MiR223-3p promotes synthetic lethality in BRCA1-deficient cancers.

Defects in DNA repair give rise to genomic instability, leading to neoplasia. Cancer cells defective in one DNA repair pathway can become reliant on remaining repair pathways for survival and proliferation. This attribute of cancer cells can be exploited therapeutically, by inhibiting the remaining repair pathway, a process termed synthetic lethality. This process underlies the mechanism of the Poly-ADP ribose polymerase-1 (PARP1) inhibitors in clinical use, which target BRCA1 deficient cancers, which is indispensable for homologous recombination (HR) DNA repair. HR is the major repair pathway for stressed replication forks, but when BRCA1 is deficient, stressed forks are repaired by back-up pathways such as alternative nonhomologous end-joining (aNHEJ). Unlike HR, aNHEJ is nonconservative, and can mediate chromosomal translocations. In this study we have found that miR223-3p decreases expression of PARP1, CtIP, and Pso4, each of which are aNHEJ components. In most cells, high levels of microRNA (miR) 223-3p repress aNHEJ, decreasing the risk of chromosomal translocations. Deletion of the miR223 locus in mice increases PARP1 levels in hematopoietic cells and enhances their risk of unprovoked chromosomal translocations. We also discovered that cancer cells deficient in BRCA1 or its obligate partner BRCA1-Associated Protein-1 (BAP1) routinely repress miR223-3p to permit repair of stressed replication forks via aNHEJ. Reconstituting the expression of miR223-3p in BRCA1- and BAP1-deficient cancer cells results in reduced repair of stressed replication forks and synthetic lethality. Thus, miR223-3p is a negative regulator of the aNHEJ DNA repair and represents a therapeutic pathway for BRCA1- or BAP1-deficient cancers.

Impaired eukaryotic translation initiation factor 2B activity specifically in oligodendrocytes reproduces the pathology of vanishing white matter disease in mice.

Vanishing white matter disease (VWMD) is an inherited autosomal-recessive hypomyelinating disease caused by mutations in eukaryotic translation initiation factor 2B (eIF2B). eIF2B mutations predominantly affect the brain white matter, and the characteristic features of VWMD pathology include myelin loss and foamy oligodendrocytes. Activation of pancreatic endoplasmic reticulum kinase (PERK) has been observed in oligodendrocytes in VWMD. PERK activation in response to endoplasmic reticulum stress attenuates eIF2B activity by phosphorylating eIF2α, suggesting that impaired eIF2B activity in oligodendrocytes induced by VWMD mutations or PERK activation exploit similar mechanisms to promote selective white matter pathology in VWMD. Using transgenic mice that allow for temporally controlled activation of PERK specifically in oligodendrocytes, we discovered that strong PERK activation in oligodendrocytes during development suppressed eIF2B activity and reproduced the characteristic features of VWMD in mice, including hypomyelinating phenotype, foamy oligodendrocytes, and myelin loss. Notably, impaired eIF2B activity induced by PERK activation in oligodendrocytes of fully myelinated adult mice had minimal effects on morphology or function. Our observations point to a cell-autonomous role of impaired eIF2B activity in myelinating oligodendrocytes in the pathogenesis of VWMD.

PERK activation preserves the viability and function of remyelinating oligodendrocytes in immune-mediated demyelinating diseases.

Remyelination occurs in multiple sclerosis (MS) lesions but is generally considered to be insufficient. One of the major challenges in MS research is to understand the causes of remyelination failure and to identify therapeutic targets that promote remyelination. Activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress modulates cell viability and function under stressful conditions. There is evidence that PERK is activated in remyelinating oligodendrocytes in demyelinated lesions in both MS and its animal model, experimental autoimmune encephalomyelitis (EAE). In this study, we sought to determine the role of PERK signaling in remyelinating oligodendrocytes in MS and EAE using transgenic mice that allow temporally controlled activation of PERK signaling specifically in oligodendrocytes. We demonstrated that persistent PERK activation was not deleterious to myelinating oligodendrocytes in young, developing mice or to remyelinating oligodendrocytes in cuprizone-induced demyelinated lesions. We found that enhancing PERK activation, specifically in (re)myelinating oligodendrocytes, protected the cells and myelin against the detrimental effects of interferon-γ, a key proinflammatory cytokine in MS and EAE. More important, we showed that enhancing PERK activation in remyelinating oligodendrocytes at the recovery stage of EAE promoted cell survival and remyelination in EAE demyelinated lesions. Thus, our data provide direct evidence that PERK activation cell-autonomously enhances the survival and preserves function of remyelinating oligodendrocytes in immune-mediated demyelinating diseases.

