BACKGROUND: Serum response factor (SRF) is a transcriptional factor that plays an important role in cell growth and differentiation for several types of cells. The expression of SRF in cholangiocarcinoma (CC) and its potential role has not been examined. The aim of this study was to determine the relationship between the expression of SRF in CC and the clinicopathological parameters, as well as patient survival. METHODS: We analyzed the expression of SRF in 84 surgically resected cases of CC (33 cases of intrahepatic CC [ICC] and 51 cases of extrahepatic CC [ECC]) by using immunohistochemistry. RESULTS: The positive expression of SRF was detected in 48.8% of the cases of CC (42.4% in ICC, 52.9% in ECC). SRF was predominantly expressed in the CC cells with intense labeling in the nucleus. A SRF expression was significantly associated with the cell proliferation rate (Ki-67 labeling index, p=0.046) and poor patient survival (p=0.002). The tumor differentiation (p=0.038), the T category (p<0.001), lymph node and distant metastasis (p<0.001, p=0.009) and nerve and vessel invasion (p=0.010, p=0.012) were also found to be significantly associated with a poor CC prognosis. CONCLUSIONS: These results suggest that the SRF may play a role in the tumor cell proliferation of CC, and its expression in tumor cells can provide additional prognostic information.
Cell Proliferation ; Cholangiocarcinoma ; Glycosaminoglycans ; Humans ; Immunohistochemistry ; Liver ; Lymph Nodes ; Neoplasm Metastasis ; Prognosis ; Serum Response Factor

Adolescent ; Adult ; Child ; Female ; Heart Failure ; blood ; Humans ; Male ; Middle Aged ; Myocardium ; metabolism ; Serum Response Factor ; metabolism

Serum response factor (SRF) is a master transcription factor of the actin cytoskeleton that binds to highly conserved CArG boxes located within the majority of smooth muscle cell (SMC)-restricted promoters/enhancers. Although most studies of SRF focus on skeletal muscle, cardiac muscle, and vascular SMCs, SRF research has recently expanded into the gastrointestinal (GI) system. Genome scale analyses of GI SMC transcriptome and CArG boxes (CArGome) have identified new SRF target genes. In addition to circular and longitudinal smooth muscle layers, SRF is also expressed in GI mucosa and cancers. In the GI tract, SRF is the central regulator of genes involved in apoptosis, dedifferentiation, proliferation, and migration of cells. Since SRF is the cell phenotypic modulator, it may play an essential role in the development of myopathy, hypertrophy, ulcers, gastric and colon cancers within the GI tract. Given the multi-functional role displayed by SRF in the digestive system, SRF has received more attention emerging as a potential therapeutic target. This review summarizes the findings in SRF research pertaining to the GI tract and provides valuable insight into future directions.
Actin Cytoskeleton ; Apoptosis ; Colonic Neoplasms ; Digestive System ; Gastrointestinal Diseases ; Gastrointestinal Tract ; Genome ; Hypertrophy ; MicroRNAs ; Mucous Membrane ; Muscle Cells ; Muscle, Skeletal ; Muscle, Smooth ; Muscular Diseases ; Myocardium ; Myocytes, Smooth Muscle ; Serum Response Factor* ; Stomach Ulcer ; Transcription Factors ; Transcriptome

RhoA is one of the main members of RhoGTPase family involved in cell morphology, smooth muscle contraction, cytoskeletal microfilaments and stress fiber formation. It has been demonstrated that RhoA modulates endothelial cell permeability by its effect on F-actin rearrangement, but the molecular mechanism of rearrangement of actin cytoskeleton remains unclear. Recent studies prove that RhoA/Rho kinase regulates smooth muscle specific actin dynamics by activating serum response factor (SRF)-dependent transcription. To further investigate the molecular mechanism of the rearrangement of vascular endothelial cell actin cytoskeleton, we explored the relationship between the activation of SRF and F-actin rearrangement induced by RhoA in human umbilical vein endothelial cells (HUVECs). HUVECs were infected with the constitutively active forms of RhoA (Q63LRhoA) or the dominant negative forms of RhoA(T19NRhoA) using retrovirus vector pLNCX-Q63LRhoA or pLNCX-T19NRhoA, the positive clone was obtained by G418 selection. The expression and distribution of SRF in normal and infected cells were evaluated by immunohistochemistry and Western blot in complete medium and in serum-free medium. The effect of F-actin polymerization was detected by Rhodamine-Phalloidine staining. Infection of PLNCX-Q63LRhoA induced F-actin rearrangement and stress fiber formation in HUVECs, as well as enhanced the expression of SRF in the nuclei. In contrast, the cells infected with T19NRhoA showed no distinct changes. With serum deprivation, the expression of SRF increased obviously in both normal and infected HUVECs, but the subcellular localization of SRF was evidently different. In HUVECs, the localization of SRF was in the nuclei after 3 d with serum deprivation, but it was redistributed outside the nuclei after 5 d with serum deprivation. In cells infected with Q63LRhoA, the immunolocalization of SRF was always in the nuclei compared with HUVECs infected with T19NRhoA, which was almost always localized in the cytoplasm. In HUVECs, the rearrangement of F-actin and formation of stress fiber increased after 3 d with serum deprivation, but appeared decreased and unpolymerized after 5 d with serum deprivation. The polymerization of F-actin and the formation of stress fiber in HUVECs infected with Q63LRhoA kept during the period of serum-free culture, whereas the rearrangement of F-actin in cells infected with T19NRhoA was not found. These results suggest that RhoA influences endothelial F-actin rearrangement in part by regulating the expression and subcellular localization of SRF.
Actins ; biosynthesis ; genetics ; Cytoskeleton ; metabolism ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; Protein-Serine-Threonine Kinases ; metabolism ; Serum Response Factor ; biosynthesis ; genetics ; Umbilical Veins ; cytology ; rho-Associated Kinases ; rhoA GTP-Binding Protein ; physiology

