Jianguo Song

Regulation of Transforming Growth Factor-β1–Induced Apoptosis and Epithelial-to-Mesenchymal Transition by Protein Kinase A and Signal Transducers and Activators of Transcription 3

Apoptosis and epithelial-to-mesenchymal transdifferentiation or transition (EMT) are crucial for normal development and body homeostasis. The alterations of these events are closely related to some pathologic processes, such as tumor formation and metastasis, fibrotic diseases of liver and kidney, and abnormal development of embryos. The mechanism that underlies the simultaneously occurring apoptosis and EMT induced by transforming growth factor-beta (TGF-beta) has not been well studied. In this report, we investigated the potential mechanism that underlies TGF-beta1-induced apoptosis and EMT. TGF-beta1-induced apoptosis and EMT were associated with the activation of protein kinase A (PKA) and signal transducers and activators of transcription 3 (STAT3). Inhibition of PKA by specific PKA inhibitor H89 or by PKA inhibitor peptide blocked STAT3 activation and suppressed TGF-beta1-induced apoptosis and EMT. Furthermore, overexpression of a phosphorylation-deficient form of STAT3, but not wild-type STAT3, produced an inhibitory effect on TGF-beta1-induced apoptosis and EMT. The results indicate that PKA is an upstream regulator for TGF-beta1-induced STAT3 activation and plays an important role in TGF-beta1-mediated apoptosis and EMT. These studies provided a new insight into the signaling mechanism underlying the apoptosis and EMT, which could be of importance in understanding some related physiologic and pathologic processes.

FOXA2 functions as a suppressor of tumor metastasis by inhibition of epithelial-to-mesenchymal transition in human lung cancers

The forkhead box transcription factor A2 (FOXA2) is an important regulator in animal development and body homeostasis. However, whether FOXA2 is involved in transforming growth factor β1 (TGF-β1)-mediated epithelial-to-mesenchymal transition (EMT) and tumor metastasis remains unknown. The present study showed that in human lung cancer cell lines, the abundance of FOXA2 positively correlates with epithelial phenotypes and negatively correlates with the mesenchymal phenotypes of cells, and TGF-β1 treatment decreased FOXA2 protein level. Consistently, knockdown of FOXA2 promoted EMT and invasion of lung cancer cells, whereas overexpression of FOXA2 reduced the invasion and suppressed TGF-β1-induced EMT. In addition, knockdown of FOXA2 induced slug expression, and ectopic expression of FOXA2 inhibited slug transcription. Furthermore, we identified that FOXA2 can bind to slug promoter through a conserved binding site, and that the DNA-binding region and transactivation region II of FOXA2 are required for repression of the slug promoter. These data demonstrate that FOXA2 functions as a suppressor of tumor metastasis by inhibition of EMT.

Retinoic acid regulates bone morphogenic protein signal duration by promoting the degradation of phosphorylated Smad1

The proper function of the bone morphogenic protein (BMP) pathway during embryonic development and organ maintenance requires its communication with other signaling pathways. Unlike the well-documented regulation of the BMP pathway by FGF/MAPK and Wnt/GSK3 signals, cross-talk between BMP/Smad and retinoic acid (RA)/RA receptor (RAR) pathways is poorly understood. Here, we show that RA represses BMP signal duration by reducing the level of phosphorylated Smad1 (pSmad1). Through its nuclear receptor-mediated transcription, RA enhances the interaction between pSmad1 and its ubiquitin E3 ligases, thereby promoting pSmad1 ubiquitination and proteasomal degradation. This regulation depends on the RA-increased Gadd45 expression and MAPK activation. During the neural development in chicken embryo, the RA/RAR pathway also suppresses BMP signaling to antagonize BMP-regulated proliferation and differentiation of neural progenitor cells. Furthermore, this cross-talk between RA and BMP pathways is involved in the proper patterning of dorsal neural tube of chicken embryo. Our results reveal a mechanism by which RA suppresses BMP signaling through regulation of pSmad1 stability.

