The present study was designed to elucidate whether the mechanism by which osthole decreases collagenI/III contents and their ratio is regulating the TGF-β/Smad signaling pathway in TGF-β1-overexpressed mouse cardiac fibroblasts (CFs). These CFs were cultured and treated with different concentrations of osthole. Our results showed that the TGF-β1 expression in the CFs transfected with that the recombinant expression plasmids pcDNA3.1(+)-TGF-β1 was significantly enhanced. After the CFs were treated with 1.25-5 μg·mL of osthole for 24 h, the mRNA and protein expression levels of collagensIand III were reduced. The collagen I/III ratio was also reduced. The mRNA and protein expression levels of TGF-β1, TβRI, Smad2/3, P-Smad2/3, Smad4, and α-SMA were decreased, whereas the expression level of Smad7 was increased. These effects suggested that osthole could inhibit collagen I and III expression and reduce their ratio via the TGF-β/Smad signaling pathway in TGF-β1 overexpressed CFs. These effects of osthole may play beneficial roles in the prevention and treatment of myocardial fibrosis.
Actins ; genetics ; Animals ; Cells, Cultured ; Collagen ; biosynthesis ; genetics ; Coumarins ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Mice ; Myocardium ; cytology ; Protein-Serine-Threonine Kinases ; genetics ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; Receptor, Transforming Growth Factor-beta Type I ; Receptors, Transforming Growth Factor beta ; genetics ; Signal Transduction ; drug effects ; Smad Proteins ; genetics ; Transforming Growth Factor beta1 ; genetics

The deubiquitinating enzyme ubiquitin specific peptidase 15 (USP15) is regarded as a regulator of TGFβ signaling pathway. This process depends on Smad7, the inhibitory factor of the TGFβ signal, and type I TGFβ receptor (TβR-I), one of the receptors of TGFβ. The expression level of USP15 seems to play vital roles in the pathogenesis of many neoplasms, but so far there has been no report about USP15 in psoriasis. In this study, immunohistochemical staining of USP15, TβR-I and Smad7 was performed in 30 paraffin-embedded psoriasis specimens and 10 normal specimens to investigate the expression of USP15, TβR-I and Smad7 in psoriasis and to explore the relevance among them. And USP15 small interfering RNA (USP15 siRNA) was used to transfect Hacat cells to detect the mRNA expression of TβR-I and Smad7. Of 30 cases of psoriasis in active stage, 28, 24 and 26 cases were positive for USP15, TβR-I and Smad7 staining, respectively. The positive rates of USP15 and Smad7 were significantly higher in psoriasis specimens than in normal skin specimens (44.1%±26.0% vs. 6.1%±6.6%, 47.2%±27.1% vs. 6.6%±7.1%), and positive rate of TβR-I (20.3%±22.2%) in psoriasis was lower than that in normal skin specimens (46.7%±18.2%). There was a significant positive correlation between USP15 and Smad7 expression, and significant negative correlations between USP15 and TβR-expression, an I d between TβR- and Smad7 expression I in psoriasis. After transfection of USP15 siRNA in Hacat cells, the expression of TβR-mRNA was up I -regulated and that of Smad7 was down-regulated. It is concluded that USP15 may play a role in the pathogenesis of psoriasis through regulating the TβR-I/Smad7 pathway and there may be other cell signaling pathways interacting with USP15 to take part in the development of psoriasis.
Adult ; Cell Line ; Female ; Gene Expression ; Humans ; Immunohistochemistry ; Keratinocytes ; cytology ; metabolism ; Male ; Middle Aged ; Protein-Serine-Threonine Kinases ; biosynthesis ; genetics ; Psoriasis ; genetics ; metabolism ; RNA Interference ; Receptors, Transforming Growth Factor beta ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; genetics ; Skin ; metabolism ; Smad7 Protein ; biosynthesis ; genetics ; Ubiquitin-Specific Proteases ; biosynthesis ; genetics ; Young Adult

Cytokine and chemokine receptors play a key role in inflammation caused by rheumatoid arthritis (RA). Two isoforms of human CC chemokine receptor R2 (CCR2), the receptor of monocyte chemoattractant protein 1 (MCP-1), have been identified but their relative expression in fibroblast-like synoviocytes (FLS) and their contribution to inflammatory responses mediated by MCP-1 or inflammatory cytokines in patients with RA remain uncertain. We examined the pattern of expression of two CCR2 isoforms upon stimulation by proinflammatory cytokines and CD40 ligation. FLS were prepared from the synovial tissues of RA patients and cultured in the presence of MCP-1, soluble CD40 ligand (sCD40L), TGF-beta, IL-1beta, IL-18, IL-15, and LPS. CCR2A and CCR2B expression was examined by immunohistochemistry, RT-PCR and western blot analysis. IL-15, TNF-alpha and MCP-1 production was determined by ELISA. Immunohistochemistry showed that CCR2A is highly expressed in RA synovium compared with OA synovium. Transcripts of both CCR2A and CCR2B were detected in FLS. Exogenous MCP-1, CD40L, TGF-beta, and IL-15 significantly increased the expression of CCR2A but not CCR2B. Exposure of FLS to sCD40L caused strong upregulation of CCR2A but not of CCR2B protein expression. MCP-1 increased the proliferation of FLS and the production of IL-15, TNF-alpha, and IL-18. Because CCR2A is the main target of regulation by cytokines and CD40 ligation, the relatively higher expression of CCR2A on the cell surface suggests that this isoform of MCP-1 receptor functions as the principal mediator of inflammatory signals in RA FLS.
Arthritis, Rheumatoid/*metabolism ; CD40 Ligand/*pharmacology ; Cells, Cultured ; Chemokine CCL2/*pharmacology ; Chemokines/biosynthesis ; Fibroblasts/*metabolism ; Humans ; Protein Isoforms ; Receptors, CCR2/*biosynthesis ; Synovial Membrane/*pathology ; Transforming Growth Factor beta/*pharmacology

OBJECTIVETo study the feasibility of osteogenic phenotype expression by human skin fibroblasts induced in polyglycolic acid (PGA) foams and the effect of tumor necrosis factor-alpha (TNF-alpha) on the expression of bone morphogenetic protein (BMP) receptors.

METHODSThe fibroblasts were isolated, purified from human skin. (1) Fibroblasts were seeded onto PGA foams. The cell-PGA complexes were cultured in RCCS for 6 weeks, in the media of TNF-alpha (50 U/ml) and BMP-2 (0.1 microg/ml). 1 d, 3 and 6 weeks later, cells and extracellular matrix were investigated by electron microscopic and histochemistry observation respectively. Secretion of osteogenic markers were analyzed by biochemical methods. (2) Fibroblasts were seeded on the glass fragments or culture flasks and treated with TNF-alpha (50 U/ml) in different usage (one-time, all-time). The RT-PCR method and the immunohistochemistry fluorescence staining were used to examine the influence of TNF-alpha on the mRNA expression and the protein expression of the type I BMP receptors at 2, 4, 6, 8 d after treatment.

RESULTSFibroblasts seeded on the PGA foams formed 3-dimensional matrix 3 weeks after seeding, which was demonstrated as osteo-tissue by tetracycline labeling and ARS staining. Cells secreted much more bone-specific alkaline phosphatase (B-AKP) and osteocalcin (OCN) into supernatant than the cells that were cultured in the media without TNF-a and BMP2. Eight days after all-time usage, the TNF-alpha (50 U/ml) increased the expression of the mRNA and protein of the type IB BMP receptor.

CONCLUSIONSFibroblasts on 3-D cell-foam structures can express osteoblastic phenotype under certain inducing conditions. The numerous fibroblasts in body would be a promising resource for cell seeds candidate of tissue- engineered bone. TNF-alpha provides the essential condition for BMP2's target effect on fibroblasts, and combined use of TNF-alpha and BMP2 is one of the regulating factors.

Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein Receptors, Type I ; biosynthesis ; genetics ; Bone Morphogenetic Protein Receptors, Type II ; biosynthesis ; genetics ; Bone Morphogenetic Proteins ; pharmacology ; Cell Culture Techniques ; methods ; Cell Differentiation ; drug effects ; Cells, Cultured ; Drug Synergism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Phenotype ; Polyglycolic Acid ; Transforming Growth Factor beta ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo explore the expression of transforming growth factor beta1 (TGFbeta1) and its type I receptors activin-like kinase 1 (ALK1) and ALK5 mRNA in the development of brain arteriovenous malformation (BAVM).

METHODSThe mRNA expressions of TGFbeta1, ALK1and ALK5 were detected with semiquantitative RT-PCR in patients with BAVM.

RESULTSThe expressions of TGFbeta1 and ALK5 mRNA increased significantly in BAVM, and their relative expression quantity were 0.777-/+0.047 and 0.585-/+0.074, respectively. However, ALK1 mRNA expression declined significantlies with a relative expression of 0.173-/+0.044 in comparison with the control group (0.720-/+0.098, P<0.01).

