Frédéric Ezan

Fibrillar collagen scoring by second harmonic microscopy: a new tool in the assessment of liver fibrosis.

BACKGROUND & AIMSnImaging of supramolecular structures by multiphoton microscopy offers significant advantages for studying specific fibrillar compounds in biological tissues. In this study, we aimed to demonstrate the relevance of Second Harmonic Generation (SHG) for assessing and quantifying, without staining, fibrillar collagen in liver fibrosis.nnnMETHODSnWe first showed the relationship between SHG signal and collagen forms over-produced and accumulated during fibrosis progression. Taking this property into consideration, we developed an innovative method to precisely quantify the fibrosis area in histological slices by scoring of fibrillar collagen deposits (Fibrosis-SHG index).nnnRESULTSnThe scoring method was routinely applied to 119 biopsies from patients with chronic liver disease allowing a fast and accurate measurement of fibrosis correlated with the Fibrosis-Metavir score (rho=0.75, p<0.0001). The technique allowed discriminating patients with advanced (moderate to severe) fibrosis (AUROC=0.88, p<0.0001) and cirrhosis (AUROC=0.89, p<0.0001). Taking advantage of its continuous gradation, the Fibrosis-SHG index also allowed the discrimination of several levels of fibrosis within the same F-Metavir stage. The SHG process presented several advantages such as a high reliability and sensitivity that lead to a standardized evaluation of hepatic fibrosis in liver biopsies without staining and pathological examination.nnnCONCLUSIONSnSecond harmonic microscopy emerges as an original and powerful tool in the assessment of liver fibrosis and offers new possibilities for the evaluation of experimental protocols. We expect that this technology could easily be applicable in the study of other fibro-proliferative pathologies.

ERK2 but not ERK1 plays a key role in hepatocyte replication: An RNAi‐mediated ERK2 knockdown approach in wild‐type and ERK1 null hepatocytes

The mitogen‐activated protein kinases (MAPKs) ERK1 and ERK2 have been implicated in various physiological events, and specific targeting of these MAPKs could affect cell proliferation in many cell types. First, to evaluate the potential specific roles of these two MAPKs, we analyzed the mitogenic response in regenerating liver after partial hepatectomy (PH) and in primary culture of hepatocytes isolated from ERK1‐deficient mice. We show that ERK1 knockout and wild‐type (wt) cells replicate with the same kinetics after PH in liver, in vivo, and in primary cultures of hepatocytes, in vitro. Indeed, Cyclin D1 and Cdk1 appear to be expressed concomitantly in knockout and wt cells, highlighting that hepatocytes progress in the cell cycle independently of the presence of ERK1. Second, we specifically abolished ERK2 expression by RNA interference in mouse and rat hepatocytes. We investigated whether small interfering RNA (siRNA) targeting ERK2 could specifically inhibit its expression and interfere with the process of replication. In ERK1‐deficient hepatocytes, silencing ERK2 expression by RNA interference and ERK2 activation by U0126 clearly demonstrate that DNA replication is regulated by an ERK2‐dependent mechanism. Furthermore, in rat wt hepatocytes, whereas ERK2 targeting inhibits late G1 and S phase progression, ERK1 silencing is devoid of any effect on cell proliferation, indicating that ERK1 cannot rescue ERK2 deficiency. Conclusion: Our results emphasize the importance of the MAPK cascade in hepatocyte replication and allow us to conclude that ERK2 is the key form involved in this regulation, in vivo and in vitro. (HEPATOLOGY 2007;45:1035–1045.)

Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.

We investigated the specific role of the mitogen‐activated protein kinase (MAPK) extracellular signal‐regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long‐term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)‐dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF‐dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2‐dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2. Conclusion: Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long‐term survival and cell cycle regulation of hepatocytes. (HEPATOLOGY 2009.)

MEK/ERK-dependent uPAR expression is required for motility via phosphorylation of P70S6K in human hepatocarcinoma cells.

Motility and invasiveness events require specific intracellular signaling cascade activations. In cancer liver cells, one of these mechanisms could involve the MAPK MEK/ERK cascade activation which has been shown over expressed and activated in hepatocellular carcinoma. To study whether the MEK/ERK cascade is involved in the motility of HCC, we examined the effect of MEK inhibitor and ERK2 silencing using monolayer wound‐healing assays and fluoroblock invasion systems. Evidence was provided that the MAPK cascade is a key transduction pathway which controls HCC cells motility and invasiveness. We could disconnect proliferation to motility using mitomycin C and we established that RNAi‐mediated inhibition of ERK2 led to strongly reduced cell motility. To improve our understanding, we analysed the regulation and the role of urokinase receptor (uPAR) in this process. We provided evidence that uPAR was under a MEK/ERK dependent mechanism and blocking uPAR activity using specific antagonist or inhibiting its expression by RNA interference which resulted in complete inhibition of motility. Moreover, we found in MAPK inhibited cultures and in uPAR silencing cells that p70S6K phosphorylation on residue Thr‐389 was significantly reduced, whereas Ser‐421/Thr‐424 phosphorylation did not change. We highlighted that the FRAP/mTOR pathway did not affect motility and Thr‐389 phosphorylation. Furthermore, we demonstrated that p70S6K inhibition by RNA interference completely inhibited hepatocarcinoma cell motility. Therefore, targeting uPAR and/or MEK/ERK/S6K by RNA interference could be a major therapeutic strategy for the future treatment of invasive hepatocarcinoma cells. J. Cell. Physiol. 212: 526–536, 2007.

