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TGFß1-induced suppression of glutathione antioxidant defenses in hepatocytes: caspase-dependent post-translational and caspase-independent transcriptional regulatory mechanisms ¹

CHRISTOPHER C. FRANKLIN^*,2,3, MARYLAND E. ROSENFELD-FRANKLIN^*,2,4, COLLIN WHITE, TERRANCE J. KAVANAGH and NELSON FAUSTO^*

^* Department of Pathology, University of Washington, Seattle, Washington, USA; and
Department of Environmental Health, University of Washington, Seattle, Washington, USA

³Correspondence: University of Washington, Department of Pathology, Box 357705, 1959 N.E. Pacific St., HSB K-088, Seattle, WA 98195-7705, USA. E-mail: cfrankli{at}u.washington.edu

SPECIFIC AIMS

Oxidative stress has been implicated in mediating TGFß1-induced hepatocyte apoptosis. The tripeptide antioxidant glutathione (GSH) plays a vital role in maintaining cellular redox status and protecting against oxidative injury in the liver. Though there is growing evidence that intracellular glutathione levels regulate cellular sensitivity to apoptotic cell death, little is known concerning the dynamic regulation of GSH biosynthesis during apoptosis. To elucidate the molecular mechanisms mediating TGFß1-induced hepatocyte apoptosis and determine whether suppression of GSH biosynthesis may play a role in TGFß1-induced apoptotic cell death, we examined TGFß1-induced apoptosis in the TAMH murine hepatocyte cell line.

PRINCIPAL FINDINGS

1. Bcl-X_L regulates TGFß1-induced apoptosis in the murine TAMH hepatocyte cell line
Treatment of TAMH cells with TGFß1 resulted in the activation of multiple caspases, which correlated with the appearance of apoptotic morphology and a loss in cell viability. TGFß1-induced apoptosis was accompanied by a decrease in Bcl-X_L protein expression, which appeared to play a role in facilitating TGFß1-induced cell death, as overexpression of Bcl-X_L prevented TGFß1-induced caspase activation and apoptotic cell death. TGFß1-induced apoptosis also required de novo protein synthesis as cycloheximide inhibited TGFß1-induced cell death.

2. Suppression of GSH biosynthesis during TGFß1-induced apoptosis: caspase-dependent cleavage and loss of the catalytic subunit of the rate-limiting enzyme in GSH biosynthesis
The rate-limiting step in GSH biosynthesis is catalyzed by glutamate cysteine ligase (GCL), a heterodimeric holoenzyme consisting of a catalytic subunit (GCLC) and a modifier subunit (GCLM). TGFß1-induced apoptosis was accompanied by the cleavage of GCLC protein from a full-length 73 kDa form to a 60 kDa form (Fig. 1 A, upper panel). In addition, TGFß1 induced a loss in total GCLC protein expression. These responses were dependent on caspase activation as Bcl-X_L overexpression (Fig. 1A , lower panel) or cycloheximide pretreatment prevented TGFß1-induced cleavage of GCLC protein and attenuated the loss of GCLC protein. TGFß1 treatment resulted in a 70–80% reduction in GCL activity in TAMH/neo cells, whereas only a slight decrease in GCL activity (20–30%) was observed in TGFß1-treated TAMH/Bcl-X_L cells (Fig. 1B ). The reduction in GCL activity in TAMH/neo cells correlated with an 80–90% decrease in intracellular GSH levels (Fig. 1C , filled bars) whereas GSH homeostasis was maintained in TAMH/Bcl-X_L cells (Fig. 1C , open bars), suggesting a causal relationship between the loss in GSH biosynthetic capacity and depletion of intracellular GSH.

View larger version (27K): [in this window] [in a new window]   Figure 1. TGFß1-induced cleavage and loss of GCLC protein, loss of GCL activity, and depletion of intracellular GSH are prevented by overexpression of Bcl-XL. A) TAMH/neo (upper panel) and TAMH/Bcl-XL (lower panel) cells were treated with TGFß1 (5 ng/mL) for the periods indicated and GCLC protein expression was assessed by immunoblot analysis. B) TAMH/neo and TAMH/Bcl-XL cells were treated with TGFß (5 ng/mL) for 48 h and analyzed for GCL enzymatic activity. Results shown are the averages ±SE of 3 experiments. C) TAMH/neo (filled bars) and TAMH/Bcl-XL (open bars) cells were treated with TGFß and total intracellular glutathione levels (GSH+GSSG) were measured. The data shown are averages ±SE of 5 experiments.

