María J. López-Zabalza

The turnover of extracellular matrix (ECM) components can generate signals that regulate several cellular functions such as proliferation, differentiation, and apoptosis. During liver injury, matrix metalloproteases (MMPs) production is enhanced and increased levels of peptides derived from extracellular matrix proteins can be generated. Synthetic peptides with sequences present in extracellular matrix proteins were previously found to induce both stimulating and apoptotic effects on several cell types including the inflammatory cells monocytes/macrophages. Therefore, in inflammatory liver diseases, locally accumulated peptides could be also important in regulating hepatic fibrosis by inducing apoptosis of hepatic stellate cells (HSC), the primary cellular source of extracellular matrix components. Here, we describe the apoptotic effect of fibronectin peptides on the cell line of human hepatic stellate cells LX‐2 based on oligonucleosomal DNA fragmentation, caspase‐3 and ‐9 activation, Bcl‐2 depletion, and accumulation of Bax protein. We also found that these peptides trigger the activation of Src kinase, which in turn mediated the increase of JNK and p38 activities. By the use of specific inhibitors we demonstrated the involvement of Src, JNK, and p38 in apoptosis induced by fibronectin peptides on HSC. Moreover, fibronectin peptides increased iNOS expression in human HSC, and specific inhibition of iNOS significantly reduced the sustained activity of JNK and the programmed cell death caused by these peptides. Finally, the possible regulatory effect of fibronectin peptides in liver fibrosis was further supported by the ability of these peptides to induce metalloprotease‐9 (MMP‐9) expression in human monocytes. J. Cell. Physiol. 230: 546–553, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company

The sustained overproduction of nitric oxide (NO) observed in inflammatory conditions can contribute to cell demise by affecting apoptosis. Nitration of tyrosine residues occurs in a range of diseases involving macrophage activation. Since NO induces apoptosis in monocytes/macrophages, we tested the hypothesis that nitration of specific proteins could result in apoptotic cell death. The peroxynitrite generator SIN-1 promoted apoptosis in monocytes based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion and accumulation of Bax and p53 proteins. We also found that the signaling pathway triggered by SIN-1 was initiated through tyrosine kinase and Rac activation and resulted in increased JNK and p38 activities. Among the tyrosine-nitrated proteins, Rac and Lyn were identified. Using specific inhibitors for different signaling and effector molecules involved in the apoptotic process we demonstrate that NO, via protein-nitration, could play an important role in controlling the inflammatory response by regulation of monocyte homeostasis.

Synthetic peptides with sequences present in extracellular matrix protein fibronectin have been described to stimulate human monocytes. We describe now that one of these peptides, FN6, induces apoptotic effects on monocytes and we investigate the molecular mechanisms involved in the regulation of this response. Incubation of monocytes with FN6 induces the activation of the small GTPase Rac. In turn, Rac mediates the increase of both JNK and p38 activities in a sustained fashion, as well as the phosphorylation levels of their respective substrates c-Jun and ATF-2. FN6 also stimulates caspases -9 and -3 and the delayed proteolysis of its substrates PARP and D4-GDI. In addition, initiator caspases -1 and -5 were activated by FN6 treatment of monocytes but, in contrast to that observed for caspases-9 and -3, this effect was not dependent on JNK or p38 activities. These kinases also mediated the increase of Bax levels, but only in some conditions Bcl-2 depletion caused by the peptide. Moreover, whereas initially only caspase-1 is involved in caspase-3 activation, later on caspase-9 seems also to participate. Therefore, we demonstrate that FN6 stimulation allows multiple, JNK and p38 -dependent and -independent interacting signals to regulate the apoptotic response in human monocytes.

The Ca(2+)-ATPase activity of the trypsin-activated CF1 presented a monophasic pattern, indicating that the active centres of the enzyme were acting with the same kinetic properties. The study of the effect of the anions cianate (OCN-) and thiocyanate (SCN-) on the ATPase activity showed the existence of cationic regulatory sites, capable of binding these modulators in a competitive way, resulting in the inhibition of the ATPase activity. Nucleotides ADP and ATP, at high concentrations, were competitive inhibitors for the substrate Ca(2+)-ATP. ATP, at low concentrations, presented an activating effect. The study of the combined effects of ATP (at low concentrations) and SCN- on ATPase activity revealed the existence of a non-competitive relationship between anions and nucleotides. The modification of CF1 with fluorescein isothiocyanate, a specific reagent that binds to amino groups of nucleotide binding centres, yielded a molar relationship FITC/CF1 = 4, both with the trypsin-treated and non treated enzyme. This specific incorporation took place on the alpha and, beta subunits of CF1, and resulted in a decrease of about 30% of the ATPase activity. These results are consistent with the existence of either three catalytic and three regulatory sites or four catalytic and two regulatory sites on CF1.