Induction of apoptosis by Smad3 and down-regulation of Smad3 expression in response to TGF-β in human normal lung epithelial cells

Yanagisawa, Kiyoshi; Osada, Hiroyuki; Masuda, Akira; Kondo, Masashi; Saito, Toshiko; Yatabe, Yasushi; Takagi, Kenzo; Takahashi, Takashi

doi:10.1038/sj.onc.1202052

Cited by 152 publications

(113 citation statements)

References 18 publications

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“…Repression of SMAD3 mRNA levels in response to TGF␤ has been noted in epithelial and mesangial cells as well as in lung fibroblasts (45)(46)(47). In light of the dependence of TGF␤ signaling intensity on the steadystate levels of its intracellular second messengers, inhibition of SMAD3 expression may thus be a negative feedback mechanism with autoregulatory function to prevent continuous SMAD signaling in response to TGF␤ stimulation.…”

Section: Discussionmentioning

confidence: 99%

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

Mori

Chen

Varga

2003

Arthritis & Rheumatism

172

115

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Objective. Scleroderma is characterized by excessive synthesis and accumulation of matrix proteins in lesional tissues. Transforming growth factor ␤ (TGF␤) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of fibroblasts; however, the underlying mechanisms are poorly understood. We undertook this study to examine the expression and function of SMADs, recently characterized intracellular effectors of TGF␤ signaling, in scleroderma fibroblasts.Methods. Primary dermal fibroblasts obtained from 14 patients with scleroderma and from 4 healthy adult volunteers were studied. Northern analysis was used to determine the expression of endogenous SMAD messenger RNA (mRNA), and Western analysis was used to determine SMAD protein expression. Intracellular compartmentalization of cellular SMAD proteins in the presence and absence of TGF␤ was studied by antibody-mediated immunofluorescence confocal microscopy. The effect of TGF␤ blockade on SMAD subcellular distribution was determined using anti-TGF␤ antibodies as well as a dominant-negative TGF␤ receptor type II (TGF␤RII) vector to disrupt TGF␤ responses. SMAD-regulated luciferase reporter expression was examined to investigate the potential functional significance of activation and nuclear accumulation of endogenous SMADs in scleroderma fibroblasts.Results. Protein and mRNA levels of SMAD3, but not of SMAD4 or SMAD7, were variably elevated in scleroderma fibroblasts compared with those from healthy controls. In sharp contrast to control fibroblasts, which displayed predominantly cytoplasmic localization of SMAD3/4 in the absence of exogenous TGF␤, in scleroderma fibroblasts SMAD3 and SMAD4 consistently showed elevated nuclear localization. Furthermore, phosphorylated SMAD2/3 levels were elevated and nuclear localization of phosphorylated SMAD2/3 was increased, suggesting activation of the SMAD pathway in scleroderma fibroblasts. Blockade of autocrine TGF␤ signaling with antibodies or by expression of dominant-negative TGF␤RII failed to normalize SMAD subcellular distribution, suggesting that elevated nuclear SMAD import was due to alterations downstream of the TGF␤ receptors. The activity of a SMADresponsive minimal promoter-reporter construct was enhanced in transiently transfected scleroderma fibroblasts.Conclusion. This study is the first to demonstrate apparently ligand-independent constitutive activation of the intracellular TGF␤/SMAD signaling axis in scleroderma fibroblasts. SMAD signaling may be a mechanism contributing to the characteristic phenotype of scleroderma fibroblasts and playing a role in the pathogenesis of fibrosis.Scleroderma is associated with fibrosis of affected tissues due to excessive synthesis and progressive accumulation of connective tissue macromolecules (1). Scleroderma fibroblasts display features of sustained activation in vitro and in vivo. In culture, these cells show elevated synthesis of collagens, fibronectin, proteoglycans, and tissue inhibitors of metalloproteinases, constitutive secretion of i...

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Section: Discussionmentioning

confidence: 99%

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

Mori

Chen

Varga

2003

Arthritis & Rheumatism

172

115

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“…The overexpression of DPC4/Smad4 in MDCK cells is sufficient to induce the activation of gene transcription, cell cycle arrest, and apoptosis [35]. It has also been shown that constitutive expression of Smad3 did not induce apoptosis, but made human normal epithelial cells more sensitive to TGF-β-induced apoptosis [36].…”

Section: Disscussionmentioning

confidence: 99%

“…JNK and p38 MAPK have been shown to mediate apoptosis induced by many stimuli [24][25][26][27][28][29][30][31][32][33][34][35][36][37]. Though it has been shown that TGF-β activates JNK and p38 signaling molecules [21][22][23], their implication in TGF-β-induced apoptosis is not clear.…”

Section: Disscussionmentioning

confidence: 99%

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

et al. 2001

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We 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.

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“…It has also been suggested that TGF-␤1 can induce apoptosis in human primary B lymphocytes (19 -21) and BL cell lines (20,(22)(23)(24)(25)(26). The induction of apoptosis by TGF-␤1 has also been observed in myeloid leukemia cells (27), gastric carcinoma cells (28), primary endometrial cells (29), primary hepatocytes and hepatoma cells (30), human cervical carcinoma cell lines (31), and human lung epithelial cell lines (32).…”

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confidence: 91%

Apoptosis Induced by TGF-β1 in Burkitt’s Lymphoma Cells Is Caspase 8 Dependent But Is Death Receptor Independent

Inman

Allday

2000

The Journal of Immunology

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TGF-β is a potent inducer of apoptosis in many Burkitt’s lymphoma (BL) cell lines. In this study, we characterize this apoptotic process in the EBV-negative BL41 cell line. Induction of apoptosis was detected as early as 8 h after TGF-β treatment, as assayed by TUNEL and poly(ADP-ribose) polymerase cleavage. FACS analysis demonstrates that this proceeds predominately from the G1, but also from the G2/M phases of the cell cycle. We observed no early detectable changes in the steady-state levels of Bcl-2 and several of its family members after TGF-β treatment. We detected cleavage of caspases 2, 3, 7, 8, and 9 into their active subunits. Consistent with the involvement of these enzymes in TGF-β-mediated apoptosis, the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(Ome)-flouromethylketone (ZVAD-fmk) blocked TGF-β-induced apoptosis and revealed a G1 arrest in treated cells. Use of specific caspase inhibitors revealed that the induction of apoptosis is caspase 8 dependent, but caspase 3 independent. Activation of caspase 8 has been shown to be a critical event in death receptor-mediated apoptosis. However, TGF-β treatment of BL41 cells was found not to affect the cell surface expression of Fas, TNF-R1, DR3, DR4, or DR5, or the steady-state expression levels of Fas ligand, TNF-R1, DR3, DR4, and DR5. Furthermore, blocking experiments indicated that TGF-β-mediated apoptosis is not dependent on Fas ligand, TNF-α, tumor necrosis-like apoptosis-inducing ligand, or TNF-like weak inducer of apoptosis signaling. Therefore, it appears that TGF-β induces apoptosis in BL cell lines via caspase 8 in a death receptor-independent fashion.

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Induction of apoptosis by Smad3 and down-regulation of Smad3 expression in response to TGF-β in human normal lung epithelial cells

Cited by 152 publications

References 18 publications

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

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

Apoptosis Induced by TGF-β1 in Burkitt’s Lymphoma Cells Is Caspase 8 Dependent But Is Death Receptor Independent

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