Alessandra Spaziani

Physical and functional interaction between HCV core protein and the different p73 isoforms

Hepatitis C virus (HCV) core protein is a structural viral protein that packages the viral genomic RNA. In addition to this function, HCV core also modulates a number of cellular regulatory functions. In fact, HCV core protein has been found to modulate the expression of the cyclin-dependent inhibitor p21WAF1/CIP1 and to promote both apoptosis and cell proliferation through its physical interaction with p53. Here, we studied the ability of HCV core to bind the p53-related p73 protein, its isoforms and its deletion mutants. We found that HCV core co-immunoprecipitated with p73 in HepG2 and SAOS-2 cells. Deletion mutational analysis of p73 indicates that the domain involved in HCV core binding is located between amino-acid residues 321–353. We also demonstrate that p73/core interaction results in the nuclear translocation of HCV core protein either in the presence of the p73 α or p73 β tumor-suppressor proteins. In addition, the interaction with HCV core protein prevents p73 α, but not p73 β dependent cell growth arrest in a p53-dependent manner. Our findings demonstrate that HCV core protein may directly influence the various p73 functions, thus playing a role in HCV pathogenesis.

Role of p38 MAPK and RNA-dependent Protein Kinase (PKR) in Hepatitis C Virus Core-dependent Nuclear Delocalization of Cyclin B1

Some hepatitis C virus (HCV) proteins, including core protein, deregulate the cell cycle of infected cells, thereby playing an important role in the viral pathogenesis of HCC. Thus far, there are only few studies that have deeply investigated in depth the effects of the HCV core protein expression on the progression through the G1/S and G2/M phases of the cell cycle. To shed light on the molecular mechanisms by which the HCV core protein modulates cell proliferation, we have examined its effects on cell cycle in hepatocarcinoma cells. We show here that HCV core protein perturbs progression through both the G1/S and the G2/M phases, by modulating the expression and the activity of several cell cycle regulatory proteins. In particular, our data provided evidence that core-dependent deregulation of the G1/S phase and its related cyclin-CDK complexes depends upon the ERK1/2 pathway. On the other hand, the viral protein also increases the activity of the cyclin B1-CDK1 complex via the p38 MAPK and JNK pathways. Moreover, we show that HCV core protein promotes nuclear import of cyclin B1, which is affected by the inhibition of both the p38 and the RNA-dependent protein kinase (PKR) activities. The important role of p38 MAPK in regulating G2/M phase transition has been previously documented. It is becoming clear that PKR has an important role in regulating both the G1/S and the G2/M phase, in which it induces M phase arrest. Based on our model, we now show, for the first time, that HCV core expression leads to deregulation of the mitotic checkpoint via a p38/PKR-dependent pathway.

Thyroid hormones regulate DNA‐synthesis and cell‐cycle proteins by activation of PKCα and p42/44 MAPK in chick embryo hepatocytes

The molecular mechanism by which thyroid hormones exert their effects on cell growth is still unknown. In this study, we used chick embryo hepatocytes at different stages of development as a model to investigate the effect of the two thyroid hormones, T3 and T4, and of their metabolite T2, on the control of cell proliferation. We observed that T2 provokes increase of DNA‐synthesis as well as T3 and T4, independently of developmental stage. We found that this stimulatory effect on the S phase is reverted by specific inhibitors of protein kinase C (PKC) and p42/44 mitogen‐activated protein kinase (p42/44 MAPK), Ro 31‐8220 or PD 98059. Furthermore, the treatment with thyroid hormones induces the activation of PKCα and p42/44 MAPK, suggesting their role as possible downstream mediators of cell response mediated by thyroid hormones. The increase of DNA‐synthesis is well correlated with the increased levels of cyclin D1 and cdk4 that control the G1 phase, and also with the activities of cell‐cycle proteins involved in the G1 to S phase progression, such as cyclin E/A‐cdk2 complexes. Interestingly, the activity of cyclin‐cdk2 complexes is strongly repressed in the presence of PKC and p42/44 MAPK inhibitors. In conclusion, we demonstrated that the thyroid hormones could modulate different signaling pathways that are able to control cell‐cycle progression, mainly during G1/S transition.

