DC-HIL is a negative regulator of T lymphocyte activation

T-cell activation is the net product of competing positive and negative signals transduced by regulatory molecules on antigen-presenting cells (APCs) binding to corresponding ligands on T cells. Having previously identified DC-HIL as a receptor expressed by APCs that contains an extracellular immunoglobulin (Ig)-like domain, we postulated that it plays a role in T-cell activation. To probe this function, we created soluble recombinant DC-HIL, which we observed to bind activated (but not resting) T cells, indicating that expression of the putative ligand on T cells is induced by activation. Binding of DC-HIL to naive T cells attenuated these cells' primary response to anti-CD3 antibody, curtailing IL-2 production, and preventing entry into the cell cycle. DC-HIL also inhibited reactivation of T cells previously activated by APCs (secondary response). By contrast, addition of soluble DC-HIL to either allogeneic or ovalbumin-specific lymphocyte reactions augmented T-cell proliferation, and its injection into mice during the elicitation (but not sensitization) phase of contact hypersensitivity exacerbated ear-swelling responses. Mutant analyses showed the inhibitory function of DC-HIL to reside in its extracellular Ig-like domain. We conclude that endogenous DC-HIL is a negative regulator of T lymphocyte activation, and that this native inhibitory function can be blocked by exogenous DC-HIL, leading to enhanced immune responses.     

The low-molecular-weight phosphotyrosine phosphatase is a negative regulator of FcgammaRIIA-mediated cell activation

Activation of human platelets by cross-linking of the low-affinity receptor for immunoglobulin G (FcgammaRIIA) is initiated by Src kinase-mediated phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) within the receptor, but the identity of the enzyme responsible for its dephosphorylation and inactivation is unknown. Here we report that the 18-kDa low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) is expressed in human platelets and undergoes subcellular redistribution upon FcgammaRIIA cross-linking. In vitro, LMW-PTP was found to efficiently dephosphorylate activated FcgammaRIIA and LAT, but not Syk or phospholipase Cgamma2. In the megakaryocytic cell line DAMI, antibody-induced phosphorylation of FcgammaRIIA was rapid and transient. The late dephosphorylation of FcgammaRIIA was dramatically delayed upon reduction of LMW-PTP expression by siRNA. Strikingly, overexpression of LMW-PTP resulted in the inhibition of antibody-induced phosphorylation of FcgammaRIIA, and caused a more rapid dephosphorylation. In addition, overexpression of LMW-PTP inhibited activation of Syk downstream of FcgammaRIIA and reduced intracellular Ca(2+) mobilization. These results demonstrate that LMW-PTP is responsible for FcgammaRIIA dephosphorylation, and is implicated in the down-regulation of cell activation mediated by this ITAM-bearing immunoreceptor.     

The murine stanniocalcin 2 gene is a negative regulator of postnatal growth

Stanniocalcin (STC), a secreted glycoprotein, was first studied in fish as a classical hormone with a role in regulating serum calcium levels. There are two closely related proteins in mammals, STC1 and STC2, with functions that are currently unclear. Both proteins are expressed in numerous mammalian tissues rather than being secreted from a specific endocrine gland. No phenotype has been detected yet in Stc1-null mice, and to investigate whether Stc2 could have compensated for the loss of Stc1, we have now generated Stc2(-/-) and Stc1(-/-) Stc2(-/-) mice. Although Stc1 is expressed in the ovary and lactating mouse mammary glands, like the Stc1(-/-) mice, the Stc1(-/-) Stc2(-/-) mice had no detected decrease in fertility, fecundity, or weight gain up until weaning. Serum calcium and phosphate levels were normal in Stc1(-/-) Stc2(-/-) mice, indicating it is unlikely that the mammalian stanniocalcins have a major physiological role in mineral homeostasis. Mice with Stc2 deleted were 10-15% larger and grew at a faster rate than wild-type mice from 4 wk onward, and the Stc1(-/-) Stc2(-/-) mice had a similar growth phenotype. This effect was not mediated through the GH/IGF-I axis. The results are consistent with STC2 being a negative regulator of postnatal growth.     

