Yanna Cao

Akt interacts directly with Smad3 to regulate the sensitivity to TGF-β-induced apoptosis

Transforming growth factor β (TGF-β) induces both apoptosis and cell-cycle arrest in some cell lines, but only growth arrest in others. It is not clear how this differential response to TGF-β is specified. Smad proteins are critical mediators of TGF-β signalling. After stimulation by TGF-β, Smad2 and Smad3 become phosphorylated by the activated TGF-β receptor kinases, oligomerize with Smad4, translocate to the nucleus and regulate the expression of TGF-β target genes. Here we report that the sensitivity to TGF-β-induced apoptosis is regulated by crosstalk between the Akt/PKB serine/threonine kinase and Smad3 through a mechanism that is independent of Akt kinase activity. Akt interacts directly with unphosphorylated Smad3 to sequester it outside the nucleus, preventing its phosphorylation and nuclear translocation. This results in inhibition of Smad3-mediated transcription and apoptosis. Furthermore, the ratio of Smad3 to Akt correlates with the sensitivity of cells to TGF-β-induced apoptosis. Alteration of this ratio changes the apoptotic, but not the growth-inhibitory, responses of cells to TGF-β. These findings identify an important determinant of sensitivity to TGF-β-induced apoptosis that involves crosstalk between the TGF-β and phosphatidylinositol-3-OH kinase (PI(3)K) pathways.

Protective Effects of Fresh Frozen Plasma on Vascular Endothelial Permeability, Coagulation, and Resuscitation After Hemorrhagic Shock Are Time Dependent and Diminish Between Days 0 and 5 After Thaw

BACKGROUND Clinical studies have shown that resuscitation with fresh frozen plasma (FFP) is associated with improved outcome after severe hemorrhagic shock (HS). We hypothesized that in addition to its effects on hemostasis, FFP has protective and stabilizing effects on the endothelium that translate into diminished endothelial cell (EC) permeability and improved resuscitation in vivo after HS. We further hypothesized that the beneficial effects of FFP would diminish over 5 days of routine storage at 4 degrees C. METHODS EC permeability was induced by hypoxia and assessed by the passage of 70-kDa Dextran between monolayers. Thrombin generation time and coagulation factor levels or activity were assessed in FFP. An in vivo rat model of HS and resuscitation was used to determine the effects of FFP on hemodynamic stability. RESULTS Thawed FFP inhibits EC permeability in vitro by 10.2-fold. Protective effects diminish (to 2.5-fold) by day 5. Thrombin generation time is increased in plasma that has been stored between days 0 and 5. In vivo data show that day 0 FFP is superior to day 5 FFP in maintaining mean arterial pressure in rats undergoing HS with resuscitation. CONCLUSION Both in vitro and in vivo studies show that FFP has beneficial effects on endothelial permeability, vascular stability, and resuscitation in rats after HS. The benefits are independent of hemostasis and diminish between days 0 and 5 of storage.

Repression of bone morphogenetic protein and activin-inducible transcription by Evi-1

Smads, key effectors of transforming growth factor (TGF)-β, activin, and bone morphogenetic protein (BMP) signaling, regulate gene expression and interact with coactivators and corepressors that modulate Smad activity. The corepressor Evi-1 exerts its oncogenic effects by repressing TGF-β/Smad3-mediated transcription, thereby blocking TGF-β-induced growth arrest. Because Evi-1 interacts with the highly conserved MH2 domain of Smad3, we investigated the physical and functional interaction of Evi-1 with Smad1 and Smad2, downstream targets of BMP and activin signaling, respectively. Evi-1 interacted with and repressed the receptor-activated transcription through Smad1 and Smad2, similarly to Smad3. In addition, Evi-1 repressed BMP/Smad1- and activin/Smad2-mediated induction of endogenous Xenopus gene expression, suggesting a role of repression of BMP and activin signals by Evi-1 in vertebrate embryogenesis. Evi-1 also repressed the induction of endogenous Smad7 expression by TGF-β family ligands. In the course of these studies, we observed Evi-1 repression of Smad transactivation even when Smad binding to DNA was kept constant. We therefore explored the mechanism of Evi-1 repression of TGF-β family-inducible transcription. Evi-1 repression did not result from displacement of Smad binding to DNA or to CREB-binding protein but from the recruitment of Evi-1 by Smad3 and CREB-binding protein to DNA. Following TGF-β stimulation, Evi-1 and the associated corepressor CtBP were recruited to the endogenous Smad7 promoter. Evi-1 recruitment to the promoter decreased TGF-β-induced histone acetylation, coincident with its repression of Smad7 gene expression. In this way, Evi-1 acts as a general Smad corepressor to inhibit TGF-β-, activin-, and BMP-inducible transcription.