CONCLUSIONThe balance of TGFbeta1 and its type I receptors ALK1 and ALK5 mRNA expressions may play important role in the development of BAVM.

Activin Receptors, Type II ; genetics ; Adolescent ; Adult ; Brain ; metabolism ; pathology ; Female ; Gene Expression ; Humans ; Intracranial Arteriovenous Malformations ; genetics ; Male ; Middle Aged ; Protein-Serine-Threonine Kinases ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Transforming Growth Factor beta ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta1 ; genetics

Liver cirrhosis is one of the major complications of hepatitis C virus (HCV) infection, but the mechanisms underlying HCV-related fibrogenesis are still not clear. Although the roles of HCV core protein remain poorly understood, it is supposed to play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to examine the role of HCV core protein on the hepatic fibrogenesis. We established an in vitro co-culture system with primary hepatic stellate cell (HSC) isolated from rats, and a stable HepG2-HCV core cell line which had been transfected with HCV core gene. The expressions of fibrosis-related molecules transforming growth factor beta1 (TGF-beta1), transforming growth factor b receptor II (TGF beta RII), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) were analyzed via histological or molecular methods. In addition, the expression levels of matrix metaloprotinase-2 (MMP-2) and collagen type I (Col I) from the co-cultured media were measured by zymogram and ELISA, respectively. The expressions of alpha-SMA, TGF-beta1, Col I, TGF beta RII and MMP-2 were significantly increased in the co-culture of stable HepG2-HCV core with HSC. Moreover, the significant increases of CTGF and TGF-beta1 in the HCV core-expressing cells were observed by either Northern or Western blot analysis. These results suggest that HCV core protein may contribute to the hepatic fibrogenesis via up-regulation of CTGF and TGF-beta1.
Actins/metabolism ; Animals ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Collagen Type I/metabolism ; Gelatinase A/metabolism ; Immediate-Early Proteins/*biosynthesis ; Intercellular Signaling Peptides and Proteins/*biosynthesis ; Liver/metabolism/*pathology ; Liver Cirrhosis/*metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Transforming Growth Factor beta/metabolism ; Research Support, Non-U.S. Gov't ; Transforming Growth Factor beta/*metabolism ; Up-Regulation ; Viral Core Proteins/genetics/*metabolism

OBJECTIVETo investigate the influence of dermal template on the expressions of signal transduction protein Smad 3 and transforming growth factor beta1 and its receptor during wound healing process in patients with deep burns.

METHODSTwenty burn patients with excision of full thickness burn in the extremities were enrolled in the study and divided into two groups, i.e. template interfering group (E, n = 20, grafting of dermal template [allogeneic acellular dermal matrix] with razor thin autoskin) and control group (C, n = 20, grafting of razor thin autoskin only). The contralateral side served as the self-control. Tissue samples from the burn wounds were harvested at 1, 2, 3 and 4 post-operative weeks (POW) for immunohistochemistry staining. The positive expression rates of TGF-beta1, TbetaRI, TbetaRII and Smad3 proteins were determined by image analysis system.

RESULTSThe positive expressions of TGFbeta1, TbetaRI, TbetaRII and signal transduction protein Smad 3 in the tissue samples in both groups could be identified during 1 approximately 4 POW, and they diminished thereafter with the process of wound healing. The expression rate of TGF-beta1 in E group was (13.08 +/- 4.65)% at 1 POW and (9.03 +/- 1.89)% at 4 POW. The positive expression rate of above indices in E group was obviously lower than that in C group in corresponding time points (P < 0.05).

CONCLUSIONThe expression levels of TGFbeta1, TbetaRI, TbetaRII and Smad 3 protein in deep burn wounds could be lowered by mixed grafting of dermal template with razor thin autoskin, which might be beneficial in ameliorating of scar hyperplasia in the burn wound.

Adolescent ; Adult ; Burns ; metabolism ; surgery ; Dermis ; transplantation ; Humans ; Middle Aged ; Receptors, Transforming Growth Factor beta ; biosynthesis ; Signal Transduction ; Skin Transplantation ; Smad3 Protein ; biosynthesis ; Transforming Growth Factor beta1 ; biosynthesis ; Transplantation, Heterologous ; Wound Healing

OBJECTIVETo study the expression of Smad4 and transforming growth factor-beta(1) (TGFbeta(1)), transforming growth factor-beta receptor II (TGFbetaRII) in cholangiocarcinoma tissue and its relationship with the biological behaviour and prognosis of the disease.

