A novel therapeutic approach to treating obesity through modulation of TGFβ signaling

Obesity results from disproportionately high energy intake relative to energy expenditure. Many therapeutic strategies have focused on the intake side of the equation, including pharmaceutical targeting of appetite and digestion. An alternative approach is to increase energy expenditure through physical activity or adaptive thermogenesis. A pharmacological way to increase muscle mass and hence exercise capacity is through inhibition of the activin receptor type IIB (ActRIIB). Muscle mass and strength is regulated, at least in part, by growth factors that signal via ActRIIB. Administration of a soluble ActRIIB protein comprised of a form of the extracellular domain of ActRIIB fused to a human Fc (ActRIIB-Fc) results in a substantial muscle mass increase in normal mice. However, ActRIIB is also present on and mediates the action of growth factors in adipose tissue, although the function of this system is poorly understood. In the current study, we report the effect of ActRIIB-Fc to suppress diet-induced obesity and linked metabolic dysfunctions in mice fed a high-fat diet. ActRIIB-Fc induced a brown fat-like thermogenic gene program in epididymal white fat, as shown by robustly increased expression of the thermogenic genes uncoupling protein 1 and peroxisomal proliferator-activated receptor-γ coactivator 1α. Finally, we identified multiple ligands capable of reducing thermogenesis that represent likely target ligands for the ActRIIB-Fc effects on the white fat depots. These data demonstrate that novel therapeutic ActRIIB-Fc improves obesity and obesity-linked metabolic disease by both increasing skeletal muscle mass and by inducing a gene program of thermogenesis in the white adipose tissues.     

Increased serum levels of transforming growth factor-α in patients with colorectal cancer

PURPOSE: This study was conducted to investigate the serum levels of transforming growth factor- in patients with colorectal cancer and to investigate the clinical significance of these levels in association with tumor stage and histologic differentiation. Also, serum levels of transforming growth factor- were measured after curative surgical resection. METHODS: Serum levels of transforming growth factor- were measured in 42 consecutive patients with colorectal cancer before surgery, in 21 patients after surgical resection (part of the 42 preoperative patients), and in 20 healthy volunteers. We used TGF- Assay. RESULTS: Serum levels of transforming growth factor- in patients with colorectal cancer were significantly higher than in the healthy control group (P=0.001). Significant elevations in serum levels of transforming growth factor- were found in 50 percent (21/42) of patients with colorectal cancer when the mean + 2 standard deviations (80.4 pg/ml) of the control group were used as the upper limit of the normal range. Serum levels of transforming growth factor- tended to decrease with increasing tumor size (n=31;r=–0.52;P=0.002). Serum levels of transforming growth factor- before surgery (89.7±44.4 pg/ml; n=21) significantly decreased to 60.3±19.8 pg/ml after surgical resections of tumors (P=0.017). Serum levels of transforming growth factor- completely decreased to the same serum levels of the control group after surgical resections in all patients who had serum levels of transforming growth factor- greater than mean + 2 standard deviations (80.4 pg/ml) of the control group preoperatively (n=11;P=0.002). CONCLUSIONS: Levels of preoperative transforming growth factor- in patients with colorectal cancer appeared to be higher than levels measured in control subjects. Serum levels of transforming growth factor- before surgery significantly decreased after surgical resections of tumors. Additional studies are warranted to determine if serum levels of transforming growth factor- may be useful as a potential biomarker in the management of patients with colorectal cancer.Supported by a grant from Ewha Womans University Mokdong Hospital, Seoul, Korea.Read at the meeting of The American Society of Colon and Rectal Surgeons, Philadelphia, Pennsylvania, June 22 to 26, 1997.     

Molecular pathogenesis of hepatocellular carcinoma: altering transforming growth factor-β signaling in hepatocarcinogenesis

Hepatocellular carcinoma (HCC) occurs subsequent to liver injury, where regenerative hepatocytes develop into a dysplastic nodule and then early HCC, supporting the multistep hepatocarcinogenesis theory. Molecular alterations such as the p53 mutation, p16 gene silencing, and AKT signaling activation are found in the late stage of HCC progression. The overexpression of some marker molecules is observed at the early stage. Transforming growth factor-β (TGF-β), a potent inhibitor of cell proliferation, is frequently overexpressed in HCC, although the role of TGF-β signaling during HCC development remains controversial. We previously reported that HCC cells show TGF-β receptor-dependent growth inhibition in response to TGF-β. Also, reduced TGF-β receptor II in HCC correlates with intrahepatic metastasis and shorter time-to-recurrence, suggesting a role of TGF-β signaling in tumor suppression. In contrast, TGF-β overexpression in HCC is known to correlate with malignant potential, suggesting a role in tumor promotion. Enhanced formation of stroma is a feature of advanced HCC, and TGF-β also promotes the proliferation of stromal fibroblasts. The microenvironment produced via tumor-stromal interactions may be the key to the modulation of the dual roles of TGF-β signaling in HCC progression.     

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β(TGF-β) signaling in vitro. METHODS: HSCs were isolated from rats by in situ perfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 h with DMEM containing 20% fetal bovine serum (FBS/ DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1, 2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscle-α-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲ procollagen (PCⅢ) in supernatants were determined by radioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively. RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2% (control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P<0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCⅢ to 84.6%, 81.5%, 75.7% or 80.7% respectively (P<0.05 vs control), with no significant difference among tetrandrine groups. RT-PCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r= -0.755, P<0.01), and both were significantly correlated with α-SMA protein expression (r= -0.938, P<0.01; r = 0.938,P<0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L) was confirmed by Western blotting as well. CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.     

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β (TGF-β) signaling in vitro. METHODS: HSCs were isolated from rats by in situ perfusion of liver and 18% Nycodenz gradient centrifugugation, and primarily cultured on uncoated plastic plates for 24 h with DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1,2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscle-α-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲ procollagen (PCⅢ) in supernatants were determined by radioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively. RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2% (control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P&lt;0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCII] to 84.6%, 81.5%, 75.7% or 80.7% respectively (P&lt;0.05 vs control), with no significant difference among tetrandrine groups. RTPCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r = -0.755, P&lt;0.01), and both were significantly correlated with α-SMA protein expression (r = -0.938, P&lt;0.01; r = 0.938, P&lt;0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L) was confirmed by Western blotting as well. CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.     

Nerve growth factor/nuclear factor-κB pathway as a therapeutic target in breast cancer

Introduction  Nuclear factor-κB pathway (NF-κB) is activated in many breast cancers. NF-кB has interactions with other pathways such as the nerve growth factor (NGF) pathway, which is involved in the survival and proliferation of breast cancer cells. NGF treatment of breast cancer cells activates NF-κB resulting in the inhibition of ceramide-induced apoptosis. NGF effects on apoptosis and cell proliferation are mediated through p75NTR and p140TrkA receptors, respectively. In this study we investigate the NGF/NF-κB pathway as a therapeutic target in breast cancer. Results  We demonstrate that p75NTR inhibitor Pep5, p140TrkA inhibitor K-252a, and NF-κB inhibitor BAY11-7085 have pro-apoptotic and anti-proliferation activities in breast cancer cells. We also show a synergy in combining the NGF receptor inhibitors with the conventional breast cancer treatments tamoxifen and taxol. Conclusion   These data suggest that NGF/NF-κB pathway is a potential therapeutic target in breast cancer.     

Cardioprotective actions of transforming growth factor-β

Transforming growth factor-beta (TGF-β) has been shown to have protective effects in experimental models of myocardial, mesenteric, and cerebral ischemia-reperfusion injury. These effects are mediated by its ability to block adhesion of neutrophils to endothelium and to preserve endothelial function in terms of physiologic release of nitric oxide (NO). TGF-β also maintains the rhythmicity of cultured cardiac myocytes and blocks the suppressive effects of interleukin-1 on their beating rate by antagonizing the pathologic induction of NO synthase. These data suggest that TGF-β will be useful clinically in treatment of both reperfusion injury and inflammatory diseases of the heart.     

miR-34a mediates oxaliplatin resistance of colorectal cancer cells by inhibiting macroautophagy via transforming growth factor-β/Smad4 pathway

