Potential conflict of interest: Nothing to report.
Chronic hepatitis B virus (HBV) infection is a major cause of liver fibrosis, eventually leading to cirrhosis and hepatocellular carcinoma. Although the involvement of the X protein of HBV (HBx) in viral replication and tumor development has been extensively studied, little is known about its possible role in the development of fibrosis. In this work we show that expression of HBx in hepatocytes results in paracrine activation and proliferation of hepatic stellate cells (HSCs), the main producers of extracellular matrix proteins in the fibrotic liver. Both human primary HSCs and rat HSCs exposed to conditioned medium from HBx-expressing hepatocytes showed increased expression of collagen I, connective tissue growth factor, α smooth muscle actin, matrix metalloproteinase-2, and transforming growth factor-β (TGF-β), together with an enhanced proliferation rate. We found that HBx induced TGF-β secretion in hepatocytes and that the activation of HSCs by conditioned medium from HBx-expressing hepatocytes was prevented by a neutralizing anti-TGF-β antibody, indicating the involvement of this profibrotic factor in the process. Conclusion: Our results propose a direct role for HBx in the development of liver fibrosis by the paracrine activation of stellate cells and reinforce the indication of antiviral treatment in patients with advanced HBV-related chronic liver disease and persistent liver replication. (HEPATOLOGY 2008.)
Cirrhosis is the end stage outcome of liver fibrosis and is highly related to the development of hepatocellular carcinoma (HCC).1, 2 Over a hundred million patients are affected worldwide by cirrhosis, being the most common non-neoplastic cause of death in the United States.1 In addition, 10,000 deaths occur in the United States every year due to HCC derived from cirrhotic livers.2, 3 Liver fibrosis results from chronic liver damage in conjunction with inflammation and extracellular matrix (ECM) production. The central mechanistic event of hepatic fibrosis is the activation, proliferation, and migration of HSCs, the predominant cell type responsible for ECM deposition.1, 2, 4 Activated HSCs display increased production of ECM components and profibrotic factors, such as collagen I, connective tissue growth factor (CTGF), and matrix metalloproteinase (MMP)-2, and show a conversion into proliferative and contractile myofibroblasts.1, 4–7 HSCs can be activated by a variety of cytokines and growth factors, including tumor necrosis factor alpha (TNFα), platelet-derived growth factor (PDGF), and transforming growth factor (TGF-β), which activates the Smad signaling pathway and stimulates ECM production and perpetuation of HSCs activation.1, 8
Development of cirrhosis and HCC is strongly associated with chronic hepatitis B virus (HBV) infection.9–11 HBV is a hepatotropic, partially double-stranded DNA virus, whose genome presents four overlapping open reading frames, coding for the hepatitis B virus surface antigen, the hepatitis B virus core protein, and the viral reverse DNA polymerase and the X protein (HBx).12, 13 The viral regulatory protein HBx exhibits pleiotropic biological effects, being able to modify different cellular functions through the regulation of a wide variety of signaling pathways.14 In the cytoplasm, HBx induces calcium mobilization and activates different mitogen-activated protein kinase pathways, including those of extracellular-regulated kinase, Jun N-terminal kinase, and p38,15–17 leading to the activation of a number of transcription factors, including nuclear factor of activated T cells, nuclear factor kappa B, or activating protein-1 (AP-1).14, 18 In addition, HBx associates with the mitochondria and regulates the response to oxidative stress and apoptosis.19, 20 In the nucleus, HBx interacts with components of the transcription machinery, including transcription factors (early growth response factor-1 [Egr-1], cyclic adenosine monophosphate-response element binding protein/activating transcription factor) and RNA-polymerase-associated factors (TATA-binding protein, human RNA polymerase II subunit 5 [RPB5]).21–25 Activation of the different cellular signaling cascades by HBx results in the transcriptional activation of a variety of genes involved in the immune response, such as TNF-α, nitric oxide synthase 2, interleukin 8, or intercellular adhesion molecule 1, and tumor development, including cyclooxygenase-2, membrane type-1 matrix metalloproteinase (MT1-MMP), vascular endothelial growth factor, c-Myc, or TGF-β1, among others.21, 26–28
Although the role of HBx in viral replication and the development of HCC has been thoroughly studied,14, 27, 29–32 its contribution to liver fibrosis is poorly understood. In this work, we describe for the first time the ability of HBx to induce paracrine HSC activation and proliferation. We show that expression of HBx in hepatocyte cell lines results in the secretion of TGF-β and that this profibrotic cytokine is necessary for the activation and increased proliferation of HSCs induced by HBx-expressing hepatocytes.
