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. 2012 Sep 14;287(38):31994-2005.
doi: 10.1074/jbc.M112.355172. Epub 2012 Aug 1.

Cystathionine protects against endoplasmic reticulum stress-induced lipid accumulation, tissue injury, and apoptotic cell death

Kenneth N Maclean  1 , Lori S Greiner, Jeffrey R Evans, Sudesh K Sood, Sarka Lhotak, Neil E Markham, Sally P Stabler, Robert H Allen, Richard C Austin, Vivek Balasubramaniam, Hua Jiang
Affiliations

Cystathionine protects against endoplasmic reticulum stress-induced lipid accumulation, tissue injury, and apoptotic cell death

Kenneth N Maclean et al. J Biol Chem. .

Abstract

Cystathionine (R-S-(2-amino-2-carboxyethyl)-l-homocysteine) is a non-proteinogenic thioether containing amino acid. In mammals, cystathionine is formed as an intermediate of the transsulfuration pathway by the condensation of serine and homocysteine (Hcy) in a reaction catalyzed by cystathionine β-synthase (CBS). Cystathionine is subsequently converted to cysteine plus ammonia and α-ketobutyrate by the action of cystathionine γ-lyase (CGL). Pathogenic mutations in CBS result in CBS-deficient homocystinuria (HCU) which, if untreated, results in mental retardation, thromboembolic complications and connective tissue disorders. Currently there is no known function for cystathionine other than serving as an intermediate in transsulfuration and to date, the possible contribution of the abolition of cystathionine synthesis to pathogenesis in HCU has not been investigated. Using both mouse and cell-culture models, we have found that cystathionine is capable of blocking the induction of hepatic steatosis and kidney injury, acute tubular necrosis, and apoptotic cell death by the endoplasmic reticulum stress inducing agent tunicamycin. Northern and Western blotting analysis indicate that the protective effects of cystathionine occur without any obvious alteration of the induction of the unfolded protein response. Our data constitute the first experimental evidence that the abolition of cystathionine synthesis may contribute to the pathology of HCU and that this compound has therapeutic potential for disease states where ER stress is implicated as a primary initiating pathogenic factor.

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Figures

FIGURE 1.
FIGURE 1.
Methionine metabolism in mammals. The transsulfuration pathway and the methionine and folate cycles are shown.
FIGURE 2.
FIGURE 2.
Hepatic steatosis induced by a methionine-choline-deficient diet is significantly attenuated in HO HCU mice. A, H and E staining from representative liver sections of C57BL/6 WT control (upper panel) and HO (lower panel) mice fed an MCD diet for 17 days. Data are representative of 5 animals per group. Scale bar denotes 200 micrometers. B, hepatic triglyceride levels from HO and C57BL/6 WT mice fed an MCD or control diet (n = 5). In this and all subsequent figures *, **, and *** denote p values of < 0.05., 0.01, and 0.001 respectively.
FIGURE 3.
FIGURE 3.
Tunicamycin induced hepatic steatosis and renal tubule damage is greatly attenuated in HO HCU mice. Representative H and E staining of liver and kidney sections from HO and C57BL/6 WT mice harvested 3 days after treatment with Tuc as described in “Materials and Methods.” Data shown are representative of n = 5 per group. Scale bar denotes 200 micrometers.
FIGURE 4.
FIGURE 4.
Daily PPG injection induces elevated plasma cystathionine in WT mice. Plasma levels in C57BL/6 WT mice of (A) cystathionine and (B) Hcy, methionine, and cysteine before and 1, 2, 3, and 4 days after treatment with the CGL inactivating compound PPG. Values are means ± S.D. at each time point (n = 7). Amino acid levels were determined as described in “Materials and Methods.”
FIGURE 5.
FIGURE 5.
PPG induced elevated cystathionine significantly protects WT mice against tunicamycin induced steatosis and liver injury. A, representative examples of whole livers dissected out of C57BL/6 WT mice from the four experimental groups. Livers from the Tuc group were all severely enlarged, yellowish, and fatty in appearance. Liver morphology appeared ostensibly normal in mice pretreated with PPG prior to tunicamycin administration. B, liver weight as an index of hepatic enlargement. C, hepatic trigycerides as an index of steatosis and D, plasma ALT as an index of liver injury were determined as described in “Materials and Methods.” Values are means ± S.D. (n = 6).
FIGURE 6.
FIGURE 6.
PPG induced elevated cystathionine significantly attenuates steatotic renal enlargement in WT mice treated with tunicamycin. A, representative examples of whole kidneys dissected out of C57BL/6 WT mice from the four experimental groups. Kidneys from the Tuc group were all severely enlarged and were encased in a relatively large amount of fat. In mice treated with PPG prior to tunicamycin administration, this effect was not apparent. B, kidney trigycerides as an index of steatosis were determined as described in “Materials and Methods.” C, kidney weight as an index of enlargement. Values shown represent means ± S.D. n = 6.
FIGURE 7.
FIGURE 7.
PPG induced elevated cystathionine significantly attenuates steatotic hepatopathy, renal tubular injury and apoptotic cell death in C57BL/6 WT mice treated with tunicamycin. H and E staining of representative sections from (A) liver and (B) kidneys from WT mice treated with Tuc in the presence and absence of elevated cystathionine induced by PPG. Tunicamycin induced hepatic steatosis and acute tubular necrosis with swelling of proximal tubular epithelial cells. These tunicamycin induced morphological alterations were virtually ablated in mice treated with PPG. Sections shown are representative of each experimental group (n = 6). C, assessment of apoptosis by TUNEL staining of representative kidney sections from WT mice treated with Tuc in the presence and absence of elevated cystathionine induced by PPG. Apoptotic cells appear dark brown. Scale bars denote 200 micrometers.
FIGURE 8.
FIGURE 8.
Cystathionine protects against ER stress induced cell death in 293AD and a23 cells. A, photomicrograph of 293AD cells treated with tunicamycin in the presence and absence of PPG. Detached floating cells were clearly visible in the tunicamycin group and were not evident in the PPG plus tunicamycin group indicating that this compound protects cells against the cytotoxic effects of tunicamycin treatment. B, relative survival of transsulfuration-positive 293AD (upper graph) and transsulfuration-deficient a23 cells (lower graph) treated with tunicamycin (5 or 10 μg/ml) in the presence or absence of either PPG (5 mm) or cystathionine (1 mm). Cell viability was assessed by LDH release assay as described in “Materials and Methods.” Shown are means ± S.D. of a typical experiment performed in triplicate that is representative of three independent experiments. The ability of cystathionine, but not PPG, to protect against ER stress-induced cell death in transsulfuration negative a23 cells indicates that cystathionine itself is directly cytoprotective and is not simply serving as a cysteine prodrug.
FIGURE 9.
FIGURE 9.
Cystathionine does not exert its cytoprotective effects by modulating the induction levels of either Grp78 or GADD153. A, Northern blot determination of Grp78 and GADD153 mRNA levels induced by 5 and 10 μg/ml tunicamycin in the presence and absence of 1 mm PPG in 293AD cells. Cells treated with 5 mm Hcy were included as a positive control for Grp78 induction. Cell treatment conditions, transfer, probe generation, and detection were performed as described in “Materials and Methods.” B, Western blotting analysis of the tunicamycin mediated induction of the UPR molecular chaperones GRP78 and GRP94 in the presence and absence of PPG (5 mm) and cystathionine (1 mm) in 293AD cells. Cell treatment conditions, transfer, antibody, and detection were performed as described in “Materials and Methods.”
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