To investigate the role and mechanism of Rac1 protein in the process of the human umbilical vein endothelial cell (HUVEC) senescence, we used hypoxia as a model for modulating HUVECs entering replicative senescence in vitro. Premature senescence of HUVECs was evidenced by detecting the SA-beta-Gal activity and PAI-1 expression. Meanwhile, cell cycle distribution and cell proliferation rate were investigated by flow cytometry assay and BrdU staining. The results indicated that the HUVECs became enlarged and flattened, both SA-beta-Gal activity and PAI-1 expression increased obviously, while cell proliferation was inhibited and G(1) phase cell cycle arresting occurred when HUVECs were treated with continued hypoxia for 96 h. Accompanied with these changes, the expression of activated Rac1 increased obviously in cells after hypoxia. All these observations suggested that endothelial senescence could be induced by continued hypoxia and it might correlate with the activity of Rac1. To further define the relationship between Rac1 and HUVEC senescence, HUVECs were transiently infected with the constitutively active form of Rac1 (V12Rac1) or dominant negative form of Rac1 (N17Rac1) using retrovirus vector pLNCX-V12Rac1 or pLNCX-N17Rac1. We observed the changes of these three kinds of HUVECs (HUVECs, N17Rac1-HUVECs, V12Rac1-HUVECs) after hypoxia for 48 h and 96 h, the expression and localization of serum response factor (SRF), which is one of the downstream signal molecules of Rac1, were also investigated. The results obtained indicated that after continued hypoxia for 48 h, HUVECs infected by V12Rac1 showed obvious senescence accompanied with SA-beta-Gal activation, PAI-1 expression increase, G(1) phase arrest and cell proliferation inhibition which were similar to HUVECs after continued 96-hour hypoxia treatment, while the senescence of HUVECs infected by N17Rac1 was significantly inhibited even if the cells were exposed to hypoxia for more than 96 h. All the results identified that the activation of Rac1 might accelerate HUVEC senescence induced by hypoxia and that inactivation of Rac1 could partly block the cell senescence. To further investigate the mechanism of HUVEC senescence induced by Rac1, we detected the expression of total SRF (tSRF) and nuclear SRF (nSRF) in these three kinds of HUVECs by immunofluorescent analysis and Western blot assay after hypoxia. The results showed that the expression of nSRF decreased obviously and the nuclear translocation of SRF was inhibited in HUVECs infected by V12Rac1 compared with those in the normal HUVECs. In contrast, the expression of nSRF increased obviously in the HUVECs infected by N17Rac1. These results suggest that activation of Rac1 accelerates endothelial cell senescence and inhibition of Rac1 activity prevents HUVECs from entering senescence induced by hypoxia, while the nuclear translocation of SRF regulated by Rac1 might play an important role in the process of senescence.
Cell Hypoxia ; Cells, Cultured ; Cellular Senescence ; physiology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Plasminogen Activator Inhibitor 1 ; genetics ; metabolism ; Serum Response Factor ; genetics ; metabolism ; beta-Galactosidase ; metabolism ; rac1 GTP-Binding Protein ; metabolism

OBJECTIVETo explore the inhibition effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on myofibroblast differentiation of MRC-5 human fetal lung fibroblasts induced by angiotensin (Ang) II.