3. Caspase-independent suppression of GCLC gene expression during TGFß1-induced apoptosis requires histone deacetylase activity and de novo protein synthesis
TGFß1 treatment also caused a rapid down-regulation of GCLC mRNA while having no effect on GCLM mRNA levels (Fig. 2 A). TGFß1 had no effect on the half-life of GCLC mRNA (4 h) (Fig. 2B ), suggesting that TGFß1 decreased GCLC mRNA levels by inhibiting GCLC gene transcription. TGFß1-induced transcriptional repression often involves the recruitment of histone deacetylases (HDACs) to the transcriptional complex. As shown in Fig. 2C , inhibition of HDAC activity with sodium butyrate or trichostatin A resulted in a reversal of TGFß1-induced down-regulation of GCLC mRNA, suggesting that this is an active repression of GCLC gene transcription. TGFß1-induced reduction in GCLC mRNA was also inhibited by cycloheximide (Fig. 2D ), indicating that de novo protein synthesis is required for this response. TGFß1-induced down-regulation of GCLC mRNA occurred before caspase activation (within 4 h) and a similar response was observed in TAMH/Bcl-X_L cells. Thus, in contrast to the caspase-dependent post-translational processing of GCLC protein, TGFß1-induced suppression of GCLC gene expression occurred independent of caspase activation.

View larger version (52K): [in this window] [in a new window]   Figure 2. TGFß1 induces the caspase-independent repression of GCLC gene expression in TAMH cells. A) Cells were incubated in the absence or presence of TGFß1 (5 ng/mL). B) Cells were treated with actinomycin D (ActD; 250 ng/mL) in the absence or presence of TGFß1 (5 ng/mL). C) Cells were treated for 8 h with TGFß1 (5 ng/mL) in the presence of increasing concentrations of sodium butyrate (Na-But; mM) or trichostatin A (TSA; uM). D) Cells were preincubated for 1 h with cycloheximide (CHX; 0.5 µg/mL) before treatment for 8 h with TGFß1 (5 ng/mL). Total RNA was isolated and GCLC, GCLM, and GAPDH mRNA were assessed by Northern blot analysis. Relative mRNA levels were quantitated and GCLC (filled bars) and GCLM (open bars) levels were normalized to GAPDH mRNA. The data presented are representative of at least 3 experiments.

CONCLUSIONS AND SIGNIFICANCE

TGFß1-induced hepatocyte apoptosis is associated with mitochondrial dysfunction characterized by a loss of mitochondrial membrane potential (_m) and the release of cytochrome c and other apoptogenic factors into the cytosol. Bcl-X_L functions at the level of the mitochondria to prevent these responses. Several reports have proposed that down-regulation of Bcl-X_L facilitates mitochondrial dysfunction during TGFß1-induced apoptosis. Our finding that ectopic overexpression of Bcl-X_L prevents TGFß1-induced cell death confirms that TGFß1 induces apoptosis by a mitochondrial-dependent mechanism and provides direct support for a model whereby down-regulation of endogenous Bcl-X_L may be required for TGFß1-induced hepatocyte apoptosis.

TGFß1-induced hepatocyte apoptosis is also associated with depletion in intracellular levels of the tripeptide antioxidant GSH. However, little is known about the mechanism(s) mediating the reduction in intracellular GSH. In this report we identify two distinct molecular mechanisms that contribute to the TGFß1-induced loss of GSH homeostasis (Fig. 3 ). We demonstrate that TGFß1-induced apoptosis is accompanied by the cleavage and loss of GCLC protein resulting in a decrease in GSH biosynthetic capacity. The correlation between TGFß1-induced depletion of intracellular GSH and the loss of GCL activity suggests a causal relationship between these two events. This is supported by our finding that GSH homeostasis is maintained in TGFß1-treated TAMH/Bcl-X_L cells when only a minimal loss in GCL activity is observed. These findings suggest a novel mechanism whereby TGFß1 induces the caspase-dependent cleavage and loss of GCLC protein, which dramatically reduces cellular GSH biosynthetic capacity. This results in the depletion of intracellular GSH, which may play a role in enhancing TGFß1-induced oxidative stress and potentiating apoptotic cell death.

