Hepatic stellate cell proliferation: A potential role of protein kinase R
Authors: Sara Ceccarelli, Nadia Panera, Anna Alisi, Valerio Nobili
Journal: Hepatology (2011)

We read with great interest the article recently published in this journal by Tarrats et al.1 In that study the authors investigated the role of tumor necrosis factor receptors (TNFRs) 1 and 2 in hepatic stellate cell (HSC) proliferation and activation. In particular, they demonstrated that TNF-α and its receptor 1 are main players in HSC proliferation and matrix metalloproteinases-9 expression, even though both TNF-α and TNFR1 were not directly involved in HSC transdifferentiation steps generating the myofibroblast phenotype. These results were confirmed both in primary mouse HSCs and in a human HSC cell line (LX-2). Noteworthy, TNF-α appeared to be involved in the induction of the tissue inhibitor of metalloproteinase-1 only in a murine model. The authors attempted to explain this discrepancy, forgetting to highlight the fact that mouse HSC are primary, whereas LX-2 are immortalized, cells. Moreover, the lack of TNFR1 inhibited HSC proliferation only upon platelet-derived growth factor (PDGF) stimulation. The authors suggested that this effect might be mediated by PI3K/AKT signaling impairment, as well as by a direct/indirect crosstalk between TNF and PDGF receptors. It is reasonable that nuclear factor kappaB (NF-κB) upstream and downstream molecules are potential mediators of suppressed PDGF-dependent proliferation, due to the absence of functioning of TNFR1. Although these NF-κB-associated mediators still remain obscure, we believe that protein kinase R (PKR) could be a potential candidate. It is well known that PKR is critical to cell proliferation. Specifically, it has been demonstrated that TNF-induced cell proliferation is suppressed in PKR-deficient cells.2 In addition, PKR has been described as being involved in PDGF signaling, although its specific role has still not been elucidated.3 Taken together, these data suggest that PKR is a possible mediator at the interface in the suggested crosstalk between PDGF and TNF receptor signaling. We analyzed the expression and/or activation of PKR in LX-2 cells treated with TNF-α (10 ng/mL) at different timepoints. As shown in Fig. 1A, TNF-α stimulation resulted in a statistically significant increase of HSC proliferation at 24 hours. Moreover, western blot analysis showed an up-regulation of PKR protein expression in TNF-α-treated cells at 48 hours and 96 hours (Fig. 1B). TNF-α effects on proliferation and PKR protein expression in LX-2 cells. (A) Proliferation rate at 24 hours was evaluated in nontreated (NT) and treated (TNF) LX-2 as bromodeoxyuridine (BrdU) incorporation performed with Delphia kit based on Europium (Eu)-labeled antibody and Envision plate reader (Perkin Elmer, Italy; generously donated by Nicolò Valenti Onlus, Italy). Results are reported as Eu counts. *P < 0.05. (B) PKR expression levels were observed in total lysates (upper panel). Actin was used as loading control (lower panel). Altogether, these results support our hypothesis that PKR might be the critical molecular link between PDGF and TNFR1 signaling pathways. The role of PKR in regulating PDGF-mediated HSC proliferation and activation, and its correlation with TNFR1, require further studies. However, the findings from the study by Tarrats et al., together with our results, add novel interesting perspectives for designing targeted molecular approaches against liver fibrogenesis. Sara Ceccarelli XX*, Nadia Panera XX*, Anna Alisi XX*, Valerio Nobili XX*, * Liver Research Unit, Bambino Gesù Children's Hospital and Research Institute, Rome, Italy.