Biomedical subjects

Scott L Friedman

Publications and source records attributed to Scott L Friedman.

62 records · Page 4Linked to original sources

PubMed2002

Discoidin domain receptor 2 interacts with Src and Shc following its activation by type I collagen.

Discoidin domain receptor 2 (DDR2) is an unusual receptor tyrosine kinase in that its ligand is fibrillar collagen rather than a growth factor-like peptide. We examined signal transduction pathways of DDR2. Here we show that DDR2 is also unusual in that it requires Src activity to be maximally tyrosine-phosphorylated, and that Src activity also promotes association of DDR2 with Shc. The interaction with Shc involves a portion of Shc not previously implicated in interaction with receptor tyrosine kinases. These results identify Src kinase and the adaptor protein Shc as key signaling intermediates in DDR2 signal transduction. Furthermore, Src is required for DDR2-mediated transactivation of the matrix metalloproteinase-2 promoter. The data support a model in which Src and the DDR2 receptor cooperate in a regulated fashion to direct the phosphorylation of both the receptor and its targets.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Cytochrome P450 2E1-derived reactive oxygen species mediate paracrine stimulation of collagen I protein synthesis by hepatic stellate cells.

To evaluate possible fibrogenic effects of CYP2E1-dependent generation of reactive oxygen species, a model was developed using co-cultures of HepG2 cells, which do (E47 cells) or do not (C34 cells) express cytochrome P450 2E1 (CYP2E1) with stellate cells. There was an increase in intra- and extracellular H(2)O(2), lipid peroxidation, and collagen type I protein in stellate cells co-cultured with E47 cells compared with stellate cells alone or co-cultured with C34 cells. The increase in collagen was prevented by antioxidants and a CYP2E1 inhibitor. CYP3A4 did not mimic the stimulatory effects found with CYP2E1. Collagen mRNA levels remained unchanged, and pulse-chase analysis indicated similar half-lives of collagen I protein between both co-cultures. However, collagen protein synthesis was increased in E47 co-culture. Hepatocytes from pyrazole-treated rats (with high levels of CYP2E1) induced collagen protein in primary stellate cells, and antioxidants and CYP2E1 inhibitors blocked this effect. These results suggest that increased translation of collagen mRNA by CYP2E1-derived reactive oxygen species is responsible for the increase in collagen protein produced by the E47 co-culture. These co-culture models may be useful for understanding the impact of CYP2E1-derived ROS on stellate cell function and activation.

Animals↗

PubMed2002

Stimulation and proliferation of primary rat hepatic stellate cells by cytochrome P450 2E1-derived reactive oxygen species.

The alcohol-inducible cytochrome P450 2E1 (CYP2E1) is expressed mainly in hepatocytes and generates reactive oxygen species (ROS). To better understand how hepatic stellate cells (HSC) become activated in the presence of oxidative stress and evaluate whether CYP2E1-derived ROS activate stellate cells, we coincubated primary stellate cells with HepG2 cells, which do (E47 cells) or do not (C34 cells) express CYP2E1. Morphologic changes and loss of lipid droplets were more apparent in the stellate cells cocultured with E47 cells. There was a more pronounced increase in alpha-smooth muscle actin (alpha-sma), intracellular and secreted collagen type I protein, and intra- and extracellular H(2)O(2) and lipid peroxidation products in stellate cells coincubated with E47 cells. Expression of collagen in stellate cells did not change when cocultured with HepG2 cells expressing a different P450, CYP3A4. Stellate cells cultured on Matrigel expressed increased alpha-sma and collagen when incubated with E47 cells. The increase in collagen production by coculture with E47 cells was prevented by antioxidants, by CYP2E1 inhibitors, and by transfected antisense CYP2E1. The addition of arachidonic acid plus ferric nitrilotriacetate (Fe-NTA), agents that potentiate oxidative stress, further induced collagen protein in the E47 coculture. Stellate cell proliferation was greater in the E47 coculture, and this was partially abrogated by catalase and vitamin E. These results show that hepatocytes containing CYP2E1 release diffusible mediators including ROS, which can activate HSC. Thus, besides perturbing the homeostasis of hepatocytes, CYP2E1-derived diffusible oxidants may also interact with stellate cells and contribute to hepatic fibrosis.