PKR is a novel functional direct player that coordinates skeletal muscle differentiation via p38MAPK/AKT pathways

Myogenic differentiation is a highly orchestrated multistep process controlled by extracellular growth factors that modulate largely unknown signals into the cell affecting the muscle-transcription program. P38MAPK-dependent signalling, as well as PI3K/Akt pathway, has a key role in the control of muscle gene expression at different stages during the myogenic process. P38MAPK affects the activities of transcription factors, such as MyoD and myogenin, and contributes, together with PI3K/Akt pathway, to control the early and late steps of myogenic differentiation. The aim of our work was to better define the role of PKR, a dsRNA-activated protein kinase, as potential component in the differentiation program of C2C12 murine myogenic cells and to correlate its activity with p38MAPK and PI3K/Akt myogenic regulatory pathways. Here, we demonstrate that PKR is an essential component of the muscle development machinery and forms a functional complex with p38MAPK and/or Akt, contributing to muscle differentiation of committed myogenic cells in vitro. Inhibition of endogenous PKR activity by a specific (si)RNA and a PKR dominant-negative interferes with the myogenic program of C2C12 cells, causing a delay in activation of myogenic specific genes and inducing the formation of thinner myofibers. In addition, the construction of three PKR mutants allowed us to demonstrate that both N and C-terminal regions of PKR are critical for the interaction with p38MAPK and Akt. The novel discovered complex permits PKR to timely regulate the inhibition/activation of p38MAPK and Akt, controlling in this way the different steps characterizing skeletal muscle differentiation.

Involvement of PI3K in HCV-related lymphoproliferative disorders.

Hepatitis C virus (HCV) core protein has been shown to deregulate cell growth and programmed cell death in hepatoma cells, but only minimal informations are available about its possible role on B‐lymphoproliferative disorders (LPDs). The aim of our work was to analyze the biological activity of HCV core protein on B‐cell proliferation. We established Wil2‐ns and Ramos B‐cell lines that stably expressed the HCV core protein. Growth curve, thymidine incorporation analysis, as well as the expression of PCNA and activated‐ERKs demonstrated that HCV core protein induced an increased growth in both cell lines. Interestingly, the HCV core protein expression determined, in our model, a downregulation of DNp73 and an upregulation of DNp63, which was essential for the maintenance of viral‐dependent effects on cell growth. Finally, we have identified phosphoinositide 3‐kinase (PI3K) as mediator of HCV core‐dependent transcriptional increase of DNp63, which in turn correlated with the increasing of lymphocyte proliferation. In primary B‐lymphocytes, derived from HCV‐related low‐grade non‐Hodgkins lymphoma patients, consistent results were obtained. These findings provide evidence for a possible pathogenetic role played by HCV core protein in HCV‐related lymphomagenesis; it could occur through the deregulation of PI3K activity, consequent activation of Akt and overexpression of DNp63. J. Cell. Physiol. 214: 396–404, 2008.

Hepatitis C virus core protein enhances B lymphocyte proliferation.

