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Activation of hepatic stellate cell in Pten null liver injury model
© The Author(s). 2016
Received: 19 January 2016
Accepted: 31 May 2016
Published: 14 June 2016
Hepatic fibrosis is a prominent pathological feature associated with chronic liver disease including non-alcoholic hepatosteatosis (NASH), and a precursor for liver cancer development. We previously reported that PTEN loss in the liver, which leads to hyperactivated liver insulin signaling results in NASH development. Here we used the same mouse model to study the progression from steatosis to fibrosis.
The Pten null livers develop progressive liver fibrosis as indicated by Sirius Red staining and increased expression of collagen I, Timp 1, SMAα, and p75NTR. Consistently, hepatic stellate cells (HSCs) isolated from Pten null livers are readily activated when compared with that from mice with intact PTEN. Deletion of AKT2, the downstream target of PTEN signal, blocked NASH development, and alleviated fibrosis. HSCs from the Pten/Akt2 double null mice are quiescent like those isolated from the control livers. Our analysis shows that the activation of HSCs does not depend on the intrinsic signals regulated by PI3K/AKT, the target of PTEN, but does depend on steatosis and injury to the liver. During the progression of liver fibrosis in the Pten null model, Wnt ligands and signaling receptor are induced, concurrent with the reduction of sFRP5, a Wnt antagonist. We showed that treatment of HSCs with Wnt receptor antagonist blocks the observed morphological changes when HSCs undergo activation in culture. This signal appears to be mediated by β-catenin, as manipulating β-catenin signaling alters marker gene expressions of HSC activation.
Wnt/β-catenin activation serves as an important mediator for fibrosis development resulting from NASH using a mouse model where NASH is mimicked by PTEN loss.
Fatty liver disease (FLD) is the most prevalent form of chronic liver disease and has become a major pandemic in developed and some developing countries. FLD is characterized by lipid accumulation in the liver resulting from sedentary lifestyle, calorie-rich diet, or alcohol consumption. Without intervention, FLD can develop into non-alcoholic or alcoholic hepatosteatosis (NASH or ASH) where fatty liver is accompanied by infiltration of inflammatory cells. Left untreated, NASH and ASH can develop into fibrosis, a common late stage chronic liver disease and a significant risk factor for cancer .
The mechanisms responsible for this fibrosis development in NASH/ASH patients are not well studied but may involve injury-repair responses and accumulation of myofibroblasts. Myofibroblasts are thought to be the cells that lay down the collagens that compose the fibrotic tissue . Of the potential sources for these myofibroblasts, activation of hepatic stellate cells (HSCs) has gained strong support . Inactive HSCs are the main storage for vitamin A. Upon injury, HSCs activate by losing their vitamin A containing lipid droplets and gaining myofibroblast characteristics. This morphological alteration is associated with increased expressions of markers such as, collagen type I (Col1a1), smooth muscle actin α (SMAα), desmin, and nerve growth factor receptor (P75NTR). Multiple stimuli and pathways have been shown to stimulate the expression of these markers in HSCs. The most prominent among these are transforming growth factor β (TGF β) and platelet-derived growth factor (PDGF) .
To investigate the molecular signals involved in fibrosis resulting from fatty liver, we used a murine model where steatosis is induced as a result of hyperactivated insulin signal, a condition that commonly occurs in NASH patients. In this mouse model (hereafter referred to as Pten null mice), Pten (phosphatase and tensin homologue deleted on chromosome 10) is deleted in the albumin-positive cell population (Pten loxP/loxP ; Alb-Cre + ). In such mice, the liver PI3K/AKT pathway responsible for transmitting insulin signal is induced without the complication of peripheral insulin resistance and hyperglycemia . The Pten null mouse model thus mimics the liver signals occurring in NASH patients where hyperinsulinemia often drives lipid synthesis in the liver. In this model, we have previously observed significant injury to the liver and ultimately liver tumor development [5–7]. In the current study, we characterize the fibrosis development in Pten null mice and validated it as an appropriate model to study the contribution of NASH to fibrosis. In addition, we investigated the mechanisms underlying the steatosis contribution to fibrosis development using this model.
PTEN levels are lower in NASH patients
Pten null mice develop progressive liver fibrosis
To confirm these gene expression changes, we stained liver sections for SMAα. In 9-month-old Pten null mice, this staining is mostly observed in areas of severe fatty liver and the ductal plates (Fig. 4b). In 12-month-old mice, SMAα staining is distributed throughout the Pten null livers, whereas the Pten/Akt2 double null and control livers have little staining for SMAα at either age. The SMAα staining is further confirmed with immunoblotting analysis showing increased SMAα in 9- and 12-month-old mice (Fig. 4c). These observations show that Pten deletion leads to progressive and severe fibrosis that is recovered when AKT2 is simultaneously lost.
