BACKGROUND: The role of Smad4 in carcinogenesis is important, because of its function as a central mediator of TGF-beta signaling. In the present study we analyzed the expressions of Smad4 mRNA and protein in human gastric cancer cell lines and tissues and we also analyzed their clinicopathological significance. METHODS: We used semi-quantitative RT-PCR for Smad4 mRNA expression in 13 cases of fresh gastric cancer tissues and two gastric cancer cell lines (MKN-28, SNU-1). We also used immunohistochemistry for Smad4 protein expression in 88 cases of formalin fixed gastric cancers tissues. RESULTS: The mRNA level of Smad4 was higher in MKN-28 cell line (intestinal type) than in the SNU-1 cell line (diffuse type). Fresh frozen gastric cancer tissues showed that the intestinal type of gastric cancer had higher Smad4 mRNA expressions than the diffuse type of gastric cancer (p<0.05). Immunohistochemical staining for Smad4 revealed that cytoplasmic and nuclear expressions of Smad4 were significantly correlated with histologic types of gastric cancer (p<0.05). That is, the intestinal type of gastric cancer showed more cytoplasmic and nuclear smad4 expressions than did the diffuse type of gastric cancer. Reduced cytoplasmic expressions and positive nuclear expressions of Smad4 were more prominent in the advanced gastric cancer than in the early gastric cancer. CONCLUSION: Taken together, we suggest that loss of Smad4 expression might be associated with the intestinal type of gastric cancer. Also reduced cytoplasmic Smad4 expressions and increased nuclear Smad4 expressions may be associated with the advanced stage of gastric cancer.
Adenocarcinoma* ; Carcinogenesis ; Cell Line ; Cytoplasm ; Formaldehyde ; Humans ; Immunohistochemistry ; RNA, Messenger ; Smad4 Protein ; Stomach Neoplasms ; Transforming Growth Factor beta

INTRODUCTION: A cleft palate is a common birth defect in humans with an incidence of 1/500 to 1/1,000 births. It appears to be caused by multiple genetic and environmental factors during palatogenesis. Many molecules are involved in palate formation but the biological mechanisms underlying the normal palate formation and cleft palate are unclear. Accumulating evidence suggests that transforming growth factor beta/bone morphogenetic proteins (TGF-beta/BMP) family members mediate the epithelial-mesenchymal interactions during palate formation. However, their roles in palatal morphogenesis are not completely understood. MATERIALS AND METHODS: To understand the roles of TGF-beta/BMP signaling in vivo during palatogenesis, mice with a palatal mesenchyme-specific deletion of Smad4, a key intracellular mediator of TGF-beta/BMP signaling, were generated and analyzed using the Osr2Ires-Cre mice. RESULTS: The mutant mice were alive at the time of birth with open eyelids and complete cleft palate but died within 24 hours after birth. In skeletal preparation, the horizontal processes of the palatine bones in mutants were not formed and resulted in a complete cleft palate. At E13.5, the palatal shelves of the mutants were growing as normally as those of theirwild type littermates. However, the palatal shelves of the mutants were not elevated at E14.5 in contrast to the elevated palatal shelves of the wild type mice. At E15.5, the palatal shelves of the mutants were elevated over the tongue but did not come in contact with each other, resulting in a cleft palate. CONCLUSION: These results suggest that mesenchymal Smad4 mediated signaling is essential for the growth of palatal processes and suggests that TGF-beta/BMP family members are essential regulators during palate development.
Animals ; Cleft Palate ; Congenital Abnormalities ; Eyelids ; Humans ; Incidence ; Mice ; Morphogenesis ; Palate ; Parturition ; Proteins ; Smad4 Protein ; Tongue ; Transforming Growth Factors

It was recently discovered that a subset of osteoblasts functions as a key component of the hematopoietic stem cells (HSC) niche in vivo, controlling HSC self-renewal and multi-lineage differentiation. Disruption of Smad4 gene specifically in osteoblasts leads to a remarkable decrease of osteoblast number and endosteal surface area. In order to elucidate if the osteoblast loss has any effect on hematopoietic activity, the bone marrow (BM) and extramedullary hematopoiesis in the osteoblast-specific Smad4 knockout mice were systematically analyzed, the proportions of mature hematocytes in BM, liver and spleen were detected by flow cytometry, the hematopoietic progenitor number in different stages was measured by colong-forming assay, CFU-S and analysis of LSK cells. The results indicated that the conditional mutant mice demonstrated normal BM hematopoiesis without sign of extramedullary hematopoiesis. Furthermore, the proportion of hematopoietic progenitor cells was normal, while cell number/body weight of the conditional knockout mice increased. It is concluded that hematopoiesis is normally maintained in osteoblast-specific Smad4 knockout mice, and osteoblast loss does not of necessity result in the decrease in BM hematopoiesis.
Animals ; Bone Marrow Cells ; cytology ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; physiology ; Mice ; Mice, Knockout ; Osteoblasts ; cytology ; physiology ; Smad4 Protein ; genetics

