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Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and Akt phosphorylation

NEERAJ K. SAXENA^*, MARK A. TITUS, XIAOKUN DING^*, JEFFREY FLOYD, SHANTHI SRINIVASAN^*, SHANTHI V. SITARAMAN^* and FRANK A. ANANIA^*,1

^* Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA; and
Division of Gastroenterology, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA

¹ Correspondence: Room 248, 615 Michael Street Atlanta, GA 30322, USA. E-mail: fanania{at}emory.edu

SPECIFIC AIMS

The goals of our study were to demonstrate that 1) leptin is a multifunctional profibrogenic cytokine and acts to increase hepatic stellate cell (HSC) proliferation and inhibits HSC apoptosis, and 2) activation of the long-form of leptin receptor (OB-Rb) conveys cross-activation of both extracellular regulated kinase (Erk) as well as Akt, two key signal transducing elements that underscore profibrogenic biological properties of proliferation and apoptosis.

PRINCIPAL FINDINGS

1. Leptin increases DNA synthesis in hepatic stellate cells
Leptin treatment of HSCs resulted in significantly higher BrdU incorporation compared with untreated HSCs in culture (Fig. 1 ). PDGF, a potent positive control for HSC mitogenesis, resulted in a 6-fold increase in BrdU incorporation whereas leptin resulted in a 4-fold increase in HSC-BrdU incorporation compared with SF conditions alone. BrdU incorporation as a result of leptin treatment was not significantly different from HSC-BrdU incorporation in the presence of serum.

View larger version (23K): [in this window] [in a new window]   Figure 1. Leptin is mitogenic for hepatic stellate cells. A) Leptin treatment increased BrdU incorporation in HSCs. HSCs were incubated for 48 h in 10% FBS/DMEM and maintained for 24 h in SF-DMEM. PDGF (30 ng/mL) served as a positive control for HSC mitogenesis. Treatments were introduced for 24 h. BrdU was added (10 µM) and HSCs were allowed to grow for an additional 24 h. Incorporation of BrdU into newly synthesized DNA was assessed as relative light units and is shown as mean ± SE of 3 independent experiments performed in triplicate. *P < 0.01 compared with untreated control cells grown in SF media. U, untreated (0.01% FBS); L, rat recombinant leptin (100 ng/mL); P, PDGF-bb (30 ng/mL). B) Leptin increased the fraction of HSCs in S-phase of the cell cycle. HSCs were synchronized by serum starvation in a medium containing 0.1% serum for 24 h. Serum-starved cells were exposed to PDGF-bb (30 ng/mL), leptin (100 ng/mL), or SF media (0.1% FBS). After 24 h, distribution of HSCs in the cell cycle was determined by flow cytometry using PI-stained nuclei. Mean values ± SE are results of 3 independent experiments performed in triplicate. *P < 0.01 compared with serum-starved conditions.

2. Leptin increases cyclin D1 protein
Immunoblot analysis was performed with quantitative densitometry, demonstrating that leptin increased cyclin D1 protein. These data were not statistically different from PDGF or in the presence of serum. Both leptin and PDGF nearly doubled HSC-cyclin D1 content vs. SF conditions. These data demonstrate that leptin is a potent HSC mitogen. D-type cyclins play a critical role in HSC cell cycle progression, especially at early G1 phase.

3. Leptin suppresses HSC apoptosis in vitro
Three approaches were used to determine whether leptin reduced apoptotic activity and whether leptin could rescue stellate cells from pretreatment with known HSC apoptotic stimuli: cycloheximide and tumor necrosis factor-regulated apoptosis inducing ligand (TRAIL). Leptin statistically reduced caspase-3 activity when compared with serum-starved cells (P<0.05) and significantly reduced caspase-3 activity induced by cycloheximide (up to 66%). Complementary data from TUNEL staining confirmed that leptin resulted in a 50% reduction in the number of apoptotic bodies counted per high-power field compared with cycloheximide. DNA fragmentation analysis corroborated these findings (Fig. 2 ). Leptin abolished DNA fragmentation associated with absolute serum starvation (L, Fig. 2 ) and cycloheximide-induced DNA fragmentation (L+C, Fig. 2 ), just as the pan-caspase inhibitor zVAD-fmk inhibited DNA fragmentation regardless of treatment.

View larger version (53K): [in this window] [in a new window]   Figure 2. Cycloheximide or TRAIL induced DNA fragmentation in HSCs was abolished by leptin, and zVAD-fmk, the pan-caspase inhibitor. HSCs were treated with TRAIL or cycloheximide for 24 h followed by leptin or zVAD-fmk for 24 h. HSCs were harvested and DNA was extracted. Fragmented DNA was analyzed by electrophoresis across a 1.2% agarose gel containing 0.1% ethidium bromide. The experiment was performed 3 times. Lyophilized apoptotic U937 cells (treated with camptothecin) were used as positive control (+) for DNA fragmentation. U, absolute serum starvation; L, leptin in SF media; C, cycloheximide in SF media; L+C, leptin plus cycloheximide; Z+C, zVAD-fmk plus cycloheximide in SF media; T, TRAIL in SF media; L+T, TRAIL and leptin in SF media; Z+T, zVAD-fmk and TRAIL in SF media.

TRAIL, a physiological relevant HSC apoptotic stimulus, was used to induce cell death. Leptin significantly rescued HSCs from the apoptotic effect of TRAIL as assessed by XTT assay and DNA fragmentation analysis (Fig. 2) . These results are consistent with those from cycloheximide-induced apoptosis and demonstrate that leptin significantly improved cell viability even at supraphysiologic concentrations of TRAIL.