α-SMA, alpha-smooth muscle actin; AP-1, activating protein-1; CTGF, connective tissue growth factor; DDR-2, discoidin domain receptor 2; DMEM, Dulbecco's modified Eagle's medium; DX, dexamethasone; ECM, extracellular matrix; Egr, early growth response factor; ELISA, enzyme-linked immunoabsorbent assay; FBS, fetal bovine serum; HBV, hepatitis B virus; HBx, hepatitis B virus X protein; HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; MMP, matrix metalloproteinase; mRNA, messenger RNA; MT1-MMP, membrane type matrix metalloproteinase 1; PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; PI3K, phosphoinositide 3 kinase, RPB5, human RNA polymerase II subunit 5; RT, reverse transcriptase; SE, standard error; Smad, small mothers against decapentaplegic; TGF-β, transforming growth factor-beta; TIMP, tissue inhibitor of metalloproteinase; TNFα, tumor necrosis factor-alpha.
Materials and Methods
Cell Lines, Primary Cells, and Culture.
We grew the hepatocyte cell lines Chang Liver (CHL; American Type Culture Collection #CCL-13) and HepG2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM streptomycin, 5 mM L-glutamine, and 100 U/mL penicillin. We generated CMX and CMO cells by stably transfecting CHL cells with the p-mouse mammary tumor virus-X or p-mouse mammary tumor virus-CAT plasmids, which drive expression of the HBV transactivator HBx or chloramphenicol acetyltransferase, respectively, in a dexamethasone (DX)-inducible manner (Fig. 1; Supplementary Fig. 1A). HBx is also expressed in lower amounts in CMX cells in the absence of DX.31, 32 Although we selected stable transfectants with hygromycin, we performed all experiments in the absence of the antibiotic 2.2.15 cells, which bear two head-to-tail copies of the HBV genome, derived from HepG2 cell line.33 We grew human primary hepatic stellate cells from human resection specimens (Sciencell, San Diego, CA)34 in DMEM 2% FBS and used for up to four passages. For experiments, we plated cells at a density of 50,000 cells/cm2 in DMEM 10% FBS and shifted to DMEM 2% FBS 24 hours later. We used the cells during the first two to three passages. The rat HSC line CFSC-2G was kindly provided by Dr. Solís Herruzo (Hospital Doce de Octubre, Madrid, Spain).35 We cultured rat HSCs in DMEM 10% FBS, 2 mM streptomycin, 5 mM L-glutamine, and 100 U/mL penicillin.
HSC Activation and Proliferation.
We grew hepatocyte cell lines in DMEM 10% FBS until 60% confluence and then shifted them to DMEM 0% FBS, for 48 hours. Where indicated, we added 1 μM DX to the culture. We grew HSCs in DMEM 10% FBS for 18 hours, shifted them to DMEM 0% FBS for 24 hours, and incubated them in conditioned media from hepatocyte cell lines for 24 hours. We used HSCs grown in the presence or absence of 10% FBS as positive and negative controls for HSC activation, respectively.
To determine the HSC proliferation rate, we seeded 6 × 105 HSCs in 10% FBS-containing medium until they attached to the plate, shifted them to 0% FBS medium for 18 hours, and incubated them for further 24 hours in conditioned medium from the different hepatocyte cell lines. Cells were detached and counted using the trypan blue-exclusion method. We confirmed results using the MTT Cell Proliferation Kit (Sigma, St. Louis, MO). For the neutralization of profibrotic factors, we added neutralizing antibodies against TGF-β1, TGF-β2, TGF-β3 (4 μg/mL), TNFα (0.03 μg/mL), or PDGF (6 μg/mL), an isotype-matched negative control (4 μg/mL), or recombinant human tissue inhibitor of metalloproteinase (TIMP)-2 (2.2 nM; all from R&D Systems, Minneapolis, MN) to the HSC culture together with the conditioned medium from the hepatocytes.
Western Blot, Zymography, and Enzyme-Linked Immunoabsorbent Assay.
