MITOCHONDRIAL CHOLESTEROL AND CANCER.

Cholesterol is a crucial component of membrane bilayers that determines their physical and functional properties. Cells largely satisfy their need for cholesterol through the novo synthesis from acetyl-CoA and this demand is particularly critical for cancer cells to sustain dysregulated cell proliferation. However, the association between serum or tissue cholesterol levels and cancer development is not well established as epidemiologic data do not consistently support this link. While most preclinical studies focused on the role of total celular cholesterol, the specific contribution of the mitochondrial cholesterol pool to alterations in cancer cell biology has been less explored. Although low compared to other bilayers, the mitochondrial cholesterol content plays an important physiological function in the synthesis of steroid hormones in steroidogenic tissues or bile acids in the liver and controls mitochondrial function. In addition, mitochondrial cholesterol metabolism generates oxysterols, which in turn, regulate multiple pathways, including cholesterol and lipid metabolism as well as cell proliferation. In the present review, we summarize the regulation of mitochondrial cholesterol, including its role in mitochondrial routine performance, cell death and chemotherapy resistance, highlighting its potential contribution to cancer. Of particular relevance is hepatocellular carcinoma, whose incidence in Western countries had tripled in the past decades due to the obesity and type II diabetes epidemic. A better understanding of the role of mitochondrial cholesterol in cancer development may open up novel opportunities for cancer therapy.

Analysis of Activity of Human Steroidogenic Acute Regulatory Protein (STARD1) Expressed in Escherichia coli Cells.

One of the main obstacles to the successful use of Escherichia coli cells for steroid transformation in biotechnological processes is inefficient transport of steroid substrates into the cells. Here, we tested the possibility of using human cholesterol transfer protein STARD1 (steroidogenic acute regulatory protein) to increase the efficiency of steroid uptake by bacterial cells. Genetic constructs were obtained for the synthesis in E. coli BL21 (DE3) cells of a truncated version of STARD1 containing protein functional domain (residues 66-285) and STARD1 (66-285)-GFP fusion protein, both carrying bacterial periplasmic targeting sequence pelB at the N-terminus. Analysis of preparations of E. coli/pET22b/STARD1-GFP cells by fluorimetry and Western blotting confirmed that the used expression system ensured the synthesis of the heterologous protein. Using fluorescence spectroscopy, it was demonstrated that the presence of STARD1 in the cells increased the efficiency of assimilation of NBD-labeled cholesterol analogues by E. coli/pET22b/STARD1 cells 1.3-1.6 times (p < 0.05) compared to the wild-type cells, thus demonstrating that human STARD1 exhibits its functional activity in bacterial cells. This opens prospects for optimizing and using a fundamentally new approach to increase the efficiency of steroid uptake by cells - the inclusion of a specific carrier protein in the cell membrane, which can expand the arsenal of methods used to obtain strains of microorganisms for synthesis.

STARD1 promotes NASH-driven HCC by sustaining the generation of bile acids through the alternative mitochondrial pathway.

BACKGROUND & AIMS: Besides their physiological role in bile formation and fat digestion, bile acids (BAs) synthesised from cholesterol in hepatocytes act as signalling molecules that modulate hepatocellular carcinoma (HCC). Trafficking of cholesterol to mitochondria through steroidogenic acute regulatory protein 1 (STARD1) is the rate-limiting step in the alternative pathway of BA generation, the physiological relevance of which is not well understood. Moreover, the specific contribution of the STARD1-dependent BA synthesis pathway to HCC has not been previously explored. METHODS: STARD1 expression was analyzed in a cohort of human non-alcoholic steatohepatitis (NASH)-derived HCC specimens. Experimental NASH-driven HCC models included MUP-uPA mice fed a high-fat high-cholesterol (HFHC) diet and diethylnitrosamine (DEN) treatment in wild-type (WT) mice fed a HFHC diet. Molecular species of BAs and oxysterols were analyzed by mass spectrometry. Effects of NASH-derived BA profiles were investigated in tumour-initiated stem-like cells (TICs) and primary mouse hepatocytes (PMHs). RESULTS: Patients with NASH-associated HCC exhibited increased hepatic expression of STARD1 and an enhanced BA pool. Using NASH-driven HCC models, STARD1 overexpression in WT mice increased liver tumour multiplicity, whereas hepatocyte-specific STARD1 deletion (Stard1ΔHep) in WT or MUP-uPA mice reduced tumour burden. These findings mirrored the levels of unconjugated primary BAs, ß-muricholic acid and cholic acid, and their tauroconjugates in STARD1-overexpressing and Stard1ΔHep mice. Incubation of TICs or PMHs with a mix of BAs mimicking this profile stimulated expression of genes involved in pluripotency, stemness and inflammation. CONCLUSIONS: The study reveals a previously unrecognised role of STARD1 in HCC pathogenesis, wherein it promotes the synthesis of primary BAs through the mitochondrial pathway, the products of which act in TICs to stimulate self-renewal, stemness and inflammation. LAY SUMMARY: Effective therapy for hepatocellular carcinoma (HCC) is limited because of our incomplete understanding of its pathogenesis. The contribution of the alternative pathway of bile acid (BA) synthesis to HCC development is unknown. We uncover a key role for steroidogenic acute regulatory protein 1 (STARD1) in non-alcoholic steatohepatitis-driven HCC, wherein it stimulates the generation of BAs in the mitochondrial acidic pathway, the products of which stimulate hepatocyte pluripotency and self-renewal, as well as inflammation.