HSCs are activated in Pten null liver
The morphological differences between the two HSC cultures are more obvious after the attachment of HSCs at day 2 of culturing (Fig. 5d). The majority of the HSC-Con cells are still round-shaped quiescent HSCs retaining vitamin A autofluorescence, whereas most HSC-Pm cells acquired spindle morphology with very little vitamin A deposits, indicating that they are activated HSCs. HSC-Dm displayed similar quiescent phenotype and retained vitamin A autoflorescence. Like reported, HSC-Con gradually changed from the round-shaped quiescent HSC phenotype to the long, stretched, spindle phenotype that resembles activated HSC from days 3 to 7 (data not shown). By day 5, majority of the HSC-Con are activated and by day 7, all cells display the elongated spindle morphology. HSCs from Pten null mice changed very little morphologically as they already acquired the spindle morphology by day 2. The cells appear to shrink with time, likely due to increasing cell density with culturing (data not shown). Consistent with the morphological changes occurring in HSC-Con culture, expressional analysis of Col1a1, Timp 1, and SMAα increased with culturing (data not shown).
Activation of HSC in Pten null mice depends on injury but not intrinsic PI3K/AKT activation
To further confirm this observation, we treated rat HSC cell line with insulin growth factor (IGF-1) to induce PI3K/AKT. In these cells, overnight starvation led to moderate downregulation of p-AKT. When IGF-1 is added, AKT phosphorylation is induced both 15 min and 1 h after the start of the treatment (Fig. 6b). Phosphorylation of AKT starts to recover after 1 h (data not shown). We evaluated the expression levels of SMAα, Col1a1, Timp 1, and desmin (Fig. 6c). The expression of most fibrotic genes did not alter significantly when p-AKT is induced in the IGF-1 treated HSCs with the exception of desmin expression. At 15 min, we observed a significant downregulation of desmin whereas this level increased after 1 h of treatment. On the other hand, treatment with PDGF and TGFβ both induced migration of the cultured HSCs but did not show significant increase on p-AKT (Fig. 6d–f). These data, together with the moderate change observed for PTEN and p-AKT expression in HSCs isolated from Pten null livers (Fig. 3a), indicate that the intrinsic signal due to PTEN loss in HSCs is an unlikely mechanism for their activation in the injured Pten null liver and that fibrosis is likely due to secondary effects, e.g., resulting from the underlying liver injury.
To test this in vivo, we induced injury in the Pten/Akt2 double null mice where injury does not occur spontaneously. Inducing injury with DDC in these mice led to robust expression of markers for fibrosis (Fig. 6g), including Col1a1, Timp 1, and SMAα. Expression of desmin and p75 NTR is also moderately induced by treatment of DDC. This observation suggests that the induction of these genes do not rely on the signals of AKT2 but do require injury.
Wnt signaling mediates HSC activation in injured steatotic Pten null liver
Finally, we used a small molecule inhibitor of Wnt/ β-catenin, ICG-001, which specifically blocks the interaction of β-catenin with its coactivator CBP to inhibit β-catenin transcriptional activity in vivo. Fibrosis in the Pten null mice becomes obvious after 6 months of age. To advance this onset of fibrosis and induce the expression of Col 1a1, desmin, and Timp 1, markers for fibrosis, we used a cohort of 1.5-month-old mice with DDC (Fig. 9g). In these DDC-treated mice, ICG-001 was given to inhibit Wnt/CBP/ β-catenin transcription [19, 20]. With the exception of Timp 1, expression of all fibrogenic markers are downregulated with ICG-001 to different extent including SMAα and p75 NTR, of which the expression did no increase with DDC treatment. Expression of Col1a1, particularly, is significantly reduced whereas the change in other makers did not reach significance. While not all markers responded in a synchronized manner to LiCl, curcumin, si-β-catenin, or ICG-001, our data is consistent with a pro-fibrogenic role of Wnt in the liver. The lack of synchrony is likely due to the nature of the markers as indicators of HSC activation rather than drivers of fibrosis. Depending on the stage of activation/differentiation of HSCs, different markers are expressed, leading to the varied response to Wnt signal-induced expression changes.