Dietary fiber enhances a tumor suppressor signaling pathway in the gut.

Objective:To determine whether sodium butyrate (NaB), a major short-chain fatty acid produced in the human gut by bacterial fermentation of dietary fiber, enhances transforming growth factor (TGF)-β signaling and potentiates its tumor suppressor activity in the gut. Summary Background Data:The molecular mechanisms by which dietary fiber decreases the risk of colon cancers are poorly characterized. TGF-β is an important tumor suppressor in the gut and has many similar biologic activities as NaB. Therefore, we hypothesized that the chemo-preventive effects of NaB are mediated in part by enhancing TGF-β signaling and its tumor suppressor function in the gut. Methods:The effects of NaB on Smad3 expression in rat intestinal epithelial (RIE-1) cells and 6 human colon cancer cell lines were examined. The effects of NaB on TGF-β-induced Smad3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) and cyclooxygenase-2 (COX-2) gene expression were also examined in RIE-1 cells. Finally, the effects of NaB and TGF-β on anchorage-independent growth were examined in Akt-transformed RIE-1 cells. Results:NaB induced Smad3 in RIE-1 cells and in 4 human colon cancer cell lines. NaB enhanced TGF-β-induced Smad3 phosphorylation and potentiated TGF-β-induced PAI-1 expression. NaB and TGF-β synergistically inhibited anchorage-independent growth of Akt-transformed RIE-1 cells. Conclusions:These results demonstrate that NaB induces Smad3 and potentiates TGF-β signaling and its tumor suppressor activity in gut epithelial cells. Our data reveal a novel molecular mechanism that may explain in part the beneficial effects of dietary fiber in decreasing the risk of colon cancers.

TGF-β Signaling in Colon Cancer Cells

Transforming growth factor-betas (TGF-βs), cytokines expressed in the colon, play important roles as tumor suppressors and tumor promoters during colorectal carcinogenesis. TGF-β signaling pathway involves activation of Smad2 and Smad3 by the type I receptor and formation of Smad2/3/4 heteromeric complexes that enter the nucleus to regulate transcription. Most human colorectal cancers are resistant to the tumor suppressor effects of TGF-β, and a subset of human colorectal cancers have mutations in Smad2 and Smad4. The purpose of this study was to determine whether Smads are required for TGF-β signaling in colon cancer cells. First, we selected a colon cancer cell line (MC-26) that has a functional TGF-β signaling pathway. We found that MC-26 cells expressed Smad2, Smad3, and Smad4 mRNAs by reverse transeription-polymerase chain reaction and confirmed that the TGF-β signaling pathway is functional using a transient transfection assay with 3TP-Lux reporter plasmid. TGF-β also inhibited cell growth and induced apoptosis in MC-26 cells. When MC-26 cells were transiently transfected with dominant-negative carboxyl-terminal truncation mutants of Smad2, Smad3, and Smad4, TGF-β-induced 3TP-Lux reporter activity was significantly reduced, suggesting that Smad2, Smad3, and Smad4 are attractive novel therapeutic targets for regulating TGF-β signaling in colorectal cancers. Because MC-26 cells express TGF-β activated Smads, have a functional TGF-β signaling pathway, and are sensitive to the growth inhibitory and apoptotic effects of TGF-β, they can serve as an excellent model to examine TGF-β signaling in colorectal cancers.