Mitochondrial cholesterol import.

All animal subcellular membranes require cholesterol, which influences membrane fluidity and permeability, fission and fusion processes, and membrane protein function. The distribution of cholesterol among subcellular membranes is highly heterogeneous and the cholesterol content of each membrane must be carefully regulated. Compared to other subcellular membranes, mitochondrial membranes are cholesterol-poor, particularly the inner mitochondrial membrane (IMM). As a result, steroidogenesis can be controlled through the delivery of cholesterol to the IMM, where it is converted to pregnenolone. The low basal levels of cholesterol also make mitochondria sensitive to changes in cholesterol content, which can have a relatively large impact on the biophysical and functional characteristics of mitochondrial membranes. Increased mitochondrial cholesterol levels have been observed in diverse pathological conditions including cancer, steatohepatitis, Alzheimer disease and Niemann-Pick Type C1-deficiency, and are associated with increased oxidative stress, impaired oxidative phosphorylation, and changes in the susceptibility to apoptosis, among other alterations in mitochondrial function. Mitochondria are not included in the vesicular trafficking network; therefore, cholesterol transport to mitochondria is mostly achieved through the activity of lipid transfer proteins at membrane contact sites or by cytosolic, diffusible lipid transfer proteins. Here we will give an overview of the main mechanisms involved in mitochondrial cholesterol import, focusing on the steroidogenic acute regulatory protein StAR/STARD1 and other members of the StAR-related lipid transfer (START) domain protein family, and we will discuss how changes in mitochondrial cholesterol levels can arise and affect mitochondrial function. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

Mitochondrial cholesterol: mechanisms of import and effects on mitochondrial function.

Mitochondria require cholesterol for biogenesis and membrane maintenance, and for the synthesis of steroids, oxysterols and hepatic bile acids. Multiple pathways mediate the transport of cholesterol from different subcellular pools to mitochondria. In steroidogenic cells, the steroidogenic acute regulatory protein (StAR) interacts with a mitochondrial protein complex to mediate cholesterol delivery to the inner mitochondrial membrane for conversion to pregnenolone. In non-steroidogenic cells, several members of a protein family defined by the presence of a StAR-related lipid transfer (START) domain play key roles in the delivery of cholesterol to mitochondrial membranes. Subdomains of the endoplasmic reticulum (ER), termed mitochondria-associated ER membranes (MAM), form membrane contact sites with mitochondria and may contribute to the transport of ER cholesterol to mitochondria, either independently or in conjunction with lipid-transfer proteins. Model systems of mitochondria enriched with cholesterol in vitro and mitochondria isolated from cells with (patho)physiological mitochondrial cholesterol accumulation clearly demonstrate that mitochondrial cholesterol levels affect mitochondrial function. Increased mitochondrial cholesterol levels have been observed in several diseases, including cancer, ischemia, steatohepatitis and neurodegenerative diseases, and influence disease pathology. Hence, a deeper understanding of the mechanisms maintaining mitochondrial cholesterol homeostasis may reveal additional targets for therapeutic intervention. Here we give a brief overview of mitochondrial cholesterol import in steroidogenic cells, and then focus on cholesterol trafficking pathways that deliver cholesterol to mitochondrial membranes in non-steroidogenic cells. We also briefly discuss the consequences of increased mitochondrial cholesterol levels on mitochondrial function and their potential role in disease pathology.

