et al., 1997, for a review). Each arterial system now supplies a functional region of the body (Fig. 7), and it is clearly important that oxygenation of each region be separately controlled. An example of circulatory control systems in action is seen in the work of DeWachter and McMahon (DeWachter and McMahon, 1996). These authors exercised individual Dungeness crabs (Cancer magister) non-stressfully using a rotating treadmill, on which the crabs walked slowly but continuously, to remain in a shaded preferred location. Each animal was fitted with pulsed-Doppler flow transducers that recorded flow through each arterial system simultaneously. During this slow sustained walking activity cardiac output increased, largely by an increase in cardiac stroke volume (Fig. 9A). The increased cardiac outflow, however, is not distributed equally among the vascular circuits; instead hæmolymph flow ceases in the hepatic arterial system leading to alimentary structures and increases in the sternal artery leading to the limbs (Fig. 9B). This indicates that, during activity, the increased cardiac output is diverted away from digestive structures and delivered preferentially to the limbs and gills. In other words, oxygen and nutrients are diverted to essential systems, precisely as occurs in mammals (reviewed in Guyton, 1996). Restitution of resting flow patterns occurs on cessation of activity. Similar shunting of haemolymph away from visceral structures and towards the limbs and gills also occurs in crustaceans exposed to hypoxia (Airriess and McMahon, 1994; B. R. McMahon, manuscript in preparation).
Human endometrial cancer cell line HEC-1-A is TGF β sensitive. In this study, a dominant-negative RII (DNRII) without the cytoplasmic kinase domain was ﬁ rst trans- fected into HEC-1-A cells for the determination of the role of TGF β signaling in their proliferation and apoptosis. Later, a EGFP or a DNRII-EGFP fusion protein was also ectopically expressed in HEC-1-A cells so that metastatic cells can be tracked in vivo. The expression of DNRII or DNRII-EGFP was detected with Western immunoblotting as shown in Figure 1A. It should be noted that because the two paired cell lines were not produced and used at the same time for various experiments, the data from the control and control-EGFP cannot be compared due to variations in assay conditions although the data from the two paired cell lines are presented in the same ﬁ gure. To determine whether the expression of DNRII or DNRII-EGFP abrogated TGF β signaling, we ﬁ rst compared the effect of TGF β to induce the phosphorylation of Smad2 (P-Smad2) in the control and DNRII cells. Figure 1B shows that the expression of both DNRII and DNRII-EGFP attenuated TGF β -induced P-Smad2 although the relative reduction of P-Smad2 was more dramatic in DNRII-EGFP cells than in DNRII cells. This is likely in part due to the lower expression level of DNRII than DNRII-EGFP as shown in Figure 1A. Consis- tently, the blockade of Smad phosphorylation also led to attenuated stimulation of a TGF β -responsive promoter activ- ity by TGF β in the both DNRII and DNRII-EGFP expressing
Bioinformatic analysis of aligned reads (hg19) was completed using Partek Genomic Suite 6.6 (PGS). Only reads that were of sufficient quality and aligned unambiguously were used. To detect ChIP-Seq peaks, the genome was divided into 200 base pair windows and midpoints of peaks within a window were counted and an empirical distribution of window counts was created. PGS uses a zero truncated binomial model to fit the distribution and calculate a false discovery rate (FDR). 29,191 peaks with a FDR less than 0.01 were scored. To identify TGFβ dependent TDG recruitment, peaks containing a greater than 2 fold scaled fold change between treatment and control were used in downstream analysis (3364 peaks). Scaled fold change compares the intensity of signal of the TGFβ sample to the control sample and is scaled by a ratio of the number of total alignments of each sample on a per chromosome basis. PGS, which uses the Gibbs motif sampler, was used for de novo motif analysis using the default parameters. Known motif analysis was conducted using the JASPAR database. CpG islands were downloaded from the ENCODE database. RefSeq (2014-10-17) was used to annotate location of a peak to a genomic feature, defining the promoter as -50 kbp to +3kbp or -5kbp to +3kbp, as indicated. Gene Ontology analysis was conducted as described in 2.6.
In our study, we present evidence for a pro- fibrotic effect of FSTL1 in liver fibrogenesis. Blocking FSTL1 with a neutralizing antibody remarkably reduced liver injury and attenuated liver fibrosis in the CCl 4 -induced liver fibrosis mouse model. The activation of primary mouse HSCs treated with the FSTL1 neutralizing anti- body was slower compared with that of the IgG- treated control group. We found that the FSTL1 mAb had no effect on HSC proliferation but reduced the migratory capacity of HSCs in vitro. We also showed that FSTL1 regulated liver fibrosis in vivo via TGF-betasignaling. Activation of TGF-betasignaling has already been shown to play a central role in regulating the progres- sion of tissue fibrosis. According to previous reports and our study results, positive regula- tion of FSTL1 on TGF-betasignaling can be rec- ognized as the main mechanism to explain the progressive effect of FSTL1 on liver fibrosis.
