The transcription factor NF-B is a pivotal regulator of inf lammatory responses. While the activation of NF-B in the arthritic joint has been associated with rheumatoid arthritis (RA), its significance is poorly understood. Here, we examine the role of NF-B in animal models of RA. We demonstrate that in vitro, NF-B controlled expression of numerous inf lammatory molecules in synoviocytes and protected cells against tumor necrosis factor ␣ (TNF␣) and Fas ligand (FasL) cytotoxicity. Similar to that observed in human RA, NF-B was found to be activated in the synovium of rats with streptococcal cell wall (SCW)-induced arthritis. In vivo suppression of NF-B by either proteasomal inhibitors or intraarticular adenoviral gene transfer of super-repressor IB␣ profoundly enhanced apoptosis in the synovium of rats with SCW-and pristane-induced arthritis. This indicated that the activation of NF-B protected the cells in the synovium against apoptosis and thus provided the potential link between inf lammation and hyperplasia. Intraarticular administration of NF-kB decoys prevented the recurrence of SCW arthritis in treated joints. Unexpectedly, the severity of arthritis also was inhibited significantly in the contralateral, untreated joints, indicating beneficial systemic effects of local suppression of NF-B. These results establish a mechanism regulating apoptosis in the arthritic joint and indicate the feasibility of therapeutic approaches to RA based on the specific suppression of NF-B.
Dynamic assembly and disassembly of microtubules is essential for cell division, cell movements, and intracellular transport. In the developing nervous system, microtubule dynamics play a fundamental role during neurite outgrowth, elongation, and branching, but the molecular mechanisms involved are unknown. SCG10 is a neuron-specific protein that is membrane-associated and highly enriched in growth cones. Here we show that SCG10 binds to microtubules, inhibits their assembly, and can induce microtubule disassembly. We also show that SCG10 overexpression enhances neurite outgrowth in a stably transfected neuronal cell line. These data identify SCG10 as a key regulator of neurite extension through regulation of microtubule instability.
Current methods for clinical estimation of total body skeletal muscle mass have significant limitations. We tested the hypothesis that creatine (methyl-d3) dilution (D3-creatine) measured by enrichment of urine D3-creatinine reveals total body creatine pool size, providing an accurate estimate of total body skeletal muscle mass. Healthy subjects with different muscle masses [n = 35: 20 men (19-30 yr, 70-84 yr), 15 postmenopausal women (51-62 yr, 70-84 yr)] were housed for 5 days. Optimal tracer dose was explored with single oral doses of 30, 60, or 100 mg D3-creatine given on day 1. Serial plasma samples were collected for D3-creatine pharmacokinetics. All urine was collected through day 5. Creatine and creatinine (deuterated and unlabeled) were measured by liquid chromatography mass spectrometry. Total body creatine pool size and muscle mass were calculated from D3-creatinine enrichment in urine. Muscle mass was also measured by magnetic resonance imaging (MRI), dual-energy x-ray absorptiometry (DXA), and traditional 24-h urine creatinine. D3-creatine was rapidly absorbed and cleared with variable urinary excretion. Isotopic steady-state of D3-creatinine enrichment in the urine was achieved by 30.7 ± 11.2 h. Mean steady-state enrichment in urine provided muscle mass estimates that correlated well with MRI estimates for all subjects (r = 0.868, P < 0.0001), with less bias compared with lean body mass assessment by DXA, which overestimated muscle mass compared with MRI. The dilution of an oral D3-creatine dose determined by urine D3-creatinine enrichment provides an estimate of total body muscle mass strongly correlated with estimates from serial MRI with less bias than total lean body mass assessment by DXA.
PPARγ agonists inhibit liver fibrosis, but the mechanisms involved are uncertain. We hypothesized that PPARγ agonists inhibit transforming growth factor (TGF)β1-activation of TGFβ receptor (TGFβR)-1 signaling in quiescent stellate cells, thereby abrogating Smad3-dependent induction of extracellular matrix (ECM) genes, such as PAI-1 and collagen-1αI. To test this, human HSC were cultured to induce a quiescent phenotype, characterized by lipid accumulation and PPARγ expression and transcriptional activity. These adipocytic HSC were then treated with TGFβ1 ± a TGFβR-1 kinase inhibitor (SB431542) or a PPARγ agonist (GW7845). TGFβ1 caused dose-and time-dependent increases in Smad3 phosphorylation, followed by induction of collagen and PAI-1 expression. Like the TGFβR-1 kinase inhibitor, the PPARγ agonist caused dose-dependent inhibition of all of these responses without effecting HSC proliferation or viability. Thus, the anti-fibrotic actions of PPARγ agonists reflect their ability to inhibit TGFβ1-TGFβR1 signaling that initiates ECM gene expression in quiescent HSC.