We lysed HSCs stimulated with hepatocyte-conditioned media for 24 hours in 2× Laemmli buffer, sonicated them, and we removed insoluble debris by centrifugation at 9500g for 5 minutes. We resolved equal amounts of total protein by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS/PAGE), and we performed western blot as described27 using antibodies raised against CTGF (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA), collagen I (1:5,000; generously provided by Dr. Detlef Schuppan, Universität Erlangen-Murnberg), MMP-2 (0.4 μg/mL, R&D Systems), alpha-smooth muscle actin (α-SMA), and α-tubulin (1:5,000; Sigma). We quantified blots using Image J (National Institutes of Health, Bethesda, MD) and normalized the expression of the different molecules to that of α-tubulin for each cell lysate. We performed zymographies essentially as described by separating equal amounts of total HSC extracts in a 10% polyacrylamide gel containing 1% gelatin.27
For TGF-β enzyme-linked immunoabsorbent assay (ELISA), we grew equal cell numbers of CHL, CMX, HepG2, and 2.2.15 cells in DMEM 10% FBS for 24 hours and then shifted them to DMEM 0% FBS, stimulated or not with 1 μm DX. After 24 hours, we centrifuged cell supernatants at 12,000g for 5 minutes and analyzed the amount of active TGF-β in the supernatant by ELISA as recommended by the manufacturer (R&D Systems). For the analysis of collagen I secretion, we incubated human HSCs with supernatant from the different hepatocyte cell lines for 24 hours and we determined the amount of collagen I in the culture medium by ELISA (Takara Bio Inc., Japan). We carried out experiments in duplicate and analyzed at least three different supernatant batches.
Immunofluorescence and Flow Cytometry.
We grew primary human HSCs and rat HSCs on poly-L-lysine–coated coverslips in DMEM 10% FBS for 18 hours, shifted them to 0% FBS for 24 hours, and stimulated them for a further 24 hours with conditioned media from the hepatocyte cell lines. After 24 hours, we fixed the cells in 4% paraformaldehyde, permeabilized them for 10 minutes with 0.1% Nonidet P40 in phosphate buffered saline and we prevented nonspecific binding by incubating for 30 minutes in TNB (0.1 M Tris-HCl pH 7.5, 0.15 M NaCl, 0.5% blocking reagent) (Roche, Basel, Switzerland). We incubated the cells sequentially with anti-α-SMA (1:500; Sigma) and an anti-mouse-Alexa488 secondary antibody (1:250; Invitrogen) for 40 minutes each. We mounted samples using Mowiol polyvinyl alcohol containing 1 μg/mL 4′,6-diamidino-2-phenylindole. For flow cytometry, we stimulated rat HSCs with the different conditioned media as above, trypsinized them, and fixed them in 4% paraformaldehyde. We permeabilized cells with 0.1% Nonidet P40 in phosphate buffered saline, blocked them with TNB, and incubated them with an anti-α-SMA antibody followed by an anti-mouse fluorescein isothiocyanate secondary antibody. We performed analysis using a FACScalibur (Becton-Dickinson, Franklin Lakes, NJ).
RNA Extraction and Polymerase Chain Reaction.
We extracted total RNA using an RNA isolation kit (Ultraspec RNA; Biotexc Laboratories, Houston, TX), according to the manufacturer's instructions. We reverse-transcribed a total of 4 μg of total RNA into complementary DNA using the GeneAmp Gold RNA Polymerase Chain Reaction (PCR) Core Kit (PerkinElmer, Foster City, CA.). We amplified HBx messenger RNA (mRNA) for 35 cycles using the following primers and conditions: sense 5′-CACCTCTCTTTACGCGGTCT-3′, antisense 5′-CCTTGAGGCCTACTTCAAAG-3′, 95°C for 30 seconds; 56°C for 45 seconds, and 72°C for 1 minute. We used primers for β-Actin (sense 5′-CCCAGAGCAAGAGAGG-3′, antisense 5′-GTCCAGACGCAGGATG-3′) to confirm both the integrity and equal loading of each complementary DNA sample. We analyzed expression of collagen I (COL1A1), CTGF, TGFB1, MMP2, TIMP1, and ACTA2/α-SMA using Taqman probes (Applied Biosystems, Foster City, CA). We amplified the hygromycin resistance gene by PCR for 30 cycles (95°C for 30 seconds; 57°C for 30 seconds, and 72°C for 30 seconds) using the following primers: sense 5′-GTAAATAGCTGCGCCGATGG-3′, antisense 5′-GGAGATGCAATAGGTCAGGC-3′.
