Mario Caputi

The urokinase plasminogen activator and its receptor. Role in cell growth and apoptosis

The urinary-type plasminogen activator, or uPA, controls matrix degradation through the conversion of plasminogen into plasmin and is regarded as the critical trigger for plasmin generation during cell migration and invasion, under physiological and pathological conditions (such as cancer metastasis). The proteolytic activity of uPA is responsible for the activation or release of several growth factors and modulates the cell survival/apoptosis ratio through the dynamic control of cell-matrix contacts. The urokinase receptor (uPAR), binding to the EGF-like domain of uPA, directs membrane-associated extracellular proteolysis and signals through transmembrane proteins, thus regulating cell migration, adhesion and cytoskeletal status. However, recent evidence highlights an intricate relationship linking the uPA/uPAR system to cell growth and apoptosis.

Alpha‐interferon and its effects on signal transduction pathways

Interferon‐α (IFNα) is a recombinant protein widely used in the therapy of several neoplasms such as myeloma, renal cell carcinoma, epidermoid cervical and head and neck tumors, and melanoma. IFNα, the first cytokine to be produced by recombinant DNA technology, has emerged as an important regulator of cancer cell growth and differentiation, affecting cellular communication and signal transduction pathways. However, the way by which tumor cell growth is directly suppressed by IFNα is not well known. Wide evidence exists on the possibility that cancer cells undergo apoptosis after the exposure to the cytokine. Here we will review the consolidate signal transducer and activator of transcription (STAT)‐dependent mechanism of action of IFNα. We will discuss data obtained by us and others on the triggering of the stress‐dependent kinase pathway induced by IFNα and its correlations with the apoptotic process. The regulation of the expression of proteins involved in apoptosis occurrence will be also described. In this regard, IFNα is emerging as a post‐translational controller of the intracellular levels of the apoptosis‐related protein tissue transglutaminase (tTG). This new way of regulation of tTG occurs through the modulation of their proteasome‐dependent degradation induced by the cytokine. Until today, inconsistent data have been obtained regarding the clinical effectiveness of IFNα in the therapy of solid tumors. In fact, the benefit of IFNα treatment is limited to some neoplasms while others are completely or partially resistant. The mechanisms of tumor resistance to IFNα have been studied in vitro. The alteration of JAK‐STAT components of the IFNα‐induced signaling, can be indeed a mechanism of resistance to IFN. However, we have recently described a reactive mechanism of protection of tumor cells from the apoptosis induced by IFNα dependent on the epidermal growth factor (EGF)‐mediated Ras/extracellular signal regulated kinase (Erk) signaling. The involvement of the Ras→Erk pathway in the protection of tumor cells from the apoptosis induced by IFNα is further demonstrated by both Ras inactivation by RASN17 transfection and mitogen extracellular signal regulated kinase 1 (Mek‐1) inhibition by exposure to PD098059. These data strongly suggest that the specific disruption of the latter could be a useful approach to potentiate the antitumour activity of IFNα against human tumors based on the new mechanistic insights achieved in the last years.

Mitogen-activated protein kinases and asthma

Mitogen‐activated protein kinases (MAPKs) are evolutionary conserved enzymes which play a key role in signal transduction mediated by cytokines, growth factors, neurotransmitters and various types of environmental stresses. In the airways, these extracellular stimuli elicit complex inflammatory and structural changes leading to the typical features of asthma including T cell activation, eosinophil and mast cell infiltration, as well as bronchial hyperresponsiveness and airway remodelling. Because MAPKs represent an important point of convergence for several different signalling pathways, they affect multiple aspects of normal airway function and also significantly contribute to asthma pathophysiology. Therefore, this review focuses on the crucial involvement of MAPKs in asthma pathogenesis, thus also discussing their emerging role as molecular targets for anti‐asthma drugs.

Molecular mechanisms underlying airway smooth muscle contraction and proliferation: implications for asthma.

Airway smooth muscle (ASM) plays a key role in bronchomotor tone, as well as in structural remodeling of the bronchial wall. Therefore, ASM contraction and proliferation significantly participate in the development and progression of asthma. Many contractile agonists also behave as mitogenic stimuli, thus contributing to frame a hyperresponsive and hyperplastic ASM phenotype. In this review, the molecular mechanisms and signaling pathways involved in excitation-contraction coupling and ASM cell growth will be outlined. Indeed, the recent advances in understanding the basic aspects of ASM biology are disclosing important cellular targets, currently explored for the implementation of new, more effective anti-asthma therapies.

