Antonio Porcellini

Differential regulation of E2F1 apoptotic target genes in response to DNA damage

E2F1, a member of the E2F family of transcription factors, in addition to its established proliferative effect, has also been implicated in the induction of apoptosis through p53-dependent and p53-independent pathways. Several genes involved in the activation or execution of the apoptotic programme have recently been shown to be upregulated at the transcriptional level by E2F1 overexpression, including the genes encoding INK4a/ARF, Apaf-1, caspase 7 and p73 (refs 3–5). E2F1 is stabilized in response to DNA damage but it has not been established how this translates into the activation of specific subsets of E2F target genes. Here, we applied a chromatin immunoprecipitation approach to show that, in response to DNA damage, E2F1 is directed from cell cycle progression to apoptotic E2F target genes. We identify p73 as an important E2F1 apoptotic target gene in DNA damage response and we show that acetylation is required for E2F1 recruitment on the P1p73 promoter and for its transcriptional activation.

An Oscillatory Switch in mTOR Kinase Activity Sets Regulatory T Cell Responsiveness

There is a discrepancy between the inxa0vitro anergic state of CD4(+)CD25(hi)FoxP3(+) regulatory T (Treg) cells and their inxa0vivo proliferative capability. The underlying mechanism of this paradox is unknown. Here we show that the anergic state of Treg cells depends on the elevated activity of the mammalian target of rapamycin (mTOR) pathway induced by leptin: a transient inhibition of mTOR with rapamycin, before Txa0cell receptor (TCR) stimulation, made Treg cells highly proliferative in the absence of exogenous interleukin-2 (IL-2). This was a dynamic and oscillatory phenomenon characterized by an early downregulation of the leptin-mTOR pathway followed by an increase in mTOR activation necessary for Treg cell expansion to occur. These data suggest that energy metabolism, through the leptin-mTOR-axis, sets responsiveness of Treg cells that use this information to control immune tolerance and autoimmunity.

Smooth Muscle Cell Proliferation Is Proportional to the Degree of Balloon Injury in a Rat Model of Angioplasty

BACKGROUNDnA variable degree of smooth muscle cell (SMC) proliferation after balloon injury has been reported in previous rat studies. It is unknown whether balloon injury induces c-fos expression and whether it is related to the degree of vascular injury in vivo. Therefore, we tested the hypothesis that proportional increases in neointimal formation and c-fos expression might be present after different degrees of balloon dilation.nnnMETHODS AND RESULTSnAngioplasty of the carotid artery was performed with a balloon catheter. Vascular injury was evaluated at 0, 0.5, 1.0, 1.5, and 2 atm (n = 6 for all). In 40 additional rats, total RNA dot blots were performed to assess the effect of various degrees of balloon injury on c-fos expression. SMC proliferation proportional to the increases of inflation pressure was found between 0 and 2 atm with neointimal areas of 0.002 +/- 0.002, 0.069 +/- 0.014, 0.128 +/- 0.043, 0.190 +/- 0.010, and 0.255 +/- 0.041 mm2, respectively. When the degree of SMC proliferation (neointima and neointima/media ratio) was plotted against balloon inflation pressure, a linear relation was observed (r = .733, P < .001 and r = .755, P < .001, respectively). An increase in c-fos expression proportional to the degree of injury was found 30 minutes after injury.nnnCONCLUSIONSnNeointimal proliferation produced by balloon injury is related to balloon inflation pressure, supporting the concept of an SMC proliferative response proportional to the degree of injury. The increase in SMC proliferation is associated with a proportional increase in the early expression of the c-fos nuclear proto-oncogene.

Requirement for cAMP-PKA Pathway Activation by M Phase-Promoting Factor in the Transition from Mitosis to Interphase

Cell cycle progression in cycling Xenopus egg extracts is accompanied by fluctuations in the concentration of adenosine 3′,5′-monophosphate (cAMP) and in the activity of the cAMP-dependent protein kinase (PKA). The concentration of cAMP and the activity of PKA decrease at the onset of mitosis and increase at the transition between mitosis and interphase. Blocking the activation of PKA at metaphase prevented the transition into interphase; the activity of M phase-promoting factor (MPF; the cyclin B-p34cdc2 complex) remained high, and mitotic cyclins were not degraded. The arrest in mitosis was reversed by the reactivation of PKA. The inhibition of protein synthesis prevented the accumulation of cyclin and the oscillations of MPF, PKA, and cAMP. Addition of recombinant nondegradable cyclin B activated p34cdc2 and PKA and induced the degradation of full-length cyclin B. Addition of cyclin A activated p34cdc2 but not PKA, nor did it induce the degradation of full-length cyclin B. These findings suggest that cyclin degradation and exit from mitosis require MPF-dependent activation of the cAMP-PKA pathway.