METHODSThe study was divided into 2 step: (1) MRC-5 human fetal lung fibroblasts was induced for 48 h at different dose of Ang II and at different time point by 100 nmol/L Ang II. Then the expression of collagen type I and α-smooth muscle actin (α-SMA) were mesaured by western blot. (2) MRC-5 human fetal lung fibroblasts were divided into 4 group: (1) control, (2) Ang II, (3) Ang II+Ac-SDKP, (4) Ang II+8-Me-cAMP (a specific activator of Epac). The α-SMA expression was observed by immnocytochemical stain. The protein expression of collagen type I, α-SMA, serum response factor (SRF), myocardin-related transcription factor (MRTF)-A, exchange protein directly activated by cAMP (Epac) 1, 2 were measured by Westen blot.

RESULTSMyofibroblast differentiation could be induced by Ang II from MRC-5 cells with a dose- and time-dependent manner. The up-regulation of SRF and MRTF-A were observed in MRC-5 cells induced by Ang II and accompanied with collagen I and α-SMA increased. Pre-treatment with 8-Me-cAMP or Ac-SDKP could attenuated all this changes induced by Ang II, and promoted the expression of Epac1.

CONCLUSIONAc-SDKP can inhibit the myofibroblast differentiation of MRC-5 cells induced by Ang II via Epac1 activating.

Actins ; Angiotensin II ; Cell Differentiation ; drug effects ; Collagen ; Collagen Type I ; Cyclic AMP ; analogs & derivatives ; Fetus ; cytology ; Fibroblasts ; cytology ; Guanine Nucleotide Exchange Factors ; Humans ; Lung ; cytology ; Myofibroblasts ; drug effects ; Oligopeptides ; pharmacology ; Serum Response Factor ; Trans-Activators

BACKGROUND/AIMS: Smooth muscle cells (SMCs) characteristically express serum response factor (SRF), which regulates their development. The role of SRF in SMC plasticity in the pathophysiological conditions of gastrointestinal (GI) tract is less characterized. METHODS: We generated SMC-specific Srf knockout mice and characterized the prenatally lethal phenotype using ultrasound biomicroscopy and histological analysis. We used small bowel partial obstruction surgeries and primary cell culture using cell-specific enhanced green fluorescent protein (EGFP) mouse lines to study phenotypic and molecular changes of SMCs by immunofluorescence, Western blotting, and quantitative polymerase chain reaction. Finally we examined SRF change in human rectal prolapse tissue by immunofluorescence. RESULTS: Congenital SMC-specific Srf knockout mice died before birth and displayed severe GI and cardiac defects. Partial obstruction resulted in an overall increase in SRF protein expression. However, individual SMCs appeared to gradually lose SRF in the hypertrophic muscle. Cells expressing low levels of SRF also expressed low levels of platelet-derived growth factor receptor alpha (PDGFRalphalow) and Ki67. SMCs grown in culture recaptured the phenotypic switch from differentiated SMCs to proliferative PDGFRalphalow cells. The immediate and dramatic reduction of Srf and Myh11 mRNA expression confirmed the phenotypic change. Human rectal prolapse tissue also demonstrated significant loss of SRF expression. CONCLUSIONS: SRF expression in SMCs is essential for prenatal development of the GI tract and heart. Following partial obstruction, SMCs down-regulate SRF to transition into proliferative PDGFRalphalow cells that may represent a phenotype responsible for their plasticity. These findings demonstrate that SRF also plays a critical role in the remodeling process following GI injury.
Animals ; Blotting, Western ; Fluorescent Antibody Technique ; Gastrointestinal Tract ; Heart ; Humans ; Mice ; Mice, Knockout ; Microscopy, Acoustic ; Muscle Cells ; Muscle, Smooth* ; Myocytes, Smooth Muscle ; Parturition ; Phenotype* ; Plastics ; Polymerase Chain Reaction ; Primary Cell Culture ; Receptors, Platelet-Derived Growth Factor ; Rectal Prolapse ; RNA, Messenger ; Serum Response Factor*

OBJECTIVETo investigate whether N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) can inhibit the differentiation of pulmonary fibroblasts into myofibroblasts by regulating Rho-associated coiled-coil forming protein kinase (ROCK) pathway mediated by transforming growth factor-β1 (TGF-β1).

METHODSPrimary culture of pulmonary fibroblasts was performed by trypsinization method. Four generations of pulmonary fibroblasts were divided into control group, TGF-β-induced differentiation group, Y-27632 treatment group, and Ac-SDKP treatment group. The intracellular distributions of ROCK, serum response factor (SRF), and α-smooth muscle actin (α-SMA) were observed by confocal laser scanning microscopy. The protein expression of ROCK, SFR, α-SMA, and type I and type III collagen in pulmonary fibroblasts was measured by Western blot. The mRNA expression of ROCK, SFR, and α-SMA was measured by real-time quantitative PCR.