METHODSThe expressions of Smad4, TGFbeta(1) and TGFbetaRII were detected by immunohistochemical technique in 47 specimens of cholangiocarcinoma and the normal bile duct tissue adjacent to the tumor. The expressions of Smad4, TGFbeta(1) and TGFbetaRII were compared with the clinical stages and pathological grades of the patients.

RESULTSThe expression of TGFbeta(1) was positive in 36 cholangiocarcinomas (76.6%), which was higher than that in the normal tissue adjacent to the lesion. The positive expressions of Smad4 and TGFbetaRII were 14 (29.8%) and 28 (59.6%) in the carcinoma tissues, respectively (P < 0.05). The expression of TGFbeta(1) was related to the clinical stage, metastasis of lymph node and liver of the tumor (P < 0.05), but not with the histological grade (P > 0.05). There was positive correlation between TGFbetaRII expression and the clinical stage (P < 0.05), but no correlation between the TGFbetaRII expression and histological grade or metastasis of lymph node and liver (P > 0.05). The expression of Smad4 was associated with the histological grade, clinical stage and metastasis of lymph node and liver (P < 0.05).

CONCLUSIONSThe expressions of Smad4, TGFbeta(1) and TGFbetaRII correlate with the histological grading, clinical staging and metastasis of the lymph node and liver in cholangiocarcinoma. Combined detection of Smad4, TGFbeta(1) and TGFbetaRII may be helpful in the determination of the malignant degree and the prognosis of this disease.

Adult ; Aged ; Bile Duct Neoplasms ; metabolism ; pathology ; Bile Ducts, Intrahepatic ; Biomarkers, Tumor ; biosynthesis ; Cholangiocarcinoma ; metabolism ; secondary ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Neoplasm Metastasis ; Neoplasm Staging ; Protein-Serine-Threonine Kinases ; Receptors, Transforming Growth Factor beta ; biosynthesis ; Smad4 Protein ; biosynthesis ; Transforming Growth Factor beta1 ; biosynthesis

OBJECTIVETo observe the effect of octreotide (OCT) on inhibiting hepatocellular carcinoma (HCC) and investigate its mechanisms.

METHODSNude mice bearing xenografts in situ were treated with OCT or saline control for 7 weeks since tumor implantation. The immunohistochemistry for somatostatin receptor 2 (SSTR2), cMet, transforming growth factor beta1 (TGFbeta1), phospho-Smad2, Smad4 and Smad7 was performed. SSTR2 and Smad4 mRNA expression was measured by semi-quantitative RT-PCR.

RESULTSAfter OCT treatment, the mean tumor weight in mice given OCT (0.17 +/- 0.14 g) was significantly lower than that of the control group (0.53 +/- 0.06 g). The inhibition rate of tumor was 67.9%. mRNA and protein expression of SSTR2, Smad4 in tumor cells of the treatment group were significantly more than that of the control group. cMet expression in OCT group was remarkably lower than that in control group. Between two groups, the expression of TGFbeta1, phospho-Smad2 and Smad7 were not remarkably different. In addition, phospho-Smad2 expression in HCC was significantly less than that of the normal hepatic cell.

CONCLUSIONOCT can inhibit the growth of HCC xenografts markedly. The mechanisms of OCT-induced inhibition effect may be related to up-regulating SSTR2 expression, down-regulating cMet, and recovering the function of TGFbeta/Smads-induced antitumor. In addition, the decreased expression of phospho-Smad2 may be an important feature of Bel7402 cells.

Animals ; Antineoplastic Agents, Hormonal ; therapeutic use ; Humans ; Liver Neoplasms, Experimental ; drug therapy ; metabolism ; pathology ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Octreotide ; therapeutic use ; Proto-Oncogene Proteins c-met ; biosynthesis ; Receptors, Somatostatin ; biosynthesis ; Smad2 Protein ; biosynthesis ; Transforming Growth Factor beta ; biosynthesis

Angiotensin II ; pharmacology ; Animals ; Cells, Cultured ; Cyclooxygenase 2 ; biosynthesis ; genetics ; Kupffer Cells ; cytology ; metabolism ; NF-kappa B ; metabolism ; Platelet-Derived Growth Factor ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-sis ; Rats ; Rats, Wistar ; Receptors, Angiotensin ; biosynthesis ; genetics ; Transforming Growth Factor beta ; biosynthesis ; genetics