ESPE Abstracts (2019) 92 FC13.5

SGPL1 Deficiency Leads to Downregulation of Key Enzymes Within the Steroidogenic Pathway

Avinaash Maharaj1, Eirini Meimaridou2, Jack Williams1, Tülay Güran3, Debora Braslavsky4, Louise Metherell1, Rathi Prasad1


1Centre for Endocrinology, William Harvey Research Institute, John Vane Science Centre, Queen Mary, University of London, Charterhouse Square, London, United Kingdom. 2School of Human Sciences, London Metropolitan University, London, United Kingdom. 3Marmara University, School of Medicine, Department of Paediatric Endocrinology and Diabetes, Istanbul, Turkey. 4Centro de Investigaciones Endocrinológicas "Dr. Cesar Bergadá" (CEDIE) – CONICET – FEI – División de Endocrinología, Hospital de Niños "Ricardo Gutiérrez,", Buenos Aires, Argentina


Background: SGPL1 deficiency is associated with a pathological accumulation of sphingolipid intermediates and a multi-systemic condition incorporating primary adrenal insufficiency. Sphingolipid intermediates such as ceramide, sphingosine and sphingosine 1-phosphate are postulated to act as modulators of the steroidogenic pathway, often acting as second messengers altering downstream expression of steroid responsive transcriptional elements. Ceramide and sphingosine are largely inhibitors of steroidogenesis with sphingosine acting as an endogenous antagonist to steroidogenic factor 1 (SF-1) maintaining it in an inactive conformation whilst acute activation of S1P signalling in H295R cells leads to increased transcription of STAR and hormone sensitive lipase (HSL) culminating in an increase in cortisol production. Pathological accumulation of these sphingolipid intermediates as seen in SGPL1 deficiency may therefore have negative implications for the steroidogenic cascade.

Objective and Hypotheses: Investigation of the impact of SGPL1 deficiency on steroidogenesis using patient derived human dermal fibroblasts and RNA-seq interrogation of the differential expression of steroidogenic genes in an SGPL1-KD human adrenocortical cell line.

Methods: 1. Primary cell cultures of human dermal fibroblasts were established from skin biopsies of two patients with SGPL1 mutations (Patient1 - p.F545del; Patient 2 - p.S65Rfs*6G) and primary adrenal insufficiency. The steroidogenic capacity of cultured fibroblasts was explored using a precursor substrate, progesterone, as a stimulator of cortisol accumulation. Culture media from treated and untreated cells were subjected to cortisol measurement (ELISA). STAR expression was quantified by RT-qPCR. 2. Lentiviral shRNA mediated KD of SGPL1 in a human adrenocortical cell line (H295R) with subsequent RNA-seq interrogation.

Results: Wild-type (control) dermal fibroblasts showed a significant cortisol response after progesterone stimulation (P<0.001). In comparison Patient 1 fibroblasts were significantly less responsive to stimulation (P<0.05) and Patient 2 cells were unresponsive to stimulation with no cortisol production (P<0.001). Concurrently, mRNA expression of STAR was significantly reduced (5-fold change, P<0.001) in patient 2 fibroblasts compared to control. Differential gene expression data from RNA-seq revealed functional enrichment of genes for the metabolism of steroids (FDR 0.045) and cortisol synthesis and secretion (FDR 0.0496). Further interrogation revealed significant downregulation of steroidogenic genes, with reduced transcript levels of STAR, CYP21A2 and CYP11B1 in the SGPL1-KD H295R.

Conclusion: Our results are in keeping with a prominent role for sphingolipids in modulating the acute phase of steroidogenesis, suggesting that alterations in sphingolipid metabolism due to SGPL1 deficiency negatively impact the expression of genes responsible for steroid hormone biosynthesis.

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