RESULTSCompared with the control group, the pulmonary fibroblasts stimulated by TGF-β1 had a lot of α-SMA antibody-labeled myofilaments in parallel or cross arrangement, as observed by confocal laser scanning microscopy, and the mRNA and protein expression of ROCK, SRF, and α-SMA and protein expression of type I and type III collagen increased significantly after 6, 12, and 24 h of stimulation (P < 0.05). Compared with the TGF-β1-induced differentiation group, the Y-27632 treatment group and Ac-SDKP treatment group had significantly decreased mRNA and protein expression of ROCK, SRF, and α-SMA and protein expression of type I and type III collagen at the same time point (P < 0.05).

CONCLUSIONAc-SDKP can inhibit the differentiation of pulmonary fibroblasts into myofibroblasts and the synthesis of collagen in rats by regulating the ROCK pathway mediated by TGF-β1. That may be one of the mechanisms by which Ac-SDKP acts against (silicotic) pulmonary fibrosis.

Actins ; metabolism ; Animals ; Animals, Newborn ; Cell Differentiation ; drug effects ; Cells, Cultured ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Fibroblasts ; cytology ; drug effects ; Lung ; cytology ; drug effects ; Myofibroblasts ; cytology ; drug effects ; Oligopeptides ; pharmacology ; Rats ; Rats, Wistar ; Serum Response Factor ; metabolism ; Transforming Growth Factor beta ; pharmacology ; rho-Associated Kinases ; metabolism

OBJECTIVETo study the therapeutic mechanisms of Danshen injection in intrahepatic cholestasis of pregnancy (ICP).

METHODThe experimental serum was collected from 6 patients with ICP before therapy when in hospital. Endothelial cells were isolated from human umbilical veins chosen among the 10 normal pregnancy according to Jaffe's method. Serum of 20% concentration of ICP and different concentration of Danshen injection were cocultured with human vascular endothelial cells (HUVECs) for 24 hours. MTT method was applied to measuring the vitality of HUVECs. The expression value of vascular endothelial growth factor (VEGF) of different groups was detected by immunocytochemistry and enzyme-linked immunosorbent assay (ELISA) respectively.

RESULTCompared with ICP group, the value of MTT and VEGF in the Danshen injection therapeutic groups were increased with the increase of concentration and moreover, 8 g x L(-1) of Danshen injection was the optical concentration. By immunocytochemistry, the expression of VEGF in HUVECs in the Danshen injection therapeutic groups was also enhanced with the increase of concentration.

CONCLUSIONDanshen injection can protect HUVECs against the injury of ICP serum and promote the expression of VEGF. Danshen injection improves HUVECs by increasing the value of VEGF.

Cells, Cultured ; Cholestasis, Intrahepatic ; blood ; Coculture Techniques ; Cytoplasm ; metabolism ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Female ; Humans ; Injections ; Plants, Medicinal ; chemistry ; Pregnancy ; Pregnancy Complications ; blood ; Protective Agents ; administration & dosage ; isolation & purification ; pharmacology ; Salvia miltiorrhiza ; chemistry ; Serum ; physiology ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the effects of the Chinese herbal medicine of Longbixiao (LBX) Capsule on the expressions of TGF-beta1 and Smoothelin in human prostatic stromal cells cultured in vitro.

METHODSBlood serum medicated with LBX was incubated with the stromal cells isolated from men with benign prostatic hyperplasia (BPH) and cultured in vitro. The mRNA expression levels of TGF-beta1 and Smoothelin were detected by real-time RT-PCR and other relevant techniques.

RESULTSIn the high and low concentration groups, the gene relative expressions of TGF-beta1 were (0.158 +/- 0.020) and (0.169 +/- 0.020) , while those of Smoothelin were (0.035 +/- 0.007) and (0.036 +/- 0.007) respectively, both significantly decreased in comparison with the control group(P < 0.01).

CONCLUSIONLBX reduces the mRNA expressions of TGF-beta1 and Smoothelin in human prostatic stromal cells and can be used in the treatment of BPH.

Animals ; Capsules ; Cells, Cultured ; Cytoskeletal Proteins ; genetics ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Gene Expression ; drug effects ; Humans ; Male ; Muscle Proteins ; genetics ; Prostatic Hyperplasia ; pathology ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction ; Serum ; chemistry ; Stromal Cells ; drug effects ; metabolism ; pathology ; Transforming Growth Factor beta1 ; genetics