Regulatory T cell proliferative potential is impaired in human autoimmune disease

Human CD4+CD25highCD127−FoxP3+ regulatory T (Treg) cells suppress immune responses in vitro and in vivo. Reduced suppressive function and/or number of peripheral Treg cells has been previously reported in autoimmune disorders. Treg cells represent the most actively replicating compartment within the CD4+ cells in vivo, but they are hyporesponsive to classical T cell receptor (TCR) stimulation in vitro, a condition that is secondary to their overactive metabolic state. Here we report that proliferation of Treg cells after TCR stimulation is impaired in subjects with relapsing-remitting multiple sclerosis (RRMS) because of altered interleukin-2 (IL-2) secretion and IL-2 receptor (IL-2R)–signal transducer and activator of transcription 5 (STAT5) signaling. This is associated with decreased expression of the forkhead box P3 (FoxP3) 44- and 47-kDa splicing forms, overactivation of S6 ribosomal protein (a downstream target of the mammalian target of rapamycin, mTOR) and altered activity of the cyclin-dependent kinase inhibitor p27 (p27kip1) and extracellular signal–related kinases 1 and 2 (ERK1/2). The impaired capacity of Treg cells to proliferate in RRMS correlates with the clinical state of the subject, where increasing disease severity is associated with a decline in Treg cell expansion. These results suggest a previously unrecognized mechanism that may account for the progressive loss of Treg cells in autoimmune disease.

Glycolysis controls the induction of human regulatory T cells by modulating the expression of FOXP3 exon 2 splicing variants

Human regulatory T cells (Treg cells) that develop from conventional T cells (Tconv cells) following suboptimal stimulation via the T cell antigen receptor (TCR) (induced Treg cells (iTreg cells)) express the transcription factor Foxp3, are suppressive, and display an active proliferative and metabolic state. Here we found that the induction and suppressive function of iTreg cells tightly depended on glycolysis, which controlled Foxp3 splicing variants containing exon 2 (Foxp3-E2) through the glycolytic enzyme enolase-1. The Foxp3-E2–related suppressive activity of iTreg cells was altered in human autoimmune diseases, including multiple sclerosis and type 1 diabetes, and was associated with impaired glycolysis and signaling via interleukin 2. This link between glycolysis and Foxp3-E2 variants via enolase-1 shows a previously unknown mechanism for controlling the induction and function of Treg cells in health and in autoimmunity.

The Proteomic Landscape of Human Ex Vivo Regulatory and Conventional T Cells Reveals Specific Metabolic Requirements

Human CD4(+)CD25(hi)Foxp3(+)CD127(-) Treg and CD4(+)CD25(-)Foxp3(-) Tconv cell functions are governed byxa0their metabolic requirements. Here we report axa0comprehensive comparative analysis between exxa0vivo human Treg and Tconv cells that comprises analyses of the proteomic networks in subcellular compartments. We identified a dominant proteomic signature at the metabolic level that primarily impacted the highly-tuned balance between glucose and fatty-acid oxidation in the two cell types. Exxa0vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured inxa0vitro, Treg cells engaged both glycolysis and FAO to proliferate, while Tconv cell proliferation mainly relied on glucose metabolism. Our unbiased proteomic analysis provides a molecular picture of the impact of metabolism on exxa0vivo human Treg versus Tconv cell functions that might be relevant for therapeutic manipulations of these cells.

Role of the angiotensin II AT2-subtype receptors in the blood pressure-lowering effect of losartan in salt-restricted rats

Objective The aim of this study was to evaluate the potential role of the angiotensin II (Ang II) AT2 receptors (AT2) in the control of blood pressure (BP) in the rat and the effects of AT2 receptors on BP during AT1 receptor (AT1) antagonism. Methods The study was performed in 52 Sprague–Dawley rats, which were preliminarily salt-restricted (SR) to enhance circulating and tissue renin–angiotensin system activity. To explore whether AT2 plays a role in BP regulation, the BP effects of the selective AT2 and AT1 receptor antagonists PD123319 (PD) (50 μg/kg/min) and losartan (Los) (10 mg/kg/day), were studied. Seven rats were used as a control group. To define whether AT2 plays a role in the BP response observed during AT1 antagonism, 17 Los treated rats were divided into two groups: seven were treated with both antagonists (Los + PD) and 10 rats received Los + vehicle. The effects of both drugs were also studied in bilaterally nephrectomized rats (NX). All treatments were maintained for 1 week. Results Los reduced BP significantly in both intact (P < 0.001) and NX (P < 0.05) rats, while PD increased BP in intact and NX rats (both P < 0.001). In the Los + PD group BP levels were significantly higher (P < 0.001 vs Los and Los + vehicle, P = ns vs pretreatment), while vehicle infusion did not modify the BP response to Los. Conclusion The results show that in salt-restricted rats AT2 blockade offsets the BP-lowering effect of losartan and suggest that AT2 receptors contribute to the hypotensive effects of losartan. Thus, AT1 receptor antagonists such as losartan, which are becoming widely used in the clinical treatment of hypertension, may reduce BP not only by blockade of AT1 receptors, but also through the stimulation of AT2 receptors by the excess of angiotensin II.

NIDDM Associated With Mutation in Tyrosine Kinase Domain of Insulin Receptor Gene

A population of 103 patients with non-insulin-dependent diabetes mellitus (NIDDM) was screened for mutations in the tyrosine kinase domain of the insulin receptor gene. Patient genomic DNAs corresponding to exons 17–21 of the insulin receptor gene have been amplified by polymerase chain reaction and analyzed by denaturing gradient gel electrophoresis (DGGE). One patient was identified with an altered pattern of mobility of exon 20 in the DGGE assay. Direct sequence of amplified DNA showed a single nucleotide substitution in the codon 1152 (CGG – – > CAG), resulting in the replacement of Arg with Gln. Two bands appeared in the sequence of exon 20 of the insulin receptor (nucleotide position 3584), indicating that this patient was heterozygous for the mutation. Insulin binding to intact erythrocytes from the patient was in the normal range. Although autophosphorylation of the purified insulin receptor also seemed normal, its kinase activity toward the exogenous substrate poly Glu:Tyr (4:1) was undetectable. This mutation may impair insulin receptor kinase and contribute to insulin resistance in this patient.

The Differential Response of Protein Kinase A to Cyclic AMP in Discrete Brain Areas Correlates with the Abundance of Regulatory Subunit II

Abstract: We analyzed the expression and relative distribution of mRNA for the regulatory subunits (RIα, RIIα, and RIIβ) and of 150‐kDa RIIβ‐anchor proteins for cyclic AMP (cAMP)‐dependent protein kinase (PKA) into discrete brain regions. The subcellular distribution of both holoenzyme and free catalytic subunit was evaluated in the same CNS areas. In the neocortex and corpus striatum high levels of RIIβ paralleled the presence of specific RII‐anchoring proteins, high levels of membrane‐bound PKA holoenzyme, and low levels of cytosolic free catalytic activity (C‐PKA). Conversely, in brain areas showing low RIIβ levels (cerebellum, hypothalamus, and brainstem) we found an absence of RII‐anchoring proteins, low levels of membrane‐bound holoenzyme PKA, and high levels of cytosolic dissociated C‐PKA. Response to cAMP stimuli was specifically evaluated in the neocortex and cerebellum, prototypic areas of the two different patterns of PKA distribution. We found that cerebellar holoenzyme PKA was highly sensitive to cAMP‐induced dissociation, without, however, a consistent translocation of C‐PKA into the nucleus. In contrast, in the neocortex holoenzyme PKA was mainly in the undissociated state and poorly sensitive to cAMP. In nuclei of cortical cells cAMP stimulated the import of C‐PKA and phosphorylation of cAMP‐responsive element binding protein. Taken together, these data suggest that RIIβ (whose distribution is graded throughout the CNS, reaching maximal expression in the neocortex) may represent the molecular cue of the differential nuclear response to cAMP in different brain areas, by controlling cAMP‐induced holoenzyme PKA dissociation and nuclear accumulation of catalytic subunits.