BMP2 inhibits TGF-β-induced pancreatic stellate cell activation and extracellular matrix formation

Activation of pancreatic stellate cells (PSCs) by transforming growth factor (TGF)-β is the key step in the development of pancreatic fibrosis, a common pathological feature of chronic pancreatitis (CP). Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have anti-fibrogenic functions, in contrast to TGF-β, in the kidney, lung, and liver. However, it is not known whether BMPs have an anti-fibrogenic role in the pancreas. The current study was designed to investigate the potential anti-fibrogenic role of BMPs in the pancreas using an in vivo CP model and an in vitro PSC model. CP was induced by repetitive intraperitoneal injections of cerulein in adult Swiss Webster mice. The control mice received saline injections. Compared with the control, cerulein injections induced a time-dependent increase in acinar injury and progression of fibrosis and a steady increase in inflammation. Cerulein injections also induced increases of the extracellular matrix (ECM) protein fibronectin and of α-smooth muscle actin (α-SMA)-positive stellate cells (PSCs). The mice receiving cerulein injections showed increased BMP2 protein levels and phosphorylated Smad1 levels up to 4 wk and then declined at 8 wk to similar levels as the control. In vitro, the isolated mouse and human PSCs were cultured and pretreated with BMP2 followed by TGF-β treatment. BMP2 pretreatment inhibited TGF-β-induced α-SMA, fibronectin, and collagen type Ia expression. Knocking down Smad1 with small-interfering RNA reversed the inhibitory effect of BMP2 on TGF-β-induced α-SMA and fibronectin expression. Thus, BMP2 opposes the fibrogenic function of TGF-β in PSCs through the Smad1 signaling pathway.

Nanoparticle Improved Stem Cell Therapy for Erectile Dysfunction in a Rat Model of Cavernous Nerve Injury

PURPOSE Recently intracavernous injection of stem cells has garnered great interest as a potential treatment of erectile dysfunction. However, most stem cells are washed out immediately after intracavernous injection. The goal of this study was to investigate using NanoShuttle™ magnetic nanoparticles to maintain stem cells in the corpus cavernosum after intracavernous injection, thereby improving stem cell therapy of erectile dysfunction in an animal model. MATERIALS AND METHODS Adipose derived stem cells were magnetized with NanoShuttle magnetic nanoparticles to create Nano-adipose derived stem cells. A total of 24 rats underwent bilateral cavernous nerve crush and were randomly assigned to 3 groups, including adipose derived stem cells, Nano-adipose derived stem cells and Nano-adipose derived stem cells plus magnet. Cells were tracked at days 1, 3, 5 and 9 after intracavernous injection. Another 40 rats with bilateral cavernous nerve crush were randomly assigned to 4 groups, including bilateral cavernous nerve crush, bilateral cavernous nerve crush plus adipose derived stem cell intracavernous injection, bilateral cavernous nerve crush plus Nano-adipose derived stem cell intracavernous injection and bilateral cavernous nerve crush plus Nano-adipose derived stem cell intracavernous injection plus magnet. Functional testing and histological analysis were performed 4 weeks after intracavernous injection. RESULTS In the in vitro study 1) NanoShuttle magnetic nanoparticles were successfully bound to adipose derived stem cells and 2) Nano-adipose derived stem cells migrated toward the magnet. In the in vivo study 1) cell tracking showed that Nano-adipose derived stem cells were successfully retained in the corpus cavernosum using the magnet for up to 3 days while most adipose derived stem cells were washed out in other groups by day 1 after intracavernous injection, and 2) intracavernous pressure/mean arterial pressure, and αSMA (α-smooth muscle actin) and PECAM-1 (platelet endothelial cell adhesion molecule 1) expression in the Nano-adipose derived stem cell group was significantly higher than in the other groups. CONCLUSIONS Magnetization of adipose derived stem cells with NanoShuttle magnetic nanoparticles kept adipose derived stem cells in the corpus cavernosum and improved adipose derived stem cell therapy of erectile dysfunction in an animal model.

TGF-β repression of Id2 induces apoptosis in gut epithelial cells

Transforming growth factor-β (TGF-β) regulates epithelial tissue homeostasis by activating processes that control cell cycle arrest, differentiation and apoptosis. Disruption of the TGF-β signaling pathway often occurs in colorectal cancers. Earlier, we have shown that TGF-β induces apoptosis through the transcription factor Smad3. Affymetrix oligonucleotide microarrays were used to identify TGF-β/Smad3 target genes that regulate apoptosis in rat intestinal epithelial cells (RIE-1). We found that TGF-β repressed the expression of the inhibitor of differentiation (Id) gene family. Knockdown of Id1 and Id2 gene expression induced apoptosis in RIE-1 cells, whereas overexpression of Id2 attenuated TGF-β-induced apoptosis. TranSignal Protein/DNA arrays were used to identify the hypoxia-inducing factor-1 (HIF-1) as a downstream target of TGF-β. HIF-1 is a basic helix-loop-helix protein, and overexpression of Id2 blocked HIF-1 activation by TGF-β. Furthermore, knockdown of HIF-1 blocked TGF-β-induced apoptosis. Thus, we have identified HIF-1 as a novel mediator downstream of Id2 in the pathway of TGF-β-induced apoptosis.

