Functional characterization of the SDR42E1 reveals its role in vitamin D biosynthesis

Abstract

Vitamin D deficiency poses a widespread health challenge, shaped by environmental and genetic determinants. A recent discovery identified a genetic regulator, rs11542462, in the SDR42E1 gene, though its biological implications remain largely unexplored. Our bioinformatic assessments revealed pronounced SDR42E1 expression in skin keratinocytes and the analogous HaCaT human keratinocyte cell lines, prompting us to select the latter as an experimental model. Employing CRISPR/Cas9 gene-editing technology and multi-omics approach, we discovered that depleting SDR42E1 showed a 1.6-fold disruption in steroid biosynthesis pathway (P-value = 0.03), considerably affecting crucial vitamin D biosynthesis regulators. Notably, SERPINB2 (P-value = 2.17 × 10^-103), EBP (P-value = 2.46 × 10^-13), and DHCR7 (P-value = 8.03 × 10^-09) elevated by ∼2-3 fold, while ALPP (P-value <2.2 × 10^-308), SLC7A5 (P-value = 1.96 × 10^-215), and CYP26A1 (P-value = 1.06 × 10^-08) downregulated by ∼1.5-3 fold. These alterations resulted in accumulation of 7-dehydrocholesterol precursor and reduction of vitamin D₃ production, as evidenced by the drug enrichment (P-value = 4.39 × 10^-06) and total vitamin D quantification (R² = 0.935, P-value = 0.0016) analyses. Our investigation unveils SDR42E1's significance in vitamin D homeostasis, emphasizing the potential of precision medicine in addressing vitamin D deficiency through understanding its genetic basis.

Keywords: CRISPR/Cas9; HaCaT; Multi-omics; SDR42E1; Steroidogenesis; Vitamin D biosynthesis.

Fig. 1

Transient protein and transcript expression of SDR42E1 in gene-edited and wild-type HaCaT cells. a, Immunoblotting of protein extracts from wild-type HaCaT cells (lane 1) and cells with transient overexpression of wild-type SDR42E1-HA (44 kDa; lane 2, indicated by a red arrow) was performed using rabbit SDR42E1-tag polyclonal antibody at a 1:1000 dilution. Ponceau-S Red staining (PonS) served as a loading control. b, HaCaT cells with wild-type SDR42E1 expression (green) were stained overnight with rabbit αHA-tag polyclonal antibody at a 1:100 dilution, with nuclear localization revealed by Hoechst (blue). Cells were transiently transfected with a 5-μg SDR42E1-HA tagged plasmid for 24 h c, WB analysis of immunoprecipitated SDR42E1 in whole protein lysate derived from the gene-edited HaCaT cells of clone 32 (SDR42E1-KO-32) revealed the absence of a 44 kDa band corresponding to SDR42E1 (highlighted by a red arrow) using rabbit SDR42E1 polyclonal antibody (PA5-53156, Invitrogen). Before the IP experiments, one-tenth of total lysates were subjected to the respective WB as input controls using an anti-beta-actin mouse antibody (A5441, Sigma, 1:5000 dilution). Ctrl-Cas9 is an untargeted sgRNA-Cas9 vector in HaCaT as a negative control. d, RT-qPCR analysis revealed significantly decreased SDR42E1 transcript expression in SDR42E1-KO-32 compared to controls (Ctrl-Cas9). The relative expression level of SDR42E1 was normalized by the internal control β-actin. Data represent the mean ± standard deviation of three replicates, with similar results and significant differences relative to Cas9 control were analyzed by t-test with p < 0.0001 (****).

