Conclusion: These findings establish SDR42E1 as a key modulator of vitamin D-related pathways and highlight its potential as a therapeutic target for addressing vitamin D deficiency and associated pathologies, including cancer.
Results: Integrated transcriptomic and proteomic analyses revealed significant dysregulation of sterol absorption and metabolism (fold change (FC) = 1.8, P = 0.007) and cancer-related signaling pathways (FC = −1.7, P = 0.02). Notably, key differentially expressed genes included upregulated LRP1B and ABCC2 , alongside downregulated WNT16 and SLC7A5 . Proteomic profiling confirmed alterations in cell proliferation-related proteins, including reduced ALDOA expression (FC = −0.37, P = 0.0005). Functionally, SDR42E1 deficiency reduced cell viability by 53% (P = 0.0001), an effect reversed by transient SDR42E1 overexpression with restoring ABCC2 expression.
Methods: This study investigates the role of SDR42E1 , a gene encoding a short-chain dehydrogenase/reductase enzyme, in vitamin D regulation and sterol metabolism. Using CRISPR/Cas9 gene-editing, we generated an SDR42E1 knock-in model in HCT116 colorectal cells, which exhibit high endogenous SDR42E1 expression, harboring a nonsense variant associated with vitamin D deficiency.
Introduction: Vitamin D is a pleiotropic hormone essential for bone health and overall physiological function. Despite its significance, vitamin D deficiency remains widespread and is often influenced by genetic factors.
1 Introduction
Vitamin D is a vital fat-soluble nutrient crucial for calcium and phosphorus homeostasis, bone health, and immune function. Despite the availability of dietary sources and sunlight exposure (1), deficiencies can arise due to impaired absorption and metabolism. These processes are orchestrated by a network of critical proteins, like ATP-binding cassette B member 2 (ABCB2) and solute carrier family proteins, including SLC7A5 (2, 3). In circulation, vitamin D binds to vitamin D-binding protein (VDBP) and undergoes hydroxylation in the liver by cytochrome P450 enzymes such as CYP2R1, CYP11A1, and CYP27A1, which hydroxylate vitamin D, followed by activation in the kidney by CYP27B1. The vitamin D receptor (VDR) mediates its biological effects, while CYP24A1 regulates its inactivation (4). Both VDR and CYP24A1 influence these processes, although key mechanistic gaps remain incompletely understood (5).
Short-chain Dehydrogenase/Reductase 42E member 1 (SDR42E1), a member of the short-chain dehydrogenase/reductase enzyme family plays a potentially significant role in lipid and steroid metabolism (6–8), potentially functioning as an oxidoreductase and steroid delta-isomerase (9, 10). The largest genome-wide association study (GWAS) on vitamin D deficiency (11), identified a nonsense variant (rs11542462) on chromosome 16q23 that replaces glutamine with a termination codon at position 30 (p.Q30*), producing a truncated, non-functional SDR42E1 enzyme (12–14). Interestingly, this variant correlates with elevated serum levels of 8-dehydrocholesterol (8-DHC) and 7-dehydrocholesterol (7-DHC), precursors in vitamin D synthesis (15). Lately, our in silico studies identified these sterols, along with vitamin D 3 and 25-hydroxyvitamin D (25(OH)D), as potential substrates of the SDR42E1 (9); however, its precise role in vitamin D metabolism remains unclear.
Our recent research, utilizing clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene-editing technology in skin keratinocytes, demonstrated that SDR42E1 depletion disrupts the steroid biosynthesis, leading to accumulation of 7-DHC and a concomitant reduction in vitamin D levels. Additionally, SDR42E1 mRNA showed elevated expression in intestinal epithelial cells and the analogous colorectal HCT116 cell line, underscoring its putative role in vitamin D homeostasis within the gastrointestinal tract (16). Building on these observations, this study seeks to elucidate the functional role of SDR42E1 in regulating intestinal vitamin D absorption and sterol metabolism. To achieve this, we employed CRISPR/Cas9-mediated gene editing in HCT116 cells to introduce a nonsense variant of SDR42E1 previously associated with vitamin D deficiency. Comprehensive transcriptomic and proteomic analyses were then performed to characterize downstream molecular alterations of SDR42E1 disruption. Through this integrative approach, we aim to advance mechanistic understanding of SDR42E1’s contribution to vitamin D homeostasis and to explore its broader implications in metabolic regulation and disease pathogenesis.
2 Materials and methods
2.1 Cell culture
... continue reading