Proteins samples were extracted in RIPA buffer (1% TritonX-100, 15 mmol/L NaCl, 5 mmol/L EDTA, and 10 mmol/L Tris-HCl (pH 7.0) (Solarbio, China) supplemented with a protease and phosphatase inhibitor cocktail (Sigma) and then separated by 10% SDS-PAGE, followed by electrophoretical transfer to a PVDF mem- brane. After soaking with 8% milk in PBST (pH 7.5) for 2 h at room temperature, the mem- branes were incubated with the following pri- mary antibodies: anti-Smad2, anti-E-cadherin (E-cad), α-SMA, Fibronectin (FN), Vimentin (Vi) and anti-GAPDH (Cell signaling). Immunode- tection was performed by enhanced chemilu- minscence detection system (Millipore) accord- ing to the manufacturer’s instructions. The house-keeping gene GAPDH was used as the internal control.
inhibited the proliferation of SMCC-7721 cells and restrained colony formation, whereas the TINAGL1-overexpressing HCC-LM3 cells grew faster and produced numerous colo- nies compared to the control cells. In addition, si-TINAGL1 inhibited HCC cell migration in vitro, while TINAGL1 had the opposite effects (Figure 3E). Consistent with this, TINAGL1 silencing and overexpression respectively decreased and increased DNA synthesis as measured in terms of EdU uptake (Figure 3F). Finally, TINAGL1 also decreased apoptosis in the HCC cells, while si-TINAGL1 induced cell apoptosis (Figure 3G). Taken together, TINAGL1 promotes the proliferation, survival, and metastasis of HCC cells in vitro.
Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF- beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and - 7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-betasignaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were
Lungs were inflation fixed in 10% formalin solution in saline, all five lobes were paraffin imbedded; and lung sections were stained with haematoxylin and eosin stain (H&E). 40X images of the H&E-stained sections were obtained on a CRi Pannoramic whole slide scanner which creates an image without showing the slide name (unless inquired). The severity of fibrosis was first blindly assessed on scanned images by two investigators using modified Ashcroft scoring system . Up to 150 fields at 10X magnification covering whole lobes were scored using Panoramic View program with “preview tracking history” function turned on, and the average score for each lung was calculated. The means±SD were then calculated for each experimental group. The extent of fibrosis was then assessed by measuring lung tissue surface area. Five representative 10X images from each lobe (devoid of airways or blood vessels) were captured, and the lung tissue surface area was measured using ImageJ program as follows. The total area was first quantified using the Analyze-Measure function. The white area (representing empty spaces) was then se- lected (with brightness set at 240) under the Image – Adjust – Color Threshold function, and was quanti- fied using the Analyze-Measure function. The lung tissue surface area was then calculated by subtracting the white area from the total area and expressed as a fraction of the total area. Finally the histologic score of fibrosis was calculated by multiplying the Ashcroft score by fraction of the lung tissue surface area for each lung, analogous to previously described approach [32, 33]. The means±SD were then calculated for each experimental group.
Previous studies have indicated that TGF- β and its cognate receptors are important regulators during early tooth development (Pelton et al., 1990; Heikinheimo et al., 1993; Chai et al., 1994, 1999). Specifically, endogenous TGF- β signals through its cognate recep- tors to exert negative control on proliferation of dental epithelium to regulate the overall growth during early tooth development. Until recently, however, the mechanism of negative regulation on prolif- eration of dental epithelium by TGF- β is not well understood. Studies of our group and others have shown that the expression of Smad2 and Smad3 are present in dental epithelium during early stages (the lamina and the bud stage) of tooth development (Dick et al., 1998; Flanders et al., 2001; Ito et al., 2001). Significantly, using the PS2 antibody, we have demonstrated that phosphorylated Smad2 is present in the nuclei of inner enamel epithelial cells starting at the late bud stage, indicating the active role of Smad2 in regulating tooth development. Functional analysis demonstrates that the effective- ness of TGF- β signaling is highly sensitive to the level of Smad gene expression (Ito et al., 2001). The spatial and temporal distribution of Smad2 and Smad3 match precisely with the distribution of TGF- β ligand and its cognate receptors during early tooth development, thus demonstrating the important regulatory function of these intracellular signaling molecules. The expression of Smad2 and Smad3 is also detected in the dental mesenchyme at the late bud and early cap stage, suggesting that both TGF- β and activin have active roles in regulating cranial neural crest derived cells as well as epithelial- mesenchymal interaction during tooth development. Interestingly, analysis of tooth development in activin receptor II and Smad2 mutants show that incisors and mandibular molars fail to develop but maxillary molars develop normally, suggesting regional specific requirement for Smad2-mediated activin signaling in regulating tooth development (Ferguson et al., 1998, 2001).
