Jean-Claude Scimeca

Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP

In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP‐stimulated MAP kinase activation. We show that cAMP stimulates p44mapk independently of these events. Secondly, we studied the role of B‐Raf in this process. We observed that NGF, PMA and cAMP induce the phosphorylation of B‐Raf as well as an upward shift in its electrophoretic mobility. We show that B‐Raf is activated following NGF and PMA treatment of PC12 cells, and that it can phosphorylate and activate MEK‐1. However, cAMP inhibits B‐Raf autokinase activity as well as its ability to phosphorylate and activate MEK‐1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B‐Raf in vitro. Further, we show that B‐Raf binds to p21ras, but more important, this binding to p21ras is virtually abolished with B‐Raf from PC12 cells treated with CPT‐cAMP. Hence, these data indicate that the PKA‐mediated phosphorylation of B‐Raf hampers its interaction with p21ras, which is responsible for the PKA‐mediated decrease in B‐Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP kinase through the activation of an unidentified MEK kinase and/or the inhibition of a MEK phosphatase.

Essential role of calcium in the regulation of MAP kinase phosphatase-1 expression.

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a dual-specificity protein phosphatase encoded by an immediate-early gene responsive to growth factors and stress. The MKP-1 protein selectively inactivates MAP kinases in vitro by dephosphorylation of the regulatory Thr and Tyr residues. Little is known on the mechanisms that regulate MKP-1 gene expression. Here, we demonstrate that Ca2+ is both necessary and sufficient for the induction of MKP-1 gene expression. Treatment of Rat1 fibroblasts with the Ca2+ chelating agent BAPTA completely suppressed serum-induced MKP-1 expression in a dose- and time-dependent manner. The inhibitory effect of BAPTA was observed at the level of the protein and the mRNA. Importantly, Ca2+ chelation blocked the induction of MKP-1 expression in response to all stimuli tested and in different cell types. Increasing the intracellular concentration of Ca2+ with the ionophore A23187 was sufficient to induce MKP-1 mRNA and protein expression in rat fibroblasts. We also provide evidence that activation of MAP kinases is not an absolute requirement for induction of the MKP-1 gene. Exposure of rat fibroblasts to A23187 induced MKP-1 expression without activating the JNK and p38 MAP kinase pathways. Also, inhibition of the ERK pathway with the selective MEK inhibitor PD98059 did not interfere with serum-stimulated MKP-1 mRNA expression. These results will help define the regulatory mechanisms that govern MKP-1 gene transcription in target cells.

Intermolecular transphosphorylation between insulin receptors and EGF-insulin receptor chimerae.

The insulin receptor, a glycoprotein consisting of two extracellular alpha‐ and two transmembrane beta‐subunits, is thought to mediate hormone action by means of its tyrosine‐specific protein kinase activity. To explore the mechanism of insulin receptor phosphorylation we have used NIH3T3 cells transfected with two receptor constructs: one encoding a chimeric receptor composed of the extracellular domain of the human EGF receptor and the cytosolic domain of the human insulin receptor beta‐subunit, and a second construct encoding a kinase‐defiecient human insulin receptor. Stimulation of these cells with EGF induced tyrosine autophosphorylation of the EGF‐insulin receptor chimera (150 kd) and tyrosine phosphorylation of the beta‐subunit of the kinase‐deficient insulin receptor (95 kd). The phosphopeptides of the autophosphorylated cytoplasmic domain of the EGF‐insulin receptor chimera were comparable to those of the transphosphorylated beta‐subunit of the kinase‐deficient insulin receptor and of the wild‐type human insulin receptor. When immunoaffinity purified EGF‐insulin receptor hybrids and kinase‐deficient insulin receptors were used in a cell lysate phosphorylation assay, it was found that addition of EGF produced 32P‐labeling of both receptor species. We conclude that EGF acting directly through the EGF‐insulin receptor chimera causes transphosphorylation of the kinase‐deficient insulin receptor. These data support the notion that autophosphorylation of the insulin receptor may proceed by an intermolecular mechanism.

Gallium modulates osteoclastic bone resorption in vitro without affecting osteoblasts

Background and purpose:  Gallium (Ga) has been shown to be effective in the treatment of disorders associated with accelerated bone loss, including cancer‐related hypercalcemia and Pagets disease. These clinical applications suggest that Ga could reduce bone resorption. However, few studies have studied the effects of Ga on osteoclastic resorption. Here, we have explored the effects of Ga on bone cells in vitro.

Structure, chromosome localization, and tissue distribution of the mouse twik K+ channel gene

We have recently discovered a new class of potassium channels with two pore‐forming domains and four membrane‐spanning domains. When heterologously expressed, these channels produce time‐ and voltage‐independent currents that classify them as background or leak channels. TWIK (for andem of P domains in a eak nwardly rectifying + channel) was the first member of this family to be cloned. Here, we describe the genomic organization of TWIK in the mouse. The coding sequence as well as the untranslated sequences are contained in three exons. The twik gene (or KCNK1) has been mapped to chromosome 8, consistent with its localization to 1q42–43 in human. The twik gene is expressed in virtually all mouse tissues. It is most abundantly expressed in brain and moderately in other organs such as kidney. The level of expression is increased in brain and kidney from neonate to adult animals, but the TWIK message is also detected during embryogenesis, as early as day 7 post conception.