Transforming growth factor-β1 induces epithelial-to-mesenchymal transition and apoptosis via a cell cycle-dependent mechanism

Apoptosis and epithelial-to-mesenchymal transition (EMT) have been implicated in a variety of biological processes, such as embryonic development, fibrosis and tumor progression. Transforming growth factor-β (TGF-β) can induce simultaneously both EMT and apoptotic response of epithelial cells. However, the underlying mechanism of these biological events remains not well understood. In the present study, we show that TGF-β1 induces apoptosis and EMT in AML-12 cells in a cell cycle-related manner, in which apoptosis and EMT took place at G2/M and G1/S phases, respectively. TGF-β1-induced apoptosis was correlated with different extent of caspase activation at different cell cycle phases. Interestingly, increased phosphorylation of protein kinase D (PKD) can be observed in G1/S phase in response to TGF-β1, and inhibition of PKD by inhibitor or by small interference RNA blocked EMT but not apoptosis. Our data suggest a previously unrecognized role of cell cycle state in the regulation of TGF-β-induced EMT and apoptosis, and demonstrate that PKD is involved in the TGF-β1-induced EMT.

The involvement of p38 MAPK in transforming growth factor β1-induced apoptosis in murine hepatocytes

ABSTRACTWe reported in this manuscript that TGF-β1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-β1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-β1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-β1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-β1-mediated gene expression and apoptosis.

Signal transducer and activator of transcription 3 (STAT3) regulates adipocyte differentiation via peroxisome-proliferator-activated receptor γ (PPARγ)

Background information. STAT3 (signal transducer and activator of transcription 3) is an important transcription factor involved in many biological events, including apoptosis, tumorigenesis, angiogenesis and epithelial‐to‐mesenchymal transition. However, no direct evidence for a role of STAT3 in 3T3‐L1 adipocyte differentiation has been reported.

Histone deacetylase 1 is required for transforming growth factor-β1-induced epithelial–mesenchymal transition

UNLABELLED Epithelial-mesenchymal transition (EMT) has been implicated in embryonic development, fibrosis, and tumor metastasis. Histone deacetylases (HDACs) also play important roles in the control of various physiological and pathological events. However, whether HDACs are involved in the control of EMT in liver cells remains unidentified. Three structurally unrelated HDAC inhibitors completely suppress transforming growth factor-beta1 (TGF-beta1)-induced EMT in AML-12 murine hepatocytes and primary mouse hepatocytes. Expression of a dominant-negative mutant of HDAC1 but not HDAC2 or downregulation of HDAC1 but not HDAC2 by RNAi suppressed TGF-beta1-induced EMT. In addition, both HDAC inhibitor TSA and HDAC1 RNAi blocked cell migration. Overexpression of HDAC1 in invasive hepatocellular carcinoma (HCC) samples was detected. Further study showed that the mRNA levels of ZO-1 and E-cadherin were downregulated during TGF-beta1-induced EMT, and HDAC1 can downregulate the promoter activities of ZO-1 and E-cadherin. CONCLUSIONS our results demonstrate that HDAC1 is required for TGF-beta1-induced EMT and cell migration in hepatocytes. Its high expression levels in majority of invasive HCC samples suggest that, by promoting EMT, HDAC1 can be related with the invasiveness of HCC. The data also suggest that the repression of transcription of ZO-1 and E-cadherin by HDAC1 may be involved in TGF-beta1-induced EMT.

Ceramide induces caspase-dependent and -independent apoptosis in A-431 cells.

