DNA Synthesis In Hepatic Stellate Cells Research Articles

Effect of natural interferon α on proliferation and apoptosis of hepatic stellate cells

Inhibition of the proliferation of hepatic stellate cells (HSC) is clinically important for the control of liver fibrosis and cirrhosis. Interferons are now frequently used for chronic viral hepatitis because of their anti-viral activity. However, patients treated with interferons exhibit a regression of liver fibrosis even if viral eradication is not achieved, indicating that interferon itself has anti-fibrotic activity. Herein, we show the anti-proliferation and pro-apoptotic activity of natural interferon α against HSC. We found that interferon α inhibited serum-stimulated [(3)H]thymidine incorporation of HSC in a dose-dependent manner, with a significant reduction at more than 100U/ml. Interferon α also attenuated PDGF-BB-stimulated DNA synthesis of HSC. Although the molecular mechanism behind these phenomena has not been defined, we found that interferon α triggers the apoptosis of HSC treated with low-dose tumor necrosis factor α, as determined by the Alamar blue assay, morphology, and DNA ladder formation. Furthermore, interferon α decreased inhibitor of caspase-activated DNase (ICAD) levels, which may augment tumor necrosis factor α-induced cell death signals. Thus, interferon α regulates the number of myofibroblastic hepatic stellate cells and may clinically contribute to the regression of human liver fibrosis.

The GABAB Receptor Inhibits Activation of Hepatic Stellate Cells

Angiotensin II (Ang II) plays an important role in the activation of hepatic stellate cells (HSCs). In this study it was found that expression of the GABA(B) receptor was elevated in HSCs treated with Ang II. We attempted to elucidate the mechanism of the GABA(B) receptor in HSCs activation. First, the target gene (GABA(B) receptor) was screened by gene chip in HSCs treated with Ang II. Second, the biological function of the GABA(B) receptor was analyzed by MTT, cell-cycle assay, real-time PCR, and western blot. The methods of MTT and cell-cycle assay were used to evaluate the effect of the GABA(B) receptor on proliferation and DNA synthesis of HSCs. Expression of ECM, TGF-beta1, and alpha-SMA was analyzed by real-time PCR and western blot. The GABA(B) receptor's specific agonist CGP35348 inhibited the activation of HSCs, which could be partially reversed by the GABA(B) receptor's antagonist. Our in-vitro results demonstrated that the GABA(B) receptor could inhibit HSCs activation.

Effect of angiotensin II and angiotensin II type 1 receptor antagonist on the proliferation, contraction and collagen synthesis in rat hepatic stellate cells

Angiotensin II (Ang II) is a very important vasoactive peptide that acts upon hepatic stellate cells (HSCs), which are major effector cells in hepatic cirrhosis and portal hypertension. The present study was aimed to investigate the effects of Ang II and angiotensin II type 1 receptor antagonist (AT(1)RA) on the proliferation, contraction and collagen synthesis in HSCs. HSC-T6 rat hepatic stellate cell line was studied. The proliferation of the HSC cells was evaluated by MTT colorimetric assay while HSC DNA synthesis was measured by (3)H-thymidine incorporation. The effects of angiotensin II and AT(1)RA on HSCs contraction were studied by analysis of the contraction of the collagen lattice. Cell culture media were analyzed by RT-PCR to detect secretion of collagen I (Col I), collagen III (Col III) and transforming growth factor beta1 (TGF-beta1) by enzyme linked immunosorbent assay. HSC was harvested to measure collagen I, collagen III and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression. Ang II ((1 x 10(-10) - 1 x 10(-4)) mol/L) stimulated DNA synthesis and proliferation in HSCs compared with untreated control cells. AT(1)RA inhibited angiotensin II induced proliferation of HSCs. A linear increase in the contractive area of collagen lattice correlated with the concentration of angiotensin II (1 x 10(-9) - 1 x 10(-5) mol/L) and with time over 48 hours. AT(1)RA blocks angiotensin II induced contraction of collagen lattice. Col I, Col III and TGF-beta1 levels of the Ang II group were higher than those of control group and this increase was downregulated by AT(1)RA. The mRNA expressions of Col I, Col III and TIMP-1 were higher in HSCs from the Ang II group than the control group and downregulated by AT(1)RA. Angiotensin II increased DNA synthesis and proliferation of HSCs in a dose-dependent manner, stimulated the contraction of HSCs dose- and time-dependently. Angiotensin also promoted excretion of Col I, Col III and TGF-beta1 levels and stimulated Col I, Col III and TIMP-1 expression in HSCs. Angiotensin acts via the angiotensin II receptor because all of these effects are blocked by angiotensin II type 1 receptor antagonist.

Expression and function of fibroblast growth factor (FGF) 9 in hepatic stellate cells and its role in toxic liver injury

Hepatic injury and regeneration of the liver are associated with activation of hepatic stellate cells (HSC). Fibroblast growth factors (FGFs) and their receptors are important regulators of repair in various tissues. HSC express FGFR3IIIc as well as FGFGR4 and different spliced FGFR1IIIc and FGFR2IIIc isoforms which differ in the presence or absence of the acid box and of the first Ig-like domain. Expression of FGF9, known to be capable to activate the HSC FGFR2/3-isoforms, was increased in HSC in liver slice cultures after exposition to carbon tetrachloride, as an acute liver injury model. FGF9 significantly stimulated 3-H thymidine incorporation of hepatocytes, but failed to induce DNA synthesis in HSC despite the fact that FGF9 induced a sustained activation of extracellular signal-related kinases (ERK) 1/2. FGF9 induced an increased phosphorylation of Tyr436 of the fibroblast growth factor receptor substrate (FRS) 2, while phosphorylation of Tyr196 which is required for efficient Grb2 recruitment remained unchanged. Our findings suggest that HSC FGF9 provide a paracrine mitogenic signal to hepatocytes during acute liver injury, while the autocrine FGF9 signaling appears to be not sufficient to induce cell proliferation.

Loss of TGF-β dependent growth control during HSC transdifferentiation

Liver injury induces activation of hepatic stellate cells (HSCs) comprising expression of receptors, proliferation, and extracellular matrix synthesis triggered by a network of cytokines provided by damaged hepatocytes, activated Kupffer cells and HSCs. While 6 days after bile duct ligation in rats TGF-β inhibited DNA synthesis in HSCs, it was enhanced after 14 days, indicating a switch from suppression to DNA synthesis stimulation during fibrogenesis. To delineate mechanisms modulating TGF-β function, we analyzed crosstalk with signaling pathways initiated by cytokines in damaged liver. Lipopolysaccharide and tumor necrosis factor-α enhanced proliferation inhibition of TGF-β, whereas interleukin-6, oncostatin M, interleukin-1α, and interleukin-1β did not. Hepatocyte growth factor (HGF) counteracted TGF-β dependent inhibition of DNA synthesis in quiescent HSCs. Since expression of c-met is induced during activation of HSCs and HGF is overrepresented in damaged liver, crosstalk of HGF and TGF-β contributes to loss of TGF-β dependent inhibition of DNA synthesis in HSCs.

Phospholipase D and extracellular signal-regulated kinase in hepatic stellate cells: effects of platelet-derived growth factor and extracellular nucleotides

