Differential metabolic consequences of fumarate hydratase and respiratory chain defects

Abstract

Defects of the oxidative ATP production pathway lead to an amazing variety of disease phenotypes, ranging from childhood encephalomyopathies to hereditary tumor formation. A key enzyme of tricarboxylic cycle, fumarate hydratase (FH), is involved in encephalopathies, but also in leiomyoma formation, and occasionally also in various types of cancer. MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and NARP (neuropathy ataxia retinitis pigmentosa) are progressive neurological disorders, caused by mitochondrial DNA mutations and respiratory chain (RC) deficiency. These diseases lead to disability and premature death, but not to tumorigenesis. We studied the cellular consequences of FH and RC deficiencies, aiming to identify general responses to energy metabolism defect and those specific for FH-deficiency, suggestively connected to tumorigenesis. Unlike in RC deficiency, the FH-deficient diploid human fibroblasts showed no signs of oxidative stress, but had a reduced redox state with high glutathione levels. The cytoplasmic FH isoform, previously described, but with an unknown function, was completely lacking in all FH-deficient lines. Fumarate was increased in two of our FH-lines, but accumulation of HIF-1alpha was not detected. Glycolysis was induced in both MELAS and in FH-deficiency. Accumulation of fumarate in primary fibroblasts did not activate a hypoxia response, suggesting that hypoxia activation due to fumarate accumulation may be a tissue-specific response. The lack of cytoplasmic form of FH and the reduced redox environment were typical for all FH-mutant lines, and their role in FH-related tumorigenesis requires further attention.

Downregulation of SRF–FOS–JUNB pathway in fumarate hydratase deficiency and in uterine leiomyomas

“…The FH-specific networks identified in primary fibroblasts were then tested in FH-deficient leiomyoma transcriptome data sets, to study the relevance of fibroblast data to leiomyoma formation. Although FH-deficient fibroblasts were diploid with normal growth behavior (Raimundo et al, 2008), but showed low FH activity similar as FH-deficient leiomyomas, networks common to these cells and leiomyomas are excellent candidates to be involved in FHrelated leiomyoma formation. We show here that FHdeficient fibroblasts and leiomyomas show widespread downregulation of the SRF-regulated transcripts and also of active SRF, known to participate in smooth muscle cell differentiation (Pipes et al, 2006).…”

“…The patient cell lines have been described in detail in Raimundo et al (2008). In short, four FH-deficient fibroblast lines from patients with recessive FH deficiency were used-FH-1, homozygous for the E362Q mutation (Bourgeron et al, 1994); FH-2, homozygous for the Q376P mutation (Remes et al, 2004); FH-3 is a compound heterozygote with InsK477/ R233H (Loeffen et al, 2005); and FH-4 is a compound heterozygote with delV387 and Q386H changes in one allele, and P369S in the other (Maradin et al, 2006).…”

“…cDNA synthesis and real-time amplification were performed using standardized Assays-onDemand probes (Applied Biosystems; Foster City, CA, USA) as previously described (Raimundo et al, 2008). The expression of each transcript was normalized to the expression of GAPDH.…”

“…Nuclear extracts were prepared according to Wang and Semenza (1995) and modified as in Raimundo et al (2008).…”

“…FH defects lead to hereditary leiomyomatosis and renal cell cancer, and have been reported in other tumors, such as ovary adenocarcinomas, Leydig cell tumors and cerebral cavernomas (Tomlinson et al, 2002;Alam et al, 2003;Carvajal-Carmona et al, 2006;Ylisaukko-Oja et al, 2006;Campione et al, 2007;Refae et al, 2007). Several hypotheses have been proposed for the mechanisms how FH deficiency leads to tumor formation, including fumarate accumulation inducing a hypoxic response, oxidative stress and decreased differentiation of FHdeficient progenitor cells (Eng et al, 2003;Pollard et al, 2003;Gottlieb and Tomlinson, 2005;Raimundo et al, 2008). However, the primary cellular signaling consequences of FH deficiency in diploid cells and the mechanisms how those lead to tumor formation remain unknown.…”