HBx Expression in Hepatocytes Induces Paracrine Activation of HSCs.
Liver fibrosis is characterized by the production of collagen I, CTGF, and MMP-2 by activated HSCs.1, 5 To determine whether HBx expression in hepatocytes could result in the paracrine activation of HSCs, we used an assay in which we studied the presence of these activation markers in HSCs after their exposure to conditioned media from hepatocyte cells expressing HBx (CMX cells) or the whole viral genome (2.2.15 cells). We analyzed the expression of collagen I and CTGF by western blot in protein extracts from human primary HSCs and the rat HSC line CFSC-2G grown in the presence of conditioned medium from the different hepatic cell lines. Western blot analysis showed that incubation of both human and rat HSCs with conditioned medium from CMX cells increased their expression of collagen I and CTGF, compared to those incubated with conditioned medium from the parental cell line CHL (Fig. 2) or the mock transfectant (CMO; Supplementary Fig. 1B). Incubation of HSCs with medium from DX-treated cells resulted in collagen I repression due to the action of the glucocorticoid itself on collagen I synthesis (our unpublished data and Ref.36) and thus we did not use it in these experiments. Medium from 2.2.15 cells, which harbor the whole HBV genome, induced collagen I and CTGF expression in HSCs, when compared to the parental HepG2 cells (Fig. 2). Moreover, we detected an increase in collagen I secretion in the culture medium of HSCs grown in the presence of supernatant from HBx-expressing cells (Fig. 2G; Supplementary Fig. 1C). Interestingly, we were unable to detect collagen I in the culture supernatant of the hepatocyte cell lines (data not shown), indicating that the source of the secreted collagen I are the activated HSCs. To further confirm the activation state of HSCs after exposure to HBx-induced factors, we analyzed the expression of α-SMA, a main sign of HSC activation, by immunofluorescence (Fig. 3A), western blot (Fig. 3B,C), quantitative reverse-transcriptase PCR (Fig. 3D; Supplementary Fig. 1E), and flow cytometry (Fig. 3E). Conditioned medium from CMX cells, DX-stimulated CMX cells, and 2.2.15 cells was able to induce both α-SMA protein and mRNA expression in HSCs, compared to CHL cells, treated or not treated with DX, and HepG2 cells, respectively (Fig. 3).
Moreover, human primary HSCs stimulated with conditioned medium from CMX and 2.2.15 cells showed increased expression of the active form (62 kDa) of MMP-2 by gel zymography (Fig. 4A). We observed further increase in active MMP-2 expression after exposure of HSCs to conditioned medium from dexamethasone-stimulated CMX cells, which express higher amounts of HBx (Fig. 1). We obtained similar findings when we quantified MMP-2 expression by western blot (Fig. 4B). Interestingly, we also observed an upregulation of TGF-β expression by HSCs after culturing them in the presence of supernatants from HBx-expressing cells CMX and 2.2.15, compared to their respective parental cell lines CHL and HepG2 (Fig. 4C,D; Supplementary Fig. 1B). Again, conditioned medium from DX-stimulated CMX cells, but not CHL cells, enhanced the expression of TGF-β by HSCs. We corroborated the activation status of HSCs exposed to soluble factors from HBx-expressing hepatocytes by analyzing the expression of a variety of activation markers in HSCs stimulated with conditioned medium from the different hepatocyte cell lines by quantitative reverse-transcriptase PCR. As shown in Fig. 5, exposure of HSCs to conditioned medium from CMX (Fig. 5A) or 2.2.15 (Fig. 5B) cells resulted in significant increase of collagen I (COL1A1), CTGF, TGF-β1, MMP-2, TIMP-1, and α-SMA (ACTA2) mRNAs, compared to CHL and HepG2, respectively. Taken together, these results strongly suggest that HBx is capable of inducing the secretion of soluble mediators that result in the paracrine activation of HSCs.
HSCs Show Increased Proliferation in Response to Conditioned Medium from HBx-Expressing Cells.