RNAi-mediated MEK1 knock-down prevents ERK1/2 activation and abolishes human hepatocarcinoma growth in vitro and in vivo

The mitogen‐activated protein kinases MEK/ERK pathway regulates fundamental processes in malignant cells and represents an attractive target in the development of new cancer treatments especially for human hepatocarcinoma highly resistant to chemotherapy. Although gene extinction experiments have suggested distinct roles for these proteins, the MEK/ERK cascade remains widely considered as exhibiting an overlap of functions. To investigate the functionality of each kinase in tumorigenesis, we have generated stably knock‐down clones for MEK1/2 and ERK1/2 isoforms in the human hepatocellular carcinoma line HuH7. Our results have shown that RNAi strategy allows a specific disruption of the targeted kinases and argued for the critical function of MEK1 in liver tumor growth. Transient and stable extinction experiments demonstrated that MEK1 isoform acts as a major element in the signal transduction by phosphorylating ERK1 and ERK2 after growth factors stimulation, whereas oncogenic level of ERK1/2 phosphorylation appears to be MEK1 and MEK2 dependent in basal condition. In addition, silencing of MEK1 or ERK2 abolished cell proliferation and DNA replication in vitro as well as tumor growth in vivo after injection in rodent. In contrast, targeting MEK2 or ERK1 had no effect on hepatocarcinoma progression. These results strongly corroborate the relevance of targeting the MEK cascade as attested by pharmacologic drugs and support the potential application of RNAi in future development of more effective cancer therapies. Our study emphasizes the importance of the MEK/ERK pathway in human hepatocarcinoma cell growth and argues for a crucial role of MEK1 and ERK2 in this regulation.

MAPK signaling in cisplatin-induced death: predominant role of ERK1 over ERK2 in human hepatocellular carcinoma cells

Hepatocellular carcinoma treatment by arterial infusion of cis-diamminedichloroplatinum-II (cisplatin) exhibits certain therapeutic efficacy. However, optimizations are required and the mechanisms underlying cisplatin proapoptotic effect remain unclear. The mitogen-activated protein kinase (MAPK) pathway plays a key role in cell response to cisplatin and the functional specificity of the isoform MAPK/ERK kinase 1 and 2 (MEK1/2) and ERK1/2 could influence this response. The individual contribution of each kinase on cisplatin-induced death was thus analyzed after a transient or stable specific inhibition by RNA interference in the human hepatocellular carcinoma cells Huh-7 or in knockout mice. We demonstrated here that ERK1 played a predominant role over ERK2 in cisplatin-induced death, whereas MEK1 and MEK2 acted in a redundant manner. Indeed, at clinically relevant concentrations of cisplatin, ERK1 silencing alone was sufficient to protect cells from cisplatin-induced death both in vitro, in Huh-7 cells and ERK1(-/-) hepatocytes, and in vivo, in ERK1-deficient mice. Moreover, we showed that ERK1 activity correlated with the induction level of the proapoptotic BH3-only protein Noxa, a critical mediator of cisplatin toxicity. On the contrary, ERK2 inhibition upregulated ERK1 activity, favored Noxa induction and sensitized hepatocarcinoma cells to cisplatin. Our results point to a crucial role of ERK1 in cisplatin-induced proapoptotic signal and lead us to propose that ERK2-specific targeting could improve the efficacy of cisplatin therapy by increasing ERK1 prodeath functions.

Protease profiling of liver fibrosis reveals the ADAM metallopeptidase with thrombospondin type 1 motif, 1 as a central activator of transforming growth factor beta†‡

During chronic liver disease, tissue remodeling leads to dramatic changes and accumulation of matrix components. Matrix metalloproteases and their inhibitors have been involved in the regulation of matrix degradation. However, the role of other proteases remains incompletely defined. We undertook a gene‐expression screen of human liver fibrosis samples using a dedicated gene array selected for relevance to protease activities, identifying the ADAMTS1 (A Disintegrin And Metalloproteinase [ADAM] with thrombospondin type 1 motif, 1) gene as an important node of the protease network. Up‐regulation of ADAMTS1 in fibrosis was found to be associated with hepatic stellate cell (HSC) activation. ADAMTS1 is synthesized as 110‐kDa latent forms and is processed by HSCs to accumulate as 87‐kDa mature forms in fibrotic tissues. Structural evidence has suggested that the thrombospondin motif‐containing domain from ADAMTS1 may be involved in interactions with, and activation of, the major fibrogenic cytokine, transforming growth factor beta (TGF‐β). Indeed, we observed direct interactions between ADAMTS1 and latency‐associated peptide‐TGF‐β (LAP‐TGF‐β). ADAMTS1 induces TGF‐β activation through the interaction of the ADAMTS1 KTFR peptide with the LAP‐TGF‐β LKSL peptide. Down‐regulation of ADAMTS1 in HSCs decreases the release of TGF‐β competent for transcriptional activation, and KTFR competitor peptides directed against ADAMTS1 block the HSC‐mediated release of active TGF‐β. Using a mouse liver fibrosis model, we show that carbon tetrachloride treatment induces ADAMTS1 expression in parallel to that of type I collagen. Importantly, concurrent injection of the KTFR peptide prevents liver damage.

