression of betaARK1 or betaARK2 resulted in the progressive attenuation of the alpha1BAR-mediated response on polyphosphoinositide (PI) hydrolysis. However, coexpression of betaARK1 or 2 at low levels did not significantly impair the PI response mediated by the truncated alpha1BAR mutant T368, lacking the C terminus, which is involved in agonist-induced desensitization and phosphorylation of the receptor. Similar attenuation of the receptor-mediated PI response was also observed for the wild type alpha1BAR, but not for its truncated mutant, when the receptor was coexpressed with beta-arrestin 1 or beta-arrestin 2. Despite their pronounced effect on phosphorylation of the alpha1BAR, overexpression of GRK5 or GRK6 did not affect the receptor-mediated response. In conclusion, our results provide the first evidence that betaARK1 and 2 as well as arrestin proteins might be involved in agonist-induced regulation of the alpha1BAR. They also identify the alpha1BAR as a potential phosphorylation substrate of GRK5 and GRK6. However, the physiological implications of GRK5- and GRK6-mediated phosphorylation of the alpha1BAR remain to be elucidated.

Muscarinic receptor M1 and phosphodiesterase 1 are key determinants in pulmonary vascular dysfunction following perinatal hypoxia in mice.

Perinatal adverse events such as limitation of nutrients or oxygen supply are associated with the occurrence of diseases in adulthood, like cardiovascular diseases and diabetes. We investigated the long-term effects of perinatal hypoxia on the lung circulation, with particular attention to the nitric oxide (NO)/cGMP pathway. Mice were placed under hypoxia in utero 5 days before delivery and for 5 days after birth. Pups were then bred in normoxia until adulthood. Adults born in hypoxia displayed an altered regulation of pulmonary vascular tone with higher right ventricular pressure in normoxia and increased sensitivity to acute hypoxia compared with controls. Perinatal hypoxia dramatically decreased endothelium-dependent relaxation induced by ACh in adult pulmonary arteries (PAs) but did not influence NO-mediated endothelium-independent relaxation. The M(3) muscarinic receptor was implicated in the relaxing action of ACh and M(1) muscarinic receptor (M(1)AChR) in its vasoconstrictive effects. Pirenzepine or telenzepine, two preferential inhibitors of M(1)AChR, abolished the adverse effects of perinatal hypoxia on ACh-induced relaxation. M(1)AChR mRNA expression was increased in lungs and PAs of mice born in hypoxia. The phosphodiesterase 1 (PDE1) inhibitor vinpocetine also reversed the decrease in ACh-induced relaxation following perinatal hypoxia, suggesting that M(1)AChR-mediated alteration of ACh-induced relaxation is due to the activation of calcium-dependent PDE1. Therefore, perinatal hypoxia leads to an altered pulmonary circulation in adulthood with vascular dysfunction characterized by impaired endothelium-dependent relaxation and M(1)AChR plays a predominant role. This raises the possibility that muscarinic receptors could be key determinants in pulmonary vascular diseases in relation to "perinatal imprinting."

The lymphoproliferative defect in CTLA-4-deficient mice is ameliorated by an inhibitory NK cell receptor.

T-cell responses are regulated by activating and inhibiting signals. CD28 and its homologue, cytotoxic T-lymphocyte antigen 4 (CTLA-4), are the primary regulatory molecules that enhance or inhibit T-cell activation, respectively. Recently it has been shown that inhibitory natural killer (NK) cell receptors (NKRs) are expressed on subsets of T cells. It has been proposed that these receptors may also play an important role in regulating T-cell responses. However, the extent to which the NKRs modulate peripheral T-cell homeostasis and activation in vivo remains unclear. In this report we show that NK cell inhibitory receptor Ly49A engagement on T cells dramatically limits T-cell activation and the resultant lymphoproliferative disorder that occurs in CTLA-4-deficient mice. Prevention of activation and expansion of the potentially autoreactive CTLA-4(-/-) T cells by the Ly49A-mediated inhibitory signal demonstrates that NKR expression can play an important regulatory role in T-cell homeostasis in vivo. These results demonstrate the importance of inhibitory signals in T-cell homeostasis and suggest the common biochemical basis of inhibitory signaling pathways in T lymphocytes.

Inhibition of death receptor signals by cellular FLIP.

