A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death.

Although cardiac cytosolic cyclic 3',5'-adenosine monophosphate (cAMP) regulates multiple processes, such as beating, contractility, metabolism and apoptosis, little is known yet on the role of this second messenger within cardiac mitochondria. Using cellular and subcellular approaches, we demonstrate here the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1), and show a protective role for sACt against cell death, apoptosis as well as necrosis in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3(-)) and Ca(2+), sACt produces cAMP, which in turn stimulates oxygen consumption, increases the mitochondrial membrane potential (ΔΨm) and ATP production. cAMP is rate limiting for matrix Ca(2+) entry via Epac1 and the mitochondrial calcium uniporter and, as a consequence, prevents mitochondrial permeability transition (MPT). The mitochondrial cAMP effects involve neither protein kinase A, Epac2 nor the mitochondrial Na(+)/Ca(2+) exchanger. In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3(-) rescued the sensitization of mitochondria to Ca(2+)-induced MPT. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism and cell death in the heart, which is independent of cytosolic cAMP signaling. Our results might have implications for therapeutic prevention of cell death in cardiac pathologies.

Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALK signaling in anaplastic large cell lymphomas.

The majority of anaplastic large cell lymphomas (ALCLs) express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, which is oncogenic due to its constitutive tyrosine kinase activity. Transformation by NPM-ALK not only increases proliferation, but also modifies cell shape and motility in both lymphoid and fibroblastic cells. We report that the Rac1 GTPase, a known cytoskeletal regulator, is activated by NPM-ALK in ALCL cell lines (Karpas 299 and Cost) and transfected cells (lymphoid Ba/F3 cells, NIH-3T3 fibroblasts). We have identified Vav3 as one of the exchange factors involved in Rac1 activation. Stimulation of Vav3 and Rac1 by NPM-ALK is under the control of Src kinases. It involves formation of a signaling complex between NPM-ALK, pp60(c-src), Lyn and Vav3, in which Vav3 associates with tyrosine 343 of NPM-ALK via its SH2 domain. Moreover, Vav3 is phosphorylated in NPM-ALK positive biopsies from patients suffering from ALCL, demonstrating the pathological relevance of this observation. The use of Vav3-specific shRNA and a dominant negative Rac1 mutant demonstrates the central role of GTPases in NPM-ALK elicited motility and invasion.

B-ind1, a novel mediator of Rac1 signaling cloned from sodium butyrate-treated fibroblasts.

Sodium butyrate is a multifunctional agent known to inhibit cell proliferation and to induce differentiation by modulating transcription. We have performed differential display analysis to identify transcriptional targets of sodium butyrate in Balb/c BP-A31 mouse fibroblasts. A novel butyrate-induced transcript B-ind1 has been cloned by this approach. The human homologue of this transcript contains an open reading frame that codes for a protein of 370 amino acids without known functional motifs. In transfected cells, the B-ind1 protein has been found to potentiate different effects of the small GTPase Rac1, such as c-Jun N-terminal kinase activation and transcriptional activity of nuclear factor kappaB (NF-kappaB). In addition, we have demonstrated that B-ind1 forms complexes with the constitutively activated Rac1 protein. To investigate the role of B-ind1 in Rac1 signaling, we have constructed several deletion mutants of B-ind1 and tested their ability to affect the activation of NF-kappaB by Rac1. Interestingly, the fragment encoding the median region of human B-ind1 acted as a dominant-negative variant to block Rac1-mediated NF-kappaB activity. These data define B-ind1 as a novel component of Rac1-signaling pathways leading to the modulation of gene expression.

Estrogen induction of the cyclin D1 promoter: involvement of a cAMP response-like element.