Actins↗

PubMed2002

Phosphorylation of retinoid X receptor suppresses its ubiquitination in human hepatocellular carcinoma.

Retinoid X receptor alpha (RXRalpha) has emerged as an important nuclear receptor involved in hepatocarcinogenesis, because its ligand suppresses the development of hepatocellular carcinoma (HCC) in both experimental and clinical studies. We have demonstrated that phosphorylation of RXRalpha at serine 260 interferes with its function and delays its degradation in cultured human HCC, leading to enhanced cellular proliferation. Here, we show that in normal liver and in nonproliferating hepatocyte cultures, RXRalpha is unphosphorylated and highly ubiquitinated, rendering it sensitive to proteasome-mediated degradation. On the other hand, phosphoserine 260 RXRalpha is resistant to ubiquitination and proteasome-mediated degradation in both human HCC tissues and a human HCC cell line, HuH7. In these tissues and cells, serine 260 is phosphorylated by mitogen-activated protein (MAP) kinase. In proliferating normal hepatocytes, similar to HCC cells, RXRalpha is also phosphorylated at serine 260 and resistant to ubiquitin-mediated degradation by proteasome, but this ubiquitination of RXRalpha is differentially regulated between HCC cells and normal hepatocytes. In proliferating hepatocytes, 9-cis retinoic acid (9cRA), a ligand to RXRalpha, suppresses MAP kinase-mediated phosphorylation and thereby enhances ubiquitination of RXRalpha, whereas it fails to exert these effects in HCC cells. In conclusion, switching of the ubiquitin/proteasome-dependent degradation of RXRalpha by phosphorylation at serine 260 may be responsible for the aberrant growth of HCC and its suppression by retinoids.

Carcinoma, Hepatocellular↗

PubMed2002

Leptin in hepatic fibrosis: evidence for increased collagen production in stellate cells and lean littermates of ob/ob mice.

Leptin is a 16-kd hormone that mediates a range of metabolic effects by using a transduction pathway from the long form of the leptin receptor, OB-R(L,) through Janus kinase-signal transducer and activator of transcription (Jak-Stat) signaling components. Leptin is produced by hepatic stellate cells (HSCs) but only following their "activation." Because activation of stellate cells is a central event in the fibrotic response to liver injury, we hypothesized that leptin may directly stimulate fibrogenesis in activated stellate cells via OB-R(L). We analyzed leptin receptors and their signaling partners in a stellate cell line (HSC-T6) as well as in primary stellate cell isolates. We also examined the effect of leptin on stellate cell expression of alpha(2)(I) collagen messenger RNA (mRNA) levels by ribonuclease protection analysis (RPA). Finally, we examined the role of leptin in in vivo fibrogenesis by inducing a wounding response in ob/ob mice, which lack functional leptin. HSC-T6 and culture-activated stellate cells expressed OB-R(L). Scatchard analysis verified specific binding of leptin to HSCs, with an association constant (K(d)) equal to 660 +/- 5.8 pmol/L. Exposure of HSCs to leptin resulted in significant increases in alpha(2)(I) collagen mRNA expression. Transient transfection with a promoter reporter construct showed a 3-fold increase in alpha(2)(I) collagen transgene activity. Leptin stimulated activation of Stat3 in activated HSCs. Finally, lean animals, but not ob/ob littermates, had significant fibrosis as assessed by picrosirius red staining and abundant alpha-smooth muscle actin staining. In conclusion, these results indicate that leptin is profibrogenic in activated HSCs and can signal via the Jak-Stat pathway. Up-regulation of leptin signaling in liver injury could contribute to enhanced fibrogenesis, particularly in states in which leptin levels are high.

Actins↗

PubMed2002

Synergistic induction of apoptosis by acyclic retinoid and interferon-beta in human hepatocellular carcinoma cells.