The widely expressed protein Fas is a member of the tumour necrosis factor receptor family which can trigger apoptosis. However, Fas surface expression does not necessarily render cells susceptible to Fas ligand-induced death signals, indicating that inhibitors of the apoptosis-signalling pathway must exist. Here we report the characterization of an inhibitor of apoptosis, designated FLIP (for FLICE-inhibitory protein), which is predominantly expressed in muscle and lymphoid tissues. The short form, FLIPs, contains two death effector domains and is structurally related to the viral FLIP inhibitors of apoptosis, whereas the long form, FLIP(L), contains in addition a caspase-like domain in which the active-centre cysteine residue is substituted by a tyrosine residue. FLIPs and FLIP(L) interact with the adaptor protein FADD and the protease FLICE, and potently inhibit apoptosis induced by all known human death receptors. FLIP(L) is expressed during the early stage of T-cell activation, but disappears when T cells become susceptible to Fas ligand-mediated apoptosis. High levels of FLIP(L) protein are also detectable in melanoma cell lines and malignant melanoma tumours. Thus FLIP may be implicated in tissue homeostasis as an important regulator of apoptosis.

Critical roles of the nuclear receptor PPARbeta (peroxisome-proliferator-activated receptor beta) in skin wound healing.

The PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma belong to the nuclear hormone receptor superfamily. While all three receptors are undetectable in adult mouse interfollicular epidermis, PPARbeta expression and activity is strongly re-activated by inflammatory stimuli during epidermal injury. The pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) stimulates transcription of the PPARbeta gene via an activator protein-1 site in its promoter and it also triggers the production of PPARbeta ligands in keratinocytes. This increase of PPARbeta activity in these cells up-regulates the expression of integrin-linked kinase and 3-phosphoinositide-dependent kinase-1, which phosphorylates protein kinase B-alpha (Akt1). The resulting increase in Akt1 activity suppresses apoptosis and ensures the presence of a sufficient number of viable keratinocytes at the wound margin for re-epithelialization. Together, these observations reveal that PPARbeta takes on multiple roles and contributes favourably to the process of wound closure.

The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity.

Proteins secreted from adipose tissue are increasingly recognized to play an important role in the regulation of glucose metabolism. However, much less is known about their effect on lipid metabolism. The fasting-induced adipose factor (FIAF/angiopoietin-like protein 4/peroxisome proliferator-activated receptor gamma angiopoietin-related protein) was previously identified as a target of hypolipidemic fibrate drugs and insulin-sensitizing thiazolidinediones. Using transgenic mice that mildly overexpress FIAF in peripheral tissues we show that FIAF is an extremely powerful regulator of lipid metabolism and adiposity. FIAF overexpression caused a 50% reduction in adipose tissue weight, partly by stimulating fatty acid oxidation and uncoupling in fat. In addition, FIAF overexpression increased plasma levels of triglycerides, free fatty acids, glycerol, total cholesterol, and high density lipoprotein (HDL)-cholesterol. Functional tests indicated that FIAF overexpression severely impaired plasma triglyceride clearance but had no effect on very low density lipoprotein production. The effects of FIAF overexpression were amplified by a high fat diet, resulting in markedly elevated plasma and liver triglycerides, plasma free fatty acids, and plasma glycerol levels, and impaired glucose tolerance in FIAF transgenic mice fed a high fat diet. Remarkably, in mice the full-length form of FIAF was physically associated with HDL, whereas truncated FIAF was associated with low density lipoprotein. In human both full-length and truncated FIAF were associated with HDL. The composite data suggest that via physical association with plasma lipoproteins, FIAF acts as a powerful signal from fat and other tissues to prevent fat storage and stimulate fat mobilization. Our data indicate that disturbances in FIAF signaling might be involved in dyslipidemia.

A concerted kinase interplay identifies PPARgamma as a molecular target of ghrelin signaling in macrophages.