Li et al discovered through reverse transcription- polymerase chain reaction (RT-PCR) that HOXD9 was highly expressed in cervical cancer cells, but not in normal cervical cells. 12 In esophageal squamous cell carcinoma (ESCC), Liu 10 detected the expression of HOXD9 protein in samples through IHC and found that over 60% of ESCC cells were stained for HOXD9 protein to varying degrees. Japanese scholars ﬁ rst discovered through PCR and IHC that HOXD9 is highly expressed in gliomas. They investigated the HOXD9 function in glioma cell lines, and their results suggested that silencing HOXD9 could effectively suppress the proliferation of U87 glioma cells and promote cell cycle arrest and apoptosis. 11 A similar phenomenon was observed in glioma stem cells. In a study on liver cancer, HOXD9 was highly expressed in invasive hepatocellular carcinoma cells. In vitro experiments demonstrated that the over-expression of HOXD9 could remarkably enhance the migratory and invasive capacities of liver cancer cells and promote their epithelial – mesenchymal transition. 18 Concerning the mechanisms, the interaction of HOXD9 with the promoter region of ZEB1 to promote its transcription has been sug- gested. Bao et al 19 discovered through a bioinformatic ana- lysis that the HOXD gene family is speci ﬁ cally upregulated in human lung squamous carcinoma, including HOXD9. In another glioma study, 20 it was discovered that miR-205 could downregulate HOXD9, suppress epithelial – mesenchymal transition in tumor cells, and inhibit growth of human glioma. However, the relationship between HOXD9 and GC has not
T cells with the mimotope peptide in the presence or absence of the 2H6 cells with or without mAbs against TGF-β (R&D Systems) or control IgG (Pierce Biotechnology). Anti–TGF-β partially reversed the suppression, and control IgG had little effect (Supplemental Figure 3). This suggests that although the mechanism of the sup- pression is not totally dependent on soluble cytokines, TGF-β may be important for the suppression. In the second set of experiments, we cultured BDC2.5 T cells with the mimotope peptide in the pres- ence or absence of the 2H6 cells expressing a dominant-negative form of TGF-β receptor type II (TGF-βDNRII). Therefore, these 2H6 T cells were not able to bind TGF-β and trigger the signaling transduction in the cells. In this culture system, 2H6 TGF-βDNRII T cells were either in direct cell contact with BDC2.5 T cells (cocul- ture) or separated from BDC2.5 T cells (Transwell). As shown in Figure 3C, the lack of TGF-β receptor on 2H6 cells resulted in loss of their ability to suppress BDC2.5 T cells in coculture. However, the lack of TGF-β receptor did not cause 2H6 cells to lose the abil- ity to produce TGF-β (data not shown). These data indicate that the signaling induced by TGF-β upon binding the TGF-β receptor is important for the suppression mediated by 2H6 T cells.
The clinical background of subjects revealed a history of cardiovascular diseases, ranging from hyperlipidemia to heart failure, and that some patients were on cardiovascular therapy, such as telmisartan, simvastatin, and isosorbide dinitrate. Additionally, the decreased blood pressure was also suspected to lead to the decrease in urinary TGF- β 1 in diabetic nephropathy patients. Although these conditions are commonly observed in the kidneys in a pathological state, TGF- β 1 concentration decreases when the blood pressure, especially SBP, decreases. 32,39
Cells were lysed using RIPA buffer (Beyotime, Shanghai, China) for protein extraction, followed by measurement of protein concentration using BCA Protein Assay Kit (Beyotime). Cell lysates were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and transferred to poly- vinylidene ﬂ uoride membranes. After blocking with 5% non- fatty milk, the membrane was incubated overnight with anti-G3BP1 antibody (1:1000) at 4 °C. GAPDH (MultiSciences, Hangzhou, China) was used as an internal control. Subsequently, the membrane was incubated with goat anti-rabbit IgG secondary antibody (Thermo Fisher Scienti ﬁ c, USA). Other antibodies included: anti-TGF β 1 antibody (1:2000), anti-TGF β 2 antibody (1:1000), anti- Smad2 antibody (1:2000), anti-phospho-Smad2 (1:1000), anti-Smad3 antibody (1:1500), anti-phospho-Smad3 (1:2000). All the aforementioned antibodies were purchased from Abcam Inc. (Cambridge, MA, USA).