HSC proliferation is another feature of liver fibrosis.1 To determine whether HBx could contribute to the progression of the disease by increasing the number of HSCs in addition to their activation, we analyzed the proliferative capacity of HSCs in the presence of the conditioned media from the different hepatic cell lines. As shown in Fig. 6, conditioned medium from CMX and 2.2.15 cells had a positive effect on HSC proliferation, compared to CHL or HepG2 cells. Furthermore, HSCs grown in the presence of conditioned medium from DX-stimulated CMX cells showed a higher proliferation rate than that obtained with unstimulated CMX, CMO, or CHL cells (Fig. 6; Supplementary Fig. 1F), suggesting that the proliferative response in the HSCs is enhanced as the HBx expression in the hepatocytes increases. Our results demonstrate that HBx-expressing hepatocytes secrete soluble factors capable of inducing both HSC activation and proliferation.
TGF-β Mediates the Paracrine Activation of HSCs by HBx-Expressing Hepatocytes.
To identify the soluble factors responsible for the paracrine activation of HSCs by HBx-expressing hepatocytes, we incubated HSCs with conditioned media from the different hepatic cell lines in the presence of neutralizing antibodies directed against major profibrotic factors, including TGF-β, PDGF, and TNFα. The anti-TGF-β blocking antibody prevented the increase in collagen I and CTGF expression in both human (Fig. 7A,B) and rat HSCs (Fig. 7C,D) exposed to CMX-conditioned and 2.2.15-conditioned medium. Incubation with anti-TNFα, anti-PDGF, or a control antibody had little effect on the expression of these markers by HSCs (Fig. 7A,B). Moreover, neutralization of TGF-β activity prevented the induction of MMP-2 expression in HSCs stimulated with conditioned medium from CMX, CMX/DX, and 2.2.15 cells (Fig. 7E), confirming the involvement of TGF-β in the paracrine activation of HSCs by HBx.
To determine whether HBx was inducing the secretion of TGF-β in hepatocytes, we analyzed the culture supernatant of the different hepatocyte cell lines by ELISA. CMX and 2.2.15 cells showed increased TGF-β secretion compared to their control cell lines, CHL, CMO, and HepG2, respectively (Fig. 7F; Supplementary Fig. 1D). Further induction of HBx expression in CMX cells by treatment with dexamethasone resulted in increased TGF-β production by these cells, indicating that increased HBx expression results in enhanced TGF-β secretion.
To study whether TGF-β was also mediating the paracrine enhancement of HSC proliferation induced by HBx, we performed HSC proliferation experiments in the presence of neutralizing antibodies directed against TGF-β, PDGF, or TNFα. Incubation of HSCs with the anti-TGF-β blocking antibody abolished the increase in the proliferation rate induced by conditioned medium from 2.2.15 and DX-treated CMX cells (Fig. 8A,B; black bars), whereas it had no effect on basal HSC proliferation. In contrast, neutralizing antibodies raised against PDGF and TNFα showed little or no effect on HSC proliferation (Fig. 8A; hatched and gray bars), indicating that TGF-β activity was necessary for the paracrine effect of HBx on HSC activation and proliferation. Interestingly, blockade of MMP-2 activity with recombinant TIMP-2 prevented the induction of HSC proliferation by supernatants from HBx-expressing cells (Fig. 8C), suggesting the involvement of this metalloproteinase in the enhancement of HSC proliferation by HBx.
Chronic infection by HBV is a major cause of liver fibrosis and eventually leads to the development of cirrhosis and HCC.9 Although the active involvement of HBx in viral replication and the progression of the disease toward HCC have been well documented,14, 27, 29–32, 37, 38 no direct evidence has been provided for the role of this viral transactivator in the development of liver fibrosis. In this work, we present different lines of evidence supporting the ability of HBx to induce paracrine HSC activation and proliferation, suggesting a direct implication for the virus in the development and progression of liver fibrosis.