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cytoprotective agent in several organ systems but its roles in liver fibrosis are unclear. We studied the roles of HB-EGF in experimental liver fibrosis in mice and during hepatic stellate cell (HSC) activation. Thioacetamide (TAA; 100 mg/kg) was administered by intraperitoneal injection three times a week for 4 weeks to wild-type HB-EGF(+/+) or HB-EGF-null (HB-EGF(-/-)) male mice. Livers were examined for histology and expression of key fibrotic markers. Primary cultured HSCs isolated from untreated HB-EGF(+/+) or HB-EGF(-/-) mice were examined for fibrotic markers and/or cell migration either during culture-induced activation or after exogenous HB-EGF (100 ng/ml) treatment. TAA induced liver fibrosis in both HB-EGF(+/+) and HB-EGF(-/-) mice. Hepatic HB-EGF expression was decreased in TAA-treated HB-EGF(+/+) mice by 37.6% (P<0.05) as compared with animals receiving saline alone. HB-EGF(-/-) mice treated with TAA showed increased hepatic α-smooth muscle actin-positive cells and collagen deposition, and, as compared with HB-EGF(+/+) mice, TAA-stimulated hepatic mRNA levels in HB-EGF(-/-) mice were, respectively, 2.1-, 1.7-, 1.8-, 2.2-, 1.2- or 3.3-fold greater for α-smooth muscle actin, α1 chain of collagen I or III (COL1A1 or COL3A1), transforming growth factor-ß1, connective tissue growth factor or tissue inhibitor of metalloproteinase-1 (P<0.05). HB-EGF expression was detectable in primary cultured HSCs from HB-EGF(+/+) mice. Both endogenous and exogenous HB-EGF inhibited HSC activation in primary culture, and HB-EGF enhanced HSC migration. These findings suggest that HB-EGF gene knockout in mice increases susceptibility to chronic TAA-induced hepatic fibrosis and that HB-EGF expression or action is associated with suppression of fibrogenic pathways in HSCs.

Integrin expression and function in the response of primary culture hepatic stellate cells to connective tissue growth factor (CCN2).

Production of connective tissue growth factor (CCN2, also known as CTGF) is a hallmark of hepatic fibrosis. This study examined early primary cultures of hepatic stellate cells (HSC) for (i) CCN2 regulation of its cognate receptor integrin subunits; and (ii) interactions between CCN2 and integrin α(5)ß(1), heparan sulphate proteoglycans (HSPG) or fibronectin (FN) in supporting cell adhesion. HSC were isolated from healthy male Balb/c mice. mRNA levels of CCN2 or α(5), ß(1), αv or ß(3) integrin subunits were measured in days 1-7 primary culture HSC, and day 3 or day 7 cells treated with recombinant CCN2 or CCN2 small interfering RNA. Interactions between CCN2 and integrin α(5)ß(1), HSPG or FN were investigated using an in vitro cell adhesion assay. Co-incident with autonomous activation over the first 7 days, primary culture HSC increasingly expressed mRNA for CCN2 or integrin subunits. Addition of exogenous CCN2 or knockdown of endogenous CCN2 differentially regulated integrin gene expression in day 3 versus day 7 cells. Either full length CCN2 ('CCN2(1-4)') or residues 247-349 containing module 4 alone ('CCN2(4)') supported day 3 cell adhesion in an integrin α(5)ß(1) - and HSPG-dependent fashion. Adhesion of day 3 cells to FN was promoted in an integrin α(5) ß(1)-dependent manner by CCN2(1-4) or CCN2(4), whereas FN promoted HSPG-dependent HSC adhesion to CCN2(1-4) or CCN2(4). These findings suggest CCN2 regulates integrin expression in primary culture HSC and supports HSC adhesion via its binding of cell surface integrin α(5)ß(1), a novel CCN2 receptor in primary culture HSC which interacts co-operatively with HSPG or FN.