Prognostic value of p53 in non-small cell lung cancer: Relationship with proliferating cell nuclear antigen and cigarette smoking

p53 mutations are among the most frequent genetic alterations reported in human lung cancer. Although the prognostic value of altered p53 expression is still debated, it is accepted widely that estimation of the proliferation rate has an important prognostic role. Moreover, an association between certain types of human lung cancers and tobacco use is well known. Drawing from this background, we investigated the immunohistochemical expression of mutant oncogenic p53 protein, and related it to the smoking history of 61 patients with non-small cell lung carcinoma (NSCLC) and to the expression pattern of proliferating cell nuclear antigen (PCNA), which is considered to be an important negative prognostic factor in several neoplasms. We found p53 overexpression in 22 (36.1%) specimens, including 16 squamous carcinomas (41%) and six (27.2%) adenocarcinomas. PCNA nuclear staining was detected in 98.4% of the specimens, and a significantly higher PCNA expression score was found in all of the p53-positive samples. When the patient survival time was compared, p53 accumulation had a statistically significant negative prognostic value (P < .001). This was supported by a Kaplan-Meier survival percentage plot of immunohistochemically p53-undetectable specimens and p53-detectable specimens. These latter patients had a greatly reduced survival time. A relationship was established between p53 immunohistochemical detection and the smoking history of the patients. None of the specimens from the nonsmoking patients expressed immunohistochemically detectable p53 protein. Altered p53 expression was detected in 40.7% of smoking patients. Our findings support the hypothesis of involvement of p53 mutations in tobacco-induced carcinogensis and indicate that altered p53 expression plays an important prognostic role in NSCLC in smokers.

Endothelin-1 Induces Proliferation of Human Lung Fibroblasts and IL-11 Secretion Through an ET A Receptor-Dependent Activation of Map Kinases

Endothelin‐1 (ET‐1) is implicated in the fibrotic responses characterizing interstitial lung diseases, as well as in the airway remodeling process occurring in asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal human lung fibroblasts (NHLFs), the ET‐1 receptor subtypes, and the intracellular signal transduction pathways involved in the proliferative effects of this peptide. Therefore, cells were exposed to ET‐1 in the presence or absence of an overnight pre‐treatment with either ETA or ETB selective receptor antagonists. After cell lysis, immunoblotting was performed using monoclonal antibodies against the phosphorylated, active forms of mitogen‐activated protein kinases (MAPK). ET‐1 induced a significant increase in MAPK phosphorylation pattern, and also stimulated fibroblast proliferation and IL‐6/IL‐11 release into cell culture supernatants. All these effects were inhibited by the selective ETA antagonist BQ‐123, but not by the specific ETB antagonist BQ‐788. The stimulatory influence of ET‐1 on IL‐11, but not on IL‐6 secretion, was prevented by MAPK inhibitors. Therefore, such results suggest that in human lung fibroblasts ET‐1 exerts a profibrogenic action via an ETA receptor‐dependent, MAPK‐mediated induction of IL‐11 release and cell proliferation.

Effect of blood gas derangement on QTc dispersion in severe chronic obstructive pulmonary disease: evidence of an electropathy?

Cardiac arrhythmias are common in patients with respiratory failure from chronic obstructive pulmonary disease (COPD). Several factors may be potentially arrhythmogenic in these patients, including hypoxemia and hypercapnia, acid-base disturbances, cor pulmonale and the use of digitalis, methylxanthines, and sympathomimetic drugs. The aim of this study was to examine the effect of hypoxemia and hypercapnia on QTc dispersion (QTcD) in COPD patients, and to evaluate the effect of a partial correction of one of these pro-arrhythmic factors, the hypoxemia, on Qtc dispersion, as QTcD has been proposed as a marker of heterogeneous repolarization and, hence of ventricular electrical instability. We showed that in 15 hypoxemic/hypercapnic COPD patients, compared to 20 controls, the QTcD was significantly higher (49.7 +/- 10.6 vs. 22.9 +/- 9.8 ms; P = 0.0001); furthermore, after only 24 h of oxygen therapy, and hence after a partial correction of hypoxemia, there was a significant reduction in QTcD in COPD patients (49.7 +/- 10.6 vs. 36.3 +/- 10.1 ms; P = 0.018). The data of the present study suggest that the increase in QTcD may be an early marker of a blood gas mediated electropathy in COPD patients.