The orphan nuclear receptor, NOR-1, is a target of beta-adrenergic signaling in skeletal muscle

beta-Adrenergic receptor (beta-AR) agonists induce Nur77 mRNA expression in the C2C12 skeletal muscle cell culture model and elicit skeletal muscle hypertrophy. We previously demonstrated that Nur77 (NR4A1) is involved in lipolysis and gene expression associated with the regulation of lipid homeostasis. Subsequently it was demonstrated by another group that beta-AR agonists and cold exposure-induced Nur77 expression in brown adipocytes and brown adipose tissue, respectively. Moreover, NOR-1 (NR4A3) was hyperinduced by cold exposure in the nur77(-/-) animal model. These studies underscored the importance of understanding the role of NOR-1 in skeletal muscle. In this context we observed 30-480 min of beta-AR agonist treatment significantly and transiently increased expression of the orphan nuclear receptor NOR-1 in both mouse skeletal muscle tissue (plantaris) and C2C12 skeletal muscle cells. Specific beta(2)- and beta(3)-AR agonists had similar effects as the pan-agonist and were blocked by the beta-AR antagonist propranolol. Moreover, in agreement with these observations, isoprenaline also significantly increased the activity of the NOR-1 promoter. Stable exogenous expression of a NOR-1 small interfering RNA (but not the negative control small interfering RNA) in skeletal muscle cells significantly repressed endogenous NOR-1 mRNA expression and led to changes in the expression of genes involved in the control of lipid use and muscle mass underscored by a dramatic increase in myostatin mRNA expression. Concordantly the myostatin promoter was repressed by NOR-1 expression. In conclusion, NOR-1 is highly responsive to beta-adrenergic signaling and regulates the expression of genes controlling fatty acid use and muscle mass.     

Activin B receptor ALK7 is a negative regulator of pancreatic beta-cell function

All major cell types in pancreatic islets express the transforming growth factor (TGF)-beta superfamily receptor ALK7, but the physiological function of this receptor has been unknown. Mutant mice lacking ALK7 showed normal pancreas organogenesis but developed an age-dependent syndrome involving progressive hyperinsulinemia, reduced insulin sensitivity, liver steatosis, impaired glucose tolerance, and islet enlargement. Hyperinsulinemia preceded the development of any other defect, indicating that this may be one primary consequence of the lack of ALK7. In agreement with this, mutant islets showed enhanced insulin secretion under sustained glucose stimulation, indicating that ALK7 negatively regulates glucose-stimulated insulin release in beta-cells. Glucose increased expression of ALK7 and its ligand activin B in islets, but decreased that of activin A, which does not signal through ALK7. The two activins had opposite effects on Ca(2+) signaling in islet cells, with activin A increasing, but activin B decreasing, glucose-stimulated Ca(2+) influx. On its own, activin B had no effect on WT cells, but stimulated Ca(2+) influx in cells lacking ALK7. In accordance with this, mutant mice lacking activin B showed hyperinsulinemia comparable with that of Alk7(-/-) mice, but double mutants showed no additive effects, suggesting that ALK7 and activin B function in a common pathway to regulate insulin secretion. These findings uncover an unexpected antagonism between activins A and B in the control of Ca(2+) signaling in beta-cells. We propose that ALK7 plays an important role in regulating the functional plasticity of pancreatic islets, negatively affecting beta-cell function by mediating the effects of activin B on Ca(2+) signaling.     

The Gas6/Axl system: a novel regulator of vascular cell function

Signal transduction downstream of tyrosine kinases has become an increasingly important area of study in cardiovascular biology. In this review, we consider the experimental evidence pointing to significant roles for the Axl receptor tyrosine kinase and its ligand, Gas6, in the vasculature. An introduction to the Gas6/Axl system and a discussion of its discovery is followed by a summary of the data regarding expression of Gas6/Axl in injured arteries. We conclude by discussing mechanisms by which Gas6/Axl signaling may impact on the response of blood vessels to injury, thereby contributing to the development of atherosclerosis and/or restenosis.     