An MLCK‐dependent window in late G1 controls S phase entry of proliferating rodent hepatocytes via ERK‐p70S6K pathway

We show that MLCK (myosin light chain kinase) plays a key role in cell cycle progression of hepatocytes: either chemical inhibitor ML7 or RNA interference led to blockade of cyclin D1 expression and DNA replication, providing evidence that MLCK regulated S phase entry. Conversely, inhibition of RhoK by specific inhibitor Y27632 or RhoK dominant‐negative vector did not influence progression in late G1 and S phase entry. Inhibition of either MLCK or RhoK did not block ERK1/2 phosphorylation, whereas MLCK regulated ERK2‐dependent p70S6K activation. In addition, DNA synthesis was reduced in hepatocytes treated with p70S6K siRNA, demonstrating the key role played by the kinase in S phase entry. Interestingly, after the G1/S transition, DNA replication in S phase was no longer dependent on MLCK activity. We strengthened this result by ex vivo experiments and evidenced an MLCK‐dependent window in late G1 phase of regenerating liver after two‐thirds partial hepatectomy. In conclusion, our results underline an MLCK‐dependent restriction point in G1/S transition, occurring downstream of ERK2 through the regulation of p70S6K activation, and highlighting a new signaling pathway critical for hepatocyte proliferation. (HEPATOLOGY 2006;44:152–163.)

The knock-down of ERCC1 but not of XPF causes multinucleation.

Excision repair cross complementing gene 1 (ERCC1) associated with xeroderma pigmentosum group F (XPF) is a heterodimeric endonuclease historically involved in the excision of bulky helix-distorting DNA lesions during nucleotide excision repair (NER) but also in the repair of DNA interstrand crosslinks. ERCC1 deficient mice show severe growth retardation associated with premature replicative senescence leading to liver failure and death at four weeks of age. In humans, ERCC1 is overexpressed in hepatocellular carcinoma and in the late G1 phase of hepatocyte cell cycle. To investigate whether ERCC1 could be involved in human hepatocyte cell growth and cell cycle progression, we knocked-down ERCC1 expression in the human hepatocellular carcinoma cell line Huh7 by RNA interference. ERCC1 knocked-down cells were delayed in their cell cycle and became multinucleated. This phenotype was rescued by ERCC1 overexpression. Multinucleation was not liver specific since it also occurred in HeLa and in human fibroblasts knocked-down for ERCC1. Multinucleated cells arose after drastic defects leading to flawed metaphase and cytokinesis. Interestingly, multinucleation did not appear after knocking-down other NER enzymes such as XPC and XPF, suggesting that NER deficiency was not responsible for multinucleation. Moreover, XPF mutant human fibroblasts formed multinucleated cells after ERCC1 knock-down but not after XPF knock-down. Therefore our results seem consistent with ERCC1 being involved in multinucleation but not XPF. This work reveals a new role for ERCC1 distinct from its known function in DNA repair, which may be independent of XPF. The role for ERCC1 in mitotic progression may be critical during development, particularly in humans.

The complexity of ERK1 and ERK2 MAPKs in multiple hepatocyte fate responses

Recent reports suggest that extracellular signal‐regulated kinase (ERK1) and ERK2 mitogen‐activated protein kinases (MAPK) may direct specific biological functions under certain contexts. In this study, we investigated the role of early and sustained epidermal growth factor (EGF) stimulation on long‐term hepatocyte differentiation and the possible role of ERK1 and ERK2 in this process. We demonstrate a long‐term survival and an elevated level of differentiation up to 3 weeks. The differentiation state of hepatocytes is supported by sustained expression of aldolase B, albumin, and the detoxifying enzymes CYP1A2, 2B2, and 3A23. Similarly to freshly isolated cells, cultured hepatocytes also retain the ability to respond to 3‐methylcholanthrene (3MC) and phenobarbital (PB), two known CYP inducers. In addition, we show evidence that continuous MAPK/ERK kinase (MEK) inhibition enhances the level of differentiation. Using RNA interference approaches against ERK1 and ERK2, we demonstrate that this effect requires both ERK1 and ERK2 activity, whereas the specific ERK1 knockdown promotes cell survival and the specific ERK2 knockdown regulates cell proliferation. In conclusion, we demonstrate that early and sustained EGF stimulation greatly extends long‐term hepatocyte survival and differentiation, and that inhibition of the ERK1/2 MAPK pathway potentiates these pro‐survival/pro‐differentiation phenotypes. We clearly attest that specific ERK1 and ERK2 MAPKs determine hepatocyte survival and proliferation, respectively, whereas dual inhibition is required to stabilize a highly differentiated state. J. Cell. Physiol. 227: 59–69, 2012.