Carotenoids are the precursors for vitamin A and are proposed to prevent oxidative damage to cells. Mammalian genomes encode a family of structurally related nonheme iron oxygenases that modify double bonds of these compounds by oxidative cleavage and cis-to-trans isomerization. The roles of the family members BCMO1 and RPE65 for vitamin A production and vision have been well established. Surprisingly, we found that the third family member, β,β-carotene-9',10'-oxygenase (BCDO2), is a mitochondrial carotenoid-oxygenase with broad substrate specificity. In BCDO2-deficient mice, carotenoid homeostasis was abrogated, and carotenoids accumulated in several tissues. In hepatic mitochondria, accumulated carotenoids induced key markers of mitochondrial dysfunction, such as manganese superoxide dismutase (9-fold), and reduced rates of ADP-dependent respiration by 30%. This impairment was associated with an 8- to 9-fold induction of phosphor-MAP kinase and phosphor-AKT, markers of cell signaling pathways related to oxidative stress and disease. Administration of carotenoids to human HepG2 cells depolarized mitochondrial membranes and resulted in the production of reactive oxygen species. Thus, our studies in BCDO2-deficient mice and human cell cultures indicate that carotenoids can impair respiration and induce oxidative stress. Mammalian cells thus express a mitochondrial carotenoid-oxygenase that degrades carotenoids to protect these vital organelles.

The uptake of dietary lipids from the small intestine is a complex process that depends on the activities of specific membrane receptors with yet unknown regulatory mechanisms. Using both mouse models and human cell lines, we show here that intestinal lipid absorption by the scavenger receptor class B type 1 (SR-BI) is subject to control by retinoid signaling. Retinoic acid via retinoic acid receptors induced expression of the intestinal transcription factor ISX. ISX then repressed the expression of SR-B1 and the carotenoid-15,15'-oxygenase Bcmo1. BCMO1 acts downstream of SR-BI and converts absorbed beta,beta-carotene to the retinoic acid precursor, retinaldehyde. Using BCMO1-knockout mice, we demonstrated increased intestinal SR-BI expression and systemic beta,beta-carotene accumulation. SR-BI-dependent accumulation of beta,beta-carotene was prevented by dietary retinoids that induced ISX expression. Thus, our study revealed a diet-responsive regulatory network that controls beta,beta-carotene absorption and vitamin A production by negative feedback regulation. The role of SR-BI in the intestinal absorption of other dietary lipids, including cholesterol, fatty acids, and tocopherols, implicates retinoid signaling in the regulation of lipid absorption more generally and has clinical implications for diseases associated with dyslipidemia.

Individuals with PTEN mutations have Cowden syndrome (CS), associated with breast, thyroid, and endometrial neoplasias. Many more patients with features of CS, not meeting diagnostic criteria (termed CS-like), are evaluated by clinicians for CS-related cancer risk. Germline mutations in succinate dehydrogenase subunits SDHB-D cause pheochromocytoma-paraganglioma syndrome. One to five percent of SDHB/SDHD mutation carriers have renal cell or papillary thyroid carcinomas, which are also CS-related features. SDHB-D may be candidate susceptibility genes for some PTEN mutation-negative individuals with CS-like cancers. To address this hypothesis, germline SDHB-D mutation analysis in 375 PTEN mutation-negative CS/CS-like individuals was performed, followed by functional analysis of identified SDH mutations/variants. Of 375 PTEN mutation-negative CS/CS-like individuals, 74 (20%) had increased manganese superoxide dismutase (MnSOD) expression, a manifestation of mitochondrial dysfunction. Among these, 10 (13.5%) had germline mutations/variants in SDHB (n = 3) or SDHD (7), not found in 700 controls (p < 0.001). Compared to PTEN mutation-positive CS/CS-like individuals, those with SDH mutations/variants were enriched for carcinomas of the female breast (6/9 SDH versus 30/107 PTEN, p < 0.001), thyroid (5/10 versus 15/106, p < 0.001), and kidney (2/10 versus 4/230, p = 0.026). In the absence of PTEN alteration, CS/CS-like-related SDH mutations/variants show increased phosphorylation of AKT and/or MAPK, downstream manifestations of PTEN dysfunction. Germline SDH mutations/variants occur in a subset of PTEN mutation-negative CS/CS-like individuals and are associated with increased frequencies of breast, thyroid, and renal cancers beyond those conferred by germline PTEN mutations. SDH testing should be considered for germline PTEN mutation-negative CS/CS-like individuals, especially in the setting of breast, thyroid, and/or renal cancers.

SUMMARYCarotenoids and their metabolites are widespread and exert key biological functions in living organisms. In vertebrates, the carotenoid oxygenase BCMO1 converts carotenoids such as ,-carotene to retinoids, which are required for embryonic pattern formation and cell differentiation. Vertebrate genomes encode a structurally related protein named BCDO2 but its physiological function remains undefined. Here, we show that BCDO2 is expressed as an oxidative stress-regulated protein during zebrafish development. Targeted knockdown of this mitochondrial enzyme resulted in anemia at larval stages. Marker gene analysis and staining for hemoglobin revealed that erythropoiesis was not impaired but that erythrocytes underwent apoptosis in BCDO2-deficient larvae. To define the mechanism of this defect, we have analyzed the role of BCDO2 in human cell lines. We found that carotenoids caused oxidative stress in mitochondria that eventually led to cytochrome c release, proteolytic activation of caspase 3 and PARP1, and execution of the apoptotic pathway. Moreover, BCDO2 prevented this induction of the apoptotic pathway by carotenoids. Thus, our study identifying BCDO2 as a crucial protective component against oxidative stress establishes this enzyme as mitochondrial carotenoid scavenger and a gatekeeper of the intrinsic apoptotic pathway.