MAMLD1 promotes cholesterol uptake through the transcriptional regulation of SCARB1 in mouse testicular cell lines

To the Editor: Mastermind like domain containing 1 (MAMLD1, Online Mendelian Inheritance in Man [OMIM] #300120) is a causative gene of 46,XY disorders of sex development (DSDs). Previous studies have suggested that MAMLD1 may cause 46,XY DSDs by affecting testosterone (T) synthesis in Leydig cells. However, the mechanism by which MAMLD affects T biosynthesis is unknown. In this study, we identified five patients with 46,XY DSDs and aimed to explore the relationship between MAMLD1 variants and 46,XY DSDs. Overall, we found that MAMLD1 plays a novel role in regulating the transcription of scavenger receptor class B, member 1 (SCARB1; OMIM#601040), thereby affecting cholesterol homeostasis in mouse Leydig cell lines. These results offer insights into the mechanisms of action of MAMLD1 in 46,XY DSDs. This study was conducted in accordance with the ethical standards of the responsible committee on human experimentation and with the Declaration of Helsinki of 1975, as revised in 2000. This study was approved by the Ethics Committee of the Beijing Children’s Hospital of Capital Medical University’s Ethics Committee (No. 2019-k-156). Written informed consent for participation in the study was obtained from the children’s parents. The clinical characteristics and MAMLD1 genotypes of the five 46,XY DSD patients are shown in Supplementary Table 1, https://links.lww.com/CM9/C772. The variants in all five patients were inherited from their mothers without DSD characterization. Four of the five MAMLD1 variants (p. R7Q, p. L217X, p. R481X, and p. Q502Afs*7) have not been previously reported. To determine the subcellular localization of MAMLD1, mouse Leydig tumor cell line (MLTC-1) cells were transiently transfected with a wildtype (WT)-MAMLD1 fusion protein expression vector (WT-MAMLD1-Flag). Immunofluorescence staining with both anti-MAMLD1 and anti-Flag antibodies showed colocalization in the nucleus in a diffuse pattern [Supplementary Figure 1, https://links.lww.com/CM9/C772]. Next, we examined the localization of MAMLD1 in MLTC-1 cells. The missense variants included the newly identified MT-R7Q-Flag and three previously reported variants (MT-A446P-Flag, MT-P567S-Flag, and MT-P359S-Flag), all diffusely located in the nucleus. The three truncated variants examined in this study (MT-R381X-Flag, MT-R481X-Flag, and MT-Q502Afs*7-Flag) were also diffusely localized in the nucleus [Supplementary Figure 1, https://links.lww.com/CM9/C772]. These observations demonstrate that the MAMLD1 variants tested did not alter their subcellular localization. To investigate the role and mechanism of MAMLD1 in regulating T cell production, we chose five genes (Scarb1, Hsd3b1, Cyp17a1, Cyp11a1, Apoe) related to T cell biosynthesis to validate the correlation between MAMLD1 and T cell synthesis. Real-time quantitative polymerase chain reaction (RT-qPCR) results showed that depletion of MAMLD1 decreased the mRNA levels of Scarb1, Hsd3b1, and Cyp11a1, but increased the mRNA level of Apoe [Supplementary Figure 2A, https://links.lww.com/CM9/C772]. In addition, overexpression of WT-MAMLD1-Flag in MLTC-1 cells led to upregulated Scarb1 mRNA levels, but not Hsd3b1, Cyp17a1, or Cyp11a1 mRNA levels [Supplementary Figure 2B, https://links.lww.com/CM9/C772]. These results suggest that MAMLD1 regulates Scarb1 expression. Given the importance of SCARB1 in modulating cholesterol metabolism, we measured the level of the protein scavenger receptor class B, type I (Srb1) encoded by Scarb1 in Mamld1-KD or MAMLD1-overexpression MLTC-1 cells. The results showed that Mamld1-KD downregulated Sr-b1 expression, and overexpression of WT-MAMLD1-Flag increased Sr-b1 expression [Supplementary Figure 2C–F, https://links.lww.com/CM9/C772]. These data suggest that MAMLD1 regulates the expression of SCARB1 at the transcriptional level. To elucidate the molecular basis of the transcriptional regulation of SCARB1 by MAMLD1, we performed a dual-luciferase reporter assay to examine whether MAMLD1 could activate the transcription of the promoter region of SCARB1 (−869 bp to +237 bp). To avoid interference from endogenous MAMLD1, we selected murine Leydig epithelioid cell line (TM3) cells with low expression of Mamld1 for this experiment. Our results showed that cotransfection with MAMLD1 rather than the empty vector resulted in pronounced activation of promega Luciferase Reporter Vector 3 (pGL3)-SCARB1 luciferase in TM3 cells [Supplementary Figure 3A, https://links.lww.com/CM9/C772]. Next, we examined whether the MAMLD1 variants in our cohort affected the ability to transactivate SCARB1. The results showed that the truncated variants (MT-R481X-Flag and MT-Q502Afs*7-Flag) led to the loss of transactivation function of SCARB1, and missense variants (MT-R7Q-Flag, MT-A446P-Flag, P567S-Flag, and