Cloning and Use of a Coral 36B4 Gene to Study the Differential Expression of Coral Genes Between Light and Dark Conditions

This paper aims to validate reference genes for gene expression studies between light and dark conditions in the scleractinian coral Stylophora pistillata for future gene expression studies of the “light-enhanced calcification” phenomenon. For this purpose, we cloned, sequenced, and characterized a candidate reference gene, the 36B4 gene from the coral S. pistillata, and validated 36B4 and β-actin as reference genes. To illustrate the future applications of these reference genes, we tested the dark and light expression of two photosynthetic genes (Rubisco and D1 protein of the photosystem II) and two genes encoding proteins involved in calcium transport for coral calcification (a calcium ATPase and a calcium channel). Results show that both photosynthetic genes are enhanced during the light when standardized against 36B4 and β-actin, whereas the two genes encoding proteins involved in calcium transport are not differentially expressed between light and dark conditions. The characterization of a coral 36B4 and the establishment of such valid reference genes will be useful for future gene expression studies between diverse conditions (aposymbiotic/symbiotic, stress/control, light/dark conditions) in scleractinian corals.

RANKL Treatment Releases the Negative Regulation of the Poly(ADP-Ribose) Polymerase-1 on Tcirg1 Gene Expression During Osteoclastogenesis†

The Tcirg1 gene encodes the osteoclast‐specific a3 isoform of the V‐ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP‐1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis.

Molecular effects of gallium on osteoclastic differentiation of mouse and human monocytes.

We had previously reported that gallium (Ga) inhibited both the differentiation and resorbing activity of osteoclasts in a dose-dependent manner. To provide new insights into Ga impact on osteoclastogenesis, we investigated here the molecular mechanisms of Ga action on osteoclastic differentiation of monocytes upon Rankl treatment. We first observed that Ga treatment inhibited the expression of Rankl-induced early differentiation marker genes, while the same treatment performed subsequently did not modify the expression of late differentiation marker genes. Focusing on the early stages of osteoclast differentiation, we observed that Ga considerably disturbed both the initial induction as well as the autoamplification step of Nfatc1 gene. We next demonstrated that Ga strongly up-regulated the expression of Traf6, p62 and Cyld genes, and we observed concomitantly an inhibition of IκB degradation and a blockade of NFκB nuclear translocation, which regulates the initial induction of Nfatc1 gene expression. In addition, Ga inhibited c-Fos gene expression, and subsequently the auto-amplification stage of Nfatc1 gene expression. Lastly, considering calcium signaling, we observed upon Ga treatment an inhibition of calcium-induced Creb phosphorylation, as well as a blockade of gadolinium-induced calcium entry through TRPV-5 calcium channels. We identify for the first time Traf6, p62, Cyld, IκB, NFκB, c-Fos, and the calcium-induced Creb phosphorylation as molecular targets of Ga, this tremendously impacting the expression of the master transcription factor Nfatc1. In addition, our results strongly suggest that the TRPV-5 calcium channel, which is located within the plasma membrane, is a target of Ga action on human osteoclast progenitor cells.

Gallium as a potential candidate for treatment of osteoporosis.

Gallium (Ga) is a semi-metallic element that displays antitumor, antiresorptive, anti-inflammatory and immunosuppressive properties. Among all these properties, antitumor properties were the most extensively applied and have shown efficacy in treatment of Pagets disease, myeloma and hypercalcemia in cases of malignancy. By contrast, no clinical trials have been conducted in prevention and/or treatment of osteoporosis. In this article I focus on Ga effects on bone tissue and cells, as well as on molecular mechanisms governing Ga internalization into cells. Eventually, the potential of Ga as an antiosteoporotic agent is discussed.

Phenylarsine oxide stimulates a cytosolic tyrosine kinase activity and glucose transport in mouse fibroblasts

In the present report we further approach the mechanism by which insulin and phenylarsine oxide (PAO), a trivalent arsenical compound, regulate glucose transport in mouse fibroblasts (NIH3T3). First, we show that PAO is a powerful stimulatory agent on glucose transport. Second, at least three series of observations indicate that this action of PAO is not mediated through the insulin receptor: (i) the same effect of PAO is observed in NIH3T3 and in transfected cells expressing 6 x 10(6) insulin receptors, while the effect of insulin is markedly increased in the transfected cells; (ii) PAO does not affect the tyrosine phosphorylation of the insulin receptor; (iii) the tyrosine kinase activity of the insulin receptor toward exogenous substrates is not increased by PAO. Since PAO appears to act on glucose transport by a different mechanism than insulin, we have compared the effect of PAO and insulin on tyrosine phosphorylation of cellular proteins. Using Western blot analysis we did not detect common substrates in PAO- and insulin-treated cells. However, we found in cell extracts from both PAO- and insulin-treated cells a 50-kDa protein that is immunoprecipitated by antiphosphotyrosine antibody. In addition, PAO activates a cytosolic tyrosine kinase capable of poly(Glu/Tyr) phosphorylation. As a whole, our data suggest that the 50-kDa protein found in cells incubated with PAO and insulin could be the convergence point of the insulin and PAO signaling pathways.