Paolo Rosini

Bcl-2 Phosphorylation by p38 MAPK: identification of target sites and biologic consequences.

The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser87 and Thr56 as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38α knock-out mice (p38α-/- MEF), whereas they occur within 12 h of serum withdrawal in p38α+/+ MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.

A conserved AU-rich element in the 3′ untranslated region of bcl-2 mRNA is endowed with a destabilizing function that is involved in bcl-2 down-regulation during apoptosis

The control of mRNA stability is becoming recognized as a crucial point of gene expression regulation. A common element responsible for mRNA decay modulation is the adenine‐ and uracil‐rich element that is found in the 3’ untranslated region of numerous mRNAs subjected to fast expression changes in response to various stimuli. Previously we identified a post‐transcriptional regulation level for the antiapoptotic bcl‐2 gene, which could be involved in t(14;18) lymphoma‐associated bcl‐2 overexpression. Here we demonstrate that bcl‐2 mRNA is endowed with an adenine‐ and uracil‐rich element (ARE) characterized by high evolutionary conservation not only among all chordates examined, but even between chordates and the nematode Caeno‐rhabditis elegans (ced‐9 gene). As for other well‐established destabilizing AREs, the insertion of the bcl‐2 ARE downstream from stable β‐globin mRNA causes an enhanced decay of the β‐globin transcript, which proves its functional role. This possibility is corroborated by the fact that the pathway leading to the modulating activity of bcl‐2 ARE is influenced by PKC, since the addition of DAG and TPA markedly attenuated the bcl‐2 ARE destabilizing potential. Conversely, it is noteworthy that when C2‐ceramide is added to the culture medium as the apoptotic agent, the β‐globin transcript harboring the bcl‐2 ARE undergoes a dramatic increase in decay. This observation clearly indicates that the destabilizing function of bcl‐2 ARE is enhanced by apoptotic stimuli and suggests that this element could be involved in a post‐transcriptional mechanism of bcl‐2 down‐regulation during apoptosis. The half‐life of the mRNA of bcl‐2 in Jurkat cells is prolonged by PKC stimulation and shortened by C2‐ceramide addition, strongly supporting the view that bcl‐2 mRNA stability plays a physiological role in modulating bcl‐2 expression, particularly in its down‐regulation during apoptosis. Thus, this element becomes a new candidate for mediating those bcl‐2 gene expression changes— from apoptosis‐associated down‐regulation to tumor‐associated overexpression—observed thus far that profoundly influence single cell fate and tissue ho‐meostasis. Schiavone, N., Rosini, P., Quattrone, A., Donnini, M., Lapucci, A., Citti, L., Bevilacqua, A., Nicolin, A., Capaccioli, S. A conserved AU‐rich element in the 3* untranslated region of bcl‐2 mRNA is endowed with a destabilizing function that is involved in bcl‐2 down‐regulation during apoptosis. FASEB J. 14, 174–184(2000)

Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity

Exploitation of the biologic activity of neurotrophins is desirable for medical purposes, but their protein nature intrinsically bears adverse pharmacokinetic properties. Here, we report synthesis and biologic characterization of a novel class of low molecular weight, non-peptidic compounds with NGF (nerve growth factor)-mimetic properties. MT2, a representative compound, bound to Trk (tropomyosin kinase receptor)A chain on NGF-sensitive cells, as well as in cell-free assays, at nanomolar concentrations and induced TrkA autophosphorylation and receptor-mediated internalization. MT2 binding involved at least two amino-acid residues within TrkA molecule. Like NGF, MT2 increased phosphorylation of extracellular signal-regulated kinase1/2 and Akt proteins and production of MKP-1 phosphatase (dual specificity phosphatase 1), modulated p38 mitogen-activated protein kinase activation, sustained survival of serum-starved PC12 or RDG cells, and promoted their differentiation. However, the intensity of such responses was heterogenous, as the ability of maintaining survival was equally possessed by NGF and MT2, whereas the induction of differentiation was expressed at definitely lower levels by the mimetic. Analysis of TrkA autophosphorylation patterns induced by MT2 revealed a strong tyrosine (Tyr)490 and a limited Tyr785 and Tyr674/675 activation, findings coherent with the observed functional divarication. Consistently, in an NGF-deprived rat hippocampal neuronal model of Alzheimer Disease, MT2 could correct the biochemical abnormalities and sustain cell survival. Thus, NGF mimetics may reveal interesting investigational tools in neurobiology, as well as promising drug candidates.

Nuclear localization of the nerve growth factor (NGF) receptor, TRK-A in liver cells

the expression of MAT2A (p<O.OOl), a gene whose induction is associated with dedifferentiation of liver cells. In vitro studies showed that primary rat hepatocytes express WTl when stimulated with TGFbeta or when the cells undergo dedifferentiation in culture. Moreover, WTl down-regulates HNF-4 and up-regulates MAT2A, thus promoting reversion of hepatocytes to a more immature phenotype. Conclusions: we show that WTl, a developmental-restricted factor, is induced by TGFbeta and is reexpressed in the cirrhotic liver. WTl appears to have a negative effect on hepatocellular differentiation. facilitating the progression of liver cirrhosis to hepatic insufficiency.