Getting to the heart of the matter: CCN2 plays a role in cardiomyocyte hypertrophy

Connective tissue growth factor (CTGF/CCN2) is overexpressed in diabetes. Diabetic rats possess myocardial and cardiomyocyte hypertrophy. In a recent report, Wang and colleagues (Am J Physiol Cell Physiol. 2009 Jul 22. [Epub ahead of print]) show that CCN2 directly mediates cardiomyocyte hypertrophy as well as that induced by high glucose and fatty acid. CCN2 acted via the TrkA receptor. These data are the subject of this commentary, and emphasize that CCN2 may be an excellent target for therapy in diabetes.     

The Administration of an α-MSH Analogue Reduces the Serum Release of IL-1α and TNFα Induced by the Injection of a Sublethal Dose of Lipopolysaccharides in the BALB/c Mouse

The injection of α-MSH or of one of its analogues ([Nle₄-D.Phe₇] α-MSH_4–10) reduced, in vivo, the release of two cytokines (IL-1α and TNFα) involved in inflammation. The inflammatory state was induced in BALB/c mice by intraperitoneal injection of a sublethal dose of lipopolysaccharides (LPS). The assay of these cytokines by ELISA showed a reduction of 20% with α-MSH and between 30 and 60% with the α-MSH analogue. The α-MSH or the analogue was administered in one of two ways: intravenously or subcutaneously. The most efficient method seemed to be the subcutaneous one because it improved the activity 10,000 times more than the intravenous method. Moreover, the analogue induced a regression of mortality in the animals treated by the intravenous method. Our results show that α-MSH and one of its analogues inhibit IL-1α and TNFα, and can be used as anti-inflammatory molecules.     

Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity

Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the prosurvival IGF-I/AKT/FOXO3 pathway by redox activation of the stress kinases p38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes, IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that, in astrocytes exposed to oxidative stress by hydrogen peroxide (H₂O₂), p38 activation did not inhibit AKT (protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and more sustained in astrocytes than in neurons either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H₂O₂-exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury.     

Estimating recent growth in the cuttlefish Sepia officinalis: are nucleic acid-based indicators for growth and condition the method of choice?

A laboratory calibration study was undertaken with juvenile Sepia officinalis (80-85 g initial wet weight) to investigate the effects of different food rations and different starving intervals on RNA/dry weight (DW) ratios and RNA/DNA ratios in cephalopod mantle muscle at two different temperatures. The digestive gland index was also used as an additional indicator of recent growth. High food rations and low temperature went along with high RNA/DW ratios and high RNA/DNA ratios. Starving resulted in a linear decline in growth performance and a concomitant decrease in RNA/DW and RNA/DNA ratio, with RNA/DNA ratios representing the growth data better. RNA/DNA ratios decreased faster at higher temperatures. A fluorimetric assay for nucleic acid analysis was optimized for cephalopod mantle tissues and yielded reproducible RNA/DNA ratios with a relative variance below 10%. Thus, it may be possible to use this estimator of recently encountered feeding regime for the evaluation of mortality rates of early teuthid paralarvae to eventually support stock management. Also, log relative digestive gland weight showed a strong relationship with starving time, but, surprisingly, not with temperature. Data from the two temperatures analyzed could be combined to form a common regression line of relative digestive gland index with starving time. This indicator for recent growth might be especially suitable for large specimens with a well-developed digestive gland.     

Bone Cell-autonomous Contribution of Type 2 Cannabinoid Receptor to Breast Cancer-induced Osteolysis

The cannabinoid type 2 receptor (CB2) has previously been implicated as a regulator of tumor growth, bone remodeling, and bone pain. However, very little is known about the role of the skeletal CB2 receptor in the regulation of osteoblasts and osteoclasts changes associated with breast cancer. Here we found that the CB2-selective agonists HU308 and JWH133 reduced the viability of a variety of parental and bone-tropic human and mouse breast cancer cells at high micromolar concentrations. Under conditions in which these ligands are used at the nanomolar range, HU308 and JWH133 enhanced human and mouse breast cancer cell-induced osteoclastogenesis and exacerbated osteolysis, and these effects were attenuated in cultures obtained from CB2-deficient mice or in the presence of a CB2 receptor blocker. HU308 and JWH133 had no effects on osteoblast growth or differentiation in the presence of conditioned medium from breast cancer cells, but under these circumstances both agents enhanced parathyroid hormone-induced osteoblast differentiation and the ability to support osteoclast formation. Mechanistic studies in osteoclast precursors and osteoblasts showed that JWH133 and HU308 induced PI3K/AKT activity in a CB2-dependent manner, and these effects were enhanced in the presence of osteolytic and osteoblastic factors such as RANKL (receptor activator of NFκB ligand) and parathyroid hormone. When combined with published work, these findings suggest that breast cancer and bone cells exhibit differential responses to treatment with CB2 ligands depending upon cell type and concentration used. We, therefore, conclude that both CB2-selective activation and antagonism have potential efficacy in cancer-associated bone disease, but further studies are warranted and ongoing.     

GPR35 is a novel lysophosphatidic acid receptor

GPR35 is a rhodopsin-like G protein-coupled receptor identified in 1998. It has been reported that kynurenic acid, a tryptophan metabolite, may act as an endogenous ligand for GPR35. However, the concentrations of kynurenic acid required to elicit the cellular responses are usually high, raising the possibility that another endogenous ligand may exist. In this study, we searched for another endogenous ligand for GPR35. Finally, we found that the magnitude of the Ca²⁺ response induced by 2-acyl lysophosphatidic acid in the GPR35-expressing HEK293 cells was markedly greater than that in the vector-transfected control cells. Such a difference was not apparent in the case of 1-acyl lysophosphatidic acid. 2-Acyl lysophosphatidic acid also caused the sustained activation of RhoA and the phosphorylation of extracellular signal-regulated kinase, and triggered the internalization of the GPR35 molecule. These results strongly suggest that 2-acyl lysophosphatidic acid is an endogenous ligand for GPR35.     