Urokinase-mediated posttranscriptional regulation of urokinase-receptor expression in non small cell lung carcinoma.

The urokinase‐type plasminogen activator (uPA) and its cellular receptor (uPAR) are involved in the proteolytic cascade required for tumor cell dissemination and metastasis, and are highly expressed in many human tumors. We have recently reported that uPA, independently of its enzymatic activity, is able to increase the expression of its own receptor in uPAR‐transfected kidney cells at a posttranscriptional level. In fact, uPA, upon binding uPAR, modulates the activity and/or the level of a mRNA‐stabilizing factor that binds the coding region of uPAR‐mRNA. We now investigate the relevance of uPA‐mediated posttranscriptional regulation of uPAR expression in non small cell lung carcinoma (NSCLC), in which the up‐regulation of uPAR expression is a prognostic marker. We show that uPA is able to increase uPAR expression, both at protein and mRNA levels, in primary cell cultures obtained from tumor and adjacent normal lung tissues of patients affected by NSCLC, thus suggesting that the enzyme can exert its effect in lung cells. We investigated the relationship among the levels of uPA, uPAR and uPAR‐mRNA binding protein(s) in NSCLC. Lung tissue analysis of 35 NSCLC patients shows an increase of both uPA and uPAR in tumor tissues, as compared to adjacent normal tissues, in 27 patients (77%); 19 of these 27 patients also show a parallel increase of the level and/or binding activity of a cellular protein capable of binding the coding region of uPAR‐mRNA. Therefore, in tumor tissues, a strong correlation is observed among these 3 parameters, uPA, uPAR and the level and/or the activity of a uPAR‐mRNA binding protein. We then suggest that uPA regulates uPAR expression in NSCLC at a posttranscriptional level by increasing uPAR‐stability through a cellular factor that binds the coding region of uPAR‐mRNA.

Effects of hydrogen peroxide on MAPK activation, IL-8 production and cell viability in primary cultures of human bronchial epithelial cells.

The airway epithelium is continuously exposed to inhaled oxidants, including airborne pollutants and cigarette smoke, which can exert harmful proinflammatory and cytotoxic effects. Therefore, the aim of our study was to investigate, in primary cultures of human bronchial epithelial cells (HBEC), the signal transduction pathways activated by increasing concentrations (0.25, 0.5, and 1 mM) of hydrogen peroxide (H2O2), as well as their effects on IL‐8 production and cell viability. The reported results show that H2O2 elicited, in a concentration‐dependent fashion, a remarkable increase in phosphorylation‐dependent activation of mitogen‐activated protein kinases (MAPKs), associated with a significant induction of IL‐8 synthesis and a dramatically enhanced cell death. Pre‐treatment of HBEC with MAPK inhibitors was able to significantly inhibit the effects of H2O2 on IL‐8 secretion, and to effectively prevent cell death. Therefore, these findings suggest that MAPKs play a key role as molecular transducers of the airway epithelial injury triggered by oxidative stress, as well as potential pharmacologic targets for indirect antioxidant intervention.

CDC2-related kinase PITALRE phosphorylates pRb exclusively on serine and is widely expressed in human tissues

Mammalian cell cycle progression is regulated by sequential activation and inactivation of cyclin‐dependent kinases (cdks). Recently, several new members of the cdk family were cloned, and some of these were shown to complex with different cyclins and to be active at discrete stages of the cell cycle. PITALRE, a new member of this family, was cloned by our laboratory and was shown to be able to phosphorylate pRb protein in vitro. In the current work, we found that PITALRE kinase activity phosphorylated pRb at sites similar to those phosphorylated by the CDC2 kinase, which itself is known to mimic, in vitro, the in vivo phosphorylation of pRb. Phosphorylation of pRb by the PITALRE‐associated kinase activity was on Ser residues exclusively. Moreover, we investigated the expression pattern of PITALRE in normal human tissues, using immunohistochemical techniques so as to gain additional data on the characteristics of this new cdk family member. The protein was widely expressed, although a different tissue distribution and/or level of expression was found in various organs. Some specialized tissues such as blood, lymphoid tissue, ovarian cells, and the endocrine portion of the pancreas showed a high expression level of PITALRE. The specific expression pattern found suggests that PITALRE may be involved in specialized functions in certain cell types. J. Cell. Physiol. 172:265–273, 1997.