Abstract: During malignant transformation, a growing body of mutations accumulate in cancer cells which not only drive cancer progression but also endow cancer cells with high immunogenicity. However, because one or multiple steps in cancer-immunity cycle are impaired, anti-cancer immune response is too weak to effectively clear cancer cells. Therefore, how to restore robust immune response to malignant cells is a hot research topic in cancer therapeutics ﬁ eld. In the last decade, based on the deeper understanding of cancer immunity, great signs of progress have been made in cancer immunotherapies especially immune checkpoint inhibitors (ICIs). ICIs could block negative immune co- stimulatory pathways and reactivate tumor-in ﬁ ltrating lymphocytes (TILs) from exhausted status. ICIs exhibit potent anti-cancer effect and have been approved for the treatment of numerous cancer types. Parallel with durable and effective tumor control, the actual response rate of ICIs is unsatisfactory. Although a subset of patients bene ﬁ t from ICIs treatment, a large proportion of patients show primary or acquired resistance. Previously intensive studies indicated that the ef ﬁ cacy of ICIs was determined by a series of factors including tumor mutation burden, programmed death ligand-1 (PD-L1) expression, and TILs status. Recently, it was reported that transforming growth factor-beta (TGF- β ) signaling pathway participated in cancer immune escape and ICI resistance. Concurrent TGF- β blockade might be a feasible strategy to enhance the ef ﬁ cacy of immunotherapy and relieve ICI resistance. In this mini- review, we summarized the latest understanding of TGF- β signaling pathway and cancer immunity. Besides, we highlighted the synergistic effect of TGF- β blockade and ICIs. Keywords: immunotherapy, immune checkpoint inhibitor, PD-1, PD-L1, TGF- β , tumor immune microenvironment, tumor in ﬁ ltrating lymphocyte
Transforming growth factor β (TGF-β) is a cytokine with immune and growth inhibitory properties [50,51]. Early in carcinogenesis TGF-β functions as a tumor suppressor by inhibiting cell growth whereas late in carcinogenesis TGF-β is thought to function as a tumor promoter by stimulating invasion and metastasis [52,53]. TGF-β is expressed by various tumor types including cervical cancer [54-56]. During cervical carcinogenesis, evidence exists that overexpression of TGF-β is accompanied with decreased sensitivity for the growth-limiting effect of TGF-β [57-59]. Alterations in TGF-β signaling can be caused by inactivating mutations in TGF-β signaling components. In particular inactivation of TβR-II is frequently observed in colon cancer with microsatellite instability (MSI) and inactivation of the Smad4 tumor suppressor gene in pancreatic and non MSI colon cancers [60-62]. In cervical cancer, inactivating mutations of TGF-β receptor and Smad4 have been reported mainly in cell lines at low incidence [63,64]. Alterations in TGF-β signal transduction may also be due to HPV E7 oncoproteins that bind to Smad proteins (e.g Smad2, Smad3 and Smad4) and subsequently interfere with Smad transcriptional activity by blocking binding of Smad3 protein to its DNA target sequence [65,66]. In addition, E7 may interfere with TGF-β signal transduction by blocking TGF-β suppression of c-myc transcription via pRb . Inhibition of TGF-β induced suppression of c-myc transcription and inhibition of Smad DNA binding activity by E7 oncoproteins was associated with resistance to the antiproliferative effect of TGF-β [66,67]. Other mechanisms that could influence TGF-β insensitivity are defects in the TGF-β pathway downstream of Smad signaling and crosstalk of TGF-β -Smad signaling with other pathways [68,69]. TGF-β signaling can activate MAPK signaling directly, independently of Smad, and pathways like MAPK, PI3K and Wnt can influence the outcome of TGF-β signaling by crosstalk [69- 71]. Many of these pathways are involved in the regulation of cell growth and cell death. This interplay of TGF-β with other pathways could enhance carcinogenesis. For example, the invasive growth of Ras-transformed epithelial cells in vitro depends on intact autocrine TGF-β signaling .