Evidence for Structural Dissimilarity in the Neurotransmitter Binding Region of Purified Acetylcholine Receptors from Human Muscle and Torpedo Electric Organ

Acetylcholine receptor (AChR) purified from human skeletal muscle affinity-alkylated with bromoacetyl[methyl-3H]choline bromide ([3H]BAC) in mildly reducing conditions to yield a specifically radiolabeled polypeptide, Mr 44,000, the alpha-subunit. The binding of [125I]alpha-bungarotoxin to AChR was completely inhibited by affinity-alkylation, indicating that the human AChR's binding site for alpha-bungarotoxin is closely associated with the alpha-subunit's acetylcholine binding site. Structures in the vicinity of the alpha-bungarotoxin binding sites of AChRs from human muscle and Torpedo electric organ were compared by varying the conditions of alkylation. Under optimal conditions of reduction and alkylation, both human and Torpedo AChR incorporated BAC in equivalence to the number of alpha-bungarotoxin binding sites. However, with limited conditions of reduction but sufficient BAC to alkylate 100% of the alpha-bungarotoxin binding sites of human AChR, only 71% of the Torpedo AChR's binding sites were alkylated. In optimal conditions of reduction but with the minimal concentration of BAC that permitted 100% alkylation of the human AChR's alpha-bungarotoxin sites, only 74% of the Torpedo AChR's binding sites were alkylated. These data suggest that the neurotransmitter binding region of human muscle AChR is structurally dissimilar from that of Torpedo electric organ, having a higher binding affinity for BAC and an adjacent disulfide bond that is more readily accessible to reducing agents.     

Hormone-dependent processing of the avian progesterone receptor

Avian progesterone receptor exists as two forms, A and B, with molecular weights of 79,000 and 110,000 daltons, respectively. The origin and significance of these two forms is an area of active investigation and debate. Monoclonal antibodies produced against these two forms were used to examine receptor stability in cytosol and changes in the receptor forms induced by hormone binding. The lability of hormone binding at elevated temperatures is well documented. Analysis by Western blotting showed the receptor was stable in freshly prepared oviduct cytosol for 2 hr at 37°C, while hormone binding was lost within 30 min. However, loss of receptor through degradation was seen when cytosol was prepared from frozen tissue or when homogenization was excessive. Progesterone was injected into diethylstilbestrol-stimulated chicks to examine, in vivo, effects of hormone treatment on receptor forms in the cytosol and nuclear fractions. Progesterone treatment caused a time- and dose-dependent conversion of the A receptor to a form (A′) with a slower electrophoretic mobility. The cytosolic progesterone receptor was divided equally between the B and A forms, while the nuclear receptor was predominantly A′. The amount of nuclear receptor was consistently less than cytosolic receptor. Receptor phosphorylation was analyzed by incubating tissue minces with [³²P]orthophosphate with or without progesterone followed by immune isolation of receptor forms. Progesterone treatment caused a time-dependent increase in cytosol receptor phosphorylation which was evident after 5 min of treatment. This phosphorylation was observed with both the A and B receptor forms. The results indicate that receptor phosphorylation is a very early event during progesterone action.     

Role of TARP interaction in S-SCAM-mediated regulation of AMPA receptors

Scaffolding proteins are involved in the incorporation, anchoring, maintenance, and removal of AMPA receptors (AMPARs) at synapses, either through a direct interaction with AMPARs or via indirect association through auxiliary subunits of transmembrane AMPAR regulatory proteins (TARPs). Synaptic scaffolding molecule (S-SCAM) is a newly characterized member of the scaffolding proteins critical for the regulation and maintenance of AMPAR levels at synapses, and directly binds to TARPs through a PDZ interaction. However, the functional significance of S-SCAM–TARP interaction in the regulation of AMPARs has not been tested. Here we show that overexpression of the C-terminal peptide of TARP-γ2 fused to EGFP abolished the S-SCAM-mediated enhancement of surface GluA2 expression. Conversely, the deletion of the PDZ-5 domain of S-SCAM that binds TARPs greatly attenuated the S-SCAM-induced increase of surface GluA2 expression. In contrast, the deletion of the guanylate kinase domain of S-SCAM did not show a significant effect on the regulation of AMPARs. Together, these results suggest that S-SCAM is regulating AMPARs through TARPs.     

Intracellular calcium release mediated by two muscarinic receptor subtypes

Four subtypes of muscarinic acetylcholine receptor (mAChR) were stably expressed in neuroblastoma-glioma hybrid cells (NG108-15). By combining fluorescent indicator dye (fura-2) studies with electrophysiological measurements it is shown that stimulation of mAChR I and mAChR III readily leads to release of calcium from intracellular stores and to associated conductance changes, whereas stimulation of mAChR II and mAChR IV exerts no such effect. Dose-response curves describing the amplitude or the delay of the calcium rise induced by acetylcholine suggest that the apparent affinity of mAChR III for its agonist is higher by about one order of magnitude than that of mAChR I. Ionic substitution experiments and current fluctuation analysis indicate that calcium activates a K⁺-specific conductance of ‘small’ single-channel amplitude similar to the SK type [1]. Furthermore, an outward current (M current) suppressed by activation of mAChR I and mAChR III has a single-channel amplitude corresponding to a conductance of approximately 3 pS.