HB-EGF protects the lungs after intestinal ischemia/reperfusion injury.

BACKGROUND: Acute respiratory distress syndrome continues to be a major source of morbidity and mortality in critically-ill patients. Heparin binding EGF-like growth factor (HB-EGF) is a biologically active protein that acts as an intestinal cytoprotective agent. We have previously demonstrated that HB-EGF protects the intestines from injury in several different animal models of intestinal injury. In the current study, we investigated the ability of HB-EGF to protect the lungs from remote organ injury after intestinal ischemia/reperfusion (I/R). METHODS: Mice were randomly assigned to one of the following groups: (1) sham-operated; (2) sham+HB-EGF (1200 microg/kg in 0.6 mL administered by intra-luminal injection at the jejuno-ileal junction immediately after identification of the superior mesenteric artery); (3) superior mesenteric artery occlusion for 45 min followed by reperfusion for 6 h (I/R); or (4) I/R+HB-EGF (1200 microg/kg in 0.6 mL) administered 15 min after vascular occlusion. The severity of acute lung injury was determined by histology, morphometric analysis and invasive pulmonary function testing. Animal survival was evaluated using Kaplan-Meier analysis. RESULTS: Mice subjected to intestinal I/R injury showed histologic and functional evidence of acute lung injury and decreased survival compared with sham-operated animals. Compared with mice treated with HB-EGF (I/R+HB-EGF), the I/R group had more severe acute lung injury, and decreased survival. CONCLUSION: Our results demonstrate that HB-EGF reduces the severity of acute lung injury after intestinal I/R in mice. These data demonstrate that HB-EGF may be a potential novel systemic anti-inflammatory agent for the prevention of the systemic inflammatory response syndrome (SIRS) after intestinal injury.

Ethanol-mediated expression of connective tissue growth factor (CCN2) in mouse pancreatic stellate cells.

Activated pancreatic stellate cells (PSC) play a central role in the pathogenesis of pancreatic fibrosis, a common feature of chronic pancreatitis which is often caused by excessive alcohol consumption. In view of the central role of connective tissue growth factor (CCN2) in fibrosis, we investigated the mechanisms by which CCN2 is regulated in PSC following their exposure to ethanol or acetaldehyde. Primary cultures of PSC from Balb/c mice were treated with 0-50 mM ethanol or 0-200 microM acetaldehyde in the presence or absence of 4-methylpyrazole (4MP; an inhibitor of alcohol dehydrogenase), diallyl sulfide (DAS; an inhibitor of cytochrome P4502E1) or anti-oxidant catalase or vitamin D. CCN2 production, assessed by reverse-transcriptase polymerase chain reaction to measure CCN2 mRNA levels or by fluorescence activated cell sorting to assess CCN2 protein, was enhanced in a dose-dependent manner by ethanol or acetaldehyde. In the presence of 4MP, DAS, or the anti-oxidants vitamin D or catalase, there was a substantial decrease in the ability of ethanol to stimulate CCN2 mRNA expression and a concomitant decrease in CCN2-positive PSC. Accumulation of reactive oxygen species in PSC after exposure to ethanol was verified by loading the cells with dichlorofluorescin diacetate and showing that there was a stimulation of its oxidized fluorescent product, the latter of which was diminished in the presence of catalase or vitamin D. These results show the production of acetaldehyde and oxidant stress in mouse PSC are the cause of increased CCN2 mRNA and protein production after exposure of the cells to ethanol. The potential therapeutic effects of inhibitors of ethanol metabolism or anti-oxidants in alcoholic pancreatitis may arise in part through their ability to attenuate CCN2 production by PSC.

Over-expression of C/EBP-alpha induces apoptosis in cultured rat hepatic stellate cells depending on p53 and peroxisome proliferator-activated receptor-gamma.

Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our previous studies, CCAAT enhancer binding protein-alpha (C/EBP-alpha) has been shown to be involved in the activation of HSCs and to have a repression effect on hepatic fibrosis in vivo. However, the mechanisms are largely unknown. In this study, we show that the infection of adenovirus vector expressing C/EBP-alpha gene (Ad-C/EBP-alpha) could induce HSCs apoptosis in a dose- and time-dependent manner by Annexin V/PI staining, caspase-3 activation assay, and flow cytometry. Also, over-expression of C/EBP-alpha resulted in the up-regulation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and P53, while P53 expression was regulated by PPAR-gamma. In addition, Fas, FasL, DR4, DR5, and TRAIL were studied. The results indicated that the death receptor pathway was mainly involved and regulated by PPAR-gamma and p53 in the process of apoptosis triggered by C/EBP-alpha in HSCs.

A selective BCL-XL PROTAC degrader achieves safe and potent antitumor activity.

B-cell lymphoma extra large (BCL-XL) is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-XL inhibitors, such as ABT263, as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-XL proteolysis-targeting chimera (PROTAC), that targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-XL-dependent leukemia and cancer cells but considerably less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing appreciable thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-XL.

Adipokines Deregulate Cellular Communication via Epigenetic Repression of Gap Junction Loci in Obese Endometrial Cancer.

Emerging evidence indicates that adipose stromal cells (ASC) are recruited to enhance cancer development. In this study, we examined the role these adipocyte progenitors play relating to intercellular communication in obesity-associated endometrial cancer. This is particularly relevant given that gap junctions have been implicated in tumor suppression. Examining the effects of ASCs on the transcriptome of endometrial epithelial cells (EEC) in an in vitro coculture system revealed transcriptional repression of GJA1 (encoding the gap junction protein Cx43) and other genes related to intercellular communication. This repression was recapitulated in an obesity mouse model of endometrial cancer. Furthermore, inhibition of plasminogen activator inhibitor 1 (PAI-1), which was the most abundant ASC adipokine, led to reversal of cellular distribution associated with the GJA1 repression profile, suggesting that PAI-1 may mediate actions of ASC on transcriptional regulation in EEC. In an endometrial cancer cohort (n = 141), DNA hypermethylation of GJA1 and related loci TJP2 and PRKCA was observed in primary endometrial endometrioid tumors and was associated with obesity. Pharmacologic reversal of DNA methylation enhanced gap-junction intercellular communication and cell-cell interactions in vitro. Restoring Cx43 expression in endometrial cancer cells reduced cellular migration; conversely, depletion of Cx43 increased cell migration in immortalized normal EEC. Our data suggest that persistent repression by ASC adipokines leads to promoter hypermethylation of GJA1 and related genes in the endometrium, triggering long-term silencing of these loci in endometrial tumors of obese patients. SIGNIFICANCE: Studies reveal that adipose-derived stem cells in endometrial cancer pathogenesis influence epigenetic repression of gap junction loci, which suggests targeting of gap junction activity as a preventive strategy for obesity-associated endometrial cancer.

Aberrant enhancer hypomethylation contributes to hepatic carcinogenesis through global transcriptional reprogramming.

Hepatocellular carcinomas (HCC) exhibit distinct promoter hypermethylation patterns, but the epigenetic regulation and function of transcriptional enhancers remain unclear. Here, our affinity- and bisulfite-based whole-genome sequencing analyses reveal global enhancer hypomethylation in human HCCs. Integrative epigenomic characterization further pinpoints a recurrent hypomethylated enhancer of CCAAT/enhancer-binding protein-beta (C/EBPß) which correlates with C/EBPß over-expression and poorer prognosis of patients. Demethylation of C/EBPß enhancer reactivates a self-reinforcing enhancer-target loop via direct transcriptional up-regulation of enhancer RNA. Conversely, deletion of this enhancer via CRISPR/Cas9 reduces C/EBPß expression and its genome-wide co-occupancy with BRD4 at H3K27ac-marked enhancers and super-enhancers, leading to drastic suppression of driver oncogenes and HCC tumorigenicity. Hepatitis B X protein transgenic mouse model of HCC recapitulates this paradigm, as C/ebpß enhancer hypomethylation associates with oncogenic activation in early tumorigenesis. These results support a causal link between aberrant enhancer hypomethylation and C/EBPß over-expression, thereby contributing to hepatocarcinogenesis through global transcriptional reprogramming.

