Tissue formation and organ homeostasis are achieved by precise coordination of proliferation and differentiation of stem cells and progenitors. While deregulation of these processes can result in degenerative disease or cancer, their molecular interplays remain unclear. Here we show that the switch of human pluripotent stem cell (hPSC) self-renewal to differentiation is associated with the induction of distinct cyclin-dependent kinase inhibitors (CDKIs). In hPSCs, Activin/Nodal/TGFβ signaling maintains CDKIs in a poised state via SMAD2/3-NANOG-OCT4-EZH2-SNON transcriptional complex. Upon gradual differentiation, CDKIs are induced by successive transcriptional complexes between SMAD2/3-SMYD2 and developmental regulators such as EOMES, thereby lengthening the G1 phase. This, in turn, induces SMAD2/3 transcriptional activity by blocking its linker phosphorylation. Such SMAD2/3-CDKI positive feedback loops drive the exit from pluripotency and stepwise cell-fate specification that could be harnessed for producing cells for therapeutic applications. Our study uncovers fundamental mechanisms of how cell-fate specification is interconnected to cell-cycle dynamics and provides insight into autonomous circuitries governing tissue self-formation.
Humans ; Smad2 Protein/genetics* ; Smad3 Protein/genetics* ; Cell Differentiation ; Pluripotent Stem Cells/metabolism* ; Signal Transduction ; Octamer Transcription Factor-3/genetics* ; Enhancer of Zeste Homolog 2 Protein/genetics* ; Nanog Homeobox Protein/genetics* ; Phosphorylation

This study aimed to investigate the potential mechanism and the compatibility significance of Tanyu Tongzhi Formula in treating atherosclerosis(AS) in mice based on the transforming growth factor-β(TGF-β)/Smad2/3 signaling pathway. Eight C57BL/6J mice were as assigned to a normal control group and fed a regular diet, while 35 ApoE~(-/-) mice of the same strain were fed a high-fat diet for 8 weeks to establish an AS model. The model mice were randomly divided into a model group, a Tanyu Tongzhi group(18.2 mg·kg~(-1)), a Huatan(phlegm-resolving) group(10.4 mg·kg~(-1)), and a Quyu(blood stasis-resolving) group(7.8 mg·kg~(-1)), with 8 mice in each group. Except for the normal group, all other groups continued to be fed a high-fat diet for 8 weeks to maintain the AS model, and then the mice were treated by gavage for 8 weeks. Plasma levels of total cholesterol(TC), triglycerides(TG), low-density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C), interleukin-1β(IL-1β), and interleukin-18(IL-18) were measured using enzyme-linked immunosorbent assay(ELISA). Hematoxylin and eosin(HE) staining, oil red O staining, and Russell-Movat pentachrome staining were performed to observe the pathological changes in the aortic tissue. The proportions of aortic plaque area, lipid-stained area, collagen fibers, and elastic fibers were calculated. Immunofluorescence was used to detect the protein expression levels of matrix metalloproteinase 2(MMP2) and tissue inhibitor of metalloproteinases 2(TIMP2). Western blot was used to detect the protein expression levels of TGF-β1, TGF-β2, Smad2/3, and Smad7 in aortic tissue. Real-time fluorescence quantitative PCR(RT-qPCR) was used to measure the mRNA expression levels of TGF-β receptor(TGF-βR), TGF-β1, Smad2/3, Smad7, intercellular adhesion molecule-1(ICAM-1), and vascular cell adhesion molecule-1(VCAM-1) in aortic tissue. The results showed that compared with the normal control group, the model group had increased plasma TC and LDL-C, significantly decreased HDL-C, and significantly elevated plasma IL-1β and IL-18 levels. The model group also exhibited an increased proportion of aortic plaque area, lipid-stained area, and collagen fiber area, along with significantly upregulated MMP2 and downregulated TIMP2 expression in the aortic arch. Additionally, the expression levels of TGF-βR, TGF-β1, and p-Smad2/3 proteins and mRNA in the aortic tissue were significantly elevated, while Smad7 expression was decreased. Compared with the model group, the Tanyu Tongzhi group showed significantly reduced plasma TC and LDL-C levels, significantly increased HDL-C levels, and significantly decreased plasma IL-1β and IL-18 levels. The Tanyu Tongzhi group also exhibited a significant reduction in aortic plaque size and severity, a significant downregulation of MMP2 expression in the aortic arch, and significantly decreased ICAM-1 and VCAM-1 mRNA expression levels. Moreover, the Tanyu Tongzhi group demonstrated significantly reduced expression levels of TGF-β1 and p-Smad2/3 proteins and mRNA in the aortic tissue, and an increased expression level of Smad7 protein to varying degrees. Compared with the Tanyu Tongzhi group, the Quyu group had significantly higher LDL-C levels and elevated plasma IL-1β and IL-18 levels. The Huatan group showed upregulated MMP2 expression and downregulated TIMP2 expression in the aortic arch. In conclusion, Tanyu Tongzhi Formula, which is composed based on the pathogenesis of phlegm and blood stasis, maintains vascular homeostasis by primarily regulating lipid metabolism and controlling inflammatory factors through the Huatan group, and maintaining vascular wall permeability, inhibiting plaque development, and stabilizing plaques through the Quyu group. The mechanism of action may involve inhibiting TGF-β1 expression in the aorta, reducing Smad2/3 phosphorylation, and simultaneously increasing Smad7 expression.
Animals ; Atherosclerosis/metabolism* ; Signal Transduction/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Mice, Inbred C57BL ; Male ; Transforming Growth Factor beta/genetics* ; Humans ; Homeostasis/drug effects* ; Aorta/metabolism* ; Smad2 Protein/genetics* ; Smad3 Protein/genetics*

