NOV/CCN3 promotes maturation of cerebellar granule neuron precursors.

A body of evidence points to the matricial CCN proteins as key regulators of organogenesis. NOV/CCN3, a founder CCN member, is expressed in the developing central nervous system but its functions during neural development have not been studied yet. Here we describe the pattern of NOV expression during rat cerebellar postnatal development and show that NOV expression increases during the second postnatal week, a critical period for the maturation of granule neuron precursors (GNP). NOV transcripts are specifically produced by Purkinje neurons and NOV protein localises extracellularly in the molecular layer and the inner part of the external granule layer, at a key position to control GNP proliferation and migration. In vitro, NOV reduces Sonic Hedgehog-induced GNP proliferation through beta3 integrins and stimulation of GSK3-beta activity whereas NOV stimulates GNP migration through distinct RGD-dependent integrins. These findings identify a new paracrine role of NOV in the development of cerebellar granule neurons.

NOVH increases MMP3 expression and cell migration in glioblastoma cells via a PDGFR-alpha-dependent mechanism.

Nephroblastoma overexpressed gene (NOV) is highly expressed in the nervous system. We investigated its biological activity by expressing the human NOV gene (NOVH) in a human glioblastoma cell line that is negative for NOVH and by analyzing four clones with different levels of NOVH expression. There was no difference in cell proliferation between the NOVH-expressing cell lines, but there was increased cell adhesion and migration that correlated with increasing NOVH expression. Gene expression profiling was used to investigate the mechanisms by which NOVH expression regulated cell activity. We identified two induced genes in NOVH-expressing cells that are involved in cell migration: matrix metalloprotease (MMP)3 and platelet-derived growth factor receptor (PDGFR)-alpha. Our studies show that PDGFR-alpha induced MMP3 gene expression and increased cell proliferation and cell migration upon stimulation by platelet-derived growth factor (PDGF)-AA. We also show that the induction of MMP3 in cells expressing NOVH is potentiated by either cell density, serum, or PDGF-BB. Thus, expression of NOVH in glioblastoma cells triggers a cascade of gene expression resulting in increased cell adhesion and migration.

Characterization of human NOV in biological fluids: an enzyme immunoassay for the quantification of human NOV in sera from patients with diseases of the adrenal gland and of the nervous system.

Immunochromatography has shown that human NOV (NOVH), a member of the CCN (CTGF/CYR61/NOV) family, forms a physiological complex with fibulin-1 in blood. We developed an enzyme immunoassay specific for NOVH and showed for the first time that the concentration of NOVH differs in each of these biological fluids. The normal concentration of NOVH circulating in the blood is 350-400 ng/ml, but this concentration varies with age. By using sera from patients with adrenal gland diseases we found that in vivo ACTH or glucocorticoids are not responsible for the high concentration of NOVH in this endocrine gland. However, the NOVH concentration was significantly modified in malignant adrenocortical tumors, but not in benign adrenocortical tumors. The concentration of NOVH was significantly decreased in patients suffering from astrocytomas or multiple sclerosis, two diseases of the nervous system. Thus, NOVH is a potentially useful marker for the diagnosis of these diseases.

Solubilization, reconstitution and molecular properties of the triiodothyronine transport protein from rat erythrocyte membranes.

Triiodothyronine (T3) transport through the mammalian erythrocyte membrane is mediated by a transport system related to the aromatic amino acid transport system T. The T3-binding component of this transport system could be photolabeled with [125I]T3 as a 52-kD protein, and subsequently solubilized with non-ionic detergents. Upon purification by ion-exchange chromatography, the photolabeled 52-kD protein solubilized with octylglucoside (OG) resolved into several peaks, suggesting charge heterogeneity of labeled proteins. The saturable [125I]T3 binding to rat erythrocyte membranes was completely inhibited by non-ionic detergents at concentrations about 20 times lower than those that solubilized membrane. Therefore, detergent-free proteoliposomes were generated from the detergent-soluble extracts by treatment with a polystyrene adsorbent. Proteoliposomes prepared from OG-soluble extract contained the highest specific activity of T3 binding. The Kd of the T3 binding sites (4.5 nmol/l) and the competitive inhibition constant of tryptophan (120 mumol/l) were similar to those for native membranes. The photolabeling of the 52-kD protein in these proteoliposomes was prevented by tryptophan and T4, but not by leucine or the D-isomer of T3, corresponding to the transport specificity of system T. The 52-kD protein solubilized with OG from native membranes was partially purified by ion-exchange chromatography. The 52-kD protein was detected by photoaffinity labeling in the purified fraction only after addition of erythrocyte membrane phospholipids to generate proteoliposomes. This indicates that the association of 52-kD protein with phospholipids is critical for T3 binding.