OBJECTIVETo clarify the relationship between the loss of expression of the deleted in pancreatic carcinoma locus 4 (DPC4) proteins and the pathogenesis of biliary tract carcinoma.

METHODS71 primary biliary tract carcinoma (BTCa), including 38 common bile duct (CBD) carcinomas, 18 gallbladder carcinomas, 15 hilar bile ducts (HBD) carcinomas were examined by immunohistochemical staining. In addition, the CBD carcinomas were divided into two groups: tumors with metastasis (M(+) group, 27 cases) and tumors without metastasis (M(-) group, 11 cases).

RESULTSThe frequency of loss of the expression of DPC4 protein was 32.8% in BTCa, 47.3% in CBD carcinoma, 11% in gallbladder carcinoma, 13% in HBD carcinoma. Comparison of the frequency of loss expression of DPC4 was significant statistical difference in CBD carcinoma versus gallbladder carcinoma and HBD carcinoma (P < 0.01). The frequency of loss expression of DPC4 was 48.1% in the M(+) group and 45.4% in the M(-) group.

CONCLUSIONThere are a close relationship between pathogenesis of BTCa and inactivation of DPC4 and different frequencies of DPC4 gene alternation in various locations of the biliary tract, which are not significantly increased with tumor metastasis in BTCa.

Bile Duct Neoplasms ; Biliary Tract ; Carcinoma ; DNA-Binding Proteins ; metabolism ; Humans ; Pancreatic Neoplasms ; Smad4 Protein ; Trans-Activators

The effect of melatonin on juveniles with cardio fibrosis is poorly understood. We investigated whether HDACs participate in the anti-fibrotic processes regulated by melatonin during hypertrophic remodeling. Abdominal aortic constriction (AAC) was employed in juvenile rats resulting in pressure overload-induced ventricular hypertrophy and melatonin was subsequently decreased via continuous light exposure for 5 weeks after surgery. AAC rats displayed an increased cross-sectional area of myocardial fibers and significantly elevated collagen deposition compared to sham-operated rats, as measured by HE and Masson Trichrome staining. Continuous light exposure following surgery exacerbated the increase in the cross-sectional area of myocardial fibers. The expression of HDAC1, HDAC2, HDAC3, HDAC4 and HDAC6 genes were all significantly enhanced in AAC rats with light exposure relative to the other rats. Moreover, the protein level of TNF-α was also upregulated in the AAC light exposure groups when compared with the sham. However, Smad4 protein expression was unchanged in the juveniles' hearts. In contrast, beginning 5 weeks after the operation, the AAC rats were treated with melatonin (10 mg/kg, intraperitoneal injection every evening) or vehicle 4 weeks, and sham rats were given vehicle. The changes in the histological measures of cardio fibrosis and the gene expressions of HDAC1, HDAC2, HDAC3, HDAC4 and HDAC6 were attenuated by melatonin administration. The results reveal that melatonin plays a role in the development of cardio fibrosis and the expression of HDAC1, HDAC2, HDAC3, HDAC4 and HDAC6 in cardiomyocytes.
Animals ; Collagen ; Constriction ; Fibrosis* ; Gene Expression ; Heart ; Histone Deacetylases ; Hypertrophy ; Injections, Intraperitoneal ; Melatonin* ; Myocytes, Cardiac ; Rats* ; Smad4 Protein

BACKGROUNDTransforming growth factor beta (TGF-beta) plays an essential role in the regulation of normal physiologic processes of cells. TGF-beta has been shown to regulate several mitogen-activated protein kinases (MAPK) pathways in several epithelial cells. However, the effects of TGF-beta on soft tissue sarcoma are seldom reported. Our previous studies suggested that there should be some other signal transduction pathways besides Smads, which are important to regulate the growth of human embryonal rhabdomyosarcoma (RMS) cells. In the present study, we examined the expression and functional relations of extracellular signal-regulated kinase 2 (ERK2) and Smad4 in human RMS tissue and a RMS cell line, RD.