Mitochondrial dysfunction in NPC1-deficiency is not rescued by drugs targeting the glucosylceramidase GBA2 and the cholesterol-binding proteins TSPO and StARD1.

Niemann-Pick type C disease (NPCD) is a rare neurodegenerative disorder most commonly caused by mutations in the lysosomal protein Niemann-Pick C1 (NPC1), which is implicated in cholesterol export. Mitochondrial insufficiency forms a significant feature of the pathology of this disease, yet studies attempting to address this are rare. The working hypothesis is that mitochondria become overloaded with cholesterol which renders them dysfunctional. We examined two potential protein targets-translocator protein (TSPO) and steroidogenic acute regulatory protein D1 (StARD1)-which are implicated in cholesterol transport to mitochondria, in addition to glucocerbrosidase 2 (GBA2), the target of miglustat, which is currently the only approved treatment for NPCD. However, inhibiting these proteins did not correct the mitochondrial defect in NPC1-deficient cells.

ASMase is required for chronic alcohol induced hepatic endoplasmic reticulum stress and mitochondrial cholesterol loading.

BACKGROUND & AIMS: The pathogenesis of alcohol-induced liver disease (ALD) is poorly understood. Here, we examined the role of acid sphingomyelinase (ASMase) in alcohol induced hepatic endoplasmic reticulum (ER) stress, a key mechanism of ALD. METHODS: We examined ER stress, lipogenesis, hyperhomocysteinemia, mitochondrial cholesterol (mChol) trafficking and susceptibility to LPS and concanavalin-A in ASMase(-)(/-) mice fed alcohol. RESULTS: Alcohol feeding increased SREBP-1c, DGAT-2, and FAS mRNA in ASMase(+/+) but not in ASMase(-/-) mice. Compared to ASMase(+/+) mice, ASMase(-/-) mice exhibited decreased expression of ER stress markers induced by alcohol, but the level of tunicamycin-mediated upregulation of ER stress markers and steatosis was similar in both types of mice. The increase in homocysteine levels induced by alcohol feeding was comparable in both ASMase(+/+) and ASMase(-/-) mice. Exogenous ASMase, but not neutral SMase, induced ER stress by perturbing ER Ca(2+) homeostasis. Moreover, alcohol-induced mChol loading and StARD1 overexpression were blunted in ASMase(-/-) mice. Tunicamycin upregulated StARD1 expression and this outcome was abrogated by tauroursodeoxycholic acid. Alcohol-induced liver injury and sensitization to LPS and concanavalin-A were prevented in ASMase(-/-) mice. These effects were reproduced in alcohol-fed TNFR1/R2(-/-) mice. Moreover, ASMase does not impair hepatic regeneration following partial hepatectomy. Of relevance, liver samples from patients with alcoholic hepatitis exhibited increased expression of ASMase, StARD1, and ER stress markers. CONCLUSIONS: Our data indicate that ASMase is critical for alcohol-induced ER stress, and provide a rationale for further clinical investigation in ALD.

Hypothalamic pregnenolone mediates recognition memory in the context of metabolic disorders.

Obesity and type 2 diabetes are associated with cognitive dysfunction. Because the hypothalamus is implicated in energy balance control and memory disorders, we hypothesized that specific neurons in this brain region are at the interface of metabolism and cognition. Acute obesogenic diet administration in mice impaired recognition memory due to defective production of the neurosteroid precursor pregnenolone in the hypothalamus. Genetic interference with pregnenolone synthesis by Star deletion in hypothalamic POMC, but not AgRP neurons, deteriorated recognition memory independently of metabolic disturbances. Our data suggest that pregnenolone's effects on cognitive function were mediated via an autocrine mechanism on POMC neurons, influencing hippocampal long-term potentiation. The relevance of central pregnenolone on cognition was also confirmed in metabolically unhealthy patients with obesity. Our data reveal an unsuspected role for POMC neuron-derived neurosteroids in cognition. These results provide the basis for a framework to investigate new facets of POMC neuron biology with implications for cognitive disorders.

Distinctive functioning of STARD1 in the fetal Leydig cells compared to adult Leydig and adrenal cells. Impact of Hedgehog signaling via the primary cilium.

STARD1 stimulates cholesterol transfer to mitochondrial CYP11A1 for conversion to pregnenolone. A cholesterol-binding START domain is guided by an N-terminal domain in a cell selective manner. Fetal and adult Leydig cells (FLC, ALC) show distinct Stard1 regulation. sm- FISH microscopy, which resolves individual molecules of Stard1 mRNA, shows uniformly high basal expression in each FLC. In ALC, in vivo, and cultured MA-10 cells, basal Stard1 expression is minimal. PKA activates loci asynchronously, with delayed splicing/export of 3.5 kb mRNA to mitochondria. After 60 min, ALC transition to an integrated mRNA delivery to mitochondria that is seen in FLC. Sertoli cells cooperate in Stard1 stimulation in FLC by delivering DHH to the primary cilium. There PTCH, SMO and cholesterol cooperate to release GLI3 to activate the Stard1 locus, probably by directing histone changes. ALC lack cilia. PKA then primes locus activation. FLC and ALC share similar SIK/CRTC/CREB regulation characterized for adrenal cells.

STARD1 and NPC1 expression as pathological markers associated with astrogliosis in post-mortem brains from patients with Alzheimer's disease and Down syndrome.

Alzheimer´s disease (AD) is a progressive neurodegenerative disorder of complex etiology, while Down syndrome (DS) is considered a genetically determined form of AD. Alterations in cholesterol homeostasis have been linked to AD although the role in this association is not well understood. Increased expression of STARD1 and NPC1, which are involved in intracellular cholesterol trafficking, has been reported in experimental AD models but not in patients with AD. Here we analyzed endolysosomal/mitochondrial cholesterol homeostasis, expression of NPC1 and STARD1 and correlation with pathological markers of AD in cortex and hippocampus from post-mortem brains from patients with AD and DS. NPC1 expression was observed in hippocampus from patients with AD and DS. Moreover, STARD1 expression increased in hippocampus and cortex from patients with AD and DS, respectively, and its immunoreactivity discriminated controls from AD or DS with a better accuracy than Aß42. Hippocampal areas stained with the recombinant GST-PFO probe showed increased mitochondrial cholesterol within astrocytes of brains from patients with AD and DS-brains compared to controls. Lysosomal cholesterol accumulation within hippocampal astrocytes was higher in DS than in AD. These data revealed increased intracellular cholesterol loading in hippocampus from patient with AD and DS and suggest that STARD1 could be a potential pre-clinical marker associated with early stages of AD pathology.

A new role for 14-3-3 protein in steroidogenesis.

Steroidogenic acute regulatory protein (STARD1) is regulated by phosphorylation and 14-3-3 protein binding. STARD1 is a key player in cholesterol transport in mitochondria, and its regulation is not fully understood. Tugaeva et al. provide novel insights on the site-specific phosphorylation and subsequent 14-3-3-dependent regulation of STARD1 function. These results may help us understand the mechanism behind the regulation of steroidogenesis. Comment on: https://doi.org/10.1111/febs.15474.

Mechanisms of cholesterol metabolism imbalance in a PA-induced non-alcoholic fatty liver disease cell model / 中国药科大学学报

@#Liver cholesterol metabolism disorder plays an important role in the development of non-alcoholic fatty liver disease (NAFLD).In order to reveal the molecular mechanism of cholesterol homeostasis imbalance induced by saturated fatty acids, HepG2 cells were stimulated with palmitic acid (PA).Lipids accumulation was analyzed by Oil Red O staining, intracellular triglyceride and cholesterol quantification.The level of genes and proteins related to cholesterol homeostasis was measured by RT-qPCR and western blotting.Additionally, intracellular bile acids and mitochondrial oxysterols were detected by LC-MS/MS.The results demonstrated that intracellular lipids such as TG and TC were significantly increased in the model with PA stimulation.Although no significant difference was detected in genes related to cholesterol synthesis and uptake, the protein expression of ABCG5 and LXRα were significantly down-regulated, indicating a decrease in cholesterol efflux.Meanwhile, the gene expression of STARD1 and CYP7B1, which are responsible for bile acid alternative synthesis, were markedly enhanced, along with a significant increase of cholesterol and 27-OHC in mitochondria and CDCA in cells.These results suggested that PA overload may disrupt cholesterol homeostasis by inhibiting cholesterol efflux and promoting bile acids synthesis.

Thirty-Eight-Year Follow-Up of Two Sibling Lipoid Congenital Adrenal Hyperplasia Patients Due to Homozygous Steroidogenic Acute Regulatory (STARD1) Protein Mutation. Molecular Structure and Modeling of the STARD1 L275P Mutation.

Objective: Review the impact of StAR (STARD1) mutations on steroidogenesis and fertility in LCAH patients. Examine the endocrine mechanisms underlying the pathology of the disorder and the appropriate therapy for promoting fertility and pregnancies. Design: Published data in the literature and a detailed 38-year follow-up of two sibling LCAH patients. Molecular structure and modeling of the STARD1 L275P mutation. Setting: University hospital. Patients: Patient A (46,XY female phenotype) and patient B (46,XX female) with LCAH bearing the L275P mutation in STARD1. Interventions: Since early-age diagnosis, both patients underwent corticoid replacement therapy. Patient A received estrogen therapy at pubertal age. Clomiphene therapy was given to Patient B to induce ovulation. Pregnancies were protected with progesterone administration. Main Outcome Measures: Clinical and molecular assessment of adrenal and gonadal functions. Results: Both patients have classic manifestations of corticosteroid deficiency observed in LCAH. Time of onset and severity were different. Patient A developed into a female phenotype due to early and severe damage of Leydig cells. Patient B started a progressive pubertal development, menarche and regular non-ovulatory cycle. She was able to have successful pregnancies. Conclusions: Understanding the molecular structure and function of STARD1 in all steroidogenic tissues is the key for comprehending the heterogeneous clinical manifestations of LCAH, and the development of an appropriate strategy for the induction of ovulation and protecting pregnancies in this disease.

STARD6 is expressed in steroidogenic cells of the ovary and can enhance de novo steroidogenesis.

STARD6 is a member of the StAR-related lipid transfer (START) domain family of proteins whose function thus far remains obscure. While it recently was shown to facilitate steroidogenesis in a cell-free setting, it has not been localized to steroidogenic cells of normal reproductive tissues. In a recent microarray study, we detected STARD6 mRNA in cultured porcine ovarian granulosa cells which are steroidogenic. In the present study, we examined regulation of STARD6 mRNA in porcine granulosa cultures, and found that it was not regulated by cyclic AMP, but it was reduced by combined knockdown of the transcription factors GATA4 and GATA6. We detected both STARD6 mRNA and protein in fresh granulosa cells and whole antral follicles and different stage corpora lutea of pig. The highest levels were discovered in the mid-luteal phase corpus luteum. Immunolocalization within ovarian tissues indicated robust STARD6 immunoreactivity in steroidogenic cells of the corpus luteum. Relatively lesser amounts of STARD6 signal were found in granulosa cells, theca cells, and oocytes. To test the ability of STARD6 to facilitate de novo steroidogenesis, non-steroidogenic COS-1 cells were co-transfected with components of the P450 cholesterol side-chain cleavage system, enabling them to make pregnenolone, and STARD6. STARD6 increased pregnenolone production by two- to three-fold over the empty vector control. In summary, STARD6 is found in the pig ovary, exhibits the strongest expression in highly steroidogenic luteal cells, and significantly enhances pregnenolone production in transfected COS cells independent of cyclic AMP treatment. Collectively, these findings indicate that STARD6 may contribute to steroidogenesis in ovarian cells, but also suggests other cellular functions that require cholesterol trafficking.

Macrophage mitochondrial damage from StAR transport of 7-hydroperoxycholesterol: implications for oxidative stress-impaired reverse cholesterol transport.

StAR family proteins in vascular macrophages participate in reverse cholesterol transport (RCT). We hypothesize that under pathophysiological oxidative stress, StARs will transport not only cholesterol to macrophage mitochondria, but also pro-oxidant cholesterol hydroperoxides (7-OOHs), thereby impairing early-stage RCT. Upon stimulation with dibutyryl-cAMP, RAW264.7 macrophages exhibited a strong time-dependent induction of mitochondrial StarD1 and plasma membrane ABCA1, which exports cholesterol. 7α-OOH uptake by stimulated RAW cell mitochondria (like cholesterol uptake) was strongly reduced by StarD1 knockdown, consistent with StarD1 involvement. Upon uptake by mitochondria, 7α-OOH (but not redox-inactive 7α-OH) triggered lipid peroxidation and membrane depolarization while reducing ABCA1 upregulation. These findings provide strong initial support for our hypothesis.

Steroidogenic acute regulatory protein expression in the central nervous system.

Locally produced neurosteroids are proposed to have many functions in the central nervous system. The identification of the steroidogenic acute regulatory protein in steroid-producing neural cells provides a new tool to understand the sites, regulation, and importance of their synthesis.