DIETARY FIBER ENHANCES TGF-β SIGNALING AND GROWTH INHIBITION IN THE GUT

Dietary fiber intake links to decreased risk of colorectal cancers. The underlying mechanisms remain unclear. Recently, we found that butyrate, a short-chain fatty acid produced in gut by bacterial fermentation of dietary fiber, enhances TGF-β signaling in rat intestinal epithelial cells (RIE-1). Furthermore, TGF-β represses inhibitors of differentiation (Ids), leading to apoptosis. We hypothesized that dietary fiber enhances TGF-βs growth inhibitory effects on gut epithelium via inhibition of Id2. In this study, Balb/c and DBA/2N mice were fed with a regular rodent chow or supplemented with a dietary fiber (20% pectin) and Smad3 level in gut epithelium was measured. In vitro, RIE-1 cells were treated with butyrate and TGF-β(1), and cell functions were evaluated. Furthermore, the role of Ids in butyrate- and TGF-β-induced growth inhibition was investigated. We found that pectin feeding increased Smad3 protein levels in the jejunum (1.47 ± 0.26-fold, P = 0.045, in Balb/c mice; 1.49 ± 0.19-fold, P = 0.016, in DBA/2N mice), and phospho-Smad3 levels (1.92 ± 0.27-fold, P = 0.009, in Balb/c mice; 1.83 ± 0.28-fold, P = 0.022, in DBA/2N mice). Butyrate or TGF-β alone inhibited cell growth and induced cell cycle arrest. The combined treatment of butyrate and TGF-β synergistically induced cell cycle arrest and apoptosis in RIE-1 cells and repressed Id2 and Id3 levels. Furthermore, knockdown of Id2 gene expression by use of small interfering RNA caused cell cycle arrest and apoptosis. We conclude that dietary fiber pectin enhanced Smad3 expression and activation in the gut. Butyrate and TGF-β induced cell cycle arrest and apoptosis, which may be mediated by repression of Id2. Our results implicate a novel mechanism of dietary fiber in reducing the risk of colorectal cancer development.

Bone morphogenetic protein signaling protects against cerulein-induced pancreatic fibrosis.

Bone morphogenetic proteins (BMPs) have an anti-fibrogenic function in the kidney, lung, and liver. However, their role in chronic pancreatitis (CP) is unknown. The aim of this study was to define the anti-fibrogenic role of BMP signaling in the pancreas in vivo under CP induction. Mice with a deletion of BMP type II receptor (BMPR2+/−) were used in this study in comparison with wild-type mice. CP was induced by repetitive cerulein injection intraperitoneally for 4 weeks, and the severity of CP was evaluated. Pancreatic stellate cells (PSCs) were isolated from the mice and treated with BMP2 and TGF-β in vitro, and extracellular matrix protein (ECM) production was measured. Smad and mitogen-activated protein kinase (MAPK) signaling was also evaluated. BMPR2+/− mice revealed a greater pancreatic fibrosis, PSC activation and leukocyte infiltration after CP induction compared to wild-type mice (P<0.05). Under CP induction, phospho (p)Smad1/5/8 was elevated in wild-type mice and this effect was abolished in BMPR2+/− mice; pSmad2 and pp38MAPK were further enhanced in BMPR2+/− mice compared to wild-type mice (P<0.05). In vitro, BMP2 inhibited TGF-β-induced ECM protein fibronectin production in wild-type PSCs; this effect was abolished in BMPR2+/− PSCs (P<0.05). In BMPR2+/− PSCs, pSmad1/5/8 level was barely detectable upon BMP2 stimulation, while pSmad2 level was further enhanced by TGF-β stimulation, compared to wild-type PSCs (P<0.05). BMPR2/Smad1/5/8 signaling plays a protective role against cerulein-induced pancreatic fibrosis by inhibiting Smad2 and p38MAPK signaling pathways.