OBJECTIVE: To investigate the intervention of melatonin (MT) in the expression of circadian genes in patients with pulmonary fibrosis and to analyze the mechanism by which it alleviates the progression of pulmonary fibrosis. METHODS: By utilizing the Gene Expression Omnibus (GEO) database, we identified differentially expressed circadian genes between patients with pulmonary fibrosis and controls. We analyzed the correlation between circadian genes and pulmonary function as well as genes related to pulmonary fibrosis. A bleomycin-induced mouse model of pulmonary fibrosis (BLM group) was constructed to observe the expression differences of PER2 and CRY2 by sequencing and immunohistochemical staining in the BLM group and after MT intervention (BLM+MT group). Hematoxylin and eosin (HE) staining and Masson staining were used to observe the effects of MT on fibrosis. We used Western blot to detect the expression of P-smad2/3 in lung epithelial cells induced by transforming growth factor β (TGF-β). Reverse transcription quantitative real-time PCR technology was employed to investigate the rhythmic expression changes of circadian genes in the control group, TGF-β group, and TGF-β+MT group. Finally, luzindole, a MT receptor antagonist, was used to intervene in TGF-β+MT group, and Western blot was used to explore the receptor dependence of MT in alleviating TGF-β-induced epithelial-mesenchymal transition. RESULTS: (1) Analysis of the GEO dataset (GSE) revealed a negative correlation between circadian genes PER2 and CRY2 and the expression of TGF-β, and a positive correlation with pulmonary function indicators in patients. (2) Transcriptome sequencing analysis of lung tissue in BLM group found that the expression of PER2 and CRY2 was significantly reduced compared with the normal group. Histopathological staining results showed that the lung tissue structure of the normal group was intact and clear, with thin alveolar septa; in the BLM group, there was a large increase in collagen fibers and disordered alveolar structure; compared with the BLM group, the BLM+MT group had reduced collagen fiber proliferation and inflammatory cell infiltration; the expression of PER2 and CRY2 in the BLM group was lower than in the normal group, and the expression in the BLM+MT group was increased compared with the BLM group. (3) In vitro lung epithelial cell experiments with TGF-β intervention showed that compared with the control group, the expression of P-smad2/3 increased in the TGF-β group, and MT intervention inhibited the inducing effect of TGF-β on P-smad2/3, while intervention with the MT receptor antagonist reversed this phenomenon. The results indicated that MT could inhibit the activation of the TGF-β pathway, and this process was dependent on MT receptors. (4) The 48-hour rhythm experiment in lung epithelial cells showed that the mRNA rhythm of PER2 and CRY2 in the TGF-β+MT group was close to 24 hours and showed a trend towards restoring the rhythm of the control group, while the addition of the MT receptor blocker tended to make the rhythm duration and amplitude of both groups approach that of the TGF-β group. CONCLUSION MT, by binding to its receptors, can restore the periodic expression of the circadian genes PER2 and CRY2, thereby inhibiting the activation of the TGF-β classical pathway and suppressing the pathological process of epithelial-mesenchymal transition in pulmonary fibrosis. This finding provides new molecular targets and potential therapeutic strategies for the treatment of pulmonary fibrosis.
Melatonin/pharmacology* ; Animals ; Mice ; Pulmonary Fibrosis/chemically induced* ; Bleomycin ; Humans ; Transforming Growth Factor beta/metabolism* ; Period Circadian Proteins/metabolism* ; Smad3 Protein/genetics* ; Disease Models, Animal ; Lung/pathology* ; Cryptochromes/metabolism* ; Smad2 Protein/genetics* ; Epithelial Cells/metabolism* ; Mice, Inbred C57BL