Fibrosis is the result of repeated injury and wound healing cycles in which the hepatocytes are eventually substituted by extracellular matrix (ECM), particularly collagen, secreted by HSCs in response to profibrotic stimuli.1, 2 In this regard we observed that HSCs incubated in the presence of conditioned medium from HBx-expressing hepatocytes increased their expression of collagen I and α-SMA. This effect was accompanied by induction of CTGF and MMP-2, which are known to mediate some of the profibrotic effects of TGF-β, including the induction of HSC proliferation, adhesion, and collagen synthesis.39 Thus, although HBx may not be able to induce the synthesis of ECM by the infected hepatocytes, it promotes the release of soluble mediators capable of activating HSCs to produce ECM components and related factors, thereby contributing to the development of fibrosis in HBV chronically infected patients. Interestingly, similar results have been reported for hepatocytes bearing the hepatitis C virus nonstructural genes 3 to 5a, which were able to induce the paracrine activation of HSCs in a TGF-β-dependent manner.40
HSCs grown in the presence of conditioned medium from HBx-expressing cells showed increased proliferation, confirming the secretion of HSC-activating soluble factors by the hepatocytes carrying the viral transactivator. Whereas mild HBx expression in the hepatic cell lines was enough to increase collagen I and CTGF synthesis in HSCs, induction of MMP-2 expression and HSCs proliferation was best observed in the presence of conditioned medium from dexamethasone-stimulated CMX cells, which show the highest HBx levels. Interestingly, we have previously shown that HBx induces the expression of both MMP-2 and its activator MT1-MMP in hepatocytes.27 Since a direct link between MMP-2 expression and HSC proliferation has been reported41, 42 and the MMP-2 inhibitor TIMP-2 prevents the increase in HSC proliferation promoted by HBx (Fig. 8C), direct and paracrine induction of MMP-2 by the viral transactivator may contribute to the enhanced proliferation of HSCs.
The role of TGF-β as a major inducer of liver fibrosis has been well established.39 TGF-β production in hepatocytes is known to be induced by HBx through the activation of the transcription factor Egr.21 In addition, HBx is also able to amplify the Smad signaling cascade in hepatocytes in response to TGF-β by direct binding to Smad homolog 4 (Smad4) and to protect the hepatocyte from the proapoptotic effects of TGF-β by activating the PI3K/Akt pathway.43, 44 Here we identify TGF-β as the main factor responsible for the paracrine activation of HSCs by HBx. Not only did HBx directly induce TGF-β secretion in hepatocytes, but it also indirectly activated HSCs to produce it, leading to perpetuation of HSC activation.1 Furthermore, blockade of TGF-β activity with a neutralizing antibody prevented the induction of collagen I, CTGF, and MMP-2, indicating the involvement of this profibrotic factor in the paracrine activation of HSCs by HBx. Interestingly, anti-TGF-β antibodies were also able to block the paracrine stimulation of HSC proliferation promoted by HBx in both human and rat HSCs. TGF-β has been described to have a negative effect on the proliferation of freshly isolated HSCs, an effect that is rapidly lost after initial cell culture.45 Although our human primary cells were used for very few passages, they were not freshly isolated and thus a positive effect of the anti-TGF-β antibody on HSC proliferation was unlikely. Indeed, anti-TGF-β prevented the increase in HSC proliferation induced by HBx, but had no effect on basal HSC proliferation. Different factors may account for the effect of anti-TGF-β antibodies on HBx-induced HSC proliferation. First, it should be noted that HBx-expressing hepatocytes secrete a variety of growth factors apart from TGF-β that may have an impact on HSC proliferation,26, 27 and thus the net effect of neutralizing TGF-β activity is not necessarily comparable to the effect of adding or not recombinant TGF-β to quiescent HSCs. Furthermore, TGF-β regulates the expression of soluble factors that are mitogenic for HSC, such as CTGF or collagen I, which induces HSC proliferation by activating discoidin domain receptor-2 (DDR-2),41 and therefore inhibition of TGF-β activity could result in an impaired proliferative response. Finally, TGF-β is necessary for the paracrine induction of MMP-2 expression by HBx. Since the increase in HSC proliferation induced by the viral transactivator is MMP-2-dependent, as shown by the inhibitory effect of TIMP-2, TGF-β may indirectly influence cell proliferation by regulating the expression of MMP-2.
It is noteworthy that the profibrogenic effect of HBx described in this work takes place through a paracrine action on HSCs activation and proliferation. Taken together, our results provide evidence of the effect that HBx expression in hepatocytes has on surrounding cells, particularly HSCs, and suggests that the HBx viral transactivator contributes actively to the development of fibrosis in patients chronically infected by HBV. Regarding practical application in the clinical setting, our findings reinforce the indication of antiviral treatment, mainly with nucleoside/nucleotide analogs, even in patients with advanced HBV-related chronic liver disease and persistent liver replication.
We thank Dr. D. Shuppan, Dr. F. Sánchez-Madrid, and Dr. J.A Solís-Herruzo for providing critical reagents for the completion of this work. We also thank Dr. R. Bataller and Dr. P. Sancho-Brau for their help during the first steps of the project.