Fig. 2

Extensive alterations in gene expressions in the SDR42E1 knockout model. a, A PCA plot demonstrates the clustering of three biological replicates of wild-type HaCaT controls (C; in rose) and SDR42E1 homozygous knockouts (Hom, in blue), through the major principal components of the regularized log-transformed counts. b, An MDS plot illustrates the correlation between log₂ fold change and the mean of the normalized counts in SDR42E1 knockouts, with significant DEG highlighted in blue (adjusted P-value <0.05). c, A volcano plot depicts significant gene expression changes in SDR42E1 knockouts compared to wild-type controls. The X-axis displays the log₂ fold change (FC), with upregulated genes to the right and downregulated to the left, while the Y-axis represents the false discovery rate (FDR). Points represent individual genes with detectable expression changes, meeting the criteria of an adjusted P-value <0.05 and a Log₂FC > 1, with the top 20 most significantly altered genes labeled. d, A cluster heatmap shows the Z-scores of regularized log-transformed counts for the top 100 DEG, with blue indicating lower and red indicating higher expression, highlighting the distinct separation of sample conditions. The X- and Y-axes are labeled with sample names and DEGs, respectively. All analyses were conducted and visualized using R/DESeq2 and Pheatmap.

Fig. 3

Extensive alterations in gene pathways in the SDR42E1 knockout model. a, A dot plot illustrates enriched KEGG pathways for DEG. The Y-axis represents the KEGG pathways; the X-axis represents the ratio of the genes enriched in the KEGG pathway. A KEGG pathway diagram, enhanced via the R/Pathview package, shows the expression profiles of genes involved in b, steroid biosynthesis and c, in steroid hormone biosynthesis. Red indicates genes upregulated, while green denotes genes downregulated by the SDR42E1 knockout. d, A bar plots of signature drugs associated with SDR42E1 knockout through DSigDB. The color and size of dots and bars reflect the significance and count of DEG linked to KEGG pathways and drugs, respectively. P-value adjusted (p. adjust) < 0.05 was used as the threshold to select KEGG terms.

Fig. 4

Extensive alterations in protein expressions and pathways in the SDR42E1 knockout model. a, A PCA plot reveals the clustering of three biological replicates of wild-type HaCaT controls (C; in rose) and SDR42E1 homozygous knockouts (Hom, in blue), based on the major components of regularized log-transformed counts. b, A heatmap displays the Z-scores of log-transformed counts for the top 100 differentially expressed proteins, using blue to indicate lower and red for higher expression, highlighting clear separation between samples. Axes are labeled with sample names and proteins. c, A volcano plot compares the proteomic data of homozygous SDR42E1 knockout cells to wild-type HaCaT controls. The X-axis shows log₂ fold change (FC), with significant upregulation to the right (greater than 1) and downregulation to the left (less than −1). The Y-axis shows the −log of the false discovery rate (FDR), marking significance below 0.05. The top 20 significantly altered proteins are highlighted. d, A GSE dot plot presents pathway enrichment analysis of the differentially expressed proteins in SDR42E1 homozygous knockouts. These analyses were conducted and visualized using R/Limma, Pheatmap, and ClusterProfiler.

Fig. 5

Decreased vitamin D levels in the SDR42E1 knockout model. Vitamin D levels in the SDR42E1 knockout model was measured with a vitamin D ELISA assay. Compared to wild-type HaCaT cells, the knockout model showed significantly reduced vitamin D levels across three replicated experiments (**; P-value <0.01).

Fig. 6

Potential role of SDR42E1 in vitamin D biosynthesis and regulation. The pathway illustrates the influence of SDR42E1 absence in vitamin D skin synthesis from 7-DHC upon solar ultraviolet B (UVB) exposure. To boost vitamin D levels, the body increases the conversion of 8-DHC or cholesterol to 7-DHC by upregulating enzymes EBP or DHCR7. Intestinal absorption of vitamin D is also improved by the upregulation of ABCB1. The liver then enhances the conversion to 25(OH)D by upregulating CYP27A1 or CYP3A4, and the kidneys increase activation to 1,25-dihydroxyvitamin D via CYP27B1 upregulation, regulating related-gene expressions via vitamin D receptor (VDR)/retinoid-X receptor (RXR) complex. The inactivation and secretion process, usually facilitated by the CYP24A1 enzyme, is also diminished. Red indicates proteins upregulated, while green denotes proteins downregulated by the SDR42E1 absence. (?) indicates our proposed SDR42E1 involvement in vitamin D biosynthesis. 2D chemical structures obtained from PubChem: https://pubchem.ncbi.nlm.nih.gov. Generated with BioRender.com.