The peroxisome proliferator-activator receptor PPARgamma plays an essential role in vascular biology, modulating macrophage function and atherosclerosis progression. Recently, we have described the beneficial effect of combined activation of the ghrelin/GHS-R1a receptor and the scavenger receptor CD36 to induce macrophage cholesterol release through transcriptional activation of PPARgamma. Although the interplay between CD36 and PPARgamma in atherogenesis is well recognized, the contribution of the ghrelin receptor to regulate PPARgamma remains unknown. Here, we demonstrate that ghrelin triggers PPARgamma activation through a concerted signaling cascade involving Erk1/2 and Akt kinases, resulting in enhanced expression of downstream effectors LXRalpha and ABC sterol transporters in human macrophages. These effects were associated with enhanced PPARgamma phosphorylation independently of the inhibitory conserved serine-84. Src tyrosine kinase Fyn was identified as being recruited to GHS-R1a in response to ghrelin, but failure of activated Fyn to enhance PPARgamma Ser-84 specific phosphorylation relied on the concomitant recruitment of docking protein Dok-1, which prevented optimal activation of the Erk1/2 pathway. Also, substitution of Ser-84 preserved the ghrelin-induced PPARgamma activity and responsiveness to Src inhibition, supporting a mechanism independent of Ser-84 in PPARgamma response to ghrelin. Consistent with this, we found that ghrelin promoted the PI3-K/Akt pathway in a Galphaq-dependent manner, resulting in Akt recruitment to PPARgamma, enhanced PPARgamma phosphorylation and activation independently of Ser-84, and increased expression of LXRalpha and ABCA1/G1. Collectively, these results illustrate a complex interplay involving Fyn/Dok-1/Erk and Galphaq/PI3-K/Akt pathways to transduce in a concerted manner responsiveness of PPARgamma to ghrelin in macrophages.

The δ-Opioid Receptor Affects Epidermal Homeostasis via ERK-Dependent Inhibition of Transcription Factor POU2F3.

Neuropeptides and their receptors are present in human skin, and their importance for cutaneous homeostasis and during wound healing is increasingly appreciated. However, there is currently a lack of understanding of the molecular mechanisms by which their signaling modulates keratinocyte function. Here, we show that δ-opioid receptor (DOPr) activation inhibits proliferation of human keratinocytes, resulting in decreased epidermal thickness in an organotypic skin model. DOPr signaling markedly delayed induction of keratin intermediate filament (KRT10) during in vitro differentiation and abolished its induction in the organotypic skin model. This was accompanied by deregulation of involucrin (IVL), loricrin, and filaggrin. Analysis of the transcription factor POU2F3, which is involved in regulation of KRT10, IVL, and profilaggrin expression, revealed a DOPr-mediated extracellular signal-regulated kinase (ERK)-dependent downregulation of this factor. We propose that DOPr signaling specifically activates the ERK 1/2 mitogen-activated protein kinase pathway to regulate keratinocyte functions. Complementing our earlier studies in DOPr-deficient mice, these data suggest that DOPr activation in human keratinocytes profoundly influences epidermal morphogenesis and homeostasis.

Polarity and specific sequence requirements of peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor heterodimer binding to DNA. A functional analysis of the malic enzyme gene PPAR response element.

The malic enzyme (ME) gene is a target for both thyroid hormone receptors and peroxisome proliferator-activated receptors (PPAR). Within the ME promoter, two direct repeat (DR)-1-like elements, MEp and MEd, have been identified as putative PPAR response elements (PPRE). We demonstrate that only MEp and not MEd is able to bind PPAR/retinoid X receptor (RXR) heterodimers and mediate peroxisome proliferator signaling. Taking advantage of the close sequence resemblance of MEp and MEd, we have identified crucial determinants of a PPRE. Using reciprocal mutation analyses of these two elements, we show the preference for adenine as the spacing nucleotide between the two half-sites of the PPRE and demonstrate the importance of the two first bases flanking the core DR1 in 5'. This latter feature of the PPRE lead us to consider the polarity of the PPAR/RXR heterodimer bound to its cognate element. We demonstrate that, in contrast to the polarity of RXR/TR and RXR/RAR bound to DR4 and DR5 elements respectively, PPAR binds to the 5' extended half-site of the response element, while RXR occupies the 3' half-site. Consistent with this polarity is our finding that formation and binding of the PPAR/RXR heterodimer requires an intact hinge T region in RXR while its integrity is not required for binding of the RXR/TR heterodimer to a DR4.

Atopic dermatitis-like disease and associated lethal myeloproliferative disorder arise from loss of Notch signaling in the murine skin.

BACKGROUND: The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth. METHODOLOGY AND PRINCIPAL FINDINGS: To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia. SIGNIFICANCE: Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.

Coexpression of the T-cell receptor constant alpha domain triggers tumor reactivity of single-chain TCR-transduced human T cells.

Transfer of tumor antigen-specific T-cell receptors (TCRs) into human T cells aims at redirecting their cytotoxicity toward tumors. Efficacy and safety may be affected by pairing of natural and introduced TCRalpha/beta chains potentially leading to autoimmunity. We hypothesized that a novel single-chain (sc)TCR framework relying on the coexpression of the TCRalpha constant alpha (Calpha) domain would prevent undesired pairing while preserving structural and functional similarity to a fully assembled double-chain (dc)TCR/CD3 complex. We confirmed this hypothesis for a murine p53-specific scTCR. Substantial effector function was observed only in the presence of a murine Calpha domain preceded by a TCRalpha signal peptide for shuttling to the cell membrane. The generalization to a human gp100-specific TCR required the murinization of both C domains. Structural and functional T-cell avidities of an accessory disulfide-linked scTCR gp100/Calpha were higher than those of a dcTCR. Antigen-dependent phosphorylation of the proximal effector zeta-chain-associated protein kinase 70 at tyrosine 319 was not impaired, reflecting its molecular integrity in signaling. In melanoma-engrafted nonobese diabetic/severe combined immunodeficient mice, adoptive transfer of scTCR gp100/Calpha transduced T cells conferred superior delay in tumor growth among primary and long-term secondary tumor challenges. We conclude that the novel scTCR constitutes a reliable means to immunotherapeutically target hematologic malignancies.

Physiology of GLP-1--lessons from glucoincretin receptor knockout mice.

GLP-1 has both peripheral and central actions, as this hormone is secreted by gut endocrine cells and brainstem neurons projecting into the hypothalamus and other brain regions. GLP-1 has multiple regulatory functions participating in the control of glucose homeostasis, beta-cell proliferation and differentiation, food intake, heart rate and even learning. GLP-1 action depends on binding to a specific G-coupled receptor linked to activation of the adenylyl cyclase pathway. Analysis of mice with inactivation of the GLP-1 receptor gene has provided evidence that absence of GLP-1 action in the mouse, despite this hormone potent physiological effects when administered in vivo, only leads to mild abnormalities in glucose homeostasis without any change in body weight. However, a critical role for this hormone and its receptor was demonstrated in the function of the hepatoportal vein glucose sensor, in contrast to that of the pancreatic beta-cells, although absence of both GLP-1 and GIP receptors leads to a more severe phenotype characterized by a beta-cell-autonomous defect in glucose-stimulated insulin secretion. Together, the studies of these glucoincretin receptor knockout mice provide evidence that these hormones are part of complex regulatory systems where multiple redundant signals are involved.

Long-lasting antidiabetic effect of a dipeptidyl peptidase IV-resistant analog of glucagon-like peptide-1.

Glucagon-like peptide-1(7-37) (GLP-1) is the most potent insulinotropic hormone characterized thus far. Because its activity is preserved in non-insulin-dependent diabetes mellitus (NIDDM) patients, it is considered a potential new drug for the treatment of this disease. One limitation in its therapeutic use is a short half-life in vivo (5 minutes), due in part to a fast degradation by the endoprotease dipeptidylpeptidase IV (DPPIV). Recently, it was reported that GLP-1 became resistant to DPPIV when the alanine residue at position 8 was replaced by a glycine (GLP-1-Gly8). We report here that this change slightly decreased the affinity of the peptide for its receptor (IC50, 0.41 +/- 0.14 and 1.39 +/- 0.61 nmol/L for GLP-1 and GLP-1-Gly8, respectively) but did not change the efficiency to stimulate accumulation of intracellular cyclic adenosine monophosphate (cAMP) (EC50, 0.25 +/- 0.05 and 0.36 +/- 0.06 nmol/L for GLP-1 and GLP-1-Gly8, respectively). Second, we demonstrate for the first time that this mutant has an improved insulinotropic activity compared with the wild-type peptide when tested in vivo in an animal model of diabetes. A single injection of 0.1 nmol GLP-1-Gly8 in diabetic mice fed a high-fat diet can correct fasting hyperglycemia and glucose intolerance for several hours, whereas the activity of 1 nmol GLP-1 vanishes a few minutes after injection. These actions were correlated with increased insulin and decreased glucagon levels. Interestingly, normoglycemia was maintained over a period that was longer than the predicted peptide half-life, suggesting a yet undescribed long-term effect of GLP-1-Gly8. GLP-1-Gly8 thus has a markedly improved therapeutic potential compared with GLP-1, since it can be used at much lower doses and with a more flexible schedule of administration.