Hepatic fibrosis is a pathological condition that follows chronic liver disease, including NASH and ASH, and is a precursor to liver cancer development. In this study, we explored the molecular link between NASH and fibrosis. We demonstrated that Pten null model is a relevant model for studying fibrosis development because (1) liver disease progresses similarly in humans and Pten null mice, (2) the Pten null mice develop progressive and severe fibrosis from 6 months on, and (3) HSCs implicated in fibrosis development in humans are also activated in Pten null mice. Enhanced expression of Col1a1, Timp 1, SMAα, and p75NTR suggests the involvement of HSCs. HSCs isolated from Pten null liver are fully activated with spindle morphology and low vitamin A autofluorescence, confirming the severe fibrosis phenotype that was observed in vivo.
After demonstrating the relevance of the Pten null mice model, we utilized these mice to investigate the signaling pathways that drive fibrosis development. We found that (1) activation of HSC is associated with steatosis and injury but not directly caused by alterations of PTEN/PI3K/AKT signaling, (2) blocking Wnt signaling significantly attenuated the ability of the quiescent HSCs to become activated, and (3) activation of Wnt signaling induces genes associated with activated HSCs. Taken together, (1), (2), and (3) suggest that Wnt activation resulting from steatosis induced liver injury activates HSCs and thus induces hepatic fibrosis.
In humans, fibrosis occurs in patients with viral infections, ASH, and NASH, as well as biliary and other diseases that lead to injury of liver parenchymals . In animal experiments, fibrosis development is typically induced by inducing injury to liver parenchymals . Injuries are typically induced using physical approaches such as ductal ligation and chemical approaches such as carbon tetrachloride treatment, choline deficient diet, alcoholic feedings, or ligation of the bile duct to cause acid buildup to damage the liver parenchymal [22, 23]. A similarity among these models and also a significant clinical interest is damage to hepatocytes and attenuation of their proliferation. In this study, rather than directly injuring the animal subjects using physical or chemical means, we used a NASH model where fat accumulation in the liver is a consequence of hyperactivated insulin signal, mimicking human NASH conditions . Liver injury in the Pten null mice results from fatty liver disease much as obesity-induced fatty liver often leads to NASH in humans. We show here that NASH is accompanied by deposition of collagen fibers and activation of HSCs, and preventing NASH resolves these conditions. This study, thus, provides experimental proof that NASH does lead to fibrosis development.
AKT, the downstream kinase of PTEN, has been shown to control multiple fibrogenic genes in various tissue/cell types. In mouse skin, loss of PTEN induces the expression of fibrogenic genes . Sustained expression of wild type PTEN in cultured rat HSC inhibited morphological changes associated with HSC activation . Chronic activation of PI3K also induced accumulation of collagens [10, 11]. Thus, we were surprised to find that PTEN and AKT2 signals did not change significantly in HSCs isolated from the Pten null livers. (Pten deletion is liver-cell specific in Pten null mice. Pten is not deleted in HSCs.) Our observations suggest that the effect of AKT2 on fibrogenesis is likely secondary to its role in steatosis and the subsequent liver injury.
Wnt signaling has recently been reported to be important for HSC activation [13, 26]. Liver HSCs express a number of different receptors for Wnt . During culturing and activation of HSCs, the expression of Wnt receptors and ligands is induced . The Wnt antagonist DKK has been found to enhance the transcriptional activity of an adipogenic gene including peroxisomal proliferation activated receptor (PPARγ) . Upregulation of PPARγ maintains the lipid droplets and allows the HSCs to remain quiescent with many lipid droplets present in each cell whereas Wnt blocks DKK signal, allowing activation of HSCs [14, 26]. Our study shows that this signal is likely relevant to the steatosis-induced fibrosis as several Wnt ligands and receptors are induced and sFRP5 is inhibited in the injured Pten null liver . Whether Wnt activation is a direct consequence of steatosis or requires injury to occur remains to be determined.
In summary, our data established that the liver Pten null model displays progressive fibrosis prior to cancer development. The fibrosis is a result of steatosis induced by PTEN loss, suggesting that the liver-specific Pten null mouse is a relevant model for studying the progression of liver cancer co-developed with fatty liver injury-induced fibrosis. Our data further indicates that the Wnt signal pathway likely mediates steatosis-induced fibrosis.
Targeted deletion of Pten (Pten null) and Pten/Akt2 double mutant mice (Dm) were reported previously [5, 6]. Control (Con) animals are Pten loxP/loxP ; Alb-Cre − . Experiments were conducted according to IACUC guidelines of the University of Southern California. Fasting glucose were measured in overnight (16 h) fasted mice. 3,5-dietoxycarbonyl-1,4 dihydrocollidine (DDC, 0.1 % w/w diet) treatment was performed in 3-month-old mice for 5 weeks. Male animals of C57BL/6 and J129svj background from the same breeding colony were used for all experiments. For 1.5-month-old mice used for ICG-001 study, DDC (0.05 % w/w diet) were given in three doses using a 2-day off 1 day on protocol. ICG-001 (5 mg/kg per day in saline) is delivered using a mini-osmotic pump that dispenses at 1 μL/h delivery rate. All experiments were conducted according to IACUC guidelines of the University of Southern California (Protocol #11162).