METHODSRD cells and normal human primary skeletal myoblasts (Mb) were treated with TGF-beta1 to establish the expression profile of ERK2 at the mRNA and protein levels detected by RT-PCR and immunofluorescence. Immunohistochemistry was used to detect the expression of ERK2 and Smad4 in 50 tissue specimens of human RMS and 23 specimens of normal skeletal muscles. Follow-up of specimens was performed 6 months to 70 months later.

RESULTSRD cells and human RMS tissues showed the higher expression of ERK2 and Smad4 than the normal control, either the protein level or the mRNA level. And, exogenous TGF-beta1 stimulation can lead to higher expression of ERK2 and its nuclear translocation, so TGF-beta1 can also activated MAPK (ERK2) pathway, resulting in a sustained activation of ERK2 for at least 2 hours. Immunohistochemistry analysis, however, showed that there was no correlation between ERK2 and Smad4 protein. The overexpression of ERK2 and Smad4 had no indicative effects on histological subtypes, histological grading, gender, age, and prognosis.

CONCLUSIONSIn RMS, signaling of TGF-beta1 from cell surface to nucleus can also be directed through the MAPK (ERK2) pathway besides the TGF-beta1/Smads pathway. The activation of ERK2 by TGF-beta1 may be Smad4 independent. Moreover, there may be some other tanglesome relationships between the TGF-beta1/Smads pathway and the MAPK pathway which takes part in the development, invasion and metastasis of tumor cells.

Cells, Cultured ; Humans ; Mitogen-Activated Protein Kinase 1 ; physiology ; Muscle, Skeletal ; metabolism ; RNA, Messenger ; analysis ; Rhabdomyosarcoma ; metabolism ; Signal Transduction ; Smad4 Protein ; physiology ; Transforming Growth Factor beta1 ; pharmacology

PURPOSE: Chromosomal instability of chromosome 18 and inhibition of the transforming growth factor beta (TGF-beta) signaling pathway, which is mediated through Smad4, play important roles in the tumorigenesis of colon cancer. This study evaluated the value of the expression of chromosome 18 monosomy in colon cancer as a prognostic factor and its correlations with the expressions of Smad4 and TGF-beta receptor II proteins. METHODS: We analyzed the rate of the expression of chromosome 18 monosomy in 66 colon cancers with using chromogenic in situ hybridization (CISH) and we evaluated its value as a prognostic factor by determining its correlation with the pathologic factors and immunohistochemical expressions of Smad4 and TGF-beta receptor II proteins. RESULTS: Of the 66 colon cancers, monosomy of chromosome 18, as determined by CISH, was observed in 18 cases (27.3%), and the decreased expression of Smad4 and TGF-beta receptor II proteins was observed in 30 cases (45.5%) and 25 cases (37.9%), respectively. The monosomy of chromosome 18 and the decreased expression of Smad4 proteins showed statistically significant correlations with the histologic differentiation, the presence of tumor emboli, the nodal status and the stage. The decreased expression of TGF-beta receptor II proteins had statistically significant correlations with the histologic differentiation, the T-stage, the nodal status and the stage. The monosomy of chromosome 18 showed a statistically significant correlation with the decreased expression of Smad4 and TGF-beta receptor II proteins. CONCLUSION: These results suggested that chromosome 18 monosomy may have prognostic value for colon cancer.
Carcinogenesis ; Chromosomal Instability ; Chromosomes, Human, Pair 18* ; Colon* ; Colonic Neoplasms* ; In Situ Hybridization ; Monosomy* ; Receptors, Transforming Growth Factor beta* ; Smad4 Protein ; Transforming Growth Factor beta*

OBJECTIVETo investigate the expressions of transforming growth factor-beta(1) and Smad4 in the prostatic tissue of rat models of chronic nonbacterial prostatitis (CNP), and to explore the mechanisms of CNP and its fibrosis.

METHODSSixty 6-month-old SD rats were randomly allocated into three groups of equal number: normal control, 30 d CNP model and 45 d CNP model, the models made by castration + high-dose intramuscular injection of estradiol benzoate. The expressions of TGF-beta1 and Smad4 in the prostatic tissue were detected by immunohistochemistry and Western blot.