We have previously provided evidence suggesting that phosphatidic acid, possibly derived from the hydrolysis of phosphatidylcholine by phospholipase D (PLD), is involved in platelet-derived growth factor (PDGF)-mediated increases in extracellular signal-regulated kinase (ERK) activity and DNA synthesis in rat hepatic stellate cells (HSC), the primary fibrogenic cells of the liver. A recent study has shown the presence of P2Y nucleotide receptors on HSC that are coupled to contraction and synthesis of the matrix component, alpha1-procollagen, leading to the suggestion that they may represent a new therapeutic target in the treatment of liver fibrosis. However, although extracellular nucleotides have been shown to stimulate both PLD and ERK, and to elicit proliferation of fibrogenic cells outside the liver, their effect on these parameters in HSC have not yet been investigated. PLD activity was determined by [3H]choline release and [3H]phosphatidylbutanol production, ERK activity by Western blotting, and DNA synthesis by [3H]thymidine incorporation. We report here, for the first time in HSC, that extracellular nucleotides stimulate PLD activity and a sustained activation of ERK. However, in contrast to PDGF, nucleotides had negligible effects on DNA synthesis. Moreover, the effects of PDGF and nucleotides on PLD and ERK were not additive, suggesting activation of the same PLD isoform and pool of ERK. The data demonstrate that nucleotide-stimulated PLD and ERK activities are not coupled to DNA synthesis in HSC. Instead, these responses may be linked to other phenotypic changes associated with activated HSC such as increases in contraction, motility, or extracellular matrix deposition.

Meso-Dihydroguaiaretic Acid fromMachilus thunbergiiDown-RegulatesTGF-β1 Gene Expression in Activated Hepatic Stellate Cells via Inhibition of AP-1 Activity

meso-dihydroguaiaretic acid (DGA), naturally occurring in plants such as Machilus thunbergii and Myristica fragrans, exhibits a neuroprotective effect and also exerts cytotoxicity to certain cancer cells. Activated hepatic stellate cells (HSCs) play an important role in liver fibrogenesis through the production of transforming growth factor beta1 (TGF-beta1) after injuries. TGF-beta1 mediates the deposition of extracellular matrix and the inhibition of collagenase activity in the liver. This study has investigated the inhibitory effect of DGA on the activation of rat HSCs in culture and TGF-beta1 production from HSCs. The level of alpha-smooth muscle actin (alpha-SMA), a representative marker of stellate cell transdifferentiation, was decreased upon treatment of activated HSCs with DGA (1 - 10 microM). Immunoblot analysis revealed that DGA inhibited the expression of TGF-beta1 in activated HSCs. Consistently, DGA down-regulated the transactivation of the TGF-beta1 promoter linked to the luciferase reporter gene in HSCs. Promoter deletion analysis revealed that the region located between -731 bp and -323 bp in the TGF-beta1 promoter, which is comprised of AP-1 response elements, conferred the inhibition of TGF-beta1 expression by DGA. DGA also inhibited AP-1-mediated gene transactivation in HSCs to a comparable extent, indicating that down-regulation of the TGFbeta1 gene by DGA might result from its inhibition of AP-1 activity. We found in addition that DGA inhibited DNA synthesis in HSCs stimulated by platelet-derived growth factor. The data provide evidence that DGA directly inhibits activation of HSCs and down-regulates TGF-beta1 gene expression through inhibition of AP-1 activity.

IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells (HSC) but DNA synthesis and proliferation in rat liver myofibroblasts (rMF)

Several lines of evidence suggest a role of insulin-like growth factor I (IGF-I) in the regulation of apoptosis. Up to now its impact on many specific cells is unknown. We therefore studied the effect of IGF-I on two similar mesenchymal matrix-producing cell types of the liver, the hepatic stellate cells (HSC) and the myofibroblasts (rMF). The present study aimed to reveal the influence of IGF-I on cell cycle and apoptosis of HSC and rMF and to elucidate responsible signaling. While IGF-I significantly increased DNA synthesis in HSC, cell number decreased and apoptosis increased. In rMF IGF-I also increased DNA synthesis, which is, however, followed by proliferation. Blocking extracellular signal regulating kinase (ERK) revealed that in HSC, bcl-2 upregulation and bax downregulation are effected downstream of ERK, whereas downregulation of NFκB and consecutive of bcl-xL is mediated upstream. In the rMF upregulation of both, the antiapoptotic bcl-2 and bcl-xL is mediated upstream of ERK. The expression of the proapoptotic bax is not regulated by IGF-I in rMF. The studies demonstrate a completely different effect and signaling of IGF-I in two morphologically and functionally similar matrix-producing cells of the liver.

Effects of endothelin-1 on hepatic stellate cell proliferation, collagen synthesis and secretion, intracellular free calcium concentration.

To explore the effects of endothelin-1(ET-1) on hepatic stellate cells (HSCs) DNA uptake, DNA synthesis, collagen synthesis and secretion, inward whole-cell calcium concentration ([Ca2+]i) as well as the blocking effect of verapamil on ET-1-stimulated release of inward calcium (Ca2+) of HSC in vitro. Rat hepatic stellate cells (HSCs) were isolated and cultivated. 3H-TdR and 3H-proline incorporation used for testing DNA uptake and synthesis, collagen synthesis and secretion of HSCs cultured in vitro; Fluorescent calcium indicator Fura-2/AM was used to measure [Ca2+]i inward HSCs. ET-1 at the concentration of 5X10(-8) mol/L, caused significant increase both in HSC DNA synthesis (2,247+/-344 cpm, P<0.05) and DNA uptake (P<0.05) when compared with the control group. ET-1 could also increase collagen synthesis (P<0.05 vs control group) and collagen secretion (P<0.05 vs control group). Besides, inward HSC [Ca2+]i reached a peak concentration (422+/-98 mol/L, P<0.001) at 2 min and then went down slowly to 165+/-51 mol/L (P<0.01) at 25 min from resting state (39+/-4 mol/L) after treated with ET-1. Verapamil (5 mol/L) blocked ET-1-activated [Ca2+]i inward HSCs compared with control group (P<0.05). Fura-2/AM loaded HSC was suspended in no Ca2+ buffer containing 1 mol/L EGTA, 5 min later, 10(-8) mol/L of ET-1 was added, [Ca2+]i inward HSCs rose from resting state to peak 399+/-123 mol/L, then began to come down by the time of 20 min. It could also raise [Ca2+]i inward HSCs even without Ca2+ in extracellular fluid, and had a remarkable dose-effect relationship(P<0.05). Meanwhile, verapamil could restrain the action of ET-1(P<0.05). Actions of ET-1 on collagen metabolism of HSCs may depend on the transportation of inward whole-cell calcium.

Characterization of hepatocyte-derived mitogenic activity on hepatic stellate cells.

Hepatic stellate cells (HSC) are located in close proximity to hepatocytes in Disse's space. Hepatocyte derived factors have earlier been implicated in the paracrine regulation of HSC proliferation. The aim of the present study was to further characterize this mitogenic activity of the parenchymal cell conditioned medium (PCcM). Primary rat HSC were cultured for 4 days. DNA synthesis was measured by 3H-thymidine incorporation. TGFbeta1 immunoreactivity was quantified by ELISA. PCcM was obtained from hepatocytes cultured in medium without serum or hormones for two days. Incubation of 4-day-old HSC on plastic surface with PCcM for 2 days increased DNA synthesis, while no effect was seen in HSC cultured on Matrigel. Heat-, acid-, and protease-treatment of PCcM abolished its stimulatory effect. Size fractionations with spin columns indicated that the stimulatory effect was contained in the fractions of a molecular size between 30 and 100 kD. The addition of LY 294002, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, dose-dependently inhibited the PCcM induced increase in DNA synthesis to about 9% of the control values. The specific MAP kinase (MAPK) inhibitor, PD 98059 only suppressed the PCcM induced DNA synthesis to 35% of control cultures at the highest dose (10 microM). DNA content in the cultures was not affected by either blocker. HSC seemed to produce immunoreactive TGFbeta1. However, addition of latency-associated peptide (LAP), a potent TGFbeta1 blocker, stimulated DNA synthesis to a much less extent than PCcM. The factor(s) that stimulate DNA synthesis in HSC from hepatocytes are most likely protein(s) with a molecular size between 30-100 kD. These factor(s) rely more on PI3-K than on MAPK for their mitogenic effect and are probably not acting via TGFbeta1 inhibition.