TGF-ß signal rewiring sustains epithelial-mesenchymal transition of circulating tumor cells in prostate cancer xenograft hosts.

Activation of TGF-ß signaling is known to promote epithelial-mesenchymal transition (EMT) for the development of metastatic castration-resistant prostate cancer (mCRPC). To determine whether targeting TGF-ß signaling alone is sufficient to mitigate mCRPC, we used the CRISPR/Cas9 genome-editing approach to generate a dominant-negative mutation of the cognate receptor TGFBRII that attenuated TGF-ß signaling in mCRPC cells. As a result, the delicate balance of oncogenic homeostasis is perturbed, profoundly uncoupling proliferative and metastatic potential of TGFBRII-edited tumor xenografts. This signaling disturbance triggered feedback rewiring by enhancing ERK signaling known to promote EMT-driven metastasis. Circulating tumor cells displaying upregulated EMT genes had elevated biophysical deformity and an increase in interactions with chaperone macrophages for facilitating metastatic extravasation. Treatment with an ERK inhibitor resulted in decreased aggressive features of CRPC cells in vitro. Therefore, combined targeting of TGF-ß and its backup partner ERK represents an attractive strategy for treating mCRPC patients.

Effects of adrenomedullin gene overexpression on biological behavior of hepatic stellate cells.

AIM: To investigate the effects of adrenomedullin (AM) gene overexpression on the biological characteristics of human hepatic stellate cells (hHSCs) by stable transfection. METHODS: hHSCs which express low basal levels of AM were stably transfected with an expression construct containing rat AM gene or with an empty expression vector. Expression of AM in hHSCs was determined by reverse transcription (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA). Cell proliferation was evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and immunocytochemistry. RT-PCR and Western blot were used to test the expression of procollagen types I and III. Protein expressions of interstitial collagenase (MMP-1), gelatinase (MMP-2) and tissue inhibitors of matrix metalloproteinases-2 (TIMP-2) were assessed by Western blot. RESULTS: Two cell clones (A-2, A-8) transfected with the AM gene expressed higher levels of AM mRNA (non-transfected group: 0.86+/-0.11, empty vector group: 1.01+/-0.11, A-2 clone group: 1.44+/-0.08 and A-8 clone group: 1.36+/-0.05) and protein (12.31+/-0.17, 12.35+/-0.12, 12.56+/-0.06 and 12.62+/-0.07) (P<0.05). AM gene overexpression had inhibitory effects on cell proliferation of hHSCs (29.6%, 30.9%, 18.9% and 21.8%, respectively. P<0.05) and expression of procollagen type I (0.58+/-0.1, 0.48+/-0.11, 0.3+/-0.06 and 0.31+/-0.07 at mRNA level) (0.27+/-0.07, 0.3+/-0.06, 0.14+/-0.05 and 0.13+/-0.05 at protein level) (P<0.05) and procollagen type III (0.17+/-0.04, 0.15+/-0.03, 0.1+/-0.02 and 0.09+/-0.02 at mRNA level) (0.22+/-0.04, 0.2+/-0.03, 0.11+/-0.04 and 0.13+/-0.03 at protein level) (P<0.05). Compared with cells non-transfected (TIMP2: 2.77+/-0.03, MMP-2: 0.5+/-0.04, MMP-1: 0.49+/-0.07) and transfected with empty vector (TIMP2: 2.79+/-0.04, MMP-2: 0.48+/-0.03, MMP-1: 0.45+/-0.09), these two clones had lower expression levels of TIMP2(A-2 clone group: 2.7+/-0.02 and A-8 clone group: 2.71+/-0.02) (P<0.05) and MMP-2(A-2 clone group: 0.15+/-0.05 and A-8 clone group: 0.13+/-0.04) (P<0.05) but displayed a higher expression level of MMP-1(A-2 clone group: 0.68+/-0.06 and A-8 clone group: 0.81+/-0.09) (P<0.05). CONCLUSION: AM gene exerts negative influence to some extent on hHSCs by inhibiting proliferation and production of extracellular matrix (ECM) in addition to inducing MMP-1 expression.

