Glutathione levels discriminate between oxidative stress and transforming growth factor-beta signaling in activated rat hepatic stellate cells

Reactive oxygen species are implicated in the pathogenesis of several diseases, including Alzheimer's disease, multiple sclerosis, human immunodeficiency virus, and liver fibrosis. With respect to liver fibrosis, we have investigated differences in antioxidant enzymes expression in stellate cells (SCs) and parenchymal cells from normal and CCl(4)-treated rat livers. We observed an increase in the expression of catalase in activated SCs. Treatment with transforming growth factor-beta (TGF-beta) increased the production of H(2)O(2). Treatment with catalase decreased TGF-beta expression. Addition of H(2)O(2) resulted in increased TGF-beta production. 3-Amino-1,2,4-triazole abolished the capacity of SCs to remove H(2)O(2). A paradoxical increase in capacity was observed when the cells were pretreated with diethyl maleate. Treatment with 3-amino-1, 2,4-triazole increased TGF-beta production. A paradoxical decrease of TGF-beta production was observed with diethyl maleate. Treatment of the cells with N-acetylcysteine resulted in increased TGF-beta production. TGF-beta decreased the capacity of the SCs to remove H(2)O(2.) An increase in the capacity to remove H(2)O(2) was observed when TGF-beta was removed by neutralizing antibodies. In conclusion, our results suggest: 1) a link between cellular GSH levels and TGF-beta production and 2) that cellular GSH levels discriminate whether H(2)O(2) is the result of oxidative stress or acts as second messenger in the TGF-beta signal transduction pathway.     

Inhibition of platelet-derived growth factor receptor tyrosine kinase and downstream signaling pathways by Compound C

AMP-activated protein kinase (AMPK) has been implicated in anti-proliferative actions in a range of cell systems. Recently, it was observed that Compound C, an inhibitor of AMPK, also reduced the cell viability in human diploid fibroblasts (HDFs). Compound C-induced growth arrest was associated with a decrease in the cell cycle regulatory proteins, such as proliferating cell nuclear antigen, phosphorylated pRB, cyclin-dependent protein kinases (Cdk 2 and 4), cyclins (D and E), and the Cdk inhibitors (p21, p16, and p27). Therefore, the present study examined the molecular mechanism of the antiproliferative effects of Compound C. Although Compound C inhibited serum-induced phosphorylation of Akt and its substrate, glycogen synthase kinase-3β, it did not affect the Akt activity in vitro. Compound C significantly inhibited the receptor tyrosine phosphorylation and the activity of downstream signaling molecules, such as p85 phosphoinositide 3-kinase, phospholipase C-γ1, and extracellular signal-regulated kinase 1/2, induced by platelet-derived growth factor (PDGF) but not by epidermal growth factor- and insulin-like growth factor. In vitro growth factor receptor tyrosine kinase activity profiling revealed the IC₅₀ for PDGF receptor-β (PDGFRβ) to be 5.07 μM, whereas the IC₅₀ for the epidermal growth factor receptor and insulin-like growth factor receptor was ≥ 100 μM. The inhibitory effect of Compound C on PDGFRβ and Akt was also observed in AMPKα₁/α₂-knockout mouse embryonic fibroblasts, indicating that its inhibitory effect is independent of the AMPK activity. The inhibitory effect of Compound C on cell proliferation and PDGFRβ tyrosine phosphorylation was also demonstrated in various PDGFR-expressing cells, including MRC-5, BEAS-2B, rat aortic vascular smooth muscle cells, and A172 glioblastoma cells. These results indicate that Compound C can be used as a potential antiproliferative agent for PDGF- or PDGFR-associated diseases, such as cancer, atherosclerosis, and fibrosis.     

MARCKS is a downstream effector in platelet-derived growth factor-induced cell motility in activated human hepatic stellate cells