Beta(2)-microglobulin as a negative growth regulator of myeloma cells

High beta(2)-microglobulin (beta(2)m) levels in myeloma correlate with poor prognosis. We hypothesized that beta(2)m may affect myeloma cell growth and survival. In this study, we examined the in vitro effects of beta(2)m on myeloma cells. Primary myeloma cells freshly isolated from patients and myeloma cell lines were used, cultured in the presence of beta(2)m, and monitored for growth and survival. Beta(2)m suppressed the growth of primary tumour cells and myeloma cell lines (ARK-RS, ARP-1, RPMI-8226, U266, ARH-77 and IM-9). High concentrations of beta(2)m induced apoptosis and cell cycle arrest. Beta(2)m-induced apoptosis was dependent on activation of a caspase cascade, inhibited by interleukin 6, and did not involve the surface death receptors, as receptor-neutralizing antibodies had no inhibitory effect. Beta(2)m-induced growth arrest was associated with downregulation of cyclins A and D2. Surprisingly, anti-beta(2)m antibodies did not block the effect of beta(2)m but were synergistic with beta(2)m, resulting in 90% growth inhibition and 70% apoptosis of myeloma cells. Whereas beta(2)m treatment resulted in slight upregulation of surface beta(2)m and major histocompatibility complex class I alpha-chain expression, treatment of myeloma cells with anti-beta(2)m antibodies alone or with beta(2)m resulted in significant downregulation of surface beta(2)m and class I molecules, suggesting that class I molecules may be involved in signal transduction. Our data demonstrate that beta(2)m plays an important role in regulating the growth and survival of myeloma cells in vitro and warrants further investigation to delineate the mechanisms of beta(2)m and anti-beta(2)m antibody-induced growth regulation of myeloma cells.     

Platelet endothelial cell adhesion molecule-1 is a negative regulator of platelet-collagen interactions

The functional importance of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) in platelets is unclear. Because PECAM-1 represents a newly assigned immunoglobulin-ITIM superfamily member expressed on the surface of platelets, it was hypothesized that it may play an important regulatory role in modulating ITAM-bearing receptors such as collagen (GP)VI receptor and FcgammaRIIA. To examine the functional role of PECAM-1 in regulating platelet-collagen interactions, 2 different approaches were applied using recombinant human PECAM-1-immunoglobulin chimeras and platelets derived from PECAM-1-deficient mice. Stimulation of platelets by collagen-, (GP)VI-selective agonist, collagen-related peptide (CRP)-, and PECAM-1-immunoglobulin chimera induced tyrosine phosphorylation of PECAM-1 in a time- and dose-dependent manner. Activation of PECAM-1 directly through the addition of soluble wild-type PECAM-1-immunoglobulin chimera, but not mutant K89A PECAM-1-immunoglobulin chimera that prevents homophilic binding, was found to inhibit collagen- and CRP-induced platelet aggregation. PECAM-1-deficient platelets displayed enhanced platelet aggregation and secretion responses on stimulation with collagen and CRP, though the response to thrombin was unaffected. Under conditions of flow, human platelet thrombus formation on a collagen matrix was reduced in a dose-dependent manner by human PECAM-1-immunoglobulin chimera. Platelets derived from PECAM-1-deficient mice form larger thrombi when perfused over a collagen matrix under flow at a shear rate of 1800 seconds(-1) compared to wild-type mice. Collectively, these results indicate that PECAM-1 serves as a physiological negative regulator of platelet-collagen interactions that may function to negatively limit growth of platelet thrombi on collagen surfaces.     

Reticulon 4B (Nogo-B) is a novel regulator of hepatic fibrosis

Nogo-B, also known as Reticulon 4B, plays important roles in vascular injuries. Its function in the liver is not understood. The aim of this study was to characterize Nogo-B in liver fibrosis and cirrhosis. Nogo-B distribution was assessed in normal and cirrhotic human liver sections. We also determined the levels of liver fibrosis in wild-type (WT) and Nogo-A/B knockout (NGB KO) mice after sham operation or bile duct ligation (BDL). To investigate the mechanisms of Nogo-B's involvement in fibrosis, hepatic stellate cells were isolated from WT and NGB KO mice and transformed into myofibroblasts. Portal pressure was measured to test whether Nogo-B gene deletion could ameliorate portal hypertension. In normal livers, Nogo-B expression was found in nonparenchymal cells, whereas its expression in hepatocytes was minimal. Nogo-B staining was significantly elevated in cirrhotic livers. Fibrosis was significantly increased in WT mice 4 weeks after BDL compared with NGB KO mice. The absence of Nogo-B significantly reduced phosphorylation of Smad2 levels upon transforming growth factor β (TGF-β) stimulation. Reconstitution of the Nogo-B gene into NGB KO fibroblasts restored Smad2 phosphorylation. Four weeks after BDL, portal pressure was significantly increased in WT mice by 47%, compared with sham-operated controls (P = 0.03), whereas such an increase in portal pressure was not observed in NGB KO mice (P = NS). CONCLUSION: Nogo-B regulates liver fibrosis, at least in part, by facilitating the TGFβ/Smad2 signaling pathway in myofibroblasts. Because absence of Nogo-B ameliorates liver fibrosis and portal hypertension, Nogo-B blockade may be a potential therapeutic target in fibrosis/cirrhosis.