SCG10 is a neuronal growth-associated protein that is concentrated in the growth cones of developing neurons. SCG10 shows a high degree of sequence homology to the ubiquitous phosphoprotein stathmin, which has been recently identified as a factor that destabilizes microtubules by increasing their catastrophe rate. Whereas stathmin is a soluble cytosolic protein, SCG10 is membrane-associated, indicating that the protein acts in a distinct subcellular compartment. Identifying the precise intracellular distribution of SCG10 as well as the mechanisms responsible for its specific targeting will contribute to elucidating its function. The main structural feature distinguishing the two proteins is that SCG10 contains an NH 2 -terminal extension of 34 amino acids. In this study, we have examined the intracellular distribution of SCG10 in PC12 cells and in transfected COS-7 cells and the role of the NH 2 -terminal domain in membrane-binding and intracellular targeting. SCG10 was found to be localized to the Golgi complex region. We show that the NH 2 -terminal region (residues 1-34) was necessary for membrane targeting and Golgi localization. Fusion proteins consisting of the NH 2 -terminal 34 amino acids of SCG10 and the related protein stathmin or the unrelated protein, -galactosidase, accumulated in the Golgi, demonstrating that this sequence was sufficient for Golgi localization. Biosynthetic labeling of transfected COS-7 cells with [ 3 H]palmitic acid revealed that two cysteine residues contained within the NH 2 -terminal domain were sites of palmitoylation.
The peroxisome proliferator-activated receptors (PPARs) impart diverse cellular effects in biological systems. Because stellate cell activation during liver injury is associated with declining PPARgamma expression, we hypothesized that its expression is critical in stellate cell-mediated fibrogenesis. We therefore modulated its expression during liver injury in vivo. PPARgamma was depleted in rat livers by using an adenovirus-Cre recombinase system. PPARgamma was overexpressed by using an additional adenoviral vector (AdPPARgamma). Bile duct ligation was utilized to induce stellate cell activation and liver fibrosis in vivo; phenotypic effects (collagen I, smooth muscle alpha-actin, hydroxyproline content, etc.) were measured. PPARgamma mRNA levels decreased fivefold and PPARgamma protein was undetectable in stellate cells after culture-induced activation. During activation in vivo, collagen accumulation, assessed histomorphometrically and by hydroxyproline content, was significantly increased after PPARgamma depletion compared with controls (1.28 +/- 0.14 vs. 1.89 +/- 0.21 mg/g liver tissue, P < 0.03). In isolated stellate cells, AdPPARgamma overexpression resulted in significantly increased adiponectin mRNA expression and decreased collagen I and smooth muscle alpha-actin mRNA expression compared with controls. During in vivo fibrogenesis, rat livers exposed to AdPPARgamma had significantly less fibrosis than controls. Collagen I and smooth muscle alpha-actin mRNA expression were significantly reduced in AdPPARgamma-infected rats compared with controls (P < 0.05, n = 10). PPARgamma-deficient mice exhibited enhanced fibrogenesis after liver injury, whereas PPARgamma receptor overexpression in vivo attenuated stellate cell activation and fibrosis. The data highlight a critical role for PPARgamma during in vivo fibrogenesis and emphasize the importance of the PPARgamma pathway in stellate cells during liver injury.
Peroxisome proliferator-activated receptor-␥ (PPAR␥) agonists have been shown to have significant therapeutic benefits such as desirable glycemic control in type 2 diabetic patients; however, these agents may cause fluid retention in susceptible individuals. Since PPAR␥ is expressed selectively in distal nephron epithelium, we studied the mechanism of PPAR␥ agonist-induced fluid retention using male Sprague-Dawley rats treated with either vehicle or GI262570 (farglitazar), a potent PPAR␥ agonist. GI262570 (20 mg/kg/day) induced a plasma volume expansion. The plasma volume expansion was accompanied by a small but significant decrease in plasma potassium concentration. Small but significant increases in plasma sodium and chloride concentrations were also observed. These changes in serum electrolytes suggested an activation of the renal mineralocorticoid response system; however, GI262570-treated rats had lower plasma levels of aldosterone compared with vehicle-treated controls. mRNA levels for a group of genes involved in distal nephron sodium and water absorption are changed in the kidney medulla with GI262570 treatment. In addition, due to a possible rebound effect on epithelial sodium channel (ENaC) activity, a low dose of amiloride did not prevent GI262570-induced fluid retention. On the contrary, the rebound effect after amiloride treatment potentiated GI262570-induced plasma volume expansion. This is at least partially due to a synergistic effect of GI262570 and the rebound from amiloride treatment on ENaC␣ expression. In summary, our current data suggest that GI262570 can increase water and sodium reabsorption in distal nephron by stimulating the ENaC and Na,KATPase system. This may be an important mechanism for PPAR␥ agonist-induced fluid retention.
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