Human liver samples
Human liver samples biopsied from fibrosis patients were obtained from Pennsylvania State University. All patient information was removed, and all experiments were conducted according to IRB guidelines of Pennsylvania State University.
Gene expression data base GSE37031 was downloaded from NCBI website. All patient information were reported in the original publication for the database . The data set was then analyzed for expressions of PTEN in NASH (n = 8) vs. non-NASH (n = 7) patients. Immunohistochemical staining images were obtained from the Human Protein Atlas website (http://www.proteinatlas.org/).
Rat HSCs  were cultured in 1 g/L glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), penicillin (50 units/ml), and streptomycin (50 μg/ml) at a 37 °C, 5 % CO2 incubator. For treatment with 0.1 μg/ml mIGF, 50 ng/ml PDGF, or 4 ng/ml TGFβ, cells were serum starved for 24 h.
Primary hepatic stellate cell isolation
HSCs were isolated using OptiPrep gradient ultracentrifugation . HSC fraction was collected from the medium and 1.034 interface of OptiPrep gradients, and cultured and morphological changes of HSCs are monitored using both light and fluorescent microscopy to monitor for vitamin A autofluorescence at indicated time points. RNAs were isolated for marker analysis to monitor the activation of HSCs. In experiment using lithium chloride (LiCl3), LiCl3 (10 mM) was added to the day 1 culture and incubated for 2 days before the collection of RNA. In experiments using curcumin, 10-μM curcumin was added to day 1 culture and RNAs were collected 2 days after the addition of curcumin. Frizzled-related protein 1 (sFRP1) (R&D systems, Minneapolis, MN) is used at 40 ng/ml added to day 3 HSC culture and morphology followed for 7 days.
Liver sections were stained with hematoxylin and eosin (H&E) for morphology and Sirius Red to visualize fibrotic fibers. Sirius Red staining is quantified using Image J. Three animals per groups were quantified, and three randomly chosen areas per animal were assessed. Anti-smooth muscle actin α (SMA α) (Sigma-Aldrich) was used in indirect immunohistochemistry to further confirm the buildup of extracellular matrix.
Total RNA (2 μg) from liver tissues was used for qPCR analysis for gene expression. Primers used are SMA α, desmin, Col1a1, and p75NTR as previously indicated . Primers for Timp 1 are: 5′-CAGTAAGGCCTGTAGCTGT GC 5′-CTCGTTGATTTCGGGGAAC. GAPDH were detected for internal controls.
Protein lysates (40 μg) were loaded from each sample for electrophoresis using polyacrylamide gels. Membranes were probed with antibodies for PTEN, p-AKT, p-GSK3β, and GSK3β (cell signaling). α-Actin (Sigma) protein expression is used as loading controls.
Data were subjected to Student’s t tests for two sample comparisons. In cases of more than two groups, multivariate ANOVA was used to determine the statistical differences followed by pairwise comparison using Fischer’s LSD test. p ≤ 0.05 is considered to be statistically significant. Data are presented as mean ± SEM.
This work was supported by R01CA154986-01, a pilot grant from the USC Liver Center program grant P30DK48522 as well as funding from Zumberg foundation (BLS). BLS also acknowledges NIDDK R21 DK075928-02 and R01DK084241-01. We would also acknowledge funding from NIH grants R01-AA020753 (to KA) and R24-AA12885 Non-parenchymal Liver Cell Core (to KA and JW). We also acknowledge support from USC Norris Comprehensive Cancer Center Support Grant P30 CA014089. NIH 1R01CA166161-01A1 and NIH 1R01 HL112638-01 are gratefully acknowledged (MK). We thank Dr. Bryan Stiles for his help with editing this manuscript.
LH, GJ, and BLS study concept and design and drafting of the manuscript. VM, JW and FF acquisition of data. ZP, KA, BLS and CBR analysis and interpretation of data; statistical analysis. MK provide key reagents. KA, CBR and BLS critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
All experiments were conducted according to IACUC guidelines of the University of Southern California (Protocol #11162). All patient information was removed, and all experiments were conducted according to IRB guidelines of Pennsylvania State University.
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