We investigated the ceramide‐induced apoptosis and potential mechanism in A‐431 cells. Ceramide treatment causes the round up and the death of A‐431 cells that is associated with p38 activation and can be observed in 10 h. Short‐time ceramide treatment‐induced cell death is not associated with the typical apoptotic phenotypes, such as the translocation of phosphatidylserine (PS) from inner layer to outer layer of the plasma membrane, loss of mitochondrial membrane potential, DNA fragmentation, caspase activation, and PARP or PKC‐δ degradation. SB202190, a specific inhibitor of p38 mitogen‐activated protein (MAP) kinase, but not caspase inhibitor, blocks the cell death induced by short‐time ceramide treatment (within 12 h). Whereas neither inhibition of p38 MAP kinase nor inhibition of caspases blocks cell death induced by prolonged ceramide treatment. Moreover, incubation of cells with ceramide for a long time (over 12 h) results in the reduction of proportion of S phase accompanied with typical apoptotic cell death phenotypes that are different from the cell death induced by short‐time ceramide treatment. Our data demonstrated that ceramide‐induced apoptotic cell death involves both caspase‐dependent and caspase‐independent signaling pathways. The caspase‐independent cell death that occurred in relatively early stage of ceramide treatment is mediated via p38 MAP kinase, which can progress into a stage that is associated with changes of cell cycle events and involves both caspase‐dependent and ‐independent mechanisms. J. Cell. Physiol. 199: 47–56, 2004© 2003 Wiley‐Liss, Inc.

TGF-|[beta]|1 suppresses apoptosis via differential regulation of MAP kinases and ceramide production

AbstractSerum deprivation induces apoptosis in NIH3T3 cells, which is associated with increased intracellular ceramide generation and with the activation of p38 mitogen-activated protein (MAP) kinase. Treatment of cells with transforming growth factor-β1 (TGF-β1) activated the extracellular signal regulated kinases 1 and 2 (ERK1/ERK2), inhibited the serum deprivation-induced p38 activation and the increase in intracellular ceramide formation, leading to the stimulation of cell proliferation and the suppression of apoptosis. Inhibition of p38 MAP kinase by SB203580 significantly reduced the serum-deprivation-induced apoptosis. Overexpression of p38 increased the cell apoptosis and reduced the antiapoptotic effect of TGF-β1. Inhibition of ERK1/ERK2 by PD98059 completely inhibited the TGF-β1-stimulated proliferation and partially inhibited the antiapoptotic effects of TGF-β1. Neither SB203580 nor PD98059 has obvious effect on TGF-β1-mediated inhibition of the increased ceramide generation. Serum-deprivation-induced apoptosis in NIH3T3 cells can also be blocked by broad-spectrum caspase inhibitor. TGF-β1 treatment has an inhibitory effect on caspase activities. Our results indicate that ceramide, p38, and ERK1/ERK2 play critical but differential roles in cell proliferation and stress-induced apoptosis. TGF-β1 suppresses the serum-deprivation-induced apoptosis via its distinct effects on complex signaling events involving the activation of ERK1/ERK2 and the inhibition of p38 activation and increased ceramide generation.

Insulin Receptor Substrate-1 Suppresses Transforming Growth Factor-β1–Mediated Epithelial-Mesenchymal Transition

We investigated the regulatory effect of insulin receptor substrate-1 (IRS-1) on transforming growth factor-beta1 (TGF-beta1)-induced epithelial-mesenchymal transition (EMT). TGF-beta1-induced EMT and cell migration in A549 cells are associated with a decrease in IRS-1 tyrosine phosphorylation and protein levels. Tissue microarray analysis of human lung carcinoma shows a correlation between IRS-1 protein levels and E-cadherin protein levels. High IRS-1 levels coexist with high E-cadherin levels, whereas low IRS-1 levels coexist with low E-cadherin levels, implying a possibility that IRS-1 protein levels may be linked with EMT. Surprisingly, overexpression of IRS-1 in A549 cells completely blocked TGF-beta1-induced EMT and cell migration, inhibited TGF-beta1-mediated expression of snail and slug genes, and abolished TGF-beta1-mediated repression of E-cadherin promoter activity. In contrast, IRS-1 knockdown by RNAi increased the expression of snail and slug genes and induced EMT. Inhibition of protein tyrosine phosphatase with sodium vanadate, which greatly increased the levels of tyrosine-phosphorylated IRS-1, suppressed TGF-beta1-induced actin remodeling and cell morphologic changes. These results show for the first time that TGF-beta1 induces EMT through mechanisms involving the modulation of IRS-1 signaling, and that IRS-1 functions as a critical EMT suppressor that suppresses TGF-beta1-induced EMT via inhibition of snail and slug expression.