To explore the influence for combination of nourishing yin and tonifying yang sequential therapy (NYTYST) with Western medicine in treating anovulatory infertility rats with diminished ovarian reserve (DOR) based on TGF-β1/Smads signaling pathway. 
 Methods: A total of 40 female rats were randomly divided into 5 groups, a normal control group, a model group, a Western medicine group, a NYTYST group and a combination group (n=8 in each group). The DOR model was established through orally taking tripterygium pill for continuous 2 weeks. The normal control group and the model group were treated with saline for 10 days. The Western medicine group was treated with hormone replacement therapy (HRT) and ovarian stimulation. The NYTYST group was treated with nourishing yin herbs in proestrus and tonifying yang herbs in late estrus and the combination group was treated with Chinese herb and Western drugs for 10 days. HE staining was used to observe histopathologic changes in ovary. Expression levels of transforming growth factor β1 receptor (TGF-β1R) in rats ovarian were detected by immunohistochemistry. Expression levels of Smad2, Smad3 and Smad7 protein in rat ovarian were detected by Western blot.
 Results: Compared with the control group, the numbers of developing follicles, mature follicles and corpus luteum were decreased , while atrefic follicles were increased significantly in the model group (P<0.01); the levels of TGF-β1R, Smad2 and Smad3 were decreased significantly, while Smad7 was increased significantly (P<0.01). Compared with the model group, the numbers of developing follicles, mature follicles and corpus luteum, Smad2 and Smad3 expression were increased, while atrefic follicles and Smad7 were decreased significantly in the treatment group (P<0.05 or P<0.01). The numbers of developing follicles and corpus luteum in the combination group was superior to the Western medicine group (P<0.05). Compared with the Western medicine group, the levels of TGF-β1R, Smad2 and Smad3 were increased significantly, while Smad7 was decreased significantly in the combination group (P<0.05 or P<0.01). 
 Conclusion: NYTYST combined with Western medicine can improve the function of ovaries reserve by up-regulation of TGF-β1R, Smad2 and Smad3 while down-regulation of Smad7 in DOR rats.
Animals ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Gene Expression Regulation ; drug effects ; Infertility ; therapy ; Medicine, Chinese Traditional ; Ovarian Reserve ; drug effects ; Rats ; Signal Transduction ; drug effects ; Smad2 Protein ; genetics ; metabolism ; Smad3 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

OBJECTIVETo detect the expression of important proteins associated with transforming growth factor-beta (TGF-beta)/Smad signaling pathway in Peutz-Jeghers syndrome (PJS) and investigate the correlation of these proteins to LKB1 gene expression.

METHODSThe expression and localization of LKB1, TGFbeta1 and pSmad2 proteins in 20 PJS polyp samples and normal intestinal mucosal tissues were detected with immunohistochemical staining.

RESULTSThe expressions of LKB1, TGFbeta1 and pSmad2 were lower in PJS polyps than in normal mucosa, and the differences in LKB1 and TGFbeta1 proteins were significantly different between them (P<0.05). In PJS polyps, positive correlations were found between LKB1 and TGFbeta1 and between TGFbeta1 and pSmad2 expressions.

CONCLUSIONTGFbeta/Smad pathway is probably subjected to the regulation by LKB1 and may play a role in the occurrence of PJS.

Humans ; Immunohistochemistry ; Peutz-Jeghers Syndrome ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Signal Transduction ; Smad2 Protein ; genetics ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism

The peroxisome proliferator activated receptor (PPAR)gamma agonist is used as antidiabetic agent with antihyperglycemic and antihyperinsulinemic actions. Beyond these actions, antifibrotic effects have been reported. We examined antifibrotic effects of PPARgamma agonist and interaction with angiotensin receptor antagonist in the unilateral ureteral obstruction (UUO) model. After UUO, mice were divided to four groups: no treatment (CONT), pioglitazone treatment, L158809 treatment, and L158809+ pioglitazone treatment. On day 14, CONT mice showed severe fibrosis and all treated mice showed decreased fibrosis. The immunohistochmistry of PAI-1, F4/80 and p-Smad2 demonstrated that their expressions were increased in CONT group and decreased in the all treated groups compared to CONT. PAI-1 and p-Smad2 determined from Western blotting, among treated groups, was decreased compared to CONT group. The expression of TGF-beta1 from real time RT PCR showed markedly increased in the CONT group and decreased in all treated groups compared to CONT. These data suggest the pioglitazone inhibited tubulointerstitial fibrosis, however, the synergism between pioglitazone and L158809 is not clear. Considering decreased expression of PAI-1 and TGF-beta/Smad2 in the treated groups, PAI-1 and TGF-beta are likely linked to the decreased renal tubulointerstitial fibrosis. According to these results, the PPARgamma agonist might be used in the treatment of renal fibrotic disease.
*Angiotensin Receptor Antagonists ; Animals ; Antigens, Differentiation/metabolism ; Disease Models, Animal ; Fibrosis ; Hypoglycemic Agents/pharmacology ; Kidney/metabolism/*pathology ; Male ; Mice ; Mice, Inbred C57BL ; PPAR gamma/*agonists ; Plasminogen Activator Inhibitor 1/metabolism ; Smad2 Protein/metabolism ; Thiazolidinediones/pharmacology ; Transforming Growth Factor beta1/genetics/metabolism ; Ureteral Obstruction/metabolism/pathology

OBJECTIVE: To explore the mechanism of enalapril for renal interstitial fibrosis by observing the effect of enalapril on the expression of transforming growth factor-beta1(TGF-beta1), p-Smad2/3 and Smad7 in renal tissuess of unilateral urethral obstruction (UUO) rat model. METHODS: Thirty female Sprague-Dawley(SD) rats were randomly subdivided into a sham-operated group, a model group and an enalapril treated group. UUO model was induced by ligating the left ureter of rats. All rats were sacrificed 14 days after UUO. Pathological changes of the renal tissue were observed by HE and Masson staining, the protein expressions of Collagen I (ColI), TGF-beta1, p-Smad2/3 and Smad7 were detected by immunohistochemical staining,and the mRNA expressions of TGF-beta1 and Smad7 were detected by RT-PCR. RESULTS: The renal interstitial damage index, the relative Collagen area and the expression of ColI in the model group significantly increased(P<0.01). Enalapril reduced these indexes. The protein and mRNA expressions of TGF-beta1 and the protein expressions of p-Smad2/3 were low in the sham-operated group, but were strongly positive in the model group, and enalapril could decrease the expressions of TGF-beta1 and p-Smad2/3(P<0.01). The protein and mRNA expressions of Smad7 in the model group were less than that in the sham-operated group(P<0.01),and enalapril could improve the expressions of Smad7(P<0.01). CONCLUSION Enalapril could inhibit the renal interstitial fibrosis by affecting TGF-beta1, p-Smad2/3 and Smad7 of TGF-beta/smads pathway in the renal tissues of UUO rats.
Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Enalapril ; pharmacology ; Female ; Fibrosis ; prevention & control ; Kidney ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Smad2 Protein ; genetics ; metabolism ; Smad3 Protein ; genetics ; metabolism ; Smad7 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Urethral Obstruction ; complications

OBJECTIVETo investigate the changes in transforming growth factor beta 1 (TGF-beta1)/Smads signaling pathway in rats with chemical hepatocarcinogenesis.

METHODSFresh diethylnitrosamine (DENA) solution was administered in SD rats to induce hepatocellular carcinoma (HCC). The protein expressions of TGF-beta1, phosphorylated Smad2, Smad4 and Smad7 were detected in these rats with immunohistochemistry, and the mRNA expression of Smad4 was evaluated with RT-PCR.

RESULTSCirrhotic nodules occurred in the rats 8 weeks after DENA treatment, and HCC nodules were found 16 weeks after the treatment. In the normal liver tissue, very low levels of TGF-beta1 and Smad4 expressions, low Smad7 expression and high phosphorylated Smad2 expression were detected. The development of liver cirrhosis was accompanied by increased expressions of TGF-beta1, Smad4 and Smad7 but at 8 weeks after DENA treatment, the expression of phosphorylated Smad2 was significantly decreased, followed then by gradual increment till nearly the normal level. Twenty-two weeks after DENA treatment, Smad4 expression in liver tissue decreased markedly as compared with the levels at 8 and 16 weeks. The expressions of Smad4 and phosphorylated Smad2 in the HCC tissue was significantly lower than those in normal liver tissue.

CONCLUSIONHepatocarcinogenesis involves very complex mechanisms, can can be related partially to the decreased Smad4 and phosphorylated Smad2 expression and TGFbeta1 and Smad7 overexpression in advanced stage of liver cirrhosis.