AIM To investigate whether micro RNA(mi R)-34 a mediates oxaliplatin(OXA) resistance of colorectal cancer(CRC) cells by inhibiting macroautophagy via the transforming growth factor(TGF)-β/Smad4 pathway.METHODS miR-34 a expression levels were detected in CRC tissues and CRC cell lines by quantitative real-time polymerase chain reaction. Computational search, functional luciferase assay and western blotting were used to demonstrate the downstream target of miR-34 a in CRC cells. Cell viability was measured with Cell Counting Kit-8. Apoptosis and macroautophagy of CRC cells were analyzed by flow cytometry and transmission electron microscopy, and expression of beclin I and LC3-II was detected by western blotting.RESULTS Expression of miR-34 a was significantly reduced while expression of TGF-β and Smad4 was increased in CRC patients treated with OXA-based chemotherapy. OXA treatment also resulted in decreased mi R-34 a levels and increased TGF-β and Smad4 levels in both parental cells and the OXA-resistant CRC cells. Activation of macroautophagy contributed to OXA resistance in CRC cells. Expression levels of Smad4 and miR-34 a in CRC patients had a significant inverse correlation and overexpressing mi R-34 a inhibited macroautophagy activation by directly targeting Smad4 through the TGF-β/Smad4 pathway. OXA-induced downregulation of miR-34 a and increased drug resistance by activating macroautophagy in CRC cells.CONCLUSION miR-34 a mediates OXA resistance of CRC by inhibiting macroautophagy via the TGF-β/Smad4 pathway.     

Transforming growth factor-β during carcinogenesis: the shift from epithelial to mesenchymal signaling

Transforming growth factor-β (TGF-β) activates not only TGF-β type I receptor (TβRI) but also c-Jun N-terminal kinase (JNK), changing unphosphorylated Smad3 to its phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). While the TβRI/pSmad3C pathway inhibits growth of normal epithelial cells, JNK/pSmad3L-mediated signaling is involved in invasion by activated mesenchymal cells. During sporadic human colorectal carcinogenesis, TGF-β signaling confers a selective advantage on tumor cells by shifting from the TβRI/pSmad3C pathway characteristic of mature epithelial cells to the JNK/pSmad3L pathway, which is more characteristic of the state of flux shown by the activated mesenchymal cells. JNK acts as a regulator of TGF-β signaling by increasing the basal level of pSmad3L available for action in the nuclei of the invasive adenocarcinoma, in the meantime shutting down TGF-β-dependent nuclear activity of pSmad3C. Loss of epithelial homeostasis and acquisition of a migratory, mesenchymal phenotype are essential for tumor invasion. From the viewpoint of TGF-β signaling, a key therapeutic aim in cancer would be restoration of the lost tumor suppressor function observed in normal colorectal epithelial cells at the expense of effects promoting aggressive behavior of the adenocarcinoma. Specific inhibitors of the JNK/pSmad3L pathway might prove useful in this respect. In the case of molecularly targeted therapy for human cancer, pSmad3L and pSmad3C could be assessed as biomarkers to evaluate the likely benefit from specific inhibition of the JNK/pSmad3L pathway.     

Impact of high glucose and transforming growth factor-β on bioenergetic profiles in podocytes

Diabetic nephropathy is the most common cause of chronic renal failure in industrialized countries. Depletion of podocytes plays an important role in the progression of diabetic glomerulopathy. Various factors in the diabetic milieu lead to serious podocyte stress driving the cells toward cell cycle arrest (p27(Kip1)), hypertrophy, detachment, and apoptosis. Mitochondria are responsible for oxidative phosphorylation and energy supply in podocytes. Recent studies indicated that mitochondrial dysfunction is a key factor in diabetic nephropathy. In the present study, we investigated metabolic profiles of podocytes under diabetic conditions. We examined oxygen consumption rates (OCRs) and oxidative phosphorylation complex activities in murine podocytes. Cells were exposed to high glucose for 48 hours, cultured for 10 passages under high-glucose conditions (30 mmol/L), or incubated with transforming growth factor-β (5 ng/mL) for 24 hours. After prolonged exposure to high glucose, podocytes showed a significantly increased OCR at baseline and also a higher OCR after addition of oligomycin, indicating significant changes in mitochondrial energy metabolism. Higher OCRs after inhibition of respiration by rotenone also indicated changes in nonmitochondrial respiration. Podocytes stimulated with a proapoptotic concentration of transforming growth factor-β displayed similar bioenergetic profiles, even with decreased citrate synthase activity. In all tested conditions, we found a higher cellular nicotinamide adenine dinucleotide content and changes in activities of respiratory chain complexes. In summary, we provide for the first time evidence that key factors of the diabetic milieu induce changes in glucose metabolism and mitochondrial function in podocytes.     