HCV chronic infection leads to liver diseases and also to a wide range of extrahepatic disorders including benign, but pre-lymphomatous forms (mixed cryoglobulinemia) to frank hematological neoplasia (non-Hodgkins lymphoma). Recent data showed the involvement of p53 superfamily members in the pathogenesis of different lymphatic malignancies. In fact, tymomas and a subset of non-Hodgkins lymphomas (NHLs) express high levels of p63. Thus, we analyzed whether alterations in p53 superfamily gene expression are observable in B lymphocytes isolated from HCV-infected patients with and without lymphoproliferative disorders. We showed, by real-time PCR, a significant induction of DNp63 mRNAs in B lymphocytes obtained from HCV-positive low grade non-Hodgkins lymphoma patients. Since our current understanding of HCV proteins emphasizes the ability of the HCV core protein to deregulate the expression and activity of p53-related proteins, we established different B lymphocyte cell lines (Wil2-ns, Daudi and Ramos) stably expressing HCV core protein, in order to investigate the possible involvement of the viral protein in the upregulation of DNp63 in B lymphocytes. The analysis of p63 family transcripts showed no transcriptional changes for the p63 TA isoforms, whereas an increase (>5 times) of DNp63 mRNA occurred. In all cell lines, this abnormal expression was associated with a significant increase of cell proliferation that was specifically inhibited by silencing DNp63 mRNA. These findings suggest a pathogenetic role of the HCV core in HCV-related lymphomagenesis, through the induction of DNp63s pro-proliferative effects.

OXYSTEROLS INFLUENCE HEPATIC CELLS HOMEOSTASIS

gastric mucosa due to H. pylori infection may result in decreased numbers of ghrelin producing cells and lower plasma ghrelin levels [7,9]. In contrast, there was not significant difference in plasma ghrelin levels between H. pylori infected patients and H. Pylori uninfected patients in our study; 32,56±21,97 and 34,04±20,59 respectively (p:0,75), similar with another Turkish study [8]. In conclusion, according to our results, there is not a relation between plasma ghrelin levels and H. pylori infection. These conflicting results may be related to different H. pylori strains, as in Turkey the prevalence of the strains, such as CagA, that cause atrophic gastritis is lower [11]. Moreover; differences in studied populations in terms of race, sample size, nutritional status and habits can also affect results. Decreasing plasma ghrelin levels in H. pylori infected subjects can be compensated by other extra gastric ghrelin producing tissues, convenient to our results. All together, further studies are needed to demonstrate the association between H. pylori infection and plasma ghrelin levels. References: [1] Date Y, Kojima M, Hosoda H. Ghrelin, a novel growth hormonereleasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology. 2000 Nov;141(11):4255-61. [2] Dass NB, Munonyara M, Bassil AK. Growth hormone secretagogue receptors in rat and human gastrointestinal tract and the effects of ghrelin. Neuroscience. 2003;120(2):443-53. [3] Ueno H, Yamaguchi H, Kangawa K. Ghrelin: a gastric peptide that regulates food intake and energy homeostasis. Regul Pept. 2005 Mar 15;126(1-2):11-9. [4] Zhang ZW, Patchett SE, Farthing MJ. Role of Helicobacter pylori and p53 in regulation of gastric epithelial cell cycle phase progression. Dig Dis Sci. 2002 May;47(5):987-95. [5] Nwokolo CU, Freshwater DA, O’Hare P. Plasma ghrelin following cure of Helicobacter pylori. Gut. 2003 May;52(5):637-40. [6] Isomoto H, Ueno H, Nishi Y. Circulating ghrelin levels in patients with various upper gastrointestinal diseases. Dig Dis Sci. 2005 May;50(5):833-8. [7] Isomoto H, Nakazato M, Ueno H. Low plasma ghrelin levels in patients with Helicobacter pylori-associated gastritis. Am J Med. 2004 Sep 15;117(6):429-32. [8] Gokcel A, Gumurdulu Y, Kayaselcuk F, Serin E. Helicobacter pylori has no effect on plasma ghrelin levels. Eur J Endocrinol. 2003 Apr;148(4):423-6. [9] Ukkola O. Ghrelin and insulin metabolism. Eur J Clin Invest. 2003 Mar;33(3):183-5.106. [10] Caminos JE, Seoane LM, Tovar SA. Influence of thyroid status and growth hormone deficiency on ghrelin. Eur J Endocrinol. 2002 Jul;147(1):159-63. [11] Aydin F, Kaklikkaya N, Ozgur O. Distribution of vacA alleles and cagA status of Helicobacter pylori in peptic ulcer disease and non-ulcer dyspepsia. Clin Microbiol Infect. 2004 Dec;10(12):1102-4.