Effect of retinoid kappa receptor alpha (RXRalpha) transfection on the proliferation and phenotype of rat hepatic stellate cells in vitro.

OBJECTIVE: To study the effect of retinoid kappa receptor alpha (RXRalpha) transfection plus treatment with the RXRalpha ligand, 9-cis-RA, on the proliferation and phenotype of platelet-derived growth factor (PDGF) activated hepatic stellate cells (HSCs). METHODS: PDGF activated rat hepatic stellate cells were transfected with eukaryotic expression vector pcDNA3.1- human RXRalpha, and confirmed by Western blot. Proliferation of transfected HSC was assayed by bromodeoxyuridine (BrdU) incorporation as well as MTT, and the phenotype (alpha-smooth muscle actin, desmin) was observed by immunocytochemistry with image analysis. RESULTS: Transfection of the RXRalpha gene and treatment with ligand 9-cis-RA of PDGF-activated HSCs extended the increased expression of RXRalpha protein for at least 168 hours. Cell proliferation and expressions of alpha- smooth muscle actin (alpha-SMA) and desmin were blocked, compared with groups of sham-transfected, PDGF-activated, no transfection, no ligand treatment, and irrelevant ligand treated HSCs. CONCLUSION: Transfection with the RXRalpha gene followed by 9-cis-RA ligand treatment will inhibit the proliferation and reverse the phenotype of activated HSC.

[Expression of CCAAT/enhancer-binding protein in cultured rat hepatic stellate cells and its significance].

OBJECTIVE: The expression of C/EBPalpha protein and mRNA during automatically activation process in primary cultures of HSCs were observed in order to explore its possible association with the proliferation and activation of HSCs. METHODS: Immunocytochemistry, Western blot and RT-PCR were used to evaluated the expression of C/EBPalpha protein and mRNA; as well as the expression of alpha-SMA, Desmin, MMP2, type I procollagen (alpha1). The eukaryotic vector harboring the full length cDNA of C/EBPalpha was transfected into activated HSC, then immunocytochemistry was applied to confirm the transfection and evaluate the effect of transfection on the proliferation of HSC by calculating the PCNA-positive cells. The morphological changes of HSC were observed by use of phase-contrast microscope. RESULTS: Constitutive expression of mRNA and protein of C/EBPalpha were detected in primarily cultured HSCs, and the protein was seen in both nuclei and cytoplasm with the latter being dominant. Their expression levels reached highest at day 2 of the culture, then decreased gradually when continually cultured to the day 4, 7, 10, on the other hand, the expression of alpha-SMA, MMP2 and ColI(alpha1) increased steadily. Transient transfection was verified by the fact that much more and stronger C/EBPalpha stain was observed in transfected HSCs than in void-vector transfected cells. In C/EBPalpha gene transfected HSCs, the number of PCNA-positive cells dramatically decreased compared with the void-vector transfected cells 24h after transfection. In addition, the C/EBPalpha gene transfected HSCs died 36 h after transfection, a few surviving cells became longer and thinner in morphology, however the void-vector transfected cells almost all remained alive. CONCLUSIONS: C/EBPalpha was likely involved in the HSCs activation, and over-expressed C/EBPalpha by transfection had inhibitory influence on the proliferation of cultured rat HSCs.

Regulation of hepatic stellate cells by connective tissue growth factor.