pathway, Neurotrophin signaling pathway, MAPK signaling pathway and so on. PPARs act as nuclear receptor and its activation induces a decrease in neuronal death by prevention of oxidative or inflammatory mechanisms implicated in cerebral in- jury . Neurotrophins are a family of trophic fac- tors involved in differentiation and survival of neural cells, which have now been shown to mediate both positive and negative survival signals, by signalling through the Trk and p75 neurotrophin receptors, re- spectively [34, 35]. TGF-βs regulate a wide spectrum of cellular functions such as proliferation, apoptosis, differentiation and migration. TGF-betasignaling is a molecular mechanism which limit neuroinflammation, and activate TGF-beta in the peri-infarct cortex and preserve brain function during the subacute period after stroke [11, 36]. The MAP-kinase family mem- bers are known to be stimulated after cerebral ische- mia and were thought to regulate signal transduction, gene expression and metabolism. Based on the above information, we hypothesize that these differentially expressed miRNAs serve as mediators of the brain’s response to FNS that leads to endogenous neuropro- tection. On the other hand, our research investigated the role of miR-29c-3p in the neuroprotection in- duced by FNS after ischemic injury and we found that miR-29c-3p attenuated ischemic neuronal death by negatively regulating apoptotic proteins Birc2 and Bak1 associated with the PI3K-Akt signaling pathway . Our finding expand the understanding of miR- NAs associated with ischemic cerebral disease and may provide a basis for novel therapeutic strategies aimed at enhancing tissue and cell survival in the is- chemic stroke.
Such reports have reveal that the incidence among non-Hispanic whites is 20% higher than for African Americans and roughly double the rate for Hispanics and Asian Americans; in contrast, the rates of change are highest among Asian Americans. In some US populations it has been reported that breast cancer incidence rose by 1.1% per year between 1993–1997 among non-Hispanic whites, 2.1% among Hispanics, and 4.6% among Asians, while it declined by 0.3% for African Americans. Surveillance data for Asian-American women are consistent with studies of migrant populations showing that when women migrate from low- to high-risk countries and visa versa, their risk and the risk in successive generations change to approximate the levels in the destination country (Kliewer and Smith 1995). Further, a population-based case–control study of Asian migrants to California and Hawaii showed higher risk associated with extended residence in the United States (Ziegler et al. 1993). For U.S. born Asian women, the study showed higher risk for those with more U.S.-born grandparents, an indicator of the possible effects of acculturation. The relative risk associated with migration changed only slightly after controlling for menstrual and reproductive factors, providing an illustration that other factors contribute to migration effects (Wu et al. 1996). As a result the investigation of hormonally active compounds in commercial products and pollutants is a priority.
determined by radioreceptor binding inhibition assay and direct assessment of transforming growth factor biological activity correlated with Northern blot analysis. Although gradients in the expression of the TGFs were present, equivalent binding was observed in the different intestinal cell populations when assessed with 125I-TGFbeta and 125I-EGF (TGF alpha). No EGF transcripts were detected in any intestinal cell population, suggesting that the true ligand of the EGF receptor was TGF alpha. IEC-6 cells expressed both TGF alpha and TGFbeta transcripts. In addition to the transcripts identified in the primary intestinal cells, this cell line contained an additional larger TGF alpha transcript (4.8 kb) and smaller TGFbeta transcripts (2.2 and 1.8 kb). TGF alpha and […]
SnoN (Ski novel protein; Skil – Mouse Genome Informatics) is a member of the Ski family of proteins that contains both pro- oncogenic and anti-oncogenic activities in human cancer (Deheuninck and Luo, 2009; Jahchan and Luo, 2010; Jahchan et al., 2010; Pan et al., 2009). It is a critical regulator of transforming growth factor- (TGF-) signaling (Deheuninck and Luo, 2009; Jahchan and Luo, 2010; Luo, 2004). TGF-, signaling through the Smad proteins, is a potent inhibitor of epithelial cell proliferation and acts to suppress tumor development at the early stages of carcinogenesis (Bierie and Moses, 2006; Chen and Wang, 2009; Heldin et al., 2009; Tian and Schiemann, 2009). SnoN interacts with Smad2, Smad3 and Smad4 in both the cytoplasm and nucleus to repress their ability to activate TGF- target genes, thereby blocking the cytostatic response of TGF- (Deheuninck and Luo, 2009; Jahchan and Luo, 2010; Krakowski et al., 2005). This ability of SnoN to antagonize the tumor suppressor activity of TGF- is likely to be responsible for its pro-oncogenic activity. In addition, SnoN is also a potent activator of p53 (Trp53 – Mouse Genome Informatics). SnoN expression is significantly upregulated in response to cellular stress, and this high level of SnoN can induce stabilization and activation of p53, leading to increased senescence and apoptosis (Pan et al., 2009). This ability might account for the anti-tumorigenic activity of SnoN (Deheuninck and Luo, 2009; Jahchan and Luo, 2010; Pan et al., 2009).