Acyclic retinoid, a synthetic retinoid analog, as well as interferon alfa (IFN-alpha) and IFN-beta induce apoptosis in hepatocellular carcinoma (HCC) cells and are used clinically in the prevention of HCC. Here, we show that acyclic retinoid acts synergistically with IFNs in suppressing the growth and inducing apoptosis (as characterized by DNA fragmentation and chromatin condensation) in 5 human HCC cell lines (JHH7, HuH7, PLC/PRF/5, HLE, and HLF). This synergism was only observed when cells were pretreated with the acyclic retinoid, whereas natural retinoic acids (all-trans and 9-cis retinoic acid) were ineffective. This promotion may be due to up-regulation of type 1 IFN receptor (IFNR) expression by the retinoid. Accordingly, incubation with antitype 1 IFNR antibody abolished the synergy. Enhanced IFNR expression was accompanied by increased expression and DNA-binding activity of STAT1, an intracellular signal transducing molecule of IFNR, and increased induction of 2', 5'-oligoadenyl-5'-triphosphate synthetase, which is a target gene of STAT1. Acyclic retinoid did not have any effects on the growth of normal human hepatocytes (Hc) probably because of a lack of IFNR and STAT1 up-regulation. In conclusion, these results provide a rationale for combined biochemoprevention of HCC using acyclic retinoid and IFN-beta.

Antineoplastic Agents↗

PubMed2002

Elevated expression of tyrosine kinase DDR2 in primary biliary cirrhosis.

BACKGROUND: Primary biliary cirrhosis (PBC) is characterized by chronic progressive destruction of small intrahepatic bile ducts with portal inflammation and cholestasis, leading to fibrosis. METHODS: We utilized a novel restriction analysis system to profile the expression of tyrosine kinases (TKs). This methodology targets a conserved sequence present in the majority of human TKs, and exploits the known restriction map of the TK cDNA sequences. We isolated mRNA from biliary epithelial cell (BEC)-enriched cell fractions, amplified the TK transcripts using degenerative primers, and identified specific TKs by restriction enzyme digest analysis and then performed in situ hybridization. RESULTS: BEC-enriched samples from PBC livers displayed marked expression of discoidin domain receptor-2 (DDR2), whereas, non-diseased livers showed no detectable DDR2. Furthermore, in situ hybridization of PBC livers revealed that DDR2 is expressed in the small bile duct epithelial regions as well as in fibroblasts/stromal cells of fibrotic regions. A similar pattern was observed in livers of primary sclerosing cholangitis (PSC), although the amount of small ducts that were positively stained was lower than in PBC. Furthermore, cirrhotic livers of patients with other diseases, including alcoholic liver disease and chronic hepatitis C, DDR2 transcripts were noted only within fibrotic lesions and the degree of intensity was much lower than in PBC and PSC. CONCLUSIONS: DDR2, a TK that is stimulated by fibrillar collagens that accumulate in cirrhotic livers, is present at elevated levels in the small bile ducts of PBC patients. DDR2 is part of a positive feedback loop in which its enhanced expression leads to enhanced deposition of fibrillar collagens (types I andIII). These fibrillar collagens can also provide binding sites for immune mediators, such as cytokines and chemokines. Therefore, unusually high DDR2 expression in the bile ducts of PBC patients could contribute to duct injury by altering local cytokine levels and thereby increasing immune-mediated damage.

Biliary Tract↗

PubMed2001

Discoidin domain receptor 2 regulates fibroblast proliferation and migration through the extracellular matrix in association with transcriptional activation of matrix metalloproteinase-2.

Discoidin domain receptor 2 (DDR2) is a tyrosine kinase receptor expressed in mesenchymal tissues, the ligand of which is fibrillar collagen. We have compared DDR2 signaling in skin fibroblasts derived from DDR2(-/-) and DDR2(+/-) mice. Proliferation of DDR2(-/-) fibroblasts was significantly decreased compared with DDR2(+/-) cells. DDR2(-/-) fibroblasts exhibited markedly impaired capacity to migrate through a reconstituted basement membrane (Matrigel) in response to a chemotactic stimulus, which correlated with diminished matrix metalloproteinase-2 (MMP-2) activity by gelatin zymography and diminished MMP-2 transcription of a minimal MMP-2 promoter. In contrast, a lack of DDR2 had no effect on cell motility or alpha-smooth muscle actin or vinculin expression. Additionally, expression of type I collagen was greatly reduced in DDR2(-/-) cells. Stable reconstitution of either wild-type DDR2 or constitutively active chimeric DDR2 in DDR2(-/-) cells by retroviral infection restored cell proliferation, migration through a reconstituted basement membrane (Matrigel), and MMP-2 levels to those of DDR2(+/-) fibroblasts. These data establish a role for DDR2 in critical events during wound repair.

Animals↗