4. Leptin-induced proliferation of HSCs requires phosphorylation of OB-Rb
AG490, a chemical inhibitor of Jak2 kinase activity, and SOCS-3, a physiological inhibitor of OB-Rb phosphorylation, were used to determine whether leptin induced HSC proliferation. Either chemical inhibition by AG490 or overexpression of SOCS-3 abolished leptin-induced HSC proliferation as assessed by BrdU incorporation. Except for leptin-induced HSC proliferation, SOCS-3 alone, empty vector alone, or AG490 alone did not result in enhanced HSC proliferation over 0.1% FBS alone.

5. Leptin increases phosphorylation of both extracellular regulated kinase (ERK) and protein kinase B (Akt)
To determine potential intracellular signaling mechanisms responsible for the mitogenic and anti-apoptotic effects of leptin, immunoprecipitation of phosphorylated signal transduction factors (Erk and Akt) were examined. Additional studies were performed to examine whether leptin resulted in increased phosphorylation of Stat1, Stat 5, p38, or JNK over the same course. We failed to find significant increases in phosphorylation of Stats 1 and 5 or stress-activated protein kinases (SAPKs), p38, or JNK. Phosphorylation of Stat3 did occur in leptin-treated stellate cells. Leptin increased Erk and Akt phosphorylation, which was maximal at 3 h treatment and detected for up to 24 and 36 h.

6. Inhibition of OB-Rb phosphorylation by SOCS-3 abolishes leptin-induced phosphorylation of Akt and Erk needed for leptin induced proliferation
We determined whether inhibition of OB-Rb phosphorylation would prohibit Akt and Erk phosphorylation, and consequently determined whether leptin-mediated HSC proliferation was Akt and Erk dependent. Overexpression of SOCS-3 abolished phosphorylation of Akt and Erk, which was not restored by leptin. Conversely, the presence of leptin or leptin and the empty vector resulted in phosphorylation of both Akt and Erk.

CONCLUSIONS AND SIGNIFICANCE

We undertook a series of experiments using the established model of primary, activated stellate cells in tissue culture, and analyzed the influence of leptin on HSC mitogenesis and apoptosis. We have demonstrated that leptin promotes stellate cell mitogenesis and cell survival, two seminal events thought to promote liver fibrosis upon chronic stimuli resulting in liver injury. Previous and current data provide cogent cellular and molecular evidence that leptin plays a unique role in development of liver fibrosis. We report here that leptin-induced phosphorylation of the long-form of leptin receptor via Jak2 kinase activation results in phosphorylation of Erk and Akt, two key signal transduction elements associated with cell growth. Abolishing leptin-induced phosphorylation by SOCS-3 overexpression or pharmacologic inhibition prevents Erk and Akt phosphorylation, but SOCS-3 clearly blocks HSC proliferation. Leptin appears to promote liver fibrosis by activating known extracellular matrix response genes and to perpetuate the fibrogenic process by directly inducing stellate cell proliferation and resistance to a timely death by apoptosis.

These results support a critical role for leptin in liver fibrosis. Leptin has been found to increase gene expression associated with a net increase in liver extracellular matrix, and is now shown to promote HSC proliferation and inhibit HSC apoptosis. Leptin, therefore, perpetuates three critical biological events associated with an increase in extracellular matrix deposition during chronic liver injury. These results also provide molecular evidence to explain the absence of fibrosis in rodent-induced fibrosis models that lack leptin production or leptin signaling via OB-Rb.

These data substantiate that whereas multiple alternatively spliced leptin receptor isoforms exist, only the long-form (OB-Rb) initiates signaling. Our results are consistent with OB-Rb signal transduction resulting in Stat3 phosphorylation, which induces feedback inhibitor SOCS-3, as well as phosphorylation of Tyr₉₈₅, which results in activation of Erk via SH2 domain-containing adaptor protein (growth factor receptor binding-2). To our knowledge, our data represent the first report that leptin-mediated HSC signaling activates the PI3-kinase-Akt pathway, well established as a potent cell survival transduction cascade. These data not only support the multiplicity of leptin’s signaling capabilities, but extend it to the hepatic stellate cell. Since previous work regarding leptin signal transduction has focused on the central nervous system and adipose tissues, we propose that the hepatic stellate cell may be a useful model that may provide a better understanding of leptin signal transduction.

Recent reports link insulin receptor substrates as potential adaptor molecules for OB-Rb/Jak2 activation and PI3-kinase. Nonalcoholic fatty liver disease is a major health problem and may account for a significant number of patients with "cryptogenic" cirrhosis. These conditions are associated with increased serum leptin levels and insulin resistance.

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic diagram for leptin-induced HSC profibrogenic mechanisms induced by Jak activation and OB-Rb phosphorylation. Leptin signals as any gp130 cytokine primarily by Jak2 phosphorylation of several key tyrosine residues. OB-Rb phosphorylation promotes HSC proliferation and survival by Erk and Akt activation. SOCS-3, a feedback inhibitor of OB-Rb signaling, and AG490, a chemical inhibitor of Jak2 kinase activity, both block HSC proliferation and survival by blocking phosphorylation of Erk or Akt. HSC survival was inhibited by PI3-kinase inhibitor LY294002. HSC proliferation was blocked by MAPK inhibitor PD98059, PI3-kinase inhibitor LY294002, as well as pharmacologic and biologic OB-Rb signal blockade. Leptin could not suppress apoptosis in the presence of LY, indicating that Akt is highly protective against HSC apoptosis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1847fje;

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This Article Full Text (PDF) All Versions of this Article: 18/13/1612 04-1847fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SAXENA, N. K. Articles by ANANIA, F. A. Search for Related Content PubMed PubMed Citation Articles by SAXENA, N. K. Articles by ANANIA, F. A.