RESULTSCompared with the normal controls, the 30 d and 45 d CNP rat models showed a significantly increased expression of TGF-beta1 and decreased expression of Smad4 (P < 0.05), even more significantly in the 45 d than in the 30 d group. And the expression of TGF-beta1 was negatively correlated with that of Smad4 in the CNP rat models.

CONCLUSIONTGF-beta1 and Smad4 may be involved in the pathogenesis of CNP, and prostatic fibrosis may make the condition difficult to cure.

Animals ; Disease Models, Animal ; Male ; Prostate ; metabolism ; Prostatitis ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Smad4 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism

Loss of transforming growth factor (TGF)-beta signaling has been implicated in malignant transformation of various tissues. Smad4 plays a central role in the signal transduction of TGF-beta. Deletion or mutation of Smad4 has been described in a number of cancers. This study was aimed to investigate a potential role of Smad4 in leukemia including its expression and location in blast cells. The mononuclear cells were separated from bone marrow of leukemia patients. The samples, blast cells of which were more than 90% in mononuclear cells, were selected. The expression and location of Smad4 protein were analyzed by immunohistochemistry methods. The results showed that the Smad4 protein located mainly in nucleus, part of this protein located in cytoplasma, the expressions of Smad4 were not detected in 6 out of 9 ALL patients, in 7 out of 24 AML patients and in 1 out of 2 CML patients; these leukemia patients, in whose cells the expression of Smad4 was not detected, included one L1 and one L3, four L2, one M0, one M1, two M2a, one M3a, one M4b, one M6 and one CML. In conclusion, the Smad4 protein was mainly in nucleus, the deletion or functional change of Smad4 may related with the pathogenesis of human AML.
Humans ; Leukemia, Myeloid, Acute ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Signal Transduction ; Smad4 Protein ; biosynthesis ; genetics ; Transforming Growth Factor beta ; biosynthesis ; genetics

OBJECTIVETo observe the effect of Tangke Decoction (TD) on the expression of TGF-beta1/Smad4 of rats with early diabetes and to explore the effect and mechanism of TD against the renal injury induced by diabetes.

METHODSSD rats were randomly divided into the normal control group (n = 12), the model group (n = 10), the Chinese herbs prevented group (n =10), the Chinese herbs treated group (n = 10), and the Western medicine control group (n = 10). TD (18 mg/kg) was given by gastrogavage to rats in the Chinese herbs prevented group immediately after successful modeling for 12 weeks, once daily. At the 4th week of successful modeling, rats in the rest 4 groups were administered by gastrogavage. Equal volume of normal saline was given to rats in the model group and the normal control group. Benazepril suspension (1 mg/kg) was administered by gastrogavage to rats in the Western medicine control group for 8 weeks, once daily. TD (18 mg/kg) was given by gastrogavage to rats in the Chinese herbs treated group for 8 weeks, once daily. The body weight, kidney weight, index of kidney weight, fasting blood sugar, 24 h urinary albumin excretion rate were examined after experiment. The pathological changes of the renal tissue were observed by HE staining, Masson staining, and electron microscope. The expression of renal transforming growth factor-beta1, (TGF-beta1) and Smad4 were detected using immunohistochemical assay.

RESULTSCompared with the normal control group, the body weight of rats decreased significantly; the kidney weight, index of kidney weight, blood sugar, 24 h urinary protein excretion, the urinary albumin excretion rate,TGF-beta1 and Smad4 expression increased significantly in the model group (all P < 0.01). Compared with the model group, the aforesaid indices were improved in each treatment group with statistical difference (P < 0.05, P < 0.01). Compared with the Western medicine control group, the kidney weight, index of kidney weight, blood sugar, 24 h urinary protein excretion, and the urinary albumin excretion rate were obviously improved in the Chinese herbs prevented group (P < 0.01). The renal pathological changes were most obvious in the model group significantly, but they were improved in all treatment groups.

CONCLUSIONTD could obviously improve the symptoms of diabetes and down-regulate the expression of renal TGF-beta1 and Smad4 of early diabetic nephropathy rats, which suggested that TD had certain preventive effect on early diabetic nephropathy.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; therapeutic use ; Kidney ; metabolism ; Male ; Rats ; Rats, Wistar ; Smad4 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism