Abstract
BackgroundMutation in a tuberous sclerosis gene (TSC1 or 2) leads to continuous activation of the mammalian target of rapamycin (mTOR). mTOR activation alters cellular including vitamin A metabolism and retinoic acid receptor beta (RARβ) expression. The goal of the present study was to investigate the molecular connection between vitamin A metabolism and TSC mutation. We also aimed to investigate the effect of the FDA approved drug rapamycin and the vitamin A metabolite retinoic acid (RA) in cell lines with TSC mutation.MethodsExpression and activity of vitamin A associated metabolic enzymes and RARβ were assessed in human kidney angiomyolipoma derived cell lines, primary lymphangioleiomyomatosis (LAM) tissue derived LAM cell lines. RARβ protein levels were also tested in primary LAM lung tissue sections. TaqMan arrays, enzyme activities, qRT-PCRs, immunohistochemistry, immunofluorescent staining, and western blotting were performed and analysed. The functional effects of retinoic acid (RA) and rapamycin were tested in a scratch and a BrDU assay to assess cell migration and proliferation.ResultsMetabolic enzyme arrays revealed a general deregulation of many enzymes involved in vitamin A metabolism including aldehyde dehydrogenases (ALDHs), alcohol dehydrogenases (ADHs) and Cytochrome P450 2E1 (CYP2E1). Furthermore, RARβ downregulation was a characteristic feature of all TSC-deficient cell lines and primary tissues. Combination of the two FDA approved drugs -RA for acute myeloid leukaemia and rapamycin for TSC mutation- normalised ALDH and ADH expression and activity, restored RARβ expression and reduced cellular proliferation and migration.ConclusionDeregulation of vitamin A metabolizing enzymes is a feature of TSC mutation. RA can normalize RARβ levels and limit cell migration but does not have a significant effect on proliferation. Based on our data, translational studies could confirm whether combination of RA with reduced dosage of rapamycin would have more beneficial effects to higher dosage of rapamycin monotherapy meanwhile reducing adverse effects of rapamycin for patients with TSC mutation.
Highlights
Mutation in a tuberous sclerosis gene (TSC1 or 2) leads to continuous activation of the mammalian target of rapamycin. mTOR activation alters cellular including vitamin A metabolism and retinoic acid receptor beta (RARb) expression
In our previous study of LAM, apart from detecting morphological abnormalities in mitochondria and suppression of ROS production, we identified downregulation of the proliferation suppressor nuclear receptors of the retinoic acid gene family both retinoic acid receptor (RAR) and retinoid x receptor (RXR) as well as several miRNA-s that regulate RAR expression including miR29b [6]
To investigate the involvement of the enzyme cascades associated with vitamin A metabolism in TSC deficient cells, human enzyme profiler arrays (RT2 PCR) were used to compare mRNA levels of specific enzymes in the human kidney angiomyolipoma cell line S102 (TSC2-/-) and its control S103 (TSC2+/+)
Summary
Mutation in a tuberous sclerosis gene (TSC1 or 2) leads to continuous activation of the mammalian target of rapamycin (mTOR). mTOR activation alters cellular including vitamin A metabolism and retinoic acid receptor beta (RARb) expression. Mutation in a tuberous sclerosis gene (TSC1 or 2) leads to continuous activation of the mammalian target of rapamycin (mTOR). MTOR activation alters cellular including vitamin A metabolism and retinoic acid receptor beta (RARb) expression. We aimed to investigate the effect of the FDA approved drug rapamycin and the vitamin A metabolite retinoic acid (RA) in cell lines with TSC mutation. The above diseases are caused by the mutation of tumour suppressor genes tuberous sclerosis 1 or 2 (TSC or TSC2) [1]. RAR and RXR are receptors of retinoic acid (RA), a metabolite of vitamin A [7] Both classes of nuclear receptors have three subtypes (a, b, and g) and in-patient derived LAM cell lines RARb mRNA expression was found significantly reduced [6].
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