Connective tissue growth factor (CTGF/CCN2) regulates cell proliferation, differentiation, adhesion, chemotaxis, migration, apoptosis and extracellular matrix production. Through these diverse actions, CTGF/CCN2 plays a major role in important physiological and pathophysiological processes such as embryogenesis, implantation, angiogenesis, chondrogenesis, tumorigenesis, differentiation, wound healing and fibrosis. Whereas hepatic levels of CTGF/CCN2 are usually low, elevated levels of hepatic CTGF/CCN2 occur in patients with liver fibrosis and in experimental animal models of liver fibrosis. In fibrotic liver, CTGF/CCN2 is produced by multiple cell types but its sustained expression by and action on hepatic stellate cells is particularly important because these cells assume an activated phenotype during fibrosing injury and are principally responsible for the excessive production of fibrillar collagens, a process that is driven by CTGF/CCN2. Through its direct actions and interactions with other molecules such as fibronectin or transforming growth factor beta-1, CTGF/CCN2 promotes proliferation, survival, migration, adhesion, and extracellular matrix production in activated hepatic stellate cells, thereby promoting hepatic fibrogenic pathways. This review focuses on the regulation of hepatic stellate cell function by CTGF/CCN2.

Over-expressed microRNA-27a and 27b influence fat accumulation and cell proliferation during rat hepatic stellate cell activation.

Hepatic stellate cells (HSCs) activation is an initial event in liver fibrosis. MicroRNAs (miRNAs) have been found to play essential roles in cell differentiation, proliferation, and fat metabolism. In this study, we showed that down-regulation of two over-expressed miRNAs, miR-27a and 27b allowed culture-activated rat HSCs to switch to a more quiescent HSC phenotype, with restored cytoplasmic lipid droplets and decreased cell proliferation. Mechanistically, retinoid X receptor alpha was confirmed to be the target of miR-27a and 27b. These results indicated a new role and mechanism of miR-27a and 27b in regulating fat metabolism and cell proliferation during HSCs activation.

Melatonin prevents H2O2-induced activation of rat hepatic stellate cells.

Considerable evidence shows therapeutic effects of melatonin on liver injury and the involvement of hepatic stellate cells (HSCs) in vivo. In the present studies, we investigate the protective effect of melatonin on H2O2-induced activation of HSCs in vitro. Compared with that in control HSCs, synthesis of collagen type I was increased in H2O2-treated cells. Melatonin pretreatment significantly inhibited the above effects of H2O2 in HSCs. CCAAT/enhancer-binding protein alpha (C/EBP-alpha), which could partially reverse the phenotype of activated HSCs, augmented in HSCs pretreated with melatonin. Moreover, secretion of the most important fibrotic cytokine transforming growth factor beta 1 (TGF-beta1) diminished in melatonin-pretreated HSCs. These results suggest that melatonin prevents H2O2-induced activation of HSCs and that the mechanism involves, at least in part, differential regulation of TGF-beta1 and C/EBP-alpha gene expression.

Adrenomedullin regulates expressions of transforming growth factor-beta1 and beta1-induced matrix metalloproteinase-2 in hepatic stellate cells.

Adrenomedullin (AM), a peptide isolated from human pheochromocytoma, can be produced and secreted by various types of cells including hepatic stellate cells (HSCs), and its possible role in HSCs is not clear now. In the present study, the interactive regulation between transforming growth factor (TGF)-beta1 and AM and the effect of AM on TGF-beta1-induced matrix metalloproteinase (MMP)-2 expression in HSCs were investigated. TGF-beta1 and AM inhibited gene transcript level mutually (real-time reverse transcription-polymerase chain reaction). AM suppressed the protein expression level of TGF-beta1 (Western blot), but TGF-beta1 might have no effect on AM secretion level. MMP-2 protein expression in HSCs was increased in response to TGF-beta1, and upregulation of MMP-2 expression stimulated with TGF-beta1 was suppressed by AM in dose-dependent manner (Western blot). AM decreased the phosphorylation level of extracellular signal-regulated kinase (ERK) in HSCs treated with TGF-beta1, and TGF-beta1-induced MMP-2 expression was suppressed by adding Mitogen-activated protein Kinase/ERK (MEK) inhibitor U(0126) (Western blot)(.) Our results suggest that AM may intervene the activation of HSCs by inhibiting TGF-beta1 production and TGF-beta1-induced MMP-2 expression; AM may suppress the upregulation of MMP-2 expression induced by TGF-beta1 partially through ERK pathway.