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic diagram of the transcriptional and post-translational pathways that contribute to TGFß1-induced suppression of GSH biosynthesis in murine hepatocytes. TGFß1-induced post-translational processing of GCLC protein is dependent on caspase activation and de novo protein synthesis as Bcl-XL overexpression and cycloheximide (CHX) prevent GCLC cleavage, respectively. Transcriptional suppression of GCLC gene expression is also dependent on de novo protein synthesis but occurs independent of caspase activation. Our data suggest that induction of a transcriptional repressor (TR) recruits a histone deacetylase (HDAC) to the transcriptional complex and suppresses GCLC gene expression.

TGFß1 also caused a rapid down-regulation of GCLC mRNA. This reduction in GCLC mRNA is due to a transcriptional mechanism as TGFß1 did not induce the post-transcriptional destabilization of GCLC mRNA. Furthermore, our findings suggest that a newly synthesized HDAC-dependent transcriptional repressor mediates TGFß1-induced inhibition of GCLC gene transcription. Whereas TGFß1 induces the caspase-dependent cleavage of GCLC protein, suppression of GCLC gene expression occurs independent of caspase activation and apoptotic cell death. The caspase-independent nature of this transcriptional repression suggests that suppression of GCLC expression and loss of GSH homeostasis may also be involved in TGFß1-induced responses that do not involve apoptotic cell death, such as fibrosis and growth inhibition.

Although the down-regulation of GCLC mRNA likely contributes to the reduction in GCLC protein during TGFß1-induced apoptosis, suppression of GCLC gene expression alone cannot account for the dramatic loss of GCLC protein in the absence of protein turnover. This is highlighted by the ability of TGFß1 to efficiently down-regulate GCLC mRNA in TAMH/Bcl-X_L cells while causing only a slight decrease in GCLC protein expression. Even though the correlation between GCLC cleavage and loss of GCLC protein suggests that caspase-mediated cleavage of GCLC may be required for subsequent degradation during TGFß1-induced apoptosis, GCLC is cleaved to a stable 60 kDa fragment during TNF- and Fas-induced apoptosis in TAMH cells. Thus, an additional TGFß1-induced signal is required for the rapid loss in total GCLC protein expression. TGFß1 is known to target multiple proteins for ubiquitination and proteasome-mediated degradation and it is possible that a similar mechanism mediates TGFß1-induced degradation of GCLC. It is interesting to speculate that while TGFß1 treatment might lead to a gradual loss of GCLC protein in the absence of caspase activation due to suppression of GCLC gene transcription, caspase-mediated cleavage of GCLC may function to ensure its rapid degradation during TGFß1-induced apoptotic cell death.

In summary, we have identified two distinct molecular mechanisms that contribute to TGFß1-induced suppression of the GSH antioxidant defense system in hepatocytes. Both caspase-dependent post-translational and caspase-independent transcriptional events appear to mediate TGFß1-induced suppression of GCLC expression and GSH biosynthesis. Our findings are consistent with a model in which TGFß1 blocks de novo synthesis of GSH by inducing the caspase-mediated cleavage and loss of GCLC protein and preventing the resynthesis of GCLC by suppressing GCLC gene expression (Fig. 3) . These findings highlight the potential importance of the GSH antioxidant defense system in regulating TGFß1-induced responses in hepatocytes. As ROS production plays a primary role in initiating TGFß1-induced hepatocyte cell death, a reduction in GSH biosynthetic capacity and subsequent depletion of intracellular GSH may promote apoptotic cell death by sensitizing cells to the damaging effects of TGFß1-induced oxidative stress.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0867fje; doi: 10.1096/fj.02-0867fje

² These authors contributed equally to this work.

⁴ Current address: ZymoGenetics Inc., Seattle, WA 98102, USA.

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