Effects of thyroidectomy, insulin, and phospholipids on cyclic AMP phosphodiesterase in rat adipocyte plasma membranes.

The effect of thyroid status on the cyclic nucleotide phosphodiesterase in adipocyte plasma membranes has been studied. In euthyroid rat fat cells, about 7% of the total cAMP phosphodiesterase was found in the plasma membrane. Thyroidectomy doubled both the enzyme's total activity in this fraction, and its specific activity (60 versus 37 pmol/min/mg) compared to euthyroid rat plasma membrane. In membranes from thyroidectomized rats, phosphodiesterase hydrolyzed cAMP with a single Km of 2 microM, whereas in euthyroid rat membranes, Lineweaver Burk plots were non-linear, with apparent Kms of 0.5 and 5 microM. This phosphodiesterase activity was insensitive to exogenous guanine nucleotides and calcium. In vivo injection of triiodothyronine restored phosphodiesterase activity in plasma membranes from thyroidectomized rats to the values obtained for euthyroid rats. Centrifugation on a 10 to 45% sucrose density gradient of the plasma membrane fractions gave two main peaks of phosphodiesterase activity which hydrolyzed the cAMP in adipocyte plasma membranes from both euthyroid and thyroidectomized rats. The distribution profiles for these activities were very similar in the two plasma membrane preparations. The peaks of phosphodiesterase and 5'nucleotidase activity coincided. Thyroidectomy raised the phosphodiesterase activity of these two peaks, particularly of the first. The cAMP phosphodiesterase activities in both hypothyroid and control plasma membrane preparations were also sensitive to insulin and were activated by phospholipase A2 and three anionic phospholipids. Thyroid hormones therefore regulate the degradation of cAMP in plasma membranes by a mechanism which seems different from the one involved in the action of insulin, and is independent of the membrane phospholipid composition.

Endogenous ADP-ribosylation of Gs subunit and autonomous regulation of adenylate cyclase.

Although cholera toxin induces a marked stimulation of adenylate cyclase activity in rat adipocyte plasma membranes, the holotoxin induces only a slight increase of cyclic AMP accumulation in intact cells. A similar apparent anomaly is seen with pertussis toxin, which has been shown to inhibit the Gi subunit of adenylate cyclase, and has a greater effect on cAMP accumulation and lipolysis than the activation by cholera toxin of the Gs subunit. To understand better the way in which these bacterial toxins are modifying the adipocyte cells, we prepared adipocyte plasma membranes and submitted them to ADP-ribosylation by cholera and pertussis toxins. During the incubation of control cells, we found endogenous ADP-ribosylation of Gs as a result of sustained stimulation of Gi by adenosine. Our results point to a possible homoeostatic system in which the autonomous adjustment of the basal activity of Gs as a function of that of Gi, under the control of feedback inhibitory ligands, ensures a steady production of cAMP within the cell.

Autophosphorylation of the insulin receptor in rat adipocytes is modulated by thyroid hormone status.

The effect of thyroid status on the in vitro autophosphorylation of the insulin receptors was studied in triton-solubilized adipocyte plasma membranes obtained from normal and thyroidectomized rats. Thyroidectomy results in an increase (two to three times) of the in vitro insulin-dependent phosphorylation of the insulin beta-subunit receptor. Phosphorylation occurred on tyrosine residues. In vivo injection of triiodothyronine to thyroidectomized rats restored plasma membranes autophosphorylation of the beta-subunit to the values obtained for control euthyroid rats. This effect was independent of the number and affinity of the insulin receptors, which were not modified regardless of thyroid status.