Estrogens induce cell proliferation in target tissues by stimulating progression through the G(1) phase of the cell cycle. Induction of cyclin D1 expression is a critical feature of the mitogenic action of estrogen. We have determined a region between -96 and -29 in the cyclin D1 promoter that confers regulation by estrogens in the human mammary carcinoma cells MCF-7. This region encompasses a unique known transcription factor binding site with a sequence of a potential cAMP response element (CRE-D1). The induction is strictly hormone dependent and requires the DNA binding domain as well as both AF-1 and AF-2 domains of the estrogen receptor (ER) alpha. Destruction of the CRE-D1 motif caused complete loss of estrogen responsiveness. Both c-Jun and ATF-2 transactivated the cyclin D1 promoter in transient transfection experiments, and a clear additional increase was detected when ER was cotransfected with either c-Jun or with c-Jun and ATF-2 but not with ATF-2 alone. Furthermore, the expression of a dominant negative variant of c-Jun, TAM67, completely abolished the induction of the cyclin D1 promoter both in the absence and presence of ER. We show that ATF-2 homodimers and ATF-2/c-Jun heterodimers, but not c-Jun homodimers, were able to bind the CRE of the cyclin D1 promoter. To interpret these results, we propose a mechanism in which ATF-2/c-Jun heterodimers bind to the CRE-D1 element and mediate the activation of cyclin D1 promoter by the ER. This mechanism represents a pathway by which estrogens control the proliferation of target cells.

Sodium butyrate induces G2 arrest in the human breast cancer cells MDA-MB-231 and renders them competent for DNA rereplication.

When exposed to sodium butyrate (NaBut), exponentially growing cells accumulate in G1 and G2 phases of the cell cycle. In the human breast cancer cell line MDA-MB-231, an arrest in G2 phase was observed when the cells were released from hydroxyurea block (G1/S interface) in the presence of NaBut. The inhibition of G2 progression was correlated with increased contents both of total p21(Waf1) and of p21(Waf1) associated with cyclin A and with an inhibition of cyclin A- and B1-associated histone H1 kinase activities measured in cell lysates, as well as with dephosphorylation of the RB protein. A decrease in the cell contents of cyclins A and B1 was also observed but this decrease was preceded by p21(Waf1) accumulation. When NaBut was removed from the culture medium of cells blocked in G2 phase, p21(Waf1) level decreased and, instead of proceeding to mitosis, these cells resumed a progression toward DNA rereplication. These results suggest that the induction of p21(Waf1) by NaBut leads to the inhibition of the sequential activation of cyclin A- and B1-dependent kinases in this cell line, resulting in the inhibition of G2 progression and rendering the cells competent for a new cell division cycle.

Direct inhibition of the expression of cyclin D1 gene by sodium butyrate.

In the mouse fibroblasts BP-A31 as well as in the human epidermoid carcinoma cells KB-3-1, both cyclin D1 mRNA and protein contents decreased rapidly during incubation with sodium butyrate. The decrease of cyclin D1 mRNA was not prevented by cycloheximide indicating that protein synthesis is not required for the inhibition of the expression of cyclin D1 gene by sodium butyrate. The 973 bp region upstream of the human cyclin D1 gene conferred inhibition of the expression of an indicator gene in transiently transfected cells. An 11 base-pair segment situated within this region, with a strong homology to the butyrate-response consensus element identified in butyrate-inducible promoters, also caused an inhibition of transcription under these conditions, indicating that cyclin D1 expression is inhibited by butyrate at the transcriptional level.

Activation of p21ras is not sufficient to ensure a complete G1 phase of the cell division cycle in mouse fibroblasts.

In the mouse BP-A31 fibroblasts, mRNAs coding the three isoforms (Ha, Ki, N) of ras are expressed, and there are no activating mutations in the codons 12, 13 or 61. We have produced a subline (Ras2) expressing an oestrogen-inducible v-Ha-ras gene. The contribution of v-Ha-ras to the overall p21ras-GTP content was evaluated by metabolic labelling with 32P. Surprisingly, p21ras-GTP complexes were predominant in the serum-deprived BP-A31 cells as well as in the Ras2 cells. The excess of p21ras-GTP was not due to the lack of the GTPase activating protein. In transient transfection experiments, the serum response element (SRE)-directed CAT was expressed in serum-deprived BP-A31 cells, and insulin caused a further two- to threefold increase in CAT activity. A dominant negative ras mutant (Ha-Ras Asn-17) cancelled both the basal and insulin-induced CAT expression in the BP-A31 but not in the Ras2 cells. Expression of v-Ha-ras in Ras2 cells did not relax their growth factor-dependence and oestradiol had only a minor mitogenic effect. We conclude that p21ras activation does not ensure a complete cell division cycle in these cells, and does not entirely account for the transduction of the mitogenic signal initiated by insulin.