MT-P359S-Flag) led to reduced transactivation function [Supplementary Figure 3B, https://links.lww.com/CM9/C772]. Since SR-B1 is the major receptor of high-density lipoprotein (HDL) on the surface of steroidogenic cells, which is mainly responsible for the uptake of cholesterol esters from HDL, we next examined whether Mamld1-KD affects the cholesterol uptake ability of Leydig cells using a Dil-HDL uptake assay. The results showed that depletion of Mamld1 markedly reduced cholesterol uptake compared with that in control cells [Figure 1A, B]. Finally, we used RNA interference to inhibit Mamld1 expression in MLTC-1 cells and verified that depletion of Mamld1 affected the T secretion of MLTC-1 cells [Figure 1C, D].Figure 1: Mamld1-KD perturbs cholesterol uptake and testosterone synthesis of MLTC-1 cells. (A and B) Dil-HDL uptake assays were performed to evaluate cholesterol uptake of the cells using live-cell confocal microscopy. The experiments were repeated at three different times. Scale bars: 200 pixels. (C and D) the knockdown efficiency of si-Mamld1-1 was validated by Western blotting, and the level of testosterone in culture media was measured with the enzyme-linked immunosorbent assay. The experiment was repeated at five different times. si-NC: Negative control group, si-Mamld1: Knockdown group. Data are shown as mean ± standard error of the mean (two-tailed t-test: * P <0.01; † P <0.001). MLTC: Mouse Leydig tumor cell-1; Dil: 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; MW: Molecular weight; NC: Negative control; HDL: High-density lipoprotein; KD: Knockdown.Our study revealed that MAMLD1 transcriptionally regulates SCARB1 expression and subsequently affects cholesterol homeostasis in MLTC-1 cells. The SR-B1-mediated “selective” uptake pathway is an important source of cholesterol for steroidogenic cells. Cholesterol source preferences vary among species, tissues, and cells. In rat adrenal glands and ovaries, cholesterol is preferentially taken from plasma lipoprotein, while it mainly relies on endogenous de novo synthesis under normal physiological states in rat mature Leydig cells.[1] However, the expression of SR-B1 in Leydig cells can be significantly increased by long-term stimulation of hCG (approximately a 20-fold increase), suggesting that Leydig cells can obtain cholesterol through the “selective” uptake pathway when the endogenous cholesterol esters are depleted. During the critical period of human embryonic sexual development, in order to meet male sexual development demands, human fetal Leydig cells need to produce a large amount of T under the stimulation of hCG, which results in the peak of T secretion at 11–17 weeks after embryo formation and promotes male sexual differentiation.[2] The “selective” uptake pathway mediated by SR-B1 uptakes cholesterol into cells, whereas the lipoprotein particles remain outside the cells, which is a high-capacity, physiologically regulated cholesterol transport system compared with other cholesterol-derived pathways. We speculate that this pathway may be used to meet cholesterol requirements during the critical period of male embryonic sexual development. Recent studies have shown that decreased T synthesis in human azoospermia and oligospermia patients results from autophagy-induced downregulation of SR-B1 in Leydig cells.[3] In addition, a clinical study showed a strong correlation between HDL cholesterol and serum T levels in middle-aged and older men.[4] These studies support a correlation between HDL levels mediated by SR-B1 and T production. The role of SR-B1 in the testis may be underestimated, and the phenotypic differences between Mamld1-KO mice and 46,XY DSD patients carrying MAMLD1 variants may be due to species and tissue differences in SR-B1 expression. This study also has some limitations. First, due to financial and technical constraints, the research mainly focuses on in vitro cell analysis and lacks in vivo validation. In vivo studies could reinforce the conclusions and further clarify the physiological relevance of MAMLD1-mediated SCARB1 regulation. Second, Co-Immunoprecipitation (Co-IP) or pull-down experiments were not conducted to explore the potential interactions between MAMLD1 and other transcription factors or coactivators involved in SCARB1 transcription. In conclusion, we identified five patients carrying four novel MAMLD1 variations, which expands the variation spectrum of the MAMLD1 gene in 46,XY DSDs. We demonstrated that MAMLD1 can transcriptionally regulate SCARB1 and affect the cholesterol uptake ability of testicular Leydig cells by regulating the expression of SR-BI, thereby affecting the biosynthesis of testosterone. Funding This work was supported by grants from the Beijing Natural Science Foundation (No. 7204260) and the Beijing Hospitals Authority Youth Program (No. QML20201205). Conflicts of interest None.