Animals ; Diethylnitrosamine ; Liver Neoplasms, Experimental ; chemically induced ; metabolism ; pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad2 Protein ; metabolism ; Smad4 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

Total saponins of Panax notoginseng (PNS) have been shown to ameliorate renal interstitial fibrosis. Ginsenoside Rg1, a panaxatriol saponin, is one of the major active molecules from PNS. The present study was undertaken to investigate the effect of ginsenoside Rg1 on renal fibrosis in rats with unilateral ureteral obstruction (UUO). The rats were randomly divided into 3 groups: sham-operation (n=15), UUO (n=15) and UUO with ginsenoside Rg1 treatment (n=15, 50 mg per kg body weight, intraperitoneally (i.p.) injected). The rats were sacrificed on Days 7 and 14 after the surgery. Histological examination demonstrated that ginsenoside Rg1 significantly inhibited interstitial fibrosis including tubular injury as well as collagen deposition. alpha-smooth muscle actin (alpha-SMA) and E-cadherin are two markers of tubular epithelial-myofibroblast transition (TEMT). Interestingly, ginsenoside Rg1 notably decreased alpha-SMA expression and simultaneously enhanced E-cadherin expression. The messenger RNA (mRNA) of transforming growth factor-beta1 (TGF-beta1), a key mediator to regulate TEMT, in the obstructed kidney increased dramatically, but was found to decrease significantly after administration of ginsenoside Rg1. Further study showed that ginsenoside Rg1 considerably decreased the levels of both active TGF-beta1 and phosphorylated Smad2 (pSmad2). Moreover, ginsenoside Rg1 substantially suppressed the expression of thrombospondin-1 (TSP-1), a cytokine which can promote the transcription of TGF-beta1 mRNA and the activation of latent TGF-beta1. These results suggest that ginsenoside Rg1 inhibits renal interstitial fibrosis in rats with UUO. The mechanism might be partly related to the blocking of TEMT via suppressing the expression of TSP-1.
Actins ; biosynthesis ; Animals ; Cadherins ; biosynthesis ; Collagen Type I ; genetics ; metabolism ; Fibronectins ; genetics ; metabolism ; Ginsenosides ; pharmacology ; Immunohistochemistry ; Male ; Nephritis, Interstitial ; drug therapy ; genetics ; metabolism ; pathology ; Panax notoginseng ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Smad2 Protein ; biosynthesis ; Thrombospondin 1 ; biosynthesis ; genetics ; Transforming Growth Factor beta1 ; biosynthesis ; genetics ; Ureteral Obstruction ; metabolism ; pathology

OBJECTIVETo investigate the expressions of phosphorylated Smad2 (P-Smad2) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) in hepatocellular carcinoma (HCC) tissues.

METHODSThe expressions of P-Smad2 and PTEN were detected using Envision immunohistochemical technique in 31 cases of HCC tissues, 25 cases of HCC adjacent liver tissues and 13 cases of non-hepatocellular carcinoma tissues.

RESULTSThe positive expression and staining intensity of PTEN in the cytoplasm of HCC cells (64.5%, 4.19+/-3.31) was significantly lower than those of the cells of the cancer adjacent tissues and non-cancerous tissues (96.0%, 7.88+/-0.93; 100%, 7.77+/-0.93). The staining intensity of PTEN in the cytoplasm of Edmondson pathologic grade III HCC cells was lower than those of the Edmondson grade I. The expression of PTEN was negatively correlated with intrahepatic vascular cancer thrombi (r=-0.43) and the expression of PTEN in the nuclei or cytoplasm of liver cells was negatively correlated with the liver disease progressions (r=-0.34). The positive rate and expression intensity of phosphorylated Smad2 in nuclei of HCC cells were the same as those in cancer adjacent and non-tumor liver tissues. The expression was mostly in the nucleus and cytoplasm of Edmondson grade I HCC cells, cancer adjacent liver tissue cells and non-tumor liver tissues, but its expression was only in the nuclei of Edmondson grade II and III HCC cells. The phosphorylated Smad2 expression appeared in the nuclei and in the cytoplasm of liver cells and it was positively correlated with the severity of the tumor pathology (r=0.22). Spearman correlation analysis revealed a significant inverse correlation between PTEN and phosphorylated Smad2 in HCC tissues (r=-0.73).

CONCLUSIONSThe aberrant expressions of PTEN and phosphorylated Smad2 and their interaction may play an important role in the pathogenesis of hepatocellular carcinoma.

Adult ; Aged ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Female ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; Oxidative Phosphorylation ; PTEN Phosphohydrolase ; metabolism ; Smad2 Protein ; metabolism