Myristoylated alanine-rich protein kinase c substrate (MARCKS) has been suggested to be implicated in cell adhesion, secretion, motility and mitogenesis through regulation of the actin cytoskeletal structure. In the present study, a possible link between MARCKS and the platelet-derived growth factor (PDGF) signaling pathway was investigated in activated human hepatic stellate cells (hHSC), critical regulators of hepatic fibrogenesis. PDGF-BB stimulation resulted in a bi-directional movement of MARCKS that coincided with the phosphorylation of MARCKS and the activation of both PKCepsilon and PKCalpha. Biochemical inhibition of PKC kinase activity and small interfering RNA (siRNA) against PKCepsilon demonstrated that PKCepsilon is indispensable for PDGF-BB-induced MARCKS phosphorylation and cell migration. Immunoprecipitation studies revealed an association between MARCKS and the PDGFbeta-receptor, while the PDGFbeta-receptor and PKCalpha associated with focal adhesion kinase (FAK). Transient transfection with MARCKS DNA plasmid remarkably reduced PDGF-BB stimulated cell motility. In contrast, siRNA against MARCKS increased cell migration in RNAi treated cells in comparison to the scrambled control cells. In conclusion, the present study indicates that MARCKS play a major key role in PDGF-BB-induced chemotaxis in activated hHSC.     

Tricin inhibits proliferation of human hepatic stellate cells in vitro by blocking tyrosine phosphorylation of PDGF receptor and its signaling pathways

4',5,7-Trihydroxy-3',5'-dimethoxyflavone (Tricin), a naturally occurring flavone, has anti-inflammatory potential and exhibits diverse biological activities including antigrowth activity in several human cancer cell lines and cancer chemopreventive effects in the gastrointestinal tract of mice. The present study aimed to investigate the biological actions of tricin on hepatic stellate cells (HSCs) in vitro, exploring its potential as a treatment of liver fibrosis, since HSC proliferation is closely related to the progression of hepatic fibrogenesis in chronic liver diseases leading to irreversible liver cirrhosis and hepatocellular carcinoma. Tricin inhibited platelet-derived growth factor (PDGF)-BB-induced cell proliferation by blocking cell cycle progression and cell migration in the human HSC line LI90 and culture-activated HSCs. It also reduced the phosphorylation of PDGF receptor β and the downstream signaling molecules ERK1/2 and Akt, which might be due to its tyrosine kinase inhibitor properties rather than inhibition of the direct binding between PDGF-BB and its receptor. Our findings suggest that tricin might be beneficial in HSC-targeting therapeutic or chemopreventive applications for hepatic fibrosis.     

Leptin facilitates proliferation of hepatic stellate cells through up-regulation of platelet-derived growth factor receptor

In the present study, we investigated the effect of leptin on proliferation of hepatic stellate cells (HSCs) in vitro. Proliferation of 3-day cultured rat HSCs was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the nuclei. The percentages of BrdU-positive cells were increased in the presence of PDGF-BB (5 ng/ml) for 8h as expected. Co-incubation with leptin (10-100 nM) potentiates this PDGF-dependent increase in BrdU positive cells in a dose-dependent manner. Messenger RNA for PDGF receptor alpha and beta subunits was increased almost 2- to 3-fold by incubation with leptin for 6h. Further, pre-incubation with leptin for 6h enhanced PDGF-induced increases in phospho-p44/42 MAP kinase and phospho-Akt levels in a dose-dependent manner. In the same condition, however, leptin per se did not increase phospho-STAT 3 and phospho-p44/42 MAP kinase levels. Instead, leptin increased phospho-Akt levels in HSCs within 30 min, suggesting that the phosphatidylinositol 3 kinase (PI3K)/Akt pathway is involved in the mechanism by which leptin accelerates the proliferation of HSCs. In conclusion, the present study clearly indicated that leptin potentiates PDGF-dependent proliferative responses of HSCs in vitro.     

Reduced tyrosine phosphorylation in polyamine-starved cells.

Onset of cell proliferation is associated with enhanced turnover of the polyamines putrescine, spermidine, and spermine, particularly evident in the massive increase in the activity of the rate-limiting enzyme in their production, ornithine decarboxylase (ODC). The physiological functions of these polyamines, however, have remained unclear. Here we report that treatment of LSTRA cells for 2-18 h with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, decreased the amount of phosphotyrosine in several cellular substrates including the T cell protein tyrosine kinase p56lck. No reductions in the amount of p56lck, overall synthesis of protein and DNA, or cell viability were observed until much later. DFMO did not affect the catalytic activity of p56lck in vitro and the activity of p56lck immunoprecipitated from DFMO-treated cells was unaltered. Addition of putrescine, the reaction product of ODC, completely reversed the effect of DFMO on tyrosine phosphorylation. Finally, we provide evidence that polyamines reduce the activity of cellular protein tyrosine phosphatases toward endogenous substrates. Our results suggest that polyamines may influence the extent of tyrosine phosphorylation during cell proliferation and malignant transformation, perhaps by modulating the rate of dephosphorylation of specific target proteins.     

Evidence for the platelet-derived growth factor-stimulated tyrosine phosphorylation of the platelet-derived growth factor receptor in vivo. Immunopurification using a monoclonal antibody to phosphotyrosine

The addition of platelet-derived growth factor (PDGF) to intact BALB/c 3T3 cells results in the rapid (less than 1 min), dose-dependent phosphorylation of a number of proteins that could be isolated by a monoclonal antiphosphotyrosine antibody. The predominant tyrosinephosphorylated protein shared many characteristics with the PDGF receptor, including its molecular weight (170,000), isoelectric point (pI of about 4.2), its binding to DEAE-cellulose, and its pattern of binding to lectins. This 170-kDa protein, labeled with [35S] methionine, was substantially purified from PDGF-stimulated cells using the monoclonal anti-phosphotyrosine antibody but was not significantly immunopurified from unstimulated cells. At 37 degrees C, phosphorylation of the 170-kDa protein was maximal by 5-10 min of exposure to PDGF, and thereafter decreased rapidly. However, at 4 degrees C, the phosphorylation continued to increase after 3 h of exposure to PDGF. Subsequently, shifting the cells from 4 to 37 degrees C resulted in an additional rapid burst of tyrosine phosphorylation. Among the other PDGF-stimulated molecules, the most prominent and consistently observed was a cytosolic, acidic (pI of about 4.2), 74-kDa protein. These findings indicate that the action of PDGF in vivo is associated with the rapid and transient tyrosine phosphorylation of several membrane and cytosolic proteins; the most prominent of these proteins, isolated by monoclonal antibody to phosphotyrosine, is likely to be the PDGF receptor. The use of this antibody provides a new approach for purification of the PDGF receptor.     

The tyrosine phosphatase SHP-1 inhibits proliferation of activated hepatic stellate cells by impairing PDGF receptor signaling

The dimerization and auto-transphosphorylation of platelet-derived growth factor receptor (PDGFR) upon engagement by platelet-derived growth factor (PDGF) activates signals promoting the mitogenic response of hepatic stellate cells (HSCs) due to liver injury, thus contributing to the development of hepatic fibrosis. We demonstrate that the tyrosine phosphatases Src homology 2 domain-containing phosphatase 1 and 2 (SHP-1 and SHP-2) act as crucial regulators of a complex signaling network orchestrated by PDGFR activation in a spatio-temporal manner with diverse and opposing functions in HSCs. In fact, silencing of either phosphatase shows that SHP-2 is committed to PDGFR-mediated cell proliferation, whereas SHP-1 dephosphorylates PDGFR hence abrogating the downstream signaling pathways that result in HSC activation. In this regard, SHP-1 as an off-switch of PDGFR signaling appears to emerge as a valuable molecular target to trigger as to prevent HSC proliferation and the fibrogenic effects of HSC activation. We show that boswellic acid, a multitarget compound with potent anti-inflammatory action, exerts an anti-proliferative effect on HSCs, as in other cell models, by upregulating SHP-1 with subsequent dephosphorylation of PDGFR-β and downregulation of PDGF-dependent signaling after PDGF stimulation. Moreover, the synergism resulting from the combined use of boswellic acid and imatinib, which directly inhibits PDGFR-β activity, on activated HSCs offers new perspectives for the development of therapeutic strategies that could implement molecules affecting diverse players of this molecular circuit, thus paving the way to multi-drug low-dose regimens for liver fibrosis.     

The A-type receptor for platelet-derived growth factor mediates protein tyrosine phosphorylation, receptor transmodulation and a mitogenic response.

A clonal human glioma cell line, U-343 MGa 31L, which expresses the A-type but not the B-type receptor for platelet-derived growth factor (PDGF), was used in a functional study of the A-type receptor. PDGF-AA induced, in a dose- and time-dependent manner, phosphorylation on tyrosine residues of the receptor in metabolically labelled cells. The optimal dose was around 30 ng/ml; at 100 ng/ml, phosphorylation was maximal at 15 min and had almost returned to the control level after 60 min. The phosphorylation on tyrosine residues of the PDGF A-type receptor was stimulated by PDGF-AA, PDGF-AB and PDGF-BB; these isoforms also stimulated [3H]thymidine incorporation into U-343 MGa 31L cells. In addition, activation of the A-type PDGF receptor induced transmodulation of the epidermal growth factor receptor.     

Disruption of transforming growth factor-beta signaling by curcumin induces gene expression of peroxisome proliferator-activated receptor-gamma in rat hepatic stellate cells

Activation of hepatic stellate cells (HSC), the major effectors of hepatic fibrogenesis, is coupled with sequential alterations in gene expression, including an increase in receptors for transforming growth factor-beta (TGF-beta) and a dramatic reduction in the peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The relationship between them remains obscure. We previously demonstrated that curcumin induced gene expression of PPAR-gamma in activated HSC, leading to reducing cell proliferation, inducing apoptosis and suppressing expression of extracellular matrix genes. The underlying molecular mechanisms are largely unknown. We recently observed that stimulation of PPAR-gamma activation suppressed gene expression of TGF-beta receptors in activated HSC, leading to the interruption of TGF-beta signaling. This observation supported our assumption of an antagonistic relationship between PPAR-gamma activation and TGF-beta signaling in HSC. In this study, we further hypothesize that TGF-beta signaling might negatively regulate gene expression of PPAR-gamma in activated HSC. The present report demonstrates that exogenous TGF-beta1 inhibits gene expression of PPAR-gamma in activated HSC, which is eliminated by the pretreatment with curcumin likely by interrupting TGF-beta signaling. Transfection assays further indicate that blocking TGF-beta signaling by dominant negative type II TGF-beta receptor increases the promoter activity of PPAR-gamma gene. Promoter deletion assays, site-directed mutageneses, and gel shift assays localize two Smad binding elements (SBEs) in the PPAR-gamma gene promoter, acting as curcumin response elements and negatively regulating the promoter activity in passaged HSC. The Smad3/4 protein complex specifically binds to the SBEs. Overexpression of Smad4 dose dependently eliminates the inhibitory effects of curcumin on the PPAR-gamma gene promoter and TGF-beta signaling. Taken together, these results demonstrate that the interruption of TGF-beta signaling by curcumin induces gene expression of PPAR-gamma in activated HSC in vitro. Our studies provide novel insights into the molecular mechanisms of curcumin in the induction of PPAR-gamma gene expression and in the inhibition of HSC activation.     

Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases

Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites.     

Transforming growth factor-beta1 downregulation of Smad1 gene expression in rat hepatic stellate cells

Smads are intracellular signaling molecules of the transforming growth factor-beta (TGF-beta) superfamily that play an important role in the activation of hepatic stellate cells (HSCs) and hepatic fibrosis. Excepting the regulation of Smad7, receptor-regulated Smad gene expression is still unclear. We employed rat HSCs to investigate the expression and regulation of the Smad1 gene, which is a bone morphogenetic protein (BMP) receptor-regulated Smad. We found that the expression and phosphorylation of Smad1 are increased during the activation of HSCs. Moreover, TGF-beta significantly inhibits Smad1 gene expression in HSCs in a time- and dose-dependent manner. Furthermore, although both TGF-beta1 and BMP2 stimulate the activation of HSCs, they have different effects on HSC proliferation. In conclusion, Smad1 expression and phosphorylation are increased during the activation of HSCs and TGF-beta1 significantly inhibits the expression of the Smad1 gene.     

Actors of the tyrosine kinase receptor downstream signaling pathways in amphioxus

SUMMARY One of the major goals of evo-developmentalists is to understand how the genetic mechanisms controlling embryonic development have evolved to create the current diversity of bodyplans that we encounter in the animal kingdom. Tyrosine kinase receptors (RTKs) are transmembrane receptors present in all metazoans known to control several developmental processes. They act via the activation of various cytoplasmic signaling cascades, including the mitogen-activated protein kinase (MAPK), the PI3K/Akt, and the phospholipase C-γ (PLCγ)/protein kinase C (PKC) pathways. In order to address the evolution of these three pathways and their involvement during embryogenesis in chordates, we took advantage of the complete genome sequencing of a key evolutionarily positioned species, the cephalochordate amphioxus, and searched for the complete gene set of the three signaling pathways. We found that the amphioxus genome contains all of the most important modules of the RTK-activated cascades, and looked at the embryonic expression of two genes selected from each cascade. Our data suggest that although the PI3K/Akt pathway may have ubiquitous functions, the MAPK and the PLCγ/PKC cascades may play specific roles in amphioxus development. Together with data known in vertebrates, the expression pattern of PKC in amphioxus suggests that the PLCγ/PKC cascade was implicated in neural development in the ancestor of all chordates.     

Identification of endoglin in rat hepatic stellate cells: new insights into transforming growth factor beta receptor signaling

Transforming growth factor beta (TGF-beta) signaling is mediated by the cell surface TGF-beta type I (ALK5), type II, and the accessory type III receptors endoglin and betaglycan. Hepatic stellate cells (HSC), the most profibrogenic cell type in the liver, express ALK5, TbetaRII, and betaglycan. To monitor the expression of betaglycan in HSC, we used the commercially available antibody sc-6199 in Western blot analysis. This antibody, raised against a peptide mapping at the carboxyl terminus of the human betaglycan, is claimed to be specific for betaglycan, although it is known that the C-terminal domain is highly conserved in type III receptors. Proteins recognized in HSC by sc-6199 did not match the characteristic migration pattern of betaglycan. Moreover, the determined molecular weight (M(r) 160) and the observed reductant sensitivity after treatment with dithiothreitol resemble those of a closely related type III receptor, endoglin (CD105). Endoglin, a disulfide-linked homodimer, is an accessory component of the TGF-beta receptor complex and mainly expressed on endothelial cells. The presence of endoglin in HSC of rat liver was confirmed by molecular cloning of the endoglin cDNA and immunocytochemistry. The reactivity of sc-6199 with both auxiliary TGF-beta receptors (betaglycan and endoglin) from rats was demonstrated by Western blot and immunocytochemical analysis of cells heterologously expressing these proteins. Furthermore, Northern and Western blotting revealed that both betaglycan and endoglin genes are differentially regulated in HSC and in transdifferentiated myofibroblasts (MFB). By surface labeling and immunoprecipitation experiments, we show that endoglin is found in significant amounts exposed at the plasma membrane of HSC and MFB, which is a pivotal prerequisite for binding of and signaling in response to TGF-beta. In conclusion, we hypothesize that TGF-beta signals in HSC and MFB are tuned by two different interconnected signaling pathways, as it was previously demonstrated for endothelial cells.     

Thyrotropin and insulin-like growth factor I regulation of tyrosine phosphorylation in FRTL-5 cells. Interaction between cAMP-dependent and growth factor-dependent signal transduction.

Pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiated the mitogenic response to insulin-like growth factor I (IGF-I) (Tramontano, D., Moses, A. C., Veneziani, B. M., and Ingbar, S. H. (1988) Endocrinology 122, 127-132; Takahashi, S.-I., Conti, M., and Van Wyk, J. J. (1990) Endocrinology 126, 736-745). The present study was undertaken to determine whether this synergism between TSH and IGF-I in FRTL-5 cells was correlated with changes in tyrosine phosphorylation of intracellular proteins. Tyrosine phosphorylation in intact cells was determined by gel electrophoresis and immunoblotting using monospecific anti-phosphotyrosine antibodies. Cells were preincubated for up to 24 h with TSH, dibutyryl cAMP, forskolin, or cholera toxin and then incubated for an additional 1 min in the absence or presence of IGF-I. As reported by others, IGF-I rapidly increased tyrosine phosphorylation of a 175-kDa protein as well as a less intense band of 90-100 kDa. Pretreatment for 6-12 h with either TSH or other agents that elevate intracellular cAMP potentiated the IGF-I-dependent tyrosine phosphorylation of the 175-kDa substrate by 3-5-fold. Since TSH did not increase IGF receptor number of kinase activity, the effect of TSH is assumed to be exerted at a step distal to IGF receptor tyrosine kinase. Surprisingly, IGF-I-independent tyrosine phosphorylation was also increased by pretreatment with TSH. When intact cells were analyzed TSH produced a time- and concentration-dependent increase in tyrosine phosphorylation of a prominent 120-125-kDa substrate and less prominent 100- and 80-kDa substrates. Assays using Triton X-100-soluble extracts incubated with MgCl2, ATP, and orthovanadate demonstrated that TSH pretreatment increased tyrosine phosphorylation over that observed in untreated cells. In this cell-free assay, TSH pretreatment enhanced the phosphorylation of multiple substrates. These studies suggest that a cAMP stimulus that initiates a trophic effect can be propagated indirectly through multiple pathways including enhancement of tyrosine phosphorylation.