The role of transforming growth factor-�� in suppression of hepatic metastasis from colon cancer

Background

The role of transforming growth factor-β (TGF-β) in the development of hepatic metastasis from colon cancer is not clearly elucidated. The aim of this study was to determine the role of TGF-β in the development of such metastasis.

Methods

Two human colon cancer cell lines were utilized: FET-α cells (intact TGF-β inhibitory response), and CBS cells (defects in TGF-β inhibitory response caused by a deficiency in type II receptor activity). The ability of these cell lines to metastasize was analysed in an orthotopic colon cancer mouse model.

Results

FET-α cells did not metastasize to the liver, but showed lung metastasis in 10% of the animals, whereas CBS cells gave rise to metastasis in 65%. Following the elimination of TGF-β activity by transfection and overexpression of dominant negative type II receptor, FET-α cells demonstrated liver and lung metastasis in 70% of the animals. Similarly, after the restoration of type II receptor activity by ectopic expression, CBS cells formed metastasis in fewer (10%) animals.

Conclusions

The results of our study demonstrate for the first time that TGF-β displays selective metastasis suppressor activity. These abnormal pathways can serve as selective targets for future development of targeted therapies.     

Transforming growth factor-β signaling is constantly shaping memory T-cell population

The long-term maintenance of memory T cells is essential for successful vaccines. Both the quantity and the quality of the memory T-cell population must be maintained. The signals that control the maintenance of memory T cells remain incompletely identified. Here we used two genetic models to show that continuous transforming growth factor-β signaling to antigen-specific T cells is required for the differentiation and maintenance of memory CD8⁺ T cells. In addition, both infection-induced and microbiota-induced inflammation impact the phenotypic and functional identity of memory CD8⁺ T cells.Infectious diseases pose a significant public health burden, accounting for nearly one-fifth of annual deaths worldwide. Vaccines remain the most effective way to prevent infectious diseases. Functionally sustained memory T cells are the ideal cell population to be generated by T-cell–based vaccines. Considerable efforts have been made to elucidate the mechanisms that mediate the establishment of long-lived immunologic memory (1–6); however, the signals that control the differentiation and maintenance of memory T cells remain incompletely identified.During the early stages of an immune response, proinflammatory cytokines IL-12 and type I IFN promote the expansion of effector CD8⁺ T cells by sustaining the expression of the high-affinity IL-2 receptor CD25 (7, 8). In addition to its role in T-cell proliferation, IL-2 also functions as a differentiation factor for effector CD8⁺ T cells by promoting the differentiation of short-lived effector cells [SLECs; IL-7Rα⁻KLRG1⁺ (killer cell lectin-like receptor subfamily G, member 1)] and inhibiting the differentiation of memory precursor effector cells (MPECs; IL-7Rα⁺KLRG1⁻) (9–12). Furthermore, IL-10 and IL-21 signals promote MPEC differentiation through a STAT3-dependent mechanism (13, 14). During the late stages of an immune response, IL-15 and IL-7 are required to maintain the population of memory CD8⁺ T cells (15, 16); however, after the clearance of an infection, whether memory CD8⁺ T cells require any additional signals to maintain their phenotypic and functional identity remains unknown.Recent findings have revealed that effector and memory CD8⁺ T cells display nearly endless diversity based on the expression of surface and intracellular molecules that serve as the markers of antigen-experienced T cells (17). Thus, it is conceivable that memory CD8⁺ T cells might not be a fixed cell lineage, but instead represent an active differentiation state. Even in the absence of cognate antigens, memory CD8⁺ T cells may constantly receive diverse environmental signals; however, how memory T cells maintain their relatively stable characters under such circumstances remains unexplored.Here we show that TGF-β signaling to CD8⁺ T cells controls the differentiation of memory T cells at both early and late stages. By deleting TGF-β receptor in antigen-specific T cells at different time points following an acute infection, we demonstrate that during the effector phase of an immune response, TGF-β restrains the inflammatory signals associated with the infection. At the memory phase, both the TGF-β signal and the basal inflammation induced by microbiota cooperate to shape the memory T-cell population. Taken together, our findings show that continuous TGF-β signaling is required to maintain the identity of memory CD8⁺ T cells following acute infections.